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SERIES EDITOR

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Life Sciences Department
The Natural History Museum, London, UK
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EDITORIAL BOARD
M. G. BASÁÑEZ R. E. SINDEN
Professor in Parasite Epidemiology, Immunology and Infection Section,
Department of Infectious Disease Department of Biological Sciences,
Epidemiology Faculty of Medicine Sir Alexander Fleming Building, Imperial
(St Mary’s Campus), Imperial College, College of Science, Technology and
London, London, UK Medicine, London, UK

S. BROOKER D. L. SMITH
Wellcome Trust Research Fellow and Johns Hopkins Malaria Research
Professor, London School of Hygiene and Institute & Department of Epidemiology,
Tropical Medicine, Faculty of Infectious Johns Hopkins Bloomberg School of Public
and Tropical, Diseases, London, UK Health, Baltimore, MD, USA

R. B. GASSER R. C. A. THOMPSON
Department of Veterinary Science, Head, WHO Collaborating Centre for
The University of Melbourne, Parkville, the Molecular Epidemiology of Parasitic
Victoria, Australia Infections, Principal Investigator,
Environmental Biotechnology CRC
N. HALL (EBCRC), School of Veterinary and
School of Biological Sciences, Biomedical Sciences, Murdoch University,
Biosciences Building, University of Murdoch, WA, Australia
Liverpool, Liverpool, UK
X. N. ZHOU
R. C. OLIVEIRA Professor, Director, National Institute of
Centro de Pesquisas Rene Rachou/ Parasitic Diseases, Chinese Center for
CPqRR - A FIOCRUZ em Minas Gerais, Disease Control and Prevention, Shanghai,
Rene Rachou Research Center/CPqRR - People’s Republic of China
The Oswaldo Cruz Foundation in the State
of Minas Gerais-Brazil, Brazil
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CONTRIBUTORS

Vahab Ali
Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences,
Indian Council of Medical Research, Agam-kuan, Patna, India
J. Kevin Baird
Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia, and Centre for Tropical
Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, United
Kingdom
Michael J. Bangs
Public Health and Malaria Control Department, International SOS, PT Freeport Indonesia,
Kuala Kencana, Indonesia
John R. Barta
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph,
Ontario, Canada
Damer Blake
Royal Veterinary College, Hatfield, United Kingdom
H. David Chapman
Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, United States
Iqbal R.F. Elyazar
Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia
Robin B. Gasser
Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia
Peter W. Gething
Spatial Ecology and Epidemiology Group, Department of Zoology, University of Oxford,
Oxford, United Kingdom
Arthur Gruber
Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São
Paulo, Brazil
Simon I. Hay
Spatial Ecology and Epidemiology Group, Department of Zoology, University of Oxford,
Oxford, United Kingdom
Mark Jenkins
Animal Parasitic Diseases Laboratory, Agricultural Research Service, USDA, Beltsville,
Maryland, United States
Aaron R. Jex
Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia

ix
x Contributors

Rita Kusriastuti
Directorate of Vector-Borne Diseases, Indonesian Ministry of Health, Jakarta, Indonesia
Tomoyoshi Nozaki
Department of Parasitology, National Institute of Infectious Diseases, Tokyo, and Graduate
School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan
Florian Roeber
Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia
Marianne E. Sinka
Spatial Ecology and Epidemiology Group, Department of Zoology, University of Oxford,
Oxford, United Kingdom
Nicholas C. Smith
Queensland Tropical Health Alliance Laboratory, Faculty of Medicine, Health and
Molecular Sciences, James Cook University, Cairns, Queensland, Australia
Xun Suo
National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural
University, Beijing, China
Asik Surya
Directorate of Vector-Borne Diseases, Indonesian Ministry of Health, Jakarta, Indonesia
Siti N. Tarmidzi
Directorate of Vector-Borne Diseases, Indonesian Ministry of Health, Jakarta, Indonesia
Fiona M. Tomley
Royal Veterinary College, Hatfield, United Kingdom
Winarno
Directorate of Vector-Borne Diseases, Indonesian Ministry of Health, Jakarta, Indonesia
CHAPTER ONE

Iron–Sulphur Clusters, Their


Biosynthesis, and Biological
Functions in Protozoan Parasites
Vahab Ali*, Tomoyoshi Nozaki†,{,1
*Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of
Medical Research, Agam-kuan, Patna, India

Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan
{
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan
1
Corresponding author: e-mail address: nozaki@nih.go.jp

Contents
1. Introduction 3
2. Variation and Features of Fe–S Clusters 4
2.1 Discovery of Fe–S clusters 4
2.2 Heterogeneity of Fe–S clusters 4
2.3 Physicochemical features and analytical methods of Fe–S clusters 10
2.4 Biochemical features of Fe–S clusters 11
3. General Biological and Physiological Roles of Fe–S Proteins in Prokaryotes
and Eukaryotes 12
3.1 Roles of Fe–S proteins in bacteria 12
3.2 Roles of Fe–S proteins in eukaryotes 15
4. General Source of Iron and Sulphur for Fe–S Cluster Biosynthesis 17
4.1 Acquisition and transport of iron 17
4.2 Acquisition, biosynthesis, and transport of L-cysteine 19
5. Four Systems for Fe–S Cluster Biogenesis in Prokaryotes and Eukaryotes 20
5.1 Catalytic reaction in Fe–S cluster biosynthesis 21
5.2 ISC machinery 21
5.3 SUF machinery 29
5.4 NIF machinery 32
5.5 CIA machinery 34
5.6 Mechanism of repair of Fe–S clusters 38
6. Genetic Disorders by a Defect of Fe–S Cluster Biogenesis 38
6.1 Friedreich’s ataxia 39
6.2 Sideroblastic anaemia 40
6.3 X-linked sideroblastic anaemia and ataxia (XLSA/A) 40
6.4 Other genetic disorders 41

Advances in Parasitology, Volume 83 # 2013 Elsevier Ltd 1


ISSN 0065-308X All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-407705-8.00001-X
2 Vahab Ali and Tomoyoshi Nozaki

7. Outline of Conservation, Unique Distribution, and Diversity of Fe–S Cluster


Biogenesis Machineries in Protozoan Parasites 41
8. Fe–S Cluster Biogenesis in Protozoan Parasites 42
8.1 Entamoeba 42
8.2 Giardia 47
8.3 Trichomonas 50
8.4 Leishmania 52
8.5 Trypanosoma 55
8.6 Plasmodium 57
8.7 Cryptosporidia 60
8.8 Microsporidia 61
8.9 Blastocystis 63
9. Regulation of Fe–S Protein Biosynthesis under Stress Conditions 65
9.1 Factors that affect Fe–S clusters 65
9.2 Stress-dependent regulation of ISC and SUF systems 66
9.3 Stress-dependent response and regulation of Fe–S cluster biosynthesis
in parasitic protozoa 67
10. Unsolved Questions and Future Perspectives 69
10.1 Acquisition and secondary loss of the machineries for Fe–S cluster
biosynthesis 69
10.2 Origins of individual machineries and crosstalk between the organellar and
cytosolic compartments 70
10.3 Cooperation of two cytosolic machineries in Entamoeba and Blastocystis:
SUF/NIF and CIA systems 71
10.4 Significance of compartmentalization of Fe–S cluster biosynthesis
in mitochondrion-related organelles (MROs) 71
10.5 New strategy for the identification of Fe–S cluster-containing proteins 72
10.6 NIF and SUF systems as drug target 72
Acknowledgements 73
References 74

Abstract
Fe–S clusters are ensembles of sulphide-linked di-, tri-, and tetra-iron centres of a variety
of metalloproteins that play important roles in reduction and oxidation of mitochondrial
electron transport, energy metabolism, regulation of gene expression, cell survival, nitro-
gen fixation, and numerous other metabolic pathways. The Fe–S clusters are assembled
by one of four distinct systems: NIF, SUF, ISC, and CIA machineries. The ISC machinery is a
house-keeping system conserved widely from prokaryotes to higher eukaryotes, while
the other systems are present in a limited range of organisms and play supplementary
roles under certain conditions such as stress. Fe–S cluster-containing proteins and the
components required for Fe–S cluster biosynthesis are modulated under stress condi-
tions, drug resistance, and developmental stages. It is also known that a defect in Fe–S
proteins and Fe–S cluster biogenesis leads to many genetic disorders in humans, which
indicates the importance of the systems. In this review, we describe the biological and
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 3

physiological significance of Fe–S cluster-containing proteins and their biosynthesis in


parasitic protozoa including Plasmodium, Trypanosoma, Leishmania, Giardia, Trichomo-
nas, Entamoeba, Cryptosporidium, Blastocystis, and microsporidia. We also discuss the
roles of Fe–S cluster biosynthesis in proliferation, differentiation, and stress response
in protozoan parasites. The heterogeneity of the systems and the compartmentalization
of Fe–S cluster biogenesis in the protozoan parasites likely reflect divergent evolution
under highly diverse environmental niches, and influence their parasitic lifestyle and
pathogenesis. Finally, both Fe–S cluster-containing proteins and their biosynthetic
machinery in protozoan parasites are remarkably different from those in their mamma-
lian hosts. Thus, they represent a rational target for the development of novel chemo-
therapeutic and prophylactic agents against protozoan infections.

1. INTRODUCTION
Iron–sulphur (Fe–S) proteins are involved in many central biological
functions such as enzymatic catalysis, electron transport, photosynthesis,
nitrogen fixation (NIF), and the regulation of gene expression (Beinert
et al., 1997; Lill and Muhlenhoff, 2006). They are found in all domains
of life: Archaea, Bacteria and Eukarya. The number of proteins containing
Fe–S clusters that are present in eukaryotes is generally much higher than in
prokaryotes due to the complexity of the eukaryotic lifestyle such as envi-
ronmental response and development (Py and Barras, 2010). The assembly
of Fe–S clusters in vitro occurs spontaneously under favourable conditions
when sufficient amounts of free iron and sulphide are available. However,
as these substances are toxic to cells in vivo (Balk and Lobreaux, 2005;
Johnson et al., 2005; Lill and Muhlenhoff, 2006, 2008; Rouault and
Tong, 2008; Vickery and Cupp-Vickery, 2007; Xu and Moller, 2008), their
concentrations have to be tightly regulated. Thus, Fe–S cluster synthesis
does not occur chemically and requires enzymes and cofactors. The assembly
of Fe–S clusters is a complex process involving many different systems made
up of numerous specific proteins (e.g. >100 in Escherichia coli) that are wide-
spread across the life (Lill, 2009). In eukaryotes, Fe–S clusters biosynthesis
involves three major systems: the ISC (iron–sulphur cluster), SUF (sulphur
utilization factors), and CIA (cytosolic iron–sulphur cluster assembly)
machineries. The ISC and SUF machineries are found only in the mito-
chondria and the plastids, respectively. The ISC machinery is considered
to be a house-keeping system and is widely distributed from prokaryotes
to eukaryotes. In contrast, the SUF system plays a role particularly under
stress conditions such as iron deprivation and oxidative conditions.
4 Vahab Ali and Tomoyoshi Nozaki

Furthermore, the maturation of Fe–S cluster proteins in the cytoplasm and


the nucleus depends on the CIA machinery, which is essential and ubiqui-
tous in all eukaryotes (Lillig and Lill, 2009). The NIF machinery is a unique
system present in limited lineages of microorganisms such as microaerophilic
bacteria, cyanobacteria, nitrogen-fixing bacteria, and unicellular protozoa
such as Entamoeba histolytica (Ali et al., 2004). In this chapter, we review
our current understanding of Fe–S cluster biogenesis in general, and the
conservation and/or unique acquisition and secondary loss of four biosyn-
thetic systems in the representative parasitic protozoa. Finally, we discuss
perspectives and possible exploitations of the research outcomes of Fe–S
cluster biogenesis in parasitic protozoa.

2. VARIATION AND FEATURES OF Fe–S CLUSTERS


2.1. Discovery of Fe–S clusters
The Fe–S clusters were first discovered in the early 1960s, when enzymes
with characteristic electron paramagnetic resonance (EPR) signals were
purified. Some of the first Fe–S proteins that were discovered include plant
and bacterial ferredoxins and respiratory complexes I–III from bacteria and
the mitochondria. In the late 1960s, chemical reconstitution was devised to
assemble Fe–S clusters into apo-proteins in vitro, which led to the view that
the Fe–S clusters can assemble spontaneously on proteins (Malkin and
Rabinowitz, 1966). However, genetic, biochemical, and cell biological
studies in the 1990s provided ample evidence that the maturation of Fe–S
proteins in living cells in vivo is catalyzed by enzymes, but does not occur
chemically.

2.2. Heterogeneity of Fe–S clusters


Many proteins depend on covalently or non-covalently bound cofactors for
their function. Organic cofactors include nucleotides (e.g. FMN and FAD),
vitamins (biotin, pantothenate and folate), and metal–organic compounds
(haem and molybdenum cofactors). The common inorganic cofactors
include metal ions (Mg2þ, Zn2þ, Mn2þ, Cu1þ/2þ, and Fe2þ/3þ). Among
them, Fe–S clusters are considered to be the oldest and most versatile
inorganic cofactors. The chemically simple Fe–S clusters are the rhombic
[2Fe–2S] and the cubane [4Fe–4S] types, which contain iron (Fe2þ/3þ)
and sulphide (S2). Representative types of Fe–S clusters and their functions
are summarised in Table 1.1.
Table 1.1 Representative Fe–S cluster-containing proteins
Type and name Type of Fe–S
Group of protein Source Function (reaction) cluster
1 Simple Fe–S proteins
[2Fe–2S] Ferredoxin, Cyanobacteria, Photosynthetic reduction of NADP, nitrite, [2Fe–2S]
plant type Clostridia, Protozoa, and thioredoxin
Chloroplasts
[3Fe–4S] Ferredoxin Bacteria, e.g., Electron transfer [3Fe–4S]
Desulfovibrio gigas
[4Fe–4S] Ferredoxin Bacteria, e.g., Bacillus, Electron transfer [4Fe–4S]
Desulfovibrio spp.
High-potential Fe–S Photosynthetic Anaerobic electron transport [4Fe–4S]
protein (HiPIP) bacteria, e.g.,
Chromatium vinosum
7Fe Ferredoxin Azotobacter vinelandii Storage of Fe [4Fe–4S] þ
[3Fe–4S]
8Fe Ferredoxin Anaerobic bacteria, Electron transfer 2[4Fe–4S]
e.g., Clostridium
pasteurianum
Continued
Table 1.1 Representative Fe–S cluster-containing proteins—cont'd
Type and name Type of Fe–S
Group of protein Source Function (reaction) cluster
2 Membrane-bound electron transfer proteins
NADH ubiquinone Aerobic bacteria, NADH þ Hþ þ UQ þ 4Hþ
in ! NAD
þ 2[2Fe–2S] þ 6
þ
oxidoreductase mitochondria þUQH2 þ 4Hout [4Fe–4S]
(complex I)
Succinate Aerobic bacteria, Succinate þ UQ ! Fumarate þ UQH2 [2Fe–2S] þ [3Fe–
dehydrogenase mitochondria 4S] þ [4Fe–4S]
(complex II)
UQH2: cytochrome c Aerobic bacteria, UQH2 þ CytCox ! UQ þ CytCred [2Fe–2S]
reductase (complex III) mitochondria
Cytochrome b6/f Cyanobacteria, PQH2 þ PCox ! PQ þ PCred [2Fe–2S]
complex chloroplasts
[NiFe] hydrogenase Bacteria, e.g. E. coli, H2 þ Menaquinone ! Menaquinol [4Fe–4S] þ
(respiratory) C. vinosum; [3Fe–4S]
A. vinelandii
Gylcerol phosphate E. coli Glycerol phosphate þ Menaquinone ! 2[4Fe–4S] þ [2Fe–
dehydrogenase Glyceraldehyde phosphate þ Menaquinol 2S]
(anaerobic)
Fumarate reductase Saccharomyces cerevisiae Fumarate þ Menaquinol ! Succinate þ Menaquinone [2Fe–2S] þ [3Fe–
4S] þ [4Fe–4S]
3 Soluble Fe–S enzymes
NAD(P)H-glutamate E. coli, plants Glutamine þ 2-Oxoglutarate þ NAD(P)H 2[4Fe–4S]
synthase ! 2Glutamate þ NAD(P)þ
Ferredoxin-glutamate Plants Glutamate þ 2-Oxoglutarate þ 2Fdox ! [4Fe–4S] þ
synthase 2Glutamate þ 2 Fdred [2Fe–2S]
Pyruvate: Euglena gracilis Pyruvate þ CoA þ NADPþ ! [4Fe–4S]
NADP þ oxidoreductase Acetyl CoA þ CO2 þ NADPH
Pyruvate:ferredoxin Cyanobacteria, Pyruvate þ CoA þ Fdox ! Acetyl CoA þ CO2 þ Fdred 2–3[4Fe–4S]
oxidoreductase Clostridia, Protozoa
[NiFe] hydrogenase Desulfovibro spp. H2þ CytCox ! 2Hþ þ CytCred 2[4Fe–4S] þ [3Fe–
(cytochrome c reducing) 4S]
[NiFe] hydrogenase Hydrogen bacteria, H2 þ NADþ ! Hþ þ NADH 3–4[4Fe–4S] þ
(NAD reducing) e.g. A. eutrophus, [2Fe–2S]
Nocardia opaca
4 Hydroxylases and dioxygenases
Ferredoxin (P-450 Bacteria, e.g. Electron transfer from flavoprotein reductase to [2Fe–2S]
reducing) Pseudomonas putida; cytochrome P-450
mitochondria
Continued
Table 1.1 Representative Fe–S cluster-containing proteins—cont'd
Type and name Type of Fe–S
Group of protein Source Function (reaction) cluster
5 Enzymes with the molybdopterin cofactor
Xanthine oxidase Milk Xanthine þ O2 > Urate þ H2 O2 þ O2  2[2Fe–2S]
Ferredoxin-nitrate Cyanobacteria, e.g. NO3  þ Fdred þ 2Hþ ! NO2  þ Fdox þ H2 O 2[2Fe–2S]
reductase Plectonema boryanum
6 Enzymes containing sirohaem
Ferredoxin: sulphite Bacteria, plants SO3 2 þ 6Fdred ! S2 þ 6Fdox 2[4Fe–4S]
reductase
7 Proteins with catalytic Fe–S or mixed-metal clusters
[Fe] hydrogenase Anaerobic bacteria, 2Hþ þ 2e ! H2 H cluster þ 2–4
e.g. C. pasteurianum [4Fe–4S]
Carbon monoxide Photosynthetic CO þ H2O > CO2 þ 2e þ 2Hþ 7[4Fe–4S] þ Ni–
dehydrogenase bacteria, e.g. [Ni–3Fe–4S] þ
Rhodospirillum rubrum [4Fe–4S] C-cluster
Carbon monoxide Acetogenic and CH3–[CP] þ CO þ CoA ! CH3 CO–CoA þ [CP] 7[4Fe–4S] þ Ni–
dehydrogenase (acetyl methanogenic [Ni–4Fe–4S] þ
CoA synthase) bacteria, e.g. [4Fe–4S] A-cluster
C. thermoaceticum,
Methanothrix soehngenii
Mo nitrogenase Nitrogen-fixing N2 þ 8e þ 10Hþ ! 2NH4 þ þ H2 P clusters, Fe–Mo
bacteria, e.g. [8Fe–7S]
Rhizobium, Azotobacter
8 Enzymes with nonredox Fe–S clusters
Aconitase (aconitate Bacteria, cytoplasm, Citrate ! Isocitrate [4Fe–4S]
hydratase) mitochondria
DNA endonuclease III E. coli Apurinic and apyridimic endonuclease [4Fe–4S]
L-Serine dehydratase Peptostreptococcus Serine > Pyruvate þ NH4þ [4Fe–4S]
asaccharolyticus
9 Regulatory proteins
Ferredoxin: thioredoxin Cyanobacteria, Thioredoxin þ Fdred ! Thioredoxinred þ Fdox [4Fe–4S]
reductase chloroplasts
Names of proteins, their sources (organisms or intracellular localizations), functions (reactions) and types of Fe–S clusters are shown.
10 Vahab Ali and Tomoyoshi Nozaki

Nitrogenase, carbon monoxide dehydrogenase (CODH), and hydroge-


nase are good examples to show the heterogeneity of Fe–S clusters. Nitro-
genase, which fixes atmospheric nitrogen gas (N2) as ammonia, is composed
of the heterotetrameric iron–molybdenum (FeMo)-cofactor-associated pro-
tein ([8Fe–7S], 7Fe:Mo:9S:homocitrate:X cluster), that is transiently associ-
ated with the homodimeric Fe protein ([4Fe–4S]). The FeMo protein binds
to a substrate and reduces Hþ and N2 to H2 and ammonia, while the Fe pro-
tein receives electrons from ferredoxin, hydrolyses ATP, and reduces the
FeMo protein. The Fe protein has a P-cluster, which is a putative electron
transfer centre (Burgess and Lowe, 1996; Howard and Rees, 1996) and
undergoes dramatic conformational changes between at least two oxidative
states (PN-cluster to POX-cluster) (Peters et al., 1997).
The metabolism of CO by CODH/acetyl CoA synthetase is mediated by
two different clusters responsible for different reaction mechanisms (Ragsdale
and Kumar, 1996). The reversible oxidation of CO to CO2 is catalysed by the
C-cluster, which is an Ni–[3Fe–4S] cluster of CODH, while the reversible
reaction of CO with coenzyme A (CoA) is catalysed by the A-cluster (Ni–
[4Fe–4S]) on acetyl CoA synthetase. Hydrogenases catalyse the reversible
reduction of protons to hydrogen. Two types of clusters that differ in metal
binding are known in hydrogenases. Iron-only hydrogenases (Fe-
hydrogenase) have the active site H-cluster composed of a [4Fe–4S] cluster
bridged to a binuclear iron centre. The second type of cluster in Ni–Fe-
hydrogenases contains a binuclear cluster of iron and nickel in part bridged
by sulphides (Volbeda et al., 1995). The existence of various Fe–S clusters in
nature, from the simplest cluster with broad non-specific properties to sophis-
ticated clusters with highly specific and efficient catalytic properties, suggests
that Fe–S clusters have evolved into various transitional states under selective
pressures such as changes in atmospheric conditions (Rees and Howard, 2003).

2.3. Physicochemical features and analytical methods


of Fe–S clusters
Iron is usually coordinated in a tetrahedron by sulphurs from inorganic sul-
phide and cysteine thiol groups of the proteins. Side chains of other amino
acids (His, Asp, Arg, Ser) are also known to coordinate iron. Sulphides gen-
erally bridge two or three irons, while in some cases more irons are bound to
sulphides. The two simplest Fe–S clusters are [2Fe–2S] and [4Fe–4S]. They
have versatile electrochemical properties with reduction potentials ranging
from over 400 to below 400 mV (Beinert, 2000), which is a range larger
than any other simple redox cofactors. Moreover, these simple clusters are
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 11

sometimes chemically integrated as part of non-redox catalysis, such as in


aconitase (Beinert et al., 1996) (Table 1.1; also see Section 8.4.1), where
the cluster serves as a Lewis acid. These physicochemical properties are
the basis of a wide range of distribution of the Fe–S clusters in a multitude
of functions (Rees and Howard, 2003). Another example of the adaptation
of [4Fe–4S] clusters is also often found in some proteins where one iron can
be lost with ease to form a [3Fe–4S] cluster under the influence of protein
structure or oxygen. The [4Fe–4S] cluster is oxygen sensitive and labile,
while the [3Fe–4S] cluster is more stable against oxygen than [4Fe–4S].
Depending on the overall oxidation state of the Fe–S cluster, iron may har-
bour unpaired electrons, and the resulting electron spin can be detected by
EPR spectroscopy. This biophysical technique provides valuable informa-
tion on the type, the oxidation state, and the electronic environment of
the clusters (Moulis et al., 1996). Other biophysical methods to study the
structure and properties of the Fe–S clusters include Mössbauer, extended
X-ray absorption fine structure, magnetic circular dichroism, electron
nuclear double resonance spectroscopy, nuclear magnetic resonance, and
resonance Raman spectroscopy. They provide specific information on
the electronic and magnetic properties of the Fe–S clusters in their particular
environment. In UV–Vis spectrophotometric measurements, Fe–S proteins
exhibit typical absorption maxima around 420 nm ([4Fe–4S] clusters) and
320, 410 and 560 nm ([2Fe–2S] clusters). These spectral properties give rise
to the dark brown colour of purified Fe–S holo-proteins.

2.4. Biochemical features of Fe–S clusters


The robust and unique physicochemical properties of Fe–S clusters confer
diverse biochemical abilities on Fe–S cluster proteins. First, Fe–S clusters
mediate electron transfer due to the properties of accessing various redox
states. Second, Fe–S clusters mediate redox catalysis. Fe–S clusters can reach
very low redox potentials and thereby reduce redox-resistant substances.
Third, Fe–S clusters are involved in non-redox catalysis. Small compounds
are allowed to bind to accessible ferric sites with extensive Lewis acid prop-
erties. Fourth, Fe–S clusters are able to regulate gene expression. This is due
to the reversible inter-conversional properties of Fe–S clusters to be exqui-
site sensors of several redox- or iron-related stresses (Beinert, 2000;
Fontecave, 2006; Kiley and Beinert, 2003) (see Section 3.1.5).
Although the robustness of Fe–S clusters is highly valuable to life, oxygen
poses a threat to Fe–S proteins and, consequently, to the organisms relying
12 Vahab Ali and Tomoyoshi Nozaki

on them (Imlay, 2006). Fe–S-containing dehydratase from E. coli is a good


example to show the high sensitivity of Fe–S clusters against oxygen. Fe–S
clusters in E. coli dehydratase directly react with univalent oxidants such as
hydrogen peroxide and peroxynitrite, leading to inactivation of dehydratase
with concomitant loss of iron. The oxidised and liberated iron can cause a
Fenton reaction, which produces highly toxic reactive oxygen species
(ROS) in the cell. Furthermore, ROS leads to deleterious effects on
DNA and other macromolecules. Therefore, while Fe–S clusters play essen-
tial roles in various biological processes (see Section 3), they make proteins
and organisms that harbour them susceptible to oxidative stress.

3. GENERAL BIOLOGICAL AND PHYSIOLOGICAL ROLES


OF Fe–S PROTEINS IN PROKARYOTES
AND EUKARYOTES
The biological functions of Fe–S proteins are diverse in all domains of
life. Fe–S proteins play major roles in electron transfer, respiration, photosyn-
thesis, substrate binding and activation, iron and cluster storage, regulation of
gene expression, and enzymatic activities, sulphur donation, disulphide reduc-
tion, structural modification, and regulation of metabolic pathways.

3.1. Roles of Fe–S proteins in bacteria


3.1.1 Electron transfer
The electron transfer by Fe–S clusters employs the property of the iron ions
of the Fe–S clusters to switch between reduced (ferrous Fe2þ) and oxidised
(ferric Fe3þ) states. The ability to delocalise electron density over iron atoms
makes Fe–S clusters ideal for their role in mediating biological electron
transport. Fe–S clusters are major components in the photosynthetic and
respiratory electron transport chains. They define the electron transport
pathways in numerous membrane-bound and soluble redox enzymes, and
constitute the redox-active centres in ferredoxins, one of the largest classes
of mobile electron carriers in biology. The clusters involved in electron
transfer contain a [2Fe–2S], [3Fe–4S], [4Fe–4S], or [8Fe–7S] core unit with
cysteinate generally completing tetrahedral S coordination at each iron site.
Aspartate, histidine, serine, or backbone amide ligation at a unique iron site
is occasionally encountered in clusters that function in electron transport.
These ligands are likely to play a role in modifying redox potential, gating
electron transport, or coupling proton and electron transport (Johnson et al.,
2005). Although the vast majority of electron transferring Fe–S clusters are
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 13

one-electron carriers, the double-cubane [8Fe–7S] cluster that is found only


in nitrogenase has the potential to act as a two-electron carrier.

3.1.2 Substrate binding and activation


Fe–S clusters serve as a substrate-binding site for a wide variety of redox and
non-redox enzymes in which Lewis acid-assisted enzyme catalysis is used for
performing the catalytic reactions. A site for substrate binding and activation
can be established in three different ways. First, a non-cysteinyl residue of
the protein side chain binds to a unique iron site of an Fe–S cluster. For
instance, the amino and carboxylate groups of methionine in S-adenosyl
methionine (SAM) are used to facilitate reductive cleavage and generate
the 50 -deoxyadenosyl radical in case of the radical-SAM family of Fe–S
enzymes (Cheek and Broderick, 2001). The radical-SAM super-family
comprises more than 60 enzymes that catalyse radical reactions in DNA pre-
cursors, vitamins, cofactors, antibiotics, herbicide biosynthesis and degrada-
tion pathways. Second, a heterometal can be incorporated in the Fe–S
cluster for achieving the active configuration of the enzymes, for example,
the Ni–[Ni–4Fe–4S] cluster in CODH (also see Section 2.2) (Dobbek et al.,
2001). Third, a substrate-binding metal site is bound to an iron of a [4Fe–4S]
cluster via a bridging cysteinyl residue. For instance, the dinickel centre is
attached to form the functional form of the acetyl CoA synthase active site
and a di-iron centre is attached to form the Fe-hydrogenase active site
(Peters et al., 1998).

3.1.3 Iron and cluster storage


Ferredoxins containing two [4Fe–4S] clusters were shown to play an essen-
tial role in iron homeostasis and in iron storage in Clostridium. This is
further supported by the presence of polyferredoxins containing up to
12 [4Fe–4S] clusters in tandemly repeated [8Fe] ferredoxin-like domains
in methanogenic archaea (Johnson et al., 2005). They are presumed to serve
as a store of Fe–S clusters.

3.1.4 Structural integrity


Fe–S clusters control protein structure. This was demonstrated by an Fe–S
cluster-driven protein reorganization in response to solvent effects and cys-
teine substitutions, as well as by the ability of designed and unstructured
minimal synthetic peptides to correctly assemble Fe–S clusters. For instance,
[4Fe–4S] clusters of DNA repair enzymes, endonuclease III and MutY, are
redox inactive and control the structure of a protein loop essential for
14 Vahab Ali and Tomoyoshi Nozaki

recognition and repair of damaged DNA, similar to zinc in zinc-finger pro-


teins (Guan et al., 1998; Kuo et al., 1992).

3.1.5 Regulation of gene expression


The sensitivity of Fe–S clusters to oxidative damage is exploited to sense
intracellular or environmental conditions, and hence regulates gene expres-
sion in prokaryotes. For instance, transcription factors (gene or operon
repressors) such as IscR, SoxR, and FNR are regulated at the transcriptional
level in oxidative or iron-deficient conditions. It was shown that a single
promoter upstream of the iscR gene directs expression of the four iscRSUA
genes in an operon (Schwartz et al., 2001), and IscR directly represses the
iscR promoter (Schwartz et al., 2001). Analysis of IscR by EPR showed that
the anaerobically isolated protein indeed contains a [2Fe–2S] cluster that is
able to undergo reversible oxidation and reduction (Schwartz et al., 2001).
The Fe–S cluster is important for the repressor function of IscR because iscS
or hscA mutations, which abolish Fe–S cluster biogenesis (see Section 5.2),
led to nearly constitutive expression of downstream genes in the operon
(Schwartz et al., 2001). In addition, the expression of the iscRSUA
operon was induced by hydrogen peroxide and by 2,20 -dipyridyl-mediated
iron starvation, and the induction was dependent upon the presence of an
intact proximal iscR gene (Outten et al., 2004). It was also demonstrated that
in E. coli, hydrogen peroxide induces the expression of a set of genes via the
transcriptional activator, OxyR (Zheng et al., 2001). It was further shown
that OxyR directly regulates expression of genes in the SufABCDSE operon
(see also Sections 3.2.1 and 9.2) (Lee et al., 2004b; Outten et al., 2004). In
addition, OxyR and Fur were also shown to mediate up-regulation of the
Suf operon induced by nitrosylated glutathione (GSH) or nitric oxide
known as nitrosative stress in bacteria (Kim et al., 2002; Mukhopadhyay
et al., 2004).

3.1.6 Regulation of enzymatic activity


Fe–S clusters are implicated in the regulation of enzymatic activity in
response to external stimuli, with the restoration of activity requiring cluster
assembly or repair. For instance, in the case of DNA glycosylase, involved in
DNA repair, and aconitase, it was shown that when the [4Fe–4S] cluster in
the holo-form of the enzyme is misassembled, oxidised, damaged, or
removed, its enzymatic activity is lost (Tang and Guest, 1999). A similar
observation was also made for the [2Fe–2S] cluster in mammalian
ferrochelatases, the terminal enzyme of haem biosynthesis (Wu et al., 2001).
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 15

3.1.7 Disulphide reduction


[4Fe–4S] clusters in disulphide reductases, such as ferredoxin:thioredoxin
reductase in chloroplasts and heterodisulphide reductase in methanogenic
archaea, were shown to be involved in disulphide reduction in two sequen-
tial steps, in which one-electron transfer is involved in a formation of an
intermediate with two thiolate ligands at a unique iron site (Dai et al.,
2000; Duin et al., 2002).

3.1.8 Sulphur donor


Fe–S clusters may also serve as a sulphur donor by its reductive cleavage.
For example, a [2Fe–2S] cluster of biotin synthase is reduced in each
catalytic cycle to provide sulphur for the conversion of dethio-biotin to
biotin. After each catalytic cycle, the cluster needs to be reconstituted
to regain the enzymatic activity (Berkovitch et al., 2004; Jameson et al.,
2004; Ugulava et al., 2001).

3.2. Roles of Fe–S proteins in eukaryotes


Besides the functions demonstrated in bacteria as mentioned above, addi-
tional functions were reported in eukaryotes.

3.2.1 Cellular iron homeostasis and gene expression regulation


It was reported that the mitochondrial ISC machinery (see Section 5.2) and
the export machinery (see Section 5.2.6) have a strong influence on the
uptake, intracellular distribution, and utilization of iron in yeasts (Kaplan
et al., 2006; Lill and Kispal, 2000). The roles of frataxin (FXN) (see
Section 4.1.4) and Atm1 (see Section 5.2.6) in iron homeostasis had been
implicated before their specific functions in Fe–S clusters metabolism were
identified. In yeast, the major regulatory factors controlling iron acquisition
and intracellular iron distribution in response to different iron levels in the
environment are the transcription factors Aft1 and Aft2 (Blaiseau et al., 2001;
Rutherford et al., 2001; Yamaguchi-Iwai et al., 1996). Aft1 was identified as
a major iron-responsive transcription factor, while Aft2 was identified when
its deletion was found to be associated with mild disturbance of iron homeo-
stasis. Upon iron depletion, Aft1 is translocated from the cytosol to the
nucleus, via interaction with importin, and transcriptionally activates iron
regulon (Ueta et al., 2003). Importantly, Aft1 and Aft2 interact with
Fe–S clusters or their precursors that are synthesised in the mitochondria
and transported to the cytosol via Atm1 (see Section 5.2.6).
16 Vahab Ali and Tomoyoshi Nozaki

In contrast to prokaryotes, in which expression of Fe–S proteins is reg-


ulated at the transcriptional level via transcription factors such as SoxR,
IscR, FNR, and OxyR (see Section 3.1.5), in eukaryotes, gene expression
is regulated in a more complex manner. For instance, in mammals, iron reg-
ulatory protein 1 (IRP1) is known to be regulated at a post-transcriptional
level (Lill and Muhlenhoff, 2006; Lill et al., 2006). IRP1 appears to have a
dual role in the cell. As an Fe–S holo-protein, it functions as a cytosolic
aconitase. When the Fe–S cluster is lost, apo-IRP1 functions as an RNA-
binding protein, which binds to the RNA stem-loop structures, called iron
regulatory elements (IRE), of specific mRNAs encoding proteins involved
in iron uptake, utilization, storage, and export. As a consequence of the
IRP1–IRE binding, target mRNAs are stabilised and thus protected from
degradation, which results in enhanced protein synthesis, whereas for
non-target mRNAs, the stem-loop structures at the 50 -untranslated end
block efficient translation by the ribosomes and lead to a decrease in protein
synthesis (Walden et al., 2006).

3.2.2 Photosynthesis
Fe–S clusters are essential components for photosynthesis, which is the pro-
cess unique to algae and plants. Iron of Fe–S clusters is engaged in the transfer
of an electron from water to NADPH, which is used to reduce carbon diox-
ide to form sugars (Raven and Falkowski, 1999). It is also involved in nitro-
gen and sulphur/sulphate reduction and assimilation, for example, by nitrite
reductase and sulphite reductase (Krueger and Siegel, 1982; Lancaster et al.,
1979). Both mitochondrial and photosynthetic electron transport chains
contain a number of proteins that require Fe–S clusters as a cofactor for their
activity. The photosynthetic electron transport chain contains three major
complexes known as photosystem I (PSI), photosystem II (PSII) and cyto-
chrome b6/f complex. Iron is present in all of these complexes and is the
most important redox-active metal ion for the photosynthetic electron
transport chain, both quantitatively and qualitatively (Bar-Ness et al.,
1992; Yehuda et al., 1996). Iron is present in both haem and non-haem
forms in PSII. In contrast, in the cytochrome b6 complex, iron is present
as haem and as [2Fe–2S] Rieske-type clusters. PSI contains one [4Fe–4S]
cluster each in PsaA and PsaB and one [2Fe–2S] cluster in ferredoxin
(Raven and Falkowski, 1999). Other plastidial proteins that contain Fe–S
clusters include ferredoxin–thioredoxin reductase (4Fe–4S), sulphite reduc-
tase (4Fe–4S), nitrite reductase (4Fe–4S), and glutamate synthase (3Fe–4S)
(Balk and Lobreaux, 2005).
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 17

3.2.3 tRNA biosynthesis


It is known that bacterial IscS plays a crucial role in providing sulphur for
thio modification of tRNA (Kambampati and Lauhon, 1999; Mueller,
2006) (see Section 5.2.4 and 8.5.3). Recently in yeast and human, thio mod-
ification was also demonstrated in both mitochondrial and cytosolic tRNAs
with two uridine nucleosides in positions 34 and 35. The modification of
uridine in the wobble position 34 seems to be present in all living organisms
and essential for viability as it increases the decoding efficiency of tRNAs
(Bjork et al., 2007). Mitochondrial and cytosolic Nfs1 are responsible for
thio modification of mitochondrial and cytoplasmic tRNAs in yeast
(Muhlenhoff et al., 2004; Nakai et al., 2004). Similarily, thiouridine mod-
ification of tRNA (tRNAS) by Nfs1 also occurs in the nucleus (Nakai et al.,
2004). Despite the previous notion that thio modification of tRNA can
occur in both the mitochondria and the cytosol, thio modification of tRNA
was shown to be dependent on mitochondrial Nfs1. In addition, IscU1 and
IscU2, the scaffold components of the mitochondrial ISC assembly (see
Section 5.2), and the components of cytosolic CIA machinery, cluster-
deficient protein 1 (Cfd1), nucleotide-binding protein 35 (Nbp35) and
CIA machinery protein 1 (Cia1) (see Section 5.5) were shown to be needed
for cytosolic tRNA thio modification (Nakai et al., 2007). Thus, thio mod-
ification of cytosolic tRNA requires the entire Fe–S protein assembly
apparatus.

4. GENERAL SOURCE OF IRON AND SULPHUR


FOR Fe–S CLUSTER BIOSYNTHESIS
4.1. Acquisition and transport of iron
4.1.1 Iron acquisition via the plasma membrane
Iron is indispensable for all living cells, and thus, organisms have multiple
pathways to scavenge iron. The ferric iron (Fe3þ) is largely insoluble and
can react with hydrogen peroxide, a by-product of respiration, generates
highly toxic hydroxyl radicals, and thus is toxic to the cell at physiological
pH under aerobic conditions. Hence, iron is usually present in a protein-
bound form in the cell. Some bacteria and eukaryotes possess surface
receptors that specifically bind transferrin or lactoferrin and facilitate the
internalization of iron–protein complexes to scavenge iron (Blanton
et al., 1990; Steverding et al., 1995). Subsequent acidification of endosomes
induces conformational changes in the complex with a consequent release of
iron (Sipe and Murphy, 1991). Yeast and other bacteria possess reductases
18 Vahab Ali and Tomoyoshi Nozaki

that reduce transferrin- or lactoferrin-bound Fe3þ to ferrous (Fe2þ) iron, a


soluble ion that is more readily internalised (Deneer et al., 1995).

4.1.2 Siderophores
Bacteria and fungi acquire iron by utilization of secreted siderophores,
which are low-molecular weight iron-chelating compounds and can effec-
tively compete with the host proteins for iron. Bacteria incorporate iron–
siderophore complexes through specific membrane receptors (Nikaido,
1993; Schryvers and Stojiljkovic, 1999; Visca et al., 2002). Bacteria lacking
siderophores also utilise specific receptors for transferrin or lactoferrin for the
iron acquisition (Braun and Braun, 2002; Braun and Hantke, 2011; Kishore
et al., 1991).

4.1.3 Iron transport to the mitochondria and the plastids


Major metabolites can cross the mitochondrial outer membrane easily.
However, the inner membrane can be crossed only with specialised carriers,
specially known as mitochondrial carrier family proteins (Nury et al., 2006).
The high affinity mitochondrial carriers, Mrs3 and Mrs4, have been pro-
posed to mediate iron transport in yeast (Muhlenhoff et al., 2003; Zhang
et al., 2005, 2006). However, as these two transporters were demonstrated
not to be essential for iron transfer to the mitochondrial matrix, other trans-
porter is likely present. In vertebrates, two isoforms of mitoferrin were
reported for iron transport (Li et al., 2002). In human, a homologue of yeast
Mrs3/4 is ubiquitously expressed and responsible for the iron transport to
the mitochondria (Li et al., 2002).
In contrast, iron uptake and transport across the plastid (chloroplast) mem-
branes is not well understood. In Arabidopsis thaliana, a membrane-spanning
iron transporter, permease in chloroplasts 1 (PIC1), was identified. PIC1 con-
tains four predicted a-helices, is targeted to the inner envelope membrane,
and displays homology with cyanobacterial permease-like proteins. Knock-
out mutants of PIC1 grew only heterotrophically and were characterised by a
chlorotic and dwarfish phenotype reminiscent of iron-deficient plants. The
mutants revealed severely impaired chloroplast development and a striking
increase in ferritin clusters. In addition, pic1 mutants showed differential reg-
ulation of genes and proteins related to iron stress or transport, photosynthe-
sis, and Fe–S cluster biogenesis. Furthermore, PIC1 and its cyanobacterial
homologue mediated iron accumulation in an iron uptake-defective yeast
mutant. These data suggest that PIC1 functions in iron transport across the
inner envelope of chloroplasts (Duy et al., 2007, 2011).
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 19

4.1.4 Frataxin
In yeast, FXN is essential for providing iron to the mitochondrial ISC sys-
tem, while in the bacterial system, the SUF proteins were found to be nec-
essary for ferric siderophore acquisition (Barras et al., 2005; Gerber et al.,
2003; Nachin et al., 2003; Ramazzotti et al., 2004). Human FXN is present
in multiple forms in the cell after mitochondrial import and processing. The
size variation of FXN was documented in haematopoietic, muscle, cardiac,
and non-haematopoietic cells, suggesting tissue-specific processing during
mitochondrial transport (Condo et al., 2007). The suggested major functions
of FXN include, in general, iron and haem metabolism, ISC assembly, oxi-
dative phosphorylation, and protection against oxidative stress (Bulteau
et al., 2004; Gakh et al., 2002; Kumanovics et al., 2008; Martelli et al.,
2007; Radisky et al., 1999).
FXN functions as a metallochaperone during Fe–S cluster biosynthesis as
shown by its in vitro ability to bind ferrous iron with micromolar-binding
affinity. FXN also directly binds Isu in an iron-dependent manner with a
submicromolar binding affinity and stimulates [2Fe–2S] cluster production
(Stemmler et al., 2010). In yeast, it was shown that FXN interacts with Isu
(and Nfs1) in vitro and that FXN depletion was associated with a defect in the
de novo Fe–S cluster formation on Isu (Gerber et al., 2003). The strong
sequence conservation of FXN and Isu orthologues across species indicates
a conserved mechanism for Fe(II) delivery and Fe–S cluster production in
eukaryotes. Cellular distribution of FXN changes upon stress response. In
adult human tissues, FXN is localised to the mitochondria under normal
non-stress conditions, while under oxidative stress, cytosolic FXN accumu-
lates, which is caused by damage on the Fe–S cluster by ROS (Martelli et al.,
2007). A donor of iron for Fe–S cluster biogenesis in the mitochondria is still
debated and direct in vivo demonstration of interaction of IscU/IscA with
FXN is required. In humans, mutations affecting FXN expression or func-
tion resulted in Friedreich’s ataxia (FRDA), an autosomal recessive neuro-
and cardio-degenerative disorder that represents the most common
inherited ataxia in humans, affecting 1 in 30,000–50,000 individuals
(Bencze et al., 2006) (see Section 6.1).

4.2. Acquisition, biosynthesis, and transport of L-cysteine


L-Cysteine is obtained via several independent routes. First, L-cysteine is
de novo synthesised from sulphide and L-serine in bacteria, fungi, protozoa
(i.e. Entamoeba, Trichomonas, Trypanosoma, and Leishmania), and plants.
20 Vahab Ali and Tomoyoshi Nozaki

Second, L-cysteine is synthesised from L-methionine by reverse trans-


sulphuration in mammals. Third, L-cysteine can be directly obtained by
scavenging via amino acid transporters from the environmental milieu or
after degradation of ingested proteins in lysosomes and phgosomes.
Cysteine biosynthesis occurs in bacteria, yeast, plants and some protozoa,
in which L-serine and sulphide are converted into L-cysteine via O-
acetylserine or O-phosphoserine. O-Acetylserine or O-phosphoserine
reacts with sulphide, which is provided directly or by reduction of sulphate,
to form L-cysteine. The cysteine biosynthetic pathway was previously
described in details in the previous volume (Nozaki et al., 2005). In contrast,
animals require L-homocysteine, which is derived from L-methionine via
the reverse trans-sulphuration pathways as a source of sulphur to produce
L-cysteine via cystathionine (Ali and Nozaki, 2007).
Cysteine permease/transporters have been identified in bacteria and
eukaryotes (During-Olsen et al., 1999; Kosugi et al., 2001), and their role
in cysteine transport was confirmed in yeast. L-Cysteine is apparently trans-
ported not by a specific permease, but by multiple permeases with a broad
specificity, and all of these permeases are active under different sets of growth
conditions. However, a specific cysteine transporter (Yct1p, YLL05wp)
with a high affinity, was identified and characterised by genetic and bio-
chemical analysis (Kaur and Bachhawat, 2007). A novel plasma membrane
cystine transporter from Candida glabrata (CgCYN1), which belongs to the
amino acid permease family, but has no similarity to known plasma mem-
brane or lysosomal cystine transporters from human and yeast was reported
recently (Yadav and Bachhawat, 2011). L-Cystine uptake by CgCYN1 was
energy dependent and inhibited by L-cystine, but not by other amino acids
including L-cysteine. However, structurally similar amino acids, for exam-
ple, cystathionine, lanthionine, and selenocystine, also inhibited transport,
confirming the cystine specificity. Cysteine or cystine transporter has not
been demonstrated in protozoan parasites, while it was demonstrated that
L-cysteine is specifically transported in Trypanosoma cruzi epimastigotes
(Canepa et al., 2009).

5. FOUR SYSTEMS FOR Fe–S CLUSTER BIOGENESIS


IN PROKARYOTES AND EUKARYOTES
Fe–S cluster assembly has been discovered in all living organisms
examined so far. Four major systems, ISC, SUF, NIF, and CIA, are known
to be involved in the process, and two to three of these systems are present in
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 21

each organism. Saccharomyces cerevisiae is the best characterised organism to


study Fe–S clusters machinery among eukaryotes (Lill and Muhlenhoff,
2006; Lill et al., 2006, 2012; Muhlenhoff and Lill, 2000).

5.1. Catalytic reaction in Fe–S cluster biosynthesis


In the ISC, SUF, and NIF systems from bacteria, lower eukaryotes, plants,
and humans, Nif S- and Nif S-like proteins, namely, IscS (Nfs1) and Suf S
(Csd, Nfs2), are responsible for the catalysis of initial donation of sulphur/
sulphide for the assembly of Fe–S clusters. These enzymes catalyse a cysteine
desulphurase reaction: the mobilization of sulphane sulphur (S ) or sulphide
(S2) from L-cysteine with concomitant production of L-alanine and in the
presence of reducing agents (Flint, 1996; Flint et al., 1996; Mihara and Esaki,
2002; Mihara et al., 1997, 1999). The cysteine desulphurase activity of SufS
and IscS is enhanced by SufE in bacteria and plants, or Isd11 in yeast and
other eukaryotes, respectively. The reaction catalysed by cysteine des-
ulphurase produces a substrate-ketimine intermediate from L-cysteine and
pyridoxal phosphate. The sulphur atom of this intermediate is then attacked
by the catalytic cysteine residue, resulting in the formation of a cysteine per-
sulphide residue at the active side of the enzyme, with a concomitant release
of L-alanine. The substrate sulphur atom of the cysteine persulphide residue
can be subsequently transferred to different biomolecules, for building of
Fe–S clusters (Mihara et al., 2000).
These catalytic enzymes can also act on L-cysteine sulphinate and
L-selenocysteine as substrates, releasing SO2 and selenium, respectively
(Mihara et al., 1999, 2000). The mechanisms of degradation of L-selenocysteine
and L-cysteine sulphinate differ from that of L-cysteine desulphuration (Mihara
et al., 2000). Furthermore, genetic studies revealed that the IscS mutant con-
tained reduced activities of several soluble and membrane-bound Fe–S proteins
(Djaman et al., 2004; Schwartz et al., 2000). The assembly of Fe–S clusters and
transfer to apo-proteins was achieved in vitro using IscS, cysteine, dithiothreitol
and ferrous ammonium sulphate (Kurihara et al., 2003).

5.2. ISC machinery


The first system for Fe–S cluster assembly, designated as ISC, is required for
the generation of the majority of cellular Fe–S proteins and thus performs a
general house-keeping biosynthetic function in all ISC-possessing organisms
from bacteria to higher eukaryotes (Fig. 1.1A).
22 Vahab Ali and Tomoyoshi Nozaki

A Bacterial ISC machinery

Fe2+
CyaY Apo
- S-SH HscA
IscS IscU
HS-S- Sulphur e- HscB
Fdx Fdx
Cys Ala
Holo

IscA Bacterial
IscR
Fe–S
proteins

iscR iscS iscU iscA
ISC operon

Fe–S cluster

B Bacterial SUF machinery

SufA

Apo
Fe2+
-S-SH sulphur
HS-S- SufE SufU
SufS -S-SH
HS-S- (CsdB) e-
ATP Holo
Cys Ala
SufC Bacterial
Fe–S
SufD proteins
SufB

ATP

Fe–S cluster

Figure 1.1 Schematic diagrams of bacterial ISC (A) and SUF (B) systems. Grey and black
circles indicate sulphur and iron, respectively. Only representative [2Fe–2S] clusters are
shown. An electron is depicted by ‘e’. ‘Apo’ and ‘Holo’ indicate the apo- and holo-forms
of Fe–S cluster-containing proteins. Note that IscR is the transcriptional repressor of the
ISC operon.

5.2.1 Genes encoded by ISC operon in bacteria


In prokaryotes, the proteins involved in the ISC system are encoded by the
ISC operon, which consists of iscR–iscS–iscU–iscA–hscB–hscA–fdx–orf3
genes. The two central proteins of the ISC machinery are IscS, cysteine des-
ulphurase, and IscU, a scaffold, which are homologous in function to NifS
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 23

and NifU, respectively (see Section 5.4). Genes homologous to iscSUA,


hscB/hscA and ddx are widely distributed in nature and can be found in
all bacterial and archae-bacterial genomes that have been sequenced so
far, often in the same arrangement as in E. coli. The operon has been shown
to be essential for their viability (Tokumoto et al., 2004).

5.2.2 Mechanism of Fe–S cluster synthesis by ISC system


The conserved Cys328 of IscS participates in a nucleophilic attack on the
sulphur atom of a PLP-bound cysteine substrate to form a cysteine per-
sulphide residue, as revealed by structural studies (Cupp-Vickery et al.,
2003; Kaiser et al., 2003). Furthermore, mutational analysis of residues pre-
sent in a loop (Ser323Ala, Ser326Ala, Leu333Ala and Ser336Ala) close to the
catalytic residue Cys328 demonstrated that these residues are necessary for
in vivo enzymatic activities of Fe–S cluster-containing proteins synthesised
by the ISC system (Lauhon et al., 2004).
Sulphur transfer from IscS to IscU is initiated by the attack of the con-
served cysteine residue, Cys63 of IscU on the Sg atom of the cysteine per-
sulphide residue transiently produced on IscS (Agar et al., 2000a). IscU and
SufU (Isu in eukaryotes) also possess three conserved cysteine residues,
which have similar functions, in a C-X24–26-C-X42–43-C arrangement.
However, IscU and SufU show distinguishable structural properties. IscU
lacks an 18–21-amino acid insertion between the second and third con-
served cysteine residues of SufU. IscU has a conserved histidine in place
of lysine in SufU, and possesses, unlike SufU, the LPPVK motif that is
required for interaction with HscA chaperone (Cupp-Vickery et al.,
2004a; Ramelot et al., 2004). Hence, dissimilar to SufU, IscU requires
HscA/HscB cochaperones for Fe–S cluster assembly or transfer.
IscR is a Fe–S protein in itself and a transcription factor to regulate the
transcription of the ISC operon. Hence, IscR regulates ISC assembly
machinery by a feedback mechanism (see Sections 3.1.5 and 3.2.1). IscA
serves as an alternative scaffold. In Synechocystis, a [2Fe–2S] cluster was
assembled in vitro between the two protomers of the IscA dimer and ligated
by two conserved cysteine residues, Cys110 and Cys112, of both protomers
(Wollenberg et al., 2003). In E. coli, thioredoxin reductase system promotes
iron binding on IscA, but prevents it on IscU. On the other hand, Fe–S clus-
ter assembly on IscU was promoted in the presence of iron-loaded IscA,
IscS, and L-cysteine (Ding et al., 2005). These data reinforce the view that
IscA is capable of binding to the intracellular free iron and providing it for
the IscS-mediated ISC assembly on IscU in cells in which the accessible free
24 Vahab Ali and Tomoyoshi Nozaki

iron content is probably restricted. HscB and HscA showed high degrees of
homology to the molecular chaperones DnaJ/Hsp40 and DnaK/Hsp70,
respectively. Therefore, HscA and HscB likely assist the maturation of
Fe–S proteins. By site-directed mutational analysis, two cysteine residues
in IscU were shown to be essential for binding with HscB, but not with IscS.
The ferredoxin-containing [2Fe–2S] cluster encoded by the fdx gene is
assumed to serve as a reductant at some point during the maturation of
Fe–S clusters (Jung et al., 1999; Kakuta et al., 2001). The role of orf3 remains
to be demonstrated. A general working model of the ISC system in bacteria
is shown in Fig. 1.1A.

5.2.3 ISC system in the mitochondria


In eukaryotes, the ISC assembly machinery in the mitochondria is required
for the maturation of virtually all cellular Fe–S proteins, that is, mitochondrial
as well as cytosolic proteins. The proteins involved in the ISC system are
produced in the cytosol and transported to the mitochondria in yeasts and
human (Gerber et al., 2004; Li et al., 2006; Lill and Muhlenhoff, 2006,
2008; Muhlenhoff and Lill, 2000; Muhlenhoff et al., 2003; Netz et al.,
2007; Tong and Rouault, 2000, 2006). A current working model of the
ISC assembly machinery in mitochondria is shown in Fig. 1.2. The biogen-
esis of Fe–S clusters in the mitochondria involves the synthesis of a transient
Fe–S cluster on the scaffold proteins Isu1 and Isu2 (Nif U/Suf U homo-
logue), and the transfer and incorporation of Fe–S clusters into target
apo-proteins, as seen in the bacterial ISC system. However, the process of
Fe–S cluster biogenesis in the mitochondria is more complex than that in
bacteria, requiring mitochondria-specific components. The process starts
with the membrane potential-dependent import of ferrous iron into the
mitochondria, which is facilitated by the carrier proteins Mrs3 and Mrs4
and unknown factors. Sulphur is released from L-cysteine by the cysteine des-
ulphurase complex, Nfs1/Isd11 complex, in the form of sulphane sulphur.
The reduction of sulphur needs electron transfer chain from NADH to fer-
redoxin (Yah1) via ferredoxin reductase (Arh1).
The Nfs1/Isd11 complex further interacts with IscU (Isu1/2) through a
direct interaction between Nfs1 and Isu1/2. Iron binding to Isu1 is accom-
plished by FXN (Yfh1, see Section 4.1.4; homologous to CyaY in bacteria),
and the iron binding is enhanced with the Nfs1–Isu1 complex (Bencze et al.,
2006; Gerber et al., 2003; Layer et al., 2006; Wang and Craig, 2008). It was
also shown in bacteria that CyaY regulates Fe–S cluster biosynthesis by
inhibiting IscS in an iron-dependent manner (Adinolfi et al., 2009).
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 25

Mitochondrial ISC machinery

Mitochondrion
IscA1
/A2 Apo
Isd11
ATP
Nfs1
Sulphur Isu1/2 Ssq1(HscA, Hsp70)
(IscS)
(IscU ) Jac1 (HscB), Grx5
e-
Mge1(GrpE)
Cys Ala
Fdx GSH

e- (Yah1) Frataxin Holo


Fdx (Yfh1/Cya Nfu
reductase Y) Mitochondrial
(Arh1)
e - NADH Fe–S proteins
Mrs3/4 ?

Cytosol
Fe–S cluster
Fe2+
Figure 1.2 A schematic diagram of mitochondrial ISC system. A transfer of sulphur/
sulphane is depicted by small arrows. Synonyms (in bacteria or other eukaryotes) are
shown in parentheses. ‘?’ depicts an unknown iron transporter. See the legend of
Fig. 1.1 for other symbols.

5.2.4 Isd11
Isd11 is unique to eukaryotes, absent in prokaryotes, and required for the
biogenesis of Fe–S proteins in both the mitochondria and the cytosol.
Isd11 functions closely with the a-proteobacterium-derived IscS. It was
demonstrated that Isd11 is present in all five eukaryotic supergroups, includ-
ing some of the hydrogenosomal and mitosomal lineages (Richards and van
der Giezen, 2006) (see Section 8). These data indicate that the eukaryotic
ancestor invented Isd11 as a functional partner to IscS, which supports
the premise of a single shared a-proteobacterial endosymbiotic ancestry
for all eukaryotes.
Isd11 is also localised to the nucleus as well as the mitochondria in mam-
malian cells. Thus, Isd11 is considered to be a unique subunit of mitochondrial
ISC machinery (Adam et al., 2006; Wiedemann et al., 2006). It forms a com-
plex with cysteine desulphurase (Nfs1) (Wiedemann et al., 2006). Isd11 seems
to stabilise Nfs1 in the mitochondria and mediate the interaction of Nfs1 with
other proteins (Adam et al., 2006). The Isd11 mutant yeast showed severe
defect in the Fe–S protein activities, and accumulation of iron in the mitochon-
dria, indicating that Isd11 is essential for cell viability (Adam et al., 2006).
26 Vahab Ali and Tomoyoshi Nozaki

It was also shown that suppression of Isd11 enhanced the binding activity
of IRP1 (see Section 3.2.1) to the iron-responsive element, increased the
protein levels of iron regulatory protein 2 and resulted in abnormal punctate
ferric iron accumulations in the cell. Isd11 of the Nfs1/IscU complex also
interacts with FXN, as shown in co-immunoprecipitation and immunoflu-
orescence assays (Shan et al., 2007). In addition, Isd11 was also shown to be
involved in thiolation of both cytoplasmic and mitochondrial tRNA (Paris
et al., 2010).

5.2.5 Additional proteins required for the cluster transfer in the


mitochondria
Several additional proteins are involved in the later stage of the Fe–S cluster
transfer: the Hsp70 (homologous to bacterial HscA), Ssq1 (the co-chaperone
of Hsp70), Jac1 (homologous to bacterial HscB), Mge1 (the nucleotide
exchange factor, homogolous to bacterial GrpE) and glutaredoxin, Grx5.
Jac1 facilitates the ATP-dependent binding of Ssq1 to Isu1/2 and stimulates
the ATPase activity of Ssq1. Ssq1p interacts with a specific conserved PVK
sequence on Isu1p (Hoff et al., 2002, 2003), which is located on an exposed
loop between two a-helices. The carboxyl-terminal region of Jac1 is suffi-
cient for interaction with Isu1. Isu1/Jac1 complex is targeted to Ssq1
(Andrew et al., 2006) and this targeting is suggested to be important under
the conditions where need for Fe–S cluster biogenesis is high or in organisms
lacking specialised Hsp70 for Fe–S cluster biogenesis.
Glutaredoxins (Grx) are a group of thioltransferases that reduce
disulphide bonds or catalyse reversible protein glutathionylation or
deglutathionylation (Herrero and de la Torre-Ruiz, 2007). A mitochondrial
monothiol glutaredoxin, Grx5, also catalyses the reduction of disulphide
bonds in proteins with a concomitant conversion of GSH to GSH-
disulphide. It has been recently demonstrated that dimeric monothiol Grx
can coordinate a [2Fe–2S] cluster via the cysteine residue of the active side
of each monomer and the cysteines of two GSH molecules (Picciocchi et al.,
2007). Grx5 was shown to be required for Fe–S cluster biogenesis in yeast
and zebrafish (Rodriguez-Manzaneque et al., 2002; Wingert et al., 2005).
Two chloroplastic monothiol Grx in plants were also shown to ligate a
[2Fe–2S] cluster in vitro (Bandyopadhyay et al., 2008), suggesting that Grx
may function in general as a scaffold in the organellar Fe–S cluster assembly
and transfer. Grx5 assists Fe–S protein maturation by serving as a transient
Fe–S cluster binding site before the cluster is inserted into apo-proteins
(Bandyopadhyay et al., 2008). Recently, in the nitrogen-fixing bacterium
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 27

Azotobacter vinelandii, it has been shown in vitro that HscA and HscB are
required for efficient ATP-dependent [2Fe–2S] cluster transfer from IscU
to Grx5, reinforcing the general role of Grx5 in the storage and transport
of the [2Fe–2S] cluster assembled on IscU with the help of HscA and HscB
(Shakamuri et al., 2012). However, this hypothesis has not been confirmed
in vivo (Lill et al., 2012). Mge1 is the nucleotide exchange factor involved in
the ATP/ADP exchange, which triggers a conformational change of the
binding domain of Ssq1 from a closed to an open state, thus leading to dis-
assembly of the Ssq1–Isu1 complex.

5.2.6 The ISC export apparatus


5.2.6.1 Transport of Fe–S clusters
Fe–S clusters synthesised in the mitochondria are also exported to the cytosol
for the activation of cytosolic Fe–S apo-proteins. The currently known
components of the specific export apparatus are: ATP-binding cassette
(ABC) transporter, Atm1, which is localised on the mitochondrial inner
membrane, Erv1, that is localised in the mitochondrial intermembrane
space, and GSH, which is in the mitochondria and the cytoplasm
(Fig. 1.3). Atm1 exports Fe–S clusters per se or an unknown form of iron
from the mitochondria to the cytosol and is a key component of the
Fe–S cluster export machinery (Csere et al., 1998; Pondarre et al., 2006;
Savary et al., 1997). As shown for the yeast ABCB7-like homologue
(Pondarre et al., 2006), Atm1 exposes its carboxyl-terminal ATP-binding
domain to the mitochondrial matrix and spans the inner membrane with
six trans-membrane segments. An analysis of ATM3 (plant Atm1 homo-
logue) mutant plants indicated that the export of iron requires not only
ATM3 but also one or two additional factors for the assembly of the
Fe–S clusters as well as molybdenum cofactors (Moco) in the cytosol
(Bernard et al., 2009). It was also shown that Atm1 plays a crucial role in
the regulation of cellular iron uptake because Fe–S clusters or precursors
transported by Atm1 interact with the iron-responsive transcription factors,
Aft1 and Aft2 (see Section 3.2.1). Disruption of the ISC assembly in the
mitochondria or the ISC export machinery caused the translocation of
Aft1 and Aft2 into the nucleus and the induction of the iron regulon
(Ueta et al., 2003).
As described above, in A. thaliana, biosynthesis of Moco also depends on
ATM3 (Teschner et al., 2010). The first step of Moco assembly takes place in
the mitochondria, in which a pterin precursor is formed. This precursor is
exported from the mitochondria via ATM3, and subsequent steps occur in
28 Vahab Ali and Tomoyoshi Nozaki

Mitochondrial ISC export machinery

Apo Holo
ISC assembly
Mitochondrial
Fe–S proteins

GSH
ATP
Atm1 Mia40
Erv1
e-
GSH
FMN
e- e- e-
Tah18 NADPH
Dre2 FAD
Cytosol ?
CIA machinery

Nar1 Apo

e-
Cfd1 Cfd

Nbp35 Nbp35
Cia1
Fe2+
Nar1
Holo

Cytosolic/
nuclear
Fe–S cluster Fe–S proteins

Figure 1.3 A schematic diagram of ISC export machinery and CIA system. See the
legends of Figs. 1.1 and 1.2 for other symbols.

the cytosol, including a sulphur insertion. Thus, analysis of ATM3 in plants


and protozoa may help the understanding of the precise role of the mito-
chondrial ABC transporters in Fe–S clusters and Moco biosynthesis as the
Moco biosynthesis and Moco-containing enzymes are lacking in yeast
(Balk and Pilon, 2010).

5.2.6.2 Reduction and oxidation of Fe–S clusters in the ISC export system
Erv1, FAD-binding sulphhydryl oxidase, catalyses the formation of dis-
ulphide bridges (Lee et al., 2000) and is involved in the disulphide
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 29

formation-driven transport of proteins into the inter-membrane space by


reoxidizing the import component Mia40 (Allen et al., 2005; Rissler
et al., 2005; Terziyska et al., 2005). Mia40 is an oxidoreductase that catalyses
oxidative protein folding in the mitochondria. Mia40 has a characteristic
conserved CPC motif, which can donate the disulphide bond to the sub-
strate (Banci et al., 2009). Mia40, in cooperation with Erv1, promotes
the formation of two disulphide bonds in the substrate protein. Thus, the
disulphide bond formation pathway is based on a relay of reactions involving
disulphide transfer from Erv1 to Mia40 and from Mia40 to the substrate pro-
teins, ensuring the efficiency of oxidative folding in the inter-membrane
space (Bottinger and Becker, 2012). An electron derived from the sul-
phhydryl oxidation is presumed to be passed by Erv1 onto either molecular
oxygen or cytochrome c (Thorpe and Coppock, 2007). Thus, Erv1 appears
to play two key roles: oxidation of Fe–S clusters and drainage of electrons
produced via Fe–S cluster transport. Although GSH is necessary to coordi-
nate Fe–S cluster binding, it remains unknown how GSH helps a cluster
transfer from the mitochondria to the cytoplasm.

5.3. SUF machinery


5.3.1 Catalytic components of SUF machinery in bacteria
The second machinery involved in Fe–S cluster biogenesis is known as the
SUF system (Fig. 1.1B). In bacteria, the SUF gene cluster comprises sufA,
B, C, D, S, and E genes (Takahashi and Tokumoto, 2002), which are regu-
lated by the Fe2þ-dependent repressor, Fur. SufS (also called CsdB) is a
cysteine desulphurase, whereas SufE is an activator of cysteine desulphurase.
Suf S and Suf E form a complex, in which SufE accepts sulphane sulphur
from Suf S.

5.3.2 Scaffold components of SUF machinery in bacteria


There are three apparently redundant scaffold components: SufA, SufBCD
and SufU. SufA serves as a scaffold component, and binds to the sulphurated
form of SufE exclusively via the three conserved cysteine residues, Cys50,
Cys114 and Cys116, as demonstrated by site-directed mutagenesis. The
reaction consists of a transfer of S atoms from persulphide/polysulphide spe-
cies on SufE to the three conserved cysteines of SufA.
In E. coli, it has been shown that the SufBC2D complex can assemble a
[4Fe–4S] cluster and transfer it to apo-proteins in vitro. This complex assem-
bles an Fe–S cluster through the mobilization of sulphur from the SufSE
complex and the FADH2-dependent reductive mobilization of iron
30 Vahab Ali and Tomoyoshi Nozaki

(Wollers et al., 2010). SufC is an ABC ATPase and forms a complex with
SufB and SufD. In E. coli, the carboxyl-terminal a-helices of SufD interact
with SufC (Wada et al., 2009).
SufU, (Isu in eukaryotes) dissimilar to IscU, serves as an alternative scaf-
fold protein. SufU possesses three conserved cysteine residues in a C-X24–26-
C-X42–43-C arrangement. However, SufU has an 18–21-amino acid inser-
tion between the second and third conserved cysteine residues, which is
missing in IscU. In addition, SufU lacks the conserved histidine (replaced
with lysine), and the LPPVK motif that is required for interaction of IscU
with HscA chaperone (Cupp-Vickery et al., 2004b; Ramelot et al.,
2004), suggesting lack of interaction with HscA/HscB chaperones of SufU
for Fe–S cluster assembly. SufU can assemble the [2Fe–2S] cluster and trans-
fer it to the apo-proteins. In contrast, SufA can hold both [2Fe–2S] and
[4Fe–4S] clusters and transfer them to the apo-proteins (Gupta et al.,
2009; Tan et al., 2009).

