GROWING TRYPANOSOMES IN THE LABORATORY

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1 Published by the International Laboratory for Research on Animal Diseases Volume 2 Number 1 Growing trypanosomes in the laboratory A closer look at the trypanosome lifecycle Cultivating trypanosomes in vitro Research applications Preliminary immunization trials Plans for 1984 ILRAD holds conference on ruminant immunology Summary of presentations GROWING TRYPANOSOMES IN THE LABORATORY Trypanosomes were first described over 300 years ago. In 1680, the Dutch naturalist Antony van Leeuwenhoek wrote his English colleague Robert Hooke that he had seen numerous small animals (probably Trypanosma theileri) moving rapidly in fluid taken from the gut of a horsefly. By the 1840s, scientists in several European countries were recording their observations of trypanosomes. The generic term Trypanosoma was first used in Paris in In 1857, David Livingstone suspected that the African livestock disease 'nagana' was carried by wild animals and transmitted to domestic stock by tsetse flies. Interest in trypanosomes increased in the 1890s when David Bruce proved that 'nagana' was caused by trypanosomes which were indeed carried by wild animals and transmitted by tsetse. The parasite species he identified was later named T brucei. By the early 1900s, scientists were studying trypanosomes in several African countries. They observed the parasites in many wild and domestic animals and described the lifecycles of the three most important trypanosome species affecting livestock T brucei, T congolense and T vivax. In 1902, trypanosomes were first observed in human patients suffering from sleeping sickness. (This brief summary of early research on trypanosomes was taken from C A Hoare, The trypanosomes of mammals, Oxford: Blackwell, 1972.) Although many important discoveries were made during the first half of the twentieth century, research on trypanosomiasis was severely limited because the parasites could only be studied by passing them continuously through their tsetse fly vectors and mammalian hosts. The two subspecies of T brucei which cause human sleeping sickness (T b rhodesiense and T b gambiense) and the subspecies which infects livestock (T b brucei) can be raised in laboratory animals, but some isolates of T congolense are difficult to maintain in laboratory animals and T vivax parasites can normally only be raised in ruminant livestock (cattle, sheep and goats) and in the wild animals which are their natural hosts. It is difficult, expensive and time consuming to maintain trypanosomes in this way, and objections are raised on humanitarian grounds. Also, when trypanosomes are raised in animals it is impossible to study the specific features or developmental aspects of the parasites in isolation from the complex and changing environment provided by the host. It is also impracticable to produce the large numbers of parasites required to support field

