Veterinary Parasitology, 18 (1985) 1 6 7 - 1 8 2 167 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

AFRICAN TRYPANOSOMIASIS IN CATTLE: WORKING WITH NATURE'S SOLUTION

Max Murray and S°J. Black

International Laboratory for Research on Animal Diseases (ILRAD),

P.O. Box 30709,

Nairobi, Kenya

ABSTRACT

Murray, Max and Black, S.J. 1985. African Trypanosomiasis in cattle:

with nature's solution. Vet. Parasitol., 18: 167-182.

Working

Both acquired and innate resistance to African trypanosomiasis can occur in cattle. The former raises the possibility of a vaccine against tsetse- transmitted metaeyclic trypanosomes which have been shown to have a smaller repertoire of variable antigens than bloodstream parasites. The latter pro- vides two further avenues of approach: firstly, trypanotolerant breeds are being increasingly exploited and improved by conventional management and breed- ing methods including embryo transfer; secondly, research is being carried out into the factors associated with their innate resistance, i.e., the control of trypanosome growth, the development of effective immune responses and resistance to anaemia. If the mechanisms underlying these factors are identified it might be possible by immunisation, by specific drug treatment or by transfection of appropriate genes to produce highly productive cattle resistant to trypano- somiasis.

INTRODUCTION

Vast humid and semi-humid areas of Africa are held captive by tsetse flies

and the trypanosomes which they transmit. Tsetse infest approximately

I0 million km 2 of Africa, representing 37% of the continent and affecting 37

countries (FAO-WHO-OIE, 1983). Much of the best watered and most fertile land

is infested with tsetse, while large areas of good grazing, most notably in

the subhumid zone, could be immediately utilised by pastoralists if trypano-

somiasis could be controlled. Currently, about 30% of the 147 million cattle

in countries affected by tsetse are exposed to infection (Murray and Gray,

1984). The limited data available indicate that the overall situation is

deteriorating and that, since the 1950s, tsetse have continued to spread

(MacLennan, 1980). Thus, with the steadily increasing human population in

Africa, there is mounting pressure on tsetse-free pasturage and increasing need

to utilise the areas currently infested.

Several factors contribute to the magnitude of the problem. First of all,

0304-4017/85 /$03 .30 © 1985 Elsevier Science Publishers B.V.

168

the complexity of the disease itself. In cattle, three species of trypanosome,

Trypanosoma congolense, T. vivax and T. brucei cause the disease, either

individually or jointly. These trypanosomes are transmitted cyclically by

several different species of tsetse, each of which is adapted to different

climatic and ecological conditions (Ford, 1971). While tsetse are not the only

vectors of African trypanosomes, cyclical transmission of infection represents

the most important problem because, once the fly becomes infected, it remains

infective for a long period in contrast to the ephemeral nature of non-cyclical

transmission. At the same time, trypanosomes infect a wide range of hosts

including wild and domestic animals. The former, particularly the wild Bovidae

and Suidae, do not suffer from severe clinical disease but become carriers and

constitute an important reservoir of infection. The success of the trypanosome

as a parasite is to a large extent due to the ability to undergo antigenic

variation, i.e., change a single glycoprotein (Cross, 1975) which covers the

pellicular surface, thereby, enabling evasion of host immune responses and the

establishment of persistent infections. Added to the complexity of multiple

variable antigen types (VATs) expressed during a single infection, each trypano-

some species comprises an unknown number of different strains or serodemes, all

capable of elaborating a different repertoire of VATs (Van Meirvenne et al.,

1977). For these reasons, no vaccine is available for use in the field.

The outcome of infection is variable. Many cattle die after an acute or

chronic illness, or if non-fatal, trypanosomiasis may cause poor growth, weight

loss, low milk yield, reproductive failure and a reduced capacity for work.

However, some animals appear to be able to cope with the infection. Thus,

certain breeds of cattle and many species of wildlife possess the ability to

survive and be productive in tsetse-infested areas, without the aid of trypano-

cidal drugs, where other animals rapidly succumb to the disease (Murray et al.,

1982). In addition, cattle which survive infection, with or without the aid of

trypanocidal drug treatment, appear to become increasingly resistant to sub-

sequent challenge (Murray et al., 1982).

In seeking new methods to control African trypanosomiasis, we believe that

the existence of bovids which show a wide range of susceptibility provide a

unique opportunity to examine the factors which promote resistance to the

disease.

