Advances in Parasitology, Volume 117

David Rollinson
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VOLUME ONE HUNDRED AND SEVENTEEN

ADVANCES IN
PARASITOLOGY
 SERIES EDITOR
D. ROLLINSON
Life Sciences Department
The Natural History Museum,
London, United Kingdom
d.rollinson@nhm.ac.uk
EDITORIAL BOARD
T. J. C. ANDERSON
Department of Genetics, Texas
Biomedical Research Institute,
San Antonio, TX, United States
M. G. BASÁÑEZ
Professor of Neglected Tropical
Diseases, Department of Infectious
Disease Epidemiology, Faculty of
Medicine (St Mary’s Campus),
Imperial College London,
London, United Kingdom
D. D. BOWMAN
Director Cornell CVM MPS—Veterinary
Parasitology, Professor of Parasitology,
C4-119 VMC, Dept Micro & Immunol,
CVM Cornell University, Ithaca,
NY, United States
R. B. GASSER
Faculty of Veterinary and Agricultural
Sciences, The University of Melbourne,
Parkville, VIC, Australia
A. L. GRAHAM
Professor of Ecology & Evolutionary Biology,
Co-Director of the Global Health Program,
Princeton University, Princeton,
NJ, United States
J. KEISER
Head, Helminth Drug Development Unit,
Department of Medical Parasitology and
Infection Biology, Swiss Tropical and Public
Health Institute, Basel, Switzerland
J. R. STOTHARD
Department of Tropical
Disease Biology
Liverpool School of Tropical
Medicine, Liverpool, United Kingdom
russell.stothard@lstmed.ac.uk
K. KING
Department of Zoology,
University of Oxford,
Oxford, United Kingdom
M. G. ORTEGA-PIERRES
Professor of the Department of Genetics
and Molecular Biology,
Centro de Investigación y de
Estudios Avanzados IPN,
Mexico City, Mexico
D. L. SMITH
Johns Hopkins Malaria Research
Institute & Department of Epidemiology,
Johns Hopkins Bloomberg School
of Public Health, Baltimore,
MD, United States
R. C. A. THOMPSON
Head, WHO Collaborating Centre
for the Molecular Epidemiology
of Parasitic Infections, Principal
Investigator, Environmental
Biotechnology CRC (EBCRC),
School of Veterinary and Biomedical
Sciences, Murdoch University,
Murdoch, WA, Australia
X.-N. ZHOU
Professor, Director, National Institute of
Parasitic Diseases,
Chinese Center for Disease Control
and Prevention, Shanghai,
People’s Republic of China
VOLUME ONE HUNDRED AND SEVENTEEN

ADVANCES IN
PARASITOLOGY
Edited by

DAVID ROLLINSON
Life Sciences Department
The Natural History Museum,
London, United Kingdom

RUSSELL STOTHARD
Department of Tropical
Disease Biology
Liverpool School of Tropical
Medicine, Liverpool, United Kingdom
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Contents

Contributors vii

1. The microscopic five of the big five: Managing zoonotic diseases

within and beyond African wildlife protected areas 1
Anya V. Tober, Danny Govender, Isa-Rita M. Russo, and Jo Cable
1. Introduction 2
2. The ‘Microscopic Five’ 4
3. Challenges of BTb control at the wildlife-livestock interface: The South
African case study 10
4. Drivers of disease: The Kruger National Park case study 13
5. Disease knowledge gaps and lessons learnt from African protected areas 28
6. Communities and conservation 33
7. Conclusions 35
Acknowledgements 37
References 37

2. Improving translational power in antischistosomal drug

discovery 47
Alexandra Probst, Stefan Biendl, and Jennifer Keiser
1. Filling the drug pipeline for schistosomiasis 48
2. Evaluating the importance of S. mansoni isolate origin for early
antischistosomal drug discovery 49
3. The S. mansoni mouse model for drug efficacy testing 51
4. Infection intensity of the patent S. mansoni mouse model 54
5. Pharmacokinetic/pharmacodynamic (PK/PD) relationship of selected drugs 56
6. Concluding remarks 68
Acknowledgements and funding 69
References 69

3. Unique thiol metabolism in trypanosomatids: Redox

homeostasis and drug resistance 75
Vahab Ali, Sachidananda Behera, Afreen Nawaz, Asif Equbal,
and Krishna Pandey
1. Introduction 77
2. Trypanothione metabolism 80

v
vi Contents

3. Effector proteins of the antioxidant defence: Old and new actors 90

4. Trypanothione metabolism is linked to cysteine, polyamine,
and pentose phosphate pathway 103
5. The role of redox active compounds and their mechanism
in parasites survival 110
6. Role of thiol metabolism in drug resistance 118
7. Anti-parasitic potential of molecules targeted against redox metabolism 123
8. Unsolved questions and future prospects 131
Acknowledgements 132
References 132
Contributors

Vahab Ali
Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry,
ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna,
Bihar, India
Sachidananda Behera
Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry,
ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna,
Bihar, India
Stefan Biendl
Swiss Tropical and Public Health Institute, Department of Medical Parasitology and
Infection Biology; University of Basel, Basel, Switzerland
Jo Cable
School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom
Asif Equbal
Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry,
ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna;
Department of Botany, Araria College, Purnea University, Purnia, Bihar, India
Danny Govender
SANParks, Scientific Services, Savanna and Grassland Research Unit, Pretoria; Department
of Paraclinical Sciences, University of Pretoria, Onderstepoort, South Africa
Jennifer Keiser
Swiss Tropical and Public Health Institute, Department of Medical Parasitology and
Infection Biology; University of Basel, Basel, Switzerland
Afreen Nawaz
Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry,
ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna,
Bihar, India
Krishna Pandey
Department of Clinical Medicine, ICMR-Rajendra Memorial Research Institute of Medical
Sciences (RMRIMS), Patna, Bihar, India
Alexandra Probst
Swiss Tropical and Public Health Institute, Department of Medical Parasitology and
Infection Biology; University of Basel, Basel, Switzerland
Isa-Rita M. Russo
School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom
Anya V. Tober
School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom

vii
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 CHAPTER ONE

The microscopic five of the big

five: Managing zoonotic diseases
within and beyond African wildlife
protected areas
Anya V. Tobera,∗, Danny Govenderb,c, Isa-Rita M. Russoa,†,
and Jo Cablea,†
a
School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom
b
SANParks, Scientific Services, Savanna and Grassland Research Unit, Pretoria, South Africa
c
Department of Paraclinical Sciences, University of Pretoria, Onderstepoort, South Africa
∗
Corresponding author: e-mail address: tobera@cardiff.ac.uk

Contents
1. Introduction 2
2. The ‘Microscopic Five’ 4
2.1 Bovine tuberculosis 7
2.2 Rift Valley fever 7
2.3 Brucellosis 8
2.4 Cryptosporidiosis 8
2.5 Schistosomiasis 9
3. Challenges of BTb control at the wildlife-livestock interface: The South African
case study 10
3.1 Control in livestock 10
3.2 Control in wildlife 10
4. Drivers of disease: The Kruger National Park case study 13
4.1 Past and present disease management 13
4.2 Kruger National Park’s current adaptive management approach 14
4.3 Environmental drivers of disease transmission 15
4.4 Anthropogenic drivers of disease transmission: Wildlife-livestock-human
interface 23
5. Disease knowledge gaps and lessons learnt from African protected areas 28
6. Communities and conservation 33
7. Conclusions 35
Acknowledgements 37
References 37

†
Authors contributed equally to this work.

Advances in Parasitology, Volume 117 Copyright #2022 Elsevier Ltd 1

ISSN 0065-308X All rights reserved.
https://doi.org/10.1016/bs.apar.2022.05.001
2 Anya V. Tober et al.

