Problems of current control methods
Towards development of a vaccine
ILRAD's research approaches to improved trypanosomiasis control
Diagnosing Infections with Monoclonal Antibodies that Detect Parasite Antigens
Diagnosing Infections with Nucleic Acid Probes that Detect Parasite DNA
Drug Resistance in Trypanosomes
Cultivating Trypanosomes in the Laboratory
The Process of Endocytosis in Trypanosomes
Molecular Genetics of Trypanosomes
Genetic Control of the Differentiation Process
Host immune responses to infection
Pathogenesis in Trypanosomiasis
Genetics of host resistance to trypanosomiasis
Mapping the genes associated with trypanotolerance
Trypanosomiasis, which occurs across more than a third of Africa, is arguably the most important livestock disease on the continent. Like East Coast fever, trypanosomiasis is caused by a protozoan parasite (Figure 14). In Africa, most trypanosome parasites are transmitted by tsetse flies. The parasites infect a variety of domestic and wild animals as well as humans. The wide occurrence of this disease in people and their livestock is a great constraint to agricultural and economic development and the consequent improvement of people's lives.
Figure 14. Light micrograph of trypanosomes, protozoan parasites that cause animal trypanosomiasis, in mammalian blood.
The tsetse fly (Glossina) vector of trypanosomiasis occurs only in Africa (Figure 15). Tsetse transmit two species of trypanosomes.Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense.that cause trypanosomiasis in people, known as sleeping sickness, and four species.Trypanosoma brucei brucei, Trypanosoma congolense, Trypanosoma simiae and Trypanosoma vivax. that cause serious forms of trypanosomiasis in cattle, sheep, goats, pigs and horses.
Figure 15. A tsetse fly feeding on an artificial membrane at ILRAD. Africa is the only continent in which this fly. recognized by a long proboscis and the habit of resting with its wings folded scissor-like over its back. occurs. The tsetse fly inhabits over a third of the continent, where it exposes some 30% of Africa's 150 million cattle to the risk of infection with trypanosomes, which give rise to the debilitating and frequently fatal disease trypanosomiasis.
In parts of Africa and Asia, where camels are an important resource, these animals also suffer from trypanosormasis. The camel disease is most commonly caused by Trypanosoma evansi, a species not transmitted by tsetse flies but by other biting flies. Increasing evidence suggests that in large areas of Asia and South America, Trypanosoma evansi poses a threat to other livestock as well, especially cattle, domestic buffalo and pigs. Trypanosoma vivax, although usually transmitted by the tsetse fly, also exists in the absence of this vector in the Caribbean and South America. The extent of the problem of non-tsetse-transmitted trypanosomiasis is not clear, but it is potentially a serious threat to livestock in many parts of the world.
Tsetse flies occur in 37 countries of Africa, an area extending over 10 million square kilometres (Figure 16). The risk of trypanosomiasis in much of this area precludes farmers from keeping cattle and small ruminants, and this fact largely accounts for Africa's low livestock productivity: the continent produces one-seventieth of the animal protein per hectare that Europe produces. The impact of trypanosomiasis is even greater than these figures suggest because many of the areas inhabited by tsetse flies are potentially the most agriculturally productive in Africa. Thirty percent of Africa's cattle population, which totals 147 million, as well as comparable numbers of small ruminants, are at risk from the disease. Annual losses in meat production alone are estimated at US$5 billion. This economic deprivation is exacerbated by losses in milk yields, tractive power, waste products that provide natural fuel and fertilizer, and secondary products such as hides. In addition, 50 million people are currently exposed to the risk of contracting human trypanosomiasis.
Figure 16. The tsetse fly and the trypanosome parasites it carries together make some 10 million square miles of potential grazing land in Africa inhospitable for livestock. Note that little overlap exists between Africa's cattle-production and tsetse-infested areas except in regions of West and Central Africa where a few indigenous cattle breeds exist that are able to tolerate trypanosome infection. Tsetse-infested areas are reported to be expanding in many parts of tropical Africa.
The trypanosomes that cause disease in livestock and humans also infect some wildlife species, which serve as a source, or reservoir, of infection for flies that may then in turn infect domestic animals and people. Many wild animals carry trypanosomes with no apparent ill effects (Figure 17). In humans and most domestic livestock, however, where such a harmless relationship with trypanosomes and their vectors has not evolved, the pathogenic effects of infection are severe.
Figure 17. The African Cape buffalo, which, like several other species of African wildlife, can be infected with trypanosomes while manifesting no clinical disease. Infected wild animals are a source of infection for flies that may in turn infect domestic animals and people.
For several days following infection with trypanosomes, animals show no signs of disease. One to two weeks later, susceptible animals develop intermittent fever and anaemia. In most endemic areas of Africa, cattle are repeatedly bitten by tsetse flies carrying different kinds of trypanosomes. In addition, in most areas livestock must daily forage for food and walk long distances for water. Under these stressful conditions, infected animals often continue to deteriorate for months before dying.
