Genetic Resistance to African Trypanosomiasis

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1 THE JOURNAL OF INFECTIOUS DISEASES VOL. 149, NO. 3 MARCH by The University of Chicago. All rights reserved /84/ $0100 Genetic Resistance to African Trypanosomiasis Max Murray, J. C. M. Trail, C. E. Davis, and S. J. Black From the International Laboratory for Research on Animal Diseases and the International Livestock Centre for Africa, Nairobi, Kenya; and the University of California Medical Center, San Diego, California Genetic resistance to African trypanosomiasis occurs in certain breeds of livestock, many species of wildlife, some strains of mice, and possibly humans. The term trypanotolerance is used to describe this trait, which in domestic livestock is best exhibited by the indigenous West African taurine breeds of cattle, N'Darna and West African Shorthorn, that have been in Africa for 5,000 to 7,000 years. Confirmation not only that these breeds are genetically resistant to trypanosomiasis but also that they are at least as productive as other indigenous breeds is currently leading to their increased use in livestock-development projects in tsetse-infested areas. Trypanotolerance appears to be related to the control of parasitemia, a capacity associated with an event that regulates parasite growth and determines how rapidly the immune response is triggered. Identification of the factors that regulate this event and definition of their genetic basis may have important implications for the development of novel strategies for control of African trypanosomiasis. Genetic resistance to infectious diseases is being given increasing attention in livestock-development programs. This is particularly the case in developing countries, where conventional diseasecontrol measures do not exist or cannot be implemented because of a lack of finance or of trained manpower. These circumstances are exemplified by tsetse-transmitted African trypanosomiasis (sleeping sickness). At present, tsetse infest rvten million km 2 of Africa, affecting 38 countries [1]. It is estimated that seven million km 2 of this area would otherwise be suitable for livestock or mixed agricultural development. Currently, rv30070 of the 147 million cattle in countries that are affected by tsetse are exposed to the disease [1]. The extent of exposure to the disease with regard to other domesticated species including sheep, goats, pigs, horses, donkeys, and camels is probably similar, but is less well documented. Human trypanosomiasis occurs in a tsetse-infested belt of some four million krrr', with rv45 million persons at risk [2]. Together with onchocerciasis (river blindness), human trypanosomiasis is a major cause of depopulation in large areas of Africa. There are many factors that contribute to the This address was presented at the 21st annual meeting of the Infectious Diseases Society of America, held in Las Vegas, Nevada, on October 27 and 28, Please address requests for reprints to Dr Max Murray, International Laboratory for Research on Animal Diseases, P. O. Box 30709, Nairobi, Kenya. magnitude of this disease problem in Africa, possibly the most important relating to the methods that are currently available for control. Because of the phenomenon of antigenic variation exhibited by each of the species that causes the disease in domestic livestock (Trypanosoma congolense, Trypanosoma vivax, and Trypanosoma bruceii and in humans (Trypanosoma rhodesiense and Trypanosoma gambiense), no vaccine is available for field use. At present, control methods involve either tsetse control with residual or nonresidual insecticides or the use of trypanocidal drugs. However, the number of effective drugs available both for humans and domestic livestock is extremely limited, and because of this the possibility of the development of widespread drug resistance looms large. Although these control measures can be highly effective in certain situations if properly applied, their net impact at the continental level is small. As a result of this situation, major efforts are being made by many laboratories throughout the world to develop new or alternative methods for the control of trypanosomiasis. At the International Laboratory for Research on Animal Diseases (llrad), we have been paying particular attention to genetic resistance to trypanosomiasis as a possible solution to the problem. It has long been recognized that certain breeds of domestic livestock, as well as many species of wildlife, possess the ability to survive and be productive in tsetseinfested areas without the aid of treatment with 311