Abstract
African protected areas strive to conserve the continent’s great biodiversity with a
targeted focus on the flagship ‘Big Five’ megafauna. Though often not considered,
this biodiversity protection also extends to the lesser-known microbes and parasites
that are maintained in these diverse ecosystems, often in a silent and endemically sta-
ble state. Climate and anthropogenic change, and associated diversity loss, however,
are altering these dynamics leading to shifts in ecological interactions and pathogen
spill over into new niches and hosts. As many African protected areas are bordered by
game and livestock farms, as well as villages, they provide an ideal study system to
assess infection dynamics at the human-livestock-wildlife interface. Here we review
five zoonotic, multi-host diseases (bovine tuberculosis, brucellosis, Rift Valley fever,
schistosomiasis and cryptosporidiosis)—the ‘Microscopic Five’—and discuss the
biotic and abiotic drivers of parasite transmission using the iconic Kruger National
Park, South Africa, as a case study. We identify knowledge gaps regarding the impact
of the ‘Microscopic Five’ on wildlife within parks and highlight the need for more
empirical data, particularly for neglected (schistosomiasis) and newly emerging (cryp-
tosporidiosis) diseases, as well as zoonotic disease risk from the rising bush meat
trade and game farm industry. As protected areas strive to become further embedded
in the socio-economic systems that surround them, providing benefits to local com-
munities, One Health approaches can help maintain the ecological integrity of eco-
systems, while protecting local communities and economies from the negative
impacts of disease.

1. Introduction
As we enter the sixth mass extinction, protecting the world’s bio-
diversity has never been more critical. Protected areas, including national
parks, cover over 18.8 million km2 and are at the forefront of a global effort
to safeguard biodiversity (Chape et al., 2003). Managers of these protected
areas must strike a balance between protecting the ecological integrity of
ecosystems and preventing exploitation of local resources while promoting
their use in education and recreation (Chape et al., 2003). If managed
correctly, protected areas can be beneficial to wildlife conservation and
the country’s economy through promoting ecotourism and creating local
employment opportunities (Cheung, 2012; Spies et al., 2018). However,
the management of protected areas is challenging, particularly in the
Anthropocene era of human mediated global change, and increased
emergence and re-emergence of infectious diseases (reviewed by Cable
et al., 2017). These diseases can reduce fitness, alter wildlife population
Managing zoonoses in African protected areas 3

structure/size and even alter ecosystem function (Holdo et al., 2009; Prins
and Weyerhaeuser, 1987; Scott, 1988). Therefore, to effectively manage
wildlife populations and ecosystems, it is essential to understand the threats
posed by pathogens and the diseases they cause.
Of the 3881 terrestrial and marine national parks in the world, almost half
are in sub-Saharan Africa, with terrestrial parks here covering 1 million
km2 (4% of the total land area; Chape et al., 2003; Muhumuza and
Balkwill, 2013). These parks aim to conserve Africa’s unique and iconic eco-
systems ranging from open savannas and grasslands to dense forest. This vari-
ety of habitats supports high levels of biodiversity, drawing numerous
tourists who aspire to spot the ‘Big Five’ megafauna: African buffalo (here-
after referred to as buffalo), lion, African elephant (hereafter referred to as
elephant), rhinoceros and leopard (Dube and Nhamo, 2019). However,
hidden and often forgotten biodiversity within protected areas includes
pathogens, which modulate animal abundance, fitness and behaviour
(Gómez and Nichols, 2013). It is crucial to better understand drivers for past
and current wildlife disease outbreaks within protected areas, to find new
approaches to predict and prevent future outbreaks. A review of all infec-
tious wildlife diseases within protected areas would be too large a task.
Instead, we focus on five diseases referred to here as the ‘Microscopic
Five’, which are important at the human-livestock-wildlife interface due
to their broad host range and zoonotic potential. These interface diseases
would all benefit from a ‘One Health’ approach to management (Fawzy
and Helmy, 2019; Innes et al., 2020; Webster et al., 2016). We therefore
purposefully included high profile diseases (bovine tuberculosis (BTb),
Rift Valley fever and brucellosis) as well as neglected diseases (cryptosporid-
iosis and schistosomiasis) for study. Using Kruger National Park, one of the
most researched parks in Africa (van Wilgen et al., 2016), we will review the
key factors that can influence outbreaks and transmission of the ‘Microscopic
Five’ within and around protected areas (Fig. 1). By focusing on a select
group of pathogens within a specific park our intention is to highlight drivers
of disease common among many protected areas and the importance of
considering all infectious diseases in wildlife management plans. We will first
give a brief introduction to the ‘Microscopic Five’ and then give examples of
the environmental and anthropogenic factors driving the dynamics of these
diseases within and around Kruger National Park. We will then discuss the
key knowledge gaps and future challenges for managing the ‘Microscopic
Five’ and other important diseases and touch on different management
approaches followed in various parks.
4 Anya V. Tober et al.

Big Five Microscopic Five

Extreme
weather events
Co-infections

Reservoir hosts

Landscape
heterogeneity

Mixing at
waterholes

Translocation
Illegal wildlife trade Permeable fences

Fig. 1 The ‘Big Five’ and ‘Microscopic Five’, and the drivers of disease at the wildlife-
livestock-human interface. Arrows represent anthropogenic drivers from beyond
Kruger National Park. Created with Microsoft PowerPoint (version 2109) and Adobe
Photoshop (2021).

2. The ‘Microscopic Five’

The ‘Big Five’ are undoubtedly one of the biggest attractions for tour-
ists visiting South Africa’s protected areas (Dube and Nhamo, 2019). To
conserve these and other wildlife, and to reduce transmission of infectious
diseases among wildlife, domestic animals and humans, we focus on the
lesser known ‘Microscopic Five’. These comprise zoonotic diseases caused
by pathogens that have multiple hosts, including humans, and are of partic-
ular importance at the human-livestock-wildlife interface. Although we
focus on five specific diseases, there are many more of importance within
protected areas (Table 1) but by highlighting a distinct few we aim to raise
the profile of all infectious diseases and possible drivers. The first three of the
‘Microscopic Five’ (bovine tuberculosis, brucellosis and Rift Valley fever)
are high profile or state-controlled diseases in South Africa and any out-
breaks must be reported to the World Organisation of Animal Health
(OIE). All three of these diseases are trade-sensitive diseases and may change
the trading status of a country and its ability to trade on the global market.
The remaining two (schistosomiasis and cryptosporidiosis) are neglected in
Table 1 Some diseases of large herbivores within Kruger National Park which may pose a threat to livestock and/or humans.
Spatial Known
Transmission Transmission distribution in susceptible
Disease. Pathogen modes routes Drivers KNP hosts Reservoir hosts
Bacteria
Anthrax Bacillus anthracis Vector, Ingestion of Calcium soil Northern and Most Maintained
environmental, contaminated content, drought central regions mammals in environment
direct contact, vegetation or including
fomites carcasses humans
Bovine Tb Micobacteria bovis Aerosol Respiratory tract Wildlife/livestock South, central, Cattle, Cape buffalo
interface, host moving north buffalo,
density humans
Virus
Foot and mouth Aphtovirus Aerosol, Respiratory tract Wildlife/livestock North, central, Cloven Cape buffalo
fomites interface, host south hooved
density animals
African horse Orbivirus Mosquito Cutaneous Introduced horses, Central Zebra, Culicoides
sickness vector, direct penetration season domestic mosquito,
contact horses possibly zebra
Rift Valley fever Phlebovirus Mosquito Cutaneous Climate change, Higher in Cattle, Aedes mosquito
vector, direct penetration drought, rainfall south and buffalo
contact central regions Cape buffalo
Continued
Table 1 Some diseases of large herbivores within Kruger National Park which may pose a threat to livestock and/or humans.—cont’d
Spatial Known
Transmission Transmission distribution in susceptible
Disease. Pathogen modes routes Drivers KNP hosts Reservoir hosts
Protozoa
Bovine Brucella abortus Direct contact Ingestion of Host density North, central, Cattle, Cape buffalo
brucellosis infected south buffalo, wild
discharges during animals,
birth, milk, mucus humans
membranes
Cryptosporidiosis Cryptosporidium Environmental Faecal-oral via Dependant on Unknown Wild Unknown
spp. contamination of species and host animals,
food and water range humans,
domestic
animals
Piroplasma
Corridor disease Theileria parva Tick vector Cutaneous Wildlife/livestock North, central, Cattle Cape Buffalo
penetration interface south
Babesiosis Babesia spp. Tick vector Cutaneous Unknown Rhinoceros, Unknown
penetration lions
Digenea
Fascioliasis Fasciola spp. Environmental Contact with Dependant on Unknown Ruminants Unknown
infected water species and host
range
Schistosomiasis Schistsoma spp. Environmental Contact with Dependant on Unknown Wild Unknown
infected water species and host animals,
range humans
Managing zoonoses in African protected areas 7

comparison, particularly in wildlife. By including these in the ‘Microscopic

Five’, we aim to bring greater attention to overlooked yet highly important
diseases (see WHO, 2020). In the following account, we briefly cover each
of the ‘Microscopic Five’ discussing their host specificity, transmission
pathways and known impacts on wildlife, livestock and humans.