5.3.3 Plastidial (chloroplastic) SUF system


In organisms other than well-studied bacteria such as E. coli (Takahashi and
Tokumoto, 2002) and Erwinia chrysanthemi (Nachin et al., 2001), the SUF
system is also found in the chloroplasts of plants. The components and reac-
tions involved in the Fe–S cluster assembly in the plastids are shown in
Fig. 1.4. The plastids including the apicoplasts, found in the phylum
Apicomplexa, have evolutionarily arisen via one to a few rounds of endo-
symbiosis of an ancestor of the extant cyanobacteria, which possess the SUF
system and lack the ISC system (Takahashi and Tokumoto, 2002). Plastids
are thus expected to possess the SUF proteins for the house-keeping role of
Fe–S clusters assembly in the organelle. The plastids generate Fe–S clusters
for their own requirements, whereas the mitochondria provide the clusters
for the cytosolic and nuclear Fe–S proteins (Balk and Pilon, 2010). In
A. thaliana, all the components of SufABCDSE are conserved and involved
in Fe–S cluster biogenesis in the plastids along with plant-specific compo-
nents such as cytochrome b6/f complex, [2Fe–2S] cluster-containing ferre-
doxin, PSI, [4Fe–4S] cluster-containing ferredoxin–thioredoxin reductase
and proteins involved in nitrogen assimilation pathways (nitrite reductase,
glutamate synthatase, and sulphite reductase) (Ye et al., 2006). These
plant-specific components in the SUF system may explain the ability of
plants to carry out Fe–S cluster assembly in the presence of oxygen.
In the chloroplast stroma, chloroplastic (Cp) NifS (also known as
AtNFS2 or CpSuf S), CpSuf E, and CpIscA form a 600-kDa complex
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 31

Plastidial SUF machinery


Cytoplasm
Fe2+ Plastid
Photo e- e-
system I
Fdx NADPH e-
? Nfu

Apo
e- ATP ADP
SufA
Sulphur
HS-S- SufE IscA? SufB SufD
Grx
HS-S- NFS2 SufC SufC
(CpNifS/CpSufS)
Holo
FADH2
ATP ADP
Ala Cys FAD+

Figure 1.4 A schematic diagram of plastidial SUF system. ‘?’ depicts an unknown iron
transporter. See the legends of Figs. 1.1 and 1.2 for other symbols. Interaction of IscA
in vivo not confirmed.

(Ye et al., 2006). CpNif S is closest to Suf S among three Nif S-like proteins
in E. coli. CpSufE (At4g26500) is an essential protein and forms a hetero-
tetrameric subcomplex with CpNif S. CpSufE1 stimulates cysteine des-
ulphurase activity 40-fold and increases the substrate affinity of CpNif S
towards cysteine. An essential cysteine residue, Cys65, in CpSufE, which
is composed of the SufE- and BolA-like domains, is likely the acceptor site
for the intermediate sulphur, but is not required for binding to CpNif S.
The BolA-like domain of CpSuf E is also presumed to interact with Grx
in the chloroplasts (Huynen et al., 2005). The components, their localiza-
tion, and function of the SUF system in plants were reviewed (Balk and
Lobreaux, 2005).
Similar to bacteria, in the Arabidopsis chloroplasts, SufB was shown to
interact with SufC (Xu et al., 2005), which in turn interacts with Suf D,
as demonstrated by a yeast two-hybrid system and bimolecular fluorescence
studies (Xu and Moller, 2004). It was shown using isolated chloroplasts that
the Suf BCD complex requires ATP for Fe–S assembly, as in E. coli (Wollers
et al., 2010). Both Arabidopsis Suf B and SufC showed ATPase activity (Xu
and Moller, 2004; Xu et al., 2005). In addition, plant SufB and SufC partially
complemented the respective E. coli mutants.
32 Vahab Ali and Tomoyoshi Nozaki

It is important to note that the mitochondrial ISC and plastidial SUF


machineries synthesise their Fe–S clusters independently. The inhibition
of Suf S (NFS2) by RNA interference in A. thaliana caused defects in PSI
and decreased the levels of all tested chloroplast Fe–S proteins. However,
no defect was seen in the amount and functions of the mitochondrial
Fe–S proteins (Balk and Pilon, 2010; Ye et al., 2006).

5.4. NIF machinery


The NIF system was discovered first in the nitrogen-fixing bacterium
A. vinelandii and is thought to be responsible for the assembly of the Fe–S
clusters as well as Fe–Mo clusters, required for activation of nitrogenase,
which is the enzyme responsible for the conversion of nitrogen gas to
ammonia (see review by Rees and Howard, 2000). The main components
of the NIF system are Nif S and Nif U. The operon of the NIF gene cluster
was identified ( Jacobson et al., 1989) and NifS was identified as a cysteine
desulphurase in A. vinelandii (Zheng et al., 1993). The NIF machinery is
present in bacteria such as nitrogen-fixing bacteria, non-diazotropic
e-proteobacteria such as Campylobacter jejuni and Helicobacter pylori (Olson
et al., 2000), and a protozoan E. histolytica (Ali et al., 2004). The character-
ization of the NIF system in non-diazotrophs enforced a view on the
premise that the NIF system is required for Fe–S cluster assembly of nitro-
genase. All the organisms that possess the NIF system are microaerophilic or
anaerobic and restricted to limited lineages.

5.4.1 NifS and NifU


NifS is the homodimeric pyridoxal phosphate-containing catalytic compo-
nent with cysteine desulphurase activity. Nif S releases the sulphur atom
from L-cysteine, which involves a formation of cysteine-PLP ketimine
adducts with a subsequent nucleophilic attack by the thiolate anion of
Cys325 on the sulphur of the substrate cysteine. An intermediate
persulphide is formed in the reaction to be incorporated as a transient
[2Fe–2S] cluster on the scaffold component, Nif U (Yuvaniyama et al.,
2000). The specific role of Nif S in the maturation of nitrogenases is
supported by the fact that NifS is co-transcribed with a CysE homologue
that encodes serine O-acetyltransferase, which catalyses the rate-limiting
step in cysteine biosynthesis.
Nif U is a homodimeric scaffold protein on which the Fe–S clusters are
preassembled before insertion into nitrogenase. Nif U holds a [2Fe–2S] clus-
ter in each protomer and possesses three domains, that is, the amino-terminal,
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 33

central, and carboxyl-terminal domains. The amino-terminal domain is sim-


ilar to IscU of the ISC system and contains three conserved cysteine residues,
Cys35, Cys62 and Cys106, which provide a labile rubredoxin-like ferric-
binding site or a transient [2Fe–2S] cluster. This domain also possesses
the LPPVK (or LPPEK in A. vinelandii Nif U) motif responsible for the
IscU–cochaperone interaction (see below) (Hoff et al., 2002; Johnson
et al., 2005). However, the pentapeptide motif is not conserved in other
organisms. The central domain of A. vinelandii Nif U contains a permanent,
redox-active [2Fe–2S] cluster ligated by Cys137, Cys139, Cys172 and
Cys175 residues with sequence homology to bacterioferritin-associated
[2Fe–2S] ferredoxin from E. coli (Agar et al., 2000b; Yuvaniyama et al.,
2000). It was presumed that the domain helps in the formation and/or release
of the transient Fe–S clusters formed on the amino- and carboxyl-terminal
domains by serving as an iron donor for the Fe–S cluster assembly (Agar
et al., 2000b). The carboxyl-terminal domain of Nif U as well as Nif U-like
proteins in eukaryotes, termed as Nfu or Cnf U, contains two conserved cys-
teine residues, Cys272 and Cys275, and can assemble a labile [2Fe–2S] cluster
for the transfer to nitrogenases (Agar et al., 2000b). The [2Fe–2S] cluster was
demonstrated in the purified Nfu protein from Synechocystis (Tong et al.,
2003; Yabe et al., 2004). The [4Fe–4S] cluster was also verified on the human
Nfu protein by absorption, analytical, and Mössbauer studies (Tong et al.,
2003). Furthermore, genetic and biochemical studies indicate that both
the amino- and carboxyl-terminal domains are involved in the assembly of
a [4Fe–4S] cluster on apo-nitrogenase (Dos Santos et al., 2004). A recent
study further explained that Nif U sequentially assembles a labile [2Fe–2S]
cluster in the amino-terminal IscU-like domain, and labile [4Fe–4S] clusters
in the carboxyl-terminal Nfu-like domain, and that these labile [4Fe–4S]
clusters are rapidly transferred to apo-nitrogenase (Smith et al., 2005). Thus,
Nif U can serve as a scaffold for Fe–S cluster assembly per se. Nif S and Nif U
are uniquely present in E. histolytica among parasitic protozoa (Ali et al., 2004;
van der Giezen et al., 2004) (see Section 8.1).

5.4.2 Alternative scaffold NifIscA


Nif
IscA shows considerable sequence similarity to IscA, and its encoding
gene is located immediately upstream of nifU in A. vinelandii (Johnson
et al., 2005). NifIscA is an alternative scaffold protein that is involved in
the assembly of transient Fe–S clusters (Krebs et al., 2001; Morimoto
et al., 2006) or the delivery of iron to U-type scaffold proteins as a metal-
lochaperone (Ding et al., 2004). Spectroscopic studies showed that
34 Vahab Ali and Tomoyoshi Nozaki

A. vinelandii NifIscA is a homodimeric protein and can assemble [2Fe–2S]


and [4Fe–4S] clusters between the two protomers, mediated by Nif S
(Krebs et al., 2001). The possibility that NifIscA is exclusively involved in
the Fe–S cluster formation of nitrogenase was not appreciated until the dis-
covery of IscA because there was no phenotype associated with the inacti-
vation of NifiscA. Nevertheless, the conservation of three cysteines in both
IscA and NifIscA was interpreted to indicate that this family of proteins play
an analogous role in Fe–S cluster biosynthesis.

5.5. CIA machinery


5.5.1 Four identified components in CIA machinery
The additional cytosolic machinery for the Fe–S cluster assembly was first
identified in yeast and named as the cytosolic iron-sulphur cluster assembly
(CIA) machinery (Lill and Muhlenhoff, 2005; Roy et al., 2003). The CIA
machinery was shown to be required for the maturation of cytosolic and
nuclear Fe–S proteins (Hausmann et al., 2005). The CIA system is present
in most eukaryotes sequenced to date (Hausmann et al., 2005), located in
both the cytosol and nucleus, and expressed at comparatively low levels
(Balk et al., 2004; Roy et al., 2003). To date, functional evidence for the
assembly and maturation of four cytosolic and nuclear Fe–S proteins have
been demonstrated in yeast (Netz et al., 2007; Sharma et al., 2010).
The main components elucidated till date are the soluble P-loop
NTPases (ATPases or GTPases), Cfd1 and Nbp35 (homologous to bacterial
ApbC and yeast Ind1), the iron-only hydrogenase-like protein, nuclear
architecture-related protein 1 (Nar1) and the seven-bladed WD40 repeat
protein, Cia1, all of which work in a coordinated fashion for the transfer
of a Fe–S cluster to apo-proteins (Fig. 1.3). First, an Fe–S cluster is transiently
assembled on the P-loop NTPases Cfd1 and Nbp35, which serve as a scaf-
fold. In vitro reconstitution studies showed that an oligomeric complex con-
sisted of Nbp35 and Cfd1 assembles labile Fe–S clusters on Nbp35. Thus,
these cysteine residues likely serve as a transient scaffold for the Fe–S cluster
before transfer to apo-proteins (Broderick, 2007; Netz et al., 2007). This
step essentially requires mitochondrial Nfs protein. Unlike the mitochon-
drial Isu1 scaffold, Cfd1 and Nbp35 do not directly interact with a
sulphur-donating protein such as Nfs1 (Biederbick et al., 2006;
Muhlenhoff et al., 2004; Nakai et al., 2001). Instead, the Cfd1 and
Nbp35 complex interacts with Nar1 for the transfer of its clusters.
The Fe–S clusters on the Cfd1 and Nbp35 scaffold are subsequently
transferred to an apo-protein by Nar1 and Cia1. Nar1, in contrast to the
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 35

Nbp35 and Cfd1 complex, possesses two interacting Fe–S clusters, and
interacts with the Nbp35–Cfd1 complex. Cia1 also makes a complex with
Nar1, holds the Fe–S cluster and is proposed to be involved in the final trans-
fer of the cluster to apo-proteins. Important aspects of the CIA machinery
such as the source of sulphide and iron, and detailed mechanisms of the Fe–S
cluster assembly remain unclear. The four components of the CIA machin-
ery are described in the following section.

5.5.2 Cytosolic Fe–S cluster-deficient protein 1


Cfd1 (Cfd1p in yeast) was the first protein shown to be involved in the cyto-
solic Fe–S cluster assembly (Roy et al., 2003). Cfd1p shares similarities with,
and falls into, the subfamily of P-loop ATPases, which include Nif H of
nitrogenase (Dean et al., 1993; Leipe et al., 2002). Nif H itself is a Fe–S pro-
tein; however, the cluster-coordinating residues of Nif H are not conserved
in Cfd1p. Cfd is more closely related to Mrp from bacteria, which is also
involved in the Fe–S cluster assembly (Skovran and Downs, 2003). Cfd1p
functions to make an iron available for the cytosolic Fe–S cluster assembly.
However, it remains unknown whether iron is provided from the mito-
chondria or acquired from other cellular pools (Roy et al., 2003). By analogy
to known metallochaperones, the metal-binding element of an iron chap-
erone is likely at, or near, the protein surface (Arnesano et al., 2002). Cfd1p
possesses the CX2CX2C motif exposed on the surface and this motif is
predicted to be involved in metal binding (Roy et al., 2003). Cellular frac-
tionation of yeast showed that >90% of Cfd1 is present in the cytoplasm
while about 10% is in the membrane-rich fraction. It was shown that cyto-
solic aconitase in IRP1-transformed cells contained 80% [4Fe–4S] and 20%
[3Fe–4S] clusters (Brown et al., 2002), while Cfd1p-deficient IRP1-
transformed strain showed no or little detectable [3Fe–4S]- or [4Fe–4S]-
containing cytosolic aconitase activity, suggesting that Cfd1p is essential
for de novo assembly of the Fe–S clusters on aconitase in the cytosol (Roy
et al., 2003).

5.5.3 Nucleotide-binding protein 35


Nbp35p is a soluble protein located in both the cytosol and the nucleus, but
not the mitochondria (Hausmann et al., 2005). Nbp35 belongs to the Mrp/
Nbp35 subgroup of the P-loop NTPases that share the highest sequence
similarity with members of other subgroups involved in NIF, metal inser-
tion, and bacterial cell division (Lutkenhaus and Sundaramoorthy, 2003).
A feature of Nbp35 is an approximately 50-a.a.-long amino-terminal
36 Vahab Ali and Tomoyoshi Nozaki

extension containing four conserved cysteine residues, which Mrp lacks, and
is likely a binding site for either a metal (e.g. zinc) or the Fe–S cluster (Leipe
et al., 2002). Nbp35 indeed carries the Fe–S cluster at the amino-terminal
domain. Cluster integration of Nbp35p depends strictly on the mitochon-
drial ISC assembly and export machineries. Thus, maturation of Nbp35p
occurs in a similar manner to other cytosolic Fe–S proteins. However,
Nbp35p differs from similar NTPases yeast Cfd1p and bacterial ApbC in that
Nbp35p, but not the latter, stably binds to iron or the Fe–S cluster (Roy
et al., 2003; Skovran and Downs, 2003).
As predicted by the striking structural similarities between Nbp35p
and Cfd1p, the two proteins are, at least in part, functionally and genet-
ically interchangeable. Cfd1 and Nbp35 have a genetic interaction.
Nbp35p overproduction exacerbated the growth defect of Cfd1p-
deficient cells (Hausmann et al., 2005). It was shown that Nbp35p stably
binds iron in vivo. As described above, Nbp35 does not directly interact
with sulphur-donating protein, cytosolic Nfs1. Instead, mitochondrial
Nfs1, together with other mitochondrial ISC and ISC exports compo-
nents, is required for the cytosolic Fe–S cluster formation by the CIA sys-
tem (Netz et al., 2007). Hence, Cfd1 and Nbp35 appear to receive the
sulphur moiety from mitochondrial Nfs1. Cfd1 and Nbp35 form a stable
complex in vivo, which suggests that their cooperation is of functional
importance. The Cfd1–Nbp35 complex binds up to three [4Fe–4S] clus-
ters, one at the amino terminus of Nbp35 and one each in the carboxyl-
terminal regions of both Nbp35 and Cfd1. These clusters are labile and
can be rapidly transferred to target [Fe–S] apo-proteins. A recent muta-
tional study of Cfd1 and Nbp35 has shown that two central cysteine res-
idues (CPXC) of the carboxyl-terminal motif are crucial for the
coordination of the labile [4Fe–4S] clusters and the formation of the
Cfd1–Nbp35 heterotetramer complex, as well as their functions as scaf-
fold proteins and the viability of the cells. In contrast, the proximal cys-
teine residues are dispensable, despite their evolutionary conservation
(Netz et al., 2012).

5.5.4 Nuclear architecture-related protein 1


Nar1 (or Nar1p in yeast) is an Fe–S protein containing two [4Fe–4S] clusters
(Horner et al., 2002; Nicolet et al., 2002) and acts at the late stage of the
cytosolic Fe–S biogenesis, after the assembly of the Fe–S cluster on the
Cfd1–Nbp35 scaffold complex. The Cfd1- and Nbp35-bound Fe–S clusters
are transferred to Nar1, which binds two Fe–S clusters and thereby is
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 37

converted into a functional CIA component. Nar1 has high sequence sim-
ilarity with bacterial iron-only hydrogenase found in virtually all organisms
including those that are not known to produce or consume hydrogen such as
yeast and humans (Balk et al., 2004; Horner et al., 2002). Both the [4Fe–4S]
clusters on Nar1 are essential for function and cell viability (Urzica et al.,
2009). At the amino terminus, Nar1p carries a ferredoxin-like domain with
four conserved cysteine residues that may bind a [4Fe–4S] cluster (Balk et al.,
2004), while the carboxyl-terminal part contains four conserved cysteine
residues that, in iron-only hydrogenases, hold a unique H-cluster. This moi-
ety consists of two sub-clusters, a cubane [4Fe–4S] cluster and a binuclear
[2Fe] centre bridged by a cysteine sulphur (Nicolet et al., 2000). This
H-cluster forms the catalytic centre of iron-only hydrogenases (Nicolet
et al., 2000).
The closest human homologue of Nar1p is NARF, which is a nuclear
protein that binds to prenylated lamin A in the nucleus (Barton and
Worman, 1999). IOP1 is the second closest mammalian homologue of
Nar1p (Song and Lee, 2007), which regulates the expression of the hypoxia-
inducible factor that represents the global mediator of the mammalian
response to hypoxia. Neither IOP1 nor NARF possesses hydrogenase activ-
ities (Huang et al., 2007). Nar1 is involved in the transfer of the Fe–S cluster
from the scaffold complex to the target apo-proteins. Nar1p is predomi-
nantly localised in the cytosol, where it interacts specifically with Cia1
(see Section 5.5.5), which is mainly located in the nucleus (Balk et al.,
2004). Depletion of Nar1p in the cytosol caused defects in the enzymatic
activity of the cytosolic Fe–S proteins. Mitochondrial Fe–S proteins, on
the other hand, exhibited normal activity and showed wild-type efficiencies
in the Fe–S cluster assembly (Balk et al., 2004).

5.5.5 CIA machinery protein 1


Cia1 acts after the assembly of the Fe–S clusters on Nbp35 and Nar1, and
thus, likely plays a role in the final incorporation of the Fe–S clusters into
target proteins. Cia1 belongs to the large family of WD40 proteins and
exhibits a seven-bladed propeller structure. These proteins function in rather
diverse processes and generally act as protein-interaction devices (Smith
et al., 1999). It was shown that Cia1 interacts with Nar1 in vivo (Balk and
Lobreaux, 2005). Depletion of Cia1 impaired maturation of cytosolic and
nuclear Fe–S proteins, but did not reduce iron binding to Nbp35 or
Nar1, suggesting that Cia1 acts in the transfer of Fe–S cluster from
Nbp35 to apo-proteins (Balk and Lobreaux, 2005).
38 Vahab Ali and Tomoyoshi Nozaki

5.5.6 Dre2 and Tah18


Dre2 contains a [4Fe–4S] cluster and a [2Fe–2S] cluster (Zhang et al., 2008).
These clusters are stable even after prolonged exposure to air, suggesting that
they play structural and catalytic roles in the protein. Depletion of Dre2
impaired cytosolic, but not mitochondrial, Fe–S cluster biogenesis, although
Dre2 was found partially localised in the mitochondrial inter-membrane
space (Zhang et al., 2008). It is possible that Dre2 is likely involved in an
early step in the cytosolic Fe–S cluster biogenesis, possibly worked with
association of the ISC export system to deliver a substrate necessary for
the Fe–S cluster formation on Cfd1 and Nbp35. Recently, a new essential
flavoprotein Tah18 was discovered in yeast. Tah18 and Dre2 form a com-
plex and is part of an electron transfer chain functioning in an early step of
the cytosolic Fe–S protein biogenesis. Electrons are transferred from
NADPH via the FAD- and FMN-containing Tah18 to the Fe–S clusters
of Dre2. This electron transfer chain is required for the assembly of the tar-
get, but not the scaffold Fe–S proteins, suggesting a need for the reducing
power in the generation of the stably inserted Fe–S clusters. It was also
shown that human Ndor1–Ciapin1 proteins can functionally replace yeast
Tah18–Dre2 (Netz et al., 2010).

5.6. Mechanism of repair of Fe–S clusters


In general, Fe–S clusters are sensitive to oxidative stress. For instance,
[4Fe–4S]2þ clusters of dehydratases are rapidly damaged by univalent oxi-
dants, including hydrogen peroxide, superoxide, and peroxynitrite. The loss
of an electron destabilises the cluster, causing it to release its catalytic iron
atom and converting the cluster initially to an inactive [3Fe–4S]1þ form
(Djaman et al., 2004). Chaperones such as HscA and HscB (Hsp70- and
Jac1-type chaperones, respectively), encoded in the ISC gene cluster in
E. coli, which are necessary for the Fe–S cluster biogenesis, may also be
involved in the repair of damaged Fe–S clusters (Schilke et al., 1999).

6. GENETIC DISORDERS BY A DEFECT OF Fe–S CLUSTER


BIOGENESIS
Several genetic diseases have been attributed to mutations in genes
involved in the Fe–S clusters assembly. Although many genes have been
proven responsible for diseases in humans, such as FRDA, microcytic anae-
mia, X-linked sideroblastic anaemia, cellular ataxia, xeroderma pigmenta-
tion, Fanconi anaemia, colon cancer, erythropoietic anaemia, and diseases
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 39

due to the disturbance of the complexes I and II (Lill and Muhlenhoff, 2008;
Ye and Rouault, 2010), only a few examples are described here. For other
genetic disorders caused by Fe–S cluster biogenesis, one may refer to the
above-mentioned reviews.

6.1. Friedreich’s ataxia


FRDA is a neurodegenerative disease, which is caused by a mutation in the
FXN gene that is involved in the transport of iron. Numerous reports have
indicated the roles of FXN, such as that of an iron chaperone (Bulteau et al.,
2004), an iron donor for haem biosynthesis (Yoon and Cowan, 2004), an iron
storage gene (Gakh et al., 2002), an iron donor for the Fe–S cluster repair and a
regulator of the iron export (Bulteau et al., 2004; Radisky et al., 1999). How-
ever, the main role of FXN is currently believed to be supplying iron in a
bioavailable form for mitochondrial Fe–S cluster biosynthesis.
FRDA is the most common form of inherited ataxia in humans. FRDA
involves the central and peripheral nervous systems and the heart, and is
characterised by a progressive gait and limb ataxia, a lack of tendon reflexes
in the legs, loss of position sense, dysarthria, and leg weakness (Campuzano
et al., 1996). Neurons in the dorsal root ganglia are adversely affected, and
hypertrophic cardiomyopathy is found in almost all patients (De Biase et al.,
2007). FRDA is common before the age of 25, and most commonly in
puberty. So far, little success has been achieved in the treatment of FRDA
(Ye and Rouault, 2010). The excess iron accumulation is observed in the
mitochondria of cardiac myocytes and neurons of affected tissues (Rotig
et al., 2002; Seznec et al., 2005). The activities of Fe–S proteins such as
aconitase and succinate dehydrogenase are reduced in FRDA patient cells.
Excess iron is imported to and accumulated in the mitochondria, which
leads to iron deficiency in the cytosol, activation of the iron regulatory pro-
teins and the impairment of cellular iron homeostasis (Huang et al., 2009; Li
et al., 2008).
It was observed that about 2% of FRDA patients have point mutations in
the FXN gene, whereas 98% have alterations in the FXN gene such as a mul-
tiple insertion of unstable GAA trinucleotides in the first intron (Campuzano
et al., 1996). Normal alleles contain 6–34 of the GAA repeats, whereas the
alleles of patients contain 66–1700 repeats. The expansion of the GAA
repeat appears to cause gene silencing by allowing heterochromatin forma-
tion to silence transcription or to inhibit transcription of the FXN gene due
to the formation of a persistent DNA–RNA hybrid (Wells, 2008).
40 Vahab Ali and Tomoyoshi Nozaki

6.2. Sideroblastic anaemia


A mutation in Grx (GLRX5) was discovered to be responsible for micro-
cytic sideroblastic anaemia (SA) in one human male (Camaschella et al.,
2007). The GLRX5 mutation was associated with severe SA, insulin-
dependent diabetes and cirrhosis with high levels of blood transferrin satu-
ration, serum ferritin, bilirubin, liver transaminases, urinary iron and liver
iron concentrations. The patient was successfully treated with iron chelation
(Camaschella et al., 2007). SA is characterised by the presence of ring
sideroblasts, which represent erythroid precursor cells due to the iron over-
load in the mitochondria.
The SA patient has a homozygous A294G mutation in the third nucle-
otide of the last codon of GLRX5 exon 1. This substitution does not change
the encoded amino acid (glutamine), but interferes with the correct splicing
and removal of intron 1 and drastically reduces the amount of the grx5 tran-
script (Camaschella et al., 2007). GLRX5 is involved in the Fe–S cluster bio-
synthesis in the mitochondria as a scaffold protein, the GLRX5 mutation
likely results in a defect in the Fe–S cluster biogenesis in multiple organs
and tissues. Interestingly, however, pathologic phenotypes are not devel-
oped in all tissues, but restricted to erythrocytes and haematopoietic cells
(Ye and Rouault, 2010).

6.3. X-linked sideroblastic anaemia and ataxia (XLSA/A)


Mutations of the ABCB7 gene in humans cause hereditary X-linked
sideroblastic anaemia and ataxia (XLSA/A) (Lill and Kispal, 2001). The
ABCB7 gene encodes the human homologue of Atm1, which is involved
in the export of the Fe–S clusters or their precursors synthesised in the mito-
chondria to the cytosol (see Section 5.2.6). The mutations in the human
ABCB7 gene generally occur in trans-membrane segments, indicating the
importance of the integrity of the membrane-spanning regions. In plants,
cyclic pyranopterin monophosphate (cPMP), a Moco synthesis intermedi-
ate, was shown to be accumulated in the mitochondria of an ATM3 mutant
(Teschner et al., 2010). ATM3 was proposed to be a cPMP transporter.
Similar SA phenotypes are attributable to mutations of the related genes
glycine (Solute carrier family 25, member 38; SLC25A38) and thiamine
(SLC19A2) transport genes (Guernsey et al., 2009), a gene encoding an
ABC transporter for the export of Fe–S clusters to the cytoplasm (ABCB7)
and a gene involved in haem synthesis (ALAS2) (Bergmann et al., 2010). It is
predicted that SLC25A38 provides glycine for haem biosynthesis, while
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 41

SLC19A2 provides thiamine for pyruvate dehydrogenase. ABCB7 exports a


haem or haem precursor to the cytosol that conveys a signal regarding the
status of the mitochondria to the cytosolic/nuclear compartment (Ye and
Rouault, 2010).

6.4. Other genetic disorders


In Sjögren’s syndrome, IscA, an alternative scaffold protein, was identified as
an autoantigen (Cozar-Castellano et al., 2004). However, it remains
unknown how IscA is associated with the disease. It was also demonstrated
that mitochondrial respiratory chain enzymes, particularly succinate dehy-
drogenase (complex II), function as a tumour suppressor (Gottlieb and
Tomlinson, 2005). The impaired function of complex II leads to an accu-
mulation of succinate, which inhibits propyl hydroxylase in the cytosol.
Defects in complex I (NADH-ubiquinone oxidoreductase) are associated
with a number of mitochondrial diseases, including mitochondrial
encephalomyopathy, lactic acidosis, stroke-like episodes, Leigh syndrome
and Leber’s hereditary optic atrophy (see DiMauro, 2004 for details).
Inherited mutations in ATP-dependent DNA helicase XPD are found in
cluster-coordinated residues in patients and shown to be associated with the
disease known as Xeroderma pigmentation, Cockayne syndrome, and tri-
chothiodystrophy (Andressoo et al., 2006). XPD and related proteins, such
as FancJ, show a striking similarity to archae-bacterial helicase, an Fe–S pro-
tein. Similarly, FancJ mutations are associated with Fanconi anaemia patients
and defects in DNA glycosylase (MutY homologue), and are perhaps asso-
ciated with colon cancer (Lukianova and David, 2005; Peterlongo et al.,
2006; Rouault, 2012; Ye and Rouault, 2010).

7. OUTLINE OF CONSERVATION, UNIQUE DISTRIBUTION,


AND DIVERSITY OF Fe–S CLUSTER BIOGENESIS
MACHINERIES IN PROTOZOAN PARASITES
All protozoan parasites analysed so far are known to possess one to
three independent machineries for the biosynthesis of Fe–S clusters
described in Section 5. The majority of protozoan parasites are similar in that
they possess the ISC system including the ISC export and cytosolic CIA
machineries. However, there are a few exceptions: E. histolytica lacks the
ISC and SUF systems, and instead possesses the NIF system along with lim-
ited components of the CIA system. Plasmodium and Blastocystis possess both
ISC and SUF systems, as well as the CIA machinery. In other parasitic
42 Vahab Ali and Tomoyoshi Nozaki

protozoa reviewed in this chapter, the ISC and CIA systems are conserved as
seen in the higher vertebrates.
Although Plasmodium and Cryptosporidium belong to the same phylum
Apicomplexa, they are remarkably different in Fe–S cluster biogenesis. Plas-
modium species conserve the apicoplast-compartmentalized SUF system and
the mitochondrion-compartmentalized ISC system, while Cryptosporidium
lacks the SUF system and retains only a limited number of proteins for
the ISC system. In contrast to Apicomplexa, at least two species of trypano-
somes and four species of Leishmania that belong to the order Kinetoplastida
and are discussed in this review are similar in the Fe–S cluster biogenesis
machineries. Blastocystis is also very unique in that the SUF system is
uniquely localised in the cytoplasm, while the ISC system is localised in
the mitochondrion-related organelle [MRO or mitochondrion
(mitochondrial)-like organelle], similar to the case in Giardia and Cryptospo-
ridium, and in Trichomonas (the hydrogenosomes, instead of the mitosomes).
It is of importance that among the ISC, SUF, and NIF systems, the lowest
number of components is required in the NIF system which is sensitive to
oxygen and retained only in some prokaryotes, Entamoeba, and Mastigamoeba
(Gill et al., 2007), the latter two of which are exposed to very low oxygen
pressure. In contrast, it is presumed that the SUF system is required and
retained in the organisms that encounter high oxygen pressure as in the case
for bacteria, plants, and Plasmodium.

8. Fe–S CLUSTER BIOGENESIS IN PROTOZOAN


PARASITES
8.1. Entamoeba
Entamoeba is the anaerobic/microaerophilic intestinal protozoan that lacks
the aerobic mitochondria and, instead, possesses the highly divergent
MRO, called mitosomes. The mitosomes is incapable of oxidative phos-
phorylation and energy generation is solely dependent on glycolysis and fer-
mentation in the cytoplasm (Ali and Nozaki, 2007).

8.1.1 Fe–S clusters proteins in Entamoeba


A majority, if not all, of the characterised Fe–S proteins as well as the ones
predicted in silico from the genome database apparently contain [4Fe–4S]2þ
clusters except Nif U, which contains permanent [2Fe–2S]. [4Fe–4S] pro-
teins include ferredoxins (Reeves et al., 1977), pyruvate:ferredoxin oxido-
reductase (Pineda et al., 2010), and NADPH-dependent oxidoreductase
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 43

( Jeelani et al., 2010). For instance, each of two isotypes of NADPH-


dependent oxidoreductase (EhNO1 and 2) has the CX2CX4CX3CP and
CX3CX3CX3C motifs, suggestive of two [4Fe–4S] clusters are present
per subunit ( Jeelani et al., 2010). Among four ferredoxins in the database,
EhFd1 appears to possess one each of [4Fe–4S] and [3Fe–4S], while three
other ferredoxin contain two [4Fe–4S] clusters. Although only a limited
number of Fe–S proteins containing [4Fe–4S] or [3Fe–4S] clusters have
been characterised so far, Fe–S proteins containing [2Fe–2S] clusters remain
largely uncharacterised. In other anaerobic/microaerobic protozoa, Giardia
intestinalis and Trichomonas vaginalis, ferredoxins are known to hold [2Fe–2S]
clusters (Ali and Nozaki, 2007).

8.1.2 NIF system


E. histolytica is unique among parasitic protozoa in that it possesses only the
NIF (or NIF-like) system among the NIF, ISC, and SUF systems as a sole
and non-redundant system for the biosynthesis of all Fe–S proteins. No
orthologous proteins involved in the ISC and SUF systems in other organ-
isms are present in E. histolytica (Tables 1.2 and 1.3). Amino acid comparison
and phylogenetic analyses indicate that both a catalytic component, NifS and
a scaffold component, NifU (EhNifS and EhNifU, respectively) showed a
close kinship to orthologues from e-proteobacteria, suggesting that both
of these genes were likely acquired by lateral gene transfer from an ancestor
of e-proteobacteria (Ali et al., 2004; Van Der Giezen et al., 2004). EhNifS
expressed in E. coli is present as a homodimer, and shows cysteine des-
ulphurase activity with a very basic optimum pH, compared with NifS from
other organisms. EhNifU protein is a tetramer and contains one stable [2Fe–
2S]2þ cluster per monomer, revealed by spectroscopic and iron analyses (Ali
et al., 2004). Since E. histolytica does not possess nitrogenase activity and is
incapable of NIF, the presence of the NIF system and the lack of other sys-
tems in this organism reinforce the premise that the NIF system is not spe-
cific for the Fe–S cluster formation of nitrogenase, but is also involved in the
Fe–S cluster assembly of both [2Fe–2S] and [4Fe–4S] clusters for non-
nitrogenase proteins, as proposed for the NIF-like system in H. pylori
(Olson et al., 2000). In vivo complementation of a temperature-dependent
growth defect of the E. coli isc/suf mutant strain by expression of the
E. histolytica NIF-like system, that is, EhNifS and EhNifU, indicates that
the NIF-like system plays an interchangeable role in the Fe–S cluster assem-
bly, but only under anaerobic conditions (Ali et al., 2004). It is worth noting
Table 1.2 Genome wide analysis of the components of ISC system
Organisms ISC system
IscR IscS IscU IscA Nfu HscA HscB Fdx Mge1 Isd11 Frataxin ATM1 Erv Grx5
E. coli þ þ þ þ þ þ þ þ þ  þ   
S. cerevisiae  þ þ þ þ þ þ þ þ þ þ þ þ þ
H. sapiens  þ þ þ þ þ þ þ þ þ þ þ þ þ
E. histolytica      þ þ þ      
G. intestinalis  þ þ þ þ þ þ þ þ     þ
T. vaginalis  þ þ þ þ þ þ þ þ þ þ þ a
 
Trypanosoma spp.  þ þ þ þ þ þ þ þ þ þ þ þ þ
Leishmania spp.  þ þa þa þ þ þa þ þ þa þ þ þa þ
Plasmodium spp.  þ þ þ þ þ þ þ þ þ þ þ þ þ
C. parvum  þ þ   þ þ þ þ  þ þ þ 
Microsporidia  þ þ   þ þ þ  þ þ þ þ þ
B. hominis  þ þ þ þ a
þ þ þ þ  þ þ þ þ
Note: ‘þ’ denotes that a putative gene is annotated in the database or the gene was previously described. ‘þa’ denotes that the corresponding gene is annotated as ‘hypo-
thetical protein’ in the database, and we manually annotated the gene based on our blast search, which showed a significant similarity to the ortholog from S. cerevisiae,
E. coli, H. pylori, or A. thaliana (>30% amino acid identity). ‘’ denotes that the organism does not have an orthologous gene based on previously published data, or our
survey, which shows no significant similarity to the ortholog from S. cerevisiae, E. coli, H. sapiens, or A. thaliana (<30% a.a. identity). ‘’ denotes that the organism does not
have a homologous gene, but that either a partial sequence or a conserved domain is present in the database.
Table 1.3 Genome wide analysis of the components of SUF and CIA systems
Systems SUF system CIA system
Components
Organisms Fur SufS SufA SufB SufC SufD SufE Nar1 Nbp Cfd Cia1 Dre2
E. coli þ þ þ þ þ þ þ þ þ þ þ 
S. cerevisiae        þ þ þ þ þ
A. thaliana  þ þ þ þ þ þ þ þ þ þ þ
H. sapiens        þ þ þ þ þ
E. histolytica        þ þ þ  
G. intestinalis        þ þ þ  
T. vaginalis        þ þ þ  
Trypanosoma spp.        þ þ þa þa þ
Leishmanias spp.        þ þ þ þ a

Plasmodium spp.  þ þ þ þ þ þ þ þ þ þ þ
C. parvum        þ þ þ þ 
Microsporidia        þ a
þ þ þ 
B. hominis    þ þ   þ þ þ þ 
Note: ‘þ’ denotes that a putative gene is annotated in the database or the gene was previously described. ‘þa’ denotes that the corresponding gene is annotated as ‘hypo-
thetical protein’ in the database, and we manually annotated the gene based on our blast search, which showed a significant similarity to the ortholog from S. cerevisiae,
E. coli, H. sapiens, A. thaliana (>30% amino acid identity). ‘’ denotes that the organism does not have an orthologous gene based on previously published data, or our
survey showing no significant similarity to the ortholog from S. cerevisiae, E. coli, H. sapiens, or A. thaliana (<30% a.a. identity) using homologs. ‘’ denotes that the
organism does not have a homologous gene, but that either a partial sequence or a conserved domain is present in the database.
46 Vahab Ali and Tomoyoshi Nozaki

that the free-living amoebozoan protozoon Mastigamoeba balamuthi also pos-


sesses the NIF system instead of the ISC system (Gill et al., 2007).

8.1.3 Localization of NIF system


It is generally accepted that all forms of the MROs ranging from the aerobic
mitochondria to highly reduced organelles such as hydrogenosomes and
mitosomes contain the ISC system for Fe–S cluster biogenesis. How-
ever, since E. histolytica lacks the ISC system, it remains as an open question
whether the NIF system, which substitutes the ISC system in this organism, is
also compartmentalised to the mitosomes. Recent immuno-electron micro-
scopic and biochemical studies have suggested that Nif S and Nif U are pre-
dominantly present in the cytoplasm, but a relatively small amount may also
be present in the mitosomes (Maralikova et al., 2010). This conclusion was
based on (1) the quantitation of gold particles per area on immuno-electron
micrographic images, showing an approximately 10-fold concentration of
Nif S and Nif U within mitosomes, compared with that in the cytosol and
(2) the quantitation of NifS and NifU in the soluble and pellet fractions of
lysates in immunoblot analysis. However, as the vast majority of EhNif S
and EhNif U are localised in the cytoplasm (Dolezal et al., 2010), it has not
been unequivocally demonstrated that the NIF system is concentrated to
mitosomes. A recent study by Tachezy and his colleague, however, clearly
demonstrated that neither Nif S nor Nif U is enriched in the organellar frac-
tion, suggesting that the previous conclusion may be erroneous (personal
communication). It is also of importance to note that the non-haem, non-
sulphur, iron-containing protein rubrerythrin, which is involved in oxygen
detoxification, is also localised in mitosomes (Maralikova et al., 2010). These
conflicting data need to be further verified with independent methods. It is
also worth mentioning that the M. balamuthi genome contains two isotypes of
Nif S and Nif U, and one isotype of each component has an amino-terminal
mitosome targeting transit peptides, and is localised in the mitosomes, while
the other isotype is localised in the cytoplasm (Nývltová et al., 2013). The
previously published report on the EST database did not identify the putative
amino-terminal mitosome-targeting peptides on Nif proteins (Gill et al.,
2007).

8.1.4 CIA system


In spite of the presence of the cytosolic NIF system for the Fe–S cluster
assembly, a set of genes encoding proteins involved in the CIA system,
namely, Nbp35, Cfd1 and Nar1, are present, while Cia1 and Dre2 are
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 47

missing in E. histolytica, which is also the case in G. intestinalis and T. vaginalis


(Table 1.3). A biochemical study using recombinant proteins as well as
in silico modelling analysis showed that Nbp35 and Cfd1 make a stable com-
plex and hold 3 [4Fe–4S] clusters for the transfer to apo-proteins (Anwar, S.
Ali, V. and Nozaki, T., unpublished). The significance of the fact that three
anaerobic protozoa, that is, E. histolytica, G. intestinalis and T. vaginalis, lack
both Cia1 and Dre2, which are relatively well conserved among other pro-
tozoan parasites except Blastocystis sp. (Stensvold et al., 2007), which lacks
Dre2, is not well understood. Since Cia1 is known to be involved in the final
transfer of an Fe–S cluster to apo-proteins, it is puzzling how Nar1 transfers a
nascent cluster to an apo-protein in the absence of Cia1. It is also unknown
how the CIA components work with the NIF system (see Section 10.3).

8.2. Giardia
G. intestinalis (synonymous to G. lamblia and G. duodenalis) is an anaerobic or
microaerophilic protozoan, similar to Entamoeba, and resides on the surface
of the epithelia of the small intestine. Giardia lacks the aerobic mitochondria,
and instead possesses mitosomes (Tovar et al., 2003). Energy generation is
solely dependent on glycolysis and fermentation (Adam, 1991, 2001;
Upcroft and Upcroft, 2001). For general reviews on epidemiology, clinical
manifestations and treatment of giardiasis, as well as energy metabolism and
drug resistance of G. intestinalis, one may consult previous reviews (Ali and
Nozaki, 2007; Thompson and Monis, 2012).

8.2.1 Fe–S proteins in Giardia


Only a few Fe–S proteins were either biochemically characterised or
predicted from the genome database of G. intestinalis (Tovar et al., 2003;
Jerlstrom-Hultqvist et al., 2010; Franzen et al., 2009; Thompson and
Monis, 2012), including ferredoxin, pyruvate ferredoxin oxidoreductase,
IscU, and IscA.

8.2.2 ISC system


G. intestinalis possesses the mitosomal ISC system, but lacks the SUF and
NIF systems. Among protozoan parasites, the ISC system was first demon-
strated in G. intestinalis and T. vaginalis (Tachezy et al., 2001). Immunoflu-
orescence assay and cellular fractionation showed that both IscS and IscU are
localised in the mitosomes (Tachezy et al., 2001; Tovar et al., 2003). The
robust phylogenetic analysis confirmed that G. intestinalis and T. vaginalis
IscS have a common ancestor with the orthologous proteins in the aerobic
48 Vahab Ali and Tomoyoshi Nozaki

eukaryotes (Emelyanov, 2003). Incorporation of de novo synthesised radio-


active Fe–S clusters into recombinant apoferredoxin was demonstrated
in vitro using the mitosome-enriched fraction (Tovar et al., 2003).
A recent proteomic analysis of the purified mitosomes (Jedelsky et al.,
2011), together with genome survey, demonstrated that mitosomes pos-
sesses all the key components of the ISC system (Table 1.2). These include
the catalytic component, IscS and the scaffold component, IscU-, IscA-,
Nfu-like protein, HscA, HscB, ferredoxin and Mge1. Nfu-like protein
and IscA may serve as an alternative molecular scaffold (Jedelsky et al., 2011).
Two IscA-like proteins, IscA1 (Isa1) and IscA2 (Isa2) are known to be
present in at least most of the aerobic eukaryotic mitochondria (Vinella
et al., 2009). In general, IscA1 is known to be present in bacteria as well
as the mitochondria, while IscA2 is exclusively in the mitochondria. It is
considered that IscA1 is involved in the transient coordination of Fe–S clus-
ters, while IscA2 serves as an iron donor (Ding et al., 2004; Song et al.,
2009). G. intestinalis mitosomes, as well as T. vaginalis hydrogenosomes, con-
tain only IscA2, but lack IscA1. The presence of a single IscA protein in
mitosomes and hydrogenosomes may be associated with the lifestyle of
G. intestinalis and T. vaginalis under anaerobic conditions. IscA1 may be lost
during evolution of anaerobic adaptation, suggesting that IscA1 may be an
oxygen-resistant scaffold of Fe–S clusters only the aerobic mitochondria
need. Alternatively, G. intestinalis IscA2 may have retained (or gained) dual
functions to perform functions of both IscA1 and IscA2.
Interestingly, Isd11 is missing in the genome and the mitosome proteome.
Isd11 interacts with and stabilises IscS in yeast and humans (Shi et al., 2009;
Wiedemann et al., 2006). The absence of Isd11 in G. intestinalis, Cryptosporid-
ium parvum (Section 8.7) and Blastocystis sp. (Section 8.9), as well as in bacteria
(Richards and van der Giezen, 2006), indicates that Isd11 is not an essential
component for the function of bacterial and mitochondrial IscS. FXN is also
missing in the genome and mitosome proteome. It is unexpected because
FXN is generally present in all eukaryotes possessing the mitochondrial
ISC system, including mitosome-containing organisms such as Encephalitozoon
cuniculi (Goldberg et al., 2008) and C. parvum (Abrahamsen et al., 2004) (see
below).
The proteome (Jedelsky et al., 2011) also revealed that the G. intestinalis
mitosomes contain monothiol glutaredoxin, Grx5, which has the conserved
CGFS motif, and may also serve as an alternative scaffold for the Fe–S cluster
export from the mitosome, as demonstrated in yeast (Rada et al., 2009). Grx5
catalyses the reduction of disulphide bonds in proteins using GSH. Although
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 49

the exact role of Grx5 is not very clear in this parasite, it is likely involved in
the transfer and coordination of the [2Fe–2S] cluster assembled transiently on
IscU in a GSH-dependent manner, and the subsequent delivery of the cluster
on to the apo-proteins at the final step. In addition, although GSH has not
been demonstrated and is believed to be lacking in G. intestinalis (Brown
et al., 1993), the genome analysis indicates the presence of genes encoding
two key enzymes required for GSH synthesis: glutamate cysteine ligase and
GSH synthase (Rada et al., 2009). Thus, GSH may be synthesised in
G. intestinalis at low concentrations that were not detectable in the previous
studies or under unknown conditions, for example, encystation (Brown et al.,
1993). G. intestinalis retains Mge1, similar to T. vaginalis, while it is missing in
E. histolytica. Erv1 is missing in G. intestinalis, similar to E. histolytica and
T. vaginalis. A partial sequence corresponding Atm1 gene was identified in
the G. intestinalis and T. vaginalis genomes.

8.2.3 Transport of Isc proteins to mitosomes


G. intestinalis IscU contains the amino-terminal region that is rich in
the hydroxylated and basic amino acids, which is reminiscent of the
mitochondrial-targeting presequence that is required for the targeting to
the organelles, while IscS lacks the transit peptide (Tachezy et al., 2001;
Tovar et al., 2003). These data suggest that the mitosomal protein import does
not depend solely on the amino-terminal transit peptide (Henze and Martin,
2003; Tovar et al., 2003). It was also demonstrated that the amino-terminal
transit sequence derived from G. intestinalis IscU is sufficient for the targeting
of a-tubulin to the hydrogenosomes in T. vaginalis (Dolezal et al., 2005).
Similarly, both G. intestinalis IscU and ferredoxin were mislocalised to the
cytoplasm when the amino-terminal presequence was removed (Dolezal
et al., 2005). Although it is generally believed that the amino-terminal transit
peptide plays an important role in the transport of mitosomal proteins, there
are also conflicting reports suggesting that the amino-terminal transit peptide
does not play a significant role in the mitosomal and hydrogenosomal trans-
port (Dolezal et al., 2005). The truncated amino- or carboxyl-terminal halves
of G. intestinalis IscS were still delivered to the hydrogenosomes when ectop-
ically expressed in T. vaginalis, suggesting that G. intestinalis IscS contains at
least two distinct targeting signals at the amino- and carboxyl halves (Dolezal
et al., 2005). It was also demonstrated in T. vaginalis that the amino-terminal
targeting signal of the several abundant hydrogenosomal proteins, such as
pyruvate ferredoxin oxidoreductase, IscA and ferredoxin, is not required
for import into hydrogenosomes (Zimorski et al., 2013). These data also
50 Vahab Ali and Tomoyoshi Nozaki

demonstrated that the mitosomes in G. intestinalis and the hydrogenosomes in


T. vaginalis share mechanisms of protein targeting to some extent, despite the
fact that their constituents and physiological roles are largely different. These
data support the notion that the mitosomes, hydrogenosomes, and mito-
chondria represent different forms of the same fundamental organelle having
evolved under distinct selection pressure (Dolezal et al., 2005).

8.2.4 CIA system in Giardia


As in E. histolytica and T. vaginalis, Nar1, Nbp35, and Cfd are present, but
Cia1 and Dre2 are missing in the genome. However, these components are
not yet characterised in G. intestinalis.

8.3. Trichomonas
T. vaginalis is a flagellated anaerobic or microaerophilic protozoan, which
resides on the surface of the epithelia in the urogenital tract of both sexes
and can cause vaginitis in women and urethritis and prostatitis in men.
T. vaginalis causes trichomoniasis, a common sexually transmitted human
infection, with 170 million cases occurring annually worldwide
( Johnston and Mabey, 2008). Trichomonas lacks the aerobic mitochondria,
like Entamoeba and Giardia, and possesses one form of MRO, called hydro-
genosomes, in which ATP, carbon dioxide, and hydrogen gas are produced
(Hrdy et al., 2004; Putz et al., 2006). Previous reviews on epidemiology,
energy metabolism, disease, treatment, and mechanism of the metronidazole
resistance should be consulted (Ali and Nozaki, 2007; Johnston and Mabey,
2008; Schwebke and Burgess, 2004).

8.3.1 Fe–S proteins, Fe–S cluster biogenesis proteins, and


hydrogenosomal proteins in T. vaginalis
The various Fe–S proteins were previously characterised or predicted based
on the genome database of T. vaginalis (Dolezal et al., 2007; Sutak et al.,
2004). Genome-wide analysis of T. vaginalis predicted 12 genes encoding
the proteins involved in the ISC assembly or maturation (Carlton et al.,
2007; Tachezy et al., 2001). Recently, the proteome analysis of the
T. vaginalis hydrogenosomes identified 569 proteins including IscU, IscA,
ferredoxin, Hsc20 (HscB), GrpE (Mge1), and two proteins of hydrogenase,
HydF and HydG (Schneider et al., 2011). However, both FXN- and Isd11-
like proteins are missing in the hydrogenosome proteome despite the fact
that they contain the putative amino-terminal mitochondrial-targeting pres-
equence (Schneider et al., 2011).
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 51

8.3.2 ISC system


Similar to G. intestinalis, T. vaginalis possesses genes for the ISC system
including the export and CIA machineries, but lacks those for the SUF
and NIF systems (Table 1.2). The genome of T. vaginalis contains two IscS
homologues (TvIscS1 and 2), which had been previously identified
(Tachezy et al., 2001). It was considered that TvIscS1 may be a pseudogene
or encodes only a truncated protein because it is shorter than TvIscS2 and
lacks the carboxyl-terminal part present in TvIscS2. In addition, the poten-
tial amino-terminal targeting sequence of TvIscS1 lacks a typical arginine
residue at 2 position, which was assumed to be the cleavage site by
hydrogenosomal processing peptidase (Tachezy et al., 2001). It was shown
that TvIscS2 is expressed, translocated to the hydrogenosomes, and involved
in biosynthesis of Fe–S proteins in T. vaginalis (Sutak et al., 2004). Although
in yeast and humans, IscS was also shown to be targeted to the nucleus (Land
and Rouault, 1998; Nakai et al., 2001), TvIscS2 was not detected in the
nucleus of T. vaginalis (Sutak et al., 2004). A nuclear targeting motif
RRRPR, which was verified experimentally in yeast (Nakai et al.,
2001), is highly conserved in all mitochondrial IscS homologues, but absent
in IscS from T. vaginalis and G. intestinalis (Sutak et al., 2004).
A phylogenetic analysis of IscS showed a common eubacterial ancestor,
suggesting a common origin of the ISC system for the Fe–S cluster biogen-
esis in T. vaginalis, G. intestinalis, and Plasmodium (Tachezy et al., 2001).
Unlike in E. histolytica and G. intestinalis, Isd11 and FXN are present in
the T. vaginalis genome. T. vaginalis Isd11 has 47% amino acid identity with
the yeast orthologue (Richards and van der Giezen, 2006), but its function-
ality, such as the association with IscS/Nfs1p and the enhancement of cys-
teine desulphurase, needs to be verified. T. vaginalis FXN contains all the
conserved residues implicated in iron binding and appears to retain the nec-
essary structures such as a typical a-b-sandwich motif (Dolezal et al., 2007).
T. vaginalis FXN also contains the amino-terminal targeting peptide.
T. vaginalis FXN can partially rescue defects in the haem and Fe–S cluster
biosynthesis in the FXN-deficient yeast strain (Dolezal et al., 2007). For
unknown reasons, FXN (Dolezal et al., 2007) and Isd11, both of which con-
tain a putative amino-terminal presequence (Richards and van der Giezen,
2006), were missing in the hydrogenosome proteome (Schneider
et al., 2011).
Although IscS, IscU, IscA, Fdx, Hsc20 (HscB) and GrpE (Mge1), which
are involved in the ISC system, were identified by proteomic analysis
(Schneider et al., 2011), the scaffold components (IscU, IscA) of the ISC
52 Vahab Ali and Tomoyoshi Nozaki

system in T. vaginalis have not yet been characterised and need attention
because they likely play different roles in the Fe–S cluster assembly.

8.3.3 ISC export and CIA machineries


Similar to E. histolytica and G. intestinalis, Nar1, Nbp35 and Cfd are present,
but Cia1 and Dre2 are missing in the genome. It is important to understand
how Nar1 can transfer the Fe–S clusters to apo-proteins without Cia1 in the
anaerobic or microaerophilic protozoan. Furthermore, two additional pro-
teins Atm1 and Erv1 that are found in the aerobic mitochondria and are
involved in the ISC export machinery are also absent from the proteome
(Schneider et al., 2011), which is consistent with the previous finding based
on the genome (Carlton et al., 2007). Thus, it remains unknown how the
Fe–S clusters that are assembled in the hydrogenosomes, are transported to
the cytosol in the absence of any known export machinery (Table 1.2).
However, a recent genome analysis showed that Atm1 may be present in
T. vaginalis, in contrast to G. intestinalis and E. histolytica (Ali, V. et al.,
unpublished).

8.4. Leishmania
Leishmania is a genus of intracellular protozoan species that replicates within
phagolysosomes of macrophages in mammals. They have a complex life cycle
involving both mammalian and invertebrate hosts. The major Leishmania spe-
cies include Leishmania donovani, Leishmania infantum, Leishmania braziliensis,
Leishmania major, and Leishmania mexicana, the first two of which mainly cause
visceral leishmaniasis (Kala azar), the third mucocutaneous leishmaniasis, and
the last two, cutaneous leishmaniasis, respectively, in around 350 million peo-
ple annually throughout the world (Antoine et al., 1998).

8.4.1 Fe–S proteins in Leishmania species


Promastigotes, the extracellular insect-stage parasites, utilise glucose as a pri-
mary energy source, while intracellular amastigotes, the vertebrate stage par-
asites, depend primarily on amino acids and fatty acids as their carbon source
(Naderer and McConville, 2008). It was shown that the increased mito-
chondrial activity in amastigotes plays a crucial role in the survival of the par-
asite in the host cells (McConville et al., 2007; Naderer and McConville,
2008). The single reticulated mitochondrion in Leishmania, Trypanosoma
brucei, and T. cruzi, having the traditional tricarboxylic acid (Kreb’s) cycle
coupled with oxidative phosphorylation, produces energy mainly through
the electron transport consisting of complex I (NADH dehydrogenase),
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 53

II (succinate dehydrogenase), III (cytochrome c reductase) and IV (cyto-


chrome c oxidase), embedded in the inner membrane of the mitochondria
(Cermakova et al., 2007; Hellemond et al., 2005; Panigrahi et al., 2008), all
of which contain one or more Fe–S cluster-containing proteins (Sazanov,
2007; Sazanov and Hinchliffe, 2006; Sun et al., 2005).
While most of these Fe–S proteins are involved in the electron transport
chain, where Fe–S proteins are associated with the redox processes, [4Fe–4S]
cluster-containing mitochondrial aconitase plays a unique catalytic role in
the Kreb’s cycle. Aconitase catalyses the elimination of a molecule of water
from citrate to form cis-aconitate, and then the rehydration of cis-aconitate
with the same eliminated water molecule to form isocitrate. In this case, one
special iron atom in the cluster is centrally involved in the coordination of
the water molecule that is first eliminated from citrate and then used to rehy-
drate cis-aconitate.

8.4.2 ISC system


A genome-wide search using the Leishmania genome database at NCBI/
DDBJ/Sanger and EuPath DB showed the presence of the ISC system
including the export machinery in L. donovani, L. infantum, L. major, and
L. braziliensis (Ivens et al., 2005). While all the genes involved in the ISC
system, except for IscR, which is a [2Fe–2S]-containing transcriptional
repressor in bacteria, are present in the Leishmania genomes, their localiza-
tion in the genome (e.g. chromosomes) varies among species. For instance,
iscS and isd11 genes have been mapped to chromosome 27 and 33, respec-
tively, in all these species, while iscU and iscA genes have been found on
chromosome 35 in L. donovani, L. infantum, and L. major, and on chromo-
some 34 in L. braziliensis. Genes encoding FXN involved in the iron storage
and transfer for the Fe–S cluster assembly are located on chromosomes
25 and 29 of L. donovani, L. infantum, L. major and L. braziliensis. Surpris-
ingly, while hscA gene is found in the all four species of Leishmania on chro-
mosome 30, a partial sequence corresponding to hscB (dnaJ) gene is present
on chromosome 36 in these species. The components for the export
machinery, Atm1, Erv1p, and Grx5 are also present in L. donovani, and
L. infantum, L. major, and L. braziliensis (Table 1.2) and their respective genes
were mapped to chromosome 32, 15, and 01, respectively.

8.4.3 Nature and interaction of ISC system


L. donovani IscS and IscU appear to be present as a single protein in both drug
(sodium stibogluconate and amphotericin B)-sensitive and -resistant strains,
54 Vahab Ali and Tomoyoshi Nozaki

as shown by immunoblot analysis. L. donovani IscS and IscU were confirmed


to be localised in the mitochondria of promastigotes by cellular fractionation
using digitonin and immunofluorescence imaging. Recombinant
L. donovani IscS showed in vitro cysteine desulphurase activity and an ability
to transfer sulphur to IscU (K. P. Singh et al., unpublished). Spectrophoto-
metric scanning of the in vitro reconstituted Fe–S cluster on the recombinant
L. donovani IscU showed that IscU holds a [2Fe–2S] cluster. It was also
found that the activities of Fe–S proteins such as aconitase in L. donovani clin-
ical isolates from sodium stibogluconate unresponsive patients were
up-regulated, compared to those in the isolates derived from drug-
responsive patients (Ali et al., unpublished), suggesting that the activity of
Fe–S cluster biogenesis or Fe–S proteins may be linked to drug metabolism
and resistance against anti-leishmanial drugs.
Recombinant L. donovani Isd11 enhanced cysteine desulphurase activity
and formed a stable complex with IscS (Amir et al., unpublished). Although
IscS and IscU are expected to form a complex, a stable complex of
L. donovani IscS and IscU was not detected by either immunoprecipitation
or pull down assay (K. P. Singh et al., unpublished). This is likely due to
transient interaction between IscS and IscU during cluster assembly. How-
ever, the formation of the IscS/IscU complex was shown in other systems,
for example, yeast and bacteria (Yuvaniyama et al., 2000). Similarly, it was
shown that the interaction between Nif S and Nif U is transient for the trans-
fer of reduced persulphide residue on cysteine 328 of Nif S to NifU, and that
the Nif S–Nif U complex dissociates immediately after the cluster formation
on IscU is completed (Yuvaniyama et al., 2000).
Interaction of L. donovani IscU and FXN was confirmed by co-purification,
although it was not directly demonstrated in the parasite (K.P. Singh et al.,
unpublished). Again, interaction between IscU and FXN and between IscU
and IscS/Isd11 during cluster assembly may be transient.

8.4.4 CIA system


All the components of the CIA machinery were identified in all four species,
L. donovani, L. infantum, L. major and L. braziliensis. Cfd1, Nbp35, Nar1 and
Cia1 are present on chromosomes 26, 21, 5, and 10, respectively, similarly in
the four species. L. braziliensis orthologues of IscS, Isd11, Nbp35, Nar1 and
Cfd1 are 24–60-a.a. shorter than those in other species. Although they have
high (70–80%) mutual amino acid identity between species, the apparent
length difference may suggest that their interaction during complex forma-
tion may also differ.
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 55

8.5. Trypanosoma
Trypanosoma belongs to the order Kinetoplastida, together with Leishmania,
which is a monophyletic group of flagellated protozoa. Both T. brucei and
T. cruzi require at least two obligatory hosts including mammals and insects
to complete their life cycle. Trypanosomes are responsible for various diseases,
including the deadly human disease called sleeping sickness, caused by T. brucei,
and Chagas disease, caused by T. cruzi. T. brucei is extracellular throughout its
life cycle and relies exclusively on glycolysis, which occurs in glycosomes
(Coustou et al., 2003; Tielens and van Hellemond, 2009), in the bloodstream
trypomastigote form, the mammalian stage, but relies on the mitochondrial res-
piration in the procyclic trypomastigote form, the insect stage, which parasitises
the tsetse fly vector. The mitochondrion of the procyclic trypomastigote stage
contains the cytochrome c-carrying respiratory complexes, the alternative ter-
minal oxidase (TAO), acetate:succinate CoA transferase, and the incomplete
Krebs cycle. Due to its reliance on glycolysis and substrate-level phosphoryla-
tion, the bloodstream stage represses many functions of the mitochondrion,
including oxidative phosphorylation (Coustou et al., 2003; Hannaert et al.,
2003; Tielens and van Hellemond, 2009). However, since neither the TAO
(Chaudhuri et al., 2006) nor the ATPase complex, which are still functional
in the bloodstream stage, contain any Fe/S clusters, the demand for these cofac-
tors dramatically drops in the mitochondrion in the bloodstream trypomastigote
stage, as compared to the procyclic trypomastigote stage (Alfonzo and Lukes,
2011; Tielens and van Hellemond, 2009; see Section 8.5.4).

8.5.1 Fe–S proteins in T. brucei


T. brucei has various Fe–S proteins such as NADH dehydrogenase subunit,
NADH:ubiquinone oxidoreductase of complex I, Rieske Fe–S protein,
aconitase of the tricarboxylic acid cycle, ferredoxin, and complexes II and
III of the respiratory chain in the procyclic trypomastigote form. Moreover,
a number of nuclear and cytosolic proteins, including the ribosomal protein
Rli1, primase Pri2 and xanthine oxidoreductase, depend on Fe–S clusters for
their functions (Alfonzo and Lukes, 2011).

8.5.2 ISC system


The whole genome analysis (Berriman et al., 2005) revealed that both T. brucei
and T. cruzi retain the complete ISC system, the export machinery and the
CIA system, but lack the SUF and NIF systems (Tables 1.2 and 1.3), similar
to Leishmania. It was demonstrated that IscS and IscU are localised to the
56 Vahab Ali and Tomoyoshi Nozaki

mitochondria and essential for the Fe–S cluster formation and consequently
the viability of the procyclic stage parasites of T. brucei (Smid et al., 2006). Fur-
thermore, repression of IscS and IscU by RNA interference affected the mat-
uration of Fe–S proteins not only in the mitochondria but also in the cytosol.
Thus, a crucial part of the Fe–S cluster assembly is localised to the mitochon-
drion of T. brucei (Smid et al., 2006). It was also demonstrated that Isd11 is
essential for the Fe–S cluster assembly of both mitochondrial and cytosolic
proteins in the procyclic trypomastigotes of T. brucei, as in other eukaryotes
(Adam et al., 2006; Goldberg et al., 2008; Shi et al., 2009; Wiedemann
et al., 2006). IscA (Isa) functions as an alternative scaffold for the assembly
of Fe–S clusters and provides iron for their assembly. It was demonstrated that
two isotypes of Isa, Isa1 and Isa2, which are abundantly present in the pro-
cyclic form, are required for the Fe–S cluster assembly of mitochondrial
aconitase, fumarase and succinate dehydrogenase, and are thus indispensable
for growth in T. brucei (Long et al., 2011).

8.5.3 Significance of Fe–S cluster biogenesis in thiolation of tRNA


Involvement of the ISC system in thiolation of tRNA in trypanosomes has
been demonstrated (Paris et al., 2009). Due to a complete lack of the tRNA
genes in the mitochondrial genome of T. brucei, all tRNAs needed for mito-
chondrial translation need to be imported from the cytosol into the mito-
chondria. The modified nucleotide 2-thiouridine (s2U) was shown to act
as a negative determinant for the import of mitochondrial tRNA in Leish-
mania tarentole (Kaneko et al., 2003). However, the inhibition of IscS led to a
concomitant decrease in tRNAGlu thiolation in T. brucei, but it had no effect
on the distribution of this tRNA species, suggesting that s2U is not a negative
determinant for tRNA import in T. brucei (Paris et al., 2009). It was shown
that Isd11 forms a stable complex with IscS and IscU and is localised in the
mitochondria of T. brucei (Paris et al., 2009). It was also shown that Isd11
functions for the thiolation of both cytoplasmic and mitochondrial tRNAs.
These data indicate that Isd11 plays an important and essential role in tRNA
thiolation as well as the Fe–S cluster assembly and the maturation of Fe–S
proteins in general in T. brucei. In contrast, Mtu1 is an essential factor that
is exclusively for the mitochondrial tRNA thiolation, but not the Fe–S clus-
ter assembly of Fe–S proteins in general (Paris et al., 2010).

8.5.4 Developmental stage variation of Fe–S cluster biogenesis


in T. brucei
During the transition from the bloodstream to the procyclic stage, the single
mitochondrion of T. brucei makes remarkable morphological alteration from
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 57

a highly reduced vesicle-like mitochondrion to a cristae-rich reticulated


organelle. This transition requires almost instant mitochondrial activation
and concomitantly the up-regulation of the mitochondrial Fe–S cluster bio-
genesis (Alfonzo and Lukes, 2011). Remarkably, the overall metabolic
changes observed in the Fe–S cluster-impaired trypanosomes mimics those
which occur during stage conversion. Consequently, it was proposed that
the function of the Fe–S cluster assembly machinery is critical for the
inter-stage changes in the T. brucei life cycle (Alfonzo and Lukes, 2011).
As described earlier (Section 8.5.2), two isotypes of IscA (Isa), Isa1 and
Isa2, are required for the Fe–S cluster assembly of a variety of mitochondrial
enzymes necessary for the procyclic form. In contrast, in the bloodstream
form, neither Isa1 nor Isa2 is essential as double knock-down did not affect
either the activity of cytosolic Fe–S proteins (e.g. ferredoxin) or growth of
the bloodstream trypomastigotes (Long et al., 2011).

8.5.5 Transport of proteins involved in Fe–S cluster biosynthesis


to the mitochondria in T. brucei
It was shown that the amino-terminal transit peptides of FXN from a plant,
A. thaliana, and a diatom, Thalassiosira pseudomonas, interchangeably function
in the import of FXN to the mitochondria of T. brucei (Long et al., 2008c).
Furthermore, plant and diatom frataxins were targeted and processed in the
mitochondria in a T. brucei strain where endogenous FXN was repressed
(Long et al., 2008c). In addition, plant, diatom and human frataxins rescued
a lethality of T. brucei FXN deficiency (Long et al., 2008b,c). It was also
suggested that trypanosome FXN functions primarily only in Fe–S cluster
biogenesis and protection against ROS, and not in iron storage (Long
et al., 2008a).

8.5.6 CIA machinery


All the components of the CIA machinery were identified in T. brucei. Cfd1,
Nbp35, Nar1 and Cia1 are present on chromosome 10, 7, 10 and 8, respec-
tively. Cia1 and Cfd1 are annotated as ‘hypothetical protein’ and nucleotide-
binding protein 2, respectively, in the TriTryp genome database (Ali et al.,
unpublished).

8.6. Plasmodium
Plasmodium is a genus of apicomplexan parasites responsible for malaria affect-
ing a wide range of vertebrates including humans. There are an estimated 500
million infections, with 1–2 million deaths occurring annually in humans.
There are four species of Plasmodium that cause human malaria: Plasmodium
58 Vahab Ali and Tomoyoshi Nozaki

falciparum, Plasmodium ovale, Plasmodium vivax, and Plasmodium malariae. Clin-


ical signs include fever, chills, headache, diarrhoea, anaemia, pulmonary and
renal dysfunction and neurological disorders. Human Plasmodium species share
the similar complex life cycle consisted of sexual reproduction in the insect
(mosquito) vector and asexual replication in the human host. They possess
two essential endosymbiont-derived organelles: the mitochondria and the
apicoplast (non-photosynthetic plastid). The apicoplast of Plasmodium is
reduced, contains only small genome of 35 kb (Wilson et al., 1996), and inca-
pable of photosynthesis despite its cyanobacterial origin. The mitochondria of
Plasmodium are unique in that their carbon and energy metabolism are rem-
odelled through reductive evolution. The Plasmodium mitochondria contain a
minimal 6 kb genomic DNA (Vaidya et al., 1989, 1993) and lack mitochon-
drial pyruvate dehydrogenase and Hþ-translocating NADH dehydrogenase
(complex I, NDH1). In the insect stages, oxidative phosphorylation occurs
in the mitochondria by using 2-oxoglutarate as an alternative means of entry
into the tricarboxylic acid cycle and a single-subunit flavoprotein as an alter-
native NADH dehydrogenase (NDH2). In contrast, in the human blood
stage, mitochondrial enzymes are down-regulated and parasite energy metab-
olism relies mainly on glycolysis (Mogi and Kita, 2010).
8.6.1 Fe–S proteins in Plasmodium
Fe–S proteins are distributed to three compartments in Plasmodium: the
mitochondria, the apicoplasts and the cytosol (Seeber, 2002; Kumar et al.,
2011). The major Fe–S proteins include mitochondrial ferredoxin, ferre-
doxin:NADPþ reductase, succinate dehydrogenase, aconitase, fumarate
hydratase in the mitochondria; and plant-type ferredoxin, ferredoxin:
NADPþ reductase, proteins of PSI (Fx, FA, and FB), NADH dehydrogenase,
lipoic acid synthase, IspG and IspH, involved in isoprenoid biosynthesis,
SufU and SufA in the apicoplasts (Seeber, 2002; Vollmer et al., 2001).
The abundant electron acceptor proteins Fx, FA and FB of PSI and the FrxB
subunit of NADH dehydrogenase all contain Fe–S clusters (Yu et al., 1997)
and are encoded by the chloroplast genome.
8.6.2 Two Fe–S biogenesis systems in Plasmodium
Fe–S cluster biogenesis occurs both in the mitochondria and in the
apicoplast in Plasmodium (Seeber, 2002; van Dooren et al., 2006).
P. falciparum possesses both the ISC and SUF systems (Wilson, 2002)
(Table 1.3). It was suggested that the ISC system in the mitochondria plays
a house-keeping role, while the SUF system localised in the apicoplast func-
tions under oxidative stress and iron-deficient conditions, as in prokaryotic
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 59

systems (Seeber, 2002). The two independent Fe–S pathways are required in
these compartments, as apicoplast- and mitochondria-targeted proteins are
transported to the organelles across the double to quadruple membranes of
the mitochondria and the apicoplast, respectively, in an unfolded state, and
in an apo-form, and subsequently assembled with Fe–S clusters inside the
respective organelles (Lim and McFadden, 2010).

