GENETIC RESISTANCE TO PARASITIC DISEASES AFRICAN TRYPANOSOMIASIS

MAX MURRAY, SCOTLAND, J.C.M. TRAIL*, and S.J. BLACK**, KENYA

Glasgow University Veterinary School, Bearsden Road, Bearsden, Glasgow G61 1QH, Scotland

international Livestock Centre for Africa (ILCA), P.O. Box 46847, Nairobi, Kenya

**International Laboratory for Research on Animal Diseases (ILRAD), P.O. Box 30709, Nairobi, Kenya.

SUMMARYIncreasing attention is being given to the use of cattle

that are innately resistant to trypanosomiasis (a trait termed trypanotolerance) in livestock development projects in tsetse- infested areas of Africa. At the same time, research is being carried out into the factors associated with innate resistance. These include the capacity to control trypanosome growth and the development of effective immune responses. It would appear that the rate of parasite multiplication is regulated by the availability of host-derived growth factors, while the rate of parasite destruction is controlled by the efficiency of the host antibody response. Host antibody production, in turn, is at least partly controlled by a parasite-induced response which influences the final stages of plasma cell maturation and antibody secretion. 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 which are resistant to trypanosomiasis.

INTRODUCTIONVast humid and semi-humid areas of Africa are held captive

by tsetse flies and the trypanosomes they transmit to animals and also to man. With the rapid growth of the human population, there is mounting pressure on tsetse-free pastures and farmlands for increased production. Many of these tsetse-free areas are in the drier regions of Africa where the ecology is fragile and where problems of overgrazing emphasise the need to bring into full pro­duction the more favourable agricultural areas currently underutil­ised due to tsetse infestation. Several factors contribute to the magnitude of the trypanosomiasis problem in Africa, one of the most important relating to the methods currently available for control. Because of the phenomenon of antigenic variation exhibited by each of the three species of trypanosome that cause the disease in domestic livestock, namely, Trypanosoma congolense, T.vivax and T.brucei, no vaccine is available for field use. Thus, control is dependent on the limited number of trypanocidal drugs that are available or on the reduction of tsetse populations by means of residual or non-residual insecticides. Both tsetse control

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(MacLennan, 1980) and trypanocidal drugs (Trail et al., 1985) can be highly effective if properly applied, but because of lack of trained manpower, the cost of implementation, as well as the massive area involved, the net effect at the continental level is small and there is an urgent need to improve current methods of control and to develop new ones.

As reported at the Congress in Madrid (Murray & Trail, 1982), increasing consideration is now being given to the use of trypano- tolerant breeds of domestic livestock in tsetse-infested areas of Africa. Trypanotolerance, i.e., the ability to survive and to be productive in tsetse-infested areas, without trypanocidal drug treatment or with reduced requirement for treatment, is recognised mainly in the indigenous taurine breeds of cattle in West and Central Africa, namely the N'Dama and West African Shorthorn.While there is also evidence that significant differences in resis­tance to trypanosomiasis occur among various Box indicus types (Cunningham, 1966; Monirei et al., 1982; Njogu et al., 1985), most Box indicus types in tsetse-infested areas require regular treat­ment or are found only on the fringes of fly belts. Imported breeds cannot be maintained even in areas of low tsetse risk without intensive drug therapy.

CURRENT USE OF TRYPANOTOLERANT CATTLETrypanotolerant breeds of cattle form an important component

of livestock production and of the socio-economic infrastructure in many tsetse—infested areas of West and Central Africa, but in Africa as a whole they represent only about 5% (8 of 147 million) of the total cattle population in the 37 countries where tsetse occur (ILCA, 1979). Failure to use these breeds more extensively can be attributed to the assumption that, because of their small size, they were not productive and to the view that their "tolerance" was locally acquired. However, these premises have not been substan­tiated by more recent investigations (reviewed at the previous Congress by Murray & Trail, 1982) which have established that trypanotolerant breeds are more productive than previously believed and that trypanotolerance is a true innate characteristic. With this knowledge, African Governments and International Agencies are now recognising that trypanotolerant breeds of cattle have immediate potential for utilising tsetse-infested humid and semi-humid regions to meet the increasing demand for food in Africa. An example of this has been the establishment in 1985 of the International Trypanotolerance Centre in The Gambia.