2.1 Bovine tuberculosis

Bovine tuberculosis (BTb) is caused by the bacterium Mycobacterium bovis
and predominantly infects bovines, such as African buffalo (Syncerus caffer
caffer) and cattle (Bos taurus), yet most warm-blooded animals including
humans can be infected (Ayele et al., 2004). Transmission mainly occurs
through inhalation of infectious particles, which is particularly problematic
when livestock are kept at high densities (Ayele et al., 2004). Though
thought to have spilled over from cattle to buffalo in the early 1960s in
South Africa (Bengis et al., 1996), buffalo now serve as the primary main-
tenance host for BTb within Kruger National Park and Hluhluwe-iMfolozi
Park, spilling over into various species of wildlife and livestock (Michel et al.,
2006). Although BTb is a controlled disease within South Africa, its control
is becoming increasingly challenging due to the presence of wildlife reser-
voirs, difficulty in controlling disease in communal herds and lack of prac-
tical control options in wildlife (see Section 3). The WHO estimated
147,000 new cases of zoonotic Tb in humans in 2016 with 12,500 deaths
globally but mostly in Africa (Sichewo et al., 2019b). Humans can become
infected through drinking unpasteurised milk, eating undercooked meat and
via aerosols inhaled from infected cattle (DAFF, 2016; Sichewo
et al., 2019a).

2.2 Rift Valley fever

Rift Valley fever (RVF) is caused by a zoonotic, vector borne virus predom-
inantly spread by Aedes mosquitoes (Clark et al., 2018). The virus was first
reported in South Africa in 1950 and subsequent outbreaks have occurred
sporadically every 7–11 years infecting mainly domestic livestock but also
a range of wild mammals and humans (Beechler et al., 2015a; M
etras
et al., 2015). Human infection occurs mainly through direct contact with
blood or tissue from infected animals or through consuming unpasteurised
milk but can also result from an infected mosquito bite. Symptoms vary from
mild, flu-like to severe haemorrhagic fever that can be fatal (Clark et al.,
2018). Over 4000 human cases and around 1000 deaths have been reported
8 Anya V. Tober et al.

in the last 20 years, predominantly in Africa and Saudi Arabia (Petrova et al.,
2020). Little is known about how the pathogen is maintained during
inter-epidemic periods. One suggestion is vertical transmission from mos-
quitoes to their ova, which has been demonstrated with Aedes mosquitoes
under laboratory-controlled conditions (Romoser et al., 2011). Another
possibility is that it is maintained in wild animal populations (Beechler
et al., 2015a; see Section 4.3.4). Commercial vaccines are available for live-
stock but there is currently no licensed human vaccine (Petrova et al., 2020).

2.3 Brucellosis
Brucellosis, caused by bacteria of the Brucella genus, is ranked among the
most economically important zoonotic diseases globally. Although it is an
OIE notifiable disease, outbreaks are thought to be greatly under-reported
in Africa (McDermott et al., 2013). The species of medical and veterinary
importance are Brucella abortus, Brucella melitensis and B. suis (see Ducrotoy
et al., 2017). Infection in humans can lead to a debilitating illness known
as ‘Mediterranean’ or ‘undulant’ fever and is commonly misdiagnosed as
malaria (Ducrotoy et al., 2017; Godfroid et al., 2011). Human infection
occurs through direct contact with or consumption of an infected animal.
Consumption of un-pasteurised milk causes most human infections, while
human to human transmission is rare (Godfroid, 2018). Several wildlife spe-
cies have been reported as seropositive for this disease and African buffalo are
thought to be a reservoir for B. abortus (see Godfroid et al., 2013). Infection
can cause abortions in livestock reducing farm productivity, however the
effects of the disease on wildlife are largely unknown and may differ between
species (Gorsich et al., 2015). Vaccines are available for livestock and small
ruminants but not yet for humans (Ducrotoy et al., 2017).

2.4 Cryptosporidiosis
Cryptosporidiosis, caused by several species of the protozoan
Cryptosporidium genus, can lead to severe diarrhoea in humans and animals
globally. Infectious diarrhoea is a major cause of death in children under five
in Africa and Cryptosporidium is second only to rotavirus as a contributor to
this disease (Kotloff et al., 2013; Squire and Ryan, 2017). Transmission
occurs through the faecal oral route via close contact with infected humans,
animals or contaminated food and water (Innes et al., 2020). Currently there
are at least 40 recognised species with varying host specificities but the most
important two species infecting humans and livestock are C. hominus and
Managing zoonoses in African protected areas 9

C. parvum. The latter is the predominant cause of diarrhoea in young calves

and is the most important zoonotic species. Cryptosporidium parvum is more
genetically diverse than C. hominus with several subtypes with differing host
specificities, therefore an integrated genotyping approach has been advo-
cated to differentiate these subtypes (Innes et al., 2020). Cryptosporidium
species have been identified in a range of wildlife, yet most studies focus
on humans and livestock (Zahedi et al., 2016). C. parvum, C. ubiquitum
and C. bovis were recently identified in wildlife within Kruger National
Park in elephant (Loxodonta africana), buffalo (Syncerus caffer) and impala
(Aepyceros melampus; see Samra et al., 2011). Oocysts of Cryptosporidium
spp. have also been detected in zebra (Equus zebra), buffalo and wildebeest
(Connochaetes gnou) faeces in Mikumi National Park, Tanzania (Mtambo
et al., 1997). There is currently no available vaccine for cryptosporidiosis
yet there is potential to develop one for cattle (Innes et al., 2020).

2.5 Schistosomiasis
Schistosomiasis is a waterborne, zoonotic disease of veterinary and medical
importance, caused by digenean parasites of the genus Schistosoma.
Schistosomiasis is a major public health threat with an estimated 207 million
people infected and 779 million people at risk globally, with 90% of these
infections in Africa (Steinmann et al., 2006). Like all digeneans, schistosomes
have an indirect lifecycle. They require an intermediate freshwater snail host
within which they reproduce asexually ultimately producing cercariae,
which are free-swimming larval stages that subsequently infect a definitive
mammalian host (Cribb et al., 2003). Definitive animal or human hosts can
become infected with schistosomiasis by entering infested waters—the
water-borne larvae burrow through the skin of the new host (Cribb
et al., 2003). There are at least 12 known schistosome species in Africa of
which 5 are known to infect humans (S. haematobium, Schistosoma
mansoni, S. intercalatum, S. guineensis and S. mattheei). Schistosoma mattheei
is of note as although predominantly a parasite of cattle, it has also been
found in wildlife and humans where it is known to hybridise with
S. haematobium (see Pitchford, 1961). The other species infect a wide range
of domestic and wild animals including cattle, horses, buffalo, baboons,
zebra, hippopotamus and rodents (Standley et al., 2012). Traditionally, mal-
acological monitoring programmes have only targeted snail species known
to harbour human infecting schistosomes, but a wider approach is clearly
needed as we become aware of wider host ranges (Pennance et al., 2021)
10 Anya V. Tober et al.

that are likely to shift with increasing environmental stressors. There is

currently no vaccine for schistosomiasis and the main control strategy for
humans is preventative chemotherapy, improved water, sanitation and
hygiene and snail control (WHO, 2022).

3. Challenges of BTb control at the wildlife-livestock

interface: The South African case study
South Africa has been challenged with the control of BTb since the
disease was first reported in the country in 1880, initially focusing on live-
stock, and now including control in wildlife (DAFF, 2016).

