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Trypanosomiasis


Towards improved control of the parasites
Antigenic variation: the genetic basis
Antigenic variation: synthesis and maintenance of the surface antigens
Cultivation of trypanosomes in vitro
Detailed studies of the parasite lifecycle
Towards improved host responses
Immune responses
Other host responses
The local skin reaction
The role of the lymphatic system
Control of parasites in the bloodstream
Effects of trypanosomiasis on the central nervous system
Resistance to trypanosomiasis in livestock and wildlife
Epidemiology
Characterization of trypanosome populations
Studies on tsetse flies as trypanosome vectors
Epidemiological survey at the Kenya coast
Collaborative epidemiology network

Trypanosomiasis is a serious, often fatal, parasitic disease which occurs in large areas of Africa, Latin America, the Middle East and Asia. It affects most species of domestic livestock, many types of wild animals, and man. In Africa, trypanosomiasis is found over approximately one-third of the continent, as shown in Figure 14.

Figure 14. Major cattle-production areas and tsetse-infested zones in Africa. There is little overlap except in the regions of West and Central Africa where trypanotolerant N'Darna and West African Shorthorn cattle are kept.

Trypanosomiasis is caused by single-celled parasites which primarily invade the bloodstream. Different species of trypanosome infect different host species. The most important trypanosomes in economic terms are the tsetse-transmitted species which infect cattle, sheep and goats in Africa—Trypanosoma congolense, T vivax and T brucei sspp. Trypanosomiasis research at ILRAD focuses on these three species.

In Africa, trypanosomes are usually transmitted by tsetse flies (Glossina spp) and occasionally by other biting flies. About 30 species and subspecies of tsetse are found in Africa. All of these are capable of transmitting trypanosomiasis, but some are more important vectors than others. In many parts of the world, trypanosomiasis is transmitted by other biting flies or occasionally sexually. Trypanosome species which are not transmitted by tsetse flies, such as T evansi cause significant livestock disease in Africa, Asia and certain areas of Latin America.

Figure 15 summarizes the lifecycle of the three main African trypanosome species. The parasites are ingested by tsetse flies when they feed on the blood of infected animals. They undergo a cycle of development in various locations within the fly depending on the trypanosome species. All three species finish this phase of their development as metacyclic forms in the tsetse mouth parts or salivary glands.

Figure 15. Life cycle of T b brucei, T congolense and T vivax. Heavy outlines indicate parasite forms with surface coats consisting of variable glycoprotein antigens. Light outlines indicate uncoated forms which are not infective to mammals. T b brucei develops in the tsetse midgut, proventriculus and salivary glands, where metacyclic forms occur which are infective to mammals. T congolense develops in the tsetse midgut, proventriculus and mouth parts, where infective metacyclic forms are produced. T vivax develops in the tsetse mouthparts.

The metacyclic trypanosomes are transmitted into an animal's skin when an infected tsetse feeds. In the case of T b brucei and T congolense, a prominent swelling, called a chancre, develops at the site where the parasites are introduced. The trypanosomes develop in the chancre: in T congolense infections a local reaction form can be distinguished at this stage. The parasites then invade the local lymph vessels and move into the bloodstream where they undergo further development. The infection which follows is characterized by successive waves of parasitaemia, as parasite populations multiply rapidly and die off. T vivax and T b brucei parasites may also invade the connective tissues, and, in later stages of the disease, T b brucei may be found in the central nervous system. In the human disease, African sleeping sickness, T b rhodesiense or T b gambiense parasites invade the central nervous system, causing death.

Trypanosomiasis in livestock causes anaemia, poor growth, infertility, and abortion. Many infected animals die. Others may eventually eliminate the parasites and recover, but their growth and productivity are often reduced.

At present, trypanosomiasis is controlled primarily by spraying tsetse resting and breeding sites with insecticide and by treating livestock regularly with trypanocidal drugs. However, neither of these approaches is completely effective in areas of heavy infestation. Further problems include the repeated use of the few drugs available, which may lead to drug resistance, and the possibility of environmental pollution due to large-scale insecticide spraying.

The goal of ILRAD's trypanosomiasis program is to develop effective, safe and economic methods to control the disease. This could be by immunological, chemical or genetic means. Research concentrates on the development of control measures which interfere with the parasites themselves or improve the responses of infected animals. Scientists are also studying aspects of trypanosomiasis epidemiology at a field site near the Kenya coast and in a collaborative animal production research network at sites in several African countries.