INNATE RESISTANCE TO TRYPANOSOMIASIS: ASSOCIATED PARAMETERS

The large herds of wild Bovidae that still roam the tsetse-infested

forests and savannas of Africa attest to the ability of these animals to co-

exist with and serve as a major food source for tsetse (Weitz, 1963). Wild

Bovidae emerged in Africa some 20 to 40 million years ago towards the end of

the Oligocene epoch (Leakey and Lewin, 1977). It is likely that tsetse

169

originated earlier than this during the late Palaeozoic or early Mesozoic eras

if it is accepted that the four fossil species from the Oligocene shales at

Florissant in Colorado are Glossina (Ford, 1971). In the absence of

palaeontological evidence, thoughts on the evolution of African trypanosomes can

only be hypothetical.

What factors allow wild Bovidae to co-exist with tsetse and trypanosomes?

Some wild Bovidae possess a variety of "tricks" that interfere with tsetse

feeding, e.g., skin twitching in the impala or the deadly accuracy with which

oryx can kill tsetse with their horns. However, analyses of tsetse blood meals

show that wild animals act as a major food source, indicating that "tricks" are

not always sufficient to prevent tsetse attack and that host resistance is

probably the most important factor in their co-existence with tsetse and

trypanosomes.

Several experimental investigations have now established that many species

of wild Bovidae exhibit marked innate resistance to the parasite and the

disease (Murray et a~., 1982). Recently, we have confirmed this in African

buffalo (Syncerus caffer), oryx (Oryx beisa), eland (Taurotragus oryx) and

waterbuck (Kobus defassa), which were reared in captivity and had not been

previously infected with trypanosomes. Following infection by tsetse tran-

smission or needle inoculation with T. congolense, T. vivax or T. brucei, these

animals exhibited no clinical signs of any pathogenic effects caused by the

infection. In contrast to trypano-susceptible cattle, only low, barely

detectable and transient parasitaemia and small temporary reductions in red

blood cell concentrations were recorded. An exception to this picture occurred

in African buffalo infected with T. vivax: red blood cell values were not

affected despite levels of parasitaemia corresponding to those found in cattle

which showed a significant drop in the number of red blood cells.

Similarly with cattle, an association can also be seen between the duration

of exposure to tsetse and the degree of resistance exhibited to trypanosomiasis.

As early as 1906, Pierre observed the ability of indigenous taurine breeds of

cattle in West Africa to survive and be productive in tsetse-infested areas.

This trait has been termed trypanotolerance and the taurine breeds involved are

the N'Dama and the West African Shorthorn. Only N'Dama and West African

Shorthorn survive in large numbers without trypanocidal drug treatment in areas

where significant tsetse risk exists (ILCA, 1979) whereas, most Bos indicus

types require treatment or are found only on the fringes of fly belts, and

exotic breeds cannot be maintained even in areas o£ low tsetse risk without

intensive drug therapy.

These field observations have now been confirmed by several experimental

studies in which cattle never previously exposed were infected with trypanosomes

by syringe inoculation or by tsetse transmission. N'Dama, the breed most

170

thoroughly investigated, were found to be significantly more resistant to

trypanosomiasis than Zebu and imported exotic breeds, in terms of productivity

and survival (Murray et al., 1982). The difference between the breeds is

associated with the fact that the prevalence, level and duration of para-

sitaemia are significantly less in trypanotolerant cattle (Fig. 1), despite

all breeds being equally susdeptible to the establishment of infection.

Correspondingly, the trypanotolerant breeds develop less severe anaemia

(Fig. 1).

Trypanoly t lc an t ibody response

Trypenotolerant 0 0 1 9 2 9 6 2 4 2 4 1 2

- - Trypanosuscept)b l l 0 0 1 2 1 2 0 -- --

m > S x l O s 6

i!. 1" J o t O 4 3 'E

2

l o 2 t x

= 2 0

2 4 6

Weeks a f ter infect ion

Fig. l. Parasitaemia and PCV in a trypanotolerant ( ) and a trypanosusceptible steer (o) infected with T. co~golense. Associated with the capacity to control parasitaemia and resist anaemia, the trypanotolerant animal de- veloped a higher and more prolonged antibody response: reciprocal titre of the highest dilution of serum that caused lysis of the majority of trypanosomes.

Fig. 2. Slender, intermediate and stumpy forms of T. br~cei. Giemsa.