3.1 Control in livestock

Early BTb surveillance included the introduction of tuberculin skin testing
in cattle in 1905, followed by its declaration as a notifiable disease in 1911
and the initiation of the Division of Veterinary Services BTb scheme in 1969
(DAFF, 2016; Michel et al., 2019). This scheme focused on compulsory
testing of commercial cattle herds suspected to be infected, with slaughter
of positive individuals, quarantine and disinfection of farms. Initially, great
progress was made, reducing prevalence to 0.04% by 1991 (1.1 million cattle
tested); however, the number of tests have since declined due to budget cuts
and a decreased workforce (DAFF, 2016; Michel et al., 2019). Current prev-
alence in communal livestock is variable (<0.5% to >15%) (Musoke et al.,
2015; Sichewo et al., 2019b).
In 2021 the national cattle herd was estimated at 12 million, consisting of
commercial dairy herds (20%) and beef and dual-purpose herds (80%)
(DAFF, 2021). Testing of cattle is no longer compulsory and current control
of BTb is guided by the Interim BTb Manual from the Department of
Agriculture, Forestry and Fisheries (DAFF), South Africa, which proposes
the use of four testing programmes (Table 2; DAFF, 2016). All programmes
are voluntary apart from the infected herd program, which can be enforced
by the Animal Diseases Act, 1984 (Act No. 35 of 1984) (DAFF, 2016). The
approved test is the cervical intradermal tuberculin (CIT) test (DAFF, 2016).

3.2 Control in wildlife

The control of BTb in wildlife is becoming increasingly important as many
farms switch from livestock to game farming, and wild buffalo reservoirs
hinder control efforts in cattle (Michel et al., 2019). Bovine Tb has been
Managing zoonoses in African protected areas 11

Table 2 Four levels of Bovine Tb surveillance programmes in South Africa.

Surveillance herd
programme
Maintenance herd
programme
Infected herd programme
Diagnostic testing
programme (individuals)
One off survey used by state officials to determine the
prevalence of BTb within an area or by a stock owner
conducting a self-assessment
To join this programme, herds are required to
undergo two consecutive tests with 100% negative
results at least 3 months apart. These BTb free herds
are then tested every 2 years. If an individual tests
positive, then the entire herd is moved to the infected
herd programme
Compulsory programme for herds that have tested
positive with the CIT test, as well as those detected
from meat and milk inspection, post-mortems or
clinical cases. These herds are placed under
quarantine and kept under supervision of a state
veterinarian, who will order the slaughter of infected
animals. The rest of the herd is tested every 3 months
and is only let out of quarantine once the herd has
undergone two consecutive negative tests
Individual cattle destined to be imported or exported.
Imported cattle are kept in quarantine and must
undergo a compulsory CIT test. Before export, cattle
must also receive a comparative CIT test—a
requirement for many importing countries

identified in 21 different wildlife species in South Africa, including most

recently giraffe (Hlokwe et al., 2019). The current control scheme is focused
on domestic cattle and although some tests have been adjusted for use in buf-
falo, this is not the case for other wildlife species. The Buffalo Veterinary
Procedural Notice (VPN) was published in 2017 outlining the procedures
for disease testing, movement and contingency planning for disease out-
breaks in buffalo (DAFF, 2017). The buffalo VPN states that for movement
purposes, buffalo must have a negative CIT test as outlined in the manual for
cattle. Importantly, the interpretation of CIT has been based on cattle
thresholds due to the lack of species-specific cut-off values for African buf-
faloes. The gamma interferon test is also an effective diagnostic tool for buf-
falo but is not approved by DAFF for movement purposes. There is
currently no guidance on control of BTb in other wildlife species and there
are limited verified diagnostic tests in these species (DAFF, 2017).
12 Anya V. Tober et al.

Kruger National Park and Hluhluwe-iMfolozi Park are the only two
parks within South Africa that contain buffalo herds maintaining BTb yet
they have adopted different control approaches. Bovine Tb was first
detected in Hluhluwe-iMfolozi Park in 1986 and a test and cull disease pro-
gramme was initiated in 1999. This programme involved a mobile capture
unit to corral buffalo in different areas of the park, test them by means of
the CIT test and culling positive individuals. Between 1991 and 2006,
4733 buffalo were tested, with herd prevalence ranging from 2.3% to
54.7%. Subsequent, data analysis suggested that the programme was effective
at reducing BTb prevalence, particularly in areas with intensive test and cul-
ling operations (Le Roex et al., 2016). Kruger National Park took a different
approach to managing BTb in its buffalo population after the disease was
detected in this host species in 1990. They aimed to breed disease free buffalo
from Foot and Mouth Disease infected parents within the park in order to
conserve the genetic pool of Kruger buffalo in an ex-situ population
(Laubscher and Hoffman, 2012). This approach, which used dairy cows
as foster parents for buffalo calves initially, and later switched to having
the buffalo mothers rear their young, was highly successful and also popular
with farmers, eventually shifting from a few government funded projects to
hundreds of private buffalo breeding farms (Laubscher and Hoffman, 2012).
Additionally, Kruger National Park did extensive BTb monitoring surveys
between 1993 and 2007, to assess the spread and impact of BTb in herds, and
determine if the disease was having population level effects. Since it entered
the park, BTb has been detected in 12 spill-over species (Michel et al., 2006)
and remains a concern in low density species, such as wild dog and black
rhinoceros (Higgitt et al., 2019).
With the disease currently not shown to be affecting population recruit-
ment or growth in buffalo, the real concern becomes spill-over to other
hosts and therefore finding an effective vaccine that limits disease severity
and spill-over is a priority. Currently there is only one registered vaccine
for BTb control. The BCG vaccine is predominantly used in humans but
has yielded promising results for use in domestic cattle (Arnot and
Michel, 2020). However, when trialled in wild buffalo within the Kruger
National Park, the BCG vaccine protection was insufficient and did not
limit bacterial shedding (De Klerk et al., 2010). This was thought to have
resulted from priming with environmental non-TB mycobacteria, which
has been shown to reduce the protective efficacy of the BCG vaccine
(Brandt et al., 2002; De Klerk et al., 2010). Importantly similar studies in
badgers in the UK found the BCG vaccine to be effective in limiting disease
Managing zoonoses in African protected areas 13

severity (and therefore bacterial load; Chambers et al., 2011), meaning

that defining the clinical end point for vaccine efficacy trials is important.
Another vaccination trial in buffalo is currently underway, testing both
BCG and DNA-sub-unit vaccines.

4. Drivers of disease: The Kruger National Park

case study
4.1 Past and present disease management
Kruger National Park first opened as the Sabi Game Reserve in 1898
(10,364 km2) as a response to campaigns for the conservation of wild animals
subjected to uncontrolled hunting and to the 1896 rinderpest epidemic
(Mabunda et al., 2003). In 1926, the Sabi Game Reserve was combined with
the Singwitsi Reserve (5000 km2 region named after the Shingwedzi River)
and later renamed Kruger National Park. James Stevenson-Hamilton, who
was appointed warden in 1902, was tasked with managing the aftermath of
the rinderpest epidemic which, along with previous hunting activities, dec-
imated the game population, leaving elephant and white rhinoceros
(Ceratotherium simum) locally extinct (Mabunda et al., 2003). The rinderpest
epidemic also severely affected buffalo, eland (Tragelaphus oryx) and greater
kudu (Tragelaphus strepsiceros; hereafter referred to as kudu), whereas wilde-
beest and zebra were unaffected (Stevenson-Hamilton, 1957). The first
60 years of park management (1900–1960) focused on protecting, preserv-
ing, and propagating, aiming to increase game numbers through introduc-
tions of large herbivores, provision of water sources and culling of predators
(Venter et al., 2008).
Colonel J.A.B Sanderberg took over from Stevenson-Hamilton as
Warden in 1946 and 8 years later the first case of anthrax was confirmed
in the north of the park (Mabunda et al., 2003). This was followed by
repeated outbreaks in 1959–60, 1970 and 1990–91, and outbreaks in the
central part of the park in 1993 and 1999 (Bengis et al., 2003; De Vos
and Bryden, 1996). The 1959–60 outbreak lasted just 4 months and yet
within this time over 1000 mammals died: kudu, waterbuck (Kobus ellip-
siprymnus) and roan (Hippotragus equinus) being the most affected (Pienaar,
1961). Simultaneously, BTb likely entered the park, transmitted from cattle
to buffalo on the southern border, although it was not detected in the park
until 30 years later (Bengis et al., 1996). At this time, park management
shifted to a ‘management by intervention’ approach and the next 30 years
(1960–1990) focused on measuring, monitoring and manipulation
14 Anya V. Tober et al.