However, it must be emphasised that trypanotolerance is not a stable characteristic and that its degree varies between individ­uals as well as breeds and that it can be affected by a variety of environmental factors, including the degree of trypanosomiasis risk and the type of management (Murray & Trail, 1982). Therefore, in order to realise the full potential of trypanotolerant breeds of cattle, it is necessary to identify and quantify the environmental factors which influence the stability of the trait, e.g., previous exposure, age, degree of tsetse challenge, the stress of pregnancy, lactation, work, poor nutrition, intercurrent disease, etc., so

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that the appropriate management measures can be instituted. In addition, breeding policies aimed at improving production and re­sistance are needed. The increases in output of food and fibre per animal that have been achieved through breeding in high performance livestock have come from the application of techniques defined in the 1930's and 1940's and are based on careful performance record­ing routines. In order to realise the foregoing objectives the International Livestock Centre for Africa (ILCA) and the Internat­ional Laboratory for Research on Animal Diseases (ILRAD) are co­ordinating a network of national epidemiological and productivity studies on trypanotolerant livestock in nine countries in West and Central Africa. Future breeding policies will be aided by the knowledge that frozen embryos can be transferred successfully from N'Dama to Boran cattle (Jordt et al., in press).

FROM THE FIELD TO THE LABORATORYIn searching for novel methods to control trypanosomiasis,

we believe that an important approach could be to identify the mechanisms underlying increased resistance to trypanosomiasis, in addition to improving trypanotolerant breeds with management tech­nology that is currently available. Resistance to trypanosomiasis is associated with two inter-related processes, firstly, the rate at which parasites switch from dividing to non-dividing forms, an event probably connected with the superior capacity of more resis­tant animals to control the height of parasitaemia and, secondly, antibody responses which cause parasite elimination and which are greater in more resistant animals (Murray & Trail, 1982; Shapiro & Murray, 1982; Murray & Black, 1985; Akol et al., in press). There is also evidence that cattle belonging to more resistant breeds acquire resistance to infection more quickly (Desowitz, 1959;Murray & Trail, in press). While many of the experiments described in the following review were conducted in genetically homogeneous strains of mice, the findings in most cases corresponded to those observed in the more limited studies that were possible in cattle.Regulation of parasite growth

In cattle, T .congolense, T.vivax 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 parasit- aemia reached reflects the duration of the growth phase and can vary between breeds and individuals infected with parasites derived from the same clone. In the case of T.brucei, 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.brucei parasite. Exponential phase trypanosomes are slender, 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, have few or no members in the S, G2 and M stages of the cell cycle (Shapiro et al., 1984), are relatively non-infective for mammals,

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move towards aerobic pathways of carbohydrate metabolism and, have a high capacity to be cyclically-transmitted by tsetse. The studies suqqest 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 T.vivax (Shapiro et al., 1984) and T.congolense (Nantulya et al., 1978) suggest that these parasites might have a similar 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 splenectomised mice, although anti­body responses are retarded in the latter (Black et al■, 1985). Parasite differentiation also occurs at the same rate in lethally- irradiated, and lethally-irradiated and spleen cell reconstituted mice but only the latter produce antibody.

Parasite population growth rates are faster and differen­tiation rates slower in susceptible (C3H/He) as compared to resis­tant mice (C57B1/6) infected with T.brucei (Black et al., 1983a). Reciprocal experiments in cattle show that T.brucei parasites differentiate more rapidly to stumpy forms in more resistant animals and breeds. Similarly, in wild Bovidae which limit T.brucei parasitaemia, parasites, when they become detectable in the blood, are almost always stumpy forms. These findings indicate that control of T.brucei parasitaemia in vivo 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 aqainst T.brucei are stimulated by fragments derived from senescent stumpy form organisms but not by actively dividing parasites (Sendashonga & Black, 1982) .