8.6.3 SUF system


In the P. falciparum genome, six genes involved in the SUF system are pre-
sent. The SufB gene alone stands on the plastid genome, while the other
five genes (SufA, C, D, S and E) are encoded by a nuclear genome. The SufC
gene is located on chromosomes 14, while the other members of the SUF
genes (SufA, D, S and E) are present on the other chromosomes. All the five
proteins encoded by the nuclear genome possess the putative plastid
targeting sequence (Kumar et al., 2011).
While the exact role of the Suf BCD complex in the Fe–S cluster assembly
remains unknown (Ye et al., 2006), ATPase activity and the localization in
the apicoplasts of P. falciparum SufC and Suf B were demonstrated (Kumar
et al., 2011; Nachin et al., 2003), as observed for A. thaliana plastidial Suf B
(Moller et al., 2001; Xu et al., 2005). Although P. falciparum SufB contains
transmembrane domains and is annotated as a membrane subunit of an ABC
type transporter, it was predicted to be a non-integral membrane protein.
It was also demonstrated that P. falciparum Suf B and SufC interact and likely
make a stable complex in vitro, demonstrated by co-purification using affinity
column chromatography (Kumar et al., 2011). P. falciparum Suf B and Suf D
are assumed to interact with each other, as seen for the E. coli Suf C2 and
Suf D2 complex, in which the carboxyl-terminal a-helices of Suf D interact
with SufC (Wada et al., 2009). Molecular modelling and protein docking
analysis of P. falciparum Suf B and SufC based on the crystal structure of
E. coli SufD and SufC predicted additional interaction sites that lie within
the internal region of Suf B. These interactions may be mediated by the internal
b-strands in P. falciparum Suf B (Kumar et al., 2011). SufA- and Nfu-like pro-
teins (Table 1.2) are assumed to function as the scaffold for Fe–S cluster assem-
bly either alone or with Suf B. The source of iron, the form in which iron
enters the apicoplast as well as the iron transporter remain unknown (Lim
and McFadden, 2010).
In the erythrocytic stage, Fe–S proteins in the P. falciparum apicoplasts are
constantly exposed to oxygen stress, attributable to haem degradation in the
60 Vahab Ali and Tomoyoshi Nozaki

digestive vacuoles and respiration in the mitochondria. Thus, it is conceiv-


able that it is probably beneficial for P. falciparum to retain the SUF system in
the apicoplast despite the fact that it has lost photosynthesis, which produces
high levels of reactive oxidative intermediates. However, transcription of
SufB and SufC genes was not up-regulated upon oxidative stress; neither
was the amount of SufC protein (Outten et al., 2004). Therefore, it remains
unknown whether the physiological roles of the SUF system in the
apicoplasts is associated with stress response.

8.6.4 ISC system


The IscS system and the genes involved in the system were first described in
P. falciparum in the early 2000s (Ellis et al., 2001; Seeber, 2002). The amino-
terminal mitochondrial targeting sequence of IscS was then shown to target
IscS-fused GFP to the mitochondrion (Sato et al., 2003). The scaffold com-
ponents (IscU and IscA), molecular chaperones [Ssq1 (HscA, mitochondrial
Hsp70) and Jac1 (HscB)], an iron carrier (FXN) and the export machinery
(Atm1 and Erv1) were discovered (Seeber, 2002). Our recent analysis indi-
cated additional genes (Isd11, Grx5, and Mge1) involved in Fe–S cluster
assembly in the mitochondria (Table 1.2). All the components of the CIA
system are apparently present in P. falciparum (Table 1.3). Thus, altogether,
P. falciparum apparently possesses the complete ISC system for Fe–S cluster
biogenesis in the mitochondria and the cytoplasm. The components of
the ISC, export, and CIA systems in P. falciparum have not been well
characterised so far and need more attention.

8.7. Cryptosporidia
C. parvum and Cryptosporidium hominis are gut-dwelling intracellular parasites
that infect both humans and animals, and belong to the order Apicomplexa,
together with Plasmodium and Toxoplasma gondii. In AIDS patients,
C. parvum is an opportunistic pathogen, causing significant morbidity and
mortality by inducing chronic diarrhoeal disease (Chen et al., 2002).

8.7.1 Fe–S proteins in Cryptosporidium


Fe–S proteins so far characterised in Cryptosporidium are very limited: ferre-
doxin, Grx5, IscU and pyruvate:NADPH oxidoreductase (Abrahamsen
et al., 2004; Xu et al., 2004). However, a previous study and our genomic
analyses showed that Cryptosporidium possesses both the ISC and CIA
machineries, but lacks the SUF system (Ali et al., unpublished; LaGier
et al., 2003).
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 61

8.7.2 ISC system and CIA machinery


The proteins involved in the Fe–S cluster assembly in C. parvum have not
been well characterised. It was shown that C. parvum IscS and IscU fused
with GFP were targeted to the mitochondria in yeast (LaGier et al.,
2003). Our genome-wide survey indicated that molecular chaperones,
Ssq1 (HscA) and Jac1 (HscB), ferredoxin, Mge1 and FXN are present,
whereas the scaffold components (IscA and Nfu) and Isd11 are missing
(Table 1.2). IscU is a sole scaffold component for Fe–S cluster biogenesis.
The export machinery (Atm1, Erv1 and Grx5) is likely present, although
the identity to yeast or mammalian orthologue is below 30% and full-length
genes are not found in the genome database. Genes involved in the CIA
machinery (Nar1, Nbp35, Cfd1, and Cia1) are also present (Table 1.3).
However, neither the SUF nor NIF system is present in C. parvum
(Table 1.3).

8.8. Microsporidia
Microsporidia are intracellular parasites that infect a variety of animals
including humans (Franzen and Muller, 2001). The Microsporidia were
previously classified as protozoa, but are now considered to belong to the
class of fungi (Baldauf et al., 2000; Hirt et al., 1999; Keeling et al., 2000;
Van de Peer et al., 2000). As highly specialised parasites, they are
characterised by a number of unusual adaptations, many of which are
manifested as extreme reduction at the molecular, biochemical and cellular
levels (Burri et al., 2006; Tsaousis et al., 2008).

8.8.1 Fe–S proteins in Microsporidia


The major characterised Fe–S proteins in Microsporidia include ferredoxin,
ferredoxin-NADPþ oxidoreductase (FNR), IscU, Nar1 and Rli1p
(Goldberg et al., 2008; Katinka et al., 2001).

8.8.2 ISC system


Based on the complete genome sequence of E. cuniculi (Katinka et al., 2001),
the major components involved in the ISC system are present in the
genome. Fe–S cluster biosynthesis is the only apparent function of the
mitosomes in Microsporidia (Goldberg et al., 2008), similar to the case in
G. intestinalis. The proteins involved in the ISC assembly and export
(IscS, Isd11, IscU, Ssq1, Jac1, ferredoxin, FXN, Grx5, Atm1 and Erv1) were
identified, while IscA, Nfu and Mge1 are missing (Burri et al., 2006;
Goldberg et al., 2008) (Tables 1.2 and 1.3). The proteins involved in the
62 Vahab Ali and Tomoyoshi Nozaki

protein and metabolite import to the mitosomes (TOM70, TIM22,


TOM40, Imp2, mitochondrial (mitosomal) Hsp70 and ATP/ADP trans-
porters) were also present in the genome (Burri et al., 2006). None of com-
ponents of the SUF and NIF systems were identified.
Several proteins involved in the ISC system in E. cuniculi and
Trachipleistophora hominis were characterised, such as IscS, Isd11, IscU, ferre-
doxin, mitochondrial Hsp70 and Grx5 (Goldberg et al., 2008). T. hominis IscS
(Nfs) and Isd11 that were ectopically expressed and co-isolated with the E. coli
membranes showed cysteine desulphurase activity (Goldberg et al., 2008).
T. hominis IscU was shown to serve as a scaffold protein (Burri et al., 2006).
Expression of E. cuniculi Yfh1 (FXN) and Grx5 rescued the growth defect
of the respective yeast Fe–S cluster mutants, confirming functional inter-
changeablility. In E. cuniculi, IscS, IscU and ferredoxin co-localised with the
mitochondrial Hsp70. However, in T. hominis, IscS and mitochondrial
Hsp70 co-localised in the mitosome, while the major pool of IscU and ferre-
doxin are in the cytosol (Goldberg et al., 2008). Localization of these proteins is
still under debate because some of the antibodies used in the study were raised
against heterologous proteins. These studies suggested that the biosynthesis of
Fe–S clusters likely occurs at least in part in mitosomes.

8.8.3 Mitosomes in Microsporidia


Like other MROs in E. histolytica, G. intestinalis, T. vaginalis and C. parvum,
the microsporidian mitosome lacks its own genome and requires the trans-
port machinery for the import of the nuclear-encoded mitosomal proteins as
described earlier. In addition, the microsporidian mitosome is highly
reduced in both physical size and biochemical properties. The number of
putative mitosomal proteins identified in all microsporidian species was
around 20–30 (Katinka et al., 2001; Williams and Keeling, 2005). This is
in remarkable contrast to the number of proteins estimated to be present
in the yeast mitochondria, 800–1000 (Sickmann et al., 2003; Truscott
et al., 2003). The microsporidian mitosomes lost the capacity for ATP pro-
duction through oxidative phosphorylation. Instead of exporting ATP
synthesised in the mitochondria, as seen in aerobic organisms, the micro-
sporidian mitosomes incorporate ATP via a novel ATP/ADP exchanger
(nucleotide transporter, EcNTT3) (Tsaousis et al., 2008) and utilise it for
the protein quality control via chaperones.
A number of the microsporidian mitosomal proteins are known to be
transported to mitosomes, including ferredoxin, FXN, Imp2, mitochondrial
G3PDH and IscU in Antonospora locustae and E. cuniculi (Burri et al., 2006).
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 63

Interestingly, a majority of mitosomal proteins including mitochondrial


Hsp70, IscS, IscU, Atm1, Erv1 and mitochondrial G3PDH lack the canonical
amino-terminal mitochondrial targeting sequence, and were not recognised
by the yeast mitochondrial import machinery (Burri et al., 2006). These data
suggest that there may be at least two independent import mechanisms.

8.8.4 CIA machinery


All the components of the CIA system except Dre2 are apparently present in
Microsporidia (Table 1.3). The short truncated sequence that shows homol-
ogy to Dre2 is present in the genome. This protein has the conserved
domain known as CIAPIN1, and is annotated as cytokine-induced anti-
apoptosis inhibitor. However, none of the proteins have been characterised
biochemically in Microsporidia.

8.9. Blastocystis
Blastocystis is a highly prevalent anaerobic unicellular stramenopile found in
the intestinal tract of humans and various animals (Tan, 2008). Blastocystis is
the only stramenopile known to cause infections in human (Arisue et al.,
2002), although its pathogenicity in humans is still controversial. Despite
common characteristics of stramenopiles, including the presence of at least
one flagellum for motility, Blastocystis is exceptional and has no flagellum.

8.9.1 Fe–S proteins in Blastocystis


The presence of ISC system in Blastocystis sp. was first identified in the
mitochondrion-like organelle (MLO, synonymous to MRO) (Stechmann
et al., 2008). The Blastocystis sp. MLOs contain cristae, a mitochondrial
genome, and the membrane potential, but appear to lack classical aerobic
respiratory chain. Blastocystis sp. MLOs instead has an incomplete tricarbox-
ylic acid cycle and an incomplete oxidative phosphorylation chain, and
function in the complete absence of oxygen (Tan, 2004). Complexes
I and II of the electron transport chain, but not complexes III and IV or
F1Fo ATPases, are present. Thus, Blastocystis MLOs have the metabolic
properties of aerobic and anaerobic mitochondria and of hydrogenosomes
(Tan, 2004, 2008), and are convergently similar to related organelles in
the ciliate Nyctotherus ovalis (Boxma et al., 2005; Stechmann et al., 2008).
Fe–S cluster-containing proteins present in Blastocystis sp. are mainly
localised in the MLOs: iron-only [FeFe]-hydrogenase, pyruvate:ferredoxin
oxidoreductase, ferredoxin, Grx5, aconitase, NADH:ubuiquinone oxidore-
ductase (complex I), and succinate dehydrogenase (Stechmann et al., 2008).
64 Vahab Ali and Tomoyoshi Nozaki

8.9.2 ISC and CIA systems in Blastocystis


EST and genome analysis of Blastocystis sp. revealed mitochondrial charac-
teristics, including iron-only [FeFe] hydrogenase, pathways for amino acid
and fatty acid metabolism, ISC biogenesis, and incomplete tricarboxylic acid
cycle and an incomplete respiratory chain. It has been shown that Blastocystis
IscS, IscU and FXN proteins were co-localised with the mitochondrial
marker Mitotracker by immunofluorescence analysis (Tsaousis et al., 2012).
Blastocystis sp. FXN, IscS and IscU were ectopically expressed in the
T. brucei lines where endogenous trypanosomal FXN, IscS or IscU was
repressed by RNA interference (Tsaousis et al., 2012). Blastocystis sp.
FXN rescued the growth defect of the FXN-repressed T. brucei line. How-
ever, Blastocystis sp. IscU failed to rescue the growth defect of the
corresponding T. brucei line (Tsaousis et al., 2012). Furthermore, while
Blastocystis sp. IscS alone failed to rescue the growth defect of the IscS-
repressed T. brucei line, but partially restored the growth defect in the pres-
ence of Isd11 of Phaeodactylum tricornutum. However, no Isd11 homologue
was identified in Blastocystis ESTs, nor could it be amplified by degenerate
PCR (Tsaousis et al., 2012).
The Blastocystis sp. ESTs generated revealed genes encoding the catalytic
and scaffold components (IscS and Isa2), FXN, ferredoxin, mitochondrial
Hsp70 and Grx5 (Stechmann et al., 2008). The recent proteome analysis also
demonstrated that the scaffold components (IscU and IscA2), Ssq1
(mHSP70, HscA) and Jac1 (Hsp40, HscB), Mge1, and FXN, the export
machinery (Grx5, Atm1, and Erv1) are present (Denoeud et al., 2011). It
was shown that Blastocystis sp. Isa2 partially rescued the growth defect and
loss of enzymatic activities in the TbIsa1/2-repressed T. brucei line (Long
et al., 2011).The components of the CIA machinery (Nar1, Nbp35,
Cfd1, and Cia1) were also identified in the Blastocystis sp. genome
(Denoeud et al., 2011).

8.9.3 SUF system in Blastocystis


The cytosolic SUF system has been recently described in Blastocystis sp.
(Tsaousis et al., 2012), suggesting an alternative or complementary Fe–S
cluster machinery in this organism. It was demonstrated that functional
SufBC proteins are present and interact with each other in the cytoplasm
(Tsaousis et al., 2012), also demonstrated by immunofluorescence and
immuno-electron microscopy. Blastocystis sp. apparently has adapted to
the partially oxidative environment in its parasitic lifestyle by acquiring
the SUF system from a non-pathogenic archaeal member of the human
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 65

microbiome to synthesise Fe–S clusters under oxygen stress. However, a


genome-wide survey revealed that all the remaining components of the
SUF system other than SufB and SufC are missing in Blastocystis sp.
SufC and SufB are fused in Blastocystis sp. and assumed to have been
acquired by lateral gene transfer from an archaeon related to the Met-
hanomicrobiales. Blastocystis sp. SufCB contains the domains homologous
to the proteins encoded in the sufCB operon of some bacteria and archaea.
Blastocystis sp. SufCB has similar biochemical properties to its prokaryotic
homologues. Genetic analysis using E. coli strains defective for the ISC
and SUF biogenesis (iscAU- and sufCD-disrupted) revealed that expression
of SufCB partially decreased the expression of both hmpA and iscR genes,
whose expression is repressed by Fe–S cluster-bound (holo) NsrR and IscR,
indicating that in the absence of the functional endogenous ISC and SUF
scaffolds, Blastocystis sp. SufCB is able to partially restore the NsrR and IscR
maturation by assembling their Fe–S clusters in the heterologous organism.
It was also shown that expression of SufBC was up-regulated under oxygen
stress, whereas the expression level of the ISC components and several Fe–S
proteins remained constant under these conditions (Tsaousis et al., 2012).
Furthermore, physical and biochemical properties, such as the [4Fe–
4S]2þ cluster and the flavin binding, of the recombinant Blastocystis sp.
SufCB, produced as inclusion body and refolded, were verified by UV spec-
trum, circular dichroism, EPR and iron and sulphur composition analysis.
Blastocystis sp. SufCB enhanced the cysteine desulphurase activity of the
E. coli SufSE complex, similar to the E. coli SufBC2D complex (Wollers
et al., 2010), despite the fact that Blastocystis sp. apparently lacks other
SUF proteins including SufS and SufE. Blastocystis SufCB also exhibited
ATP-hydrolyzing properties comparable to the E. coli SufBC2D complex.
Finally, the ability of Blastocystis SufCB to transfer its Fe–S cluster to E. coli
aconitase B, a (4Fe–4S) protein, was also verified.

9. REGULATION OF Fe–S PROTEIN BIOSYNTHESIS


UNDER STRESS CONDITIONS
9.1. Factors that affect Fe–S clusters
Since iron and sulphur are highly reactive chemical species, Fe–S clusters can
be easily affected and damaged under various stress conditions. Fe–S proteins
are one of the major targets of oxidative and nitrosative compounds that
cause displacement of the iron atoms of the cluster and consequent malfunc-
tion of the proteins. Univalent oxidants, including superoxide, hydrogen
66 Vahab Ali and Tomoyoshi Nozaki

peroxide and peroxynitrite are also physiological oxidants that inactivate


Fe–S proteins. In addition, during oxidative stress the release of iron
may further stimulate the formation of the hydroxyl radical. The loss of
an electron destabilises the cluster, causes a release of its catalytic iron atom,
and converts the cluster to an inactive [3Fe–4S]1þ form in a reaction:
Enz  [4Fe–4S]2þ þ H2O2 ! [3Fe–4S]þ þ 2OH þ Fe3þ.

9.2. Stress-dependent regulation of ISC and SUF systems


As described in Section 3.1.5, Fe–S clusters are exquisite sensors of oxygen
and oxygen radicals in stress-responsive transcription factors (Outten et al.,
2004). In bacteria and higher plants, the SUF system is believed to play a
specific role in Fe–S cluster biosynthesis under stress (Outten et al., 2004)
(see Section 5.3). Under oxidative stress and iron starvation conditions, both
isc and suf operons are induced (Lee et al., 2004a; Outten et al., 2004; Zheng
et al., 2001). The isc operon is negatively regulated by IscR, which loses its
repressor activity upon oxidative stress. The induction of the isc operon by
oxidative stress and iron starvation reflects a failure in the formation of the
Fe–S cluster in the IscR repressor in the regulatory system designed to sense
perturbations in the overall Fe–S cluster status of the cell (Schwartz et al.,
2001; Vecerek et al., 2007) (also see Section 3.1.5).
The suf operon also adapted for Fe–S cluster synthesis under conditions
of iron starvation and oxidative stress. The suf operon is regulated by OxyR,
integration host factor, Fur and IscR. OxyR and Fur are optimised to sense
hydrogen peroxide stress and iron starvation. It is known that hydrogen
peroxide-stressed E. coli activates the OxyR regulon, which includes the suf
operon. Oxidation of iron by hydrogen peroxide impedes the function of
Fur, the regulatory protein, and leads to disruption of iron homeostasis
(Vecerek et al., 2007). Hydrogen peroxide also reacts with iron cofactors of
metalloproteins and thus damages covalent polypeptides (Anjem et al.,
2009). Although both the isc and suf operons are induced in response to oxi-
dative stress and iron starvation, a deletion of the suf operon renders the cell
more sensitive to iron starvation than a deletion of the isc operon. It has been
observed that the ISC proteins cannot function effectively to synthesise Fe–S
clusters when iron is limited. Thus, the physiological roles of the suf and isc
operons diverge under iron starvation conditions, explaining the requirement
for two separate Fe–S cluster assembly operons in E. coli (Outten et al., 2004).
Iron depletion also caused derepression of the SUF system through both
the inactivation of Fur and the activation of IscR. The dissociation of Fur
from IscR is necessary to act as an activator under oxidative stress
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 67

(Lee et al., 2008). Therefore, the activation of the suf operon by IscR occurs
only under conditions where Fur loses its repressor activity, and those con-
ditions at the same time can convert IscR to an ironless activator form.

9.3. Stress-dependent response and regulation of Fe–S cluster


biosynthesis in parasitic protozoa
9.3.1 NIF system in Entamoeba
In E. histolytica, which possesses the NIF and CIA machinery and lacks the ISC
and SUF systems (see Section 8.1), there is no evidence suggesting the oxida-
tive stress-dependent regulation or anti-oxidative stress response of the Fe–S
cluster biosynthesis. Although E. histolytica NifS and NifU were capable of
complementing the defect in the biosynthesis of all species (e.g. [2Fe–2S],
[4Fe–4S]) of Fe–S clusters and the growth defect caused by the ISC and
SUF deletion in E. coli, the E. histolytica NIF system, was functional only under
anaerobic conditions, suggesting that the E. histolytica NIF system is sensitive
to oxidative stress (Ali et al., 2004; see Section 8.1.2). Since E. histolytica utilises
oxygen (Takeuchi et al., 1979), ROS is produced intracellularly. Thus, there
must be mechanisms that protect E. histolytica from the damages on Fe–S clus-
ters caused by ROS. It remains necessary to identify proteins and systems that
protect Fe–S clusters and the NIF system from oxidative stress. There is no
evidence that expression of the NIF system is affected under oxidative stress
conditions as mRNA expression of NifS and NifU genes remained unchanged
under L-cysteine deprivation, while a few iron flavoproteins were upregulated
(Husain et al., 2011). A sensor of oxidative stress, for example, Fe–S protein,
and a transcriptional regulator also remain unknown.
Unlike the reversible inactivation by ROS, nitric oxide causes an irre-
versible inactivation of Fe–S proteins, probably by forming adducts with sul-
phur atoms of cysteine residues involved in Fe–S cluster binding. In
E. histolytica, inactivation of Fe–S proteins and upregulation of the NIF sys-
tem by sodium nitroprusside were demonstrated (Santi-Rocca et al., 2012).
Activation of the endoplasmic reticulum stress-sensing mechanism upon
treatment with nitric oxide appears to reflect lost or damaged Fe–S clusters.
However, it awaits further investigations to demonstrate whether Fe–S pro-
teins and the NIF system per se are directly involved in anti-nitrosative and
anti-oxidative mechanisms.

9.3.2 SUF system in Blastocystis and Apicomplexa


In Blastocystis, the streamlined cytosolic SUF system, consisted of only
SufBC, was up-regulated under oxidative stress, whereas the expression
level of the ISC components and several Fe–S proteins remained constant
68 Vahab Ali and Tomoyoshi Nozaki

(Tsaousis et al., 2012) (see Section 8.9.3). These data suggest that the cyto-
solic SUF system in Blastocystis sp. functions for the oxidative stress defence.
However, similar to Blastocystis sp., Entamoeba, Giardia, and Trichomonas also
reside in only the anaerobic or microaerophilic niche, but apparently do not
require the cytosolic SUF system. As described earlier (Section 8.9), Blast-
ocystis sp. appears to have acquired the cytosolic SUF machinery by lateral
gene transfer, as E. histolytica for the NIF machinery, from bacteria during
adaptation to the metabolic requirements of its distinctive anaerobic lifestyle.
It remains unknown, hence, how only Blastocystis sp., but not other intes-
tinal or vaginal anaerobic protozoa described above, gained the cytosolic
SUF system, although they also had a chance to acquire the SUF system
by lateral gene transfer.
It looks reasonable that Plasmodium retained the SUF system in the
apicoplast even after it had lost photosynthesis, which produces high levels
of oxidative stress. The compartmentalised SUF system is beneficial and
probably essential for malaria parasites because Fe–S proteins in the
apicoplasts are sensitive to ROS generated by degradation of haem in the
digestive vacuoles and from the electron transport chain during oxidative
phosphorylation in the mitochondria. The SUF system is also present in
the related apicomplexan protozoa, including T. gondii, Neospora and Eimeria
(data not shown), but missing in Cryptosporidium. These data are consistent
with the hypothesis that the plastidial SUF system was present in the ances-
tral Apicomplexa and lost in a lineage containing Cryptosporidium.
Although it was implied in bacteria that the ISC and SUF systems have
preferential roles in the stress response, that is, defence mechanisms against
oxidative stress versus iron starvation, respectively (see Section 9.2), the specific
roles of the ISC and SUF systems in Apicomplexa including Plasmodium and
Blastocystis sp. are not well understood. Are the apicoplasts in apicomplexan
protozoa more prone to iron deficiency? Is the acquisition and retention of
the SUF system in the cytoplasm of Blastocystis sp. also causally connected with
the stress response against iron starvation? As described earlier, E. coli employs
the ISC system for house-keeping functions, while it utilises the SUF system
only as an emergency backup.

9.3.3 ISC system as a non-redundant Fe–S cluster biosynthesis


in other protozoa
Other protists, Giradia, Trichomonas, Leishmania, Trypanosoma and Micro-
sporidia, are also exposed to oxidative and nitrosative stress from their
own respiration and the host immune systems. However, they possess only
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 69

the ISC system for Fe–S cluster biogenesis. One can argue that the ISC sys-
tem is catalytically more efficient than the SUF system, and thus, predom-
inantly maintained in a variety of lineages, even in organisms that harbour
highly reduced MROs.
The ISC system in the mitochondrial matrix is shielded from the external
environment by three membranes, that is, the plasma membrane and the
mitochondrial double membranes. Since membranes are only semi-
permeable to hydrogen peroxide, the mitochondria in the organisms that
live in aerobic habitats protect themselves from exogenous hydrogen perox-
ide. The mitochondria also have their own scavengers, such as GSH and
Grx, which are strangely missing in T. vaginalis, to ensure low hydrogen per-
oxide concentrations in the mitochondrial matrix (Koehler et al., 2006;
Petrova et al., 2004). Hydrogen peroxide is efficiently detoxificated in
the cytosol by catalases and peroxidases to reduce the cytoplasmic hydrogen
peroxide concentrations compared to that present outside the cell (Jang and
Imlay, 2010). Again, these detoxification enzymes of hydrogen peroxide in
the cytosol are not present in the anaerobic or microaerophilic protozoa.
These multilayered protections against ROS should rigorously protect the
ISC system from externally and internally produced ROS in all the ISC
system-dependent organisms lacking the SUF system (Jang and Imlay,
2010). However, it remains unknown in individual parasites how these mul-
tilayers are composed.

10. UNSOLVED QUESTIONS AND FUTURE PERSPECTIVES


10.1. Acquisition and secondary loss of the machineries
for Fe–S cluster biosynthesis
The distribution of the ISC, SUF, NIF, and CIA systems largely depend upon
evolutionary positions, that is, subdomains or lineages in the eukaryotic tree,
of individual parasites. It is plausible that the oxygen tension, or anaerobicity,
of the niche each parasite resides may have affected the acquisition and loss of
the Fe–S cluster biogenesis systems. For example, the oxygen-sensitive NIF
system has been retained only in Entamoeba and Mastigamoeba. It is generally
accepted that the ancestor of the mitochondria and MROs arose only once
in the evolution of eukaryotes, as a consequence of endosymbiosis of
a-proteobacteria in a methanogenic archaeon. Therefore, it is conceivable
that the last eukaryotic common ancestor (LECA) possessed the ISC system
in the mitochondria and the SUF system in the cytosol.
70 Vahab Ali and Tomoyoshi Nozaki

Important fundamental questions related to the establishment of Fe–S clus-


ter biogenesis in Entamoeba include (1) How (i.e. through lateral gene transfer
from or endosymbiosis of e-proteobacteria), when and from what organism did
Entamoeba acquire the NIF system? (2) When was the mitosomal ISC system
lost and what drove this secondary loss: the unnecessity of the ISC system under
anaerobic conditions (passive loss) or the disadvantages of metabolic/genetic
interactions between the ISC system and the newly gained metabolites/
pathways (active negative selection)? (3) Why did not a similar replacement
of the ISC system with the NIF system occur in other lineages? Genomic
and proteomic analysis of various anaerobic organisms that belong to other taxa
should clarify the premise that the acclimation to oxidative stress or the loss of
defence against oxidative stress influenced the retention of one of the three
major Fe–S cluster biosynthetic machineries in the individual species. (4)
How and when was the cytosolic SUF machinery, which the LECA was pre-
sumed to possess, lost during the eukaryotic evolution? This question is not
restricted to Entamoeba, but common for all eukaryotic organisms that lack
the SUF system.

10.2. Origins of individual machineries and crosstalk between


the organellar and cytosolic compartments
The plastids produce Fe–S clusters for their own necessity, whereas the
mitochondria produce them for the cytosolic as well as mitochondrial
Fe–S proteins. The general localization of the SUF system to the plastids
and the ISC system to the mitochondria in eukaryotes reflects their inher-
itance from their cyanobacterial and a-proteobacterial ancestors, respec-
tively, whereas the CIA machinery might have been passed on from the
archaeal lineage, where Nbp35-like proteins are found (Boyd et al.,
2009). The mitochondria ISC machinery is required for the biogenesis of
Fe–S clusters in the cytoplasm and nucleus as well as the mitochondria. This
is consistent with the view that the mitochondria are enslaved to the regu-
lation by the host nucleus, as a consequence of the establishment of endo-
symbiosis between a-proteobacterial and the ancestral archae. As the
cytosolic Fe–S biosynthesis by the CIA machinery depends on the ISC
machinery in the mitochondria and MROs in general (except Entamoeba),
the CIA machinery needs to be regulated via signalling from the mitochon-
drial ISC system. However, it remains unknown how the signal transduces
between the mitochondrial ISC and the cytosolic CIA systems.
It also remains totally unknown whether the Fe–S biosynthetic machin-
eries in the mitochondria and the plastids communicate. It seems unlikely
that Fe–S clusters are transported from the mitochondria to the plastids or
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 71

vice versa across 4–6 layers [two mitochondrial and 2–4 plastidial (primary
and secondary plastids)] of membranes (Ralph et al., 2004). Moreover,
nuclear-encoded Fe–S proteins are imported into the apicoplasts in an
unfolded state (Tonkin et al., 2008a,b; van Dooren et al., 2002). It was also
shown in A. thaliana that suppression of SufS (NFS2) caused defect only in
chloroplast Fe–S proteins, but not mitochondrial Fe–S proteins (Balk and
Pilon, 2010; Ye et al., 2005). Altogether, the Fe–S cluster biogenesis in
the plastids does not rely on the mitochondrial ISC machinery, and the
mitochondrial ISC and plastidial SUF machineries function independently.
It also remains unknown whether the CIA and the plastidial SUF machin-
eries physically or genetically interact, although the current understanding of
the SUF system is that it works totally independently from the mitochondrial
ISC and cytosolic CIA systems.

10.3. Cooperation of two cytosolic machineries in Entamoeba


and Blastocystis: SUF/NIF and CIA systems
It remains unknown how two cytosolic machineries of Fe–S cluster biosynthe-
sis function in a coordinated (or in an independent) fashion. In E. histolytica, the
NIF and CIA machineries coexist in the cytoplasm, while in Blastocystis sp., the
SUF and CIA systems are present in the cytoplasm. If the NIF or SUF system
plays a redundant role similar to that of the CIA machinery, one of the two
systems could have been lost by natural selection during evolution. Therefore,
they likely have non-overlapping and indispensable roles. It remains totally
unknown how these two systems cross-talk; for example, how do the CIA
components work with the NIF system in E. histolytica? More specifically,
how does NifS or an unknown catalytic component donate sulphur to the
Cfd1–Nbp35 complex? How are the two sets of the scaffold components,
NifU and Nar1/Cia1, engaged in the specific roles without competing for
the substrates? Further biochemical and genetic studies of the NIF and CIA sys-
tems should shed light on these important questions. What is the carrier of iron
in the organisms such as E. histolytica and G. intestinalis, in which FXN is absent?
Are there universal iron carriers for both the NIF and CIA systems? It is also
important to identify and characterise the iron and electron donors of the
SUF and CIA machineries in the protozoan parasites, which may lead to a dis-
covery of new molecules involved in Fe–S clusters biogenesis.

10.4. Significance of compartmentalization of Fe–S cluster


biosynthesis in mitochondrion-related organelles (MROs)
A common denominator of the functions of the mitochondria and MROs
appears to be Fe–S cluster biosynthesis with an exception of E. histolytica.
72 Vahab Ali and Tomoyoshi Nozaki

The hydrogenosomes in T. vaginalis and the MLO (MRO) in Blastocystis sp.


are involved in both ATP generation and Fe–S cluster biogenesis, while the
MROs from other parasitic protozoa apparently lost an ability of ATP gen-
eration. It remains unknown why these mitochondrion-remnant organelles
still retain machinery involved in Fe–S cluster biosynthesis, even after they
lost ATP generation by oxidative phosphorylation, Hþ-ATPase, and the
membrane potential across the inner membrane. Since the MRO-targeting
sequences are promiscuous in anaerobic protozoa including G. intestinalis
and T. vaginalis (see Sections 8.2 and 8.3), and apparently lacking in
E. histolytica (see Section 8.1), prediction of organelle-localised proteins is
unreliable. Thus, a highly sensitive and unbiased proteomic analysis of highly
purified MROs by conventional physical separation, immunoprecipitation
and FACS-based separation of organelles is needed to demonstrate all the
components involved in the synthesis, transport and maturation of Fe–S
clusters in MROs.

10.5. New strategy for the identification of Fe–S


cluster-containing proteins
When machineries for Fe–S cluster biogenesis are inhibited or inactivated,
expression of genes encoding the Fe–S apo-proteins is often up-regulated for
compensation of the decrease or loss of functional proteins. Thus, creation of
the parasite strains in which the core components of the ISC, SUF, NIF, or
CIA machinery is repressed by gene silencing, RNA interference, and gene
knockout approaches, combined with RNA-seq or DNA microarray-based
expression analyses, of these strains, should lead to the identification of the
proteome of the Fe–S cluster-containing proteins. For instance, repression
of either NifS or NifU in the non-redundant NIF system in E. histolytica
should in theory help to identify genes encoding for proteins containing
Fe–S clusters. Such approaches may also lead to the discovery of additional
components and regulators involved in Fe–S cluster biosynthesis or genetic
interactions between the different Fe–S cluster biosynthesis systems.

10.6. NIF and SUF systems as drug target


Since the NIF system is non-redundant and essential for the Fe–S cluster
biogenesis in Entamoeba and absent in humans and mammals, the NIF system
represents an ideal target for new anti-amoebic drugs. The SUF system in
Plasmodium and Blastocystis is also absent in mammals and can be a drug target
unless the other system, that is, the ISC system, can complement the roles
Iron–Sulphur Cluster Biogenesis in Protozoan Parasites 73

under in vivo conditions. However, for the reasons described earlier


(Section 10.2), the ISC system is not likely to be able to complement the
defect of the plastidial SUF system in Apicomplexa, reinforcing the premise.
We ought to be cautious, however, because the E. histolytica NIF system can
functionally complement, under anaerobic conditions, defects caused by
double disruption of the ISC and SUF systems in E. coli, suggesting the inter-
changeable functions of these systems to some extent (Ali et al., 2004). Sim-
ilarly, the growth defect of a SUF-deficient E. coli strain was rescued by the
isc genes, also suggesting that interchangeable nature of these systems
(Takahashi and Tokumoto, 2002; Tokumoto et al., 2004). Thus, it is ques-
tionable whether targeting the SUF system solely elicits sufficient cytotox-
icity against these organisms. However, since some proteins in the SUF
system are structurally divergent from the counterpart in the human ISC sys-
tem, these proteins can be potentially explored to design drugs against Plas-
modium and Blastocystis.
The Plasmodium apicoplasts harbour a plant-type FNR/ferredoxin redox
system, which works like the non-photosynthetic FNR/ferredoxin systems
(Rohrich et al., 2005; Seeber et al., 2005). In photosynthetic plastids, ferre-
doxin receives electrons from PSI and the reduced ferredoxin transfers elec-
trons to FNR to produce NADPH from NADPþ for the Calvin cycle. In
the non-photosynthetic plastids, the ferredoxin redox system operates in the
reverse direction. The FNR catalyses electron transfer from NADPH to fer-
redoxin, which acts as a reductant for various reactions (Rohrich et al.,
2005). By virtue of the uniqueness of the plant-type FNR/ferredoxin
redox system, together with other unique metabolic pathways in the
apicoplast of Plasmodium, the FNR/ferredoxin redox system represents an
attractive drug target (Seeber et al., 2005; Lim and McFadden, 2010). Abun-
dant structural information on the FNR and ferredoxin in Cyanobacteria
and plants should facilitate rational drug designing of inhibitors against
the Plasmodium enzymes (Binda et al., 1998; Morales et al., 1999; Serre
et al., 1996). Since the FNR/ferredoxin redox system is involved in the
Fe–S cluster biosynthesis in plastids, its importance is further implicated
in the insertion of Fe–S clusters into several apicoplast enzymes (Ralph
et al., 2004; Seeber et al., 2005).

ACKNOWLEDGEMENTS
We thank Amir Zaidi, Shadab Anwar, Krishn Pratap Singh, Ghulam Jeelani, and other
members of our laboratories for genome analysis and discussions. This review and the
work necessary for the review were supported in part by a grant from the Department of
74 Vahab Ali and Tomoyoshi Nozaki

Science and Technology and Indian Council of Medical Research, India (DST/INT/JSPS/
P-117-2011) to V. A., a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science, and Technology (MEXT) of Japan (23117001,
23117005, 23390099); a Grant-in-Aid on Bilateral Programs of Joint Research Projects
and Seminars from Japan Society for the Promotion of Science; a Grant-in-Aid on
Strategic International Research Cooperative Program (SICP) from Japan Science and
Technology Agency; a grant for research on emerging and re-emerging infectious diseases
from the Ministry of Health, Labour, and Welfare (MHLW) of Japan (H23-
Shinkosaiko-ippan-014); a grant for research to promote the development of anti-AIDS
pharmaceuticals from the Japan Health Sciences Foundation (KHA1101); and by Global
Center of Excellence Program (Global COE for Human Metabolomic Systems Biology)
from MEXT, Japan to T. N.

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CHAPTER TWO

A Selective Review of Advances


in Coccidiosis Research
H. David Chapman*,1, John R. Barta†, Damer Blake{, Arthur Gruber},
Mark Jenkins}, Nicholas C. Smith||, Xun Suo#, Fiona M. Tomley{
*Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, United States

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada
{
Royal Veterinary College, Hatfield, United Kingdom
}
Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
}
Animal Parasitic Diseases Laboratory, Agricultural Research Service, USDA, Beltsville, Maryland, United
States
||
Queensland Tropical Health Alliance Laboratory, Faculty of Medicine, Health and Molecular Sciences, James
Cook University, Cairns, Queensland, Australia
#
National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University,
Beijing, China
1
Corresponding author: e-mail address: dchapman@uark.edu

Contents
1. Introduction 95
2. Taxonomy and Systematics 96
2.1 The genus Eimeria Schneider 1875: A melting pot of biologically diverse
coccidia 97
2.2 Molecular identification and characterization of Eimeria and related
coccidia 99
2.3 Conclusions 104
3. Genetics 105
3.1 Markers employed in genetic studies 105
3.2 Cross-fertilization and genetic recombination 105
3.3 Genetic linkage analyses 106
4. The ‘Omics’ Technologies 108
4.1 Genomics 108
4.2 Transcriptomics 113
4.3 Proteomics 116
4.4 The future 119
5. Transfection 120
5.1 Transfection construct design 120
5.2 Transient transfection 121
5.3 Stable transfection 121
5.4 PiggyBac-based forward genetic system 122
5.5 Stably transfected Eimeria as a vaccine vector and beyond 123

Advances in Parasitology, Volume 83 # 2013 Elsevier Ltd 93


ISSN 0065-308X All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-407705-8.00002-1
94 H. David Chapman et al.

6. Oocyst Biogenesis 124


6.1 Veil and WFBs 124
6.2 Oocyst wall proteins 126
6.3 Formation of the oocyst wall 126
7. Host Cell Invasion 128
7.1 Parasite surface proteins 129
7.2 MIC proteins are adhesins and many function as multi-protein
complexes 130
7.3 Host glycan recognition by MIC proteins contributes to host
and tissue tropism 131
7.4 Regulated secretion of microneme and rhoptry organelles 132
7.5 AMAs and formation of the MJ 133
8. Immunobiology 133
8.1 Innate responses to primary infection 134
8.2 Acquired immunity 138
8.3 Maternal immunity 138
8.4 Immunological research 139
9. Diagnosis and Identification 140
9.1 Traditional methods 140
9.2 Early molecular methods 141
9.3 Methods based on DNA amplification by PCR 142
9.4 LAMP 146
9.5 Morphological diagnosis revisited 148
9.6 Conclusions 148
10. Control 149
10.1 Chemotherapy 149
10.2 Vaccination 151
10.3 Strategies for the control of coccidiosis 153
10.4 Natural products 154
11. Conclusions 154
Acknowledgement 155
References 155

Abstract
Coccidiosis is a widespread and economically significant disease of livestock caused by
protozoan parasites of the genus Eimeria. This disease is worldwide in occurrence and
costs the animal agricultural industry many millions of dollars to control. In recent years,
the modern tools of molecular biology, biochemistry, cell biology and immunology
have been used to expand greatly our knowledge of these parasites and the disease
they cause. Such studies are essential if we are to develop new means for the control
of coccidiosis. In this chapter, selective aspects of the biology of these organisms, with
emphasis on recent research in poultry, are reviewed. Topics considered include taxon-
omy, systematics, genetics, genomics, transcriptomics, proteomics, transfection, oocyst
biogenesis, host cell invasion, immunobiology, diagnostics and control.
Review of Coccidiosis Research 95

1. INTRODUCTION
Coccidiosis is caused by protozoan parasites of the apicomplexan
genus Eimeria that occur in many vertebrate and invertebrate hosts. This dis-
ease is a major cause of mortality, poor performance and lost productivity in
domestic livestock. The parasites have an oral-faecal life cycle involving
three phases: schizogony (also known as merogony), gametogony and spo-
rogony (or sporulation). The infective transmission stage is the oocyst which
contains, when sporulated, four sporocysts each containing two sporozoites.
Following ingestion, the sporozoites are released and penetrate epithelial
cells of the intestine. This is followed by schizogony, an asexual phase of
multiplication involving several repeated generations, and gametogony,
which results in the production of a new generation of oocysts that are passed
out in the faeces. The third phase of the life cycle, sporogony, occurs in the
external environment and results in the formation of a new generation of
oocysts. Seven species are recognized in the fowl that vary according to
the number of generations of schizogony, physical characteristics such as
the size of the oocyst, and biological characteristics such as site of develop-
ment in the intestine, pathogenicity and immunogenicity. A similar number
of species have been described from the turkey. In poultry, the life cycle is
completed in about 7 days but in ruminants it may be longer. Both schizog-
ony and gametogony can cause pathology because the cells in which the par-
asites develop are functionally impaired and eventually destroyed. The
extent of such destruction is determined by the numbers of infective oocysts
ingested, which in turn depends upon the extent to which sporulation is suc-
cessful. This requires warmth, oxygen and moisture, factors often not lac-
king in commercial livestock production.
It is in the poultry industry that coccidiosis is of the greatest economic
significance because modern production methods involve the rearing of
large numbers of birds in confinement at high stocking densities, often on
built-up litter. For example, a modern broiler house may contain as many
as 20–50,000 birds under one roof at a stocking density of one bird per
0.08 m2 and as many as 10 houses may be present at one farm. Furthermore,
both the broiler industry and the turkey industry tend to be restricted geo-
graphically; for example, in the United States, one of the largest areas of pro-
duction is confined to a few counties in northwest Arkansas. Thus the
commercial conditions under which poultry are raised provide ideal condi-
tions for parasite transmission. Fortunately, control of coccidiosis can be
96 H. David Chapman et al.

achieved either by the inclusion of drugs in the feed (prophylactic chemo-


therapy) or by vaccination and, because of such control measures, coccidiosis
is less a problem today than in the past (Chapman, 2003). Nevertheless, the
resilience of the oocyst ensures the continued presence of these organisms
wherever poultry are reared. The expansion of the poultry industry has pro-
vided a major source of protein to feed the growing human population and
any disease that limits this production, such as coccidiosis, will have the
potential to affect human health. There is a continued need, therefore,
for both basic and applied research into all aspects of the biology of these
organisms (Shirley and Lillehoj, 2012).
An example of basic research is the utilization of genomics and proteo-
mics to elucidate molecular aspects of invasion of host cells by sporozoites.
The apical complex of coccidia, found in motile stages of the life cycle (spo-
rozoites and merozoites), contains several sub-cellular organelles including
paired rhoptries, micronemes and dense bodies that secrete proteins
involved in the invasion process. Another example is the investigation of
biochemical events involved in the formation of the wall of the oocyst. Pro-
gress has also been made in understanding the role of CD4/CD8 lympho-
cytes and cytokines in inflammatory responses to infection; cell-mediated
immune mechanisms have been studied utilizing murine Eimeria as a model.
Molecular techniques have been utilized to develop new methods for iden-
tifying the species that infect the fowl. Much applied research has been
undertaken to demonstrate efficacy of various products for the control of
coccidiosis but in general this has not been published in peer-reviewed sci-
entific journals. In this review, some aspects of coccidiosis research in poul-
try is provided by the following contributors: taxonomy and systematics
(Barta); genetics (Blake); ‘omics’ technologies (Blake, Tomley, Gruber);
transfection (Suo, Blake); oocyst biogenesis (Smith); host cell invasion
(Tomley); immunology (Smith); diagnostics ( Jenkins, Gruber) and control
(Chapman).

2. TAXONOMY AND SYSTEMATICS


The taxonomy and systematics of eimeriid coccidia and Eimeria species
infecting vertebrates is, at best, problematic. This confounding state has
arisen because of numerous poorly described species, lack of type deposition
(even of micrographs or drawings) and improper taxonomic methods.
Misidentification is a general difficulty in studies concerned with the biology
of Eimeria. Classical original descriptions of the species that infect poultry
Review of Coccidiosis Research 97

were based upon isolates that have since been lost and, thus, there are no
surviving type specimens. A few defined laboratory strains still exist
(although not type specimens in the taxonomic sense), for example, the
‘Houghton’ and ‘Weybridge’ strains of various species, but others, such as
most ‘Beltsville’ strains, have been lost. Ideally, molecular studies should
be based upon strains derived from a single oocyst and identified using
key parasitological characters. Difficulties with the interpretation of DNA
analysis from inadequately described strains of Eimeria have been addressed
by Williams et al. (2010).
Confounding this taxonomic history is frequent application of the
‘different-host–different-parasite’ mindset. For Eimeria species in some host
groups, this principle may be justified because of relatively strict host spec-
ificities of these parasites; however, in some coccidia that infect passerine
birds (Isospora species in particular), host specificity may not be nearly as strict
and there may be many Isospora species that may ultimately be synonymized.
To further complicate matters, parasites in the genus Atoxoplasma Garnham
1950 may actually all be members of either the genus Isospora (and perhaps
synonymized with previously described Isospora species) or the genus Lank-
esterella (e.g. Barta et al., 2005; Merino et al., 2006).

2.1. The genus Eimeria Schneider 1875: A melting pot


of biologically diverse coccidia
The apicomplexan suborder Eimeriorina (Léger, 1911) is home to the
eimeriid coccidia (family Eimeriidae Minchin 1903) that includes the
Eimeria species and related monoxenous or facultatively heteroxenous
coccidia infecting vertebrates. A recent taxonomic definition of the family
Eimeriidae (see Upton, 2000) included apicomplexan parasites with the fol-
lowing features: ‘Homoxenous or facultatively homoxenous; merogony,
gamogony and formation of oocysts all in the same host; in vertebrates or
invertebrates’. Clearly this is a broadly applicable definition for inclusion
of parasites into this family. The definition of the genus is likewise permis-
sive: ‘oocysts with four sporocysts, each with two sporozoites’. Not surpris-
ingly, the number of Eimeria species that have been described to date exceeds
1200 and this number continues to grow. The frequently observed strict host
specificity of many Eimeria species and the infection of many hosts with mul-
tiple Eimeria species means that there remain probably tens of thousands of
undescribed Eimeria species infecting birds, herbivorous or omnivorous
mammals, reptiles, amphibia and, perhaps, even fish.
98 H. David Chapman et al.

Whilst taxonomically useful (and conservative), this rather broad defini-


tion of the genus bears part of the responsibility for one of the fundamental
taxonomic difficulties with the genus Eimeria—it is not a monophyletic tax-
onomic group. There is considerable agreement that species currently
assigned to the genus Eimeria do not form a monophyletic group (e.g.
Jirků et al., 2002, 2009) whether assessed systematically using morphological
or molecular characters. Instead, the genus Eimeria is paraphyletic or poly-
phyletic. When analyzed using complete or partial nuclear 18S rDNA
sequences, the phylogenetic analysis generated a consensus tree (Fig. 2.1)
in which members of the Toxoplasmatinae (species of Cystoisospora,
Neospora, Hammondia and Toxoplasma) formed a well-supported monophy-
letic group that had a sister group relationship with the other coccidia,
including Eimeria species. The large clade of eimeriid coccidia that includes
all Eimeria species for which 18S rDNA sequence data have been obtained
includes many genera other than Eimeria, including Goussia, Caryospora,
Lankesterella, Atoxoplasma, Isospora and Cyclospora. Even the genera currently
included in the large family Eimeriidae are not found within a monophyletic
grouping; members of the heteroxenous family Lankesterellidae are found
within the clade that contains all species belonging to the Eimeriidae.
The tissue coccidia (Toxoplasma and its relatives—Toxoplasmatinae) and
all of the early branching coccidia (Hyaloklossia and Goussia spp. plus
E. tropidura) possess valvular sutures on their sporocysts and do not have
Stieda bodies (see Fig. 2.2). Eimeria arnyi from a snake and E. ranae from frogs
were found in a well-supported branch that arose near the base of the
coccidia that possess Stieda bodies in their oocysts (when sporocysts are
formed). The next branching clade consisted of: (1) Eimeria species of mar-
supials (E. trichosuri); (2) Eimeria spp. of cranes (E. gruis and E. reichenowi) and
(3) a clade containing Caryospora spp. and Lankesterella spp. This sister group
to these parasites was a large collection of Eimeria species from a wide variety
of hosts interrupted by the inclusion of a well-supported clade of
Cyclospora spp.
The genus Eimeria was observed to be polyphyletic with at least four
independent lineages of Eimeria species. Caryospora and Lankesterella species
formed a monophyletic clade as has been described previously (Barta et al.,
2001). Avian Isospora and Atoxoplasma species formed a well-supported
monophyletic clade; the mixing of Atoxoplasma and Isospora species gives
additional support for the questioning the validity of the genus Atoxoplasma.
Giving some support to the concept of host specificity, Eimeria species fre-
quently formed well-supported clades of parasites that parasitized the same
Review of Coccidiosis Research 99

or closely related definitive hosts such as Eimeria spp. infecting swine, rabbits
or galliform birds. Among the Eimeria species infecting galliform birds, the
cecal coccidia of chickens and turkeys (i.e. E. necatrix, E. tenella and
E. adenoeides) are frequently found to form a monophyletic group of parasites
(e.g. Miska et al., 2010; Fig. 2.1) to the exclusion of the other Eimeria species
infecting the chicken. This repeated observation, and the failure to find
E. necatrix or E. tenella in wild jungle fowl (Fernando and Remmler,
1973), present the possibility that these two species of Eimeria infecting
domestic chickens may have arisen from a host transfer from some other gal-
liform host (Barta et al., 1997).

2.2. Molecular identification and characterization of Eimeria


and related coccidia
Multiple, distinct rDNA copies were described decades ago for the malar-
ial parasites, Plasmodium species (e.g. McCutchan et al., 1988; Nishimoto
et al., 2008). In these haemosporinid parasites, up to three independently
evolving paralogous rDNA copies were described that frequently demon-
strated more sequence variation between paralogous within a single para-
site species than between homologues among different species.
Interestingly, paralogous rDNA copies were expressed differentially in dif-
ferent life cycle stages. Until recently, the presence of paralogous rDNA
copies had not been observed in Eimeria species and thus the nuclear
18S rDNA locus was considered a relatively useful and stable genetic target
for species-level identification and for molecular phylogenetics. However,
Vrba et al (2011) recently showed that a single-oocyst-derived line of
E. mitis contained two paralogous types of nuclear 18S rDNA that had
considerable sequence variation (i.e. 1.3–1.7% sequence diversity between
paralogous compared with 0.3–0.6% sequence diversity among homo-
logues). Recent work with a number of single-oocyst-derived lines of sev-
eral Eimeria species infecting turkeys indicates that paralogous 18S rDNA
copies exist within the nuclear genome of at least some of these parasites as
well. For example, single-oocyst clonal lines of E. meleagrimitis, from
which polymerase chain reaction (PCR)-amplified, near-complete nuclear
18S rDNA amplicons were cloned and sequenced, demonstrated consid-
erable sequence diversity. Two paralogous groups of sequences were
obtained; each group of sequences had mean intraspecific sequence diver-
sities of about 0.5% but the mean interspecific variation between these
paralogous exceeded 2.7%. By the way of comparison, the mean interspe-
cific variation for the 18S rDNA between the widely accepted species
Figure 2.1 Maximum likelihood consensus tree resulting from the analysis of complete or near complete nuclear 18S rDNA sequences. For
each taxon in the tree, the definitive host (DH), if known, and the GenBank Accession number for the sequence used to generate the tree are
included. Percentage bootstrap supports for major clades are presented as numbers located at nodes on the tree. Horizontal branch lengths
are proportional to hypothesized evolutionary change with the scale indicating 10% hypothesized sequence variation.
102 H. David Chapman et al.

Figure 2.2 Photomicrographs of sporulated oocysts of coccidia that each possess two
sporocysts, containing four sporozoites. (A) An avian Isospora sp. that possess Stieda
bodies at the apex of each sporocyst in the oocyst (arrows) and that belongs to the fam-
ily Eimeriidae. (B) Cystoisospora felis, a coccidium infecting felids that has an oocyst with
no Stieda bodies on its sporocysts and that belongs to the family Sarcocystidae.

E. tenella and E. necatrix is only 1.1%. Clearly, such paralogous loci can
confound both molecular phylogenetics and the taxonomic pursuits of
identification and characterization.
Recognition of intragenomic polymorphisms among 18S rDNA in
Eimeria has important ramifications for use of other regions of the rDNA
gene array for molecular diagnostics and/or characterization. For example,
the internal transcribed spacer (ITS) regions have been used to characterize
species and strains of Eimeria. However, the ITS-1 and ITS-2 regions are
likely subject to both intragenomic (e.g. Blake et al., 2006; Vrba et al.,
2011) and intraspecific (e.g. Barta et al., 1998) variations that may limit
the utility any resulting ITS sequences or molecular diagnostic methods
based on these sequences.
Ogedengbe et al. (2011a) have recently demonstrated that another
genetic locus, the mitochondrial cytochrome c oxidase subunit I gene
(cox-1, COI), is a much more reliable gene for molecular species delimitation
of Eimeria species and other coccidia than nuclear 18S rDNA sequences. The
recognition of intragenomic polymorphism of the 18S rDNA locus (e.g.
Vrba et al., 2011) may explain the superiority of the COI locus for species
delimitation and, potentially, identification. The COI genetic target has
additional features that argue for its use in the molecular characterization
and identification of coccidia: (1) COI is found in multiple copies within
the mitochondria of coccidia making it a good PCR target; (2) COI
Review of Coccidiosis Research 103

demonstrates sufficient DNA sequence variability (2–3% sequence diver-


sity between Eimeria spp.) that 500–800 bp fragments will provide sufficient
data for most identification purposes; (3) COI is sufficiently divergent from
the COI of most hosts that parasite-specific PCR primers can be readily
developed, permitting the use of this locus for parasites located within host
tissues; (4) COI is located on a genome derived from an endosymbiotic pro-
karyote meaning that the resulting COI protein coding sequences are free of
introns and can be assessed relatively easily for contiguous open reading
frames (ORFs) as an internal check for correct PCR amplification
(Ogedengbe et al., 2011a) and finally, (5) intraspecific variation among spe-
cies is relatively modest (usually <0.2%) compared with interspecific varia-
tion (usually >1.5%). However, the use of COI sequences for parasite
identification and/or species delimitation is not without its problems. Com-
pared with the 18S rDNA locus, relatively few COI sequences have been
deposited to public sequence databases, limiting the use of this locus for
identification purposes until more reference sequences become available.
In addition, PCR amplification of COI fragments from mixed parasite
DNA templates (such as litter or mixed species samples, e.g. Schwarz
et al., 2009) can generate hybrid amplicons (e.g. GenBank Accession num-
ber FJ236441). In our experience, up to 5% of COI fragments amplified by
PCR from DNA samples containing multiple Eimeria spp. may be hybrid
molecules generated during the PCR process. For Eimeria spp. of chickens
or other well-sampled taxonomic groups (such as Plasmodium spp. and
related haemosporinid parasites), this is not a major issue. In such cases,
hybrid sequences are easily detected using a simple BLAST search because
of the availability of reliable reference sequences in the public sequence data-
bases. Examining pairwise alignments of the top BLAST hits will show
sequence divergence restricted to only one portion of the new sequence
when aligned with sequences from one Eimeria sp. and with a different por-
tion of the molecule when aligned with a second Eimeria species. However,
for Eimeria or Isospora species from less well-sampled hosts, hybrid sequences
may be problematic. To avoid the influence of such hybrids, direct sequenc-
ing of PCR products is recommended—messy or ambiguous sequences
resulting from such direct sequencing likely indicates multiple species within
the sample and biological purification should be attempted (biological
enrichment or cloning of the parasite). Alternately, after cloning PCR frag-
ments from such a sample, multiple clones must be sequenced so that both
repeated COI sequences (likely valid amplicons) and potential hybrids can
be identified.
104 H. David Chapman et al.

For the well-accepted Eimeria species infecting chickens it was frequently


assumed that lack of immunological cross-protection in the host was evi-
dence in support of identification of particular species. For example, chickens
immunized against Eimeria maxima through natural infection would be
expected to be protected against subsequent challenge with the same species.
However, it has been shown conclusively that immunologically distinct
strains of E. maxima can arise both spontaneously and in response to selective
immunological pressure. Despite a near complete lack of immunological
cross-protection, the Guelph and M6 strains of E. maxima have identical
COI sequences (Ogedengbe et al., 2011a) demonstrating again the utility
of this genetic locus for species identification, even when classical methods
of species identification such as immunological cross-protection can fail.

2.3. Conclusions
The taxonomic mess that is the current genus Eimeria is likely to be resolved
slowly through the application of appropriate nuclear or mitochondrial
sequence analysis coupled with phenotypic characters. Ideally, monotypic
reference strains of Eimeria species and other coccidia of veterinary or med-
ical importance should be characterized biologically (minimally oocyst
dimensions as described by Bandoni and Duszynski, 1988), but optimally
including solid descriptions of endogenous development during experimen-
tal infections as well as molecularly (preferentially obtaining both nuclear
18S rDNA and mitochondrial COI sequences).
Such combined morphological and molecular characterizations have
been accomplished with single Isospora sp. oocysts (Dolnik et al., 2009).
However, even with both molecular and morphological data available, tax-
onomic decisions are ultimately opinions of the various authors involved.
For example, although there were significant differences for two strains of
turkey Eimeria in oocyst dimensions and 2.3% sequence divergence at the
COI locus over 767 bp, Poplstein and Vrba (2011) concluded that these par-
asites were simply variants of a single species, E. adenoeides because of some
immunological cross-protection of these parasites in turkeys. Clearly further
studies are warranted even for parasites that have been described for many
years and that infect agriculturally important animals.
Finally, the taxonomic breadth of the genus Eimeria is clearly too broad.
It is anticipated that the genus Eimeria will be divided into several genera,
each containing fewer, but biologically more homogenous, species with
each new genus.
Review of Coccidiosis Research 105

3. GENETICS
Studies with Eimeria genomes can be divided into physical and non-
physical categories including genomic, transcriptomic and proteomic or
fundamental and applied genetics, respectively. Key among the features that
underpin such studies is the haploid state of the eimerian genome through-
out the majority of the life cycle. Thus, clonal parasites can be isolated by
passage of a single sporozoite or sporocyst and each cloned parasite line
may be considered homologous at all loci prior to fertilization and zygote
formation (Chapman and Rose, 1986; Shirley and Harvey, 1996). Nonethe-
less, the brief sexual phase of the life cycle facilitates chromosomal segrega-
tion and genetic recombination ( Jeffers, 1976) and supports classical genetics
studies where a departure from the anticipated Mendelian phenotypic ratio
of inheritance informs on genotype (Sturtevant, 1913). More recently,
molecular biology has revolutionized our understanding of genomes and
their associated biology, providing new tools for genetics-led research and
creating the ‘omics’ disciplines.

3.1. Markers employed in genetic studies


Variation between and within Eimeria species has been investigated using
several phenotypic and genotypic tools as reviewed elsewhere (Beck
et al., 2009). Briefly, phenotypic tools have included differential isoenzyme
migration through starch gels or by isoelectric focusing, resistance or suscep-
tibility to defined chemotherapeutic exposure, precocious development and
the ability to escape strain-specific immune killing (Blake et al., 2005; Jeffers,
1976; Shirley et al., 1989; Smith et al., 1994a–d). Genotypic tools, including
random amplification of polymorphic DNA polymerase chain reaction
(RAPD-PCR), restriction fragment length polymorphism (RFLP), ampli-
fied fragment length polymorphism (AFLP) and gross chromosomal size
polymorphism as revealed by pulsed field gel electrophoresis (PFGE), have
also been used to define inter- and intraspecific variation (Blake et al., 2011a;
Fernandez et al., 2003a; Shirley, 2000).

3.2. Cross-fertilization and genetic recombination


Early studies with coccidial parasites suggested the absence of genetic recom-
bination, however, molecular, genetic and microscopic studies have now
demonstrated numerous crossover events and recombination nodules,
106 H. David Chapman et al.

respectively (Blake et al., 2011b; Canning and Anwar, 1968; del Cacho
et al., 2005; Shirley and Harvey, 2000). Almost 40 years ago, the first evi-
dence of genetic exchange during multi-clonal infection revealed a capacity
for cross-fertilization, independent segregation and the consequential pro-
duction of hybrid progeny ( Jeffers, 1974). In this example, concurrent infec-
tion of E. tenella strains resistant to the anticoccidial drugs amprolium or
decoquinate yielded a population resistant to both compounds. However,
the efficiency of cross-fertilization remains unclear, as the proportion of
hybrid progeny within specific crosses has been calculated to vary between
0.05% and 39.5% (Blake et al., 2004; Joyner and Norton, 1975). Proportions
of hybrid progeny, based on comparison of oocyst output in the presence or
absence of deleterious selection, depend on the underlying genetic com-
plexities of the trait (i.e. the number of contributing genetic loci). In addition,
proportions are affected by the timing of selection within the life cycle and
the magnitude of the doses of oocysts given, which can result in parasite
‘crowding’ within the intestine (Blake et al., 2004; Williams, 2001).

3.3. Genetic linkage analyses


The inheritance of polymorphic genetic markers can be used to infer a
genetic map based upon comparisons of recombination frequencies between
markers. Key assumptions include a random (independent) association
between markers on separate chromosomes and a correlation between
recombination rate and physical distance for markers linked on the same
chromosome. For the Eimeria species, early linkage studies included the cre-
ation and selection of multi-drug resistant hybrid lines by cross-fertilization,
in which it was noted that close physical linkage of contributing loci may be
an explanation for incompatible resistance combinations ( Joyner and
Norton, 1978). The development of sequence-based genetic markers per-
mitted the development of more sophisticated linkage analyses, including
the derivation of genetic linkage maps and whole genome association
(WGA) studies.
Genetic linkage maps have been produced for two Eimeria species. In the
first genome-wide linkage study, a cross was made between an attenuated
E. tenella Wisconsin line (selected for precocious development within
125 h) and a Weybridge line selected to become resistant to the anticoccidial
drug arprinocid (Shirley and Harvey, 2000). The hybrid component of the
progeny of this cross, capable of replication within 125 h in the presence of
arprinocid, were recovered and amplified by selective in vivo passage and
Review of Coccidiosis Research 107

used to derive a panel of 22 hybrid clonal progeny lines by single sporocyst


cloning. Using RAPD-PCR, RFLP, AFLP and full karyotype PFGE, 443
parent-specific genetic markers were generated and mapped against all
22 clones. Using the free linkage software Map Manager QT (Manly and
Cudmore, 1997), 16 linkage groups were created, representing a genetic
genome size of 653 centimorgans (cM). Intriguingly, 53% of all the
polymorphic markers scored were clustered into just three linkage groups,
indicating the absence of dividing recombination events within all 22 cloned
progeny. At the time of publication, this was attributed to possible bias intro-
duced by AFLP, however, the subsequent description of a segmented
genome structure for E. tenella now suggests pronounced hot and cold spots
of genetic recombination (Blake et al., 2011b; Ling et al., 2007). More
recently, a genetic map was created for E. maxima using a similar strategy
with the selectable traits resistance to the drug robenidine and escape from
strain-specific immune killing (Blake et al., 2011b). Using a larger panel of
647 genetic markers, generated exclusively by AFLP, linkage analysis cre-
ated a map made up of 13 major linkage groups representing a genetic
genome size of 2883.9 cM.
The first WGA study on E. tenella (Shirley and Harvey, 2000) made the
key assumption that each parent-specific genetic marker is inherited in a 1:1
ratio in the absence of deleterious selection. Thus, a genetic marker distantly
linked to a locus under selection, or located on a separate chromosome,
should persist within the hybrid progeny irrespective of selection. Represen-
tation of a more closely linked marker should be reduced within the progeny
that survive selection, while a marker that is linked most intimately should be
severely under-represented or lost altogether. Clonal lines drawn from a
hybrid population under selection should conform to the same rules,
resulting in linkage disequilibrium. Following combined selection for resis-
tance to arprinocid and the ability to reproduce within 125 h, all genetic
markers incorporated into the E. tenella genetic map were found to persist
in the anticipated 1:1 ratio with the exception of linkage groups mapped to
chromosomes 1 and 2, whose inheritance correlated with drug resistance
and precocious ability, respectively (Shirley and Harvey, 2000). The number
and size of loci mapped in this study would have been influenced by the bio-
logical factors described above, as well as practical factors including the num-
ber of genetic markers and independent clones.
While the number of genetic markers available can be adjusted with rel-
ative ease, the effort required to isolate and amplify clonal lines of Eimeria
places an inherent limit on the number of clones that can be handled in
108 H. David Chapman et al.

any one laboratory. Working with an uncloned hybrid progeny population


under selection, instead of a finite panel of clones, can provide a massive
increase in mapping power. Working on this hypothesis, hybrid
E. maxima populations, selected for resistance to robenidine and the ability
to escape strain-specific immune killing, were used in the first population-
based WGA strategy with Eimeria species to map loci that encode strain-
specific immunoprotective antigens (Blake et al., 2011a). Briefly,
E. maxima isolates are commonly characterized by antigenic diversity such
that immunization with one strain can induce apparently complete immune
protection against homologous challenge but incomplete protection against
challenge by an antigenically distinct strain (Smith et al., 2002). In one
extreme example, immunization of inbred Line C White Leghorn chickens
(maintained at the Institute for Animal Health, UK) with the Houghton (H)
or Weybridge (W) E. maxima strains yields no statistically significant cross-
protection. WGA scrutiny of this phenotype, using the uncloned progeny of
multiple crosses between the H and W E. maxima strains before and after
W strain-specific immune selection, identified six distinct genetic loci whose
inheritance correlated absolutely with the immune phenotype (Blake et al.,
2011a). Subsequent fine mapping of two of these loci identified immune
mapped protein-1 and confirmed apical membrane antigen 1 (AMA1) as
partially immunoprotective antigens. The immunoprotective capacity of
three of the remaining four loci was also demonstrated using bacterial arti-
ficial clones (BACs) that covered each mapped W strain locus. Transient
transfection with whole BAC DNA was used to genetically complement
the H strain, and transferred the ability to induce a protective immune
response against the W strain (Blake et al., 2011a).

4. THE ‘OMICS’ TECHNOLOGIES


4.1. Genomics
Three distinct DNA genomes have been defined in eimerian parasites, local-
ized to the nucleus, the mitochondrion and the apicoplast organelle. Addi-
tionally, a double stranded RNA genome associated with virus-like particles
has been commonly found in Eimeria species (Han et al., 2011; Lee and
Fernando, 2000; Shirley, 2000). Using PFGE, the nuclear genomes of all
avian Eimeria species investigated to date have been estimated to contain
between 50 and 60 Mb DNA (Blake et al., 2011b; Shirley, 2000). The
E. tenella genome is the best characterized to date, featuring a GC content
Review of Coccidiosis Research 109

of 53% and two major ribosomal gene clusters on chromosomes 10 and


12 (500 copies of the 5S rDNA and 140 copies of the 18S–5.8S–28S
rDNA, respectively) (Shirley, 2000). Complete mitochondrial genome
sequences have been assembled for all seven species that infect the chicken,
and comprise 6200 bp (6148–6407 bp) DNA with 65% A þ T content
(Lin et al., 2011; Liu et al., 2012). The relatively high A þ T content influ-
ences codon usage and, consequently, amino acid content of the three pro-
tein coding genes cox-1, cox-3 and cytB. Sequencing of the larger plastid
genome identified 35 kb DNA with a similarly high A þ T content
(Cai et al., 2003). Double stranded RNA virus genomes have been iden-
tified within many isolates of chicken Eimeria species ranging in size from
1.7 to >7.4 kb. Hybridization studies suggest the existence of multiple,
genetically distinct viral strains or species (Lee and Fernando, 2000;
Lee et al., 1996).