(Mabunda et al., 2003). Fencing of the park was ordered by the National
Department of Agriculture in order to prevent the spread of disease to
surrounding livestock, such as foot and mouth (FMD) endemic in buffalo
(Bengis et al., 2003). Fence construction started in the early 1960s with
the western boundary followed by the eastern boundary in the late 1960s,
by 1980 all boundaries of the park were enclosed. The fences (over
360 km in length and 65 km in width) restricted movement of wildlife lead-
ing to increased numbers of large herbivores, such as elephant and buffalo,
which were subsequently controlled by culling operations and in the early
1970s, a certified abattoir was built within the park to optimise use of the
culled meat (Mabunda et al., 2003). From 1990 to 2010 management
shifted again to focus on integration, innovation and internationalisation.
The severe drought of 1992–93 followed by the February floods in 2000
as well as the catastrophic wildfire in September 2001, which killed both
people and animals within the park, were indicative of the need for manage-
ment to become more adaptive to the increasingly unpredictable environ-
ment (Mabunda et al., 2003). Since 1995, Kruger has used a strategic
adaptive management approach, which involves management decisions
and actions guided by research and monitoring while learning from unex-
pected events or outcomes. This approach also aims to maximise heteroge-
neity of the park and led to its expansion across national boundaries creating
the Greater Limpopo transfrontier conservation area (GLTFCA) spanning
the Limpopo (Mozambique), Kruger (South Africa) and Gonarezhou
(Zimbabwe) National Parks. A portion of fences of approximately 45 km
was removed between Limpopo and Kruger in 2002 (Caron et al., 2016;
Venter et al., 2008).

4.2 Kruger National Park’s current adaptive management

approach
In the past, most management issues in Kruger National Park were focused
within the park boundaries; however, since the recognition that threats and
drivers to biodiversity conservation often occur outside of the footprint of
the National Parks, management issues are extending beyond the park
boundaries and becoming more socio-economic in nature (Venter et al.,
2008). The creation of the Greater Limpopo transfrontier conservation area
shifted the park from being single use for wildlife to a multi-use park, sharing
its land with communities and their livestock. The park’s current strategic
adaptive management aims to increase understanding of complex ecosystems
and broader societal needs of local communities. This process is guided by
Managing zoonoses in African protected areas 15

setting appropriate thresholds of potential concern (TPC), a set of adaptive

management goals and endpoints that define upper and lower levels of
acceptable change, enabling management to determine how much a system
can be allowed to fluctuate before it becomes a concern and requires man-
agement action. Although TPCs prove useful for simple metrics like invasive
plants and river flows, they have proven more challenging for complex sys-
tems such as disease where drivers and responders are not always known
(Gaylard and Ferreira, 2011; Venter et al., 2008).
Kruger National Park’s 2018–28 management plan includes a disease
management programme as a supporting objective to the higher-level
objective of biodiversity conservation. This programme acknowledges
endemic wildlife diseases within the park as a key component of biodiversity
yet highlights the need to prevent and mitigate the spread of disease at the
wildlife-livestock-human interface and limit the introduction or impact of
novel infectious diseases (Spies et al., 2018).

4.3 Environmental drivers of disease transmission

4.3.1 Spatial heterogeneity and the north/south divide
Topography, climate, geology and the associated soil and vegetation patterns
can exert a bottom-up control on ecosystems. The combination of these
abiotic factors can influence fire patterns and animal behaviours, as well as
disease dynamics (Venter et al., 2003).
Kruger National Park lies within part of the north-eastern South African
lowveld, which generally has plains of low to moderate relief with some low
mountains and hills. The geology of the park can be crudely divided into
granite plains on the west and basalt plains on the east, separated by a
north-south strip of sedimentary rock (Venter et al., 2003). Rainfall in
the park increases along a north to south gradient with annual mean rainfall
of 350 mm in the northeast to 750 mm in the southwest. Geology and rainfall
have influenced the difference in soil and vegetation types between the
north and the south of the park. The south generally consists of deeper
and more diverse soil types with predominantly open canopy acacia tree
bushveld and savannah with a well wooded area in the southeast. In contrast,
the north tends to have less diverse, thinner soils with a higher calcium con-
tent. Vegetation is dominated by mopane trees with rare lowveld riverine
forest occurring along the rivers in the northeast and sandveld vegetation
type in the northwest (Gertenbach, 1983; Spies et al., 2018). The northern
most section of the park is unique as it contains a varied assemblage of rock
formations with associated soil and vegetation types. It also contains the only
16 Anya V. Tober et al.

true floodplain in Kruger (Venter et al., 2003). For management purposes,

Kruger National Park has been partitioned into 35 landscapes depending
on geomorphology, vegetation, soil, climate types and associated fauna
(Gertenbach, 1983; Venter et al., 2003). A social-economic gradient exists
along the northern and southern boundaries of the park. Dense peri-urban
to urban developments lie along the southwestern border, including sugar-
cane plantations, forestry and the nearby city of Mbombela (previously
known as Nelspruit; Fig. 2). The central and north-western boundaries
are buffered by private nature reserves and community subsistence farming,
and further north becomes more rural with large agricultural areas and poor
villages with limited economic opportunities (Spies et al., 2018). Wildlife
densities also differ across the park with megaherbivore (elephant and
buffalo) densities higher in the north than the south (Fig. 2).
This ecological heterogeneity within the park can create spatial hetero-
geneity in disease dynamics. A park wide survey of RVF in buffalo in 1998
showed significantly higher seroprevalence of buffalo herds in the south and
central regions of the park compared to the north (Beechler et al., 2015a).
This was attributed to lower rainfall and different vegetation in the north
leading to less suitable breeding habitats for mosquito vectors (Beechler
et al., 2015a). Brucellosis prevalence in buffalo was significantly associated
with park section and soil type (Gorsich et al., 2015). Buffalo captured on
the resource poor granitic soils were twice as likely to be seropositive for
brucellosis compared to those on the resource rich basaltic soils (Gorsich
et al., 2015). Moreover, buffalo on granitic soils had higher prevalence in
the southern section of the park compared to the central section (Gorsich
et al., 2015). This was attributed to nutrient poor vegetation in the south-
western granitic soils and general lower body condition of buffalo in the
south of Kruger National Park (Caron et al., 2003; Gorsich et al., 2015).
The effect of brucellosis infection was also dependant on the seasonal het-
erogeneity of the park, brucellosis infection was significantly associated with
lower body condition but only in the dry season (Gorsich et al., 2015).
Knowledge of this heterogeneity of different disease dynamics and how
the landscape and environment affect this is of great importance and can help
target monitoring and management of diseases within the park.

4.3.2 Climate change and severe weather events

Africa is considered one of the most vulnerable areas to global climate
change (Serdeczny et al., 2017). Average temperature readings from the
Skukuza weather station in Kruger National Park have shown a 2 °C
Managing zoonoses in African protected areas 17

Fig. 2 Megaherbivore (African buffalo and elephant) density across Kruger National
Park and fence breakages (red cross) from damage causing animals (DCAs). Elephant
cause most breakages enabling diseased buffalo to escape. Foot and mouth (FMD) vet-
erinary control zones and nearby villages are also shown. Map produced by the Skukuza
GIS Office.
18 Anya V. Tober et al.