2 312 Murray et al these beliefs have not been substantiated by more recent investigations. The present report reviews the evidence for the genetic basis of resistance to African trypanosomiasis, evaluates the productivity of trypanotolerant breeds, and discusses the possible underlying mechanisms of this trait. Figure 1. N'Dama (Bas taurus) bull at a ranch in The Gambia, West Africa. trypanocidal drugs, whereas other breeds rapidly succumb to the disease [3]. This trait, which has been termed trypanotolerance, is generally attributed to the taurine (humpless) breeds of cattle in West and Central Africa, the N'Dama (figure 1) and the West African Shorthorn. It is thought, on the basis of rock paintings and engravings, that the taurine Hamitic longhorn breed, from which the N'Dama as well as the Texas Longhorn breeds are descended, arrived in the Nile Delta from the Near East at about 5,000 B.C., while the taurine Shorthorn cattle were introduced into the same area between 2,750 and 2,500 B.C. [4]. On the other hand, Bos indicus (humped) cattle (figure 2), which are the most prevalent type of indigenous cattle in Africa and are regarded as highly susceptible to trypanosomiasis, did not become numerous in Africa until after the Arab invasion (A.D. 669), although they were recognized in Egypt between 2,000 and 1,500 B.C. Despite the fact that the capacity of N'Dama and West African Shorthorn cattle to survive in tsetse-infested areas is well established, these breeds represent only about eight million (5OJo) of the total cattle population of 147 million in the 38 countries where tsetse occur [3]. Failure to exploit these breeds is the result of two main factors. First, it was thought that their trypanotolerance was not innate but was limited to local trypanosome populations, and that their "tolerance" would therefore break down if they were moved to distant tsetseinfested locations where different trypanosome strains existed. Second, it was generally assumed that because they were physically smaller than other breeds, they were unproductive. However, Evidence in Livestock for Genetic Resistance to Trypanosomiasis Cattle. In an early account of West African livestock, Pierre [5] recorded the ability of certain cattle to survive in tsetse-infested areas. Subsequently, in both field investigations and experimental studies the resistance of the N'Dama and West African Shorthorn breeds was increasingly recognized, although these early reports could not always evaluate the relative contribution of innate and acquired resistance, since the history of the animals was not known [6]. More recently, however, it has been clearly demonstrated that interbreed differences in resistance to trypanosomiasis reflect differences in innate characteristics. Studies using animals that had never been previously exposed to trypanosomes confirmed that N'Dama cattle were significantly more resistant than were Zebu cattle to experimental challenge with wild-caught tsetse [6, 7], to exposure in the field [8, 9], and to inoculation of trypanosomes with a syringe [10, 11]. These results were assessed in terms of both productivity and survival of the cattle. The difference in susceptibility between the two breeds was demonstrated by the fact that the prevalence, level, and duration of parasitemia were significantly less in the N'Dama Figure 2. Kenya. Boran (Bas indicus) cow at ILRAD, Nairobi,

3 Resistance to African Trypanosomiasis 313 en w ;l;i 0 en 0 z<q. > IX: ~ < :i w < ~ iii < IX: <Q. --' --' w o~ N'DAMA ~~ --,- ~ ZEBU ~ o- w IX: ~ 0 3 w :ol: 0 o > <Q II I I II I WE EKS OF CHALLENGE Figure 3. Comparison of 10 N'Dama and 10Zebu cattle after exposure to a natural challenge of Glossina morsitanssubmorsitans tsetse flies. TOPt mortalities of N'Dama and Zebu cattle. The single N'Dama death was attributable to anthrax. Middle, mean weekly parasitemic scores. Shaded bars represent N'Dama cattle; white bars represent Zebu cattle. The level of the first peak of parasitemia + 1 SO is shown for N'Darna (e) and Zebu (0) cattle. Bottom, packed red-cell volume values for N'Darna (e) and Zebu (0) cattle are given. (Reprinted with permission from Baker et al [12].) than in the Zebu cattle. Correspondingly, the N'Dama breed developed anemia that was less severe than that seen in the Zebu (B indicus) breed (figure 3). Further evidence that trypanotolerance has a genetic basis and is not based merely on resistance acquired to local trypanosome populations has been provided by the successful establishment of trypanotolerant cattle from West Africa in distant tsetse-infested areas of West and Central Africa, where other breeds could previously not survive; for example, the introduction of West African Shorthorn (1904) and N'Dama cattle (1920) into Zaire (formerly Belgian Congo) and more recently, N'Dama cattle into the Central African Republic, Gabon, and the People's Republic of the Congo [3]. Trypanotolerant breeds of cattle may also be resistant to several other important infectious diseases. N'Dama and West African Shorthorn cattle have been found to be resistant to streptothricosis [13, 14] and are thought to be more resistant to helminthiasis than are other breeds [12]; N'Dama cattle have also been reported to be more resistant to tick-borne diseases, including