The life cycle of the single-celled trypanosome parasite is complex. In both the tsetse fly vector and the mammalian host, trypanosomes undergo a series of transformations into different forms (Figure 18). The tsetse fly ingests trypanosomes when it feeds on an animal infected by the parasite. In the fly, the trypanosomes differentiate into several forms, culminating the metacyclic form, which is able to infect mammalian hosts. When the infected fly next feeds, these metacyclic trypanosomes are injected into the skin along with tsetse saliva. In the animal, the parasites differentiate into a bloodstream form specially adapted to live in mammalian blood. The bloodstream parasites multiply by binary fission and enter the animal's lymphatic and blood circulation. As flies feed on animals infected with the parasite, they take up blood containing trypanosomes, which then completes the life cycle.
Figure 18. The life cycle of Trypanosoma brucei. Infection begins when trypanosomes are injected into the blood of a mammal by a tsetse fly as it feeds on the animal. In the animal host, slender forms of the parasites (a) multiply by binary fission until large parasite numbers build up in the blood. The trypanosomes then transform first into intermediate forms (b) and then into stumpy forms (c), the latter of which are able to infect tsetse flies.
Stumpy forms of the parasites are ingested by a tsetse fly as it feeds on an infected animal. In the midgut of the tsetse fly, procyclic forms (d) arise and undergo division, after which the parasites enter the proventriculus and later the salivary glands of the fly, where they assume epimastigote forms (e) and undergo further division. Finally, metacyclic forms (f) arise in the salivary glands. Metacyclics are able to infect mammals, and the life cycle is repeated.
Note that the mitochondrion is inactive in the slender forms, begins to become active in the stumpy forms and is fully active in forms that occur in the tsetse fly. Parasite forms that live in the mammalian bloodstream (slender, intermediate and stumpy) have a glycoprotein surface coat, shown here in blue. This surface coat of the parasites disappears in the procyclic forms that arise in the midgut of the tsetse fly and is later reformed in the metacyclic forms in the tsetse salivary glands. (After Vickerman, 1979.)
Three methods are currently used to control trypanosomiasis: administration of drugs to treat or prevent the disease, use of methods to reduce populations of the flies that transmit the disease, and keeping indigenous livestock breeds that are resistant to the disease. To date, each of these methods, while remaining very useful, has drawbacks.
Only a few drug compounds have been developed for treating trypanosomiasis and those that are available may produce unpleasant or fatal side-effects. Furthermore, all trypanocidal compounds now available have been widely used for many years, leading to the development of parasite resistance to the drugs. There are no signs that new and improved drugs will be produced in the near future.
The methods used to control populations of the tsetse vector include spraying areas with insecticides to kill the flies, clearing bush hospitable to the fly and killing flies with targets and traps. Although the tsetse fly is apparently sensitive to low levels of modern insecticides, application of such compounds over large areas is environmentally damaging. The felling of forests and clearance of bush inhabited by tsetse flies effectively rids areas of tsetse, but such destruction of diminishing natural resources is unacceptable in most areas. The targets and traps developed over the last several decades to kill tsetse have been demonstrated to reduce fly populations to low levels but only in some types of areas and only for some species of tsetse. Moreover, to be efficacious this control method demands regular target and trap maintenance and the active participation of livestock-keeping communities where the traps are deployed.
Another control method for trypanosomiasis relies on use of N'Dama (Bos taurus) and other cattle breeds indigenous in West Africa that are able to tolerate infection with trypanosomes (Figure 19). This genetic ability to resist the pathogenic effects of infection is called 'trypanotolerance'. (Livestock that are susceptible to trypanosomiasis are called 'trypanosusceptible' or, in this report, `susceptible').
Figure 19. Some of the N'Dama (Bos taurus) cattle transferred to ILRAD from the Gambia as frozen embryos and born in 1984. Indigenous to West Africa, N'Dama cattle tolerate infections with trypanosomes. Studies on the immune responses of these cattle to trypanosome infection are an important part of ILRAD's research into improving immune responses in cattle breeds such as the Boran that are susceptible to trypanosomiasis.
It is believed that mechanisms to resist the disease evolved in trypanotolerant breeds because of long exposure to the parasite: these cattle were probably introduced to the continent as long as five to seven thousand years ago. The more widespread humped cattle breeds, such as the Zebu (Bos indicus), which are susceptible to trypanosomiasis, appeared in Africa in large number only about thirteen hundred years ago. Highly productive, genetically improved European (Bos taurus) cattle breeds introduced in this century are extremely susceptible to trypanosomiasis.
Use of trypanotolerant livestock, while making livestock rearing possible in tsetseaffected areas, has two main drawbacks. First, many farmers believe these breeds are less productive than others. (Although the meat production of trypanotolerant livestock compares favourably with that of other breeds, the amount of milk. a critically important food in rural Africa. produced by trypanotolerant cattle is relatively poor; in addition, trypanotolerant animals raised under traditional farming practices are typically small in size and therefore are not ideal for draught work.) Second, few such animals are available. In spite of their importance in tsetse-infected areas where other livestock cannot survive, trypanotolerant cattle constitute only 5% of the total cattle population in the 37 countries where tsetse occur (Figure 20). Although the numbers of trypanotolerant animals on the continent are now increasing, they are doing so only slowly.