4.1.1 Nuclear karyotype


Oocysts of the Eimeria species that infect poultry are characterized by
extremely tough walls which are resistant to chemical, detergent,
enzymatic- and temperature-based disruptions, in contrast to those that
infect mammalian hosts such as the rat (Kurth and Entzeroth, 2008). Until
recently, the oocyst wall has prevented morphological analysis of eimerian
karyotypes and chromosome replication, hindering microscopic charac-
terization (del Cacho et al., 2001). However, the recent development of
a protocol based upon oocyst incubation in a hydrochloric acid/ethanol
solution followed by multiple freeze–thaw cycling has permitted the
release and spread of intact chromosomes, confirming previous PFGE-
based estimates of a 14 chromosome karyotype for all species that have
been investigated (del Cacho et al., 2005; Shirley, 1994a). Using PFGE,
the E. tenella and E. maxima karyotypes have been shown to range from
1 to >7 Mb and 2 to >6 Mb, respectively (Blake et al., 2011b;
Shirley, 2000). Comparison between E. tenella strains has revealed variable
sizes for chromosomes 1–4 and 11 (Sheriff et al., 2003; Shirley, 2000). Var-
iation by as much as 5% for chromosome 1 between extreme examples has
been used as a genetic marker in linkage analyses (Shirley and Harvey,
2000). The molecular basis for the observed polymorphism remains
unclear, although the highly repetitive nature of the E. tenella genome is
likely to encourage non-homologous genetic recombination, resulting
in length polymorphisms.
110 H. David Chapman et al.

4.1.2 Genome sequencing


The E. tenella Houghton strain was the first eimerian to be subjected to
nuclear genome sequencing. A combination of Sanger, 454 and Illumina
sequencing technologies was used to create a whole genome assembly rep-
resenting 94% of the complete nuclear genome in 4682 contiguous
sequences (contigs). While high frequency of repetitive sequences has hin-
dered further assembly of the genome, sequencing large insert genomic
DNA libraries has permitted assembly of 90% of the genome into 1720
supercontigs. Public access to the consensus assembly is available through
the Wellcome Trust Sanger Institute website, GeneDB (www.genedb.
org) and EUPathDB (www.eupathdb.org). Additionally, chromosome 1
of E. tenella has been fully sequenced and assembled. The identification of
telomeric-like repeats at each end of the assembly suggests representation
of the majority of the chromosome, including 85% of the predicted
1.05 Mb sequence with a small number of sequence gaps (Ling et al.,
2007). Comparison with a chromosome 1 HAPPY map (mapping based
on the analysis of approximately HAPloid DNA samples using PCR) sup-
ported the validity of the assembly and provided a tool to anchor and order
the sequence contigs (Ling et al., 2007). More recently, a cloned line derived
from the E. maxima Houghton strain has been sequenced, providing the first
genomic resource for a species that parasitises the mid-intestine (Blake et al.,
2012). Sanger sequencing combined with 454 sequencing yielded 13-fold
genome coverage and a consensus assembly representing 74% of the
nuclear genome in 12,852 contigs (publically available through EmaxDB,
www.emaxdb.org). Nuclear genome sequences for the Houghton strains
of all seven species of Eimeria of the chicken, and for additional strains of
E. tenella, are now being generated using multiple next-generation sequenc-
ing technologies and during 2013 all of these sequences will be available for
public scrutiny within both the GeneDB and the EuPathDB databases.
Gross genomic comparison among apicomplexan parasites reveals a rel-
atively conserved genome size for coccidial parasites, significantly larger than
the haemosporids and piroplasms (Table 2.1). While genome size varies sig-
nificantly among the Apicomplexa, the predicted number of protein coding
genes varies less dramatically (from largest to smallest fold difference:
genome ¼ eightfold, gene number ¼ twofold; Table 2.1). Broader com-
parison with most other apicomplexan parasites reveals a negatively corre-
lated association between gene density and genome size, likely to be
underpinned by a core gene set essential to parasite function and survival
irrespective of genome size. Furthermore, the multi-host lifestyles of the
Table 2.1 Apicomplexan parasites: A current genetics and genomics summary
Genome size Chromosome Predicted Gene density Recombination rate
Organism Strain (Mb) no. proteins (genes Mb1) (kb cM1) Referencea
Eimeria tenella Houghton 55.0 14 8786 160 264b Blake et al.
(2011b)
Eimeria maxima Houghton 57.5 14 Not known Not known 60–120 Blake et al.
(2011b)
Toxoplasma ME49 63.0 14 7993 127 104 Khan et al.
gondii (2005)
Neospora NCLiv 61.0 14 7082 116 na na
caninum
Plasmodium 3D7 23.3 14 5538 238 17 Su et al. (1999)
falciparum
Babesia bovis T2Bo 8.1 4 3706 458 na na
Theileria parva Muguga 8.3 4 4082 492 4.6 Katzer et al.
(2011)
Cryptosporidium Iowa II 9.1 8 3805 418 10-56 Tanriverdi et al.
parvum (2007)
a
Reference used to derive the rate of genetic recombination.
b
Expected to drop should additional markers be included as described previously for T. gondii (Khan et al., 2005; Sibley et al., 1992).
na, not available.
Data derived from EuPathDB (http://eupathdb.org/eupathdb/, accessed 3 July 2012).
112 H. David Chapman et al.

hemoparasites and piroplasms might indicate a requirement for a larger core


gene set. While total chromosome number remains stable within the larger
genomes, the relative rate of genetic recombination exhibits a strong neg-
ative correlation (Table 2.1), possibly indicating a minimum requirement
for recombination, irrespective of genome size.

4.1.3 Genome structure


The Eimeria species are protozoan eukaryotic organisms. Each eimerian
genome is represented by a series of chromosomes including telomeres
and centromeres (del Cacho et al., 2005). Transmission electron microscopy
studies have revealed a constant telomere length of 32 nm for E. tenella and a
consistent centromere index per chromosome among several strains,
although figures vary between chromosomes, thereby providing distinctive
identifiers (del Cacho et al., 2005). Intronic sequences are common within
most coding regions and there is proteomic evidence for alternative splicing
(Lal et al., 2009). Unusually, among apicomplexan parasites, sequences
related to eukaryotic transposable elements are readily identified. It has
been suggested that these are most similar to non-LTR LINE-like
retrotransposons (long terminal repeat and long interspersed nuclear
elements) (Ling et al. 2007). Most strikingly, the sequencing and assembly
of E. tenella chromosome 1 revealed an unusual segmented structure to
the chromosome, which contains three repeat-rich segments flanked by four
repeat-poor segments (GenBank Accession number AM269894). The
repeat-rich segments contain large numbers of simple sequence repeats
and LINE-like elements as well as highly variable A þ T content, CpG to
GpC ratio and second-order Markov entropy, prompting the identifier
‘feature-rich’ (R-segment) (Ling et al., 2007). In contrast, the repeat-poor
segments exhibited less variation in all measures, prompting the identifier
‘feature-poor’ (P-segment). Whole genome HAPPY mapping now suggests
that the segmental organization described from chromosome 1 is also present
throughout the rest of the E. tenella genome (Lim et al., 2012).
Preliminary characterization of predicted gene structures within the
R- and P-segments suggests shorter coding sequences with larger exons
and less numerous introns in the latter. RFLP comparison of E. tenella geno-
mic DNA revealed polymorphism between the Houghton, Weybridge and
Wisconsin strains for all four of the R-segment probes tested, but none of the
four P-segment probes, suggesting a higher rate of genome evolution within
the feature-rich regions. Comparative studies with the E. maxima genome
Review of Coccidiosis Research 113

suggests that R and P regions are also present throughout the genome of this
species (Blake et al., 2011b).

4.1.4 Repetitive sequences


Eimerian genes are known to feature large numbers of repetitive DNA
sequences ( Jenkins, 1988) and early reports of numerous trinucleotide
GCA repeats (and alternative frame permutations CAG, AGC, TGC,
GCT and CTG) are supported by more systematic studies of published
EST data and the E. tenella chromosome 1 assembly. Almost 3000 simple
repeat units were identified on chromosome 1 and more than 60% of these
were GCA-based (Ling et al., 2007). Other common repeats included a
telomere-like heptamer AGGGTTT, representing nearly 20% of the repeats
on chromosome 1, and the palindromic octamer TGCATGCA, which has
been described previously within several other apicomplexan genomes. As
noted above, simple sequence repeats were confined largely to the
R-segments in the E. tenella chromosome 1 assembly, where triplet repeats
and AGGGTTT represent 14% of the sequence. Both EST and genomic
analyses identified frequent triplet repeats in putative coding regions, a fea-
ture confirmed by recent proteomic analysis (Lal et al., 2009; Ling et al.,
2007; Shirley, 2000). Importantly, triplet repeats do not interfere with
the coding frame in expressed sequences, unlike heptamer and octomer
repeats. To date, no functional association has been made with these trans-
lated repetitive sequences, although they have been hypothesized to play a
role in genome evolution and diversification as possible hotspots of recom-
bination (Ling et al., 2007).
Other repetitive sequences include multiple putative transposable
sequences and arrays of tandemly repeated 5S and 18S–5.8S–28S ribosomal
genes (both discussed above). The high copy number of the rDNA arrays
and the ITS regions (ITS-1 and ITS-2) has promoted their use as targets
for molecular diagnostics and phylogenetic analyses, although it is important
to note the existence of polymorphism between copies within a single
genome (Blake et al., 2006; Vrba et al., 2011).

4.2. Transcriptomics
Systematic descriptions of four Eimeria species transcriptomes have been
reported in addition to small numbers of targeted cDNA sequences from
the same and other species. By far the most thoroughly characterized has
been E. tenella, with more than 50,000 publically available expressed
sequence tag (EST) sequences (Amiruddin et al., 2012; Chen et al., 2008;
114 H. David Chapman et al.

Klotz et al., 2007; Novaes et al., 2012; Wan et al., 1999). E. acervulina and
E. maxima are both well represented (e.g. Dong et al., 2011; Miska et al.,
2008; Novaes et al., 2012; Schwarz et al., 2010) and E. brunetti has been sam-
pled (Aarthi et al., 2011). Sequences have frequently been derived from the
most easily accessed oocyst and sporozoite life cycle stages, although second-
generation merozoites have commonly been prioritized given their rele-
vance to coccidiosis caused by E. tenella (Amiruddin et al., 2012; Miska
et al., 2008; Novaes et al., 2012; Schwarz et al., 2010). Key stages in the
eimerian life cycle waiting to be sampled include the gametocytes and devel-
oping intracellular schizonts.

4.2.1 Full-length cDNA sequences


Analysis of 433 full-length cDNA sequences from E. tenella Houghton strain
second-generation merozoites has provided the most detailed study of tran-
script structure for the Eimeria species. At 1647 bp the average transcript
length was longer than described for Toxoplasma gondii or Cryptosporidium
parvum (range 441–3083 bp), including average 50 untranslated region
(UTR), ORF and 30 UTR sizes of 342, 867 and 438 bp, respectively
(Amiruddin et al., 2012). The longer transcript length was possibly
influenced by the high frequency of simple sequence repeats. Alignment
of translation initiation sequences proximal to each predicted start codon
identified a consensus Kozak sequence of (G/C) AAAATGG. Usage analysis
identified 10 under-represented codons, UAU, UGU, GUA, CAU, AUA,
CGA, UUA, CUA, CGU and AGU, in line with previous reports for
E. tenella (Amiruddin et al., 2012; Ellis et al., 1993). Simple sequence repeats
were again found to be common throughout many of the full-length
sequences, most commonly translating as poly-glutamine tracts (CAG) or
related equivalents following frameshifts and repeat degradation.

4.2.2 Transcript identification and inter-species comparison


Significant EST and open reading frame expressed sequence tag
(ORESTES) cDNA datasets exist for E. tenella, E. acervulina and
E. maxima in the public domain. In a recent study, 48,361 ORESTES
and EST sequences derived from a series of E. tenella zoite and oocyst stages
at multiple developmental time points were collated and assembled into
8700 contiguous and singleton sequences (Novaes et al., 2012; Rangel
et al., 2013). Comparison with the 8786 putative protein coding sequences
predicted from the E. tenella genome sequence (http://www.genedb.org;
Table 2.1) suggested a good coverage, although the absence of sequences
Review of Coccidiosis Research 115

derived from schizont and gametocyte stages indicates distinct gaps imposed
by the difficulty in obtaining suitable parasite material. Comparison with
other coccidian parasites including Neospora caninum and T. gondii
highlighted a conserved genome-wide gene density (as discussed above;
Table 2.1). Consideration of the individual sequence read distribution across
these assemblies prompted the authors to hypothesize that each life cycle
stage is likely to be characterized by a small number of highly expressed
genes, supplemented by a larger number of genes expressed at a much lower
level (Novaes et al., 2012). The application of RNAseq technologies is now
starting to significantly improve transcriptome coverage and gene predic-
tion, as has been described for N. caninum (Reid et al., 2012), and may con-
firm this hypothesis.
Equivalent ORESTES analyses for E. acervulina and E. maxima resulted
in 3413 and 3426 assembled cDNAs, respectively (Novaes et al., 2012;
Rangel et al., 2013), overlapping with many of the 1029 and 1380 unique
contiguous and singleton EST sequences derived in other notable studies for
these species (Miska et al., 2008; Schwarz et al., 2010). E. brunetti is at present
the only other eimerian parasite whose transcriptome has been systematically
sampled, being represented by 269 unique contiguous and singleton EST
sequences (Aarthi et al., 2011). Comparison with E. tenella cDNA sequences
identified putative homologues for between 19% and 32% of these unique
expressed sequences, with more than 47% of all sequences sharing no signif-
icant similarity to currently available annotated cDNA sequences derived
from any organism. The appearance of such a large number of unknown
putative coding sequences was anticipated and has been a common feature
of many apicomplexan genomes (Reid et al., 2012; Schwarz et al., 2010).
The number of putatively genus- and species-specific expressed sequences
is consistent with the specialized life cycle and exquisitely restricted host
and tissue range of these parasites. Nonetheless, transcripts encoding homo-
logues of key apicomplexan invasion-relevant proteins including several
microneme proteins (MICs) and glideosome components have been readily
identified in at least three Eimeria species (Novaes et al., 2012; Schwarz et al.,
2010). Similarly, surface antigen (SAG) transcripts have been identified
within the E. tenella, E. acervulina and E. maxima transcriptomes.
Hierarchical clustering of transcriptomic data derived from each of the
Eimeria species sampled to date has revealed a highly conserved expression
profile between life cycle stages and a strong correlation with stage order
within the life cycle. Thus, transcripts derived from sporozoites were most
likely to be conserved within the sporulated oocyst transcriptome
116 H. David Chapman et al.

(Novaes et al., 2012). Similarly, close associations have been identified


between the sampled zoite stages and between oocyst datasets collected at
different stages of sporulation.
Functional transcriptomic studies using custom designed cDNA arrays
with or without a suppression subtractive hybridization step have been
undertaken with E. tenella and E. maxima. Examples include the association
of monensin resistance with up-regulation of transcripts involved with cyto-
skeletal rearrangement and energy metabolism and a panel of 32 differentially
expressed transcripts associated with precocious parasite development (Chen
et al., 2008; Dong et al., 2011). Screening a cDNA panel derived from mul-
tiple purified zoite and unpurified chicken intestine/schizont samples against
a genome tiling array has been used to identify coding sequences within
genetic loci mapped by association with susceptibility to strain-specific
immune killing in E. maxima (Blake et al., 2011a).

4.3. Proteomics
Before the advent of genome sequencing and annotation, the processes by
which specific proteins could be identified, characterized and analyzed
were time-consuming, laborious and low throughput, usually limited to
the study of one or two individual proteins at a time. Methods for direct
analysis of known polypeptide sequences often utilized mass spectrometry
(MS), for example, to identify specific features of the protein such as pro-
teolytic cleavage sites. However, generation of de novo protein sequence
was most commonly achieved by chromatographic analyses of peptides
following chemical treatment of purified protein to progressively remove
amino acids, usually from the N-terminus. Over the past decade, huge
advances in MS instrumentation and the availability of well-annotated
genomes has allowed the burgeoning of high-throughput proteomics
technologies. Complex mixtures of proteins can be fragmented into small
peptides by enzymatic digestion or chemical degradation, and subjected in
parallel to high-energy MS that generates large numbers of individual spec-
tra from which peptide sequences can be directly inferred. These experi-
mentally derived sequences are then mapped in silico onto databases of
predicted proteins derived from annotated genomes, allowing the
unequivocal identification of full coding sequences. This type of approach
is well suited to high-throughput experiments in which hundreds or thou-
sands of individual proteins can be identified. While it is possible to analyze
very complex mixtures, such as a whole cell lysate, it is usual to carry a
Review of Coccidiosis Research 117

protein separation technique prior to MS to reduce sample complexity and


aid the downstream in silico analysis. For parasites, a number of approaches
have been taken including one- or two-dimensional gel electrophoresis,
and in-line liquid chromatography (LC; recently reviewed by Wastling
et al., 2012).

4.3.1 Studies using MALDI-MS


High-throughput proteomics technologies emerged in parasitology at the
beginning of the twenty-first century and were readily adopted by the
coccidian research community. Initial studies focused on polypeptide spots
excised from polyacrylamide gels following their separation by two-
dimensional electrophoresis (2DE). Spots were subjected to in-gel digestion,
usually with trypsin, and protein identifications made by generating peptide
mass fingerprints, acquired by matrix-assisted laser desorption/ionization
(MALDI) MS. Initial proteomes were derived for tachyzoites of T. gondii
(Cohen et al., 2002) and N. caninum (Lee et al., 2003) and for sporozoites
and second-generation merozoites of E. tenella (de Venevelles et al.,
2004; Liu et al., 2009). A major disadvantage of MALDI-MS is, however,
that unambiguous protein identifications can be made only if high-quality
gene annotations are available, which was not the case for these parasites
at the time. Therefore, MALDI data were generally supplemented by
re-analysis of selected protein spots using tandem MS to generate de novo
peptide sequence data. As an adjunct to proteomics studies, de Venevelles
et al. (2004) and Liu et al. (2009) probed 2DE blots of sporozoite or mer-
ozoite proteins with hyperimmune chicken sera and identified 50 and
85 spots, respectively, that were immunogenic, numbers that are in agree-
ment with several earlier studies using Western blotting (Sutton et al., 1989;
Tomley, 1994; Xie et al., 1992).
A different proteomics approach, aimed at elucidating the proteome of
purified microneme organelles from E. tenella sporozoites (Bromley et al.,
2003), used a post-source modification of MALDI, termed chemically
assisted fragmentation, which improves fragmentation efficiency and sim-
plifies interpretation of the spectrum. Briefly, a negatively charged group
is coupled to the N-terminus of tryptic peptides so that formation of a pos-
itively charged ion requires the introduction of two protons, one of which
resides in the peptide backbone, where it can resonate and assist fragmenta-
tion. After fragmentation, only y-ions retain a positive charge, which sim-
plifies the spectrum and allows it to be used to generate de novo peptide
sequences. Using this method, 37 of 96 spots excised from 2DE gels were
118 H. David Chapman et al.

successfully identified, which included proteins known to reside in the


micronemes (EtMIC1, 2, 3) as well as several novel proteins that had not
previously been linked to this organelle. In a similar type of study, de
Venevelles et al. (2006), used a combination of MALDI and tandem MS
to analyze peptides derived from partially purified refractile bodies (RB)
of E. tenella sporozoites. As well as two proteins known to reside in the
RB (the aspartyl proteinase Eimepsin and the antigen SO7), 30 additional
putative RB proteins were identified including a hydrolase, a subtilisin
and a lactate dehydrogenase.

4.3.2 Studies using high-energy MS


Advances in MS instrumentation, especially the use of high-energy,
collision-induced dissociation-based, tandem MS, has revolutionized the
field of proteomics, allowing high-throughput identification of very large
numbers of tryptic peptides obtained from gel spots, gel slices or LC frac-
tions. Using a range of complementary approaches (2DE, gel-LC linked
to tandem MS and multi-dimensional protein identification technology,
MuDPIT), whole cell proteomes with a high level of coverage have now
been obtained for four developmental stages of E. tenella (Lal et al.,
2009). In addition, there are also high-quality proteomes available for var-
ious sub-cellular fractions including the rhoptry organelles of E. tenella
(Oakes et al., 2013).
The most comprehensive proteomics study of an eimerian is that of Lal
et al. (2009) who generated proteomes for four life cycle stages of E. tenella
(unsporulated oocysts, sporulated oocysts, sporozoites and second-
generation merozoites) resulting in the unequivocal identification of 1868
proteins, which represents almost 30% of the likely total number of
E. tenella proteins. A total of 288 proteins were conserved between sporo-
zoites and merozoites, but not found in unsporulated oocysts, suggesting
these are linked to zoite-specific functions such as attachment, invasion
and egress. These included proteins known to localize to the microneme,
and rhoptry secretory organelles and proteins associated with the
‘glideosome’, which drives zoite motility and the ‘moving junction’ (MJ)
structure that is formed at the host–parasite interface during invasion.
Importantly, it was noted that stage-specific variants of key molecules
involved in the formation of the MJ are expressed, such as AMAs and
rhoptry neck proteins (RONs), suggesting strongly that sporozoites and
merozoites assemble the MJ in a stage-specific manner, something
that has since also been shown to occur in T. gondii (Fritz et al., 2012a,b)
Review of Coccidiosis Research 119

and has been confirmed again in the rhoptry sub-cellular proteome of


E. tenella (Oakes et al., 2013). Additional protein differences between
the zoite stages included differential expression of the GPI-linked families
of SAGs, as also shown in the transcriptome analyses (Novaes et al., 2012).
The multi-stage proteome indicates that energy production throughout
most of the developmental cycle is linked strongly to gluconeogenesis
and glycolysis, with the mannitol cycle present in oocysts and sporozoites,
but not in merozoites, consistent with in-depth biochemical analysis of this
pathway throughout the E. tenella life cycle (Allocco et al., 1999). It was
also apparent that proteins linked to oxidative phosphorylation were
expressed at the highest levels in the merozoite, suggesting a metabolic
shift to use oxygen to mobilize energy production during the asexual
phases of growth and replication. There is also a higher abundance of
proteins linked to transcription, protein synthesis and nucleotide metabo-
lism in the merozoite, compared to the sporozoite, which is consistent
with observations in the transcriptome and likely to be related to the
massive replication that has taken place within the schizont, just prior to
merozoite release.

4.4. The future


Progress in the development of novel molecular technologies applicable to
nucleotide sequencing, epigenetics and proteomics support increased
understanding of eimerian parasites, their close relatives and their host inter-
actions. Improved sequencing technologies facilitating cheaper, more effec-
tive, genome sequencing and assembly are now supporting the extension of
such studies to a rapidly increasing range of coccidial parasites. Next-
generation DNA and RNA sequencing strategies also have the potential
to revolutionize genetic mapping and our biological understanding of select-
able parasite phenotypes. Importantly, many such resources have been made
publically available for the Eimeria species. Genome sequences and predicted
protein datasets are freely available through EUpathDB (http://eupathdb.
org/eupathdb/), GeneDB (http://www.genedb.org/Homepage/Etenella)
and EmaxDB (http://www.genomemalaysia.gov.my/emaxdb/). Comple-
mentary resources including annotated transcript assemblies and genetic
maps can be accessed through the Eimeria Transcript database (Rangel
et al., 2013) (http://www.coccidia.icb.usp.br/eimeriatdb/) and NCBI
Map Viewer (http://www.ncbi.nlm.nih.gov/projects/mapview/), respec-
tively. The production of novel and improved datasets will impact upon
120 H. David Chapman et al.

the development of new diagnostic and anticoccidial control strategies and


provide tools with which questions of evolutionary and population biology
may be interrogated.

5. TRANSFECTION
Transfection refers to the introduction of exogenous DNA or RNA
into cells by chemical, biological or physical means. Through transfection,
the recipient cell can gain a new genetic trait, and some of the introduced
DNA can be integrated into the genome of the recipient cell. In
apicomplexan parasites, such as Toxoplasma and Plasmodium, plasmid-
mediated transient and stable transfection systems were established in the
early 1990s, but owing to the difficulty of completing the life cycle of Eimeria
in vitro, and the lack of regulatory DNA sequences, genetic manipulation has
lagged behind that of other protozoan parasites (Hao et al., 2007; Kelleher
and Tomley, 1998; Shi et al., 2008). The first stable transfection system was
developed in Eimeria in 2008, 10 years after the first report of a transient
transfection system in this genus of parasite (Clark et al., 2008; Kelleher
and Tomley, 1998).

5.1. Transfection construct design


Transfection constructs are usually based on pre-constructed, commercially
available plasmids, and contain elements including regulatory and signal
sequences. Promoters of both constitutive genes and stage-specific genes,
together with their homologous or heterologous 30 UTR sequences, have
been used successfully to drive expression of reporter genes in eukaryotic
cells, including Eimeria. Using enhanced yellow fluorescent protein (eyfp
marker gene) as a reporter, it was shown that the E. tenella microneme pro-
tein 1 (EtMIC1) promoter drives EYFP expression in sporulated but not
unsporulated oocysts, as would be expected (Yin et al., 2011). Another study
showed that three different promoter sequences originating from E. tenella
could function effectively not only in other species of Eimeria but also in
T. gondii (Kurth and Entzeroth, 2009; Zou et al., 2009). Similarly, promoters
of the ‘housekeeping’ tubulin gene and the differentially regulated surface
antigen gene (sag1) of T. gondii, were effective in driving the expression
of the EYFP marker gene in E. tenella (Zou et al., 2009). As genetic tools
are well developed for T. gondii, the mutual recognition of these promoter
sequences in Eimeria and Toxoplasma suggests that some promoter sequences
from T. gondii could be utilized directly in Eimeria and that T. gondii could be
Review of Coccidiosis Research 121

used as a novel transfection system for Eimeria-rooted vectors. This has the
potential to help improve our understanding of Eimeria spp. through the
development of both forward and reverse genetic technologies.
Several signal sequences are known to exhibit conserved activity in
apicomplexan parasites. For example, parasitophorous vacuole targeting sig-
nal sequences of T. gondii GRA8 and Plasmodium falciparum repetitive inter-
spersed family proteins have been found to function effectively in transfected
E. tenella and successfully target EYFP to the parasitophorous vacuole in
E. tenella (Shi et al., 2009; Yin et al., 2011). Furthermore, the nucleus-
targeting signal of the H5N1 subtypic avian influenza virus nuclear protein
also exhibits conserved functionality in eukaryotic cells, supporting nuclear
targeting when incorporated into an E. tenella transfection construct (Yin
et al., 2011).

5.2. Transient transfection


In transient transfection, introduced exogenous DNA does not integrate
into the genome of the cell, but is transcribed into mRNA and is subse-
quently translated to protein. Transient transfection is an efficient tool to
identify regulatory and signal sequences of genes and to screen for genes
associated with certain phenotypes. For Eimeria, transfection of sporozoites
has been achieved by electroporation with plasmids; PCR amplified DNAs
or fragmented genomic templates that encode the exogenous DNA, flanked
by Eimeria-specific regulatory sequences (Hao et al., 2007; Kelleher and
Tomley, 1998; Liu et al., 2008). The efficiency of transient expression is usu-
ally low but this has been overcome by the use of restriction enzyme-
mediated integration (REMI), which boosts transfection efficiency about
200-fold (Liu et al., 2008). Very high transfection efficiency has been
achieved in E. tenella sporozoites by using cytomix-buffered REMI and
the AMAXA nucleofection system (Clark et al., 2008).

5.3. Stable transfection


Stable transfection refers either to the permanent expression of the gene of
interest through the integration of the transfected DNA into the nuclear
genome, or the maintenance of a transfected plasmid as an extra chromo-
somal replicating episome within the cell. Stable transfection has been dif-
ficult to achieve in Eimeria because of the inability to transfect oocysts or
sporocysts, the absolute requirement for in vivo amplification and selection,
and the poor survival of sporozoites in the acidic environment of the host
122 H. David Chapman et al.

stomach. The latter has been overcome by gavaging birds with sodium
bicarbonate to neutralize the acidic barrier (Clark et al., 2008). Stable trans-
fection systems have also been established for E. tenella by cloacal inoculation
of sporozoites, combined with in vivo drug selection and/or fluorescence
activated cell sorting (FACS) (Clark et al., 2008; Yan et al., 2009). To date,
the mutated dihydrofolate reductase–thymidylate synthase gene is the only
drug-mediated selection marker available for the transfection of Eimeria
(Clark et al., 2008; Yan et al., 2009). The mutated gene confers resistance
to pyrimethamine, a drug used to potentiate the action of the sul-
phonamides. Drug selection, together with FACS of fluorescence reporter
proteins and the high transient transfection efficiency using REMI, contrib-
uted to the success in establishing stable transfection in Eimeria.
Integration of a transfection construct into the Eimeria genome seems to
occur at random during the production of stably transfected Eimeria as
detected by Southern blotting and plasmid rescue (Yan et al., 2009). Quan-
titative real-time PCR analysis of insertion rate post-transfection showed an
average persistence of four copies of the tandem YFP reporter cassette per
genome from the first round of replication after electroporation and REMI.
After two further cycles of in vivo amplification with both FACS and pyri-
methamine selection, an average of 10 copies per genome were detected and
remained relatively stable through five further unselected generations. In
contrast, when REMI was not used, only a single copy of the relevant
reporter gene was detected per genome in first or second-generation trans-
fected parasite populations (Clark et al., 2008). Serial selection of fluorescent
mCitrine-transfected oocysts by FACS did not notably increase copy num-
ber, although tightened gating and FACS with sporocysts in place of oocysts
increased both the expression rate and the copy number to 2–3 per genome
(Clark et al., 2008).

5.4. PiggyBac-based forward genetic system


PiggyBac is a cut-and-paste transposon that is useful for transgenesis and inser-
tional mutagenesis and has been used for stable transfection in a wide variety of
organisms. This new molecular technology has been used successfully to
achieve targeted insertional mutagenesis in Eimeria (Su et al., 2012). Using
REMI, E. tenella sporozoites were electroporated with a mix containing
the restriction enzyme AscI, an AscI-linearized helper plasmid containing
the transposase gene, and an uncut donor plasmid containing the eyfp gene.
The eyfp gene was flanked by Eimeria-specific regulatory sequences that
Review of Coccidiosis Research 123

were further flanked by piggyBac inverted terminal repeats (ITRs). Subse-


quently, electroporated sporozoites were inoculated into chickens via the
cloacal route and transfected progeny oocysts expressing eyfp were sorted
by flow cytometry. A stable eyfp expressing population was obtained by suc-
cessive in vivo passaging and FACS selection (Su et al., 2012). Locus-specific
PCR and genome walking revealed that the ITR-restricted sequence was
successfully targeted into TTAA sites, with about seven copies per genome
(Su et al., 2012). Both reverse and forward genetic tools will hopefully allow
an in-depth analysis of Eimeria basic biology. PiggyBac-mediated efficient
TTAA targeted mutations should be an attractive tool for genetic manipula-
tion of Eimeria.

5.5. Stably transfected Eimeria as a vaccine vector and beyond


The feasibility of using genetically modified Eimeria as a vaccine vector has
been studied using model antigens such as EYFP. It was found that
E. tenella expressing EYFP stimulated both humoral and cell-mediated
immunity to the expressed protein, and that antigen compartmentalization
affects the magnitude of the immune response with microneme-targeted
EYFP stimulating a higher IgA response than cytoplasm-targeted EYFP
(Huang et al., 2011). In another study, vaccination of specific pathogen-
free chickens with a population of E. tenella expressing Campylobacter
jejuni antigen A caused a significant reduction in bacterial load following
challenge with C. jejuni compared with unvaccinated and wild-type
E. tenella vaccinated controls (Clark et al., 2012). Thus, it has been dem-
onstrated that transfected Eimeria parasites can successfully express foreign
antigens that may stimulate immunity against a target pathogen. However,
to provide complete protection, co-expression of adjuvant antigens and/or
cytokines may be necessary (Guangwen Yin and Xun Suo, unpublished
observations).
Transfection of Eimeria species is still limited by the inability to transfect
oocysts and sporocysts, the difficulty of obtaining single-sporocyst-derived
recombinant clones, and the obligate requirement of in vivo amplification
and selection of stably transfected parasites (Clark et al., 2008; Shi et al.,
2008; Yan et al., 2009). It is difficult to maintain a large number of mutated
clones as a mutated Eimeria library, which needs manpower, facilities and
financial support. Nevertheless, high transfection efficiencies (Clark et al.,
2008; Hanig et al., 2012) will boost the advance of both reverse and for-
ward genetic systems in this important group of parasites. The interchange
124 H. David Chapman et al.

and development of Eimeria transfection constructs between laboratories in


countries including China, France, Germany, Japan and the UK promises
rapid development over the coming years. More advanced genetic tools
established in other protozoa, such as Toxoplasma, Plasmodium, Leishmania
and Trypanosoma may eventually be applied to research with Eimeria.

6. OOCYST BIOGENESIS
One of the defining features of the coccidia is the oocyst. There are
three crucial milestones in oocyst production: first, merozoites undergo rapid,
asexual division within the intestine, amplifying dramatically the total number
of parasites poised to develop into microgametes or macrogametes; second,
microgametes fertilize the macrogametes and third, the macrogametes mobi-
lize specialized organelle – wall forming bodies (WFBs) – to generate the
oocyst wall, one of the most remarkable biological structures known.
The oocyst wall encapsulates and protects coccidian parasites as they exit
their definitive host in faeces and, subsequently, in the harsh, external world,
while they undergo meiosis to produce infectious sporozoites. Thus, the
oocyst is the endpoint of sexual reproduction. It is also notoriously resilient,
resisting both mechanical and chemical damage and tolerating changes in
humidity and temperature for months, if not years (reviewed by Belli
et al., 2006; Fritz et al., 2012a). This resilience is critical for transmission
of coccidian parasites from host to host, via ingestion of contaminated food
or water.

6.1. Veil and WFBs


The formation of the oocyst wall proceeds via an orderly release of the con-
tents of: first, the veil forming bodies; second, wall forming bodies type 1
(WFB1) and third, wall forming bodies type 2 (WFB2) (Ferguson et al.,
2003). The contents of the veil forming bodies are undescribed but form
a loose outer veil that appears to provide a temporary scaffold or frame
around the developing oocyst wall (Ferguson et al., 2000, 2003). It is lost
before the oocyst is excreted in the faeces and, therefore, plays no role in
protecting the parasite in transit from host to host (Ferguson et al., 1975;
Pittilo and Ball, 1980).
The release of the contents of the WFBs appears to be controlled by the
rough endoplasmic reticulum/Golgi apparatus (Ferguson et al., 2003). Once
a zygote has formed, WFB1 migrate to the periphery of the parasite, align
and disaggregate rapidly, before appearing to merge together to form the
Review of Coccidiosis Research 125

Figure 2.3 Immunofluorescent images of macrogametocytes and early oocysts of


E. maxima within the intestine of a chicken 144 h post-infection. (A) An early-stage
macrogametocyte—type 1 and type 2, wall forming bodies (WFBs) are indistinguish-
able. (B) A mid-stage macrogametocyte—type 1 and type 2, WFBs are distinguishable
by size (WFB1s are larger). (C) A cluster of mature gametocytes showing peripheral
alignment of WFBs, with disaggregation of some type 1 WFBs evident. (D) Early
oocysts—the outer wall has formed and disaggregation of type 2 WFBs is evident. Host
nuclei are stained with 40 ,6-diamidino-2-phenylindole (blue); WFBs and oocyst walls are
stained with antibodies to affinity-purified gametocyte antigens (green). Image supplied
by Professor D.J.P. Ferguson (University of Oxford, UK).

outer layer of the bi-layered oocyst wall (Fig. 2.3). This outer layer may ini-
tially be as thick as 600 nm but quickly compacts to 200 nm or less (Ferguson
et al., 2003). Not long after the outer layer forms, WFB2 are also transferred
to the parasites surface, by the endoplasmic reticulum, and also align, disag-
gregate and also appear to fuse together to form the inner layer of the oocyst
wall (Ferguson et al., 2003). This layer is less electron-dense than the outer
layer and more consistent in size, being around 40 nm in most species exam-
ined (reviewed by Belli et al., 2006). The inner and outer layers are, at first,
separated by a 40 nm zone, which shrinks as the wall compacts. However,
the two layers never fuse together and are readily separated in the laboratory
(Monné and Hönig, 1954).
126 H. David Chapman et al.

6.2. Oocyst wall proteins


Gas chromatography and MS analyses of the oocyst walls of E. maxima and
E. tenella indicate that both layers are dominated by protein (>90%) with
surprisingly low levels of carbohydrate and lipids (Mai et al., 2009). Thus,
an understanding of the structure and characteristics of proteins that com-
prise the oocysts wall is essential for genuine understanding of how the wall
forms and why it is so robust. Only a small number of oocyst wall proteins
have been identified and the origin of all of these can be traced back to the
WFBs in macrogametes (reviewed in detail by Mai et al., 2009). These pro-
teins can be grouped into, essentially, three groups.
First, there is a 22 kDa antigen in the macrogametocytes of E. tenella,
which is found in WFB2 and the inner layer of the oocyst wall (Krücken
et al., 2008). This 22 kDa protein is dominated by histidine and proline res-
idues. As yet, no information is available about if or how this protein is
incorporated into the oocyst wall, though its involvement in stabilizing
the oocyst wall via cross-links between histidine and catechols, as seen in
insect cuticles, is a distinct possibility (Krücken et al., 2008).
Second, there is a family of nine large (174–190 kDa), cysteine-rich pro-
teins that localize to WFB1 of macrogametocytes and the outer wall of the
Cryptosporidium oocyst (Spano et al., 1997; Templeton et al., 2004). It is
thought that these ‘OWPs’ form disulphide bridges and matrices within
the oocyst wall (Spano et al., 1997). The recent discovery of seven OWPs
in Toxoplasma, with at least some of these localized to the outer oocyst wall
(Possenti et al., 2010), supports the idea that OWPs are involved in wall for-
mation. Recently, two OWP homologues have been found in Eimeria, at
least one of which appears to localize to WFB1 in macrogametes, further
evidence that OWPs are destined for the outer wall (Walker, 2009).
Third, there are numerous tyrosine-rich proteins, ranging in size from
8 to 31 kDa, in the inner wall of the Eimeria oocyst; all of these are derived from
precursor proteins of 56 and 82 kDa (GAM56 and GAM82) from WFB2 in
macrogametes of several species of Eimeria (Belli et al., 2003a, 2009). It has also
been discovered very recently that, although the Toxoplasma genome contains
no direct homologues of either GAM56 or GAM82, the oocyst wall of Toxo-
plasma contains up to six tyrosine-rich proteins (Fritz et al., 2012a).

6.3. Formation of the oocyst wall


The role of tyrosine-rich proteins in the formation of the Eimeria oocyst wall
has been studied in some depth, the result being the proposal of a two-step
Review of Coccidiosis Research 127

model: in step 1, precursor proteins found in WFBs are processed by


gametocyte-specific proteases into smaller, tyrosine-rich proteins and in step
2, peroxidases and/or oxidoreductases catalyze cross-linking of these pro-
teins via their tyrosine residues, resulting in extensive dityrosine matrices
within the oocyst wall. There is some evidence for both of these proposed
reaction steps.
It has been known for more than two decades that two proteins –
GAM56 and GAM82 – dominate the protein profile of gametocytes
(Wallach et al., 1989). Both GAM56 and GAM82 are processed into small,
tyrosine-rich proteins, as demonstrated in two ways: (i) antibodies to
GAM56 and GAM82 react with proteins of these sizes in gametocytes
but react with proteins of 8–31 kDa in oocysts (Belli et al., 2003a,b,
2009); and (ii) N-terminal sequencing of these wall proteins shows that they
are ‘cleaved’ from GAM56 or GAM82 at specific points (Belli et al., 2003a).
It has been discovered recently, using an in vitro assay, that the degradation of
GAM56 into smaller proteins is largely dependent on subtilase-like serine
protease activity (Katrib et al., 2012). There are at least six subtilase-like
enzymes in the genome of E. tenella, and at least three of these are expressed
specifically in gametocytes. Thus, assembly of the oocyst wall may follow a
mechanism that is similar to that involved in the assembly of the cuticle of
nematodes (Page and Winter, 2003; Thacker et al., 2006).
After formation of the numerous, small, tyrosine-rich derivatives of
GAM56 and GAM82, peroxidases or oxidoreductases are predicted to cat-
alyze their cross-linking via dityrosine bond formation. There is substantial
circumstantial evidence indicating that oocyst walls are rich in dityrosine
bonds. First, the oocysts exhibit a vivid blue autofluorescence between
the ultraviolet excitation wavelengths of 330 and 385 nm (Fig. 2.4), which
is characteristic of dityrosine cross-linking (Belli et al., 2006). Second,
dityrosine levels have been measured in the oocyst wall of E. maxima and
found to be remarkably high (Belli et al., 2003a), begging the conclusion
that their generation within the oocyst wall is a deliberate, enzymatically
catalyzed process, initiated by the parasite (Belli et al., 2006). It has been
shown that the WFBs of E. maxima embody a highly focused region of
peroxidase activity (Belli et al., 2003a, 2006) and, while neither an endog-
enous peroxidase or oxidoreductase has yet been isolated from Eimeria, it
has been established that exogenous peroxidases can induce dityrosine
cross-linking of a truncated version of GAM56 in vitro (Mai et al., 2011).
And, an oxidoreductase has been found in the oocyst wall of T. gondii
(Fritz et al., 2012a).
128 H. David Chapman et al.

Figure 2.4 Sporulated and unsporulated oocysts of E. maxima showing characteristic


UV autofluorescence (blue).

The concept that dityrosine cross-linking constitutes a critical feature of


the structure of the oocyst wall helps to explain the resilience of oocysts –
dityrosine cross-linking, leading to the formation of structural matrices,
sclerotization and quinone tanning, is widespread in nature, almost always
in association with the construction of protective coatings such as inverte-
brate egg shells, cuticles, cell walls, glues and cements (reviewed by Belli
et al., 2006). Moreover, it might be predicted that interfering with this pro-
cess is a way to limit the transmission of coccidian parasites. The subunit vac-
cine, CoxAbic®, may be an example of this. This vaccine contains GAM56
and GAM82 from E. maxima and laboratory experiments have shown that
immunization of broiler breeder hens with this vaccine stimulates the pro-
duction of protective IgY (¼IgG) antibodies that are transferred to offspring
chicks via the egg yolk (Wallach et al., 2008). There are two potential expla-
nations for this: (1) the antibodies ‘protect’ GAM56 and GAM82 from pro-
teolysis and, thereby, deprive the parasite of the tyrosine-rich building
blocks it needs to form the oocyst wall; and/or (2) the antibodies interfere
with dityrosine bond formation (Sharman et al., 2010).

7. HOST CELL INVASION


Apicomplexans, including all species of Eimeria, are highly successful
obligate intracellular parasites. Unlike many microorganisms that rely on
host-cellular pathways such as phagocytosis or pinocytosis for invasion,
Review of Coccidiosis Research 129

apicomplexans invade host cells rapidly and forcefully in a highly regulated,


parasite-driven process (reviewed by Santos and Soldati-Favre, 2011). Initial
interaction with host cells can occur with the parasite in any orientation but
commitment to invasion requires that the apical pole of the parasite makes
irreversible contact with the host cell surface. This initiates formation of a
MJ, a tight focus of constriction between the parasite and host cell mem-
brane, which migrates towards the posterior end of the parasite as invasion
proceeds (reviewed by Besteiro et al., 2011). The parasite synthesizes spe-
cialized molecular complexes at the parasite–host interface, which are essen-
tial for gliding movement (the actinomyosin-dependent glideosome) and
formation of the MJ. These complexes are assembled by regulated secretion
of microneme (MIC) and rhoptry (RON/ROP) proteins, and interact with
the parasite motor, localized in the pellicle, and with specific receptors on
the host cell surface. As the parasite propels itself forwards, surface-bound
adhesion complexes are released by proteolysis. Other proteins, derived
largely from the rhoptries, are secreted into the host cell where they contrib-
ute to the formation of a parasitophorous vacuole and its associated mem-
brane, and modify the host intracellular environment (reviewed by
Boothroyd and Dubremetz, 2008).

7.1. Parasite surface proteins


In common with many groups of protozoa, the surfaces of Eimeria sporozo-
ites and merozoites are coated with glycosylphosphatidylinositol (GPI)-
anchored proteins that are collectively referred to as surface antigens or SAGs
(Gurnett et al., 1990; Tabarés et al., 2004). In T. gondii and species of Plas-
modium, GPI-anchored proteins are implicated in the early stages of parasite
attachment, prior to apical re-orientation. This requires interaction with
sulphated glycosaminoglycans on the surface of host cells. Preliminary data
indicate that several E. tenella SAGs are able to bind a variety of cultured cells
(F. Tomley and C. Subramaniam, unpublished observations) suggesting that
they too are involved in this initial non-specific binding step.
Examination of EST sequences from E. tenella identified 37 potential
GPI-linked variant SAGs encoded by multi-gene families and differentially
expressed between sporozoites and second-generation merozoites (Tabarés
et al., 2004). GPI-anchored proteins in higher eukaryotes are often found
within membrane structures called lipid rafts, which are detergent-resistant
microdomains involved in signal transduction, membrane trafficking and
molecular sorting. Potential lipid rafts were identified on the surface of
130 H. David Chapman et al.

Eimeria invasive stages by staining for the lipid-raft marker flotillin-1 (del
Cacho et al., 2007). However flotillin-1 was most prominent at the apical
end of sporozoites, whereas E. tenella SAGs are expressed over the entire
sporozoite surface (Tabaré et al., 2004).
Recent transcriptome, proteome and genome data indicate that E. tenella
expresses up to 80 different SAG proteins, and confirms that these are dif-
ferentially regulated during the life cycle such that second-generation mer-
ozoites are coated with more complex mixtures of SAGs than either
sporozoites or first-generation merozoites (Lal et al., 2009; Novaes et al.,
2012; A. Reid, Wellcome Trust Sanger Institute, personal communication;
F. Tomley, unpublished observations). Their surface location suggests that
SAGs may induce potent immune responses that can, for example, block
sporozoite invasion of cultured cells (Brothers et al., 1988). The
co-expression by merozoites of highly polymorphic SAGs could render
anti-SAG immune responses ineffective against these stages. A study of
10 E. tenella merozoite-expressed SAGs showed that three of these induced
an increase in nitric oxide production, IL-1b and IL-10 transcription, and
induced a decrease in IL-12 and interferon-g (IFN-g) transcription in
chicken macrophages (Chow et al., 2011). This indicates that at least a subset
of SAGs has the ability to modulate chicken innate and adaptive immune
responses, which may suppress cell-mediated immunity and also contribute
to the marked pro-inflammatory responses and associated pathology seen
during E. tenella infection.

7.2. MIC proteins are adhesins and many function


as multi-protein complexes
The repertoire and broad functions of coccidian MIC proteins, including
those from Eimeria species, has been reviewed extensively, and the reader
is referred to recent articles for more details (Carruthers and Tomley,
2008; Cowper et al., 2012). Most MICs comprise modular arrangements
of protein domains that share homology with adhesins from higher eukary-
otes and it is the specific binding of these domains to host cell glycans that
establish irreversible apical attachment. Across the Apicomplexa there are
many orthologous MIC proteins although the precise arrangement of
domains is not always conserved and there are some, such as the sialic
acid-binding MAR domains and galactose-binding Apple/PAN domains,
discussed below, that are restricted to the coccidia.
MIC proteins often associate to form multivalent heteromeric com-
plexes, which assemble within the endoplasmic reticulum before being
Review of Coccidiosis Research 131

trafficked to the micronemes. Each MIC complex contains an ‘escorter’ pro-


tein that possesses a transmembrane domain and a short cytoplasmic tail. This
facilitates targeting to the micronemes and allows the microneme complex
to interact with the underlying glideosome ( Jewett and Sibley, 2003). Some
MIC complexes are conserved across different genera, whereas others are
not. The T. gondii complex of TgMIC2/MIC2AP, which is essential for
gliding motility, host cell attachment and invasion (Huynh and
Carruthers, 2006), is orthologous to the E. tenella complex of EtMIC1/
MIC2. The introduction of the E. tenella complex into tachyzoites of
T. gondii can partially complement for loss of endogenous TgMIC2/
M2AP, indicating conservation of function (Huynh et al., 2004). However,
the T. gondii TgMIC1/4/6 complex, in which TgMIC6 is the escorter for
the MAR-domain containing TgMIC1 and Apple-domain containing
TgMIC4, is not replicated in E. tenella. The MAR-domain containing
EtMIC3 (Labbé et al., 2005) has not been found in a complex, but instead
is secreted directly from the micronemes onto the host cell surface (Lai et al.,
2011). The Apple-domain containing EtMIC5 forms a complex with
EtMIC4, which is presumed to act as an escorter, but which also bears
adhesive thrombospondin-like domains that have the potential to bind host
receptors (Periz et al., 2005, 2007).

7.3. Host glycan recognition by MIC proteins contributes


to host and tissue tropism
One of the most intriguing biological questions is why there are such huge
differences in host and tissue tropisms across members of the coccidia.
T. gondii, for example, invades virtually any nucleated cell and infects almost
all warm-blooded vertebrates, whereas each species of Eimeria infects only a
single host, replicates only in epithelial cells, and is often restricted to very
specific regions of the intestine. Recent studies on the binding of coccidian
MIC proteins to host glycans, particularly sialic acid and galactose, are now
shedding light on this issue (Cowper et al., 2012; Lai et al., 2011; Marchant
et al., 2012). Generally, there is a direct correlation between host range and
the possession of a wide repertoire of MIC proteins expressing variant
domains that are capable of binding a broad range of oligosaccharide epi-
topes. MAR domains, which are found in MICs across the coccidia, bind
a range of sialyl groups but evidence from carbohydrate microarrays, atomic
structure, and cell binding studies, reveals that those from T. gondii and
E. tenella are differentially equipped for binding. Thus E. tenella MAR
domains bind a limited range of structures, with a strong in vivo preference
132 H. David Chapman et al.

for a2,3-linked sialic acid and an absence of binding to any N-glycolated


sialyl structures (which are not found in the chicken). In contrast, MAR
domains from T. gondii MICs are more divergent, and bind a variety of oli-
gosaccharides including a2,9-linked sialic acid and N-glycolylated deriva-
tives (Lai et al., 2011). Very recent studies on carbohydrate recognition
by Apple domains, also found in MICs across the coccidia, indicates an addi-
tional contribution to host range and tissue tropism conferred by differential
recognition of galactose, another sugar that is widely distributed in animal
tissues, commonly forming b1,3 or b1,4 linkages to a preceding glucose
or galactose. While both T. gondii and E. tenella Apple domains bind gal-
actosylated structures, there is a marked preference by T. gondii for
Galb1,3GalNAc, commonly found on gangliosides which are prevalent
on many host cell surfaces (Marchant et al., 2012). The precise binding pref-
erences of E. tenella Apple domains have not yet been elucidated, but pre-
liminary data indicate that these bind predominantly to b1,4-linked
galactose and do not recognize the more common b1,3 linkages (Cowper
et al., 2012).

7.4. Regulated secretion of microneme and rhoptry organelles


MIC proteins are discharged onto the parasite surface at an early stage in
invasion. The physiological trigger that induces microneme secretion is
not known, but treating parasites with agents that cause a rise in intracellular
free calcium induces rapid secretion and this can be inhibited by treatment
with intracellular calcium chelating agents (Bumstead and Tomley, 2000;
Carruthers and Sibley, 1999; Wiersma et al., 2004). Blocking calcium release
channels by treating parasites with IP3 inhibitors or ryanodine, or blocking
activity of cyclic GMP-dependent kinase or calcium-dependent protein
kinase, all interrupt the regulated exocytosis of MICs and prevent parasite
attachment and invasion of host cells (Dunn et al., 1996; Lourido et al.,
2010; Schubert et al., 2005; Wiersma et al., 2004).
ROP proteins are also secreted in a regulated manner and, while the
exact mechanisms are unknown, it is hypothesized that the initial signal
comes via the cytoplasmic tails of membrane-bound MIC complexes, fol-
lowing their interaction with a host cell receptor. The process is complicated
because rhoptry secretion occurs in two separate ‘waves’. Proteins that reside
within the anterior neck portion of the rhoptry (RONs) are secreted early in
invasion and are critical for the formation and maintenance of the MJ
(Alexander et al., 2005; Lebrun et al., 2005). However, proteins from the
Review of Coccidiosis Research 133

posterior bulb of the rhoptry (ROPs) are secreted slightly later, once the par-
asitophorous vacuole is formed. In T. gondii, several ROP proteins are
known to be potent virulence factors that modify and subvert host cell sig-
nalling pathways (Bradley and Sibley, 2007). The current model for differ-
ential secretion of RONs and ROPs in T. gondii is that a membrane-bound
MIC complex containing the escorter protein TgMIC8 triggers release of
the RONs (Kessler et al., 2008). Interaction of RONs with AMA1 then
triggers the release of ROPs (Tyler and Boothroyd, 2011). Regulation of
secretion of ROP/RON proteins in Eimeria, where there is no defined
orthologue of TgMIC8, has not been determined.

7.5. AMAs and formation of the MJ


The MJ was visualized many years ago (Aikawa, 1978) and is a key structure
that provides an anchor, against which the parasite can generate a force that
allows forward movement into the parasitophorous vacuole. A key compo-
nent is AMA1, a transmembrane protein secreted by the micronemes onto
the parasite surface, where it interacts with secreted RONs to initiate for-
mation of the MJ (Alexander et al., 2005; Lebrun et al., 2005). Collaboration
between proteins that are secreted from different sub-cellular organelles
requires a remarkable degree of orchestration. It has emerged recently that
while AMA1 remains anchored on the parasite surface, a RON complex is
secreted into the host cell and then one of them, RON2, becomes inserted
into the host cell membrane and interacts directly with AMA1 (Tyler and
Boothroyd, 2011). Both AMA1 and the RON protein repertoire are con-
served in Eimeria indicating that the mechanism for forming the MJ is likely
to be conserved in different coccidia (Blake et al., 2011a,b; Jiang et al., 2012;
Lal et al., 2009; Oakes et al., 2013). It is worthy of note, however, that
E. tenella expresses stage-specific variants of AMA1 and several of the
RON proteins, which suggests that each zoite stage assembles a different
set of gene products with which to build the MJ (Lal et al., 2009; Oakes
et al., 2013).

8. IMMUNOBIOLOGY
It is 50 years since Elaine Rose and Peter Long published, ‘Immunity
to four species of Eimeria in fowls’, sparking a seminal year in research into
the immunology of poultry coccidiosis. By the end of 1962, it was known
that: (a) even a single infection with various species of Eimeria confers solid
134 H. David Chapman et al.

resistance to reinfection and this is increased further after a second infection


(Rose and Long, 1962); (b) immunity is expressed against the very early
asexual stages of infection such that, although penetration of epithelial cells
by sporozoites may occur, subsequent development is blocked (Pierce et al.,
1962); (c) immunity is not confined to early stages of parasite development
but also affects later stage merozoites and sexual stages (Rose, 1963); (d) in
general, immunity to one species confers no protection against other species
(Rose and Long, 1962), though it was shown subsequently that some degree
of cross-protection can occur between closely related pairs of Eimeria, such as
E. maxima and E. brunetti (Rose, 1967a), and E. tenella and E. necatrix (Rose,
1967b) and (e) B cells and the antibodies they produce play little, if any, role
in resistance to reinfection, implicating cell-mediated responses in acquired
immunity (Long and Pierce, 1963). However, in reviewing this remarkable
year, Rose (1963) noted that, ‘The way in which an animal which has expe-
rienced an infection with a species of Eimeria subsequently prevents the
development of that species within its body is not yet understood’. That
statement is equally valid today.
Immunity to Eimeria is complex, multifactorial and influenced by host
and parasite, with different elements playing greater or lesser roles in three
different types or stages of immunity: innate resistance to primary infection;
acquired immunity to reinfection and maternal immunity. Many host/
parasite combinations have been used to dissect the immunobiology of coc-
cidiosis, with significant insights being gained through the use of murine
models due to advantages connected with the availability of murine immu-
nological reagents, in-depth fundamental understanding of the murine
immune system and technologies to disrupt immune response genes in mice.
In this review, key similarities rather than differences in the immunobiology
of coccidial infections will be emphasized.

8.1. Innate responses to primary infection


Primary infections with the majority of Eimeria species, in poultry and
rodents, are self-limiting; asexual reproduction proceeds via a pre-set num-
ber of cycles of schizogony prior to differentiation into gametocytes, subse-
quent sexual reproduction and production of oocysts. This can make it
challenging to demonstrate a role for the immune system in resistance to pri-
mary infection. Nevertheless, it has been shown that increased oocyst excre-
tion, by different Eimeria species, is a consistent feature of primary infection
in immunodeficient hosts (Klesius and Hinds, 1979; Long and Rose, 1970;
Review of Coccidiosis Research 135

Mesfin and Bellamy, 1979; Rose, 1970; Rose and Hesketh, 1979, 1986;
Rose and Long, 1970; Schito and Barta, 1997; Schito et al., 1996;
Stockdale et al., 1985). Moreover, there is one parasite and host pairing –
E. vermiformis in the mouse – where the patent period of primary infection
is clearly increased in susceptible mice, almost certainly due to a relaxation of
immune pressure on the parasite that allows additional generations of schiz-
onts to develop (Rose and Millard, 1985; Rose et al., 1984, 1985; Schito
et al., 1996). Thus, the E. vermiformis murine model of coccidiosis has proved
particularly significant for our understanding of immunological resistance to
primary infection.
Studies with E. vermiformis have demonstrated that resistance to primary
infection is associated with more rapid inflammatory responses including
increased granulocyte numbers (Ovington et al., 1990), enhanced generation
of free oxygen radicals (Ovington et al., 1990), increased Natural Killer (NK)
cell activity (Smith et al., 1994a), earlier production of pro-inflammatory
cytokines such as IFN-g, tumour necrosis factor (TNF) and others
(Ovington et al., 1995; Wakelin et al., 1993), and faster T cell responses
(Rose et al., 1990; Wakelin et al., 1993). Corollaries of these observations
also exist for other rodent (Dkhil et al., 2011; Rausch et al., 2010; Rose
and Hesketh, 1982; Rose et al., 1979a; Schito and Barta, 1997), chicken
(Hong et al., 2006a,b; Kim et al., 2008, 2010; Rose et al., 1979a;
Rothwell et al., 1995, 2000, 2004; Yun et al., 2000) and turkey (Gadde
et al., 2011) coccidioses. However, of all these immunological parameters,
only two – lymphocytes and IFN-g – appear to be indispensible for resistance.
Severe combined immunodeficient mice, which are deficient in both
T and B cells, are highly susceptible to primary infection with
E. vermiformis, producing many more oocysts and harbouring parasites for
far longer than immunocompetent mice (Schito et al., 1996). Studies in
B cell-deficient mice (Smith and Hayday, 1998) and bursectomized chickens
(Long and Pierce, 1963; Rose and Hesketh, 1979), suggest that B cells play a
minor, though consistent, role in this resistance. Since deficiencies in antigen
presentation also increase susceptibility (Smith and Hayday, 1998, 2000),
this relatively minor role may be via the ability of B cells to act as antigen
presenting cells rather than anything to do with antibodies. Experiments
with congenitally athymic (nude) mice, however, show that T cells play a
critical role in resistance to primary infections with E. vermiformis; infected
nude mice excrete many more oocysts over a much extended patent period
(Rose et al., 1984, 1985). Other murine Eimeria (Klesius and Hinds, 1979;
Mesfin and Bellamy, 1979; Rose and Hesketh, 1986; Stockdale et al., 1985),
136 H. David Chapman et al.

as well as E. nieschulzi in rats (Rose et al., 1979b), also produce many more
oocysts in athymic animals but without any affect on patency. Experiments
with thymectomized chickens have generated inconsistent data, probably
because of the difficulty in completely removing all T cells (Rose and
Long, 1970).
Depletion of specific T cell subsets, either via antibodies with appropri-
ate, rigorous, confirmatory adoptive transfer experiments (Rose et al., 1988,
1992), or via the deletion of specific genes from mice (Roberts et al., 1996;
Smith and Hayday, 1998, 2000), show that CD4 þ, and not CD8þ, T cells
are the critical T cell subset mediating resistance to primary infection with
E. vermiformis. Moreover, protective effects appear to be ab T cell-specific
(Roberts et al., 1996; Smith and Hayday, 1998), though gd T cells may play
an important role in preventing immunopathology (Roberts et al., 1996)
rather than in contributing to the control of the parasite (Roberts et al.,
1996; Rose et al., 1996). Immune CD8 þ mesenteric lymph node cells have
also been shown to be capable of suppressing immunopathology in
E. falciformis infections (Pogonka et al., 2010). However, similar effects
are not seen in infections with Eimeria papillata (Schito et al., 1998) and
depletion studies in chickens infected with E. acervulina or E. tenella are
somewhat equivocal, possibly confounded by relatively small numbers of
experimental chickens in each treatment group and by inefficient depletion
of CD4þ T cells (Trout and Lillehoj, 1996).
The most important role for CD4þ T cells in mediating resistance to
primary infection with E. vermiformis is most likely as the source of IFN-
g. Mice treated with an antibody to IFN-g (Rose et al., 1991a) or IFN-g
gene-knockout mice (Smith and Hayday, 2000) are highly susceptible to
E. vermiformis, suffering prolonged patency, high levels of excretion of
oocysts and increased mortality. This is not so evident in infections of
IFN-g knockout mice with E. papillata where patency is not affected; in
this case, NK cells are the likely source of IFN-g (Schito and Barta,
1997). How IFN-g is controlling the parasite is not known; generation of
free oxygen radicals (Ovington et al., 1995), reactive nitrogen intermediates
(Ovington et al., 1995; Smith and Hayday, 2000) and interference with
tryptophan metabolism (Schmid et al., 2012) can all be ruled out. However,
it is known that the effects of IFN-g are mediated via the host cell rather than
a direct effect on parasites (Rose et al., 1991b).
Infections with E. pragensis or E. falciformis indicate an additional role for
IFN-g in the immunobiology of coccidiosis. Depletion of IFN-g using
Review of Coccidiosis Research 137

monoclonal antibodies has little apparent effect on parasite load but has a
significant affect on weight loss during primary and secondary infection with
E. pragensis (Rose et al., 1991a). Similarly, IFN-g receptor knockout mice
infected with E. falciformis suffer severe intestinal immunopathology and
weight loss mediated via Th17 pathways, involving the cytokines, IL17
and IL-22 (Stange et al., 2012). Thus, IFN-g may have an important immu-
noregulatory role in response to infection with Eimeria, helping to keep
intestinal inflammation in check.
Mice deficient in granulocyte and NK cell function are more susceptible
to primary infection with E. vermiformis (Rose et al., 1984). However, T cell-
mediated control of infection with E. vermiformis does not require
co-operation with granulocytes (Rose et al., 1989) and experiments with
E. papillata indicate that increased susceptibility to primary infection may
be due more to participation of NK cells than granulocytes in resistance
(Schito and Barta, 1997). However, the effects on oocyst excretion and pat-
ent period are relatively modest compared to those of lymphocyte deficiency
(Schito et al., 1996) and a role for NK cells in innate resistance is not
supported by results obtained with E. vermiformis (Rose et al., 1995;
Smith et al., 1994a).
Free oxygen radicals appear to play no role in resistance to E. vermiformis
since quenching of their activity in vivo actually leads to reduced, not
enhanced, oocyst activity (Ovington et al., 1995). Moreover, treatment
of mice with agents designed to enhance macrophage activity, including free
oxygen radical generation, leads to increased oocyst excretion (Smith and
Ovington, 1996), as does treatment with TNF (Ovington et al., 1995). Sim-
ilarly, reactive nitrogen intermediates, despite their temporal association
with resistance to poultry coccidia (Allen and Fetterer, 2002), also enhance
oocyst production in E. vermiformis (Ovington et al., 1995). These are, at first
glance, puzzling results in light of the well-established anti-protozoal effects
of TNF, free oxygen radicals and reactive nitrogen intermediates (see
Ovington and Smith, 1992). However, with our current knowledge about
involvement of an oxidative reaction in oocyst wall assembly (Belli et al.,
2006; Mai et al., 2009, 2011) it makes some sense, indicating that perhaps
Eimeria actually subjugates the host’s oxidative burst to assist it in construc-
tion of its oocysts. Intriguingly, a related proposal has been put forward
recently – it appears that E. falciformis also subverts IFN-g-induced
indoleamine 2,3-dioxygenase activity to help drive microgamete develop-
ment (Schmid et al., 2012).
138 H. David Chapman et al.

8.2. Acquired immunity


Acquired immunity to Eimeria is even more enigmatic than innate resistance
to primary infections. All that can be said with any certainty is that immunity
to reinfection with Eimeria is remarkably effective and is T cell dependent
(this has been realized for more than 30 years; Rose et al., 1979b), and
that B cells (and, therefore, antibodies) are not involved in acquired immu-
nity since bursectomized birds (Rose and Hesketh, 1979) and mice lacking
B cells (Rose et al., 1984; Smith and Hayday, 1998) are perfectly capable of
developing immunity to reinfection. It has proven almost impossible to
correlate any immune parameter with immunity to reinfection because
the expression of that immunity in experimental settings, at least, is so rapid
and efficient. However, studies using gene-knockout mice have proved
extremely useful in determining which factors may play a role. Thus,
as for primary infection, CD4þ ab T cells are crucial for immunity to
reinfection with E. vermiformis (Roberts et al., 1996; Smith and Hayday,
1998, 2000). Similar, though less definitive data were also obtained for
secondary infections with E. papillata (Schito et al., 1998). However, in con-
trast to primary infection, IFN-g plays no role in this acquired immunity
(Rose et al., 1991a; Schito and Barta, 1997; Smith and Hayday, 2000).
Contrarily, some studies demonstrate that CD8 þ T cells can be used
to transfer immunity (e.g. to E. falciformis; Pogonka et al., 2010) or that
depletion of CD8þ T cells can increase, very slightly, susceptibility to
E. vermiformis (Rose et al., 1992). Evidence from poultry experiments
(Trout and Lillehoj, 1996) is more difficult to interpret because experiments
showing an increase in oocyst excretion in secondary infection of birds
depleted of CD8 þ T cells did not include a concomitant primary infection
control, making it hard to assess how significant the increased oocyst pro-
duction really was. More, and more sophisticated, analyses of acquired
immunity to Eimeria are required to resolve the mechanism(s) that are
operating.

8.3. Maternal immunity


The immune system of young animals is ‘uneducated’ rendering them more
susceptible to infectious disease. Protection against infection during this vul-
nerable period is provided via transfer of antibodies from mother to young.
In chickens, this occurs via the egg yolk; indeed, the ability of hens to trans-
fer remarkable quantities of IgY (¼IgG) antibodies to their hatchlings has
long been appreciated, including in regard to the transfer of antibodies that
Review of Coccidiosis Research 139

protect chicks from infection with E. tenella (Rose and Long, 1971) or
E. maxima (Rose, 1972). In many of the progeny from hens deliberately
infected with high doses of E. maxima, this maternal immunity can be abso-
lute (i.e. result in the complete absence of oocysts in the faeces of chicks), at
least during the first week post-hatching (Smith et al., 1994b). Maternal anti-
body levels (in egg yolk or chicks) are correlated with protection (Smith
et al., 1994b). Moreover, maternal immunity induced by E. maxima confers
partial protection against E. tenella, possibly via cross-recognition of con-
served proteins (or, at least, epitopes) in different Eimeria species (Smith
et al., 1994c), an idea lent further credibility by the ability of maternal immu-
nization with conserved macrogametocyte proteins to protect hatchlings
against multiple species of Eimeria (Wallach et al., 1995, 2008).
The effectiveness of maternal, antibody-mediated immunity to Eimeria
appears contradictory to the body of evidence, reviewed above, indicating
that antibodies play only a minor role in resistance to Eimeria. However,
there is actually no shortage of (often overlooked) evidence, dating back
over 40 years, showing that antibodies can protect against infection with
Eimeria. Thus, for example, sera taken 2 weeks after infection with
E. maxima can be transferred to naı̈ve birds and, as for maternal immunity,
protect some of them almost completely against infection (Rose, 1972,
1974). The protection conferred by these convalescent sera was later dem-
onstrated to be correlated tightly with levels of parasite-specific IgG
(Wallach et al., 1994). Additionally, it was demonstrated 40 years ago
(Rose, 1972) and, again, more recently (Lee et al., 2009a,b) that Eimeria-
specific antibodies purified from egg yolks of immunized hens can be used
to transfer passive immunity against several species of poultry coccidia; the
protection can be achieved via injection or oral delivery of the antibodies.
Immune sera can even partially protect highly susceptible T cell-deficient
animals (Rose and Hesketh, 1979). Thus, antibodies certainly can protect
against Eimeria but the effect must be described as variable – from absolute
to negligible even if similar immunization regimens are used (Wallach et al.,
1994). Why this is so is anything but clear. Maternal immunization,
however, does appear to be a phenomenon that can be harnessed to control
poultry coccidiosis (Smith et al., 1994b; Wallach et al., 2008).

8.4. Immunological research


Lamentably, in the decade since the retirement of Elaine Rose, research
into the immunobiology of coccidiosis has declined significantly
140 H. David Chapman et al.

(notwithstanding the efforts of researchers at the United States Department


of Agriculture to understand the role of various feed additives and Eimeria
profilin in boosting immune responses to Eimeria; reviewed by Lillehoj
and Lee, 2012). This has been exacerbated by the declines, even disappear-
ance, of the coccidiosis research programmes at the UK’s Institute for Ani-
mal Health and at the University of Technology, Sydney in Australia, as well
as a distinct lack of new commercial research into vaccines. It is doubly
lamentable because immunity to Eimeria, whether innate, acquired or
maternal, is remarkable among all parasites in its effectiveness. Moreover,
coccidiosis appears to be an excellent model to study the molecular basis
of gut immunopathology. The gut epithelium is the first point of contact
with the host for many pathogens but studies of this interaction have proven
challenging experimentally, with many models employing delivery of path-
ogens via subcutaneous, intravenous or intraperitoneal injection. Infection
with poultry or murine Eimeria, being largely confined to the intestine is,
therefore, an exceptional model for genuine study of gut immunology.
Hopefully, recent promising and innovative insights into unravelling the
complexity of the host/Eimeria inter-relationship (Blake et al., 2011a,b;
Stange et al., 2012) will be followed up and exploited fully.

