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Editorial Board

W. H. R. Lumsden Department of Genitourinary Medicine, Royal Infirmary,


Edinburgh EH3 9YW, UK

P. Wenk Tropenmedizinisches Institut, Universitat Tubingen, D7400 Tubingen 1,


Wilhelmstrasse 3 1, Federal Republic of Germany

C. Bryant Department of Zoology, Australian National University, G.P.O. Box 4,


Canberra, A.C.T. 2600, Australia

Lord Soulsby Department of Clinical Veterinary Medicine, University of


Cambridge, Madingley Road, Cambridge CB3 OES, UK

K. S. Warren Director for Science, Maxwell Communication Corporation, 866 Third


Avenue, New York, N.Y. 10022, USA

J. P. Kreier Department of Microbiology, College of Biological Sciences, Ohio State


University, 484 West 12th Avenue, Columbus, Ohio 43210-1292, USA

M. Yokogawa Department of Parasitology, School of Medicine, Chiba University,


Chiba, Japan

C. Combes Laboratoire de Biologie Animale, UniversitC de Perpignan, Avenue de


Villeneuve, 66025 Perpignan Cedex, France
Advances in
PARASITOLOGY
Edited by

J. R. BAKER
Cambridge, England

and

R. MULLER
International Institute of Parasitology
St Albans. England

VOLUME 31

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CONTRIBUTORS TO VOLUME 31

J. ALEXANDER, Department of Immunology, University of Strathclyde. The


Todd Centre, 31 Taylor Street, Glasgow G4 ONR, U K

B. J. BRABIN,Liverpool School of Tropical Medicine, Pembroke Place,


Liverpool L3 5QA, U K

L. BRABIN,Liverpool School of Tropical Medicine, Pembrokr Place, Liver-


pool L3 SQA, U K

B. GOTTSTEIN,Institute of Parasitology, University of Zurich, CH-8057


Zurich, Switzerland

M . Ho, Department of Microbiology and Infectious Diseases, Health Sciences


Centre, University of Calgary, Calgary, Alberta, Canada T2N IN4

W. M. HOMINICK, Department of Biology, Imperial College of Science,


Technology and Medicine at Silwood Park, Ascot, Berkshire SL5 7PY. UK

I. POPIEL,Paravax Inc., 2301 Research Boulevard, Suite 110, Fort Collins,


Colorado 80526. USA

D. G. RUSSELL,Washington University School of Medicine, Molecular


Microbiology Department, 660 South Euclid Street, Box 8230, St Louis,
Missouri 63110. USA

G. A. SCHAUB,Department of Special Zoology and Parasitology, Ruhr


University, Universitatstrasse 15O/ND, 0-4630 Bochum, Germany

N. J. WHITE,Wellcome-Mahidol University, Oxford Tropical Medicine Re-


search Programme, Faculty of Tropical Medicine, Mahidol University,
Bangkok, Thailand

V
PREFACE

This volume of Advances in Parasitology, somewhat unusually, has a


predominance of protozoological papers. The volume starts with a timely
and topical review by Loretta and Bernard Brabin, both now at the
Liverpool School of Tropical Medicine, which includes subjects both hel-
minthological and protozoological-parasitic infections in women-and
concentrates on the World Health Organization’s six major tropical diseases:
onchocerciasis, filariasis, schistosomiasis, malaria, African trypanosomiasis
and leishmaniasis. As the authors write in their introduction, “the import-
ance of parasitic diseases in women and their consequences have not been
fully appreciated” and, at a time when the significance of the role played by
the female half of the human race in national economies is belatedly being
recognized (particularly in the so-called developing countries), this compre-
hensive and scholarly review is especially relevant.
The second chapter is purely protozoological. Nick White and May Ho
review current knowledge of, and ideas about, the pathophysiology of
malaria. Sadly, due to the resurgence of malaria as one of the major scourges
of the warmer parts of the world as a result of the continuing spread of drug
resistant Plasmodium fulciparum, this topic too is relevant and timely. It is
now 20 years since the late Professor Brian Maegraith expounded his views
in Volume 10 on the pathogenesis of malaria, and in that 20 years a vast
amount of work has been done on this topic. As a result, our understanding
of the processes involved has been considerably deepened. A good pro-
portion of this new knowledge has emanated from the Wellcome-Mahidol
University, Oxford Tropical Medicine Research Programme, of which Dr
White is one of the leading investigators. The review by him and Dr Ho
brings together this newer work, examines it critically, and integrates it with
the earlier, pioneering studies of Professor Maegraith and others.
The third chapter, by James Alexander of Glasgow University and David
Russell of Washington University, is a transatlantic co-operation which
deals comprehensively and in depth with the relationship between Leish-
mania parasites and their host macrophages. This intriguing relationshipa
parasite invading and thriving within the very cells which are supposed to
destroy it-has long fascinated parasitologists and, again, the last few
decades have seen an explosion of knowledge concerning the means by
which the parasite manages to exploit its would-be enemies. Doctors
Alexander and Russell have been in the forefront of this work, and in this
wide-ranging review they not only summarize and discuss it, but seek also to
vii
...
Vlll PREFACE

relate the new understanding to the prospects for improved therapy and
vaccination.
Gunter Schaub, now at the Ruhr University of Bochum but until recently
at the Albert-Ludwigs University in Freiburg, reviews a rather neglected
aspect of parasitic protozoology-the effects of trypanosomatids on their
insect hosts. Dr Schaub discusses not only the well-known “two-host’’
trypanosomatids of medical and veterinary importance ( Trypanosoma and
Leishmania) but also those much less studied members of the family which
parasitize insects only, a topic to which he has himself contributed greatly.
Most of these organisms are examples of the truly “successful” parasites,
those which cause little or no harm to their hosts, but one, Blastocrithida
triatomae, causes considerable damage to its hosts (reduviid bugs, the
vectors of Trypanosoma cruzi) and Dr Schaub discusses the possibility of its
being used as an agent of biological control of the bugs in areas where
Chagas disease is endemic (biological control of insects is also dealt with in
the last chapter of this volume).
Bruno Gottstein then reviews the immunology and immunodiagnosis of
infection with Echinococcus multilocularis. Dr Gottstein is at the University
of Zurich, which under Professor J. Eckert is undoubtedly the leading world
centre for research into this organism. Although E . multilocularis does not
have such widespread economic importance as E. granulosus, principally
because the latter utilizes ubiquitous domestic herbivores as intermediate
hosts, when alveolar echinococcosis does occur in man it is one of the most
serious parasitic diseases known. Variation within the genus Echinococcus
has already been reviewed by Drs Thompson and Lymberry in Volume 27,
but the present review outlines some very elegant work on the differential
diagnosis of the two species carried out in Zurich. Recent progress has
focused on early diagnosis of pre-clinical cases in endemic areas and this,
together with new treatment procedures, has dramatically changed the
prognosis of the disease. New purified antigens are now available, including
what may prove to be the first commercially available recombinant helminth
antigen, and the exciting prospects for a vaccine are outlined.
The final chapter, by Irene Popiel of Paravax Inc. and William Hominick
of Imperial College, London, is a continuation of that by James Petersen in
Volume 24 which reviewed the mermithids as biological control agents. The
present contribution deals mainly with the other nematode groups-the
allantonematids, steinernematids and heterorhabditids-but also briefly
reviews recent information on the mermithids in the ensuing seven years.
Following sections on morphology and taxonomy and on the biology of the
groups, stressing the population dynamics and environmental limitations,
there is a stimulating discussion of their commercial status and prospects for
the future. The techniques of mass propagation, production and storage of
PREFACE ix

commercially produced species are described, as is their efficacy in the field.


It is clear that for some species of pest insects, nematodes can provide a
welcome alternative to chemical insecticides.
J. R. BAKER
R. MULLER
Parasitic Infections in Women and their Consequences

LORETTA BRABIN AND BERNARD J. BRABIN

Liverpool School of Tropical Medicine, Pembroke Place, Liverpool


L3 5 Q A , UK

I. Introduction .............................................. I
11. Epidemiologi r Sex Differences in Parasite Prevalence, Densit
and Clinical Disease Mani .......................... 2
A. Helminthic infections ...................................... 2
B. Protozoal infections ........................ 5
111. Evidence for Sex Differences Attributed to Exposure . . . . . . . . . . . . . . 9
A. Behavioural observations ............................... 9
B. Immunological observations . . . . . . . . . . . .......... 14
C. Effect of sex on the host immune response to chemotherapy . . . . . 20
IV. Evidence for Sex Differences Attributed to Hormonal and Genetic Factors . . . . 23
ntributing to sexual dimorphism in human
....................................... 24
parasitic disease contributing to sexual
26

............ 31
32
33
........... 31
C. Infection of the foetus and n ........................... 39
D. Foetal and infant immunity ........................... 49
VII. Conclusions ................................... 56
A. Maternakhild health .......................................... 51
B. Vaccine development . . . . . . . .................... 51
C. Drug treatment ..................................... 58
59
............. 60
.......................................... 60

I. INTRODUCTION

The importance of parasitic diseases in women and their consequences has


not been fully appreciated. The pattern and results of infection are likely to
be different in women because (i) exposure to infective vectors is related to
ADVANCES IN PARASITOLOGY VOL. 31 Cop.vrighr 0 1992 Academic Press Limited
ISBN 0-12-03 I73 I - I A11 righrs o/reprodurrion in any /orm reserved
2 L. BRABIN A N D B. J. BRABIN

behaviour and work patterns of males and females, which are frequently
distinct; (ii) immunity to infection and response to treatment may differ
between the sexes; (iii) pregnancy alters susceptibility to infection and risk of
disease which can lead to deterioration in maternal health; (iv) infections
during pregnancy frequently influence the outcome of pregnancy; and (v)
maternal immune status relates to the development of infant immunity.
These factors are integral to our awareness of how control of parasitic
diseases in communities can be influenced by an understanding of the
pattern of infection in women.
The infections reviewed in this chapter are primarily those identified by
the Special Programme for Research and Training in Tropical Diseases
(World Health Organization) as being of particular public health concern
(onchocerciasis, filariasis, schistosomiasis, malaria, African trypanosomiasis
and leishmaniases), although reference to other infections is made where
relevant.

EVIDENCE
11. EPIDEMIOLOGICAL FOR SEX DIFFERENCES
IN PARASITE
PREVALENCE, DENSITY
AND CLINICAL
DISEASE
MANIFESTATIONS

A. HELMINTHIC INFECTIONS

I, Lymphatic jilariasis

A review of sex differences in susceptibility to lymphatic filariasis presented


data from 53 studies from Africa, south-east Asia, the Indian subcontinent
and the Americas (Brabin, L., 1990a). Forty-three studies found lower mean
prevalences of infection in females than in males. When classified by age and
sex, prevalence was consistently lower in women of reproductive age, and this
difference was statistically significant in 16 of the 33 studies for which data
were available. Fewer studies had data on microfilarial densities by age and
sex but, of those available, densities were similar in both sexes in the
youngest and the oldest age groups, but were lower in females in their
reproductive years. The difference held true over a wide geographical area,
and was observed for both Bancroftian and Brugian filariasis, irrespective
of periodicity. Clinical manifestations of disease were also lower in females
than in 'males. In areas endemic for Wuchereria bancrofti, more cases of
hydrocele were detected in men than of lymphoedema in women, and both a
history of acute lymphangitis (Kazura et al., 1984) and filarial fevers (Weller
et al., 1983) were more common in males. In areas endemic for Brugia malayi
elephantiasis was less frequent in females and, in general, age of onset and
peak prevalence occurred at a later age in women.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 3

One problem facing the interpretation of prevalence data is the relative in-
sensitivity of blood slide examination in detecting low density parasitaemias.
In Tonga, for example, 68% and 74%, respectively, of men and women aged
21-50 years were positive by a sensitive filtration method, but only 33% of
males and 26% of females by blood slide examination (Desowitz and
Hitchcock, 1974). If women are less exposed, or have lighter infections,
lower prevalence in the reproductive age may reflect the insensitivity of
blood slide techniques. A similar problem is faced when interpreting the
observed plateau effect in prevalence curves, i.e. a flattening of the curve
with increasing age, which is thought to indicate belated development of
host resistance (Piessens and Partano, 1984). This latter problem has been
addressed by two recent papers which analysed microfilarial frequency
distributions by fitting various statistical models (Das et al., 1990; Grenfell et
al., 1990). One finding was that a large proportion of observed microfilariae-
negative individuals may be truly negative, due to the absence of adult
worms or the presence of unmated adults only, rather than the result of an
inaccurate blood sampling process. The models also provided indirect
evidence for the operation of density-dependent limitations on parasite
burden, as reflected in microfilarial counts which might be attributable to
acquired immunity. Mechanisms acting to reduce microfilarial infection in
women of reproductive age may differ from those operating to diminish
microfilaraemia with increasing age.

2. Onchocerciasis

Microfilarial density is a more accurate measurement of infection with


Onchocerca volvulus than is prevalence. This is because, in areas of moderate
to high endemicity, reinfection constantly occurs and there is no evidence of
a protective immune response against reinfection. Lower mean microfilarial
densities have been reported in females than males in a number of studies.
Fig. 1 shows the geometric mean microfilarial densities for 65 Onchocerciasis
Control Programme (OCP) villages representing all levels of endemicity
(Kirkwood et al., 1983a) and seven Guatemalanjncas, of which five were
hyperendemic (Brandling-Bennett et al., 198I). The difference in worm loads
between males and females is seen from the age of five years onwards in
West Africa, and from a younger age in Guatemala. Using a “force of
infection” model to study the age-specific epidemiological trends during a
period of vector control in the OCP in the Volta river basin area, there was
generally good agreement in predictions of microfilarial load in skin snips
from 23 villages, but trends for females were less well predicted (Remme et
al., 1986). A wide divergence was seen in the 2&30 years old pre-control
group, where microfilarial load was lower than expected. A review of factors
4 L. BRABIN AND B. J . BRABIN

affecting the differential susceptibility of males and females to onchocerciasis


(Brabin, L., 1990b) considered that: (i) the most marked sex differences were
seen when transmission rates were high; (ii) in highly endemic areas, worm
burdens were lower in females from early in childhood; and (iii) in hypo-
endemic regions, microfilarial densities were similar in both sexes and
probably accurately reflected exposure to infective vectors.

1- -1
0- 5- 10- 15- 20- 25- 30- 35- 40- 45- 50- 55- 60- 65-

Age (years)

FIG. I . Geometric mean microfilarial density by age and sex in 65 villages in West
Africa and in seven Guatemalan fincas (estimated from Kirkwood et al., 1983a;
Brandling-Bennett et al., 1981). Reproduced with permission from Brabin, L.
(1990b), Acta Leidensia 59, 413426.

Ocular lesions and blindness are less frequent in females. The severity of
ocular onchocerciasis is known to be related to the intensity of infection
(Thylefors and Brinkman, 1977) and the community microfilarial load
(CMFL) has been developed as an index of the community level of infection
(Remme et a/.,1989). In 33 West African savanna villages, mean microfilar-
ial loads in the anterior chamber of the eye and in the cornea showed a linear
relationship with the CMFL and the relationship appeared to be the same
for both sexes. None the less, the prevalence of posterior segment lesions was
higher in males-a difference which remained statistically significant after
correction for intensity of infection. With a different approach, Kirkwood et
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 5

al. (1983b) found that ocular lesions were more common in males after
applying a logistic regression analysis to data from 53 OCP villages in order
to control for sex and duration of exposure at a given microfilarial load. The
results indicated that males were about I .5 times more likely to be blind than
females of the same age and same level of infection (P c 0.001). One
disadvantage of this study was that it did not control for non-onchocercal
eye disease, but it does suggest that community level indicators may obscure
differential sex risks.

B. PROTOZOAL INFECTIONS

1. Trypanosoma cruzi infection

Although there appears to be no sex difference in parasitaemia arising from


T. cruzi infection (Hoff et al., 1979), there is evidence from selected
populations that certain lesions, such as apical aneurysm, associated with
chronic heart disease occur less frequently in women (Oliveira et al., 1981).
In longitudinal studies in Castro Alves, Brazil, abnormal electrocardiogram
(ECG) tracings were significantly more frequent in seropositive men than
women despite similar age-specific rates of seroreactivity (Maguire et al.,
1983). When the same population was followed up after nine years to assess
the development of ECG abnormalities in seronegative and seropositive
individuals, it was found that seropositive individuals developed abnormal
ECGs twice as frequently as did those who were seronegative (Mota et al.,
1990). While there was little difference between seronegative males and
females in the rate of development of an ECG alteration ( 1 1.2 and 14.2 per
1000 person-years (PY), respectively), amongst seropositive persons the rate
for males was 32.3/1000 PY compared to 2 1 . 1 / 1000 PY for females. Ven-
tricular conduction defects developed more frequently in seropositive males
( 1 1.2/1000PY) than in seropositive females (9/1000 PY), as did frequent or
multiform ventricular extrasystoles (4.8 vs 2.3/ 1000 PY, respectively). There
was no difference in age-adjusted mortality rates by sex and seropositivity
but, in seropositive individuals with an initial abnormal or borderline ECG,
the mortality rate at age 4&59 years was 20.4/1000PY in males compared to
11.2/1000PY in females. Megaoesophagus was also detected more often in
men living in Castro Alves (Mota et al., 1984).
Few longitudinal studies of Chagas disease have examined a large,
representative sample of the population. In Goiinia, central Brazil, a
population-based case-control study was undertaken amongst unskilled
workers (Zicker et al., 1990). This study also found that the risk of ECG
alterations of any kind was greater in males, as was the risk of left anterior
6 L. BRABIN A N D B. J. BRABIN

hemiblock. These associations were stronger among seropositive than


among seronegative subjects.

2. Visceral leishmaniasis

From very early, sex differences in visceral leishmaniasis were noted and a
number of studies of kala-azar in India addressed the question of why more
cases were diagnosed in boys than in girls (Balasubramanian, 1920-1 92 1;
Cunningham and Pundit, 1924-1925). Poor case detection was suspected in
a society where women’s mobility was restricted by seclusion in purdah
(Turkhud et al., 1925-1926). Napier and Das Gupta (1931-1932) showed
that both the age and sex distributions of kala-azar were affected by distance
from a dispensary. In the Bihar epidemic in 1977-1978 the ma1e:female ratio
of confirmed reported cases of kala-azar was 5.5:l (Thakur, 1984). Given
that family clustering of infection is characteristic of Indian kala-azar
(Michael, 1925-1926; Nandy et al., 1988), it would be surprising if females
were not frequently exposed and more cases in girls were not found by active
case detection.

TABLE1 Age an2 sex distribution of visceral leishmaniasis cases in Kenya, south
Ethiopia and Sudan (Jahn et al., 1986; Ayele and Ali. 1984; Van Peenen and Reid,
1963, respectively)

Kenya South Ethiopia Sudan


Age group Age group
( vears ) Males Females Males Females (years) Males Females
0-9 35 23 4 1 0-10 50 40
10-19 42 28 10 4 11-20 21 24
20-29 13 10 5 4 21-30 21 15
30-39 5 4 3 1 > 30 21 6
>40 0 4 2 0

Totals 95 69 24 10 1I9 85

In Kenya a 60% preponderance of male cases was observed by Southgate


and Oriedo (1962) and here there were no problems with case detection
because house-to-house investigations of whole populations were con-
ducted. In all the East African foci visceral leishmaniasis has been found to
predominate in males (Archibald and Mansour, 1937; Baruffa, 1965; Hoog-
straal and Heyneman, 1969). Table 1 records a preponderance of males in
every age group during the Khor Falus epidemic in the Sudan (Van Peenen
and Reid, 1963), in South Ethiopia (Ayele and Ah, 1984) and in Kenya
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 7

(Jahn et al., 1986). In an outbreak following floods in south Sudan among


the Nuer tribe, who moved to Khartoum for treatment, 57% of 99
confirmed cases were males (De Beer et al., 1990).
In China, where both Mediterranean visceral leishmaniasis and Indian-
type kala-azar occur, cases in male patients exceeded those in females by
about 60% in 1 1 of 12 provinces (Leng Yan-Jia, 1982).

3. Malaria

Malaria parasite rates are often not reported by sex and there is a general
belief that parasite prevalence in males and non-pregnant females is similar.
This was not the finding of the Garki project, where lower parasite rates and
densities were found for all age groups of females above 4 years of age for
both Plasmodium falciparum and P . malariae (Molineaux and Gramiccia,
1979). In children aged 4 years and under, parasite rates were similar for
both sexes. Spleen and parasite indices for males aged 5-14 years were
significantly higher than those in females in another large study (4500
subjects) in Pattukkotai, south-east India (Russell et al., 1938). Several
smaller studies have shown higher parasite densities in infant girls (McGre-
gor, I. A., 1964; Hendrickse et al., 1971). Considering that mortality from
malaria is greatest in children under 5 years old in holo- and hyperendemic
areas, any advantage to females in parasite clearance appears to be slight.
Females experience increased susceptibility to malaria in their first preg-
nancy (Brabin, B. J., 1983; Brabin, B. J. et al., 1988) and malaria-associated
anaemia has more serious consequences in adolescent girls and women
(Brabin, B. J., 1990). In an early study, higher rates of albuminuria were
noted in females in Surinam although rates of P . malariae and P. falciparum
nephritis were higher in males (Van der Kuyp, 1950). Since this was a
hospital-based study, the results may be due to case selection bias.
Sex differences are apparent in clinical syndromes associated with malaria,
such as hyper-reactive malarious splenomegaly and Burkitt’s lymphoma. In
Papua New Guinea, where hyper-reactive malarious splenomegaly occurs
with varying degrees of severity (Brabin et al., 1989), higher spleen rates
have been found in women. In a total village survey, Crane and Pryor (1971)
observed a higher spleen rate (males 72%; females 88%) and larger average
enlarged spleen size in women, and this confirmed similar results from
earlier, non-random samples from the Sepik region (Mackerras and Aber-
deen, 1945; Peters, 1960). Comparable data were reported by Schofield
(1962) in two complete village surveys and, more recently, from Madang
(Brabin, L., 1988).
Burkitt’s lymphoma, a cancer associated with both malaria and Epstein-
Barr virus (EBV) infection, has been observed more frequently in boys than
8 L. BRABIN AND B. J. BRABIN

girls in Uganda and Papua New Guinea. In the Mengo district of Uganda
the incidence rate among males was 1.5 that of females. Incidence was higher
in females under 5 years of age but was considerably higher in males over 15
years (Morrow et al., 1976). In the Lango and Acholi districts the incidence
rate was twice as high in males and the ma1e:female ratio was higher in the
younger age groups (Morrow et a[., 1977). In Papua New Guinea, Burkitt
and O’Conor (1 96 1) reported a male predominance of 2 to 1 but noted that
this was also the approximate ratio of male to female patients in general
hospital admissions. In a later study the ratio was noted to be 1.8: 1 (Reay-
Young and Chir, 1974). In contrast, in North Mara, Tanzania, from 1964 to
1983 the male:female ratio was similar overall but showed a marked
difference in the 6-7 years age group, where there was a large excess of
female cases and the ma1e:female ratio was 0.29:l (Geser and Brubaker,
1985).
The factors which affect the age and sex distribution of Burkitt’s lym-
phoma and how both malaria and EBV affect the distribution of cases are
not clear. In North Mara females had higher geometric mean titres (GMT)
for EBV but it is of interest that males bearing the X-linked lymphopro-
liferation gene are predisposed to EBV infection, and in them a variety of
lymphoproliferative diseases develop, such as malignant lymphomas and
malignant mononucleosis (Barnabei et al., 1982). One explanation for sex
differences may be related to differential susceptibility to EBV infection
rather than to malaria. The higher frequency of Burkitt’s lymphoma in boys
than in girls, which is the most frequent observation from endemic areas, has
posed a dilemma for the hypothesis that malaria causes Burkitt’s lymphoma
since sex differences in malaria prevalence are not recognized (Geser et al.,
1989). In North Mara, the sex ratio of Burkitt’s lymphoma and malaria were
said to be similar (although malaria antibody data by sex and age were not
presented), and there appeared to be no conflict with the possibility of a
causal role for malaria (Geser et al., 1989). This problem will not be resolved
until the questions of sex differences in malaria prevalence, and whether this
is affected by the level of malarial endemicity, are addressed.

4. African trypanosomiasis

Most of the data published on African trypanosomiasis were obtained from


mass surveys. Although the number of diagnosed cases is often reported
separately for males and females, difficulties in diagnosing sleeping sickness
parasitologically and the use of active and passive case detection methods
have not permitted a detailed comparison of prevalence data by age and sex.
African trypanosomiasis has been considered a disease of which the relative
risk was greatest for males, especially in areas endemic for the rhodesiense
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 9

form of T. brucei,* and under stable conditions (Veatch, 1946; Robertson,


1963; Scott, 1970). In a sample of 145 patients in Central Nyanza during the
epidemic of 1964, females were equally commonly infected with peak
infection rates occurring at 20-29 years (Willett, 1965). In the north
Luangwa Valley, Zambia, 22 female and 38 male cases of rhodesiense
sleeping sickness were diagnosed by active case detection and from hospital
admissions (Boatin et al., 1986). The highest number of cases in women (7)
was in those aged 20-29 years, while most male cases (1 7) were seen at 40-59
years. In a parasitological survey in Kwamouth, Zaire, two-thirds of the
3500 resident ethnic group were examined in a house-to-house survey
(Henry et al., 1982). Table 2 shows the distribution of gambiense sleeping
sickness by sex and age. In almost every age group more females than males
were positive, and peak prevalence of infection in women again occurred in
those aged 20-29 years (8.8%). Thirty-two female and 30 male cases were
parasitologically diagnosed in village surveys on the Ivory Coast (Felgner et
al., 1981); the peak age of infection was 20-29 years in both sexes.

TABLE2 Prevalence of parasitologicall-v diagnosed cases of gambiense sleeping sick-


ness by sex and age in Kwamouth, Zaire (Henry et al., 1982)

Males Females
Age group (years) Number" Percentage Number Percentage
CL-4 158 1.3 180 2.8
5-9 209 0.5 186 2.6
Is14 20 1 2.0 168 4.8
15-19 124 4.0 107 4.7
2&29 I22 5.0 136 8.8
3w9 163 8.0 225 8.0

a Total examined in each age group.

111. EVIDENCE
FOR SEX DIFFERENCES
ATTRIBUTED
TO EXPOSURE

A. BEHAVIOURAL OBSERVATIONS

Exposure is affected by the division of labour, age, family size and labour
requirements, economic and social status, but womens' activities are usually
delineated from male activities. The importance of documenting patterns of
This terminology has been used to avoid commitment to the precise taxonomic status of the
names rhodesiense and gambiense [eds].
10 L. BRABIN AND B. J. BRABIN

exposure is linked to the interpretation of prevalence data because it should


be known whether observed sex differences are the result of differential
exposure, or reflect different immunological responses to infection.

1. Helminthic infections

Studies on human immunity to helminthic infections are complicated by the


fact that adult worms continue to survive for many years, during which
period the host is constantly reinfected. Some immunity to superinfection
may develop, but investigations have to be based on the level of the worm
burden, as reflected by egg output, rather than on clearance of infection.
Changes in the worm burden may reflect not only the state of immunity, but
also changes in the degree of exposure. This leads to difficulties in interpret-
ing age-specific prevalence or intensity curves in terms of immunity or to
comparing differences in worm burden in males and females. The difficulties
are well illustrated by water contact studies for schistosomiasis, which show
not only that the nature and degree of water contact patterns are complex
but that infected water sources may be visited by different groups and for a
variety of purposes (Chandiwana and Christensen, 1988). Whereas, for
schistosomiasis, a determined effort has been made to measure water contact
and to relate this to age- and sex-specificprevalence and intensity curves and
to a number of immunological indicators (Butterworth, A. E. et al., 1984;
Hagan et al., 1985), this has not been the case for other helminthic diseases.
For several of these diseases, distinct sex differences have been observed in
epidemiological studies, and these have been attributed to differences in
exposure. The evidence is largely anecdotal. As noted by Bundy (1988), sex-
related behaviour and cultural differences are apparent in most human
communites and it is generally possible-though this may require some
ingenuity-to identify gender-related practices to which differences in ex-
posure can be ascribed. What is merely a plausible association is often
presented as causality. Surprisingly, many associations have been accepted
without question.

( a ) Lymphaticjlariasis. Although exposure must be an important factor


influencing the epidemiology of lymphatic filariasis, investigators have not
agreed on whether the evidence for lower microfilarial densities in women
favoured an entomological or an immunological explanation. Murray
(l948), in an area of south-east Asia endemic for W . bancrofti, claimed that
clothing did not account for the lower microfilarial rates in women. Nor did
occupational exposure, since mosquitoes caught in the centre of the village
had a mugh higher infectivity rate than those collected at the edge or far
away. A completely contrary view was taken by McCarthy and Fitzgerald
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 11

(1956), both in regard to clothing and transmission site. Similar disagree-


ments surrounded the interpretation of sex differences in filariasis prevalence
in Pakistan (Barry et al. 1971; Wolfe and Aslamskhan, 1972). In Africa,
where night-biting Anopheles gambiae and An.funestus are the main vectors,
it seems unlikely that one sex would be consistently exposed more than the
other (Jordan, 1960; Ripert et al., 1982)-although several ingenious expla-
nations have been devised (McFadzean, 1954; Brunhes, 1975). Controlling
for socio-economic differences did not affect the pattern of lower microfilar-
ial densities in women of reproductive age in two Kenyan villages (Wijers
and Kinyanjui, 1977).
Differences in clinical manifestations according to sex may, in part, be due
to greater difficulty in examining females (Wolfe and Aslamkhan, 1972). In
areas where W . bancrofti is endemic the difference in clinical disease is due
largely to the frequency of hydrocele in men. Cases of genital involvement
may have been missed in some studies, but external genital involvement, for
unknown reasons, is rare.

( 6 ) Onchocerciasis. Behavioural factors are not considered to be a


satisfactory explanation for all the sex differences observed in connection
with onchocerciasis (Brabin, L., 1990b), although entomological indices of
transmission and exposure have been used to support such arguments.
Observations that villages located close to breeding sites were more heavily
infected than second- or third-line villages contributed substantially to the
principle that man-vector contact was the most important determinant of
infection (Rolland and Balay, 1969). Subsequently, the division of labour in
West Africa was characterized as one in which male farming was predomi-
nant and took men into high transmission zones, whereas women and
children remained in the peridomestic areas of the village where infectivity
rates were lower (Remme et al., 1986). Such a general argument is quite
refutable, and the principle of first-line villages also has been recently
challenged (De Sole et al., 1991). In a study of three onchocerciasis foci in
West Africa, it was found that the geographical distribution of prevalence
and intensity of onchocercal infection in the community could be very
different from what would be expected on the basis of demographic and
entomological information. This illustrates that entomological indices can-
not completely explain the distribution of onchocerciasis between villages
nor bet.ween sub-groups and individuals within villages.

(c) Schistosomiasis. Water contact studies on schistosomiasis have


allowed some estimation of infection rates in males and females in relation
to water use. How this relates to the development of resistance in either sex,
and whether resistance differs according to sex, are difficult to assess since, in
12 L. BRABIN A N D B. J. BRABIN

many areas, water contact by males decreases with age whereas it remains
more constant for females. Increased susceptibility of young girls was
implied in a study of Schistosoma mansoni in Machakos, Kenya, where girls
less than 9 years of age were judged to have less water contact than boys but
higher mean egg counts of S. mansoni (arap Siongok et al., 1976). In
Zanzibar, in a high prevalence area for S. haematobium, schistosomiasis-
related morbidity was measured in schoolchildren by ultrasound (Hatz et al.,
1990). A striking feature of the results was the relatively low risk of uropathy
among females when compared with egg counts and haematuria. The highest
proportion of uropathy and haematuria was in girls aged M years, the
reverse of the situation for boys of the same age. The lowest proportion of
female uropathy and haematuria cases occurred in women of reproductive
age (26-40 years). Menstruation did not appear to influence the predictive
potential of microhaematuria. By contrast, in an area of low endemicity
studied by Hatz et al. (1990) (Mauritius), the most severe uropathy was
found in adult women. No sex difference was found in the incidence of
squamous cell carcinoma of the bladder in Zimbabwe, but these obser-
vations could not be correlated with exposure since data were retrospectively
based on medical records (Thomas, J. E. et al., 1990).
The availability of macrofilaricidal drugs permits quantitative studies of
reinfection rates and water contact which can be analysed by sex. In The
Gambia, girls had significantly higher reinfection rates of S. haematobium
following treatment with praziquantel than boys of the same age range, and
these differences remained after controlling for levels of exposure and
eosinophil counts (Hagan et al., 1985). In women ( > 15 years), intensities of
infection following treatment were 100 times lower than in men, although
levels of exposure were only five-fold less (Wilkins et al., 1987). Differences
were not observed in 72 schoolchildren in Tanzania (Hatz et al., 1990), but
water contact studies were not done. These results seem to indicate differ-
ences in the immune response of females of different ages as well as
differences due to sex.

2. Protozoal infections

Under highly endemic conditions, exposure to a number of protozoal


infections is likely to be high for both sexes. Little difference in exposure
would be expected in highly malarious areas or in regions endemic for
Chagas disease, where the vector of T. cruzi, the triatomine bug, is ubiqui-
tous in houses. For diseases where vectors are localized to specific breeding
sites or vegetation, explanation for sex differences is often made in terms of
man-vector contact.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 13

( a ) African trypanosomiasis. Under non-endemic conditions for rhode-


siense sleeping sickness, transmission generally takes place in the bush or
gardens, at a distance from village communities and clinical cases are
detected more frequently in males (Mulligan and Potts, 1970). In situations
where tsetse invade village compounds, as might happen in an epidemic, all
ages and both sexes are potentially at equal risk of infection (Robertson and
Baker, 1958; Scott, 1959; Morris, 1960; Buyst, 1977). In areas endemic for
gambiense sleeping sickness, the classical man-fly transmission cycle in both
endemic and epidemic conditions, and the presence of the parasite in
domestic animals, increase the risk of transmission in the proximity of
village settlements (WHO, 1986). To explain why females were more
frequently diagnosed with sleeping sickness in Zaire, Henry et al. (1982)
claimed that females were more exposed to infective tsetse during their
agricultural activities, but the activities of males and females of different age
groups were not clearly documented. Similar, and at times contradictory,
explanations have been given in other studies (Frezil, 1981). No anthropolo-
gical investigation has been undertaken to support these explanations.

( 6 ) Visceral leishmaniasis. In Kenya, herding activities and the proximity


of termite hills have been considered to increase vector contact in males.
High infection rates were found in boys aged 4-9 years (Southgate and
Oriedo, 1962; Wijers, 1963), and this was attributed to their herding
duties (McKinnon and Fendall, 1956). Subsequently this view was revised
(McKinnon, 1963) since the Baringo tribe studied were pastoralists and from
the age 5 years onwards girls herded with boys till they married. Higher
infection rates in men were attributed to the time they spent in the vicinity of
termite hills at times of peak sandfly biting activities, while women were
protected by smoke from fires as they cooked (Southgate, 1974). More
recent studies in the Machakos and Baringo districts of Kenya have cast
doubt on the role of termite hills in transmission (Ho et af., 1982).

(c) Cutaneous leishmaniasis. In Central and South America it is clear


that males and females are not equally exposed to infective vectors, and
“chiclero’s ulcer” (vector Lutzomyia olmeca ofmeca) and “pian-bois” (major
vector Lu. umbratilis) are more commonly seen in men. Forest workers
clearing lands for plantation are at high risk, and in some areas few persons
who penetrate the forest escape infection (Lainson and Shaw, 1978). Espun-
dia is more common in males, possibly because this is primarily a forest
disease, although it has been recorded in an area where peridomiciliary
transmission was suspected (Marsden, 1986). In Bolivia, cutaneous leishma-
niasis was reported to be higher in males aged 10-30 years at Los Yungas
14 L. BRABIN AND B. J . BRABIN

(Desjeux et al., 1974). and soldiers have been particularly affected in French
Guyana (Dedet et al., 1989).
Females are at risk in some areas. Since the 1980s, the migration of entire
families to new residential areas in endemic areas of Panama has increased
infection rates (Arias, 1988). In Brazil, cutaneous leishmaniasis is now
observed close to metropolitan regions in areas where forest was cleared
many years previously. In one study area 35 km from Rio de Janeiro city
centre, cases due to Leishmania braziliensis braziliensis were found frequently
in women and children (Oliveira-Net0 et al., 1988). Similarly, in the north
Amazon jungle, villages are likely to be well inside the forest, bringing the
total population into a risk zone (Guerra, 1988). In Costa Rica, cases of
cutaneous leishmaniasis reported by province show an equal distribution
between the sexes (Hidalgo, 1988).
From regions outside the Americas, detailed data on sex differences are
limited. In a retrospective study of cutaneous leishmaniasis in Meta Abo,
Ethiopia, similar rates were found for all ages and both sexes (Wilkins,
1972). However, of 33 patients with diffuse cutaneous leishmaniasis studied
by Bryceson (1969), 21 were male and 12 female. In Khartoum, up to 1975,
51 cases of mucocutaneous leishmaniasis had been reported, all in adult
males (El Safi, 1988). Of 9657 cases of cutaneous leishmaniasis reported in
Khartoum province between September 1986 and March 1987, 61% were
males and 39% females (El-Safi and Peters, 1991). To what extent these
differences were due to exposure, or to failure of females to report for
treatment, cannot be assessed.

B. IMMUNOLOGICAL OBSERVATIONS

Understanding the distribution of infection within communities is greatly


facilitated by the use of immunodiagnostic tests, some of which have only
recently been applied in field studies. Serological tests provide additional
information on past exposure. Tests to detect specific antigens present in
sera are useful for identifying active infection. Specific tests of T cell
reactivity, in addition to skin testing, are increasingly being used in field
studies. The use of immunodiagnostic testshas shown how complex the host
response following exposure may be, and it provides a means to differentiate
male and female host response when applied to well-defined population
samples.

1. Helminthic infections

( a ) LymphaticJilariasis. While there is no direct evidence that resistance


of humans to filariasis is due to protective immunity, indirect support for
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 15

this concept is available. For example, the prevalence and levels of immuno-
globulin (Ig) G antibodies to the sheath of B. rnalayi and W . bancrofti micro-
filariae are much higher in sera from amicrofilaraemic than from microfilar-
aemic donors (Piessens et al., 1980). It has been observed that immigrants
developed IgM antibodies to microfilariae soon after arrival in a filariasis
endemic area but did not develop IgG antibodies to the same antigen
preparation until they had lived continuously in the area for many months
(Piessens et al., 1987). In a study by Kurniawan et al. (l990), it was assumed
that those with low levels of ( < 1 : 100) IgG antimicrofilarial antibodies were
“underexposed”. In this study, “exposed” individuals were divided into
three categories: those in whom no objective evidence of infection could be
detected; those with filarial antigens present in the sera; and those who were
microfilaraemic. The purpose of the study was to compare antigen recog-
nition patterns of defined groups of amicrofilaraemic persons with similar
degrees of exposure, and it was conducted in Indonesia with 81 adult
immigrant volunteers. Comparison of the immunological response by sex in
a representative population is undocumented, but it would be of interest (i)
to determine the proportion and age distribution of amicrofilaraemic
females, (ii) to define the characteristics of females with active infection, and
(iii) to ascertain the proportion of those individuals who are pregnant.

( h ) Onchocerciasis. A study was undertaken by Ward et al. (1988) to


compare the immune responses of infected individuals living in an area
endemic for onchocerciasis with those of persons who were free of infection
despite continued exposure. The infected group contained a significantly
higher proportion of male subjects than did the infection-free group,
although all had been resident for a long period in the area. Despite having
less parasitic-specific serum antibodies, the infection-free individuals showed
greater lymphocyte responsiveness, especially of interleukin-2 production to
0. volvulus antigen, than did infected subjects. Whether such differential T
cell responsiveness to parasite antigen actually relates to susceptibility or to
resistance is not certain, and, clearly whether female subjects are more likely
to exhibit enhanced T cell responsiveness to 0. volvulus antigen needs to be
confirmed in a more representative sample.

2 . Protozoal infections

( a ) Malaria. In Thailand sero-epidemiological findings were specifically


used to show the bias of case detection rates reported from malaria clinics
(Ettling et al., 1989). Although a predominance of male cases was treated in
malaria clinics, a random sample of villages from the area showed similar
exposure rates in males and females as judged by the proportion seropositive
16 L. BRABIN AND B. J. BRABIN

by indirect fluorescent antibody (IFA) or enzyme-linked immunosorbent


assay (ELISA), concentration of antibodies in ELISA units, and rates of
seroconversion up to the age of 30 years. The results suggested that females
with malaria went elsewhere for treatment, although malaria clinics are the
best source of appropriate, rapid and inexpensive treatment.
In a study to explore the relationship between malarial infection and
Burkitt’s lymphoma, IgG, IgM and IgA levels were measured in sera from
Burkitt’s lymphoma patients and from neighbourhood controls matched for
sex and age (Nkrumah et al., 1979). All three classes of immunoglobulins
were present in significantly lower amounts in the sera from Burkitt’s
lymphoma patients than in controls. Levels of IgG antibodies specific for P.
falciparum and P . malariae were similar, suggesting that both groups had
been equally exposed, but IgM antibodies specific for P. falciparum were
significantly lower in Burkitt’s lymphoma patients. Ziegler et al. (1972) had
also observed low total IgM levels in Burkitt’s lymphoma patients in
Uganda. These studies did not report IgM levels in males and females
separately, but there is evidence to show that the humoral immune response
is more active in females (Grossman, 1989), and this might offer some
protection against Burkitt’s lymphoma.
In Papua New Guinea, hyper-reactive malarious splenomegaly occurs
more frequently in females and is associated with high levels of total and
malaria-specific IgM, but malaria-specific IgG profiles are similar to those
observed in males (Brabin, B. J. et ai., 1989). Patients with hyper-reactive
splenomegaly are frequently aparasitaemic and seem to be more efficient at
clearing peripheral parasitaemia, although anaemia may be a serious result
of increased splenic activity.
It was noted above (Section TI.B.3) that, in the Garki project, fewer
females over 5 years old had parasitaemias with either P. falciparum or P .
malariae infections than males, and those who were infected had lower levels
of parasitaemia (Molineaux and Gramiccia, 1979). In this study it was also
shown that there was a positive correlation within persons between the level
of immunity to P. falciparum and the level of immunity to P. malariae
(Molineaux et al., 1980). This was not considered to relate to current
exposure since there was a negative association between P. falciparum
parasitaemia and several serological indices of the immune response. Although
the difference could be due to past exposure, they considered it was more
probably related to constitutional factors such that persons (and by impli-
cation, perhaps, the sex) with a weaker immunity to one would have a
weaker immune response to the other.

(6) T. cruzi. Age-specific serological testing in Castro Alves, Brazil


showed no sex difference, suggesting that exposure was similar in both sexes,
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 17

which would not explain a lower prevalence of cardiomyopathy in women


(Mott et al., 1976). That exposure was at least as high in women as in men
was further supported by observations showing that females over the age of
10 years had higher geometric mean antibody titres than males.

(c) Visceral leishmaniasis. Although the interpretation of a positive


leishmanin skin test is debatable, it still provides a useful epidemiological
measure of immunological stimulation and the degree of exposure of a
community. Fig. 2 shows the distribution of leishmanin testing by age and
sex in Voo in Kenya in 1961 (Southgate and Oriedo, 1967) which should be
compared with the proportional risk ratios calculated for parasitological
case detection in Voo (Table 3) between 1957 and 1961 (Southgate, 1964).
The proportion of males with positive skin tests was higher for each group,
suggesting that males were more exposed to infection. However, positivity
increased with age for both sexes and older women continued to be exposed,
although Table 3 suggests that their risk of developing a clinical infection
was slight.

I I I I I I I I I #

0-4 5-9 10-14 15-19 20-29 30-39 40-49 50-59 60+


Age (years)
FIG.2. Distribution (70)of positive reactions to a leishmanin skin test in Voo,
Kenya, by age and sex (data from Southgate and Oriedo, 1967).

In Kivaa, Machakos (Kenya), 25% of household contacts of four patients


had positive skin tests, compared to 3.8% in non-infected households (Ho et
al., 1982). Kala-azar occurred in homesteads with and without adjacent
termite mounds, suggesting that the microfocus of infection may have been
the household rather than sandfly breeding sites.
Among 144 cases of 164 detected in Baringo district, Kenya, a male
predominance of 57% was observed (Jahn et al., 1986) using an ELISA to
18 L. BRABIN A N D B. J. BRABIN

detect leishmanial antibodies, with parasitological confirmation. The


authors thought that herding activities did not account for differences in
exposure since most children attended school, and the spread of cases bore
no relationship to the distribution of termite hills. This was not a popu-
lation-based sample.

TABLE
3 Proportional risk ratios by age and sex for visceral leishmaniasis in Voo,
Kenya (1957-1961) (Southgate, 1964)

Age group (years) Males Females


(r9 7.0 6.3
1&19 10.1 4.2
20-29 21.5 4.4
3&39 14.8 2.6
4w9 11.8 -

s(r59 10.9 -
> 60 6.3 ~

An outbreak of visceral leishmaniasis was recently reported in the upper


West Nile region of southern Sudan, in an area which was previously
thought to be non-endemic for visceral leishmaniasis (Perea et al., 1991). In a
10 day period, 100 cases were identified at Ler hospital and the overall male:
female ratio was 4.5: 1. However, seroprevalence studies in the general
population found 40 (20%) of 198 women seropositive compared to 26
(16%) of males. Women older than 15 years had a higher prevalence (28%)
than men in the same age group (18%) (relative risk = 1.58). It is not yet
known whether seropositive individuals are likely to develop symptomatic
or subclinical infections, or will remain asymptomatic and eventually lose
their antibodies. The excess male cases identified in hospital studies may
reflect the fact that men use hospital services more than women, or that
males are more likely to develop symptomatic infections than females.
Overall, these observations support the suggestion that exposure to
infection is higher for males, but it is not clear under what circumstances
exposure is most likely to occur. At the same time there is some evidence that
exposed females are less likely to develop clinical symptoms than exposed
males. Immune susceptibility, as well as exposure, probably accounts for
observed patterns of infection (Ashford, 1988). Susceptibility may be the
result of predisposing factors, such as intercurrent infection (Busuttil, 1974)
or malnutrition (Cerf et al., 1987; Harrison et al., 1986), and sex differences
could be associated with the predisposing factors rather than directly with
leishmanial infection.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 19

( d ) Cutaneous leishmaniasis. For cutaneous, as for visceral, leishmania-


sis, there is increasing evidence that host immune response, as well as
exposure, modifies the pattern of infection. In a study from Iran (Nadim and
Faghih, 1968), it was noted that skin lesions in children mostly healed in 3
months, but in some individuals healing time was longer and extended up to
8 months. A recent investigation of zoonotic cutaneous leishmaniasis in the
Al-Ahsa oasis in south Arabia found that the distribution of active lesions
was bimodal, due to an outstanding number of cases in non-Saudi immi-
grant males in their twenties (Dye et al., 1989). After excluding non-Saudis, a
comparison of the sex ratios for active and past infections showed no
significant difference: 59% (93/157) of active and 54% (131/242) of past
cases were males. However, the size of scars in males was significantly
smaller. Wirtzum et al. (1979) reported on the reaction to inoculation with a
frozen vaccine against Leishmania tropica in 39 soldiers of the Israeli
Defence Forces (including 18 women). Men had significantly more ulcers
than women and developed them more quickly, whereas in women nodular
lesions tended to be more protracted. The authors concluded that vaccine
batch and host factors such as age and sex could play a part in determining
the nature of the lesion following inoculation. In Jericho, Naggan et al.
(1 972) reported that vaccination against cutaneous leishmaniasis was much
in demand because of the high incidence of the disease and the high average
number of sores and scars in women. In six of the 40 girls, vaccination ulcers
showed signs of spreading.

TABLE4 Skin test reactions and total and specific IgE antibody levels by sex in
Venezuelan patients with cutaneous leishmaniasis (Lynch et al., 1987)

Skin test reaction Total IgE Specific IgE


Mean Percentage Percentage
Sex diameter (rnrn) > lOmm Log,, Mean positive
Male 12.0 57.9 3.447 3.71 20.0
Female 16.3 66.0 3.202 2.15 3.4

That females have a heightened cellular immune response to leishmania1


antigens was demonstrated by Lynch et al. (1987) in Venezuela, in an
outbreak of cutaneous leishmaniasis thought to be due to Leishmania
mexicana. Skin test reactions were significantly larger in females than males
(Table 4) and a greater proportion of females had a reaction which was
> 10 mm in diameter. Conversely, a higher proportion of males were found
to be positive for specific IgE antibody (20 vs. 3.4%), and mean levels of
20 L. BRABIN AND B. J. BRABIN

both specific and total IgE were significantly higher in males. The authors
suggested that elevated total IgE levels may be associated with deficiencies in
T cell function. The presence of detectable levels of specific IgE antibody
seemed to depend on the cellular immune reactivity of the individual rather
than on clinical status.

( e ) African trypanosomiasis. Several serological diagnostic tests have


been developed for gambiense and rhodesiense sleeping sickness and mass
surveys have been undertaken for gambiense infection. These studies con-
firm parasitological observations that infection rates are higher in women.
Table 5 shows seropositivity to an IFA test by age and sex in three endemic
areas in the Congo (vector:Gfossina pafpafispafpafis)(Frtzil, I98 I). From
the age of 20 years onwards, more women were seroprevalent than men in
Mbomo and Ngabe, which are established foci of infection, whereas in
Niari, where infection rates were low, seroprevalence was similar for adults
of both sexes. In the two endemic villages clinical symptoms were also more
frequent and severe in females. Exposure was said to account for these
differences, but this was not clearly demonstrated. In a village in the Congo,
where 1536 of 1700 inhabitants were examined, seroprevalence by IFA test
was 65% with a significantly higher positivity rate in females (Noireau et af.,
1988). In Bender state, Nigeria, in 670 volunteers tested by the card
agglutination test, seroprevalence was also higher in females (Edeghere et
af., 1989). Most of the infections diagnosed by IFA test in Zaire and the
Congo were not confirmed parasitologically and were considered to be
asymptomatic or silent infections (Frezil, 1981; Henry et af., 1981).

C. EFFECT OF SEX ON THE HOST IMMUNE RESPONSE TO CHEMOTHERAPY

Recent studies using the S . rnansoni-mouse system indicate that the schisto-
somicidal compound, praziquantel, may depend for its efficacy upon the
humoral immune status of the host (Brindley and Sher, 1987; Flisser et af.,
1989; Piper et al., 1990), although it is not yet known whether immune-
facilitated drug action is also a feature of schistosomiasis in humans
(Mitchell, 1990) nor how far it is directed against a limited subset of
antigens. Immunosuppression is known to reduce the efficacy of experi-
mental chemotherapy for several parasitic diseases, including malaria (Lwin
et af., 1987), trypanosomiasis (Frommel, 1988), onchocerciasis (Bianco et af.,
1986) and visceral leishmaniasis (Iwobi et af., 1991). Clinical studies have
suggested a link between poor efficacy of chemotherapy against severe
cerebral malaria and low levels of antimalarial antibody (Doenhoff et af.,
1991). Difficulties in treating diseases like diffuse cutaneous leishmaniasis
may reflect poor T cell immune responsiveness to parasite antigens (Doen-
hoff et al., 1991).
TABLE
5 Seroprevalence (indirectjuorescent antibody test) by age and sex in three areas of Gambian sleeping sickness in Congo ( F r k d .
1981)

Age group (years)


04 5-9 10-19 2 20 Total
Sex No." Percentage No. Percentage No. Percentage No. Percentage No. Percentage
Mbomo
Males 63 3.2 104 5.7 108 3.7 166 6.0 441 5.0
Females 73 4.1 104 1.9 101 4.0 224 1 5.6b 502 8.8'

Ngabe
Males 178 2.8 252 8.3 376 8.0 40 1 12.7 1207 8.9
Females 216 2.8 26 1 6.1 382 11.8 463 20.7b 322 12.3b

Niari
Males 809 0.7 969 0.9 121 1 4.3 I687 5.9 4676 3.5
Females 805 0.1 933 1.4 I189 2.2b 2232 5. I 5159 3.0

a Number examined; xz test of significance.


P < 0.01.
P < 0.025.
22 L. BRABIN A N D B. J. BRABIN

The sex of the host may affect the immune response to chemotherapy and
this may be expressed both in relation to toxicity (or adverse reactions) and
efficacy. This issue is difficult to address on the basis of current knowledge
since drug trials systematically exclude female subjects because of pregnancy
risks. There are also few experimental data available and, although Goble
and Konopka (1973) demonstrated the influence of sex on chemotherapy in
several systems, the finding that some drugs were more effective in males and
others in females could not be explained. Some observations on Indian kala-
azar are suggestive of a poorer treatment response in males. Post kala-azar
dermal leishmaniasis (PKDL) is more commonly observed in males (Acton
and Napier, 1927-1928; Napier and Das Gupta, 1931-1932). In 1000
consecutive cases of PKDL seen in Calcutta, 80% were males in the second
and third decade of life (Sen Gupta, 1956). In one study of the Bihar
epidemic (India) a large number of cases had previously received inadequate
treatment and all but two responded when managed on controlled thera-
peutic regimens (Aikat et al., 1979). Table 6 shows that the male:female
ratio of cases presenting without previous treatment was similar. Of patients
giving a history of treatment, most were males. This might suggest either
that males were more likely to discontinue treatment or that females
responded better to therapy. From Table 7 it is seen that the number of
untreated males and females was similar in all age groups, indicating that the
discrepancy in treated patients was not due to any one age category of
females failing to present for treatment.

TABLE
6 Male :female ratio of kala-azar cases in Bihar, India, grouped in relation to
previous treatment and method of diagnosis (Aikat et al., 1979)

Treatment history Diagnosis Male :female ratio


Untreated Bone marrow 33: 30
Treated Bone marrow 23:7
Untreated Clinical/aldehyde test 25:23
Treated Clinical/aldehyde test 6:2

TABLE
7 Age and sex distribution of treated and untreated kala-azar cases in Bihar,
India (Aikat et al., 1979)

Age group (years) 0-10 11-20 21-30 > 30


Sex" M F M F M F M F
Treated 10 3 16 3 3 3 - -

Untreated 33 31 12 11 9 6 4 5

M , male; F. female.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 23

In summary, the pattern of infection for a number of parasitic diseases is


not the same in males and females. For some, the differences are observed in
parasite rates, but in others the spectrum of clinical disease is different.
Differentials cannot be accounted for by exposure alone since there are many
situations where exposure, as demonstrated by immunological indices, is
judged to be similar for both sexes, but where females-at least non-
pregnant females-appear to be at a biological advantage. One exception is
African gambiense trypanosomiasis, where infection rates may be higher in
women of reproductive age, suggesting increased susceptibility or loss of
asymptomatic status during pregnancy.
In the next section, the role of sex hormones and genetic influences on sex
differentials are reviewed.

Iv. EVIDENCE
FOR SEX DIFFERENCES
ATTRIBUTED
TO HORMONAL
AND
GENETICFACTORS

Results of experimentation in vitro suggest several immune mechanisms


through which sexual dimorphism may be expressed and some of these
studies are summarized below. How these mechanisms operate in relation to
specific parasitic antigenic challenge has been little explored-partly because
sex differentials have not been systematically investigated and partly because
immunity to many parasitic diseases is, in general, insufficiently understood.
Several authors suggest that hormonal factors account for sex differences to
helminthic infections (Raccurt et af., 1988; Remme et al., 1989) or that
pregnancy-associated mechanisms may be operative which reduce infection
in women of reproductive age in comparison to men of the same age group
(Brabin, L., 1990a). For protozoal infections it is now known that primi-
gravidae are more susceptible to malaria, but the mechanisms involved are
not understood, although some authors have suggested that hormonal
changes during pregnancy are responsible. Serum glucocorticoid levels have
been observed to increase linearly during gestation and to be significantly
higher in primigravidae with a patent infection than in multigravidae
(Vleugels et al., 1987). Women with patent infections exhibited higher
serum values before, during and after the patent episode (Vleugels et al.,
1989), and this was taken to indicate a causal relation between cortisol and
loss of immunity during infection. Other authors postulate that resistance to
malaria in multigravidae is the result of immune stimulation during the first
pregnancy (see Section V).
Because mechanisms of sexual dimorphism in humans are poorly under-
stood, in Section A below consideration is given firstly to studies in v i m
which indicate general immunological mechanisms through which sexual
dimorphism may be expressed, and secondly to animal studies in which
24 L. BRABIN AND B. J. BRABIN

immune mechanisms to specific parasitic diseases are experimentally mani-


pulated.

A. GENERAL IMMUNE MECHANISMS CONTRIBUTING TO SEXUAL DIMORPHISM


IN HUMAN STUDIES

In females the humoral immune response is more active than in males


(Grossman, 1989). Circulating levels of the major immunoglobulin classes
(IgG, IgM and IgA) are higher in females than in males of the same age and
physiological condition (Butterworth, M. B. et al., 1967). In a study of three
Gambian villages, Rowe et al. (1968) found that mean IgM levels in males
ranged between 56% and 83% of female means in the age group 10-50
years. Females have been noted to mount higher antibody responses to polio
(Ainbender et al., 1968), Escherichia coli (Michaels and Rogers, 1971), and
microorganisms such as measles (Patty er al., 1976), rubella (Spencer et al.,
1977), brucella (Rhodes et al., 1969a) and hepatitis B (London and Drew,
1977).
Females are reported by some authors to have decreased cell-mediated
immunity compared to males (Inman, 1978). In one study measuring
effector cell activity in a system for antibody-mediated cell-dependent
immune lympholysis, no difference was found in activity between males and
females c 12 or >63 years old. At 16-49 years, females had significantly
lower effector levels than males of the same age or females >63 years old
(Kovithavongs et al., 1974). Oestrogen receptors have been reported to be
present on OKT-8 (CD-8, Ts/Tc) cells (Cohen et al., 1983) and oestrogen
treatment can suppress or deplete the functional activity of human T cells in
vitro (Paavonen et al., 1981). In females, T-suppressor cells may be less
active than in males, which may explain the elevated immunoglobulin levels
in females compared with males. Down regulation of antigen-specific T cell
reactivity and stimulation of B cell function may increase the risk of
autoimmune disease but protect against virulent infection. Alternatively, the
effects of sex steroids at the level of the thymic (and possibly splenic)
epithelium can modulate the release of thymic hormones, influencing effec-
tor lymphocyte function (Grossman, 1989) since lymphocytes undergoing
blastogenic transformation possess receptors for oestrogens as well as
receptors for other steroids, growth hormone and prolactin. Adult effector T
cells may not possess sex steroid receptors although they have receptors for
glucocorticoids. Thus, while sex steroids regulate the development of lym-
phocyte classes, glucocorticoids may control the final outcome of their
response.
Others consider females to have a more active cell-mediated response than
males (Grossman, 1989), and this is further suggested by the reportedly
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 25

reduced incidence of tumours in females and better resistance against viral


and parasitic infections (Ansar Ahmed et af., 1985). This advantage is
modified during pregnancy when, to prevent rejection of foreign foetal
tissues, elevated levels of oestrogen and progesterone function to depress the
cell-mediated response. Natural killer cell activity is reported to be sup-
pressed in negative correlation with oestrogen levels (Gabrilovac et af.,
1988), and killer cell levels are significantly reduced during pregnancy and
significantly increased I month post partum (Asari et al., 1989). Natural
killer cell activity is also decreased during the peri-ovulatory period (Sulke et
al., 1985). It has been suggested (Shomer and Toder, 1990) that, at low
physiological levels, hormones such as progesterone, oestrogen, adreno-
corticotropin (ACTH), luteinizing hormone and prolactin may activate
mononuclear activity while, at high pharmacological levels, they are sup-
pressive. Pregnancy may also produce qualitative changes in serum levels of
antibody and complement such that the opsonizing ability of antibodies
from pregnant and non-pregnant women differ.
Grossman (1989) suggested that an underlying mechanism for sexual
dimorphism is the hormonal microenvironment during the early stages of
foetal life, which might promote differences in the development of immune
effector cells in later life. He summarized results of a number of studies
showing that sex steroids can act at the level of stem cells, pre-T and pre-B
lymphocytes, as well as on adult T cells and cells of the monocyte-
macrophage system, and that both foetal thymic tissue and foetal thymus
blast lymphoid cells contain receptors for oestrogen. If sexual dimorphism
results only from variations in the endogenous hormonal environment, it
should become apparent following puberty. If it is apparent in the pre-
pubertal period, this suggests that a genetic component is also operational.
The interaction of genetic and hormonal factors is likely to be complex.
This can be seen in relation to a non-parasitic disease-juvenile rheumatoid
arthritis, for which there is substantial evidence of onset before 5 years of
age. Grossman (1989) noted that there is clearly a human leucocyte antigen
(HLA) genetic component responsible, since some forms of the disease are
more prevalent in females, indicating that development of the disease
requires the disease-producing genotype. He suggested, however, that
expression of disease is brought about through hormonal interactions.
In the following section animal studies have been reviewed in some detail
because they give insight into possible mechanisms by which sex hormones
contribute to sexual dimorphism in the immune response to specific parasitic
infections. In addition, genetic studies have advanced in recent years and
complement earlier detailed reviews of experiments on sex differences in
animal models (Solomon, 1969; Goble and Konopka, 1973; Alexander and
Stimson, 1988).
26 L. BRABIN AND B. J. BRABIN

B. SPECIFIC IMMUNE MECHANISMS TO PARASITIC DISEASE CONTRIBUTING TO


SEXUAL DIMORPHISM IN ANIMAL STUDIES

1. Hormonal factors

( a ) Helmin thic infections

( i ) Strongyloides. In a number of studies manipulation of hormonal


levels has reversed the animals’ normal state of susceptibility. Larval output
in the faeces of male C57BL/6 mice infected with S. ratti was greater than
that from females but could be markedly reduced by orchidectomy. Ovari-
ectomy had no antiparasitic effect in female mice but testosterone treatment
of both orchidectomized males and normal females increased their suscepti-
bility to infection (Kiyota et al., 1984).

( i i ) Lymphatic Jilariasis. Experiments with jirds (Meriones unguiculatus)


have shown non-pregnant females to be less susceptible to infection than
male animals (Ash, 1971). It was suggested that resistance to Brugiupahangi
infection derived from the absence of testicular lymphatics, a preferential
anatomical site for the development of adult filariae. Significant microfilar-
aemias occurred only in animals harbouring considerable worm burdens in
the testes. Recently it has been demonstrated that testosterone has an
important regulatory role in susceptibility of C57BL/6 mice to B. pahangi,
similar to that reported for jirds (Nakanishi et al., 1989a). In orchidecto-
mized male mice the percentage of worms recovered was significantly lower
than that from control males and similar to that from female mice.
Ovariectomy of female mice had no marked effect on susceptibility. Perito-
neal cell responses showed a significant increase in the number of lympho-
cytes, macrophages and eosinophils in orchidectomized male mice, but no
effect in ovariectomized female mice.
In experiments to clarify the role of macrophages and eosinophils on the
expression of differential susceptibility to B. pahungi infection, Nakanashi et
al. (l989b) treated male and female mice with carbon to block macrophage
function and promethazine, a histamine type I receptor antagonist, to block
eosinophil function. The recovery of worms from the carbon-treated female
mice was 12-fold higher than that from controls and similar to that from
males. Promethazine treatment had little effect on the recovery rate of
worms, indicating that macrophages, rather than eosinophils, were express-
ing the sex differences in susceptibility to B. pahangi. Since macrophages have
several functions, it was not known whether there were sex differences in
macrophages functioning as effector cells in killing larvae, or as regulatory
cells through the production of interleukin 1 or colony-stimulating factor.
Although testosterone is clearly implicated as a suppressive factor which
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 27

increases male susceptibility, available evidence does not suggest that oestro-
gen increases resistance in females. A study by Wesley (1973) found that
resistance in females could be attributed to lack of androgen rather than
presence of oestrogen. In another study, older female jirds (retired breeders)
were found to be more susceptible than young female jirds (Devereux and
Ash, 1978). Declining oestrogen levels in old female jirds would be unlikely
to increase susceptibility. One interpretation of the data would be that
hormonal changes during pregnancy have an additional effect on adult
worms or depress parasitaemia. This would explain the higher parasitaemias
seen in post-reproductive jirds and does not conflict with the conclusion that
testosterone increases susceptibility. Although increased oestrogen levels
during pregnancy are immunosuppressive, the role of progesterone, levels of
which are also increased during pregnancy, is not fully understood.