Figure 20. Countries in West and Central Africa where indigenous cattle breeds exist that tolerate infection with trypanosomes. These so-called 'trypanotolerant' cattle breeds include the N'Dama, the savannah West African Shorthorn and the dwarf West African Shorthorn. (Based on 1980 data from the Food and Agriculture Organization of the United Nations [Rome], the International Livestock Centre for Africa [Addis Ababa] and the United Nations Environment Programme [Nairobi].)
The most cost-effective way of controlling infectious diseases is through immunization. Successful vaccination against a pathogen exploits the ability of animals to control disease-causing organisms by mounting an immune response. When an animal is infected with a parasite, the host's immune system recognizes molecules exposed on the surface of the parasite and targets an immune response to those molecules, called antigens (antibody generators). This leads to the destruction and removal of the pathogen by 'effector' components of the immune system.
Most vaccines contain one or more of these accessible molecules purified from the parasite. These stimulate an immune response without having to expose the animal host to infection with the whole organism. The immune system 'remembers' these antigens and, if the animal is exposed to the pathogen, is primed and ready to control the infection. This response is faster and qualitatively superior to the response elicited following primary exposure to the antigen. In this way, vaccinated animals are able to eliminate parasites quickly and efficiently and stop the development of disease.
Results of cell and molecular biology studies of trypanosomes at ILRAD and elsewhere over the years have established that a defense mechanism known as 'antigenic variation' has evolved in trypanosomes to enable them to avoid elimination by the immune system. A mammalian host can usually make good immune responses to the first wave of invading trypanosomes by producing antibodies against antigenic glycoprotein molecules displayed on the surface of the parasites. However, before all the parasites can be eliminated, trypanosomes develop with different surface molecules not recognized by the animal's initial immune response. This second wave of trypanosomes multiplies rapidly until the animal produces new antibodies against the second kind of glycoprotein displayed on the parasite surface, but then parasites appear displaying yet another glycoprotein molecule, and so the process continues, the parasite population always keeping a step ahead of the host's immune system (Figure 21).
Figure 21. Diagram illustrating the process of antigenic variation, which enables trypanosomes to survive attack by the immune system of an animal host.
Trypanosomes are able to express many kinds of glycoproteins, designated 'variable surface glycoproteins', or VSGs. The VSGs are a major cell product of the trypanosome, accounting for 10% of all the proteins synthesized by the parasite. (It is estimated that between 300 and 1,000 trypanosome genes code for VSGs.) Each of the many different strains of trypanosomes displays a particular set of VSGs. Further complicating the matter, genetic recombination among trypanosome populations may occur in the field, increasing the potential for antigenic diversity. Clearly, a conventional type of vaccine, which primes an animal's immune system against only one or a few antigens, will not be broadly effective against trypanosomiasis. With this in mind, ILRAD scientists are researching other methods, including new vaccine applications, for controlling the disease.
Research into tsetse vector control and the development of new drugs is being conducted by several other institutes. ILRAD's trypanosomiasis program specifically conducts research in four areas: epidemiology, trypanosome biology, host resistance and drug applications. The epidemiology project conducts research in diagnosis and chemotherapy and maintains the Laboratory's link with the African Trypanotolerant Livestock Network.
The aim of the trypanosomiasis program is to fulfill two broad objectives. The first is to meet increasingly pressing requirements for immediate improvements in existing control methods. This may be accomplished in two ways: by gaining a more complete understanding of the epidemiology of trypanosomiasis, through development of improved tests for detecting infection and identifying parasites, and by improving and making better use of current control methods, particularly the employment of drug therapy and trypanotolerant livestock.
The second objective of the program is to translate long-term research into practical and sustainable trypanosomiasis control through the development of novel control methods. Four strategies drive ILRAD's trypanosomiasis research. (1) Identification of molecular mechanisms. parasite components or physical or chemical processes. that could, through intervention with new trypanocidal agents, be manipulated to disrupt parasite development or to make the parasites more vulnerable to the defences of the mammalian host. (2) Development of new therapeutics, other than trypanocides, on which to base treatment and disease management. (3) Improvement of the immune responses of susceptible cattle to trypanosome infection through a better understanding of the natural resistance to trypanosomiasis displayed by trypanotolerant cattle. This work will also involve a search for genetic markers that one day may be used to identify highly resistant animals for use in livestock breeding programs. (4) Identification of trypanosome antigens that, by inducing protective immune responses in cattle, are potential material on which to base vaccines.
Trypanosomiasis is just one of several factors that reduce cattle productivity. Better diagnosis of trypanosomiasis would help researchers to define the disease problem more precisely, to better understand the epidemiology of trypanosomiasis (the effect of the disease on livestock populations) and to compare the impact of implementing various trypanosomiasis control programs. Improved diagnostic techniques. accurate, simple and inexpensive. have long been needed to detect trypanosomes in both the mammalian host and the tsetse fly vector.
An antigen-trapping enzyme-linked immunosorbent assay (ELISA) developed at ILRAD uses monoclonal antibodies for the identification of parasite components in blood (Figure 22). This ELISA has been shown to be sensitive and reliable in the detection of the three major tsetse-transmitted trypanosome species that infect livestock, as well as in the detection of T. evansi infections in camels and pigs.