2 studies or immunization trials. Because of these problems, scientists have been working for decades to develop laboratory techniques which will support trypanosome populations in vitro. F G Novy and W J MacNeal first described laboratory cultures of trypanosomes in 1903: the parasites were found in 'condensation water' which collected in test tubes at the base of nutrient blood agar slopes. For many years, it has been possible to grow non infective forms of T brucei and T congolense in various culture media, equivalent to forms which develop in the gut of the tsetse fly. Scientists have gone on to develop techniques to support these and other trypanosome species at different stages of their developmental cycle. Ultimately, the goal has been to maintain the parasites through all the transitions that make up their complete lifecycle, with substantial successes first achieved in the mid 1970s. A CLOSER LOOK AT THE TRYPANOSOME LIFECYCLE Trypanosomes pass through several different stages during their lifecycle. Changes in the parasites' digestion and respiration systems enable them to adapt to life in different locations in tsetse flies and mammals. On examination, the different stages of development are distinguished primarily by changes in parasite shape, by the presence or absence of a thick coat of variable surface antigen and by shifts in the position and length of the flagellum which enables the parasite to move. The surface coat is a particularly important feature, composed of a dense layer of identical glycoproteins. In the course of an infection, trypanosomes can display a large number of different antigens on their surface coats, thus evading antibodies produced by the host. The developmental stages vary among different trypanosome species. When a tsetse fly feeds on an animal infected with T b brucei, the parasites become established in the fly's midgut and lose the thick surface antigen coat they displayed in the mammalian bloodstream. These forms are not infective to mammals. They migrate to the salivary glands and change to animal infective metacyclic forms, which regain their coat of surface antigen. The metacyclic trypanosomes initiate a new infection when the tsetse next feeds. They enter the skin, multiply and migrate into the bloodstream where they multiply further, die off and multiply again, causing successive waves of parasitaemia. The parasites which multiply in the bloodstream are long and slender, while those which die out have a short, stumpy shape. The short, stumpy forms appear to be adapted to ingestion by a tsetse fly: when a fly feeds on blood containing these forms the trypanosome lifecycle is completed. When ingested by tsetse, T congolense parasites also enter the flies' midgut and lose their coat of surface antigen. From the gut, they move to the tsetse mouth parts, rather than the salivary gland, and change into metacyclic form, which again display a surface coat. When the infected fly bites another animal, the parasites enter the skin, where they multiply, and then migrate into the bloodstream. They seem to develop into rapidly multiplying and senescent forms, but without the obvious changes in shape observed in T b brucei. Uncoated insect forms of T vivax develop entirely in the mouth parts of tsetse flies. When an animal is bitten by an infected fly, metacyclic trypanosomes are injected in the skin and move to the bloodstream where they again display a coat of variable surface antigen. Like the other two trypanosome species, T vivax parasites develop in the bloodstream from rapidly dividing to senescent forms. CULTIVATING TRYPANOSOMES IN VITRO Early efforts to cultivate trypanosomes in the laboratory concentrated on subspecies of T brucei Although T b brucei is probably less important in livestock than T vivax or T congolense, a great deal of research had focused on this species because T b rhodesiense and T b gambiense cause disease in humans. Thus the basic biology and developmental cycle of this species were relatively well understood. Research interest focused primarily on the bloodstream forms of the parasite, with their

3 changing coats of surface antigen. However, bloodstream parasites maintained in culture flasks quickly transformed to non infective, uncoated forms similar to those that develop in the midgut of tsetse flies. Knowing that T b brucei bloodstream forms can invade the connective tissue of infected cattle, scientists at ILRAD developed several cultures based on cells similar to those found in bovine connective tissue. By the end of 1976, a system had been developed which supported a population of T b brucei bloodstream parasites for over a year. The parasites could not be distinguished from long, slender bloodstream forms raised in animals: they kept their surface antigen coats and remained infective. In the following year, bloodstream forms of a second T b brucei strain were successfully maintained in culture and a number of clones were established. By 1979, ILRAD's cell biologists were maintaining bloodstream forms of four T b brucei strains in culture, and a total of 42 clones had been established. The in vitro cultivation methods developed at ILRAD were adopted widely for T brucei research in laboratories all over the world. ILRAD scientists also succeeded in maintaining populations of T b brucei through their complete lifecycle. When the temperature of the culture was reduced from 37 to 25 C, bloodstream forms changed to uncoated insect forms which were no longer infective for mice. Eventually metacyclic forms appeared which were again infective. Within 8 days of raising the temperature to 37 C, these metacyclic parasites changed back to bloodstream forms, completing the lifecycle. This achievement made it possible to study in much greater detail the different structural forms of the parasites, the mechanisms controlling their development, and the variety of antigenic types displayed by the surface coat. Beginning in 1980, considerable research effort focused on extending the culture techniques developed for T b brucei to the propagation of T congolense and T vivax parasites in vitro. Noninfective insect forms of T congolense could be raised in culture systems similar to those developed for T b brucei, but preliminary investigations showed that bloodstream forms of T congolense need very different culture conditions to survive. A significant achievement in 1980 was the development of an in vitro culture system, using bovine endothelial cells, which supported the growth of bloodstream forms of T congolense for long periods. In 1981, scientists at the University of Edinburgh developed an in vitro system to cultivate T congolense metacyclics. Using a modified version of this system, cell biologists at ILRAD have established six lines of metacyclic producing cultures from three clones and three uncloned stocks: these have been maintained over 9 months. To complete the parasite lifecycle, T congolense metacyclics are transferred to cultures containing bovine endothelial cells as feeder layers. When incubated under these conditions at 28 C, the trypanosomes appear to change to bloodstream forms by the 7th day. They are coated with surface antigens and are capable of infecting goats and mice.