( i i i ) Schistosomiasis. Increased male susceptibility and mortality have


been noted in acute schistosomal infections (Solomon, 1969). The import-
ance of hormonal factors in modulating helminth infection has been demon-
strated by Knopf (1982) in rats, animals which are normally insusceptible to
schistosomiasis. Delay in elimination of juvenile worms and maturation of
juvenile to adult worms in rats were two consequences of thyroidectomy,
supporting the hypothesis that host hormones can interfere with completion
of the S. mansoni life cycle. Since thyroid hormone and growth hormone
(GH) are thought to enhance synergistically the proliferative activity of
lymphoid tissue, a mediatory role for G H was thought possible. It is
noteworthy that sex differences in the pattern of GH secretion have been
observed in adult rats (Grossman, 1989), which might contribute to differ-
ences in worm survival between male and female hosts. It has been
suggested that, in females, oestrogens and progestins are responsible for a
female pattern of elevated basal G H release accompanied by reduced
pulsatile G H release while, in males, the reverse pattern of G H release is
present. Differences in male and female G H secretory patterns have been
shown to alter steroid metabolism in the liver (Mode et al., 1982).
For helminthic infection, apart from the effect of hormones on the
acquired immune system, testosterone is thought to increase dermal collagen
content, making cutaneous tissue more susceptible to penetration by third
stage larvae. This has been interpreted as an action-modulating natural or
innate immunity (Alexander and Stimson, 1988).

(6) Protozoal infections

(i) Cutaneous leishmaniasis. A number of studies indicate that hormonal


factors influence the response to cutaneous leishmaniasis. Male mice devel-
oped non-healing ulcerated lesions (Alexander, 1988) or showed signifi-
28 L. BRABIN A N D B. J. BRABIN

cantly higher liver parasite burdens (Mock and Nacy, 1988) than females. In
the latter study testosterone treatment of female BALB/c mice resulted in an
88% increase in the number of liver amastigotes. It was suggested that
testosterone modulation of L. major infection could be due to a direct effect
of the hormone or to an indirect effect on cell-mediated immunity-over and
above susceptibility due to genetic factors. Exacerbation of infection with
Leishmania mexicana amazonensis in hamsters treated with testosterone was
also observed by Arcay ( 1 985). In one set of experiments using gonadecto-
mized mice with some males having silastic tubing implants containing
oestrogen, it seemed that oestrogen may determine the comparatively
greater resistance of female DBA/2 mice to L . mexicana (Alexander, 1988).
Other experiments, by contrast, showed relatively greater resistance to
infection with L. major in male mice (De Tolla et al., 1981; Giannini, 1986;
Alexander, 1988). Conflicting results may be the result of variations in
experimental procedure. Gonadectomy, for example, reduces sex hormone
levels, but does not totally deplete sex hormones due to compensation by
extra-gonadal tissue (Ansar Ahmed et al., 1985).

( i i ) T. cruzi. Resistance to T. cruzi depends on the phagocytic capacity of


macrophages for intracellular amastigotes. Nicol et al. (1965) have shown
that when the reticuloendothelial system of mice is stimulated by oestrogen,
body defences are increased, as demonstrated by increased phagocytosis. In
a study by Kierszenbaum et al. (1974), parasitaemias in mice in which the
reticuloendothelial system had been stimulated by diethylstilbestrol were
considerably lower than those in a control group and the survival time of the
stimulated mice was longer. This contrasted with experiments by Goble and
Konopka (1973), in which the course of T. cruzi infection was not altered
in mice receiving diethylstilbestrol in amounts compatible with hormone
therapy during the 2 weeks after infection.

2. Hormonal changes induced by pregnancy

Pregnant hosts show several alterations in the immune response, and


hormonal factors which are not present in the non-pregnant state have been
considered to relate to the immunosuppression of pregnancy. Although
most attention has been paid to immunological mechanisms, non-immuno-
logical factors may also be important. This has been emphasized for a non-
parasitic infection in a study by Kita et al. (1989) on typhoid infection of
mice. This study suggested that non-specific mechanisms, such as soluble
serum factors, are affected by hormone action, and the authors quoted
results from their studies of gonoccocal infection demonstrating that the
effect of interleukin 1 on thymocyte cells was suppressed by oestrogen. For
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 29

mouse typhoid they suggest that progesterone enhances non-specific resist-


ance by increasing the influx of peritoneal cells after infection, while
oestrogen affects the acute inflammatory response. It is probable that
parasitic infections in the pregnant host are also modulated by a series of
hormonal changes, most of which still need to be delineated.
In humans, plasma corticosteroids are raised in late pregnancy. Most of
this increase is in the protein-bound fraction because there is a three-fold
increase in corticosteroid-binding globulin. Unbound cortisol levels are also
raised (Smith et al., 1980), as are ACTH levels (Carr et al., 1981). In
pregnancy, ACTH levels were found to be lower than those in normal
ovulatory women, and this suggested a direct suppression of ACTH by
oestrogens and/or progesterones. It has been suggested that plasma cortico-
steroid levels regulate the effector part of immunity against several pathogens
since they decrease the number, as well as the activity, of circulating
lymphocytes and monocytes. Van Zon et al. (1982) suggested that there was
a relation between the level of plasma corticosterone and the loss of malaria
immunity in the murine model. Mice that lost immunity during pregnancy
exhibited higher plasma corticosterone levels than those with persisting
immunity, whereas adrenalectomy before pregnancy not only blocked
maternal corticosterone production but also prevented loss of immunity.
Given that spontaneous recrudescences are rare in non-pregnant immune
mice, these results indicated a pregnancy-associated suppression of effector
function of immunity. In mice, recrudescence of parasitaemia occurred in
association with elevated corticosterone values towards the end of preg-
nancy. In human malaria, however, increased parasite rates are observed
early in pregnancy (Brabin, B. J., 1983; Brabin, B. .I. et al., 1990a).

3. Genetic factors

(a) Importance of innate or natural resistance. Attempts to explain sexual


dimorphism on the basis of sex chromosone genes have been equivocal.
IgM, but not IgG, has been correlated with the number of X chromosomes
in both animal and human studies (Rhodes et al., 1969b; Grundbacher,
1972). However, for other authors, such an association was not readily
apparent (Adinolfi et al., 1978). The following examples of genetic mechan-
isms influencing susceptibility concern protozoal infections.

( i ) African trypanosomiasis. The role of sex chromosome genes has been


explored for trypanosomiasis, a disease in which parasitaemias in mice are
usually lower in females. Pinder (1984) found C57BL/6 mice of both sexes to
be partially resistant to T. congolense, but parasitaemias were higher in
males. When inheritance of resistance was investigated, female mice showed
30 L. BRABIN AND B. J. BRABIN

the same segregation as males, but the range of parasitaemia was always
about 2 log,, values lower in females, except when the F1 generation was
back-crossed to BALB/c, when parasitaemias in both male and female
progeny were indistinguishable. Greenblatt and Rosenstreich (1 984) also
showed that the longer any strain of mice survived, the greater was the
difference in survival time between male and female mice exposed to
infection with the rhodesiense form of T. brucei. Their investigations
suggested that an X-linked gene could not account for the differences
observed between resistant (C57BI/6) and susceptible (BALB/c) mice, but
the mechanism responsible was not identified.

(ii) T. cruzi. Sex-related differences in survival from infection with T.


cruzi have been observed, dependent upon the mouse strain and parasite
isolate used (Hauschska, 1947; Chapman et al., 1975). In one study (Rivera-
Vanderpas et al., 1983) involving F344 rat hosts, which are highly suscept-
ible to T. cruzi and experience high parasitaemias, all males died while all
females survived with the total disappearance of parasitaemia. Wrightsman
et al. (1984) considered that survival was influenced by genes outside the H-2
complex although Trischmann (1983) found that the magnitude of differ-
ence between male and female survival in BALB/c mice depended signifi-
cantly on the H-2 haplotype. Among female mice, mortality ranged from
14% to 96% according to the H-2 locus present.

( b ) Hormonal modulation of genetic background. Various mouse strains


differ in their sensitivity to hormone action. It has been suggested that
susceptibility to oestrogen- and testosterone-mediated depression of the
delayed hypersensitivity response are inherited as dominant traits, although
this may not apply to oestrogen-mediated enhancement of the antibody
response (Carlsten et al., 1989).
Genetic differences in testosterone and complement levels are associated
with the mouse H-2 system, and the H-2’ end of the H-2 locus has been
implicated. An influence of the major histocompatibility antigens (MHC) H-
2 locus on the amount of oestrogen receptor in the mouse uterus has been
described (Palumbo and Vladutiu, 1979) and could imply a genetic influence
on such mechanisms as the oestrogen-enhanced clearance of antibody-
coated cells by splenic macrophages. Although the hormonal system may be
influenced by the HLA haplotype, several studies indicate that the H-2
system is modulated by the hormonal system with a selective effect on the
lymphoid cell population (Ivanyi et al., 1972). Antigen presentation capabi-
lity is thought to be superior in females. Presenting spleen cells obtained
from normal female mice or from female mice with testosterone implants
have been compared to those of normal or castrated male mice. It was found
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 31

that cells from male and androgen-treated female mice presented a soluble
antigen (KLH) less efficiently than cells from normal female or castrated
mice (Weinstein et al., 1984). In conjunction with evidence that lymphocytes
from female mice are more reactive than those of male mice to cell-
associated allo-antigens, it seems that there are major sex-associated differ-
ences linked to functions known to be regulated by MHC-encoded glyco-
proteins.

( i ) Malaria. In murine malaria, the influence of sex on host resistance to


Plasmodium has been ascribed to the superior erythropoietic system in
female mice (Stevenson et al., 1982). More recently, it has been suggested
that the expression of genes controlling resistance are modulated by sex
hormones (Wunderlich et al., 1988). Castration of male mice increased their
survival, but female mice became more susceptible to P . chabaudi following
testosterone treatment. Increased survival was considered to be dependent
on three factors: mouse strain (controlled by non-H-2 genes) and the H-2
complex genes, which in turn are affected by testosterone. The mechanism
by which survival was improved by testosterone was unknown since testos-
terone did not accelerate growth and multiplication within the erythrocytes.
Others (Stevenson and Skamene, 1985) suggested that female mice belonging
to susceptible strains compensate for genetic susceptibility (exhibited as
fulminant parasitaemia and minimal splenomegaly) by preventing the conse-
quences of overwhelming shock.

(ii) Cutaneous leishmaniasis. Failure of female DBA/2 mice to develop


ulcerating lesions following infection with L. mexicana suggested a predis-
posing genetic or hormonal mechanism (Alexander, 1988). Resistance to
leishmaniasis is thought to be under genetic control, and the expression of
resistance in females suggests the action of sex hormones on the gene
product.

( c ) The strength of the antigenic challenge. Schuurs and Verheul (1990)


have noted that another mediating factor in the relationship between sex
hormones and the immune reaction is the strength of the antigenic challenge.
Thus, if hormonal influence is based on a “minor” genetic trait, a strong
immunogenic challenge would hardly be affected by sex hormones.

v. EVIDENCE
FOR SEX DIFFERENCES
RESULTING
FROM IMMUNE
STIMULATIONDURING HUMANPREGNANCY

The effect of parity on maternal immunity to infection has not been


systematically investigated, although there is evidence that infection during
32 L. BRABIN AND B. J. BRABIN

pregnancy increases maternal immunity (Brabin, B. J., 1985a). This principle


may be particularly relevant to chronic parasitic infections that may relapse
or recrudesce during pregnancy and is one explanation for lower prevalence
figures for parasitic infections or their complications in females of child-
bearing age compared to males. There is some evidence to support this
suggestion for P. falciparum infection.
Among adult women with established immunity to malaria who are living
under holoendemic conditions, multigravidae are less susceptible than primi-
gravidae to P. fakiparum recrudescences (Brabin, B. J., 1983). The lower
prevalence in multigravidae may depend on the acquistion of age-dependent
immunity or the development of parity-specific immunity. The short inter-
pregnancy period in these populations makes it unlikely that age-dependent
immunity is a sufficient explanation for the observed parity difference in
malaria prevalence. Van Zon el al. (1985) have demonstrated that a
previously existing malaria immunity to P. berghei in mice is improved after
pregnancy and depends on reinforced anti-parasitic immune reactions devel-
oping in relation to a pregnancy-dependent immunosuppressive period.
Table 8 shows the mean specific IFA titres for pregnant women screened
at their first antenatal visit in western Kenya, grouped by parity and age
class (Brabin, B.J., 1984). Age was accurately known because of birth
registration and mission records in the study area. A regression linear-parity
model fitted to these data with age and parity as factors shows a significant
parity effect (P < 0.05). Williams (1973) has suggested that malaria infection
in pregnancy provides an opportunity to develop antibodies to antigens
unique to the placental form of the parasite. Late pregnancy sera obtained
from the subjects whose IFA titres are reported in Table 8 showed enhanced
recognition of schizont-specific polypeptides of P. fakiparum compared to
recognition patterns observed for sera collected early in gestation (Brabin, B.
J. and Perrin, 1985).

VI. PARASITIC
INFECTION
AND PREGNANCY
OUTCOME

Monitoring the level of parasitic infection in non-pregnant females is


important because virtually all women in developing countries, where these
diseases are endemic, will become pregnant. Sexual dimorphism itself can be
regarded as a mechanism for ensuring that women are better prepared for
the physiological stress of pregnancy, which in turn should assure repro-
ductive success (Grossman, 1989). This advantage is required because
women marry young and child-bearing starts in adolescence when infection
rates for many parasites are high. Parasitic infection in pregnancy may lead
to maternal malnutrition and deterioration in the disease state of the
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 33

mother, cause pre-term delivery and intrauterine foetal growth retardation,


placental and perinatal infection, and possibly long-term effects on infant
immunity.

TABLE 8 Mean age specific indirect fluorescent antibody titres (log,,) by age and
parity in pregnant women at jirst ante-natal visit in western Kenya (Nangina)

Parity group
Age group (years) P O P 1-3 P 4-6 >P 6
< 18 3.31 f 0.08" 3.54 f 0.90 - -

(Wb (7)
19-20 3.49 f 0.50 3.60 f 0.50 - -

(27) (27)
21-22 3.1 1 3.42 f 0.57 4.01 -
(2) (22) (1)
23-24 2.80 3.77 f 0.62 3.81 -
(3) (12) (3)
25-26 - 3.24 f 0.70 3.01 -

(7) (3)
> 26 - 3.33 f 0.40 3.70 & 0.62 3.68 0.58
(6) (14) (12)

Standard deviation.
Numbers in parentheses are number of patients screened.

A. MATERNAL MORBIDITY

Several important parasitic infections in pregnancy have not been


thoroughly investigated, and their effects on maternal morbidity are difficult
to assess. Prevalence studies at delivery or in relation to gestational age are
available for P.fulcipurum malaria only. Case reports and hospital studies,
some of which have been summarized by Macleod (1988), give no indication
of the epidemiological importance of these infections and their compli-
cations in women living in endemic areas. Anaemia in women has a high
prevalence in less developed countries (Royston, 1982) and must be con-
sidered one of the more serious maternal complications of several parasitic
diseases in pregnancy.

1. Helminthic infections

Acute maternal helminthic infection is rare in endemic areas, and although


chronic helminth infections like schistosomiasis and filariasis may cause
34 L. BRABIN A N D B. J. BRABIN

peritonitis, pelvic inflammatory disease and infertility (Mcfalls and Mcfalls,


1984), such conditions are considered unusual. Kain and Keystone (1988)
reported a patient with recurrent hydatid disease (Echinococcusgranulosum)
during pregnancy, to whom a normal child was born. Pregnant women also
suffer occasionally from disseminated strongyloidiasis (Benirschke and
Kaufmann, 1990).
There are several interactions between intestinal helminthic infections and
nutritional status (Stephenson and Holland, 1987). Ancylostoma duodenale
and Necator americanus increase iron loss leading to anaemia; S. haemato-
bium causes haematuria (Malimood, 1966), and polyparasitism and chronic
infection can have cumulative haematological and nutritional consequences,
frequently resulting in a high prevalence of iron deficiency anaemia in
nulliparae. Infections of hookworm, trichuris and schistosomiasis can, in
decreasing order of severity, induce anaemia. Wickramasuriya (1937), in a
consecutive series of 2384 patients, found the incidence of eclampsia to be
three times, and that of pre-eclampsia to be 18 times, more common in those
infected with hookworm. St George (1976) suggested a relationship between
hypertensive disease, anaemia and hookworm infection amongst parturients
in Trinidad and Tobago. Amenorrhoea is also reported as a consequence of
heavy hookworm infection and chronic anaemia (Crompton and Stephen-
son, 1990). Investigations of helminthic infections in selected groups of
pregnant refugees in Thailand, USA and England have not shown signifi-
cant morbidity or anaemia (Roberts et al., 1985; D’Alauro et al., 1985;
Constantine et al., 1988). The high levels of health care following migration,
nutritional rehabilitation and lack of re-exposure were thought to have
played a significant role in reducing maternal and perinatal complications in
one of these studies (Roberts et al., 1985).
The effect of pregnancy on onchocercal dermatitis is currently being
studied in Ette, Anambra state, Nigeria, following observations which
suggested severe and rapid exacerbation of skin lesions with increasing
gestational age in two young, anaemic women (U. Amazigo, personal
communication). Fig. 3 shows skin lesions with severe papular and pustular
eruptions in one of these women at 24 weeks gestation. At 16 weeks, this had
been a localized, mild dermatitis. At 32 weeks gestation, pachydermia of the
skin around the buttocks and shoulder area was apparent, with further
degeneration of the dermatitis. Deterioration in onchocercal dermal lesions
could arise from an accumulation of eosinophil proteins in tissue surround-
ing parasites (Mackenzie et al., 1987), resulting from altered macrophage
function during pregnancy. Steward (1987) has suggested that a low-affinity
antibody response in malnourished patients favours the production of
antigen-antibody complexes and type I11 hypersensitivity reactions.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 35

FIG. 3. Onchodermatitis in a pregnant woman at 24 weeks gestation.

2. Protozoal infections

There is'convincing evidence that amoebiasis due to Entamoeha histolytica is


more severe during pregnancy (Abioye, 1973), with many cases in tropical
countries probably resulting from the reactivation of infection. Enteric
blood loss may be an important complication. There is a single case report
from Kenya of an exacerbation of cutaneous leishmaniasis due to L . tropica
during pregnancy (Mibrahtu et al., 1988).
36 L. BRABIN A N D B. J. BRABIN

Chronic infections with T. cruzi are not associated with severe morbidity.
Polyhydramnios and varicose veins were complications observed in sero-
positive pregnant women in Argentina (Hernandez-Matteson et al., 1983).
African trypanosomiasis is traditionally regarded as a severe and acute
infection in pregnancy, although it is now apparent that asymptomatic
seropositive women may have uncomplicated pregnancies (Lapierre and
Coste, 1963). African trypanosomiasis is frequently associated with anaemia
as a result of immunological mechanisms or splenomegaly, as is visceral
leishmaniasis.
Severe anaemia in pregnant women living in malarious areas is associated,
in several reports, with increased risk of pre-term delivery and perinatal
mortality. These reports have been summarized by B. J. Brabin (1991a).
Chronic splenomegaly is an important complication of malaria in women
living under holoendemic conditions (Topley, 1968; Brabin, B. J. et al.,
1988). Spleen rates of 25% are not uncommon in these areas and in coastal
Papua New Guinea spleen rates in women of over 50% have been reported
by several investigators over the past 40 years (Metselaar, 1956; Brabin, L.,
1988). Table 9 shows the relation between haematological indices and spleen
size in pregnant women from rural Madang, Papua New Guinea. Anaemia
increases with increasing spleen size, as does red cell folacin concentration.
The high red cell folate concentration may be related to the chronic
reticulocytosis of malaria, as reticulocytes have high concentrations of
folate, although this may not be the only mechanism to explain this
observation (Brabin, B. J., in press). The higher free erythrocyte protopor-
phyrin and lower mean corpuscular haemoglobin concentration with
increasing spleen size suggest that iron deficiency increases with the develop-
ment of splenomegaly. Iron deficiency is almost universally present in
malaria-endemic areas and may be causally associated with malaria
(McGregor, I. A., 1988).
During pregnancy in malaria-endemic areas, spleen rates and size increase
(Brabin, B. J. et al., 1988). This has important consequences for maternal
and foetal health. In view of the increased requirements for iron and folate
during pregnancy, the additional burden of splenomegaly can result in the
progressive development of iron deficiency and haemolysis with advancing
gestation. In Madang, Papua New Guinea, the prevalence of severe anaemia
( < 8 g/dl) in the last trimester was 44% in primigravidae and 29% in
multigravidae for those who had no antenatal care (Brabin, B. J. et al.,
1990b). Such women with severe anaemia have an increased mortality risk
during pregnancy, especially if associated with post parturn haemorrhage or
puerperal sepsis. Other complications of malaria in pregnancy include
hypoglycaemia and cerebral malaria, although under holoendemic con-
ditions these are very uncommon. Renal insufficiency is a rare complication.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 31

The risks and severity of malaria in pregnant women are discussed in detail
by B. J. Brabin (1991a).

TABLE9 Haematological indices and spleen size in pregnant women in rural Madang,
Papua New Guinea

Spleen size (cm)a

0 1-3 4-6 >6

Haemoglobin (g dl-') 8.9 f 1.5 8.6 f 1.5 8.4 f 1.4 8.3 f 1.8
(1 26) (55) (35) (30)
Haematocrit (YO) 29.6 f 4.3 29.4 f 4.7 28.3 & 5.3 28.3 f 4.7
(120) (52) (27) (27)
Free erythrocyte proto- 34.2 f 15.4 31.5 f 12.2 37.2 f 16.4 40.3 f 21.2
porphyrin ( l g dl- ') (1 20) (51) (35) (28)
Mean corpuscular haemo- 30.5 f 3.5 29.4 f 2.2 30.2 5.0 28.9 f 3.1
globin concentration (g dl) (1 19) (52) (27) (27)
Red cell folacin 1641 f 744 1821 f 1067 2340 f 842 2245 f 685
(nmol 1- l ) (63) (36) (17) (14)
Reticulocytes (YO) 2.1 f 2.0 1.8 f 2.5 1.9 f 1.7 3.1 f 4.0
(52) (22) (16) (16)
~~

Spleen size, distance palpable below costal margin. Numbers in parentheses are numbers of
a
patients examined (women examined at first antenatal visit).

B. FOETAL GROWTH RETARDATION

Recent evidence suggests that foetal growth retardation is an important


consequence of some parasitic infections during pregnancy in less developed
countries and contributes significantly to the problem of low birth weight
and the risk of perinatal death. Placental involvement following infection
with several parasites is described in Section V1.C.

1. Helminthic infections

In a prospective study of 14914 pregnant women in Guatemala, 20% of


this urban, clinically healthy, working class population were found to have
helminthic infections, predominantly Ascaris lumbricoides (14.5%) and Tri-
churis trichiura (3.9%) (Villar et al., 1989). Mothers infected with helminths
tended to have lower pre-pregnancy weight and height and a higher
-=
prevalence of anaemia (haemoglobin 11 g/dl). There was a significant trend
of increased foetal growth retardation when more than one species of
38 L. BRABIN AND B. J. BRABIN

parasite was present, from 20.5% in uninfected women to 23.2% with two or
more species. Short, presumably malnourished, women had a significantly
greater risk of foetal growth retardation. As there is suggestive evidence that
malnutrition influences the pathogenicity of intestinal parasites in humans
(Crouch, 1982; Crompton, 1989, these findings support the hypothesis
that foetal growth retardation is related both to parasitic burden and to
nutritional status.'

2 . Protozoal infections

( a ) Entamoeba histolytica, Giardia lamblia, Trichomonas hominis. In the


Guatemalan study by Villar et al. (1989), these three infections were
associated with foetal growth retardation. E. histolytica increased the risk of
foetal growth retardation among women of short stature, as did Giurdia
among underweight mothers. The increased risk associated with T. hominis
was considered unusual since this parasite is thought to be non-pathogenic.
Together, helminthic and protozoal infections were considered to account
for 10% of foetal growth retardation among chronically malnourished
women (maternal height 1.47 m or less).

( b ) T. cruzi. Foetal growth retardation was found in offspring of preg-


nant mice infected with T . cruzi (Carlier et al., 1987). Confirmation of this
association in human studies is still required.

( c ) Malaria. There are several studies which demonstrate that placental


malaria is associated with a reduction in birth weight and that this effect is
greatest in primigravidae (Brabin, B. J., 1983; McGregor, I. A., 1984). The
relative risk of low birth weight associated with primiparity has been shown
to correlate significantly with the malaria parasite rate at delivery (Brabin,
B. J., 1991b). There is some evidence that the increased incidence of foetal
growth retardation associated with placental malaria may relate more to the
severity of maternal anaemia than to a direct effect of placental parasitiza-
tion. Studies in Africa, India and Papua New Guinea have reported that
anaemic women are at greater risk of delivering low birth weight babies
(primarily due to foetal growth retardation) (McGregor, M. W., 1963; Ojo,
1965; Harrison and Ibeziako, 1973; Reinhardt, 1978; Osuhor, 1982; Oppen-
heimer et al., 1986; Bhargava et al., 1987; Fleming, 1989; Brabin, B. J. et al.,
1990b). Similar observations have been made in industrialized countries
where malaria is not endemic (primarily due to pre-term delivery) (Lieber-
mann et al., 1988). Recent evidence from Papua New Guinea indicates that,
under holoendemic conditions for malaria, it is principally in primigravidae
that anaemia is associated with low birth weight (Brabin, B. J. et al., 1990b).
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 39

In this study, 65% of babies were low birth weight (<25OOg) and born to
primigravidae anaemic at booking ( < 8 g/dl). Of these, three-quarters were
growth retarded and the rest were premature. This association of maternal
malaria anaemia and low birth weight will be influenced by the prevalence of
iron deficiency. There is very little information on the effect of maternal iron
deficiency on outcome of pregnancy in these anaemic populations. Maternal
iron deficiency has been associated with an increased risk of low birth weight
in a non-malarious area (Whiteside et a!., 1968).
Table 10 shows data from the Papua New Guinea study (Brabin, B. J. et
al., 1990b) which illustrate how the risk ratio for low birth weight increases
in primigravidae with decreasing levels of maternal haemoglobin. In this
same population, women with evidence of iron deficiency at booking (free
erythrocyte protoporphyrin (FEP) > 35 pg/dl) had a significantly increased
risk of delivering a low birth weight baby (relative risk 4.1, 95% confidence
limits 1.2-14.0). Maternal anaemia and iron deficiency were not associated
with increased risk in multigravidae. The population attributable risk of low
birth weight in primiparae associated with anaemia at booking in this
population would be 43.5% (haemoglobin < 9 g/dl) and 50.0% (haemo-
globin < 8 g/dl). The population attributable risk of low birthweight associ-
ated with iron deficiency at booking in primigravidae (FEP >35pg/dl)
would be 55.9%.

TABLE10 Relative risk for low birth weight at Alexishafen, Papua New Guinea, in
primiparae according to maternal haemoglobin at booking and delivery

Relative risk'
At booking At delivery
Haemoglobin ( g dl- ') (n = 48) (n = 71)

<11.0 0.33 (0.03-3.67) 0.90 (0.27-3.00)


< 10.0 0.89 (0.24-3.25) 1.64 (0.624.31)
<9.0 2.48 (0.768.1 1) 2.39 (0.90-6.35)
c 8.0 3.67 (1.09-12.36) 2.63 (0.87-7.94)

'Numbers in parentheses = 95% confidence limits.

C. INFECTION OF THE FOETUS AND NEWBORN INFANT

In areas endemic for parasitic infections, prevalence of these infections is


sometimes high in young infants. Some of these infections are acquired
perinatally rather than in utero. The placenta generally serves as an effective
barrier, although recent evidence for malaria indicates that while congenital
40 L. BRABIN A N D B. J. BRABIN

symptomatic malaria (i.e., in utero transmission) is rare, cord parasitaemia


(perinatal transmission) is not uncommon. Where possible, the use of the
term congenital should indicate whether infection was acquired in utero or
perinatally. Foetal pathology present in the newborn clearly indicates that in
utero infection has occurred. For asymptomatic infants, whether trans-
mission was in utero or perinatal is often uncertain.

1, Helminthic infections

The importance of in utero or perinatal transmission as a survival strategy


for helminthic parasites in animal experimental systems has been reviewed
by Shoop (1991). In this strategy, incoming larvae are arrested at the
development stage (hypobiosis) and during pregnancy or lactation, probably
due to hormonal stimulus, they reactivate and advance to the uterus and
mammary glands. In humans, vertical transmission has been less readily
demonstrated, and hormonal changes in pregnancy may, in contrast, serve
to inhibit vertical transmission.

( a ) Onchocerciasis. Robles (1919) found an onchocercal nodule on a 2


months old child, and Strong et al. (1934) saw six children aged from 4
months to 1 year with nodules. Since the prepatent period is considered to
range from about 7 months to 2 years, it is not easy to account for these
observations, although female worms, without fertilization, can probably
induce the formation of small nodules even though the patient does not have
microfilariae (WHO, 1987). One of the two pregnant women studied by U.
Amazigo (personal communication) (Section VI. A. 1) gave birth to an
infant who, at 9 weeks old, showed evidence of pruritis-the earliest
symptom of onchocercal dermatitis-on one thigh, and whose skin snip was
positive for microfilariae. Although Brinkmann et al. ( 1976) suggested that
congenital infection was common, this conclusion has not been accepted by
other authors. Prost and Gorim de Ponsay (1979) argued that infection in
utero was rare in the Upper Volta and was not related to maternal
microfilarial load. Their study took place in an area where transmission had
been interrupted by vector control, and re-infection before or early in
pregnancy could not occur.

( h ) Other helminthic infections. Cort (1921) reported cases of infection in


utero with hookworm and S. japonicum, and noted the difficulties of finding
schistosomes by placental sectioning and the failure of physicians to look
systematically for infection in very young infants. He was surprised that
congenital infection did not occur more frequently, given the migratory
phase of a number of helminths. More recent cases of placental schisto-
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 41

somiasis have been described by Sutherland et al. (1 965) and Bittencourt et


al. (1980), but these authors also experienced difficulties in finding eggs by
histological sectioning. Using sodium hydroxide digestion to examine 322
placentas, Renaud et al. (1972) found schistosome eggs in 72. In the four
cases studied by Bittencourt et al. (1980), there was no evidence of foetal
involvement. Placental parasitization resulted in three still births, and in the
fourth case the apparently normal infant died at 3 months without an
autopsy being performed. None of these studies indicated under what
epidemiological conditions placental infection is likely to occur.
Intra-uterine death due to hookworm infection was commonly observed
by Wickramasuriya (1937). Of 648 consecutive still births examined, hook-
worm accounted for 34%. The immediate cause of death appeared to be
placental insufficiency and not placental infection. Reports from areas where
A . duodenale is endemic suggest that human lactogenic hookworm infections
occur (Schad, 1990). Evidence for lactogenic infection with N . americanus is
thought to be lacking (Schad, 1990), although there is a single report of
transmammary transmission (Setasuban et al., 1980). Infants may be
infected at an early age with S. fuelleborni (Barnish and Ashford, 1989),
although the examination of milk has only rarely revealed the presence of
Strongyloides (Brown and Girandeau, 1977). Infection in utero with A .
lumbricoides has been reported (Chu et al., 1972). Trichinella spiralis is
present in the placenta of women with acute trichinosis and can be passed to
the infant by means of breast milk (Salzer, 1916). Four T . spiralis larvae
were found in the diaphragm of a foetus by Kuitunen-Ekbaum (1941).
Invasion of the embryo by Enterobius vermicularis was described by
Mendoza et al. (1987).

2. Protozoal infections

( a ) African trypanosomiasis. African trypanosomiasis is regarded as an


acute condition in pregnancy which gives rise to abortion and still births
(Duboz, 1984). On the basis of experiments in which pregnant mice were
infected, Loke (1978) considered abortions to be most frequent when
infection occurred early in pregnancy and to result from febrile attacks in the
mother, not from transplacental infection. Although sleeping sickness can be
severe in pregnancy, there is increasing evidence from areas endemic for the
gambiense infection that infected women can carry apparently normal preg-
nancies through to term delivery. This is illustrated in Tables 11-13, which
relate cases of congenital transmission to the clinical state of the pregnant
mother. Asymptomatic mothers are those who may have had mild, non-
specific indicators of disease, such as headache, but who otherwise remained
well throughout pregnancy. Mildly symptomatic mothers had symptoms
TABLE
1 1 Review of cases of children congenitally infected with African trypanosomiasis born to asymptomatic mothers"

Case Maternal age


no. Reference Parasite Country (years) Comments
1 Kazyumba et al. gam biense Zaire 23 Diagnosed 5 days before delivery. Treatment
( 1 978) postponed. Mother and child blood slide and
IFAT positive. Cord positive. Child untreated
for 6 weeks at which time serum IFAT was
1/80 and C S F positive, but no clinical
cerebral involvement
2 Chambon gam biense Cameroun 28 Mother interrupted treatment 3 years before.
(1933) Child presented at 5 days, apparently well.-
Treated and remained well 4 years later
3 Lingham et al. gambiense Zaire/ - Mother infected in Zaire; delivered in London.
(1985) London Parasitaemia and antibody negative till para-
sites found in bone marrow 2 years later.
Child untreated till motor development
ceased at 18 months. Severe mental retar-
dation
4 Burke et al. gambiense Zaire 25 Mother diagnosed in routine blood survey on
( 1974) plantation at delivery. Normal CSF. No
lymphatic enlargement. Baby appeared
normal but blood slide positive
5 Woodruff et al. gambiense Zaire/ - Mother lived in Zaire, delivered in England.
( 1 982) England Mother diagnosed 3 years post-natally with
no symptoms. Child first showed evidence of
mental retardation at 2 years. Mother appar-
ently did not infect a second child born 2
years after the congenital case

a An infection is termed congenital if the infected infant of an infected mother had never been in an endemic area, or if the parasite was found in the

newborn within 5 days of birth.


Abbreviations: CSF. cerebrosuinal fluid: IFAT. indirect fluorescent antibodv test.
TABLE
12 Review of cases of children congenitally infected with African trypanosomiasis born to mildly symptomatic mothersa

Case Maternal age


no. Reference Parasite Country bears) Comments
6 Sina et al. gam biense Cameroun 22 Mother symptomatic near end of pregnancy. No
( 1979) CSF involvement in mother or child at birth.

7 Sina et al.
( 1979)
gam biense Cameroun - 30 Both treated and well 7 months later
Fever at 5 months gestation, but normal pro-
gression of pregnancy. Diagnosed and-treated
at 8 months. Twins born with cords and peri-
pheral blood films positive. Treated and
apparently well 4 months later
8 Darre et al. gambiense Chad/Cameroun -
b
Infection in expatriate mother at about 6 months
( 1 937) France gestation, with a febrile event. Febrile epi-
sodes increased in frequency post-natally.
Child severely mentally retarded from birth
9 Triolo et al. gambiense Cameroun 25 Febrile episodes during pregnancy associated
(1985) with trypanosomiasis and malaria. Child born
with hepatomegaly. Mother and child treated
with good response
10 Kalanda gambiense Zaire 25 Mother diagnosed with first stage infection at 8
(1991) months gestation. Twins born, apparently
well. Thick smear of twin I positive at birth,
but serological results in twin 2 not positive
till 6 weeks. Mother and twins were treated
and yielded normal results at 6 months

a See footnote a to Table 11.


Primigravida.
TABLE13 Review of cases of children congenitally infected with Ajrican trypanosomiasis born to severely symptomatic mothers"

Case Maternal age


no. Reference Parasite Country (years) Comments
11 Lowenthal rhodesiense Zambia 25 Mother stuporous at 20 weeks gestation. Blood
(1971) smear positive. Cerebrospinal fluid (CSF) in-
volvement. Mother moved from endemic to
non-endemic area before pregnancy and his-
tory suggested she was asymptomatic at start
of pregnancy. Treated while pregnant and
child apparently healthy at birth
12 Traub et al. rhodesiense Zambia 31 Mother previously interrupted treatment. Symp-
(1978) toms at 30 weeks, Caesarian section at 36
weeks. Mother died. Child not treated till 30
days post-natally. Parasites in CSF. Immu-
noglobulin M absent. Child responded to
treatment. No comment on whether mentally
retarded
13 Sina et al. gambiense Cameroun 20 Clinical symptoms several months before deliv-
(1979) ery. Mother's condition poor and child died
in neo-natal period
14 Triolo et al. gambiense Cameroun 25 Post-natal (3 days) admission to hospital in
(1985) weak condition. Child blood slide and CSF
positive, in convulsive state and died. Mother
responded to treatment
15 Triolo et al. gambiense Cameroun 28 Mother presented with clinical symptoms at
(1985) delivery. Died when treatment started. Child
born with heavily infected CSF and died at 3
days of age

a See footnote a to Table 11.


PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 45

associated with early disease such as headache, giddiness, general malaise,


asthenia, lack of appetite and febrile attacks, but were ambulant. Severe
symptoms were those associated with the late-stage disease. Some women
may remain asymptomatic for many years; such cases have been docu-
mented by Lapierre and Coste (1963) and I. A. McGregor (1987). Pro-
gression to a symptomatic condition during pregnancy seems to be associ-
ated with other factors. In case no. 12 (Table 13), for example, the mother
had previously interrupted her treatment and this may have precipitated a
relapse during pregnancy.
The view that congenital infection is rare has been challenged by investi-
gators in Cameroun where six hospital cases of congenital transmission were
found between 1968 and 1984, which is almost as high as the total number of
cases ever cited elsewhere (Triolo et al., 1985). The low overall number is
partly due to a number of other suspected congenital cases which could not
be proved because infection was not parasitologically diagnosed in the first 5
days of life (Aubert, 1915; Van der Branden, 1934; Daveloose, 1934). To
concentrate attention on classification of cases is likely to obscure the public
health significance of asymptomatically infected mothers, who give birth to
apparently normal babies in whom neurological sequelae and illness may
not develop until the second year of life. This is clearly illustrated by cases
nos 3 and 5 (Table ll), whose mothers delivered in England. Had these
children been delivered in an endemic area, congenital transmission would
have been denied because the relationship between maternal and childhood
infection cannot be established retrospectively. Moreover, in these two
examples, it was several years before infection of the mothers could be
confirmed, even serologically. In contrast, when maternal infection was
severe, as in cases nos 14 and 15 (Table I3), cerebrospinal fluid involvement
of the infants was evident at birth. Table 14 shows that both serologically
and parasitologically diagnosed infection rates were higher in infants aged
CL24 months than in children 2 4 4 8 months old in Congo and Zaire (Frezil,
1981; Henry et al., 1982), which may indicate the proportion of congenitally-
infected infants as well as that of children infected after birth.

( b ) Visceral leishmaniasis. Congenital transmission of visceral leishmani-


asis from non-immune mothers has been demonstrated in experimental
animals (Nuwayri-Salti and Fallah Kansa, 1985) and, rarely, in humans
(Low and Cooke, 1926; Banerji, 1955; Nyakundi et al., 1988; Yadav et al.,
1989). Transmission is presumably due to parasitized leucocytes crossing the
placenta. In three well-documented congenital cases it is interesting to note
that infection with kala-azar during pregnancy did not necessarily lead to
severe illness in the mother. One Kenyan mother, infected early in preg-
nancy, delivered prematurely, but it was her tenth pregnancy and she had a
46 L. BRABIN A N D B. J. BRABIN

history of abortions (Nyakundi et al., 1988). The child remained in hospital


and was not diagnosed with kala-azar until 4 months of age. An Indian
mother with a double-peaking fever from the sixth month of gestation had
an uneventful delivery and recovered spontaneously afterwards (Yadav et
al., 1989). The child was found to have kala-azar at 8 months of age.
Another mother, infected in India and delivering in England, was ill
throughout pregnancy but delivered at term (Low and Cooke, 1926).
Although sickly from 2 weeks after birth, the spleen of the baby was not
enlarged at 6 months and did not become markedly enlarged till 10-12
months of age.

TABLE14 Gambiense sleeping sickness in children aged 0-4 years assessed by indirect
fluorescent antibody test in the Congo and parasitologically in Zaire (Frbzil, 1981;
Henry et al., 1982)

Age group (months)


0-24 24-28
Region No. examined Percentage No. examined Percentage
Congo Mbomo 36 8.3 100 2.0
Ngabe 61 4.9 333 2.4
Zaire Kwamouth 101 3.0 237 1.7

Depending on endemic conditions and the level of acquired immunity in


women, infections in pregnancy must occur. If maternal infection resolves
without specific treatment, and splenomegaly in the child has a late onset,
recognition of congenital cases may be difficult, and they could occur more
frequently than at present realized. Hindle (1928) considered that congenital
infections were not infrequent in Chinese kala-azar. He reported a child,
diagnosed with “spleen disease” by a local practitioner at 2 months of age,
who had gross splenomegaly and a positive spleen puncture at 4 months. He
did not consider that the child could have been exposed to infected sandflies
because the transmission season had ended 2 months before the child was
born. The child’s mother had no obvious clinical infection. Hindle (1928)
suggested that what was locally described as “milk spleen” was infantile
kala-azar.

( c ) T. cruzi. It is noteworthy that most cases of congenital infection with


T. cruzi have been reported as occurring in offspring of women who had not
lived in an endemic area for some years (Azogue et al., 1985; Bittencourt et
al., 1985aJ. In Bahia, Brazil, studies of congenital cases have been largely
based on samples and autopsy data from urban hospitals where a high
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 47

proportion of patients originated from rural areas but had lived for a
number of years in the city. The migration history of the mother of one
congenital case is probably similar to that of many urban women: she moved
to an urban area at 13 years of age, lived for a few years in an area of low
transmission, and finally moved to a non-endemic area where, 9 years later,
aged 33 years, she gave birth to congenitally-infected twins in her seventh
pregnancy (Hoff et al., 1978). The occurrence of congenital infection in
urban areas is of concern because transmission can occur by vertical
infection even in areas where vector control has interrupted direct trans-
mission through triatomine bugs although, if congenital infection occurs
only sporadically, this may not represent a serious threat to control. The
incidence of congenital infection has been estimated to vary between 2% and
10% (Bittencourt et al., 1985a). There is little information on its frequency
in endemic areas, but confirmed cases appear to be rare (Mott et al., 1976).
Although there is little evidence to question the assumption that immunity
to T. cruzi infection is lifelong, relevant information on how frequently
persons exposed to infective metacyclic forms of the parasite under natural
conditions are infected is not available. Nor is it known whether immune
response mechanisms in infected individuals living in non-endemic areas
differ from those living in places where exposure to reduviid vectors is
common, and whether this would alter maternal immunity during preg-
nancy.
The factors leading to congenital infection have never been clearly
delineated. Bittencourt et al. (1988) have suggested that parasitaemic
episodes can occur repeatedly during pregnancy. Of 77 women chronically
infected with Chagas disease, on whom xenodiagnosis was performed during
pregnancy, 10 ( 1 1 YO)had three or more positive diagnoses, and one of these
mothers gave birth to a congenitally-infected baby. It was considered that
the more frequent or persistent the parasitaemia, the more probable was
congenital infection. Whether parasitaemic episodes occur more frequently
in pregnant than in non-pregnant women requires further investigation. In
this study the same patients were taken as a control group after delivery, and
no significant difference in incidence rates of parasitaemia was observed.
These women were urban residents and ideally the results of the study need
to be compared with those of pregnant and postnatal women delivering in
an endemic area.
Besides frequency of parasitaemia, virulence of the infective strain,
duration of parasitaemia (a characteristic of different strains), gestational
stage when parasitaemia occurs, maternal age, and parity are also likely to
influence the timing or severity of congenital infection. Azogue et al. (1989,
in Bolivia, found that most congenital cases occurred in newborn infants
weighing 100&2500g, with a gestational age of 26-37 weeks. In a series
48 L. BRABIN A N D B. J. BRABIN

studied by Bittencourt (1988) in Brazil, the highest proportion of congenital


cases was found at 19-26 weeks gestation in conceptuses weighing c 1000 g.
Bittencourt et al. (1985b) showed that enzymatically indistinguishable
strains of T. cruzi could behave differently in respect to degrees of severity of
infection and prognosis. Several congenital cases have been reported in
which the child was believed to have been infected late in pregnancy, since
IgM was found in the serum within a few days of delivery. In these cases the
babies were asymptomatic (Szarfmann et al., 1975; Bittencourt et al., 1985a).

( d ) Malaria. The passage of malaria parasites across the placenta has


been demonstrated for all species of human malaria (Covell, 1950). Congeni-
tal malaria with peripheral parasitaemia may be symptomatic or asympto-
matic and, in a proportion of cases, only cord parasitaemia occurs.

( i ) Symptomatic congenital malaria. Babies who acquire malaria in utero


may be stillborn with evidence of intense placental infection, with parasites
in the foetal spleen and brain (Ballantyne, 1902; Wickramasuriya, 1937).
These findings suggest that transplacental infection occurs chiefly with dense
placental infection or in untreated cases of severe P . falciparum infection. In
those cases with intra-uterine transmission, the causes of intra-uterine death
relate to massive infection of the placenta, persistently high fever and,
possibly, a failure of successful placentation. Miscarriage early in pregnancy
is well documented. Epidemiological analysis of still birth and miscarriage
rates (reviewed by Brabin, B. J., 1991a) suggests that malaria during
pregnancy in semi-immune women may have an important effect in increas-
ing the risk of still birth, particularly in primigravidae. Late foetal infection
may result in symptomatic infection of the newborn, with jaundice in the
first 24-48 hours. Splenomegaly is a characteristic feature of these cases, a
fact noted by Laveran (1902).
Kortmann ( 1972) summarized reports for semi-immune African women
and in none of these was the peripheral blood infection rate in the babies
greater than 0.7% at birth. This indicates that symptomatic malaria from
infections in utero must be very uncommon in endemic areas. Clinical
episodes of malaria in perinatally-infected babies may be delayed until
several weeks after birth (Thompson et al., 1977). Transmission is thought to
relate to mechanical damage of the placenta at delivery. In endemic areas,
these infections would be masked by the high attack rates of primary
infection.

( i i ) Asymptomatic perinatal malaria. Recent studies have documented


that cord parasitaemia occurs more frequently than formerly realized. High
cord infection rates ( 1 50/,-55?h) have been reported by several investigators
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 49

(Le Van Hung, 1951; Kortmann, 1972; Reinhardt et al., 1978; Marshall,
1983; Ezeoke, 1985). Infants born to these mothers are at higher risk of low
birth weight (Kortmann, 1972), although this may be because a higher
proportion were primigravidae. In such cases, parasitaemia appears to clear
rapidly or within a few weeks of birth (Kortmann, 1972; Diallo et al., 1983)
and symptomatic malaria does not occur. Several studies have shown a zero
cord infection rate despite high malaria prevalence in mothers. The reasons
for this are unknown.

D. FOETAL AND INFANT IMMUNITY

I. Helminthic infections

( a ) Schistosomiasis
( i ) Passive immunity. Maternal transfer of antibody, resulting in the
capacity to resist challenge by a primary infection with cercariae, has been
demonstrated in newborn rats (Knopf and Coghlan, 1989). In human
studies, only a proportion of children born to mothers with detectable
schistosomal antibodies are antibody positive. In the West Indies, no
antibodies to S. mansoni were found in half of the newborn children of
seropositive mothers (Lees and Jordan, 1968). Of 31 seropositive newborn
infants, antibodies had waned by the age of 6 months. In Burundi, the
presence of schistosomal circulating antigens (CSA) in 24 of 26 cord sera
was associated with a lack of significant antibody titres in the newborn
infants (Carlier et al., 1980). It is not known how children with or without
detectable antibodies at birth differ in their subsequent immune response to
schistosomiasis.

( i i ) Foetal sensitization. In Burundi, Carlier et al. (1980) found CSA to


S. mansoni in 24 of 25 maternal and cord sera, and this was significantly
different from the prevalence in the control group of men and non-pregnant
women of the same age range (18-36 years) and from the same area (or
family). No significant difference was found between men and women in the
control group. No detail of the level of endemicity or parity of mothers was
given. The authors suggested that such high CSA levels could be related
to the depression of the S. mansoni humoral response during pregnancy,
although pregnancy-associated depression of humoral immunity is not
observed in several other infections (Brabin, B. J., 1985a). An alternative
explanation might be a hormonal effect on, or in association with, altered
cell-mediated immunity.
In Burundi, it was also observed that controls had significantly higher
levels of schistosomal circulating immune complexes than mothers or
50 L. BRABIN A N D B. J. BRABIN

newborn infants and it is possible that the placenta acts as a site for
dissociation of antigen-antibody complexes (Loke, 1982). Foetal exposure
to free or bound schistosomal antigens would influence the degree of antigen
exposure and might influence the subsequent development of either toler-
ance or sensitization. Both these effects have been reported for schisto-
somiasis in human (Lewert and Mandolitz, 1969) and animal studies (Uhr et
al., 1957; Hang et al., 1974).
Children (aged 7-36 months) of infected mothers were studied in Brazil
for delayed hypersensitivity reactions to S. mansoni antigens (Camus et af.,
1976). Delayed hypersensitivity was detected in about half the children. In a
later study sensitization was demonstrated in 17 of 25 newborn children of
mothers with positive delayed skin reactions to S. mansoni (Tachon and
Borojevic, 1978). Camus et al. (1976) considered that delayed hypersensiti-
vity reactions indicated sensitization to CSA. Neither of these studies
defined maternal characteristics or the epidemiological conditions under
which sensitization occurred, although the second study took place in an
area where transmission of S. mansoni had been interrupted 4 years pre-
viously. Sensitization may also have occurred postnatally from antigens in
breast milk, although this was not investigated.

(iii) Postnatal sensitization. Early references have been made to infection


in humans with S. juponicum following suckling (Fujinami and Nakamura,
191 1; Narabayashi, 1914). M antigen was identified in six of 24 milk samples
from Brazilian mothers infected with S. mansoni (Santoro et al., 1977). The
precipitating band was never found in normal milk, nor in the sera of normal
or infected mothers. Two hypotheses were proposed to explain its presence:
either free M antigen is derived from blood and is detected in milk after
concentration in the mammary gland, or M antigen may be complexed in the
blood circulation with specific antibodies. Dissociation of these immune
complexes may occur in the mammary gland or during the process of milk
delipidation.
A study of delayed hypersensitivity responses in the infants of 35 mothers
infected with S . mansoni, of whom 25 (group A) were breast-fed and 10
(group B) bottle-fed, confirmed sensitization in 14 infants from group A and
two from group B (Eissa et al., 1988). Some of the group B infants had been
breast-fed for short periods.

(6) Filariasis

( i ) Passive immunity. Filaria-specific maternal antibodies are known to


cross the placenta and can be detected in cord sera (Dissanayake et al.,
1980); they may persist for up to 6 months (Dutta et al., 1976). It is not
known whether these antibodies are protective.
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 51

( i i ) Foetal sensitization. Unlike the situation in schistosomiasis, filarial


antigens have not been demonstrated in human cord blood or infant sera,
and in animal experiments B. pahangi antigen was not detected in sera from
offspring of infected mothers (Weil et al., 1990), although this may have been
due to the high molecular weight (105-1 10 kDa) of the antigen identified. It
is noteworthy that the transfer of soluble microfilarial antigens could not be
demonstrated in mothers suffering from two other filarial infections-loiasis
and mansonellosis-in Gabon (Van Hoegaerden and Akue, 1986).
In contrast, antifilarial antibodies of isotypes which do not normally cross
the placenta (IgM and IgE) have been found in cord sera (Dissanayake et al.,
1980; Weil et al., 1983), which suggests that offspring of filaria-infected
mothers are sensitized immunologically. The epidemiological conditions to
which the mothers were exposed are unclear, but in the Sri Lankan study
(Dissanayake et al., 1980) it appeared that some mothers may have made
only short visits to endemic areas and were not necessarily resident there. In
studies such as this, it is important to clarify whether mothers are living
under conditions where continuous transmission of larval parasites occurs,
since such factors may affect the ability to detect antigen in the presence of
antibody, especially if the amount of antigen released is small. It is thought
that when high levels of antigen production exist-resulting in antigen
excess-free antigen and/or small persisting immune complexes (with
unbound epitopes) are likely to be present in the circulation (Harnett et al.,
1990). In addition, antigenaemia at delivery may not reflect antigen circu-
lation in early pregnancy.

( i i i ) Postnatal sensitization. In a hospital study of 123 pregnant women


in Andhra Pradesh, India, 5% of the women had microfilaraemia (Rao et
al., 1984). Microfilariae were found in the cords of four babies of 12 positive
mothers who delivered at night, using a Nuclepore@ concentration tech-
nique.
Petralanda et al. (1988) detected parasite antigens in seven of 13 milk
samples from Venezuelan women infected with 0. volvulus. A non-competi-
tive ELISA was used with any of five different monoclonal antibodies raised
against 0. volvulus, B. malayi and W , bancrofti, all of which react with 0.
volvulus antigens. Levels of microfiladermia correlated with antigen levels in
breast milk. With the methods used it was not possible to ascertain whether
the epitope detected by monoclonal antibody 10 was present in milk in the
form of immune complexes with excess free antigenic epitopes, or whether
parasite components in breast milk were, in fact, anti-idiotypic antibodies.
In vitro, breast milk from these donors suppressed the response of mono-
nuclear cells to mitogens, but also activated a population of non-specific
suppressor cells. Some mitogens appeared to be of parasite origin. Parasite
components could induce either non-specific suppression or specific toler-
52 L. BRABIN AND B. J. BRABIN

ance to onchocercal antigens. The authors suggested that the existence of a


state of immune tolerance could explain why clinically apparent systemic
immune complex disease is rare in onchocerciasis patients, in whom high
levels of immune complexes are present in sera.

2. Protozoal infections

( a ) Malaria

( i ) Passive immunity. A recent review of malaria parasite rates in infants


and passively acquired immunity to malaria has drawn attention to evidence
for the lack of efficacy of passively acquired maternal antibody in reducing
susceptibility to malaria infection in infants (Brabin, B. J., 1990). Studies of
age-specific patterns of malarial antibodies in infants indicated that antibody
prevalence was markedly reduced by antimalarial drug use, whereas in
African infants unprotected from malaria the prevalence of antibody to P.
fulciparum decreased slightly after birth, but not to below about 70%
seropositivity. Because the attack rate is so high in holoendemic areas, it
seems that children mount their own immune response early in infancy,
indicated by the increase in antibody titres in the first few months of life
(Brabin, B. J., 1990). High parasite and spleen rates occur in the first few
months of life in many endemic areas (if drug use is low) and it seems
probable that malaria recovery rates in infancy are not constant, but are
governed by immunological mechanisms which operated during pregnancy
and in utero. These differences may determine whether a child develops
clinical disease in infancy or childhood.

( i i ) Foetal sensitization. In individual case reports of congenital malaria,


increased concentrations of antigen-specific IgM have been observed in
neonatal blood collected a few weeks after delivery, and malaria-specific
IgM has been detected at the time of birth following a primary infection in
late gestation (Thomas and Chit, 1980). Prenatal sensitization to malaria
antigens, but without congenital parasitaemia, would be expected to result in
a foetal immune response (Brabin, B. J., 1985b). This may be due to the
transplacental passage of soluble malaria antigen (Druilhe et al., 1976).
However, I. A. McGregor (personal communication), using the same simple
immunodiflusion technique, could not demonstrate soluble malaria antigens
in the peripheral blood of a large number of babies 3-24 hours old who were
born to mothers with heavy injections and with detectable soluble malaria
antigen in their blood.
Sub-clinical foetal splenomegaly may also occur, although the evidence
for this in humans is indirect (Bruce-Chwatt, 1956; Corkill et al., 1989). A
direct effect on spleen weight in the offspring of infected pregnant mice has
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 53

been reported (Oduola et al., 1982). In a series of studies by Desowitz (1973)


and Desowitz and Raybourne (1985) in rats and by Harte and Playfair
(1983) in mice, evidence for prenatal immune priming to malaria was
reported. The studies by Harte and Playfair (1983) demonstrated that
offspring of infected female mice were specifically refractory to vaccination
and that specific maternally-derived IgG interfered with protection from
challenge infections with P . yoelii. These results contrast with those of
Desowitz (1973) and Desowitz and Raybourne (1989, who demonstrated
enhanced responsiveness to vaccination in offspring of female rats infected
with P . berghei. These differing results may relate to differences in the timing
of the maternal infection or the amount of specific passively-acquired
maternal IgG, which may act to block priming. Recent studies on lympho-
cytes obtained from cord blood of Papua New Guinean and Gambian
newborn infants suggested that they had been primed in utero (Desowitz,
1988; Greenwood, 1991).

( h ) T . cruzi

( i ) Passive immunity. Prenatal transfer of maternal antibodies to T, cruzi


has been demonstrated. Amongst 5 1 seropositive mothers in Bahia, Brazil,
21 (46%) had seropositive infants (Miles et al., 1975). This was in contrast
to the report of Breniere et al. ( 1 983), in which transmission of antibodies
occurred for all infected mothers. No evidence yet shows that infants with T.
cruzi antibodies are passively protected.
In animal models, immunity transmitted from mother to young in T.
Iewisi and T. cruzi infections is thought to stem from milk (Culbertson, 1939;
Kolodny, 1939). Miles (1972) demonstrated significant protection in young
suckled by mothers recovered from the acute phase of infection with the
Peru strain of T. cruzi. In contrast, young mice fed by females suffering from
the primary phase of infection had no demonstrable antibody levels and
were very susceptible. Trypanosomes occurred frequently in milk from
infected mice during the primary acute phase, but in low numbers. Bitten-
court et al. (1988) did not observe milk parasitism in urban women with
Chagas disease, although five mothers were parasitaemic at the time of
sample collection. Milk or colostrum (56 single samples) was collected and
22 mothers were sampled twice. The samples were usually collected in the
first month post partum, but a few were not collected until several months
post partum. These authors concluded that children of chronically infected
mothers were unlikely to become infected during breast feeding.

( i i ) Foetal sensitization. The suggestion that intra-uterine infection with


T. cruzi induces the synthesis of antibodies to foetal heart muscle (Szarfman
et al., 1975) is now considered doubtful. However, sensitization in utero was
54 L. BRABIN A N D B. J. BRABIN

reported by Eloi-Santos et al. (1989), who demonstrated that cord blood


mononuclear cells from children born of three mothers infected with T. cruzi
exhibited strong proliferative responses against idiotypes expressed on
antibodies with specificity for T. cruzi epimastigote antigens. They predicted
that idiotypes expressed on transferred maternal antibodies could influence
the development of morbidity in children. The characteristics of those
mothers in whom transplacental transfer of antibodies occurs may relate to
the risk of sensitization.
Family clustering of infection has been noted in Castro Alves, Brazil,
where 10% (20/203) of households had five or more seropositive individuals.
The rate of seropositivity and median age of seropositives varied according
to the serological status of the parents (Mott et al., 1976). The authors
suggested that the immunological response of children of seropositive
mothers differed from that of children of seronegative mothers, and that
various maternal-foetal immunological interactions might account for this.
Family clustering of cardiovascular disease was also observed in Goitna,
central Brazil (Zicker et al., 1990). An association between a sibling history
of heart disease and left anterior hemiblock was considered consistent with
studies suggesting a genetic component in the determinant of Chagas heart
disease, but could also indicate maternal-foetal sensitization. Longitudinal
studies in Castro klves also indicated that the risk of development of heart
disease followed a bimodal pattern, with peaks at age 10-19 years and 30-39
years (Mota et al., 1990). The authors suggested that some individuals
develop cardiac abnormality after a long latent period. Although there is no
evidence at present to support an explanation in terms of early clinical
disease manifestation following foetal sensitization, such a possibility cannot
be excluded.

( c ) African trypanosomiasis

( i ) Passive immunity. Antibodies have been detected in animals born to


infected mothers. Offspring of uninfected rats who were fostered by females
immunized against the gambiense form of T. brucei had high titres of
agglutinating and phagocytosis-promoting activities in their sera, but were
not protected against a challenge infection (Takayanagi et al., 1978); some
delay in the onset of parasitaemia and extended survival were observed.
Offspring, of immune females, who were infected after birth and then
received colostrum from control females, showed low agglutinating and
phagocytosis-promoting activity in their sera, but 50% were protected, as
shown by delayed parasitaemia and extended survival time. It was concluded
that IgG antibodies passing through the placenta of immunized females were
more effective than IgA antibodies from colostrum, in which no comp-
lement-dependent activity was demonstrable. These results conflict with
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 55

those of Whitelaw and Urquhart (1989, who demonstrated that young mice
suckled by mothers infected with T. brucei were immune to homologous
trypanosome challenge. The immunity was considered to be transmitted in
colostrum or milk, since mice born of infected mothers and transferred at
birth to normal foster mothers were susceptible to challenge. It is difficult to
relate these observations to human infection because placentation in rodents
is of the haemoendothelial type and the majority of immune transfer from
mother to offspring occurs after birth (Brambell, 1970).
In humans the amount of maternal antibody available to the neonate
depends on the time available for production and placental transfer and,
therefore, on the gestational time at onset of primary or recurrent infections
in pregnancy (Brabin, B. J., 1985a). Transplacental immunity is thought to
be less protective against infections caused by organisms eliciting maternal
antibodies that are predominantly of the IgM class (Miller and Stiehm,
1983), and Loke (1978) has observed that maternal protective antibodies
may not be able to penetrate the blood-brain barrier and prevent invasion of
the cerebrospinal fluid. IgM antibodies may be more effective in the control
of peripheral parasitaemia than IgG (Campbell et al., 1978), but less
effective than IgG class antibodies in reaching tissue parasites (Sacks et al.,
1980). It has been suggested that there are, in human sera, natural trypano-
cidal IgM antibodies which account, at least in part, for the occurrence of
natural immune defence mechanisms in the refractory host. Trypanocidal
activity and agglutinins to T. equiperdum could not be detected in cord blood
or newborn sera, but were present in the sera of individuals from 6 months
of age till late in life (Verducci et al., 1989). This was noted to be in
accordance with the significant appearance of IgM at 4 6 months after birth.
These observations suggest that the neonate may be relatively unprotected
from infection during early infancy.

( i i ) Foetal sensitization. No information is available on the risk of


parasite antigens reaching the foetus, and little is known of the long-term
prognosis for children born to asymptomatic seropositive mothers.

( d ) Leishmaniases

( i ) Passive immunity. Transplacental passage of maternal antibodies to


the newborn offspring has not been studied, and most evidence suggests that
antibody does not play a primary role in control of leishmania1 infection,
although an antibody-mediated mechanism may contribute to the develop-
ment of acquired immunity (Scott er al., 1986; Sacks er al., 1987; Saravia er
al., 1989). None the less, it cannot be assumed that passively acquired
maternal antibody has no protective function for the neonate. It is note-
worthy that a new form of visceral leishmaniasis has emerged in the
56 L. BRABIN AND B. J. BRABIN

reclaimed desert areas of Xinjiang autonomous region in China, which is


characterized by a short incubation period (2-3 months), is seen predomi-
nantly in children less than 1 year of age, and is considered to be a zoonosis
(Xu Zhi-Biao, 1989); 20% (46/225) of cases occurred in infants 1-6 months
old, suggesting an absence of passive immunity. This may be because adult
immunity in this newly established focus is not well developed. In other
provinces of China, the proportion of children infected at less than 1 year of
age, with either Mediterranean or Indian visceral leishmaniasis, ranged from
0.3% to 10.6% of cases.

( i i ) Foetal sensitization. Prenatal sensitization has been demonstrated in


experimental animal infection. Herman et al. (1982) observed that offspring
of female hamsters infected with L. donovani responded better to immuniza-
tion with amastigotes than did offspring of non-infected hamsters. Sensitiza-
tion through nursing could not be shown.
The aberration in cellular immunity which results in diffuse cutaneous
leishmaniasis (DCL) is suggestive of a state of immune tolerance, although
this explanation was not accepted by Bryceson (1970) because of the return
of cell-mediated immunity following treatment. Since cure of DCL presents
a major problem, immune tolerance, due to the congenital absence of the
lymphocyte clone specifically able to respond, or to induction of tolerance
following transplacental passage of antigen, remains a possibility. The fact
that cell-mediated immune responses are detectable for many years follow-
ing recovery from infection suggests that parasites, or antigens, persist for a
very long time (Modabber, 1989). This view is supported by the appearance
of cases of recrudescent leishmaniasis in patients suffering from human
immunodeficiency virus infection. Changes in cellular immunity during
pregnancy may increase the risk of parasite products crossing the placenta to
sensitize the foetus. In immunocompromised women, it may also increase
the risk of placental parasitization and congenital transmission.