Figure 22. (a) Diagram of steps involved in using an antigen-trapping enzyme-linked immunosorbent assay (ELISA) developed at ILRAD to detect trypanosome antigens in blood from livestock. A multi-well plastic plate is coated with a particular monoclonal antibody. The plate is washed, a test sample of bovine serum is added and incubated on the plate for a short time and then the plate is washed again. If the antigen that the coating antibody recognizes is in the sera, the antigen is trapped. To reveal the trapped antigen, unbound components of the sera are washed off the plate and the same antibody that was used to coat the plate, now labelled with an enzyme, is added to the plate. The labelled antibody binds to the trapped antigen, forming an enzyme-containing complex. This complex is revealed by adding an appropriate indicator system, which changes the colour of the sample according to the amount of antigen present. (b) This assay may also be carried out using test tubes rather than a mult-iwell plastic plate.
In 1990, the antigen-trapping ELISA for use in tsetse-transmitted animal trypanosomiasis continued to be evaluated in the field. The validation exercise is being jointly conducted by ILRAD, the Food and Agriculture Organization of the United Nations, the International Atomic Energy Agency and ten national agricultural research institutions (Figure 23). With the support of the World Health Organization, ILRAD and six national research institutes also continued to investigate how effectively the antigen-trapping ELISA can be used to diagnose human trypanosomiasis.
Figure 23. Map showing the location of 15 institutions that are evaluating ILRAD's antigen-trapping enzyme-linked immunosorbent assay kits to diagnose animal and human trypanosomiasis. Filled-in dots: diagnostic kits for animal trypanosomiasis; circles: kits for human trypanosomiasis (sleeping sickness).
A workshop is planned for May 1991 in which the results of the continent-wide evaluation will be considered and decisions taken concerning the future development and deployment of the system. During the year, ILRAD personnel visited a few national laboratories to solve minor technical problems that occasionally arose with use of the assay.
At ILRAD, staff continued to find evidence of the efficacy and sensitivity of the antigen-trapping ELISA. It was shown in 1990 to be capable of revealing parasite antigens in goats and cattle that were carrying chronic T. congolense infections undetectable using the microhaematocrit centrifugation method.
In studies conducted within the African Trypanotolerant Livestock Network in Gabon this year, N'Dama cattle that showed no parasitaemia (appearance of parasites in the blood) were found by use of the ELISA to be carrying trypanosome antigen. It was further discovered that the growth rates and packed cell volume levels (the proportion of red blood cells in blood used to estimate anaemia) of these antigenaemic but non-parasitaemic animals were similar to growth rates and packed cell volume levels of animals free of both parasite antigens and parasites. This finding highlights the potential value of antigendetection as distinct from parasite-detection systems when attempting to select highly trypanosome-resistant cattle among less resistant animals in the field. The antigen-trapping ELISA is also proving to be a valuable tool in laboratory studies of the basis of trypanotolerance.
Work continued during the year on cloning the T. congolense antigen recognized by a diagnostic monoclonal antibody used in the ELISA. Characterization of a T. vivax-specific antigen has now reached the sequencing stage. The objective in this research area is to make defined antigens available for development of an inhibition antigen-trapping ELISA, which may be even more sensitive than the existing technology.
Several parasite species-specific monoclonal antibodies have now been raised at ILRAD to forms of trypanosomes that occur in the tsetse fly. The efficacy of these monoclonal antibodies in detecting fly infections will be determined over the coming year.
Many DNA PROBES have been developed at ILRAD and elsewhere over the last several years. Rather than detecting the antigenic products of trypanosome genes, as the ELISA does, these probes detect the genes themselves. DNA probes are superior to the other available diagnostic techniques in terms of specificity, sensitivity and ease of use when large numbers of samples need to be examined for parasite detection and characterization. Most of the DNA probes developed to date are tagged with radioisotopes so that their hybridization with genetic material obtained from parasites can be visualized.
A panel of species- and subspecies-specific DNA probes has been developed at ILRAD and used to detect and characterize trypanosome samples collected from experimental infections of tsetse flies and laboratory animals as well as from animals in the field. Methods developed at ILRAD to optimize the collection of blood from animals and materials from tsetse flies were tested and shown to be useful for the proper storage of samples isolated in the field before transportation to the laboratory for analysis.
Although use of DNA probes to detect parasites has great advantages over more conventional detection methods, the technique also has disadvantages, the greatest being its reliance on radioisotope labelling. Because of the hazards, short shelf-lives and high costs and operational difficulties involved in use of radioisotopes, alternative ways of labelling DNA probes are now being explored. ILRAD staff aim to develop probes that are both safer than the radiolabelled probes now widely used in advanced laboratories and easier to use in the field.
The livestock industry in Africa has traditionally depended heavily on trypanocidal drugs both to prevent and to treat trypanosomiasis. Drug treatment currently relies on the salts of three compounds. isometamidium, homidium and diminazene. which are closely related chemically and have been used for more than 30 years. When rationally applied, these drugs have effectively controlled the disease, but reports indicate that the incidence of parasite resistance to the compounds is increasing.