9. DIAGNOSIS AND IDENTIFICATION


9.1. Traditional methods
Diagnosis of coccidiosis in poultry flocks continues to rely on necropsy and
the examination of birds for intestinal lesions in different areas of the gut.
Because of the site-specificity of invasion, the presence of lesions can provide
insight into which species of coccidia is/are responsible for clinical symp-
toms. Diagnosis may be corroborated by microscopic analysis of shape
and size of Eimeria oocysts shed in faeces from infected birds. Additional
criteria classically used to characterize Eimeria species include pre-patent
period, minimum sporulation time, tissue location of parasitic forms and
immunological specificity. However, definitive identification of a particular
Eimeria species based on morphological and pathological criteria can be
tedious, requires highly qualified personnel and may be confounded by
the overlapping features observed in different Eimeria species (Long and
Joyner, 1984).
While lesion site and aspect, and oocysts size and shape, are features often
sufficient to corroborate clinical signs of coccidiosis, there are instances
when knowing precisely which Eimeria species is/are present would be
Review of Coccidiosis Research 141

helpful in managing the disease. For example, a preponderance of


E. acervulina in a litter sample might indicate increased drug resistance in this
species. This information would be useful in choosing alternative strategies,
such as switching to another anticoccidial compound known to be effective
against E. acervulina, or to a live Eimeria oocyst vaccine. If, on the other hand,
E. mitis or E. brunetti were present, then using a vaccine that contains only
E. acervulina, E. maxima and E. tenella would not be a particularly useful con-
trol strategy.

9.2. Early molecular methods


9.2.1 Starch gel electrophoresis
In the 1970s, a biochemical approach to identification of Eimeria spp. was
developed that involved starch gel enzyme electrophoresis of enzymes, such
as lactate dehydrogenase and glucose phosphate isomerase obtained from
oocyst homogenates (Rollinson, 1975; Shirley, 1975; Shirley and
Rollinson, 1979). The technique was employed to examine field isolates
of E. tenella obtained from around the world and was used to distinguish
mixtures of at least three species when present in one sample (Chapman,
1982; Shirley et al., 1989). The former study showed for the first time that
E. praecox, a species difficult to identify because of the absence of diagnostic
lesions, had a high incidence (74% of samples) in broiler flocks. While an
interesting laboratory tool for investigation of phenomena such as intraspe-
cific variation, starch gel electrophoresis is a time-consuming technique that
requires large numbers of oocysts. In addition, protein variability is limited
by evolutionary constraints, thus limiting the observed phenotypic poly-
morphism. Due to these limitations, this technique has been superseded
by DNA-based methods for identification of Eimeria spp. in the field.

9.2.2 DNA hybridization


DNA hybridization was the first DNA-based technique proposed for the
molecular discrimination of Eimeria parasites (Shirley, 1994b). A typical pro-
tocol consisted of genomic DNA digestion with different restriction
enzymes, separation through agarose gel electrophoresis, blotting and
hybridization with DNA probes composed of repetitive regions. The final
result was a DNA fingerprinting comprising multiple band profiles. Similarly
to enzyme variation detection, this approach also required large numbers of
parasites and was highly time demanding. Also, the method was inherently
unable to deal with mixed samples, since overlapping band profiles are not
informative.
142 H. David Chapman et al.

9.3. Methods based on DNA amplification by PCR


Molecular techniques, primarily the PCR, have been developed to over-
come the limitations of morphological examination and the aforementioned
molecular techniques. Since primers can be designed to specifically amplify
DNA of any single Eimeria species, samples containing multiple species can
be properly diagnosed. Also, the high level of amplification permits the use
of low numbers of oocysts. An excellent review of early efforts to develop
PCR assays for Eimeria is available (Morris and Gasser, 2006); here we pro-
vide an update of work in this area.

9.3.1 RAPD fingerprinting


The first PCR-based diagnostic assays developed for Eimeria relied on the
use of RAPD. This technique is based on DNA amplification using arbitrary
primers under low stringency. Under this condition, primers can anneal in
multiple sites of the target genomic DNA, thus producing DNA fingerprints
that permit differentiation of polymorphic populations. Because no previous
knowledge of the nucleotide sequence is required, RAPD was employed
largely for parasite discrimination at a time when very few genomic
sequences were available. Several groups succeeded in developing RAPD
assays for species and strain differentiation of poultry Eimeria (MacPherson
and Gajadhar, 1993; Procunier et al., 1993; Shirley and Bumstead, 1994).
However, given the low stringency of the reaction, RAPD typically suffered
from a low reproducibility, especially among different laboratories, and was
superseded by more reliable and specific PCR assays. Two main approaches
have been employed by different groups to develop such specific assays:
(1) the use of ITS1 and ITS2 and (2) the conversion of anonymous RAPD
markers into Sequence-Characterized Amplified Region (SCAR) markers.

9.3.2 PCR assays directed to specific targets


A specific PCR assay directed to E. tenella 5S rDNA was reported over
20 years ago (Stucki et al., 1993). This pioneer work was followed by assays
capable of detecting and differentiating the seven Eimeria species of domestic
fowl using ITS1 (Schnitzler et al., 1998, 1999) and ITS2 rDNA (Gasser et al.,
2005) as targets. ITS1 and ITS2 are intervening sequences that are post-
transcriptionally excised from the rRNA precursor. Unlike 18S, 5.8S and
28S rRNAs, ITS1 and ITS2 are not subjected to an appreciable selective
pressure, and have undergone sufficient divergence among Eimeria species
to allow design of species-specific primers (Lew et al., 2003). A number
Review of Coccidiosis Research 143

of assays based on amplification of ITS1 and ITS2 rDNA have been devel-
oped, and used to determine the species of Eimeria present in poultry litter
(Hamidinejat et al., 2010; Haug et al., 2007, 2008; Jenkins et al., 2006a; Lew
et al., 2003). As a word of caution in using ITS sequences, Lew et al. (2003)
found sufficient variation on ITS1 of different E. maxima isolates to require
the design and use of two distinct sets of specific primers for this species.
Combined with rapid techniques for extracting high-quality DNA from
oocysts, ITS1-specific PCR was found to provide a more accurate picture
of Eimeria distribution at poultry farms than traditional morphometric anal-
ysis (Hamidinejat et al., 2010; Haug et al., 2008).
Primers directed to conserved ribosomal DNA sequences (18S, 5.8S,
28S), flanking either the ITS1 or ITS2 regions, have also been used in com-
bination with denaturing polyacrylamide gel electrophoresis or capillary
electrophoresis (CE) for species discrimination (Cantacessi et al., 2008;
Gasser et al., 2005; Morris et al., 2007a,b). The latter method relies upon
identifying species-specific peaks in CE chromatograms that have been
established using pure cultures (Gasser et al., 2005). Using this approach,
one group has identified a genetic variant of E. maxima, and new operational
taxonomic units in oocysts isolated from poultry operations (Cantacessi
et al., 2008; Morris et al., 2007b). By employing primers directed to con-
served regions (28S, 5.8S) flanking the ITS1 sequence, these authors iden-
tified genetic variants that would have gone unnoticed using ITS1-specific
primers. Although assays based on ITS1 and ITS2 are highly sensitive due to
the large number of rDNA repeats (Vrba et al., 2010), variation in the
sequence may prevent primer binding. Nevertheless, ITS1 is still being used
as a target for the development of diagnostic assays for Eimeria parasites of
other hosts, including 4 species pathogenic for turkeys (Cook et al.,
2010) and 11 species that infect the domestic rabbit (Oliveira et al., 2011).
As an alternative to ITS1 and ITS2, Fernandez et al. (2003a) used RAPD
to develop SCAR markers for each Eimeria species of domestic fowl. In
developing this assay, the DNA sequences of individual RAPD markers
were determined and used to design longer primers, which were then tested
under highly stringent conditions for species-specific amplification of
Eimeria DNA. By combining a set of seven SCAR markers, Fernandez
et al. (2003b) developed a multiplex PCR assay that permits the simulta-
neous discrimination of all Eimeria species infecting chickens in a single-tube
reaction (Fig. 2.5). An Eimeria SCAR database containing 151 SCARs is
publicly available on the web (Fernandez et al., 2004; http://www.
coccidia.icb.usp.br/eimeriaScardb), and SCAR markers have been used
144 H. David Chapman et al.

Figure 2.5 Agarose gel electrophoresis of multiplex PCR products using DNA samples
of E. acervulina (lane 1), E. brunetti (lane 2), E. tenella (lane 3), E. mitis (lane 4), E. praecox
(lane 5), E. maxima (lane 6), E. necatrix (lane 7), a mixture of the seven Eimeria species
(lane 8) and a control with no starting DNA (lane 9). Molecular size markers (lane M) in
base pairs are indicated on the left Reproduced from Fernandez et al. (2003b) with per-
mission from Cambridge University Press.

by other groups to determine the Eimeria species composition on poultry


farms in different regions of the world (Carvalho et al., 2011a,b;
Ogedengbe et al., 2011b), and for the development of quantitative PCR
assays (Blake et al., 2008; Vrba et al., 2010). The advantage of SCAR marker
technology, unlike assays based on ITS1- and ITS2-PCR, is that highly con-
served SCAR marker sequences are available as targets of amplification
(Blake et al., 2008; Fernandez et al., 2004; Vrba et al., 2010). This avoids
the problem of false negative reactions due to poor annealing of primer
to target DNA because of variation in the target sequence. The drawback
to SCAR technology is that it may be less sensitive than assays based on
ITS1 and ITS2, which are found in multiple copies in the Eimeria genome
(Vrba et al., 2010).
Finally, strain differentiation still lacks the variety of molecular markers
already available for other apicomplexan parasites. In this direction, it is
worth mentioning that species-specific sets of microsatellite markers for
E. acervulina, E. maxima and E. tenella have been developed in Brazil
(A. Gruber and S. Fernandez, unpublished observations). This work has
been deposited as a patent (Espacenet, patent BRPI0702051). These
Review of Coccidiosis Research 145

microsatellite markers allowed for the differentiation of both field samples


and commercial vaccines lines.
Despite the enormous impact of PCR-based methods to detect and dis-
criminate Eimeria species, some drawbacks still persist. Oocysts are the most
accessible stage of the life cycle, and the obvious choice as a source of Eimeria
DNA. Since the oocyst wall is remarkably resistant to chemical agents,
mechanical disruption with glass beads is the most common method to
extract DNA. However, DNA yield does not correlate linearly with the
number of oocysts (Fernandez et al., 2003b), due to decreased efficacy of
mechanical disruption in low-concentration suspensions. Despite some
authors having proposed alternative chemical treatments to disrupt the
oocyst wall (Haug et al., 2007; Zhao et al., 2001), this step remains the most
important limiting factor for good sensitivity. Beside faeces, litter is another
important source of Eimeria samples; however, a drawback of utilizing poul-
try litter as a source of Eimeria DNA is the presence of PCR inhibitors.
Although different extraction techniques have been developed to recover
DNA from oocysts (Carvalho et al., 2011a; Haug et al., 2007; Zhao
et al., 2001), the adequate removal of inhibitory substances from litter is
often difficult. With the goal of controlling for false negative reactions
due to PCR inhibition, a technique has been developed based on
co-amplification of ITS1 sequences and an Eimeria species-specific internal
standard ( Jenkins et al., 2006b). Using gel electrophoresis, the target and
internal standard PCR products can easily be distinguished from each other
by acrylamide gel electrophoresis (Fig. 2.6). In the event of inhibition, a sec-
ond extraction of DNA is undertaken with the goal of removing inhibitors.

9.3.3 Quantitative PCR assays


Early assays using ITS1, ITS2 or SCAR markers relied on qualitative assays
in which identification of amplification products has been obtained by aga-
rose or polyacrylamide gel electrophoresis. Over the past 5 years, a number
of assays using quantitative real-time PCR (qPCR) amplification of ITS1,
ITS2 or SCAR markers have been developed. Blake et al. (2008) utilized
primers that were specific for SCAR markers of E. acervulina, E. necatrix
and E. tenella, and for a mic gene of E. maxima. The authors reported a sen-
sitivity varying from 1 to 10 genomes. Another group utilized ITS1 targets
to amplify E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella DNA
isolated from pure cultures and field samples, and achieved an assay sensitiv-
ity that was between 10 and 100 oocysts (Kawahara et al., 2008). A qPCR
assay using primers conserved among various protozoa and melting curve
146 H. David Chapman et al.

Figure 2.6 Detection of Eimeria species oocysts using ITS1-PCR and internal standard (IS).
Ea, E. acervulina; Eb, E. brunetti; Ema, E. maxima; Emi, E. mitis; En, E. necatrix; Et, E. tenella. kbp,
fX174 HaeIII DNA standards. *Target band for each species of Eimeria. Reproduced from
Jenkins et al. (2006a,b) with permission from the American Association of Avian Pathologists.

analysis could detect E. acervulina in a mixture of oocysts (Lalonde and


Gajadhar, 2011).
Multiplexing real-time PCR, using one of four different upstream
primers conserved between two species, and a conserved downstream
primer, in combination with species-specific TaqMan probes, was used to
analyze all Eimeria species in poultry litter (Morgan et al., 2009).
A greater number of species were identified than those revealed by micros-
copy. Another group reported the development of FAM-labelled TaqMan
species-specific probes, targeted to microneme 1 gene of E. maxima and
SCAR markers of the remaining species (Vrba et al., 2010). An assay sensi-
tivity of a single sporulated oocyst has been claimed. In a study using only
E. acervulina, qPCR directed to SCAR markers was applied to DNA
extracted from oocysts obtained from cloacal swabs, and compared with
oocyst counts from individual faecal droppings (Velkers et al., 2010). The
authors concluded that qPCR of cloacal swabs might be useful for determin-
ing the prevalence and identity of Eimeria oocysts in litter. A similar
approach using high-resolution melting curve analysis and qPCR directed
to ITS1 sequences was found capable of identifying all seven Eimeria species
in pure oocysts cultures (Kirkpatrick et al., 2009).

9.4. LAMP
While qPCR is superior to conventional PCR in that it eliminates the need
for gel electrophoresis and provides quantitative results, samples must be run
Review of Coccidiosis Research 147

on a fairly expensive real-time apparatus. In fact, the complexity of DNA


extraction from the oocyst, associated with the need for expensive
thermocycling and electrophoresis equipment, severely limit the use of
molecular assays in poultry farms. To overcome these limitations, an
Eimeria-specific PCR-based technique that utilizes loop-mediated isother-
mal amplification (LAMP) technology has been developed (Barkway et al.,
2011). Since the enzyme is isothermal, the reaction can be performed in a
simple heat block or water bath, without the need for thermocyclers. Also,
detection can be made with intercalating dyes using the naked eye for obser-
vation (Fig. 2.7), thus eliminating the requirement for electrophoresis.
Finally, instead of using oocysts, the proposed protocol employs mucosal tis-
sues collected post-mortem as samples, and DNA extraction by a simple
boiling method. Altogether, the method addresses the several limitations

Figure 2.7 Loop-mediated isothermal amplification (LAMP) specific for E. tenella.


Application to a purified genomic DNA dilution series revealed a limit of detection
of between 1 and 10 E. tenella genomes using agarose gel electrophoresis (A) or
hydroxynaphthol blue as a visual indicator (B, blue: positive, pink: negative). ve,
no template negative control.
148 H. David Chapman et al.

of conventional molecular assays and may become a mainstream cost-


effective tool for the diagnosis of Eimeria infection in poultry flocks.

9.5. Morphological diagnosis revisited


One of the key features of morphological diagnosis based on oocyst shape
and size is the inherent subjectivity of the method and the requirement of
skilled personnel. An early attempt to differentiate species of Eimeria in
the fowl utilized a computerized image-analysis system (Kucera and
Reznický, 1991). The method used two measurements, length and width
of oocysts, which restricted the ability to differentiate all seven species
due to the overlap of these characters. To address these limitations,
Castañón et al. (2007) have reported the development of COCCIMORPH,
a system that implements a framework for feature extraction, shape charac-
terization and automated classification of chicken Eimeria oocyst images.
The system employs a classifier trained with thousands of oocyst images
of all species of chicken Eimeria. COCCIMORPH provides a public web
frontend (www.coccidia.icb.usp.br/coccimorph) that permits users to
upload oocyst images and obtain a reliable diagnosis in real-time. The
Bayesian classifier showed an overall correct species assignment of 85.7%,
with individual rates varying from 74.9% for E. necatrix to 99.2% for
E. maxima. While this system still has many limitations to be widely used
as a mainstream diagnostic system, it represents a proof of principle that mor-
phology may have gained a revival in the era of digital image processing and
pattern recognition methods. COCCIMORPH breaks a classical paradigm,
as it does not require sample transportation to a reference laboratory, and
photomicrographs sent through the Internet are sufficient to obtain species
diagnosis. While not competing with the accuracy, sensitivity and quantita-
tive nature of modern PCR-based methods, morphological diagnosis based
on digital image processing might represent a near-zero cost alternative to be
used by technicians in poultry farms that do not harbour expensive molec-
ular biology facilities.

9.6. Conclusions
Field diagnosis of Eimeria infection in poultry will continue to rely on the
identification of intestinal lesions and microscopic examination of faecal
droppings and litter for Eimeria oocysts. However, several molecular assays
that can detect and differentiate all seven Eimeria species of the chicken are
now available, and are being used either in a research setting to study the
Review of Coccidiosis Research 149

epidemiology of avian coccidiosis, or by private companies to monitor the


purity of vaccine lines. Information gleaned from molecular assays can assist
in managing disease by allowing informed decisions on which anticoccidial
compounds or live oocyst vaccines should be used in particular poultry
farms. The development of real-time qPCR represents a step forward
towards quantitative diagnosis of Eimeria. Also, isothermal amplification
assays with colorimetric detection (e.g. LAMP), that avoids a requirement
of expensive equipment, and the development of more robust image
processing software, may provide low-cost alternatives for species diagnosis
in poultry farms.

10. CONTROL
10.1. Chemotherapy
A truly landmark contribution to poultry science was the demonstration, in
1948, that it was possible to control coccidiosis by the continuous inclusion
of an anticoccidial drug (sulphaquinoxaline) in the feed of chickens
(Grumbles et al., 1948; reviewed by Chapman, 2009). The principle
involved (prevention or prophylaxis) has had a profound impact on our abil-
ity to grow chickens and turkeys under intensive conditions. Indeed it is
possible that the modern poultry industry could never have developed to
its present extent without the advent of drugs to control coccidiosis. Today,
anticoccidial drugs are incorporated routinely into the feed of broiler
chickens and turkeys for this purpose (Chapman, 2001, 2008). For example,
data available for the United States indicates that the use of anticoccidial
drugs in broiler flocks varied from 70% to 98% depending upon the season
(AgriStats, Inc., Fort Wayne, IN, USA). In Western Europe, 91% of com-
plexes use an anticoccidial drug (C. Bostvironnois, Elanco Animal Health,
personal communication). Drug usage is similarly extensive in other major
poultry producing regions around the world. Although there may be sea-
sonal variation in the use of drugs, it is clear that chemotherapy as a means
of control is widespread. The long-term outlook for such a heavy reliance
upon chemotherapy is often stated to be uncertain because of the widespread
development of drug resistance, a problem first recognized in the 1950s, and
a concomitant lack of new drug discovery. Furthermore, some anticoccidials
have been banned (in the EU) and others are said to be under threat
(McDonald and Shirley, 2009). So far, however, such considerations do
not seem to have led to a decline in drug use.
150 H. David Chapman et al.

Anticoccidial drugs fall into two categories, the synthetic compounds


(produced by chemical synthesis and popularly known as ‘chemicals’) and
the ionophore antibiotics, which are by-products of bacterial fermenta-
tion. Synthetic drugs were the first to be discovered and comprise a diverse
array of molecules that are absorbed into the blood stream of the host and
kill developing parasites in epithelial cells of villi in the intestine. One of
the oldest synthetic drugs, nicarbazin, is also one of the most successful and
is still used widely today (Chapman, 1994a). Ionophores have a different
mode of action from synthetic drugs since they are able to destroy motile
stages of the Eimeria life cycle (sporozoites and merozoites) in the gut
lumen or following cell penetration (Smith and Strout, 1979). Since the
introduction of monensin in 1972, ionophores have been the most widely
used anticoccidial drugs for the control of coccidiosis (Chapman, 2001).
The mode of action and discovery of monensin, together with matters
of importance to the poultry industry such as toxicity, pharmacology,
residues and resistance to this drug, has been reviewed recently
(Chapman et al., 2010).
Despite the availability of a dozen or so anticoccidial drugs it may be sur-
prising to know that for the majority the mode of action against coccidia is
not known (Chapman, 1997). In one case, the discovery of a biochemical
pathway unique to Eimeria (the mannitol cycle) enabled the mode of action
of nitrophenide, a drug briefly used in the 1950s, to be elucidated (Schmatz
et al., 1989). Unfortunately, resistance to this drug developed quickly, a fate
shared by most other synthetic compounds. Diclazuril and decoquinate are
synthetic drugs to which resistance can also develop. Diclazuril has recently
been shown to induce ultrastructural changes in merozoites and cause
disruption of transmembrane potential in the mitochondrion (Zhou et al.,
2010). It is not clear if this reflects a true mode of action or is just a
consequence of cell death. Decoquinate, like other members of the quino-
lone family, is known to act against the electron transport chain of coccidia
(reviewed by Chapman, 1997). This drug has recently been shown to cause
chromosomal rearrangements during meiosis in oocysts of E. tenella
(Del Cacho, et al., 2006). The mode of action of ionophores involves dis-
ruption of ion transport across the parasite cell membrane (reviewed by
Chapman, et al., 2010) and resistance has been much slower to develop.
Evidence has been obtained that resistance may be due to changes in the flu-
idity of the cell membrane of sporozoites (Wang, et al., 2006). A recent study
suggests that monensin is also able to interrupt invasion of host cells by spo-
rozoites (del Cacho et al., 2007).
Review of Coccidiosis Research 151

Most drugs are no longer as effective as when they were first introduced
due to the development of drug resistance. For example, one recent report
indicated that 68% and 53% of field isolates of E. acervulina from chicken
flocks in the EU were resistant to the synthetic drug diclazuril, and the ion-
ophore monensin, respectively (Peek and Landman, 2006). Similar reports
of resistance have been reported worldwide. In the turkey, drug resistance
has also been shown to be widespread (Rathinam and Chapman, 2009).
Details of the emergence of resistance in the 1970s to decoquinate have been
provided retrospectively (Williams, 2006). Although many surveys have
been published indicating the extent of resistance, little research has been
conducted on the mechanisms involved. Biochemical, genetic and applied
aspects of resistance have been reviewed (Chapman, 1997).
An early insight was that use of low concentrations of certain drugs in the
feed did not necessarily prevent the acquisition of immunity (Grumbles
et al., 1948). It is now known that most drugs are effective in the field
because they only partially suppress parasite development, allowing birds
to acquire natural immunity as a consequence of exposure to parasites that
escape drug action (Chapman, 1999). An advantage of immunity develop-
ment is that it allows the safe withdrawal of drugs several weeks before the
birds are sold with considerable savings in the cost of medication and reduc-
tion of the risk of potential drug residues in poultry meat.

10.2. Vaccination
Vaccination as a means to control coccidiosis has a long history (see
Chapman, 2003; Williams, 2002a) but it is only in the past 20 years or so
that this has proved a practical method for the control of coccidiosis in com-
mercial broiler flocks, principally because it has proved feasible to vaccinate
chicks in the hatchery by spraying birds with controlled numbers of oocysts
within enclosed cabinets. This involves considerable cost savings compared
with traditional methods of vaccination which were carried out on the farm
by trained personnel. Most commercially available vaccines comprise live
oocysts and vary according to the number of species of Eimeria included,
the numbers of oocysts present, and whether or not they are attenuated.
Vaccines containing all species that infect the chicken are used mainly to
immunize egg laying stock whereas vaccines containing fewer species (usu-
ally E. acervulina, E. maxima and E. tenella) are used in broilers. The first vac-
cines comprised populations of wild-type oocysts that were potentially
pathogenic, but more recently, vaccines containing attenuated parasites
152 H. David Chapman et al.

which have reduced pathogenicity but retain immunogenicity, have been


introduced.
The purpose of vaccination with live oocysts is to provide an early initial
stimulus of the immune response. After placement of birds on litter, new
vaccinal oocysts are shed in the faeces and, following sporulation, these
are capable of re-infecting the flock. Secondary exposure to vaccinal oocysts
and wild-type oocysts present in the litter is thought to induce protective
immunity. Development of immunity takes several weeks and some cases
of vaccination failure occur because birds are overwhelmed with exposure
to wild-type virulent oocysts before they have had time to develop an
immune response. It is obviously important that vaccination is undertaken
carefully because any chicks that are not exposed to vaccinal oocysts may be
vulnerable to potentially high numbers of virulent oocysts when placed on
litter. The objective of vaccination is to induce sufficient immunity to pre-
vent chronic infestation while still allowing sufficient Eimeria to accumulate
such that a full immune response to the local Eimeria species will develop.
Several reviews have been published that are concerned with various aspects
of vaccination (Chapman, 2000; Chapman et al., 2002; Shirley et al., 2005;
Vermeulen et al., 2001; Williams, 2002b). Guidelines have been developed
to facilitate the worldwide adoption of consistent standard procedures
for determining the efficacy and safety of live anticoccidial vaccines
(Chapman et al., 2005).
Vaccination can also be achieved by in ovo injection of sporulated oocysts
into the embyronating egg (Weber et al., 2004). This is carried out in the
hatchery using complex machines that are also able to deliver other poultry
vaccines. Surprisingly, little research has been published that explains how
the oocysts, when injected into the egg, are able to establish a patent infec-
tion in the gut of the developing embryo. It is possible that infection results
from exposure to oocysts present in the eggshells post-hatch. As with other
methods of vaccination, secondary exposure to infection via the litter fol-
lowing placement of birds on the litter is necessary for a protective immune
response.
Another approach to vaccination involves immunizing hens with
affinity-purified antigens from the WFBs of macrogametocytes of
E. maxima (Sharman et al., 2010; Wallach et al., 2008). The nature of these
antigens has already been described in the section on oocyst biogenesis
above. Maternal antibodies pass via the egg to the newly hatched chick
and provide passive protection of limited duration. This is the only subunit
vaccine currently employed against any protozoan parasite. As in the case of
Review of Coccidiosis Research 153

live oocyst vaccines, however, full protective immunity requires exposure to


potentially pathogenic coccidia in the litter.
Considerable research has been undertaken utilizing molecular technolo-
gies to identify antigens capable of inducing an immune response but in most
cases only partial protection has been achieved and none of the vaccine candi-
dates have been proven in commercial applications (Shirley and Lillehoj, 2012).

10.3. Strategies for the control of coccidiosis


Meetings aimed at poultry producers, poultry veterinarians and other pro-
fessional groups with an interest in the poultry industry, often include pre-
sentations concerned with ‘strategies’ for the control of coccidiosis.
Unfortunately, data that can be tested rigorously are rarely presented, partly
due to the difficulty in designing reproducible trials under field conditions.
Thus the almost universal presence of Eimeria in poultry flocks negates the
inclusion of an ‘uninfected’ group in any controlled study. Strategies may
involve drugs, vaccines or both. In the case of drugs, different products,
often with different modes of action, may be used in the different feeds that
are provided during the life of a flock of birds. Often referred to as ‘shuttle’
programmes, one such employed in the United States involves inclusion of
nicarbazin or a nicarbazin/narasin mixture in the first feed and monensin or
salinomycin in the second; however, there are numerous variations
(Chapman, 2001). In subsequent flocks, different drugs may be used, for
which the term ‘rotation programme’ has been coined. Use of such
programmes is widespread and may be considered an appropriate strategy
for the control of coccidiosis although rarely is evidence of long-term effi-
cacy (compared with other approaches) available.
Vaccination, and its integration with chemotherapy in control
programmes, is an alternative strategy. Evidence has been obtained that par-
tial restoration of sensitivity to drugs may occur following the use of vaccines
comprising drug-sensitive strains of Eimeria. This phenomenon has been
demonstrated for the ionophores, monensin and salinomycin, and the syn-
thetic drug, diclazuril (Chapman, 1994b; Jenkins et al., 2010; Peek and
Landman, 2006). Based upon these observations, a yearly rotation pro-
gramme has been proposed in which use of ionophores is alternated in suc-
cessive flocks with vaccination (Chapman et al., 2010); such programmes are
commonly used in the United States. There is a need for more evidence to
support the notion that rotation programmes involving vaccines and drugs
prolong the life of the latter in the field.
154 H. David Chapman et al.

10.4. Natural products


There is considerable current interest in the use of so-called ‘natural prod-
ucts’ which may include plant extracts, probiotics and so on, to reduce prob-
lems caused by coccidiosis (e.g. Allen, 2003, 2007; Faber et al., 2012;
Giannenas et al., 2012; Lee et al., 2011). For example, the antimalarial
artemisinin, a product extracted from the herb Artemisia annua, was shown
to have a deleterious effect upon macrogametocytes of E. tenella by affecting
the expression of an enzyme sarcoplasmic–endoplasmic reticulum calcium
ATPase (del Cacho et al., 2010). Improved resistance to E. acervulina was
observed when the diet of chickens was supplemented with garlic metabo-
lites (Kim et al., 2012). There are many other examples in the literature.
Most natural products, however, are said not to specifically target the parasite
but ‘improve gut health’ or act as ‘enhancers’ of some aspect of immune sys-
tem function. All natural products contain undefined chemicals that, neces-
sarily, will have to be evaluated for safety and toxicity before being
acceptable for registration authorities. There is often very little scientific
literature that supports the claims made for these products and, as far as is
known, none are used commercially at present probably because of unreal-
istic dietary inclusion rates and failure to demonstrate efficacy under con-
trolled conditions.

11. CONCLUSIONS
In this review, we have considered selective aspects of research con-
cerned with the apicomplexan parasites of the genus Eimeria which cause the
disease, coccidiosis, of domestic livestock. The emphasis has been on poul-
try, where coccidiosis has been shown to have an enormous economic
impact. Fortunately, control of coccidiosis in poultry has been achieved,
by a combination of improved management, the prophylactic use of drugs,
and vaccination. Nevertheless, we should not be complacent because the
parasite has not been eradicated from commercial facilities where animals
are reared and is still capable of causing production losses.
In recent years, many research projects, and publications that result, have
used the modern tools of molecular biology, biochemistry, cell biology and
immunology to expand greatly our knowledge of these parasites and the dis-
ease they cause. Such studies are essential if we are to develop new means for
the control of coccidiosis. Past success was achieved by research funded and
conducted by universities, government agencies and private industry.
Review of Coccidiosis Research 155

In recent years, however, a number of government funded organizations


have terminated their coccidiosis programmes and private industry has been
reluctant to allocate funds to the animal health sector due to the enormous
costs involved in drug discovery and vaccine development. Few university
departments have the facilities to undertake costly coccidiosis research. It is
evident, therefore, that both from a practical and research perspective con-
trol of coccidiosis is at a crossroads. In the future, the ever expanding human
population will become increasingly dependent upon a source of cheap pro-
tein of which poultry will necessarily be an important component. Hope-
fully, this will not be compromised by the ubiquitous parasites of the
genus Eimeria.

ACKNOWLEDGEMENT
We would like to thank Thilakar Rathinam for help in preparing the figures.

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CHAPTER THREE

The Distribution and Bionomics


of Anopheles Malaria Vector
Mosquitoes in Indonesia
Iqbal R.F. Elyazar*,1, Marianne E. Sinka†, Peter W. Gething†,
Siti N. Tarmidzi{, Asik Surya{, Rita Kusriastuti{, Winarno{,
J. Kevin Baird*,}, Simon I. Hay†, Michael J. Bangs}
*Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia

Spatial Ecology and Epidemiology Group, Department of Zoology, University of Oxford, Oxford,
United Kingdom
{
Directorate of Vector-Borne Diseases, Indonesian Ministry of Health, Jakarta, Indonesia
}
Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford,
United Kingdom
}
Public Health and Malaria Control Department, International SOS, PT Freeport Indonesia, Kuala Kencana,
Indonesia
1
Corresponding author: Iqbal RF Elyazar

Contents
1. Introduction 175
2. Assembling a National Database of Anopheles Mosquitoes Susceptible
to Plasmodium spp. Infections, Host Preference, Bionomics and Insecticide
Susceptibility in Indonesia 176
3. Infectivity of Anopheles Mosquitoes to Plasmodium in Indonesia 177
4. The Distribution of Anopheles Malaria Vectors in Indonesia 178
5. Malaria Vectors in Indonesia: Plasmodium spp. Infections, Host Preferences, Larval
and Adult Bionomics and Insecticide Susceptibility 178
5.1 Anopheles (Cellia) aconitus Dönitz 178
5.2 Anopheles (Cellia) balabacensis Baisas 182
5.3 Anopheles (Anopheles) bancroftii Giles 184
5.4 Anopheles (Anopheles) barbirostris van der Wulp 186
5.5 Anopheles (Anopheles) barbumbrosus Strickland & Chowdhury 189
5.6 Anopheles (Cellia) farauti Laveran species complex 191
5.7 Anopheles (Cellia) flavirostris (Ludlow) 193
5.8 Anopheles (Cellia) karwari James 195
5.9 Anopheles (Cellia) kochi Dönitz 197
5.10 Anopheles (Cellia) koliensis Owen 199
5.11 Anopheles (Cellia) leucosphyrus Dönitz 201
5.12 Anopheles (Cellia) maculatus Theobald species subgroup 203
5.13 Anopheles (Anopheles) nigerrimus Giles 206
5.14 Anopheles (Cellia) parangensis (Ludlow) 208
5.15 Anopheles (Cellia) punctulatus Dönitz 209

Advances in Parasitology, Volume 83 # 2013 Elsevier Ltd 173


ISSN 0065-308X All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-407705-8.00003-3
174 Iqbal R.F. Elyazar et al.

5.16 Anopheles (Anopheles) sinensis Wiedemann 211


5.17 Anopheles (Cellia) subpictus Grassi species complex 213
5.18 Anopheles (Cellia) sundaicus Rodenwaldt species complex 216
5.19 Anopheles (Cellia) tessellatus Theobald 219
5.20 Anopheles (Cellia) vagus Dönitz 221
6. Anopheles Susceptibility to Insecticides 238
6.1 Anopheles aconitus 239
6.2 Anopheles barbirostris 240
6.3 Anopheles farauti s.l. 240
6.4 Anopheles kochi 240
6.5 Anopheles koliensis 240
6.6 Anopheles maculatus 241
6.7 Anopheles subpictus s.l. 241
6.8 Anopheles sundaicus s.l. 241
6.9 Anopheles vagus 242
7. Outlook for Indonesian Challenges to Malaria Vector Control 242
8. Conclusions 244
Acknowledgements 244
References 245

Abstract
Malaria remains one of the greatest human health burdens in Indonesia. Although
Indonesia has a long and renowned history in the early research and discoveries of
malaria and subsequently in the successful use of environmental control methods to
combat the vector, much remains unknown about many of these mosquito species.
There are also significant gaps in the existing knowledge on the transmission epidemi-
ology of malaria, most notably in the highly malarious eastern half of the archipelago.
These compound the difficulty of developing targeted and effective control measures.
The sheer complexity and number of malaria vectors in the country are daunting. The
difficult task of summarizing the available information for each species and/or species
complex is compounded by the patchiness of the data: while relatively plentiful in one
area or region, it can also be completely lacking in others. Compared to many other
countries in the Oriental and Australasian biogeographical regions, only scant informa-
tion on vector bionomics and response to chemical measures is available in Indonesia.
That information is often either decades old, geographically patchy or completely lac-
king. Additionally, a large number of information sources are published in Dutch or
Indonesian language and therefore less accessible. This review aims to present an
updated overview of the known distribution and bionomics of the 20 confirmed malaria
vector species or species complexes regarded as either primary or secondary (inciden-
tal) malaria vectors within Indonesia. This chapter is not an exhaustive review of each of
these species. No attempt is made to specifically discuss or resolve the taxonomic
record of listed species in this document, while recognizing the ever evolving revisions
in the systematics of species groups and complexes. A review of past and current status
of insecticide susceptibility of eight vector species of malaria is also provided.
Anopheles Malaria Vector Mosquitoes in Indonesia 175

1. INTRODUCTION
An integrated approach to interventions against mosquito vectors of
malaria has become increasingly important for those nations aiming for elim-
ination of malaria transmission or a significant reduction of infection risk
(World Health Organization, 2007b). Such evidence-based strategies for
vector control require detailed knowledge of the identity, distribution
and bionomics of the primary malaria vectors within the target area
(Zahar, 1994). Recent work by the Malaria Atlas Project (www.map.ox.
ac.uk), defining the spatial distributions of the dominant vector species of
human malaria worldwide (Hay et al., 2010), has begun to address the need
for geographical species-specific information, including a detailed review
of the bionomics of these primary vectors in the Asia-Pacific region (Hay
et al., 2010; Sinka et al., 2011). On a national scale, however, and despite
a long history of study of the important Anopheles, no contemporary
systematic review of this mosquito genus has been undertaken in Indonesia.
This chapter, therefore, closely examines both the past and current state
of knowledge of many of the anopheline malaria vectors present in this
environmentally diverse archipelago.
The main arsenal for adult mosquito control consists of applying long-
lasting, residual insecticides, either on bednets or applied/sprayed directly
onto the walls within human dwellings (World Health Organization,
2010). Unfortunately, the continuous exposure of mosquitoes to these
chemicals has resulted in measurable physiological resistance, and in some
instances significant behavioural avoidance amongst a number of studied
malaria vectors species (Najera and Zaim, 2003). Physiological resistance
refers to the ability of a mosquito to tolerate doses of insecticide which
would normally prove lethal to the majority (>98%) of individuals in a
local population of the same species, whilst behavioural avoidance relates
to the tendency of mosquitoes to avoid contact with the insecticide-
treated surface, either as a result of contact ‘irritancy’, spatially active
repellency, or as a combination of both (World Health Organization,
1963). Monitoring the insecticide-resistance profile of a population
of medically important Anopheles species is essential for better design
and implementation of an evidence-based vector control policy (World
Health Organization, 1992). Until now, no contemporary review of the
insecticide-resistance patterns amongst Indonesian anophelines vectors
has been published.
176 Iqbal R.F. Elyazar et al.

2. ASSEMBLING A NATIONAL DATABASE OF ANOPHELES


MOSQUITOES SUSCEPTIBLE TO PLASMODIUM SPP.
INFECTIONS, HOST PREFERENCE, BIONOMICS AND
INSECTICIDE SUSCEPTIBILITY IN INDONESIA
A systematic search and review of published and unpublished entomo-
logical literature from online and library sources was used to assemble a data-
base of the distribution of Indonesian Anopheles, their natural infection with
human malaria parasites, bionomics and frequency of insecticide resistance.
Visits were made to university and Ministry of Health library resources to
search for more obscure or offline/unpublished information. Searches were
completed on 31 December 2011. Once a relevant data source was identi-
fied, information was extracted into an Excel worksheet including an unique
identification record of each source, year of source, location (region, island,
province, district, sub-district and specific locality such as village), species
and species identification method used (morphological and molecular
based), physiological measures (mating status, parity, age-grading, blood-
feeding preference, etc.), the sporozoite and oocyst rate (using midgut
and salivary gland dissections, circumsporozoite immunological assays and
molecular-based tests). Based on the presence of oocysts and/or sporozoites,
each record was classified into two ‘susceptibility’ categories: infected (mid-
gut oocysts) or infective (presence of salivary gland sporozoites). When
a mosquito was found to be infected but not necessarily infectious, it was
classified as a ‘suspect’ vector, whereas those identified as infectious were
classified as an incriminated or ‘confirmed’ malaria vector (Swellengrebel
et al., 1919; Warrell and Gilles, 2002). Additional data recorded for the
number found positive for the presence of human blood (i.e. human blood
index, HBI) were also searched. Larval and adult bionomic data were
included focusing on blood-feeding behaviour and activity patterns, pre-
dominant resting sites of adults and aquatic habitats for immature stages.
These data were stratified into western and eastern sectors of the Indonesian
archipelago for descriptive purposes. Western and eastern sectors of Indone-
sia are biogeographically distinct regions of the archipelago, demarked by a
series of different transecting lines including Wallace’s and Weber’s Lines
near the centre of the nation (surrounding the island of Sulawesi; shown
in Figs. 3.1–3.21; Wallace, 1863; Weber, 1890).
Finally, a database of the vector insecticide susceptibility status was
assembled identifying those sources that reported an insecticide susceptibility
Anopheles Malaria Vector Mosquitoes in Indonesia 177

test, including the method (bioassay, biochemical, molecular), the insecti-


cide (active ingredient) tested, doses (percent concentrations) used, number
of mosquitoes assayed and the mosquito mortality following exposure. The
insecticides in the database included the six currently recommended for
indoor residual spraying by the Indonesian Vector Control Program
(VCP), primarily pyrethroid and carbamate class chemicals (Departemen
Kesehatan, 2010), plus other insecticide classes used historically such as
organophosphates and organochlorine compounds.

3. INFECTIVITY OF ANOPHELES MOSQUITOES


TO PLASMODIUM IN INDONESIA
From the reviewed and compiled literature, a total of 74 sources were
used to extract 1266 records reporting Plasmodium spp. infections (sporozo-
ites or oocyst stages) for 29 Anopheles species found in Indonesia between
1919 and 2010 (Table 3.1). These data indicate the presence of 20 Anopheles
species confirmed as primary or secondary (incidental) malaria vectors in the
country. No records of naturally occurring infectious stages (sporozoites)
were found for the remaining nine species, despite four species, including
Anopheles annularis, Anopheles hyrcanus, Anopheles indefinitus and Anopheles
umbrosus being reported as suspected vectors in Indonesia (Table 3.2).
The confirmed malaria vectors are not uniformly distributed across the
archipelago. Twelve species are located in the western portion of the country
and 13 species in the eastern region of Indonesia with some overlap across
both areas: Anopheles balabacensis, Anopheles flavirostris, Anopheles nigerrimus,
Anopheles subpictus and Anopheles sundaicus were reported as natural vectors
in both regions. The distribution of malaria vectors amongst the main islands
is also not uniform (Fig. 3.1), with Java and Sulawesi appearing to contain
the greatest number of reported malaria vectors (eight species), followed by
Sumatra (six species), Papua (at least five species) and the Lesser Sundas archi-
pelago (five species). Only two species were confirmed as malaria vectors in
Kalimantan. No data on the infectivity of Anopheles species on Maluku were
identified but at least two species present in the island chain (Anopheles farauti
and Anopheles punctulatus) are known to be efficient vectors elsewhere (Papua).
A map of the distribution of the Anopheles malaria vectors in Indonesia is
provided (Fig. 3.1) which illustrates species by principal islands or island
groups from Sumatra in the west to Papua in the east. Sulawesi and the Lesser
Sundas archipelago lie in the centre of Indonesia and are between two major
zoogeographical lines (Wallace’s and Weber’s Lines drawn to demark the
178 Iqbal R.F. Elyazar et al.

Oriental and Australasia Regions) based on unique and overlapping fauna


distributions in the region.

4. THE DISTRIBUTION OF ANOPHELES MALARIA


VECTORS IN INDONESIA
A total of 259 sources, published from 1917 to 2011, have docu-
mented the presence of 20 Anopheles malaria vector species in Indonesia rep-
resenting 755 independent sites. A greater number of sites in western
Indonesia reported vectors present than in eastern Indonesia (66% vs.
34%), no doubt reflecting the relatively higher number of investigations
in the far more densely populated western sector. Over the seven main
islands in Indonesia, the greatest number of sites where vectors have been
found were on Java (41%; 311 sites) with the least found on Papua (4%;
32 sites). Anopheles vagus was reported from the greatest number of indepen-
dent sites (46%; 349 sites) across Indonesia, while Anopheles bancroftii was the
most restricted (1%; 7 sites in Papua, 1 in Maluku).
For each species, an individual map has also been generated indicating
geo-referenced locations of occurrence and where malaria infectious mos-
quitoes have been recorded (Figs. 3.2–3.21). These records were then over-
laid to the Plasmodium falciparum malaria endemicity map in Indonesia that
was produced in an earlier publication (Elyazar et al., 2011a). The endemic-
ity maps defined five land categories with areas colour shaded accordingly:
no malaria risk area (light grey, where PfAPI ¼ 0 per 1000 pa), unstable
transmission risk area (medium grey, where PfAPI < 0.1 per 1000 pa),
low risk area (light red, PfPR2–10 < 5%), intermediate risk area (medium
red, 5% < PfPR2–10 < 40%) and high risk area (dark red, PfPR2–10 > 40%).
Using these geo-referenced records and endemicity map, distribution maps
were produced for each species or species complex. The presentation of spe-
cies is alphabetical rather than geographical or by taxonomic affinities.

5. MALARIA VECTORS IN INDONESIA: PLASMODIUM


SPP. INFECTIONS, HOST PREFERENCES, LARVAL
AND ADULT BIONOMICS AND INSECTICIDE
SUSCEPTIBILITY
5.1. Anopheles (Cellia) aconitus Dönitz
An. aconitus is a member of the Funestus Group (Garros et al., 2005). This
species is broadly distributed throughout the Indonesian archipelago,
Anopheles Malaria Vector Mosquitoes in Indonesia 179

Figure 3.1 A map of the distribution of primary Anopheles malaria vectors in Indonesia.

although relative densities and frequency vary dramatically. A total of 132


sources reported the presence of An. aconitus at 325 independent sites
(Fig. 3.2). The species has been most commonly reported from Java (197
sites) and extends across the archipelago as far east as Timor-Leste and the
Maluku Islands, but it seems absent from Papua (the Indonesian half of
New Guinea Island). Using an enzyme-linked immunosorbent assay
(ELISA) to detect the parasite circumsporozoite protein, Barodji et al.
(2007) found only one specimen amongst 1432 tested in Central Java having
malaria (P. falciparum) sporozoites. Over a 20-year period, the U.S. Naval
Medical Research Unit No. 2 (NAMRU-2) in Jakarta detected sporozoite
(P. falciparum and Plasmodium vivax) positive An. aconitus only from Central
Java Province (Bangs and Rusmiarto, 2007). No other infective specimens
have been reported from the other main islands. This species is reputed to be
a major vector on Java, but generally only when present in high human-
biting densities (Kirnowardoyo, 1988).
The adult females are predominantly zoophilic, with a greater presence
in cattle and other outdoor animal shelters than human habitations (Barodji,
1983a; Barodji et al., 1992; Chow et al., 1959; Joshi et al., 1977; Mardiana
et al., 2005; Yunianto et al., 2004). The combined proportion of mosquitoes
that contained human blood resting in cattle shelters was 2.9% (94/3185)
(Chow et al., 1959; Joshi et al., 1977; Noerhadi, 1960; World Health
Organization and Vector Biology and Control Research Unit 2 Subunit
180 Iqbal R.F. Elyazar et al.

Figure 3.2 Anopheles aconitus distribution in Indonesia. The blue stars indicate the
records of infectious An. aconitus mosquitoes found. The yellow dots show 325 records
of occurrence for this species between 1917 and 2011. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al.,
2011a). The database of distribution of An. aconitus in Indonesia was acquired from
the references: Adrial (2003), Adrial and Harminarti (2005), Adrial et al. (2000), Alfiah
et al. (2008), Atmosoedjono et al. (1993), Atmosoedjono et al. (1975), Bang et al. (1982),
Barbara et al. (2011), Barodji (1983b,c), Barodji (1986), Barodji (2003), Barodji et al.
(2003), Barodji et al. (1984a), Barodji et al. (2007), Barodji et al. (1986a), Barodji et al.
(1992), Barodji et al. (1989a), Barodji et al. (1984b), Barodji et al. (1986b), Barodji et al.
(1998/1999), Barodji and Supratman (1983), Barodji et al. (1989b), Blondine et al. (2000),
Boesri et al. (1996a), Boesri et al. (2004), Boesri and Boewono (2006), Boewono and Nalim
(1989, 1991), Boewono et al. (1991), Boewono and Ristiyanto (2004, 2005), Boewono
et al. (2005), Brug and Bonne-Wepster (1947), Buono (1987), Citroen (1917), Dasuki and
Supratman (2005), Garjito et al. (2004b), Hadi et al. (2006), Hafni (2005), Handayani
and Darwin (2006), Hasan (2006), Hoedojo (1992, 1995), Idris-Idram et al. (1998/1999),
Ikawati et al. (2006), Ikawati et al. (2004), Isfarain and Santiyo (1981), Jastal et al.
(2002), Jastal et al. (2001), Kaneko et al (1987), Kazwaini and Martini. (2006),
Kirnowardoyo (1977), Kirnowardoyo and Supalin (1982), Kirnowardoyo and Supalin
(1986), Kirnowardoyo and Yoga (1987), Kurihara (1978), Lee et al. (1984), Lestari et al.
(2000), Lien et al. (1975), Mangkoewinoto (1919), Mardiana et al. (2002), Mardiana and
Sukana (2005), Mardiana et al. (2005), Mardihusodo et al. (1988), Marjiyo (1996),
Martono (1988a,b), Marwoto et al. (1992a), Munif (1990, 1994, 2004), Munif et al.
(2007), Munif et al. (2003), Munif et al. (1994), Nalim (1980), Nalim (1980/1981), 1985,
1986, Nalim and Boewono (1987), Nalim et al. (2000), Nalim and Tribuwono (1983),
Ndoen et al. (2010), Noor (2002), Ompusunggu et al. (2006), Ompusunggu et al.
(1994a), Pranoto and Munif (1993), Pranoto (1989), Pribadi et al. (1985), Raharjo et al.
(2007), Raharjo et al. (2006), Ramadhani et al. (2005), Saleh (2002), Schuurman and
Huinink (1929), Self et al. (1976), Sigit and Kesumawati (1988), Soekirno et al. (2006a),
Anopheles Malaria Vector Mosquitoes in Indonesia 181

Semarang, 1978), while those captured in human settlements was only


slightly higher at 6.7% (1004/14,811) (Chow et al., 1959; Garret-Jones,
1964; Joshi et al., 1977; Sundararaman et al., 1957; Walch and Sardjito,
1928; World Health Organization and Vector Biology and Control
Research Unit 2 Subunit Semarang, 1978) (Table 3.3). A stronger
exophagic (outdoor biting/blood feeding) habit is commonly reported in
Java (Barodji et al., 1992; Boesri and Boewono, 2006; Chow et al., 1959;
Ikawati et al., 2004; Joshi et al., 1977; Kirnowardoyo, 1977; Munif,
2004; Munif et al., 2007; Yunianto et al., 2002, 2004), whereas a stronger
endophagic (indoor biting) behaviour has been shown along the southern
coastal zone of western Java (Stoops et al., 2009b) and West Sumatra Prov-
ince (Adrial, 2003). Females typically reach their peak blood-feeding activity
in the second quarter of the night (Barodji et al., 2007; Boesri and Boewono,
2006; Joshi et al., 1977; Stoops et al., 2009b), after which blood-fed females
are generally found resting outdoors (Alfiah et al., 2008; Barodji et al., 1992,
2007; Boesri and Boewono, 2006; Boewono and Ristiyanto, 2005;
Boewono et al., 1991; Chow et al., 1959; Joshi et al., 1977;
Kirnowardoyo, 1977; Munif et al., 2007; Yunianto et al., 2004) in shaded
animal shelters (Boewono et al., 1991; Chow et al., 1959; Joshi et al., 1977;
Kirnowardoyo, 1977; Munif et al., 2007), rock crevices (Alfiah et al., 2008),
earthen pits (Alfiah et al., 2008) and river banks (Boesri and Boewono, 2006)
to complete their gonotrophic cycle.
The characteristic larval habitats of An. aconitus have been comprehen-
sively described in Indonesia. Larvae are most commonly found in sunlit,
exclusively fresh water, often clear in appearance, stagnant or slow flowing
(Takken et al., 1990) and either natural- or man-made habitats (Table 3.4). Nat-
ural water collections include marshes (Sudomo et al., 2010; Swellengrebel and
Swellengrebel-de Graaf, 1919a), streams (Mangkoewinoto, 1919; Stoops et al.,

Soekirno et al. (2006b), Stoops et al. (2009a), Stoops et al. (2008), Stoops et al. (2009b),
Sudomo et al. (2010), Sukowati et al. (2001), Sundararaman et al. (1957), Suparno
(1983), Susana (2005), Suwarto et al. (1987), Suwasono et al. (1993), Swellengrebel
(1921), Swellengrebel and Rodenwaldt (1932), Swellengrebel and Swellengrebel-de Graaf
(1920), Syafruddin et al. (2010), Tarore (2010), Tativ and Udin (2006), Trenggono (1985),
Van Hell (1952), Vector Biology and Control Research Unit (1979b), Verdrager and
Arwati (1975), Widiarti (2005), Widiarti et al. (2005a), Widiarti et al. (2005b), Widiarti
et al. (2001), Widiastuti et al. (2006), Widjaya et al. (2006), Widyastuti et al. (2003),
World Health Organization and Vector Biology and Control Research Unit 2 Semarang
(1977), Yoga (1991), Yudhastuti (2009), Yunianto (2002), Yunianto et al. (2002) and
Yunianto et al. (2004).
182 Iqbal R.F. Elyazar et al.

2007; Swellengrebel and Swellengrebel-de Graaf, 1919a) and river beds (Boesri
and Boewono, 2006; Boewono and Ristiyanto, 2005; Mangkoewinoto, 1919;
Swellengrebel, 1916) and man-made sources most commonly include rice
fields (Adrial, 2003, 2008; Boesri and Boewono, 2006; Boesri et al., 1996b;
Joshi et al., 1977; Mangkoewinoto, 1919; Munif et al., 2007; Ndoen et al.,
2010; Stoops et al., 2007, 2008; Sundararaman et al., 1957; Swellengrebel
and Swellengrebel-de Graaf, 1919a), fish ponds (Adrial, 2008; Swellengrebel
and Swellengrebel-de Graaf, 1919a) and irrigation ditches (Boesri and
Boewono, 2006; Joshi et al., 1977; Mangkoewinoto, 1919; Munif et al.,
2007). A positive correlation between An. aconitus larval densities and
phase of rice production has been observed with larval peak abundance occur-
ring early in the growing season, around six weeks after rice planting
(Kirnowardoyo, 1988; Munif et al., 2007). This species is widely dispersed
in the environment and can be found from the coastal plain (Ndoen et al.,
2010; Stoops et al., 2007) to hilly areas (Joshi et al., 1977; Mangkoewinoto,
1919; Ndoen et al., 2010; Soemarlan and Gandahusada, 1990; Stoops et al.,
2007; Sundararaman et al., 1957) up to altitudes of 1000 m above sea level
(asl) wherever suitable larval habitats exist (Sundararaman et al., 1957).

5.2. Anopheles (Cellia) balabacensis Baisas


An. balabacensis is a member of the Leucosphyrus Subgroup, within the
Leucosphyrus Complex (Sallum et al., 2005), a subgroup which includes
several very important vectors of human malaria in forest fringe areas of
Southeast Asia, including the Southeast Asian mainland, Philippine Islands,
Brunei, Malaysian Borneo and Indonesia (Sinka et al., 2011). Thirty-four
sources reported the presence of An. balabacensis from 43 independent sites
on Java, Kalimantan, Sulawesi and Lesser Sundas with this species was most
commonly reported from Java (30 sites) (Fig. 3.3). An. balabacensis has been
found infected with P. falciparum sporozoites in Kalimantan (Harbach et al.,
1987). Both P. falciparum and P. vivax infections were also detected in East
(Kenangan) and South Kalimantan (Salaman) and Central Java (Magelang
and Purworejo [Menoreh Hills]) (Bangs and Rusmiarto, 2007). The pres-
ence of P. vivax sporozoites has also been reported from Central Java
(Adrial et al., 2000).
The degree of anthropophily amongst female An. balabacensis appears to
depend on location. Low levels of anthropophilic behaviour have been
observed in hilly areas of Central Java (Alfiah et al., 2008), while in the
mountainous areas of Lombok Island in the Lesser Sundas a high degree
Anopheles Malaria Vector Mosquitoes in Indonesia 183

Figure 3.3 Anopheles balabacensis distribution in Indonesia. The blue stars indicate
the records of infectious An. balabacensis mosquitoes found. The yellow dots
show 43 records of occurrence for this species between 1987 and 2010. Areas were
defined as no risk (light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission
(medium grey, where PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%),
intermediate risk (medium red, 5% < Pf PR2–10 < 40%) and high risk (dark red,
PfPR2–10  40%) (Elyazar et al., 2011a). The database of distribution of An. balabacensis
in Indonesia was acquired from the references: Adrial et al. (2000), Alfiah et al. (2008),
Aprianto (2002), Ariati (2004), Barodji et al. (2003), Barodji and Sularto (1993), Boesri et al.
(2004), Boewono and Ristiyanto (2005), Buono (1987), Effendi (2002), Handayani and
Darwin (2006), Harbach et al. (1987), Ikawati et al. (2006), Ikawati et al. (2004), Lestari
et al. (2000), Maekawa et al. (2009a), Maekawa et al. (2009b), Marjiyo (1996), Noor
(2002), Pranoto and Munif (1993), Raharjo et al. (2007), Santoso (2002), Sukmono
(2002), Sukowati et al. (1987), Susana (2005), Suwasono et al. (1997), Suwasono et al.
(1993), Syafruddin et al. (2010), Tarore (2010), Ustiawan and Hariastuti (2007),
Wardana (2010), Widiastuti et al. (2006) and Yunianto et al. (2002).

of anthropophily was noted (Maekawa et al., 2009b) (Table 3.3). Females


have been reported to mostly bite outdoors in Central Java (Boewono
and Ristiyanto, 2005; Ikawati et al., 2006; Suwasono et al., 1993, 1997;
Yunianto et al., 2002) and Lesser Sundas (Maekawa et al., 2009b), and
mostly feeding indoors in eastern Kalimantan (White, 1983). The feeding
activity also varies by location with peak biting normally occurring during
the second quarter of the night in Java and Lesser Sundas (Adrial, 2000;
Adrial et al., 2000; Barodji et al., 2003; Harbach et al., 1987; Ikawati
et al., 2006; Kirnowardoyo, 1988; Lestari et al., 2007; Maekawa et al.,
2009b; Raharjo et al., 2007; Sukowati et al., 1987; Suwasono et al.,
184 Iqbal R.F. Elyazar et al.

1993, 1997; Ustiawan and Hariastuti, 2007; Yunianto et al., 2002) and in the
third quarter of the night in Kalimantan (Boewono and Ristiyanto, 2005;
Kirnowardoyo, 1988; White, 1983). After blood feeding, An. balabacensis
generally exits houses soon afterwards to rest outdoors (Alfiah et al., 2008;
Barodji et al., 2003; Lestari et al., 2007) in shaded locations such as cattle shel-
ters (Boesri and Boewono, 2006; Boewono and Ristiyanto, 2005; Ikawati
et al., 2006; Lestari et al., 2007; Widiastuti et al., 2006), under trees
(Alfiah et al., 2008; Boewono and Ristiyanto, 2005; Harbach et al., 1987;
Kirnowardoyo, 1991; Sukowati et al., 1987; Suwasono et al., 1993;
Widiastuti et al., 2006; Yunianto et al., 2002), on embankments at heights
up to 1 m above ground level (Alfiah et al., 2008; Boewono and Ristiyanto,
2005; Lestari et al., 2007) and inside ground pits (Alfiah et al., 2008).
An. balabacensis larvae are found almost exclusively in shaded habitats
containing fresh, often clear water (Takken et al., 1990) in both natural- and
man-made habitats (Table 3.4) including stream-side rock pools
(Kirnowardoyo, 1988; Maekawa et al., 2009a; Pranoto and Munif, 1993;
White, 1983), pools found under shrubs or low trees (Boewono and
Ristiyanto, 2005; Kirnowardoyo, 1988; Lestari et al., 2007; Pranoto and
Munif, 1993; Raharjo et al., 2007; White, 1983; Yunianto et al., 2002), river
banks (Lestari et al., 2007; Suwasono et al., 1993), puddles, muddy (turbid) ani-
mal wallows, hoof prints and tyre tracks. This species is usually found associated
with hilly, forested terrain (Lestari et al., 2007; Pranoto and Munif, 1993;
Suwasono et al., 1993, 1997; White, 1983) up to 700 m asl (Suwasono
et al., 1997).

5.3. Anopheles (Anopheles) bancroftii Giles


An. bancroftii was reported from only seven sources and at only eight inde-
pendent sites from eastern Indonesia (Fig. 3.4): one site in Seram Island,
Maluku and seven sites in Papua (New Guinea Island). Five of the six ref-
erences were published before the 1960s. The single contemporary source
documented its presence in Jayapura, Papua in 2008 (Yamtama et al., 2008)
and An. bancroftii has also been encountered, but infrequently, in human-
landing collections in Timika, southern Papua (Bangs, Personal
communication, 2012). An. bancroftii was found in unusually high abun-
dance during a 1-year study in the late 1920s in Tanah Merah, in a remote
jungle environment in southern Papua. Seventy percent of approximately
10,100 collected Anopheles mosquitoes were morphologically identified as
An. bancroftii. In this high vector density area, this species was found infected
Anopheles Malaria Vector Mosquitoes in Indonesia 185

Figure 3.4 Anopheles bancroftii distribution in Indonesia. The blue stars indicate the
records of infectious An. bancroftii mosquitoes found. The yellow dots show eight
records of occurrence for this species between 1929 and 2008. Areas were defined
as no risk (light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey,
where PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk
(medium red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar
et al., 2011a). The database of distribution of An. bancroftii in Indonesia was acquired
from the references: Brug and Bonne-Wepster (1947), De Rook (1929), Elsbach (1938),
Swellengrebel and Rodenwaldt (1932), Van den Assem (1959) and Yamtama et al. (2008).

with malaria oocysts (3%, 29/1199) (De Rook, 1929). The role of
An. bancroftii in malaria transmission has been confirmed in Papua with
the identification of two mosquitoes harbouring malaria sporozoites
amongst 982 dissected in Merauke in 1957 (Van den Assem and Bonne-
Wepster, 1964). Likewise, it has been confirmed a malaria vector in the
neighbouring country of Papua New Guinea (PNG) that shares a border
with Papua, Indonesia (Cooper et al., 2009). No infective An. bancroftii have
been reported from Maluku (Table 3.2). This species has not been consid-
ered a very important malaria vector (Swellengrebel and Rodenwaldt, 1932)
despite reports of high human blood indices from specimens captured on a
bednet (Walch and Sardjito, 1928) (Table 3.3). It also appears to be partially
endophilic, with Van den Assem reporting the presence of many blood-fed
females resting inside huts in southern Papua yet none having advanced
ovarian development (Van den Assem, 1959), suggesting that females
likely leave their daytime indoor resting site the following evening post
blood meal.
186 Iqbal R.F. Elyazar et al.

Table 3.4 shows gravid females and immature stages of An. bancroftii
prefer shaded habitats with fresh, clear and still to slow running water
(Russell et al., 1946). Larvae are typically found in natural habitats, such
as marshes (Koesoemowinangoen, 1953), pools associated with creeks and
rivers (Taylor, 1943), ground pools (Taylor, 1943) or man-made habitats
including heavily shaded irrigation ditches (Koesoemowinangoen, 1953).

5.4. Anopheles (Anopheles) barbirostris van der Wulp


An. barbirostris is a member of the Barbirostris Group (Sinka et al., 2011),
made up of at least 12 species. It is a taxonomically complex assemblage that
is broadly distributed throughout the Indonesian archipelago and much of
south and Southeast Asia. An. barbirostris is currently regarded as a complex
of three to five sibling species with unclear distributions and vector status in
Southeast Asia (Paredes-Esquivel et al., 2009). In Indonesia, 119 sources have
reported the presence of An. barbirostris at 330 independent sites and it was
commonly reported from Java (140 sites), Sumatra (74 sites) and Sulawesi
(55 sites) (Fig. 3.5). The species complex has a wide distribution, extending
from Sumatra, Java, Bali, Kalimantan, Sulawesi and throughout the Lesser
Sunda Island chain to Timor (O’Connor and Sopa, 1981). An. barbirostris
has been documented in Maluku (Buru Island) but no reliable/confirmed
records of its presence in Papua (New Guinea) have been found.
An. barbirostris is medically important (malaria and filariasis) in the eastern
part of Indonesia and Sulawesi. The role of An. barbirostris as a malaria vector
was first reported in 1939 by Machsoes who examined 1041 mosquitoes in
South Sulawesi and found 30 (2.9%) with sporozoites (Machsoes, 1939). In
the early 1990s, Marwoto et al. (2002), Marwoto et al. (1992a) and Sukowati
et al. (2001) confirmed the infection of An. barbirostris with both P. falciparum
and P. vivax from specimens collected in the Lesser Sunda Island group
(Lombok and Flores) and northern Sulawesi. Both P. falciparum and
P. vivax infections were also detected in northern Sulawesi (Meras and
Tomohon), Flores (Korowuru and Tilang) and Adonara Island in the eastern
Lesser Sundas (Bangs and Rusmiarto, 2007). Cooper et al. (2010) also
detected sporozoite infective mosquitoes in neighbouring Timor-Leste
(Timor Island). This species complex has not been incriminated as a malaria
vector outside of Sulawesi and Lesser Sunda Island chain. In addition, this
species is an important vector of lymphatic filariasis in Sulawesi (Brugia
malayi) and eastern Lesser Sundas (Brugia timori) (Lim et al., 1985). Although
An. barbirostis is commonly found in Sumatra and Java, the most plausible
Anopheles Malaria Vector Mosquitoes in Indonesia 187

Figure 3.5 Anopheles barbirostris distribution in Indonesia. The blue stars indicate the
records of infectious An. barbirostris mosquitoes found. The yellow dots show 330 records
of occurrence for this species between 1918 and 2011. Areas were defined as no risk (light
grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where PfAPI < 0.1
per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium red, 5% <
PfPR2–10 < 40%) and high risk (dark red, PfPR2–10 > 40%) (Elyazar et al., 2011a). The database
of distribution of An. barbirostris in Indonesia was acquired from the references: Adrial
(2003, 2008), Adrial and Harminarti (2005), Adrial et al. (2000), Alfiah et al. (2008),
Atmosoedjono et al. (1993), Atmosoedjono et al. (1975), Bahang et al. (1981), Barbara et al.
(2011), Barodji et al. (2003), Barodji et al. (2007), Barodji et al. (1992), Barodji et al. (2004a),
Barodji et al. (2004b), Barodji et al. (1994), Barodji et al. (1998/1999), Barodji et al. (1996),
Blondine et al. (1994), Boesri (1994b), Boesri et al. (2004), Boewono et al. (1997b), Boewono
and Ristiyanto (2004, 2005), Brug (1931), Brug and Bonne-Wepster (1947), Buono, 1987,
Collins et al. (1979), Dasuki and Supratman (2005), Dharma et al. (2004), Djenal et al.
(1987), Fryauff et al. (1997), Gandahusada (1979), Garjito et al. (2004a), Garjito et al.
(2004b), Gundelfinger et al. (1975), Handayani and Darwin (2006), Hasan (2006), Idris-
Idram et al. (2002), Idris-Idram et al. (1998/1999), Idris et al. (2002), Ikawati et al. (2006),
Ikawati et al. (2004), Isfarain and Santiyo (1981), Iyana (1992), Jastal et al. (2002), Jastal
et al. (2003), Kaneko et al. (1987), Kazwaini and Martini (2006), Kurihara (1978), Lee et al.
(1983), Lee et al. (1984), Lestari et al. (2000), Lien et al. (1975), Maekawa et al. (2009a),
Maekawa et al. (2009b), Mangkoewinoto (1919), Mardiana et al. (2002), Mardiana and
Sukana (2005), Mardiana et al. (2005), Marjiyo (1996), Marwoto (1995), Marwoto et al.
(2002), Marwoto et al. (1992a), Munif (1990, 1994, 2004), Munif et al. (2007), Munif et al.
(2003), Nalim (1980a,b), Nalim (1982), Nalim (1985), Nalim and Boewono (1987), Nalim
et al. (2000), Nalim and Tribuwono (1983), Ndoen et al. (2010), Noor (2002), Nurdin et al.
(2003), Ompusunggu et al. (2006), Ompusunggu et al. (1994a), Partono et al. (1973), Priadi
et al. (1991), Raharjo et al. (2007), Raharjo et al. (2006), Ramadhani et al. (2005),
Schuurman and Huinink (1929), Self et al. (1976), Shinta et al. (2003), Sigit and
Kesumawati (1988), Soekirno et al. (2006a), Stoops et al. (2009a), Stoops et al. (2008),
Stoops et al. (2009b), Sudomo et al. (2010), Sukowati et al. (2005b), Sukowati et al. (2001),
(Continued)
188 Iqbal R.F. Elyazar et al.

reason it is not important as a malaria vector is due to its strong zoophilic


behaviour.
The first evidence that An. barbirostris in Indonesia is a complex of species
was based on analysis of mtDNA Cytochrome Oxidease I gene (COI) in
which three putative species were formally designated W, X and Z (form
Y was identified from Thailand) (Satoto, 2001). More recently, the molec-
ular phylogeny of An. barbirostris in Indonesia (COI and ITS2 data) has rev-
ealed several sympatric but distinct species clades exist in Java and Sumatra,
the precise distribution, biology and vector status of each and control impli-
cations have yet to be determined (Paredes-Esquivel et al., 2009).
Zoophilic and anthropophilic from of An. barbirostris have been reported
in Indonesia (Lien et al., 1977), behavioural traits which can greatly influ-
ence their capacity to transmit pathogens (Table 3.3). An. barbirostris females
are often found resting outdoors (Adrial, 2008; Barodji et al., 1992; Idris-
Idram et al., 1998/1999; Munif et al., 2007; Ompusunggu et al., 2006)
and are more common amongst cattle shelters than human settlements, espe-
cially in Java (Barodji et al., 1992, 2007; Ikawati et al., 2006; Maekawa et al.,
2009b; Mardiana and Sukana, 2005; Mardiana et al., 2002, 2005; Munif
et al., 2007; Takken et al., 1990; Walch and Sardjito, 1928). The HBI varies
depending on the source location of the mosquitoes with 12.6% (42/332)
from animal shelter resting collections containing human blood (Chow
et al., 1959; Noerhadi, 1960) and 20% (2/10) from indoor collections
(Walch and Sardjito, 1928). When biting humans, An. barbirostris typically
feeds outdoors (Adrial, 2008; Garjito et al., 2004b; Ikawati et al., 2006;
Jastal et al., 2001; Maekawa et al., 2009b; Mardiana and Sukana, 2005;
Munif et al., 2007; Ompusunggu et al., 1994a, 1996; Stoops et al.,
2009b; Widjaya et al., 2006) but the biting behaviour and activity of this
species will vary depending on geographic location. For example, in western
Java and central Sulawesi, females are more frequently found biting during
the first half of the night (Garjito et al., 2004b; Jastal et al., 2003; Stoops et al.,

Figure 3.5—cont’d Sukowati et al. (2002), Sulaeman (2004), Sundararaman et al.