increase from 1977 to 2018 with a maximum temperature increase of 0.5 °C

per decade (Dube and Nhamo, 2019). Kruger National Park has suffered
numerous droughts in 1966–67, 1982–83, 1991–92, 1995–96 and most
recently in 2015–17 (Staver et al., 2019). The most severe drought on record
in 1991–92 had a mean total rainfall of 235.6 mm compared to the 534 mm
long-term mean annual rainfall and the number of days with rain within this
period (24.2) was significantly less than the mean annual total (48.3;
Zambatis and Biggs, 1995). This was followed by a severe flood event in
1996. Certain disease outbreaks within the Kruger National Park, such as
anthrax and foot and mouth disease (FMD) have occurred after a dry period
(Pienaar, 1961).
Drought not only affects resource availability and body condition, but
also the behaviour and movement of animals, including buffalo, which in
turn can alter disease transmission (Cross et al., 2004; Staver et al., 2019).
Combining buffalo behavioural association data with disease models
predicted that dry conditions facilitate increased spread of BTb within
buffalo populations due to increased herd switching (Cross et al., 2004).
The 2015–17 drought forced buffalo to move north to areas where the
drought was less severe (Staver et al., 2019). Movements like this could lead
to the transmission of diseases into new areas of the park and is likely to have
played a role in the spread of BTb northwards through the park (Michel
et al., 2006).
Drought particularly affects the dynamics of water borne diseases such
as schistosomiasis and RVF carried by vectors (snails and mosquitoes res-
pectively) reliant on water or moisture with populations that fluctuate
depending on climate variations (Cribb et al., 2003; Romoser et al.,
2011). Floods and prolonged wet periods have been associated with out-
breaks of RVF. South Africa has experienced three major RVF epidemics
(1950–51, 1973–75 and 2008–11). Epidemiological data from the 2008 to
2011 epidemic was modelled to quantify spatial and temporal environmental
factors associated with disease incidence (M
etras et al., 2015). Initial years
saw the incidence of RVF increase with increased vegetation density and
presence of wetlands. However, for 2010 and 2011, of which 2010 was
the longest lasting outbreak, the strongest risk factor was temperature. For
2010, the risk of RVF increased by a hazard ratio of 15.7 in areas between
25 and 32 °C and by 44.35 in areas over 35 °C compared with those below
25 °C (M
etras et al., 2015). This is important for Kruger National Park as
the average annual temperature in the south of the park was 32.3 °C and
is set to increase (Dube and Nhamo, 2019).
Managing zoonoses in African protected areas 19

Modelling the RVF outbreaks in Kruger National Park from 2008 to

2011 suggested that soil saturation index anomalies exceeding the long-term
mean by 20%, followed by a sudden rainfall event could be a reliable pre-
dictor for outbreaks. When tested on previous outbreaks, the model success-
fully predicted 90% of outbreaks more than 1 month before they occurred
(Williams et al., 2016). Other factors such as vegetation density and
increased temperature are also important risk factors (M
etras et al., 2015).

4.3.3 Water sources

Water is often a focal point for wildlife-livestock interaction, particularly
rivers which run between protected areas and communal farmlands (Kock
et al., 2014). Such interaction can enable spread of diseases between wildlife
within the park and livestock on the borders (Miguel et al., 2013; Pienaar,
1961). Within Kruger National Park, five perennial rivers (Sabi-Sands,
Crocodile, Olifants, Letaba and Luhuvu) run through the park as well as sev-
eral seasonal rivers, natural pans and wetlands (Mabunda et al., 2003; Pienaar
et al., 1997). The Sabi River runs parallel to the fenced south-western bor-
der between Kruger National Park and the adjacent communal lands of
Bushbuckridge. Landscape resistance maps for cattle and buffalo resource
utilisation were used to model dispersal of these animals within these two
areas. Contact risk between buffalo and cattle was significantly higher in
the dry season and was concentrated along the Sabi River at the weaker parts
of the fence. Contact risk was more widespread and closer to villages in the
wet season, yet still highest along the river (Kaszta et al., 2018). Water
sources can also increase the permeability of nearby fences to wildlife move-
ments. Interviews with fence maintenance workers in Kruger National Park
reported that fences damaged by flooding and predation were higher in areas
with rivers compared to those without. Furthermore, reports of kudu cross-
ing the fences were significantly higher in areas with rivers, although this was
not observed for other wildlife (elephant, buffalo, warthog (Phacochoerus
africanus) and impala) in the park ( Jori et al., 2011).
Contact at water sources has been attributed to disease outbreaks, such as
FMD and anthrax (Miguel et al., 2013; Pienaar, 1961). The Limpopo River
runs between the northern edge of Kruger National Park and adjacent com-
munal land of Pezvi in Zimbabwe, both within the GLTFCA. Satellite data
from collared individual cattle from Pezvi and buffalo from Kruger National
Park showed that the two species shared 16.9% of habitat with most contacts
occurring less than 500 m from the riverbed. These contacts increased during
the dry season suggesting that contact is driven by resource availability
20 Anya V. Tober et al.

(Miguel et al., 2013). Incidence of FMD antibodies in cattle was higher in

sites with high buffalo contact suggesting spread of the infection from buffalo
to cattle (Miguel et al., 2013). Water points within protected areas are also a
source for interaction between different wildlife species, particularly in dry
seasons when water sources are limited. The anthrax outbreaks which
occurred in the northern section of Kruger National Park between 5
June and 11 October 1960 were all associated with natural and artificial
water points where large numbers of animals aggregated around the
remaining available water sources during the dry season (Pienaar, 1961).
Water sources also provide habitats for parasite-harbouring vectors such
as freshwater snails and mosquitoes. Freshwater snails harbour a huge num-
ber of digenean parasites some of which can cause diseases of veterinary and
medical importance, such as schistosomiasis and fascioliasis. Although fresh-
water snails within Kruger National Park have been surveyed several times
in the past (De Kock and Wolmarans, 1998; De Kock et al., 2002;
Wolmarans and De Kock, 2006), the diversity and distribution of digenean
parasites hosted by these snails has not yet been studied. The original surveys
identified the intermediate host snails for both schistosomes (Bulinus
africanus, B. globosus, Bradyidius tropicus, B. forskali, Biompharia pfeifferi) and
fasciolids (Lymnea columella, L. natalensis; see (De Kock and Wolmarans,
1998; De Kock et al., 2002), therefore it is important to explore the hidden
digenean diversity within these snails. Within Kruger National Park, schisto-
some species have been detected in several animals including baboons, zebra,
warthog, giraffe, kudu, wildebeest, buffalo (S. mattheii; see Beechler et al.,
2017; Pitchford et al., 1974) and hippopotamus (S. hippopotami and
S. edwardiense; see Pitchford and Visser, 1981). Animals sampled near man
made dams had higher S. mattheei infection rates and egg outputs than those
at natural water sources, suggesting perennial exposure and transmission at
these sites (Pitchford et al., 1974).