4 Bloodstream forms of T b brucei which have been maintained in culture for over a year. These parasites appear identical to the long, slender forms raised in animals. The surface antigens of these bloodstream forms are identical to those displayed by the metacyclic parasites from which they are derived. Using this system, 100 to 200 million parasites can be produced daily, yielding 20 to 30 times more surface antigen than could be produced by culturing the metacyclic forms. Experience through the years has shown that T vivax is the most difficult of the three major African trypanosome species to cultivate in vitro. Then in 1982, scientists from the Swiss Tropical Institute, working with colleagues at ILRAD, devised a culture system which could maintain bloodstream forms of T vivax. By mid 1983, ILRAD scientists developed a system to maintain T vivax parasites throughout their entire lifecycle. Bloodstream forms taken from infected mice are placed in plastic flasks with a culture medium (Eagle's MEM), feeder layers of bovine fibroblasts and Matrex Gel Green A beads. When incubated for 3 days at 25 C, the parasites change to noninfective, uncoated insect forms and attach themselves to the beads. By day 10 to 12 and onwards, many detach themselves from the beads, become loosely attached to the surface of the feeder layer cells and multiply. From days 12 to 21, a small proportion (0.5 to 1.0%) of the parasites change to metacyclic forms which are again infective. The mixed populations of insect and metacyclic forms are moved to a different medium (RPMI 1640) which contains feeder layer cells derived from meadow vole (Microtus montanus) embryos. When incubated in this medium, the metacyclic parasites change back to bloodstream forms and continue to multiply for long periods.

5 Sections of T congolense parasites photographed through an electron microscope. (a) Section of a bloodstream form raised in a mouse. The arrow indicates the thick coat of variable surface antigen. (b) Section of an insect form derived from bloodstream forms and propagated in culture. Note that the surface antigen coat is absent (arrow). This form is noninfective to mammals. (c) Section of a metacyclic form propagated in culture. The coat of variable surface antigen has reappeared and the parasite is now infective to mammals. Recently, attention has focused on the biochemical factors which promote parasite growth in vitro. The goal is to develop culture systems which do not require the addition of host serum or cells, making it possible to produce large numbers of trypanosomes inexpensively.

6 Insect forms of T vivax became attached to the surface of Matrix Green Gel A beads when maintained for 3 days under appropriate culture conditions. These parasites lack surface coats and are noninfective to mammals. RESEARCH APPLICATIONS In vitro studies have yielded important information about trypanosome biology and the parasite lifecycle. For one thing, considerable attention has focused on the coat of surface antigens displayed by metacyclic and bloodstream trypanosomes because changes in the surface coat appear to be the primary mechanism enabling the parasites to evade the normal antibody responses of infected hosts. One important issue is whether host antibodies are necessary to stimulate the parasite to change its antigenic coat. In a few of the cloned populations of T b brucei bloodstream forms maintained in vitro at ILRAD, a small proportion of the parasites displayed a new antigen type on their surface coat. So this important phenomenon antigenic variation had occurred in the absence of host antibodies. Another question concerns the development of trypanosomes in the mammalian bloodstream. It has been known for some time that T b brucei parasites change from rapidly dividing to nondividing forms in the bloodstream, and scientists have postulated that other trypanosome species may develop in a similar way. In 1982, cell biologists at ILRAD observed short slender bloodstream forms of T vivax which divided rapidly in culture, elongated slender forms which did not divide and short, tadpole like forms which appeared to be dying off. The short slender forms are more easily maintained in vitro, while the elongated parasites transform more readily to the uncoated insect stage, suggesting that the longer forms are adapted for transmission to tsetse flies.