(1957), Suparno (1983), Susana (2005), Suwasono et al. (1993), Swellengrebel (1921),
Swellengrebel and Rodenwaldt (1932), Swellengrebel and Swellengrebel-de Graaf
(1920), Syafruddin et al. (2010), Tarore (2010), Tativ and Udin (2006), Trenggono
(1985), Ustiawan and Hariastuti (2007), Widiarti et al. (1993), Widiastuti et al. (2006),
Widjaya et al. (2006), Widyastuti and Widiarti (1996), Widyastuti et al. (1995), World
Health Organization and Vector Biology and Control Research Unit 2 Semarang
(1977), Yunianto et al. (2002) and Yunianto et al. (2004).
Anopheles Malaria Vector Mosquitoes in Indonesia 189

2009b), but elsewhere these mosquitoes will typically reach biting peaks
during the third quarter of the night (24:00–03:00) (Garjito et al., 2004b;
Munif et al., 2007; Ompusunggu et al., 1994a, 1996; Widjaya et al., 2006).
The preferred larval habitat of An. barbirostris is sunlit water bodies con-
taining exclusively fresh, often clear water, with varying amounts of emer-
gent aquatic vegetation to (Table 3.4) (Takken et al., 1990) include lagoons
(Marwoto et al., 1992b; Ompusunggu et al., 1994b; Shinta et al., 2003),
marshes (Adrial, 2008; Boesri, 1994b; Church et al., 1995; Garjito et al.,
2004b; Sudomo et al., 2010; Widjaya et al., 2006), pools (Boewono and
Ristiyanto, 2005; Garjito et al., 2004a; Jastal et al., 2003; Nurdin et al.,
2003; Ompusunggu et al., 1994b, 1996, 2006; Shinta et al., 2003), slow run-
ning streams (Adrial, 2008; Church et al., 1995; Maekawa et al., 2009a;
Mardiana and Sukana, 2005; Miyagi et al., 1994; Ompusunggu et al.,
1994a,b), along river banks (Boewono and Ristiyanto, 2005; Marwoto
et al., 1992b; Nurdin et al., 2003), springs (Munif et al., 2007) and various
man-made habitats, such as rice fields (Adrial, 2008; Boewono and
Ristiyanto, 2005; Church et al., 1995; Garjito et al., 2004a,b; Idris-Idram
et al., 1998/1999; Jastal et al., 2003; Mardiana and Sukana, 2005;
Mardiana et al., 2002; Marwoto et al., 1992b; Miyagi et al., 1994; Munif
et al., 2007; Ndoen et al., 2010; Ompusunggu et al., 1994a, 1996;
Sekartuti et al., 1995a; Widjaya et al., 2006), fish ponds (Garjito et al.,
2004a; Sekartuti et al., 1995a), drainage ditches (Barodji et al., 2007;
Church et al., 1995; Garjito et al., 2004a; Idris-Idram et al., 1998/1999;
Mardiana and Sukana, 2005; Munif et al., 2007) and wells (Church et al.,
1995). An. barbirostris is broadly dispersed from the coastal plain (Jastal
et al., 2003; Marwoto et al., 1992a; Ndoen et al., 2010; Ompusunggu
et al., 1994a) to hilly terrain (Jastal et al., 2003; Ndoen et al., 2010;
Ompusunggu et al., 1994a) at altitudes up to 2000 m asl (Hoedojo, 1989).

5.5. Anopheles (Anopheles) barbumbrosus Strickland &


Chowdhury
An. barbumbrosus was documented by 13 sources at 63 independent sites in
Indonesia (Fig. 3.6). This species has been reported from almost all of the main
islands, excluding Papua, and most commonly from Sulawesi (45 sites). This
species can often be mistaken for An. barbirostris. Reid (1968) considers its dis-
tribution to be limited to western part of Indonesia (Sumatra and Java) and pen-
insular Malaysia, Thailand, India and Sri Lanka. It has been suggested that this
species is replaced by a very similar and closely related species, An. vanus, in Kali-
mantan, Sulawesi, Maluku and possibly the western tip of Papua. Nevertheless,
190 Iqbal R.F. Elyazar et al.

Figure 3.6 Anopheles barbumbrosus distribution in Indonesia. The blue stars indicate the
records of infectious An. barbumbrosus mosquitoes found. The yellow dots show 63 records
of occurrence for this species between 1932 and 2010. Areas were defined as no risk (light
grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where PfAPI < 0.1
per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium red, 5% <
PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al., 2011a). The data-
base of distribution of An. barbumbrosus in Indonesia was acquired from the references:
Bahang et al. (1981), Brug and Bonne-Wepster (1947), Buono (1987), Garjito et al. (2004a),
Idris-Idram et al. (1998/1999), Kurihara (1978), Marwoto et al. (2002), Nurdin et al. (2003),
Sulaeman (2004), Swellengrebel and Rodenwaldt (1932), Syafruddin et al. (2010), Tarore
(2010) and Van Hell (1952).

Van Hell reported a single An. barbumbrosus female containing sporozoites


amongst 21 specimens collected from South Sulawesi in 1952 (unknown if
the infection was a human malaria parasite or other primate plasmodia) (Van
Hell, 1952). No other reports are known describing the presence of sporozoites
in this species (Nurdin et al., 2003; Sekartuti et al., 1995b). To date, this species
is only regarded as a secondary malaria vector in Sulawesi (Table 3.2) and is typ-
ically found in low abundance regard human blood feeding (Bahang et al.,
1981; Garjito et al., 2004a; Marwoto et al., 2002; Sulaeman, 2004).
Like the majority of species in the subgenera Anopheles, An. barbumbrosus
shows a marked zoophilic tendency. Sulaeman reported greater numbers of
females resting in cattle shelters than human settlements (54% vs. 46%;
n ¼ 83) and a ratio of indoor to outdoor human biting of 1:6 (Sulaeman,
2004), indicating much greater exophagy. There are no known reports
on the HBI or any evidence of preferential resting habits in Sulawesi or
elsewhere.
Anopheles Malaria Vector Mosquitoes in Indonesia 191

The immature stages of An. barbumbrosus prefer a variety of habitats


including both partially shaded and sunlit fresh and slowly running water,
grass-fringed streams to stagnant water pools (Table 3.4; Takken et al.,
1990). These include natural water collections along river banks
(Nurdin et al., 2003), clear streams emerging from jungle areas
(Koesoemowinangoen, 1953; Russel et al., 1943) open grassy ravines
(Bonne-Wepster and Swellengrebel, 1953; Koesoemowinangoen, 1953)
and man-made water collections, such as rice fields (Bonne-Wepster and
Swellengrebel, 1953; Koesoemowinangoen, 1953).

5.6. Anopheles (Cellia) farauti Laveran species complex


The An. farauti complex comprises the largest complex of sibling species
(8 members) within the Punctulatus Group (Cooper et al., 2009; Sinka
et al., 2011), seven of which have been identified on the island of New
Guinea (Cooper et al., 2009). An. farauti s.s. has the widest geographic dis-
tribution of any member in the group but is restricted to the coastal areas.
Papua has been shown to contain at least five of the sibling species based on
molecular analysis (Bangs, Personal communication, 2012), including An.
hinesorum (¼An. farauti 2), a confirmed malaria vector in PNG (Cooper
et al., 2009). Unfortunately, the vast majority of studies on An. farauti s.l.
occurred before the advent of molecular (DNA) analysis techniques that
provide the ability to differentiate isomorphic (morphological identical) spe-
cies in the complex (Cooper et al., 2002). Fifteen sources were found
reporting the presence of An. farauti s.l. at 31 independent sites in Indonesia
(Fig. 3.7). Of these, 19 sites were located in Papua, where the role of this
complex in malaria transmission has been well known since the mid-
1950s when Metselaar reported a sporozoite rate of 0.8% (8/1023) near Jaya-
pura (Metselaar, 1956). This species sporozoite positive (P. falciparum and
two P. vivax variants) was found in both southern (Mapurujaya, Tipuka,
Timika, Atuka) and northern (Arso, Armopa) areas of mainland Papua from
1987 through 1999 (Bangs and Rusmiarto, 2007). Evidence of sporozoite
infection in An. farauti s.s. (P. falciparum and P. vivax) has also been reported
from Gag Island, the western-most locality in Papua (east of Halmahera
Island, northern Maluku Island chain where it is also present and regarded
a malaria vector). The complex appears to exist at relative low densities in
southern Papua (<5% of collections) compared to other members in the
Punctulatus Group (Lee et al., 1980; Van den Assem, 1959), but it can be
found in high abundance in northern Papua (>50% of collections) (Sari
et al., 2004; Slooff, 1964).
192 Iqbal R.F. Elyazar et al.

Figure 3.7 Anopheles farauti s.l. distribution in Indonesia. The blue stars indicate the
records of infectious An. farauti s.l. mosquitoes found. The yellow dots show 31 records
of occurrence for this species between 1945 and 2010. Areas were defined as no risk (light
grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where PfAPI < 0.1
per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium red, 5% <
Pf PR2–10 < 40%) and high risk (dark red, Pf PR2–10  40%) (Elyazar et al., 2011a). The data-
base of distribution of An. farauti s.l. in Indonesia was acquired from the references: Bangs
et al. (1993b), Brug and Bonne-Wepster (1947), Knight (1945), Kurihara (1978), Lee et al. (1980),
Metselaar (1956), Mulyadi (2010), Pranoto and Munif (1994), Rozeboom and Knight (1946), Sari
et al. (2004), Slooff (1964), Soekirno et al. (1997), Sutanto et al. (2003), Syafruddin et al. (2010)
and Van den Assem (1959).

The behaviour of An. farauti s.l. appears to vary by geographical location


and presumably by sibling species (note that most work on this species was con-
ducted before it was known to be a complex of sibling species). For example, a
study conducted in the coastal areas of northwestern Papua (Sorong) (Pranoto
and Munif, 1994) found the human biting ratio between indoor and outdoor
collections was 1:8, suggesting a strong exophagic tendency in that location;
whereas a longitudinal study in northeastern Papua (near Jayapura) reported
an indoor:outdoor human biting ratio of 1:3 and hence moderate or little pref-
erence in biting location (Slooff, 1964). On the coast of northwest Papua
(Pranoto and Munif, 1994) and on the northeast side of the island (Entrop near
Jayapura), biting activity peaked early in the evening hours whereas at a site
35 km away (Arso), biting was more commonly observed between the second
or third quarter of the night (Slooff, 1964). Resting behaviour may also vary by
both location and sibling species, with females from the coastal northwest of
Papua showing a preference to rest indoors immediately after feeding but
Anopheles Malaria Vector Mosquitoes in Indonesia 193

leaving the house before dawn (Pranoto and Munif, 1994). Conversely, those
in the northeast, showed a strong exophilic behaviour, with high numbers of
newly blood fed females collected in exit traps during the evening compared to
those remaining indoors (Slooff, 1964).
An. farauti s.l. larvae prefer sunlit habitats with fresh or brackish water
(Takken et al., 1990), depending on the sibling species (Table 3.4). The pri-
mary vector species in the complex, An. farauti sensu stricto, is restricted to the
coastal zones and generally prefers brackish habitats, often tolerating high
salinity levels. The larval stages of this species complex have been found in
a variety of natural habitats, including marshes, ponds and lagoons with emer-
gent vegetation (Hoedojo, 1989; Koesoemowinangoen, 1953; Lee et al.,
1980; Pranoto and Munif, 1994; Slooff, 1964; Van den Assem, 1961), large
and small streams with grassy margins and floating wood and other natural
debris (Church et al., 1995), along river banks (Hoedojo, 1989) or temporary
man- and animal-made habitats, such as borrow pits, pig-gardens, garden
pools and pools along river and stream margins (Knight, 1945; Lee et al.,
1987; Pranoto and Munif, 1994; Van den Assem, 1961), fishponds
(Pranoto and Munif, 1994) and ditches (Church et al., 1995; Pranoto and
Munif, 1994). An. farauti has also been observed in container habitats such
as discarded cans, drums, coconut shells and open canoes, as well as holes
in coral pits, wells and carb holes (Lee et al., 1987). This species complex is
found from the coastal plain (Church et al., 1995; Lee et al., 1980; Van
den Assem, 1961) to hilly and mountainous terrain (Metselaar, 1959;
Van den Assem, 1961) to altitudes up to 2250 m asl (Cooper et al., 2009;
Metselaar, 1959; Takken et al., 1990).

5.7. Anopheles (Cellia) flavirostris (Ludlow)


An. flavirostris is a member of the Minimus Subgroup (Chen et al., 2003)
and was previously considered a subspecies of the Minimus Complex;
however, molecular investigations have confirmed An. flavirostris as a valid
species. Moreover, Sinka et al. (2011) now regard all previous records of
An. minimus reported from Indonesia, the Philippines and Sabah, Malaysia
as invalid and misidentifications of An. flavirostris; therefore, data presented
here include An. minimus records. An. flavirostris was reported from
46 sources at 119 independent sites across Indonesia, most commonly from
central and southern Sulawesi (39 sites) and Java (30 sites) (Fig. 3.8),
followed but also Sumatra, Kalimantan and the Lesser Sunda Islands exten-
ding to Timor-Leste (Cooper et al., 2010). An. flavirostris has been found
194 Iqbal R.F. Elyazar et al.

infected with P. falciparum sporozoites on Sulawesi (Van Hell, 1952) and


Java (Wigati et al., 2006). In many locations in Java (Handayani and
Darwin, 2006; Lestari et al., 2000; Mardiana et al., 2002; Ndoen et al.,
2010; Stoops et al., 2009a), Lesser Sundas (Barbara et al., 2011;
Maekawa et al., 2009a; Marwoto et al., 1992a) and Sulawesi (Marwoto
et al., 2002), it has generally been reported in low abundance (<5% of
all Anopheles species collected).

Figure 3.8 Anopheles flavirostris distribution in Indonesia. The blue stars indicate the
records of infectious An. flavirostris mosquitoes found. The yellow dots show 119 records
of occurrence for this species between 1932 and 2011. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al.,
2011a). The database of distribution of An. flavirostris in Indonesia was acquired from
the references: Alfiah et al. (2008), Arianti (2004), Atmosoedjono et al. (1993), Barbara
et al. (2011), Barodji et al. (2003), Barodji et al. (1992), Barodji et al. (2004b), Barodji
et al. (1998/1999), Barodji et al. (1996), Boesri et al. (2004), Boesri and Boewono (2006),
Boewono and Ristiyanto (2004, 2005), Brug and Bonne-Wepster (1947), Dasuki and
Supratman (2005), Gandahusada (1979), Handayani and Darwin (2006), Harbach et al.
(1987), Isfarain and Santiyo (1981), Jastal et al. (2003), Kaneko et al. (1987), Kazwaini
and Martini (2006), Lestari et al. (2000), Lien et al. (1975), Maekawa et al. (2009a),
Maekawa et al. (2009b), Mardiana et al. (2002), Marwoto et al. (2002), Marwoto et al.
(1992a), Mulyadi (2010), Ndoen et al. (2010), Noor (2002), Stoops et al. (2009a), Stoops
et al. (2008), Stoops et al. (2009b), Sudomo et al. (2010), Sukowati et al. (1987),
Sukowati et al. (2001), Suwasono et al. (1993), Swellengrebel and Rodenwaldt (1932),
Syafruddin et al. (2010), Trenggono (1985), Van Hell (1952), Wigati et al. (2006) and
Yunianto et al. (2002).
Anopheles Malaria Vector Mosquitoes in Indonesia 195

An. flavirostris is typically zoophilic (Sinka et al., 2011). Greater relative


numbers of An. flavirostris mosquitoes were captured at cattle shelters than
human settlements (inside homes) in Central Java (Barodji et al., 2003;
Soekirno et al., 1983). Another study in Java reported 9% of mosquitoes
contained human blood and suggested low anthropophily (Alfiah et al.,
2008), but as only a small number of mosquitoes (n ¼ 33) were examined
and the data presented did not differentiate between indoor and outdoor
collections such a conclusion is potentially questionable (Table 3.3).
There are some apparent, albeit minor, variations in the biting habits
of An. flavirostris depending on location. For example, Barbara et al.
(2011) reported a ratio of indoor/outdoor human biting in western
Sumba Island of 1:1.2, indicating no clear preference for feeding location,
whereas an indoor/outdoor ratio of between 1:1.5 and 2.9 was reported
from Flores Island (Barodji et al., 1998/1999). In all cases, the general
tendency appears to be towards exophagy, although this preference
appears weak indicating a more ‘opportunistic’ biting habit. In coastal
Flores, biting activity has been shown to peak in the second quarter of
the night, yet in the interior of the island activity can peak during the third
quarter of the night (Barodji et al., 1998/1999). In Flores, An. flavirostris
appears endophilic, preferring to rest indoors after feeding (Barodji et al.,
1998/1999).
The immature stages of An. flavirostris are often found in shaded habitats
containing fresh and clear water (Table 3.4; Takken et al., 1990) that can
include springs (Barodji et al., 1999/2000; Jastal et al., 2003; Lestari et al.,
2007), shaded grassy edges of clear, slow-flowing small streams (Barodji
et al., 1998/1999; Barodji et al., 2007; Hoedojo, 1989; Jastal et al., 2003;
Koesoemowinangoen, 1953; Lestari et al., 2007; Maekawa et al., 2009a;
Ompusunggu et al., 1994b), pools (Barodji et al., 1999/2000; Jastal et al.,
2003; Lestari et al., 2007; Ompusunggu et al., 1994b), rice fields (Ndoen
et al., 2010; Stoops et al., 2008) and irrigation ditches (Van Hell, 1952). This
species can be found from the coastal plains (Stoops et al., 2007) to the hill
areas (Barbara et al., 2011; Maekawa et al., 2009a; Ndoen et al., 2010) up to
600 m asl (Bonne-Wepster and Swellengrebel, 1953; Swellengrebel and
Swellengrebel-de Graaf, 1919b).

5.8. Anopheles (Cellia) karwari James


Anopheles karwari is a member of the Maculatus Group and the second scarc-
est species reported here from Indonesia, present at only 36 independent sites
196 Iqbal R.F. Elyazar et al.

and identified by only seven sources (Fig. 3.9), four of which were published
prior to 1985. Sumatra had the highest number of sites, with others reported
from Java, Kalimantan, Sulawesi and Papua. An. karwari is apparently absent
from the Lesser Sundas and Maluku Island chains and infective females
have only been reported from Papua (near Jayapura) (Metselaar, 1956). Very
little is known about the bionomics of this species in Indonesia because
of its infrequent and patchy occurrence in collections. It is presumed to
be primarily zoophilic.
An. karwari larvae are found in natural- and man-made shaded habitats
containing fresh water (Table 3.4), such as marshes (Koesoemowinangoen,
1953; Taylor, 1943), small, slow-moving streams (Church et al., 1995;
Koesoemowinangoen, 1953; Swellengrebel, 1921), seepages (Church et al.,
1995; Taylor, 1943), ground and rock pools (Taylor, 1943; Van den
Assem, 1961), springs (Church et al., 1995; Koesoemowinangoen, 1953)
and irrigation canals associated with rice cultivation (Church et al., 1995;
Koesoemowinangoen, 1953; Swellengrebel, 1921; Taylor, 1943).

Figure 3.9 Anopheles karwari distribution in Indonesia. The blue stars indicate the
records of infectious An. karwari mosquitoes found. The yellow dots show 36 records
of occurrence for this species between 1932 and 2005. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al.,
2011a). The database of distribution of An. karwari in Indonesia was acquired from
the references: Brug and Bonne-Wepster (1947), De Rook (1929), Kaneko et al. (1987),
Metselaar (1957), Self et al. (1976), Susana (2005) and Swellengrebel and Rodenwaldt
(1932).
Anopheles Malaria Vector Mosquitoes in Indonesia 197

5.9. Anopheles (Cellia) kochi Dönitz


An. kochi is widely dispersed across Indonesia and is found on nearly all of the
main islands except for New Guinea (Papua). Foley et al., 2000 reported the
apparent introduction (likely by aircraft) to southern Papua but there have
been no evidence this species has established itself on the island. It has been
documented from 88 sources and 253 independent sites (Fig. 3.10). Publi-
shed accounts of its role in malaria transmission has only been confirmed in
Nias Island off the western coast of northern Sumatra (Boewono et al.,
1997a). Studies in Java (Barodji et al., 2007; Boewono and Ristiyanto,
2005; Lestari et al., 2007; Stoops et al., 2009b) and Sulawesi (Sekartuti
et al., 1995b) have been unable to detect the presence of sporozoites in
An. kochi. However, this species positive CSP-ELISA for P. falciparum
and P. vivax was found in northern Sulawesi and for P. vivax in Central Java
(Bangs and Rusmiarto, 2007).
An. kochi generally appears in low densities in human-landing collections
(Adrial, 2003; Alfiah et al., 2008; Arianti, 2004; Barbara et al., 2011; Barodji
et al., 2007; Garjito et al., 2004b; Harbach et al., 1987; Hasan, 2006;
Hoedojo, 1992, 1995; Idris-Idram et al., 1998/1999; Lee et al., 1983,
1984; Marwoto et al., 2002; Ndoen et al., 2010; Raharjo et al., 2006;
Ramadhani et al., 2005; Stoops et al., 2009b; Yunianto et al., 2004), possibly
reflecting a zoophilic feeding behaviour throughout much of its range
(Table 3.3). Indeed, females appear more common in cattle shelters than
human habitation (Adrial, 2003; Barodji et al., 1992, 2007; Garjito et al.,
2004b; Munif et al., 2007; Sulaeman, 2004). In western and eastern Java
from 554 females captured resting in cattle’s shelters, 15% contained human
blood (Chow et al., 1959; Noerhadi, 1960) and only 2.8% of 287 mosqui-
toes contained human blood from indoor collections in homes (Alfiah et al.,
2008; Walch, 1932). Human-landing catches in northern Sumatra recorded
an indoor/outdoor ratio of 1:6 (Idris et al., 2002), whereas a near equal dis-
tribution (1:1.2) was reported in Java (Stoops et al., 2009b) and 1:8 ratio in
central Sulawesi (Sulaeman, 2004), indicating a general tendency for
exophagy. An. kochi reach their peak blood-feeding activity during the first
half (second quarter) of the night (Chow et al., 1959). Their resting habits
depend on location for example, they appear more exophilic in Central Java
(Barodji et al., 1992) and endophilic in southern Java (Chow et al., 1959;
Soekirno et al., 1983).
An. kochi larvae prefer sunlit habitats with either fresh or brackish running
or stagnant, often muddy (turbid) water (Table 3.4; Takken et al., 1990).
198 Iqbal R.F. Elyazar et al.

Figure 3.10 Anopheles kochi distribution in Indonesia. The blue stars indicate the
records of infectious An. kochi mosquitoes found. The yellow dots show 253 records
of occurrence for this species between 1918 and 2011. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al.,
2011a). The database of distribution of An. kochi in Indonesia was acquired from the
references: Adrial (2003), Adrial et al. (2000), Alfiah et al. (2008), Arianti (2004),
Atmosoedjono et al. (1993), Barbara et al. (2011), Barodji et al. (2003), Barodji et al.
(2007), Barodji et al. (1992), Boesri et al. (2004), Boesri and Boewono (2006), Boewono
et al. (1997b), Boewono and Ristiyanto (2004, 2005), Brug (1931), Brug and Bonne-
Wepster (1947), Buono (1987), Dasuki and Supratman (2005), Dharma et al. (2004),
Fryauff et al. (2002), Gandahusada (1979), Garjito et al. (2004a), Garjito et al. (2004b),
Harbach et al. (1987), Hasan (2006), Hoedojo (1992, 1995), Idris-Idram et al. (2002), Idris
et al. (2002), Ikawati et al. (2006), Ikawati et al. (2004), Jastal et al. (2002), Jastal et al.
(2001), Kaneko et al. (1987), Knight (1945), Kurihara (1978), Lee et al. (1983), Lee et al.
(1984), Lestari et al. (2000), Lien et al. (1975), Maekawa et al. (2009b), Mangkoewinoto
(1919), Mardiana et al. (2002), Marjiyo (1996), Marsaulina (2002, 2008), Marwoto et al.
(2002), Marwoto et al. (1992a), Mulyadi (2010), Munif (1990, 1994), Munif et al. (2007),
Munif et al. (2003), Nalim et al. (2000), Ndoen et al. (2010), Noor (2002), Priadi et al.
(1991), Raharjo et al. (2007), Raharjo et al. (2006), Ramadhani et al. (2005), Schuurman
and Huinink (1929), Self et al. (1976), Sigit and Kesumawati (1988), Stoops et al.
(2009a), Stoops et al. (2008), Stoops et al. (2009b), Sudomo et al. (2010), Sukowati et al.
(1987), Sukowati et al. (2001), Sulaeman (2004), Suparno (1983), Susana (2005),
Suwasono et al. (1993), Swellengrebel (1921), Swellengrebel and Rodenwaldt (1932),
Swellengrebel and Swellengrebel-de Graaf (1920), Syafruddin et al. (2010), Tativ and
Udin (2006), Ustiawan and Hariastuti (2007), Van Hell (1952), Van Peenen et al. (1975),
Widiastuti et al. (2006), Widyastuti et al. (1997), World Health Organization and Vector
Biology and Control Research Unit 2 Semarang (1977), Yunianto et al. (2002) and
Yunianto et al. (2004).
Anopheles Malaria Vector Mosquitoes in Indonesia 199

Swellengrebel and Swellengrebel-de Graaf specifically noted this species as ‘[a]


true dirty water breeder’ because of its apparent preference for muddy habitats
(Swellengrebel and Swellengrebel-de Graaf, 1919a). Larval habitats also
include natural- and man-made sites such as marshes (Adrial, 2003;
Swellengrebel and Swellengrebel-de Graaf, 1919a; Taylor, 1943), springs
(Bonne-Wepster and Swellengrebel, 1953; Koesoemowinangoen, 1953;
Lestari et al., 2007; Noerhadi, 1960; Swellengrebel and Swellengrebel-de
Graaf, 1920), rice fields (Boewono and Ristiyanto, 2005; Bonne-Wepster
and Swellengrebel, 1953; Idris et al., 2002; Koesoemowinangoen, 1953;
Marsaulina, 2008; Noerhadi, 1960; Stekhoven and Stekhoven-Mayer,
1922; Swellengrebel and Swellengrebel-de Graaf, 1919a), ponds (Adrial,
2003; Bonne-Wepster and Swellengrebel, 1953; Idris et al., 2002;
Koesoemowinangoen, 1953; Mangkoewinoto, 1919; Sudomo et al., 2010;
Swellengrebel and Swellengrebel-de Graaf, 1919a), pools (Swellengrebel
and Swellengrebel-de Graaf, 1920), buffalo wallows (Bonne-Wepster and
Swellengrebel, 1953; Swellengrebel and Swellengrebel-de Graaf, 1919a),
wells (Taylor, 1943) and ditches (Bosh, 1925; Idris-Idram et al., 1998/
1999; Koesoemowinangoen, 1953; Mangkoewinoto, 1919; Swellengrebel
and Swellengrebel-de Graaf, 1919a). This species can be found from the
coastal plain (Mangkoewinoto, 1919; Stoops et al., 2007, 2009b) to hilly loca-
tions (Mangkoewinoto, 1919; Ndoen et al., 2010; Stoops et al., 2007) at alti-
tudes up to 1100 m asl (Brug, 1931). In western Java, more An. kochi
mosquitoes were found nearer coastal locations than upland areas (99% vs.
1%; n ¼ 88) (Stoops et al., 2009b).

5.10. Anopheles (Cellia) koliensis Owen


An. koliensis is a member of the diverse Punctulatus Group (Sinka et al.,
2011) which also includes the primary malaria vectors, An. punctulatus
and An. farauti complexes (Cooper et al., 2009; Rozeboom and Knight,
1946). An. koliensis occurrence has been reported from 12 sources covering
15 independent sites which were all located in Papua (Fig. 3.11). Unfor-
tunately, the vast majority of these studies took place before the advent of
molecular (DNA) analysis techniques provide the ability to differentiate
species accurately within the group because of the occurrence of over-
lapping and variable (polymorphic) morphological characters between
species (Cooper et al., 2002). Metselaar dissected 1748 mosquitoes col-
lected in Jayapura, and found 11 containing malaria sporozoites (0.63%)
(Metselaar, 1956). Pribadi et al. (1998) reported a P. vivax sporozoite rate
200 Iqbal R.F. Elyazar et al.

Figure 3.11 Anopheles koliensis distribution in Indonesia. The blue stars indicate the
records of infectious An. koliensis mosquitoes found. The yellow dots show 15 records
of occurrence for this species between 1946 and 2008. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10 > 40%) (Elyazar et al.,
2011a). The database of distribution of An. koliensis in Indonesia was acquired from
the references: Anthony et al. (1992), Bangs et al. (1993a), Bangs et al. (1996), Brug and
Bonne-Wepster (1947), Lee et al. (1980), Metselaar (1956), Pribadi et al. (1998),
Rozeboom and Knight (1946), Sari et al. (2004), Slooff (1964), Sutanto et al. (2003)
and Yamtama et al. (2008).

of 0.3% by CSP-ELISA in the Mimika area, southern Papua and an ento-


mological inoculation rate (EIR) of 0.17 infective bites/person/night. An.
koliensis appears particularly abundant in settlement areas near sago palm
and swamp forests (Lee et al., 1980; Slooff, 1964). This species has been
found infected in many locations in northern and southern Papua
(Bangs and Rusmiarto, 2007).
Lee et al. (1980) observed that due to a lack of abundance of large animals
such as cattle, buffaloes or horses in Papua, the human population is the pri-
mary host for this vector. Indeed, a high proportion of mosquitoes con-
taining human blood (74%; 126/170) have been reported from outdoor
collections (Slooff, 1964). However, before this species can be designated
as anthropophilic a well-designed host-choice experiment should be under-
taken. The feeding behaviour of An. koliensis varies depending on location.
A human indoor/outdoor biting ratio of 1:1.1 was reported from Arso,
Papua, whereas a ratio of 1:4 was found in Entrop, Papua, suggesting a
Anopheles Malaria Vector Mosquitoes in Indonesia 201

exophagic habit in some areas. In Arso, An. koliensis was the most common
vector species found biting indoors between the second and third quarters of
the night with biting occurring mainly in the first quarter of the night out-
doors. In contrast, early-biting was seen both indoors and outdoors in
Entrop. After indoor blood feeding, this species usually leaves the house very
soon afterwards to rest outdoors (Slooff, 1964).
The larval stages of An. koliensis can be found in mostly sunlit temporary
and semi-permanent sunlit habitats such as ground pools in grassland and
along the edge of jungles (Church et al., 1995; Lee et al., 1980; Van den
Assem, 1961), ditches (Anthony et al., 1992; Lee et al., 1980; Slooff,
1964), riverside ponds (Lee et al., 1980) and occasionally in pig ruts and
wallows (Anthony et al., 1992; Bangs et al., 1996) (Table 3.4). In some loca-
tions, it is often closely associated with An. farauti (Lee et al., 1987; Slooff,
1964). This species can also be found in temporary pools such as shallow
earth drains, footprints and wheel ruts, the typical habitat of An. punctulatus.
This species can be found from lowland areas (Church et al., 1995; Van
den Assem and Van Dijk, 1958) to the highlands, up to 1700 m asl
(Metselaar, 1959).

5.11. Anopheles (Cellia) leucosphyrus Dönitz


An. leucosphyrus is a member of the Leucosphyrus Subgroup (Rattanarithikul
et al., 2006). It is considered to be of epidemiological importance for malaria
transmission in forested areas of Sumatra (McArthur, 1951), reflecting its
preferred habitat. Within the Leucosphyrus complex, An. leucosphyrus is a
sister species to An. balabacensis and more recently Anopheles latens (Sallum
et al., 2007), the primary vector of zoonotic Plasmodium knowlesi between
monkeys and humans in Sarawak, Malaysia (northern Borneo) and possibly
elsewhere in Kalimantan (Indonesian Borneo). The separation of these
two species was derived from earlier cytogenetic evidence (Baimai
et al., 1988) and eventually DNA analysis (Sallum et al., 2005). An. latens
appears to be restricted to the island of Borneo. Due to confusion and
potential misidentification, Sinka et al. (2011) suggested that much of
the published literature on ‘An. leucosphyrus’ should be treated with cau-
tion, specifically where referring to An. leucosphyrus in locations other
than Sumatra.
In this current study, An. leucosphyrus was reported from eight sources at
47 independent sites including Sumatra (25 sites) and Kalimantan (16 sites)
(Fig. 3.12). However, in light of the issues raised by Sinka et al. (2011) and
202 Iqbal R.F. Elyazar et al.

the clear genetic differentiation between An. latens and An. leucosphyrus,
occupying very similar environmental conditions and the existence of
An. latens in Malaysian Borneo, these latter data cannot be confirmed. How-
ever, we suggest molecular identification should be conducted on An.
leucosphyrus specimens collected from Indonesian Kalimantan to confirm
the presence of absence of these species beyond the State of Sarawak,
Malaysia.
The bionomic information for An. leucosphyrus remains limited. Walch
(1932) found that in areas where cattle are scarce, 101 of 102 An. leucosphyrus
mosquitoes collected indoors contained human blood. However, this find-
ing may be bias sampling as the same experimental design was not repeated
in areas where cattle or other alternative blood sources were abundant.
Therefore, the conclusion of human host preference may not be valid
for all localities. Limited information exists on the vector status of

Figure 3.12 Anopheles leucosphyrus distribution in Indonesia. The blue stars indicate
the records of infectious An. leucosphyrus mosquitoes found. The yellow dots show
47 records of occurrence for this species between 1932 and 2004. Areas were defined
as no risk (light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium
grey, where PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate
risk (medium red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10 > 40%)
(Elyazar et al., 2011a). The database of distribution of An. leucosphyrus in Indonesia
was acquired from the references: Brug and Bonne-Wepster (1947), Harbach et al.
(1987), Idris-Idram et al. (1998/1999), Isfarain and Santiyo (1981), Kaneko et al. (1987),
McArthur (1951), Suparno (1983) and Swellengrebel and Rodenwaldt (1932).
Anopheles Malaria Vector Mosquitoes in Indonesia 203

An. leucosphyrus (via detection of natural malaria sporozoite infections). Har-


bach et al. in the 1980s did report a single sporozoite positive An. leucosphyrus
(tested by NAMRU-2) collected from southern Kalimantan (Harbach et al.,
1987) but the species identification is now in question and would need to be
confirmed.
Similar to all members of the complex, An. leucosphyrus prefers shaded
larval habitats within or very near forested environments and containing
fresh water (Table 3.4; Bonne-Wepster and Swellengrebel, 1953; White,
1983). Larval sites include marshes (Swellengrebel and Swellengrebel-de
Graaf, 1920), small streams (Swellengrebel and Swellengrebel-de Graaf,
1920; Taylor, 1943), seepage springs (Swellengrebel and Swellengrebel-
de Graaf, 1920), jungle pools (Mangkoewinoto, 1919; Swellengrebel
and Swellengrebel-de Graaf, 1920; Taylor, 1943), ground depressions
(Swellengrebel and Swellengrebel-de Graaf, 1920), fishponds
(Swellengrebel and Swellengrebel-de Graaf, 1920; Taylor, 1943), wheel
ruts (Swellengrebel and Swellengrebel-de Graaf, 1920) and hoof prints
(Swellengrebel and Swellengrebel-de Graaf, 1920).

5.12. Anopheles (Cellia) maculatus Theobald species


subgroup
An. maculatus s.l. belongs to the larger Maculatus Group comprised of several
subgroups in the Southeast Asian region (Harbach, 2004). The precise
relationship of the Indonesian populations remains to be clarified and
may represent as an yet undescribed species in the subgroup. Occurrence
data have been reported by 93 sources from 188 independent sites
(Fig. 3.13) throughout much of western and central Indonesia. The most
common sites were located on Java (86 sites) where this species group is
encountered relatively often in collections; elsewhere in Indonesia its biting
densities are typically very low. There is no evidence of this species being
present in Maluku or Papua. Plasmodium spp. infections of An. maculatus
have been reported in Indonesia, particularly from eastern (Venhuis,
1941) and Central Java (Wigati et al., 2006). Likewise, CSP-ELISA positive
P. falciparum and P. vivax specimens were also observed in three localities
near Jogyakarta (Kokap, Purworejo and Banjarmangu), in Central Java
and one locality in southern Sumatra (Tenang) (Bangs and Rusmiarto,
2007). This species is considered a major malaria vector in the Menoreh
Hills of Central Java (Barcus et al., 2002; Lestari et al., 2000; Wigati
et al., 2006).
204 Iqbal R.F. Elyazar et al.

Figure 3.13 Anopheles maculatus s.l. distribution in Indonesia. The blue stars indicate
the records of infectious An. maculatus s.l. mosquitoes found. The yellow dots show
188 records of occurrence for this species between 1918 and 2011. Areas were defined
as no risk (light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey,
where PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk
(medium red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar
et al., 2011a). The database of distribution of An. maculatus s.l. in Indonesia was acquired
from the references: Adrial (2003, 2008), Adrial et al. (2000), Alfiah et al. (2008), Aprianto
(2002), Ariati (2004), Atmosoedjono et al. (1993), Barbara et al. (2011), Barodji et al. (2003),
Barodji et al. (2007), Barodji et al. (1992), Barodji et al. (2004b), Barodji and Sularto (1993),
Barodji et al. (1998/1999), Blondine (2004), Blondine (2005), Blondine et al. (2003), Blondine
and Widiarti (2008), Boesri et al. (2004), Boesri and Boewono (2006), Boewono and
Ristiyanto (2004, 2005), Boewono et al. (2005), Boewono et al. (2004), Brug and Bonne-
Wepster (1947), Dasuki and Supratman (2005), Effendi (2002), Gandahusada (1979),
Garjito et al. (2004b), Handayani and Darwin (2006), Idris-Idram et al. (2002), Idris et al.
(2002), Ikawati et al. (2006), Ikawati et al. (2004), Iyana (1992), Jastal et al. (2002), Jastal
et al. (2001), Kaneko et al. (1987), Kirnowardoyo et al. (1991, 1992), Lee et al. (1984),
Lestari et al. (2000), Lien et al. (1975), Maekawa et al. (2009a), Maekawa et al. (2009b),
Mangkoewinoto (1919), Mardiana et al. (2002), Mardiana and Sukana (2005), Mardiana
et al. (2005), Marjiyo (1996), Marwoto et al. (2002), Marwoto et al. (1992a), Munif and
Pranoto (1994, 1996), Munif et al. (2007), Munif et al. (2003), Ndoen et al. (2010), Noor
(2002), Ompusunggu et al. (1994a), Pranoto and Munif (1993), Priadi et al. (1991),
Raharjo et al. (2007), Raharjo et al. (2006), Ramadhani et al. (2005), Santoso (2002), Self
et al. (1976), Setyawati (2004), Stoops et al. (2008), Stoops et al. (2009b), Sukmono
(2002), Sukowati et al. (2001), Sulaeman (2004), Suparno (1983), Susana (2005),
Suwasono et al. (1997), Suwasono et al. (1993), Swellengrebel (1921), Swellengrebel and
Rodenwaldt (1932), Swellengrebel and Swellengrebel-de Graaf (1920), Syafruddin et al.
(2010), Ustiawan and Hariastuti (2007), Van Hell (1952), Waris et al. (2004), Widiarti
et al. (2005a), Widiarti et al. (2005b), Widiastuti et al. (2006), Widyastuti et al. (2004),
Wigati et al. (2006), World Health Organization and Vector Biology and Control
Research Unit 2 Semarang (1977), Yudhastuti (2009), Yunianto et al. (2002) and
Yunianto et al. (2004).
Anopheles Malaria Vector Mosquitoes in Indonesia 205

Female An. maculatus are considered primarily zoophilic throughout


most of their range and are regularly reported as more prevalent in cattle
shelters than in human habitation (Adrial, 2008; Barodji et al., 2003,
2007; Boesri and Boewono, 2006; Boewono and Ristiyanto, 2005; Jastal
et al., 2001; Lestari et al., 2000; Mardiana et al., 2002; Noerhadi, 1960;
Noor, 2002; Ompusunggu et al., 1996; Pranoto and Munif, 1993;
Raharjo et al., 2007; Ramadhani et al., 2005; Venhuis, 1941). It has been
found biting humans both indoors (Adrial, 2008) and outdoors (Adrial,
2008; Barodji et al., 2003, 2007; Boewono and Ristiyanto, 2005; Ikawati
et al., 2006; Lestari et al., 2000; Munif et al., 2007; Pranoto and Munif,
1993; Ramadhani et al., 2005; Stoops et al., 2009b; Suwasono et al.,
1997). Blood-feeding activity varies by location but in most areas
An. maculatus generally tends to bite during the first half of night (Adrial,
2008; Barodji et al., 2003; Boesri and Boewono, 2006; Boewono and
Ristiyanto, 2005; Ikawati et al., 2006; Lestari et al., 2000; Raharjo et al.,
2007; Stoops et al., 2009b; Suwasono et al., 1997); however, an increased
biting density has been observed to occur near the early morning (dawn)
hours in Central Java (Boesri and Boewono, 2006; Suwasono et al.,
1997; Yunianto et al., 2002). After feeding indoors, An. maculatus typically
leaves the house to rest outdoors (Chow et al., 1959; Munif et al., 2007) in or
near cattle shelters (Barodji et al., 2003; Boesri and Boewono, 2006;
Handayani and Darwin, 2006; Lestari et al., 2000; Pranoto and Munif,
1993; Raharjo et al., 2007), natural ground pits and amongst bushes/low
vegetation (Handayani and Darwin, 2006), under shaded plants
(Boewono and Ristiyanto, 2005; Chow et al., 1959; Lestari et al., 2000),
under moist banks of small streams (Sundararaman et al., 1957) and in
earthen overhangs in cliff sides (Lestari et al., 2000).
The larvae of An. maculatus prefer habitats that are sunlit, containing fresh
and clear water (Table 3.4; Takken et al., 1990). Larval habitats include
stream-side rock pools (Adrial, 2008; Bonne-Wepster and Swellengrebel,
1953; Lestari et al., 2000; Pranoto and Munif, 1993), along margins of small,
slow-moving streams (Boesri and Boewono, 2006; Boewono and
Ristiyanto, 2005; Maekawa et al., 2009a; Mardiana and Sukana, 2005;
Ompusunggu et al., 1994b; Takken et al., 1990; Venhuis, 1941;
Yunianto et al., 2002), drying river beds (Russel et al., 1943), ground seep-
ages (Bonne-Wepster and Swellengrebel, 1953; Sundararaman et al., 1957;
Taylor, 1943), small pools and puddles containing turbid water
(Swellengrebel and Swellengrebel-de Graaf, 1920), natural springs (Boesri
and Boewono, 2006; Bonne-Wepster and Swellengrebel, 1953; Lestari
206 Iqbal R.F. Elyazar et al.

et al., 2000; Munif et al., 2007; Noerhadi, 1960; Raharjo et al., 2007;
Sundararaman et al., 1957; Swellengrebel and Swellengrebel-de Graaf,
1920; Yunianto et al., 2002), rice fields (Adrial, 2008; Mangkoewinoto,
1919; Mardiana and Sukana, 2005; Noerhadi, 1960; Ompusunggu et al.,
1994b; Sundararaman et al., 1957; Swellengrebel and Swellengrebel-de
Graaf, 1919c, 1920), ponds (Lestari et al., 2000; Swellengrebel and
Swellengrebel-de Graaf, 1920; Taylor, 1943) and ditches (Mardiana and
Sukana, 2005; Pranoto and Munif, 1993; Swellengrebel and
Swellengrebel-de Graaf, 1920; Takken et al., 1990). This species can be
found from the coastal plain (Jastal et al., 2001; Mardiana et al., 2002;
Ndoen et al., 2010; Swellengrebel and Swellengrebel-de Graaf, 1920) to
hilly areas (Chow et al., 1959; Lestari et al., 2000; Mangkoewinoto,
1919; Ndoen et al., 2010; Sundararaman et al., 1957; Swellengrebel and
Swellengrebel-de Graaf, 1919a, 1920) at altitudes up to 1100 m asl
(Brug, 1931).

5.13. Anopheles (Anopheles) nigerrimus Giles


An. nigerrimus is a member of the Hyrcanus Group. The presence of this spe-
cies has been reported in Indonesia by 32 sources at 91 independent sites
(Fig. 3.14). It appears more common on Sulawesi (43 sites) followed by
Sumatra, Java and Kalimantan. No evidence was found of An. nigerrimus
occurrence on the eastern islands of the Lesser Sundas or Papua and only
one report from Maluku that is likely a misidentification (O’Connor and
Sopa, 1981). An. nigerrimus is a confirmed malaria vector in Indonesia
with the first evidence of Plasmodium infection reported by Overbeek
from Palembang, South Sumatra in 1940 (Overbeek, 1940). This species
has been found infected in Sihepeng, northern Sumatra (Bangs and
Rusmiarto, 2007).
The host preference for this species is unclear. Only one study was found
to report HBI and they found only a low proportion of females (7%) con-
tained human blood amongst 236 examined from animal shelter collections
in eastern Java (Chow et al., 1959), however this could be the result of sam-
pling bias. An. nigerrimus appears to rest in cattle shelters in preference to
human habitations (Gandahusada, 1979; Garjito et al., 2004a). Where
human biting occurs, it tends to be exophagic (Boesri, 1994b; Boewono
et al., 1997b; Gandahusada, 1979; Garjito et al., 2004a; Idris et al., 2002;
Idris-Idram et al., 1998/1999). An. nigerrimus has been found to bite unusu-
ally early in the evening compared to most other malaria vectors, peaking
Anopheles Malaria Vector Mosquitoes in Indonesia 207

Figure 3.14 Anopheles nigerrimus distribution in Indonesia. The blue stars indicate the
records of infectious An. nigerrimus mosquitoes found. The yellow dots show 91 records
of occurrence for this species between 1932 and 2008. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10 > 40%) (Elyazar et al.,
2011a). The database of distribution of An. nigerrimus in Indonesia was acquired from
the references: Atmosoedjono et al. (1993), Bahang et al. (1981), Boesri (1994b), Boewono
et al. (2002b), Boewono et al. (1997b), Brug and Bonne-Wepster (1947), Buono (1987),
Gandahusada (1979), Gandahusada et al. (1983), Garjito et al. (2004a), Hasan (2006),
Idris-Idram et al. (2002), Idris-Idram et al. (1998/1999), Idris et al. (2002), Isfarain and
Santiyo (1981), Kaneko et al. (1987), Kirnowardoyo et al. (1991, 1992), Lien et al. (1975),
Marsaulina (2008), Nalim et al. (2000), Saleh (2002), Sigit and Kesumawati (1988),
Stoops et al. (2008), Supalin (1981), Suparno (1983), Swellengrebel and Rodenwaldt
(1932), Tativ and Udin (2006), Trenggono (1985), Van Hell (1952), Van Peenen et al.
(1975) and Widjaya et al. (2006).

during first quarter of the night in Sulawesi (Garjito et al., 2004a). When it is
found biting indoors (northern Sumatra), An. nigerrimus usually exits imme-
diately after feeding to rest outdoors (Idris et al., 2002).
An. nigerrimus larvae prefer sunlit habitats containing fresh and clear still
or slow running water (Table 3.4; Takken et al., 1990). Their larval sites
include lake margins (Chow et al., 1959), marshes (Koesoemowinangoen,
1953), pools (Idris-Idram et al., 1998/1999), rice fields (Idris et al., 2002;
Koesoemowinangoen, 1953; Sekartuti et al., 1995a), irrigation channels
(Koesoemowinangoen, 1953) and fishponds (Idris et al., 2002). This species
has been found along the coastal plain to hilly environments at altitudes up to
700 m asl (Stoops et al., 2007).
208 Iqbal R.F. Elyazar et al.

5.14. Anopheles (Cellia) parangensis (Ludlow)


Anopheles parangensis is a member of the Pyretophorus Series, an assemblage
of mosquitoes that represent important vectors in both Asia and Africa.
The presence of this species was reported by 12 sources at 42 independent
sites (Fig. 3.15), most commonly from Sulawesi (40 sites). One record,
published in the early 1930s, indicated its presence on Ternate, Maluku
(Swellengrebel and Rodenwaldt, 1932). The first record of An. parangensis
from Sumatra was reported by O’Connor and Sopa (1981) but with no
details on location. In 2005, this species was found in concrete pools on
Simeulue Island, Aceh, northern Sumatra (Sudomo et al., 2010). Where
present, the density of this species was lower than other biting Anopheles
species in central and southeast Sulawesi (<1%) (Bahang et al., 1981;
Garjito et al., 2004b; Widjaya et al., 2006) but higher in northern Sulawesi
(60%) (Marwoto et al., 2002). P. falciparum sporozoites have been

Figure 3.15 Anopheles parangensis distribution in Indonesia. The blue stars indicate the
records of infectious An. parangensis mosquitoes found. The yellow dots show
42 records of occurrence for this species between 1932 and 2011. Areas were defined
as no risk (light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey,
where PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk
(medium red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10 > 40%) (Elyazar
et al., 2011a). The database of distribution of An. parangensis in Indonesia was acquired
from the references: Bahang et al. (1981), Brug and Bonne-Wepster (1947), De Rook (1929),
Garjito et al. (2004a), Garjito et al. (2004b), Jastal et al. (2003), Marwoto (1995), Marwoto
et al. (2002), Nurdin et al. (2003), Sudomo et al. (2010), Swellengrebel and Rodenwaldt
(1932) and Widjaya et al. (2006).
Anopheles Malaria Vector Mosquitoes in Indonesia 209

detected by NAMRU-2 in An. parangensis from Sulawesi, near Manado


(Marwoto et al., 1996) and an EIR of 0.1 infective bites/person/night
was reported from the same locality during the epidemiological investiga-
tion (Marwoto et al., 2002).
The host preference of this species is poorly known in Indonesia and
there is no known study examining the presence of human blood in this
species. Widjaya et al. (2006) observed greater numbers of An. parangensis
females resting in cattle shelters than in houses (95% vs. 5%; n ¼ 78) in central
Sulawesi. However, in northern Sulawesi, only 41% (n ¼ 7594) of resting
An. parangensis were collected from cattle shelters, together with ratio
of 1:1.6 indoor to outdoor human-landing captures (Marwoto et al.,
2002), indicating a stronger tendency for exophagic behaviour. The larval
stages are found in sunlit habitats containing either fresh or coastal
brackish water (Table 3.4; Koesoemowinangoen, 1953) including marshes
(Koesoemowinangoen, 1953; Nurdin et al., 2003), pools (Bonne-Wepster
and Swellengrebel, 1953; Rodenwaldt, 1925) or man-made habitats, such
as fish ponds (Jastal et al., 2003; Nurdin et al., 2003; Rodenwaldt, 1925)
and ground puddles (Bonne-Wepster and Swellengrebel, 1953).

5.15. Anopheles (Cellia) punctulatus Dönitz


An. punctulatus is one of 12 members of the Punctulatus Group (Sinka et al.,
2011) which also includes the malaria vectors An. farauti s.l. and An. koliensis
(Rozeboom and Knight, 1946). An. punctulatus occurrence data were
extracted from 18 sources and 46 independent sites in Indonesia
(Fig. 3.16). The two reported locations were Papua (23 sites) and Maluku
(21 sites). In Papua, this species is a proven malaria vector of P. falciparum,
P. vivax and P. malariae (Anthony et al., 1992; Bangs et al., 1996;
Metselaar, 1956) and is an important vector in neighbouring PNG
(Cooper et al., 2009). This species has been found infected with
P. falciparum and P. vivax in both southern and northern Papua, from coastal
and lowland inland areas (Armopa, Timika, Arso, Mapurujaya and Tipuka)
and highland (Obio, near Wamena and Oksibil Valley) locations (Bangs and
Rusmiarto, 2007).
An. punctulatus was reported responsible for a malaria outbreak in the high-
lands of Papua in 1989 at an elevation of 1260 m asl (Bangs et al., 1996) where
it was the predominant species (98%; n ¼ 2577) biting humans. This species
has also been implicated in transmission in the central highlands of Papua
during a period of extreme drought period (Bangs and Subianto, 1999).
210 Iqbal R.F. Elyazar et al.

Figure 3.16 Anopheles punctulatus distribution in Indonesia. The blue stars indicate the
records of infectious An. punctulatus mosquitoes found. The yellow dots show 46 records
of occurrence for this species between 1929 and 2011. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al.,
2011a). The database of distribution of An. punctulatus in Indonesia was acquired from
the references: Anthony et al. (1992), Bangs et al. (1993b), Bangs et al. (1996), Brug and
Bonne-Wepster (1947), De Rook (1929), Kurihara (1978), Lee et al. (1980), Metselaar
(1956), Mulyadi (2010), Pribadi et al. (1998), Rozeboom and Knight (1946), Sari et al.
(2004), Slooff (1964), Suprapto (2010), Sutanto et al. (1999), Swellengrebel and
Rodenwaldt (1932), Syafruddin et al. (2010) and Yamtama et al. (2008).

An. punctulatus usually bites humans outdoors (Van den Assem and Van Dijk,
1958) but when indoor feeding does occur, peak activity is normally before
midnight (second quarter) (Bangs et al., 1996; Lee et al., 1980). After feeding,
this species will typically rest outdoors, including the exterior surfaces of house
walls and amongst surrounding vegetation (Lee et al., 1980; Slooff, 1964).
An. punctulatus larval sites are routinely sunlit containing fresh, clear or tur-
bid water (Table 3.4; Takken et al., 1990). Larvae have been sampled from
freshwater coastal marshes (Takken et al., 1990), low-lying riverine areas
(Takken et al., 1990), riverside pools (Lee et al., 1980), grasslands (Takken
et al., 1990), along jungle edges (Takken et al., 1990), pools (Lee et al.,
1980; Russel et al., 1943; Takken et al., 1990; Van den Assem, 1961; Van
den Assem and Bonne-Wepster, 1964; Van den Assem and Van Dijk,
1958), ground depressions and shallow drainage around houses (Anthony
Anopheles Malaria Vector Mosquitoes in Indonesia 211

et al., 1992), rock pools in drying stream beds (Bonne-Wepster and


Swellengrebel, 1953; Church et al., 1995), earthen drains (De Rook,
1929), footprints (Slooff, 1964; Takken et al., 1990), ditches (Anthony
et al., 1992; Lee et al., 1980), pig ruts (Anthony et al., 1992), pits with grey
turbid water (De Rook, 1929; Swellengrebel and Swellengrebel-de Graaf,
1919a) and wheel prints (Slooff, 1964; Takken et al., 1990). Lee et al.
(1987) found the most commonly recorded habitats are man-made depres-
sions (wheel ruts, road site ditches, footprints) holding water temporarily
and exposed to direct sunlight. The water is commonly without vegetation
and may be clear to muddy. Larvae have been found in water of nearly
42  C, indicating a tolerance to high temperatures (Van den Assem, 1961;
Van den Assem and Van Dijk, 1958). This species is found in the lowlands
(Lee et al., 1980; Van den Assem and Van Dijk, 1958) and in hilly and moun-
tainous terrain (Anthony et al., 1992; Slooff, 1964; Van den Assem and
Bonne-Wepster, 1964) at 1500 asl or higher (Anthony et al., 1992).

5.16. Anopheles (Anopheles) sinensis Wiedemann


Anopheles sinensis is a member of the Hyrcanus group of mosquitoes
(Harbach, 2004) and is the second member (An. nigerrimus) of the group that
is a confirmed malaria vector in Indonesia. A total of 13 sources reported the
presence of this species from 32 independent sites across Sumatra, Kaliman-
tan and Sulawesi (Fig. 3.17). An. sinensis was most commonly reported from
Sumatra (30 sites). Boewono et al. (1997a) first documented the mosquito,
including specimens with Plasmodium sporozoites, amongst 1614 examined
by head–thorax dissections in Nias, northern Sumatra. An. sinensis normally
appears in low densities compared to other Anopheles mosquito populations
(<1%) in both northern Sumatra (Idris et al., 2002; Lien et al., 1975) and
eastern Kalimantan (Buono, 1987). Although this species appears to play
a relatively minor role in malaria transmission in Indonesia, it is still recog-
nized as a primary vector in Korea and central/northern China
(Harrison, 1973).
The host preferences of An. sinensis in Indonesia is poorly known but is
assumed to be mostly zoophilic. However, studies examining the impact of
cattle presence on human feeding found that in areas with cattle, 83% of
females (n ¼ 381) contained human blood, whereas 90% (n ¼ 102) contained
human blood in areas where cattle were scarce (Walch, 1932). The author
concluded that this species remained anthropophilic even in the presence of
abundant alternative hosts. However, those mosquitoes examined were
212 Iqbal R.F. Elyazar et al.

Figure 3.17 Anopheles sinensis distribution in Indonesia. The blue stars indicate the
records of infectious An. sinensis mosquitoes found. The yellow dots show 32 records
of occurrence for this species between 1931 and 2005. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al.,
2011a). The database of distribution of An. sinensis in Indonesia was acquired from
the references: Boewono et al. (1997b), Brug (1931), Buono (1987), Fryauff et al. (2002),
Idris et al. (2002), Iyana (1992), Kaneko et al. (1987), Kirnowardoyo et al. (1991), Lien
et al. (1975), Nalim (1982), Supalin et al. (1979), Suparno (1983) and Susana (2005).

collected from indoor collections only therefore likely providing a bias sam-
pling. An. sinensis shows exophagic human biting behaviour in northern
Sumatra (Nias Island) where a 1:1.3–3.5 indoor to outdoor ratio was
reported (Boewono et al., 1997b). The feeding activity has been shown
to peak in the first quarter of night (Ave Lallemant et al., 1932) and with
a flight range of up to 1500 m (Ave Lallemant et al., 1932). We found no
information reporting the specific resting habits of An. sinensis in Indonesia;
however, elsewhere this species appears to mostly rest outdoors in animal
shelters after feeding (Beasles, 1984).
The larval stages of An. sinensis are typically found in sunlit habitats con-
taining fresh clear or stagnant water (Table 3.4; Takken et al., 1990). There is
some evidence of larvae being found in saline or brackish water
(Swellengrebel and Swellengrebel-de Graaf, 1919a, 1920) and it has also been
reported from rice fields where the water temperature exceeded 41  C, indi-
cating a tolerance to high temperatures (Beasles, 1984). Larvae have been
Anopheles Malaria Vector Mosquitoes in Indonesia 213

found along lake margins (Bonne-Wepster and Swellengrebel, 1953;


Mangkoewinoto, 1919; Takken et al., 1990), marshes (Bonne-Wepster and
Swellengrebel, 1953; Koesoemowinangoen, 1953; Mangkoewinoto, 1919;
Swellengrebel and Swellengrebel-de Graaf, 1919a, 1920; Takken et al.,
1990; Taylor, 1943), pools (Mangkoewinoto, 1919; Schuurman and
Huinink, 1929; Swellengrebel and Swellengrebel-de Graaf, 1920; Taylor,
1943), small streams (Bonne-Wepster and Swellengrebel, 1953; Schuurman
and Huinink, 1929; Swellengrebel and Swellengrebel-de Graaf, 1919a,
1920; Takken et al., 1990; Taylor, 1943), borrow pits (Bonne-Wepster
and Swellengrebel, 1953; Takken et al., 1990), fish ponds (Swellengrebel
and Swellengrebel-de Graaf, 1920; Taylor, 1943), rice fields (Brug, 1931;
Koesoemowinangoen, 1953; O’Connor, 1980; Stekhoven and Stekhoven-
Mayer, 1922; Swellengrebel, 1916; Swellengrebel and Swellengrebel-de
Graaf, 1919a,c, 1920; Takken et al., 1990; Walch, 1924), wells (Taylor,
1943) and irrigation ditches (Bonne-Wepster and Swellengrebel, 1953;
Koesoemowinangoen, 1953; Mangkoewinoto, 1919; Swellengrebel and
Swellengrebel-de Graaf, 1919a, 1920; Takken et al., 1990). An. sinensis has
been recorded in the lowlands (Swellengrebel and Swellengrebel-de Graaf,
1920) and in hilly terrain (Stekhoven and Stekhoven-Mayer, 1924) at altitudes
up to 1100 m asl (Brug, 1931).

5.17. Anopheles (Cellia) subpictus Grassi species complex


Subpictus Complex is a member of Pyretophorus Series (Harbach, 2004)
and is by far the most widely distributed species in Indonesia, being found
from Sumatra to Papua. The Subpictus Complex has been described as con-
sisting of at least four sibling species in India: A, B, C and D (Suguna et al.,
1994). Species A, B and D are generally found in fresh water habitats, while
species B appears restricted to coastal brackish water (Sinka et al., 2011). The
presence of this species complex in Indonesia was documented by 72 sources
at 204 independent sites and was more commonly reported from Java
(74 sites) than any other island (Fig. 3.18). The role of An. subpictus as a
malaria vector in Indonesia (Vector Biology and Control Research Unit)
was first confirmed in the late 1920s when Soesilo found 32 mosquitoes with
sporozoite infections amongst 164 collected (Soesilo, 1928). Sporozoite pos-
itive females have also been reported from the Sikka area (Flores Island) in
the eastern Lesser Sundas (Marwoto et al., 1992a), Sulawesi (Marwoto et al.,
1996, 2002; Van Hell, 1952) and western Lombok (Dasuki and Supratman,
2005). Results from sporozoite CSP-ELISA have found this species infected
214 Iqbal R.F. Elyazar et al.

Figure 3.18 Anopheles subpictus s.l. distribution in Indonesia. The blue stars indicate the
records of infectious An. subpictus s.l. mosquitoes found. The yellow dots show 204
records of occurrence for this species between 1932 and 2011. Areas were defined
as no risk (light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey,
where PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk
(medium red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar
et al., 2011a). The database of distribution of An. subpictus s.l. in Indonesia was acquired
from the following references: Adrial (2003, 2008), Adrial and Harminarti (2005), Ariati
et al. (2007), Arwati et al. (2007), Atmosoedjono et al. (1993), Barbara et al. (2011),
Barodji et al. (1992), Barodji et al. (2004a), Barodji et al. (2004b), Barodji et al. (1998/
1999), Barodji et al. (1999/2000), Barodji et al. (1996), Boesri et al. (2004), Boesri and
Boewono (2006), Brug and Bonne-Wepster (1947), Collins et al. (1979), Dasuki and
Supratman (2005), Dharma et al. (2004), Garjito et al. (2004b), Gundelfinger et al.
(1975), Hoedojo (1992, 19950), Idris-Idram et al. (2002), Idris et al. (2002), Isfarain and
Santiyo (1981), Jastal et al. (2003), Jastal et al. (2001), Kazwaini and Martini (2006),
Kurihara (1978), Lee et al. (1983), Lee et al. (1984), Maekawa et al. (2009a), Maekawa
et al. (2009b), Mardiana et al. (2002), Marjiyo (1996), Marwoto (1995), Marwoto et al.
(2002), Marwoto et al. (1992a), Mogi et al. (1995), Nalim et al. (2000), Ndoen et al.
(2010), Noor (2002), Nurdin et al. (2003), Ompusunggu et al. (1994a), Sari et al. (2004),
Self et al. (1976), Shinta et al. (2003), Sigit and Kesumawati (1988), Soekirno et al.
(2006a), Soekirno et al. (1997), Soekirno et al. (2006b), Stoops et al. (2009a), Stoops
et al. (2008), Stoops et al. (2009b), Sudomo et al. (2010), Sukowati et al. (2001),
Sukowati et al. (2002), Sulistio (2010), Sundararaman et al. (1957), Susana (2005),
Swellengrebel and Rodenwaldt (1932), Syafruddin et al. (2010), Tjitra et al. (1987),
Ustiawan and Hariastuti (2007), Utari et al. (2002), Van Hell (1952), Van Peenen et al.
(1975), Widiarti et al. (2005b) and Yudhastuti (2009).
Anopheles Malaria Vector Mosquitoes in Indonesia 215

in northern (Briet et al., 2003) and southern Sulawesi (Selayar Island) and the
Lesser Sundas (Lombok, Flores and Adonara) (Bangs and Rusmiarto, 2007).
An. subpictus s.l. are generally zoophilic throughout most of their range
(Sinka et al., 2011). The proportion of mosquitoes collected from indoor
and outdoor sampling on Java and Sulawesi revealed only 15% (of 2093)
containing human blood (Chow et al., 1959; Collins et al., 1979; Issaris
and Sundararaman, 1954; Noerhadi, 1960; Sundararaman et al., 1957;
Walch, 1932; Walch and Sardjito, 1928). Overall, females tend to be cap-
tured more often from cattle shelters than human houses (Adrial, 2003,
2008; Adrial and Harminarti, 2005; Dasuki and Supratman, 2005; Garjito
et al., 2004b; Mardiana et al., 2002; Noor, 2002) also suggesting stronger
zoophilic tendencies. Where An. subpictus are recorded feeding on humans,
they are generally found feeding outdoors but this can vary depending on
geographic location (Adrial, 2008; Adrial and Harminarti, 2005; Barodji
et al., 1999/2000; Garjito et al., 2004b; Noor, 2002; Ompusunggu et al.,
1994a, 1996; Sukowati et al., 2000; Sundararaman et al., 1957), for example,
in western Java and northern Sulawesi where indoor biting has been
recorded (Issaris and Sundararaman, 1954; Marwoto et al., 2002; Stoops
et al., 2009b). An. subpictus has been shown to bite primarily during the
second half of the night (Adrial, 2008; Adrial and Harminarti, 2005;
Garjito et al., 2004b; Hoedojo, 1992), although in the Lesser Sundas and
Sulawesi, this species is reported to be active during the first half of the night
(Barodji et al., 1999/2000; Collins et al., 1979; Ompusunggu et al., 1994a,
1996; Sukowati et al., 2000).
A number of studies have reported An. subpictus females as endophilic
(Adrial, 2003; Adrial and Harminarti, 2005; Barodji et al., 1999/2000;
Collins et al., 1979; Dasuki and Supratman, 2005). Resting sites include
bed nets (Adrial, 2003; Adrial and Harminarti, 2005), hanging clothes
(Adrial, 2003; Adrial and Harminarti, 2005), interior wall surfaces (Adrial,
2003; Adrial and Harminarti, 2005; Issaris and Sundararaman, 1954) and
ceiling (Issaris and Sundararaman, 1954). However, in western Sumatra, this
species has been found resting outdoors (Adrial, 2008), in bushes and under
shaded trees.
An. subpictus larvae are common in sunlit aquatic habitats containing
either fresh or brackish water (Table 3.4; Takken et al., 1990), including
tidal lagoons and coastal blocked freshwater rivers and streams (Adrial,
2003; Adrial and Harminarti, 2005; Barodji et al., 1999/2000; Garjito
et al., 2004b; Hoedojo, 1992; Marwoto et al., 1992b; Ompusunggu et al.,
1994a,b, 1996; Sekartuti et al., 1995a; Soekirno et al., 1983; Sundararaman
216 Iqbal R.F. Elyazar et al.

et al., 1957; Takken et al., 1990), marshes (Adrial, 2003; Adrial and
Harminarti, 2005; Jastal et al., 2003; Takken et al., 1990), pools (Bonne-
Wepster and Swellengrebel, 1953; Church et al., 1995; Idris-Idram et al.,
1998/1999; Sekartuti et al., 1995a; Soekirno et al., 1997; Sudomo et al.,
2010), rocky streams (Adrial, 2008; Ompusunggu et al., 1994b), mangrove
forests (Takken et al., 1990), springs (Barodji et al., 1998/1999; Shinta et al.,
2003), rice fields (Darling, 1926; Dharma et al., 2004; Idris-Idram et al.,
1998/1999; Soekirno et al., 1983; Sundararaman et al., 1957; Takken
et al., 1990), fish ponds (Adrial, 2003; Ariati et al., 2007; Idris-Idram
et al., 1998/1999; Jastal et al., 2003; Maekawa et al., 2009a; Sukowati
et al., 2000; Takken et al., 1990), borrow pits (Church et al., 1995), drains
(Church et al., 1995), furrows in gardens (Church et al., 1995), water tanks
(Adrial, 2008; Adrial and Harminarti, 2005; Barodji et al., 1998/1999;
Church et al., 1995; Mardiana et al., 2002), buffalo wallows (Adrial,
2003; Adrial and Harminarti, 2005), brackish ponds (Van den Assem and
Van Dijk, 1958), seaweed ponds (Ariati et al., 2007; Maguire et al., 2005;
Takken et al., 1990) and irrigation ditches (Idris-Idram et al., 1998/1999;
Miyagi et al., 1994; Stoops et al., 2008; Takken et al., 1990). This species
can be found primarily across coastal plains (Ariati et al., 2007; Collins
et al., 1979; Jastal et al., 2003; Marwoto et al., 2002; Miyagi et al., 1994;
Ndoen et al., 2010; Ompusunggu et al., 1994b; Soekirno et al., 1983;
Stoops et al., 2009b; Utari et al., 2002) and much less so in hilly terrain
(Ndoen et al., 2010; Stoops et al., 2007; Utari et al., 2002) up to 700 m asl
(Utari et al., 2002).