171

Thus, a l l a v a i l a b l e e v i d e n c e now i n d i c a t e s t h a t r e s i s t a n c e t o t r y p a n o -

s o m i a s i s i s d e t e r m i n e d by a t l e a s t two main c h a r a c t e r i s t i c s of t h e h o s t ,

1) t h e a b i l i t y (o r i n a b i l i t y ) t o r e g u l a t e p a r a s i t e p o p u l a t i o n e x p a n s i o n and,

2) t h e c a p a c i t y (o r i n c a p a c i t y ) t o r e s i s t anaemia .

FROM THE FIELD TO THE LABORATORY

It is our intention to consider in this paper, mechanisms which control

the degree of anaemia and the level of parasitaemia with a view to introducing

these traits into susceptible cattle or enhancing their expression in

relatively resistant animals. In search of mechanisms pertinent to the

regulation of parasitaemia, many of the experiments were conducted in gene-

tically homogeneous strains of mice. The experimental approaches used with

these mice would not have been possible in genetically heterogeneous breeds of

cattle.

Control of anaemia

Trypanosome-derived haemolytic factors and trypanosome-induced immune

complexes have been shown to alter the surface of erythrocytes leading to their

sequestration and destruction in the mononuclear phagocytic system (Murray,

1979). Thus, the degree of red cell damage is likely to be related to the

number of lysed or degenerating parasites present in the blood, in part because

"haemolytic factors" arise from degenerating organisms (Tizard et al., 1978)

and in part because antibody responses, which result in immune complex forma-

tion, are stimulated by degenerating organisms (Sendashonga and Black, 1982).

Haemolytic activity has been shown to be generated on lysis of T.

congolense, T. vivax and T. brucei parasites [Murray, 1979). The nature of the

haemolytic activity has not been extensively investigated but has been

speculated to include trypanosome-derived neuraminidase, phospholipase enzymes

and perhaps products of trypanosome autolysis, e.g., free fatty acids (Tizard

et al., 1978; Esievo et al., 1982). We have detected haemolytic activity in

plasma collected from trypanosome-infected cattle and laboratory animals and

hence it is present in excess of possible plasma inhibitors. Identification of

the components of trypanosomes responsible for haemolytic activity might lead

to the development of chemical or immunological blocking agents, e.g.

antibodies.

In addition to the number of parasites in the blood, the degree of anaemia

which develops in infected animals may also relate to the efficiency of

erythropoietic responses, the sensitivity of red cells to haemolytic factors,

the presence of inhibitory factors in plasma or alterations of the mononuclear

phagocytic system leading to a failure to discriminate between healthy and

altered red blood cells. A series of erythrokinetic and ferrokinetic studies

172

c a r r i e d o u t i n N'Dama and Zebu e x p e r i m e n t a l l y i n f e c t e d w i t h t r y p a n o s o m e s

i n d i c a t e d t h a t t h e l e s s s e v e r e a n a e m i a i n t h e N'Dama r e f l e c t e d t h e i r c a p a c i t y

t o c o n t r o l p a r a s i t a e m i a and c o u l d n o t be a t t r i b u t e d t o d i f f e r e n c e s i n i n n a t e

e r y t h r o p o i e t i c r e s p o n s e s ( D a r g i e e t a l . , 1979 ) . The o t h e r p o s s i b l e m e c h a n i s m s

w h i c h m i g h t c o n t r o l a n a e m i a h a v e n o t b e e n i n v e s t i g a t e d .

R e c e n t s t u d i e s i n o u r l a b o r a t o r y h a v e shown t h a t , i n c o n t r a s t t o Bo ran and

e x o t i c c a t t l e , c e r t a i n s p e c i e s o f w i l d B o v i d a e do n o t show any s i g n i f i c a n t o r

p r o l o n g e d c h a n g e s i n t h e i r r e d c e l l m a s s , i r r e s p e c t i v e o f t h e d e g r e e o f p a r a -

s i t a e m i a o r t h e number o f p a r a s i t a e m i c waves t h a t a r i s e . The b a s i s o f t h i s

p o s s i b l e r e s i s t a n c e t o a n a e m i a i s c u r r e n t l y b e i n g i n v e s t i g a t e d as i t c o u l d l e a d

t o new a p p r o a c h e s t o t h e c o n t r o l o f a m a j o r p a t h o g e n i c f e a t u r e o f t h e d i s e a s e .

Control of parasitaemia

Parasitaemia appears to be limited by two inter-related processes, firstly,

physiological responses which influence the rate at which parasites switch from

dividing to non-dividing forms and, secondly, antibody responses which cause

parasite elimination.