7 T vivax bloodstream forms raised in a mouse. The preparation consists of elongated forms (L), short slender forms (S) and tadpole shaped forms (T). Different stages of T b brucei, T congolense and T vivax are being characterized biochemically using in vitro culture systems. Interest focuses on how trypanosomes absorb nutrients and convert them into energy. These functions are based on different mechanisms at different stages of parasite development, adapted to environmental factors in the tsetse vector and mammalian host. Interventions may be identified which can block changes in trypanosome metabolism, thus disrupting the parasite lifecycle. Present efforts to control trypanosomiasis often rely on regular treatment with trypanocidal drugs. Only a few compounds are available to livestock producers and drug resistance and cross resistance are increasingly serious problems. Yet no new trypanocidal drug his been brought out on the market for many years. One obstacle has been the lack of inexpensive, yet reliable procedures to screen new compounds. Beginning in 1982, ILRAD scientists have used the culture system which supports bloodstream forms of T b brucei to measure the activity of trypanocidal drugs. Ten promising trypanocidal drugs have been screened using simple tests developed in a collaborative project with the Kenya Trypanosomiasis Research Institute (KETRI) and a Kenya Government project funded by the German (Federal Republic) Geselleschaft für Technische Zusammenarbeit (GTZ). When drugs are added to trypanosomes in culture, their trypanocidal activity is measured along with any toxic side effects on the bovine feeder layer cells. Tests are now being developed to screen the effectiveness of trypanocidal drugs against T congolense and T vivax PRELIMINARY IMMUNIZATION TRIALS Recent studies both in vitro and in vivo indicate that metacydic forms of T congolense display a much smaller number of surface antigens than the bloodstream forms which develop in mammals. This smaller repertoire of surface antigens appears to remain constant when the parasites are transmitted back and forth between tsetse flies and cattle.

8 Thus considerable attention is being given to the analysis of protective host responses against metacyclic trypanosomes and to the possibility of immunizing livestock against this form of the parasite. Immunization trials have been initiated at ILRAD using metacyclic forms of T congolense produced in vitro and then killed and broken down by ultra sonication. In the first experiment, conducted in 1983, eight goats were inoculated with material derived from metacyclic forms of one T congolense serodeme (a group of closely related parasite populations) plus adjuvant, while another six goats were injected with adjuvants alone as controls. After immunization, blood samples were collected from all the animals. Tsetse flies infected with the same T congolense serodeme were allowed to probe these blood sample, and afterwards the blood was inoculated into mice. The blood taken from the goats used as controls did not neutralize the metacyclics transmitted by the infected flies: all mice inoculated with blood taken from the controls developed infections within 9 days. By contrast, none of the mice which were inoculated with blood taken from immunized goats became infected, showing that the blood from the immunized goats had neutralized the parasites transmitted by the tsetse flies. In the next experiment, the eight immunized goats, the six goats inoculated with adjuvants only and five other goats with no pretreatment were all exposed to tsetse flies infected with the same T congolense serodeme. None of the immunized goats became infected, while all the controls developed infection by the 16th day after exposure to the infected flies. The immunized goats were rechallenged with tsetse 2 months later and again they all resisted infection. These results show, for the first time, that trypanosome metacyclics produced in vitro and killed by ultra sonication can stimulate protective immunity against challenge with parasites of the same serodeme. To evaluate whether this approach would be effective in a field situation, parasites must be collected from the field, characterized and compared to see how many serodemes are present and whether immunization with one is likely to confer protection against others. In vitro derived T congolense metacyclics of different stocks and clones are now being compared at ILRAD by indirect immunofluorescence staining. PLANS FOR 1984 Trypanosomes raised in the laboratory are used increasingly in a number of research areas. This work involves collaboration between ILRAD's cell biologists and specialists in molecular biology, immunology, immuno parasitology, entomology and pathology. Among other projects, work in 1984 will concentrate on: 1. characterization of T congolense metacyclics derived in vitro from parasites isolated in different field locations 2. development of techniques to cultivate T vivax and T b brucei metacyclics in vitro, aimed at producing larger numbers of parasites at this important stage of development 3. improvement of techniques for screening the effectiveness of trypanocidal drugs and detecting drug resistance in different trypanosome strains. In the long term, the goal is to use trypanosome culture systems to identify vital metabolic pathways or parasite components which might be susceptible to intervention, either by immunization or by therapeutic means. ILRAD HOLDS CONFERENCE ON RUMINANT IMMUNOLOGY Immune responses come into play whenever an animal is challenged by disease whether the infection is caused by a virus, a bacterium or a parasite. In most cases, the immune system blocks the infection or eventually overcomes it so the animal stays healthy, or become ill but recovers.