heartwater (Cowdria ruminantiumi. anaplasmosis, and babesiosis [4]. The view that trypanotolerant breeds of cattle, because of their smaller size, are less productive than larger, more susceptible breeds has not been confirmed. A recent survey of the status of trypanotolerant livestock in 18 countries in West and Central Africa examined all the indices of productivity that could be found for various regions, management systems, and levels of tsetse challenge [3]. The survey strongly indicated that in areas of no or low tsetse-challenge, the productivity of trypanotolerant cattle relative to other indigenous breeds was much higher than had previously been assumed. Ranch herds of Zebu cattle were estimated to yield a mean productivity index (total weight of a one-year-old calf plus the liveweight equivalent of milk produced per year per 100 kg of cow body-weight) of 38.6 kg. This compared with 37.1 kg for ranch herds of trypanotolerant cattle in low tsetse-challenge areas; thus, the productivity of Zebu cattle was only 4010 higher than that of the N'Dama and West African cattle. Furthermore, data directly comparable between breeds were not available in many areas because the level of tsetse challenge was such that cattle of other than the trypanotolerant breeds did not survive. It must be emphasized that the level of trypanotolerance can be influenced by several factors that affect the host and its environment. One of the most important factors is the severity of the tsetse challenge to which the animals are exposed. Thus, as the level of challenge rises, productivity falls, and in high-risk situations even N'Dama cattle can be severely affected by trypanosomiasis, as evidenced by stuntedness, emaciation, spontaneous abortion, and even death. Stressors (exertion, pregnancy, parturition, lactation, suckling), intercurrent disease, and poor nutrition have all been identified as additional factors that can reduce the level of trypanotolerance [12]. On the other hand, re-

4 314 Murray et al cent evidence indicates that cattle (of both trypanotolerant and trypanosusceptible breeds) that survive trypanosomiasis with or without the aid of chemotherapy gradually become more resistant to infection [12]. This finding has probably contributed to the widespread belief that the resistance of trypanotolerant breeds is largely the result of immunity acquired to local trypanosome strains and that tolerance.disappears if cattle are moved to distant locations. Although exposure to new trypanosome strains will undoubtedly lead to infection, the superior genetic resistance of the trypanotolerant breeds ensures that their chances of survival and of acquiring resistance in new locations will be significantly greater than those of trypanosusceptible breeds. The possibility that significant genetic resistance to trypanosomiasis might have developed in other regions of Africa and in other breeds of cattle has not been given serious consideration. Most observers believe that if it had it would have been well documented by this time. However, resistance to trypanosomiasis has been reported in local cattle of the Koalib Hills in the Nuba Mountains in Sudan [15]; these cattle had the appearance of West African Shorthorn. In addition, we have found differences in resistance to trypanosomiasis in certain B indicus types in Kenya, and similar results have been reported in Upper Volta [16]. However, since the animals in these studies had all been previously exposed to trypanosomiasis, it is not possible to assess the relative contribution of innate and acquired resistance to their susceptibility status. Although critical comparative studies of interbreed differences in resistance and productivity remain to be carried out, the degree of genetic resistance in such B indicus types is probably considerably less than that in the recognized trypanotolerant breeds. Nevertheless, it appears that the development of B indicus breeds that have a significant degree of resistance is a long-term possibility and could be an important objective in certain situations. Sheep and goats. Although the capacity of indigenous breeds of sheep and goats to survive in tsetse-infested areas is well recognized throughout Africa, experimental evidence that this reflects an innate capacity to control the parasite and resist the effects of the disease is poorly documented. In West Africa it is generally accepted that the indigenous Djallonke sheep and Dwarf West African goats are trypanotolerant; experimental evidence has been presented to substantiate the trypanotolerance of the Djallonke sheep [17]. However, the susceptibility status of Dwarf goats is far from clear; there are reports demonstrating that they can be highly susceptible to experimental infection [12]. There is also evidence that indigenous breeds of sheep and goats in Eastern Africa are more resistant to trypanosomiasis than are imported exotic breeds [12]. We have