Towards improved control of the parasites

In the course of an infection, successive generations of trypanosomes display different antigens on their surface coats. These antigens are variable surface glycoproteins (VSGs). Trypanosomes with different VSGs are called variable antigen types (VATs). Trypanosomes in the tsetse fly are not coated with variable antigens until they develop into the metacyclic forms which are transmitted to mammals. Metacyclic trypanosomes are thought to display a limited number of VSGs, while the parasites which develop in the bloodstream may display very large numbers, thus evading the immune responses of the host.

In 1982, research on trypanosomes concentrated on the genetic basis of antigenic variation and on the structure and synthesis of the variable antigens. Other studies focused on the identification of specific variable antigens and on their role in the course of infection. Techniques developed earlier to cultivate and maintain trypanosomes in vitro were extended as progress continued towards the goal of maintaining all three major trypanosome species on a continuous basis. This technology has made it possible to study the different stages of the trypanosome lifecycle in detail as well as the mechanisms involved in the transition from one stage to the next. The goal of all these projects is to identify factors which could be manipulated to disrupt parasite development.

Antigenic variation: the genetic basis

Antigenic variation involves switching on and off the expression of a large family of genes which code for different VSGs. Molecular biologists at ILRAD are studying the changes which occur when different genes are expressed. This work is being carried out in collaboration with Cambridge University's Molteno Institute, the Université Libre de Bruxelles and the Vrije Universiteit Brussel.

The expression of different VSGs during the course of infection is associated with rearrangements in the parasite DNA. Two types of gene rearrangement have been observed in T b brucei. Sometimes the expression of a VSG gene is accompanied by the appearance of a duplicated copy of the gene at a new location in the genome. The extra copy disappears when the gene is no longer expressed. This type of rearrangement has been termed 'expression-linked duplication'. When scientists at ILRAD cloned parasites from one T b brucei stock at different times, they also found three different VSG genes which were not duplicated when their antigens were expressed. This indicates that a second type of gene rearrangement can occur in association with antigenic variation. The different type of gene rearrangement is related to the specific VSG gene being expressed, not to general differences between typanosome stocks.

Scientists have been studying four VSG genes from the ILTaR 1 antigen repertoire. Of these, only one (ILTat 1.1) undergoes rearrangement involving expression-linked duplication. There are four distinct copies of the ILTat 1.3 gene and three of the ILTat 1.4 gene in each member of a closely related group of trypanosome clones, and the number of copies of these genes does not vary with expression. One copy of the ILTat 1.3 gene has been characterized in detail and compared with known ILTat 1.3 messenger RNA. Beyond a homologous region, the two sequences differ completely, indicating that this copy gene cannot code for a normal VSG.

Rearrangements have been detected in the genomic environment of some, but not all, copies of these two genes. For those copies which do undergo rearrangement, ILRAD scientists have found that clusters of enzymes cut the DNA at a site which is probably at the end of a chromosome. Figure 16 shows eight restriction enzyme fragments of DNA during a period of treatment with the enzyme Bal 31 exonuclease. Two fragments (12.0 and 7.2 kilobases) have been digested by the enzyme, while the others have not. This indicates a natural double-strand break in the DNA at this site.

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Figure 16. Bal 31 digestion of T b brucei nuclear DNA: 40 µg of DNA was digested with 13 units of Bal 31 enzyme in 600 mM NaCl, 12mM CaCl2,, 12 mM MgCl2, 20 mM Tris-HC1 pH 8 and 1 mM EDTA. The reaction was incubated at 30°C and aliquots were taken at times shown by pipetting into water-saturated phenol. Each DNA was precipitated with ethanol and subsequently digested with Aval restriction endonuclease. The digested DNAs were electrophoresed in a 0.8% agarose gel and transferred to nitrocellulose paper. The plasmid pcBC1 containing the complete coding sequence for VSG ILTat 1.3 was nick-translated and hybridized. The figure shows the results of the blotting experiments: only fragments 12.0 and 7.2 kilobases were digested by the enzyme.