4.3.4 Reservoir hosts

As seen with the ‘Microscopic Five’, most pathogens can infect more than
one host (Cleaveland et al., 2001). This is true for 77% of known livestock
pathogens and 60% of known human pathogens (Cleaveland et al., 2001;
Haydon et al., 2002). Some hosts can act as reservoir hosts, also known as
maintenance hosts (Ashford, 1997; Haydon et al., 2002; Swinton et al.,
2002). Essentially, reservoir hosts can maintain the pathogen in the absence
of cases in other species, and with a high enough prevalence that parasites can
spill over into another host species. Identifying reservoir hosts is crucial to
Another random document with
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otra alguna,
ueys vna perfeçion jamas
oyda,
ueys una discreçion, qual fue
ninguna,
de hermosura y graçia
guarnescida?
¿ueys la que está domando a
la fortuna
y a su pesar la tiene alli
rendida?
la gran doña Leonor Manuel
se llama,
de Lusitania luz que al orbe
inflama.
Doña Luisa Carrillo, que en
España
la sangre de Mendoça ha
esclareçido:
de cuya hermosura y graçia
extraña,
el mismo amor, de amor está
uençido,
es la que a nuestra Dea ansi
acompaña
que de la uista nunca la ha
perdido:
de honestas y hermosas claro
exemplo,
espejo y clara luz de nuestro
templo.
¿Ueys una perfeçion tan
acabada
de quien la misma fama está
embidiosa?
¿ueys una hermosura más
fundada
en graçia y discreçion que en
otra cosa,
que con razon obliga a ser
amada
porque es lo menos de ella el
ser hermosa?
es doña Eufrasia de Guzman
su nombre,
digna de inmortal fama y gran
renombre.
Aquella hermosura
peregrina
no uista en otra alguna sino en
ella,
que a qualquier seso apremia
y desatina,
y no hay poder de amor que
apremie el della,
de carmesí uestida y muy más
fina
de su rostro el color que no el
de aquella,
doña Maria de Aragon se
llama,
en quien se ocupará de oy
más la fama.
¿Sabeys quién es aquella
que señala
Diana, y nos la muestra con la
mano,
que en graçia y discreçion a
ella yguala,
y sobrepuja a todo ingenio
humano,
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y en gala,
sería (segun es) trabajo en
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doña Ysabel Manrique y de
Padilla,
que al fiero Marte uenze y
marauilla.
Doña Maria Manuel y doña
Ioana
Osorio, son las dos que estays
mirando
cuya hermosura y graçia sobre
humana,
al mismo Amor de amor está
matando:
y esta nuestra gran Dea muy
vfana,
de ueer a tales dos de nuestro
uando,
loallas, segun son es
escusado:
la fama y la razon ternan
cuydado.
Aquellas dos hermanas tan
nombradas
cada una es una sola y sin
segundo,
su hermosura y graçias
extremadas,
son oy en dia un sol que
alumbra el mundo,
al biuo me paresçen
trasladadas,
de la que a buscar fuy hasta el
profundo:
doña Beatriz Sarmiento y
Castro es una
con la hermosa hermana qual
ninguna.
 El claro sol que ueys
resplandeçiendo
y acá, y allá sus rayos ya
mostrando,
la que del mal de amor se está
riendo,
del arco, aljaua y flechas no
curando,
cuyo diurno rostro está
diziendo,
muy más que yo sabré dezir
loando,
doña Ioana es de Çarate, en
quien vemos
de hermosura y graçia los
extremos.
Doña Anna Osorio y Castro
está cabe ella
de gran valor y graçia
acompañada,
ni dexa entre las bellas de ser
bella,
ni en toda perfeçion muy
señalada,
mas su infelize hado vsó con
ella
de una crueldad no vista ni
pensada,
porque al ualor, linaje y
hermosura
no fuesse ygual la suerte, y la
uentura.
Aquella hermosura
guarnecida
de honestidad, y graçia sobre
humana,
que con razon y causa fue
escogida
por honra y prez del templo de
Diana,
contino uençedora, y no
uençida
su nombre (o Nimphas) es
doña Iuliana,
de aquel gran Duque nieta y
Condestable,
de quien yo callaré, la fama
hable[1256].
Mirad de la otra parte la
hermosura
de las illustres damas de
Valençia,
a quien mi pluma ya de oy
mas procura
perpetuar su fama y su
excelençia:
aqui, fuente Helicona, el agua
pura
otorga, y tú, Minerua, enpresta
sçiençia,
para saber dezir quién son
aquellas
que no hay cosa que ver
despues de vellas.
Las cuatro estrellas ved
resplandesçientes
de quien la fama tal ualor
pregona
de tres insignes reynos
desçendientes,
y de la antigua casa de
Cardona,
de la vna parte Duques
exçelentes,
de otra el trono, el sçeptro, y la
corona,
del de Segorbe hijas, cuya
fama
del Borea al Austro, al Euro se
derrama.
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España,
el fin de la beldad y
hermosura,
el coraçon real que le
acompaña,
el ser, valor, bondad sobre
natura,
aquel mirar que en verlo
desengaña,
de no poder llegar alli criatura:
doña Anna de Aragon se
nombra y llama,
a do por el amor, cansó la
fama.
Doña Beatrix su hermana
junto della
vereys, si tanta luz podeys
miralla:
quien no podré alabar, es sola
ella,
pues no ay podello hazer, sin
agrauialla:
a aquel pintor que tanto hizo
en ella,
le queda el cargo de poder
loalla,
que a do no llega
entendimiento humano
llegar mi flaco ingenio, es muy
en vano.
 Doña Françisca d'Aragon
quisiera
mostraros, pero siempre está
escondida:
su vista soberana es de
manera,
que a nadie que la vee dexa
con vida:
por esso no paresçe. ¡Oh
quién pudiera
mostraros esta luz, que al
mundo oluida,
porque el pintor que tanto hizo
en ella,
los passos le atajó de
meresçella.
A doña Madalena estays
mirando
hermana de las tres que os he
mostrado,
miralda bien, uereys que está
robando
a quien la mira, y biue
descuydado:
su grande hermosura
amenazando
está, y el fiero amor el arco
armado,
porque no pueda nadie, ni aun
miralla,
que no le rinda o mate sin
batalla.
Aquellos dos luzeros que a
porfia
acá, y allá sus rayos uan
mostrando,
y a la exçelente casa de
Gandia,
por tan insigne y alta
señalando,
su hermosura y suerte sube oy
dia
muy más que nadie sube
imaginando:
¿quién uee tal Margareta y
Madalena,
que tema del amor la horrible
pena?
Quereys, hermosas
Nimphas, uer la cosa,
que el seso más admira y
desatina?
mirá una Nimplia más que el
sol hermosa,
pues quién es ella, o él jamas
se atina:
el nombre desta fenix tán
famosa,
es en Valençia doña Cathalina
Milan, y en todo el mundo es
oy llamada
la más discreta, hermosa y
señalada.
Alçad los ojos, y vereis de
frente
del caudaloso rio y su ribera,
peynando sus cabellos, la
exçelente
doña Maria Pexon y
Çanoguera
cuya hermosura y gracia es
euidente,
y en discreçion la prima y la
primera:
mirad los ojos, rostro
cristallino,
y aquí puede hazer fin uuestro
camino.
Las dos mirad que están
sobrepujando,
a toda discreçion y
entendimiento,
y entre las más hermosas
señalando
se uan, por solo vn par, sin par
ni cuento,
los ojos que las miran
sojuzgando:
pues nadie las miró que biua
essento:
¡ued qué dira quien alabar
promete
las dos Beatrizes, Vique y
Fenollete!
Al tiempo que se puso alli
Diana,
con su diuino rostro y
excelente
salió un luzero, luego una
mañana
de Mayo muy serena y
refulgente:
sus ojos matan y su uista
sana,
despunta alli el amor su flecha
ardiente,
su hermosura hable, y
testifique
ser sola y sin ygual doña Anna
Vique.
Bolued, Nimphas, uereys
doña Teodora
Carroz, que del valor y
hermosura
la haze el tiempo reyna y gran
señora
de toda discreçion y graçia
pura:
qualquiera cosa suya os
enamora,
ninguna cosa nuestra os
assegura,
para tomar tan grande
atreuimiento,
como es poner en ella el
pensamiento.
Doña Angela de Borja
contemplando
uereys que está (pastores) en
Diana,
y en ella la gran dea está
mirando
la graçia y hermosura
soberana:
Cupido alli a sus pies está
llorando,
y la hermosa Nimpha muy
ufana,
en uer delante della estar
rendido
aquel tyrano fuerte y tan
temido.
De aquella illustre cepa
Çanoguera,
salio una flor tan extremada y
pura,
que siendo de su edad la
primauera,
ninguna se le yguala en
hermosura:
de su excelente madre es
heredera,
en todo quanto pudo dar
natura,
y assi doña Hieronyma ha
llegado
en graçia y disceçion al sumo
grado.
¿Quereys quedar (o
Nimphas) admiradas,
y uer lo que a ninguna dió
uentura:
quereys al puro extremo uer
llegados
ualor, saber, bondad y
hermosura?
mirad doña Veronica
Marradas,
pues solo uerla os dize y
assegura
que todo sobra, y nada falta
en ella,
sino es quien pueda (o piense)
meresçella.
Doña Luysa Penarroja
uemos
en hermosura y graçia más
que humana,
en toda cosa llega los
estremos,
y a toda hermosura uençe y
gana:
no quiere el crudo amor que la
miremos
y quien la uió, si no la uee, no
sana:
aunque despues de uista el
crudo fuego
en su vigor y fuerça buelue
luego.
Ya ueo, Nimphas, que
mirays aquella
en quien estoy continuo
contemplando,
los ojos se os yran por fuerça
a ella,
que aun los del mismo amor
está robando:
mirad la hermosura que ay en
ella,
mas ued que no çegueys
quiçá mirando
a doña Ioana de Cardona,
estrella
que el mismo amor está
rendido a ella.
Aquella hermosura no
pensada
que ueys, si uerla cabe en
nuestro uaso:
aquella cuya suerte fue
estremada
pues no teme fortuna, tiempo
o caso,
aquella discreçion tan
leuantada,
aquella que es mi musa y mi
parnaso:
Ioanna Anna, es Catalana, fin
y cabo
de lo que en todas por
estremo alabo.
 Cabe ella está un estremo
no uicioso,
mas en uirtud muy alto y
estremado,
disposiçion gentil, rostro
hermoso,
cabellos de oro, y cuello
delicado,
mirar que alegra, mouimiento
ayroso,
juyzio claro y nombre
señalado,
doña Angela Fernando, aquien
natura
conforme al nombre dio la
hermosura.
Vereys cabe ella doña
Mariana,
que de ygualalle nadie está
segura;
miralda junto a la exçelente
hermana,
uereys en poca edad gran
hermosura,
uereys con ella nuestra edad
ufana,
uereys en pocos años gran
cordura,
uereys que son las dos el
cabo y summa
de quanto dezir puede lengua
y pluma.
Las dos hermanas Borjas
escogidas,
Hippolita, Ysabel, que estays
mirando,
de graçia y perfeçion tan
guarnesçidas,
que al sol su resplandor está
çegando,
miraldas y uereys de quantas
uidas
su hermosura siempre ua
triumphando:
mirá los ojos, rostro, y los
cabellos,
que el oro queda atras y
passan ellos.
Mirad doña Maria
Çanoguera,
la qual de Catarroja es oy
señora,
cuya hermosura y graçia es de
manera,
que a toda cosa uençe y la
enamora:
su fama resplandeçe por do
quiera
y su uirtud la ensalça cada
hora,
pues no ay qué dessear
despues de uella,
¿quién la podrá loar sin
offendella?
Doña Ysabel de Borja está
defrente
y al fin y perfeçion de toda
cosa,
mira la graçia, el ser, y la
exçelente
color más biua que purpurea
rosa,
mirad que es de uirtud y graçia
fuente,
y nuestro siglo illustre en toda
cosa:
al cabo está de todas su
figura,
por cabo y fin de graçia y
hermosura.
La que esparzidos tiene sus
cabellos
con hilo de oro fino atras
tomados,
y aquel diuino rostro, que él y
ellos
a tantos coraçones trae
domados,
el cuello de marfil, los ojos
bellos,
honestos, baxos, uerdes, y
rasgados,
doña Ioana Milan por nombre
tiene,
en quien la uista pára y se
mantiene,
Aquella que alli ueys, en
quien natura
mostró su sçiençia ser
marauillosa,
pues no ay pasar de alli en
hermosura,
no ay más que dessear a una
hermosa:
cuyo ualor, saber, y gran
cordura
leuantarán su fama en toda
cosa,
doña Mençia se nombra
Fenollete,
 a quien se rinde amor y se
somete.