VII. CONCLUSIONS

Loke (1982) noted that, before his review of the transplacental transmission
of parasites, the approach to this subject was concerned with the frequency
with which maternal infections affected the foetus and their pathological
consequences for the child. His own viewpoint was that of a reproductive
immunobiologist interested in the immune competence of the placenta and
the immunomodulation of the foetal response. The present review has
concentrated primarily on factors affecting immunity in both non-pregnant
and pregnant women and on the chronicity of several infections which are
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 57

carried through successive pregnancies with adverse consequences for


mother and child. It considers risks to women of reproductive age to differ
from men of equivalent years. Ensuring a reduction in the parasitic burden
of women of reproductive age is the first step in reducing foetal infection and
sensitization, and in influencing the pathological pattern of parasitic disease
in communities living in endemic areas.
The practical and research implications of considering women as a special
risk group for complications of parasitic infection are considerable, and
should lead to the development and implementation of improved control
strategies.

A. MATERNAL-CHILD HEALTH

Maternakhild health services have concentrated almost exclusively on the


prevention of obstetric complications. The control of infectious disease in
women must also be included, both to improve maternal health, especially to
correct anaemia, and to reduce perinatal and neonatal mortality. To do this
effectively health interventions will be necessary throughout a woman’s life
and not simply during pregnancy-although the antenatal and postnatal
periods represent times when women are most likely to come into contact
with the health services.
Assessment of the immunological profiles of mothers and their progeny is
needed. Using a raised cord serum IgM value as an indicator of congenital
infection is not sufficiently sensitive. Recently, Reyes et al. (1990) have
shown that, using recombinant deoxyribonucleic acid techniques, individual
antigens to T. cruzi can be used to detect IgG specificities in cord blood
which are shed during the acute phase of infection and are present in infected
newborn infants, but not in their mothers or in uninfected infants. Detection
of IgM and newly synthesized foetal IgG may be used to discriminate
congenitally-infected infants from non-infected ones-and such methods
could be used for other parasitic diseases.
Protection from clinical disease in children may differ from protection
from infection. For malaria this division has been described as antitoxic and
antiparasitic immunity (Playfair et al., 1990). It may be possible to dis-
tinguish these two forms of immunity by biochemical and biological charac-
terization of antibody profiles of mothers and their newborn children
(Greenwood, 1991).

B. VACCINE DEVELOPMENT

It is important to relate evidence for sex differences in immunity to research


on vaccine development for the following reasons.
58 L. BRABIN AND B. I. BRABIN

(i) The immune response of non-pregnant females to vaccination may be


different from that of males.
(ii) Vaccine efficacy may vary in children born to mothers with different
levels of acquired immunity because maternal immunity probably regulates
the response of offspring to parasite products. For example, a vaccine giving
partial protection against schistosomiasis now seems a feasible objective.
Most people exposed to natural cercarial challenge are born of schistosome-
infected mothers. Children born with antigen and/or anti-Id responsive T
cells may have established a regulatory suppressor immune response to
certain antigens of the organisms'with which the mother was infected (Colley
and Colley, 1989; Eloi-Santos et al., 1989). Foetal exposure may thus affect
an individual's response to eventual prophylactic immunization against that
organism. Vaccination development strategies need to consider the potential
differences that a given vaccine might have on endemic and non-endemic
populations, or on populations where immune responses have been altered
by prior vector control-and how these variations affect maternal immunity.
(iii) Protecting infants by vaccinating mothers may be the optimum
strategy for preventing perinatal and early childhood mortality and mor-
bidity associated with low birth weight and infection.

C. DRUG TREATMENT

The immune response of non-pregnant females to drug treatment may differ


from that of males.
Licensing of drugs for use in pregnant women becomes increasingly
difficult, and no medical treatment is generally recommended for asympto-
matic or minimally symptomatic patients with parasitic infections (Macleod
and Lee, 1988). IvermectinB at present cannot be administered to pregnant
women with onchocerciasis nor to women during the first 3 months of
lactation. Follow-up studies of babies of mothers inadvertently treated
during pregnancy detected no increased risk of miscarriage, still birth or
congenital infection in treated compared to untreated mothers (Pacquk et
af., 1990). Treating patients after delivery is a useful policy for women living
under sanitary conditions and infrequently infected (Villar et al., 1989), but
may be less acceptable for pregnant women living in conditions under which
they are frequently reinfected. While care should always be taken to ensure
safety of treatment for pregnant women, excess caution should not prevent
treatment of pregnant women if maternal infection represents a significant
risk to the health of the baby.
If treatment must be excluded during pregnancy, alternative strategies
should ensure that pre-pregnant women receive treatment, especially girls
who have never been pregnant and who therefore have little contact with
PARASITIC INFECTIONS IN WOMEN AND THEIR CONSEQUENCES 59

primary health care facilities. For example, at present the World Health
Organization recommends that patients serologically diagnosed as infected
with African trypanosomiasis should be followed up without treatment in
endemic situations, but in epidemic conditions treatment should be given if
at least two different serological test systems give positive results (WHO,
1986). The advisability of presumptive treatment of asymptomatic non-
pregnant women seropositive for sleeping sickness needs to be assessed. This
would involve establishing the relative risk of serologically positive, but
parasitologically negative, mothers infecting their children, and the risk of
recrudescence of infection during pregnancy.
Achieving optimal iron supplementation to reduce anaemia during preg-
nancy is difficult due to a high level of non-compliance in many areas
(WHO, 1990). Programmes should aim to improve haemoglobin levels
through iron supplementation and treatment of infectious diseases which
contribute to anaemia in non-pregnant women, especially girls aged 5-1 5
years. Cultural proscriptions against drug usage during pregnancy would
not apply to this group.

D. SOClAL FACTORS AND STRATEGIES FOR WOMEN’S HEALTH

This review has been primarily biological in its approach to parasitic


infections in women. This is not to underestimate the social causes which
contribute to a high level of parasitic infection in women, or the social
consequences, seen as chronic poor health and, sometimes, stigma. A
biological emphasis is needed, however, to encourage prioritization by
research bodies and disease control programmes to collect data on patterns
of infection in males and females separately, and to develop intervention
programmes which aim to reduce morbidity in non-pregnant women,
increase the health of pregnant women, and improve pregnancy outcome.
The social implications of implementing such programmes are considerable,
and success requires attention to factors which increase risk of exposure,
affect case detection and prevent access to, and completion of treatment in,
females (Brabin, L., 1991a, 1991b). Underlying these is a lack of knowledge
about signs and symptoms and appropriate treatment for infectious diseases
which women may not consider detrimental to their health or pregnancy
outcome. Conversely, some infections are feared for their potential effects on
fertility (Brieger et al., 1987).
Health planners need to recognize that parasitic infection has serious
consequences in pregnant women and primary health care services must be
better equipped to detect and manage infections during pregnancy and
between pregnancies. Disease control programmes and researchers should
appraise current approaches for case detection, drug treatment and vacci-
60 L. BRABIN AND B. J. BRABIN

nation, to find schedules which optimally protect mother and child. They
should seek implementation of programmes understood by women and
operable within the framework of cultural constraints on them (Holland,
1989).

ACKNOWLEDGEMENTS

Part of the research for this review was funded by a grant from the UNDP/
World Bank/WHO Special Programme for Research and Training in Tropi-
cal Diseases, World Health Organization, Geneva, to Dr L. Brabin. We
thank Dr Uche Amazigo, University of Nigeria, Nsukka, for the photo-
graph of onchodermatitis in a pregnant woman, and Dr H. J. Van der Kaay
for permission to reproduce Fig. 1.

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The Pathophysiology of Malaria

NICHOLAS J. WHITE

Wellcome-Mahidol University, Oxford Tropical Medicine Research Pro-


gramme, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thai-
land; Wellcome Trust Clinical Research Unit, Centre for Tropical Diseases,
Cho Quan Hospital, Ho Chi Minh City, Vietnam; NuBeld Department of
Clinical Medicine, John Radclifle Hospital, University of Oxford, Oxford, UK

and

MAY HO

Wellcome-Mahidol University, Oxford Tropical Medicine Research Pro-


gramme, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thai-
land; Department of Microbiology and Infectious Diseases, Health Sciences
Centre, University of Calgary, Calgary, Alberta, Canada

I. Introduction ............................................ 84
11. Animal Models ...................................... 85
111. Human Malaria ........................ ............... 86
A. Clinical features ........................................ 86
B. Causes of death and permanent sequelae .............................. 88
IV. Pathogenesis .......................... 93
A. Sequestration ...................................................... 93
B. Reduced red cell deformability 95
C. Cytoadherence .................................. 96
D. Putative endothelial cytoadherence receptors .......................... 98
E. Parasite cytoadherence ligands . . . . . . . . . . ............... 101
.................................. I02
V. Parasite Virulence Factors ................................... 105
A. Multiplication ........................... ............. 105
B. Synchronicity .................................... I08
110
110
A. Animal studies ................................................ 110
................. 112
C. Human studies ............................................. 112

ADVANCES IN PARASITOLOGY VOL. 31 Cnpyrighr 0 1992 Academic Press Limired


ISBN 0-12-031731-1 All rights o/reprodurrion in any /orm reserved
84 N. J. WHITE AND M. HO

VII. Pathophysiology of Vital Organ Dysfunction 1 I4


A. Cardiovascular abnormalities . . . . . . . . 1 I4
B. Algid malaria and septicaemia ....................................... I15
C. Pulmonary oedema ............................................
D. Blood flow and metabolism .....................................
E. Cerebral malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
F. Capillary permeability . . . 122
G. Fluid space changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
H. Electrolyte changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
I. Endocrine dysfunction
J. Renal impairment . . . .
K. Gastrointestinal dysfunction ......................................... 129
L. Liver dysfunction .................................... 130
M. Hypoglycaemia . .
N. Lactic acidosis . . . . . . . . . . . . . . . .
0. Skeletal muscle abnormalities ........................................ 135
P. Anaemia.. ......................................................... 136
Q. Bone marrow function
R. Blackwater fever . . . . . .
S. Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
T. Coagulation.. ...................................................... 139
U. Splenic function . . . . . . . . . . . . . . . . .

W. Immune dysfunction ................................................ 144


X. Complement . . . . .
Y. Pregnancy . . . . . . .
VIII. Conclusion ............................
.............................................. 149

I. INTRODUCTION

Plasmodium vivax, P. malariae and P. ovale are phylogenetically associated,


and lie relatively close to the simian malarias ( P . knowlesi, P. fragile) on the
evolutionary tree. They very rarely cause fatal disease in man. P. falciparum,
which may cause malignant tertian malaria, is a major cause of death in the
tropics. This parasite is closest phylogenetically to the malaria parasites of
birds ( P . gallinaceum, P. lophurae), and is far from the benign human
malarias (Waters, A. P., et al., 1991). It is thought to be a recent evolution-
ary acquisition of man. The propensity of P . falciparum to kill its host has
been considered evidence of a lack of parasitic sophistication-a debatable
point. In fact, P . falciparum is an extremely successful parasite infecting
approximately 200 million people, of whom the vast majority are unaware of
its presence in their bloodstream. Overall, falciparum malaria is the most
important parasitic disease of man (Wernsdorfer and McGregor, 1988).
The resurgence of malaria in the past two decades has stimulated a
THE PATHOPHYSIOLOGY OF MALARIA 85

considerable amount of scientific and medical research. Since the review by


Maegraith and Fletcher in 1972 our understanding of pathophysiological
mechanisms in malaria has advanced considerably in areas such as the
pathogenesis of metabolic dysfunction, the molecular processes involved in
cytoadherence, and the causes of anaemia. However, in other areas progress
has been slow. We do not know why coma occurs, or what causes
pulmonary oedema in severe malaria, and we do not fully understand
blackwater fever. There is still no good in vitro correlate of immunity. Much
of the recent research has been conducted either in animal models or with
cultured P. fakiparum parasites. The relevance of the observations, and the
hypotheses they generate, to disease in man still needs to be established in
many cases.

11. ANIMAL
MODELS

Certain features of severe malaria are common to all infected species


(Olsson, 1967). The metabolic consequences of hypoglycaemia and lactic
acidosis occur in the terminal phase of avian, rodent, simian and human
malarias (Sadun et af., 1965; White, 1985). It appears reasonable, therefore,
to extrapolate from observations in the animal to the disease in man (Ehrich
et al., 1984). Unfortunately, there is no good animal model for cerebral
malaria (Yoeli, 1976). Although sequestration occurs in several of the
animal malarias (e.g. P.fragile and P . coatneyi in rhesus monkeys), the hosts
do not become unconscious at low peripheral parasitaemias. Cerebral
symptoms are not a feature of rhesus monkeys infected with P . knowlesi, or
Aotus or Saimiri monkeys infected with P. falciparum (two commonly used
models). Nervous system dysfunction occurs in certain rodent models, but
the clinical manifestations (focal signs, coma only in the terminal stages)
and the main pathological findings (vasculitis with mononuclear cell aggre-
gation and infiltration, little or no sequestration) are different from those of
human cerebral malaria (Polder et af., 1983; Thumwood et al., 1988). Several
hypotheses concerning the pathophysiology of coma in falciparum malaria
and, unfortunately, several suggested treatments for severe malaria, have
been based on animal models of dubious relevance to human disease. For
example, the use of steroids in cerebral malaria derived largely from
observations on rhesus monkeys infected with P . knowlesi (Maegraith and
Fletcher, 1972), and the use of cyclosporin was based on observations on
rodents infected with P. berghei (Grau et af., 1987a). Both treatments were
subsequently found to be harmful in man.
86 N. J. WHITE AND M. HO

111. HUMAN
MALARIA

Immunity to malaria develops slowly. In areas of intense transmission of P.


falciparum, the infant is inoculated repeatedly with sporozoites in the first
months of life, but, for a variety of reasons which include the passive
transfer of malarial immunity and a less favourable intra-erythrocytic
environment (a high haemoglobin F content), severe disease is rare at this
age (Bruce-Chwatt, 1952; Pasvol et al., 1976). Most deaths occur between
the ages of one and four years (Greenwood et al., 1987). Thereafter the
infection becomes progressively less severe, and by the time adulthood is
reached falciparum malaria is largely asymptomatic (premunition). The rate
at which this process occurs is determined by the intensity of malaria
transmission and thus the number of infections. Age may be an independent
factor; i.e., for the same exposure adults develop immunity more rapidly
than do children (Baird et al., 1991). In areas where transmission is low,
uneven or highly seasonal, then symptomatic disease is seen at all ages. The
principal manifestation of severe malaria in these circumstances is cerebral
malaria (Brewster et al., 1990), whereas severe anaemia in young children is
more prominent in areas of intense stable transmission.
Several factors are responsible for the slow acquisition of immunity to
malaria. Malaria parasites exhibit considerable antigenic diversity, and
readily undergo antigenic variation. Most, or possibly all, naturally acquired
infections consist of several discrete genotypic and antigenic parasite clones.
In addition to this diverse antigenic array, the host immune system is
activated in a non-specific manner, while malaria antigen-specific immune
responses are suppressed (Ho et al., 1986Fan “immunological smoke
screen”. The precise immunological processes and other host defence mech-
anisms responsible for the control of falciparum malaria infections have not
been elucidated, but their failure contributes to the development of severe
disease with heavy parasite burdens and potentially lethal vital organ
dysfunction. Why some infections are more severe than others is at present
largely unknown. A list of possible factors is given in Table 1. It should be
noted that although a variety of cellular immune defects has been docu-
mented, it is not yet known whether these are a cause or an effect of severe
malaria. Indeed nearly all the factors listed.in this table are controversial.

A. CLINICAL FEATURES

Uncomplicated malaria is a febrile illness associated with headache, muscu-


lar discomfort, weakness and malaise. These features are non-specific,
resembling influenza, and are common to the four human malarias caused
by P . falciparum, P. vivax, P . malariae and P . ovale. As the untreated
THE PATHOPHYSIOLOGY OF MALARIA 87

TABLE1 Possible factors determining the severity of falciparum malaria

Factor Reference
Genetic
Protective erythrocyte abnormalities (surface receptors, Miller (1988)
cytoskeleton, haemoglobin type, enzymes, availability
of reticulocytes), HLA type Hill et al. (199

Age
Haemoglobin F Pasvol et al. (1 976)
Transfer of maternal immunity McGregor and
Pregnancy Wilson (1988)
WHO (1990)

Nutrition
Protective effects
Malnutrition Edington ( 1967)
Low iron Murray et al. (1975)
Deficiencies of riboflavin, a-tocopherol, Thurnham et al.
essential amino acids (1983)

Host defence
Humoral immunity Tharavanij et al. ( 1984)
Cellular immunity Druilhe et al. (1983)
Phagocytic function Ward et al. (1984)
Failure to augment splenic function rapidly?

Other diseases/conditions
Concomitant or previous infection impairing host defence?
Anaemia
Drug addiction

Parasite factors
Cytoadherence Ho et al. (1991a)
Rosetting Carlson et al.
Multiplication capacity? (1990b)
Antigenic variation?
Sporozoite inoculum/viability
Environmental/social factors
Location
Availability and use of antimalarial drugs
Family respanse to illness
Competence and resources of primary health care worker
Competence and resources of referral centre
88 N. J. WHITE AND M. HO

infection becomes synchronized, the fever becomes periodic with pyrexial


spikes every one, two or three days associated with chills or rigors. Synchro-
nization occurs earlier with P. vivax and P . ovule than with P . falciparum.
The untreated infection continues for weeks or months in non-immune
patients, but only P. falciparum produces fulminant disease.
Severe falciparum malaria is a multi-system disease. The clinical features
reflect the pattern of vital organ involvement. Coma is the most prominent
manifestation (Khan, 1945) (Fig. 1). Loss of consciousness may be sudden
following a seizure, or gradual (White and Looareesuwan, 1987bi.e. over a
period of hours, often preceded by a period of delirium. The neurological
signs are usually symmetrical and suggest diffuse encephalopathy. Retinal
haemorrhages can be seen by direct ophthalmoscopy in approximately 15%
of comatose patients (Kayembe et al., 1980; Looareesuwan et al., 1983a;
-=
WHO, 1990), but papilloedema is rare ( 1 YO).Convulsions are generalized
in most cases and often herald the onset of cerebral malaria; they are
particularly common in children. Most patients recover consciousness
within 3 days (children recover more quickly than adults). Jaundice is more
common in adults than children with severe malaria. Anaemia develops
rapidly in all age groups. Shock may occur in severe malaria, but most
patients are warm and well perfused with sinus tachycardia and blood
pressure at the lower end of the normal range. Abnormal ventilatory
patterns may be associated with extensor or flexor posturing. Sustained
hyperventilation (Kussmaul’s breathing) is a worrying finding indicating
metabolic acidosis if the chest examination is normal, or aspiration pneu-
monia or pulmonary oedema when there are abnormal chest signs. Vomiting
is common, particularly in children. Enlargement of the liver and spleen is
usual and the spleen is often palpable, but lymphadenopathy is not a feature
of malaria. A bleeding diathesis resulting from disseminated intravascular
coagulation occurs in approximately 5% of patients with severe malaria.

B. CAUSES OF DEATH AND PERMANENT SEQUELAE

1. Fatal malaria in adults

P . vivax, P . malariae and P . ovule kill very rarely during acute infections.
Occasional patients debilitated from other diseases may die, and rupture of
the enlarged spleen may cause death from haemorrhage if immediate surgery
is not available (Covell, 1955). In most adults who die from severe falci-
parum malaria there is multiple vital organ dysfunction and an adequate
explanation for the fatal outcome is apparent to the attending clinician. The
principal causes of death are pulmonary oedema, acute renal failure, and
metabolic acidosis with circulatory failure (WHO, 1986, 1990). Although
THE PATHOPHYSIOLOGY OF MALARIA 89

FIG. 1 . (a) Cerebral malaria. Opisthotonus in a comatose Gambian child. (b)


Disconjugate gaze in a child with cerebral malaria.
90 N. J. WHITE AND M. HO

any of these manifestations may occur in isolation, they more commonly


coexist, and it is often difficult to apportion causality. Coma is usual, but not
invariable. In the majority of adult cases, nervous system dysfunction does
not appear to contribute directly to death (White and Looareesuwan, 1987),
although fatal aspiration pneumonia may follow convulsions. Spontaneous
respiratory arrest may occur with cerebral malaria, commonly following a
sequence of hyperventilation, cyclical (Cheyne-Stokes), and then ataxic
breathing patterns. This is more common in children. Renal failure during
or after acute falciparum malaria is an important cause of death in adults.
Patients with severe malaria may die from secondary complications; aspi-
ration pneumonia, spontaneous Gram-negative septicaemia, or infective
complications of intensive therapy. Deaths from antimalarial drug toxicity,
bleeding, or cardiac dysrhythmias also occur but are rare. Deaths from acute
pulmonary oedema and renal failure may happen after several days treat-
ment when parasitaemia has fallen, or even cleared, but most fatalities occur
within 3 days of admission to hospital during the acute phase of the disease.
Many laboratory measurements have been correlated with outcome. The
more readily measurable poor prognostic variables are hypoglycaemia,
hyperlactataemia, raised cerebrospinal fluid (CSF) lactate, leucocytosis,
acidosis and raised serum concentrations of urea, creatinine and the trans-
aminases. High parasite counts are associated with an increased risk of fatal
outcome. In the classic studies from Kuala Lumpur, counts over 100 000 per
pl were associated with increased mortality, and 50% of those with counts
over 500000 per pl died (Field and Niven, 1937; Field, 1949). However,
there is considerable variability. Parasite counts in semi-immune African
children can be well over 100 000 per p1 without symptoms or signs of severe
disease, whereas non-immune patients may die with very low peripheral
parasitaemias. Recent studies have shown that staging of parasite develop-
ment in the peripheral blood film adds considerably to the prognostic
sensitivity and specificity of the parasite count (N. J. White, unpublished
observations).

2. Fatal malaria in children

Falciparum malaria is a major cause of infant death and morbidity in the


tropics (McGregor et al., 1956; Greenwood et al., 1987). Children with
severe malaria have a slightly lower overall mortality rate than adults.
Definitions of cerebral malaria have varied considerably but, of the interpre-
table published series in which chloroquine or quinine was used for treat-
ment, the overall mortality rate from cerebral malaria in adults was 21.5%
(141/655) compared with 13.8% (230/1383) in children (P= 0.009). The
causes of death in children are often different from those in adults. Vital
organ dysfunction is less evident. Acute renal failure is very rare in young
THE PATHOPHYSIOLOGY OF MALARIA 91

children, whereas renal impairment contributes to over half the adult deaths
(White et al., 1987a; Molyneux et al., 1989b; WHO, 1990). Pulmonary
oedema is also unusual. In contrast, hypoglycaemia and lactic acidosis with
terminal circulatory failure and aspiration pneumonia are more common in
young children. Many children die suddenly in the acute phase of severe
malaria without a clear explanation (unpublished observations). There is
primary respiratory arrest without circulatory failure. Of the 26 deaths in a
series of 180 Gambian children with severe malaria, no clear explanation
was evident in 14 children, four of whom died with primary respiratory
arrest (N. J. White and D. Waller, unpublished observation). The possibility
that this phenomenon results from brainstem compression- secondary to
raised intracranial pressure has been suggested by recent findings that
opening pressures at lumbar puncture are usually raised in children with
cerebral malaria (Newton et al., 1991; Waller et al., 1991). This is in contrast
to the findings in adults where opening pressures are normal in 80% of cases
(Fig. 11) and are significantly lower in fatal cases than in survivors (Warrell
et al., 1988). Some of the neurological findings in children with cerebral
malaria have been attributed to the development of a tentorial pressure
cone, but the specificity of these interpretations needs to be confirmed
(Section VII.F.2). Nevertheless, the observations are important because of
the possibility of providing specific treatment (e.g. with mannitol) which
would reduce intracranial pressure.
In areas of stable intense transmission (hyper- or holoendemicity),
anaemia becomes more common than cerebral malaria as a presentation of
severe malaria in children. The case specific mortality rate is lower. In a
recent large series of over 600 severely ill children reported from The
Gambia, overall mortality was approximately 7% in children with severe
anaemia compared with 16% in cerebral malaria (Brewster et al., 1990). The
causes of death in children with severe malarial anaemia are also different.
Some children have relatively low parasite counts, and in these the patho-
physiological processes relate largely to severe anaemia, whereas in others
both severe malaria and severe anaemia coexist. High output heart failure is
an important and potentially lethal manifestation of severe anaemia. Many
children die suddenly before or during blood transfusion. Twenty-four hour
electrocardiographic recordings suggest that cardiac arrhythmias are prob-
ably not a cause of “unexplained deaths” in acute cerebral malaria (F.
Nosten and N. J. White, unpublished observations), but this has not been
studied in severe malarial anaemia.

3. Permanent sequelae

Approximately 10% of children, but less than 5% of adults, surviving


cerebral malaria have obvious permanent residual neurological sequelae
92 N. J. WHITE AND M. HO

(Molyneux et al., 1989b; Brewster et al., 1990). In over half the cases there is
hemiparesis (Fig. 2), but cortical blindness and clinical evidence of more
diffuse brain damage are also common. In half the children discharged from
hospital with a neurological deficit, there is full recovery within 6 months
(Brewster et al., 1990). The possibility that cerebral malaria causes more
subtle permanent defects such as slight motor impairment or mild intellec-
tual retardation in survivors has not been explored.
(a)

FIG.2. (a) Right hemiparesis following cerebral malaria (courtesy of Dr J. Craw-


ley). (b) Diffuse cortical damage 6 months after cerebral malaria.
THE PATHOPHYSIOLOGY OF MALARIA 93

Carotid angiography has been performed in 1 1 cases of post-cerebral


malaria stroke. Four showed large vessel obstruction and one segmental
narrowing, but the remaining six were normal (Collomb et al., 1967;
Sanohko et al., 1968; Omanga et al., 1983). Persistent neurological deficit is
associated with protracted hypoglycaemia, seizures, prolonged and pro-
found coma, and coexistent severe anaemia during the acute phase of
cerebral malaria. There is no evidence that large intracerebral haemorrhages
occur, so presumably cerebral infarction is responsible for the neurological
deficit, but the mechanisms responsible have not been elucidated. Obviously
the increased cerebral metabolic demands associated with seizures, hypo-
glycaemia, reduced cerebral perfusion pressure and cerebral arterial oxygen
transport may all combine to compromise the fragile balance between blood
supply and metabolic demands, particularly in vulnerable “watershed” areas
of limited vascular reserve such as the parieto-occipital region (Sections
VII.D.1 and VI1.T).

IV. PATHOGENESIS

The pathology of uncomplicated malaria is determined by the destruction of


parasitized erythrocytes, the intravascular liberation of parasite and host
products at merogony (schizogony)*, and the host reaction to this process.
In severe falciparum malaria there is a greater parasite burden and sequest-
ration in the microcirculation of the vital organs. These two factors account
for the lethal potential of this parasite.

A. SEQUESTRATION

All stages of parasite development are seen in blood smears taken during
infections with P. vivax, P . malariae and P . ovale, but the peripheral blood in
P. fakiparum malaria rarely contains pigmented trophozoites or meronts
(schizonts).* The intravascular sequestration of erythrocytes containing
these mature forms of the parasite is an essential pathophysiological feature
of falciparum malaria (Luse and Miller, 1971). The degree of vascular
sequestration varies between organs, being greatest in the brain in patients
with cerebral malaria and least in the skin (Li et al., 1983; MacPherson et al.,
1985). In patients who die without developing cerebral malaria, sequest-
ration is significantly less in the brain (Pongponratn et al., 1991). These
findings suggest a relationship between the organ distribution of sequest-
ration and pathology.
* Throughout this review, the etymologically more consistent terms meront and merogony have
been used instead of schizont and schizogony (eds).
94 N. J. WHITE AND M. HO

FIG.3. Cerebral venule packed with parasitized erythrocytes and pigment in a fatal
case of cerebral malaria (courtesy of Professor M.Aikawa).
Within a few years of Laveran’s discovery of the malaria parasite in the
blood of a febrile patient in Algeria, pathological observations from Italy
(Marchiafava and Bignami, 1894) recorded the extraordinary discrepancy
between the microscopical appearance of the peripheral blood and that in
the cerebral vessels of patients dying from cerebral malaria. The capillaries
and venules in the brain were packed with erythrocytes containing mature
forms of the parasite and abundant brown-black pigment, which were not
seen in ante-mortem blood samples (Fig. 3). These findings were confirmed
in pathological reports on the soldiers dying from falciparum malaria in
Macedonia during the First World War of 1914-1918 (Dudgeon and Clarke,
1917, 1918; Gaskell and Miller, 1920). It was suggested that the parasitized
erythrocytes had difficulty traversing the capillary bed and, as a result, blood
flow was obstructed. Initially it was thought that thrombus formation
occurred (Dudgeon and Clarke, 1917), altho.ugh the authors also conceded
that microvascular obstruction by parasitized erythrocytes might be revers-
ible (Dudgeon and Clarke, 1918). Later pathological studies have concluded
that widespread thrombus does not occur in fatal cerebral malaria, and have
favoured the concept of “plugging” (i.e. obstruction) of small vessels by
masses of parasitized erythrocytes (Gaskell and Miller, 1920; Spitz, 1946)
(Section VI1.T).
The mechanism of microvascular obstruction was investigated in a series
THE PATHOPHYSIOLOGY OF MALARIA 95

of studies by Knisely and colleagues (1941). They studied rhesus monkeys


infected with the lethal kra monkey parasite, P. knowlesi (which does not
sequester), and also recorded observations in patients with falciparum
malaria. The microcirculation was directly visualized and cinematographic
recordings were made of flow in the mesenteric vessels of the monkeys and
the bulbar conjunctiva in man. During infection the erythrocytes were seen
to agglutinate and eventually to form what was delicately described as a
“thick muck-like sludge”. Pathological events in severe malaria were inter-
preted as resulting from ischaemia, hypoxia, and any subsequent toxic
effects resulting from release of unidentified materials from the “sludge”.
Although microvascular thrombus deposition appears to be unusual, large
vessel occlusion may occur rarely in childhood cerebral malaria leading to
stroke (Brewster et af., 1990).
In the last 20 years investigations have focused first on reduced red cell
deformability, and more recently on the specific adherence (cytoadherence)
of infected erythrocytes to vascular endothelium.

B. REDUCED RED CELL DEFORMABILITY

Red cells containing malaria parasites do not pass through micropore filters
as easily as unparasitized erythrocytes. This suggests that these infected
erythrocytes are less deformable than normal cells, and might therefore not
pass as easily through capillary beds (Miller, L. H. et af., 1971, 1972; Lee, M.
V. et al., 1982). In normal microcirculatory flow, red cells (diameter 7-8 pm)
must undergo considerable deformation in their passage through the capil-
lary (diameter 3 4 p m ) . Capillary blockage does occur when red cells are
unusually rigid, as in sickle cell crisis, but the clinical features and organ
distribution of vascular obstruction in this condition are most unlike those
of severe malaria. The reduced deformability of red cells infected with P.
falciparum is directly proportional to the maturity of the parasite (Cranston
et al., 1984); the older, and larger, the parasite, the more rigid is the infected
cell. There is increased expression of phosphatidylserine and phosphatidyl-
ethanolamine and reduced phosphatidylcholine on the outer leaflet of the
trophozoite-infected erythrocyte membrane. Cells containing meronts also
have reduced sphingomyelin in the outer leaflet (Maguire et al., 1991). These
abnormalities of phospholipid distribution, which may result from depletion
of adenosine triphosphate, oxidative stress and alterations in the cytoskele-
ton, influence the surface properties of the infected erythrocyte. They are
associated with increased phagocytic clearance and adherence to monocytes
and endothelial cells (Section VII.P.4). Several other factors contribute to
reduced red cell deformability: increased membrane stiffness, increased
cytoplasmic viscosity resulting from changes in membrane permeability
96 N. J. WHITE AND M. HO

(Dunn, 1969; Kutner et al., 1983), reduced surface area-to-volume ratio


(increased sphericity), and principally the rigidity of the parasite itself (Nash
et al., 1989). However, reduced erythrocyte deformability alone does not
explain the phenomenon of sequestration nor, therefore, the severity of
severe falciparum malaria. It does not explain the concentration of parasit-
ized erythrocytes in venules which are downstream from the site of minimum
vascular cross-sectional area (i.e. the mid capillary), nor the precise parasite
stage specificity of sequestration. If a rigid red cell, or an aggregate of cells
such as a “rosette”, irreversibly blocks flow by becoming stuck in a small
blood vessel, then the tail of erythrocytes stacked behind the obstructing cell
should have a similar proportion of parasitized cells to that in the peripheral
blood (White, 1985). This is analogous to a car accident causing a traffic
jam. In addition, reduced red cell deformability does not explain preferential
sequestration in the cerebral microvasculature, which has similar internal
dimensions to the vessels in other organs.

C. CYTOADHERENCE

The principal event causing sequestration and impeding microcirculatory


flow appears to be the cytoadherence of parasitized erythrocytes (PRBC) to
vascular endothelium (Luse and Miller, 1971; Raventos-Suarez et al., 1985).
Cytoadherence is a specific process, in that it occurs only in capillaries
and post-capillary venules, and involves only erythrocytes containing the
more mature stages of the parasite, viz. trophozoites and meronts. The cyto-
adherent properties of P . fakiparum appear to be modulated by the spleen
(Hommel et al., 1983). Infected erythrocytes do not cytoadhere in splenecto-
mized saimiri monkeys, but the property is regained after several asexual
cycles of a cloned line following transfusion into monkeys with intact spleens
(David et al., 1983). It is not known how this modulation takes place. At the
ultrastructural level, electron-dense, knob-like protrusions of the erythro-
cytic membrane are seen at the points of contact between the PRBC and
endothelial cells (MacPherson et al., 1985) (Fig. 4). These knobs were
considered essential for cytoadherence by facilitating the initial attachment
of the infected erythrocyte to the vascular endothelial cell, and by concen-
trating the parasite ligands at a particular site. In the last few years, the
importance of the knobs has been disputed. Two clones of knobless (K -)
laboratory-adapted parasites (Biggs et al., 1989; Udomsangpetch ef al.,
1989), and one clinical isolate (S. Semoff, B. Singh and M. Hommel,
unpublished data), have been shown to cytoadhere in vitro in the absence of
knobs, but probably through the same molecular mechanism (see later) as
the knobby (K+) parasites (Biggs et al., 1990). Since the laboratory-adapted
K - cytoadherent parasites were obtained after many cycles of selection
from a K + isolate, it is possible that K + and K - parasites coexist in
THE PATHOPHYSIOLOGY OF MALARIA 97

isolates from natural infections. Parasitism would continuously favour the


selection of K + organisms if they were able to form a more stable union
with host cells, and thus evade splenic clearance. The relevance of these
experimental findings remains to be determined, since cytoadherence under
the static conditions of the assays in vitro may require a less durable
interaction than that of infected erythrocytes exposed to the dynamics of the
host circulation and immune response in vivo. Raventos-Suarez et al. (1985)
have investigated microvascular obstruction by P.falciparum and the role of
knobs in cytoadherence using the isolated perfused rat mesocaecum. When
human erythrocytes infected with P.falciparum were added to the perfusate,
blood flow was seen to slow and then finally stop, as a result of cytoadher-
ence to the vascular endothelium. Only K + erythrocytes cytoadhered and
blocked flow. Ultrastructural studies of human tissues from fatal malaria
cases have not shown cytoadherence independent of knobs.

FIG.4. Cytoadherence between parasitized erythrocyte (PE) and cerebral vascular


endothelial cell (E) showing knobs at the points of attachment (arrowed) (courtesy of
Professor M. Aikawa) ( x 38 000).
98 N. J. WHITE AND M. HO

D. PUTATIVE ENDOTHELIAL CYTOADHERENCE RECEPTORS

The stage and host cell specificity of cytoadherence suggest that the inter-
action between PRBC and endothelial cells involves specific parasite ligands
and host receptors. The quest for these proteins has proved more difficult
and confusing than expected originally, and has relied heavily on the use of
models in vitro. The search began with the observation that PRBC adhere to
cultured human umbilical vein endothelial cells (HUVEC) in vitro (Udeinya
et al., 1981) with the same stage and host cell specificity as observed with
sequestration in vivo. This model proved difficult and unpredictable. A
number of normal cell types and continuous cell lines have subsequently
been shown to have cytoadherent properties, of which the human amelano-
tic melanoma cell line C32 (American Type Culture Collection, no.
CRL1585) (Schmidt et a/., 1982) has been the most extensively employed. In
addition, putative receptor proteins have been purified and studied either
immobilized on plastic or in their soluble forms. Although such studies
provide definitive information regarding cytoadherence to a particular
molecule, it has been argued that the immobilized or free receptor molecule
in vitro may not have the same tertiary structure as that expressed on the cell
surface in vivo. One approach to circumvent this problem has been to
transfect COS cells with plasmids carrying the complementary deoxyribo-
nucleic acid (cDNA) for the receptors. The receptor under investigation is
then expressed on the otherwise antigen-free surface of the COS cell.

1. Thrombospondin
The first molecule identified as a potential receptor for cytoadherence was
thrombospondin (TSP), an adhesive glycoprotein produced by activated
platelets and involved ubiquitously in cell-to-cell interactions (Tandon et al.,
1989). Using purified TSP, Roberts et al. (1985) showed that PRBC adhered
selectively to TSP in a dose-dependent manner, but not to other adhesive
proteins such as fibronectin and von Willebrand’s factor. Cytoadherence
was specifically inhibited by anti-TSP monoclonal antibodies and soluble
TSP, and occurred under both static and shear-flow conditions (Rock et al.,
1988). However, further work has revealed that while TSP may contribute to
cytoadherence, it is not sufficient to mediate the process alone. PRBC do not
adhere to every melanoma cell line which secretes TSP (Panton et al., 1987)
and anti-TSP antibodies neither bind, nor inhibit cytoadherence, to C32
melanoma cells (Ockenhouse et al., 1989a).

2. CD36

The second molecule to be implicated in cytoadherence was the leucocyte


differentiation antigen CD36, a membrane glycoprotein (molecular mass
THE PATHOPHYSIOLOGY OF MALARIA 99

88 kDa). A monoclonal antibody to C36,OKM5, was shown to inhibit and .


reverse the cytoadherence of PRBC to a number of target cells in vitro
including epithelial cells and C32 melanoma cells (Barnwell et al., 1985;
Panton et al., 1987). PRBC have been shown subsequently to adhere
selectively to purified CD36 immobilized on plastic. The purified protein
specifically and competitively inhibits the cytoadherence of PRBC to the
C32 cells and endothelial cells (Barnwell et al., 1989; Ockenhouse et al.,
1989a). Furthermore, although binding to purified TSP and purified CD36
are highly correlated (Hasler et al., 1990), PRBC adhere directly to CD36-
transfected COS cells in the absence of TSP (Oquendo et al., 1989). CD36 is
also expressed on the surface of monocytes and platelets, and PRBC have
also been shown to cytoadhere to these cells (Ockenhouse and Chulay, 1988;
Ockenhouse et al., 1989b). Peripheral blood monocytes are triggered by this
process to produce oxidative metabolites which are toxic to intra-erythro-
cytic parasites (Ockenhouse et al., 1984).
The current concept with regard to the role of CD36 and TSP in
cytoadherence has been summarized by Barnwell et al. (1989). The two
molecules could interact by the association of soluble TSP in plasma with
exposed parasite ligands. The TSP-ligand complex would then interact with
cell-bound CD36. Alternatively, plasma or cellular TSP may associate with
the cell surface membrane receptor before interaction with parasite ligands.
There is some evidence that CD36 acts as the natural receptor for TSP (Asch
et al., 1987). However, cytoadherence to CD36 may occur completely
independently of TSP, as has been shown with CD36-transfected COS cells
(Oquendo et al., 1989). This adherence is not inhibited or reversed by anti-
TSP antibody, and TSP does not adhere to the CD36-transfected COS cells.

3. ICAM-I

A third candidate receptor molecule, the intercellular adhesion molecule 1


(ICAM-1) or CD54, has been identified by Berendt et al. (1989). This
glycoprotein (also molecular mass = 88 kDa) has a well established role in
mediating cellular immune responses by acting as a ligand for lymphocyte
function antigen 1 (LFA-1). ICAM-1 is also the natural receptor for
rhinovirus attachment. Binding of parasitized erythrocytes to CD36
or ICAM-1 expressed on transfected COS cells can be inhibited by their
respective monoclonal antibodies. A schematic diagram of the possible
molecular interactions involved in cytoadherence is shown in Fig. 5.

4. Clinical correlates

To determine the relative importance of the three receptor molecules in vivo,


the cytoadherence of parasite isolates taken directly from patients with acute
1 00 N. J. WHITE AND M. HO

FIG. 5. Schematic diagram of the molecular interactions involved in cytoadherence


between the parasitized erythrocyte (PRBC) and endothelial cells. The knob protru-
sions could bear specific ligands for thrombospondin (TSP-R, and TSP, respect-
ively), CD36 and ICAM-I. These three ligands could be expressed as separate
molecular entities (A) or as a single composite cell surface molecule (B). The
endothelial cell is also shown secreting TSP, which is then bound to endothelial cell
components (TSP-RE). TSP could also derive from platelets or other cells and act as
a bridge between the receptor (TSP-R,) and TSP-RE or CD36. Cytoadherence may
also take place independently of TSP with direct interaction between CD36 and/or
ICAM-I and their PRBC ligands. (Reproduced with kind permission from Howard
and Gilladoga, 1989.)

falciparum malaria has been examined in several studies. Binding to TSP


(Sherwood et al., 1987), C32 melanoma cells (Marsh et al., 1988; Ho et al.,
1991a) and purified CD36 (Ockenhouse et al., 1991b) was directly pro-
portional to parasitaemia. When parasite isolates were compared at a fixed
parasitaemia (i.e. binding was “normalized”), a range of intrinsic cytoadher-
ent capabilities among different isolates was evident. In the case of cytoad-
herence to C32 melanoma cells and purified CD36, the degree of binding was
positively correlated with biochemical indicators of disease severity in adult
Thai patients (Ho et al., 1991a; Ockenhouse et al., 1991b), but there was no
correlation between cytoadherence and the presence of cerebral symptoms
either in this series (Ho et al., 1991a) or in a separate study of Gambian
children (Marsh et al., 1988). Indeed, adults with cerebral malaria tended to
THE PATHOPHYSIOLOGY OF MALARIA 101

have lower melanoma cell binding rates than other patients with severe
disease. These findings support the hypothesis that CD36 mediates sequest-
ration in vital organs other than the brain but question the role of CD36 in
mediating cerebral sequestration. In immunohistochemical studies of human
tissues using the monoclonal antibody OKM5, CD36 can be demonstrated
on vascular endothelium in sections of lung, kidneys and liver (Knowles et
af., 1984), but not in the brain (A. Berendt, personal communication).
However, using a different monoclonal antibody, CD36 was detected on
cerebral vascular endothelium (Barnwell et af., 1989). This suggests that a
different receptor epitope of CD36 may be expressed in the cerebral
microvasculature.
In contrast to the results with CD36, cytoadherence of freshly isolated P .
,fakiparum to purified ICAM- I , and to a sub-clone of the C32 melanoma cell
line bearing ICAM- I but not CD36, was generally low in one study and bore
no quantitative relationship to any clinical manifestations of malaria (Ock-
enhouse et af., 1991b). When both CD36 and ICAM-I were expressed
together, as on the surface of the C32 melanoma cells, there was preferential
binding to CD36.
The weight of evidence suggests that CD36 is the most important of the
candidate receptor molecules thus far identified. However, the true role of
these molecules, and others perhaps yet undiscovered, in P . fakiparum
sequestration will undoubtedly require more investigation. Further studies
must also take into account the distribution and density of the endothelial
ligands on different tissues, in order to reconcile the variability in end organ
damage seen in patient populations of differing age and background immu-
nity.

E. PARASITE CYTOADHERENCE LIGANDS

There is relatively little information on the parasite ligands involved in


cytoadherence. Although a number of parasite-derived proteins has been
detected on infected erythrocytes (Howard, 1987; Hommel and Semoff,
1988), it has been assumed that only protein(s) which protrude from the
surface of the membrane would be likely to mediate cytoadherence. So far
only P . falciparum erythrocyte membrane protein (PWMPI) has been shown
unequivocally to have this property (Leech et af., 1984). PfEMPl consists of
a family of molecules (molecular mass 240-260 kDa) in which only the
higher molecular mass variants are associated with cytoadherence
(Magowan et af., 1988).
A similar molecule of molecular mass = 270 kDa, called sequestrin, has
recently been demonstrated on the surface of infected erythrocytes using
anti-isotypic antibodies raised against OKM8, a monoclonal antibody
specific for the putative endothelial receptor CD36 (Ockenhouse et af.,
102 N. J. WHITE A N D M. HO

1991a). This finding further strengthens the hypothesis that CD36 is the
receptor for the parasite ligand on vascular endothelium. The definitive
proof of the role of these parasitized red cell surface proteins in cyto-
adherence awaits the production of specific isotypic antibodies and/or the
cloning of the genes encoding these antigens.
Two other molecules have been proposed as the cytoadherence parasite
ligand, although the evidence supporting them is far less convincing. The
155 kDa ring-infected erythrocyte antigen (RESA) antigen, which is trans-
ferred from the merozoite to the erythrocyte membrane during invasion,
was thought initially to be entirely submembranous, but recent evidence
suggests that part of the molecule is exposed on the exterior of the red cell as
the parasite matures. RESA could therefore have a role in cytoadherence.
The RESA antigen has been shown to have cross-reactive epitopes with
band 3 protein (Holmquist et al., 1988), the human erythrocyte anion
transporter, and this too has been implicated as a ligand for cytoadherence
(Winograd and Sherman, 1989). Presumably, changes in the erythrocyte
cytoskeleton which occur as a result of parasitization expose previously
hidden host molecules (neoantigens) on the cell surface. At present there is
considerably less evidence to support a role for RESA or modified band 3 in
cytoadherence than for PfEMPl, but the situation is far from resolved.
Regardless of the eventual identity of the cytoadherent ligand, a conserved
component must be present since all P. falciparum parasites causing natural
infections cytoadhere. In addition, there must be a strain-variable compo-
nent since inhibition or reversal of cytoadherence by immune sera occurs in
a strain-specific manner (Udeinya et al., 1983; Singh et al., 1988). Indeed, the
cytoadherence surface proteins show antigenic variation within cloned
parasite lines in a manner analogous to the schizont-infected cell antigen
(SICA) of P. knowlesi. The constant and variant components could be either
closely associated molecules or different epitopes on the same molecule. This
ability of the parasite to vary the surface antigenicity of the cytoadherent
protein is obviously important for its survival as it helps to evade host
recognition and thus parasite removal.

F. ROSETTING
Non-parasitized erythrocytes will agglutinate around red cells containing
mature forms of the parasite in vitro (David et al., 1988; Udomsangpetch et
al., 1989b). This phenomenon is termed rosetting and may sometimes be
seen in fresh blood samples (Ho et al., 1991). It shares many characteristics
with the properties of cytoadherence. Rosetting occurs only with species of
Plasmodium which also exhibit cytoadherence (Handunnetti et al., 1989).
Both phenomena occur with mature stages of P . falciparum and begin after
approximately 26 h of intra-erythrocytic development (David et al., 1988).
THE PATHOPHYSIOLOGY OF MALARIA 103

Rosetting can be reversed by immune sera which also reverse cytoadherence


(David et al., 1988). The antigens responsible for rosetting and cytoadher-
ence are both very protease-sensitive (Udomsangpetch et al., 1989b). Both
properties are maximal at acidic pH, but rosetting is inhibited by heparin
and EGTA*, a calcium chelator, whereas cytoadherence is not (Carlson et
al., 1990a). Interestingly, rosetting is inhibited by one anti-CD36 mono-
clonal antibody (OKM8) but not by another (OKM5). In the perfused rat
mesocaecum model, human erythrocytes containing mature P . falciparum
parasites of known rosetting lines (K+ R + ) cause more microvascular
obstruction than infected cells from cytoadherent but non-rosetting lines
(K+ R-). They cause greater resistance to microvascular flow, and the
erythrocytes aggregate readily in the larger venules (Kaul et al., 1991).
There have been several recent studies of rosetting in parasite isolates
obtained from patients with acute falciparum malaria. Rosette formation
varied considerably: whereas all fresh isolates cytoadhered to some extent,
not all isolates showed rosetting (Wahlgren et al., 1990). Rosetting is
associated with cerebral malaria. Parasites obtained from Gambian children
with cerebral malaria showed a significantly greater degree of rosetting than
those from children with uncomplicated disease (Carlson er al., 1990b).
Patients with cerebral malaria also lacked antibodies which could inhibit
rosette formation in vitro, whereas these antibodies were present in about
20% of immune sera. No significant correlation between rosetting and
biochemical indices of disease severity was seen in a smaller population of
adult patients in Thailand (Ho et al., 1991b) but, as in the Gambian series,
rosette formation tended to be greater in patients with cerebral malaria. In
this latter series there was a significant inverse correlation between rosetting
and cytoadherence for a given isolate. Cytoadherence was not increased
when rosetting was partially inhibited by heparin, which suggests that the
inverse relationship is not the result of steric hindrance. Cytoadherence and
rosette formation properties are probably intrinsic to the parasites, with
individual parasite isolates having greater propensity for one or the other
(Ho et al., 1991b). This is consistent with the observation that isolates from
patients with cerebral malaria have increased rosetting properties, but
adhere poorly to C32 melanoma cells in vitro (Marsh et al., 1988; Ho et al.,
1991a). Whether the inverse relationship between cytoadherence and rosette
formation holds true in vivo remains to be determined. However, it is likely
that once a parasitized erythrocyte becomes encased in a rosette there would
be a considerable physical obstacle to cytoadherence-so in larger venules
the two processes must be to some extent mutually exclusive.
The evidence so far linking rosette formation to the pathogenesis of
cerebral malaria is suggestive but not conclusive. Unlike cytoadherence,

* Ethyleneglycol-bis-(P-aminoethylether)-~,,N,llr,K-tetraacetic
acid.
104 N. J. WHITE AND M. HO

rosettes have not been prominent findings in any histological studies of


clinical or post-mortem tissues in falciparum malaria. Significant rosette
formation on the arterial side of the microcirculation appears unlikely, as
arteriolar obstruction is not a pathological feature of severe malaria.
Presumably the low pH and shear forces on the venous side tend to favour
rosetting (Howard and Gilladoga, 1989), and this can be seen ex vivo in the
perfused rat mesocaecum model (Kaul et al., 1991), but if complete vascular
obstruction occurs one would still expect the tail of blood into the capillary

(a)

FIG.6. (a) Mechanical separation using micropipettes of a red cell containing a


mature Plasmodium falciparum parasite (left) and an uninfected erythrocyte (right).
These are the adhesive properties that cause rosetting (courtesy of Dr G. Nash). (b)
Hypothetical scheme for the role of rosettes in sequestration. At point A the vessel
diameter is sufficient to allow passage of the formed rosette. At point B the rosette
interacts with the vessel wall. Flow may be obstructed, or the rosette could disrupt
flow allowing cytoadherence to take place (C). (Reproduced with kind permission
from Howard and Gilladoga, 1989.)
THE PATHOPHYSIOLOGY OF MALARIA 105

to reflect the distribution of parasitized erythrocytes in the circulation. In


fact, the majority of the erythrocytes sequestered in the cerebral vessels in
cerebral malaria are parasitized; i.e., sequestration is selective. The cell-to-
cell adhesive forces involved in rosetting have been shown experimentally to
be considerable and capable of withstanding intravascular shear forces (Fig.
6). It may be that the adhesive properties of red cells containing mature
parasites (i.e. the processes that lead to rosette formation in vitro) may
contribute to a reduction in forward blood flow, which would then initiate or
“encourage” cytoadherence as a secondary phenomenon (Fig. 6b). The
passage of uninfected red cells squeezing past the sticky cytoadherent
infected cells would also be slowed by this process (static hindrance). A
circulating rosette would presumably become obstructed in the pulmonary
capillary bed or, if it traversed this successfully, it would then be vulnerable
to splenic removal. Rosetting is currently a subject of considerable interest.
Its pathophysiological role, particularly in relation to cerebral malaria, will,
hopefully, become evident over the next few years.

VIRULENCEFACTORS
V. PARASITE

A. MULTIPLICATION

Infection begins with the inoculation of malaria sporozoites from a female


anopheline mosquito probing for a blood meal. The sporozoites migrate
rapidly via the lymphatics and the bloodstream to the liver. Each sporozoite
which successfully invades a hepatocyte will subsequently develop into a
meront (schizont) containing many thousands of merozoites. For P. vivax
and P . ovule, development will be delayed in a proportion of invaded cells,
and in these hepatocytes the parasites lie dormant as hypnozoites. Months
or years later they will “awaken” and develop, and cause the relapses of
infection characteristic of these species. Each fully developed hepatic meront
of P. vivax, P. ovule and P . malariae (i.e. the benign human malaria
parasites) will burst to liberate up to 15 000 merozoites into the host’s
bloodstream, whereas the hepatic schizonts of the potentially lethal P.
fakiparum contain 30 000 or more merozoites (Garnham, 1966). Further-
more, the exoerythrocytic phase of development (liver merogony) of P.
falciparum takes an average of 5.5-7 days (and may be shorter), compared
with 7 days or longer in the benign malarias. Thus, early amplification of the
infection is greater in the potentially lethal falciparum malaria (Garnham,
1988).
The current estimates for the median P. falciparum sporozoite inoculum
are 8-15 sporozoites-but the distribution is skewed-and on occasions as
106 N. J. WHITE AND M. HO

many as 100 sporozoites may be inoculated (Rosenberg, R. and Wirtz, 1990;


Ponnudurai et al., 1991). If these estimates are correct, and they are very
difficult to prove, then between 3 x lo5 and 3 x lo6 merozoites of P.
falciparum are released into the bloodstream to start the asexual erythrocytic
cycle. This begins the symptomatic phase of the infection. The merozoites
are motile ovoid bodies structurally similar to sporozoites. They rapidly
invade passing erythrocytes, and then proceed to consume the red cell
contents, growing progressively larger through the trophozoite stage, to
become multinuclear meronts inthe last 12 h of the cycle. After 48 h of intra-
erythrocytic development, the P . falciparum meront bursts to release 18-24
daughter merozoites (estimates have ranged from 8 to 36 per red cell),
compared with 12-16 for P. vivax and 8 for P. ovale (Garnham, 1988).
Asexual multiplication in vivo is approximately 30-50% efficient during the
early phase of infection and, in the non-immune host, the parasitaemia rises
by roughly an order of magnitude every 2 days (Fairley, 1947; Kitchen,
1949). At this rate the threshold of thick film microscopical detection (ca. 50
parasitized erythrocytes per pl) is reached in three to four asexual cycles, i.e.
12-16 days after hepatic merogony. If multiplication is unchecked, poten-
tially lethal parasite burdens are reached in another three to four cycles (Fig.
7). It is obvious from this that a higher multiplication rate is associated with

014 7 10% parasitaemia


1% parasitaernia I

1012 :
.-* .-’:
I-.
I *a e
I-..

,.--.
I t 1 I
0I

1x101 i 1x6) ;
I

1010 -
,-..-,‘
’-’
Body ,i‘: ../
,,-- , 1

pbu
”7
:; ,---..-/
8 ,
.-.I

*.-. r ,.--..-.*
--
Detection limit (50pL 1) !
108 - ,-,, ! ; ,--. ’
; :
I
I .-#’
,.-.._,
, ,
,,--..-;
*-.‘

106 -
,-.,;;--*.2 ,--.*- *#J

.-- :,-.* .-.,


1. -.‘..j’
100 sporozoites ’
,--=.-/
104 - I
:
-..-.’
I

1 sporozoite
102 - MOSaUltO Erythrocytic cycle

*
blt e 0 0 0 @ 0 @ @ @
1
I
C I I ,’ I I I I

FIG.7. Plasmodium falciparum infection: expansion of the parasite burden follow-


ing different sporozoite inocula, and at two different multiplication rates in vivo ( x 6
and x 10) for the larger inoculum. (Reproduced from White and Krishna, 1989.)
THE PATHOPHYSIOLOGY OF MALARIA 107

a shorter duration of illness before reaching dangerous parasite burdens, and


that young children begin the asexual phase of parasite development at a
higher parasitaemia, as they have a smaller blood volume in which to dilute
the merozoites liberated by hepatic merogony (White and Krishna, 1989).
For example, if one assumes that all the merozoites released at merogony
invade a red cell, then the combination of a single large viable sporozoite
inoculum, or multiple inocula (e.g. 100 successful parasites) and highly
efficient asexual multiplication (15-fold per cycle) in a young child (blood
volume 500 ml) would result in potentially lethal parasitaemias (250 000 per
pl) in only four cycles (8 days) from hepatic merogony. It is not surprising,
therefore, that the length of history of children who die from cerebral
malaria can be very short (Molyneux et al., 1989b).

1. Stabilization of parasitaemia

Most untreated episodes of falciparum malaria are not fatal. The log-linear
rise in parasite numbers is checked, and the host reaches an equilibrium with
the parasite burden, and later effects its removal from the body. With
repeated infections in areas of intense transmission, the level at which
parasitaemia stabilizes falls; also, the threshold for symptoms rises (Kitchen,
1949). Eventually parasitaemias become asymptomatic, a state known as
premunition. Concurrently the risks of unrestrained parasite multiplication
to lethal burdens declines. Several factors converge to limit parasite multi-
plication. These include increased splenic clearance function, the inhibition
of merozoite invasion by antibodies, the humoral immune response with
antibody opsonization of red cells and merozoites, various antibody-inde-
pendent cellular effector mechanisms, exhaustion of more receptive erythro-
cytes (certain red cells are more susceptible to invasion than others), and
non-specific activation of host defences leading to release of cytokines and
other toxic species that directly or indirectly damage parasites (White and
Krishna, 1989). However, equilibration of the parasite population can occur
in the absence of these factors. For example, a simple feedback mathemat-
ical model in which fever (temperature >40°C) inhibits merogony (Kwiat-
kowski, 1989) illustrates this tendency to equilibrate (Kwiatkowski and
Nowak, 1991)-but also, as in other biological systems, shows fundamental
instability (chaotic dynamics) of the parasite population when multiplication
rates are high. Such a system has a tendency to “overshoot”-i.e. the
number of parasites might become very large indeed before the feedback
factor reduces the population. The overshoot may prove fatal.
The benign human malarias have an inbuilt brake on their capacity to
multiply; P . vivax and P . ovule have a predilection for young erythrocytes,
whereas P . malariae is thought to invade mature cells only (Garnham, 1988).
P . falciparum also preferentially invades younger erythrocytes (Pasvol et al.,
108 N. J. WHITE AND M. HO

1980), but can invade cells of all ages-hence the propensity for untram-
melled multiplication to reach lethal parasite burdens (Field, 1949). P .
falciparum does not develop well in erythrocytes containing haemoglobin F
(Pasvol et al., 1976, 1977), which partly explains why malaria is rarely severe
in the first few months of life.

B. SYNCHRONICITY

1. Fever patterns

The synchronicity of malaria is such a characteristic feature of the infection


that the 2 or 3 day periodicity has been enshrined in the terminology of the
disease. If the infection is not treated and reaches a host-parasite equilib-
rium, fever spikes usually occur every 1 (quotidian), 2 (tertian), or 3 days
(quartan) (Kitchen, 1949).* Classically P . malariae infection shows a quar-
tan pattern, and the other three human malarias are tertian. The fever
is caused by “sporulation” (merogony). There is some evidence that
synchronous sequestration can also cause fever (G. Q. Li, personal com-
munication). Nowadays the classical malaria fever charts are rarely Seen
because the diagnosis is made early, and appropriate treatment is given
before there is time to document the fever pattern adequately. P . malariae,
P . ovule and P . vivax tend to synchronize earlier than P . falciparum and
intermittent “paroxysms” with chills and rigors are more common in the
initial phases of these infections. Indeed P . falciparum rarely shows regular
periodicity of fever early in the development of the infection-the pattern of
temperature is more usually erratic. Infections may also segregate into more
than one brood (James, S. P. et al., 1932), i.e. bimodal or multimodal
parasite age distributions. This may cause daily fever spikes (quotidian
fever) with alternating “sporulation”, or more complex fever patterns with
multiple broods. Although widely practised once, the validity of extrapo-
lating from the fever chart to the number of infecting parasite “broods” has
not been established fully. Overall, the fever pattern is seldom useful in
diagnosis.

2 . Rigors

Synchronicity is important in the pathophysiology of malaria as many of the


symptoms of the disease are related to synchronous rupture of meronts with
* The apparent terminological contradiction (tertian for two days and quartan for three)
arises from the early malariologists’ adoption of the Roman practice of counting the first day of
a series as day 1 rather than day 0.
THE PATHOPHYSIOLOGY OF MALARIA 109

the release of cellular debris and a variety of parasite proteins, glycoproteins


and glycolipids into the circulation. This may explain why, in non-immune
subjects, the symptoms of P. vivax malaria (highly synchronous) are often
worse than those of the less synchronous P. falciparum early in uncompli-
cated infections. The rigor characteristic of established untreated infections‘
consists of shaking and shivering, a sensation of coldness, irritability and
malaise, accompanied by “gooseflesh”, tachycardia and rapidly rising tem-
perature. This is followed by a “flush phase” in which the patient feels warm,
vascular resistance falls and there is often profuse sweating. Rigors are
unpleasant, but very seldom lethal. Contrary to conventional wisdom, they
are unusual in falciparum malaria. A glycoprotein (glycolipid in murine
malaria) with many of the properties of bacterial endotoxin is released at
merogony. This induces macrophages to release tumour necrosis factor
(TNF), the pivotal agent in the cascade of cytokine release (Kwiatkowski et
al., 1989). The rigor or “paroxysm” may result from a pulse of cytokine
release which causes systemic toxicity and resetting of the hypothalamic
thermostat.

3. Distribution of parasites in the body

The synchronicity of P. falciparum malaria determines the discrepancies


between peripheral blood parasitaemia and total body parasite burden
which result from deep vascular sequestration (see later) and often perplex,
and may mislead, the examining physician (White and Krishna, 1989; Davis
et al., 1991). Synchronicity determines the proportion of P. falciparum
parasites that are circulating in the bloodstream (i.e. are relatively harmless)
compared to those more harmful forms sequestered in the microvascu-
lature-i.e. the relationship between peripheral parasitaemia and the total
parasite biomass. Pathophysiological processes induced by sequestration
and its metabolic sequelae, or by rupturing meronts (cytokine release),
are proportional to the number of these unseen mature parasites (White,
1985), and are most evident in synchronous infections at the time when
the majority of parasites are in the pathological stage (i.e. sequestered).
Synchronicity is also relevant to the therapeutic response to antimalarial
treatment. The antimalarial drugs affect principally large ring and young
trophozoite stages of parasite development (i.e. the middle third of the 48 h
erythrocytic life cycle) (Yayon et al., 1983; Zhang et al., 1986; F. ter Kuae et
al., unpublished observations). Obviously, for drugs where blood concen-
trations fluctuate widely (e.g. chloroquine, artesunate), the greater the
proportion of parasites that are at the vulnerable stage of development at the
time of peak blood antimalarial drug concentration, the greater will be the
antiparasitic effect.
110 N. J. WHITE AND M. HO

C. PARASITE STRAIN DIFFERENCES

As with all pathogens, there are important biological differences between


different strains of malaria parasites. In the era of malaria therapy of
neurosyphilis, it was well known that parasites of the same species obtained
from different geographical locations differed in their infectivity to mosqui-
toes, antimalarial drug sensitivity, and virulence. For example, the European
strains of P . falciparum were infectious to the English Anopheles mosquito
( A . atroparvus), whereas African and Indian strains were not. The European
strains were also more dangerous, with a tendency to produce severe disease
rapidly (Shute and Maryon, 1954). In experimentally infected Aotus mon-
keys there are also considerable differences in virulence between strains of P .
falciparum (Schmidt, 1978). The intrinsic ability of a strain to cytoadhere
may be one virulence factor (Ho et al., 1991a). Intrinsic multiplication
capacity is almost certainly another (although several factors determine the
multiplication rate in vivo). Antimalarial drug resistance is an obvious
advantage to the parasite, as is the ability to change rapidly its repertoire of
antigens expressed on the red cell surface. Satisfactory typing methods which
correlate parasite strains with these putative virulence factors have yet to be
developed, and the relative importance of strain differences in the patho-
physiology of falciparum malaria remains to be elucidated.