9 In a general evolutionary context, immune responses have tended to favour accommodation of a stable host/invader relationship, rather than elimination of the invader, enhancing both the host's and the invader's chances of survival and allowing continuous stimulation of the immune system. However, many populations of domestic livestock are challenged with pathogenic organisms which, although not always fatal, are severely debilitating and limit productivity. The parasites which cause trypanosomiasis and East Coast fever (ECF), ILRAD's two target diseases, have developed mechanisms to evade normal immune responses, so an infected animal often becomes chronically ill or dies. Thus a major research area at ILRAD is the study of immune responses and ways in which they can be enhanced to overcome trypanosomiasis and ECF infections. Metacyclic forms of T congolense propagated in vitro and separated from other insect forms by passage through a DE52 cellulose column. These parasites were killed and inoculated into goats. When the goats were then challenged with the same T congolense serodeme, transmitted by tsetse, they resisted infection. Over the past 10 years, scientists in many countries have learned a great deal about immune responses and how they work. The most important gains have been made in understanding the immune responses of humans and laboratory animals. Many of these findings can be applied directly to studies of immune responses in cattle and other livestock. However, the immune systems of ruminant livestock differ in some respects from those of other animals. Given the rapid increase in information about immune systems, it seemed appropriate for ILRAD to sponsor an intetuational conference where specialists in basic and applied immunology could review recent research findings with participants from developing countries who are concerned with the role of immune responses in livestock disease. A Conference on the Application of Ruminant Immunology to the Control of Bovine Diseases was held at ILRAD from 26 to 30 September It was sponsored jointly by ILRAD, the Australian Centre for International Agricultural Research (ACIAR), the Commonwealth Foundation, The Ford Foundation, The Government of the Netherlands, and May and

10 Baker Limited. Eighty six participants attended from 17 countries in Africa, Asia and Latin America. Twenty three specialists were invited to present papers, and the participants also had the opportunity to present results from their own research. During the first part of the conference, scientists presented papers on the different elements of the immune system: how they interact, their structural organization and the nature of humoral and cell mediated immune responses. Most of the information presented was based on studies of laboratory animals and man. The second part of the program focused on ruminants, specifically on the ways immune responses in ruminants provide protection against different infectious diseases. The conference concluded with a discussion on possible directions for future research. While scientists know a great deal about the structure of ruminant antibodies, more studies are required on how humoral immune responses are induced and controlled and the relevance of these responses to pathogenicity and protection against disease. In the case of cellular immune responses, there is an obvious need for better characterization of the cell populations involved and their functions, especially related to protection against disease. The conference participants also emphasized the importance of identifying innate and acquired resistant traits to specific diseases in various breeds of livestock. With the recent development of new technologies such as monoclonal antibody production, lymphocyte cloning, embryo transfer and splitting, cell fusion and transfection rapid progress can be expected in increasing our knowledge of the ruminant immune system and devising better methods of disease control. SUMMARY OF PRESENTATIONS A number of papers were presented on the characterization of different cell populations within the immune system. In mouse and man, monoclonal antibody markers are available which define monocytes, B cells, T cells and subpopulations of T cells. For cattle, monoclonal antibodies are now available which identify different cell types, but no defined markers for T cell subsets are yet available. Cells in the human and mouse immune systems have also been defined by deriving cloned lines of lymphocytes. This work has provided a great deal of valuable information on the function and specificity of lymphocyte populations and could now usefully be applied to studies in ruminants. Several papers were also presented on the major histocompatibility complex (MHC). In cattle, alloantisera have been derived which define polymorphic determinants on class I MHC antigens coded by one locus. Using antisera against murine and human MHC antigens, it has also been possible to carry out initial biochemical characterization of bovine class I and II MHC molecules. Recently, rapid progress has been made using DNA technology to determine the number of MHC genes and characterize their polymorphism in man. This has been particularly useful for class II antigens which have been notoriously difficult to define in outbred species using alloantisera. A similar approach to typing class II antigens is now being applied successfully in sheep. Alloreactive cytotoxic T cells generated in vitro from bovine peripheral blood leukocytes (PBL) were shown to exhibit potent cytotoxic activity, specific for A locus MHC antigens. Alloreactive cytotoxic cells could be propagated in vitro for periods of several months. Derivation of continuously growing cloned populations of these cytotoxic cells could potentially provide an additional tool for investigation of the bovine MHC. The role of the MHC in determining susceptibility to disease was discussed and evidence was presented that defects in immune recognition at the level of the MHC may be involved in determining susceptibility in mice. Studies in sheep are now in progress to identify possible associations between MHC products and disease resistance.