Non-immunological control of parasitaemia

In cattle, T. congolense (Fig. I), T. vivca~ and T. brucei parasites give

rise to infections characterised by an exponential phase of parasite population

expansion in the blood followed by a plateau and in most instances, a remission

phase. The height of parasitaemia reached reflects the duration of the growth

phase and can vary between breeds and individuals infected with parasites

derived from the same clone (Fig. 1). In the case of T. br~xcei, parasites

present in the blood during the exponential phase of parasitaemia differ

morphologically from those of the plateau or remission phases (Bruce et al.,

1910). This has also been found in mice even when infections are initiated

with a single T. br~cei parasite. Exponential phase trypanosomes are slender

(Fig. 2), multiply rapidly, are highly infective for mammals, have inefficient

aerobic pathways of energy utilization and are defective in their capacity to be

cyclically-transmitted by tsetse. Parasites of the plateau phase are stumpy

(Fig. 2) have few or no members in the S, G 2 and M stages of the cell cycle

(Shapiro et al., 1984), are relatively non-infective for mammals, move towards

aerobic pathways of carbohydrate metabolism and, have a high capacity to be

cyclically-transmitted by tsetse flies. The studies suggest that T. brucei

parasites switch from dividing to non-dividing forms in preparation for tsetse

transmission and that the switch or differentiation event is not reversible in

the mammal, i.e., the switch from slender to stumpy form parasites in the

mammal marks a reduction in the parasite population growth rate. More limited

studies conducted with f. vivc~. (Shapiro et al., 1984) and T. congolense

(Nantulya et al., 1978) suggest that these parasites might have a similar

173

pathway of differentiation.

The morphological transition from slender to stumpy forms of T. brucei is

accompanied by the development of the parasite mitochondrion and appears to be

independent of the host antibody response in that parasite differentiation

occurs at the same rate in intact mice, athymic mice and splenectomized mice,

although antibody responses are retarded in the latter (Black et al., in press).

Parasite differentiation also occurs at the same rate in lethally-irradiated,

and lethally-irradiated and spleen cell reconstituted mice hut only the latter

produce antibody.

Parasite population growth rates are faster and differentiation rates

slower in susceptible as compared to resistant mice infected with T. br~cei

derived from the TREU 667 stock (Black et al., 1983a). Reciprocal experiments

in cattle show that T. b~cei parasites belonging to the LUMP 227 stock

differentiate more rapidly to stt~npy forms in more resistant animals and breeds.

Similarly, in wild Bovidae which limit T. brq~cei parasitaemia, parasites, when

they become detectable in the blood, are almost always stumpy forms (unpublished

data). These findings indicate that control of T. brucei parasitaemia in vivo i

reflects physiological processes which influence parasite differentiation rates.

The contribution of the process to regulation of parasitaemia is further

emphasised by observations which suggest that antibody responses against

T. b1~cei are stimulated by fragments derived from senescent stumpy fol~n

organisms but not by actively dividing parasites (Sendashonga and Black, 1982).

A number of other studies indicate that the growth of T. br~cei populations

can be negatively or positively regulated by the host. On the one hand, human

serum high density lipoprotein has been shown to lyse T. brucei but not

T. rhodesiense parasites (Rifkin, 1978); at present, this is the only method

available to distinguish between these organisms. On the other hand, T. br~cei

parasites which induce an acute infection in mice and do not give rise to stumpy

forms, establish chronic infections in cattle characterised by recurrent waves

of parasitaemia in which stumpy forms are easily detected (Black et al., 1985b).

Parasites cloned back into mice from any parasitaemic wave in the cattle do not

give rise to stumpy forms even when the recipient mice are inoculated daily

with normal or immune bovine plasma. This suggests that cattle do not produce

molecules which stimulate parasite differentiation. Hence, the growth rates of

T. br~cei populations are more likely to be controlled by the availability of

host-derived molecules which stimulate parasite multiplication (see later).

The rate of parasite differentiation in any given host is not, however,

immutable. Differentiation of T. br~cei in mice can be accelerated by daily

injection of indomethacin or acetylsalicyclic acid (Jack et al., 1984). These

drugs are unlikely to exert their effect via inhibition of prostaglandin syn-

thesis because we have found that other more specific blockers of prostaglandin

174

synthetase, e.g., flurbiprofen and caprofen have no effect on parasite

differentiation.