Thus two copies of the ILTat 1.3 gene are both adjacent to the end of a DNA molcule. These copies might be located on unusual extra chromosomal DNA structures. To test this possibility, DNA was prepared from three trypanosome clones expressing VSGs ILTat 1.2, 1.3 and 1.4 and fractionated on sucrose density gradients. Figure 17 shows the distribution of the four copies of the ILTat 1.3 gene in one of the gradients. Copy D is located on fractions 16 and 17 and has been clearly separated from the bulk of the nuclear DNA. It appears that this copy is located on a minichromosome—a DNA molecule that is separate from, and smaller than, the bulk of the trypanosome DNA. This small molecule is present whether the ILTat 1.3 gene is expressed or not. It may be some form of intermediary in the normal nuclear gene rearrangement associated with antigenic variation.

Figure 17. Location of ILTat 1.3 genes in fractions from intact trypanosome DNA sedimented through a sucrose density gradient. Trypanosomes homogeneously expressing different VSGs were isolated by Percoll density gradient centrifugation. The trypanosomes were lysed by adding SDS to 0.5%. The lysate was incubated for 2 h with 100 mg ml–1 RNase A (previously heated to 70°C for 60 min), then for 4 h with 1 ml–1 of Pronase (preincubated at 37°C for 2 h), both at 37°C. The extremely viscous DNA solution was layered onto a 10–40% sucrose gradient in 10 mM HCI, 5mM EDTA pH 8.0 and centrifuged at 24 000 rpm for 10 h. The gradient was fractioned with an ISCO gradient fractionator (30 × 1.2 ml fractions). The position of the ILTat 1.3 genes in the gradient was determined by a Southern blot analysis of DNA from alternate fractions digested with Hincll, using pcBC1 as a probe. Tracks marked × are digests of unfractionated DNA. A, D, B and C mark the Hincil fragments from the four distinct copies of the ILTat 1.3 gene. Copy D is purified at fraction 16, while the other copies are purified at fraction 30. This shows that copy D is smaller and can be separated from the others.

Antigenic variation: synthesis and maintenance of the surface antigens

The surface antigen coat is probably the only component found in common on all African trypanosomes. The integrity of this structure is crucially important to the survival of the parasites in the host bloodstream. For this reason, biochemists at ILRAD are investigating how the VSGs are synthesized in the parasite and exported to the cell surface. A thorough knowledge of this process may suggest some form of interference which will disrupt the stability of the surface coat and render the trypanosome susceptible to host defence mechanisms.

Previous work at ILRAD and elsewhere has shown that T b brucei and T congolense VSGs contain two different types of carbohydrate (CHO) side chains, added to the protein by a process called glycosylation. The first is constructed in a region of the parasite cell called the endoplasmic reticulum (ER) shown in the simplified diagram of a trypanosome (Figure 18). It is attached to the protein by a linkage found in mammalian and other systems, and its addition can be blocked by the antibiotic tunicamycin. The other CHO side chain is linked near the end (C-terminal) of the protein by a mechanism which has not yet been described in any system and which cannot be blocked by tunicamycin. Its importance is suggested by evidence that it remains constant for all VSGs of T b brucei and T congolense.

Figure 18. Bloodstream form of T congolense. Derived from K Vickerman, 1969, Journal of Protozoology, 16: 56.

 

Figure 19. Electron micrograph of T vivax rough endoplasmic reticulum (a), smooth endopiasmic reticulum (b) and Golgi (c). Endoplasmic reticulum and Golgi fractions were isolated from total microsomes obtained from T b brucei, T congolense and T vivax and tested for glycosyltransferase activities. Trypanosomes were disrupted in 250 mM sucrose-HKMM (50 mM HEPES, 25 mM KCI, 5 mM MgSO4, 10 M MnC12; pH 7.4 at 5°C). Crude microsomes were prepared and a Golgi fraction isolated essentially after the method of Redman et al, 1975 (Journal of Cell Biology, 66: 42). Both smooth and rough endoplasmic reticulum fractions were prepared from the residual microsomal fractions according to the method of Ragland et al, 1971 (Biochemical Journal, 121: 271).

Figure 19(b).

It is more difficult to purify VSG from T vivax than from T b brucei or T congolense using available methods. However, ILRAD scientists have recently purified a VSG-like protein from a T vivax clone and are now conducting studies to determine how this molecule(s) compares biochemically and immunologically with VSG isolated from the other trypanosome species.