Because chemotherapy will play a major role in controlling trypanosomiasis in livestock for many years to come while researchers investigate other control methods, ILRAD staff members in 1987 began studies to determine ways of maintaining the long-term efficacy of the compounds now in use. The detailed information they are obtaining on how the drugs act on trypanosomes, on how the parasites develop resistance to the drugs, and on the pharmacokinetics of the drugs. how the mammalian host transports, metabolizes and excretes the compounds. is laying the groundwork for devising better ways to use the drugs in the field, including treatment strategies that militate against the advent of drug-resistant parasite strains.
To develop ways to combat drug resistance, researchers first need to know how prevalent the resistance is and how the levels of resistance vary among field populations of trypanosomes. To this end, trypanosomes obtained from sites across Africa were characterized IN VIVO. The Laboratory now has a collection of populations expressing various levels of resistance to the different trypanocides used to treat domestic livestock.
The development and assessment of an antibody-based ELISA for measuring levels of isometamidium in mammalian blood was undertaken during the year in collaboration with the University of Glasgow. With IN VITRO techniques and culture systems developed at the Laboratory, researchers are determining the levels of drug resistance in various populations of parasites. For example, two assays of sensitivity to isometamidium. a long-term viability test and a drug incubation survival test. gave encouraging results with T. vivax epimastigote (forms that occur in the tsetse fly) and bloodstream forms, respectively, that correlated with sensitivities determined in vivo.
ILRAD's trypanosomiasis program continued in 1990 to give support to the African Trypanotolerant Livestock Network, whose headquarters are in Nairobi. The program provided the Network with the services of a technologist who helped to establish tsetse control measures at two sites in Ethiopia, Ghibe and Tolley, where parasite resistance to trypanocides is a serious threat to trypanosomiasis control operations. Trypanosome isolates from the two sites were brought to ILRAD for studies of the basis of the drug resistance occurring in those areas. All the isolates examined to date are T. congolense, and 12 of 13 of them have shown significant resistance to diminazene aceturate, isometamidium chloride and homidium.
Both the scope and depth of ILRAD's research on the mechanisms by which trypanosomes evade the harmful effects of trypanocidal drugs increased in 1990. The availability of in vitro assays, of cloned parasites of differing drug sensitivities, and of methodologies to discriminate among the different PHENOTYPIC and GENOTYPIC characteristics of different clones. together with an ability to conduct studies in relevant host species. have contributed to the increasing strength of the Laboratory in this area.
Drug resistance is a complex phenomenon, with multiple factors contributing to trypanosome resistance and different mechanisms being responsible for resistance among different parasite species. In studies conducted at ILRAD in 1990, levels of drug resistance in T. congolense were shown to vary with time during both in vivo and in vitro maintenance not only among clones derived from a drug-resistant parasite but also among subclones derived from one clone. These findings suggest that parasite resistance is not a stable phenotype, that is, the level of resistance expressed in a parasite population may alter even when that population has never been exposed to the drug. On the other hand, it appears that a T. vivax cloned population has maintained its drug resistance phenotype in the absence of drug selection over a period of years in culture.
Further progress was made in characterizing an extrachromosomal genetic element earlier discovered in a multi-drug resistant stock of T. b. brucei. Over the coming year, the occurrence of this element will be examined in both drug-resistant and drug-sensitive trypanosome stocks and isolates, and the element will be SEQUENCED.
In other experiments, scientists are determining if genetic recombination in the sexual cycle in trypanosomes can transfer the mechanisms of resistance; if this is established, researchers will begin to look for associations between the resistance phenotype and various phenotypic and genotypic markers. If successful, this experiment may be expected to yield markers of the genes controlling resistance.
Underpinning much of ILRAD's research in the trypanosomiasis program is the ability to grow large quantities of trypanosomes successfully in culture (Figure 24). Trypanosomes grown in the Laboratory are used by scientists conducting studies in areas as varied as bovine immunology and genetics, trypanosome drug resistance and responses to antibodies, the differentiation process in trypanosomes, and important metabolic pathways and molecular mechanisms in the parasites. The culture systems developed at ILRAD for growing and maintaining trypanosomes have been increasingly refined over the years so that the parasites may be cultured under increasingly defined conditions.
Figure 24. A laminar flow hood and a collection of the apparatus used to cultivate trypanosome parasites in the laboratory. Much of ILRAD's trypanosomiasis research relies on the availability of trypanosome strains grown artificially, in culture, in addition to parasites developed naturally in the tsetse fly vector and mammalian host. ILRAD staff members are constantly refining parasite culture
It has been possible for some time to culture the different forms of T. b. brucei without the aid of feeder-layer cells (termed an 'axenic' culture). A recent major advance has been the development of culture systems for maintaining all life cycle stages of several T. congolense populations without feeder layers. In the coming year, the serum components essential for in vitro maintenance of T. congolense will be defined further. This work was stimulated in the past year by the discovery of biochemical differences between transferrins. molecules in the blood that transport iron essential for both parasite and animal cells. in calf and young goat serum and the subsequent finding that the goat serum appears to be the more successful supplement of growth media for axenic culture of T. congolense.
The basic biology of the trypanosome is studied at ILRAD to investigate processes that are likely targets for drug or immunological attack. Of particular interest to ILRAD staff is the process of 'endocytosis'.