VI. CYTOKINES
IN MALARIA
PATHOLOGY

The concept that the symptomatology of severe malaria may be mediated by


products of activated macrophages was first proposed by I. A. Clark et al.
(1981), based on the similarities between the features of severe malaria and
endotoxaemia. Over the subsequent years, much evidence has emerged to
support the idea that cytokines, in particular TNF, may indeed play an
important role in ctlusing some of the pathological changes that characterize
malaria.

A. ANIMAL STUDIES

1. Toxicity to the host

Most of the experimental data in support of a role for TNF in the


pathogenesis of cerebral malaria have come from studies of P . berghei
(ANKA strain) infection in CBA/Ca mice. The neurological signs which
develop in these mice result from an immunopathological reaction which is
strictly dependent on the presence of activated CD4+ T cells (Grau et al.,
1986). The syndrome is associated with elevated levels of circulating TNF,
and can be prevented by the administration of polyclonal antibodies to TNF
THE PATHOPHYSIOLOGY OF MALARIA 111

(Grau et al., 1987b,c). The treatment, however, does not influence the
parasitaemia, and protected mice eventually die of anaemia. Furthermore,
monoclonal antibodies to certain other cytokines, e.g. the combination of
anti-interleukin 3 and anti-granulocyte macrophage-CSF (anti-GM-CSF)
(Grau et al., 1988b), as well as anti-y interferon (IFN-y) (Grau et al., 1989d),
can also prevent cerebral symptoms through a reduction in TNF produc-
tion. The administration of recombinant TNF to mice infected with P .
berghei, and which are resistant to cerebral symptoms, apparently induces
the lethal neurological syndrome (Grau et al., 1989a,b,c). Interleukin (IL) 6
levels are also elevated in this model (Grau et al., 1990), but this cytokine
seems less directly involved in pathology; high IL-6 levels also occur in the
absence of cerebral pathology in animals infected with non-lethal P . yoelii
parasites, and anti-IL-6 antibody does not protect against the development
of cerebral symptoms.
The following sequence of events is proposed. In genetically susceptible
strains of mice, CD4+ T cell activation during acute malaria leads to the
production of cytokines which “upregulate” a variety of macrophage
functions, one of which is the release of TNF. Elevated TNF levels in the
circulation alter the surface properties of endothelial cells and cause the local
accumulation of leucocytes. These sequestered leucocytes in turn release
more TNF, thus amplifying the cytotoxic effects on endothelial cells, with
resultant vascular wall damage and haemorrhagic necrosis. In this murine
model, sequestration of leucocytes and monocytes is considered to be more
important than that of infected erythrocytes.
Cytokines have also been implicated in the pathogenesis of anaemia,
hypoglycaemia and pulmonary oedema in rodent models. Bone marrow
dysfunction results from TNF-induced dyserythropoiesis and erythrophago-
cytosis (Clark, I. A. and Chaudhri, 1988a; Miller, K. L. et al., 1989). If
recombinant TNF is infused into mice infected with P . vinckei, hypo-
glycaemia, midzonal liver necrosis and neutrophil adhesion in pulmonary
vessels occur (Clark, I. A. et al., 1990). These features are commonly seen in
terminal P . vinckei infection in association with high levels of circulating
TNF. Administration of TNF has also been shown to cause foetal death in
pregnant mice infected with P . vinckei (Clark, I. A. and Chaudhri, 1988b).

2. Toxicity to the parasite

In contrast to the deleterious effect of high levels of TNF on the host, small
doses of parenteral IFN-y and TNF have been shown to reduce parasitae-
mia in P . chabaudi adami (Clark, I. A. et al., 1987) and P . yoelii (Taverne et
al., 1987) infections. More recently, low doses of IL-I were found to protect
C57BL/6J mice against cerebral pathology induced by P . berghei and also to
reduce parasitaemia, although the two effects were separate (Curfs et al.,
112 N. J. WHITE AND M. HO

1990). These observations have led to the concept that low levels of
cytokines could be beneficial by exerting an indirect antiparasitic effect on
blood-stage parasites, but high concentrations of the same cytokines may act
in concert to produce toxic damage in the host. The antiparasitic effect is
known to require other serum components since recombinant T N F and
IFN-y are not directly cytotoxic to intra-erythrocytic parasites in vitro
(Taverne et al., 1987), and IL-1 has no effect when given to T cell-deficient
animals (Curfs et al., 1990). The additional toxic serum components remain
to be identified, although the killing of blood-stage P. falciparum in vitro by
serum containing T N F results mainly (> 70%) from the lipid peroxide
content (Rockett et al., 1988). These peroxides are formed by the interaction
of lipoproteins with reactive oxygen intermediates and are unaffected by
antioxidants. Malaria parasites are readily killed by free radicals (Malhotra
et al., 1988). Lipid peroxidation has the effect of stabilizing the reactive
oxygen groups, and thus creating a more stable cytotoxic molecule than
other oxygen radicals.

B. INDUCTION OF CYTOKINE RELEASE

Soluble antigens of P. falciparum, released into culture supernatants and


also found in the plasma of patients with acute malaria, have been shown to
induce T N F release from macrophages (Bate et al., 1988, 1989; Taverne et
al., 1990a,b). Two of these antigens, designated Agl and Ag7, are both
glycoproteins of molecular mass 60-70 kDa. Like bacterial lipopolysacchar-
ide (LPS), they react in the Limulus assay, and the activity of Ag7 is reduced
by polymyxin B. However, C3H/HeJ mice, which are hyporesponsive to
LPS, secrete T N F normally in response to these antigens (Riley et al., 1988),
suggesting interaction with a different macrophage receptor from LPS. They
appear to act directly on monocyte/macrophages without a requirement for
accompanying T cell activation. This is consistent with the observation that
supernatant preparations containing the two antigens do not stimulate
peripheral blood T lymphocytes during acute malaria (Theander et al.,
1986). A similar, T-independent TNF-inducing antigen (a glycolipid) has
been identified in rodent malaria parasites. Premunition probably represents
“tolerance” to these LPS-like substances-i.e. their release during malaria
infection stimulates progressively less cytokine release (Bate et al., 1990;
Playfair. et al., 1990). Thus “anti-disease” rather than “anti-infection”
immunity is induced (Riley et al., 1991).

C. HUMAN STUDIES

How relevant are these diverse observations in selected rodent models to


severe malaria in man? A positive association between plasma concen-
THE PATHOPHYSIOLOGY OF MALARIA 113

trations of TNF and mortality in severe falciparum malaria has been


observed in three studies (Grau et al., 1988b; Kern er al., 1989; Kwiatkowski
et al., 1990), but could not be confirmed in a recent study of children in Zaire
(Shaffer et al., 1991). In these studies TNF levels were correlated with several
indicators of severity, namely hypoglycaemia, hyperparasitaemia and
anaemia. Levels of IL-1 (Kwiatkowski er al., 1990), IL-6 (Kern et al., 1989)
and IL-8 (J. Friedland, personal communication) have also been shown to
be correlated with disease severity. In all these studies, there was consider-
able overlap in the range of cytokine levels in the different patient groups
(Fig. 8).

FIG.8. Relationship between plasma concentrations of tumour necrosis factor


(TNF) and disease severity in Gambian children with acute falciparum malaria.
(Reproduced with kind permission from Kwiatkowski et al., 1990.)

There are several problems with the cytokine story in human malaria.
First, cytokine levels are high in malaria due to P.vivax as well as that due to
P.fakiparum-but P. vivax does not kill. Second, there are differences
between the clinical manifestations of severe malaria and lethal septic
shock-a condition in which there is good evidence for cytokine-mediated
pathology (Parillo et al., 1990). Third, the clinical and pathological features
of so-called “cerebral malaria” in the P. berghei-infected mouse model
(where much of the evidence supporting a role for cytokines in malaria
pathology has been obtained) are quite different from those seen in human
disease. In humans, there are no haemorrhagic foci or focal intravascular
accumulations of large mononuclear cells, and endothelial cell damage is
114 N. J. WHITE AND M. HO

minimal. Furthermore, unlike P . fakiparum, P . berghei parasites do not


sequester in the brain. Indeed, use of the term “cerebral malaria” to describe
this neurological syndrome in rodents is potentially misleading. The pathol-
ogy in the P.vinckei-infected mouse model is also very different from that in
severe falciparum malaria. Third, it is now known that peak TNF produc-
tion occurs at merogony (Kwiatkowski et al., 1989) and the cytokine is then
cleared rapidly from the circulation with a half-life of 5-20 min (Michie et
al., 1988). A single measurement in blood is therefore of limited value.
Moreover, the commercial enzyme-linked immunosorbent assays used in
most studies to date measure both free biologically active TNF, as well as
TNF bound to circulating receptors. There is also evidence that the bioactive
receptor-binding moiety of TNF is an unstable non-covalently linked
oligomer that tends to form inactive mono- and polymers in solution. Both
oligo- and polymers are measured in current assays. These factors account
for the sometimes large discrepancies between the levels of immunoreactive
(usually high) and bioactive (usually absent) TNF (Petersen et al., 1989).
Finally, the pathological effects of TNF and other cytokines on the host
must also depend on the variable density of receptors on different target
organs, and individual variations in sensitivity.
TNF obviously plays a central role in cytokine-induced pathology, but
other cytokines are almost certainly involved too, and their roles may
become apparent only when assay methods become more reliable and more
widely available. The importance of cytokines in human malaria remains to
be determined. At present it seems that TNF could well be involved in the
pathogenesis of fever, hypoglycaemia and haemopoietic suppression in
malaria, is of uncertain relevance to pulmonary oedema, abortion and
circulatory failure, and is unlikely to cause coma in cerebral malaria. The
critical question of whether it kills patients directly remains to be answered.
These uncertainties may be resolved partly by a study of the therapeutic
effects of administering a monoclonal antibody against TNF to patients with
severe falciparum malaria.

OF VITALORGAN
VII. PATHOPHYSIOLOGY DYSFUNCTION

In severe malaria there is multiple organ dysfunction. Those organs with an


obligatory high metabolic rate are particularly affected.

A. CARDIOVASCULAR ABNORMALITIES

Despite intense sequestration of parasitized erythrocytes in the myocardial


microvasculature, pump function appears to be remarkably good in severe
THE PATHOPHYSIOLOGY OF MALARIA 1 I5

falciparum malaria (Sprague, 1946). Echocardiography usually indicates an


increased ejection fraction (derived from measurement of fractional shorten-
ing) (Charoenpan et al., 1990). Transiently reduced ejection fraction with
very high central venous pressure (which responded well to inotropes and
loop diuretics) has been reported (Le Camus et al., 1988), but this may have
resulted from volume overload. Most patients are admitted with an elevated
cardiac index (> 5 1 m-' min- ') with low systemic and pulmonary vascular
resistance, and low or normal right- and left-sided filling pressures (James,
M. F. M., 1985; White, 1986). Similar haemodynamic profiles are observed
in bacterial septicaemia, although hypotension is more prominent, and there
is clear evidence of a sepsis-induced depression of left and right ventricular
systolic function (Parillo et al., 1990). TNF is one of a number of circulating
low molecular weight myocardial depressant substances responsible for
these negatively inotropic effects in bacterial septicaemia. A detailed study of
ventricular performance in severe falciparum malaria would be of value in
differentiating the pathological effects of T N F in the two conditions.
Dysrhythmias are rare in severe malaria; 24-h electrocardiographic record-
ings are usually unremarkable even in ultimately lethal infections (F. Nosten
and N. J. White, unpublished observations). Blood pressure is usually at the
lower end of the normal range with sinus tachycardia. Orthostatic hypo-
tension is common (Butler and Weber, 1973; Kofi-Ekue et al., 1988) and may
be profound, particularly with high fever, even in otherwise uncomplicated
malaria. There is an associated failure of reflex cardioacceleration, suggest-
ing autonomic dysfunction. Postural hypotension is worsened by the quino-
line antimalarial drugs (W. Supanaranond et al., unpublished observations).
In very severe falciparum malaria, cardiac index and blood pressure may fall
secondary to metabolic acidosis, hypoxaemia and, in some patients, super-
vening Gram-negative septicaemia (Bygbjerg and Lanng, 1982).
Visceral perfusion is reduced in monkeys infected with P. knowlesi,
particularly at the time of merogony, and in some cases this is reversible by
a blockade (Skirrow et al., 1964). Vasoconstriction may contribute to the
reduction in vital organ perfusion in falciparum malaria, although there has
been no study in man to confirm this suggestion (Section VII.D.2).

B. ALGID MALARIA A N D SEPTICAEMIA

The rapid development of shock in severe malaria is termed algid malaria


(Sullivan, 1876; Gage, 1926). This is an enigmatic condition, and appears to
have several aetiologies. Shock may result from stress-induced gastrointesti-
nal bleeding (i.e. hypovolaemia), severe hypoxaemia and acidosis, or Gram-
negative septicaemia. Spontaneous bacterial septicaemia caused by Entero-
bacteriaceae or pseudomonads is an important cause of sudden clinical
deterioration in severe falciparum malaria (Bygbjerg and Lanng, 1982;
116 N. J. WHITE A N D M. HO

WHO, 1986). Endotoxaemia has been reported in severe malaria by several


groups, based on positivity in the Limulus lysate assay (Tubbs, 1980; Aung-
Kyaw-Zaw et al., 1988; Usawattanakul et al., 1985), but whether this
represents ‘‘leakage’’of endotoxin across the gut and past the hepatic barrier,
or measurement of the endotoxin-like malarial glycoprotein (Taverne et al.,
1990a,b), is uncertain. Septicaemia caused by Salmonella spp. is particularly
common in African children with P.falciparum infections (Mabey et al.,
1987), but this more often presents with protracted fever rather than shock.
Whether algid malaria exists as a discrete entity, i.e. primary hypotension
leading to secondary type A lactic acidosis, remains to be confirmed, but
most clinicians managing severe malaria have encountered patients who
develop hypotension rapidly and without apparent cause-and in whom
blood cultures are later shown to be sterile. The pathogenesis of this
condition is unknown, although it could result from pulse release of a large
amount of a vasoactive mediator such as T N F (Parillo et al., 1990) (Section
V1.C).

C. PULMONARY OEDEMA

Early workers considered that malaria caused pneumonia directly (Falconer,


1919), but the “intermittent pneumonia” they described was almost certainly
pulmonary oedema. Bacterial pneumonia may complicate severe falciparum
malaria (Spitz, 1946), particularly if there has been aspiration by a comatose
patient. The two conditions can be very difficult to distinguish clinically.
However, in most patients lung function and oxygenation are normal.
Adults with severe falciparum malaria may develop acute non-cardiogenic
pulmonary oedema at any stage of their illness (Bergin, 1967; Deaton, 1970;
Fein et al., 1978; Martell et al., 1979; Blanloeil et al., 1980; James, M. F. M.,
1985; Feldman and Singer, 1987; Bernadin et al., 1989; Charoenpan et al.,
1990). Pulmonary oedema commonly coexists with other vital organ dys-
function (Brooks et al., 1968). It is a particular problem of severe malaria in
pregnancy, but is rare in children. Hypoalbuminaemia, hyperparasitaemia
and renal failure are common associations. The development of frank
pulmonary oedema in malaria carries a mortality rate of approximately 80%
despite treatment. As in other circumstances in which the adult respiratory
distress syndrome occurs, the precise cause is not known (Gurman et al.,
1988). Whether increased pulmonary capillary permeability occurs more
commonly than is clinically manifest in severe malaria remains to be
determined. Certainly patients with severe malaria are very vulnerable to
fluid overload and have a low threshold for developing iatrogenic pulmon-
ary oedema (Hall et al., 1975). Hypoalbuminaemia is a contributory factor
by reducing the oncotic pressure of plasma, and thus lowering the pulmon-
ary capillary pressure threshold at which pulmonary oedema will develop.
THE PATHOPHYSIOLOCY OF MALARIA 1 I7

Ultrastructural studies of the lung have not helped to elucidate the


pathophysiology. Hyaline membrane formation in the alveoli suggests
leakage of proteinaceous fluid (Charoenpan et af.,1990). The alveolar septa1
walls may also be thickened. Cytoadherent parasitized erythrocytes may be
seen if the patient dies rapidly (Duarte et al., 1985; Corbett et al., 1989), and
large numbers of leucocytes may be seen both in the vascular lumen and
adherent to the vascular wall (Pongponratn et al., 1991). These are mainly
pigment-containing macrophages, although neutrophils are also evident
(MacPherson et al., 1985; Corbett et al., 1989). It has been suggested that
pulmonary capillary damage might be caused by release of toxic mediators
from these adherent mononuclear cells. In one ventilated patient, open lung
biopsy on the 11th day in hospital showed florid diffuse fibrosing alveolitis
(Feldman and Singer, 1987), but this is a common and non-specific sequel to
prolonged ventilation and secondary infection.

D. BLOOD FLOW AND METABOLISM

1. Cerebral bloodflow

Although the cardiac index is usually high, and systemic vascular resistance
is low in severe malaria (White, 1985), flow in some vital organs may be
reduced. In health, autoregulation ensures that blood flow to the brain
provides sufficient oxygen and substrates for metabolic demands. In 12
patients with cerebral malaria, cerebral blood flows were within the range
considered normal in healthy adults, but were considered inappropriately
low for the arterial oxygen content (i.e. oxygen supply) and the augmented
metabolic demands associated with fever and infection (Warrell et al., 1988).
The cerebral metabolic production of lactate was increased during coma, but
fell to normal on recovery of consciousness. In a separate study, CSF
concentrations of lactate were found to be elevated consistently in cerebral
malaria (White et af., 1985), and were significantly higher in fatal cases than
in survivors (Fig. 9). CSF lactate was inversely correlated with CSF
concentrations of glucose, and values also returned to normal with recovery
of consciousness. Similar findings have been reported in children (White et
al., 1987b; Molyneux et al., 1989b). These observations all indicate anaero-
bic glycolysis within the brain in cerebral malaria. This results presumably
from interference with microcirculatory flow (i.e. ischaemia causing
hypoxia), but in addition could reflect a flow-independent shift to anaerobic
respiration (e.g. inhibition of citric acid cycle activity by toxic moieties such
as cytokines or other secondary products). Obligatory anaerobic glycolysis
by the sequestered parasites also contributes to local lactate accumulation.
But coma in cerebral malaria cannot be explained simply by hypoxia;
118 N. J. WHITE AND M. HO

volunteers breathing low oxygen mixtures remain conscious with cerebral


metabolic rates for oxygen which are lower than those observed in cerebral
malaria (Kety and Schmidt, 1948). Other factors must contribute.

MeantSD 9.055.3 3.41tl.l

I4 0

.
a

12

a
10
a

8
a
CSF
Lactate
m moll1

# 6

4 a
a

--
2
NO R M A L RANGE

_c.
FATAL SURVIVORS CONVALESCENT

FIG.9. Cerebrospinal fluid lactate concentrations in cerebral malaria. (Reproduced


from White et al., 1985, with permission.)

2. Liver and skeletal muscle bloodjlow

In uncomplicated malaria, liver blood flow (LBF) is increased in parallel


with the rise in cardiac index, but in severe malaria flows are variably
reduced (Molyneux et al., 1989a) (Section VI1.L). In a recent study using
indocyanine green clearance as a measure of LBF, the median value was
THE PATHOPHYSIOLOGY OF MALARIA 1 I9


10.7 ml kg- min- in six fatal cases of falciparum malaria compared with

15.6 ml kg- min-’ in survivors. There was also a significant inverse corre-
lation between venous lactate concentrations and LBF at flows less than
’ ’.
15 ml kg- min- This suggests that reduced LBF could contribute towards
liver dysfunction and lactic acidosis in life-threatening infections (Pukrit-
tayakamee et al., in press). Recent studies of forearm blood flow and
metabolism in severe malaria indicate that a shift to anaerobic glycolysis
also occurs in skeletal muscle. However, there is no evidence that muscle
blood flow is reduced sufficiently to impair or retard intramuscular drug
absorption in severe malaria (White, 1985; Waller et al., 1991).
The increase in lactate production in these various organs cannot be
explained simply by reduction in measured total organ blood flows, and it
is unlikely to be explained solely by parasite glycolytic metabolism. This
suggests that either microcirculatory obstruction is variable or “patchy” (i.e.
low flow in some capillaries, high flow in adjacent vessels) or that the
cytoadherent parasitized erythrocytes interfere with gas and substrate
exchange and host metabolic functions but do not cause a large increase in
the resistance of the circuit. In either case it is likely that normal erythrocytes
can squeeze past the adherent parasitized red cells (White, 1986). Otherwise,
permanent ischaemic neuronal damage would be the rule rather than the
exception following cerebral malaria.

E. CEREBRAL MALARIA

1. Vascular and metabolic abnormalities

The cause of coma in severe falciparum malaria is not known. Although


there is ample evidence for disturbances of microcirculatory flow and
consequent anaerobic glycolysis, it is not clear whether this is sufficient to
cause unconsciousness. The importance of locally produced or circulating
toxic mediators (such as cytokines) is also unresolved. Ultimately it is likely
that a combination of impaired flow and metabolic derangement resulting
from the adherent highly metabolically active parasitized cells, and local
toxicity leads to a derangement of neuronal function. Whether there are
specific abnormalities of neurotransmitter release or sensitivity remains to be
determined. However, in the limited studies to date there have been no
consistent alterations in the pattern of CSF amino acids, and no therapeutic
responses to the use of benzodiazepine or opiate antagonists which would
point to involvement of a specific neurotransmitter pathway (N. J. White,
unpublished observations).
At post-mortem examination, multiple small haemorrhages are evident
120 N. J. WHITE AND M. HO

throughout the white matter of the brain (Spitz, 1946). Retinal haemor-
rhages are also commonly seen in cerebral malaria (Looareesuwan et al.,
1983a), particularly with the use of indirect ophthalmoscopy and fluorescein
angiography (E. Schulenberg, T. M. E. Davis and N. J. White, unpublished
observations) (Fig. 10). The microvascular damage in the connecting path-
ways appears to be related to sequestration rather than thrombus formation
and may contribute to nervous system dysfunction, seizures and possibly
residual sequelae.

2. Irnrnunopathology

The hypothesis that cerebral malaria results from an immune or allergic


vasculopathy has been raised by several pathologists (Rigdon and Fletcher,
1945; Schmid, 1974; Toro and Roman, 1978). The diffuse white matter
petechial haemorrhages, the glial reaction to these haemorrhages (Durck’s
granuloma) and the perivascular and parenchymatous oedema with sub-
sequent demyelination, have been graced by the terms perivenous allergic
encephalopathy and disseminated vasculomyelinopathy. In one study it was
even suggested that vascular permeability was so extreme that entire red cells
could escape through the capillary walls (00and Than, 1989).
In two recent pathological studies of human cerebral malaria (00et al.,
1987; Boonpucknavig et al., 1990), P.falciparurn antigens and immuno-
globulin G deposits have been demonstrated on the cerebral capillary
basement membrane. Although these findings were interpreted as represen-
ting immune damage, this conclusion should be treated with caution. In one
of the studies (Boonpucknavig et al., 1990), only three of the six fatal cases
had parasitized erythrocytes in the cerebral microvasculature (probably
because these patients died after being in hospital for many days), and in the
other study (00et al., 1987), half the patients had been ill for 9 or more
days. There was no evidence of associated vasculitis, and complement
deposition was seen in only one of 13 cases studied. In the ultrastructural
study of 22 fatal cases of cerebral malaria reported by MacPherson et al.
(1985), only one patient had been ill for more than 9 days and there was no
evidence of immune complex deposition. It is possible that the immunofluor-
escence studies showing immunoglobulin and malaria antigen deposition on
the vascular basement membrane are documenting terminal leakage rather
than a primary immunopathological process (Igarishi et al., 1987). The rapid
clinical course of human cerebral malaria, the lack of evidence of inflamma-
tion in pathological specimens, and the generally good prognosis in survivors
all argue against immune damage. Overall, the evidence that cerebral
malaria results from an allergic process is unconvincing.
THE PATHOPHYSIOLOGY OF MALARIA. 121

FIG. 10. (a) Retinal haemorrhages and exudates in cerebral malaria (courtesy of S.
Ward and Dr E. Schulenberg). (b) Fluorescein angiography of the retina; disruption
of the macular microvasculature in cerebral malaria (courtesy of S. Ward and
Dr E. Schulenberg).
122 N. J. WHITE A N D M. HO

F. CAPILLARY PERMEABILITY

1. Cerebral capillary permeability

On the basis of his autopsy observations, Rigdon (1944) concluded that the
primary pathological process in cerebral malaria was anoxia, that hypoxia
damaged the cerebral capillary endothelium and that this led to an increase
in capillary permeability. Maegraith and Fletcher (1972) later concluded,
from a series of studies on rhesus monkeys infected with P . knowlesi, that
increased capillary permeability. was the primary pathological process in
cerebral malaria. In terminally infected animals there was increased per-
meability to albumin labelled with ‘’’1 and fluorescein isothiocyanate, and
increased penetration of the brain by water-soluble dyes. This was rapidly
reversed by corticosteroids and the antimalarial drugs chloroquine and
mepacrine (Migasena and Maegraith, 1967; Maegraith and Fletcher, 1972).
Extravasation of plasma into the cerebral interstitium was considered to
account for cerebral oedema-a common post-mortem finding in cerebral
malaria. Reduced microcirculatory flow leading to cerebral hypoxia was
considered a secondary phenomenon attributed to local haemoconcen-
tration. It was proposed that the increase in cerebral capillary permeability
resulted from the local release of inflammatory mediators, originally thought
to be kinins (Onabanjo and Maegraith 1970a,b; Maegraith and Fletcher,
1972; Desowitz, 1987), but more recently suggested to be free oxygen
radicals (Clark, I. A. et al., 1986; Migasena and Areekul, 1987). As an
extension of this suggestion, the beneficial effects of the quinoline anti-
malarial drugs in cerebral malaria were thought to result from their anti-
inflammatory, rather than their antiparasitic, activity (Migasena and
Maegraith, 1967). The “permeability theory” was widely accepted, and
provided the theoretical basis for the use of corticosteroids and osmotic
agents to reduce cerebral oedema in human cerebral malaria.
The hypothesis that increased capillary permeability causes cerebral
malaria is no longer generally accepted. There are several reasons for this.
First, there are criticisms of the experiments on which the theory was based;
P . knowlesi infection in the rhesus monkey is clinically and pathologically
most unlike cerebral malaria in man. The identification of the vasoactive
kinins and their precursors relied on non-specific bioassays and, although a
permeability defect was identified, the blood/CSF albumin partition was
unchanged. This latter finding necessitated postulating increased bi-direc-
tional flux of albumin (i.e. exporting albumin from the CSF compartment
against a 100 : 1 concentration gradient), which is most unlikely. Second,
there is now considerable evidence from studies of human adults with
cerebral malaria (Looareesuwan et al., 1983b; Warrell el al., 1986) that
THE PATHOPHYSIOLOGY OF MALARIA 123

cerebral oedema is not a consistent or prominent feature of the disease in


life. Papilloedema is very uncommon in cerebral malaria (Looareesuwan et
al., 1983a). Opening pressures at lumbar puncture in adult cerebral malaria
are usually normal (Fitz-Hugh et al., 1944; Khan, 1945; Chipman et al.,
1966), and in fatal cases are significantly lower than in survivors (Warrell et
al., 1986). If there were significant brain swelling in all patients then, in the
absence of spinal block, compression of the subarachnoid space within the
rigid confines of the cranium would raise CSF pressure. The “anti-inflam-
matory” drug arguments cannot be sustained either. Chloroquine has no
effect on cerebral malaria caused by chloroquine-resistant P . falciparum, yet
reversal of the permeability defect in rhesus monkeys infected with P .
knowlesi was rapid (Maegraith and Fletcher, 1972). Furthermore the arteme-
sinin-related compounds (qinghaosu) are rapidly effective in cerebral
malaria, yet they are structurally unrelated to the “anti-inflammatory”
quinoline drugs. In a large prospective double-blind placebo-controlled
study, dexamethasone in moderately high doses had no effect on mortality in
cerebral malaria, but was shown to prolong coma, and was associated with
an increased incidence of superimposed bacterial infections (Warrell et al.,
1982). This was confirmed in a later study using very high doses of
corticosteroids (Hoffman et al., 1988). Finally, in a series of studies of
blood-brain barrier permeability in cerebral malaria (Warrell et al., 1986),
there was no consistent alteration in the partitioning of a variety of marker
substances (including 1251-albumin). These observations strongly suggest
that cerebral oedema is not the cause of coma in cerebral malaria (Looaree-
suwan et al., 1983b; Badibanga et al., 1986). Permeability may well be
increased in some patients. Brain swelling does occur terminally in adults
with cerebral malaria (Looareesuwan et al., 1983b), and is a common (but
not universal) finding at autopsy (Kean and Smith, 1944; Rigdon, 1944;
Spitz, 1946; Edington, 1954, 1967; Thomas, 1971; Riganti et al., 1990; T. T.
Hien, personal communication), but it does not appear to cause coma. It is
likely that cerebral oedema is an agonal phenomenon in many fatal cases.

2. Intracranial pressure

In children with cerebral malaria, lumbar puncture opening pressures are


generally lower than those observed in adults, but the “normal” range for
young healthy children is considerably lower than that in adults (Fig. 1 I).
Thus, in marked contrast to the findings in adults, the majority (80%) of
opening pressures in 40 Gambian children with cerebral malaria in one study
(Waller et al., 1991), and all those in a study of 26 Kenyan children (Newton
er al., 1991), were above the normal range. In this latter detailed prospective
I24 N. J. WHITE AND M. HO

.
..
300-
....
. .
.
200 -
..
... ...
.
.....
om

...
-
.. .
0..

100

..
0..

.. .. . .
0.

.
0- I III
Survivors Fatal cases Survivors Fatalcases
LAdUWS 1 L Chlldnn 1
FIG. 11. Opening pressures of cerebrospinal fluid (CSF) at lumbar puncture in
adults and children with cerebral malaria.

study, the clinical findings suggested rostro-caudal progression of brainstem


dysfunction in 9 of 12 fatal cases. This was interpreted as indicating central
tentorial herniation. However, herniation was also diagnosed clinically in
35% of surviving children. CSF findings were otherwise similar to those in
adults, which suggested that there was no significant increase in cerebral
capillary permeability. These findings raise the possibility that in children,
whose skulls offer relatively less space than those of adults for cranial
expansion, there may be raised intracranial pressure (RICP) without cere-
bral oedema. This could occur as a result of expansion of the intracerebral
blood volume by the sequestered parasitized erythrocytes and reflex vasodi-
latation to compensate for microvascular obstruction and local ischaemia.
When intracranial pressure is increased, blood pressure usually rises as a
reflex to maintain perfusion pressure (mean arterial pressure minus intracra-
nial pressure). However, blood pressures in children with cerebral malaria
are usually at the lower end of the normal range. A failure to maintain
THE PATHOPHYSIOLOGY OF MALARIA 125

adequate cerebral perfusion pressure will reduce cerebral blood flow and
may cause ischaemic damage. RICP and subsequent tentorial or foramen
magnum coning could explain the sudden respiratory arrests which appear
to be an important cause of death in childhood cerebral malaria. On the
other hand, the observations that lumbar puncture opening pressures are no
higher in fatal cases than in survivors (Waller et al., 1991), the frequency
with which neurological signs attributed to coning occur in survivors
(Newton et af., 1991), the lack of convincing evidence that lumbar puncture
causes neurological deterioration, and the excellent recovery in children with
very high opening pressures all argue against an important role of RICP in
the pathophysiology of cerebral malaria. These questions are unresolved at
the present time, but as RICP is a potentially treatable condition (with
osmotic agents such as mannitol), further studies are clearly needed.

3 . Systemic capillary permeability

Studies of 251-albumin disappearance and preliminary observations on


forearm swelling rates suggest that capillary permeability is increased in
falciparum malaria (Migasena and Areekul, 1987; Areekul, 1988), and that
this returns to normal on recovery. Approximately one-third of patients with
severe malaria have extravasation of albumin-bound fluorescein dye on
retinal angiography (although this is not correlated with the development of
cerebral malaria) (Fig. 10). Urinary microalbumin excretion (a measure of
renal capillary permeability) and the transcapillary escape rate for albumin
are increased in proportion to disease severity, but the magnitude of these
changes is not great (T. M . E. Davis and N. J. White, unpublished
observations). The increase in systemic permeability is seldom sufficient to
cause oedema in the acute phase of the disease, or significant albuminuria,
and probably has no pathophysiological consequence. It is likely that the
cerebral capillaries are more resistant to this process, having particularly
“tight” basement membrane junctions. Occasionally patients do develop
peripheral oedema following the acute phase of the illness, but this resolves
without specific therapy.
In summary, there is evidence for a moderate generalized increase in
capillary permeability in acute malaria, but this is usually symptomless, and
there is no convincing evidence that it causes cerebral oedema, coma or
other adverse effects in cerebral malaria,

G. FLUID SPACE CHANGES

Patients with severe malaria, particularly those who have been comatose
with high fever for more than 24h, may be significantly dehydrated on
126 N. J. WHITE AND M. HO

admission to hospital. However, following rehydration plasma volume is


increased in both moderate and severe malaria (Chongsuphajaisiddhi et al.,
1971; Davis et al., 1990d). Measurements of total body water and extracellu-
lar volume (sulphate space) have been normal (Feldman and Murphy, 1945;
Chongsuphajaisiddhi et al., 1971). Plasma renin activity, aldosterone and
antidiuretic hormone concentrations are elevated in acute malaria (Malloy
et al., 1967). These findings suggest activation of homeostatic mechanisms to
maintain an adequate circulating blood volume in the presence of genera-
lized vasodilatation and a falling haematocrit. Brooks et al. (1967) suggested
that homeostasis is incomplete, and that there is a reduction in “effective
blood volume” relative to the expanded capacitance of the dilated vascula-
ture which contributes to orthostatic hypotension in malaria (Section
VI1.A). By contrast, in the simian models used to study circulatory patho-
physiology in malaria, i.e. the rhesus monkey infected with P. knowlesi or
the sequestering tertian parasite P. coatneyi, plasma volume is contracted in
the acute phase of the disease, and this is associated with the development of
hypotension (Miller, L. H. et al., 1968).
Plasma osmolality may be reduced acutely but, together with the electro-
lyte abnormalities, this returns to normal with control of the infection.
Miller, L. H.’et al. (1967) studied the response to a water load in patients
with acute uncomplicated malaria who were mildly hyponatraemic, and had
normal glomerular filtration rates (assessed by inulin clearance) and renal
plasma flows (assessed by p-aminohippurate clearance). The responses were
variable, although urinary sodium concentrations remained low. Inappro-
priate secretion of antidiuretic hormone was suggested in this and a
subsequent study (Ogunye and Gbadebo, 1981). However, in more recent
studies the antidiuretic hormone response (measured directly) has been
found to be appropriate in acute falciparum malaria (R. E. Phillips ef al.,
unpublished observations).

H. ELECTROLYTE CHANGES

As in many infections, mild hyponatraemia and mild hypochloraemia are


common. More profound reduction in serum sodium may occur in severe
infections. This is associated with low urinary sodium and high urinary
aldosterone excretion (Brooks et al., 1967; Miller, L. H. et al., 1967). In
experimental malaria, sodium entry to the erythrocyte is increased and
sodium efflux is reduced (Dunn, 1969). Cation fluxes in other tissues have
not been studied. Infected erythrocytes also have elevated concentrations of
intracellular calcium, but concentrations in uninfected cells are within the
normal range (Krishna and Ng, 1989). Plasma concentrations of potassium
are remarkably normal in malaria, balanced between the opposing forces of
THE PATHOPHYSIOLOGY OF MALARIA 127

active haemolysis and possible tissue injury which tend to raise plasma
potassium, and activation of the renin-angiotensin system, and quinine-
induced insulin release, which tend to reduce it.

1. ENDOCRINE DYSFUNCTION

Serum thyroxine (T4) concentrations are reduced in many illnesses and


malaria is no exception. Serum concentrations remain low until after
parasite and fever clearance (Davis et al., 1990a). Very low levels may be
found in severe malaria, and in two patients with lethal infections serum T4
was unmeasurable (Davis et al., 1990a). Basal thyrotropin levels are normal,
but the response to thyrotropin releasing hormone is reduced, which
suggests depression of both pituitary thyrotroph and thyroid gland function
(Davis et af., 1990a). This pattern is commonly termed the “sick euthyroid”
syndrome. Sequestration or cytokine-mediated pathology could be respon-
sible, but the finding of elevated plasma concentrations of somatostatin
(>25 pmol 1-I) in cerebral malaria may also be relevant (Davis et al.,
1990a), as this hormone inhibits a broad range of endocrine functions. P.
falciparum has been shown to synthesize a somatostatin-like peptide in
amounts that could have a biological effect on the host (Pan et af., 1987).
Brooks et a f . (1969) studied pituitary-adrenal function in 29 adults with
acute falciparum malaria and concluded, on the basis of plasma and urinary
17 hydroxy and 17 ketosteroid concentrations and the metyrapone test, that
pituitary-adrenal function was normal. These are rather imprecise measures
and more subtle defects cannot be excluded.
Mineral homeostasis is perturbed in acute falciparum malaria (Petithory
et al., 1983; Lewis, 1987). Hypocalcaemia occurred in 36% and hypophos-
phataemia in 43% of patients in a recent series (Davis et al., 1991). Plasma
concentrations of magnesium were normal in the majority of cases. Pro-
found hypophosphataemia ( <:0.30 mmol 1- I) was associated with severe
disease, and this could be of pathological relevance. Very low phosphate
concentrations have been associated with confusion, muscle weakness,
depressed reflexes and focal neurological deficits, platelet and leucocyte
dysfunction, and reduced red cell deformability and oxygen carriage. The
major factor contributing to hypophosphataemia was a lowered renal
threshold for phosphate (Davis et al., 1991). Parathormone concentrations
were also inappropriately low for the corresponding plasma calcium levels,
indicating malaria-associated dysfunction of the parathyroid glands.

J. RENAL IMPAIRMENT

Acute renal failure is a major cause of death in adults with severe falciparum
128 N. J. WHITE AND M. HO

malaria. The presentation and recovery phase is that of acute tubular


necrosis (Sitprija et al., 1967; Stone et al., 1972; Sitprija, 1988; Lumlertgul et
al., 1989). Renal dysfunction is associated with cerebral involvement, high
parasitaemia, jaundice and haemoglobinuria (Canfield et al., 1968; WHO,
1986), and a high risk of pulmonary oedema. About half of all adult patients
with cerebral malaria will have biochemical evidence of renal impairment
(raised blood urea and serum creatinine) (White and Looareesuwan, 1987;
T. T. Hien, personal communication). Some of these will have a period of
oliguria during the acute phase (Sitprija, 1988), and about one-fifth will
become anuric. Over half the latter group will die from fulminant disease
with a combination of metabolic acidosis and pulmonary oedema. Renal
failure may also occur without an oliguric phase: i.e. urine volumes remain
normal or increase with a steadily rising blood urea and creatinine. In areas
of unstable endemicity, patients may present either with fulminant disease
and multiple organ failure (and a poor prognosis), or with established renal
failure following severe malaria or associated with haemoglobinuria. The
parasitic infection in these latter patients has been controlled by antimalarial
drugs and the subsequent management is that of the renal failure only. The
prognosis is then entirely dependent on the facilities available for dialysis. By
contrast with adults, renal failure is extremely unusual in young children
with severe malaria. Even biochemical evidence of renal dysfunction is rare,
and when present usually indicates severe dehydration.
Renal impairment, and ultimately acute tubular necrosis, in severe falci-
parum malaria presumably result from a reduction in renal microvascular
blood flow (i.e. ischaemic nephropathy). In some patients dehydration and
haemoglobinaemia (Blackburn et al., 1954) contribute to this process.
Studies of renal cortical flow using the '33Xe clearance technique (Sitprija et
al., 1977), angiography and contrast urography (Arthachinta et al., 1974)
indicate a reduction in cortical perfusion, but this is common in acute
tubular necrosis from any cause. Increased whole blood viscosity and
hypovolaemia were considered to account for these findings. Histopatholo-
gical examination shows cytoadherence in the glomerular capillaries, but to
a lesser degree than in the brain or heart.
Several pathological studies (Hartenblower et al., 1972; Bhamarapravati
et al., 1973; Futrakul et al., 1974; Boonpucknavig and Sitprija, 1979) have
also suggested that there is active glomerulonephritis, which would not be
surprising in view of the large amounts of malarial and host red cell
neoantigens confronting the glomerular filter. Immunofluorescence studies
show immunoglobulin deposition. Mesangial and endothelial cell prolifer-
ation can be seen by electron microscopy. However, the clinical and
laboratory features of the disease are most unlike acute glomerulonephritis
(Sitprija, 1988); hypertension does not occur, the urinary sediment lacks red
THE PAdHOPHYSlOLOGY OF MALARIA 129

cell casts, proteinuria is mild or absent, children are unaffected and in most
cases renal impairment is transient. It is possible that occasional patients
develop significant glomerular disease following acute falciparum malaria,
but this appears to be rare.
The relative roles of haemoglobin (Brant et al., 1951) and the large
amounts of erythrocyte and parasite debris released at merogony in the
pathogenesis of malarial renal failure are unresolved. Massive haemolysis
causing “black water” certainly causes renal failure both in acute malaria
associated with quinine treatment (Blackie, 1944), and also in some patients
with glucose-6-phosphate dehydrogenase deficiency who receive oxidant
antimalarial (or other) drugs (Section VI1.R). Originally, it was thought that
renal failure resulted from blockage of the renal tubules by haemoglobin and
related pigments, but studies of renal histology in humans argue against this
hypothesis (Maegraith and Findlay, 1944; Dukes et al., 1968). How quinine
and severe Malaria consort to induce massive haernolysis remains to be
discovered-but the end result is acute tubular necrosis. The natural history
of renal failure is determined initially by the overall severity of the infection
(approximately two-thirds of deaths associated with malaria renal failure
occur rapidly from multiple organ dysfunction), and then by the ability of
the medical facilities to conduct peritoneal or haemodialysis. Provided no
complications on dialysis ensue, most patients’ renal function will return to
normal over a period of several weeks (T. T. Hien, personal communi-
cation).

K. GASTROINTESTINAL DYSFUNCTION

Nausea and vomiting are common in malaria, particularly when fever is


high. Antimalarial drugs may be regurgitated and comatose patients may
aspirate stomach contents into the lungs with fatal consequences. In cerebral
malaria the vomitus often contains altered blood (“coffee grounds”), indi-
cating gastric or duodenal bleeding. This presumably results from stress
ulceration. Malabsorption of amino acids, sugars, fats and antimalarial
drugs have all been reported (Olsson and Johnston, 1969; Karney and Tong,
1972; Segal et al., 1974; Herzog et al., 1982; Molyneux et al., l989a)-
although oral absorption of antimalarial drugs is adequate in most patients
(White, 1985) except those who are seriously ill (and require parenteral
treatment anyway). Both sugars requiring active transport, and those
absorbed by passive diffusion, are malabsorbed in acute malaria, suggesting
impaired splanchnic perfusion (Molyneux et al., 1989a). Histological studies
show parasitized erythrocyte sequestration in the gut microvasculature
(Olsson and Johnston, 1969). Taken together these findings suggest reduced
perfusion of the intestinal microvasculature in falciparum malaria. Abdomi-
130 N. J. WHITE AND M. HO

nal pain with watery diarrhoea is a common presenting feature of acute


falciparum malaria in some areas, but is not seen in others, suggesting either
parasite strain differences or unidentified co-factors. The pathogenesis of
this condition has not been studied.

L. LIVER DYSFUNCTION

Jaundice, elevated transaminases, rapid development of hypoalbuminaemia,


prolonged coagulation indices, failure of gluconeogenesis and reduced
metabolic clearance of the antimalarial drugs all point to liver dysfunction in
malaria (Keys et al., 1950; Deller et al., 1967; Hills, 1971; Joshi, Y.K. et al.,
1986; WHO, 1990). However, these laboratory measures reflect multiple
pathology. Jaundice, which is much more common in adults, results also
from haemolysis, although this component is relatively small. Hepatocyte
injury and associated cholestasis are more important. The elevated transami-
nases may also result from skeletal muscle damage (Miller, K. D. et al.,
1989), and prolongation in prothrombin and partial thromboplastin times
from associated consumptive coagulopathy. Some adults may be deeply
jaundiced with abnormally elevated transaminases and relatively little evi-
dence of other vital organ dysfunction, but the combination of “hepatitic”
biochemical findings, renal impairment and coma indicating multi-system
failure carries a poor prognosis-as indeed it does in any condition. Acute
liver failure causing hepatic encephalopathy does not occur. Perhaps the
most sensitive marker of liver dysfunction in malaria is the impaired
elimination of drugs such as quinine (White et al., 1982), which are cleared
principally by hepatic biotransformation. Reduced metabolic clearance
parallels disease severity and returns to normal coincident with clinical
recovery.
In rhesus monkeys infected with P. knowlesi, angiography reveals vaso-
constriction of the portal tree (Skirrow et al., 1964). In severe falciparum
malaria, clearance of indocyanine green is variably reduced, whereas galac-
tose clearance is not (Molyneux et al., 1989a; Pukrittayakamee et al., in
press). Both are measures of liver blood flow, although the kinetics of
hepatic removal are different (Section VII.D.2). In uncomplicated malaria
clearance of both markers is normal or high. One interpretation of these
liver blood flow findings, as with the observations of cerebral blood flow in
cerebral malaria, is that microcirculatory obstruction is not uniform. Flow is
obstructed in some vessels, but is high in others. Parasitized red cells do
sequester in the portal and hepatic vasculature. There is often sinusoidal
dilatation with congestion of the centrilobular capillaries, slight hepatocyte
swelling, prominent Kupffer cell hyperplasia and pigment deposition, and
variable mononuclear cell infiltration (Srichaikul, 1959). There may be
THE PATHOPHYSIOLOGY OF MALARIA 131

centrizonal necrosis in some cases (Spitz, 1946; Clark, C. and Tomlinson,


1949). In the unfortunate patients who underwent liver biopsy (usually with
uncomplicated malaria), liver histology was remarkably normal (Corcoran
et al., 1953; de Brito et al., 1969).

M. HYPOGLYCAEMIA

Hypoglycaemia, commonly accompanied by lactic acidosis, is now recog-


nized as an important manifestation of falciparum malaria (Fisher, 1983;
Migasena, 1983; White et al., 1983; Kiire, 1986; Mbelepe et al., 1986; White
et al., 1987a; Currie and Kerau, 1988; Das et al., 1988; Taylor et al., 1988;
Molyneux et al., 1989a). Although hypoglycaemia may occur in any severe
infection, and is an important problem in malnourished children (Butler et
al., 1989; Bennish et al., 1990), it appears to be particularly common in
severe falciparum malaria, where it is associated with increased mortality
(Fig. 12). Hypoglycaemia arises as a result of several discrete processes.

100

80

Mortality
('A)

60

40

20

0
I I I I I
<1 1-2 2-3 A
Lactate I ~iucomratio

FIG. 12. Relationship between the ratio of admission venous plasma lactate to
glucose in 200 adults and children with severe malaria (mean and 95% confidence
interval).
132 N. J. WHITE AND M. HO

1. Iatrogenic hypoglycaemia

Quinine is an extremely potent inhibitor of pancreatic islet cell potassium


channel permeability. This effect is shared by glucose and stimulates insulin
release (Henquin et al., 1975; Henquin, 1979). Chloroquine inhibits insulin
degradation, but this does not alter glucose homeostasis significantly, and
there is no convincing evidence that chloroquine or other antimalarial drugs
(except for quinine and quinidine) cause hypoglycaemia (Phillips et al.,
1986b). Quinine stimulates insulin secretion in vitro and in vivo (White et al.,
1983; Okitolonda et al., 1986). In terms of free drug concentration, the
dextro-rotatory isomer quinidine is about half as potent a secretagogue as
quinine (Davis et al., 1990b). Insulin secretion is increased in all patients
receiving quinine treatment for falciparum malaria (White et al., 1983;
Taylor et al., 1988; Okitolonda et al., 1987; Das et al., 1988), but in most
there is also reduced peripheral tissue sensitivity to insulin (Davis et al.,
1990c), and the blood glucose is not reduced. Quinine-induced hypoglycae-
mia is very unusual on the first day of treatment. However, in quinine-
treated patients who are severely ill for a protracted period, hypoglycaemia
becomes increasingly likely. Pregnant women are particularly vulnerable to
hyperinsulinaemic hypoglycaemia because of an accelerated ketogenic res-
ponse to starvation (Metzger et al., 1982), and an amplified pancreatic islet
cell response to the secretory stimulus provided by quinine. They may
become hypoglycaemic with otherwise uncomplicated malaria. In African
children receiving parenteral quinine for severe malaria, insulin secretion is
stimulated, but hypoglycaemia results mainly from other factors (see below)
(Taylor er al., 1988). The biochemical features of quinine-induced hypogly-
caemia are raised plasma concentrations of insulin, lactate and alanine, but
low concentrations of ketone bodies (acetoacetate, P-hydroxybutyrate)
(White et al., 1987a).

2. Impaired gluconeogenesis

Severe malaria is associated with impairment of gluconeogenesis. Hypo-


glycaemia occurs in the presence of elevated plasma concentrations of the
principal gluconeogenic substrates alanine and lactate (White et al., 1983). In
patients receiving chloroquine (which does not stimulate insulin secretion),
plasma ketones, principally 3-hydroxybutyrate, are usually elevated and
plasma glycerol concentrations are normal. Plasma concentrations of tri-
acylglycerols and non-esterified fatty acids are elevated, as in other severe
infections (Angus et al., 1971; Onongbu and Onyeneke, 1983; Kawo et al.,
1990). This is the usual biochemical pattern in African children with severe
THE PATHOPHYSIOLOGY OF MALARIA 133

malaria (White et al., 1987b; Taylor et al., 1988; Molyneux et al., 1989b).
Fasting rapidly depletes hepatic glycogen in children (who have an average
12 h worth of stores) even if they are well nourished, and glycogen depletion
certainly contributes to hypoglycaemia in some cases, but not all
children are ketotic, and other factors are undoubtedly responsible.
Counter-regulatory hormone concentrations (i.e. growth hormone and cor-
tisol) are elevated (Taylor et al., 1988) and there is often concomitant lactic
acidosis. These biochemical features, together with hypoglycaemia, have
been described in other severe childhood infections (Phillips et al., 1988;
Kawo et al., 1990), but they are particularly common in severe P. falciparum
infections, occurring in approximately one-third of all children with cerebral
malaria.
In humans, clearance of the monosaccharide galactose is normal in severe
malaria (Pukrittayakamee et al., in press). This suggests that there may be a
specific defect in glycolysis (rather than a generalized reduction in glycolytic
enzyme activity) and that the biochemical locus of impaired hepatic gluco-
neogenesis may reside outside the small segment of the glycolytic pathway
between galactose and glucose.
Biochemical studies on isolated liver slices from rats with severe P.berghei
infections also suggest that there may be a specific defect in the glycolytic
pathway (P. A. H. Holloway and D. H. Williamson, personal communi-
cation). Hepatic gluconeogenesis from lactate is particularly impaired, and
cannot be explained entirely by the reduction in hepatic adenosine triphos-
phate concentrations. Hepatic ketogenesis is also reduced, but ketogenesis
from endogenous substrates shows a relative increase in hydroxybutyrate
synthesis.
Elevated plasma concentrations of the cytokine T N F are correlated with
hypoglycaemia and death in severe falciparum malaria (Grau et al., 1989a-d;
Kwiatkowski et al., 1990) (Section V1.C). T N F is a potent inhibitor of
hepatic gluconeogenesis (Evans et al., 1989), inducing many of the metabolic
derangements observed in malaria, and this cytokine could well be impli-
cated in the pathogenesis of hypoglycaemia in severe malaria.
Taken together, these findings point principally to an impairment of
hepatic gluconeogenesis which is proportional to the severity of malaria and
might be localized to specific biochemical steps in carbohydrate metabolism.
The counter-regulatory hormone response is appropriate and proteolysis
and lipolysis are unimpaired. There appears to be primary hepatocyte
dysfunction. This is compounded by reduced hepatic perfusion, increased
peripheral glucose consumption and lactic acid production, reduced or
absent glycogen stores, and-in some patients-uinine-stimulated hyper-
insulinaemia. The liver cannot clear the increased quantities of lactate and
alanine produced peripherally; hypoglycaemia and lactic acidosis result.
134 N. J. WHITE A N D M. HO

3. Glucose consumption

The overall consumption of glucose is increased in malaria (Davis et al.,


1988). The host is febrile, hypercatabolic and probably respires anaerobi-
cally in vascular territories occluded by parasitized erythrocytes. In addition,
there is an accelerated demand for glucose by the parasite which is pro-
portional to the stage of intra-erythrocytic development (Pfaller et al., 1982;
Jensen et al., 1983). Cells containing mature forms of the parasite may
consume up to 70 times as much glucose as their uninfected counterparts.
Most of this glucose is metabolized to lactate as the parasite does not have
the enzymatic machinery to complete the citric acid cycle (Sherman, 1979).
Glycolysis continues even in acid conditions that inhibit host glucose
metabolism (Sander et al., 1982). Thus, hypoglycaemia and lactic acidosis
may be much worse in the occluded microcirculation of vital organs than in
the systemic circulation overall. In the absence of precise methods for
calculating the size and synchronicity of the sequestered parasite biomass
(White and Krishna, 1989), the overall metabolic contribution of the
sequestered parasites cannot be assessed accurately. Rough calculations
suggest that, with heavy synchronous parasite burdens, parasite glycolysis
could double overall glucose turnover. This would be accommodated easily
by increased gluconeogenesis and glycogenolysis in a healthy host with
adequate glycogen stores, but in patients with severe malaria the combi-
nation of reduced supply (impaired gluconeogenesis and glycogenolysis)and
increased demand for glucose leads to hypoglycaemia. When quinine-
stimulated insulin secretion compounds the problem, hypoglycaemia often
proves difficult to correct because glucose replacement stimulates further
insulin secretion and a vicious cycle ensues. The disposition of administered
glucose in severe malaria is normal (Pukrittayakamee et al., 1991a),
although peripheral tissue insulin sensitivity is reduced (Davis et al., 1991).

N. LACTIC ACIDOSIS

Metabolic acidosis is an important cause of death in severe malaria.


Concentrations of lactate in arterial and venous blood and in CSF are
elevated in proportion to disease severity (White et al., 1983, 1985, 1987b;
Warrell et al., 1988; Molyneux et al., 1989b), and return to normal coinci-
dent with clinical recovery. Lactic acidosis is associated with hyperalaninae-
mia and hypoglycaemia. Measurement of venous or cerebrospinal fluid
lactate or the lactate/glucose ratio is a useful prognostic index (White et al.,
1986) (Fig. 12). In most patients with severe malaria, the anion gap widens
as bicarbonate falls, but hyperlactataemia is buffered. However, arterial pH
THE PATHOPHYSIOLOGY OF MALARIA 135

falls eventually if hydrogen ion production outstrips the buffering capacity


of the blood and the impaired ability of the kidney to excrete hydrogen ions.
Chemoreceptor triggering leads to increased respiratory drive. Kussmaul’s
breathing is an ominous sign in malaria. Hydrogen ion retention results
from increased production and reduced clearance of lactic acid, and in
adults this is often compounded by retention of other organic acids because
of renal failure.
Lactic acid arises from host and parasite anaerobic glycolysis (Zolg et al.,
1984). The contribution of the host in the form of L (+) lactate is the more
important. Approximately 7% of parasite lactate appears as the laevorota-
tory enantiomer D (-) lactate (Van der Jagt et al., 1990), but plasma
concentrations of this isomer are not elevated significantly in severe malaria
(S. Krishna and P. A. H. Holloway, personal communication). The healthy
liver and kidney are capable of clearing large quantities of L (+) lactic acid
from the blood and converting this either to energy (via the Krebs cycle) or
to glucose (via the Cori cycle), but in severe malaria compromised gluconeo-
genic function and reduced liver blood flow reduce lactate clearance (Section
VII.D.2). In certain circumstances (partial ischaemia), exogenous glucose
has been shown to fuel intracellular lactic acid production and worsen
acidosis (Gardiner et al., 1982), but studies in rodents and humans suggest
that glucose administration tends to ameliorate the condition in malaria
(Holloway et al., 1991; Pukrittayakamee et al., 1991). This is important
because patients with severe malaria are often treated with glucose empiri-
cally to prevent hypoglycaemia. In the young rat infected with P. berghei
lactic acidosis develops predictably as the infection worsens, and is associ-
ated with reduced gluconeogenesis and terminal hypoglycaemia (Holloway
et al., 1991). In this model lactic acidosis can be attenuated by dichloroace-
tate, an activator of the pyruvate dehydrogenase complex. This temporary
holding measure is now being evaluated in human patients.

0. SKELETAL MUSCLE ABNORMALITIES

The muscles are not painful in severe malaria, but there is biochemical
evidence of muscle damage (elevations in serum concentrations of myo-
globin and creatine kinase) which parallel disease severity (Miller, K. D. et
al., 1989). On histopathological examination, skeletal muscle is a site of vas-
cular sequestration, but muscle cell abnormalities are usually mild. Rarely,
rhabdomyolysis occurs (De Silva et al.,. 1988) and acute myoglobinuric
renal failure may develop. The quantitative contribution of skeletal muscle
(the largest tissue mass in most bodies) to the development of lactic
acidosis remains to be determined.
136 N. J. WHITE AND M. HO

P. ANAEMIA

Anaemia is an inevitable consequence of malaria, resulting from a combi-


nation of parasitized erythrocyte destruction at merogony, accelerated
removal of unparasitized red cells and ineffective erythropoiesis (Zucker-
man, 1966; Abdallah et al., 1980; Weatherall and Abdallah, 1982).

1. Oxygen delivery

Anaemia reduces the oxygen carrying capacity of blood. In order to


maintain oxygen delivery, blood flow increases and the haemoglobin oxygen
dissociation curve (ODC) is shifted to the right. The right shift in the ODC
results from increased intra-erythrocytic synthesis of 2-3-diphosphoglycerate
and causes increased unloading of oxygen at the tissues. In malaria both
compensatory mechanisms may be perturbed. There is microcirculatory
obstruction, and in rodents infected with P . yoelii there is a reduced right
shift in the ODC (Krishna et al., 1983). Oxygen carriage in humans has not
been studied directly.

2. Red cell destruction

Anaemia develops rapidly in acute malaria (Perrin et al., 1982), particularly


in severe P . falciparum infections (Fleming and Allan, 1969; Davis et al.,
1990d; Looareesuwan et al., 1991). The number of red cells lost by parasite
destruction is underestimated from the peripheral blood film because of the
unknown number of sequestered cells. Using a simple mathematical model,
Davis et al. (1990d) have analysed the differences between observed haema-
tocrit, and that predicted by labelling red cells with 'lCr to derive an
estimate of the sequestered red cell haematocrit in severe falciparum malaria,
and thus the contribution of these hidden cells to the subsequent develop-
ment of anaemia. These data gave a predicted sequestered cell haematocrit
twice that of the peripheral blood (70% vs. 35%). Plasma volume expansion
was estimated to contribute 7.5%, parasitized red cell destruction 6.3%, and
non-parasitized cell destruction 8.9% (i.e. approximately equal amounts), to
the average fall in haematocrit in the patients studied.
In acute malaria there is accelerated destruction of unparasitized erythro-
cytes associated with increased splenic clearance that persists beyond the
time of parasite clearance. Reduced survival of unparasitized red cells has
been documented both in acute malaria and following treatment (Charoen-
larp et al., 1979; Looareesuwan et al., 1987a,b). Survival is particularly short
in severe disease, when the haematocrit can fall precipitously in the acute
phase (Canfield, 1969; Phillips et al., 1986a; Looareesuwan et al., 1991).
Because of this obligatory red cell destruction, the haematocrit can be
THE PATHOPHYSIOLOGY OF MALARIA 137

considered as a “clock” of the infection. Thus, high parasitaemias without a


reduction in haematocrit in a well-hydrated patient imply rapid expansion of
the parasite population (i.e. a fulminant infection).

3. Role of antibody

The role of red cell-bound antibody (“Coombs positivity”) in the develop-


ment of anaemia has been contentious (Adner et al., 1968; Rosenberg, E. B.
et al., 1973; Facer et al., 1979; Facer, 1980; Weatherall and Abdallah, 1982;
Abdallah, 1986). Studies in Gambian children with falciparum malaria
suggested that erythrocytes were sensitized with immunoglobulin G (IgG)
and the C3 component of complement (Facer, 1980). But, when the
erythrocyte-bound antibody was measured in acute malaria, the distribution
was found to be normal with average values less than 200 molecules of
immunoglobulin per erythrocyte. There was no relationship between
changes in these numbers and the subsequent development of anaemia
(Merry et al., 1986). However, it may be argued that the red cells studied, i.e.
those remaining in the circulation, obviously represented those cells not
removed. Cells with greater antibody binding might have been cleared.
Increased splenic removal of immunoglobulin-coated red cells has been
demonstrated in malaria, even at relatively low levels of antibody sensitiza-
tion (Ho et al., 1990a). Thus a non-specifically activated phagocytic system
(Newsome, 1984) may remove cells which would otherwise be allowed to
continue in the circulation. An analogous process may contribute to short-
ened platelet survival. This increased splenic clearance function lasts for
weeks after resolution of the infection (Lee et al., 1989; Ho et al., 1990a).

4. Membrane abnormalities

Both parasitized and unparasitized erythrocytes have abnormal cell mem-


branes in malaria (Section 1V.B). There is increased surface expression of
phosphatidyl serine residues suggesting eversion of the inner surface of the
lipid bilayer (Joshi, P. et al., 1986). This process is analogous to accelerated
ageing, and may provide a target for autoantibodies normally present in
healthy subjects. Increased red cell surface expression of phosphatidyl serine
and senescent antigens, coupled with enhanced Fc receptor and filtrative
clearance processes, provide a plausible mechanism for the accelerated
removal of parasitized and unparasitized red cells.

Q. BONE MARROW FUNCTION

The bone marrow fails initially to respond to acute anaemia in falciparum


malaria. Reticulocytosis does not occur for many days, and then the
138 N. J. WHITE AND M. HO

response is often inadequate. Bone marrow, white cell and platelet produc-
tion is normal or increased. Preliminary data suggest that the erythropoietin
response in patients with normal renal function is usually appropriate (P. M.
Cotes, personal communication). Morphologically the bone marrow is
dyserythropoietic in both falciparum and vivax malaria (Srichaikul et al.,
1967; Abdallah et al., 1980; Knuttgen, 1987; Wickramasinghe et al., 1987,
1989). Iron is plentiful in the marrow, but serum iron is low and serum
ferritin is very high (Phillips et al., 1986a). In rodent malarias, cytokines,
particularly TNF, appear to contribute to bone marrow dysfunction (Clark,
I. A. and Chaudhri, 1988a; Miller, K. L. et al., 1989). In human malaria,
local cytokine release could be responsible for dyserythropoiesis, but-as in
the other situations where cytokine induced pathology is hypothesized-
definitive proof is lacking. In P.fakiparum infections there is some sequest-
ration of parasitized erythrocytes in the bone marrow, and it has been
suggested that this could cause local hypoxia (Wickramasinghe et al., 1987).