A number of other studies indicate that the growth of T.brucei 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 (non-infective for humans) but not T .rhodesiense (human infective) parasites (Rifkin, 1978); atpresent, this is the only in vitro method available to distinguish between these organisms. On the other hand, T.brucei 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., 1983b). 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.brucei populations are more likely to be con­trolled by the availability of host-derived molecules which stimu­late parasite multiplication (see later). The rate of parasite

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differentiation in any given host is not, however, immutable.fsrentiation of T .brucei in mice can be accelerated by daily in­

jection of indomethacin or acetylsalicylic acid (Jack et al., 1984). These drugs are unlikely to exert their effect via inhibition of prostaglandin synthesis because we have found that other more spec­ific blockers of prostaglandin synthetase, e.g., flurbiprofen and caprofen have no effect on parasite differentiation. Cyclic 3', 5' -adenosine monophosphate (cAMP) could play a pivotal role in the events that trigger differentiation in that intratrypanosomal cAMP varies during parasitaemia (Mancini & Patton, 1981) and theophylline (a cAMP phosphodiesterase inhibitor) blocks differentiation, elevates cAMP levels in the plasma and depresses cAMP levels in trypanosomes (Reed et al., 1985).

Another significant finding is that treatment of mice with various immunomodulators can alter parasite population growth rates (Murray & Morrison, 1979). In particular, the rate of differentia­tion of T.brucei in both intact and lethally-irradiated mice can be accelerated by pre-treatment of the hosts with dead Corynebacterium parvum 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. In addition to controlling T.brucei parasit­aemia, C .parvum has also been shown to limit T.congolense (Murray & Morrison, 1979) and T.vivax parasitaemia, particularly in more re­sistant strains of mice. The effect cannot be transferred between treated and untreated syngeneic partners by spleen cells or serum. Nevertheless, it is possible that the treatment directly or indir­ectly modifies the rate of production of trypanosome growth- stimulating factors by radio-resistant cells.

T.brucei 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., 1985).The molecules are not lipoproteins, lipids or immunoglobulins 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 multiplication.We speculate that similar growth requirements will be found with bloodstream forms of T ■vivax which require serum and fibroblasts to multiply in vitro and T.congolense which require serum and endo­thelial cells. Identification 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, thereby, increase host resistance.Regulation of parasite destruction

While parasite multiplication up to peak levels of parasit­aemia appears to be regulated by the availability of growth factors, it is the host antibody responses that are involved in the actual

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destruction of parasites leading to parasite wave remission (Murray & Urquhart, 1977). These antibody responses are directed against the glycoprotein coat covering the pellicular surface of the para­site (Cross, 1975); this is termed the variable surface glyco­protein (VSG). Studies in mice using T.brucei stocks that give rise to stumpy or degenerating parasites and T.brucei 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 & Black, 1982; Black et al., 1985). 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-immunogenic (Sendashonga & Black, 1982). These 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.brucei are triggered by frag­ments of parasites derived from senescent stumpy form organisms. Furthermore, parasite fragments may play a significant role in stimulating antibody responses against T.congolense and T.vivax, since living parasites of these species do not release VSG in vitro. 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 were further examined in resistant (C57BL/6) and susceptible (C3H/He) mice infected with T.brucei (Black et al.,1983a) or T.vivax (Mahan, 1984). The resistant mice mount rapid antibody responses which cause parasite wave remission, whereas, little or no antibody responses, either parasite specific or by­stander, are detected in the susceptible animals. Nevertheless, in susceptible mice, stumpy forms of T,brucei arise and T-vivax para- sitaemia 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 susceptibility to T.congolense (Murray & Black, 1985; Akol et al., in press). 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 generation of immunostimulatory parasite fragments. The effic­iency of immune responses against the parasites might relate to the intrinsic capacity of the animals to respond to the parasite anti­gens or be associated with other processes 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.brucei (Black et al., 1983a) , T.vivax (Mahan, 1984) and T.congolense (Morrison & Murray, 1985) parasites.

In contrast to the considerable differences in the capacity of C57B1/6 and C3H/He mice to control parasitaemia and to mount556

serologically-detectable antibody responses after infection with T.brucei parasites, both strains of mice developed similar numbers of spleen cells containing cytoplasmic immunoglobulin (Ig) (Table 1). Furthermore, isolation and disruption of the spleen cells from both strains of mice led to the release of similar quantities of IgM and IgG antibodies which reacted with exposed-VSG-epitopes on the infecting parasites.TABLE 1 B-cell responses in C57B1/6 and C3H/He mice infected with