Trypanosomes, like all organisms, need nutrients to live and grow. The parasites obtain these nutrients from their environment by taking up molecules and particles in a process known as endocytosis. Past studies using the electron microscope have shown that trypanosomes draw in molecules by way of a structure named the flagellar pocket (Figure 25). Molecules binding to the surface of the trypanosome, including antibodies, have been found also to be engulfed in this way. Therefore, in addition to changing its surface coat of proteins to avoid antibody binding, the parasite seems able to take up antibodies, and possibly in this way to neutralize their effect. It was demonstrated during the year that clearance of antibodies from the surface of the trypanosome in this way renders parasites in vitro resistant to the effects of 'complement', the collective name for a group of proteins in mammalian blood so named because they complement and amplify the action of antibodies to cause the lysis (rupture) of parasites.
Figure 25. Schematic diagram of a trypanosome of the Trypanosoma brucei group in its intermediate bloodstream form, illustrating the major organelles.
One approach to learning more about the physical organization of the endocytic pathway is to determine the functional activity of defined fractions and subcomponents of trypanosomes. In 1990, in collaboration with scientists from the European Molecular Biology Laboratory (Heidelberg), a free-flow electrophoresis technique was used to separate microsomal trypanosome fractions. The fractions obtained were characterized to some extent on the basis of demonstrations of enzymatic activity.
Progress was also made during the year in purifying and characterizing a receptor molecule of T. b. brucei that recognizes and binds to transferrin circulating in the blood of the mammalian host. The receptor appears to be much smaller than its mammalian equivalent. The possibility that trypanosomes have, and make use of, receptors to mammalian growth factors will be examined in the coming year.
Parasite enzymes that break down proteins, called proteases, appear to be involved in the endocytic process. A lysosomal thiol protease of T. congolense was purified in 1990. Antibodies to the enzyme have proved to be valuable markers of the lysosome. An important discovery made during the year was that this enzyme is better recognized by antibodies in the sera of trpyanotolerant N'Dama cattle recovering naturally from infections than in Zebu cattle that required treatment to recover from infection. Furthermore, the enzyme is detectable in the sera of cattle early in an infection and so is a possible cause of the immune and physiological dysfunctions that characterize trypanosomiasis in animals. The degree of protection afforded livestock by immunization with the purified molecule, and the possibility that it is involved in the pathogenesis of the disease, are subjects of current studies.
Much still needs to be learned of the genetics of basic functions in the trypanosome. An important goal of the trypanosomiasis program is to better understand the mechanisms involved in genetic recombination, sexual and otherwise, which would help solve important epidemiological questions concerning the extent that diversity in trypanosome populations is being generated in the field. Of particular interest is the identification of trypanosome genetic functions that could be interfered with and the identification of trypanosome genes and their protein products that are directly involved in levels of drug resistance in parasites, degrees of immunity induced by parasite antigens, degrees of virulence in parasites and the development of disease in cattle.
The technology required to transfect DNA into trypanosomes and to identify which organisms internalize exogenous genetic material continued to be developed in 1990. Application of transfection techniques led to an interesting and important finding: it was shown that it is possible to restore a degree of virulence to T. b. brucei attenuated (weakened) in culture by transfecting into the attenuated parasites DNA derived from a virulent clone. Rescue of the transfected DNA from the formerly non-virulent organisms could lead to the identification of genes that control the virulence trait in these parasites.
In the mammalian host, while adapting to conditions that occur in the tsetse fly vector, T. b. brucei change from actively dividing slender forms to non-dividing stumpy forms (Figure 26). If the parasites did not stop proliferating, all mammalian hosts would eventually be overwhelmed by parasites and the life cycle of the trypanosomes would be broken. The change to non-dividing forms perhaps ensures trypanosome survival by ensuring that some parasites are transmitted onward to the parasite's intermediate host, the tsetse fly. This change to stumpy forms is of particular interest to ILRAD since the switch may provide a clue to the regulatory genes responsible for the differentiation.
Figure 26. Light micrograph of slender and stumpy forms of Trypanosoma brucei brucei in mouse blood. The clear structural differences between the two life cycle stages is accompanied by biological and biochemical differences.
Studies are being carried out of trypanosome genes that, because they are active only in certain stages of the life cycle, might control processes of differentiation and adaptation to mammalian or insect hosts. A novel approach to identifying stage-specific genes in trypanosomes was developed during the year. This involves the use of genetic sequences normally associated with expressed RNA (ribonucleic acid), together with a genetic element widely distributed in the trypanosome genome (called a transposable element), to amplify DNA of different life cycle stages and thereby identify differentially expressed parts of the genome.
Understanding how immune responses to trypanosomes are generated in livestock may lead to an ability to manipulate those responses so that they stop the proliferation of parasites. Studies of the responses to infection of bovine B cells, which produce antibodies to trypanosomes, and of T cells, which effect and regulate various immune mechanisms, including some aspects of antibody production, were continued in 1990. Scientists also continued to study macrophages, which ingest and destroy parasites of different kinds, and which, like T cells, are involved in the regulation of bovine immune responses.
The production of large amounts of antibodies is a feature of trypanosomiasis, and the ability of antibodies to neutralize parasites carrying the particular antigen used to induce them is well documented. Therefore, research into factors affecting the generation of antibody responses, including factors leading to B-cell activation and the roles of accessory cells and soluble factors, is being undertaken by the program. This research is stimulated by the possibility that with increasing knowledge and increasing availability of 'invariant' antigens, which comprise all the molecules of the parasite other than its surface glycoproteins, it may become possible to generate antibody responses that protect livestock against challenge with a broad range of trypanosome strains.