R. BLACKWATER FEVER

This enigmatic condition (Hamilton-Fairley and Bromfield, 1934; Blackie,


1944; Maegraith, 1948, 1952; Bruce-Chwatt, 1987) remains incompletely
understood. Objectively there is haemolysis and black (“coca-cola’’ col-
oured) urine (Barratt and Yorke, 1909-1910). When severe, the patient is a
pale, slate-grey colour. Acute renal failure may supervene. Blackwater may
be present in the absence of fever (or even of malaria) when patients with
glucose-6-phosphate dehydrogenase (G6PD) deficiency take oxidant anti-
malarial drugs (Gilles and Ikeme, 1960; Chan et al., 1976). This is a
predictable and well-characterized reaction. However, many patients with
black urine have normal erythrocyte G6PD levels (WHO, 1990). In those
patients quinine (which is not itself an oxidant) and malaria somehow
conspire to produce massive haemolysis. In most cases renal failure does not
occur, blackwater is transient, and the patient recovers. Renal failure is
particularly likely in severe malaria, and in this situation the mortality is
high. Quinine does not induce haemolysis with the patients’ red cells in
v i t r e a n d there is no evidence for anti-quinine antibodies or a drug-hapten
mechanism causing antibody-mediated haemolysis (A. H. Merry, personal
communication). The role of quinine metabolites in blackwater fever has not
been explored. Renal failure presumably results from acute tubular necrosis.
Haemoglobin itself is not nephrotoxic (Brandt et al., 1951; Conn et al.,
I956), but other erythrocytic proteins and cellular material can induce renal
failure, particularly if patients are dehydrated or acidotic (Hamilton-Fairley
and Bromfield, 1934; Blackburn et al., 1954).
THE PATHOPHYSIOLOGY OF MALARIA 139

S. THROMBOCYTOPENIA

The platelet count is usually low in malaria. A moderately reduced count (ca.
100 000 PI-') is observed in symptomatic infections with all four species
of human Plasmodium (Horstmann et al., 1981; Hill et al., 1964), but much
lower counts may be recorded, particularly in severe falciparum malaria.
Several hypotheses have been proposed to account for thrombocytopenia;
disseminated intravascular coagulation (Devakul er al., 1966; Dennis et al.,
1967), antibody-mediated clearance (Kelton et al., 1983; Grau et al., 1988a;
Srichaikul et al., 1988), and enhanced aggregation (Essien, 1989).
Bone marrow megakaryocyte numbers are normal in malaria, suggesting
adequate production of platelets. Increased turnover is suggested by obser-
vations of platelet enlargement (Fajardo and Rao, 1971), and studies of the
disposition of platelets labelled with 'Cr have confirmed accelerated peri-
pheral platelet destruction and splenic pooling (Sheagren et al., 1970;
Skudowitz et al., 1973). There is usually increased coagulation cascade
activity (Pukrittayakamee et al., 1989), but full blown disseminated intravas-
cular coagulation is rare and occurs in only 5% of cerebral malaria cases
(White and Looareesuwan, 1987; WHO, 1990); it is therefore unlikely to be
the sole cause of the reduced platelet count. Increased platelet-associated
IgG has been demonstrated in both vivax and falciparum malaria (Kelton et
al., 1983; Mohanty et al., 1988). Platelets were shown to have saturable
binding sites for malarial antigens, and it was suggested that malaria-specific
IgG bound to platelet-absorbed antigen via the Fab portion of the molecule.
However, in Thailand no relationship was evident between either free or
bound platelet-directed antibodies and either the absolute platelet count or
changes in the count (S. Looareesuwan and N. J. White, unpublished
observations). Platelets taken from patients with acute malaria have shown
increased ability to aggregate (Mohanty et a[., 1988). Some workers have
reported increased plasma concentrations of the platelet-specific proteins, p-
thromboglobulin and platelet factor 4, in uncomplicated malaria (Essien,
1989), but in other studies of severe malaria there was no evidence of platelet
activation (Supanaranond et al., in press). Thus there is contradictory
evidence on both hypotheses, and no firm conclusion can be drawn on the
relative importance of immune mechanisms or activation. Platelet antibody
is unlikely to play a significant role as the platelet count rises coincident with
the resolution of symptoms in malaria, but the other proposed mechanisms
require further study.
T. COAGULATION

There is considerable laboratory evidence of disseminated intravascular


coagulation in falciparum malaria (Conrad, 1969; Jaroonvesama, 1972; Reid
140 N. J. WHITE AND M. HO

and Nkrumah, 1972; Sucharit et al., 1975; Goodall, 1981; Horstmann and
Dietrich, 1985), but the clinical importance of these laboratory findings has
been overemphasized. The coagulation cascade is certainly activated in acute
malaria (Pukrittayakamee el al., 1989). Erythrocytes containing mature
falciparum malaria parasites promote coagulation (Udeinya and Miller,
1987). There is accelerated fibrinogen catabolism (Devakul et al., 1966) with
elevated serum fibrin degradation products, but fibrinogen concentrations
are usually raised even in severe malaria as part of the acute phase response.
Thus, in most patients, synthesis exceeds consumption (Jaroonvesama, 1972;
Looareesuwan et al., 1983b; Pukrittayakamee et al., 1989). Hypofibrino-
genaemia indicates significant consumptive coagulopathy and may be associ-
ated with a bleeding diathesis. In most patients antigen related to factor VIII
and von Willebrand factor concentrations are raised, and are correlated
directly with parasitaemia and inversely with the platelet count (Horstmann
and Dietrich, 1985). Plasma concentrations of antithrombin I11 (the natural
inhibitor of thrombin) are reduced in malaria (Pukrittayakamee et al., 1989),
and antithrombin 111-thrombin complexes are increased even in uncompli-
cated malaria. These changes closely parallel disease activity. Thus acceler-
ation of coagulation cascade activity is proportional to disease severity, but
only in a relatively few patients with severe disease does disseminated
intravascular coagulopathy with consumptive coagulopathy cause signifi-
cant bleeding (Borochovitz et al., 1970). An association between dissemi-
nated intravascular coagulopathy and the development of acute pulmonary
oedema has been suggested (Punyagupta et al., 1974), but the high incidence
of bleeding in the patients studied may have been more related to coexistent
uraemia and treatment with heparin, low molecular weight dextran and
steroids.
The importance of thrombus formation in the pathology of fatal malaria
is uncertain. The pathological interpretations of autopsy findings have been
contradictory. Several authors have reported finding microthromboses with
fibrin deposition, particularly in the cerebral vessels of patients who died
with cerebral malaria (Dudgeon and Clarke, 1917; Rigdon, 1944; Thomas,
1971; Schmid, 1974; Toro and Roman, 1978; Boonpucknavig et al., 1990),
but others have considered true thrombus formation to be relatively unusual
(Spitz, 1946; MacPherson et al., 1985) or not to occur at all (Gaskell and
Miller, 1920; Edington, 1967; Janota and Doshi, 1979). These latter authors
considered that the microvascular obstruction was not caused by thrombus,
but by “plugging” with a compressed or agglutinated mass of parasitized
cells and pigment (Dhayagude and Puranare, 1943; Kean and Smith, 1944;
Arieti, 1946; Spitz, 1946; 00et al., 1987). Occasional fibrin strands may be
seen, but electron microscopy reveals a “striking absence of platelets” in the
obstructed vessels (MacPherson et al., 1985). Overall these results suggest
THE PATHOPHYSIOLOGY OF MALARIA 141

FIG. 13. (a) Ultrastructure of the spleen: a parasitized erythrocyte (PE) is adherent
to the littoral cells (LC) of the splenic sinusoid. E, uninfected erythrocyte; K, knob; M,
mitochondrion of littoral cell; P, parasite. (b) Trapping of a parasitized erythrocyte
(PE) between two splenic littoral cells (LC). E, uninfected erythrocyte; P, parasite.
(Reproduced with kind permission from Pongponratn et al., 1989.)
142 N. J. WHITE AND M. HO

that microvascular thrombus formation may occur in cerebral malaria, but


it is neither widespread nor common.

U. SPLENIC FUNCTION

1. Filtration

The spleen plays a central role in the clearance of parasitized erythrocytes


(Garnham, 1970), recognizing their loss of deformability (Cranston et al.,
1984) and opsonization with antibodies and/or complement components.
The mechanical filtration function occurs at the inter-endothelial slits in the
walls of the splenic sinuses (Weiss et al., 1986; Weiss, 1990) (Fig. 13). In the
mouse infected with P . yoelii, a blood-spleen barrier is formed by contrac-
tion of the splenic reticular fibres during the phase of rising infection. At
crisis (spontaneous resolution of the infection) the barrier is reversed. This
anatomical alteration appears to parallel the changes in clearance of parasit-
ized erythrocytes. In rats infected with P . berghei, splenic filtration function
is impaired during the period of rising parasitaemia, but then becomes
supernormal as parasitaemia is controlled (Wyler et al., 1981). Similar
changes in splenic! filtration have been observed in patients with acute
falciparum malaria, using the clearance of heat-damaged erythrocytes as a
measure of filtration function (Looareesuwan et al., 1987a). These damaged
red cells are rigid and spherocytic, and are removed rapidly by the normal
spleen. In acute falciparum malaria removal was increased in patients with
splenomegaly. In patients without palpable spleens, removal increased
rapidly after antimalarial treatment.

2. Immune mechanisms

Antibody-coated erythrocytes are removed by Fc receptor-mediated inter-


actions with splenic macrophages. This function is also increased in acute
malaria (Lee et al., 1989; Ho et al, 1990a). Red cells coated with relatively
low numbers of antibody molecules (300-500 per cell) were removed rapidly
from the circulation and were localized by 51Crlabelling to the spleen (Ho et
al., 1990a). Clearance was correlated directly with anaemia (Fig. 14) and
inversely with parasitaemia, but (unlike filtration function) it was indepen-
dent of spleen size. High levels of circulating immune complexes may
compromise splenic interactions mediated by Fc receptors, but in these
studies there was no correlation between antibody-coated red cell clearance
and the level of circulating immune complexes.
As the intra-erythrocytic parasite grows, increasing amounts of parasite
THE PATHOPHYSIOLOGY OF MALARIA 143

and host neoantigens are exposed on the surface of the red cell, and more
antibody is bound (Luzzi et al., 1991). Once the cell has sequestered, it
cannot be removed by the spleen. Thus, there is a balance between the ability
of the spleen to remove the increasingly opsonized and rigid erythrocyte, and
the parasite's capacity to export cytoadherence proteins to the red cell
surface and effectively remove itself from the circulation. Although phago-
cytic activity is generally increased in uncomplicated malaria, there is
evidence that it is compromised in severe malaria (Ward et al., 1984). In this

.
situation, it is difficult to distinguish cause from effect.
50 -
0.

.
0

.
* .
40 -
.
0.

Hematocrlt 0 .

(%) *.

.
30 - 0
R=0.791
pco.001

20 , I I ,, I

10 20 30 40 " >40
tIn (hours)

FIG. 14. Relationship between haematocrit and clearance half-time (t,,J of erythro-
cytes coated with immunoglobulin G in acute falciparum malaria. (Reproduced from
Ho et al., 1990a.)

In acute infections, failure to augment rapidly both filtration and macro-


phage clearance functions would allow unrestrained parasite multiplication
to occur and severe malaria to develop. It is not clear which of these two
functions is the more important. Increased filtration, and particularly Fc-
mediated clearance function, persist for many weeks after acute infection
(Lee et al., 1989; Ho et al., 1990a), and almost certainly contribute to the
persistent shortened red cell survival time and the anaemia following acute
malaria (Looareesuwan et al., 1987b).
There have been few detailed ultrastructural studies of the spleen in
human malaria. In one case report (Pongponratn et al., 1989) of a 13-year-
old boy who died of acute renal failure 5 days after admission to hospital,
cordal macrophage erythrophagocytosis was prominent. The phagocytosed
parasitized erythrocytes contained mainly ring forms and young tropho-
zoites. Although red cells containing more mature parasites should be
preferentially cleared by the spleen, being both less deformable and more
likely to be opsonized, they also sequester and thereby escape splenic
removal. This balance between removal of infected cells by the spleen and
their cytoadherence to vascular endothelium is critical to the pathogenesis of
144 N. J. WHITE AND M. HO

malaria, as it determines the multiplication rate in vivo, and therefore,


ultimately, disease severity (Section V.A).

V. NEUTROPHILS AND EOSINOPHILS

The macrophage is the major immunological effector cell in malaria (Talia-


ferro and Mulligan, 1937), but neutrophils too play their part. The low
peripheral blood neutrophil counts observed in acute malaria have been
attributed to a shift to the marginal pool (Dale and Wolff, 1973). Neutrophil
counts may be raised in very severe disease, indeed neutrophilia indicates a
poor prognosis (Warrell et al., 1982; Stein, 1987; Molyneux et al., 1989a;
WHO, 1990). Neutrophils, like macrophages, can ingest free merozoites and
infected erythrocytes and commonly contain malarial pigment. In vitro,
neutrophils can be shown to be activated by co-incubation with malaria
parasites, and they can kill malaria trophozoites and meronts, probably by
singlet oxygen release (Nnalu and Friedman, 1988). Evidence for neutrophil
activation in vivo comes from observations that the proportion of circulating
neutrophils which give a positive reaction with nitroblue tetrazolium is
increased (Andersen, 1971), and that plasma levels of polymorphonuclear
leucocyte elastase (a granule protein) are increased in severe malaria (R.
Clemens and S . Pukrittayakamee, personal communication). Whether neu-
trophils cause direct tissue damage, such as that causing acute pulmonary
oedema, remains to be determined. Defective neutrophil and monocyte
chemotaxis has been reported in malaria (Nielsen et al., 1986) and patients
with severe malaria are vulnerable to bacterial urinary tract infections,
respiratory infections and septicaemias (Bygbjerg and Lanng, 1982; Khar-
azmi et al., 1987). In acute malaria the eosinophil count is consistently
suppressed (Davis et al., 1991), with a rebound eosinophilia in convales-
cence. Eosinophil secretory products inhibit P . fakiparum multiplication in
vitro (Waters, L. S . et af., 1987), but whether the eosinophil contributes to
malarial immunity remains uncertain.

W. IMMUNE DYSFUNCTION

Acute malaria is associated with both activation and suppression of the


immune system (Brasseur et af., 1983; Druilhe et al., 1983). Several studies
have shown alterations in peripheral blood lymphocyte subpopulations, but
this has not led to a clearer understanding of malaria immunology. This is
probably because the peripheral blood populations may not reflect those in
the spleen and liver, where much of the immunological activity takes place.

1. Immune suppression

In acute falciparum malaria there is impaired T cell responsiveness to


THE PATHOPHYSIOLOGY OF MALARIA I45

malaria-specific antigens. The immune unresponsiveness is manifested as a


defect both in proliferation to malaria-specific antigen in vitro (Ho et al.,
1986) and, in some cases, malaria-specific antibody production in vivo
(Webster et al., 1987). In severe disease, the immune unresponsiveness
extends to unrelated antigens (Brasseur et al., 1983; Druilhe et al., 1983), and
there is a tendency to develop supervening bacterial infections. Although
autoantibodies have been detected (Greenwood, 1968), it is unlikely that
they contribute to the pathology of acute falciparum malaria. Histologically,
there is a conspicuous lack of immunological damage in all the organs
examined from patients who die of the disease (MacPherson et al., 1985).
This does not, however, rule out an important role for soluble mediators
causing tissue damage.
The mechanism of the immune suppression in acute falciparum malaria is
complex. A defect in production of IL-2 and IL-2 receptor expression in
response to malaria-specific antigens has been demonstrated (Ho et al.,
1988). This might suggest an increase in suppressor T cell activity, but
although CD8+ and adherent suppressor cells have been reported in
immune individuals (Riley et al., 1989a,b), neither has been demonstrated
during an acute infection (Ho et al., 1986, 1988). It is now clear that within
the CD4+ subpopulation of T lymphocytes there are helper-inducer and
suppressor-inducer cells which are distinguishable by additional cell surface
+
markers. Alterations in the proportions of these CD4 cell populations
may contribute to the immune abnormalities in acute falciparum malaria, as
they do in visceral leishmaniasis (Cillari et al., 1991). In most patients, T
lymphocyte function returns to normal by 4-6 weeks after therapy.

2. A ct ivation

In acute malaria there is intense cellular immune activation, with markedly


elevated levels of circulating soluble factors such as the IL-2 receptor, CD8
antigen and IFN-)I (Ho and Webster, 1990). This immunological reactivity
probably results from non-specific T cell activation, since it fails to control
the developing infection, and may result in exaggerated production of
cytokines and subsequent immunopathology. Increased numbers of y/6 T
cells have been reported in both the peripheral blood (Ho et al., 1990b) and
spleen (Bordessoule er al., 1990) of patients with acute falciparum malaria. It
is not known whether they contribute to the pathophysiology of the disease.

3. Burkitt 's lymphoma

Burkitt's lymphoma, an uncontrolled monoclonal proliferation of B lym-


phocytes, is associated with both malaria and Epstein-Barr virus (EBV)
146 N. J. WHITE AND M. HO

infection. In areas of intense malaria transmission, Burkitt’s lymphoma is


the most common malignancy in childhood. EBV infection is acquired in
infancy and is widespread in the tropics. Progression of the infection is
normally controlled by virus-specific cytotoxic T cells. The EBV-specific
cytotoxic T cell response is significantly decreased (Whittle er a]., 1984) and
there is increased proliferation of EBV-carrying lymphocytes during acute
malaria (Lam et al., 1991). The resulting increase in virus-infected B cells
predisposes to cytogenetic abnormalities and the consequent development of
malignancy.

4. Hyper-reac t ive malarial splenomegaly

Hyper-reactive malarial splenomegaly (HMS), previously known as the


tropical splenomegaly syndrome, represents another aberrant immuno-
logical response to malarial parasites. In this condition, there is hyper-
gammaglobulinaemia and massive enlargement of the spleen which leads to
hypersplenism with anaemia, leukopenia and thrombocytopenia (Crane,
198I). There is increased susceptibility to infection and increased mortality.
Although malaria parasites are rarely demonstrable in the blood, the spleen
shrinks and symptoms resolve with effective antimalarial prophylaxis. The
hypergammaglobulinaemia is believed to be the result of polyclonal B cell
activation in the absence of adequate numbers of CD8+ suppressor T cells
(Hoffman et al., 1984), which are removed by an antibody-dependent
cytotoxic mechanism (Piessens et al., 1985). In a sub-group of HMS patients
who became resistant to antimalarial treatment, rearrangements of the
immunoglobulin gene have recently been demonstrated (Bates et al., 1991).
The finding suggests that the clonal lymphoproliferation in these patients
may evolve into a malignant proliferative disorder such as chronic lympho-
cytic leukaemia.

X. COMPLEMENT

1. Activation

Complement activation in malaria is proportional to disease severity (Adam


et al., 1981; Kidwai et al., 1986). Classical pathway activation predominates
(Petchclai et al., 1977; Phanuphak et a[., 1985). Cryoglobulins and circu-
lating immune complexes are detectable, and in one study peak levels of
immune complexes and C, breakdown products (e.g. C,d) were correlated
with thrombocytopenia (Adam er al., 1981). Hypocomplementaemia has
also been documented during the paroxysms of vivax malaria relapse (i.e. at
THE PATHOPHYSIOLOGY OF MALARIA 147

the time of merogony) (Neva et al., 1974). In both falciparum and vivax
malaria, concentrations of the later components of the complement pathway
were not reduced (Neva et al., 1974; Petchclai et al., 1977). Despite these
associations, there is no convincing evidence that complement activation or
immune complexes have a pathological role in acute malaria. In murine
malaria, immune complexes inhibit Fc receptor-mediated phagocytosis, but
there is no evidence for this in humans (Ho et al., 1990a).

2. Quartan nephropathy

Chronic immune complex deposition may cause the glomerulonephritis


associated with P . mafariae infections (quartan nephropathy) (Gilles and
Hendrickse, 1963; Allison et al., 1969; Hendrickse et al., 1977). In this
condition, children living in an endemic area present with nephrotic syn-
drome which progresses remorselessly to chronic renal failure. Antibodies to
P . malariae, and in some cases malaria antigen, have been detected by renal
biopsy (Houba, 1975). The histopathological findings vary considerably, but
there is some evidence that a coarse granular pattern of immunofluorescent
staining in the glomeruli is associated with response to cytotoxic therapy,
whereas patients with a fine granular pattern or linear staining do not
respond (Hendrickse et a f . , 1971). Why quartan nephropathy develops in a
few children, whereas the vast majority of those infected have no renal
pathology, is not known.

Y. PREGNANCY

Falciparum malaria adversely affects pregnancy (Archibald, 1956; Brabin,


1983; Nosten et al., 1991; Brabin and Brabin, 1992). In areas of intense
transmission, where symptomatic disease in adults is rare, these adverse
effects are largely confined to the foetus of the first pregnancy (Jelliffe, 1968;
McGregor et al., 1983). There is intra-uterine growth retardation and a
mean reduction in the weight of babies born to primigravidae of approxi-
mately 170g (Clark, H. C., 1915; Brabin, 1983). The placenta is a site of
preferential parasite sequestration (Bray and Sinden, 1979) and commonly
contains large numbers of mature parasites and abundant pigment (and is
sometimes black in gross appearance), even when the peripheral blood film
is negative. There may also be trophoblastic thickening (Galbraith et al.,
1980; Walter et al., 1982), macrophage infiltration and perivillous fibrin
deposition (Philippe and Walter, 1985; Anagnos et al., 1986), which presum-
ably interfere with transplacental exchange of substrates and metabolites
and reduce foetal growth. By electron microscopy, erythrocytes containing
mature parasites are seen free in the vascular spaces and not adherent to the
148 N. J. WHITE AND M. HO

endothelial surfaces (M. Aikawa, personal communication). How sequest-


ration occurs in the absence of cytoadherence is unclear. Placental para-
sitization is presumably present for many weeks or months in immune
women living in areas of intense malaria transmission.
In holo- or hyperendemic areas, there is an increased incidence of
maternal anaemia associated with falciparum malaria (Fleming et al., 1969;
Gilles et al., 1969; Greenwood et al., 1989), which on occasions may be
severe (Fleming, 1989), but most women are asymptomatic. This contrasts
sharply with observations in areas of unstable transmission (Menon, 1972;
Nosten et al., 1991), where infections with P . falciparum in non-immune
pregnant women tend to be severe (Bray and Anderson, 1979; Looaree-
suwan et al., 1985). Pregnant women with cerebral malaria have a case-
specific mortality rate over twice that of non-pregnant adults. Premature
labour and foetal death are common. In mesoendemic areas even transient
exposure to malaria (i.e. promptly treated disease) is associated with a
significant reduction in birthweight (150-180 g) in the first, second and third
pregnancies (Nosten et al., 1991).
The natural immunosuppression of pregnancy and the pre-partum level of
immunity are important determinants of disease severity. There is also
evidence of malaria antigen-specific cellular unresponsiveness in pregnancy
(Riley et al., 1989b). The notion that the placenta is an “immunologically
privileged” site (like the eye, brain and testes) has been used to account for
the localization of parasites there; i.e. effector mechanisms operating else-
where in the body which kill parasites do not operate in the placenta (Riley
et al., 1989b). Abnormal steroid metabolism, notably increases in plasma-
free cortisol, have been documented in Tanzanian women with malaria in
pregnancy (Vleugels et al., 1987), but whether this accounts for pregnancy
immunodepression is uncertain. In murine models, abnormalities in steroid
metabolism are correlated with immunodepression (Van Zon et al., 1982,
1985), and cytokines appear to be important in the pathogenesis of malaria-
induced abortion, but whether these observations can be extrapolated to
humans also remains to be determined.
Pregnant women with severe malaria have an increased risk of pulmonary
oedema and hypoglycaemia (White et al., 1983; Looareesuwan et al., 1985).
There is an accelerated metabolic response to starvation (Metzger et al.,
1982), and the pancreatic P-cell response to secretagogues such as glucose
(Spellacy and Goetz, 1963) and quinine is increased (Looareesuwan et ul.,
1985; T. M. E. Davis and N. J. White, unpublished observation). As a
consequence, pregnant women are particularly vulnerable to quinine-
stimulated hyperinsulinaemic hypoglycaemia (White et al., 1983).
THE PATHOPHYSIOLOGY OF MALARIA 149

VIII. CONCLUSIQN

In the past decade detailed studies of human malaria infections in different


age groups and geographical settings have reformulated our ideas on the
pathophysiology of the disease. The roles of cytoadherence, rosetting and
cytokine release have come to the fore, whereas the parts played by immune
damage, intravascular coagulation and increased vascular permeability have
receded. Clinical investigation has taken some of the mystery out of malaria,
but we still know relatively little for certain. The next challenge is to translate
these advances in our understanding of pathophysiology into improved
treatments.

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The Interaction of Leishmania Species with
Macrophages

JAMES ALEXANDER

Department of Immunology, University of Strathclyde, The Todd Centre, 31


Taylor Street, Glasgow, G4 ONR, UK

AND

DAVID G. RUSSELL

Washington University School of Medicine, Molecular Microbiology Depart-


ment, 660 South Euclid Street, Box 8230, St Louis, Missouri 63110, USA

I. Introduction . . . . . . . . . . . . . . . 176
178
179
B. Entry into the vertebrate . . . . . . . . . . . ................... 181
111. Outer Membrane Molecules of Leishmania . . . . . . . . . . . . . . . . . . . . . 181
A. Promastigotes . . . . . . . . . ............................. 181
188
191
191
B. Characterization of the parasitophor 196
C. Intracellular trafficking and the para vacuole ................ 199
200
200
V. Macrophage Heterogeneity . . . . . . . . . . . . . ............... 202
A. Genetic control of Leishmania infection ............. 202
207
VI. Antigen Presentation and the Induction of Immunity 207
A. Parasite interference with antigen-presenting cell 208
B. Antigen complexity and antigen-presenting cell heterogeneity . . . . . . . . . . . . 209
C. Processing of parasite antigen and activation of T cells 214
VII. Lymphocyte Control of Macrophage Anti-Leishmania Activity . . . . . . . . . . . . . . . 215
A. B and T cell involvement . . . . . . . . . . . . .............. 215
B. Lymphokines and macrophage activation . . . . . . . . . . . . . . . . . . . . 220
C. Macrophage activation and parasite killing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1

ADYANCES IN PARASITOLOGY VOL. 31 Copyright 0 1992 Academic Press Limited


ISBN 0- 12-03I73 I - I A// rights of reproduction in any form reserved
176 J. ALEXANDER A N D D . G. RUSSELL

VIII. Therapy and Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222


A. Therapy . . . . . . . . . . . . . . . . . . ...........
B. Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IX. Concluding Remarks ...................................
Acknowledgements ..................... ............... 231
References ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 23 I

I. INTRODUCTION

The leishmaniases comprise a group of diseases of major and increasing


public health importance. There is a reported incidence rate of some 400 000
new cases a year and 12 000 000 cases in total throughout the world. In India
during 1977-1 978 there were an estimated 20 000 deaths as a result of visceral
leishmaniasis caused by Leishmania donovani (Modabber, 1987). The genus
consists of many species which are epidemiologically diverse and complex.
Nevertheless, all species of Leishmania are transmitted by sandfly vectors,
either of the genus Phleboromus (in the Old World) or Lutzomyia (in the
New World), and it is generally accepted that they are all obligate intracellu-
lar parasites in their vertebrate hosts, where they are reportedly found within
macrophage phagolysomes (Alexander and Vickerman, 1975; Chang and
Dwyer, 1976). Although the forms found infecting macrophages, amasti-
gotes, are for the most part morphologically identical, the diseases produced
in humans display widely different clinical manifestations. This may depend
primarily on the species of parasite initiating the infection, but could equally
depend on the general state of health or age and, more particularly, the
genetic make-up of the host (Blackwell and Alexander, 1986). The major
species and subspecies of Leishmania causing disease in humans are summar-
ized in Table 1.
Although there are several reports of Leishmania parasitizing cells other
than macrophages or even existing extracellularly in mammalian tissue
(reviewed by Ridley, 1987), it is generally acknowledged that in the ver-
tebrate host these organisms normally reside within cells of the macrophage
lineage. The first description of this particular host cell-parasite interaction
can probably be attributed to Cunningham ( I 885), who examined biopsy
material, stained with gentian violet, from a patient with “Delhi boil”. Given
the now well-documented multi-faceted role of macrophages in the immune
response, the parasite’s interplay with its host cell poses many intriguing
problems for researchers. How, for example, do leishmanias survive inter-
action with a cell, a major function of which is to kill microorganisms using
a variety of oxygen dependent and independent mechanisms? Do they evade,
inactivate or withstand macrophage products and functions? Given that
macrophages, in their role as accessory cells, play a pivotal role in the
TABLE1 Major species of Leishmania causing disease in humans'

Species Subspecies Distribution Disease Tendency to self-cure


L. donovani L.d. donovani Old World VL/PKDL No
L.d. infantum Old World VL/CL No
L.d. chagasi New World VL No
L. major Old World CL Rapid
L. tropica Old World CL/LR Slow/no
L. aethiopica Old World CL/DCL Slow/no
L . braziliensis L.6. braziliensis New World MCL/CL NO/yes
L.b. guyanensis New World CL Yes
L.b. panamensis New World CL/MCL No/yes
L.6. peruviensis New World CL Mostly
L. mexicana L.m. mexicana New World CL Variable
L.m. amazonensis New World CL/DCL/VL Variable/no
L.m. pifanoi New World CL/DCL Variablelno

aModified from Alexander and Russell (1985).


CL, Cutaneous leishmaniasis; DCL, diffuse cutaneous leishmaniasis; LR, leishmaniasis recidivans; MCL, mucocutaneous leishmaniasis; PKDL,
post-kala azar dermal leishmaniasis; VL, visceral leishmaniasis.
178 J. ALEXANDER AND D. G. RUSSELL

initiation and regulation of lymphocyte proliferation, can leishmanias alter


macrophage functions to inhibit antiparasite inflammatory responses or,
conversely, do they induce macrophages to promote counter-protection and
disease exacerbation? Does the tissue predilection of different Leishmania
species reflect macrophage heterogeneity and parasite adaption to local
microenvironments? Can macrophages be activated to kill parasites? If so,
by what biochemical means are the parasites killed and what lymphoid cells
and cytokines are involved? What biochemical and phenotypic, as well as
morphological, changes take place in adapting from the insect or cell-free
culture to the intracellular environment? Does an understanding of these
interactions allow us to combat the disease by developing new drugs and
therapies or improving the aim of existing drugs? Can important parasite
antigens be identified which could form a basis for vaccination?
In this review, therefore, we intend to update the progress that has been
made in recent years in understanding the biology of the leishmania-
macrophage interaction and how the parasite can attach to and enter the
host cell and survive in the intracellular envirionment. We shall discuss the
implications of these interactions in the induction of specific immune
responses and consequently on disease progression. We shall also indicate
those areas where future research may be of value in the rational develop-
ment of new chemical and immunological therapies and vaccines.

11. LIFECYCLESTAGES
OUTSIDE
THE MACROPHAGE

The parasites found within the sandfly are particularly important as these, or
at least a subpopulation of them, initiate natural infection, and it is against
these forms that humans must be protected. Not only does the sandfly
midgut present the parasite with a totally different environment from that
within the macrophage, but throughout the length of the gut differing
conditions induce well-characterized morphological changes (reviewed by
Killick-Kendrick, 1979, 1990).
Following an infected blood meal, amastigotes enter the gut of the female
sandfly. Macrophages have never been reported in the sandfly midgut. Not
all amastigotes survive (Franke et al., 1987) and those that do seem to go
through at least one division before developing a flagellum (Warburg et al.,
1986). Whether a subpopulation of amastigotes is pre-adapted for life in the
invertebrate is a matter for speculation. As well as amastigotes, several
promastigote types and morphologically distinct paramastigotes have been
described as the parasites migrate forward in the sandfly gut (reviewed by
Killick-Kendrick, 1979). Within the midgut, two morphologically distinct
types of promastigote have been described; the long, thin nectomonad (body
INTERACTION OF LEISHMANIA WITH MACROPHAGES 179

length > 12 pm), which is free swimming or attached by interdigitation of


the flagellum to the ciliated epithelium of the midgut, and the short broad
haptomonad (body length < 12 pm), bound by hemidesmosome attach-
ments of the flagellum to the cuticular intima of the stomodeal valve
(Killick-Kendrick et al., 1974). As the parasites migrate into the pharynx,
they become round to oval, and the kinetoplast lies posterior to the nucleus.
These forms, paramastigotes, give rise in turn to the characteristic free-
swimming promastigotes found in the proboscis, which have a short body
length (10 pm), but a comparatively long flagellum (Fig. 1). The mechanisms
by which the parasites are induced to attach or detach and migrate through
the sandfly gut are unknown. However, promastigotes have been shown to
migrate along chemical gradients (Bray, 1983a) in a manner reminiscent of
the “running and tumbling” response of flagellated bacteria (Alexander and
Burns, 1983). They may be induced to do this by sugars normally stored in
high concentrations in the sandfly crop and released into the alimentary
canal just posterior to the pharynx (Bray, 1983a). It would seem likely that
promastigotes have as yet uncharacterized lectin-like receptors which modu-
late this movement as well as attachment.

FIG. 1. Light micrograph of Leishmania major metacyclic promastigotes stained


with Giemsa’s stain. The promastigotes were obtained from the proboscis of
Phlebotomus papatasi 12 days after an infected blood meal. Bar = 10 pm.

A. THE “METACYCLIC” PROMASTIGOTE

At what stage during promastigote migration and multiplication within the


sandfly the so-called metacyclic promastigotes develop and their mode of
transmission to the vertebrate host are subjects currently under detailed
investigation (reviewed by Killick-Kendrick, 1990). Many studies in vitro
have clearly established that promastigote infectivity for macrophages is
dependent on the parasite growth cycle: rapidly dividing log phase promasti-
180 J. ALEXANDER A N D D. G. RUSSELL

gotes have low infectitivy for host cells and animals, while stationary phase
promastigotes have comparatively high infectivity. This has been demon-
strated for L. major (Sacks and Perkins, 1984, 1985; Sacks et al., 1985;
Mallinson and Coombs, 1989a,b), L. donovani (Giannini, 1974; Doran and
Herman, 1981; Howard et al., 1987), L. mexicana (Mallinson and Coombs,
1989a,b) and L. braziliensis (Kweider et al., 1987). Changes in infectivity
have been associated with differential gene expression (Meade et al., 1989),
changes in cell surface characteristics (Sacks et al., 1985; Howard et al.,
1987; Sacks and da Silva, 1987; Cooper et al., 1988; Pimenta et al., 1989) and
biochemical properties (Doran and Herman, 1981; Mallinson and Coombs,
1986, 1989a,b). Infective stationary phase promastigotes of L. major (Sacks
et al., 1985; Sacks and da Silva, 1987) and L. donovani (Howard et al., 1987;
Cooper et al., 1988) lose the ability to bind the lectin peanut agglutin (PNA).
This is associated with a developmental modification of the surface lipo-
phosphoglycan (LPG) (Sacks and da Silva, 1987), which results in a
thickening of the glycocalyx from 7 nm in log phase promastigotes to 17 nm
in infective stationary phase promastigotes (Pimenta et al., 1989). Increased
promastigote infectivity in L. braziliensis and L. mexicana may also be
associated with an upgrading of the expression of the major surface
glycoprotein gp63 (Kweider et al., 1987; Russell and Alexander, 1988).
There are also quantitative and qualitative changes in enzyme content
between log and stationary phase promastigotes of L. donovani (Doran and
Herman, 1981), L. major and L. mexicana (Mallinson and Coombs, 1986,
1989a,b). In L. mexicana, isoenzymes and the content of certain amino acids
found in stationary phase promastigotes are normally associated with
amastigotes, suggesting that these forms are already partially pre-adapted
for life in the macrophage (Mallinson and Coombs, 1986, 1989a,b).
These stationary phase infective promastigotes, metacyclic forms as they
are invariably called, have a characteristic morphology (reviewed by Killick-
Kendrick, 1990), and promastigotes of similar morphology are found in the
thoracic midgut (Sacks and Perkins, 1984, 1985) and proboscis (Fig. 1) of
sandflies (Killick-Kendrick, 1986; Killick-Kendrick et al., 1988). While
proboscis forms have always been likely candidates to initiate infections
because of their close proximity to wounds (Adler and Theodor, 1931),
numerous authors have postulated that regurgitation during feeding from
more posterior parts of the gut is equally likely to facilitate transmission
(Davies et al., 1990; Sacks and Perkins, 1984, 1985; Warburg et al., 1989).
Using a monoclonal antibody (3F12) specific for metacyclic epitopes of L.
major LPG (Sacks and da Silva, 1987), Davies and coworkers (1990) have
identified metacyclic parasites in the pharynx, oesophagus and thoracic
midgut of P. papatasi 7-10 days after infection. No parasites were ever
found in the mouthparts, although infections could be transmitted to mice at
INTERACTION OF LEISHMANIA WITH MACROPHAGES 181

the second blood meal 6 days after infection. Expression of the metacyclic
marker recognized by monoclonal antibody 3F12 was, however, strongest in
the “short-stumpy” pharyngeal forms, indicating that paramastigotes could
also infect the vertebrate host, as suggested by Walters and coworkers
(1989a,b) studying L. chagasi and L. panamensis.

B. ENTRY INTO THE VERTEBRATE

On entering the vertebrate host promastigotes may activate complement via


the alternative pathway (Bray, 1983a) or the classical pathways (Puentas et
al., 1988). This not only creates a C5a gradient along which macrophages are
chemotactically attracted, but also produces a range of factors, including
opsonins and the components of the membrane attack complex, with which
the parasite must interact before entering the host cell to become the
characteristic small (3 pm), rounded amastigote. Although a certain number
of reports suggest that stationary phase promastigotes are resistant to
complement lysis (Franke et al., 1985; Russell, 1987a), as generally are
amastigotes (Bray, 1983a; Hoover et al., 1985a), other reports contradict
these (Mallinson and Coombs, 1989b). However, the presence of minute
quantities of insect saliva greatly enhances the infectivity of L. major
promastigotes (Titus and Ribeiro, 1988). Killick-Kendrick (1990) therefore
suggested that it would be advisable for in vitro tests of promastigote
complement sensitivity to be done in the presence of saliva.

111. OUTER MOLECULES


MEMBRANE OF LEISHMANIA

The amastigote and promastigote forms of Leishmania must be equipped to


survive in, and exploit, two highly contrasting biological environments,
namely the acidic intracellular vacuole in the amastigote and the milieu of
the insect midgut. Obviously, the topography of the parasite’s surface should
reflect its particular needs in these environments, and the following section
discusses the various constituents of the amastigote and promastigote
surfaces.
A. PROMASTIGOTES

The surface of the Leishmania promastigote is dominated by two highly


abundant glycoconjugates, the surface protease gp63 and the glycolipid
LPG. There are also some other surface-exposed enzymes that have been
identified, such as an acid phosphatase and both 3‘ and 5‘ nucleotidases, and
some minor proteins. This section discusses the biology of some of the better
known components.
182 J. ALEXANDER AND D. G . RUSSELL

1. gp63 in the promastigote

Early surface iodination attempts suggested that the surface of Leishmania


promastigotes was heterogeneous in relation to externally exposed proteins.
However, Etges et al. (1985) and Colomer-Gould et al. (1985) generated
surface iodination profiles indicating that a range of Leishmania spp.
possessed an extremely abundant conserved glycoprotein, designated gp63.
Bordier and colleagues showed that gp63 was retained in the membrane by a
phosphatidylinositol anchor, analogous to the structure identified on the
variant surface glycoprotein (VSG) of Trypanosoma brucei (Bordier et al.,
1986; Etges et al., 1986a). It was estimated that the promastigote possessed
500000 copies of the protein on its surface (Bordier, 1987); the density
achieved is approximately 6 x lo3 molecules per pm (Russell and Wright,
1988), or one-tenth that of VSG on T. brucei. T. brucei possesses a lipase
capable of cleaving the phosphatidylinositol anchor (Bulow and Overath,
1986), although its biological significance in the shedding of the VSG is
still controversial. No such activity could be detected in Leishmania (D. G.
Russell and M. A. Davitz, unpublished results); gp63 is glycosylated, and
these carbohydrate additions generate considerable heterogeneity at the level
of the mature protein (Chang et al., 1986; Russell and Wilhelm, 1986). Kink
and Chang (1988) demonstrated that glycosylation was, at least in part,
tunicamycin sensitive, and have recently, in collaboration with Olafson
(Olafson et al., 1990), published the structure of the carbohydrate additions
to L. amazonensis gp63.
During transformation of promastigotes into the infective metacyclic
form (Sacks and Perkins, 1984), the surface expression of gp63 increases
(Kweider et al., 1987; Russell and Alexander, 1988). Kweider and colleagues
( 1 987) described an isoform of the protein that appeared to be specific to
metacyclic promastigotes.
The most biologically significant property of gp63 was discovered by
Etges et al. (1986b), who demonstrated that gp63 from L. major possessed
protease activity. This activity survived sodium dodecyl sulphate gel electro-
phoresis, with optimum activity, on substrates used, on the alkaline side of
neutral. The authors showed similar enzymatic activity in a variety of
Leishmania species and isolates (Bouvier eta[., 1987). Chaudhuri and Chang
(1988) studied the proteolytic activity of gp63 in L. mexicana amazonensis;
however, they determined that the protease was most active at acid pH, and
proposed a role for gp63 in degradation of the complement component C3
on the promastigote surface. They produced further data (Chaudhuri er al.,
1989) supporting their contention that gp63 was an acid protease by
measuring bovine serum albumin (BSA) degradation at different pH values.
The demonstration that gp63 is a zinc-binding metalloprotease (Jongeneel el
INTERACTION OF LEISHMANIA WITH MACROPHAGES 183

al., 1989; Bouvier et al., 1990) was supported by identification of a consensus


zinc-binding site in the deduced amino acid sequence for gp63 (Button and
McMaster, 1988). Bouvier and co-workers (1990) argued that, by homology
with other metalloproteases, residue Glu,,, must remain in an unprotonated
condition for the enzyme to function. This would be unlikely at acidic pH.
More recent analysis of the activity of L. mexicana gp63 on synthetic
substrates might help clarify this issue (Ip et al., 1990). Although a pH
optimum value for a protease is a misleading concept due to pH-induced
alterations to different substrates, it could be argued that the use of synthetic
peptides affords a more accurate picture of enzymatic activity across a pH
spectrum. Peptide AKDSSILVTKKFA was cleaved by gp63 in two places,
the second cleavage site being detected only at high enzyme to substrate
ratio. The primary cleavage site was on the amino side of the serine residue,
and the secondary cleavage site was on the amino side of threonine. Similar
results were obtained with other peptides. The pH optimum of the endo-
peptidase activity was around pH 7.5, in keeping with the results of Bordier
and colleagues (1986). The apparent K, for digestion of the peptide referred
to above was 9.4 x lop4M, which is comparable to the values for other
proteases such as trypsin and chymotrypsin and their peptidyl substrates.
The significance of this surface-exposed protease in the biology of Leish-
mania is not clear. Reports of similar surface protease activity in phylo-
genetically rather distant trypanosomatids such as Crithidia fusciculata and
Leptomonas seymouri (Bouvier et al., 1987; D. G . Russell, unpublished
results) may contribute to an understanding of the evolution of the protease,
and are discussed in the next section.

2. Molecular biology of the gp63-encoding gene loci

Button and McMaster (1988) reported the cloning and sequencing of a


genomic clone that contained the sequence for L. major gp63, and later
published a corrected sequence (Button and McMaster, 1990). The deduced
amino acid sequence from this clone contained both pre- and pro-peptide
regions, and had a hydrophobic stretch of amino acids at its carboxyl
terminus which showed the characteristics of a phosphatidylinositol-process-
ing signal peptide. Recent structural analysis of the phosphatidylinositol
anchor shows that the protein cleavage and addition of the anchor glycan
occur at an asparagine residue 25 amino acids from the COOH terminus.
Further characterization of the gp63 genes in L . major (Button et al., 1989)
revealed that the gp63-encoding genes consisted of a family of six similar
coding regions tandemly arrayed at a single chromosomal locus. Hybridiza-
tion of chromosomes separated from a variety of Leishmania species by
orthogonal field alternating gel electrophoresis (OFAGE) and pulse-field
184 J. ALEXANDER AND D. G. RUSSELL

electrophoresis showed that the genes encoding gp63 were localized on


a similar-sized chromosome unit in all species examined. Northern blot
analysis of polyA+ ribonucleic acid (RNA) from both promastigotes and
amastigotes demonstrated a 3 kilobase (kb) RNA species in both, indi-
cating that the locus was constitutively transcribed, a finding now confirmed
by protein data (Medina-Acosta er al., 1989a). In contrast, detailed analysis
of the gp63 genes in L. m. mexicana revealed that the gp63 coding regions
differed, even within the genomes of single cloned parasites. Complementary
deoxyribonucleic acid (cDNA) clones isolated from L. m. mexicana could,
by differential restriction enzyme sensitivity and sequence data, be divided
into three distinct “families” (Medina-Acosta et al., 1989b). Analysis of the
genome showed that individual gene members of each family were present as
tandemly arrayed copies of at least 6,4 and 2 members, respectively. In view
of the heterogeneity found at the level of the protein, in amastigotes in
particular, we are trying to assess the extent to which the genomic hetero-
geneity dictates the heterogeneity found in the mature protein. Interestingly,
differences in the gp63 messenger RNA (mRNA) size has been reported
between virulent and avirulent L. chagasi promastigotes (Wilson et al.,
1989), suggesting that heterogeneity at this locus may extend beyond L.
mexicana.
Finally, similar surface proteases have also been described on more
phylogenetically distant trypanosomatids (Bouvier et al., 1987; D. G. Rus-
sell, unpublished observations). If such proteins represent homologues of the
leishmania1 surface protease, their primary structure would be extremely
informative with respect to function and evolution. Hybridization of pulse-
field separated chromosomes from C. fasciculata with the L. mexicana gp63
gene probe revealed two chromosomal bands. Clones have now been isolated
from C. fasciculata, and detailed Southern blotting and sequence analysis
support the supposition that these represent gp63-related genes (Russell et
al., 1991a). The existence of gp63 homologues in Crithidia may indicate that
the functional evolution of the protease gp63 pre-dated the invasion of the
vertebrate host, although this does not preclude gp63 from now having a
function within the vertebrate host.

3. Lipophosphoglycan

Lipophosphoglycan has been identified on the surface of all Leishmania spp.


studied to date. It was originally identified as the major constituent of the
“excreted factor” or shed material present in promastigote culture medium
(El-On et al., 1979). Handman and colleagues (1984) demonstrated that the
molecule was actually a glycolipid which could be released from the
promastigote surface. Subsequent structural analysis by Turco and co-
INTERACTION OF LEISHMANIA WITH MACROPHAGES 185

workers (Orlandi and Turco, 1987; Turco et al., 1987, 1989) revealed that the
molecule was tripartite, comprising repeated phosphorylated saccharide
units linked via a mannose-rich carbohydrate core to a lysoalkylphos-
phatidylinositol lipid anchor. More recent studies in other species (McCon-
ville et al., 1987; Sacks et al., 1990) have revealed that, although LPG is
represented on all species, its structure shows considerable polymorphism
primarily in the phosphorylated saccharide repeats. For example, the repeat-
ing units of L. donovani LPG are phosphorylated disaccharides of PO,-
6Gal( IH)Manl, whereas the repeats of L . major consist of tetra, tri- and
disaccharide units containing galactose, mannose, glucose and arabinose.
One of the most intriguing observations concerning LPG has come from
the work of Sacks and colleagues on the differentiation of the promastigote
form from a non-infective dividing cell in mid log phase, to a non-dividing
infective form in stationary phase culture (Sacks and Perkins, 1984). This
attainment of infectivity as a culture passes into stationary phase has been
shown to mirror the increase in infectivity detected in promastigotes within
the sandfly vector as they migrate out of the midgut and into the proboscis
(Sacks and Perkins, 1985). This differentiation is reflected in a profound
alteration in the structure of LPG in Leishmania spp. from the Old World
(Sacks, 1990; Sacks et al., 1990). Originally, this alteration was detected in L.
major promastigotes by the loss of binding of the lectin PNA, which
conveniently allowed direct isolation of the infected forms from culture
(Sacks et al., 1985). Sacks et al. (1990) have now characterized these
differences further, and shown that LPG from metacyclic forms has under-
gone changes in both the structure and number of phosphorylated repeat
units. The number of neutral hexoses per lipid molecule is virtually doubled,
indicating that the LPG from metacyclic forms is twice the size of its log
phase counterpart. The effect of such a change on the surface topography of
the promastigote has been graphically illustrated by Pimenta et al. (1989,
1991), showing the appearance of a glycocalyx on the surface of metacyclic
forms.
This change in the LPG structure has been shown to correlate with
important alterations in the parasite’s biology. It had already been noticed
that metacyclic promastigotes of L . major showed considerably more resist-
ance to lysis by complement than their log phase counterparts (Puentas et
al., 1988). Intriguingly, activation of complement by metacyclic forms
proceeds via the classical pathway even in the absence of antibody. Sacks
and colleagues (1990) have suggested that the elongation of LPG, which is
known to act as the predominant deposition site of C3b, effectively blocks
access of the later lytic components of the complement system to the
parasite’s membrane. Recent demonstration of released CS-9 complexes that
have fallen off the promastigote surface supports this proposal (Puentas et
186 J. ALEXANDER A N D D. G. RUSSELL

af., 1991). Finally, although the development of infectivity is closely corre-


lated with the elongation of LPG in both L. donovani and L. major, attempts
to detect a similar extension in the LPG of L . mexicana, which also
demonstrates metacyclogenesis, have failed. This suggests that development
of infectivity in Leishmania promastigotes extends beyond alterations solely
in LPG structure. The structural alterations in LPG on the promastigote
may also play a role in the binding of the parasite to different regions of the
sandfly digestive track (Sacks, 1990).
Lipophosphoglycan also contributes directly to infectivity in the macro-
phage. Handman et a f . (1986) found that addition of LPG to the surface of
a strain of L . major lacking LPG prolonged the survival of the avirulent
organism within the macrophage. McNeely and Turco (1987), McNeely et
al. (1989) and Chan et al. (1989) have shown that LPG is capable of both
inhibiting the activity of protein kinase C, similar to the phenolic glycolipid
from Mycobacterium, and acting as a scavenger of free oxygen radicals. Both
these properties could significantly contribute to the infectivity of the
parasite.

4. Membrane-bound acid phosphatase

Gottlieb and Dwyer (1981) first demonstrated the existence of phosphatase


activity with an acid pH optimum associated with the plasmalemma and
flagellar pocket of L. donovani promastigotes. The authors defined two
different enzyme activities by virtue of differential tartrate sensitivity. One
acid phosphatase remained associated with the promastigote plasmalemma,
while the other appeared to be secreted into the culture medium via the
flagellar pocket (Fig. 2). The secreted enzyme consisted of a highly glyco-
sylated protein of apparent molecular weight IlCL130 kDa and carried acid
labile phosphosaccharide additions that were immunologically cross-reactive
with LPG (Bates et af., 1990; Jaffe et al., 1990; Ilg et al., 1991). In contrast,
the membrane acid phosphatase is a dimeric complex of 65 kDa and 68 kDa
subunit size. Recent isolation and N-terminal sequencing of both enzymes
by Menz and coworkers (1 99 1) revealed related but non-identical sequences,
indicating that the enzymes are the products of at least two different genes.
The number of molecules of membrane-associated acid phosphatase is
relatively low, 16 000 per cell.
The work of Glew and colleagues (Remaley et af., 1985; Das et al., 1986;
Glew et al., 1988) has indicated that the tartrate-resistant membrane-bound
acid phosphatase of L. donovani is an extremely potent inhibitor of the
neutrophil superoxide “burst”. Obviously the ability of the promastigote to
suppress generation of reactive oxygen radicals would confer a strong
selective advantage on the invading parasite. The mechanism of action of the
INTERACTION OF LEISHMANIA WITH MACROPHAGES 187

phosphatase, however, is unknown. Study of its ability to dephosphorylate a


variety of substrates indicated that it is relatively inefficient with phospho-
proteins. The authors suggest that it may function through effects on
secondary messengers, although how this might operate across the macro-
phage membrane is unclear.

FIG.2. Electron micrograph of a promastigote of Leishmania mexicana (embedded


in Lowicryl). The section was probed with monoclonal antibody L3.13 directed
against the secreted acid phosphatase, followed by gold conjugated goat anti-mouse
antibody. The label is primarily concentrated in the region of the flagellar pocket,
which has been shown by Bates et al. (1989) to be the route of exit of the
glycoprotein. ( x 33 000)

5. Other identlJied promastigote outer membrane components

Although not so fully characterized or studied, a variety of other interesting


outer membrane proteins has been identified by several groups. Employing
cytochemistry, Dwyer and Gottlieb (1983, 1984) have shown the existence of
both 3' and 5' nucleotidase activity associated with the promastigote plasma-
lemma. Zilberstein and Dwyer (1 988) have applied similar functional
approaches to identify a proton-translocating ATPase molecule that shows
I88 J. ALEXANDER A N D D. G. RUSSELL

an activity profile comparable to similar molecules found in yeast and fungi.


These workers have also identified a glucose transport protein in the promas-
tigote outer membrane (Zilberstein et al., 1986). Cloning of a candidate gene
for this transporter protein has been reported by Stack et al. (1991),
although confirmation of the functional identity of the gene product is not
yet available.
Other promastigote membrane molecules have been described, but await
functional study. The most noted of these include the gp46/M2 family of
proteins found on L. amazonensis by McMahon-Pratt and colleagues (Kahl
and McMahon-Pratt, 1987; Lohman et al., 1990). These proteins are
promastigote specific, yet are capable of generating protective immunity
against promastigote challenge. One of the members of this gene family has
recently been transfected into L. major to study gene regulation and
differential protein processing (Lebowitz et al., 1990). Another protein
family of interest is the PSA-2 glycoprotein complex of L. major (Murray, P.
J. et al., 1989). These proteins were identified in the Triton X-1140 fraction
following detergent phase separation. This family of proteins contains three
members of 94, 90 and 80 kDa, all retained in the membrane by phosphati-
dylinositol membrane anchors.

B. AMASTIGOTES

Due to the relatively inaccessible location of the amastigote form, and the
comparative difficulty of completely isolating it from contaminating host cell
proteins, study of the surface-exposed molecules on the amastigote has
lagged behind that of promastigotes. However, some recent studies have
addressed the expression of the two major promastigote surface glycoconju-
gates on the surface of the amastigotes.

1. gp63 in the amastigote

Earlier studies on the expression of gp63 throughout the parasite’s life cycle
reported conflicting findings (Fong and Chang, 1982; Chang et al., 1986;
Colomer-Gould et al., 1985). A more recent study (Medina-Acosta et al.,
1989a) has resolved this issue. Monoclonal antibodies raised against L. m.
mexicana promastigote gp63 were screened against extract from metaboli-
cally labelled amastigotes. Two of the antibodies recognized one or more
proteins in the extract. Characterization of the recovered material by
enzymatic deglycosylation and chemical peptide mapping of immunopreci-
pitated material demonstrated that it shared a common peptide backbone
with promastigote-derived gp63 (Medina-Acosta et al., 1989a). Frommel et
al. (1990) recently demonstrated that antibody raised against recombinant
FIG. 3. Two-dimensional polyacrylamide gel electrophoretogram (2D-PAGE) of surface-labelled amastigotes; from
Medina-Acosta et al. (1989a). Autoradiographs from 2D-PAGE gels run with modified amastigotes isolated from infected
mice reveal that the major protein, of parasite origin, on the surface of lesion amastigotes is a gp63. (a) The amastigotes in this
gel were covalently modified with N-hydroxysuccinimide-sulpho-biotin (NHS-biotin), washed, fractionated by 2D-PAGE,
and transferred to nitrocellulose. The membrane was then probed with 1Z5[I]streptavidin,and autoradiographed. The most
abundant polypeptide is host actin (open arrow). The other arrowed protein is amastigote gp63, in the form present on the
surface. (b) Amastigotes metabolically labelled for 3 h with 3s[S]methionine,covalently modified with NHS-biotin and then
detergent extracted. The metabolically labelled modified polypeptides were recovered with streptavidin agarose, removed with
2-mercaptoethanol and separated by 2D-PAGE. By this procedure parasite surface proteins can be discriminated from those
of host origin. The most abundant amastigote surface protein (arrowed) has been shown immunologically to be amastigote
gp63.
190 J. ALEXANDER AND D. C. RUSSELL

gp63 from Escherichia coli recognized a polypeptide expressed in L. major


amastigotes. Two-dimensional gel analysis of the total exposed proteins of
amastigotes is shown in Fig. 3.
However, differential labelling procedures of amastigote gp63 generated
different two-dimensional gel profiles. The pattern from immuno-isolated
gp63 from surface-iodinated amastigotes was appreciably simpler than gp63
from metabolically labelled amastigotes (Medina-Acosta et al., 1989a). In
addition, metabolic labelling of amastigote gp63 with [3H]myristic acid,
which is incorporated into the phosphatidylinositol membrane anchor,
revealed only the isoform of amastigote gp63, which was susceptible to surface
iodination. These results suggested that the majority of isoforms of gp63
in amastigotes were not surface exposed, and lacked stable addition of
the phosphatidylinositol membrane anchor. Analysis of amastigotes by
immunofluorescence and immunoelectron microscopy indicated that the gp63
inaccessible to surface procedures was predominantly concentrated in the
lumen of the flagellar pocket. The flagellar pocket of all trypanosomatids
appears to be a specialized area for mediating secretion and endocytosis, e.g.
the receptor-mediated endocytosis of low density lipoprotein in T. brucei
(Coppens et dl., 1987, 1988; Lee et al., 1990) and secretion of acid phospha-
tase in Leishmania (Bates et al., 1989). The function of gp63 in the flagellar
pocket may be to degrade host macromolecules, either to protect sensitive
components of the flagellar pocket membrane or for nutrition for the
amastigote.

2 . Lipophosphoglycan in the amastigote

As with gp63, the existence of LPG in the amastigote form of Leishmania


has been the subject of conflicting reports. Early studies with the aid of anti-
LPG monoclonal antibodies described the existence of LPG in and on
infected macrophages (Handman and Hocking, 1982; Handman, 1990). The
results obtained from these studies could be questioned because, firstly, they
all employed promastigotes as the infective agent and did not adequately
control for residual promastigote LPG persisting in the cell. Secondly, it is
now known that elements of the acid-labile carbohydrate repeat units on
promastigote LPG are also added to other glycoconjugates, such as the
secreted acid phosphatase (Bates et al., 1990; Jaffe et al., 1990; Ilg et al.,
1991; Steirhof et al., 1991). However, a more definitive study by Turco and
Sacks (1991) demonstrated the existence of a glycolipid with a phosphatidyl-
inositol-specific phospholipase C-sensitive membrane anchor and acid-
labile phosphosaccharide repeat units, comparable to the promastigote
LPG. Although similar in theme, the structure is noticeably different,
promastigote LPG possessing multiple small repeat units, whereas amasti-
INTERACTION OF LEISHMANIA WITH MACROPHAGES 191

gote LPG has fewer larger units that show a reduction in overall anionic
charge. This difference is supported by the description of promastigote LPG-
specific epitopes.
With respect to the biology of the amastigote LPG, while promastigote
LPG on infective L . major metacyclic forms develops a detectable glyco-
calyx, no such coat could be found on the amastigote surface (Pimenta et al.,
1991). Attempts to surface-label the LPG by ~eriodate-NaBr[~H] before
isolation yielded little recoverable label (Turco and Sacks, 1991). So,
although amastigotes must also survive exposure to serum components and
enter macrophages, the protection of the LPG glycocalyx does not appear to
have been retained. Properties of the amastigote LPG require further study.

IV. PARASITIZATION
OF THE MACROPHAGE

All Leishmania spp., regardless of the disease syndrome resulting from the
infection, parasitize members of the host’s mononuclear phagocyte system.
The problems inherent in exploiting the macrophage fall into three main
categories: first, identification and entry into the “chosen” cell type; second,
survival within a cell that has evolved to kill invading microbes; and third,
long-term survival within an antigen-presenting cell.

A. HOST CELL ENTRY

Although binding and internalization of Leishmania promastigotes can be


shown in all phagocytes, they survive only in macrophages and the less
mature monocytes. The subsequent success of a Leishmania infection is
dependent on the ability of the parasite, initially in the promastigote form
and later as the amastigote, to adhere specifically to and to enter macro-
phages (Figs 4, 5). Several publications in the early 1980s indicated that the
interaction of promastigotes with macrophages is a receptor-mediated event.
Given the required specificity of attachment, coupled with the aggressive
behaviour associated with the interaction of some ligands, such as cross-
linked immunoglobulin, the nature of receptors involved may both limit the
cell type adhered to and influence the outcome of invasion. Interpretation of
studies of the interaction between the promastigote and the macrophage are
complicated by three discrete issues. Firstly, Leishmania represents a com-
plex of different species with demonstrated differences in their surface
exposed molecules. Secondly, the promastigote population cannot be
regarded as homologous since the demonstration by Sacks and Perkins
(1984, 1985) that the parasite differentiates from a non-infective to an
infective form both in culture and during migration from the sandfly midgut
192 J. ALEXANDER AND D. G . RUSSELL

FIG.4. Scanning electron micrograph of a Leishmania mexicana promastigote


during internalization by its host cell, a macrophage. The phagocytosis “trumpet”
can be seen extending down the promastigote’s body as it enters the macrophage.
( x 5000)

to the salivary glands. As discussed, in L. major this differentiation is


accompanied by a drastic alteration in the structure of the major surface
glycoconjugate, LPG (Sacks et al., 1990). Finally, although the majority of
experiments into the promastigote-macrophage interaction have been con-
ducted in the absence of serum, in vivo the involvement of components of the
complement system cannot be ignored.
Early studies showed that, in the absence of serum, both the major surface
molecules, gp63 and LPG, of the promastigote could bind to the macro-
phage (Handman and Goding, 1985; Chang and Chang, 1986; Russell and
Wilhelm, 1986; Wilson and Hardin, 1988). Experiments conducted with
INTERACTION OF LEISHMANIA WITH MACROPHAGES 193

intact promastigotes implicated a range of receptors in the attachment


process. These included the receptors CR3, for the C3bi fragment of C3
(Blackwell et al., 1985b; Mosser and Edelson, 1985; Wozencroft et al., 1986),
the mannose fucose receptor (Blackwell et al., 1985b; Wilson and Pearson,
1986) and the advance glycosylation end product receptor (Mosser et al.,
1987). More recent studies with isolated promastigote surface moieties have
helped to elucidate some of these interactions.

FIG. 5 . Transmission electron micrograph of a Leishmania mexicana amastigote


being phagocytosed by a peritoneal macrophage in vitro. The macrophage pseudo-
podia can be clearly seen extending around and enveloping the parasite (arrows).
Bar = I l m .

Rizvi and colleagues (1988) reported that gp63 exhibited some structural
similarity to the Arg-Gly-Asp cell-binding domain of fibronectin. Peptides
based on this sequence inhibited binding of promastigotes of L. chagasi to
macrophages. In another study published shortly after, Russell and Wright
(1988) demonstrated that isolated gp63, reconstituted on to the surface of
C18-derivatized silica beads, bound to macrophages as a function of the
density of incorporated gp63 (Fig. 6). Interaction of these gp63-beads with
macrophages was blocked if the macrophages were plated on to plastic
coated with anti-CR3 monoclonal antibodies. Binding was also inhibited by
synthetic peptides based on the Arg-Gly-Asp and Ala-Gly-Asp regions of C3
194 J. ALEXANDER AND D. G. RUSSELL

and fibrinogen. The original published sequence for gp63 from L. major
contained an Arg-Gly-Asp sequence (Button and McMaster, 1988). Synthe-
tic peptides based on this region were also inhibitory to gp63/CR3 binding.
Although at that time it was proposed that binding was mediated by this
Arg-Gly-Asp region, it is now known that this region of the deduced amino
acid sequence was incorrect (Button and McMaster, 1990; Russell, 1990).
So, although it is clear that gp63 binds to CR3, the nature of the interaction
remains unknown.