La cançion del çelebrado Orpheo,

fue tan agradable a los oydos de
Felismena, y de todos los que la
oyan, que assi los tenia
suspensos, como si por ninguno
de ellos uuiera passado más de lo
que presente tenian. Pues
auiendo muy particularmente
mirado el rico aposento, con
todas las cosas que en él auia
que uer, salieron las Nymphas por
una puerta de la gran sala, y por
otra de la sala a un hermoso
jardin, cuya uista no menos
admiraçion les causó que lo que
hasta alli auian uisto, entre cuyos
arboles y hermosas flores auia
muchos sepulchros de nimphas y
damas, las quales auian con gran
limpieça conseruado la castidad
deuida a la castissima diosa.
Estauan todos los sepulchros
coronados de enredosa yedra,
otros de olorosos arrayhanes,
otros de uerde laurel. De más
desto auia en el hermoso jardin
muchas fuentes de alabastro,
otras de marmol jaspeado, y de
metal, debaxo de parrales, que
por ençima de artifiçiosos arcos
estendian todas sus ramas, los
myrthos hazian cuatro paredes
almenadas, y por ençima de las
almenas, paresçian muchas flores
de jazmin, madreselua, y otras
muy apazibles a la uista. En
medio del jardin estaua una
piedra negra, sobre quatro pilares
de metal, y en medio de ella un
sepulchro de jaspe, que quatro
Nimphas de alabastro en las
manos sostenian, entorno dél
estauan muchos blandones, y
candeleros de fina plata, muy bien
labrados, y en ellos hachas
blancas ardiendo. En torno de la
capilla auia algunos bultos de
caualleros, otros de marmol
jaspeado, y de otras diferentes
materias. Mostrauan estas figuras
tan gran tristeza en el rostro, que
la pusieron en el coraçon de la
hermosa Felismena, y de todos
los que el sepulchro veyan. Pues
mirandolo muy particularmente,
vieron que a los pies dél, en una
tabla de metal que una muerte
tenía en las manos, estaua este
letrero:

Aqui reposa doña Catalina

de Aragon y Sarmiento cuya
fama,
al alto çielo llega, y se
auezina,
y desde el Borea al Austro se
derrama:
matéla, siendo muerte, tan
ayna,
por muchos que ella ha
muerto, siendo dama,
 acá está el cuerpo, el alma
allá en el çielo,
que no la meresçio gozar el
suelo.

Despues de leydo el Epigramma,

vieron cómo en lo alto del
sepulchro estaua vna aguda de
marmol negro, con vna tabla de
oro en las vñas, y en ella estos
uersos.

Qual quedaria (o muerte) el

alto çielo
sin el dorado Apollo y su
Diana
sin hombre, ni animal el baxo
suelo,
sin norte el marinero en mar
insana,
sin flor, ni yerua el campo y sin
consuelo,
sin el roçio d'aljofar la
mañana,
assi quedó el ualor, la
hermosura,
sin la que yaze en esta
sepultura.

Quando estos dos letreros

vuieron leydo, y Belisa entendido
por ellos quién era la hermosa
Nimpha que alli estaua sepultada,
y lo mucho que nuestra España
auia perdido en perdella,
acordandosele de la temprana
muerte del su Arsileo, no pudo
dexar de dezir con muchas
lagrimas: Ay muerte, quán fuera
estoy de pensar, que me as de
consolar con males agenos!
Dueleme en estremo lo poco que
se gozó tan gran ualor y
hermosura como esta Nimpha me
dizien que tenía, porque ni estaua
presa de amor, ni nadie meresçio
que ella lo estuuiesse. Que si otra
cossa entendiera, por tan dichosa
la tuuiera yo en morirse, como a
mí por desdichada en uer, o cruda
muerte, quan poco caso hazes de
mi: pues lleuandome todo mi bien,
me dexas, no para más, que para
sentir esta falta. O mi Arsileo, o
disçreçion jamás oyda, o el más
claro ingenio que naturaleza pudo
dar. ¿Qué ojos pudieron uerte,
qué animo pudo suffrir tu
desastrado fin? O Arsenio,
Arsenio, Arsenio quan poco
pudiste suffrir la muerte del
desastrado hijo, teniendo más
ocasion de suffrirla que yo? ¿Por
qué (cruel Arsenio) no quesiste
que yo partiçipasse de dos
muertes, que por estoruar la que
menos me dolia, diera yo çien mil
vidas, si tantas tuuiera? A Dios,
bienauenturada Nimpha, lustre y
honrra de la real casa de Aragon,
Dios dé gloria a tu anima, y saque
la mia de entre tantas
desuenturas. Despues Belisa vuo
dicho estas palabras, y despues
de auer uisto otras muchas
sepulturas, muy riquissimamente
labradas, salieron por una puerta
falsa que en el jardin estaua, al
verde prado: adonde hallaron a la
sabia Feliçia, que sola se andaua
recreando: la qual los reçibio con
muy buen semblante. Y en quanto
se hazia hora de çenar, se fueron
a vna gran alameda, que çerca de
alli estaua, lugar donde las
Nimphas del sumptuoso templo,
algunos dias salian a recrearse. Y
sentados en un pradezillo,
çercado de uerdes salzes,
començaron a hablar vnos con
otros: cada vno en la cosa que
más contento le daua. La sábia
Feliçia llamó junto a si al pastor
Sireno, y a Felismena. La Nimpha
Dorida, se puso con Syluano
hazia vna parte del verde prado, y
las dos pastoras, Seluagia, y
Belisa, con las más[1257]
hermosas Nimphas, Cinthia y
Polydora, se apartaron haçia otra
parte: de manera que aunque no
estauan vnos muy lexos de los
otros, podian muy bien hablar, sin
que estoruasse vno lo que el otro
dezia. Pues queriendo Sireno,
que la platica, y conuersaçion se
conformasse con el tiempo y
lugar, y tambien con la persona a
quien hablaua, començo a hablar
desta manera: No me paresçe