confirmed this experimentally in the case of the indigenous Red Maasai sheep in Kenya; however, we have not demonstrated any differences in susceptibility between indigenous breeds of goat, such as the East African and imported breeds (author's unpublished observations). Nevertheless, there is no question that indigenous breeds of goats do survive under natural tsetse-challenge. These contradictory results might be explained by the fact that some species of tsetse exhibit definite host-feeding preferences [18]; therefore, in an area where several host species exist, certain animals will be at greater risk of tsetse attack and infection than will others. The question of whether this explanation is adequate for sheep and goats awaits further investigation. Wildlife. It is widely accepted that many species of wildlife are highly resistant to trypanosomiasis - that is, exhibit marked trypanotolerance. Findings in early experimental studies [12] have recently been confirmed in African buffalo (Syncerus caffer) (figure 4), oryxes (Oryx beisa), eland (Taurotragus oryx), and waterbuck (Kobus defassa) [19]. Following syringe inoculation or experimental tsetse-infection with T congolense, T vivax, or T brucei organisms, animals of these four species that had not previously been infected with trypanosomes, all exhibited a marked degree of resistance to trypanosomiasis. This was reflected in low, transient parasitemia and small, temporary reductions in erythrocyte levels. An exception to this picture occurred in waterbuck infected with T brucei organisms; while erythrocyte values decreased only transiently, parasitemia levels remained high, corresponding to those recorded in domestic livestock' that develop anemia. Wild bovidae, which offer a unique opportunity to study the possible mechanisms that allow the host to resist trypanosome infections, are currently being used for this purpose at ILRAD. Humans. In considering the epidemiology of human sleeping sickness, Buyst [20] argued that

5 315 Figure 4. African buffalo (Syncerus caffer) used for experiments at Kenya Veterinary Research Laboratories, Nairobi, Kenya. natural selection of people with an innate ability to cope with the infection might have occurred. Thus, many of the Bantu-speaking people are well adapted to an ecology of light woodlands with medium humidity. Such groups have a history extending over several thousand years of almost uninterrupted contact with tsetse, and presumably human-infective trypanosomes. These people tend to develop a less acute form of the disease, and there are some cases in which spontaneous selfcure is believed to have occurred [21]. In contrast, the disease syndrome in Caucasians is almost invariably very acute [22]. Also, Bantu-speaking people whose ancestors had little or no contact with human-infective trypanosomes for several centuries usually experience a more acute form of the disease, as has occurred in Zambia [21]. Similarly, Nilotic groups, who are adapted to a hot and dry environment, have had little contact with tsetse or human-infective trypanosomes until relatively recently. These groups have exhibited very acute types of sleeping sickness, both in Ethiopia and around Lake Victoria [21]. Mechanisms Underlying Trypanotolerance The greater resistance to trypanosomiasis of trypanotolerant vis a vis trypanosusceptible animals appears to be related to a more effective innate characteristic that controls and reduces levels of parasitemia. This characteristic is usually explained in terms of a superior immune response. Other factors that have been considered to contribute to trypanotolerance include the ability to prevent the development of anemia, reduced attractiveness to tsetse attack, and greater resistance to the effects of infection that is attributable to physiologic characteristics that aid survival. Immune response. Despite the belief that differences between the immune response of trypanotolerant and trypanosusceptible livestock account for the more effective control of parasitemia in trypanotolerant cattle, the published evidence is conflicting. Following renewed challenge in livestock with a history of trypanosomiasis, trypanosomes were eliminated more rapidly in N'Dama than in Zebu cattle [23]. An in vitro test that involved the use of sera from the challenged animals in order to inhibit trypanosome respiration showed that the activity of N'Dama sera was superior to that of Zebu [23]. Similarly, a serum-neutralization test demonstrated that the immune response to trypanosomes was higher in N'Dama than in Zebu cattle [24]. Further evidence that N'Dama cattle possess a more effective immune response was suggested by an association between the greater control of T brucei infections in N'Dama vis avis Zebu cattle, and by the recognition of at least one of the three common trypanosome antigens (lj x los, 1.5 X 10 5, and 3.0 x 10 5 daltons) in N'Dama cattle [25]. On the other hand, we have