Another significant finding is that treatment of mice with various

immunomodulators can modify parasite population growth rates (Murray and

Morrison, 1979). In particular, the rate of differentiation of T. b~cei in

both intact and lethally-irradiated mice can be accelerated by pre-treatment of

the hosts with dead Corynebacteri~ pa~ organisms. The effect is most

marked in strains of mice with the greatest innate resistance to the parasites

and can be boosted by administration of the bacteria prior to or during the

course of infection (unpublished data). In addition to controlling T. b~cei

parasitaemia, C. parv~ has also been shown to limit T. congolense (Murray and

Morrison, 1979) and T. viv~ parasitaemia (unpublished data), particularly in

more resistant strains of mice. The effect cannot be transferred between

treated and untreated syngeneic partners by spleen cells or serum. Neverthe-

less, it is possible that the treatment directly or indirectly modifies the

rate of production of trypanosome growth-stimulating factors by radio-resistant

cells.

T. b~cei parasites multiply in vitro when co-cultured with bovine or

mouse fibroblasts in modified Iscoves medium, supplemented with components of

foetal bovine serum (FBS) of a molecular mass range from 105 to greater than

106 daltons (Black et al., in press). The molecules are not lipoproteins and

attempts to dissociate complexes and possibly obtain a single active ingredient

have so far been fruitless. Neither mouse T-cells, B-cells nor macrophages can

substitute for the fibroblast cells which must be intact and metabolically

active to support growth. Furthermore, the presence of bovine T-cell growth

factors does not enhance the activity of FBS to stimulate parasite multipli-

cation. We speculate that similar growth requirements will be found with

bloodstream forms of T. viv~ which require serum and fibroblasts to multiply

in vitro and T. congolense which require serum and endothelial cells. Identi-

fication of the growth factors involved and the parasite receptors to which

they bind might lead to the development of efficient immune or chemoprophylactic

reagents which would limit parasite growth and induce a quick effective immune

response to eliminate the parasites.

Immunological control of parasitaemia

i. Evasion of immune responses

There is no evidence that immune responses against trypanosome common

antigens contribute to host protection. Host protective responses are effected

by antibodies directed against the surface coat antigens of the trypanosome

(Murray and Urquhart, 1977). Metacyclic trypanosomes extruded by tsetse flies

and all bloodstream parasites are covered by a glycoprotein coat which can

175

differ between parasites derived from a single organism (LeRay et al., 1978).

The repertoire of VATs of bloodstream parasites is large, is revealed slowly

during the course of infection and often differs between parasites isolated in

the same geographical region (Van Meirvenne et al., 1977). The actual number

of possible VATs in any serodeme is unknown; diversity appears to arise from

the expression of different genes which exist as basic copies within the para-

site genome and can be modified by gene conversion (Bernards, 1984). The vari-

able surface glycoprotein (VSG) is presented on the parasite surface in such a

way that only a few epitopes, which do not include common antigens, are exposed.

The bloodstream forms of trypanosomes have thus developed an extremely effici-

ent method which enables some organisms to evade ongoing in~aune responses.

In contrast, the VAT repertoire of metacyelic forms of T. brucei and

T. congolense is much more limited than that of the bloodstream parasites

(Nantulya et al., 1983; Crowe et al., 1983). Furthermore, parasites derived

from one serodeme, when cyclically transmitted through tsetse flies, appear to

revert to a similar repertoire of metacyclic VATs. This has been shown with

T. congolense and T. brucei using monoclonal antibodies against metacyelic VSGs.

Moreover, it is implied by studies in cattle which demonstrate that animals can

be immunized against tsetse-transmitted homologous (but not heterologous) sero-

domes of T. congolense by prior exposure to metacyclic parasites which were

generated in vitro or by prior infection via tsetse flies followed by trypane-

cidal drug treatment (Morrison et al., 1985). In these studies, complete

immunity to tsetse-transmitted challenge was only achieved by delaying treat-

ment to day 15 after infection. This time coincided with the appearance of

antibodies which completely neutralised tsetse-transmitted metacyclic trypano-

somes of the same serodeme. The immunity produced has been shown to last for

as long as S months. These observations raise the possibility that a vaccine

against metacyclic VSGs might be of potential value in areas which have a

limited number of serodemes, particularly since metacyclic trypanosomes can now

be cultured in vitro [Gray et al., 1981).