Having produced over 40 trypanotolerant N'Dama cattle, the first ten by implanting embryos obtained in 1983 from Gambian cows into foster mothers of an East African Zebu breed called Boran (Figure 27), ILRAD has a unique opportunity to study, under controlled circumstances, the immune responses of these animals to infection and to define the mechanisms of tolerance. A major area of research in 1990 was a comparison of the responses of N'Dama with those of susceptible Boran cattle.
Figure 27. An N'Dama calf with her Boran foster mother.
Results of this research show that the N'Dama are capable of a superior antibody response during infection and are also able to control infection better than their susceptible Zebu counterparts. The two breeds differ in the quantity, quality and specificity of antibodies they generate. The trypanotolerant N'Dama cattle produce higher levels of anti-VSG specific antibodies than the Boran, they produce more IgG1 antibodies to invariant trypanosome antigens, and they produce a greater response to two trypanosome-derived molecules, one of which may be useful in diagnosis while the other may have important implications in the control of the disease. More work is required to determine the relevance of these superior antibody responses in trypanotolerant cattle. This research, together with studies being carried out on other aspects of the bovine immune response to trypanosome infection, will determine whether the immune system plays a primary role in controlling trypanosomiasis in trypanotolerant livestock.
Antibody responses to trypanosome infection also appear to be more specific in N'Dama than in Boran cattle, where there is evidence of greater production of antibodies to irrelevant antigens during infection. Most antibody responses require the help of T cells in their generation and maintenance. T cells may be particularly important in the maturation of antibody responses. Results of comparative experiments indicate that N'Dama cattle make earlier and more persistent T-cell responses to trypanosome antigens and recognize a greater repertoire of the antigens than the susceptible Boran.
A striking and obvious feature of trypanosome infection in susceptible cattle is anaemia, although little is known of the mechanisms involved in its induction. It has been reported that there is a decreased half-life of red blood cells in cattle suffering from the disease. It is now also apparent that the capacity to maintain normal levels of red blood cells during trypanosome challenge is a feature of trypanotolerant animals and that this trait is highly heritable.
Previous work has demonstrated that during infection of susceptible cattle, macrophages, which are usually thought of as playing a protective role in infections, inflict self-harm by engulfing and destroying red and white blood cells and their progenitors in the bone marrow. Results of studies conducted in 1990 corroborated this (Figure 28). In vitro phagocytosis of red blood cells by macrophages increased in both N'Dama and Boran cattle undergoing experimental infection, but the rate of increase was significantly higher in the Boran.
Figure 28. Light micrograph of a large macrophage from the bone marrow of a calf infected with Trypanosoma vivax. Mature and immature red blood cells have been engulfed by the macrophage. A single trypanosome lies adjacent to the macrophage.
New studies were begun at ILRAD this year of the role of cytokines in trypanosomiasis. Cytokines. such as tumor necrosis factor, gamma interferon and interleukins. are released by macrophages that have been activated by infection. In a healthy animal, cytokines function as modulators of the immune system, but they may have toxic effects on cells of the animal, including those of the endocrine system, if they are produced in excess. Attempts will be made to identify parasite components that may trigger a cascade of events leading to the harmful activation of macrophages in livestock.
Hormones produced by the adrenal gland, particularly cortisones, are important in controlling the effects of stress and in protecting mammals from toxic products produced by parasites. Hormones also help regulate host molecules that may adversely affect animals when the molecules are overproduced during an infection. Work of the previous year showed that circulating levels of cortisol were depressed in susceptible cattle during trypanosome infection. Further collaborative studies with scientists from the Institute of Primate Research (Nairobi) have now shown that whereas the adrenal gland is capable of a normal response to ACTH, the response of the pituitary gland. which orchestrates the activity of many hormones, including cortisol. to the corticotropin releasing factor is compromised in infected animals. Electron microscopic examinations made in the year revealed large numbers of T. congolense parasites in the pituitary glands of challenged animals and loss of pituitary architecture (Figure 29).
Figure 29. Electron micrograph of part of the pituitary gland of a Boran cow infected with Trypanosoma congolense showing the presence of a group of trypanosomes (T) within the lumen of a blood vessel. The presence of trypanosomes within the microvasculature of the pituitary gland correlates with localized degenerative changes within the region of the glands and with pituitary dysfunction in general.
The role of cytokines and/or parasite enzymes in inducing pituitary lesions and abnormal hormonal function is still unknown. As with anaemia, these manifestations of disease may be consequences of a more fundamental dysfunction induced by trypanosomes in susceptible host livestock.
Any future methods developed to increase livestock resistance to trypanosomiasis and to decrease the pathogenic effects of infection would greatly alleviate the animal trypanosomiasis problem in Africa and in addition would help advance research of human trypanosomiasis and other trypanosome-caused diseases that occur in the tropics. Host resistance to the parasites might be increased in several ways. Vaccination to generate effective immune responses in livestock would, in addition to being of inestimable value in itself, constitute a major biological advance. An understanding of how trypanosome infection produces disease would lead to more effective treatment and would help further the development of livestock types able to resist the harmful effects of infection. An understanding of trypanotolerance would facilitate better use of trypanotolerant animals and, through genetic manipulations, might allow the transfer of trypanototerant trait(s) to cattle breeds that, although susceptible to trypanosomiasis, possess other desirable characteristics, such as good productivity.