The synthesis of trypanosome VSG involves three subcellular organelles: the protein molecules are first synthesized on the rough ER and are then transported by the smooth ER to the Golgi apparatus. The CHO side chains are added during this process. The function of the side chains is unknown, but they may play a role in bringing the glycoprotein to the membrane surface. They may also enable the Golgi apparatus to recognize the VSG. This is important because the Golgi processes and sends glycoproteins to a variety of other cellular locations.

In 1982, ILRAD biochemists purified fractions of Golgi and rough and smooth ER in order to identify which of these subcellular organelles is critical to the synthesis of the VSG. This work was carried out in collaboration with Harvard University.

Golgi and ER fractions were isolated from T b brucei T congolense and T vivax and checked for morphological purity by electron microscope examination. Figure 19(a) is an electron micrograph of a rough ER fraction from T vivax, showing spherical and flattened vesicles with irregularly arranged attached ribosomes. Clusters of free ribosomes, fragmented microtubules and other non-membrane material are also present. As shown in Figure 19(b), the smooth ER fractions from T vivax consist primarily of clear granular vesicles 30–200 nm in diameter. A few vesicles contain some flocculent material, but vesicles with attached granules are rare. All T vivax Golgi fractions contain flattened u-shaped, curved or cup-shaped cisternae with slightly expanded rims, as shown in Figure 19(c).

Figure 19(c).

The different glycosyltransferase enzymes involved in the process of glycosylation are being characterized biochemically. From total preparations of T b brucei, galactosyltransferase, mannosyltransferase and two different N-acetylglucosaminyltransferases have been characterized. Studies are now in progress to clarify the possible role of sialyl- and fucosyl-transferases using the purified ER and Golgi fractions. The glycosyltransferase activities observed so far have been remarkably similar in all fractions of the three major trypanosome species. The Golgi fractions contain the majority of the galactosyltransferase activity, followed by the smooth and then the rough ER. The dolicholdependent mannosyltransferase activity was highest for rough ER, then for smooth ER and then for Golgi.

The dolichol-independent N-acetylglucosaminyltransferase activity was similar in all the fractions tested, while the dolichol-dependent N-acetylgluco-saminyl- transferase activity was much higher for ER than for Golgi, and somewhat higher for smooth than for rough ER. This enzyme is sensitive to tunicamycin inhibition in smooth ER, but not in rough ER or in Golgi, which suggests that it may take two or more forms. It also suggests that the core carbohydrate may be added to the VSG in the smooth ER. Experiments are currently in progress to see if these membrane fractions can process a mature VSG in vitro.

Another perspective on the factors involved in the synthesis and maintenance of trypanosome VSGs is provided by the stage of development when VSG synthesis stops. This occurs when bloodstream trypanosomes change into uncoated forms in the midgut of the tsetse fly. A culture system is available in which an entire population of bloodstream trypanosomes can be induced to transform into the fly midgut stage in 2 days. The termination of VSG synthesis is being investigated by incorporating radioactive amino acids into newly synthesized protein and looking for rearrangements of the genes encoding for VSG by gene probe hybridization to Southern blots. This method is being used to analyse trypanosomes in the process of shutting off VSG synthesis, as well as trypanosomes many generations after VSG synthesis has ceased.

Cultivation of trypanosomes in vitro

Cell biologists at ILRAD have made steady progress in the development of techniques to propagate the various stages of T b brucei, T congolense and T vivax in vitro. In collaboration with other scientists, these techniques have been used to study the biological, biochemical and immunological properties of the parasites. A great deal of trypanosomiasis research is now possible which could not be carried out in the past when trypanosomes could only be maintained by continuous passage through tsetse vectors and mammalian hosts.

Beginning with T b brucei, techniques have been developed to propagate and maintain trypanosomes in cell culture throughout all the stages of their life cycle (see Figure 20). Recent work has focused on extending these techniques to the cultivation of T congolense and T vivax. Once a system for maintaining trypanosomes is proven successful on an experimental basis, it can be standardized and used to produce large numbers of parasites which are required for many different research projects.

 

T b brucei

T congolense

T vivax

Bloodstream forms (coated trypomastigotes)

yes

up to 35 days

up to 7 days

Procyclic forms (uncoated trypomastigotes)

yes

yes

yes

Epimastigotes

Yes

yes

yes

Metacyclics

Yes

yes

no

Complete lifecycle on continuous basis

Yes

no

no

Figure 20. Stages of trypanosome development which can now be maintained in vitro.