However, if metacyclic VSG populations are completely static, it is

puzzling as to how trypanosomes managed to survive on the African continent,

given that animals infected with either metacyclic or bloodstream forms of T.

brucei or T. congolense have been shown to produce antibodies which neutralise

metacyclic organisms of the same serodeme (Nantulya et al., 1984). Recent

studies suggest that the serial propagation of a T. rhodesiense serodeme through

mammals and tsetse flies can lead to variation in the metacyclic VAT repertoire

in that serodeme and hence possible evasion of responses directed against meta-

cyclic VSGs [Barry et al., 1983). Sexual recombination between trypanosomes

belonging to different serodemes (Tait, 1980), if it were accompanied by

176

crossovers within genes encoding metacyclic VSGs, could also extend the diver-

sity of metacyclic VAT types. In the case of T. vivax parasites, studies to

date show that cattle can be only partially immunised against tsetse challenge

by prior exposure to tsetse flies infected with the same serodeme followed by

trypanocidal drug treatment (unpublished data).

The feasibility of production and the efficacy of a vaccine against meta-

cyclic trypanosomes will depend on the relative stability of the metacyclic VAT

repertoire for each of the pathogenic species of trypanosomes and on the number

of serodemes which occur in the field. While initial studies at ILRAD indicate

that the number of serodemes of T. congolense, T. vivax and T. brucei is likely

to be large, it is worth noting that this may not be the case for T. gambiense

and T. rhodesiense, the human pathogens, in which only a few serodemes would

appear to be dominant (Van Meirvenne, personal communication).

Even partial protection against trypanosomes could be of considerable

economic significance by reducing the requirement for therapeutic drug treat-

ment. Under natural field conditions, the innate resistance of trypanotolerant

cattle can be supplemented by the acquisition of resistance following trypano-

some exposure (Murray et al., 1982). Furthermore, the more susceptible Bos

indicus and exotic Bos taurus breeds, maintained in endemic areas by treating

infections with trypanocidal drugs, a process similar to that used experiment-

ally to induce anti-metacyclic VSG responses, also acquire a degree of

resistance against local trypanosome populations; this is assessed by the fact

that these animals require progressively fewer chemotherapeutic treatments

(Murray et al., 1982). It might be that the use of an appropriate metacyclic

VSG vaccine might achieve similar results more readily.

The difficulties inherent in immunizing animals against the plethora of

VATs of African trypanosomos is a strong encouragement to elucidate mechanisms

by which infected animals recognise and respond to VATs arising during the

course of infection.

2. Induction of immune responses against African trypanosomes

Studies in mice using T. brucei stocks that give rise to stumpy or degene-

rating parasites and T. brueei stocks that under selected conditions do not,

suggest that only the former stimulate antibody responses even when both sets of

parasites express serologically identical VSGs (Sendashonga and Black, 1982;

Black et al., in press). The parasites which do not give rise to stumpy forms

or parasite-specific and bystander antibody responses, do not depress the

capacity of infected animals to respond to challenge with other antigens and

hence are not potently immunodepressive: on the other hand, these parasites

when lethally-irradiated do stimulate VSG specific antibody responses and hence

are not intrinsically non-i~unogenic (Sendashonga and Black, 1982). These

177

results together with studies which show that VSG is not released by living

trypanosomes (Black et al., 1982), were interpreted as showing that antibody

responses of mice against T. b~cei are triggered by fragments of parasites

derived from senescent stumpy from organisms. Furthermore, parasite fragments

may play a significant role in stimulating antibody responses against

T. c~golense and T. vivc~, since living parasites of these species do not

release VSG in vitro (unpublished). In other words, the kinetics of immune

responses might, to some extent, be controlled by non-immune processes which

regulate parasite differentiation.

The kinetics of antibody responses relative to parasite differentiation

(see Non-immunological control of parasitaemia) were further examined in

resistant (C57BL/6) and susceptible (C3H/He) mice infected with T. b~cei (Black

et al., 1983a) or T. viv~ (Mahan, 1984). The resistant mice mount rapid anti-

body responses which cause parasite wave remission, whereas, little or no anti-

body responses, either parasite specific or bystander, are detected in the

susceptible animals. Nevertheless, in susceptible mice, stumpy forms of T.

b~cei arise and T. viv~ parasitaemia reaches a plateau implying the generation

of senescent non-dividing organisms. Similar observations have been made in

mice (Morrison et al., 1978) and in cattle exhibiting differences in suscep-

tibility to T. congolenae (Fig. I). Therefore, antibody responses against

trypanosomes in susceptible mice and cattle are likely to be regulated by other

factors in addition to the kinetics of parasite differentiation and the gene-

ration of immunostimulatory parasite fragments. The efficiency of immune res-

ponses against the parasites might relate to the intrinsic capacity of the

animals to respond to the parasite antigens or be associated with other pro-

cesses which modify the capacity of responding lymphocytes to mature to effector

function. That the latter is likely to be true is implied by studies which show

that both resistant and susceptible mice mount similar antibody responses

against lethally-irradiated T. b~cei (Black et al., 1983a) T. viv~ (Mahan,

1984) and T. congolese ~Morrison and Murray, in press) parasites.