5.18. Anopheles (Cellia) sundaicus Rodenwaldt species


complex
The Sundaicus Complex belongs to the Pyretophorus Series (Sinka et al.,
2011) and represents one of the most important malaria vectors in Indonesia.
Sukowati et al. (1996, 1999) first described the cytotypes (forms) A, B and
C of the An. sundaicus complex using both cytogenetics and enzymatic anal-
ysis. Form A was collected from coastal areas in Sumatra and Java, while form
B was mainly collected in the freshwater habitats at South Tapanuli in north-
ern Sumatra (>87% of samples) with fewer found in the brackish water hab-
itats near Purworejo in Central Java (only 10% of samples). Form C was only
found at a coastal location in Asahan, northeastern Sumatra, where all three
forms were sympatric (A 48%, B 15%, C 37%) (Dusfour et al., 2004a).
An. sundaicus form D has been identified only from the Nicobar Islands
in the Indian Ocean (Nanda et al., 2004). Dusfour et al. (2007b) reported
no genetic distinction between the brackish and fresh water forms using
Anopheles Malaria Vector Mosquitoes in Indonesia 217

mitochondrial DNA markers (cytochrome oxidase I and cytochrome


b genes), suggesting they were the same species. Their ecological differences
were regarded as adaptations to the prevailing ecology of the area ranging
from strongly brackish to fresh water (Dusfour et al., 2004b). Using the same
markers validated by PCR, Dusfour et al. (2007a) and Dusfour et al. (2007b)
analysed specimens collected from Sumatra and Java and found no similarity
to sympatric forms A, B and C of Sukowati (Sukowati et al., 1999) and
proposed the presence of a new sibling species of the Sundaicus Complex
in Indonesia, designated An. sundaicus E.
The distribution of An. sundaicus s.l. has been reported throughout the
main islands of the archipelago, except Papua, from 79 sources representing
205 independent sites (Fig. 3.19). More sites reported the presence of An.
sundaicus in western Indonesia than the eastern part of the country (73%
vs. 27%). Based on these reports, the complex appears most common in
Sumatra (81 sites), followed by Java (67 sites) although this is likely
influenced by sampling frequency. It has been primarily reported from
coastal lowlands but can extend inland to slightly higher elevations, up to
altitudes of 300 m asl in western Java (Stoops et al., 2007).
The An. sundaicus complex is mainly responsible for malaria transmission
in coastal areas of Indonesia. Mangkoewinoto first identified sporozoites
amongst 31 dissected An. sundaicus s.l. in western Java in 1918
(Mangkoewinoto, 1919). Nalim et al. (2000) identified both P. falciparum
and P. vivax sporozoites in specimens collected from Lampung, southern
Sumatra. Other authors have also confirmed this species as an important
malaria vector in Java (Issaris and Sundararaman, 1954; Mangkoewinoto,
1919; Soesilo, 1928; Sundararaman et al., 1957), Sulawesi (Collins et al.,
1979; Van Hell, 1952) and the Lesser Sundas (Marwoto et al., 1992a)
(Table 3.2), and most recently in western Sumba (An. sundaicus E)
(Bangs, Personal communication, 2012). Results from CSP-ELISA have
found this species infected more often than any other species tested over
a 30-year period across Sumatra (Nias, Sihepeng, Riau/Bintan Island,
Lampung), Java (Pari Island, near Jakarta) and the Lesser Sunda Islands
(Sumbawa, Flores and Adonara Islands) (Bangs and Rusmiarto, 2007).
The females of An. sundaicus have a slightly greater tendency to bite
humans compared to domesticated animals. The compiled human blood
tests for this species revealed 54% of 5928 mosquitoes collected indoors
and outdoors from Sumatra and Java contained human blood (Collins
et al., 1979; Issaris and Sundararaman, 1954; Sundararaman et al., 1957;
Walch, 1932; Walch and Sardjito, 1928) (Table 3.3). The Sundaicus Com-
plex appears to have no clear preferential biting location, exhibiting both
218 Iqbal R.F. Elyazar et al.

Figure 3.19 Anopheles sundaicus s.l. distribution in Indonesia. The blue stars indicate
the records of infectious An. sundaicus s.l. mosquitoes found. The yellow dots show
205 records of occurrence for this species between 1917 and 2011. Areas were defined
as no risk (light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey,
where PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk
(medium red, 5% < PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar
et al., 2011a). The database of distribution of An. sundaicus s.l. in Indonesia was acquired
from the references: Adrial (2003, 2008), Adrial and Harminarti (2005), Barbara et al.
(2011), Barodji et al. (2004a), Barodji et al. (2004b), Barodji et al. (1998/1999), Barodji
et al. (1996), Blondine et al. (2004), Blondine et al. (2005), Boesri (1994a), Boewono et al.
(2002a), Boewono et al. (1997b), Brug and Bonne-Wepster (1947), Budasih (1993),
Citroen (1917), Collins et al. (1979), Dharma et al. (2004), Dusfour et al. (2007a), Dusfour
et al. (2007b), Fryauff et al. (2002), Idris-Idram et al. (2002), Idris-Idram et al. (1998/
1999), Idris et al. (2002), Isfarain and Santiyo (1981), Kaneko et al. (1987), Kazwaini and
Martini (2006), Kikuchi et al. (1997), Kirnowardoyo et al. (1993), Kirnowardoyo et al.
(1989), Kirnowardoyo et al. (1991, 1992), Kurihara (1978), Lien et al. (1975), Maekawa
et al. (2009a), Maekawa et al. (2009b), Mardiana et al. (2002), Mardiana et al. (2003),
Marjiyo (1996), Marsaulina (2002, 2008), Martono (1987), Marwoto et al. (1992a), Nalim
et al. (2000), Ndoen et al. (2010), Ompusunggu et al. (1994a), Schuurman and Huinink
(1929), Setyaningrum (2006), Shinta et al. (2003), Soekirno (1990), Soekirno et al.
(2006a), Soekirno et al. (2006b), Soemarto et al. (1980), Stoops et al. (2009a), Stoops
et al. (2008), Stoops et al. (2009b), Subagyo (2006), Sudomo et al. (2010), Sudomo et al.
(1998), Sudomo and Sukirno (1982), Sukowati et al. (2005a), Sukowati et al. (2005b),
Sulistio (2010), Sundararaman et al. (1957), Susana (2005), Swellengrebel (1921),
Swellengrebel and Rodenwaldt (1932), Swellengrebel and Swellengrebel-de Graaf (1920),
Syafruddin et al. (2010), Takagi et al. (1995), Van Hell (1952), Widiarti et al. (2005b),
Widyastuti et al. (1997), Widyastuti et al. (2004), Widyastuti and Widiarti (1992) and
Yudhastuti (2009).
Anopheles Malaria Vector Mosquitoes in Indonesia 219

endophagic and exophagic behaviours. However, a stronger exophagic


habit (>60%) has been reported from Sumatra (Adrial and Harminarti,
2005; Isfarain and Santiyo, 1981), Java (Stoops et al., 2009b;
Sundararaman et al., 1957) and western Lesser Sundas (Lombok)
(Budasih, 1993), with a slightly more endophagic habit (54%) seen in the
eastern Lesser Sundas (Barbara et al., 2011). In western Java, biting activity
has been seen to be high during both the first and last quarters of the night
(Stoops et al., 2009b), while in Central Java, feeding activity begins more
slowly, peaking during the second and third quarters of the evening
(Collins et al., 1979; Sundararaman et al., 1957). After feeding, females
may be found resting indoors on clothes, curtains and walls or outdoors,
under shaded tress, rock crevices and bushes (Adrial and Harminarti,
2005; Boesri, 1994a; Sundararaman et al., 1957). Age grading of nearly
1130 An. sundaicus s.l. mosquitoes captured in early morning resting collec-
tions indoors in Sulawesi, identified 96% as fed or gravid, suggesting most
had remained indoors after blood feeding (Collins et al., 1979).
Larvae of the Sundaicus Complex are primarily found in sunlit sites con-
taining either brackish or fresh water (Table 3.4; Dusfour et al., 2004a;
Soemarlan and Gandahusada, 1990). Sites are also generally of low acidity,
varying water depth and with the presence of vegetation (Kirnowardoyo
et al., 1991; Stoops et al., 2007), in particular floating filamentous algae. Exam-
ples include: lagoons (Adrial and Harminarti, 2005; Shinta et al., 2003;
Sudomo et al., 2010), marshes (Isfarain and Santiyo, 1981; Marsaulina,
2008; Sudomo et al., 2010), pools (Adrial and Harminarti, 2005;
Marsaulina, 2008), seasonally blocked streams (Bangs and Atmosoedjono,
1990) and man-made water collections, especially abandoned fish ponds
(Adrial and Harminarti, 2005; Isfarain and Santiyo, 1981; Marsaulina, 2008;
Sudomo et al., 2010), rice fields (Idris et al., 2002; Marsaulina, 2008;
Stoops et al., 2008) and irrigation ditches (Stoops et al., 2008).

5.19. Anopheles (Cellia) tessellatus Theobald


Anopheles tessellatus is within its own group within the Neomyzomyia Series
(Rattanarithikul et al., 2006). The presence of An. tessellatus were docu-
mented by 56 sources at 121 independent sites (Fig. 3.20). The most com-
mon sites were located on Java (39 sites). This species is found throughout
the archipelago, including isolated reports from western Papua (Sorong,
Manokwari) (O’Connor and Sopa, 1981). An. tessellatus has been confirmed
as a malaria vector in Sumatra with P. falciparum infected females identified in
220 Iqbal R.F. Elyazar et al.

Figure 3.20 Anopheles tessellatus distribution in Indonesia. The blue stars indicate the
records of infectious An. tessellatus mosquitoes found. The yellow dots show 121 records
of occurrence for this species between 1947 and 2011. Areas were defined as no risk
(light grey, where PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where
PfAPI < 0.1 per 1000 pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium
red, 5%< PfPR2–10 < 40%) and high risk (dark red, PfPR2–10  40%) (Elyazar et al., 2011a).
The database of distribution of An. tessellatus in Indonesia was acquired from the refer-
ences: Adrial et al. (2000), Atmosoedjono et al. (1993), Bahang et al. (1981), Barbara et al.
(2011), Barodji et al. (1992), Boesri et al. (2004), Boesri and Boewono (2006), Boewono
et al. (1997b), Brug and Bonne-Wepster (1947), Buono (1987), Dasuki and Supratman
(2005), Dharma et al. (2004), Djenal et al. (1987), Fryauff et al. (2002), Gandahusada
(1979), Garjito et al. (2004a), Garjito et al. (2004b), Hasan (2006), Idris-Idram et al. (2002),
Idris-Idram et al. (1998/1999), Idris et al. (2002), Isfarain and Santiyo (1981), Jastal et al.
(2002), Jastal et al. (2001), Kaneko et al. (1987), Lee et al. (1983), Lee et al. (1984),
Maekawa et al. (2009a), Mardiana et al. (2002), Mardiana and Sukana (2005), Mardiana
et al. (2005), Marjiyo (1996), Marwoto et al. (2002), Munif (1994), Munif et al. (2007), Munif
et al. (2003), Nalim (1982), Ndoen et al. (2010), Nurdin et al. (2003), Priadi et al. (1991), Self
et al. (1976), Sigit and Kesumawati (1988), Soekirno et al. (2006a), Soekirno et al. (1997),
Stoops et al. (2009a), Stoops et al. (2009b), Sudomo et al. (2010), Sukowati et al. (2001),
Sulaeman (2004), Suparno (1983), Syafruddin et al. (2010), Trenggono (1985), Van Hell
(1952), Widjaya et al. (2006) and World Health Organization and Vector Biology and
Control Research Unit 2 Semarang (1977).

Nias, northern Sumatra (Boewono et al., 1997a). No other reports were


found incriminating An. tessellatus as a malaria vector on any other of
Indonesia’s main islands.
An. tessellatus is primarily zoophilic, with the assembled records showing
that only 10% of 182 mosquitoes examined from Sumatra and Java contained
human blood (Chow et al., 1959; Noerhadi, 1960; Walch, 1932; Walch and
Anopheles Malaria Vector Mosquitoes in Indonesia 221

Sardjito, 1928). In western Lesser Sundas, where cattle are prevalent, higher
numbers of An. tessellatus were collected from cattle shelters compared to
inside houses (>90%), also suggesting stronger zoophilic tendencies. Feed-
ing behaviour varies by location with more female An. tessellatus found bit-
ing indoors (>60%) in western Java (Stoops et al., 2009b), whereas
exophagic biting appears more common in eastern Indonesia (Sulawesi
and Lombok) (Garjito et al., 2004b; Jastal et al., 2001; Maekawa et al.,
2009b; Sulaeman, 2004; Widjaya et al., 2006). Blood-feeding activity was
also seen to peak during the second quarter of the evening in Sukabumi,
western Java (Stoops et al., 2009b). In Java, females also prefer to rest out-
doors after feeding (Barodji et al., 1992; Munif et al., 2007). An. tessellatus has
been reported in greater densities in coastal compared to upland areas
(Maekawa et al., 2009b; Stoops et al., 2009b).
The larval stages of An. tessellatus can be found in shaded habitats, typ-
ically associated with slow-moving water (Table 3.4; Takken et al., 1990).
This species is usually found in fresh water, but can also tolerate relatively
high salinity (Boyd, 1949). They are also found in ground pools (Sudomo
et al., 2010), rice fields and fish ponds (Mardiana and Sukana, 2005).

5.20. Anopheles (Cellia) vagus Dönitz


An. vagus is the third species in the Indonesia list of important malaria vectors
belonging to the Pyretophorus Series. Similar to An. subpictus, it is broadly dis-
tributed throughout the main islands of the Indonesian archipelago, excluding
Papua (O’Connor and Sopa, 1981). The species is also broadly distributed
across much of Asia and it would come as no surprise if it was also a species com-
plex. The presence of this species was reported by 107 sources from 349 inde-
pendent sites (Fig. 3.21), 138 of which were found on Java, followed by 83 sites
on Sumatra. This species has been confirmed as a malaria vector (P. falciparum) in
Central Java (Purworejo, Kokap) (Wigati et al., 2006) and western Timor Island
(Kupang) (Bangs and Rusmiarto, 2007). A morphologically similar but genet-
ically different (putative) species (An. vagus genotype B) has been found infected
in neighbouring Timor-Leste on Timor Island (Cooper et al., 2010). The tax-
onomic status of this genotype remains unclear. Numerous attempts to find
Plasmodium infected An. vagus from Sumatra, Sulawesi, Maluku and other
Lesser Sunda locations has as yet failed to detect the presence of malaria parasites
(Bangs and Rusmiarto, 2007; Boesri, 1994b; Boewono and Nalim, 1996;
Cooper et al., 2010; Hoedojo, 1992, 1995; Lien et al., 1975; Marwoto
et al., 1992a; Nurdin et al., 2003; Soekirno et al., 1997).
222 Iqbal R.F. Elyazar et al.

Figure 3.21 Anopheles vagus distribution in Indonesia. The blue stars show the records of
infectious An. vagus mosquitoes found. The yellow dots show 349 records of occurrence for
this species between 1931 and 2011. Areas were defined as no risk (light grey, where
PfAPI ¼ 0 per 1000 pa), unstable transmission (medium grey, where PfAPI< 0.1 per 1000
pa), low risk (light red, PfPR2–10  5%), intermediate risk (medium red, 5% < PfPR2–10 < 40%)
and high risk (dark red, PfPR2–10  40%) (Elyazar et al., 2011a). The database of distribution of
An. vagus in Indonesia was acquired from the references: Adrial et al. (2000), Alfiah et al. (2008),
Aprianto (2002), Arianti (2004), Atmosoedjono et al. (1993), Atmosoedjono et al. (1975), Bahang
et al. (1981), Barodji et al. (2003), Barodji et al. (2007), Barodji et al. (1992), Barodji et al. (1998/
1999), Blondine et al. (1992), Blondine et al. (1996), Boesri (1994b), Boesri et al. (2004), Boesri and
Boewono (2006), Boewono and Ristiyanto (2004, 2005), Brug (1931), Brug and Bonne-Wepster
(1947), Buono (1987), Dasuki and Supratman (2005), Dharma et al. (2004), Effendi (2002),
Gandahusada (1979), Garjito et al. (2004a), Garjito et al. (2004b), Handayani and Darwin
(2006), Haryanto et al. (2002), Hasan (2006), Hoedojo (1992, 1995), Idris-Idram et al. (1998/
1999), Ikawati et al. (2006), Ikawati et al. (2004), Isfarain and Santiyo (1981), Iyana (1992),
Jastal et al. (2002), Jastal et al. (2001), Kaneko et al. (1987), Kazwaini and Martini (2006),
Kurihara (1978), Lee et al. (1983), Lee et al. (1984), Lestari et al. (2000), Lien et al. (1975),
Maekawa et al. (2009a), Mardiana et al. (2002), Mardiana and Sukana (2005), Mardiana
et al. (2005), Marjiyo (1996), Marwoto et al. (2002), Marwoto et al. (1992a), Mulyadi (2010),
Munif (1990, 1994), Munif et al. (2007), Munif et al. (2003), Nalim, 1980a,b, Nalim (1982),
Ndoen et al. (2010), Noerhadi (1960), Noor (2002), Nurdin et al. (2003), Ompusunggu et al.
(2006), Ompusunggu et al. (1994a), Partono et al. (1973), Priadi et al. (1991), Raharjo et al.
(2007), Raharjo et al. (2006), Santoso (2002), Self et al. (1976), Shinta et al. (2003), Sigit and
Kesumawati (1988), Soekirno et al. (2006a), Soekirno et al. (1997), Stoops et al. (2009a),
Stoops et al. (2008), Stoops et al. (2009b), Sudomo et al. (2010), Sukmono (2002), Sukowati
et al. (2001), Sulaeman (2004), Sundararaman et al. (1957), Suparno (1983), Susana (2005),
Suwasono et al. (1993), Swellengrebel and Rodenwaldt (1932), Syafruddin et al. (2010), Tativ
and Udin (2006), Trenggono (1985), Ustiawan and Hariastuti (2007), Van Hell (1952), Waris
et al. (2004), Widiarti et al. (1993), Widiastuti et al. (2006), Widjaya et al. (2006), Wiganti et al.
(2010), Wigati et al. (2006), Windarso et al. (2008), World Health Organization and Vector
Biology and Control Research Unit 2 Semarang (1977), Yunianto et al. (2002) and Yunianto
et al. (2004).
Anopheles Malaria Vector Mosquitoes in Indonesia 223

An. vagus is predominately a zoophilic, exophagic and exophilic species. It


is often found in very high densities compared to other local anophelines. The
combined proportion of mosquitoes having human blood was reported at
45% (820/1806) from studies in Sumatra and Java (Alfiah et al., 2008;
Chow et al., 1959; Noerhadi, 1960; Walch, 1932). In areas where cattle
are readily available hosts, An. vagus is typically found in much higher propor-
tions resting in cattle shelters rather than human structures; for example, in
Central Java (95%) (Barodji et al., 1992), Central Sulawesi (87%) (Garjito
et al., 2004b; Jastal et al., 2001) and Lesser Sundas (99%) (Maekawa et al.,
2009b). More An. vagus were captured at outdoor than indoor locations in
Java (85% of 5212 mosquitoes), Sulawesi (71% of 477) (Barodji et al.,
1992; Hasan, 2006; Stoops et al., 2009b) and Lesser Sundas (78% of 419)
(Maekawa et al., 2009b). In western Java (Stoops et al., 2009b), An. vagus
females blood fed throughout the night, whereas in eastern Java (Chow
et al., 1959), a clear peak was seen in the second quarter of night. Significantly,
more mosquitoes were found resting outdoors than indoors in Central Java
(64% vs. 36%; n ¼ 6982) (Barodji et al., 1992), in ground pits and tree poles
in ‘salak’ (Salacca zallaca) plantations (Alfiah et al., 2008), low bushes
(Handayani and Darwin, 2006), cattle shelters (Handayani and Darwin,
2006) and grassy ditches (Idris-Idram et al., 1998/1999).
An. vagus larval habitats are typically sunlit, containing fresh, stagnant, shal-
low water (Table 3.4). Natural habitats include still margins of streams (Taylor,
1943), river edges (Maekawa et al., 2009a; Schuurman and Huinink, 1929),
small pools near beaches (Lestari et al., 2007; Sudomo et al., 2010) and springs
(Noerhadi, 1960; Raharjo et al., 2007; Shinta et al., 2003). Larvae also can be
found in many man-made habitats, such as rice fields (Boewono and
Ristiyanto, 2005; Brug, 1931; Darling, 1926; Idris-Idram et al., 1998/
1999; Mardiana and Sukana, 2005; Marwoto et al., 1992b; Miyagi et al.,
1994; Sekartuti et al., 1995a), irrigation ditches (Barodji et al., 2007; Idris-
Idram et al., 1998/1999; Mardiana and Sukana, 2005) wheel ruts (Idris-
Idram et al., 1998/1999; Russel et al., 1943) and a variety of artificial con-
tainers such as tyres, drums and upturned small boats. A 12-month longitudi-
nal survey in Sukabumi, West Java recorded the presence of larvae in 464
aquatic habitats, mostly in the lowlands, close to human habitation, and con-
taining water of low salinity and warm temperatures (Stoops et al., 2007). This
species can often be found in great abundance from the coastal plain to low
hilly areas, but predominantly associated with hillside rice fields (<140 m ele-
vation) than coastal areas (95% vs. 5%) (Ndoen et al., 2010). An. vagus had
been found at altitudes up to 1100 m asl in eastern Java (Brug, 1931).
Table 3.1 Natural Plasmodium species infections of Anopheles mosquitoes in Indonesia
Oocyst detection Sporozoite detection Plasmodium infection
Number of Number of Number of Number of
Year of mosquitoes mosquitoes Oocyst mosquitoes mosquitoes with Sporozoite P. P. P. P.
Species samples examined with oocysts rate (%) examined sporozoites rate (%) falciparum vivax malariae ovale
An. aconitus 1917–2007 8827 115 1.30 17,554 15 0.09 Yes Yes ? ?
An. aitkenii 1919 2 0 0.00 – – –
An. 1918–1931 48 0 0.00 6 0 0.00
albotaeniatus
An. 1917–2007 1229 2 0.16 489 0 0.00
annularis
An. 1982–2005 – – – 2348 111 4.73 Yes Yes ? ?
balabacensis
An. 1928–2002 1119 29 2.59 983 2 0.20 ? ? ? ?
bancroftii
An. 1917–2007 6750 314 4.65 9568 91 0.95 Yes Yes ? ?
barbirostris
An. 1940–2002 19 1 5.26 22 1 4.55 ? ? ? ?
barbumbrosus
An. crawfordi 1994–2002 – – – 773 0 0.00
An. farauti 1922–1979 1093 57 5.22 1199 12 1.00 Yes Yes ? ?
An. 1938–2007 53 5 9.43 2175 2 0.09 Yes ? ? ?
flavirostris
An. hyrcanus 1918–1929 30,055 682 2.27 – – –
An. 1917–2007 2413 1 0.04 173 0 0.00
indefinitus
An. karwari 1919–1955 65 0 0.00 685 6 0.88 ? ? ? ?
An. kochi 1917–2007 7223 89 1.23 2967 1 0.03 Yes Yes ? ?
An. koliensis 1947–1994 – – – 2616 24 0.92 ? Yes ? ?
An. 1918–1986 3757 324 8.62 89 1 1.12 Yes ? ? ?
leucosphyrus
An. 1953–1964 – – – 7 0 0.00
longirostris
An. 1918–2007 1289 26 2.02 3504 6 0.17 Yes Yes ? ?
maculatus
An. 1932–1995 5074 483 9.52 3443 11 0.32 ? ? ? ?
nigerrimus
An. 1939–2002 2 0 0.00 688 6 0.87 Yes ? ? ?
parangensis
An. 1934–2007 – – – 594 0 0.00
peditaeniatus
Continued
Table 3.1 Natural Plasmodium species infections of Anopheles mosquitoes in Indonesia—cont'd
Oocyst detection Sporozoite detection Plasmodium infection
Number of Number of Number of Number of
Year of mosquitoes mosquitoes Oocyst mosquitoes mosquitoes with Sporozoite P. P. P. P.
Species samples examined with oocysts rate (%) examined sporozoites rate (%) falciparum vivax malariae ovale
An. 1917–1992 1614 1 0.06 10,501 117 1.11 Yes Yes Yes ?
punctulatus
An. sinensis 1917–1998 29,489 612 2.08 2733 1 0.04 ? ? ? ?
An. 1918–2007 32,698 130 0.40 88,784 160 0.18 Yes Yes ? ?
subpictus
An. 1917–2007 49,649 1469 2.96 56,624 169 0.30 Yes Yes ? ?
sundaicus
An. 1919–2007 2266 4 0.18 838 10 1.18 Yes ? ? ?
tessellatus
An. 1918–1921 257 16 6.23 25 0 0.00
umbrosus
An. vagus 1919–2007 10,303 2 0.02 6844 7 0.10 Yes ? ? ?
The ‘?’ mark denotes where the Plasmodium species infecting the mosquitoes is unknown. In some instances, the observed oocysts and sporozoites upon dissection might have been derived
from non-human primate or rodent hosts. ‘Yes’ indicate parasite species was identified using CSP-ELISA.
The database of susceptibility of Anopheles mosquitoes to Plasmodium spp. infections in Indonesia was acquired from the following references:
An. aconitus: Bangs and Rusmiarto (2007), Barodji et al. (2007), Boesri et al. (2004), Boesri and Boewono (2006), Boewono and Ristiyanto (2005), Bosh (1925), Doorenbos (1927, 1931),
Hoedojo (1995), Kirnowardoyo et al. (1985), Lestari et al. (2007), Lien et al. (1975), Mangkoewinoto (1919), Marwoto et al. (1992a), Nalim et al. (2000), Schuurman and Huinink (1929),
Soesilo (1935), Stoops et al. (2009b), Sundararaman et al. (1957), Swellengrebel and Rodenwaldt (1932), Swellengrebel et al. (1919) and Walch and Walch-Sordrager (1922).
An. aitkenii: Swellengrebel and Swellengrebel-de Graaf (1920).
An. albotaeniatus: Doorenbos (1927, 1931), Mangkoewinoto (1919), Swellengrebel and Rodenwaldt (1932) and Walch and Walch-Sordrager (1922).
An. annularis: Barodji et al. (2007), Boesri (1994b), Boesri and Boewono (2006), Hoedojo (1992, 1995), Kirnowardoyo et al. (1985), Lien et al. (1975), Maekawa et al. (2009b),
Schuurman and Huinink (1929), Soesilo (1935), Stoops et al. (2009b), Swellengrebel and Rodenwaldt (1932), Swellengrebel and Swellengrebel-de Graaf (1920) and Venhuis (1941).
An. balabacensis: Adrial et al. (2000), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono and Ristiyanto (2005), Harbach et al. (1987), Lestari et al. (2007), Maekawa et al.
(2009b), Pranoto and Prasetyo (1990), White (1983) and Wigati et al. (2006).
An. bancroftii: De Rook (1929), Metselaar (1956), Nurdin et al. (2003) and Van den Assem and Bonne-Wepster (1964).
An. barbirostris: Barodji et al. (2007), Boesri (1994b), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono et al. (1997a), Boewono and Ristiyanto (2005), Collins et al.
(1979), Doorenbos (1927, 1931), Gundelfinger et al. (1975), Kirnowardoyo et al. (1985), Lestari et al. (2007), Lien et al. (1975), Machsoes (1939), Maekawa et al. (2009b),
Mangkoewinoto (1919), Marwoto et al. (2002), Marwoto et al. (1992a), Marwoto et al. (1996), Nalim et al. (2000), Nurdin et al. (2003), Soesilo (1935), Stoops et al. (2009b),
Sukowati et al. (2001), Sukowati et al. (2002), Swellengrebel and Rodenwaldt (1932), Swellengrebel et al. (1919), Swellengrebel and Swellengrebel-de Graaf (1920), Van Hell
(1952), Venhuis (1941), Walch and Walch-Sordrager (1922) and Widjaya et al. (2006).
An. barbumbrosus: Nurdin et al. (2003), Sekartuti et al. (1995b) and Van Hell (1952).
An. crawfordi: Boewono and Nalim (1996), Boewono et al. (1997a), Fryauff et al. (2002) and Nurdin et al. (2003).
An. farauti: Bangs and Rusmiarto (2007), Boyd (1949), De Rook (1929), Lee et al. (1980), Metselaar (1956), Swellengrebel and Rodenwaldt (1932) and Van den Assem and Bonne-
Wepster (1964).
An. flavirostris: Barodji et al. (1998/1999), Boewono and Ristiyanto (2005), Lestari et al. (2007), Maekawa et al. (2009b), Overbeek and Stoker (1938), Stoops et al. (2009b), Sundararaman
et al. (1957), Van Hell (1952), Venhuis (1941) and Wigati et al. (2006).
An. hyrcanus: Swellengrebel and Rodenwaldt (1932).
An. indefinitus: Boewono and Nalim (1996), Kirnowardoyo et al. (1985), Maekawa et al. (2009b), Stoops et al. (2009b), Swellengrebel et al. (1919) and Walch and Walch-Sordrager
(1922).
An. karwari: Metselaar (1956), Swellengrebel and Swellengrebel-de Graaf (1920) and Van den Assem and Bonne-Wepster (1964).
An. kochi: Bangs and Rusmiarto (2007), Barodji et al. (2007), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono et al. (1997a), Boewono and Ristiyanto (2005), Bosh
(1925), Doorenbos (1927, 1931), Fryauff et al. (2002), Hoedojo (1992, 1995), Kirnowardoyo et al. (1985), Lestari et al. (2007), Lien et al. (1975), Nalim et al. (2000), Sekartuti et al.
(1995b), Soekirno et al. (1997), Soesilo (1935), Stoops et al. (2009b), Sundararaman et al. (1957), Swellengrebel and Rodenwaldt (1932), Swellengrebel et al. (1919), Swellengrebel and
Swellengrebel-de Graaf (1920), Venhuis (1941) and Walch and Walch-Sordrager (1922).
An. koliensis: Bangs et al. (1996), Church et al. (1995), Lee et al. (1980), Metselaar (1956), Pribadi et al. (1998) and Van den Assem and Bonne-Wepster (1964).
An. leucosphyrus: Bosh (1925), Doorenbos (1927), Harbach et al. (1987), Machsoes (1939), McArthur (1951), Swellengrebel and Rodenwaldt (1932), Swellengrebel and Swellengrebel-de
Graaf (1920) and Van Hell (1952).
An. longirostris: Metselaar (1956) and Van den Assem and Bonne-Wepster (1964).
An. maculatus: Barodji et al. (2007), Boesri and Boewono (2006), Boewono and Ristiyanto (2005), Doorenbos (1927, 1931), Kirnowardoyo et al. (1985), Kirnowardoyo et al. (1991),
Lestari et al. (2007), Lien et al. (1975), Maekawa et al. (2009b), Marwoto et al. (1992a), Stoops et al. (2009b), Sundararaman et al. (1957), Swellengrebel and Rodenwaldt (1932),
Swellengrebel and Swellengrebel-de Graaf (1920), Venhuis (1941) and Wigati et al. (2006).
An. nigerrimus: Bangs and Rusmiarto (2007), Boesri (1994b), Boewono and Nalim (1996), Boewono et al. (1997a), Kirnowardoyo et al. (1991), Nalim et al. (2000), Overbeek (1940),
Sundararaman et al. (1957), Swellengrebel and Rodenwaldt (1932), Van Hell (1952) and Venhuis (1941).
An. parangensis: Machsoes (1939), Marwoto et al. (2002), Marwoto et al. (1996) and Nurdin et al. (2003).
An. peditaeniatus: Boewono and Nalim (1996), Boewono et al. (1997a), Lien et al. (1975), Soesilo (1935) and Stoops et al. (2009b).
An. punctulatus: Bangs et al. (1996), Church et al. (1995), Doorenbos (1927, 1931), Lee et al. (1980), Metselaar (1956), Pribadi et al. (1998), Swellengrebel et al. (1919), Swellengrebel and
Swellengrebel-de Graaf (1920), Van den Assem and Bonne-Wepster (1964) and Walch and Walch-Sordrager (1922).
Continued
An. sinensis: Boewono and Nalim (1996), Boewono et al. (1997a), Bosh (1925), Doorenbos (1931), Fryauff et al. (2002), Lien et al. (1975), Mangkoewinoto (1919), Schuurman and
Huinink (1929), Soesilo (1935), Sundararaman et al. (1957), Swellengrebel et al. (1919), Swellengrebel and Swellengrebel-de Graaf (1920), Walch and Walch-Sordrager (1922) and Walch
(1924).
An. subpictus: Barodji et al. (1999/2000), Boesri and Boewono (2006), Collins et al. (1979), Dasuki and Supratman (2005), Gundelfinger et al. (1975), Hoedojo (1992, 1995), Issaris and
Sundararaman (1954), Machsoes (1939), Maekawa et al. (2009b), Mangkoewinoto (1919), Marwoto et al. (2002), Marwoto et al. (1992a), Marwoto et al. (1996), Nalim et al. (2000),
Nurdin et al. (2003), Sekartuti et al. (1995b), Soekirno et al. (1997), Soesilo (1928, 1935), Sukowati et al. (2001), Sundararaman et al. (1957), Swellengrebel and Rodenwaldt (1932),
Swellengrebel et al. (1919), Swellengrebel and Swellengrebel-de Graaf (1920) and Van Hell (1952).
An. sundaicus: Boewono and Nalim (1996), Boewono et al. (1997a), Bosh (1925), Collins et al. (1979), Doorenbos (1931), Fryauff et al. (2002), Isfarain and Santiyo (1981), Isfarain and
Santyo (1981), Issaris and Sundararaman (1954), Kirnowardoyo et al. (1991), Lien et al. (1975), Maekawa et al. (2009b), Mangkoewinoto (1919), Marwoto et al. (1992a), Nalim et al.
(2000), Overbeek and Stoker (1938), Soesilo (1928), Stoops et al. (2009b), Sundararaman et al. (1957), Swellengrebel and Rodenwaldt (1932), Swellengrebel et al. (1919), Swellengrebel
and Swellengrebel-de Graaf (1920), Takken et al. (1990), Van Breemen and Sunier (1919), Van Hell (1952) and Walch (1924).
An. tessellatus: Bangs and Rusmiarto (2007), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono et al. (1997a), Fryauff et al. (2002), Kirnowardoyo et al. (1985),
Machsoes (1939), Maekawa et al. (2009b), Nurdin et al. (2003), Schuurman and Huinink (1929), Soekirno et al. (1997), Soesilo (1935), Stoops et al. (2009b), Sundararaman et al.
(1957), Swellengrebel and Rodenwaldt (1932) and Venhuis (1941).
An. umbrosus: Bosh (1925), Doorenbos (1927, 1931), Kirnowardoyo et al. (1991), Mangkoewinoto (1919), Overbeek and Stoker (1938), Swellengrebel and Rodenwaldt (1932) and
Takken et al. (1990).
An. vagus: Barodji et al. (2007), Boesri (1994b), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono and Ristiyanto (2005), Doorenbos (1927, 1931), Hoedojo (1992,
1995), Kirnowardoyo et al. (1985), Lestari et al. (2007), Lien et al. (1975), Maekawa et al. (2009b), Marwoto et al. (1992a), Nurdin et al. (2003), Soekirno et al. (1997), Soesilo (1935),
Stoops et al. (2009b), Swellengrebel and Rodenwaldt (1932), Venhuis (1941), Wiganti et al. (2010) and Wigati et al. (2006).
Table 3.2 Natural sporozoite infections of Anopheles mosquitoes from the main islands in Indonesiaa
Western Indonesia Eastern Indonesia
Year of
Species samples Sumatra Java/Bali Kalimantan Sulawesi Maluku Lesser Sundas Papua
An. aconitus 1918–2007 0/15 15/17,463 – – – 0/76 –
(0.08)
An. 1918 – 0/6 – – – – –
albotaeniatus
An. annularis 1934–2007 0/107 0/283 – – – 0/99 –
An. balabacensis 1982–2005 – 4/138 (2.9) 60/1449 – – 47/761 (6.18) –
(4.14)
An. bancroftii 1954–2002 – – – – – – 2/983 (0.2)
An. barbirostris 1918–2007 0/119 0/3263 – 82/6063 (1.35) – 9/123 (7.32) –
An. 1940–2002 – – – 1/22 (4.55) – – –
barbumbrosus
An. crawfordi 1994–2002 0/773 – – – – – –
An. farauti 1953–1979 – – – – – – 12/1199 (1.0)
An. flavirostris 1941–2007 – 1/1452 (0.07) – 1/60 (1.67) – 0/663 –
An. indefinitus 1982–2007 – 0/133 – – – 0/40 –
An. karwari 1953–1955 – – – – – – 6/685 (0.88)
An. kochi 1934–2007 1/1025 (0.1) 0/1864 – 0/1 0/75 0/2 –
Continued
Table 3.2 Natural sporozoite infections of Anopheles mosquitoes from the main islands in Indonesia—cont'd
Western Indonesia Eastern Indonesia
Year of
Species samples Sumatra Java/Bali Kalimantan Sulawesi Maluku Lesser Sundas Papua
An. koliensis 1947–1994 – – – – – – 24/2616 (0.92)
An. 1938–1986 – – 1/85 (1.18) 0/4 – – –
leucosphyrus
An. longirostris 1953–1964 – – – – – – 0/7
An. maculatus 1941–2007 0/13 6/3270 (0.18) – – – 0/213 –
An. nigerrimus 1939–1995 3/555 (0.54) 0/1343 – 8/1545 (0.52) – – –
An. parangensis 1939–2002 – – – 6/688 (0.87) – – –
An. 1934–2007 0/258 0/336 – – – – –
peditaeniatus
An. punctulatus 1953–1992 – – – – – – 117/10,501
(1.11)
An. sinensis 1919–1998 1/1660 0/1072 – 0/1 – – –
(0.06)
An. subpictus 1918–2007 0/678 52/69,702 – 19/12,996 0/219 89/5189 –
(0.07) (0.15) (1.72)
An. sundaicus 1918–2007 6/1480 128/51,701 – 16/1635 – 19/1808 –
(0.41) (0.25) (0.98) (1.05)
An. tessellatus 1934–2007 10/455 (2.2) 0/283 – 0/1 0/39 0/60 –
An. umbrosus 1918–1991 0/23 0/2 – – – – –
An. vagus 1934–2007 0/92 7/6562 (0.11) – – 0/104 0/86 –
a
Sporozoite infections based on dissections only are presumed to be of human origin, but in some instances (e.g. forest/forest-fringe dwelling vectors) might have been derived from
non-human primate or rodent hosts.
The bold values emphasize the finding of natural sporozoite infections in mosquitoes which confirms the role of those species as malaria vectors.
The database of natural sporozoite infections of Anopheles mosquitoes to Plasmodium spp. infections in Indonesia was acquired from the following references:
An. aconitus: Bangs and Rusmiarto (2007), Barodji et al. (2007), Boesri et al. (2004), Boesri and Boewono (2006), Boewono and Ristiyanto (2005), Doorenbos (1931), Hoedojo (1995),
Kirnowardoyo et al. (1985), Lestari et al. (2007), Lien et al. (1975), Mangkoewinoto (1919), Marwoto et al. (1992a), Nalim et al. (2000), Soesilo (1935), Stoops et al. (2009b) and
Sundararaman et al. (1957).
An. albotaeniatus: Mangkoewinoto (1919).
An. annularis: Barodji et al. (2007), Boesri (1994b), Boesri and Boewono (2006), Hoedojo (1992, 1995), Kirnowardoyo et al. (1985), Lien et al. (1975), Maekawa et al. (2009b), Soesilo
(1935), Stoops et al. (2009b) and Venhuis (1941).
An. balabacensis: Adrial et al. (2000), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono and Ristiyanto (2005), Harbach et al. (1987), Lestari et al. (2007), Maekawa
et al. (2009b), Pranoto and Prasetyo (1990), White (1983) and Wigati et al. (2006).
An. bancroftii: Metselaar (1956), Nurdin et al. (2003) and Van den Assem and Bonne-Wepster (1964).
An. barbirostris: Barodji et al. (2007), Boesri (1994b), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono et al. (1997a), Boewono and Ristiyanto (2005), Collins et al.
(1979), Kirnowardoyo et al. (1985), Lestari et al. (2007), Lien et al. (1975), Machsoes (1939), Maekawa et al. (2009b), Mangkoewinoto (1919), Marwoto et al. (2002), Marwoto et al.
(1992a), Marwoto et al. (1996), Nalim et al. (2000), Nurdin et al. (2003), Soesilo (1935), Stoops et al. (2009b), Sukowati et al. (2001), Sukowati et al. (2002), Van Hell (1952), Venhuis
(1941) and Widjaya et al. (2006).
An. barbumbrosus: Nurdin et al. (2003), Sekartuti et al. (1995b) and Van Hell (1952).
An. crawfordi: Boewono and Nalim (1996), Boewono et al. (1997a), Fryauff et al. (2002) and Nurdin et al. (2003).
An. farauti: Bangs and Rusmiarto (2007), Lee et al. (1980), Metselaar (1956) and Van den Assem and Bonne-Wepster (1964).
An. flavirostris: Barodji et al. (1998/1999), Boewono and Ristiyanto (2005), Lestari et al. (2007), Maekawa et al. (2009b), Stoops et al. (2009b), Sundararaman et al. (1957), Van Hell
(1952), Venhuis (1941) and Wigati et al. (2006).
An. indefinitus: Boewono and Nalim (1996), Kirnowardoyo et al. (1985), Maekawa et al. (2009b) and Stoops et al. (2009b).
An. karwari: Metselaar (1956) and Van den Assem and Bonne-Wepster (1964).
An. kochi: Bangs and Rusmiarto (2007), Barodji et al. (2007), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono et al. (1997a), Boewono and Ristiyanto (2005),
Fryauff et al. (2002), Hoedojo (1992), Kirnowardoyo et al. (1985), Lestari et al. (2007), Lien et al. (1975), Nalim et al. (2000), Sekartuti et al. (1995b), Soekirno et al. (1997), Soesilo
(1935), Stoops et al. (2009b), Sundararaman et al. (1957) and Venhuis (1941).
An. koliensis: Bangs et al. (1996), Church et al. (1995), Lee et al. (1980), Metselaar (1956), Pribadi et al. (1998) and Van den Assem and Bonne-Wepster (1964).
An. leucosphyrus: Harbach et al. (1987), Machsoes (1939) and Van Hell (1952).
Continued
An. longirostris: Metselaar (1956) and Van den Assem and Bonne-Wepster (1964).
An. maculatus: Barodji et al. (2007), Boesri and Boewono (2006), Boewono and Ristiyanto (2005), Kirnowardoyo et al. (1985), Kirnowardoyo et al. (1991), Lestari et al. (2007), Lien
et al. (1975), Maekawa et al. (2009b), Marwoto et al. (1992a), Stoops et al. (2009b), Sundararaman et al. (1957), Venhuis (1941) and Wigati et al. (2006).
An. nigerrimus: Bangs and Rusmiarto (2007), Boesri (1994b), Boewono and Nalim (1996), Boewono et al. (1997a), Kirnowardoyo et al. (1991), Nalim et al. (2000), Overbeek (1940),
Sundararaman et al. (1957), Van Hell (1952) and Venhuis (1941).
An. parangensis: Machsoes (1939), Marwoto et al. (2002), Marwoto et al. (1996) and Nurdin et al. (2003).
An. peditaeniatus: Boewono and Nalim (1996), Boewono et al. (1997a), Lien et al. (1975), Soesilo (1935) and Stoops et al. (2009b).
An. punctulatus: Bangs et al. (1996), Church et al. (1995), Lee et al. (1980), Metselaar (1956), Pribadi et al. (1998) and Van den Assem and Bonne-Wepster (1964).
An. sinensis: Boewono and Nalim (1996), Boewono et al. (1997a), Fryauff et al. (2002), Lien et al. (1975), Mangkoewinoto (1919), Soesilo (1935) and Sundararaman et al. (1957).
An. subpictus: Barodji et al. (1999/2000), Boesri and Boewono (2006), Collins et al. (1979), Dasuki and Supratman (2005), Gundelfinger et al. (1975), Hoedojo (1992, 1995), Issaris and
Sundararaman (1954), Machsoes (1939), Maekawa et al. (2009b), Mangkoewinoto (1919), Marwoto et al. (2002), Marwoto et al. (1992a), Marwoto et al. (1996), Nalim et al. (2000),
Nurdin et al. (2003), Soekirno et al. (1997), Soesilo (1928, 1935), Sukowati et al. (2001), Sundararaman et al. (1957) and Van Hell (1952).
An. sundaicus: Boewono and Nalim (1996), Boewono et al. (1997a), Collins et al. (1979), Fryauff et al. (2002), Issaris and Sundararaman (1954), Kirnowardoyo et al. (1991), Lien et al.
(1975), Maekawa et al. (2009b), Mangkoewinoto (1919), Marwoto et al. (1992a), Nalim et al. (2000), Soesilo (1928), Stoops et al. (2009b), Sundararaman et al. (1957) and Van Hell
(1952).
An. tessellatus: Bangs and Rusmiarto (2007), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono et al. (1997a), Fryauff et al. (2002), Kirnowardoyo et al. (1985),
Machsoes (1939), Maekawa et al. (2009b), Nurdin et al. (2003), Soekirno et al. (1997), Soesilo (1935), Stoops et al. (2009b), Sundararaman et al. (1957) and Venhuis (1941).
An. umbrosus: Kirnowardoyo et al. (1991) and Mangkoewinoto (1919).
An. vagus: Barodji et al. (2007), Boesri (1994b), Boesri and Boewono (2006), Boewono and Nalim (1996), Boewono and Ristiyanto (2005), Hoedojo (1992, 1995), Kirnowardoyo et al.
(1985), Lestari et al. (2007), Lien et al. (1975), Maekawa et al. (2009b), Marwoto et al. (1992a), Nurdin et al. (2003), Soekirno et al. (1997), Soesilo (1935), Stoops et al. (2009b), Venhuis
(1941), Wiganti et al. (2010) and Wigati et al. (2006).
Table 3.3 Blood-feeding preference/human blood index of Anopheles malaria vectors in Indonesia
Species or species Year of Total sample giving positive reaction to Total sample contains Human blood
complexa samples blood present human blood index (%)
An. aconitus 1932–2003 17,762 1551 8.7
An. balabacensis 2003 82 8 9.8
An. bancroftii 1928 51 46 90.0
An. barbirostris 1932–2003 510 60 11.8
An. farauti s.l. 1962 20 18 90.0
An. flaviirostris 2003 33 3 9.1
An. kochi 1932–2003 841 89 10.6
An. koliensis 1962 170 126 74.1
An. leucosphyrus 1932 204 202 99.0
An. maculatus s.l. 1932–2003 425 134 31.5
An. nigerrimus 1958 236 10 7.2
An. punctulatus 1954–1962 84 67 79.8
An. sinensis 1932–1954 1157 903 78.0
An. subpictus s.l. 1932–1976 2093 309 14.8
Continued
Table 3.3 Blood-feeding preference/human blood index of Anopheles malaria vectors in Indonesia—cont'd
Species or species Year of Total sample giving positive reaction to Total sample contains Human blood
complex samples blood present human blood index (%)
An. sundaicus 1932–1976 5928 3188 53.8
An. tessellatus 1932–1960 182 19 10.4
An. vagus 1932–2003 1806 820 45.4
a
No human blood index data were found for An. barbumbrosus, An. karwari and An. parangensis.
The database of blood-feeding preference/human blood index of Anopheles malaria vectors in Indonesia was acquired from the following references:
An. aconitus: Alfiah et al. (2008), Barodji et al. (1984a), Boewono et al. (1991), Chow et al. (1959), Joshi et al. (1977), Kirnowardoyo and Supalin (1982), Kirnowardoyo
and Supalin (1986), Noerhadi (1960), Sundararaman et al. (1957), Vector Biology and Control Research Unit (1979a), Walch and Sardjito (1928), Walch (1932),
Widyastuti et al. (2003), World Health Organization and Vector Biology and Control Research Unit 2 Subunit Semarang (1978) and World Health Organization
and Vector Biology and Control Research Unit Semarang (1978).
An. balabacensis: Alfiah et al. (2008).
An. bancroftii: Walch and Sardjito (1928).
An. barbirostris: Alfiah et al. (2008), Chow et al. (1959), Noerhadi (1960), Walch and Sardjito (1928) and Walch (1932).
An. farauti s.l.: Slooff (1964).
An. flavirostris: Alfiah et al. (2008).
An. kochi: Alfiah et al. (2008), Chow et al. (1959), Noerhadi (1960) and Walch (1932).
An. koliensis: Slooff (1964).
An. leucosphyrus: Walch (1932).
An. maculatus s.l.: Alfiah et al. (2008), Noerhadi (1960) and Walch (1932).
An. nigerrimus: Chow et al. (1959).
An. punctulatus: Slooff (1964) and Walch and Sardjito (1928).
An. sinensis: Walch and Sardjito (1928) and Walch (1932).
An. subpictus s.l.: Chow et al. (1959), Collins et al. (1979), Issaris and Sundararaman (1954), Noerhadi (1960), Sundararaman et al. (1957), Walch and Sardjito (1928) and
Walch (1932).
An. sundaicus s.l.: Collins et al. (1979), Issaris and Sundararaman (1954), Sundararaman et al. (1957), Walch and Sardjito (1928) and Walch (1932).
An. tessellatus: Chow et al. (1959), Noerhadi (1960), Walch and Sardjito (1928) and Walch (1932).
An. vagus: Alfiah et al. (2008), Chow et al. (1959), Noerhadi (1960) and Walch (1932).
Table 3.4 The typical larval habitats of Anopheles malaria vectors in Indonesia
Water
Light intensity Water salinity turbidity
Water movement Habitat type
Species or
species complex Sunlit Shaded Brackish Fresh Clear Turbid Stagnant Flowing Natural Man-made
An. aconitus • • • • • • Marshes, lakes, streams, river Rice fields, fish ponds,
beds. irrigation ditches.
An. balabacensis • • • • Ground depressions, stream- Puddles, animal wallows,
side rock pools, pools under hoof prints, tyre tracks.
shrubs or low trees, river
banks, seepage.
An. bancroftii • • • • Marshes, pools associated Heavily shaded irrigation
with slow streams, creeks and ditches.
rivers, ground pools.
An. barbirostris • • • • • • Lagoons, marshes, pools, Rice fields, fish ponds,
slow running streams, river drainage ditches, wells.
banks, springs.
An. barbumbrosus • • • • • River banks, clear streams Rice fields.
emerging from jungle areas,
open grassy ravines.
An. farauti s.l. • • • • • • • Marshes, lagoons, large and Pools, fish ponds, irrigation
small streams margins and ditches, pig-wallows, garden
floating wood and other pools, tins, drums, coconut
natural debris, river banks. shells, canoes.
An. flavirostris • • • • • Springs, shaded grassy edges, Rice fields, irrigation
slow-flowing small streams, ditches, wells.
pools.
Continued
Table 3.4 The typical larval habitats of Anopheles malaria vectors in Indonesia—cont'd
Water
Light intensity Water salinity turbidity
Water movement Habitat type
Species or
species complex Sunlit Shaded Brackish Fresh Clear Turbid Stagnant Flowing Natural Man-made
An. karwari • • • • Marshes, small slow-moving Irrigation canals associated
streams, seepages, ground with rice cultivation.
pools, rock pools, springs.
An. kochi • • • • • • • • Marshes, pools, small Rice fields, fish ponds,
streams. buffalo wallows, wells,
ditches, hoof prints.
An. koliensis • • • • • Small streams, ground pools, Pig ruts and wallows, ditches.
riverside ponds, marshes.
An. leucosphyrus • • • • • Marshes, small streams, Fish ponds, wheel ruts, hoof
seepage springs, jungle pools, prints.
ground depressions.
An. maculatus s.l. • • • • Stream-side rock pools, Rice fields, ponds, ditches.
margins of small slow-
moving streams, drying river
beds, ground seepages, small
pools, springs.
An. nigerrimus • • • • Lake margins, marshes, Rice fields, irrigation
pools, small streams. channels, large borrow pits.
An. parangensis • • • • • • • Coastal marshes, pools. Fish ponds, ground
depressions.
An. punctulatus • • • • • Freshwater coastal marshes, Foot prints, ditches, pig ruts,
low-lying riverine areas, pits with grey turbid water,
riverside pools, grasslands, wheel ruts.
along jungle edges, pools,
ground depressions, rock
pools in drying stream beds,
earthen drains.
An. sinensis • • • • Lake margins, marshes, small Rice fields, borrow pits, fish
streams, bogs, slow-flowing ponds, irrigation ditches.
rivers.
An. subpictus s.l. • • • • • • • Tidal lagoons, coastal Rice fields, fish ponds,
blocked freshwater rivers and furrows in gardens, water
streams, marshes, pools, tanks, buffalo wallows,
rocky streams, mangrove brackish ponds, seaweed
forests, springs. ponds, irrigation ditches.
An. sundaicus s.l. • • • • • • Lagoons, marshes, pools, Fish ponds.
seasonally blocked coastal
streams.
An. tessellatus • • • • • • • Ground pools. Rice fields, fish ponds.
An. vagus • • • • • • Stagnant margins of streams, Rice fields, irrigation
river edges, small and ditches, wheel ruts, hoof
swallow pools near beaches, prints, artificial containers
springs. (tyres, drums, upturned small
boats).
The ‘•’ mark denote the common habitat characteristics of individual Anopheles malaria vectors.
s.l. (sensu lato) indicates the existence or possibility of more than one sibling species is represented in Indonesia, therefore, collectively expanding the natural- and man-made habitats listed.
238 Iqbal R.F. Elyazar et al.

6. ANOPHELES SUSCEPTIBILITY TO INSECTICIDES


Seventy-four sources of Anopheles insecticide susceptibility data from
Indonesia were reviewed. Table 3.5 summarizes insecticide resistance
amongst Anopheles mosquitoes in Indonesia including the six insecticides
that are currently recommended by the VCP, Indonesian Ministry of
Health, for indoor residual spraying; five pyrethroids: alpha-cypermethrin,
bifenthrin, deltamethrin, etofenprox, lambda-cyhalothrin and one carba-
mate:bendiocarb (Department Kesehatan, 2003). The assembled data of
insecticide susceptibility tests reveal that resistance to four chemicals

Table 3.5 Insecticide susceptibility of Anopheles malaria vectors in Indonesia


IMCP
Insecticide Anopheles malaria
Insecticide Class 1993 2003 resistancea vector species
Alpha- Pyrethroid No Yes Yes An. barbirostris,
cypermethrin An. sundaicus†
Bifentrin Pyrethroid No Yes Yes An. aconitus†
Cyflutrin Pyrethroid No No No
Deltamethrin Pyrethroid No Yes Yes An. aconitus†,
An. sundaicus†
Etofenprox Pyrethroid No Yes No
Lambda- Pyrethroid Yes Yes No
cyhalothrin
Bendiocarb Carbamate Yes Yes Yes An. kochi
Propoxur Carbamate No No No
Fenitrothion Organophosphate Yes No Yes An. aconitus†,
An. maculatus,
An. subpictus†,
An. sundaicus†
Malathion Organophosphate Yes No No
Pirimiphos- Organophosphate Yes No No
methyl
DDT Organochlorine Yes No Yes An. aconitus†,
An. koliensis,
An. sundaicus†
a
Based on physiological toxicity response tests or presence of kdr (knock-down resistance) gene mutation
in those species marked with †.
Anopheles Malaria Vector Mosquitoes in Indonesia 239

(alpha-cypermethrin, bifenthrin, deltamethrin and bendiocarb) has been


detected in Indonesian anophelines, specifically An. aconitus, An. barbirostris,
An. kochi and An. sundaicus. Resistance to two other insecticides that
were used prior to 2003, fenitrothion and DDT were also found in five
Anopheles species: An. aconitus, An. koliensis, An. maculatus, An. subpictus
and An. sundaicus.

6.1. Anopheles aconitus


Insecticide resistance in An. aconitus has been known since the early 1960s. In
Indonesia, it was first noted against DDT in Central Java in 1962 (Soerono
et al., 1965). Over 275 sentinel sites were subsequently established to mon-
itor the status of DDT resistance on Java between 1960s and 1970s. These
activities confirmed that DDT resistance in An. aconitus had developed across
the island over time (Joshi et al., 1977; Martono, 1988b; O’Connor and
Arwati, 1974; World Health Organization and Vector Biology and
Control Research Unit 2 Semarang, 1977). The long history of DDT use
in the rice fields of Java may have been partially (or primarily) responsible
for the swift development of resistance (O’Connor and Arwati, 1974;
Syafruddin et al., 2010). Molecular analysis conducted to assess the presence
of the insecticide-resistant allele (1014F kdr mutation) (Syafruddin et al.,
2010) associated with the resistant phenotype (to organochlorine and pyre-
throid class insecticides) yet the analysis did not detect the allele, albeit only a
small sample of six An. aconitus were tested. In contrast, in southern Sumatra,
two of three An. aconitus examined indicated the existence of the kdr gene,
but again, only a small sample size was tested preventing any definitive con-
clusions being made regarding the distribution of this allele in Indonesia
and its significance in control operations.
An. aconitus has shown resistance against both fenitrothion and
deltamethrin in Indonesia. Fenitrothion resistance was first reported in Central
Java in 1976 (Joshi et al., 1977) and reconfirmed over two decades later, via
molecular analysis of specimens collected in the same region (Widiarti et al.,
2001). The proportions of An. aconitus larvae exhibiting evidence of resistance
were 23% (n ¼ 208) in high malaria endemic areas, 19% (n ¼ 210) with
medium endemicity, and only 3% (n ¼ 199) in low endemic localities, pre-
sumably a reflection of degree of previous exposure in each respective pop-
ulation. Resistance to deltamethrin was first reported in Indonesia within An.
aconitus populations from the northern coast of Central Java in 2003 (Widiarti
et al., 2005a). A mortality rate of only 66% was detected amongst 125 An.
aconitus mosquitoes following exposure with 0.05% deltamethrin for 1 h.
240 Iqbal R.F. Elyazar et al.

The Indonesian VCP routinely currently conducts insecticide suscepti-


bility tests (standard WHO contact tube tests), mostly in Java, to bifenthrin,
bendiocarb and lambda-cyhalothrin (Winarno, Unpublished data). Resis-
tance to 0.1% concentration of bifenthrin has been documented amongst less
than 50% of tested An. aconitus mosquitoes in Central Java. Exposure to
0.05% lambda-cyhalothrin also showed evidence of resistance in western
Java with a mortality rate of 80%. Resistance has not yet been detected to
0.2% bendiocarb in Java. At the time of writing, we have found no published
evidence of resistance to alpha-cypermethrin and etofenprox.

6.2. Anopheles barbirostris


WHO bioassay tests by the Indonesian VCP reported 72–79% mortality
rates after 1-h exposure to 0.05% alpha-cypermethrin from An. barbirostris
collected in Central Java and southern Sumatra (Winarno, Unpublished
data). Tests exposing An. barbirostris from western Java, eastern Lesser Sundas
and northern Sulawesi to 0.2% bendiocarb, 0.05% deltamethrin and 0.05%
lambda-cyhalothrin showed no evidence of resistance (Winarno,
Unpublished data). To date, there are no published accounts of resistance
to etofenprox.

6.3. Anopheles farauti s.l.


No records were found on susceptibility status of this species complex to
residual chemicals. However, use of methoprene (an insect growth regula-
tor) for control of immature stages was found effective for the control of this
species (Maridana et al., 1997).

6.4. Anopheles kochi


The Indonesian VCP have tested of An. kochi for resistance to 0.75% alpha-
cypermethrin, 0.05% lambda-cyhalothrin and 0.1% bendiocarb (Winarno,
Unpublished data) of which only specimens from western Sumatra have
appeared to show low levels of resistance (mortality rate slightly above
70% after 1-h exposure) to 0.1% bendiocarb. No resistance was found to
alpha-cypermethrin in Maluku and lambda-cyhalothrin in eastern
Kalimantan.

6.5. Anopheles koliensis


DDT resistance has been reported in An. koliensis populations from Papua
since the late 1980s (Bangs et al., 1993b). Bioassay tests involving 404
An. koliensis resulted in a mortality rate of over 70% after 1-h exposure to
Anopheles Malaria Vector Mosquitoes in Indonesia 241

4% DDT. Further tests using 2-h 4% DDT exposure produced only a 67%
mortality rate within the 24-h holding period. Tests using 1% fenitrothion
and An. koliensis from same locality indicated no resistance to this chemical
(Bangs et al., 1993a). Very little is known about the current insecticide sus-
ceptibility profile of this species in Papua.

6.6. Anopheles maculatus


An. maculatus has been found to be resistant to fenitrothion with biochem-
ical assays indicating resistance in 6% of mosquitoes from Central Java and
27% of mosquitoes near Yogyakarta (Widiarti et al., 2005b). In contrast,
no resistance was observed after 1-h exposure with 0.05% deltamethrin
(Widiarti et al., 2005a) in six districts in Central Java Province and to
0.1% bendiocarb (Barodji et al., 1997) in Yogyakarta, central-south Java.
The VCP conducted bioassay tests for susceptibility to 0.75% alpha-
cypermethrin, 0.05% lambda-cyhalothrin, 0.2% bendiocarb and 4%
DDT (Winarno, Unpublished data) and reported no resistance amongst
specimens tested from all locations in southern Sumatra and both West
and Central Java. At the time of writing, no published resistance to
bifenthrin and etofenprox has been found.

6.7. Anopheles subpictus s.l.


Susceptibility tests to fenitrothion at nine sites in high malaria endemic areas
in Central Java and Yogyakarta revealed that approximately two percent of
An. subpictus larvae showed resistance to organophosphate and carbamate
active ingredients (Widiarti et al., 2005b). Molecular analysis of An. subpictus
from Lampung, southern Sumatra, also documented the existence of the kdr
mutation to organochlorine and pyrethroid class chemicals (Syafruddin
et al., 2010). Tests for susceptibility to 0.75% alpha-cypermethrin, 0.2%
bifenthrin, 0.05% lambda-cyhalothrin and 0.1% bendiocarb found no
evidence of resistance in northern Sumatra, southern Sulawesi, Maluku
and Lesser Sundas (Winarno, Unpublished data). No other published infor-
mation on resistance against other insecticides has been reported for this
species.

6.8. Anopheles sundaicus s.l.


An. sundaicus s.l. was the first species in Indonesia to be reported as having
developed resistance to insecticides and the first evidence of DDT resistance
in Central Java (Chow and Soeparmo, 1956; Martono, 1988b; Soerono
et al., 1965). Bioassay tests amongst 1070 An. sundaicus mosquitoes within
242 Iqbal R.F. Elyazar et al.

DDT indoor residual spray zones showed only 0.5–2.2% mortality rates after
exposure to 0.2–5% DDT (Chow and Soeparmo, 1956). The widespread
use of DDT in agriculture is believed partially responsible for the rapid
appearance of DDT resistance in this area including the broad scale use of
both aerial and ground-based spraying of DDT as a larvicide between
1945 and 1949 (Chow and Soeparmo, 1956). After the last application
of DDT in Indonesia in 1992 (World Health Organization, 1998), nearly
30% of 77 tested mosquitoes continued to demonstrate resistance after
1-h exposure with 4% DDT in southern Sumatra in 2008 (Winarno,
Unpublished data). Furthermore, molecular analysis of An. sundaicus adult
mosquitoes from South Lampung District in southern Sumatra found the
kdr mutation amongst 72.5% (29/40) mosquitoes examined (Syafruddin
et al., 2010). Resistant to alpha-cypermethrin and fenitrothion insecticides
have been detected in this species from the same area. Susceptibility
tests documented 18% resistance amongst 78 mosquitoes after exposure
of 0.005% alpha-cypermethrin (Winarno, Unpublished data) and biochem-
ical tests to fenitrothion revealed 6–33% of tested mosquitoes from eight
sentinel sites in Central Java were resistant in 2002 (Widiarti et al.,
2005b). To date, no published evidence of resistance bifenthrin and
etofenprox is available.

6.9. Anopheles vagus


Molecular analysis found five out of six mosquitoes of An. vagus examined
from southern Sumatra had the insecticide-resistance allele (kdr) against
pyrethroid and organochlorine class insecticides (Syafruddin et al., 2010).
The VCP conducted susceptibility tests for 0.75% alpha-cypermethrin
and 0.05% lambda-cyhalothrin (Winarno, Unpublished data) and found
no resistance amongst An. vagus specimens collected from sites in Lampung,
Kalimantan and Maluku.

7. OUTLOOK FOR INDONESIAN CHALLENGES


TO MALARIA VECTOR CONTROL
This chapter presents an updated overview of the known geographical
distribution and bionomics of 20 species or species complexes known or
suspected to transmit malaria in Indonesia. A review of the status of insec-
ticide susceptibility of eight malaria vector species also provided to illustrate
Anopheles Malaria Vector Mosquitoes in Indonesia 243

what is known in Indonesia and the general paucity of information on this


important topic.
We list here what we consider to be three challenges aimed at better
equipping vector control strategy in Indonesia:
1. Species identification: An updated keys to the anopheline fauna of
Indonesia is crucial to help in understanding the complicated nature
of malaria in Indonesia. The current keys used in much of the reviewed
literature were based on entomological/taxonomical work of decades
ago before the use of molecular species identification was available or
widely used. The variability of morphological characteristics within
and between species, the practical limitations of local keys and the
presence of cryptic species within many of the Anopheles taxa clearly
undermines the effectiveness of relying on morphological identifica-
tion techniques alone. Differences in the biological characteristics of
members of the complexes have also an important bearing on malaria
transmission dynamics. It is, therefore, imperative to determine sibling
species composition and their bionomics as well as their roles in the
transmission of malaria (World Health Organization, 2007a). The
advancement of molecular entomology tools are able to support better
understanding of anopheline species identification and the genetic
structure of anopheline populations (Collins et al., 2000). Additionally,
these molecular tools are also able to detect and distinguish the four
human Plasmodium species in individual mosquitoes or in pools of
up to one hundred mosquitoes in populations with low level parasite
infections (Benedict, 2008).
2. Vector bionomics: Knowing the local Anopheles species and adjusting the
control programme to their particular behaviour is essential if malaria
control and elimination activities in Indonesia are to be successful. In
Indonesia, relatively scant information on vector bionomics and
response to chemical measures is available, often either dated, geograph-
ically patchy or completely lacking. Understanding the feeding behav-
iour, host preference and peak feeding periods may have significant
implications for selecting the most appropriate adult vector control strat-
egies. For example, long-lasting insecticide-treated bed nets might be a
poor investment if the vectors are feeding in the early part of the night or
predominantly outdoors where most of the local human population are
unprotected during the peak transmission period. Consequently, there is
a need for implementing complementary vector control tools that can
target exophagic and early-biting vectors (Bugoro et al., 2011), such
244 Iqbal R.F. Elyazar et al.

as control of larval production and habitats, house screening and personal


protection (Elyazar et al., 2011b). Other novel methods of control
should be explored such as zooprophylaxis (host diversion) or use of spa-
tial repellents to prevent attack via a behavioural avoidance mechanism
in the vector. Integrated vector control approaches, tailor-made and site-
specific to the local ecology will therefore strengthen efforts to suppress
malaria transmitting mosquitoes (Feachem et al., 2009).
3. Insecticide susceptibility monitoring: The evolving resistance patterns of mos-
quito vectors to insecticides is one of the main challenges facing
insecticide-based malaria VCP in Asia and Indonesia (Kelly-Hope
et al., 2005; Syafruddin et al., 2010; Van Bortel et al., 2008). Resistance
to four of the six recommended insecticides has been reported in four
primary malaria vectors in the country. Therefore, comprehensive mon-
itoring of insecticide susceptibility using standardized tests against the
local main vectors species is needed to ensure the continued viability
and proper use of the chemicals currently available in the malaria control
arsenal (Kelly-Hope et al., 2005; Syafruddin et al., 2010; Van Bortel
et al., 2008).

8. CONCLUSIONS
Vector control interventions require evidence-based strategies using
accurate and current knowledge of the identity, distribution and bionomics
of the key malaria vectors in different localities in the country. This contem-
porary review of the primary anopheline malaria vectors of the Indonesian
archipelago is aimed at providing the Indonesian health authorities and other
organizations responsible for malaria control with the background and
means of better focusing their resources where vector control interventions
will be most effectively applied.

ACKNOWLEDGEMENTS
The assembly of an insecticide susceptibility test national database was possible because of the
generous assistance and collaborative spirit of the Indonesian MoH Vector Control Program.
We thank Dr. Trevor Jones for reviewing and substantially improving the manuscript. We
thank the medical entomologists at the Department of Entomology, U.S. NAMRU-2,
Jakarta, especially Saptoro Rusmiarto and the late Yoyo R. Gionar for generously sharing
their expertise. We also thank to the Library of U.S. NAMRU-2, Jakarta and the Library
of the Eijkman Institute for Molecular Biology, Jakarta for providing us free access to
their collections of scientific literature published in Dutch before 1942. This work is
dedicated to the lasting and fond memory of the late Dr. Soeroto Atmosoedjono, the
Anopheles Malaria Vector Mosquitoes in Indonesia 245

man who inspired and trained the majority of modern medical entomologists in Indonesia.
His passion finds expression in this review penned by several of his students.
Author contributions. I. E. compiled the database of Anopheles distribution, bionomics and
insecticide susceptibility status (I. E. and W.). I. E., M. E. S., P. W. G., M. J. B., J. K. B., S. I.
H. contributed to the first and subsequent drafts of the manuscript. S. N. T., A. S., R. K. and
W. provided context regarding the Indonesian vector control strategy. All authors
commented on the final submission of the manuscript.
Funding. I. E. is funded by grants from the University of Oxford—Li Ka Shing
Foundation Global Health Program and the Oxford Tropical Network. M. E. S. is
funded by a project grant from the Bill and Melinda Gates Foundation via the VECNet
consortium (http://www.vecnet.org/). M. J. B. is an independent consultant funded by
various private industries. S. I. H. is funded by a Senior Research Fellowship from the
Wellcome Trust (#095066) which also supports P. W. G. S. I. H. also acknowledges
funding support from the RAPIDD program of the Science & Technology Directorate,
Department of Homeland Security, and the Fogarty International Center, National
Institutes of Health. S. N. T., A. S., R. K. and W. are funded by the Indonesian Ministry
of Health. J. K. B. is funded by a grant Vietnam Wellcome Trust Major Overseas
Programme (#B9RJIXO). This work forms part of the output of the Malaria Atlas
Project (MAP, http://www.map.ox.ac.uk), principally funded by the Wellcome Trust,
UK. The funders had no role in study design, data collection and analysis, decision to
publish or preparation of the manuscript.
Competing interests. No competing interests are declared from any of the authors.