Genetically mediated resistance to the effects of trypanosome infection clearly exists, but the nature of the mechanisms involved and their genetic control are not yet understood. Previous work undertaken within the African Trypanotolerant Livestock Network demonstrated that trypanotolerance is heritable, as measured by an ability of trypanotolerant cattle to maintain normal red blood cell levels and to control parasitaemia while under trypanosome challenge in the field.
With the availability at ILRAD of trypanotolerant N'Dama cattle and a unique ability to assess their responses to parasite challenge under controlled circumstances, an ambitious project was initiated in 1990 to locate the genes that are responsible for trypanotolerance. As a first step, scientists began work to identify markers of trypanotolerance trait(s). Such markers will help identify trypanotolerant animals, obviating the necessity of challenging animals with trypanosomes to determine which are trypanotolerant. It is hoped that the markers will ultimately be used to identify the bovine genes that control resistance. This work currently involves both the identification of potential marker locations on the chromosomes and the generation of a population of cattle segregating trypanotolerance. These studies are being broadened through collaboration with Japanese scientists and support from the Japan International Co-operation Agency to encompass potential markers of various kinds in cattle blood.
During the year, a start was made to identify `microsatellites'. simple DNA nucleotide sequences that occur with a high frequency in the mammalian genome. Microsatellites provide a potentially powerful marker system. They were exploited in 1990 to synthesize oligonucleotides. short, single-stranded synthetic DNA molecules. that hybridize specifically with chromosome regions flanking the micros atellites. The oligonucleotides serve as primers in the polymerase chain reaction to produce labelled copies of the microsatellite regions. The length polymorphism of the copies can then be determined by reducing polyacry-lamide gel electrophoresis to give markers at known sites within the genome.
Ultimately, this approach will provide a set of linked and mapped markers for the bovine genome. Such work is labour-intensive, both in the identification of markers and their locations and in the determination of their distribution throughout the genome. However, work in developing a linkage map of the bovine genome will almost certainly involve collaboration among several advanced laboratories.
The new project to map the trypanotolerance genes requires large numbers of closely related crosses of trypanotolerant and susceptible cattle. The availability of such families will greatly simplify the search for genetic markers of trypanotolerance. Using embryo transfer expertise developed at ILRAD over the last several years (Figure 30), ILRAD staff in 1990 produced the first N'Dama-Boran cross-bred calves; by the end of the year, 75 such calves were born (Figure 31). At about one year of age, the calves will be challenged for the first time with T. congolense naturally through the bite of infected tsetse flies (rather than artificially through needle injection). Responses to the challenge, including packed cell volume levels, parasitosis and serological responses, will be recorded. As calves become available, they will be challenged monthly until all members of four full-sibling families, each family consisting of 30 cross-bred calves, have been PHENOTYPED for aspects of trypanotolerance. A second filial generation will then be produced. The animals will be examined for white blood cell antigen and biochemical polymorphisms and will be GENOTYPED with DNA markers as these are developed at ILRAD and other laboratories.
Figure 30. The splitting of a bovine embryo before implanting in a foster Boran mother.
Figure 31. The first Boran-N'Dama crossbred calves produced at ILRAD using embryo-transfer technology were born on ILRAD's Kapiti Ranch in 1990. By the end of the year, 75 Boran-N'Dama crosses had been produced. Large families of these cattle are needed for a new research project to locate the genes responsible for trypanotolerance, an ability of N'Dama and other livestock breeds indigenous in Africa to remain productive while infected with trypanosomes.
The trypanosomiasis research program made several important advances in 1990. In parasite biology, ongoing characterization of parasite molecules has facilitated the rapid identification of parasite components that dominate the immune response of the mammalian host.
Our knowledge of parasite genetics was strengthened during the year, with further information obtained on the fundamental principles of gene control. This research, in addition to highlighting parasite vulnerabilities that could be targets for novel control methods, could have broad implications for an understanding of protozoal genetics in general. Research on genetic recombination in trypanosomes received more attention this year in the knowledge that we will not completely understand the epidemiology of trypanosomiasis until we know how and to what extent parasite diversity is generated in the field.
More information was obtained on how the immune system of cattle responds to trypanosome infection. Studies begun during the year on the role that cytokines may play in both parasite control and pathogenesis are unifying efforts to understand the immune responses and the pathology in trypanosomiasis. It is clear that the latter are closely connected in trypanosomiasis and that they may, indeed, be manifestations of the same basic mechanisms.
The start of work to produce a linkage map of the bovine genome, and its application to the search for markers of trypanotolerance, is a landmark in the evolution of ILRAD's research program. The development of a relatively fine definition linkage map of the bovine genome has potential uses that go far beyond that of providing a means for understanding trypanotolerance. To fulfil its promise, however, this research area will require a strong commitment over the coming years in both financial and staff support.