Current experiments at ILRAD have shown that T-cells and B-cells of

s u s c e p t i b l e and r e s i s t a n t mice i n f e c t e d w i t h T. brucei or T. vivax a r e induced

t o mount s t r o n g and s i m i l a r DNA s y n t h e t i c r e s p o n s e s (7 to 15 t i m e s b a c k g r o u n d ) .

F u r t h e r m o r e , immunof luo rescence a n a l y s e s o f s p l e e n c e l l p r e p a r a t i o n s have

shown t h a t t h e a b s o l u t e number o f c y t o p l a s m i c I g - c o n t a i n i n g c e l l s i n t h e s p l e e n

i n c r e a s e s c o n s i d e r a b l y r e a c h i n g a peak o f 15 t o 20% o f t o t a l c e l l s i n b o t h

s t r a i n s o f mice by 7 days a f t e r i n f e c t i o n . However, whereas a n a l y s e s o f s p l e e n

s e c t i o n s r e v e a l s o r d e r l y lymphoid r e s p o n s e s a s s o c i a t e d w i t h a n t i b o d y p r o d u c t i o n

i n t h e r e s i s t a n t mice , i n s u s c e p t i b l e mice , which do no t produce d e t e c t a b l e

a n t i b o d y , t h e v a r i o u s compar tments o f t h e s p l e e n a r e d i s t o r t e d and d i s o r g a n i s e d .

E l i m i n a t i o n o f t h e p a r a s i t e s f r ~ n s u s c e p t i b l e mice w i t h B e r e n i l (Hoechs t ) or

178

Arsobal (Specia) results in recovery of splenic organisation within 24 to 48

hours and an equally rapid detection of parasite-specific and bystander anti-

body responses in the serum. Elimination of trypanosomes with Berenil has also

been shown to alleviate immunodepression rapidly in resistant mice chronically-

infected with T. bruoei (Murray et al., 1974) and to restore order in the

spleens of mice chronically-infected with T. eor~olense (Roelants et al., 1979).

The results suggest that living trypanosomes or short-lived factors from

degenerating trypanosomes cause splenic disorganisation, impair immune compet-

ence and, at least in the case of susceptible mice, interfere with the

secretion of immunoglobulin by plasma cells.

Preliminary electron microscopical studies have shown that many of the

plasma cells in trypanosome-infected mice have markedly distended cisternae of

rough-surfaced endoplasmic reticulum, a morphological feature that suggests a

disorder in immunoglobulin transport. By histochemical and immunochemical

staining these cells closely resemble Russell body-containing plasma cells. In

this respect, it is significant that Russell body-containing plasma cells (Mott

cells) appear early after infection and are a striking feature of the pathology

of African trypanosomiasis in man (Mott, 1907) and in cattle (Murray et al.,

1980). As in mice, cattle infected with trypanosomes can become depressed in

their capacity to mount antibody responses against viral and bacterial vaccines

CMorrison et al., 1985). However, malfunction of the immune system would, in

most instances, appear to be less severe than in mice. During the early stages

of infection, lymphoid organ architecture remains intact although lymphoid

organs can become depleted of cells in chronic infections (Murray et al., 1980).

An exception has been noted in highly susceptible Ayrshire cattle, in which

following infection, parasitaemia quickly reaches a high level, antibody

responses are low and transient, remission of parasitaemia is absent (Fig. i)

and splenic architecture becomes rapidly disorganised.

Microscopic channels, membrane antigens and cell secretory products which

are responsible for order in lymphoid organs, together with intracellular trans-

port mechanisms which permit Ig secretion, may be vulnerable to the same kinds

of factors which can reduce the life span of erythrocytes in trypanosome-infec-

ted susceptible animals, e.g., short-lived enzymes and enzyme substrate reaction

products. This possibility lends force to the argument that many of the

pathological features of trypanosomiasis may result from the release of break-

down products from degenerating trypanosomes. However, trypanosome-induced

disruption of lymphoid organs and inhibition of Ig secretion is greatest in the

most susceptible animals. This suggests that substrate polymorphisms or the

level of plasma-blocking factors may contribute to the control of parasitaemia

and the degree of pathology which develops.