Using the culture system established for T b brucei, two simple in vitro tests were developed in 1982 to screen the effectiveness of trypanocidal compounds. The trypanocidal activity of a compound can be expressed in terms of the minimum drug concentration required to inhibit trypanosome growth by significantly more than 50% during 24 hours' exposure. Nine compounds were screened in 1982, with results shown in Figure 21. The tests being developed also measure any toxic side-effects on the bovine feeder cells included in the culture system. These two drug-screening procedures are now being applied to T b brucei field isolates. Tests are also being developed to screen the effectiveness of trypanocidal drugs against T congolense and T vivax. This work is carried out in collaboration with the Kenya Trypanosomiasis Research Institute and the Gesellschaft für Technische Zusammenarbeit (GTZ) of the Federal Republic of Germany.

Compound (trade name)

Supplier

MIC50 (g/ml)

Diminazene aceturate (Berenil)

Farbwerke Hoechst

1.00

Isometamidium (Samorin)

May 8 Baker

10.00

Phenanthridinium bromide (Novidium)

Boots

3.00

Quinapyramine dimethylsulfate (Antrycide)

Imperial Chemical

0.10

Melarsporol B.P. (Arsobal)

La Specia

0.01

Suramin (Naganol)

Bayer

1.00

α-difluoromethylornithine

Merrell-Dow

300.00

4', 6-diamidino-2-phenylindole

Serva

1.00

Quinapyramine isethionate

May & Baker

1.00

Figure 21. Minimum inhibitory drug concentration (MIC50) required to inhibit in vitro growth of T b brucei bloodstream forms by significantly more than 50% during 24 h exposure.

The method of propagating T congolense metacyclics in vitro has been simplified and improved. Cultures are now initiated with bloodstream forms taken from infected rodents and raised in the presence of purified bovine dermal collagen. Six populations of T congolense parasites are maintained on a continuous basis, four from clones and two derived from uncloned stocks.

Work with T vivax has concentrated on two stages of parasite development: the surface-coated trypomastigote which develops in the bloodstream and the uncoated trypomastigote which is presumably equivalent to the next stage of development after the trypanosome is ingested by a tsetse fly. A culture system was tested in 1982 which maintains the coated bloodstream form for several days and a new system was developed in which coated parasites are induced to change to the uncoated form.

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Confirming results of previous research, bloodstream forms of a West African T vivax stock (Zaria Y 486 derivative) were isolated from a goat and maintained in vitro in collaboration with the Swiss Tropical Institute. The parasites multiplied in culture and showed the T vivax bloodstream-form morphology. Best results were obtained when Microtus montanus (mountain vole) whole embryo fibroblasts were used as the feeder layer. A similar effort to propagate two East African T vivax stocks (ILRAD 1875 and KETRI 2377) in culture has not yet been successful.

Bloodstream trypomastigotes from the West African T vivax stock can now be taken from infected mice, maintained in culture and induced to change to the uncoated form. After being kept at 25°C for 3 days, the parasites are no longer infective to mice; examination with the electron microscope reveals that they lack a surface coat. Using this method, 109 or more of the uncoated forms can be produced at a time.

Several interesting findings have resulted from this work. For one thing, scientists at ILRAD have now observed rapidly dividing short-slender forms of T vivax with the kinetoplast at the posterior end during periods when parasite populations are rising (see Figure 22) and non-dividing elongated-slender forms with the kinetoplast midway between the posterior end and the nucleus during periods of peak parasitaemia (see Figure 23). Short tadpole-like forms have also been observed which are possibly senescent. These can also be seen in Figure 23. This pleomorphism is more pronounced when T vivax is raised in mouse blood than in goat or cattle blood. In vitro experiments have shown that the short-slender forms are more easily maintained in culture than the elongated-slender forms. However, the elongated forms change more readily in culture into the uncoated stage.

Figure 22. Bloodstream forms of T vivax (ILRAD 1392) during rising parasitaemia in a mouse. There are many dividing forms (D). Note the position of the kinetoplast, indicated by the arrows, at the posterior end

Figure 23. Bloodstream forms of T vivax (ILRAD 1392) in a mouse at the peak of parasitaemia. Note the pleomorphism: some parasites are elongated (L) with the kinetoplast midway between the nucleus and the posterior end, while others are shorter (S), with the kinetoplast at the posterior end, or tadpole-shaped (T).