11 Several papers dealt with cell mediated responses in cattle. Both non specific killer (NK) cells and cells mediating antibody dependent cytotoxicity (ADCC) can be demonstrated in bovine leukocyte populations. NK cell activity resides in the mononuclear leukocyte population in peripheral blood, whereas ADCC in cattle is mediated most potently by neutrophils and to a lesser extent by monocytes. Three papers were presented on the structural organization and foetal ontogeny of the immune system in cattle. The ruminant foetus is capable of producing antibody to certain antigens around mid gestation and thereafter progressively acquires the capacity to respond to a wider range of antigens. In foetal lambs, removal of Peyer's patches in utero resulted in a dramatic decrease in surface Ig positive cells, which suggests a role for Peyer's patches in the maturation of B cells. The final session was devoted to presentations on immunity in ruminants to specific infectious diseases, with emphasis on the mechanisms of immunity and the antigens involved. Examples of different viral infections were presented in which either hummoral or cell mediated immune responses are believed to be effective. Although NK cell and ADCC responses are readily demonstrable during some viral infections, the degree to which they participate in immunity in vivo is still unclear. In the case of bovine babesiosis, evidence has been obtained which indicates that antibody responses to the parasite are important in immunity, and monoclonal antibodies have been used to characterize and isolate parasite antigens which could potentially be used in immunization. Monoclonal antibodies have also been used in bovine theileriosis to study the repertoire of target cells which can become infected and transformed by the parasite. Theileria sporozoites can establish infection in a range of different lymphocytes of the T and B cell lineages. These different cell types may vary in their pathogenicity in vivo. With regard to immunity against theileriosis, evidence was presented that antibody and cell mediated immune responses may be operative against the sporozoite and macroschizont infected cell respectively. In trypanosomiasis, while antibody almost certainly mediates the final destruction of the parasites, the rate of induction of the antibody response appears to be dependent on other, as yet undefined, mechanisms which alter parasite metabolism and growth. Finally, intestinal nematodes were shown to induce a complex array of antibody and cell mediated responses. Both IgA and IgE antibodies are produced, and there is a marked local increase in eosinophils and mast cells with mast cell degranulation. A striking feature during the period of parasite expulsion is a change in the quantity and quality of mucus produced by the goblet cells. A component of host response during the final expulsion phase may thus be immunoiogically non specific. Thirty papers were presented at the conference. The full text of these papers will be published in a proceedings volume. ILRAD Reports International Laboratory for Research on Animal Diseases P O Box 30709, Nairobi, Kenya telephone: Nairobi telex: 'ILRAD'