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CHAPTER FOUR

Next-Generation Molecular-
Diagnostic Tools for
Gastrointestinal Nematodes of
Livestock, with an Emphasis on
Small Ruminants: A Turning Point?
Florian Roeber, Aaron R. Jex, Robin B. Gasser1
Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia
1
Corresponding author: e-mail address: robinbg@unimelb.edu.au

Contents
1. Introduction 268
2. Economic Impact of Parasitic Diseases of Livestock 270
3. Strongylid Nematodes of Ruminant Livestock 271
3.1 Key gastrointestinal nematode species of small ruminants 273
3.2 Pathogenesis and clinical signs of disease 273
4. Some General Aspects of the Epidemiology of Gastrointestinal Nematodes
of Livestock 277
4.1 Distribution of trichostrongylids of sheep according to climate zone 279
5. The Control of Gastrointestinal Nematodes and Anthelmintic Resistance (AR) 281
6. Traditional Methods for the Diagnosis of Strongylid Nematode Infections and
Their Limitations 289
6.1 Validation of diagnostic tests 289
6.2 Diagnosis in the live animal 289
6.3 Post-mortem diagnosis 300
7. DNA-Based Methods for Specific Diagnosis of Strongylid Nematodes Infections
in Livestock: Sample Preparation, Markers and Developments 302
7.1 Sample preparation prior to PCR 303
7.2 Specific genetic markers for strongylid nematodes 304
8. Conventional PCR-Based Tools for the Identification of Species of Strongylid
Nematodes and Diagnosis of Infections 305
9. Real-Time PCR Coupled to High-Resolution Melting Analysis as a Diagnostic Tool 306
9.1 Principles of the technology 306
9.2 RT-PCR assays for the diagnosis of infections with strongylid nematodes 307

Advances in Parasitology, Volume 83 # 2013 Elsevier Ltd 267


ISSN 0065-308X All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-407705-8.00004-5
268 Florian Roeber et al.

9.3 Critical evaluation and application of a RT-PCR method to assess the


composition of strongylid nematode populations in sheep with naturally
acquired infections 311
9.4 Assessment of RT-PCR to replace larval culture (LC) and to support
conventional faecal egg count reduction testing (FECRT) of sheep 313
10. A Robotic, Multiplexed-Tandem PCR Assay for the Diagnosis of Gastrointestinal
Strongylid Nematode Infections of Sheep 314
10.1 Rationale, establishment and critical assessment of the multiplexed-
tandem PCR assay 314
10.2 Future applications of the assay and extensions 317
11. Concluding Remarks: Replacing Traditional with Molecular-Diagnostic Tools 319
Acknowledgements 321
References 321

Abstract
Parasitic nematodes of livestock have major economic impact worldwide. Despite
the diseases caused by these nematodes, some advances towards the development
of new therapeutic agents and attempts to develop effective vaccines against some
of them, there has been limited progress in the development of practical diagnostic
methods. The specific and sensitive diagnosis of parasitic nematode infections of live-
stock underpins effective disease control, which is now particularly important given the
problems associated with anthelmintic resistance in parasite populations. Traditional
diagnostic methods have major limitations, in terms of sensitivity and specificity. This
chapter provides an account of the significance of parasitic nematodes (order
Strongylida), reviews conventional diagnostic techniques that are presently used rou-
tinely and describes advances in polymerase chain reaction (PCR)-based methods for
the specific diagnosis of nematode infections. A particular emphasis is placed on the
recent development of a robotic PCR-based platform for high-throughput diagnosis,
and its significance and implications for epidemiological investigations and for use in
control programmes.

1. INTRODUCTION
The phylum Nematoda (roundworms) includes many parasites that
are of major socio-economic importance. For example, grazing ruminants
are frequently parasitized by multiple species of the order Strongylida, which
can cause significant disease, known as parasitic gastroenteritis (PGE) (Kassai,
1999). Different species of strongylid nematodes can vary considerably in
their pathogenicity, geographical distribution and susceptibility to anthel-
mintic drugs (Dobson et al., 1996). Mixed infections, involving multiple
Next-Generation Molecular-Diagnostic Tools 269

genera and species are common, and usually have a greater impact on the
host than monospecific infections. Furthermore, the species composition
of the parasites present in a host animal plays an important role in the severity
of infection (Wimmer et al., 2004). Depending on the number, species and
burden of parasitic nematodes, common symptoms of PGE include reduced
weight gain or weight loss, anorexia, diarrhoea, reduced production and, in
the case of blood-feeding species, anaemia and oedema, due to the loss of
blood and/or plasma proteins (Kassai, 1999). Therefore, the knowledge
of the nematode species present in a particular geographical area, their
biology and epidemiology have important implications for the control of
parasitism, particularly given the increasing problems of drug resistance in
strongylid nematodes of livestock (Kaplan, 2004; Sangster, 1999;
Wolstenholme et al., 2004).
Conventional coprological and immunological methods of diagnosis can
be time consuming and have limitations, in terms of their sensitivity and spec-
ificity, and often do not achieve a specific diagnosis (Gasser, 2006). In partic-
ular, in the case of mixed infections, the specific detection and identification of
parasites (and specific diagnosis of infection) can be laborious and time con-
suming using conventional methods, such as faecal egg counts (FECs) or larval
culture (LC) and differentiation (Wimmer et al., 2004). Molecular techniques
that rely on the amplification of nucleic acids, particularly those coupled to
the polymerase chain reaction (PCR) (Saiki et al., 1988), are effective for
the specific identification of parasites, and aid the diagnosis of infections
from minute amounts of target template, if suitable markers are utilized.
Such methods are likely to provide powerful alternative tools to traditional
approaches, to underpin fundamental research into parasite epidemiology
and to improve the control of parasitic disease (Gasser, 2006).
The knowledge of epidemiological factors, such as geographical and
seasonal occurrence of different species of parasitic nematodes, has major
implications for the control of parasitism and the development of better
strategic, anthelmintic treatment regimens (Barger, 1999). For instance, in
Australia, several studies have been carried out to investigate the epidemi-
ology of livestock parasites, but there is a paucity of recent, published data
for the south-eastern part of this continent; most studies were published in
the 1970s (Anderson, 1972, 1973). Furthermore, published data have been
based largely on the use of traditional diagnostic approaches, and no detailed
epidemiological investigations of gastrointestinal nematodes of sheep have
been conducted using molecular tools.
270 Florian Roeber et al.

The purpose of this chapter was to (i) concisely cover the economic
impact of the diseases of livestock caused by gastrointestinal strongylid nem-
atodes; (ii) provide a background on the biology and epidemiology of these
important nematodes, and to review current information on anthelmintic
resistance (AR); (iii) review conventional methods for the diagnosis of stro-
ngylid nematode infections and discuss their constraints; (iv) provide an
account of key molecular-diagnostic methods, with an emphasis on recent
advances through the development of robotic PCR-based technology and
its significance and implications.

2. ECONOMIC IMPACT OF PARASITIC DISEASES


OF LIVESTOCK
The livestock industry plays a major role in the economies of many
developed and developing countries. The production of livestock animals
provides food, animal products (e.g. leather, hides and wool), income,
employment, a source of organic fertilizer and biogas as well as draught
and work power (particularly important in developing countries in which
modern machinery is not affordable or available). Globally, parasite infec-
tions are associated with significant economic losses to the livestock industry
(Vercruysse and Claerebout, 2001). For example, nematode infections of
sheep were estimated, in 2006, to cost the Australian grazing industry 369
million dollars per annum, making these parasites one of the greatest con-
straints on the Australian grazing industry (McLeod, 1995; Sackett and
Holmes, 2006). The economic impact of parasitic diseases differs between
the developed and developing worlds. In the developing world, the greatest
impact of parasitic diseases relates to direct productivity losses and lost socio-
economic potential. In the developed world, on the other hand, the main
impact is in the costs of control (Hawkins, 1993; Perry and Randolph,
1999) associated with the use of anthelmintics, strategic anthelmintic
programmes and improved pasture utilization, which all limit the impact
of parasitism at the subclinical or economical levels (Corwin, 1997). How
the economic impact of parasitism should be assessed, in order to aid deci-
sions in disease control, is a topic of debate and has been reviewed in detail
(Hawkins, 1993; Perry and Randolph, 1999).
In general, to measure the effects of subclinical parasitic infections, per-
formance parameters, such as weight gain, feed conversion, forage utiliza-
tion, conception rate, calving–breeding interval, milk production and
disease resistance, are employed routinely (Corwin, 1997). Parasites can
Next-Generation Molecular-Diagnostic Tools 271

have various pathological effects on their host and may contribute to a


decreased production performance (Hawkins, 1993).
Whilst lungworms, such as species of Dictyocaulus, Protostrongylus or
Muellerius, are economically important, gastrointestinal nematodes that para-
sitize the abomasum, small and large intestines of ruminants are also responsible
for major losses to the livestock industries (Hawkins, 1993; McLeod, 1995).
Strongylid nematodes of the superfamily Trichostrongyloidea, including spe-
cies of Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus, and, to lesser
extent, Nematodirus spp. are regarded to be of great importance, particularly
in small ruminants (Berghen et al., 1993; Zajac, 2006). Other important gas-
trointestinal nematodes include species of Oesophagostomum, Chabertia and the
bovine hookworm, Bunostomum, of which the latter two genera are regarded
to be less common (Zajac, 2006). Usually, low intensities of infection do not
cause a serious hazard to the health of livestock animals and may be tolerated
(i.e. allowing the development of some immunity in the host), but as the
numbers of worms increase, subclinical disease can manifest itself by signs of
reduced appetite, weight gain, wool production and fertility, and is, therefore,
of great economic importance (Fox, 1997; Zajac, 2006). These factors affect
the herd productivity, the capacity to maintain and improve a herd (i.e. its
health and genetic potential), human nutrition, community development
and cultural issues relating to the use of livestock (Perry and Randolph,
1999). This is of particular relevance in regards to a rapidly growing human
population and an associated increase in the global demand for products of
animal origin, emphasizing the importance of maintaining and/or increasing
the productivity of the livestock industry (Nonhebel and Kastner, 2011).

3. STRONGYLID NEMATODES OF RUMINANT LIVESTOCK


The order Strongylida includes five superfamilies: the
Diaphanocephaloidea, Ancylostomatoidea, Strongyloidea, Trichostrongyloidea
and Metastrongyloidea. The Strongylida are characterized by the presence of a
copulatory bursa and are, therefore, referred to as bursate nematodes (Anderson,
2000). The first four of these superfamilies are monoxenous (single host) and
mainly inhabit the gastric and/or intestinal tracts of their vertebrate hosts. In con-
trast, members of the Metastrongyloidea are almost entirely heteroxenous
(multiple hosts), relying in their transmission on intermediate hosts, such as earth-
worms and gastropods (Anderson, 2000). Adult metastrongyloids are usually
found in the lungs of their hosts, but some species can be associated with the
vascular system external to the lung (Anderson, 2000).
272 Florian Roeber et al.

In general, with some exceptions (e.g. Nematodirus), the life cycle of the
most important gastrointestinal strongylid nematodes follows a similar pat-
tern (Fig. 4.1) (Levine, 1968). Sexually dimorphic adults are present in the
digestive tract, where fertilized females produce large numbers of eggs which
are passed in the faeces. Strongylid eggs (70–150 mm) usually hatch within
1–2 days. After hatching, larvae feed on bacteria and undergo two moults to
then develop to ensheathed third-stage larvae (L3s) in the environment (i.e.
faeces or soil). The sheath (which represents the cuticular layer shed in the
transition from the L2 to L3 stage) protects the L3 stage from environmental
conditions but prevents it from feeding. Infection of the host occurs by
ingestion of L3s (with the exception of Nematodirus spp., for which the infec-
tive L3 develops within the egg). During its passage through the stomach,
the L3 stage loses its protective sheath and has a histotrophic phase (tissue
phase), depending on species, prior to its transition into the L4 and adult

Figure 4.1 Life cycle representing gastrointestinal nematodes (order Strongylida) of


small ruminants. First-, second- and third-stage larvae (L1, L2 and L3, respectively)
are free-living in the environment. The fourth larval (L4) and adult stages (dioecious)
are parasitic in the gastrointestinal tract of the ruminant host. Adapted from Demeler
(2005).
Next-Generation Molecular-Diagnostic Tools 273

stages (Levine, 1968). Under unfavourable conditions, the larvae undergo a


period of hypobiosis (arrested development; typical for species of
Haemonchus and Teladorsagia). Hypobiotic larvae usually resume their activ-
ity and development in spring in the case of Haemonchus or autumn in the
case of Teladorsagia. This may be synchronous with the start of the lambing
season, manifesting itself in a peri-parturient increase in FECs in ewes
(Salisbury and Arundel, 1970). The peri-parturient reduction of immunity
increases the survival and egg production of existing parasites, increases
susceptibility to further infections and contributes to the contamination
of pasture with L3s when young, susceptible animals begin grazing
(Hungerford, 1990).

3.1. Key gastrointestinal nematode species of small ruminants


Of the numerous genera and species of gastrointestinal nematodes that infect
small ruminants, the key representatives known to occur in Australasia are
listed in Table 4.1. The major species responsible for most disease in grazing
sheep are Haemonchus contortus, Teladorsagia circumcincta and intestinal species
of Trichostrongylus (Besier and Love, 2003).

3.2. Pathogenesis and clinical signs of disease


Almost all sheep are infected with one or more gastrointestinal parasites, but
the degree of severity of infection and the clinical signs associated with infec-
tions may vary considerably (e.g. Donald et al., 1978). The severity of disease
is mainly influenced by factors such as the parasite species present, the num-
ber of worms present in the gastrointestinal tract, the general health and
immunological state of the host, environmental factors, such as climate
and pasture type, stress, stocking rate, management and/or diet (Kassai,
1999). Usually, three groups of animals are prone to heavy worm burdens:
(i) young, non-immune animals; (ii) adult, immuno-compromised animals;
and (iii) animals exposed to a high infection pressure from the environment
(Zajac, 2006). Nematode populations in sheep are generally over-dispersed
with only few sheep carrying heavy worm burdens, whilst the majority of
sheep harbour low numbers of worms (Barger, 1985).

3.2.1 Haemonchus contortus


H. contortus is one of the most fecund strongyle nematodes; individual
females are capable of producing thousands of eggs per day, which can lead
to rapid larval pasture contamination and associated outbreaks of
Table 4.1 The key morphological characteristics, pre-patent periods and locations in the host of the most important genera and species
of gastrointestinal nematodes infecting sheep in Australasia (Anderson, 2000; Besier and Love, 2003; Gibbons, 2010; Levine, 1968; Taylor
et al., 2007)
Morphometrics/morphology
Length Pre-patent Location in the
Family Species (mm) Features period (days) host
Trichostrongylidae Haemonchus contortus ♂ 10–20 Red pseudocoelomic fluid and white, coiled 18–21 Abomasum
♀ 18–30 uterus, giving a barber’s pole appearance.
Presence of vulvar flap depends on strain
Teladorsagia ♂ 7–8 Small head and buccal cavity 15–21 Abomasum
circumcincta ♀ 10–12 In females, a vulvar flap can be present
Trichostrongylus axei ♂ 2–6 Dissimilar spicules of unequal length 15–23 Abomasum or
♀ 3–8 stomach
T. colubriformis ♂ 4–8 Equal length spicules with triangular tip 15–23 Anterior small
♀ 5–9 intestine
T. vitrinus ♂ 4–7 Equal length spicules with sharp tips 15–23 Anterior small
♀ 5–8 intestine
T. rugatus ♂ 4–7 Dissimilar spicules with foot-like appearance 15–23 Small intestine
♀ 6–7
Cooperia curticei ♂ 4–5 Transverse striation of cuticle in all species 14–15 Small intestine
♀ 5–6 Watch-spring-like body posture and the
presence of a small cephalic vesicle are
characteristic
Molineidae Nematodirus spathiger ♂ 10–19 Small but distinct cephalic vesicle 18 Small intestine
♀ 15–29 Very long spicules ending in a spoon-
shaped terminal piece
N. filicollis ♂ 10–15 Small but distinct cephalic vesicle 18 Small intestine
♀ 15–20 Long and slender spicules with a narrow
lanceolate membrane
Ancylostomatidae Bunostomum ♂ 12–17 Anterior end is bend dorsally 40–70 Small intestine
trigonocephalum ♀ 19–26 Buccal capsule with is equipped with two
cutting plates
Chabertiidae Oesophagostomum ♂ 12–16 Have two leaf crowns and a shallow buccal 40–45 Large intestine
columbianum ♀ 14–18 capsule. Position of cervical papillae used for
species differentiation
Oe. venulosum ♂ 11–16 Cervical papillae are situated posterior to the 40–45 Large intestine
♀ 13–24 oesophagus
Chabertia ovina ♂ 13–14 Mouth is directed antero-ventrally 42–50 Large intestine
♀ 17–20 Buccal capsule is subglobular without teeth
276 Florian Roeber et al.

haemonchosis (Levine, 1968). In sheep, the pre-patent period of H. contortus


is 18–21 days; adult worms are short-lived, surviving in their hosts for only a
few months. The main pathogenic effects are caused by the L4s and adults,
which both feed on blood, causing severe anaemia which usually becomes
apparent after two weeks of infection (Baker et al., 1959). Acute disease is
usually intensity-dependent and is associated with signs of haemorrhagic
anaemia, dark-coloured faeces, oedema, weakness, reduced production of
wool and muscle mass, or sometimes sudden death. In cases of chronic dis-
ease, decreased food intake, weight loss and anaemia are most commonly
observed (Kassai, 1999). Unlike many other gastrointestinal parasites,
H. contortus is not a primary cause of diarrhoea, and its effects on a flock
are often not readily detected by routine observation (Zajac, 2006).

3.2.2 Teladorsagia circumcincta


Females of Te. circumcincta are less fecund than H. contortus, with an average
egg production of 100–200 eggs per female per day (Cole, 1986).
Teladorsagia does not feed on blood, and the main pathogenic effects are cau-
sed by its larval stages. Larval development takes place in the gastric glands,
leading to nodule formation in abomasal mucosa and extensive damage to
parietal cells, in turn causing a decrease in hypochloric acid production
(Levine, 1968). Subsequently, the increase in abomasal pH causes a failure
of pepsinogen to convert to the active form, pepsin, which results in elevated
plasma pepsinogen levels and reduced protein digestion. The severity of the
infection depends on concurrent infections, nutritional state of the host and
also the ability to develop an immunogenic response (Stear et al., 2003).
Commonly, moderate or subclinical infections occur, causing diarrhoea,
poor weight gain, weight loss and reduced wool production (Zajac, 2006).

3.2.3 Trichostrongylus species


Species of Trichostrongylus represent an important group of parasites of
grazing small ruminants. These parasites occur in the small intestine and
mainly exert their pathogenic effects in lambs and weaners, but have
also been reported to cause a significant depression of wool growth in old
animals (Donald et al., 1978). In Australia, the three most common species
are Trichostrongylus colubriformis, Trichostrongylus vitrinus and Trichostrongylus
rugatus (see Beveridge et al., 1989a). The main pathogenic effects are caused
by the exsheathed L3s of T. vitrinus, which burrow between the intestinal
villi and lead to the formation of sub-epithelial tunnels (Beveridge
et al., 1989b). Young nematodes developing in these tunnels are released
Next-Generation Molecular-Diagnostic Tools 277

10–12 days following infection. The liberation of young adults is associated


with extensive damage to the duodenal mucosa and with signs of generalized
enteritis, including haemorrhage, oedema and plasma protein loss into the
intestinal lumen, and subsequent hypoalbuminaemia and hypoproteinaemia
(Taylor et al., 2007). Infections with Trichostrongylus are often difficult to dis-
tinguish from malnutrition in the case of low-intensity infections (Taylor
et al., 2007) but, if present at high numbers, cause protracted watery diarrhoea
which stains the fleece of the hindquarters “black scours” (Levine, 1968).
Trichostrongylus axei, which inhabits the abomasum, is less common and
occurs usually in smaller numbers (Donald et al., 1978).

3.2.4 Other species of veterinary significance


Cooperia curticei, Nematodirus spathiger, N. fillicollis, Oesophagostomum ven-
ulosum are common parasites of the small and/or large intestine, whilst
Chabertia ovina and the hookworm, Bunostomum trigonocephalum, are less
common (Zajac, 2006). Individually, these species have relatively low path-
ogenicity, but may contribute to PGE in grazing small ruminants.
Nematodirus battus is of particular pathogenic significance in some temperate
areas, such as the British Isles, where the mass-hatching of infective L3s
occurs during spring, causing disease of young lambs (Taylor and
Thomas, 1986); however, this species of Nematodirus has not been reported
in Australasia.

4. SOME GENERAL ASPECTS OF THE EPIDEMIOLOGY


OF GASTROINTESTINAL NEMATODES OF LIVESTOCK
In general terms, infectious diseases can be transmitted horizontally
(from one animal to another) or vertically (from one generation to the next).
Horizontal transmission can occur either directly or indirectly (Thrusfield,
2005). In the case of gastrointestinal nematodes infecting sheep, transmission
occurs exclusively horizontally and directly and can be captured as a rela-
tionship among the parasite, host and environment (Fig. 4.2) (Levine,
1968). Various factors linked to this relationship determine the type and
severity of infection, and many interactions occur among these components.
Host-related factors include species, genotype, age, sex and immunity;
parasite-related factors include life history, duration of the histotrophic
phase, survival of larvae in the environment and their location in the
host; environmental factors include climate, weather, season, type of
vegetation and microclimate. The interactions between host and parasite
278 Florian Roeber et al.

Figure 4.2 Relationship among host, parasites and environment, and factors that affect
parasite control. Adapted from Gordon (1948), Levine (1968) and Donald et al. (1978).

mainly determine the potential for disease to occur and the pattern/course of
infection, whereas the interaction between host–environment and parasite–
environment influence disease transmission (Levine, 1968). In addition, the
relationship among host (e.g. genotype), parasites and the host microbiome
(particularly at the infection site) could play a key role in governing
parasite burdens, disease progression and also disease transmission, and
warrants detailed investigation.
Regional differences in climate have major effects on the epidemiology
of nematode infections and their geographical distribution (Beveridge et al.,
1989a; Cole, 1986; De Chaneet and Dunsmore, 1988). Because species dis-
tributions and the seasonal availability of different parasites are largely deter-
mined by their ecological needs (e.g. for the successful development of their
free-living stages), environmental conditions, in particular temperature and
relative humidity, are of major importance (Beveridge et al., 1989a;
O’Connor et al., 2006) (Table 4.2). Thus, climate impacts directly on the
distribution of parasites. However, there are other exogenous factors, such
as anthelmintic treatment regimens or host movement, which can influence
the distribution and prevalence of these parasitic nematodes in a particular
geographical environment (Blouin et al., 1995; Gordon, 1948).
Next-Generation Molecular-Diagnostic Tools 279

Table 4.2 Key features of major trichostrongylid nematodes of sheep


and environmental influences on survival
Life-cycle stage
Nematode Unembryonated Embryonated Pre-infective
species egg egg larvae Infective larvae
H. contortus High Susceptible to High Optimum
susceptibility to cold and susceptibility survival under
cold and desiccation. to cold and warm and moist
desiccation. Low hatching desiccation. conditions.
High mortality in the absence Poor survival in
<10  C. of moisture dry climates
and/or (warm or cool)
<10  C. and sub-
freezing winter.
T. colubriformis Intermediate Intermediate Susceptible Optimum
susceptibility to susceptibility to cold and survival under
cold and to cold. Low desiccation. warm or cool
desiccation. susceptibility High moist
High mortality to desiccation. mortality conditions.
<5  C. <5  C. Poor survival
over sub-
freezing
winters.
Te. circumcincta Low Low Intermediate Optimum
susceptibility to susceptibility susceptibility survival under
cold. to cold and to cold. cool moist
Intermediate desiccation. Susceptible conditions and
susceptibility to Hatching to sub-freezing
desiccation. <5  C. desiccation. winters.
High egg Poor survival
viability at under warm,
0–10  C. dry conditions.
Adapted from O’Connor et al. (2006).

4.1. Distribution of trichostrongylids of sheep according


to climate zone
H. contortus is regarded as the most important nematode species in tropical
(between 23.5N and 23.5S ) and subtropical climates (north of 23.5N
and south of 23.5S ), where usually warm temperatures and summer-
dominant or uniform rainfall favour the development of this species
280 Florian Roeber et al.

(O’Connor et al., 2006). In cool, temperate zones (between latitudes 45 and
65 ), conditions throughout most of the year are limiting for the develop-
ment of H. contortus, because its free-living stages are susceptible to low tem-
peratures and desiccation (O’Connor et al., 2006). In the United States, for
example, the climate is most suitable for H. contortus in the south-eastern to
north-eastern and mid-western states, whilst in the western part of the coun-
try cold winters and dry summers reduce the success of this species (see
Zajac, 2006). Trichostrongylus and Teladorsagia are more dominant in winter
and uniform rainfall zones, because of their resistance to desiccation and their
ability to develop at lower environmental temperatures (Beveridge et al.,
1989a; Donald et al., 1978; Young and Anderson, 1981). In Mediterranean
regions (between latitudes 30 and 40 ), cool and wet winters favour the
development of free-living stages of Teladorsagia and Trichostrongylus, whilst
hot and dry summer conditions cause a decrease in larval pasture contami-
nation (O’Connor et al., 2006). For example, in western regions of the
United States, arid summers reduce the transmission rate of most gastroin-
testinal nematodes, and PGE is usually less important than in the eastern part
of the United States (Zajac, 2006). In temperate zones (>40 ), the cooler
environment creates a perfect climate for Teladorsagia and Trichostrongylus,
and peak infections occur during summer and autumn (O’Connor et al.,
2006). In warmer temperate zones, such as in south-eastern Australia, where
milder winters prevail, peak infections occur in late winter to early spring
(Brunsdon, 1980). In the tropics and subtropics, these two genera may be
prevalent, but extend to regions with cooler, humid early spring or late
autumn conditions (O’Connor et al., 2006). The three most common intes-
tinal species of Trichostrongylus, namely T. vitrinus, T. colubriformis and
T. rugatus, may occur sympatrically, but usually one species predominates
in a particular geographical region and/or season (De Chaneet and
Dunsmore, 1988). T. vitrinus predominates in lambs in Britain (Parnell
et al., 1954), northern Europe (Eysker, 1978) or in the winter rainfall envi-
ronments of south-eastern Australia (Anderson, 1972). T. colubriformis is most
prevalent in North America (Levine, 1968), East and South Africa (Grant,
1981) and in the summer rainfall zones of eastern Australia (Southcott
et al., 1976). In uniform rainfall zones of eastern Australia and in New
Zealand, T. colubriformis and T. vitrinus occur together, but which species
predominates depends on the season (Brunsdon, 1970; Waller et al.,
1981). T. rugatus is most common in arid climates of South Africa or the
inland of Australia (De Chaneet and Dunsmore, 1988).
Next-Generation Molecular-Diagnostic Tools 281

5. THE CONTROL OF GASTROINTESTINAL NEMATODES


AND ANTHELMINTIC RESISTANCE (AR)
Various approaches are used for the control of gastrointestinal hel-
minths of livestock (Fig. 4.2). These are based on three basic principles
(Hoste and Torres-Acosta, 2011):
i. The first principle is to reduce the exposure of the host to the infective
stages (L3s), mainly achieved by strategies of grazing management (e.g.
rotational grazing, alternate grazing, pasture-spelling and/or reduced
stocking rates).
ii. The second principle targets the development of a more favourable
response of the host to gastrointestinal parasite infection (e.g. achieved
through vaccination, improved nutrition, genetic selection of hosts and
breeding for resistance).
iii. The third principle directly targets the elimination of worms from their
hosts through the administration of conventional (synthetic formula-
tions) or non-conventional (plant or mineral) anthelmintic
compounds.
Following the introduction of phenothiazines in the 1950’s, the control of gas-
trointestinal parasites has been achieved using chemical anthelmintics (Hoste
and Torres-Acosta, 2011) and still predominantly relies on the treatment with
broad-spectrum parasiticides, including the benzimidazoles (BZs), macrocy-
clic lactones (MLs) and imidazothiazoles/tetrahydropyrimidines (LVs)
(Besier and Love, 2003) (Table 4.3). Although there has been a recent break-
through with the development of a new drug, monepantel, from an alternative
compound class (amino-acetonitrile derivatives) (Kaminsky et al., 2008), suc-
cess in the discovery of new anthelmintics, more generally, has been extremely
limited over the past two decades (Kaplan, 2004).
The frequent and often uncontrolled use of these drugs has led to wide-
spread problems with AR in parasites of livestock (Taylor, M.A.,
Learmount, J., Lunn, E., Morgan, C., Craig, B.H., 2009). AR in parasites
of veterinary importance has emerged as a major bionomic and economic
problem worldwide, being currently most severe in parasitic nematodes
of small ruminants (von Samson-Himmelstjerna, 2006; Waller, 1994,
1997). For instance, in Australia, it has been proposed that the prevalence
and extent of resistance to all major classes of broad-spectrum anthelmintics
is so widespread that it threatens the profitability of the entire sheep industry
Table 4.3 Main anthelmintics used for the treatment of nematode infections in livestock; their mode of action (if known) and proposed mechanisms
of resistance
Anthelmintics Understood mode of action Proposed mechanisms of resistance References
Benzimidazoles Bind to b-tubulin and prevent the Mutations in the b-tubulin gene causing Lacey (1988)
formation of microtubules. Causes the structural changes in b-tubulin. As a Winterrowd et al.
inhibition of glucose uptake, protein secretion consequence the drug can no longer bind to (2003)
and microtubule production, leading to its target site.
starvation of the parasite.
Imidazothiazoles/ Mimic the action of acetylcholine causing Poorly understood, possible involvement Richmond and
tetrahydropyrimidines spastic paralysis of the worms. of structural changes in the nicotinic Jorgensen (1999)
Paralysed worms are expelled by normal acetylcholine receptor preventing the Robertson et al.
gut peristalsis leading to rapid removal of binding of the drug. (1999)
present worms. Also proposed have been changes in the Martin et al.
sensitivity of the receptor towards acetylcholine (2003)
which can lead to a cross-resistance with Martin and
organophosphates. Robertson (2007)
Macrocyclic lactones Causes an opening of glutamate-gated Poorly understood, possible involvement of: Blackhall et al.
(avermectins/ chloride channels (GluCl). This leads to Mutations in P-glycoprotein gene could cause a (1998)
milbemycins) an increased C1 ion influx into nerve gain-of-function leading to a more rapid Blackhall et al.
cell causing flaccid paralysis of the worm. removal of the drug from the parasite. (2003)
Selection at glutamate- and Kerboeuf et al.
y-aminobutyric-acid-gated chloride channels. (2003)
Amino-acetonitrile The hypothesized mode of action involves Full or partial loss of the gene which encodes Kaminsky et al.
derivatives a nematode-specific clade of acetylcholine the particular type of acetylcholine receptor. (2008)
receptor subunits.
Cycloocta- Binds to a presynaptic latrophilin receptor Harder et al.
depsipeptides in nematodes. (2003)
(emodepside/ Harder et al.
PFIO22A) (2005)
von Samson-
Himmelstjerna
et al. (2005)
Next-Generation Molecular-Diagnostic Tools 283

(Besier and Love, 2003). However, Larsen et al. (2006) showed that, despite
widespread problems of drug resistance, farm productivity can be
maintained or increased if sound farm management practices are put in place,
emphasizing the need for integrated approaches of worm control.
Although there is hope for new, effective anthelmintics, there is also a
major need to preserve those that we currently have at our disposal. Hence,
monitoring the drug-susceptibility and -resistance status of strongylid nem-
atode populations in livestock must be a high priority and should be an
important component of integrated management strategies. Various
methods, such as faecal egg count reduction test (FECRT) and egg hatch
and larval development assays, have been used for estimating levels of
drug-susceptibility/resistance in strongylid nematodes of small ruminants,
cattle and horses (Coles et al., 1992) (Table 4.4).
Most recent advances in the diagnosis of AR have focused on the imple-
mentation of a standardized protocol for the egg hatch test (von Samson-
Himmelstjerna et al., 2009) and the development and standardization of a
larval migration inhibition test (Demeler et al., 2010a). However, many
of these assays can be very time consuming to carry out, suffer from a lack
of reliability and sensitivity as well as reproducibility of results (Taylor et al.,
2002). Therefore, novel approaches of AR diagnosis are needed.
Molecular methods have been proposed to provide new alternatives to
the most commonly applied in vivo and in vitro techniques for the diagnosis of
AR and might be capable of overcoming some of their limitations (Beech
et al., 2011; Demeler et al., 2010a,b; von Samson-Himmelstjerna, 2006).
Critical to the development of molecular-diagnostic assays for AR is the
in-depth knowledge of the mode of action of these chemicals, their target
sites and mutations in the genome of parasitic helminths linked to a reduced
susceptibility to these drugs (Beech et al., 2011; von Samson-
Himmelstjerna, 2006; Wolstenholme et al., 2004). However, although
the molecular basis of mechanisms of AR is currently best understood for
BZ anthelmintics, concise information for other classes of broad-spectrum
anthelmintics is far less elucidated (Taylor et al., 2002). In the case of BZ, a
single-nucleotide polymorphism (SNP) at codon 200 of the b-tubulin
isotype 1 is currently believed to be most closely linked to BZ resistance
(Wolstenholme et al., 2004) and has been demonstrated in resistant strains
of H. contortus (see Geary et al., 1992), T. colubriformis (see Silvestre and
Humbert, 2002) and Te. circumcincta (see Elard and Humbert, 1999) in sheep.
At least two more SNPs at position 167 and 198 have been identified,
but appear to be less common amongst different species of trichostrongylid
Table 4.4 Summary of in vivo and in vitro tests currently used for the diagnosis of anthelmintic resistance in gastrointestinal nematodes of
livestock, and their principles and limitations
Assay Principle Comments and existing limitations References
In vivo Faecal egg Provides an estimate of anthelmintic – Does not accurately estimate the efficacy Martin et al.
count efficacy by comparing faecal egg counts of an anthelmintic to remove worms. (1985)
reduction test from sheep before and after treatment. It rather measures the effects on egg Presidente (1985)
Resistance is declared if reduction in the production by mature female worms. Coles et al. (1992)
number of eggs counted is <95% and the – Different anthelmintics require sample Jackson (1993)
lower confidence interval for the collection at different time intervals. Grimshaw et al.
percentage of reduction is below 90%. – No agreed standard for faecal egg count (1996)
method or for the calculation of McKenna (1996,
reduction. 1997, 2006)
– Results can be inconclusive due to low Taylor et al. (2002)
analytical sensitivity of the technique. Coles et al. (2006)
– Different results in repeated Miller et al. (2006)
experiments. Dobson et al.
– Does not provide species specific (2009)
information if undifferentiated. Larval Levecke et al.
culture required for further (2011)
differentiation
Controlled test Involves the infection of worm free sheep. – This test is considered the most reliable Presidente (1985)
Infective larvae (of susceptible and resistant method. Taylor et al. (2002)
strains) are inoculated together with the – Rarely used because of high costs due to
tested anthelmintic at 0.5, 1 and 2 times the labour requirements and animals that
recommended dose. need to be necropsied.
Resistance is declared if reduction in
geometric mean worm counts is less than
90% or greater than 1000 worms surviving
treatment.
In vitro Egg hatch test Known numbers of undeveloped eggs are – Was developed for benzimidazoles Le Jambre (1976)
incubated in serial dilutions of the (thiabendazole) which prevents nema- Boersema (1983)
anthelmintic. tode eggs from embryonation and Weston et al.
The percentage of eggs that hatch at each hatching. Therefore, determined ED50 (1984)
concentration is then determined and the values are variable, depending on the Borgsteede and
dose response calculated for the different counting method, by counting either Couwenberg
concentrations of the drug tested only hatched larvae or hatched larvae (1987)
(calculated as ED50 ¼ the concentration of and embryonated eggs. Kerboeuf and
drug required to kill 50% of eggs). – Different ED50 values can be deter- Hubert (1987)
mined throughout the course of infec- Martin et al.
tion, thus providing an inconclusive (1989)
result. Scott et al. (1989)
– Day-to-day variation in calculated levels von Samson-
of resistance may be observed. Himmelstjerna
– Technique lacks sensitivity to detect et al. (2009)
levels of resistance <25%.
– False-positive results can be obtained if
eggs are used that have advanced their
development past the ventral indenta-
tion stage.
– Test results can be influenced by the
water used (mainly its calcium, magne-
sium and phosphorus concentration)
and the method of preparing the
anthelmintic solution.

Continued
Table 4.4 Summary of in vivo and in vitro tests currently used for the diagnosis of anthelmintic resistance in gastrointestinal nematodes of
livestock, and their principles and limitations—cont'd
Assay Principle Comments and existing limitations References
Larval paralysis Infective third-stage larvae are incubated – Repeatability of this method is Martin and Le
test for 24 h in serial dilutions of the uncertain. Jambre (1979)
anthelmintic. Boersema (1983)
After 24 h the percentage of paralysed Geerts et al. (1989)
larvae is determined at each concentration Sutherland and
of the drug and a dose response line is Lee (1990)
plotted and compared to known reference
strains.
Larval motility Measures the motility of larval and adult – Was reported to be not suitable to detect Bennett and Pax
test nematodes after incubation together with levamisole resistance. (1986, 1987)
dilutions of anthelmintics. A motility index Coles et al. (1989)
is then calculated by a computer. Gill et al. (1991)
Migration test Adult worms are used to differentiate – Requires adult worms collected from Petersen et al.
between susceptible and resistant nematode their host at necropsy. (1997, 2000)
strains.
Worms are incubated for 30 min in
anthelmintic dilutions and then transferred
to migration chambers where their ability
to migrate through a polyamide net is
evaluated.
A dose response is plotted based on the
inhibited migratory capacity.
Larval Nematode eggs are cultured to the third- – Currently, only standardized for the Coles et al. (1988)
development larval stage in the presence of a food source detection of benzimidazole and levam- Giordano et al.
test (E. coli or yeast extract and the anthelmintic isole resistance in sheep parasitic (1988)
to be tested. nematodes. Taylor (1990)
Larvae affected by the anthelmintic fail to – Restricted in terms of labour and time Gill et al. (1995)
develop to the third-stage larvae, expressed requirements. Gill and Lacey
as LD50 (¼the concentration of the drug (1998)
required to kill 50% of larvae). Demeler et al.
(2010a, 2010b)
288 Florian Roeber et al.

nematodes (Beech et al., 2011; Wolstenholme et al., 2004). Besides the


sequence changes in b-tubulin, which are currently believed to be the major
cause of BZ resistance, more recent investigations have also suggested a link
to the drug transporter P-glycoprotein, which plays a role in the transport of
the anthelmintic away from its site of action and may also select for resistance
to MLs (Beech et al., 2011).
Based on the current knowledge of the mechanism of AR to BZ anthel-
mintics, conventional, allele-specific PCRs were developed to determine
the genotype of adult worms of H. contortus (see Kwa et al., 1994) and
Te. circumcincta (see Elard and Humbert, 1999). This work was further
extended by Silvestre and Humbert (2000) by combining the previously
described PCR assays with a RFLP procedure, which allowed the phenetic
characterization and identification of L3s of H. contortus, T. colubriformis and
Te. circumcincta. Alvarez-Sanchez et al. (2005) designed a real-time PCR
(RT-PCR) method to determine the allele frequency of b-tubulin isotype
1 (codon 200) in nematode DNA samples. As stated by the authors, the diag-
nosis of BZ resistance using this assay showed a significant agreement with
phenotypic tests, such as the egg hatch test and the faecal egg count reduc-
tion test, and allowed the analysis of allele frequencies in DNA samples from
pooled larvae (von Samson-Himmelstjerna, 2006).
Despite these developments, there has been no assessment of the suitabil-
ity of these assays for applications to field samples containing mixed species of
gastrointestinal parasites, which limits their practical utility at this stage. Fur-
thermore, all currently employed molecular assays involve adult nematodes
(only available through necropsy of the host) or infective L3s (requires cul-
turing of eggs for 7–10 days), but none of the existing assays has yet been
assessed for the detection of AR directly from (mixed populations of ) eggs,
which would significantly reduce the time required for diagnosis. In contrast
to the BZs, the molecular mechanisms associated with resistance to LEV and
ML anthelmintics are not yet understood in detail, and recent research has
suggested that in both cases multiple genes (Beech et al., 2011) are involved
in resistance and that resistance is often the result of changes in the parasite
other than the immediate drug target, including transporters and drug
metabolism (Cvilink et al., 2009). Consequently, the polygenic nature of
resistance to these anthelmintics and the lack of reliable or universal markers
represent a major obstacle to the development of molecular-diagnostic tools
for AR. No molecular test is yet available for these two groups of broad-
spectrum anthelmintics.
Next-Generation Molecular-Diagnostic Tools 289

6. TRADITIONAL METHODS FOR THE DIAGNOSIS OF


STRONGYLID NEMATODE INFECTIONS AND THEIR
LIMITATIONS
Different species of nematodes can be variable in their pathogenicity,
seasonal and geographical distribution and susceptibility towards different
anthelmintics. Therefore, the accurate diagnosis of infections and the
detailed knowledge of which parasites are present in a particular geograph-
ical environment are of major importance. Such information directly sup-
ports parasite control strategies and is of relevance for investigations into
parasite biology, ecology and epidemiology.

6.1. Validation of diagnostic tests


The validation of a diagnostic test involves the determination of basic param-
eters (Table 4.5) and can be achieved in five steps (Table 4.6) (OIE, 2004).
As a first step, a test suitable for a particular use has to be selected, developed
and optimized. Subsequently, validation parameters have to be determined,
such as analytical sensitivity and analytical specificity (Conraths and Schares,
2006). Following this initial assessment, the diagnostic sensitivity and spec-
ificity are determined by examining a larger number of samples for which
the true disease or infection status of the animals being tested is known
(determined by a ‘gold standard’). After a test has been evaluated, it may
be considered validated (Conraths and Schares, 2006). However, a contin-
uous monitoring of test performance during routine application is advisable.

6.2. Diagnosis in the live animal


Various methods are employed for the ante mortem diagnosis of gastrointes-
tinal nematode infections. The most commonly employed methods are
based on the observation of clinical signs indicative of disease, microscopic
examination of faeces from infected hosts, biochemical and/or serological
diagnostic approaches. However, widespread standardization of many labo-
ratory techniques, including FECs or LCs, does not existent, and most diag-
nostic, research and teaching facilities apply their own modifications to
published protocols (Kassai, 1999). Although these techniques are regarded
to be standard diagnostic procedures, there is a lack of detailed studies of
diagnostic performance, including the diagnostic sensitivity, specificity
and/or repeatability.
290 Florian Roeber et al.

Table 4.5 Important validation parameters used in the assessment of diagnostic


tests (Conraths and Schares, 2006; Pfeiffer, 2010; Thrusfield, 2005)
Term Definition Method of assessment
Sensitivity The proportion Assessment of these two parameters requires an
of animals with the independent, valid criterion termed a ‘gold
disease and which standard’ used to define the true disease status of
test positive. an animal.
Specificity The proportion
of animals without
the disease and
which
test negative.
Agreement The agreement in Frequently assessed by the Kappa test, which
results between measures the proportion of agreement beyond
two diagnostic that to be expected by chance.
tests, with one of
the tests being a
generally accepted
diagnostic method.
Accuracy Refers to the Depends on the number of ‘false-positives’ and
concordance ‘false-negatives’, in comparison with the true
between test results infection state as determined by the ‘gold
and the ‘true’ standard’.
clinical state.
Reliability The extent to This includes the assessment of repeatability,
which test results reproducibility, inter- and intra-assay variability.
are consistent in Repeatability assessment can be done by running
repeat the test two or more times on the same samples
experiments. in the same laboratory under the same conditions.
Additionally, the intra-assay variability (between
replicates within the same run) and inter-assay
variability (replicates between different runs) can
be assessed.
Reproducibility can be assessed in the same
manner as described before, and assessed between
different laboratories.

6.2.1 Diagnosis based on clinical signs of disease


Disease caused by gastrointestinal nematodes manifests itself in a variety of
clinical signs, including scouring, anaemia, loss of body condition and in
severe cases death (Hungerford, 1990). The type and degree of clinical
manifestation are also influenced by factors, such as the species of worms
Next-Generation Molecular-Diagnostic Tools 291

Table 4.6 Stages of test validation


Stages of test validation
1. Feasibility studies
2. Assay development and standardization
– optimization of reagents, protocols and equipment
– preliminary estimate of repeatability
– determination of analytical sensitivity and specificity
3. Determination of assay performance characteristics
– diagnostic sensitivity and specificity
– repeatability and reproducibility
4. Monitoring the validity of assay performance
5. Maintenance and enhancement of validation criteria
Adapted from Conraths and Schares (2006).

involved, the present worm burden, the plane of nutrition and reproduc-
tive/immunological status of the host animal (Hungerford, 1990; Levine,
1968). To aid the diagnosis of gastrointestinal nematode infections, a num-
ber of approaches have been developed for the interpretation of clinical signs
linked to PGE. These approaches include, for example, body condition-
(Russel et al., 1969), ‘dag’- (Larsen et al., 1994) or anaemia-scoring (van
Wyk and Bath, 2002). Although useful as indicators for PGE, with applica-
bility on a farm level, these approaches are subjective and lack specificity, as
clinical signs can relate to an extremely wide range of diseases and problems
affecting the host (van Wyk and Bath, 2002).

6.2.2 Faecal egg counts


The enumeration of eggs in faeces is the most common method for the diag-
nosis of gastrointestinal nematode infections and is routinely employed in
parasitology. The technique is inexpensive, easy to perform and does not
require specialized instrumentation, which makes it suitable for use in most
diagnostic settings and countries. Important applications of this method
include estimating infection intensity (McKenna, 1987; McKenna and
Simpson, 1987), determining the degree of contamination with helminth
eggs (Gordon, 1967), assessing the effectiveness of anthelmintics (Waller
et al., 1989), determining the breeding value of an animal when selecting
for worm resistance (Woolaston, 1992), and guiding control and treatment
decisions (Brightling, 1988).
This method involves mixing faeces with a high-density solution (e.g.
saturated sucrose, sodium chloride or sodium nitrate) to float parasite eggs
292 Florian Roeber et al.

(with the exception of trematode eggs) on the surface of the suspension. An


aliquot of this suspension is withdrawn to count the eggs present and to then
extrapolate the number to the volume of faeces used (in eggs per gram,
EPG). Different methods have been developed for this purpose, such as
the direct centrifugal flotation method (Lane, 1922), the Stoll dilution tech-
nique (Stoll, 1923), the McMaster method (Gordon and Whitlock, 1939)
and the Wisconsin flotation method (Cox and Todd, 1962), of which the
McMaster method is most widely used (Nicholls and Obendorf, 1994).
For decades, numerous modifications of these methods have been described
(Levine et al., 1960; Raynaud, 1970; Roberts and O’Sullivan, 1950;
Whitlock, 1948), and most teaching and research institutions apply their
own modifications to existing protocols (Kassai, 1999). Many of these
modifications make use of different flotation solutions, sample dilutions
and counting procedures, which achieve varying sensitivities and can com-
plicate the comparison of FECs between laboratories. These inconsistencies
make a pooled data analysis or retrospective studies of results between lab-
oratories virtually impossible. The choice of flotation solution, sample dilu-
tion and volume examined can have a significant impact on the results
obtained using this approach (Cringoli et al., 2004); similarly, biological fac-
tors can also limit the interpretation of these results, as described in the
following.
Fecundity. The biotic potential of different species of trichostrongylid
nematodes varies (Gordon, 1981), and parasite density and immune-
mediated ‘control’ by the host have been shown to influence the egg pro-
duction of female worms in different species (Rowe et al., 2008; Stear and
Bishop, 1999). The diagnostic value of FECs to estimate worm burdens
for the highly fecund nematode species, H. contortus, has been shown
(Le Jambre et al., 1971; Roberts and Swan, 1981). However, the correlation
between FECs and worm burdens is much lower for genera with a low
fecundity, such as species of Teladorsagia (Ostertagia) (Martin et al., 1985),
Trichostrongylus (Sangster et al., 1979) and Nematodirus (Martin et al.,
1985; McKenna, 1981).
Water content of faeces. Samples intended for faecal analysis can be of vary-
ing consistencies, being soft to watery (diarrhoeic) or hard and desiccated
(mostly from animals following transport and without access to food or
water) (Gordon, 1953, 1981). These aspects are of importance, as the water
content of the sample can either dilute or concentrate the numbers of eggs
determined from 1 g of faeces, depending on the actual amount of dry mat-
ter (Le Jambre et al., 2007).
Next-Generation Molecular-Diagnostic Tools 293

Storage of faeces. For practical reasons, faecal material requires proper stor-
age prior to coprological examination. Storage conditions are of importance
because they can cause a reduction in egg numbers. An artefactual reduction
in FECs occurs primarily due to hatching of eggs or biological degradation
(Nielsen et al., 2010). To circumvent this problem, different strategies, such
as chemical preservation (Whitlock, 1943), airtight storage (Rinaldi et al.,
2011) or refrigeration (Nielsen et al., 2010), have been recommended.
Additional considerations are that FECs (i) only reflect patent but not
pre-patent infections (Thienpont et al., 1986), (ii) do not provide any infor-
mation regarding male or immature worms present (McKenna, 1981) and
(iii) can be influenced by variation in times of egg excretion by adult worms
(Villanua et al., 2006), age of the worm population and/or the immunity,
age and sex of the host (Thienpont et al., 1986). Although there are mor-
phological differences between the eggs of socio-economically important
nematodes (Georgi and McCulloch, 1989), specific identification cannot
be achieved by routine microscopy (with few exceptions, such as
Nematodirus spp.) (Lichtenfels et al., 1997).
Therefore, FECs alone should not be used to make a diagnosis or guide
treatment decisions, but should be interpreted in conjunction with informa-
tion about the nutritional status, age and management of sheep in a flock
(McKenna, 2002). However, according to common practice, an FEC
of 200 EPG is regarded to indicate a significant worm burden and is
used as basis for the decision for anthelmintic treatment (www.wormboss.
com.au). The value of FEC results also depends on the hosts and/or parasite
species involved. For example, FEC results for adult cattle are of limited
diagnostic value, as they do not usually correlate with worm burden
(McKenna, 1981); FECs in cattle are usually low and require more sensitive
flotation techniques than for sheep (Mes et al., 2001); for species of Nema-
todirus, egg counts are also regarded to be of limited value, as most damage is
caused by the immature stages before egg-laying commences (McKenna,
1981). In addition, the low sensitivity of FEC techniques, being in the range
of 10–50 EPG for the McMaster technique, represents a limitation in rela-
tion to the diagnosis of AR by FECRT (Levecke et al., 2012).
Although it is unlikely that some of the current limitations of FEC will be
resolved in the near future (i.e. those which relate to the fecundity of the
nematodes), some recent developments have been made towards improving
the procedure. Attempts have been made by the World Association for the
Advancement of Veterinary Parasitology (WAAVP) to implement FEC
protocols for the assessment of AR in different species of animals (Coles
294 Florian Roeber et al.

et al., 2006). Lectin staining for the identification of H. contortus eggs (Palmer
and McCombe, 1996), computerized image recognition of strongylid eggs
(Sommer, 1996) and automated egg counting (Mes et al., 2007) are inter-
esting developments towards improved species identification and differen-
tiation. However, the suitability of the latter two approaches needs
rigorous assessment for routine applications because of their technical com-
plexity. With the development of FECPAK, a diagnostic test-kit containing
the necessary equipment for coproscopic examination (www.techiongroup.
co.nz), efforts have been made to enable sheep farmers to carry out FECs
themselves. However, the implementation of such a system requires a sig-
nificant level of cooperation by farmers, adequate training and integrated
quality assurance to ensure that correct diagnoses are made (McCoy et al.
2005). Despite the provision of training courses for farmers, many difficulties
in the differentiation of structures observed during coproscopy were
encountered for inadequately trained farm employees, limiting the value
of the results obtained (McCoy et al., 2005). The development of the FLO-
TAC egg counting method (Cringoli et al., 2010), seems to be promising.
Once validated for different host and parasite species, this method may
deliver FECs with increased sensitivity (i.e. 1 EPG) and might represent
an alternative to current flotation techniques.

6.2.3 Larval culture


To aid the identification of different nematode genera present in mixed
infections, faecal culture methods have been developed to identify first-stage
(L1) or infective L3s of strongylid nematodes. The most commonly used
methods involve the incubation of faecal material, in order to produce infec-
tive L3s for subsequent morphological differentiation. Currently, a number
of protocols have been published which differ in the temperatures, times and
media used for culture and the approach of larval recovery (Dinaburg, 1942;
Hubert and Kerboeuf, 1984; MAFF, 1986; Roberts and O’Sullivan, 1950;
Whitlock, 1956). Probably, the most widely employed protocol suggests
incubation at 27  C for 7 days (MAFF, 1986).
However, studies investigating the ecology and developmental require-
ments of various species of gastrointestinal nematodes infecting livestock
(Beveridge et al., 1989a; O’Connor et al., 2006) have shown that different
species of strongylid nematodes require different conditions, such as envi-
ronmental temperature and relative humidity, to enable adequate develop-
ment. This is particularly important to consider when LC results are used
to estimate the contribution of different species to mixed infections.
Next-Generation Molecular-Diagnostic Tools 295

One culture protocol is likely to favour the development of one species over
others (Dobson et al., 1992). For instance, Whitlock (1956) observed that
culture conditions (27  C for 7 days) are suitable for most species, but that
the free-living stages of Teladorsagia (Ostertagia) species develop better at
somewhat lower temperatures. This statement was supported by the findings
of Dobson et al. (1992) who demonstrated that the developmental success of
the infective larvae in faecal cultures was lower for Te. circumcincta than for
T. colubriformis when cultured alone or concurrently, thus indicating that
LCs are unreliable for estimating the contribution of individual species in
mixed infections. Similar observations were made by Berrie et al. (1988)
for the bovine parasites Haemochus placei, Oesophagostomum radiatum and
Cooperia pectinata. In this study, these authors observed that the recovery
of larvae of H. placei was significantly lower compared with the other two
species under the same culture conditions. Based on their findings, the
authors stated that faecal culture and subsequent larval differentiation are
unsuitable for an accurate estimation of the proportions of individual species
in mixed infections and can only be used to provide an indication of the spe-
cies present (Berrie et al., 1988).
Further variability in the results obtained from LCs have been attributed
to differences in the composition of the culture medium used, which influ-
ences the moisture, oxygen availability and/or pH that larvae encounter
during their development (Hubert and Kerboeuf, 1984; Roberts and
O’Sullivan, 1950). Therefore, it had been suggested that providing a
better-defined medium than faeces might help to obtain more consistent
results (Hubert and Kerboeuf, 1984). To further examine this hypothesis,
Hubert and Kerboeuf (1984) developed a modified method of LC using
an ‘on-agar’ approach to provide well-defined and standardized conditions.
Their results showed that the culture on agar medium led to higher recov-
eries of larvae compared with traditional faecal cultures. However, lengthy
preparation times and increased laboratory requirements appear to limit the
routine application of this method.
In addition to the variability of results related to the culture conditions
employed, the specific identification of cultured larvae provides challenges
(Fig. 4.3). For the identification of infective L3s to the species or genus level,
a number of different approaches have been described. A commonly
employed method for species differentiation involves the detection of par-
ticular morphological features of the larvae (e.g. the length of the tail sheath
extension and total body length of L3s) and their comparison with published
identification keys (Dikmans and Andrews, 1933; Gordon, 1933; MAFF,
296 Florian Roeber et al.

Figure 4.3 Third-stage larvae (L3s) of key species of gastrointestinal nematodes of


sheep, encountered following larval culture (LC).

1986; McMurtry et al., 2000; van Wyk et al., 2004). Various keys for the
identification of L3s have been published (Dikmans and Andrews, 1933;
Gordon, 1933; MAFF, 1986), and there is a substantial overlap between
the body length measurements of different species, and a substantial variabil-
ity in the length of L3s has been reported by different authors (McMurtry
et al., 2000).
Van Wyk et al. (2004) developed a simplified approach which uses the
mean length of the tail sheath extension to differentiate L3s of Teladorsagia
and/or Trichostrongylus from the larvae of Haemonchus and Chabertia and/or
Oesophagostomum. However, although useful for a differentiation of genera,
without the requirement to measure every single larva (and thus being more
time efficient), this approach has the disadvantage that it does not allow the
unequivocal differentiation of all genera. For instance, Teladorsagia and
Next-Generation Molecular-Diagnostic Tools 297

Trichostrongylus (being the most common genera in winter rainfall areas) can-
not be differentiated based on sheath extension length alone. To further
refine their differentiation, additional morphological features are required.
Lancaster and Hong (1987) proposed the presence of an inflexion (‘shoul-
der’) at the cranial extremity of Teladorsagia larvae as an informative morpho-
logical feature. However, this feature is very subtle and its detection is
subjective.
Another approach to differentiate L3s of Teladorsagia from those of
Trichostrongylus was proposed by Gordon (1933); it is based on the body
length measurements of the larvae. According to this author, and based
on the measurements of 1000 larvae of each genus, a body length ‘cut-
off’ value of 720 mm allowed reliable differentiation of these genera, with
Trichostrongylus larvae being 720 mm and those of Teladorsagia being
>720 mm. Although, practical, this technique requires the measurement
of individual larvae and does not take into account variability in the length
of developing larvae (e.g. influenced by culture conditions employed, cli-
mate/season in a particular environment, availability of appropriate food
for developing L1s and L2s and/or immune status of the host)
(McMurtry et al., 2000).
A third approach for the differentiation of Teladorsagia from
Trichostrongylus L3s was described by McMurtry et al. (2000). The latter
approach involves the treatment of cultured larvae with sodium hypochlo-
rite to exsheath the larvae and count tubercles at the posterior end of
the exsheathed L3. As claimed by the authors, this approach allows the
differentiation among populations of T. axei, T. colubriformis, T. vitrinus
and Te. circumcincta. However, the authors acknowledge that there is a degree
of variability in the number of observed tubercles and that the tails of
Te. circumcincta and T. axei lack these structures (McMurtry et al., 2000).
Although being regarded as economically ‘less important’ parasites, the dif-
ferentiation of L3s of Oesophagostomum and Chabertia, which infect the large
intestine, is not considered possible using current techniques for larval dif-
ferentiation, which makes this approach unsuitable if detailed epidemiolog-
ical information on distribution and prevalence of genera and species is
required. A less commonly used method for larval differentiation involves
the culture and morphological identification of L1s (Whitlock, 1959). This
technique has the advantage of being rapid, since the time required for the
development of the L1 stage is shorter; however, the same limitations for the
culture and identification of L3s exist for L1s and L2s (Lichtenfels
et al., 1997).
298 Florian Roeber et al.

In conclusion, although regarded as a standard diagnostic procedure, the


technique of LC coupled to larval differentiation has the disadvantage of
being time consuming and laborious, cannot readily be automated, and
the morphological identification/differentiation, based on available publica-
tions, requires substantial expertise and training (Johnson et al., 1996;
Lichtenfels et al., 1997).

6.2.4 Biochemical and immunological methods


In addition to commonly used coprodiagnostic methods, a number of bio-
chemical and immunological approaches have been developed that aim at
the specific diagnosis of infection. These methods are mainly based on
the detection and measurement of parameters (e.g. detection of elevated
serum pepsinogen and gastrin levels or circulating antibodies) that might
be indicative of parasitic infections.

6.2.5 Pepsinogen and gastrin


Chief cells of the gastric fundus produce the pro-enzyme pepsinogen, which
is converted to its active form by acid produced by parietal cells. When par-
asitized glands of the gastric mucosa are destroyed, the hydrochloric acid
production of parietal cells decreases, causing a rise in abomasal pH and
resulting in a failure to convert pepsinogen to active pepsin (Levine,
1968). Accumulating pepsinogen can escape between disrupted cell junc-
tions into the blood. Therefore, an increase in serum pepsinogen concen-
tration has been regarded to relate to mucosal damage by developing
larval stages of Ostertagia (Levine, 1968). Berghen et al. (1993) reviewed
the value and application of pepsinogen, gastrin and antibody responses as
diagnostic indicators for ostertagiasis and identified a number of potentially
limiting factors. The authors suggested that other parasitic or non-parasitic
diseases can be responsible for a moderate rise in pepsinogen concentrations
in blood, thus limiting the specificity of this approach.
Gastrin is a hormone produced by G-cells in the stomach. Gastrin stim-
ulates parietal cells to secrete acid and also stimulates pepsinogen secretion,
stomach motility and blood circulation in gastric vessels. It was suggested
that strongylid nematodes can directly stimulate G-cells, causing an
increased gastrin production (Berghen et al., 1993). However, as demon-
strated for pepsinogen, the specificity of this approach was questioned
(Berghen et al., 1993), because other parasites or factors, such as diet, lacta-
tion or abomasal lesions, can also affect gastrin levels. Furthermore, in an
experimental context, it has been shown that high infective doses need to
Next-Generation Molecular-Diagnostic Tools 299

be administered to parasite-naı̈ve calves to provoke a significant gastrin


release (Berghen et al., 1993).

6.2.6 Immunological methods of diagnosis


A wide range of immunological methods, including those that are based on
the detection of an immune response in an infected animals and those for the
detection of parasite antigens, have been developed for the specific diagnosis
of parasitic infections (e.g. Engvall and Ruitenberg, 1974; Fletcher, 1965;
Ogunremi et al., 2008). Based on the target molecule (antigen or antibody),
such immunological methods can be classified as being either direct or
indirect.
Direct immunological methods are those that provide direct evidence
of an infection and can be based on the detection of parasite antigens pre-
sent in the circulation and/or excreta from infected hosts. Parasitic
extracts have a complex composition and contain molecules which are
sometimes shared by other parasites (Cohen and Sadun, 1976). Shared
antigenic composition of closely related parasite species is a challenge,
particularly for nematodes, and often leads to cross-reactivity in immuno-
logical tests (Eysker and Ploeger, 2000; Noordin et al., 2005). Also the
presence of host materials associated with the parasite can complicate
antigen purification and can interfere with the specificity of a diagnostic
assay. Furthermore, the life-cycle stage of the parasite used as an antigen
source can influence immuno-diagnostic results, as parasites undergo sig-
nificant structural and biochemical changes during their development
(Cohen and Sadun, 1976). As an example, the antigenic composition
of larval stages differs from that of adults (Williams and Soulsby, 1970)
and can give rise to variations in diagnostic sensitivity and specificity
(McLaren et al., 1978).
Although limited research has been undertaken, to date, for livestock par-
asites, Johnson et al. (1996) described an immuno-diagnostic assay for the
quantitative detection of excretory/secretory parasite antigens in host faeces
(coproantigens). These authors evaluated the usefulness of this approach in
a murine model system using Heligmosomoides polygyrus, a trichostrongyloid
gastrointestinal nematode related to the common nematode species infecting
ruminants. The authors also suggested that the enzyme-linked immunosor-
bent assay (ELISA) was useful to specifically detect parasite antigens in the host
faeces and might have potential to detect pre-patent infections and make large
scale, rapid screening possible. The diagnostic performance of the assay was
promising under experimental conditions, but cross-reactivity, faecal
300 Florian Roeber et al.

components interfering with the reactivity and the loss of antigens in faeces
have been reported (Johnson et al., 1996).
Indirect immunological methods are usually based on the detection of
anti-parasite antibodies or cell-mediated immune responses in infected
hosts. A variety of methods have been developed and applied to the diagnosis
of nematode infections, such as the complement fixation test, indirect
immunofluorescence, indirect haemagglutination and ELISA, of which
the latter has been most commonly used (Doenhoff et al., 2004). However,
parasitic helminths possess a huge variety of antigens, and there is limited
information on which stages and antigens are actually responsible for
eliciting immune responses (Berghen et al., 1993). Antibody detection from
serum has several disadvantages, including that it cannot distinguish between
a current and past infection, which is a major challenge when evaluating the
effects of chemotherapy, does often not reflect infection intensity and some-
times achieves poor specificity, particularly in disease-endemic areas
(Doenhoff et al., 2004).
The detection of anti-Ostertagia antibodies in the serum of cattle has
been found to be useful for epidemiological and cross-sectional studies, but
only of limited utility for diagnosis on an individual animal basis (Berghen
et al., 1993). Although anti-Ostertagia antibodies are detectable in milk samples
by ELISA, there are also some limitations to this approach (Charlier
et al., 2010). The response to parasitic infections is variable among host indi-
viduals, and it has been shown that serum antibody levels can be influenced by
factors, such as milk yield, season, mastitis, the number of pregnancies of a
cow, stage of lactation and genetic constitution (Gasbarre et al., 1993;
Kloosterman et al., 1993; Sanchez et al., 2004). Also the use of bulk milk sam-
ples has been investigated, which has the advantage of being an inexpensive
and user-friendly approach (Charlier et al., 2010). However, bulk milk sam-
ples taken only a few weeks apart can show significant variation in test results,
depending on calving patterns, number of cows contributing to the milk in a
tank (i.e. dilution effect) and their relative milk yields (Pritchard, 2001).

6.3. Post-mortem diagnosis


The post-mortem diagnosis of infections is routinely employed in parasitology
to determine the number of nematode parasites present in the gastrointes-
tinal tract, for epidemiological studies or to assess anthelmintic efficacy. Gen-
erally, these techniques involve the opening and washing of the respective
parts of the GI tract and the examination of subsamples of the washes to
Next-Generation Molecular-Diagnostic Tools 301

estimate the total numbers of nematodes present. A number of different


techniques have been described (Eysker and Kooyman, 1993; MAFF,
1986; Robertson and Elliott, 1966). The differences among these techniques
exist in the counting of nematode worms from either the chyme or digesta
and/or the washes (separately or combined); the soaking or not of the organ
in water or saline (mainly used to recover immature stages); and the propor-
tion of the total volume and the number of aliquots examined (reviewed by
Gaba et al., 2006). Other differences exist in the length of the intestinal sec-
tion examined (proximal 10 m of small intestine vs. the entire length) and
the mesh size of the sieve used to remove plant debris from the washes
(McKenna, 2008). Most necropsy techniques for the estimation of the inten-
sity of infection involve the counting of worms from pooled gut contents
and washes.
Eysker and Kooyman (1993) described a method that involves three
components (contents, immediate water wash of the organ and the saline
wash after 5 h of soaking the organ). The disadvantage of this method is that
it involves more labour at necropsy, but it has the distinct advantage that
worms are separated from the bulk of the gut contents, allowing a more rapid
worm count in the laboratory. Gaba et al. (2006) assessed their approach for
H. contortus and Te. circumcincta and suggested that the estimation of infection
intensity, based on gut washes alone, is reliable. However, a prerequisite is
that abomasa are processed rapidly (within 15 min) after the death of the
sheep, as worms progressively start migrating into the contents (Gaba
et al., 2006). Gaba et al. (2006) also stated that immediate washing of the
gut is insufficient for extracting T. axei or larvae from the mucosa and, there-
fore, may distort the results if necropsy is carried out for the purpose of
assessing trichostrongylid diversity or to examine the gut for the presence
of immature larval stages.
Similarly, the choice of mesh size of the sieves used is dictated by
the purpose of the worm counting procedure, as the use of a smaller mesh
size enables a higher recovery of early L4s, but has the disadvantage that
more debris is held back in the subsample examined, resulting in a prolonged
time for counting (McKenna, 2008). Therefore, a small sieve size (e.g.
38 mm aperture) is only necessary if information about early L4s is required,
whereas, if studies are carried out to confirm AR (reflected by a reduced effi-
cacy against adult worms), also larger mesh sizes (e.g. 250 mm aperture) can
be used (McKenna, 2008).
The common practice of examining the proximal 10 m of the small
intestine is based on the observation that most intestinal Trichostrongylus
302 Florian Roeber et al.

spp. occur within the first 6 m of the small intestine (Beveridge and Barker,
1983), whereas larger amounts of plant debris are present in the more distal
part of the small intestine (McKenna, 2008). McKenna (2008) claimed that
processing only the first 10 m of the small intestine led to a recovery of <50%
of the worms located in the entire length, resulting in serious underestimates
of the total number of worms present. However, the data presented were
based on the necropsy of only 15 sheep, and a recovery of less than 50%
of the total number was observed only in a few individual sheep, whereas
in most infected sheep trichostrongylid nematodes were located in the prox-
imal 10 m of the small intestine (cf. McKenna, 2008). Therefore, it can be
concluded that the improvement of accuracy achieved by processing the
entire small intestine is marginal and involves a significant increase in the
amount of labour and time required for processing.

7. DNA-BASED METHODS FOR SPECIFIC DIAGNOSIS


OF STRONGYLID NEMATODES INFECTIONS
IN LIVESTOCK: SAMPLE PREPARATION, MARKERS
AND DEVELOPMENTS
Clearly, traditional methods (reviewed in Section 6) have limitations,
in terms of sensitivity and/or specificity. In addition, they can be time con-
suming and costly to carry out. Advances in molecular biological technol-
ogies have enabled the development of new, sensitive and specific diagnostic
methods that have found applications in the field of veterinary parasitology.
The ability to specifically identify and study parasites (irrespective of life-
cycle stage) using such tools has provided new insights into parasite system-
atics, population genetics, ecology and epidemiology, and has important
implications for the specific diagnosis, treatment and control of parasitic dis-
eases (Gasser, 2006). In particular, methods that rely on the enzymatic ampli-
fication of nucleic acids can overcome some of the limitations of traditional
approaches (Gasser, 2006). Techniques that employ the PCR (Mullis et al.,
1986; Saiki et al., 1988) can selectively amplify in vitro target DNA sequences
from complex genomes or matrices and have led to advances in many areas
of the biological sciences. PCR involves the heat denaturation of double-
stranded DNA, followed by a decrease in temperature to allow oligonucle-
otide primers to bind (¼anneal) to their complementary sequence on sense
and antisense strands of the target template. Then, the temperature is
increased again to enhance the enzymatic activity of a thermostable DNA
polymerase, which extends the complementary strands from the primer sites.
Next-Generation Molecular-Diagnostic Tools 303

These synthesis steps are usually repeated 20–40 times in an automated ther-
mal cycler, resulting in an exponential increase in target DNA copies. The
major advantage of this methodology is that it enables the study of parasite
DNA from minute amounts of template, which would otherwise be insuf-
ficient for conventional analysis. The value of this technology in the field of
diagnostic veterinary parasitology lies in its ability to specifically identify par-
asites, detect infection and analyse genetic variation, which are particularly
important, given the increasing problems of AR in parasitic nematodes
(Gasser, 2006; Gasser et al., 2008).

7.1. Sample preparation prior to PCR


The main goals of sample preparation are to (i) concentrate the target organ-
isms and, subsequently, the template for the PCR; (ii) eliminate possible
PCR inhibitors; and (iii) produce a homogenous sample for specific and sen-
sitive enzymatic amplification (Rådström et al., 2004). Complex biological
(e.g. faecal) samples can contain a wide range of inhibitory substances (e.g.
bile salts, collagen, haeme, humic acids and polysaccharides), which are
capable of reducing or preventing PCR amplification