FIG.6. Binding and phagocytosis of CIS-alkyl derivatized, reverse phase beads


coated with macrophage-binding moieties isolated from the promastigote surface;
from Russell and Wright (1988). Monolayers of human monocyte-derived macro-
phages were incubated with ligand-bearing beads for 15 min at 37°C and unbound
beads removed by washing. The gp63-coated beads bind to the surface of cultured
human macrophages, but no internalization is evident (A). However, gp63 beads
coated with IgG anti-gp63 were readily internalized (B). Similar uptake was also seen
of beads coated with both gp63 and LPG, or gp63 beads incubated with macro-
phages plated on to the extracellular matrix proteins, fibronectin or laminin. ( x 600)
INTERACTION OF LEISHMANIA WITH MACROPHAGES 195

The binding of LPG to macrophages was first demonstrated by Handman


and Goding in 1985. Analysis with the aid of a fluorescence-activated cell
sorter of LPG on the surface of a variety of cell types indicated that the
receptors for LPG were confined to phagocytes. Employing reverse phase
high-performance liquid chromatography beads as carriers, Talamas-
Rohana et al. (1990) identified the macrophage receptor(s) for LPG. Attach-
ment of LPG-beads to macrophages was blocked if the macrophages were
plated on to substrate coated with monoclonal antibody IB4 against the
common chain of the CD18 family of intergrins, CR3, p150/95 and
lymphocyte function antigen 1 (LFA- I). Differential inhibition of individual
members of the family indicated that CR3 and p150/95 were primarily
responsible for LPG binding. Macrophages isolated from a patient express-
ing leucocyte adhesion deficiency syndrome, a consequence of a genetic
lesion in the gene encoding the common flchain which leads to the absence
of all three receptors from the cell surface, failed to recognize LPG. Soluble
LPG, produced by the enzymatic cleavage of the phosphatidylinositol
membrane anchor, acted as a competitive inhibitor of LPG binding and
rough LPS binding, but did not block binding of C3bi-coated erythrocytes.
These results indicated that LPG is recognized by the binding site on CR3
and p150/95 implicated in the attachment of rough LPS, Histoplasma
capsulatum, b-glucan, and zymosan (Ross et al., 1985; Bullock and Wright,
1987; Wright and Jong, 1986; Wright et al., 1989).
Variation in the structure of these molecules between species, and during
the differentiation from non-infective to infective promastigotes, may have a
profound effect on the attachment characteristics of these molecules to
macrophages. The gp63 molecule appears to be fairly well conserved
between species. Polyclonal and monoclonal antibodies against the protein
cross-react to varying degrees across species barriers, and the sequence
content and gene organization of the family appear closely related. LPG,
however, shows greater variation. In Leishmania spp. from the Old World,
LPG undergoes extensive elongation during differentiation of promastigotes
to the metacyclic form. This alteration leads to increased complement
resistance and a thick coat is formed on the parasite surface. Under such
conditions, L. major metacyclic forms bind poorly to macrophages unless
opsonized by complement (da Silva et al., 1989). Of Leishmania spp. from
the New World, L. mexicana in particular does not share a comparable
alteration in LPG structure (D. G. Russell, unpublished results), although
some changes are apparent, and does not ‘‘lose’’ its ability to bind avidly to
macrophages. Therefore, the relative contribution or requirement for comp-
lement opsonization differs between species.
None the less, understanding the physiological significance of the different
macrophage-binding properties described above cannot be addressed with-
196 J. ALEXANDER AND D. G. RUSSELL

out reference to the situation in vivo, where infection is achieved in the


presence of tissue fluid and complement. Promastigotes of a number of
different Leishmania spp. have been shown to activate complement, leading
to the deposition of C3 fragments on the promastigote surface. However,
differences in the activation pathway have been observed, possibly as a
function of the LPG structural differences. Metacyclic promastigotes of L.
major activate complement via the classical pathway (Puentas et al., 1988), in
the absence of specific antibody, and accumulate C3b, predominantly in the
form of an ester linkage to LPG, on their surface. In contrast, stationary
phase promastigotes of L. mexicana (Russell, 1987a) and, more recently, L.
donovani (Puentas et al., 1990), have been shown to activate the complement
cascade via the alternative pathway, again accumulating C3 predominantly
as C3b. In these species the C3b is at least in part bound via an amide
linkage (resistant to nucleophilic compounds) to surface proteins such as
gp63.
Subsequent binding of promastigotes opsonized with complement will
therefore be a function of both the intrinsic macrophage-binding ligands on
the parasite surface and the deposited C3 fragments. Studies conducted on
L. major metacyclic promastigotes, in the presence of human C8-deficient
serum, indicated that complement receptor type 1 (CRl) is the principal
mediator of attachment (da Silva et al., 1989). Although this cannot be
directly equated with increased infectivity in vivo because of the use of C8-
deficient serum which is not lysis competent, it does demonstrate that all
receptors (CR3, p150, 95 and CRl) of known ligands have two important
points in common: they are all restricted to cells of the macrophage lineage,
and they are all relatively inefficient in triggering a microbicidal response
when complexed with their respective ligands, as discussed by Russell and
Talamas-Rohana ( 1989). Additional active mechanisms of survival enhance-
ment, such as the suppression of the superoxide burst by LPG or acid
phosphatase, may also be important in the initial establishment of an
infection.

B. CHARACTERIZATION OF THE PARASITOPHOROUS VACUOLE

The early work of Chang and colleagues demonstrated that Leishmania


resides in an acidic intracellular compartment, the parasitophorous vacuole
(Chang and Dwyer 1976; Chang, 1980). More recently, workers have
calculated the pH of the vacuole to lie between 4.7 and 5.2 (Antoine et al.,
1990). However, apart from the vacuole pH, very little is known about
formation and maintenance of the parasitophorous vacuole as an intra-
cellular compartment. Early electron microscopical studies of L . mexicana
INTERACTION OF LEISHMANIA WITH MACROPHAGES 197

indicated that the vacuole was capable of fusing with compartments that
appeared to be secondary lysosomes (Alexander and Vickerman, 1975).
Early histochemical studies have also shown the presence of acid phospha-
tase in the vacuole (Antoine et al., 1987); however, as the parasite itself is
known to produce a soluble acid phosphatase, the relevance of this result to
the definition of the compartment is limited. A more definitive study on the
nature of the lysosomal enzyme in the parasitophorous vacuole and in entire
macrophages infected with Leishmania has been more revealing. Prina et al.
(1990) have shown that cathepsins D, B, H and L are delivered to vacuoles
containing parasites. The studies were all carried out on relatively new
infections, initiated by L. amazonensis promastigotes, because older estab-
lished infections resulted in larger vacuoles which were technically difficult to
label. Interestingly, the total hydrolytic activity of a range of lysosomal
enzymes was unaltered in infected macrophages.
Although circumstantial evidence indicates that the parasitophorous
vacuole is lysosomal in derivation, a careful description in terms of defined
membrane protein markers and lysosomal hydrolases is lacking and is
fundamental to further analysis of the vacuole as a dynamic cellular
compartment. Employing immunofluorescence and immunoelectron mi-
croscopy, Russell et al. (1991b) have reported preliminary analysis of the
distribution of various membrane proteins and hydrolases in an established
L. mexicana infection of murine macrophages.
The markers of particular relevance for the endocytic pathway employed
were the lipid-associated membrane proteins LAMPl and LAMP2 (Chen et
al., 1985), present in both late endosomal and lysosomal compartments, and
the mannose-6-phosphate receptor which is present in the membranes of late
endosomes, but absent from lysosomal compartments (Kornfeld and Mell-
man, 1988). In addition, antibodies against the hydrolases cathepsin D and
P-glucuronidase were used. The parasitophorous vacuole was positive for
both cathepsin D and P-glucuronidase. The density of gold label for both
hydrolases within the lumen of the vacuole was similar to that of small
lysosomes around the periphery of the vacuole, indicating that a constant
supply of lysosomal enzymes is released into the vacuole. The parasito-
phorous vacuole membrane was shown by both immunofluorescence and
immunoelectron microscopy to contain the lysosomal glycoproteins LAMPl
(seen in Fig. 7) and LAMP2, and Lgp 120. In contrast, labelling with anti-
mannose-6-phosphate receptor antibody reveals an extensive concentration
of vesicles in the perinuclear space, but label appeared at low density within
the parasitophorous vacuole, which was also LAMP-positive. By accepted
convention (Kornfeld and Mellman, 1988), the compartment is late endo-
soma1 to lysosomal in nature.
198 J. ALEXANDER AND D. G. RUSSELL

(a)
INTERACTION OF LEISHMANIA WITH MACROPHAGES 199

C. INTRACELLULAR TRAFFICKING AND THE PARASITOPHOROUS VACUOLE

Access to the parasitophorous vacuole via the macrophage’s endosomal


system is of interest both from a cell biological and chemotherapeutic
viewpoint. Studies by Rabinovitch and colleagues have demonstrated the
ability of vacuoles containing freshly interiorized parasites to fuse with
endocytosed material (Shepherd et al., 1983; Rabinovitch et al., 1985),
showing that they lie within the endosome-lysosome network. The first
study detailed the trafficking of radio-iodinated p-glucuronidase and iodi-
nated mannose-BSA in macrophages derived from infected mouse bone
marrow. Uptake of both proteins was similar in infected and non-infected
macrophages. P-glucuronidase could be found in the parasitophorous
vacuole, but no mannose-BSA was detected. The second study examined
uptake and trafficking of horse radish peroxidase (HRP) in infected mouse
and rat macrophages. H R P was predominantly internalized via a mannan-
inhibitable pathway, and was detected in the parasitophorous vacuoles of
rat, but not mouse, macrophages. The authors suggested that the H R P was
delivered into secondary lysosomes that subsequently fused with parasite-
containing compartments.
In a more recent study (Russell et al., 1991b), N-acetyl glucosamine-BSA
(GluNAc-BSA) coupled to either fluorescein isothiocyanate (FITC) or gold
was added to cultures of mature macrophages infected with L. mexicana to
examine the mannose/fucose receptor endocytic pathway. Despite extended
incubation times, GluNAc-BSA did not reach the parasitophorous vacuole.
In contrast, another endocytosed ligand, a-2 macroglobulin, either deriva-
tized with gold or FITC, was readily detectable within the lumen of the
vacuole. These results indicate a hierarchy of access of different endocytosed
material to the parasitophorous vacuole. The cellular basis for this discrimi-
nation is unclear. It can be argued that either the amastigote is serving
merely as a novel probe for mature lysosomes, and uncovering normal
lysosomal behaviour, or that the amastigote actually modifies the lysosome
to its own needs, and that this partitioning is peculiar to the Leishmania-
infected macrophage.

FIG. 7. Fluorescence micrographs of a Leishmania mexicana macrophage. (a)


Preparation labelled with Hoechst dye to reveal the nuclei of the host and parasite
cells. (b) Preparation probed with rat monoclonal antibody ID4B against the
lysosome associated membrane protein LAMP 1 (see Chen et al., 1985), followed by
anti-rat IgG conjugated to fluorescein isothiocyanate. In addition to the clear
labelling of small intracellular vesicles there is a marked edge effect at the boundary
of the parasitophorous vacuole, indicating that the membrane surrounding the
vacuole contains the lysosomal protein LAMP. This has been confirmed by immuno-
electron microscopy (Russell et al., 1991b). (~4000)
200 J. ALEXANDER AND D. G. RUSSELL

Whatever the cellular basis, the results of these studies raise several
interesting questions concerning the accessibility of the parasitophorous
vacuole to endocytosed material and its relevance to the possible potentia-
tion of leishmanicidal drugs by targeting them to the parasitophorous
vacuole of infected macrophages. With respect to the latter point, Mukho-
padhyay et al. (1989) indicated that methotrexate coupled to maleylated
BSA, which it was presumed was being internalized via scavenger receptors,
was two orders of magnitude more effective than free drug in eliminating
Leishmania from an infection in vivo.

D. LEISHMANIA ANTIGENS AND THE MACROPHAGE SURFACE

Trafficking of parasite-derived material in the other direction, out of the


parasitophorous vacuole and on to the host cell surface, is of great signifi-
cance in understanding the generation of an immune response against
infected cells. Several studies reported the occurrence of Leishmania-derived
material on the surface of infected macrophages (Handman and Hocking,
1982; Williams et al., 1986; Handman, 1990). However, none conclusively
showed that the material had originated from active live amastigotes rather
than from aborted infections resulting in dead digested promastigotes. The
trafficking of parasite-derived material through the infected macrophage is
important in evaluating the ability of an infected macrophage to present
antigen to T cells, and represents a conspicuous gap in our knowledge of the
Leishmania-macrophage interaction. In addition, the ability of Leishmania
antigens to induce a CD8 response (Farrell et al., 1989; Hill et al., 1989) is
intriguing, given the current dogma concerning the cytoplasmic origin of
class I presented antigens and the fact that Leishmania never leaves its
vacuole.

E. INTRACELLULAR SURVIVAL AND MODULATION OF THE HOST CELL

The body of results discussed above indicates that Leishmania inhabits an


acidic lysosomal compartment that has unrestricted fusion with vacuoles
containing lysosomal hydrolases. It has access, possibly limited, to material
within the macrophage’s endosomal compartment, and the parasitophorous
vacuole is, at least ea,rly in the infection, capable of delivering parasite
antigens to the macrophage plasmalemma.
The exploitation of such a hostile environment has placed pressure on the
parasite to evolve a variety of solutions and strategies to ensure its survival.
Zilberstein et al. (1986) and Zilberstein and Dwyer (1988) have shown that
the amastigote proton pump functions at an acidic pH optimum and
maintains an intracellular cytoplasmic pH of 6.2. In addition, study of the
INTERACTION OF LEISHMANIA WITH MACROPHAGES 20 1

amastigote metabolism, and that of axenically cultured amastigotes grown


in acidic medium, indicated that the amastigote is acidophilic. The resistance
of the amastigote to attack by lysosomal hydrolases poses another problem.
It has been suggested that the surface protease gp63 may actively degrade
lysosomal enzymes, or that the glycolipid LPG forms a barrier glycocalyx
around the amastigote. However, surface labelling procedures indicate that
although gp63 appeared to be the most abundant exposed protein, its total
amount would be unlikely to compete adequately with, or inactivate, a
constant supply of hydrolases. However, the gp63 in the flagellar pocket,
which is probably controlled in terms of access, might function in this way,
thus protecting exposed receptors against hydrolytic attack. Electron micro-
scopical studies by Pimenta et al. (1991) demonstrated that the LPG on
amastigotes does not form the developed glycocalyx found on metacyclic
promastigotes, so the amastigote does not appear to be protected by any
carbohydrate coat. Studies on infected macrophages suggested that the
hydrolases remain active, arguing that the amastigotes are resistant to
degradation rather than surviving through deactivation of hydrolases.
There are also additional long-term requirements for amastigote survival
within the macrophage, relating to control of the antigen-presentation and
the activation response of macrophages. The ability to limit loss or to
salvage and digest its own proteins may be important in controlling the
release of potential antigens from the amastigote. Amastigotes are extremely
rich in proteases, the most extreme example being L. mexicana which has
developed specialized organelles called megasomes that have been shown to
contain a variety of cysteine proteases (Pupkis et al., 1986; North et al.,
1990). This apparent excess of proteases may be involved in the degradation
of polypeptides derived from both host and parasite. In addition, L .
donovani amastigotes have been shown to induce a down-regulation in the
surface expression of class I1 major histocompatibility complex (MHC)
molecules on the surface of infected macrophages (Reiner et al., 1987). In
contrast, the expression of class I, although less than that in controls,
showed little change. In addition to avoiding generating an immune re-
sponse, Leishmania also causes a down-regulation in the macrophage’s
response to stimulating lymphokines (Mauel, 1984). Although this dampen-
ing of response does not induce total protection from macrophage acti-
vation, it does require a higher concentration of interferon y (IFN-y) to
induce the same level of microbicidal response, both in terms of the
respiratory burst and Ia antigen expression in the murine system. It was
previously thought that Leishmania was killed primarily by the ability of
stimulated macrophages to generate reactive oxygen intermediates (Mauel et
al., 1984), although an early study by Scott et al. (1985) indicated that
additional mechanisms were required. More recent studies, however, indi-
202 J. ALEXANDER AND D. G. RUSSELL

cated that nitrous oxide intermediates were responsible for much of the
parasite killing (Green et a/., 1990). It is not yet known if Leishmania
possesses any ability to combat this response in the way that LPG acts as a
scavenger of oxygen radicals. However, the effect of both LPG and acid
phosphatase on the generation of superoxide intermediates should extend to
other expressions of macrophage activation.

V. HETEROGENEITY
MACROPHAGE

The leishmaniases include a number of diseases with a wide spectrum of


clinical manifestations. While the tissue site invaded by the parasite and the
severity of the disease undoubtedly reflect in some manner the species
initiating infection, the heterogeneity of macrophages, both within a single
host and between different individuals, no doubt also limits the severity and
site of infection. Thus, on the one hand, L. donovani and L. major (Hoch-
meyer et al., 1984) and L. major and L. mexicana (Scott and Sher, 1986)
show marked differences in their relative abilities to survive within macro-
phages of similar origin. On the other hand, both L. mexicana (Alexander,
1981) and L. donovani (Crocker et al., 1987) show marked differences in their
ability to survive within macrophages from different tissues of the same host.
Additionally macrophages from similar tissue sites, but from hosts with
different susceptibilities to infection, differ markedly in their ability to
control or permit parasite growth (Nacy et al., 1983; Crocker et al., 1984a).
These differences displayed by different individuals in macrophage resistance
to parasites may be innate and not require T cell involvement (Crocker et al.,
1984a), or they may be related to the ability to respond to activating
lymphokines (Nacy et al., 1983). Environmental conditions can also be
influential, as survival of L. major and L. mexicana within macrophages is
markedly increased by lowering the temperature of incubation, reflecting the
preference of these parasites for the cooler cutaneous tissues. However,
whatever the mechanism of macrophage responsiveness or non-responsive-
ness to infection and lymphokine-induced activation may be, it has become
increasingly apparent that generally the outcome of disease is largely under
genetic control.

A. GENETIC CONTROL OF LEZSHMANIA INFECTION

Over the past 15 years several genes controlling leishmania1 infections in


mice have been identified and mapped, and their functions have been
characterized (reviewed by Blackwell, 1988).
A noticeable feature is that, generally, different host genes recognize and
INTERACTION OF LEISHMANIA WITH MACROPHAGES 203

control infections with taxonomically distinct Leishmania spp. (Table 2).


This in part, but not wholly, reflects the particular tissue predilection of the
Leishmania spp. examined. Thus, primary cutaneous lesion growth induced
by L. mexicana is under different genetic control, Scl-2, from the growth of
the same organism in the viscera (Roberts et al., 1990). Lsh, H-2 and H-11
genes, which have been shown to influence the visceral growth of L.
mexicana, also determine the visceral growth of L. donovani (Roberts et al.,
1989). However, although parasites may occupy the same tissue site, this
need not imply they are under the same genetic controls. For example, the
cutaneous growth of L. mexicana is not influenced by Scl-1, which controls
the cutaneous growth of L. major (Blackwell et al., 1984; Mock et al.,
1985a), while the visceralization of L. major is totally independent of Lsh
gene involvement (Mock et al., 1985b).
TABLE
2 Mouse genes controlling Leishmania infection with proven or probable
macrophage involvement

L. mexicana
Gene Chromosome L. donovani L. major Primary cutaneous Metastaticlvisceral
Lsh 1 ++ - - ++
H-2 17 ++ + - ++
H-11 NK ++ ++ + ++
Scl-1 8( 1 1) - ++ + -
Scl-2 4 - - ++ + +/NK
+ +, Major influence; +, minor influence; -, no influence; NK, not known.

Nevertheless, what all these genetically regulated host-limiting factors


have in common is that they all, directly or indirectly, involve the partici-
pation of macrophages.

1. Lsh

By far the most comprehensively studied genetic control of intra-macro-


phage parasitism is that pertaining to Lsh control of L. donovani infection.
This gene, which is often referred to as “the macrophage resistance gene”,
has been mapped to a position near Zdh and In on mouse chromosome 1. It is
undoubtedly identical to the genes separately designated Zty and Bcg which
control Salmonella typhimurium and Mycobacterium bovis, respectively
(reviewed by Blackwell, 1988). Expression is T cell-independent (Bonventure
and Nickol, 1984) and can be transferred with donor haematopoietic cells in
radiation bone marrow chimaeras (Crocker et al., 1984a). Resistance is also
204 J. ALEXANDER AND D. G. RUSSELL

variably expressed in different macrophage populations, being more associ-


ated with mature resident cells (Crocker et af., 1987). Lsh gene expression is
manifest 2-3 days after infection in vitro or in vivo (Crocker et al., 1984b,
1987) as an inhibition of parasite multiplication, as well as increased
responsiveness to low-density polysaccharide (LPS) and IFN-)I as measured
by increased oxidative activity, increased production of tumour necrosis
factor and interleukin 1 (IL-I) and up-regulation of MHC class I1 expression
(Blackwell et af., 1989; Kaye and Blackwell, 1989). The gene product is yet
to be identified and recent experiments comparing induced peritoneal
macrophages infected with L. donovani from congenic Lsh' and Lshs mice
have yielded surprising results. Blackwell and coworkers have identified the
phosphorylation after 20 min of low molecular weight proteins in the Lshs
but not Lsh' macrophages. In addition to increased T cell-independent
antimicrobial activity and greatly magnified responsiveness to T cell prod-
ucts (lymphokines), Lsh' genes are associated with an enhanced ability to
mount a strong and long-lasting cell-mediated immune response. This
suggests a regulatory role in MHC class I1 controlled antigen presentation
(Kaye el af., 1988).
Lsh has also been shown to regulate the visceral growth of L. mexicana
(Roberts et af., 1989). Expression in this case was later than that demon-
strated for L. donovani; more than 8 days as opposed to 2-3 days. Resistance
against L. mexicana was also leishmanicidal as opposed to leishmanistatic,
suggesting that a different activation pathway to the T cell-independent
mechanism controlling early L. donovani infection may control this organ-
ism. The later onset of Lsh-controlled resistance to L. mexicana indicates
enhanced macrophage microbicidal activity mediated by lymphokines.

2. H-2

Between homozygous recessive Lshs mouse strains, different disease profiles


are observed when the course of L. donovani infection in the liver and spleen
is followed over 130 days (Blackwell et af.,1980). On BALB and B10 genetic
backgrounds these differences in the long-term response are controlled by
genes mapping to IE and a subregion to its left, presumably IA, in the K
end of the MHC H-2 (Blackwell, 1983). Using intravenous parasite inocula
of 5 x lo5 or more (Ulczak and Blackwell, 1983), three H-2 controlled
phenotypic patterns of infection were observed: early cure (s and r haplo-
types), cure (b haplotype), and non-cure (d, f and q haplotypes). BlOBR
(H-2k) mice displayed an intermediate or variable haplotype. At these
parasite doses cure in C57Bl. 10 mice is mediated by CD4' T cells (Ulczak et
a f . , 1988) and correlates with a positive delayed-type hypersensitivity re-
sponse and with the lymphokine-generating capacity of spleen cells from
INTERACTION OF LEISHMANIA WITH MACROPHAGES 205

curing mice (Rezai et al., 1980). Non-cure in B10.D2 mice is also associated
with the generation of CD4’ T cells (Blackwell and Ulczak, 1984). Low
infecting inocula in this non-cure mouse strain result in a cure profile, while
large doses, > lo7 amastigotes, given to the cure H-2b haplotypes result in a
variable phenotype response. Thus, for L . donovani infection in B10 H-2
congenic mice, there appear to be two determinants for the development of a
non-curing disease: a sufficiently high antigenic load and the type of class I1
molecule presented in association with the parasite antigen by the antigen-
presenting cell. The generation of T cells able to determine a non-healing
response occurs later, > 30 days after infection (Ulczak and Blackwell,
1983). Control of this response, as indicated above, has been mapped to IE
at the K end of H-2 (Blackwell, 1983). However, some non-cure haplotypes,
as well as cure haplotypes, fail to express IE molecules, suggesting that both
non-cure and cure phenotypes controlled by these haplotypes must also map
to K end genes other than IE. As both curing and non-curing responses are
mediated by CD4’ T cells, which recognize antigen in association with class
I1 molecules, IA seems to be the likely candidate.
Treating B10.D2 mice, bearing the H-2“ non-curing haplotype, with
specific monoclonal antibodies against IA or IE molecules has clarified the
involvement of these molecules in disease outcome (Blackwell and Roberts,
-=
1987). Early treatment, at 30 days, with either anti-A or anti-E resulted in
an increased liver and spleen parasite burden. After day 50, anti-A treatment
resulted in exacerbated infection, while anti-E treatment led to the resolution
of disease. These results reaffirm that L . donovani antigens presented in
association with IE class I1 molecules on the macrophage are invariably
associated with non-curing disease.
H-2, by contrast, has only a limited effect on subcutaneous L . major
infections (Howard et al., 1980a). However, despite a failure to observe any
H-2 controlled differences in primary cutaneous growth of L. mexicana, this
genetic locus did markedly influence the visceralization and metastatic
spread of this organism, with the L. donovani “non-curing” haplotypes
developing the heaviest infections with generalized metastasis (Roberts et al.,
1989).

3. H-11

Of all the genetic controls shown to influence the course of Leishmania


in mice, only H-Zl remains unmapped. Nevertheless studies with
BlO.l29(10M) mice, which carry alternative alleles at H-IZ, have demon-
strated parallel influences with all three Leishmania spp. studied (Blackwell
et al., 1985a; Roberts et al., 1989). H-ZZ is thought to exert its influence by
regulating MHC gene expression. Thus, Roberts et al. (1988) have demon-
206 J. ALEXANDER AND D. G. RUSSELL

strated: (i) reduced ability of splenic adherent cells (macrophages) isolated


from B10.129(10M) mice to present amastigote antigen to primed T cells
from infected B10 mice; (ii) reduced ability of splenic adherent cells from
uninfected or infected B 10.129(10M)mice to stimulate mixed lymphocyte
reactions across H-2 barriers; and (iii) reduced interleukin 2 (IL-2) receptor
expression in splenic T cells isolated from BlO.I29(10M) mice 3-14 days
after L. donovani infection. All of these observations may again be explained
by reduced class I1 molecule expression on macrophages from
BIO. 129(10M) mice, a conclusion supported by the observation that these
macrophages fail to up-regulate class I1 molecule expression in response to
recombinant IFN-y (Roberts et al., 1988).

4. Scl-1

Healing and non-healing responses to cutaneous L. major infection are


controlled by a single gene designated ScZ-Z (susceptibility to cutaneous
leishmaniasis), which has been mapped provisionally to mouse chromosome
8, although an alternative location on chromosome 11 is a possibility
(Blackwell et al., 1984; Mock er al., 1985a). Expression of this gene can be
transferred with donor haematopoietic cells in radiation bone marrow
chimaeras (Heward et al., 1980b). Susceptibility has been associated with a
primary macrophage defect which allows uncontrolled parasite multiplica-
tion in a skin macrophage population (Gorczinski and MacRae, 1982). This
results in differential expression of class I1 molecules in susceptible animals
and the production of CD4’ T suppressor cells, presumably TH2 cells, in
susceptible BALB/c mice.

5. Scl-2

Most laboratory mouse strains develop non-healing lesions following infec-


tion with L. mexicana (Alexander and Blackwell, 1986). However, a unique
“no lesion development” phenotype has been described for DBA/2 mice
which is under the control of a single gene, provisionally mapped to
chromosome 4 (Blackwell and Alexander, 1986; Roberts et al., 1990). This
resistance is associated mainly with female mice (Alexander, 1988a) and can
be overcome by increasing the number of parasites initiating infection.
Resistant .female mice develop strong delayed-type hypersensitivity (DTH)
reactions which are absent from susceptible male mice (H. H. K. Al-Qaisi
and J. Alexander, unpublished observations). Treating male mice with
oestrogen increases their resistance to L. mexicana infection (Alexander and
Stimson, 1988), and this appears to operate through macrophage activation
mediated via an oestrogen receptor (Gulshan et al., 1990). This results in
INTERACTION OF LEISHMANIA WITH MACROPHAGES 207

increased macrophage resistance to L. mexicana infection and increased IL- 1


production in vitro (H. H. K. Al-Qaisi and J. Alexander, unpublished
results). Epidemiologically, sex differences in L . mexicana infection have
been reported, although this may reflect relative exposure rates to the
sandfly vector. Nevertheless, among patients with active infections, healing
is closely correlated to the development of DTH, which is strongest in
females (Lynch et al., 1982).

B. RELEVANCE TO HUMANS OF GENETIC STUDIES IN MICE

Of the described genes which influence the disease phenotypes induced by


various species of Leishmania in mice, only H-11 remains unmapped. Of the
others, homology between the major histocompatibility complexes, H-2 on
mouse chromosome 17 and HLA on human chromosome 6, is already well
documented; while Lsh, Scl-1 and Scl-2 have been mapped to regions of
mouse chromosomes with known homologues in humans (reviewed by
Blackwell, 1988). Thus, using DNA probes for genes within MHC and
linked marker DNA probes for Lsh, Scl-1 and Scl-2, it should now be
possible-on the basis of family linkage analysis-to determine whether
homologous genes control the spectrum of disease phenotypes diagnosed in
humans. If polymorphism at these loci does determine the disease profile in
humans, probes should indicate susceptible individuals within a population
at risk, while characterization of the mode of action of the genes should
indicate the possible rational means for therapeutic intervention.

PRESENTATION
VI. ANTIGEN AND THE INDUCTIONOF IMMUNITY

Early studies demonstrated that the generation of protective immunity


against both experimental visceral (Skov and Twohy, 1974a,b) and cu-
taneous (Preston and Dumonde, 1976) leishmaniasis was T cell-mediated.
These observations have since been confirmed by numerous later experimen-
tal and clinical studies (reviewed by Liew, 1990). However, T lymphocytes
can recognize antigen only in association with class I (CD8'T cells) and
class I1 (CD4+ cells) MHC molecules on the surface of accessory or antigen-
presenting cells (APCs): these include macrophages. Leishmania1 antigen has
often been demonstrated on the surface of infected macrophages (Farah et
al., 1975; Berman and Dwyer, 1981; Handman and Hocking, 1982), but it
has not yet been associated with the concomitant expression of class I1
molecules, although this undoubtedly is what happens. Consequently, the
production of co-stimulatory factors, including IL-1, by the accessory cells
promotes antigen-specific lymphocyte activation (Unanue, 1984; Kaye,
208 J. ALEXANDER AND D. G. RUSSELL

1987; Unanue and Allen, 1987; Weaver and Unanue, 1990; Kaye et al.,
199I ) . The generation of antigen-specific lymphokine-producing T cell
populations, particularly those producing IFN-y, promote in their turn
macrophage leishmanicidal activity (Section VI1.B). Although a role for
CD8 T cells cannot be discounted in influencing leishmanial infections, the
+

T,I and T,2 subsets of CD4' T cells, which are dependent on class I1
molecule-associated antigen presentation, appear to control healing and
disease exacerbation, respectively (Section VI1.A).

A. PARASITE INTERFERENCE WITH ANTIGEN-PRESENTING CELL FUNCTION

The requirement for accessory cells in stimulating the proliferation of L.


major specific CD4+ T cells was demonstrated in vitro by Louis et al. (1981).
The proliferation of T cell blasts specific to the parasite occurred only in the
presence of syngeneic spleen cells and was inhibited by anti-Ia antibodies
plus complement. Although the resulting antigen-specific CD4' T cells
activated macrophages to kill intracellular parasites in vitro and adoptively
transferred to naive mice DTH reactions to leishmanial antigens (Louis et
al., I982), they also rather unexpectedly adoptively transferred disease
exacerbation. Handman et al. (1979) had previously suggested that a defect
or a deficiency in antigen presentation by macrophages could induce non-
healing disease. In many subsequent studies, adherent cell populations
(presumably macrophages) have been associated with a suppressed immune
response to leishmaniasis (Scott and Farrell, 198I ; Reiner and Malemud,
1984, 1985; Cillari et al., 1986; Murray et al., 1986). Impairment of the
immune response was invariably associated with an inability to produce IL-2
upon specific or mitogenic stimulation (Scott and Farrell, 1981; Reiner and
Finke, 1983; Cillari et al., 1986; Murray et al., 1987). Cytokine production
associated with healing and non-healing disease states is summarized in
Table 3. Obviously the presence of the parasite within a major accessory cell
population may enable it to interfere with, or thwart, normal antigen
presentation or other accessory cell functions. This has indeed been shown
to be the case in macrophages of BALB/c mice infected with L. donovani,
where synthesis of prostaglandin (PG) E,, a known down-regulator of class
I1 expression (Snyder et al., 1982), is greatly enhanced compared with
uninfected macrophaies (Reiner and Malemud, 1985; Reiner et al., 1987).
Although basal levels of class I1 molecules in infected macrophages were not
affected, expression was not up-graded following stimulation with IFN-y.
Class I molecule expression, on the other hand, was depressed below the
basal state by leishmanial infection. Inhibitors of PG synthesis reversed the
inhibition of MHC expression (Reiner et af., 1987) and administration in
INTERACTION OF LEISHMANIA WITH MACROPHAGES 209

vivo of such an inhibitor, indomethacin, was found to be therapeutic against


L. major in the BALB/c mouse (Farrell and Kirkpatrick, 1987). The ability
of macrophages infected with L. donovani to produce IL- 1 following second-
ary stimulation was also selectively inhibited (Reiner, 1987). While human
monocytes infected with L. major could not be stimulated to produce IL-I
(Crawford et al., 1989, peritoneal macrophages from highly susceptible
BALB/c mice infected with L. major in vitro did produce high levels of this
cytokine. In the mouse, IL-l is a co-stimulator of T,2 but not TH1 cells
(Weaver and Unanue, 1990), and in the BALB/c - L. major experimental
model the TH2subset is associated with disease exacerbation (Heinzel et al.,
1989; Scott et al., 1989). The inhibition of macrophage IL-I production by
L. donovani suggests that control of this parasite operates via different
immunoregulatory pathways from those concerned with L. major.

B. ANTIGEN COMPLEXITY AND ANTIGEN-PRESENTING CELL HETEROGENEITY

Whether a healing or a non-healing response is induced by the APCs may


depend either on which antigen is processed and expressed or on which
APC is doing the processing and presentation. Gorczynsky and MacRae
(1982) demonstrated that certain BALB/c subpopulations of skin macro-
phages allowed increased L. major growth and failed to stimulate T cell
proliferation. The same population could stimulate T cell proliferation,
however, if incubated with soluble antigen. This suggested that different
antigens as well as APCs could be important in stimulating either a healing
or a non-healing response. This has been confirmed by the work of Scott et
al. (1987a,b, 1988), who demonstrated that protective TH1cells were gener-
ated by a low molecular weight soluble L. major antigen while counter-
protective TH2 cells were stimulated by a high molecular weight fraction.
Supporting the notion that distinct determinants or epitopes are responsible
for particular disease states, Gorczynsky (1 986) demonstrated that protec-
tion, rather than suppression, against L. mexicana was associated with N-
linked glycoprotein. Alternatively, the way in which the antigen associates
with the accessory cell surface and the MHC class I1 molecules could be
crucial (Mitchell and Handman, 1985). Intraperitoneal vaccination with
LPG plus Corynebacterium parvurn as adjuvant promoted a protective
response against L. major, whereas the lipid-free moiety introduced under
the same conditions induced exacerbative disease. Mitchell and Handman
(1985) suggested that the lipid allowed insertion of the molecule into the
plasma membrane associated with MHC class I1 molecules and thus acti-
vated protective CD4' T cells; the lipid-free molecule, on the other hand,
would attach to specific receptors unassociated with MHC. Although
specific antigens and epitopes in association with APCs may be important in
TABLE3 Cytokine activity associated with Leishmania infection

Mouse Cvtokine
Cytokine Species Cell type strain Healing Cell stimulation activity Reference
Murine
IL-l L. donovani RPM BALB/c No -
1 Reiner ( 1 987)
IL-l L. major RPM BALB/c No -
T Cillari et al. (1989)
IL-2 L. donovani splenic BALB/c No Leishmanial lysate Ag 1 Murray, H. W. et
al. (1987)
IL-2 L. donovani splenic BALB/c No PHA 1 Reiner and Finke
( 1983)
IL-2 L. major splenic BALB/c No PHA 1 Cillari et al. (1986)
IL-2 L. major LNC BALB/c No Leishmanial crude Ag 1 Solbach et al.
(l987b)
L. major LNC C57BL/6 Yes Leishmanial crude Ag t Solbach et al.
(l987b)
I L-2 L. major splenic BALB/c No Fraction 1 Ag 1 Scott et al. (1988)
vaccine
fraction 1
I L-2 L. major splenic BALB/c Yes Leishmanial soluble Ag t Scott et al. (1988)
vaccine
fraction 9
IL-3 L. major splenic BALB/c No Leishmanial soluble Ag t Lelchuk et a[.
(1988)
I L-3 L. major splenic CBA Yes Leishmanial soluble Ag 1 Lelchuk et al.
( I 988)
IL-3 L. major splenic BALB/c Yes Leishmanial soluble Ag 1 Lelchuk et al.
irradiated ( 1 988)
IL-3 L. major splenic BALB/c No Leishmania1 soluble Ag t Lelchuk et al.
vaccine sc (1989)
IL-3 L. major splenic BALB/c Yes Leishmanial soluble Ag 1 Lelchuk et al.
vaccine iv (1989)
I L-4 L. major splenic BALB/c No Fraction 1 Ag t Scott et al. (1988)
vaccine
fraction 1
IL-4 L. major splenic BALB/c Yes Leishmanial soluble Ag 1 Scott et al. (1988)
vaccine
fraction 9
IL-5 L. major splenic BALB/c No Fraction 1 Ag Scott et al. (1988)
vaccine
fraction 1
IL-5 L. major splenic BALB/c Yes Leishmanial soluble Ag Scott et al. (1988)
vaccine
fraction 9
TNF-a L. donovani BMDM BALB/c No LPS Descoteaux and
Matlashewski
( 1989)
TNF-a L . donovani BMDM BIOLsh' Yes LPS Blackwell et al.
(1989)
TNF-a L. major LNC C3H Yes Leishmanial whole Ag t Titus et al. (1989)
TNF-a L. major LNC BALB/c No Leishmanial whole Ag 1 Titus et al. (1989)
IFN-y L. major LNC C57BL 16 Yes Leishmanial crude Ag Sadick et al. (1986)
IFN-y L. major LNC BALB/c No Leishmanial crude Ag Sadick et al. (1986)
IFN-./ L. major LNC BALB/c Yes Leishmanial crude Ag Sadick et al. (1 986)
IFN-7 L. donovani splenic BALB/c No Leishmanial lysate Ag Murray, H. W. et
al. (1 987)
IFN-y L. major splenic BALB/c No Fraction 1 Ag 1 Scott et al. (1988)
vaccine
fraction 1
IFN-y L. major splenic BALB/c Yes Leishmanial soluble Ag r Scott et al. (1988)
vaccine
fraction 9
IL-4 L. major splenic C57BL16 Yes In vivo 1 Heinzel et al.
(1989)
TABLE
3 (Continued)

Mouse Cytokine
Cytokine Species Cell type strain Healing Cell stimulation activity Reference

IL-4 L. major splenic BALB/c No In vivo t Heinzel et al.


( 1989)
IFN-y L. major splenic C57BL/6 Yes In vivo Heinzel et al.
(1989)
IFN-y L . major sp 1enic BALB/c No In vivo 1 Heinzel et al.
(1989)

Human
IL-2 L.d. chagasi Lymphocytes - No Leishmanial soluble Ag 1 Carvalho et af.
(19854
IL-2 L.d. chagasi Lymphocytes - Yes Leishmanial soluble Ag t Carvalho et al.
(1985a)
IL-2 L. donovani PBMC - No PHA 1 Cillari et al. (1988)
IL-2 L . donovani PBMC - Yes PHA 1 Cillari et al. (1988)
IL-2 L . major PBMC - Yes Leishmanial lysate Ag Passwell et al.
(1 987)
IFN-y L. mexicana PBMC - No Leishmanial lysate Ag 1 Murray, H. W. et
al. (1984)
IFN-y L. mexicana PBMC Yes Leishmanial lysate Ag Murray, H . W. ct
u1. ( 1984)
IFN-y L. bra:iliensis PBMC - No L. amazonensis r Carvalho et ul.
soluble Ag (1985b)
IFN-y L. donovani PBMC - No Leishmanial soluble Ag 1 Sacks et al. (1987)
IFN-y L. donovani PBMC - Yes Leishmanial soluble Ag f Sacks er al. (1987)
IFN-y L. braziliensis PBMC ~ No L. rnexicana pijianoi 1 Rada er al. (1987)
diffuse whole Ag
cutaneous
IFN-y L. braziliensis PBMC - No L. mexicana pijanoi t Rada et al. (1987)
muco- whole Ag
cutaneous
IFN-y L. major PBMC - Yes Leishmanial lysate Ag Passwell et al.
(1987)

Ag, Antigen; BMDM, bone marrow derived macrophage; IFN, interferon; IL, interleukin; iv, intravenous; LNC, lymph node cells; LPS, lipopolysacchar-
ide; PBMC, peripheral blood mononuclear cells; PHA, phytohaemagglutinin; RPM, resident peritoneal macrophages; TNF, tumour necrosis factor; t. up-
graded; 1, down-graded.
214 J. ALEXANDER AND D. G. RUSSELL

determining disease outcome, it is undoubtedly true that heterogeneity


within the APC population could be equally important. Thus, while sub-
cutaneous vaccination of BALB/c mice against L. major resulted in disease
exacerbation and the production of T,2 cell equivalents, the same vaccine
preparation introduced intraperitoneally or intravenously was protective
and produced T, 1 cell equivalents (Liew, 1990). Presumably different routes
of vaccination would target antigen to different APC populations. In a
similar fashion it may be significant that expression of the L. donovani
natural resistance gene Lsh, a known regulator of APC function, varies
between tissue sites (see Section V). Heterogeneity of APCs and the ability of
different subpopulations to influence the outcome of disease have been
further demonstrated by using monoclonal antibodies to block either IA
gene-coded or IE gene-coded class I1 molecules (Blackwell and Roberts,
1987). This work indicated that L. donovani antigen presented in association
with IE class I1 molecules resulted in non-healing disease.

C. PROCESSING8OF PARASITE ANTIGEN AND ACTIVATION OF T CELLS

Recently major progress has been made in understanding the mechanisms by


which APCs process and present antigen to, and stimulate, T cells (Weaver
and Unanue, 1990; Kaye et al., 1991). Electron microscopical studies using
immunological localization techniques have identified class I1 molecules on
the surface membrane, phagolysosome and peripheral endosomes in macro-
phages infected with L. donovani after stimulation by concanavalin A (Kaye
et al., 1991). Using paraformaldehyde-fixed macrophages as APCs, 2-4 h
elapsed before presentation to class I1 restricted T cells took place. Antigen
presentation to class I1 restricted T cells was inhibited by chloroquine, an
inhibitor of enzyme degradation, as well as by brefeldin A, an inhibitor of
protein transport from the endoplasmic reticulum to the Golgi apparatus
(Adorini et al., 1990). Class I1 molecules remained present both on the
plasma membrane and phagolysosome. Kaye .et al. (1991) suggested,
therefore, that presentation of L. donovani antigen by macrophages requires
newly synthesized class I1 molecules, and association of antigen with class 11
molecules probably takes place in the trans-Golgi, or in an endosome
following antigen recovery from the phagolysosome.
Antigen presentation to class I1 restricted T cells is not sufficient for full T
cell functional activation; co-stimulators are also required (Weaver and
Unanue, 1990). In the absence of these signals long-term occupancy of the T
cell receptor may lead to a state of antigen-specific unresponsiveness or
tolerance (Mueller et al., 1989). Long-term antigen-specific unresponsiveness
is common during active visceral leishmaniasis, and is associated with
reduced IFN-y and IL-2 production (Carvalho et al., 1985a). Kaye et al.
INTERACTION OF LEISHMANIA WITH MACROPHAGES 215

(1991) suggested that it was the absence of these co-stimulatory signals in


mice chronically infected with L . donovani that resulted in the failure to
provide the necessary signals to activate T cells producing IFN-y. So far, IL-
1 has been identified as a co-stimulator for activation of TH2 cells (Kurt-
Jones et al., 1987), but not TH1 cells. Although the co-stimulators for T,1
activation have still to be characterized (Weaver and Unanue, 1990),
adjuvants are capable of inducing APCs to produce these factors. Freund’s
complete adjuvant, for example, which is a potent stimulator of TH1 cells
(Grun and Maurer, 1989), can promote a protective response against PT3, a
T cell epitope derived from the primary structure of L . major gp63 (Jardim et
al., 1990). PT3 alone induces TH2 cell proliferation and disease exacerbation.

VII. LYMPHOCYTE
CONTROL
OF MACROPHAGE
ANTI-LEISHMANIA
ACTIVITY

A. B AND T CELL INVOLVEMENT

1. B lymphocytes and antibody

The bulk of clinical and experimental evidence demonstrates that immunity


to Leishmania is largely T cell-mediated and protective immunity tends to
coincide with the development of DTH. Nevertheless, clinical observations
and several studies indicate that B cells and antibodies can influence disease
progression and healing. However, whether this arm of the immune response
functions in a protective or a counter-protective manner during infection is
dependent on the experimental system examined. Clinically, non-healing
visceral and cutaneous leishmaniasis have been associated with high
immunoglobulin (Ig) levels and negative DTH, while healing or cured
individuals display strong DTH and antibody levels fall (reviewed by Turk
and Bryceson, 1971). This suggests a disease-enhancing, DTH-blocking
effect by elements of the humoral response. Evidence for this assumption has
been produced by Sacks et al. (1984) who, by treating BALB/c mice from
birth with anti-IgM and thus rendering them B cell and antibody deficient,
were able to reverse the normal non-healing response of these mice to
cutaneous infection with L. major. Paradoxically, similar treatment of C3H/
HeN mice resistant to L . major resulted in these animals developing non-
healing lesions (Scott et al., 1986). The disease progression in these B cell-
depleted BALB/c and C3H/HeN mice could be restored to normal by
adoptively transferring T cells. These results suggest that the generation of
protective T cells in resistant mice is B cell-dependent, as is the generation of
counter-protective T cells in susceptible mice, and this has been related to
the ability of B cells to present antigen (Liew, 1990). B cell depletion would
216 J. ALEXANDER AND D. G. RUSSELL

presumably allow antigen presentation by other APCs, particularly macro-


phages. Antibodies may also influence the course of infection by directly
affecting parasite-macrophage interactions. For example, parasites binding
antibody will have their surface receptors hidden and therefore will have to
be phagocytosed via the macrophage Fc receptors (Bray, 1983b). This may
inhibit normal rates of attachment (Handman and Goding, 1985) and
uptake and may lead to increased intracellular killing. The potential protec-
tive functions of antibody have also been demonstrated in vivo (Anderson et
al., 1983). A monoclonal antibody against a surface glycoprotein (M-2) of L.
mexicana was protective if inoculated with the parasite into the footpads of
BALB/c mice. Vaccination with M-2 plus adjuvant also induced protection,
which was associated with increased antibody levels (Champsi and McMa-
hon-Pratt, 1988).

2. CD8' T cells

While the studies reported above indicate that the contribution of the
humoral response to disease progress cannot be ignored, an enormous
weight of evidence continues to identify T cell immunity as the controlling
factor (Bryceson et al., 1972; Preston et al., 1972; Skov and Twohy, 1974b;
Liew et al., 1982; Sheppard et al., 1983). Moreover, those T cells conferring
protection or counterprotection primarily belong to the CD4' T cell subset
(Mitchell et al., 1981; Liew et al., 1982; Louis et al., 1982; Gorczynski, 1985).
Some evidence does, however, suggest a protective, though not a suppressor,
role for CD8' T cells. The contribution of CD8' T cells to protective
immunity was first suspected because higher numbers of antigen-specific
CD8' T cells were generated in mice resistant to L. major than were
generated in susceptible mice (Milon et al., 1986). Furthermore, adminis-
tration in vivo of anti-CD4' monoclonal antibodies increased resistance
(Titus et al., 1985; Hill et al., 1989) to L. major, while administration in vivo
of anti-CD8' monoclonal antibodies exacerbated infection (Farrell et al.,
1989). Further evidence 'suggesting a role for the CD8' T cell subset in
protection came from studies on L. donovani. An influx of CD8' T cells was
associated with the inhibition of parasite growth in hepatic nodules (McEI-
rath et al., 1988), and both CD4' and CD8' T cells had to be adoptively
transferred from euthymic litter mates to reconstitute resistance against L.
donovani in athymic BALB/c mice (Stern et al., 1988). The mode of action of
these cells in protective immunity remains to be clarified. Early indications
that infected macrophages could be destroyed by specific cytotoxic cells
(Bray and Bryceson, 1968) have yet to be convincingly substantiated (Mauel
and Behin, 1987). However, CD8' cells can produce INF-y upon specific
stimulation (Kaufmann, I988), and this product has been repeatedly shown
INTERACTION OF LEISHMANIA WITH MACROPHAGES 217

to activate macrophages to kill Leishmania parasites (for references, see


Table 4). [Please see note added in proof on p. 254.1

3. CD4' T cells

While the role of CD8' T cells in mediating anti-Leishmania activity still


awaits clarification, an abundance of evidence, derived particularly from
experimental L. major infections, indicates that susceptibility and resistance
to this parasite are mediated by at least two different CD4' T cell subsets
(reviewed by Scott, 1989; Liew, 1989; Locksley et al., 1989). These are the
TH1 and TH2 CD4' T cell subsets which are morphological identical, but
distinguishable functionally and by the cytokines they produce (Mosmann et
al., 1986; Cherwinski et al., 1987). T H 1 cells produce IFN-y and IL-2, and
mediate DTH and IgG2a antibody production, while TH2cells produce IL-4
and IL-5, and promote IgE and IgG1 production, but do not mediate DTH.
Several other cytokines including IL-3 and granulocyte-macrophage colony-
stimulating factor (GM-CSF) are produced in varying amounts by both cell
types. Evidence initially suggested that the TH1 cell was preferentially
induced in resistant mice and the TH2 subset in susceptible mouse strains
(Scott et al., 1988; Heinzel et al., 1989; Locksley et al., 1989). Using cDNA
probes, mRNA for IL-4 was found in the lymph nodes and spleen of
infected non-healing mice while mRNA for IFN-y was found in the lymph
nodes and spleen of infected resistant mice (Heinzel et al., 1989). Conversely,
intraperitoneal injections of neutralizing anti-IL4 monoclonal antibodies
protected BALB/c mice against L. major (Sadick et al., 1990), while similar
treatment with anti-IFN-y monoclonal antibodies exacerbates not only L.
major infection in C3H/He mice (Belosevic et al., 1989) but also L. donovani
infection in BALB/c mice (Squires et al., 1989). Studies at the clonal level
also identified a protective role for TH1cells and a counter-protective role for
TH2cells. T cell lines and clones derived from BALB/c mice vaccinated with
a protective low molecular weight soluble fraction of L. major could
adoptively transfer resistance to syngeneic animals, while T cell lines and
clones responsive to a non-protective antigen fraction adoptively transferred
disease exacerbation (Scott et al., 1989). The protective T cell lines and
clones were shown to secrete IFN-y and IL-2 while the counterprotective T
cell lines and clones secreted IL-4 and IL-5.
Despite this wealth of published information suggesting a protective role
for TH1 cells and a counter-protective role for TH2cells, some observations
have suggested that the demarcation in function between these T cell subsets
might not be quite so clear-cut (Muller and Louis, 1989). Although healing
and resistance are invariably associated with IFN-y and IL-2 secretion, and
susceptibility and non-healing with IL-4 and IL-5 production, IL-3, which is
TABLE4 Modification of Leishmania infection in vitro by cytokines

Protective
Cytokine Species Host cell Mouse strain function Reference
Murine
IL-3 and L. major Macrophages BALB/c No Louis et al. (1987)
GM-CSF
GM-CSF L. major RP macrophages CBA/H Yes Handman and Burgess (1 979)
BALB/c
C3H/He
CSF (M-CSF L. major RP macrophages C3H/HeN Yes Ralph et al. (1983)
and
GM-CSF)
GM-CSF L. major PE macrophages CBA No Titus et al. (1984)
M-CSF
GM-CSF L. major S macrophages BALB/c No Greil et al. (1988)
IFN-y L. major PE macrophages CBA Yes Titus et al. (1984)
L. major BM macrophages C57BL/6 Yes Titus et al. (1984)
L. enriettii PE macrophages C57BL/6 Yes Titus et al. (1984)
L. enriettii PE macrophages CBA Yes Titus et al. (1984)
IFN-7 L. donovani RP macrophages BALB/c Yes Murray, H. W. et al. (1985)
IFN-y L. major P macrophages C57BL/6 Yes Wyler et al. (1987)
IFN-y L. major RP macrophages C3H/HeN No Davis et al. (1988)
IFN-y L. major S macrophages BALB/c Yes Greil et al. (1988)
IFN-y L. major RP macrophages C3H/HeN No Belosevic et al. (1988)
IFN-y L. major RP macrophages C3H/HeN Yes Belosevic et af. (1 988)
+ IL-2
IFN-y L. major RP macrophages C3H/HeN Yes Belosevic et al. (1988)
+IL-4
IFN-y L. major RP macrophages C3H/HeN Yes Belosevic et al. (1988)
+ GM-CSF
IFN-7 L. major PE macrophages CBA Yes Liew et al. (1989)
IFN-1 L. major PE macrophages CBA No Liew et al. (1989)
+ IL-3
IFN-y L. major PE macrophages CBA No Liew et al. (1989)
+ IL-4
TNF-a L. major PE macrophages BALB/c No Bogdan et al. (1990)
TNF-a L. major PE macrophages BALB/c No Bogdan et al. (1 990)
+ IL-4
TNF-a L. major PE macrophages BALB/c Yes Bogdan et al. (1 990)
+ IFN-y
TNF-a L. major PE macrophages CBA Yes Liew et al. (1990b)
TNF-a L. major PE macrophages CBA Yes Liew et al. (l990a)
+ IFN-y
Human
GM-CSF L. donovani M D macrophages - Yes Weiser et al. (1987)
IFN-y L. donovani MD macrophages - Yes Murray et al. (1983)
IFN-y L. donovani Monocytes - Yes Hoover et al. (1986)
IFN-7 L. donovani Monocytes - Yes Hoover et al. (1985b)
L. major Monocytes - No Hoover et al. (1985b)
IFN-7 L. mexicana Monocytes Yes Murray, H. W. et al. (1984)
+
~

IFN-7 L. donovani Monocytes - Yes Murray, H. W. et al. (1988)


Pentostam
IFN-y L.m. amazonensis Monocytes - Yes Ho et 01. (1990)
+ GM-CSF
G M G F L.m. amazonensis Monocytes - Yes Ho et al. (1990)
M-CSF L.m. amazonensis Monocytes - Yes Ho et al. (1 990)
IFN-y L.m. amazonensis Monocytes - Yes Ho et al. (1990)
+ M-CSF
IFN-y L. donovani Monocytes - No Lehn et al. (1989)
+ IL-4

BM, Bone marrow; CSF,colony stimulating factor; GM, granulocyte-macrophage;IFN,interferon; IL,interleukin; M, macrophage; MD, monocyte-
derived: PE. Deritoneal exudate: RP. resident Deritoneal: S. sdeen; TNF.tumour necrosis factor.
220 J. ALEXANDER AND D. G. RUSSELL

produced by both TH1 and TH2 cells, is clearly counter-protective (for


references, see Table 3). Thus, a TH1cell line which secretes IL-3 has been
associated with disease exacerbation (Feng et al., 1988), while other studies
have shown counter-protective CD4' T cells to transfer DTH-a suppo-
sedly TH1, but not a TH2 cell, property (Liew et al., 1985a,b; Milon et al.,
1986). The reported ability of CD4' T cell subsets to transfer resistance
adoptively, but not delayed hypersensitivity (Howard et al., 1982), may
equally argue a protective role for TH2 cells. Recent work by Moll and
Rollinghoff (1990) suggests that, while resistance to infection is associated
with TH1 cells producing IFN-y, susceptibility is associated with CD4' T
cells that do not segregate into the T,1 or TH2 subsets on the basis of
lymphokine secretion.

B. LYMPHOKINES AND MACROPHAGE ACTIVATION

While INF-y and TNF-a have clearly been shown to be involved in


activating macrophages to kill Leishmania, the role of other cytokines in
either inducing macrophage activation themselves or augmenting or inhibit-
ing the effects of IFN-y and TNF-a awaits further clarification (for refer-
ences see Table 4). Thus, for example, while all the evidence indicates that
IL-3, which is produced by both T,1 and TH2 cells, does not activate
macrophages and inhibits the anti-Leishmania protection mediated by IFN-y
(Liew et al., 1989), IL-4, a product of the counter-protective TH2cell subset,
can activate macrophages (Crawford, R. M. et al., 1987) and has been
reported as both augmenting (Belosevic et al., 1988) and inhibiting (Liew et
al., 1989) the macrophage-activating function of IFN-y. Similarly, although
IL-4 has been shown to prime the macrophage respiratory burst, a function
enhanced by TNF-a but inhibited by IFN-y (Phillips et al., 1990), it has been
shown not to enhance the macrophage leishmanicidal activity mediated by
TNF-a, which is enhanced by IFN-y (Bogdan et al., 1990). These apparently
contradictory observations may be explained by the different macrophage
subpopulations used by these different groups of workers. Thus, while Liew
et al. (1989) and Bogdan et al. (1990) used peritoneal exudate macrophages,
Belosevic et al. (1988) and Phillips et al. (1990) used resident peritoneal and
bone marrow-derived macrophages. Although using different macrophage
populations, all these various groups of workers pretreated their cells with
cytokines before infecting them with L. major. When Scott et al. (1989)
incubated resident peritoneal macrophages in cytokine mixtures before and
after infection with L. major, contrasting results were obtained: cytokine
pretreatment of macrophages indicated that IL-4 inhibited macrophage
activation and parasite killing mediated by IFN-y, while treatment of
infected macrophages suggested that IL-4 enhanced IFN-y-mediated leish-
INTERACTION OF LEISHMANIA WITH MACROPHAGES 22 I

manicidal activity. The interplay between IL-4, IFN-y and TNF-a in


inducing macrophage leishmanicidal activity is indeed complex, a fact that is
further emphasized by some recent publications. What is certain is that
TNF-a and IFN-y act synergistically to activate macrophages to kill L.
major (Liew et al., 1990a,b; Bogdan et al., 1990). Whether TNF-a is capable
of inducing macrophage leishmanicidal activity alone, as has been suggested
by Liew et al. (1990b,c), is still open to question (Moll et al., 1990; Bogdan et
al., 1990) (it should be noted that these authors used different mouse
strains). IL-4, unlike IFN-y, does not promote TNF-a-mediated macro-
phage activation (Bogdan et al.,' 1990) but, like TNF-a, it does, in low
concentrations, independently act synergistically with IFN-y to promote
macrophage activation (Solbach et al., 1991). Macrophage activation pro-
duced by IL-4 and INF-y could be inhibited by antibodies to TNF-a.
Solbach et al. (1991) have therefore suggested that the IL-4 and IFN-y
together induce macrophages to release TNF-a, which then activates the
leishmanicidal activity of macrophages synergistically with IFN-y. As the
results from studies with GM-CSF are as variable as those with IL-4 (Table
4), further complex interplays between T cell products and macrophages no
doubt await elucidation.
Although the ability of cytokines to activate macrophages to kill parasites
has been well documented, cytokine-independent mechanisms involving
direct macrophage-lymphocyte contact have also been described (Sypek
and Wyler, 1988; Wyler et al., 1987). This leishmanicidal activity, which is
mediated by CD4' T cells and not CD8' T cells, does not result in host cell
lysis.

C. MACROPHAGE ACTIVATION AND PARASITE KILLING

Until recently the toxic metabolites of oxygen, superoxide (0, -), singlet
oxygen (lo,), the hydroxyl radical (OH) and most especially hydrogen
peroxide (H,O,), have been thought to be responsible for macrophage
leishmanicidal activity (Murray, H. W. 1981; Pearson et al., 1982). Evidence
for this viewpoint arose because of studies that demonstrated that amasti-
gotes of L. donovani and metacyclic promastigotes of L. major (Da Silva et
al., 1989) survived better than, and triggered the macrophage respiratory
burst only weakly compared with, log phase promastigotes; this ability
was also attributable to amastigotes having higher levels of glutathione
peroxidase, superoxide and catalase than promastigotes (Murray, H. W.,
1982; Pearson et al., 1983). Intracellular amastigotes are also capable of
down-regulating the macrophage oxygen-dependent microbicidal potential
(Buchmuller-Rouiller and Mauel, 1987). Nevertheless, there has been
increasing evidence from several studies that oxygen-independent mechan-
222 J. ALEXANDER AND D. G. RUSSELL

isms are capable of killing Leishmania; not only are L. donovani, L. mexicana
and L. major resistant to oxygen radicals (Pearson et al., 1982; Mallinson
and Coombs, 1989b), but they can be killed by macrophages deficient in the
production of oxygen metabolites (Murray, H.W. and Cartelli, 1983; Scott
et al., 1985). Recently a new mechanism of macrophage anti-leishmania1
killing has been characterized as a novel metabolic pathway synthesizing
nitric oxides [nitric oxide (NO), nitrite (NO,-) and nitrate (NO,-)] from
L-arginine (Green et al., 1990; Liew et al., 1990a,b) with L-citrulline as a co-
product. Macrophage NO-mediated killing of L. major has been shown to be
induced by TNF-a acting synergistically with IFN-y (Liew et al., 1990a).
This biochemical pathway, as well as anti-Leishmania activity, is inhibited in
the presence of D-arginine and N-monomethyl-L-arginine (James and Hibbs,
1990; Liew et al., 1990a,b). Although the exact mechanism of NO-mediated
killing is not known, it is suspected that NO reacts covalently with intracel-
lular iron leading to the inhibition of enzymes with Fe-S prosthetic groups.
Such enzymes include some involved in DNA synthesis as well as
the proximal two oxidoreductases of the mitochondria1 electron transport
system (James and Hibbs, 1990).

AND VACCINATION
VIII. THERAPY

A. THERAPY
1. Chemotherapy

There have been relatively few new developments in anti-Leishmania chemo-


therapy in recent years. The pentavalent antimonial compounds sodium
stibogluconate (Pentostam@) and meglumine antimonate (Glucantimes)
remain the primary therapeutic agents for all forms of leishmaniasis
(reviewed by Bryceson, 1987). Furthermore, these drugs are known to be
fairly toxic, their mechanism of action is poorly understood (Berman et al.,
1985), and their success rate is variable and particularly poor in immuno-
depressed individuals-most notably patients with acquired immunodefi-
ciency syndrome (AIDS) (Rizzi et al., 1988). Many workers have suggested
that the intracellular environment is a barrier against successful chemo-
therapy; however the phagocytic potential of the macrophage as well as the
confirmed lysosomal nature of the parasitophorous vacuole (Alexander and
Vickerman, 1975; Russell et al., I991b) offer intriguing possibilities for
specifically targeting drugs to the parasite. Thus, by encapsulating drugs in
liposomes (New et al., 1978; Alving and Steck, 1979) or niosomes (Hunter et
al., 1988), which are preferentially phagocytosed by the macrophages of the
INTERACTION OF LEISHMANIA WITH MACROPHAGES 223

spleen and liver, chemotherapy against visceral leishmaniasis has been


markedly improved by delivering large quantities of the drug directly to the
parasitophorous vacuole. The physical and chemical characteristics of the
drug delivery system may be important in determining successful chemo-
therapy. Small vesicular carriers, for example, have been shown to be more
effective in vivo (Carter et al., 1988) while a commonly used liposomal
constituent, phosphatidyl serine, inhibits the macrophage respiratory burst
and leishmanicidal activity, and therefore would not be a suitable carrier
component (Gilbreath er al., 1986).
Not only do amastigotes reside within phagolysosomes but they contain
many lysosomes themselves which display high activity of a variety of
enzymes, particularly proteinases (reviewed by Coombs, 1988). Thus, the
likelihood is that lysosomotropic drugs would not only accumulate in the
parasitophorous vacuole, but also in the parasite lysosomal compartments
(reviewed by Rabinovitch, 1989). By such a mechanism the lysosomotropic
amino acid esters, for example the L-amino acid methyl ester of leucine, have
been shown to concentrate in parasite lysosomes, where they are hydrolysed
to form less permeable amino acids which accumulate to cause osmotic
disruption and parasite lysis. Unfortunately these amino acid esters are also
toxic for mammalian cells and could not be used therapeutically unless their
selectivity for parasites could be increased. This might be achieved by
attaching peptides to the drug to block activity which could be reactivated
following hydrolysis by the parasite proteases (Coombs, 1988). Drugs could
be attached to substances for which macrophages have high affinity recep-
tors. Acetyl low-density lipoprotein (Hart and Lawrence, 1988) and maley-
lated BSA (Mukhopadhyay er al., 1989) have both been used in this way to
improve chemotherapy .
An alternative approach to chemotherapy, again taking advantage of the
host macrophage, would be chemically to enhance macrophage microbicidal
activity. Indeed, several chemotherapeutic agents, particularly electron car-
riers which induce the release of toxic oxygen free radicals, have been shown
to be highly effective at killing parasites in vitro, e.g., chlorpromazine
(Pearson et al., 1984). However, these drugs are only poorly effective in vivo
and often toxic. Nevertheless, a recent study has indicated the potential
feasibility of such an approach when a 50% reduction in numbers of L.
donovani amastigotes could be achieved by an in vivo macrophage-targeted
hydrogen peroxide-generating system consisting of latex beads and glucose
oxidase bound to erythrocyte ghosts (Murray, H.W. and Nathan, 1988).
However, a more acceptable approach to increasing macrophage microbici-
dal activity might be to use biological or immunological modulators in
conjunction with chemotherapeutic agents.
TABLE5 ModiJication of Leishmania infection in vivo by cytokines

Protective1
Route of Mouse therapeutic
Cvtokine Species administration strain function Exacerbative Reference
Murine
IL-2 L. donovani ip + iv BALBIc No No Murray, H. W. et al.
(1987)
IL-2 L. donovani ip + Pentostam nu/nu/BALB/c Yes No Murray, H. W. et al.
(1989)
IL-2 L.m. amazonensis sc local BALBIc No Yes Mazingue et al. (1989)
IL-3 L. major iP BALBIc No Yes Feng et al. (1988)
CBA No No
IL-3 L. major ? BALBIc No Yes Louis et al. (1 987)
I L-4 L. major sc local BALBIc Yes No Carter et al. (1989)
TNF-a L. major iv C3H Yes No Titus et al. (1989)
BALBIc Yes No
GM-CSF L. major iP BALB/c No Yes Solbach et al. (1987a)
GM-CSF L. major iP BALBIc No No Corcoran et al. (1988)
GM-CSF L. major iP BALBIc No Yes Greil et al. (1988)
IFN-y L. donovani ip, iv + im BALBIc Yes No Murray, H . W. et al.
( I 985)
IFN-7 L. major sc local BALB/c Yes No Kiderlen and Lohmann-
iv C57BL/6 Yes No Matthes (1988)
IFN-.( L. donovani ip + iv BALB/c Yes No Murray, H. W. et al.
(1987)
IFN-.( L. donovani ip + Pentostam BALB/c Yes No Murray, H. W. et a/.
(1988)
IFN-y L. donovani ip + Pentostam nu/nu BALB/c Yes No Murray, H. W. et al.
(1989)
IFN-)I L. donovani sc osmotic minipump BALB/c Yes No Murray, H. W. (1990)
TNF-a L. major sc local CBA Yes No Liew et al. (1990~)

Human
IL-2 L. aethiopica Intranodular - Yes No Akuffo et al. (1 990)
IFN-.I L . braziliensis sc local - Yes No Harms et al. (1989)
L. tropica sc local Yes No
IFN-.I L. d. chagasi +
im Pentostam - Yes No Badaro et al. (1990)
IFN-7 L. tropica sc + pentavalent - Yes No Kurkcuoglu and Tandogdu
antimony ( 1990)

CSF, Colony stimulating factor; G, granulocyte; IFN, interferon; IL, interleukin; ip, intraperitoneal; iv, intravenous; M, macrophage; sc, subcutaneous;
TNF. tumour necrosis factor.
226 J. ALEXANDER AND D. G. RUSSELL

2. Combined chemotherapy and immunotherapy

The success of any chemotherapeutic regimen is often dependent on the


potential or latent immunological response of the patient. It is also generally
accepted that successful chemotherapy of leishmaniasis in humans results in
the generation of antigen-specific T cells and delayed hypersensitivity.
However, when the patients have a defective immune response (e.g. AIDS
patients), chemotherapy is invariably ineffective (Rizzi et al., 1988). This
close association between chemotherapy and cell-mediated immunity
suggests that a dual approach to therapy could be advantageous. Several
workers have therefore examined the ability of a variety of immunomodu-
lators and adjuvants to enhance the effects of standard anti-Leishmania
drugs and have achieved notable success. Thus, synergistic activity between
muramyl dipeptide encapsulated in liposomes (Adinolfi et al., 1985) or in
Corynebacterium parvum (Haidaris and Bonventure, 1983) and pentavalent
antimonials has been demonstrated in experimental visceral leishmaniasis.
The immunopotentiator is presumed to function by non-specifically activa-
ting macrophages. It is not surprising, therefore, that more recent experi-
mental as well as clinical studies have successfully used recombinant IFN-y
as the macrophage-activating agent in this dual therapy approach to
treatment (for references see Table 5).