Trypanosoma brucei

MouseStrain

Day after infection i£>gioparasites/ml

Log-, q reciprocal antibody titre in serum*

% cytoplasmic Ig+ spleen cells

Log2 reciprocal antibody titre from lysed spleen cells*

C57B1/6 1 0 0 0.4 05 7.3 0.5 1.2 0.57 7.6 1.4 16.5 2.88 0 2.5 18.2 5.29 0 3.7 17.4 4.6

C3H/'He 1 0 0 0.2 05 7.5 0 0.8 0.87 8.0 0 17.8 3.08 8.2 0 16.3 3.89 8.1 0 15.3 3.9

* Only _IgM is shown: similar titre of IgG occurred.Estimations were made by solid-phase radio-immunoassay.

Thus, it would appear that the antibody-containing plasma cells in infected C3H/He mice had an impaired capacity to secrete Ig. Furthermore, when mice of the C3H/He and C57B1/6 strains which had been infected with T.brucei parasites for 8 days were inoculated intravenously with L - 3 5 S-methionine, immuno-precipitat- ion analysis conducted on serum samples collected 2 hours later showed that the C3H/He mice produced less labelled serum IgM and IgG than the C57B1/6 mice. Similarly, spleen cells from 8 day in­fected C3H/He mice^produced less labelled IgM and IgG in vitro when incubated with L-35 s-methionine than spleen cells from 8 day in­fected C57B1/6 mice. Nevertheless, the amounts of radiolabel incorporated into cell-associated material which could be precipi­tated with trichloroacetic acid were similar in cultured spleen cells from both strains of mice. Under the same conditions, spleen cells from uninfected C57B1/6 and C3H/He mice were found to secrete similar amounts of labelled Ig.

Electron microscopical studies showed that many of the plasma cells in infected mice exhibited an unusual intracellular morphology. In normal antibody-secreting plasma cells a regular array of flattened sacs lined by rough-surfaced endoplasmic retic­ulum (RSER) occurs. However, particularly in the highly suscept-

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ible C3H/He mice infected with T.brucei, a number of plasma cells contained irregular and distended sacs lined by RSER. Cells with this phenotype are believed to be transition stages en route to Russell body-containing plasma cells, cells frequently found in human beings (Mott, 1907) and domestic livestock (Murray et al., 1980) infected with African trypanosomiasis. While a statistical analysis of the splenic plasma cell populations has not been con­ducted as yet, a correlation is proposed between the unusual plasma cell morphology and the rate of production of Ig.

Treatment of C3H/He mice during the first 9 days after in­fection with the trypanocidal drug diminazene aceturate (Berenil, Hoechst) resulted in rapid, serologically-detectable, antibody responses against T.brucei. From this it would appear that the maintenance of non - or low rate Ig-secreting cells in infected C3H/He required the continuous presence of living T.brucei parasites or perhaps, short-lived factors from degenerating organisms.Similar results have been obtained in highly susceptible mice in­fected with T.vivax. The identification of the parasite and host components which interact and prevent or reduce Ig secretion by plasma cells in trypanosome-infected C3H/He mice could be central to the understanding of host resistance to trypanosomiasis and could permit the development of strategies to improve antibody re­sponses in host species that are highly susceptible to African trypanosomiasis.

CONCLUSIONSWith the knowledge that trypanotolerant breeds of domestic

livestock can be productive and that their resistance to trypano­somiasis is an innate characteristic, there is now considerable confidence that these animals are capable of making a contribution towards alleviating Africa's food problem. Furthermore, a major research priority is to identify the mechanisms which regulate parasite growth and allow the development of an effective immune response. It is possible to envisage increasing resistance by blocking the action of the putative trypanosome growth-promoting factor or inhibiting the induction of defective immune responses by immunisation or by specific drug treatment.

Such studies might also provide marker(s) for resistance that would allow selection of breeding stock without having to infect the animals. Resistance appears to be'inherited as an in­complete dominant trait which is not linked to the MHC type in mice or to any known MHC type or red cell antigen in cattle (reviewed by Murray et al., 1982 ; Greenblatt et al. , 1984). In the same way, identification of the mechanisms responsible for genetic resistance to trypanosomiasis might lead to the isolation of appropriate genes for transfection, a procedure that is now tech­nically feasible in mammals (Palmiter et al., 1982) and is likely to revolutionise livestock development.

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