found no demonstrable differences in specific antibody responses between N'Dama and Zebu cattle after needle inoculation with bloodstream forms of T brucei (author's unpublished observation). Similar observations have been made with trypanotolerant and trypanosusceptible cattle in Upper Volta [16], where it was also shown that there was no difference between the two breeds' in the antibody response to nondividing, irradiated T brucei. Such studies need to be extended in domestic livestock to provide more substantial information on the role of the immune response. In the search for an explanation of the mechanism(s) of trypanotolerance, experimental work has been carried out in inbred, genetically homogenous strains of mice. The experimental approaches used with these mice would not have been possible in genetically heterogenous breeds of domestic livestock. Differences in susceptibility to African trypanosomes have been demonstrated in inbred strains ofmice [12]. As was found in domestic livestock and wildlife, increased resistance and survival were associated with the control and reduction of parasitemia. Studies of F I hybrids and

6 316 Murray et at Figure 5. Giemsa stain ofslender and stumpy forms of T brucei organisms. backcrosses derived from strains of low, intermediate, and high susceptibility indicated that increased resistance was inherited as a dominant trait and was under complex genetic control [26]. Furthermore, results obtained using mice congenic at the major histocompatibility (H-2) complex suggested that resistance was not linked to H-2 haplotype [26, 27]. It has been demonstrated in mice that the rate of differentiation of T brucei from rapidly dividing slender forms to nondividing stumpy forms (figure 5) correlated with the control and reduction of parasitemia in the host [28]. Furthermore, the rate of parasite differentiation influenced the kinetics of antibody production, because antibody responses were stimulated by nondividing stumpy, but not by dividing slender forms of the parasite [28, 29]. It has been shown that the host exerts an important influence on the degree of differentiation exhibited by a given population of T brucei organisms [301. Thus, it was found in mice that two populations of T brucei organisms that were pleomorphic and monomorphic, respectively, were equally pleomorphic when inoculated into cattle [30]. When parasites were recloned back into mice, they reverted to the original phenotypes. These findings suggested that factors regulating differences in the rate of parasite differentiation might playa central role in determining host susceptibil- ity to infection. This was confirmed when parasite differentiation was compared in mouse strains of high and low susceptibility. The lower level of parasitemia displayed by the resistant mice was found to be associated with more rapid parasite differentiation and a superior antibody response [31]. As was found in cattle, no inherent differences in the immune response to irradiated trypanosomes could be demonstrated between strains of mice exhibiting different susceptibility to trypanosome infection [31]. The demonstration of a nondividing, late bloodstream form of T vivax organisms,' together with the report of a similar stage with T congolense organisms [32], indicates that a series of events similar to those described for T brucei organisms might be involved in determining host susceptibility to T vivax and T congolense organisms. As found with T brucei organisms, susceptibility in mice to T vivax and T congolense organisms has been shown to be related to the control of parasitemia and the development of more effective antibody responses. Preliminary studies with T brucei organisms suggest that regulation of parasite growth and differentiation may also be important in influencing the susceptibility of cattle and wild bovidae to infection. Not only are the factors responsible for parasite growth host related, but it is also possible to manipulate them using immunostimulants such as Corynebacterium parvum organisms. When we treated mice with C parvum organisms prior to infection with T brucei, T congolense, or T vivax organisms, trypanosome differentiation (T bruceii occurred earlier, levels of parasitemia were lower and of shorter duration, and mice survived longer. Similar results were obtained when mice infected with T brucei organisms were treated with indomethacin [33], an outcome suggesting that differentiation might be negatively controlled by prostaglandin levels. However, further work is required in this area, because we have found that other inhibitors of prostaglandin synthetase including flurbiprofen, corprufen, and ketoprofen have no such effect. The findings reviewed suggest that genetic resis- I s. Z. Shapiro, J. Naessens, B. Liesegang, S. K. Moloo, and J. Magondu, "Analysis of DNA synthesis during the life cycle of African trypanosomes by flow cytometry," manuscript in preparation.