3. Immunotherapy

Various immunopotentiators, including bacillus Calmette-Gukrin (BCG)


(Fortier et al., 1987), BCG plus killed promastigotes (Castes et al., 1989),
levamisole (Rezai et al., 1988), cyclosporin A (Bogdan et al., 1989), C.
parvum (Hill, 1987), and glucan (Cook et al., 1980), have been used to
modify the course of Leishmania infections, largely through their ability to
activate macrophages non-specifically. Not only are complex immunopoten-
tiators like BCG and C . parvum excluded from general human usage because
of potential undesirable side-effects, but experimental evidence indicates that
they may not always produce the desired therapeutic effect: BCG, for
example, has been reported as exacerbating L . mexicana lesion growth in
laboratory mice (Grimaldi et al., 1980). However, the current availability of
recombinant cytokines provides a new generation of tools with which we can
influence the immune response against Leishmania in a more controlled and
effective fashion. Consequently, there are many recent reports of workers
trying to manipulate the growth of Leishmania by local or systemic injection
of these substances (Table 5).
Results using cytokines have been variable and at times have appeared to
be contradictory. In part, this may reflect the species of parasite used, the
INTERACTION OF LEISHMANIA WITH MACROPHAGES 227

genetic background of the host, the route and dosage of cytokine adminis-
tration and the state of disease progression at the time of treatment. Of the
two CD4' T cell subsets shown to be involved in controlling Leishmania
infections, T,1 cells (which produce IFN-y, IL-2 and IL-3) have been
associated with protection, while T,2 cells (which produce IL-3, IL-4 and
IL-5) have been associated with disease exacerbation (Section VII.A.3).
Logically, one would expect that treatment with IFN-y and/or IL-2 should
induce a protective response against this organism, while IL-4 and/or IL-5
would promote disease exacerbation. Similarly, it would be expected that
cytokines which are known to promote macrophage leishmanicidal activity,
such as GM-CSF (Handman and Burgess, 1979; Weiser et al., 1987; HO et
al., 1990) and TNF-a (Liew et al., 1990a,b) would be protective in vivo, while
those generally associated with inhibiting microbicidal activity such as IL-3
(Liew et al., 1989) would exacerbate disease.
Although, as one would expect, IFN-y has been shown in numerous
studies to control the growth of Leishmania in vivo (Table 5 ) , an effect
reversed by treating mice with antibodies neutralizing IFN-y (Belosevic et
al., 1989; Squires et al., 1989), the few reports that have been published on
administration of IL-2 in vivo suggest that its role is open to question. Where-
as, for example, local administration of IL-2 to patients infected with L.
aethiopica did induce a protective response (Akuffo et al., 1990), similar
treatment of mice infected with L. amazonensis exacerbated lesion and
parasite growth (Mazingue et al., 1989). Systemic injection of IL-2, on the
other hand, had no effect on the growth of L . donovani in BALB/c mice
(Murray, H. W. et al., 1987). All studies in vivo have so far confirmed the
expected disease-exacerbating capacity of injected IL-3 (Louis et al., 1987;
Feng et al., 1988) and the protection-inducing capacity of TNF-a (Titus el
al., 1989; Liew et al., 1990c), the TNF-a effect being reversible with
neutralizing antibodies (Liew et al., 1990~).Surprisingly, when one considers
the ability of GM-CSF to activate macrophage leishmanicidal activity (Hand-
man and Burgess, 1979; Ralph et al., 1983; Ho et al., 1990), therapeutic
studies to date indicate a counter-protective role for this cytokine (Solbach
et al., 1987a; Greil et al., 1988). The exacerbative effects of GM-CSF and
IL-3 have been related to their increasing the pool of circulating monocytes
and providing additional host cells for the parasites. The role of IL-4 in
macrophage activation remains unclear and somewhat controversial (Sec-
tion'VI1.B). Its role in vivo in inducing protection or counter-protection also
awaits further clarification. Whereas, for example, treating mice with anti-
bodies neutralizing IL-4 allowed BALB/c mice to inhibit the growth of L.
major (Sadick et al., 1990), by distinct contrast treating developing lesions of
L. major locally with low concentrations of IL-4 in a slow-release matrix
promoted healing and protective immunity (Carter et al., 1989). The effects
228 J. ALEXANDER AND D. G . RUSSELL

of IL-4 injected directly into footpads infected with L. major have recently
been studied (Lezama-Davila et al., in press). Treatment at the onset of
infection promoted increased parasite growth while, by contrast, treatment
of established but still developing lesions inhibited further lesion growth and
parasite multiplication. Obviously T cellkytokine-macrophage interplay is
complex and awaits further detailed study, but the prospects for future
cytokine immunotherapy in leishmaniasis remain exciting.

B. VACCINATION

Vaccination against leishmaniasis has a long and chequered history


(reviewed by Alexander, 1988b). From as early as the 19th century, and as
recently as 1990, living organisms have been used for vaccination (Peters et
al., 1990). In the latter, most recent, study, the exacerbated L. major lesion
growth following challenge of individuals vaccinated with L. arabica, as
compared with that in controls, is extremely troubling with regard to the
development of a vaccine for use in humans. Experimental studies using
subcutaneous vaccination with heat-killed or radio-attenuated parasites
have also often resulted in disease exacerbation following challenge (Liew et
al., 1985b). In order to limit the likelihood of any candidate vaccine
enhancing the disease process following infection, it is essential that the
vaccine consists of immunologically characterized purified antigen or its
derivatives. Studies have shown that not only parasite membrane antigens
can induce protection (Handman and Mitchell, 1985; Russell and Alex-
ander, 1988), but also soluble non-membrane antigens (Scott et al., 1989).
While Scott et al. (1989) have demonstrated that distinct antigen fractions
stimulated protection and exacerbation, it is our view that the way in which
antigen is packaged and presented to the host can often determine the
outcome of disease following challenge infection. In this context we believe
that macrophages, in their antigen-presenting role, play a crucial part.
Although the recent vaccine literature is large and many antigens have been
studied, in this review we shall confine our discussion to the antigens we have
personally investigated, namely LPG and gp63.

1. Vaccination with lipophosphoglycan

Handman and Mitchell (1985) reported successful vaccination of BALB/c


mice with immunoaffinity purified L. major LPG. Interestingly, soluble LPG
that lacked the phosphatidylinositol anchor appeared to induce a disease-
exacerbating immune response. Similar protection against L. mexicana in
CBA/Ca mice was later induced with homologous LPG, also isolated by
immunoaffinity chromatography, reconstituted into liposomes (Russell and
INTERACTION OF LEISHMANIA WITH MACROPHAGES 229

Alexander, 1988). However, it is now known that LPG prepared by


immunoaffinity isolation is heavily contaminated with peptide material.
Difficulties have been experienced in obtaining evidence that LPG contains
functional T cell epitopes; therefore, the mechanism of protection and the
possible use of an LPG-based vaccine are open to question.

:s ca M ce. Sukutaneaua.

control.(6/6)
CBA/ca Mice. Intraperitoneal.

rude &.(2/6)

10
ICE. u u onwuo. W B / C Mice. Intmparitoneol.

0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 20

TIME (weekm).

FIG. 8. A compilation of data from Russell and Alexander (1988) concerning the
protective properties of liposomes with gp63 reconstituted into the bilayer. Female
BALB/c or CBA mice were inoculated with either phosphatidyl choline liposomes
(control), phosphatidyl choline liposomes with crude membrane antigens (crude Ag),
or liposomes with gp63. Inoculations were delivered by the subcutaneous or intra-
peritoneal route twice, 4 and 8 weeks before challenge with 5 x lo4 stationary phase
L. rnexicunu promastigotes. The numbers in parentheses at the end of each curve
indicate (first) the number of infected individuals and (second) the group size.
Complete protection, determined by the inability to detect lesion development, was
found in CBA mice inoculated with gp63 liposomes by both subcutaneous and
intraperitoneal routes.

2. Vaccination with gp63

We have reported that mice inoculated with liposomes reconstituted with


gp63 in the bilayer were protected against subsequent challenge with L.
mexicana promastigotes (Fig. 8) (Russell, 1987b; Russell and Alexander,
230 J. ALEXANDER AND D. G . RUSSELL

1988; Alexander and Russell, 1988). In CBA/Ca mice, under some experimen-
tal conditions, the level of protection appeared complete. Protection in
BALB/c mice, which are exquisitely sensitive to L. mexicana and show a low
T cell response to gp63, was less impressive. This result is reproducible in the
CBA/Ca system which, in contrast to the BALB/c, is a high responder for
gp63 (unpublished results). Protection can be transferred with T cells that
are predominantly of the CD4 phenotype. Since our results appeared,
several groups have published studies, mainly using the BALB/c-L. major
model, that were unable to document similar levels of protection or response
(Kahl et al., 1989; see also Handman et al., 1990). However, considerable
protection against L. major has been induced in CBA/Ca mice by means of
an oral Salmonella typhimurium vaccine expressing gp63 (Yang et al., 1990).
Furthermore, Jardim et al. (1990) have recently made considerable progress
in defining the “desirable” epitopes on gp63 by studying the protective
response induced by synthetic peptides selected on the basis of Rothbard’s T
cell epitope algorithm (Rothbard and Taylor, 1988). One peptide, PT3, from
amino acids 154-168, generated, when injected with adjuvant, a strong
protective response against L. major in BALB/c mice, L. mexicana in CBA/
Ca mice, and L . infantum in hamsters (unpublished results). Interestingly,
this peptide spans the zinc binding site of the metalloprotease and is 100%
conserved in theifour species of Leishmania sequenced to date (Button and
McMaster, 1988; Miller et al., 1990; E. Medina-Acosta and D. G. Russell,
unpublished results). The T cells responsible for immunity were CD4’ and
secreted IL-2, a characteristic ascribed to the TH1 functional T-helper cell
subset. Surprisingly, PT3 inoculated without adjuvant resulted in disease
exacerbation following challenge infection with L. major.

3. Macrophage involvement

The observations reported above collectively have led us to suggest an


essential role for macrophages in the generation of a protective response
following vaccination. We know, for example, that liposomal antigen can be
processed only by macrophages and not by B cells (reviewed by Van
Rooijen, 1990). Moreover, macrophage antigen presentation is associated
with, and up-regulated by, the products of .TH1cells which, conversely,
down-regulate B cell class I1 expression (Steeg et al., 1982; Mond et al.,
1986). TH1cell cytokine production is, in turn, inhibited by the products of
TH2 cells which up-regulate B cell class I1 expression and down-regulate
expression in macrophages (Fiorentini et al., 1989; Go et al., 1990). Interest-
ingly, BALB/c mice depleted of B cells by anti-IgM treatment developed
resistance to L. major (Sacks et al., 1984). Thus, given the correct stimu-
lation, and presumably processing preferentially via macrophages, a T, 1 cell
INTERACTION OF LEISHMANIA WITH MACROPHAGES 23 1

response and protection rather than a TH2 cell response and counter-
protection can be induced against a single T cell epitope. Therefore,
adjuvants which are potent stimulators of TH1 cell activation, namely
Freund’s complete adjuvant (Grun and Maurer, 1989) and certain non-ionic
surfactants (Brewer and Alexander, in press), have transformed a potentially
counter-protective T cell epitope, PT3, into one that inhibits disease pro-
gression (Jardim et d., 1990).

IX. CONCLUDING
REMARKS

Despite the volume of literature cited, indicating how intensely the subject is
being studied at present, this review was not intended as an encyclopaedic
compendium of all that is known about Leishmania. It was heavily slanted
towards our current understanding of the biology of the host-parasite
interaction and, as such, avoided mention of basic molecular genetics and
also parasite metabolism. Parasite metabolism was left out because the line
had to be drawn somewhere, and neither of us felt we could do this field
justice. Basic molecular genetics was omitted because it does not, as yet,
contribute greatly to our understanding of host-parasite interplay, although,
through the development of transfection, transformation and gene deletion
techniques, this area will probably include the next generation of exper-
iments.

ACKNOWLEDGEMENTS

We thank Denise Williams for her help in compiling the tables and Ellen
Anne Quinn for her secretarial assistance.

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252 J. ALEXANDER A N D D. G. RUSSELL

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254 J. ALEXANDER AND D. G. RUSSELL

Note added in proof [see pp. 2 16-2 171

One of us (D.G.R.)has recently shown that Leishmania amastigotes survive


the initial interaction with CD8+ cytotoxic T cells but are killed subse-
quently by bystander macrophages, activated as a consequence of cytokines
released by the stimulated T cell.

(Smith, L. E., Rodrigues, M. and Russell, D. G. (1991). The interaction between


CD8 + cytotoxic T-cells and Leishmania-infected macrophages. Journal of
Experimental Medicine 174, 499-505.)
The Effects of Trypanosomatids on Insects

GUNTERA. SCHAUB

Department of Special Zoology and Parasitology, Ruhr University,


0-4630 Bochum, Germany

I. Introduction ............................ 255


11. Parasitogenic Alterations of Host Behaviour 251
A. Reduction of fitness . . . . . . . . . . . . . . . . . 258
B. Modification of vector feeding behaviour ........................
111. Disturbances in Organ Systems .
A. Disturbances of the digestive tract
B. Disturbances of the Malpighi
C. Effects on the haemolymph .................................. 275
D. Effects on the cuticle ..................
E. Other affected organ systems ......................................... 281
IV. Effects on Pre-adult Development and Mortality
A. Trypanosoma infections of Triatominae ................................ 282
B. Blastocrirhidia rriafomae infections of Triatominae ...................... 284
C. Homoxenous trypanosomatids in Hymenoptera and Diptera
V. Effects on Adult Life Span and Reproduction Rate . . . . . . . . . . . .
A. Leishmania and Trypanosoma ................ 291
B. Homoxenous trypanosomatids ........................................ 294
VI. Synergistic Effects of Trypanosomatids and other Stressors . . . . . . . . . . . . . . . . . . 295
A. Sensitivity to insecticides 296
B. Starvation resistance ................................................. 297
C. Sensitivity to isolation and overcrowding .............................. 298
VII. Mechanisms of Pathogenicity . . . . . . . . . . . . . .
VIII.
Acknowledgements .......... 304
References .............................................................. 304

I. INTRODUCTION

The trypanosomatids in insects can be divided according to their life cycles


and their genus-specific developmental stages into two groups, both to be
considered in this review. Members of the genera of the first group, the
heteroxenous trypanosomatids, are transmitted by insects to vertebrates
(Leishmania, Trypanosoma, Endotrypanum) or plants (Phytomonas) and are
ADVANCES IN PARASITOLOGY VOL. 31 Copyright 0 1992 Academic Press Limited
ISBN 0-12-031731-1 A// rights of reprodurrion in any form reserved
256 G . A. SCHAUB

causative agents of important diseases (Hoare, 1972; Molyneux, 1977).


Those of the second group, the homoxenous (entomophilic) trypanosoma-
tids, have only a single host, an arthropod (Herpetomonas, Crithidia, Rhynch-
oidomonas, Blastocrithidia) or sometimes another invertebrate (Leptomonas)
(Wallace, 1966, 1979; Molyneux, 1977).* Usually they colonize the intestinal
tract of the insects, but some species of both groups also invade the
haemocoele (summarized by Molyneux et al., 1986b).
In most cases the heteroxenous trypanosomatids do not affect their
vectors; some exceptions (Leishmania spp., Tryp. cruzi, Tryp. lewisi, Tryp.
rangeli, a bat and a bird trypanosome) are mentioned by Kramer (1963),
Jenkins (1964), Brooks (1974) and Molyneux (1977, 1981, 1983). Only Tryp.
rangeli has been the object of detailed studies. Like Tryp. cruzi, the causative
agent of Chagas disease, it colonizes the intestinal tract of triatomines, but
only Tryp. rangeli additionally invades the haemocoele and salivary glands
and multiplies intra- and extracellularly in the haemocoele (D’Alessandro.
1976). Investigations with Tryp. rangeli are complicated by the high varia-
bility of strains: also rapid attenuation during culture in vitro occurs,
indicated by loss of the ability to invade the haemocoele. Therefore, some
workers have inoculated the parasite directly into the haemocoele. The
pathology of this trypanosome has been reviewed by D’Alessandro (1 976),
but additional interesting aspects were investigated later (e.g. by Aiiez,
1982, 1983, 1984; Aiiez and East, 1984; Schwarzenbach, 1987).
Since insects are important pests, any parasite of insects such as the
homoxenous trypanosomatids should be considered as a possible agent for
biological control. However, textbooks or reviews on insect pathology
usually emphasize the importance of viruses, bacteria and fungi (Steinhaus,
1949, 1963a,b; Weiser, 1977). Of the Protozoa, the Sporozoa are always
considered, and most diseased insects are infected by Microsporidia and
Gregarina and only rarely by Flagellata (Lipa and Steinhaus, 1962;
McLaughlin, 1973). The opinion of Sweetman (1958) that trypanosomatids
“do not seem to seriously interfere with reproduction or produce serious
epizootics among their hosts” is shared by most protozoologists. In later
reviews of insect pathology or trypanosomatids, some pathogenic effects of
entomophilic trypanosomatids are mentioned. Flagellatoses due to homo-
xenous trypanosomatids have been reported, for Lept. pyrrhocoris, H.
muscarum, H . swainei ahd B. caliroa (Lipa, 1963; Brooks, 1974; Molyneux,
1977, 1980b; Wallace, 1979; Henry, 1981).
* Based on the Greek term xenos = host, the term “heteroxenous” should be used for
trypanosomatids which develop in different groups of hosts and “homoxenous” for those
developing in related species; “monoxenous” should be used only for those developing in a
single species. The terms “monogenetic” and “digenetic”, which are sometimes used, are
misleading-specially the latter (see “Lexikon Biologie”, published by Herder Verlag, Frei-
burg).
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 257

In 1 9 7 6 f i v e years after the first description-Haberkorn first presented


observations showing that B. triatomae is pathogenic for Triatoma infestans,
the most important vector of Chagas disease. At his invitation, his system
has been taken over by me for standardization and the inclusion of further
vectors of Chagas disease. Since then, B. triatomae has been the object of
detailed studies to elucidate its possible application for biological control of
triatomines (results are summarized by Schaub, 1988e, 1990c; Schaub et al.,
1990a). Like Tryp. cruzi and Tryp. rangeli, B. triatomae colonizes the
intestinal tract and the Malpighian tubules, but only B. triatomae develops
drought-resistant cysts with peculiar ultrastructural adaptations (Schaub
and Pretsch, 1981; Schaub, 1983; Reduth, 1986; Reduth and Schaub, 1988;
Schaub and Losch, 1988). Interestingly, B. triatomae and Tryp. rangeli are
pathogenic or non-pathogenic to different species of triatomines; specifi-
cally, Tryp. rangeli affects species of the genus Rhodnius only.
More and more effects of trypanosomes have been recognized in other
systems in the last 10 years, making it possible to write this present review
which is concerned, for the first time, solely with pathological effects of
trypanosomatids on insects.* Instead of describing the effects in each system,
I have grouped them into five topics: behavioural alterations, disturbances
of organ systems, effects on pre-adult developmental times and mortality
rates, effects on adult life span and reproduction rate, and synergistic effects
of trypanosomatids and other stressors. This review is also intended to direct
the reader to a phenomenon which is indicated only by minor effects and
needs to receive more attention, the subpathogenic stressing of the insect
hosts. Since natural populations of ihsects rarely live under optimum
conditions, the subpathogenic stressor trypanosomatid might act synergisti-
cally with other biotic or abiotic stressors and thus harm the insect host.

IT. PARASITWENIC
ALTERATIONS
OF HOSTBEHAVIOUR

Effects of infections on host behaviour, which improve parasite transmission


and which have been elucidated in recent years in many parasite-host
systems (reviewed by Schaub, 1989c; Hurd, 1990), also occur in trypano-
somatid-insect systems. Some insects are only weakened by the infection
while others, bloodsucking insects, attack their hosts more frequently.

As this is the first review summarizing publications concerning the influence of trypano-
somatids on insects, it is possible that I have missed some publications. If any reader knows of
any such missing reports I should very much appreciate the information, so as to be able to in-
clude them in a later review.
258 G. A. SCHAUB

A. REDUCTION OF FITNESS

So far, no investigation has studied the predation rate of insects infected


with trypanosomatids, but this rate should be increased if the insects are
weakened. Such non-specific effects on behaviour, i.e. sluggish movements,
have been reported for Rhodnius prolixus infected with Tryp. rangeli
(Grewal, 1957, 1969), Pyrrhocoris apterus infected with Lept. pyrrhocoris
(Lipa, 1963), Tri. infestans infected with B. triatomae (Schaub and Schnitker,
1988), and advanced infections of Hippelates pusio with H . muscarum (Bailey
and Brooks, 1972a). Most recently, Shykoff and Schmid-Hempel (1991b)
found that worker bumble bees naturally infected with C. bombi are less
likely to forage for pollen.
The only quantitative investigation of endurance has been undertaken by
Arnqvist and Maki (1990): male water striders, naturally infected with B.
gerridis and/or C.Jlexonema, do not skate as intensively against the current
in a circular stream channel as uninfected specimens. Skating endurance is
negatively correlated with the intensity of the trypanosomatid infection, and
may adversely affect predation of food and mating. t

Such an adverse effect on the ability of males to acquire mates occurs in


natural populations. Whereas light and moderate infections lower the mate
acquisition ability only to some extent, heavy infections drastically reduce
the mating success of infected males. This effect is caused by the reduced
fitness, since the search for females and the precopulative struggle with
females, which are reluctant to mate, is energy-intensive (G. Arnqvist,
personal communication).

B. MODIFICATION OF VECTOR FEEDING BEHAVIOUR

1. General

Parasitogenic alterations of behaviour of many bloodsucking insects by


infections with trypanosomatids are not so spectacular as those induced by
helminths in their intermediate hosts, e.g. they do not include the death of
the invertebrate host, but they seem to be very efficient (Schaub, 1989~).
There are two possible mechanisms by which the number of attacks on
blood donors by bloodsucking insects could be increased. (i) Trypano-
somatids and the insect host compete for metabolites in the ingested blood,
and the depletion leads to a new attempt by the insect to ingest blood. This
possibility seems to occur in phlebotomines which are presumed to be
infected with the bat trypanosome Tryp. leonidasdeani (Williams, 1976) (see
Section V.A), and it may also be relevant in infected bugs (see Section
II.B.4). (ii) The trypanosomes interfere with the ingestion process. These
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 259

effects on bloodsucking insects are connected with disturbances of the


digestive tract, especially the foregut and the anterior midgut (see Section
II1.A). Ingestion of blood by infected sandflies, tsetse flies and bugs is often
delayed and ceases if the host makes repulsive actions. These vectors may
then attack another host. Additionally, the infected vectors often take no, or
only a small, bloodmeal and therefore become hungry earlier and attack a
new host, enhancing the chances of parasite transmission (reviewed by
Molyneux and Jefferies, 1986). The mechanisms of these effects seem to
differ slightly in the different trypanosomatid-vector systems.

2. Sandflies infected with Leishmania

In several Leishmania-sandfly systems the parasites initially colonize the


midgut and then the foregut, which can be covered by a “carpet” of attached
flagellates (Warburg et al., 1986; Kaddu et al., 1988; Killick-Kendrick et al.,
1988). The infectious stages then detach and remain lying on the “carpet” or
migrate forward. At least in infections with one species, Leish. donovani, the
pharynx of the sandfly Phlebotomus argentipes can be blocked for its entire
length with a plug of parasites (Shortt et al., 1926), similar to the develop-
ment of Bacillus pestis in the rat flea (Bacot and Martin, 1914; Holdenried,
1952). Nearly 22% of the infected flies possess blocked foreguts, and in other
specimens the lumen of the foregut is significantly narrowed by the parasites
(Smith et al., 1940). Sandflies with a partially blocked foregut can take up
only minute quantities of blood, and complete blockage excludes a further
blood meal. However, all continue trying to obtain a blood meal (Smith et
al., 1940), sometimes at different locations, but in an extreme case for 18 min
at one location (Smith et al., 1941). Probing without subsequent uptake of
blood increases the chance of transmission of the parasites compared to
infected flies which successfully engorge (Killick-Kendrick et al., 1977b).
Five out of 16 sandflies infected with Leish. mexicana amazonensis probed
repeatedly but took no blood, and a further eight flies ingested only a small
meal (Killick-Kendrick et al., 1977b). This behaviour occurs also in phlebo-
tomines infected with Leish. major, thereby explaining the occurrence of 11
separate, closely adjacent lesions in humans after 11 probings of one sandfly
(Beach et al., 1984). A single infected sandfly probed 26 times in an area 2 cm
in diameter on the arm of a volunteer; 11 small cutaneous lesions resulted,
indicating transmission of parasites (Killick-Kendrick et al., 1985). Individ-
ual evaluation of the number of probings, the occurrence of blood ingestion
and the colonization of the different intestinal regions by Leishmania showed
that uninfected sandflies and those infected with Leish. major, in which the
infection was limited to the midgut, engorged in less than 10min after the
first or second probing. Sandflies in which the established infection had
260 G. A. SCHAUB

proceeded to the cibarium region of the foregut probed at least three times-
in most cases more often-and took only a little or no blood during a period
of 15 min or more (Beach e f af., 1985).
The mechanism of the action of the parasites is still unknown. The theory
of blockage of the foregut has been called in question by Killick-Kendrick et
a f .(1977b). They emphasized that “the blockage is probably more apparent
than real, since the powerful dilator muscles of the cibarium and pharynx
would easily widen the canal” and suggested that parasites might interfere
with sensilla in the cibarium. At that time such sensilla were known only
from other bloodsucking insects, but later they were indeed described in the
proboscises of uninfected sandflies (Killick-Kendrick and Molyneux, 198 1)
and their presence in the labrum and the cibarium was suggested by Lewis
( 1 984). In a detailed scanning electron microscopical investigation, Jefferies
(1987) described in the cibarium two to five trichoid sensilla with a tapering
hair that were not chemoreceptors, but were perhaps mechanoreceptors. In
addition to the blockage and the sensilla theory, calculations of the fluid
mechanisms of blood flow suggest a third possible mechanism, as they
indicate that the attached parasites, especially those in the pharynx, are
likely to impair flow (Jefferies et af.,1986). Perhaps this results in an indirect
feedback effect on receptor functions in the anterior foregut. In a later
publication Killick-Kendrick et a f . ( 1988) described a pharynx blocked by
Leish. major and indicated the importance of a gel around the parasites,
possibly an excreted factor. This report supports the blockage theory
(Killick-Kendrick and Molyneux, 1990).

3. Tsetse .pies infected with Trypanosoma

Development of the salivarian trypanosomes in tsetse flies varies subgenus-


specifically. Some of these species, which are the causative agents of nagana
and sleeping sickness, e.g. Tryp. vivax, colonize the mouthparts only, while
others develop in the midgut and salivary glands (Hoare, 1972; Molyneux
and Ashford, 1983).
Results of investigations of the feeding behaviour of tsetse flies are
contradictory. Compared to uninfected flies, fewer flies infected with Tryp.
hrucei fed at the first probe and about two to three more probes occurred
before blood ingestion. In addition, the infected flies seemed to be more
voracious (Jenni et al., 1980). Also, tsetse flies infected with Tryp. congolense
probed significantly more times than uninfected flies, and-as in the case of
Leishmania infections-probing alone was sufficient to infect mammals
(Roberts, 1981).
These results seem to explain the observation that natural infection rates
are much lower in tsetse flies than in mammals. However, in the studies by
Moloo’s group most aspects of feeding (e.g. number of probes, ingestion
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 26 1

rate, volume of ingested blood) of Glossina morsitans morsitans, G . m.


centralis or G . palpalis gambiensis fed on mice, rabbits or goats were not
affected by infections with Tryp. congolense, Tryp. vivax or Tryp. brucei
(Moloo, 1983; Moloo and Dar, 1985; Makumi and Moloo, 1991).
Searching for explanations for these effects, the early studies of sandflies
infected with Leishmania prompted similar studies in tsetse flies. Scanning
electron microscopy demonstrated heavy colonization of the labrum and a
close association of Tryp. congolense with mechanoreceptive sensilla which
act as fluid flow meters (Molyneux et al., 1979). In addition, Tryp. brucei and
Tryp. vivax also attach to the bases of the sensilla, and sensilla hairs are
entangled in rosettes of flagellates, but the colonization density of Tryp.
brucei in the labrum is remarkably low compared with that of the other two
species (Molyneux, 1980a; Molyneux and Jenni, 1981) (Fig. la,b). Compar-
ing development of laboratory and natural infections with Tryp. congolense
and Tryp. vivax in the cibarium of Glossina, Tryp. congolense infections also
tended to be heavier (Jefferies et al., 1987). A compact layer of Tryp.
congolense in the labrum was also evident by light microscopy (Ladikpo and
Seureau, 1988) and transmission electron microscopy (ThCvenaz and
Hecker, 1980). In the latter study, hemidesmosome-like plaques in the
enlargements of flagella at the bases of the receptors indicated firm attach-
ment of parasites (Fig. Ic).
Calculation of the effect of the colonization on blood flow in labrum and
hypopharynx indicated that the reduced diameter would strongly affect the
blood flow and increase the pressure required to expel saliva (Molyneux,
1980a). The frequency or capacity of the cibarial pump cannot be increased
without limit, since increased viscosity of the blood meal in membrane
feeding experiments reduced the rate of feeding (Jenni et al., 1980). The
reduced rate of feeding can be compensated by a longer feeding period. This
has been observed in tsetse flies infected with Tryp. congolense (Roberts,
1981). Since mammals normally attempt to repel a probing tsetse fly, the
increase of feeding time increases the chance of feeding being interrupted.
At first the data indicated that the discrepancies between investigations of
the effects on feeding behaviour could be due to different colonization
densities. As stated by Molyneux and Jefferies (l986), Jefferies also found no
effects in his PhD thesis research, but only small areas of the labrum were
colonized by Tryp. congolense and Tryp. vivax and not the region with the
sensilla. 'However, in the most recent investigation by Moloo's group,
rosettes of Tryp. vivax were reported to be present in the labrum (Makumi
and Moloo, 1991).
Further detailed studies with natural infections or fresh strains of parasite
and insect host are necessary, including determination of the colonization
densities in different regions of the foregut. The mechanism of action of
salivarian trypanosomes could thus be further elucidated. So far, the dense
262 G. A. SCHAUB
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 263

colonization of the foregut and/or the interference with the sensilla seem to
be responsible for the altered feeding behaviour of infected flies (Livesey et
al., 1980). However, pathological effects on the salivary glands should also
be considered. Such effects occur in infected Gfossina(see Section 1II.E) and
it has been suggested that they are responsible for affecting the feeding
behaviour of mosquitoes infected with malaria (Rossignol et af., 1986).

4. Triatornines infected with Trypanosoma

Effects on feeding behaviour are also known to occur with triatomines after
infection with Tryp. cruzi or Tryp. rangeli (D’Alessandro and Mandel, 1969;
Aiiez and East, 1984). If uninfected larvae and adults and those which are
naturally infected with Tryp. rangeli and/or Tryp. cruzi were given an
opportunity to feed on mice, infected larvae fed less frequently (the
difference was statistically significant) than uninfected larvae (D’Alessan-
dro and Mandel, 1969). This phenomenon was also evident with infected
adults, but the difference was not statistically significant from those infected
with Tryp. cruzi. Probing behaviour of R. robustus and R . prolixus infected
with Tryp. rangefi was also affected (Aiiez and East, 1984): whereas unin-
fected bugs probed on average twice before engorging (range 1-5 probes),
infected bugs probed on average 13 times (range 2-28 probes) and for longer
periods than uninfected ones. Some of the infected bugs ingested only small
amounts or no blood at all, one of them even after 28 probes.
The mechanisms of these disturbances, e.g. the colonization of the
foregut, have not been elucidated. Effects on the salivary gland have to be
considered, because their cells can be damaged or destroyed (Schwarzen-
bach, 1987) (see Section 1II.E).
In addition, more features have to be included to elucidate the action of
the trypanosomatids on bugs. In a series of investigations of the effects of
starvation on trypanosomatid-triatomine interactions (Schaub and Boker,
1986b; Schaub, 1988b, 1990d, 1991; Schaub and Losch, 1989; Schaub et al.,
1989a), we had the impression that prolonged starvation affected the volume
of ingested blood. In our most recent study of the feeding behaviour of
FIG. 1. Sensilla (arrow heads) in the labrum of Glossina morsitans morsitans
associated with Trypanosoma parasites (P) (a, b: scanning electron micrographs; c:
transmission electron micrograph). (a) Trypanosoma (Trypanozoon) brucei. Bar =
5 pm. (b, c) Trypanosoma (Nannomonas) congolense. (b) Bar = 2 pm. (c) Hemides-
mosomal plaques (arrowheads) are present in the attachment zone of parasites to the
cuticle (C) of the labrum, and to the basal cup (B) and stalk (S) of a sensillum. Bar =
I pm. (Fig. la, b reproduced by permission from Molyneux and Jenni, 1981,
Transactions of the Royal Society of Tropical Medicine and Hygiene 75, 160-163, and
Fig. lc reproduced by permission from Thevenaz and Hecker, 1980, Acta Tropica 37,
163-175.)
264 G. A. SCHAUB

uninfected first instars of Tri. infestans with different starvation periods,


young and old first instar bugs probed more often, and especially after long-
term starvation they ingested less or no blood (G. A. Schaub, unpublished
observations). Therefore, the effects described with infected bugs can be
explained by a competition of trypanosomatids and insect host for the
ingested food and an earlier hunger response of infected bugs. This effect
could be elucidated by providing a constant food supply and making a non-
stop videotape recording of behaviour.

111. DISTURBANCES
IN ORGAN SYSTEMS

A. DISTURBANCES OF THE DIGESTIVE TRACT

In many trypanosomatid-insect systems, e.g. phlebotomines infected with


Leishmania, different regions of the host’s digestive tract are colonized by
different species (Molyneux and Ashford, 1983). Other flagellates like Tryp.
melophagium, Tryp. cruzi or B. triatomae are prevalent in all regions of the
intestine (Molyneux, 1975; Molyneux et al., 1978; Schaub and Boker, 1986a;
Schaub, 1989a; Jensen et al., 1990; Schaub et al., in press b). Often “carpets”
of flagellates cover the intestinal wall (e.g. see Paillot, 1933; Anderson, J. R.
and Ayala, 1968; Hoare, 1972; Molyneux et al., 1978; Mehlhorn et al., 1979;
Jensen et al., 1990; Schaub et al., in press b) (Fig. 2). A presumed trypano-
some of bats (Williams, 1976) and Leish. donovanican even completely block
the lumen of the posterior intestine or the pharynx, respectively, with a solid
plug of parasites, thereby also distending the oesophagus (see Section 1I.B).
The intense colonization of the intestinal tract must, presumably, interfere
with the normal function of this organ system. However, even invasion of
the haemocoele does not necessarily affect the function (Smirnoff and Lipa,
1970); larvae of the jack pine sawfly, Neodiprion swainei, naturally infected
with H. swainei, are normal with respect to movement, appetite and digestion.
Only in isolated cases have trypanosomatid-induced disturbances of
digestion been observed: in wild-caught sandflies, in which cardia, stomach
and hindgut are colonized by different species of trypanosomatids, e.g. of
toads and lizards, tbe period of blood digestion in the stomach is sometimes
increased (Ayala, 1971, 1973). The opposite effect, more rapid digestion,
seemed to occur in phlebotomines which were naturally infected, presum-
ably with a bat trypanosome (Williams, 1976). In the experimental vector
Aedes aegypti, infections with Tryp. avium may slightly accelerate the rate of
erythrocyte breakdown, thereby favouring the development of the parasite
which multiplies only in the digested, initially peripheral regions of the blood
meal (Bennett, 1970b). Perhaps one of these two phenomena explains the
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 265

observation that large, round, refractile globules or granules in the cyto-


plasm of the midgut wall of wild-caught sandflies indicated infections with
Leishmania (Johnson et al., 1963). A homoxenous trypanosomatid, Lept.
pyrrhocoris, attaches to the intestinal wall of the hemipteran Pyr. upterus
and sometimes has been found in the salivary glands and the haemocoele. If
the parasites are restricted to the gut, fluid faeces are deposited (Lipa, 1963).
In tsetse flies heavily parasitized by Tryp. congolense, the gut seems to be
affected, since it broke more easily during dissection than it did in uninfected
flies (Kaddu and Mutinga, 1983).

FIG. 2. Transmission electron micrographs showing dense colonization of the


intestinal tract of Triatoma infestans by Blastocrithidia triatomae. Bar = 2 pm. (a)
Small intestine. Flagellopodia (arrow) or flagella (arrowhead) anchor the parasites in
the microvillar border. (Reproduced by permission of Gustav Fischer Verlag from
Schaub et al., in press b, European Journal of Protistology.) (b) Hindgut. Flagellar
enlargements (arrow) anchor the parasites to the cuticular lining.
266 G. A. SCHAUB

1. Efects in the foregut

Whereas in different trypanosomatid-vector systems the feeding behaviour


is affected by the colonization of the foregut (see Section II.B), ultrastructural
alterations of the foregut have been reported for Phl. papatasi infected with
Leish. major only (Schlein et al., 1991). In an established infection the
parasites concentrate in the cardiac valve region which loses its cuticular
lining. The cuticle seems to be digested by enzymes which are secreted by the
parasites: in cultures in vitro three Leishmania spp. secreted two enzymes,
chitinase and N-acetylglucosaminidase.In addition to the cuticle the under-
lying epithelial cells also appeared to be damaged (Schlein et al., 1991).

2. Efects in the midgut

An effect on digestive enzymes has been reported by Schlein’s group only


(Schlein and Romano, 1986; Borovsky and Schlein, 1987). The initially
reduced proteolytic activity of gut homogenates from Phl. papatasi infected
with Leish. major indicates that the flagellates may inhibit enzyme produc-
tion of the sandflies, presumably by the release of glycoconjugates which are
also released into the supernatant of cultures in vitro. If such glycoconjugates
are fed to sandflies they delay the digestion of infective meals (Schlein et al.,
1990). Modulation of the digestive enzymes seems to be an important
mechanism, determining the susceptibility of a sandfly for a species of
Leishmania.
Whereas Tryp. cruzi does not affect haemoglobin crystallization in the
stomach (Pick, 1952), two other trypanosomatids of triatomines, Tryp.
rangeli and B. triatomae, strongly affect the midgut. Several effects are
evident in R . prolixus infected with Tryp. rangeli. After penetration of the
gut wall, parasites invade the gut muscles to multiply and haemocytes
accumulate at these sites (Watkins, 1971a). Depending on the intensity of
infection, only some or many parasitized muscle cells degenerate, and
eventually the gut cells are lysed. Thereby, in moderate infections-indicated
by only a slight increase of haemolymph-bugs continue to suck blood, but
the gut of larvae, not of adults, may burst. In heavy infections with a great
increase of haemolymph, gut peristalsis is reduced or absent, perhaps
because of insufficient stimuli from damaged nerves. These bugs neither
excrete nor feed. Most of these effects also occur in larvae with blocked
abdominal spiracles (Watkins, 1971a) (see Section VII).
More obvious effects can be observed in several species of triatomine bugs
infected with B. triatomae. Beakers containing infected Tri. infestans, Tri.
sordida and Dipetalogaster maxima often contain blood-red faecal drops,
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 267

compared with the normal white, yellow or dark brown drops (Schaub,
1988a; Schaub and Breger, 1988; Schaub and Meiser, 1990; Jensen et al.,
1990). Whereas in uninfected bugs the onset of digestion of haemoglobin at
the anterior end of the small intestine coincides with a colour change to
brown, infected bugs regularly have red contents in the dilated small intestine
(Schaub and Meiser, 1990). Interestingly, none of the bugs which die of
starvation has red intestinal contents (Schaub and Losch, 1989). Occa-
sionally haemolymph of Tri. infestans infected with B. triatomae is a light red
colour (Schaub, 1988a, 1990a; Schaub and Meiser, 1990; Jensen et al., 1990).
Similar effects in sheep keds infected with Tryp. melophagium were later
shown to be caused by experimental conditions resulting in blockage of the
spiracles (Nelson, 1956, 1981; Hoare, 1972).
Whereas red rectal fluid is usually deposited by healthy Anopheles ste-
phensi (Briegel and Rezzonico, 1985), in triatomines both reddening
phenomena indicate a disturbance of the function of the intestine. By starch
gel electrophoresis, the posterior intestines and the haemolymph of these
bugs were shown to possess proteins with the same migration behaviour as
marker haemoglobin (Schaub and Meiser, 1990). In additional photometric
measurements of the contents of different regions of the intestine, absorption
spectra of red stomach contents of infected and uninfected Tri. infestans, and
also of the red contents of the posterior small intestine of infected bugs,
showed the two typical haemoglobin maxima, whereas brown contents
showed neither of these maxima (G. A. Schaub, unpublished observations).
These data support the interpretation that ingested blood is not fully
digested in bugs infected with B. triatomae.
What is the mechanism of these disturbances in bugs infected with B.
triatomae? Valuable indications are offered by an ultrastructural study in
which we detected sequential steps of the damage process to the functional
subunits of the midgut, which are the extracellular membrane layers (acting
like the peritrophic membranes in other insects), the microvilli and the
epithelial cells (Figs2a, 3). These subunits are also affected by other
trypanosomatids.

( a ) Membrane systems. Usually peritrophic membranes or extracellular


membrane layers act as a barrier to parasites and provide microenviron-
ments for different digestive enzymes (Peters, W., 1982). Some salivarian
trypanosomes can penetrate the peritrophic membranes (Evans and Ellis,
1983), and in tsetse flies infected with Tryp. congolense, and also in sandflies
infected with Leish. aethiopica, the ultrastructure of the peritrophic mem-
branes seems to be disturbed (Kaddu and Mutinga, 1981, 1983). Whereas in
uninfected Phl. papatasi the peritrophic membranes disintegrate at the
posterior end, in specimens infected with Leish. major the chitin layer is also
268 G. A. SCHAUB

lysed in the anterior region (Schlein et al., 1991). These observations


might be explained by the secretion of the two enzymes, chitinase and N-
acetylglucosaminidase, which are secreted in cultures in vitro by different
trypanosomatids (Schlein et al., 1991).

(4 (b)
FIG. 3. Transmission electron micrographs of sections of small intestine of Tria-
toma infestans infected with Blastocrithidia triatomae, showing different types of
pathology. Bar = 2 p.(a) Cell with reduced microvilli. (b) Lysed epithelial cell with
parasites. The cell on the basal lamina (arrowhead) is vacuolated. (Reproduced by
permission of Cambridge University Press from Jensen et al., 1990, Parasitology 100,
1-9.)

The barrier function of the peritrophic membranes is evident.inthe species-


dependent establishment of Leishmania in phlebotomines, in which the
peritrophic membranes either disintegrate or remain intact, thus allowing
or preventing colonization (Feng, 1951), and also in their role in determining
whether early infections of other trypanosomatids are restricted to the
endoperitrophic space (e.g. Mungomba et al., 1989). Whereas Lept. lygaei in
the bug Lygaeus pandurus is closely associated with the extracellular mem-
brane layers, but not attached to the midgut epithelium, B. familiaris in the
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 269

same host attaches only to microvilli free of extracellular membrane layers


(Tieszen et al., 1986, 1989).
In contrast, in Tri. infestans infected with B. triatomae, flagella can cross
the extracellular membrane layers and reach the microvilli, and often these
layers are lacking (Mehlhorn et al., 1979; Jensen et al., 1990; Schaub er al., in
press b) (Fig. 2a). Their absence is not caused by the feeding state. Whereas
in starving adult R. prolixus extracellular membrane layers are not present
on the microvilli but develop after a blood meal (Billingsley and Downe,
1986; Billingsley, 1990), in the larvae of R. prolixus and Tri. infestans the
developmental phases of the extracellular membrane layers are not so
strictly separated (Bauer, 1981; Jensen et al., 1990). In contrast to uninfected
bugs, in the intestines of those infected with B. triatomae, fed and unfed, the
extent of regions without extracellular membrane layers is increased (Jensen
et al., 1990). Because of the variation in the production of the extracellular
membrane layers, we cannot ascertain if the layers are destroyed by B.
triatomae or if their production is disrupted. The precursor, the double
apical membrane, is developed below attached flagella, a phenomenon not
observed in B. familiaris (Tieszen et al., 1986). Since these layers and
membranes normally serve to keep separate the different digestive enzymes
(Billingsley and Downe, 1988; Ferreira et al., 1988), digestion of haemo-
globin is likely to be disturbed.

( b ) Microvilli. The apical microvilli, the second functional subunit of the


midgut, are also affected by different trypanosomatids. Their height seems to
be reduced in phlebotomines infected with Leish. amazonensis (Molyneux er
al., 1986a). Microvilli in the midgut of G. pallidipes infected with Tryp.
congolense are poorly developed or can be totally reduced (Kaddu and
Mutinga, 1983), as is also found in water-striders infected with B. gerridis
(Tieszen et al., 1983). Progressive reduction in height and number of
microvilli occurs in the small intestine and the stomach of Tri. infestans
infected with B. triatomae (Jensen et al., 1990; Schaub et al., in press b)
(Fig. 3a). These effects are not caused by the direct attachment of the
parasites, since densely colonized regions can possess well-developed micro-
villi, and in some microvilli-free regions no parasites are attached.

( c ) Intestinal cells. Not only the apical microvilli but also the body of the
intestin'al cells can be affected by the flagellates. One group of trypano-
somatids frequently destroys the cells if they are invaded for intracellular
multiplication. For example, only a mere membrane remains from the
stomach cells of the rat flea invaded by Tryp. lewisi after multiplication of
the trypanosome (Wenyon, 1926).
Members of a second group of trypanosomatids penetrate the midgut cellS
270 G . A. SCHAUB

only during invasion of the haemocoele. The penetration of Tryp. rangeli


does not occur intercellularly, but only via an intracellular route, even
through the nucleus of a cell of the intestinal wall (Schwarzenbach, 1987;
Hecker et al., 1990). The intracellular parasites are surrounded by a
membrane, presumably derived from the host, which remains around the
parasite together with a portion of extruded cytoplasm as the trypanosome
penetrates the basal lamina. The penetration pores in the cell membrane and
the basal lamina are repaired, but can be recognized by their unstructured
cytoplasm (Hecker et al., 1990). There are no ultrastructural differences
between infected and uninfected cells of infected bugs or intestinal cells of
uninfected bugs, even with high parasite densities in single cells. No
ultrastructural data are available for other flagellates of this group.
In the third group of trypanosomatids, the parasites normally insert only
their flagella into the epithelial cells, sometimes occur intracellularly and
occasionally invade the haemocoele. All three phenomena have been
observed in different species of Leishmania in phlebotomines, both experi-
mentally and in natural infections (Adler and Theodor, 1929; Adler and
Ber, 1941; Killick-Kendrick et al., 1974, 1977a; Molyneux et al., 1975;
Kaddu and Mutinga, 1981; Molyneux and Killick-Kendrick, 1987), but it is
unknown whether or not the parasites are killed after invasion (see Section
1II.C.I). There sqems to be an effect on the host cell in sandflies infected with
Leish. amazonensis (Killick-Kendrick et al., 1974), and midgut cells of
Ornithomyia avicularia invaded by Tryp. corvi have vesiculated endoplasmic
reticulum (Mungomba er al., 1989). The host cells are also affected in tsetse
flies infected with Tryp. congolense (Kaddu and Mutinga, 1983). However, in
a morphometric study of the midgut of tsetse flies infected with Tryp. brucei
only one of 12 features (relative volume of lysosomes) was significantly
affected, and therefore the authors stated that “cellular functions do not
seem to be strongly impaired” (Hecker and Moloo, 1981).
This third group also contains homoxenous trypanosomatids. Sometimes
Lept. pyraustae invades the haemocoele of corn borer larvae, but the light
colour of the intestinal epithelium when observed under the microscope is
normal (Paillot, 1933). In larvae of eye gnats (Diptera) infected with H.
muscarum, invasion of the haemocoele occurs in about half of the host
population. Ultrastructural appearance of organelles indicates that the cells
penetrated by parasites are not affected. The penetration results in a
bacterial septicaemia which kills the flagellates and the host larvae. Whereas
midgut epithelium is relatively intact in heavy infections of the intestinal
tract, its degeneration is evident after development of heavy haemocoelic
infections (Bailey and Brooks, 1972a). In Muscidae, H. muscarum colonizes
the intestine (Wallace, 1979) and is usually non-pathogenic. However, in
moribund and dead Musca domestica larvae, large numbers of flagellates
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 27 1

may occur in the haemocoele, indicating that this trypanosomatid might also
be pathogenic to this host under certain conditions (Kramer, 1961).
Only insertion of the flagellum occurs in water striders infected with B.
gerridis (Tieszen et al., 1983) and in triatomines infected with B. triatomae
(Jensen et a[., 1990; Schaub et al., in press b). B. triatomae also inserts its
flagellum into the epithelial cells of the Malpighian tubules (Schaub and
Schnitker, 1988) and into host cells co-cultivated in vitro (Reduth et al.,
1989). Penetration of the intestinal wall of R . prolixus has been postulated by
Peng (1979), based on haemocoele infection in five of 16 bugs after
experimental rectal infection. However, artefactual damage to the intestinal
wall cannot be excluded and, in seven of 16 bugs, B. triatomae was found in
the haemocoele 2-1 6 weeks after inoculation into the haemocoele. After
infection by coprophagy or feeding in vitro through a membrane, we found
no flagellates in the haemocoele of about 50 Tri. infestans (G. A. Schaub and
C . Jensen, unpublished observations). Whereas the other trypanosomatids
of this group rarely affect the intestinal cells, the cells of midguts colonized
by B. triatomae are often vacuolated or lysed (Jensen et al., 1990) (Fig. 3).
Thus, the basal lamina is freely accessible to the intestinal contents, and
cannot be a barrier to the passage of haemoglobin into the haemolymph.
Perhaps the very rare penetration by Tryp. corvi and Leish. major, cited
above, is restricted to degenerating cells in weakened insect hosts, i.e. cells
which had perhaps been affected before invasion (Mungomba et al., 1989).
This could also be the explanation for the intracellular development of Tryp.
cruzi in cells of the bug’s intestinal wall (Gomes de Faria and Cruz, 1927) or
its penetration and infection of the coelomic cavity (Lacombe, 1980). Also
the phenomenon that bacteria were found only in G. m. morsitans infected
with Tryp. brucei (see Hecker and Moloo, 1981) might be due to parasito-
genic weakening of the insect. The importance of the fitness of the host is
also shown by Herpetomonas sp. in Drosophila melanogaster (Lushbaugh
et al., 1976): the trypanosome normally develops in the lumen of the
intestinal tract, but penetrates the cells of the intestinal wall and multiplies
intracellularly if the insect has a concomitant infection with a yeast-like
fungus.
3. EfSects in the hindgut
The German term “Schorf” [scab] indicates a reaction of bees to infection
with C . mellificae (syn. Lept. apis), occurring in the dorsal part of the pylorus
and only at its end. However, Lotmar (1946) and Fyg (1954) suggested that
this scab material was of flagellate origin. The low colonization density also
argues against pathological effects. Number and size of the scabs was
affected by the type of food but not by a concomitant infection with Nosema
apis (Bahrmann, 1967).
272 G. A. SCHAUB

Another flagellate species in bees-and also other species in different


insects such as fleas, water striders or bugs-may cover the hindgut cuticle
like a “carpet” (Fyg, 1954; Molyneux and Ashford, 1975; Molyneux et al.,
1981; Zeledon ef al., 1977, 1984, 1988; Boker and Schaub, 1984; Tieszen et
al.. 1986; Zimmermann et al., 1987; Schaub et al., 1989b; Tieszen and
Molyneux, 1989). Trypanosomatids of toads and lizards, and some pre-
sumed to infect bats, can multiply so intensively that the hindgut and/or
rectal ampulla becomes noticeably distended (Anderson, J. R. and Ayala,
1968; Christensen and Telford, 1972; Williams, 1976), and masses of Tryp.
lewisi practically block the hindgut of the flea (Garnham, 1955). However,
no cytopathological effect on the rectal ampullae of fleas infected with
Leptomonas was observed (Molyneux et al., 1981); only a slight reaction of
the host at the attachment site seems to occur (Molyneux and Ashford,
1975).
Often the parasites prefer a specialized region, the so-called rectal glands
or rectal pads (Fig. 4a). Investigating the course of colonization in the
rectum of triatomines by Tryp. cruzi and B. triatomae with the scanning
electron microscope, we found that this region is preferred by both species
after initiation of the rectal infection, and it continues to be more densely
colonized (Boker and Schaub, 1984; Schaub and Boker, 1986a,b, 1987;
Schaub and Losch, 1988). In B. triatomae infections, about five interdigi-
tated layers of flagellates cover the rectal pads (Fig.4b). Even after long-
term starvation, parasites first detach from the other regions of the rectum
and last-shortly before and after death of the host-from the rectal pads
(Schaub and Boker, 1986b; Schaub and Losch, 1989).
In a variety of different insects the rectal pads seem to be involved in water
uptake, but amino acids are also absorbed from the rectal lumen (Wall and
Oschmann. 1975). The intense colonization must presumably interfere with
the function of the rectum or the rectal pads, a theory already proposed by
Lipa (1963) and by Molyneux and Ashford (1975) for infections of fleas, and
by Laugi and Nishioka (1977) for Lept. oncopelti infections of lygaeid bugs.
In the latter, some slight damage seems to occur since detached flagellates
carry the outer part of the epicuticle of the rectal pads with them, necessi-
tating constant replenishment of the epicuticle. This might be explained by
the secretion of a chitinase and N-acetylglucosaminidase which were found
in supernatants of Leptomonas, Herpetomonas and Crithidia cultures in vitro
(Schlein et a!., 1991), all species which attach to the rectal cuticle.

B. DISTURBANCES OF THE MALPIGHIAN TUBULES

Malpighian tubules are colonized by a large number of species of hetero-


xenous and homoxenous trypanosomatids, e.g. Leishmania and Tryp. theca-
FIG.4. Scanning electron micrographs of the anterior rectal wall of Triatoma infestans. Bar = 100 pm. (a) The rectum of an
uninfected bug shows the different cuticular structure of regions A-D. Region A is located around the exit of the midgut/
hindgut from which the processes of the ampullae cells are extended into the rectal lumen. The rectal pads (zone B) are clearly
separated from the narrow region C and from region D, the main part of the rectal wall. (Reproduced by permission of
Springer Verlag, Heidelberg, from Boker and Schaub, 1984, Zeitschrijt fiir Purasitenkunde 70,459469.) (b) In an established
infection of Blastocrithidia triatomae the cuticle is totally covered by a “carpet of flagellates”. (Reproduced by permission of
the Society of Protozoologists from Schaub and Boker, 1986, Journal of Protozoology 33, 266270.)
274 G. A. SCHAUB

dactyli in sandflies (Christensen and Telford, 1972; Kaddu and Mutinga,


1984; Range1 et al., 1985), Tryp. cruzi in triatomines (summarized by Schaub
and Losch, 1988), H. ampelophilae in Drosophila (Rowton et al., 1981), Lept.
pulexsimulantis in fleas (Beard et al., 1989), nearly all species of Rhynchoido-
monas in Diptera (Wallace, 1966, 1979), and C.Jlexonema in water striders
(Tieszen and Molyneux, 1989). This last species invades the host cells
without apparent effects (Tieszen and Molyneux, 1989), whereas Tryp. avium
appears to destroy the tubules of tabanids (Bennett, 1970b). In many
parasite-insect systems the Malpighian tubules are sometimes slightly hyper-
trophied; additionally, Malpighian tubules in corn borer larvae infected with
Lept. pyraustae are greyish in colour (Paillot, 1927), and in sandflies infected
with Endotrypanum schaudinni and Tri. infestans infected with B. triatomae
they are highly refractile (Shaw, 1981; Schaub and Schnitker, 1988). Oc-
casionally, in R . prolixus infected with Tryp. rangeli, the diameter of
localized areas of the Malpighian tubules is reduced to less than half the
normal, while other areas lpve expanded to twice their normal diameter
(Watkins, 1971b).
Only in the last two trypanosomatid-triatomine systems and in Tri.
sordida and D . maxima infected with B. triatomae has an increased volume
of haemolymph and/or a reduced excretion rate been observed (Grewal,
1957, 1969; Watkins, 1971b; Schaub, 1988a, 1990a; Schaub and Breger,
1988; Schaub and Schnitker, 1988; Schnitker et al., 1988); Tryp. rangeli and
B. triatomae infections have been studied in detail.
In R . prolixus infected with Tryp. rangeli, gut infections affect the
excretion of various developmental stages differently (Watkins, 1971a,b).
One month after infection the excretion rate of fifth instar larvae is reduced
by 27%, and after a further month of infection the reduction in females and
males is 53% and 6%, respectively. However, 2 months after haemocoelic
inoculation the excretion rate of females and males is reduced by 58% and
99%, respectively. Light microscopy demonstrates effects on the apical
microvillar border and the basal lamina and sometimes blockage of the
lumen of the Malpighian tubules by the uratic spheres. Compared to
uninfected bugs, the cytoplasm is coarsely granular and often contains
necrotic areas. Watkins (1971b) measured the diuresis in vitro of the entire
preparation of Malpighian tubules (free of tracheoles) and the hindgut. In
some experiments mesometathoracic ganglia were added (Watkins, 1969) as
a source of the diuretic hormone (Maddrell, 1980). Experiments with various
combinations of haemolymph and Malpighian tubules of infected and
uninfected bugs showed that tissue damage caused by Tryp. rangeli resulted
in a reduced secretion rate, even with normal haemolymph. In addition, the
ganglia from infected bugs did not contain enough diuretic hormone to
increase the secretion in Malpighian tubules from uninfected bugs. This lack
of diuretic hormone, or the presence of a chemical inhibitor in the haemo-
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 275

lymph, decreased the secretion of Malpighian tubules of uninfected bugs


maintained in the haemolymph of infected specimens.
The effect of B. triatomae on bugs is shown by a swollen abdomen, even
some days after bloodsucking. During the first 24 h after feeding, fifth instars
of Tri. infestans infected with B. triatomae excreted approximately 2.5 times
less urine (Schnitker et al., 1988). Even during dissection of long-term
infected bugs alterations are conspicuous in the upper region of the Malpig-
hian tubules, which are quite rigid and slightly widened, sometimes having
localized conspicuous swellings. The cells are filled with white concretions
and strong autofluorescence is evident with the fluorescence microscope.
Using the transmission electron microscope the cells of the slightly
widened region can be seen to possess many more concretions than normal;
the extremely swollen parts of the tubules show a reduction in the number of
basal cell interdigitations, mitochondria and microvilli, and the concretions
are much larger (Fig. 5). Normally, mitochondria and microvilli are essential
structures for fluid secretion by Malpighian tubules (Bradley, 1983). To
clarify whether or not the ultrastructural alterations, especially the increased
concretions, could be responsible for the dysfunction, we measured the
secretion rate of isolated Malpighian tubules. Surprisingly, secretion rates of
all isolated tubules were nearly identical. In addition, the storage and release
of diuretic hormone in infected bugs was sufficient to induce normal
secretion rates by Malpighian tubules of uninfected bugs. These tubules also
secreted normally when maintained in the haemolymph of infected bugs
and, therefore, no chemical inhibitor can be present.
What then might be the cause of disturbed excretion in infected bugs? One
possibility is that infection affects the ampullae or rectal reabsorption.
However, I emphasize the reduced tracheal system in infected bugs.
During measurements of isolated tubules in vitro, oxygen supply is guaran-
teed, but this is probably not so in vivo (Schaub and Schnitker, 1988). The
importance of oxygen supply is also indicated by the experiments of Watkins
(1971b) using bugs with blocked spiracles. Pathological effects were very
similar to those observed in bugs infected with Tryp. rangeli (see Section
VII). Whereas Tryp. rangeli develops inside the tracheal cells and destroys
them (Watkins, 1971a,b; Schwarzenbach, 1987), B. triatomae could act only
indirectly on the development of the trachea.

C. EFFECTS ON THE HAEMOLYMPH

Obvious effects of parasitization by trypanosomatids on the haemolymph


are only rarely reported. In R. prolixus infected with Tryp. rangeli, the
haemolymph is whitish and more copious in heavily infected bugs (Grewal,
1969), and in Pyr. apterus infected with Lept. pyrrhocoris the haemolymph is
thicker and whitish instead of the normal light green (Lipa, 1963).
276 G. A. SCHAUB

FIG. 5. Diagrammatic representation of the Malpighian tubule wall of Triatoma


infestans. Bar = 2pm. (a) Uninfected tubule; (b) slightly swollen region and (c)
extremely swollen region of tubules of bugs infected with Blastocrithidia triatomae. b,
Basal lamina; c, concretions; i, electron dense inclusions; I, lumen of tubule; m,
mitochondria; v, layered vesicles. (Reproduced by permission of Springer Verlag,
Heidelberg, from Schaub and Schnitker, 1988, Parasitology Research 75, 88-97.)