179

THE WAY AHEAD

Today

Trypanotolerant breeds of cattle are a well recognised component of live-

stock production in many areas of West and Central Africa, but in the 37

countries where tsetse occur they represent only about 5 per cent (8 of 147

million) of the total cattle population (ILCA, 1979). Failure to exploit these

breeds might be attributed to several factors. It has generally been assumed

that, because of their small size, they were unproductive. Moreover, it was

believed that their trypanotolerance was limited purely to local trypanosome

populations and would break down if they were moved to different tsetse-infested

locations. As a result, there was no initiative to revitalise these breeds

after they were decimated by the rinderpest pandemic of the late 19th century,

when the N'Dama, which are particularly susceptible to rinderpest, were very

severely affected (Murray et al., 1982).

However, more recent investigations have not substantiated the premises of

low productivity and local trypanotolerance. Firstly, it has now been estab-

lisLed that the productivity of trypanotolerant cattle relative to other

indigenous breeds is much higher than previously believed in areas of low tsetse

challenge or where tsetse don't exist (ILCA, 1979). Directly comparable data

between breeds are not available in many areas because the level of tsetse

challenge is such that breeds other than trypanotolerant ones cannot survive.

Secondly, it has now been confirmed that trypanotolerance is an innate charac-

teristic and is not solely due to resistance to local trypanosome populations

(Murray et al., 1982).

As a result, African governments and International Agencies are now

recognising that trypanotolerant breeds of cattle, in particular the N'Dama,

have immediate potential for utilising tsetse-infested humid and semi-humid

regions to meet the increasing demand for food in Africa.

F u t u r e

A major r e s e a r c h p r i o r i t y i s t o i d e n t i f y t h e mechanisms which r e g u l a t e

p a r a s i t e growth and a l low t h e development o f an e f f e c t i v e immune r e s p o n s e . In

a d d i t i o n , t h e i s o l a t i o n and c h a r a c t e r i s a t i o n o f t r y p a n o s o m e - d e r i v e d f a c t o r s

t h a t cou ld be r e s p o n s i b l e f o r t h e i n d u c t i o n o f anaemia and d e f e c t i v e immune

r e s p o n s e s t o g e t h e r w i th e l u c i d a t i o n o f t h e f a c t o r s t h a t a l low c e r t a i n a n ima l s to

r e s i s t t h e s e p a t h o g e n i c f e a t u r e s m igh t a l s o p r o v i d e new s o l u t i o n s . I t i s p o s s i -

b l e to e n v i s a g e i n c r e a s i n g r e s i s t a n c e by b l o c k i n g t h e a c t i v i t y o f the p u t a t i v e

t rypanosome g r o w t h - p r o m o t i n g f a c t o r o r i n h i b i t i n g t he i n d u c t i o n o f anaemia or

d e f e c t i v e immune r e s p o n s e s by i m m u n i s a t i o n or by s p e c i f i c drug t r e a t m e n t ,

Such s t u d i e s migh t a l s o p r o v i d e m a r k e r ( s ) o f r e s i s t a n c e t h a t would a l low

s e l e c t i o n o f b r e e d i n g s t o c k w i t h o u t h a v i n g to i n f e c t t h e a n i m a l s . R e s i s t a n c e

180

appears to be inherited as an incompletely dominant trait which is not linked

to the MHC type in mice or to any known MHC type or red cell antigen in cattle

(Murray et al., 1982). With the identification of the genes which control

parasitaemia and anaemia, it might he possible to isolate and transfer them

into the germ lines of appropriate recipients. That such an approach might be

feasible has been demonstrated recently by the identification and isolation of

growth genes and their successful transfection from one species to another

(Palmiter et al., 1982). Thus, it is now possible to envisage transfection of

genes selected for specific functions, such as trypanotolerance, growth or milk

production, with a view to producing highly productive highly resistant cattle.

In conclusion, it would appear that, through a process of rigorous natural

selection in the face of tsetse challenge, Africa has generated within the

overall cattle population a group of animals capable of making a significant

contribution towards alleviation of the continent's food problems. Study of

these cattle, together with wild Bovidae and mice, is providing insights into

the mechanisms by which more susceptible hosts might be induced to live

harmoniously with trypanosomes.

ACKNOWLEDGEMENTS

We thank members of staff at ILRAD and Dr. J. C. M. Trail of the

International Livestock Centre for Africa (ILCA) for their advice and criticism.

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ILRAD PUBLICATION NO. 280.