7 Resistance to African Trypanosomiasis 317 tance to African trypanosomiasis is associated with an event that regulates parasite growth and differentiation and that determines how rapidly the immune response is triggered. Identification of the factor(s) mediating this event may be the key to understanding the basis of genetic resistance. Anemia. One of the major features ofinfected, trypanotolerant animals is that they develop less severe anemia than do those of more susceptible breeds (figure 3). A series of erythrokinetic and ferro kinetic studies on N'Dama and Zebu cattle infected with T congolense [34] and T brucei [35] organisms showed that the severity of anemia and its underlying processes generally reflected the numbers of parasites in the blood. Thus, it appeared that the differences in anemia between N'Dama and Zebu cattle were attributable to differences in the control of parasitemia rather than to differences in innate erythropoietic responses. In contrast, major differences were found in the severity of the anemia between the relatively resistant Red Maasai sheep and the more susceptible Merino, despite similar levels of parasitemia [12]. Also, in wild animals, significant anemia rarely occurs, despite fairly high levels of parasitemia in some species- for example, in the waterbuck infected with T brucei [19]. It might be that the erythrocytes of certain species have a greater capacity than those of others to resist the pathogenic effects of trypanosomes or that resistant hosts are able to carry out a faster and more efficient erythropoietic response. Attractiveness to tsetse. Another explanation for survival in areas infested with tsetse is that certain breeds and species are rarely subjected to tsetse attack. As discussed earlier, this might be the reason why sheep and goats in tsetse-infested areas are only rarely infected and are usually in excellent condition, despite the fact that they (especially goats) are highly susceptible to experimental infection. Blood-meal analysis indicates that tsetse exhibit definite host-feeding preferences that vary among tsetse species [18]. However, preferences are affected by a large number of environmental factors, of which host availability is one of the most important [36]. Recently, significant new observations have been made on the factors that influence host attractiveness to tsetse. Although color, size, and movement have all been considered important, Vale [37] has demonstrated the overriding importance of host odor and the attractant power of carbon dioxide and acetone in bovine breath. Also, cattle showing a loss of condition are less attractive to tsetse, possibly because of changes in the composition of ruminal gases [38]. The identification and characterization of factors that attract, as well as those that repel tsetse [37], may have important implications for the development of novel strategies for the control of African trypanosomiasis. Physiologic characteristics. Trypanotolerance may also reflect a reduced susceptibility to the effects of the infection that is attributable to a number of physiologic characteristics that aid in the survival of trypanotolerant breeds. These factors include a well-developed ability to digest and metabolize food, tolerate heat, and conserve water. Unfortunately, data concerning the effect of these characteristics on trypanotolerance are not available [12]. Conclusions A rigorous process of natural selection over several thousand years has produced in Africa a unique group of animals with characteristics that permit survival in the face of tsetse challenge. The use of these animals can make a significant contribution toward the alleviation of Africa's food problems by allowing more effective development of livestock and mixed agriculture in the continent's humid and semihumid, tsetse-infested regions. This conclusion is now widely accepted, with the result that N'Dama heifers and bulls are being imported by several countries in West and Central Africa to form the nucleus of livestock-development programs in tsetse-infested areas. In addition, studies on the possibility of N'Dama embryo transfer are currently being carried out by ILRAD. If successful, such an approach would allow the rapid multiplication and dissemination of the best quality N'Dama cattle. A better understanding of genetic resistance to trypanosomiasis, especially of the factors that regulate parasitegrowth and allow the development of an effective immune response, could also produce new methods of controlling the disease. For example, techniques might be derived for enhancing resistance to trypanosomiasis through regulation of parasite growth by the use of either suitable drugs or recombinant-dna technology if the appropriate genes could be identified. Also, genetic

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