1. Eflects on the immune response

Insects possess a cellular and a humoral immune response. The inoculation of


non-virulent bacteria or fungi into the haemocoele induces a strong humoral
response, protecting against subsequent inoculations with virulent species
(Gotz and Boman, 1985). Immune proteins also appear in the haemolymph
of tsetse flies inoculated with bacteria, but not after inoculation of Tryp.
hrucei, which additionally does not induce phagocytosis or encapsulation by
haemocytes (Kaaya et al., 1986). However, an anti-trypanosomal factor is
already present in the haemolymph before inoculation, which specifically
destroys Tryp. congolense, Tryp. vivax and Tryp. brucei but not Leish. hertigi
or C . fasciculata (reviewed by Molyneux et al., 1986b; Kaaya, 1989). The
haemolymph of locusts and cockroaches, used as model systems, aggluti-
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 277

nates Tryp. brucei and Leish. hertigi in vitro, and the agglutinin titres are
increased by a prior inoculation of either trypanosomatid into the haemo-
coele (Ingram et al., 1984).
After injection of Tryp. rangeli into the haemocoele of Tri. infestans or R .
prolixus, the number of phagocytic cells increases greatly (Zeledon and de
Monge, 1966). Uninfected Tri. infestans already possess more than twice as
many haemocytes than R. prolixus, and thereby Tri. infestans can overcome
the infection. Some strains can also be controlled by R. prolixus and nearly
all by Tri. infestans (Zeledon and Blanco, 1965; D’Alessandro, 1976).
Whereas Tryp. rangeli multiplies inside the phagocytic haemocytes after
inoculation into the haemocoele, Tryp. cruzi is killed by the haemocytes of
R. prolixus (Tobie, 1968, 1970). Initially, the number of haemocytes in-
creases in R. prolixus, but to differing extents for the various haemocytic
cells (Gomez, 1967). Since all types of haemocytes are parasitized by Tryp.
rangeli (Schwarzenbach, 1987), their number is considerably reduced in old
and heavy infections (Grewal, 1957).
The prophenoloxidase system is not activated by Tryp. rangeli in R .
prolixus or in Tri. infestans. Since the intensity of this immune response is
lowered if the parasites are incubated together with a microbially derived
molecule, which normally activates the prophenoloxidase system, it was
suggested that the susceptibility of R. prolixus might be explained, at least
in part, by immune suppression. In the tissues of the refractile Tri. infestans,
agglutinating and trypanolytic factors seem to be more widely distributed
than in those of R. prolixus (Gregorio and Ratcliffe, 1991a,b).
The haemolymph of bugs infected with Tryp. cruzi has a normal appear-
ance, but implantation experiments indicate a strongly reduced cellular
immune response of infected Tri. infestans (Bitkowska et al., 1982). Since the
fluid from cultures in vitro caused similar effects, the authors suggested that
some parasites may develop in the haemocoele after suppression of the
host’s immune reactions. However, in our Tryp. cruzi-Tri. infestans system
the cellular encapsulation of pieces of nylon thread seemed to be identical in
infected and uninfected larvae (G. A. Schaub, unpublished observations),
but in bugs infected with B. triatomae, the cellular encapsulation and
melanization reactions were almost totally inhibited (G. A. Schaub, unpub-
lished observations). The latter effect may be due to the decreased concen-
tration of amino acids used for melanization (see Section 1II.D).

2. Eflects on chemical composition

The effects of trypanosomes on metabolites in the insect’s haemolymph have


been investigated only for triatomines infected with Tryp. cruzi, Tryp. rangeli
and B. triatomae (Zeledon and de Monge, 1966; Ormerod, 1967; Watkins,
278 G . A. SCHAUB

1969; Donandt, 1982; Schaub et al., 1990b). In the investigation by Zeledon


and de Monge (1966), the total concentration of free amino acids decreased
by 27% at 5-6 days after inoculation of Tryp. rangeli into the haemocoele of
R. prolixus, while it increased by 66% in uninfected bugs. Concentrations of
total proteins and carbohydrates also decreased in these infected bugs. In
Tri. infestans slight alterations occurred (Zeledon and de Monge, 1966).
In all the other studies cited above, concentrations of individual amino
acids were determined. Watkins (1969) and Donandt (1982) used the semi-
quantitative thin layer chromatography technique, whereas Ormerod ( 1967)
and Schaub et al. (l990b) used ion-exchange chromatography; only the last
method allowed analyses of the haemolymph from individual bugs by a
sensitive fluorescence detection system after post-column derivatization of
amino acids with o-phthaldialdehyde.
Ormerod (1967) compared the effects of a slightly and a highly virulent
strain of Tryp. rangeli on R . prolixus. Despite the difficulties of comparing
the data for infected and uninfected bugs (discussed by Schaub et al., 1990b),
some results were obvious. The less virulent strain produced a large increase
in the concentration per bug of aspartic acid and taurine in short- and long-
term infected bugs, and a 10-fold increase of isoleucine in those long-term
infected bugs which survived but did not moult. In infections with the lethal
strain, concentrations of alanine, glycine and isoleucine increased 10-fold,
and those of taurine and aspartate 100- and 500-fold, respectively. Further-
more, concentrations of tyrosine, phenylalanine and lysine were below the
level of detection.
Gut infections of R . prolixus with Tryp. cruzi greatly decreased the
concentrations of cysteic acid and histidine in the haemolymph of late instar
larvae (Watkins, 1969). Infections with Tryp. rangeli also decreased the
concentrations of leucine, phenylalanine and serine. In female bugs, concen-
trations of arginine and tyrosine were decreased by Tryp. ranxeli infection
and additionally, that of proline was increased by Tryp. cruzi. After
inoculation of Tryp. cruzi into the haemocoele of R . prolixus, no effect was
evident in infected larvae, but decreased concentrations of leucine and valine
and increased concentrations of proline, serine and tyrosine were found in
adults. Infections with Tryp. rangeli greatly increased concentrations of
arginine and proline in larvae and adultsi and decreased those of nearly all
the remaining amino acids.
Tryp. rangeli develops in the haemocoele of Rhodnius, but is killed $by
haemocytes in the haemocoele of other bugs; hence, Tri. phyllosoma, studied
by Donandt (1982), is not likely to be greatly affected by Tryp. rangeli. The
investigation by Donandt (1982) of short-term effects (up to 4 weeks) found
only slight differences between bugs infected with Tryp. cruzi and uninfected
bugs, and for most amino acids the infection-induced alterations were
slightly greater in bugs infected with Tryp. rangeli. During the first week
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 279

concentrations of alanine, glutamate, leucine/isoleucine, lysine, phenylala-


nine and serine were slightly reduced in infected bugs, but thereafter they
were mainly higher than in uninfected bugs. Concentrations of arginine,
asparagine and tyrosine were nearly always higher.
No consistent trend can be recognized in these three studies on bugs
infected with Tryp. cruzi or Tryp. rangeli, and only the decrease of the
concentration of tyrosine in the studies by Ormerod (1967) and Watkins
(1969) is noteworthy.
In our study we investigated the free amino acids in the haemolymph of
uninfected fourth instar larvae of Tri. infestans and in fifth instars 1 day after
ecdysis, and of those infected with B. triatomae (Schaub et al., 1990b).
About 15 and 21 weeks after infection, concentrations of the majority of
amino acids in infected fourth instar larvae were lower than those in the
respective uninfected bugs--40-80% lower for methionine, serine, threonine
and tyrosine. In fifth instar larvae a similar decrease was obvious for alanine,
arginine, histidine and tyrosine, and concentrations of aspartate, cystine/
cysteine and lysine were increased markedly by 130%, 380% and 150%,
respectively. The differences between infected and uninfected fifth instars,
which were also statistically significant in fourth instars, were undoubtedly
due to the effects of B. triatomae the lower values of alanine, arginine,
histidine and tyrosine. However, the major alteration induced by infection
with B. triatomae was the occurrence of p-alanine in infected fifth instars.
instars.

D. EFFECTS ON THE CUTICLE

Many parasites affect the colour of the cuticle of their hosts. In R. prolixus,
Pyr. apterus and Tri. infestans infected with trypanosomatids the cuticle is
often paler (Grewal, 1957; Lipa, 1963; Watkins, 1969, 1971a; Schaub, 1988a;
Schaub et al., 1990b). However, C . cimbexi, which develops in the haemo-
coele of the hymenopteran host larvae, causes no apparent alterations of
external appearance (or behaviour) of the host larvae (Lipa and Smirnoff,
1971).
The translucent and pale cuticle of R. prolixus infected with Tryp. rangeli
seems to be caused by the parasite’s multiplication in the epidermal cells
(Watkins, 1971a). The pigment granules disappear in heavy haemocoelic
infections, and periodically orange-coloured urine is excreted (Watkins,
1969). An effect on pigmentation seems also to be evident in the eyes of
infected R. prolixus. However, the fact that about 50% of infected adults
have white eyes, while only 0.2% of uninfected adults do so (Watkins, 1969),
might also be explained by a survival of tolerant or refractory bugs if the
presence of white eyes is a genetic marker.
280 G . A. SCHAUB

In infections with B. triatomae tanning of the cuticle can be totally


inhibited (Fig. 6 ) . Usually the resulting pale pink colour is only transiently
observed during the first 15 min after moulting, but the infected bug shown
in Fig.6 was photographed 2 weeks after the moult. In populations of
infected Tri. infestans, all grades from pale to the normal dark brown colour
occur. On dissecting infected bugs, we often found that the cuticle was softer
(Schaub et al., 1990b).

FIG. 6. Male Triatoma infestans infected with Blastocrithidia triatomae (left) and
uninfected (right) 2 weeks after ecdysis. (Reproduced by permission of Pergamon
Press from Schaub et al., 1990, Journal of Insect Physiology 36, 843-853.)

In bugs infected with B. triatomae, indirect effects due to intoxication or


direct effects on the substrate, enzymes or hormones involved in develop-
ment of the new cuticle are possible causes of this phenomenon. N-Acetyl-
dopamine, which is made from tyrosine, plays an important role in the
process of tanning; therefore, determining the concentration of free amino
acids in the haemolymph can indicate whether or not the substrate for
tanning is limited in infected bugs (Schaub et al., 1990b) (see Section 1II.C).
Such measurement; on haemolymph from individual bugs show great
variations in concentrations. However, the greatest variation is found in
those five amino acids which are specifically used for development of the new
ckicle, e.g. a standard deviation of 84% of the mean value of tyrosine in
infected fourth instars. Despite this variation, differences are often statisti-
cally significant (including those for tyrosine) if the concentration of amino
acids before and after moulting in infected and uninfected bugs is compared.
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 28 1

Infected bugs before moulting contain lower levels of the amino acids which
are incorporated into the cuticle.
Unfortunately, this effect can also be caused by retarded development,
which is seen normally in infected bugs. Including data from bugs which had
not changed their metabolism in preparation for the development of the new
cuticle presumably lowered the mean values obtained for infected bugs, e.g.
for tyrosine. However, three pieces of indirect evidence support the theory
that a lower concentration of tyrosine occurs and is responsible for the
reduced tanning. (i) Amino acid analysis of cultures of B. triatomae in vitro
indicates that the flagellates may compete with the bug for essential amino
acids in the food. (ii) The fat body, which presumably makes or stores most
of the amino acids needed for the new cuticle, is greatly reduced in bugs
infected with B. triatomae (see Section 1II.E). (iii) The most important
indication is that after the moult we could find detectable concentrations of
p-alanine and an accumulation of its precursor, aspartate, in infected bugs
only. There is increasing evidence that not only N-acetyl-dopamine but also
N-P-alanyl-dopamine plays an important role in sclerotization, tanning and
melanization. In mutants of Diptera, Lepidoptera and Coleoptera, inhibi-
tion of the incorporation of p-alanine prevents tanning and causes intense
melanization (discussed by Schaub et al., 1990b). Why did this melanization
not occur in our bugs? The failure of tanning seems to occur only if the
substrate for melanization is not available. This also is dopamine, which is
made from tyrosine. Together, these results strongly indicate that it is a
reduced concentration of tyrosine that is responsible for the reduced tanning
in infected bugs, and not a reduced oxygen supply due to the reduced
tracheal system (see Section 1II.B). However, possible actions on enzymes
and .hormones involved in sclerotization and tanning cannot at present be
ruled out.

E. OTHER AFFECTED ORGAN SYSTEMS

The outer appearance, but not the tanning of the cuticle, of the hymen-
opteran Caliroa cerasi is affected by an infection (Carl, 1976; Lipa et al.,
1977). The yellow spots on so-called “slug larvae” infected with B. caliroa
are caused by an effect on the mucous coating, which dries up and peels off.
The cause of the change of the colour of the larvae to dark brown or
blackish brown was not identified, but the colour indicates a disruption of
the gut during penetration of the flagellate into the haemocoele.
An obvious effect on colouration also occurs in the salivary glands of R.
prolixus; they are normally pink and become whitish in bugs infected with
Tryp. rangeli (Grewal, 1956). This might be caused by the parasites pene-
trating the cells on their way from the haemocoele into the lumen of the
282 G. A. SCHAUB

gland. In cases of severe infection the tissue is damaged and the basal
lamina is detached from the gland cells (Schwarzenbach, 1987; Hecker et al.,
1990). An opposite effect on colouration occurs with salivary glands of
infected tsetse flies, which normally have a chalky appearance, but become
brown to black in flies with very old natural infections of Tryp. brucei (Burtt,
1942, 1950), a phenomenon reported only from the Amani region of
Tanzania. In experimentally infected flies, the host membrane of the micro-
villi in the salivary gland shows a clear reaction at the attachment site of
Tryp. brucei, a clustered arrangement of intermembranous particles (Vicker-
man et al., 1988). Salivary glands of uninfected G. m. morsitans when
dissected into saline display sinuous motility, which is not seen with glands
heavily infected by Tryp. brucei (Golder et al., 1987). These changes coincide
with considerable alterations of the composition of the secretion, e.g.
reduced cholinesterase activity (Patel et al., 1982; Golder et al., 1987), which
might be the cause of the reduced feeding behaviour of infected flies (see
Section II.B.3).
In three host-parasite systems in which the parasite is highly virulent, the
fat body is considerably reduced (Smirnoff and Lipa, 1970; Watkins, 1971a;
Schaub et al., 1990b). This might explain the retarded development of sawfly
larvae infected with H. swainei, bugs infected with B. triatomae and R .
prolixus infected with Tryp. rangeli. Because the concentration of metab-
olites concerned with moulting does not increase above the critical level, the
hormonal induction of moult is not initiated (see Section 1II.D).
Since Tryp. rangeli invades the haemocoele and develops intracellularly in
all organs, they are all affected by the flagellate. In addition to the gut
cells, Malpighian tubules, haemocytes, cuticle, tracheal and epidermal cells,
salivary glands and fat body, all discussed in earlier sections, Tryp.
rangeli damages the nervous system of R. prolixus (Watkins, 1969, 1971b;
D’Alessandro, 1976).

Iv. EFFECTS
ON PRE-ADULT DEVELOPMENT
AND MORTALITY

A. TR YPANOSOMA INFECTIONS OF TRIATOMINAE

There is only one report of adverse effects of Tryp. cruzi on the larval
, developmental times of the pre-adult stages of triatomines (Reis dos Santos
and Lacombe, 1985). However, the retarded development of infected bugs
might be explained by their having been maintained in isolation (see Section
V1.C) or it might have been unique to the Tryp. cruzi-bug system used in
that study (reviewed by Schaub, 1989b). Such effects did not occur in my
system (Schaub, 1988c,d), and Juarez (1970) also reported no adverse effect
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 283

of Tryp. cruzi. Mortality rates also seemed to be unaffected (Schaub, 1988~).


In a contrary example, cited by Kramer (1963), the single dead bug in which
the body fluid contained numerous Tryp. cruzi (Wood, 1942) was presum-
ably not killed by the flagellate alone. This bug possessed a swollen
abdomen, a phenomenon known to occur in aposymbiotic bugs (see Section
VII). Tryp. cruzi seems to act as a subpathological stressor, leading to
adverse effects only if a second synergistic stressor is present (Schaub,
1989b). Under optimum feeding conditions the metabolite losses to the
parasite seem to be compensated by an increase in the number of blood
meals and/or the volume of blood ingested (Juarez, 1970).
In contrast to Tryp. cruzi, Tryp. rangefi is pathogenic to the vector and not
to the human host. It is even more deadly to Cimex than to triatomines,
killing more than 80% before they reached the adult stage (Grewal, 1957). In
the reduviid bugs R. prolixus and R. robustus, but not in Tri. infestans,
infections with Tryp. rangefi cause retardation of larval development
(Grewal, 1956, 1957; Tobie, 1965; Aiiez, 1984).
Developmental retardation has rarely been measured in this system.
Whereas uninfected first and second instar larvae of R. prolixus needed at
least 7 days after feeding before they moulted, and third, fourth and fifth
instar larvae needed 9, 10 and 16 days, respectively, infected bugs needed 8,
9, 10, 12 and 21 days (Tobie, 1965). After infection of 30 bugs of each instar,
most groups needed 10-40% more time to reach the adult stage than the
uninfected groups (Aiiez et af., 1987).
The mortality rate data from laboratory studies with Tryp. rangefi are
summarized in Table 1. The first investigation of the virulence of Tryp.
rangeli-unfortunately without control groups-demonstrated a clear dose
dependency, at least in the first instar (Grewal, 1957). The infective dose
given to group X2 and group X3 was about five and ten times higher,
respectively, than that of group XI. Only G6mez (1967) used a mixture of
culture stages and blood, with a high concentration of Tryp. rangefi, instead
of a living host for the infection of the first instars, which might explain the
extremely high mortality rate of his infected group (B in Table 1). However,
the control group also had a high mortality rate. Perhaps handling stress
caused these increased mortalities, a factor to which bugs react very
sensitively (summarized by Schaub, 1988a; Schaub and Breger, 1988).
Data in Table I indicate that the first, second and fifth instar larvae react
more sensitively to Tryp. rangefi than do the other instars. However, after
infection of 30 or 35 bugs of each instar, comparison of their instar-specific
mortality rates showed a statistically significantly higher mortality rate in the
fourth instar only (Aiiez et af., 1987). This aspect should be investigated
again with more bugs, since in all groups the number of deaths per instar
was very low, between 0 and 4. A further interesting phenomenon which
needs reinvestigation is the observation by Tobie (1965) that in the unin-
284 G. A. SCHAUB

fected groups more females than males developed, but after infection of first
instar larvae the numbers of both sexes were identical. Sex also influences
salivary gland infections; they arise in a higher proportion of males than
females.

TABLE1 Instar-specific rates of mortality' in groups of Rhodnius uninfected and


infected with Trypanosoma rangeli

Uninfected controls' Infected groups'


A B CI C2 D X I X2 X3 A B CI C2 D
Initial number of bugs
Instarb 154 100 20 25 10 34 49 74 100 100 170 105 30
L1 3 1 9 0 0 0 12 22 29 13 64 12 6 10
L2 5 1 1 0 0 0 3 8 8 1 3 1 9 1 3 8 4
L3 4 1 0 0 8 0 0 3 6 1 1 0 4 1 0 8
L4 0 3 0 0 0 3 3 -' 3 4 6 4 1 2 4
L5 6 2 5 4 0 7 12 - 10 57 14 21 9
LI-LSd 16 38 5 12 0 24 41 - 34 94 39 46 30

aMortality rate (YO)calculated for each instar from the number of dead larvae in the
respective instar and the number which entered that instar.
LI, L2, etc., first, second, etc. instar larvae.
'The same capital letter marks control and infected group data originating from one
investigation, as follows: A, Tobie (1965), R. prolixus; B, Gomez (1967), R. prolixus; C, Aiiez
(1984). CI, R. prolixus, C2, R. robustus; D, Aiiez er al. (1987), R. prolixus; X, Grewal (1957).
R. prolixus. X I , X2. X3, increasing infection rates.
Total mortality rate.
Observations discontinued.

B. BLASTOCRITHIDIA TRIA TOMAE INFECTIONS OF TRIATOMINAE

In our detailed investigations with E. triatomae, two modes of infection


were used, natural and in vitro infection. Only the first mode of infection
gives information as to whether or not the parasite may be transmitted in
natural populations. In our initial studies we infected the bugs naturally
solely by maintaining uninfected and infected animals together. In such
groups direct transmission of E . triatomae between bugs occurs, regularly by
coprophagy, but cannibalism is not excluded (Schaub et af., 1989a). Copro-
phagy seems to occur after feeding, and the rate of coprophagic transmission
of trypanosomatids is greatly reduced in populations which have been
starved for a long time (Schaub, 1988b, 1990d; Schaub et af., 1989a). Dry
faeces has to be redissolved by fresh faeces before infection is possible
(Schaub et af., 1989a; Schaub and Jensen, 1990).
TABLE
2 Instar-specij5c rates of mortality' and total infection rate in uninfected groups of triatomines and in groups exposed to
Blastocrithidia triatomae infection by coprophagy

Uninfected controls' B. triatomae coprophagy'


Instarb A B C D E F G H A B C D E F G H

L1 0 3 28 3 0 3 16 7 1 8 2 4 4 0 2 7 11
f0 f5 f 4 f 2 -+o f3 +
- 16 f 5 f2 f8 fll f4 f0 +3
- f3 f5
L2 1 3 5 3 1 8 5 0 4 9 4 5 2 1 8 1 4
f l f4 f 5 +2
- f2 f5 f 7 +O
- f4 f7 f8 f5 f26 f 13 f2 f5
L3 3 8 10 12 0 8 2 4 13 5 1 22 54 2 3 1
f3 f7 f 9 f 4 fO f4 f 3 f 3 f7 +4 f2 f12 *37 +5 f2 f2
L4 1 3 5 11 2 2 1 1 25 8 4 36 30 1 0 1
f2 f5 f 9 f 9 +3
- f2 f 2 f2 f14 f7 f7 f20 f16 f l fO 5-2
L5 5 6 6 20 21 6 0 3 75 21 38 68 83 4 1 1
+2
- f5 fll f 16 f12 f6 fO f2 f17 f l l f21 f24 f15 f 3 f2 f2
Ll-LSd 10 22 46 40 24 25 22 14 85 41 59 85 94 16 11 17
f4 f14 f 9 f 18 flO f3 f 17 f3 flO f20 f10 f13 f7 f 15 f4 f11
Infection 93 49 11 69 48 34 5 2
rate f6 f27 57 f16 f15 f 6 f7 f3

a Mortality rate (%) calculated for each instar from the number of dead larvae in the respective instar and the number which entered that instar;
mean and standard deviation of three to five groups, initially each consisting of 20-45 first instar larvae.
LI, L2, etc., first, second, etc. instar larvae.
'The same capital letter marks control and infected group data originating from one investigation, as follows: A, Schaub (1988a) Triafoma
infesfans;B, Schaub and Breger (1988) Tri. sordida; C, Schaub and Breger (1988), Tri. pallidipennis; D, G . A. Schaub (unpublished data), Tri.
spinolai; E, Schaub and Breger (1988), D. maxima; F, Schaub (1988a), R.prolixus; G, G. A. Schaub (unpublished data), R. robustus; H, G . A. Schaub
(unpublished data), R . neglectus.
Total mortality rate.
286 G. A. SCHAUB

The results from groups exposed to coprophagic infection (Table 2) show


that some species react sensitively, i.e. development is retarded and mortality
rate increased (Schaub, 1988a, 1990d; Schaub and Breger, 1988; Schaub and
Jensen, 1990; G. A. Schaub, unpublished observations). Such retarded
development is less apparent during the first three instars, and in the fourth
and fifth instar the first animals moult at the same time in all groups.
However, in most infected groups, larval development is greatly retarded in
later instars. In one of these investigations, 50% of uninfected fifth instar
bugs moulted to the adult stage at 16 weeks after the first feed of the first
instars, whereas the same proportion of infected bugs needed 22 weeks
(Fig. 7) (Schaub and Jensen, 1990).

100

-s-
-3
c
v)

U
50
c
-
VI
c
3 Bugs in control group:
r" Bugs in coprophagy groups
uninfected: o
infected: 0
10
0
15 20 25 29 34
Weeks after first feeding

FIG.7. Cumulative percentage of moults to adults plotted against age for Triatoma
infestans in uninfected populations and those infected with Blastocrithidia triatomae.
(Reproduced from Schaub and Jensen, 1990, Journal of Invertebrate Pathology 55,
17-27.)

Instar-specific mortality rate varies in the different species (Table 2). The
infection rate of Tri. infestuns and--clearly correlated therewith-the mor-
tality rate, is higher in groups given more infected bugs (Schaub and Jensen,
1990). In all sensitive species the final larval instar shows the highest
mortality rates (Table 2). This is similar to the situation with Rhodnius spp.
infected with Tryp. rangeli. Bugs often die during ecdysis in both systems.
However, this increase of mortality during ecdysis is not specific to B.
triatomae, but is correlated with the higher mortality (Schaub and Jensen,
1990), an aspect which has not been considered in the Tryp. rungeli
infections.
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 287

In another study we excluded the possibility that the pathological effects


of B. triatomae were due to our experimental design. Whereas some groups
of primarily uninfected first instar larvae had only infected late instar larvae
introduced, infected and uninfected larvae were added to other groups. Since
the pathological effects in the different groups were very similar, either the
bugs did not discriminate between faeces from infected and uninfected bugs
or they did not reject the infectious faeces (Schaub and Jensen, 1990).
In the series of investigations on coprophagic infection, the infection rates
varied greatly between the different species (Table 2). It remains to be
investigated whether the relatively low infection rate of Tri. pallidipennis was
caused by different coprophagic behaviour or whether the infected bugs
which were added to the primarily uninfected first instars excreted only low
numbers of cysts of B. triatomae. Only some adults of R . neglectus and
R . robustus were infected. This could be due to differences in coprophagic
behaviour and/or susceptibility, or to an infection in late instars. A further
important result is that only the groups of R . prolixus had relatively high
infection rates but remained unaffected. This suggested a tolerance to B.
rriaromae infections, or else a late infection. Unequivocal verification would
need infections in vitro in which the exact time of infection and infection
doses were known.
After development of isolation procedures to obtain the infectious stage of
B. triatomae-the drought-resistant cysts-bugs were infected in vitro by
feeding a mixture of blood and cyst stages through artificial membranes
(Schaub et al., 1988; Schaub, 1990a). While investigating the number of cysts
necessary to infect all bugs, different percentages of bugs of the various
groups became infected. As in the coprophagic infection experiments, the
correlation of infection rate and mortality rate was also evident after
infection in vitro (Schaub et al., in press a).
We varied different experimental features to exclude the possibility that
we had wrongly attributed synergistic effects solely to B. triatomae. Using
different infection doses ( 104-108 cysts per ml blood) for infection of first
instars-all doses were sufficient to infect all bugs-the effects were very
similar in the different groups (G. A. Schaub and B. Rohr, unpublished
observations). Presumably, this similarity was caused by high division rates
of B. triatomae resulting in a similar level of parasites 4 weeks after infection,
whether infected with lo4 or lo8 cysts per ml. Also long-lasting starvation of
bugs did hot affect the virulence of B. triatomae (Schaub, 1991). Infections of
different instars of Tri. infestans clearly show the existence of a time-lag
before pathological effects are observed (G. A. Schaub and S. Wolf,
unpublished observations). After infection of bugs of the first, second, third
or fourth instar, developmental retardation was first evident at the moults of
the third, fourth, fifth and fifth instars, respectively, but retardation was
288 C . A. SCHAUB

almost undetectable after infection of fifth instars. Variation of maintenance


temperature also showed that B. triatomae needed some time before effects
were evident. Normally maintenance was at 26°C; lower temperatures
retarded the development of Tri. infestans and increased the pathological
effects of B. triatomae. Maintenance at higher temperatures shortened the
developmental times, and more bugs reached the adult stage. Variations in
relative humidity did not influence the pathological effects (G. A. Schaub,
unpublished observations). Crowding stress acted synergistically with the B.
triatomae infection only at higher population densities than those we usually
used (Schaub, 1990b) (see Section V1.C).
Therefore, the pathological effects were caused by B. triatomae alone, and
the sensitivity of species and strains of triatomines could be compared. In
these infections in vitro, in which all first instars ingested the same number of
cysts, effects on sensitive species were similar to those obtained after
coprophagic infection (Table 3). The results clearly demonstrated that all
three species of Rhodnius are tolerant and that the Triatoma spp. and D .
maxima are susceptible and sensitive. Interestingly, exactly the opposite
groups of triatomines are affected by B. triatomae or Tryp. rangeli; the
homoxenous flagellate is pathogenic to bugs of the genus Triatoma and
Dipetalogaster, but not to Rhodnius (D’Alessandro, 1976; Aiiez, 1984;
Schaub, 1988a; Schaub and Breger, 1988; G . A. Schaub, unpublished
observations). This difference is not strain-specific; using six strains of Tri.
iqfestans (old laboratory strains or strains in the first or third laboratory
generation) and three strains of R. prolixus, only Tri. infestans was affected
by B. triatomae (Schaub, 1988a, 1990d; Schaub and Jensen, 1990; G. A.
Schaub, unpublished observations). I should emphasize that B. triatomae
affects sensitive bug species much more severely than Tryp. rangeli does its
insect hosts.
Theoretically, the tolerance of the Rhodnius spp. could be caused by the
brief larval developmental period, since mainly late instars are affected in
sensitive species. Since bugs usually need only one adequate blood meal to
induce development to the next larval instar, insufficient amount of blood or
longer starvation periods after the moult prolong the total developmental
time of larvae. Therefore, the influence of starvation was studied with R.
prolixus infected in vitro, but here again the infected and uninfected groups
did not differ in developmental times or mortality rates (G. A. Schaub,
unpublished observations). Long-lasting starvation also did not alter the
effects of B. triatomae on Tri. infestans infected in vitro, although this species
of bugs is affected (Schaub, 1991).
In natural populations selection phenomena occur. Theoretically, the
tolerance of R . prolixus to B. triatomae could also appear in “wild”
populations of the Triatoma spp. Therefore, in four generations of offspring
TABLE3 Instar-specific rates of mortality' in groups of triatomines uninfected and infected with Blastocrithidia triatomae

Uninfected con troIs B. triatomae infection


Instarb A B C D E F G H A B C D E F G H
LI 1 7 10 3 5 17 16 7 3 17 12 6 25 10 10 8
f2 +7 f3 f2 f5 f l l f16 f5 +I f2 flO f l f14 f3 f8 f5
L2 1 6 1 1 3 0 7 5 0 10 27 15 8 33 2 6 4
+1
- f7 f4 f2 fO f6 f7 fO f 7 f12 f7 f5 f15 f2 f7 f3
L3 2 7 2 1 2 3 3 2 4 5 23 12 23 22 0 1 3
+3 f12 f4 f4 f3 f6 f3 f3 f7 fll f 7 +I0 f5 fO f2 f6
L4 0 3 8 1 1 2 0 1 1 22 24 17 38 44 1 0 1
fO f3 f6 f9 f3 fO f2 f2 f18 f5 f 7 f16 f29 f2 f0 f2
L5 1 0 2 2 2 0 7 1 0 3 79 62 36 61 80 2 1 3
f2 fO f 7 f16 f6 f2 fO f2 f20 f25 f14 f20 f26 f2 f2 f4
LI-LSd 4 20 44 40 17 25 22 14 84 86 66 82 97 14 18 17
f6 f23 f6 f18 f 7 f16 f17 f3 f19 f12 f8 f15 f3 f 6 f14 f6

a Mortality rate (%) calculated for each instar from the number of dead larvae in the respective instar and the number which entered the respective

instar; mean and standard deviation of three to five groups, initially each consisting of 2 M 5 first instar larvae.
LI, L2, etc., first, second, etc. instar larvae.
The same capital letter marks control and infected group data originating from one investigation, as follows: A, Schaub (1990a), Tri. infestans;
E H , G . A. Schaub (unpublished data): B, Tri. sordida; C , Tri. pallidipennis; D,Tri. spinolai; E, D . maxima; F, R. prolixus; G , R. robustus; H,
R. neglectus.
Total mortality rate.
290 G. A. SCHAUB

of adult Tri. infestans infected with B. triatomae, larval development and


mortality rate were investigated in groups maintained together with unin-
fected bugs or those infected with B. triatomae (Schaub, 1980; Schaub and
Jensen, 1985). In comparison to the parent generation, pathological effects
were slightly reduced in the groups which had the possibility of copro-
phagic infection, but this reduction coincide with reduced infection rates.
Infection in vitro of the fourth generation showed that the sensitivity to B.
triatomae was not reduced.

C. HOMOXENOUS TRYPANOSOMATIDS IN HYMENOPTERA AND DIPTERA

Only three other species of homoxenous trypanosomatids are known to


affect the mortality rate of immature insect hosts. In a pioneer study, H .
swainei was found to increase the mortality rate of the jack-pine sawfly
(Hymenoptera) only slightly in the late larval instars (up to 20%), and this
only in larvae infected during the first two instars. In double infections with
a virus, the two parasites did not act synergistically, and when the virus
developed successfully, the flagellate was killed (Smirnoff and Lipa, 1970).
(Warburg and Ostrovska (1987) also described a detrimental effect of virus
infections on the development of Leish. major in the sandfly.) H . swainei
overwinters in the cocooned host and, in the initial study, in spring the
period of emergence and the number of emerged adults did not differ
between infected and uninfected populations (Smirnoff and Lipa, 1970). In a
later investigation H . swainei strongly affected the emergence rate: whereas
67.5% of uninfected adults emerged from cocoons (about two-thirds being
males) only 20% of infected pupae (half of them males) survived. In an
uninfected “wild” population 55% of adults emerged (Smirnoff, 1974).
In eye gnats (Diptera) infected with H . muscarum, developmental times of
larvae and pupae did not appear to be affected, but under normal rearing
conditions at 27°C only 38% of the adults emerged, compared to 69% in
uninfected populations (Bailey and Brooks, 1972b). At higher temperatures
the developmental times of uninfected and infected populations decreased,
as did most mortality rates. At lower temperatures the opposite effect
occurred (Bailey and Brooks, 1972b). (This correlation was also obvious in
Tri. infestans infected with B. triatomae.) Similarly to H . swainei, H . mus-
carum seemed to be more virulent if young larvae were infected (Bailey and
Brooks, 1972a). Since mortality of infected gnats seems to be caused by the
bacterial septicaemia, and since studies of this flagellate were undertaken
with laboratory colonies, the bacterial fauna and the importance of parasiti-
zation in “wild” populations remain to be investigated.
More striking effects are known for B. caliroa, which seemed to be
responsible for the collapse of outbreaks of a fruit-tree pest, the hymenop-
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 29 1

teran Caliroa cerasi (Carl, 1976; Lipa et al., 1977). N o laboratory study has
been performed with this species, but of 2500 collected larvae, 53% died
during rearing, usually in late larval instars, and 92% of these larvae
contained heavy flagellate infections in the haemocoele. At another locality a
mortality rate of about 40% was associated with the infection. First the
colour of the larvae changed (see Section III.E), and then they eventually
stopped feeding and died.

V. EFFECTS
ON ADULTLIFESPANAND REPRODUCTION
RATE

The effects on female fecundity can mainly be explained by a parasitogenic


reduction of the amount of material needed for egg production. In contrast
to helminth-infected intermediate hosts, there is no known example of
trypanosomatids increasing the life span of insect hosts by castration or
reduction of reproduction.

A. LEISHMANIA AND TR YPANOSOMA

Effects of Leishmania on adult sandflies have been considered only in single,


old studies and need confirmation using better rearing conditions. Single
observations that sandflies heavily infected with Leish. donovani die within a
few days (Smith et al., 1940) were confirmed in an extensive study (Smith et
al., 1941): only nine uninfected, but 77 infected, sandflies failed to take up
blood and died within the first week. According to Killick-Kendrick (1979)
and Molyneux and Killick-Kendrick (1987), two additional studies which
reported harmful effects on sandflies are not convincing. In a further
investigation of Leish. major and a saurian Leishmania, longevity of two
species of sandflies was reduced after simultaneous infection with both
parasites (Alekseev et al., 1975; Safyanova and Alexeiev, 1977). These effects
were due to infections with that species of Leishmania with which the
sandflies are not naturally infected, i.e. the saurian Leishmania affected Phl.
papatasi, the natural vector of Leish. major, and vice versa. A comparison of
infection rates of parous and gravid Phl. papatasi showed similar infected
proportions in both groups; therefore, Leish. major infections did not appear
to affect sandfly survival in the field (Yuval, 1991). However, the very rough
classification would not indicate slight effects.
In phlebotomines presumed to be infected with a bat trypanosome,
parasite and insect host seemed to compete for metabolites essential for
egg development. Thereby, gonotrophic concordance of blood ingestion
and ovarian development was disrupted and longevity must be reduced
(Williams, 1976).
292 G . A. SCHAUB

Heavy infections of tsetse flies with trypanosomes are also likely to


affect the vital characteristics of this insect. Based on the respiratory rates of
trypanosomatids and the energy content of the ingested blood, Bursell
(1981) calculated a more than 15% reduction of flight duration for infected
flies. These calculations are supported by observations by Ryan (1984)
showing significantly higher activity of infected flies and indicating that the
nutritional reserves of infected flies may be slightly lower than those of
uninfected flies.
Results of investigations of the effects of trypanosomes on life span and
reproduction rate of tsetse flies are contradictory: G . palpalis and G .
morsitans, infected with Tryp. rhodesiense, Tryp. gambiense or Tryp. brucei,
showed a tendency for greater longevity than uninfected flies (Duke, 1928;
Baker and Robertson, 1957). There was no difference in mean longevity,
number of puparia or weight of puparia of G . m. morsitans (ca. 100 or 200
individuals) fed on calves and goats, comparing uninfected flies with those
infected with Tryp. vivax, Tryp. congolense or Tryp. brucei, and observed for
63 days (Moloo and Kutuza, 1985). In a later study by this group using
Tryp. vivax and G . p . gambiensis, the 88 infected males had a statistically
significantly higher mean survival time (82 days) compared with that of
uninfected males (71 days), but opposite results (although not statistically
significant) were obtained for 100 females (99 and 102 days) (Makumi and
Moloo, 1991). The other features investigated, number and weight of
puparia, were not affected by the infection.
Two reports have described clear effects of Tryp. brucei and/or Tryp.
congolense on G . m. morsitans. In both parasite-vector systems, mortality
rate was increased within 30 days after infection, and the number of pupae
per female within the first 18 days was decreased compared to uninfected
flies. Abortion rate, mean weight and viability of pupae were unaffected
(Kaaya et al., 1987). However, only 10-12 flies were investigated. The
mortality of an initial population of 150 G . m. morsitans, comparing
uninfected flies with those infected with Tryp. congolense, was significantly
increased in the infected flies from the 10th day after infection. At the end of
the observation period ( 1 7 days after infection), 25 infected flies, but only 10
uninfected flies, had died (Nitcheman, 1988). To summarize these investi-
gations, an effect of trypanosome infection on Glossina may be present
during the initial phase of infection.
The infection of fleas with the rat trypanosome, Tryp. lewisi, caused an
initial increase in the mortality rate, corresponding to the time in which the
cells of the midgut are invaded (see Section 1II.A.l.c) (Garnham, 1955). This
effect did not occur after a second infectious feed and is attributed to the
higher sensitivity of newly emerged fleas. This interpretation remains to
be verified since it is also possible that all sensitive individuals were killed
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 293

by the first infection. Another often cited example, the pathogenicity of


Tryp. melophagium to sheep keds, was later shown to be caused by the
experimental conditions (Nelson, 1956, 1981; Hoare, 1972).
An infection with a bird trypanosome developing in the haemocoele
eventually became so profound that it was lethal to the vector (Macfie and
Thompson, 1929); however, this parasite is transmitted not by insects, but
by mites, and the suggested pathogenicity needs to be verified by an
experimental study. In the experimental vector Aedes aegypti, infections with
Tryp. avium reduced the number of eggs produced (Bennett, 1970a). Since
this effect was very strong if the birds on which the mosquitoes fed had high
parasitaemias, it could be partly due to a reduction in the quality of the
blood.
Investigations with Tryp. cruzi also led to contradictory results as to
whether or not adult triatomines are affected (reviewed by Schaub, 1989b).
Two publications mentioned reduced life expectancy of a species of Tria-
toma and R . prolixus (Carcavallo, 1970; Neves and Peres, 1975), and the
latter study also noted a reduced egg-laying period, but essential data were
not given (see Schaub, 1989b). In Tri. dimidiata, mean life spans of males
and females, as well as the hatching rate of eggs, were apparently unaffected
(Zeledon et al., 1970), as were the egg-laying period and mean adult life span
of Tri. infestans (Schaub et al., 1985). The reduced egg production of
infected Tri. infestans reported by two other authors might have been caused
by initial effects of the infection (of adults or fifth instars) or by their having
been fed in vitro with defibrinated blood (reviewed by Schaub, 1989b), both
indicating a subpathogenic effect of Tryp. cruzi (see Section VI). In our Tryp.
cruzi-Tri. infestans system a slight reduction in egg-laying rate during the
first weeks, and a slight decrease in the hatching rate, seemed to occur
(Schaub et al., 1985). Such studies are complicated by high variability and
the effects of blood ingestion and ageing. Daily counting of the number of
eggs laid showed that reproduction of infected and uninfected adults and
blood ingestion possessed a corresponding periodicity. The effects of ageing
were an even greater complication; the periods when no eggs were laid
increased with age, and egg weight and hatching rate decreased. Thus, more
detailed studies are necessary. However, the slight decreases observed in
bugs infected with Tryp. cruzi cannot influence natural populations with
good feeding opportunities.
Only two investigations compared the adult life span of R. prolixus when
uninfected or infected with Tryp. rangeli. No increase of mortality rate in the
infected group was observed during the first 3 months after infection of
adults (Tobie, 1965); in the investigation by Aiiez et al. (1987), four of 30
infected adults, but none of 10 uninfected bugs, had died by the end of the
3-month observation period.
294 G. A. SCHAUB

In R. prolixus infected with Tryp. rangeli, egg production was reduced by


about two-thirds compared to controls; in the infected bugs the percentage
of non-viable eggs increased from 5% to 27% (Watkins, 1969). This effect
seemed to be due to Tryp. rangeli and not to the intracoelomic inoculation,
since bugs infected in the same way with Tryp. cruzi showed no reduction of
egg production and only a slight increase in the percentage of non-viable
eggs.

B. HOMOXENOUS TRYPANOSOMATIDS

1, Eflects on life span

There are only a few investigations considering the effects of homoxenous


trypanosomatids on adult insects. Similarly to Tryp. cruzi, for regularly fed
bugs survival time of males and fecundity of females are nearly identical in
uninfected water striders and those with variable parasite loads of B. gerridis
and/or C . flexonema (Arnqvist and Maki, 1990). However, adult life span
was reduced by 9% to 25% in eye gnats infected with H. muscarum if the
temperature range permitted activity of the Diptera (Bailey and Brooks,
1972b). According to Lotmar (1946), A. Porter mentioned that H. vespae
killed populations of bees in Canada and Natal. Since certain diseases of
bees were unknown at that time, and since H. vespae infections of this well-
studied insect have not been reported since, it is more likely that the bees
were killed by another pathogen. Also the pathogenicity of H. bombycis,
which invades the haemocoele, remains to be reinvestigated since the alleged
lethal effect was observed after inoculation into only one butterfly (Levaditi,
1905).
More detailed information is available for Tri. infestans infected with B.
triatomae, which are greatly affected by the parasite. Individual infected
females live up to 20 weeks and males up to 27 weeks, and mean life spans of
infected females and males after moulting to the adult stage are 9 and 12
weeks, respectively. However, in uninfected Tri. infestans, the mean life
spans are 35 weeks in males and 30 weeks in females (Schaub et al., 1984,
1985; G. A. Schaub, unpublished observations).

2. Eflects on the reproduction rate

Effects on the reproduction rate have been reported in three systems. In an


initial study, H. swainei did not seem to affect the reproduction rate of the
naturally-infected jack-pine sawfly (Smirnoff and Lipa, 1970). However, in
a later study from the same group, dissection of the females and counting
of the eggs showed that fecundity of infected flies was reduced by 25%
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 295

(Smirnoff, 1974). According to Jenkins (l964), Ayroza Galvgo and


Coutinho (1941) reported that Anopheles infected with B. pessoai (syn. Lept.
pessoai) showed a pathogenic action on ovaries and gut.
As with parallel studies with Tryp. cruzi, evaluation of the reproductive
data of Tri. infestans infected with B. triatomae is complicated by the effects
of feeding and ageing, but the effects of B. triatomae are considerable. In
infected groups or pairs, the number of eggs laid per day, egg weight,
hatching rate and weight of the first instar larvae are always reduced
compared to controls (Schaub et al., 1984, 1985; G. A. Schaub, unpublished
observations), resulting in a reduction of the reproductive rate by 95%.
Recently detailed studies were made of the effects of C. bombi on bumble
bees (Shykoff and Schmid-Hempel, 1991a,b,c). Spring queens of Bombus
terrestris, but not of B. lucorum, that failed to found nests in the laboratory
had less developed ovaries than did uninfected queens (Shykoff and Schmid-
Hempel, 1991b). Some infected queens were able to found nests, but early in
the colony cycle the oviposition rate of the infected queens was reduced
(Shykoff and Schmid-Hempel, 1991~).In the laboratory, colony producti-
vity of these small colonies did not differ from that of larger colonies of
uninfected queens if enough food was offered. However, effects in the field
might well occur. Thus, overwintering of the parasite could be complicated,
since the parasite seems to overwinter only in infected queens and only large
field colonies produce queens. Interestingly, the effect of the parasites on the
queen seems to be compensated, since parasites also delay the age-dependent
ovarian development in workers. Thereby, worker-laid eggs appear later in
infected nests than in uninfected nests. After the worker bumble bees begin
egg laying, they reduce the time they invest in foraging and feeding of the
queen’s larvae. Therefore, the delayed reproduction of infected workers
increases the likelihood of the colony producing more queens (Shykoff and
Schmid-Hempel, 1991~).

V I. EFFECTS
SYNERGISTIC OF TRYPANOSOMATIDS STRESSORS
AND OTHER

In laboratory investigations insects are maintained under optimum con-


ditions. However, natural populations are often subjected to adverse biotic
and abiotic stressors. Environmental stress, especially a combination of
different stress factors, can cause adverse effects as shown, for example, for
mites infected with a weak bacterial pathogen (Lighthart et al., 1988). In
infections with those trypanosomatids which are subpathogenic and do not
obviously affect the host, the synergistic action of the trypanosomatid and
other stressors could result in recognizable effects. In infections with patho-
genic trypanosomatids, the intensity of the effects may well be increased.
296 G. A. SCHAUB

So far the effect of the most important abiotic stressors, temperature and
relative humidity, on insects infected with trypanosomatids have been
investigated only in the B. triatomae-Tri. infestans system (see section IV.B),
but the temperature steps used were too large to recognize synergistic effects.
Laboratory studies are necessary to clarify whether the significantly lower
prevalence of C. bombi in spring queens of bumble bees than in previous
summer workers is caused by reduced hibernation success of infected queens
or by loss of the parasites during the winter (Shykoff and Schmid-Hempel,
I99 1b).
Observations by Gorla's group indicate that a synergistic stress of infec-
tion and abiotic factors might act on natural populations of Tri. infestans.
The percentage of bugs infected with Tryp. cruzi is statistically significantly
lower in winter and early spring, with mean daily minimum and maximum
environmental temperatures of about 6" and 17"C, respectively, than it is in
mid spring and autumn (15" and 27°C) (Giojalas et al., 1990). However,
other possibilities, such as temperature dependency of the development of
Tryp. cruzi and the age structure of the population, could be excluded only
by a detailed study, e.g. using populations in chicken houses (Gorla and
Schofield, 1989).
Another stress factor for specimens from naturally infected populations
could be capture and transport to the laboratory. This was evident with
sandflies infected with different trypanosomatids of toads and lizards. The
highest rates of infection occurred in flies that did not survive the transport
(Ayala, 1973).
In other systems synergistic effects of the infection and a second stressor
are evident.

A. SENSITIVITY TO INSECTICIDES

The sensitivity of tsetse flies infected with Tryp. brucei to a low dose (50%
lethal dose or less) of different insecticides was tested by Golder et al. (1982,
1984). Within 48 h after topical application of endosulfan, about 50% more
infected flies than uninfected ones were dead. The increased sensitivity of
infected flies was also evident after application of a natural pyrethrum
extract, and in both studies males reacted more sensitively than females. In
addition, G. m. morsitans infected with Tryp. congolense had reduced
resistance to deltamethrine (Nitcheman, 1988). Whereas the results con-
cerning the effect of infection on longevity of flies are contradictory, these
insecticide data indicate at least a subpathological effect of the trypano-
somes.
In our Tryp. cruzi-Tri. infestans system we used different insecticides. The
effective dose which killed 50% of the populations did not differ between
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 297

uninfected and infected populations. Also, B. triatomae did not seem to


affect the sensitivity of bugs to insecticides. The long-term effect of sublethal
doses remains to be investigated (G. A. Schaub and R. Pospischil, unpub-
lished observations).

B. STARVATION RESISTANCE

The effect of short-lasting starvation of R. prolixus naturally infected with


Tryp. rangeli has been investigated by Marinkelle (1968). Starvation for 8
weeks increased the mortality rate of regularly fed, laboratory reared,
uninfected fourth and fifth instar larvae (normally 10%) only up to l8%,
but during a 6-week starvation period 87% of bugs caught in the field died.
However, the infection rates detected in dead and surviving larvae were
nearly identical and, unexpectedly, very low (7%), in contrast to 46% in the
original batch of insects. The low rate detectable in the dead bugs was
explained as an artefact due to the delay in determination of the infection
after death of the insects, since no motile forms of Tryp. rangeli could be
found 1-3 h after death of the bugs (Marinkelle, 1968). Since infection rates
and especially the feeding state of bugs caught in the field may vary,
determination of starvation resistance of such bugs offers results with only
limited value.
These difficulties were also evident in a calculation of starvation resistance
of Tri. dimidiata naturally infected with Tryp. cruzi (Vargas and Zeledon,
1985). In addition, infected and uninfected groups contained different
numbers of bugs of different developmental stages, which vary in their
capacity to survive starvation. Therefore, it was uncertain whether the lower
mean survival time in the infected group really indicated an adverse effect of
the infection on starvation resistance.
Using laboratory reared Tri. infestans, which had been infected in the first
instar with Tryp. cruzi and given their last feed in the second, third or fourth
instar, the mean starvation resistance period was reduced respectively by
3%, 14% and 17% relative to uninfected bugs, the differences between the
two latter values being statistically significant by Student’s t test (Schaub
and Losch, 1989). The respective data for bugs infected with B. triatomae,
51%, 55% and 32%, demonstrated the strong pathological effect of the
homoxenous flagellate. Whereas the most resistant stage, the fourth instar,
survived uninfected up to 432 days after the last feed in the third instar
(allowing development to the fourth instar), the last bug of this instar
infected with Tryp. cruzi died on day 331 and the last bug infected with B.
triatomae survived only to day 140. Food remnants were present in the
intestines of a higher proportion of dead bugs infected with Tryp. cruzi than
in those of uninfected bugs, and in even more larvae infected with B.
298 G. A. SCHAUB

triatomae. Thus, flagellates and bug either seem to compete for essential
metabolites whose depletion results in death, or else the flagellates excrete
toxic substances (Schaub and Losch, 1989).
Similarly to Tryp. cruzi, which does not affect survival time of regularly
fed bugs reared under optimum conditions, B. gerridis and/or C .flexonema
infections reduced starvation resistance of male water striders (Arnqvist and
Maki, 1990). Surviving males showed a statistically significant lower parasite
load than those dying of starvation during the first days. Since water striders
do not survive such long starvation periods as triatomines (mean survival
time of starved males was 5 days), their natural populations are probably
more affected than those of triatomines.

C. SENSITIVITY TO ISOLATION AND OVERCROWDING

Maintenance in isolation or in overcrowded conditions can be a stress factor


for insects, adversely affecting different life characteristics (Chauvin, 1967;
Peters, T. M. and Barbosa, 1977). The occurrence of such effects depends on
the natural life style of the species or its developmental stages. In insects
which normally live singly the stressor is grouping, and in insects which
normally live gregariously, like the triatomines, it can be either isolation or
overcrowding (Peters, T. M. and Barbosa, 1977).
Whereas it has long been known that crowding can induce outbreaks of
insect diseases (e.g. Steinhaus, 1958; further literature cited by Schaub,
1990b), the effects of isolation on insects infected with trypanosomatids have
only recently been investigated with triatomines. Reis dos Santos and
Lacombe (1985) attributed retarded development of some first instar larvae
of Tri. infestans which were infected with Tryp. cruzi to the infection and not
to an isolation effect; this effect was not obtained in our Tryp. cruzi-Tri.
infestans system (Schaub, 1988d).
In a detailed study investigating the importance of group size in Tri.
infestans, both uninfected and infected with B. triatomae, the developmental
time and mortality of larvae which were maintained in isolation or in groups
of 20, 30, 40 and 50 bugs were compared (Schaub, 1990b). In uninfected
groups only a minor proportion of isolated older larvae, but no crowded
bugs, showed delayed development, demonstrating for the first time an
isolation effect on development of uninfected triatomines. The lack of such
an effect on development of first instars (Schaub, 1988d) was confirmed in
that study. In groups infected with B. triatomae, development of isolated
bugs was more retarded than in uninfected bugs. In addition, a synergistic
action of overcrowding and infection slightly increased developmental times.
At 35-40 weeks after first feeding of first instars of the most crowded groups
(50 bugs), only about half of those infected bugs from which adults
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 299

eventually developed had moulted to adults. By that time 20% more bugs
had emerged in all the other infected groups (Schaub, 1990b).
Mean total mortality rates of about 10% in uninfected groups were
unaffected by group size. In most groups of infected bugs the mean mortality
rates were about 50%, but in the most crowded groups, consisting of 50
larvae, a higher mean mortality rate of 75% (a statistically significant
difference) was observed. This indicates a subpathological overcrowding
stress, increased by the synergistic action of the flagellate (Schaub, 1990b).
In natural populations, crowding of Tri. infestans reduces blood intake
(Schofield, 1982), but isolation is likely to be a less important stress factor
for triatomines. The more sensitive reaction of infected bugs to these
stressors implies that other stress factors may well also act synergistically
with B. triatomae and perhaps also with Tryp. cruzi (see Section VIII).

VII. MECHANISMS
OF PATHOGENICITY

The mechanism of the pathological effects seems to be clear when the


parasites multiply intracellularly in the affected organ, e.g. the intestine.
Presumably the altered behaviour of infected blood sucking insects can be
attributed to effects on salivary glands, blood flow rate, or receptor function.
In the two systems in which many effects are known in detail, R . prolixus
infected with Tryp. rangeli and Tri. infestans infected with B. triatomae, we
are far from understanding the physiological bases of the similar complex
sickness syndrome in the insect. Gomez (1967) suggested that it is not
invasion of the haemocoele which causes the pathogenicity of Tryp. rangeli,
but an effect in the intestine. In her investigation, larval mortality rate was
much higher than that observed by Grewal (1957) (see Section IV.A), but
only 2% of her fifth instar larvae which did not moult to the adult stage
possessed parasites in the haemocoele, compared to 100% (total infection
rate 54%) in the study by Grewal (1957). However, direct inoculation into
the haemocoele also harmed the bugs (Grewal, 1957, 1969; Watkins, 1969),
and Grewal (1957) emphasized that mortality “is probably not connected
with the penetration of the gut wall”. The discussion of Aiiez (1984)
indicated that invasion of the haemocoele and continuous development in all
tissues were the important pathological mechanisms. Accordingly, the high
mortality rate in the first, second and fifth instars could be caused by either
early or late invasion of the haemocoele by Tryp. rangeli (Aiiez, 1984).
Watkins (1969, 1971a,b) has already referred to two phenomena which are
similar to some effects observed in R . prolixus infected with Tryp. rangeli. In
larvae infected with this trypanosomatid, and also in those with blocked
spiracles, peristalsis of the gut is absent, the rectum is much distended,
300 G . A. SCHAUB

defaecation and feeding stop, and haemolymph volume increases. On the


other hand, some of these effects were also evident in aposymbiotic bugs of
different species of triatomines if they were fed on humans, rabbits or
guinea-pigs; larval developmental times and mortality rates were increased,
mainly in the last two instars, bugs also often died during or soon after
ecdysis, moulting deformities were common, the cuticle was pale, excretion
and digestion were disturbed and some larvae probed but did not ingest
blood (summarized by Gumpert and Schwartz, 1962; Auden, 1974). Those
few aposymbiotic Rhodnius that became adults produced no eggs (Brecher
and Wigglesworth, 1944).
Therefore, Watkins (1969, 1971a) suggested an effect of Tryp. rangeli
on the gut-colonizing symbionts which are thought to produce B-group
vitamins. She observed that cross-sections of midguts contained far fewer
symbionts than those of uninfected bugs. However, her tests with smears
made from intestinal contents on agar plates (Watkins, 1969) were not
totally convincing. Culture material of Tryp. rangeli inhibited the growth of
the intestinal bacteria originating from uninfected bugs. In addition, smears
made from contents of uninfected intestines yielded a good growth of
symbiont-like bacteria, visible at 48 h and heavy after 78 h. With material
from infected bugs, growth was first visible on the third day and then ceased.
However, this also happened when using intestinal contents from uninfected
bugs that showed deformities similar to those of infected bugs. Therefore,
independently of infections with Tryp. rangeli, intestinal bacteria may not be
viable in sick bugs.
Since the sickness syndrome of bugs infected with B. triatomae also
resembles the effects in aposymbiotic bugs, we tried to ascertain the number
of bacteria in uninfected and infected Tri. infestans, but preliminary tests
showed great variation in the number of bacteria in bugs of both groups
( G . A. Schaub and K . Fischer, unpublished observations). However, other
experiments support the interpretation that B. triatomae might interfere with
the symbionts or their supply of vitamins or other metabolites. Supplemen-
tation of blood with B-group vitamins supported the initial, but not late,
development of B. triatomae in the small intestine of young, but not old, Tri.
infestans (Jensen and Schaub, 1991). Since this supplementation also greatly
reduced the pathogenicity, competition of the bug and the flagellates for the
vitamins/metabolites or other effects on the symbionts may be the mechan-
ism of the pathogenicity of B. rriatomae (Jensen, 1987). The fact that B.
triatomae and Tryp. rangeli affect different groups of bugs might then be due
to differences in the sensitivity of the symbionts. In both systems the inter-
actions of parasites and bug symbionts need detailed investigation. This
aspect has only once been studied, in bugs infected with Tryp. cruzi
(Muhlpfordt, 1959).
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 30 1

So far we cannot decide what is the sequence of steps in the pathological


process. It is possible that anoxia inhibits the growth of symbionts, thereby
reducing the vitamin supply. However, it seems more likely that a reduced
vitamin/metabolite supply causes reduced growth of the tracheoles, resulting
in anoxia.
In both triatomine systems these questions could be answered soon,
perhaps also providing explanations applicable to other trypanosomatid-
insect systems.

VIII. CONCLUSIONS

Although many trypanosomes reach high population densities in the insect


host, strong pathological effects are observed in only a few systems (Table
4). Usually the interaction of trypanosomes and insects seems to be
balanced, and no ill effects are obvious. However, under natural conditions
insects are often exposed to adverse conditions-factors which are rarely
considered in laboratory studies-and the parasite might then act synergisti-
cally with other stressors (Schaub, 1989b) (see Section VI). Theoretical
models indicate that synergistic action with other stressors might be import-
ant in regulation of the host population (Anderson, R. M., 1979; Anderson,
R. M. and May, 1981).
According to the intensity of the effects on the insect host, trypanosoma-
tids can be classified into three groups. Trypanosomatids of the first group
reach low population densities in the insect and will not affect the host even
under adverse conditions. Since tests under adverse conditions have not
been performed with most trypanosomatid-insect systems, the majority of
trypanosomatids must be classified into this group. The second group is sub-
pathogenic, but harms the insect if other stress factors act synergistically.
This group consists of most Leishmania species and other trypanosomatids
of sandflies, the salivarian trypanosomes developing in tsetse flies, Tryp.
cruzi, B. gerridislc. jlexonema, C . bombi, Lept. pyrrhocoris and perhaps
Lept. pyraustae and B. pessoai. Most of the effects discussed in the present
review can be attributed to this group. So far only a few trypanosomatids are
known to affect the insect even under optimum conditions, and these can be
classified into the third group. They are Tryp. lewisi, Tryp. rangeli, H .
swainei, H . muscarum, B. caliroa and B. triatomae.
A better grouping will be achieved after more investigations of natural
populations like those of Arnqvist and Maki (1990) and Shykoff and
Schmid-Hempel(199 1a,b,c). Such studies should be accompanied by labora-
tory studies under exactly defined conditions including infection doses,
temperature and relative humidity, and the exclusion of factors such as
302 G . A. SCHAUB

TABLE
4 Summary of effects of trypanosomatids on insectsa

Feature assessedb
Trypanosomatid Host insect A B C D E F G H I K
Endotrypanum Sandfly +
schaudinni
Leishmania Sandfly + + +
donovani
L. major Sandfly + + +
L. braziliensis Sandfly +
L. amazonensis Sandfly + +
L. aethiopica Sandfly +
Trypanosoma sp. Sandfly + +
(of toads,
lizards)
Trypanosoma sp. Sandfly + +
(of bats)
T. congolense Tsetse fly f + f
T. brucei Tsetse fly f + + f +
T. lewisi Rat flea + +
T. avium Mosquito + +
Tabanid +
T. corvi Mosquito +
T. cruzi Triatomine + f + f +
T. rangeli Triatomine + + + + + + + + +
Bed bug +
Blastocrithidia Triatomine + + + + + + + + +
triatomae
B. gerridis Water strider +' + +' -c

B. pessoai Mosquito +
B. caliroa Hymenoptera + +
Herpetomonas Hymenoptera - + + +
swainei
H.muscarum Diptera + + +
Crithidia bombi Bumble bee + -
+ +
C. cimbexi Hymenoptera
Leptomonas Bug + + + +
pyrrhocorris
L. oncopelti Bug +
L. pyraustae Corn borer +
a References are given in the text.
bA, fitness; B, probing and engorgement; C, intestine; D, Malpighian tubules; E, haemo-
lymph; F, cuticle; G, further organs; H, larval development and mortality rate; 1, adult

-.
longevity and fecundity; K, survival, usually subpathogenically affected (synergistic stressors).
+, Affected (sometimes only slight indications for the respective effect); not affected; k,
contradictory results.
' Double infections with Crithidiujlexonemu.
EFFECTS OF TRYPANOSOMATIDS ON INSECTS 303

concomitant double infections with other pathogens. In these laboratory


studies it is very important that parasites and hosts originate from the same
locality (discussed by Boker and Schaub, 1984). Investigations of “wild”
populations for several years can provide data on the variation of the genetic
heterogeneity. Theoretically, in “wild” populations the proportion of sus-
ceptible and tolerant individuals should vary and, therefore, also the
percentage of affected insects (Anderson, R. M., 1986). Selection phenomena
require more time to develop in insects with long developmental times such
as the triatomines. Genetic heterogeneity may also be important in insect
populations which are normally less heterogeneous, the social insects.
Effects at the population level are indicated by the recent field and labora-
tory studies comparing uninfected bumble bees and those infected with C.
bombi (Shykoff, 1991; Shykoff and Schmid-Hempel, 1991a). Differences in
susceptibility result in higher parasite transmission rates between related
bees than among those which are unrelated. Assuming that all susceptible
individuals react sensitively, subpathogenic trypanosomatids also provide a
selection pressure on social insects to maintain genetic variability.
Another interesting theoretical aspect is offered by the infection rate of
natural populations with B. triatomae. Whereas all specimens of the insecti-
vorous bug Zelus leucogrammus were found to be infected by Carvalho and
Deane (1974), though no pathological effect was reported, maximum infec-
tion rates of “wild” triatomines were 5.4% in Tri. maculata and less than 1 %
in Tri. sordida and Panstrongylus megistus (publications summarized by
Schaub and Boker, 1986a; Schaub, 1988a). On the one hand, it is possible
that B. triatomae is pathogenic only in infections of insect species which are
only occasionally infected in nature. On the other hand, it is possible that the
high infection rate of Z. leucogrammus is caused by predation of infected
triatomines. The low infection rate of triatomines would then be an indicator
of virulence, since a characteristic of highly pathogenic species seems to be
a very low infection rate in the host population (Anderson, R. M., 1979).
However, Anderson, R. M. (1979) also emphasized another characteristic,
the low average parasite burden of the host; this is not the case in bugs
infected with B. triatomae.
Only B. triatomae seems to be a good candidate for biological control of
its host. Unlike viruses and bacteria, a fast action cannot be expected since
the main effects occur in late larval instars. An application of