Figure 24. Uncoated forms of T vivax (ILRAD 1392) transformed from the bloodstream form at 25°C in vitro. Under these conditions, they attach to the surface of matrix Green Gel A beads (Amicon) within 3 days. These parasites are noninfective to mammalian hosts.

When uncoated T vivax trypomastigotes were cultured with Matrex Green Gel A beads (Amicon), many of them became attached to the beads at their anterior end, as shown in Figure 24. These parasites probably attach themselves in a similar way to the mouth parts of tsetse flies. Uncoated forms of other trypanosome species do not become attached to the gel beads in culture, so this difference in behaviour might be used to identify T vivax in vitro. When uncoated T vivax trypomastigotes transform further to the shorter epimastigote forms, those parasites which were attached to beads survive longer than those which were not. The T vivax culture system is now being developed further in an effort to support transformation to the metacyclic stage and back to the bloodstream form, thus completing the parasite lifecycle.

Work was initiated in 1982 on the biochemical characterization of forms of T vivax, T congolense and T b brucei propagated in vitro. This work has been undertaken in collaboration with a visiting scientist from the University of Ibadan in Nigeria.

Detailed studies of the parasite lifecycle

Metacyclic forms of T b brucei are injected in the bite of an infected tsetse fly, and change to long-slender forms which multiply rapidly in the mammalian bloodstream. Many of the long-slender parasites differentiate into intermediate and then short-stumpy forms which do not multiply and eventually die out. The parasite population usually rises and falls in a series of waves associated with this rapid multiplication and differentiation to the senescent form.

It has been postulated that a similar differentiation from a rapidly dividing to a non-dividing bloodstream form occurs in T congolense and T vivax, though the physical changes are less obvious. Significant evidence was obtained in 1982 that such a differentiation does occur in these two trypanosome species, indicating that this is an important phenomenon which might present a target for disease control.

Different stages in the parasite lifecycle are characterized by different DNA contents. Non-dividing cells contain a baseline amount of DNA, while in an actively dividing population many cells have double the baseline DNA content in preparation for cell division. Using ILRAD's fluorescence-activated cell sorter, two actively dividing forms of T b brucei, procyclic forms equivalent to those which develop in the tsetse midgut and long-slender bloodstream forms, were shown to contain more DNA than non-dividing metacyclic and short-stumpy bloodstream forms. An analysis of early and late T vivax bloodstream populations revealed a similar transition from a dividing state to a non-dividing state with less DNA. This analysis is now being extended to T congolense.

Other studies focus on the proteins which are specific to different stages of the T b brucei lifecycle. The aim is to identify proteins which could serve as targets for immunological or chemotherapeutic attack. In 1982, proteins were examined which are specific to the long-slender and short-stumpy bloodstream forms and to the uncoated procyclic form. The protein composition of whole parasites at these different stages was analysed using two-dimensional gel electrophoresis. Several differences appeared, but the background of thousands of unchanging proteins made it difficult to detect which changes were significant. For this reason, the nuclei of trypanosomes in the three stages are being purified and analysed using the same procedure.

Many physiological processes similar to trypanosome differentiation are mediated through the action of a limited set of structurally related regulatory proteins which depend on calcium. Recently, ILRAD scientists detected one of these, calmodulin (CaM), in T vivax. This protein is now being purified and analysed biochemically. Preliminary evidence suggests that it is smaller than vertebrate CaM. Scientists elsewhere have found that CaM isolated from T b brucei is also smaller than vertebrate CaM. In addition, CaM-like activities in T b brucei, T congolense and T vivax do not cross react well with mammalian CaM when tested by radioimmunoassay. These are important findings because if CaM in fact plays a role in the differentiation process and if trypanosome CaM differs biochemically from the mammalian protein, then it may be possible to treat animals to inhibit this molecule and disrupt the parasite lifecycle without interfering with the host.

Trypanosome respiration and energy production systems have also been shown to change at different stages of the parasite lifecycle. Procyclic trypanosomes possess a fully functional mitochondrion and their respiration depends in part on the conventional cylochrome electron transport system. Bloodstream forms, however, appear to lack cytochromes; their energy is produced by glycolysis using an L-glycerophosphate oxidase (GPO) transport system. Studies in vitro have shown that respiration depends entirely on the -GPO system in bloodstream forms of T b brucei, T congolense and T vivax. The respiration of T b brucei procyclics raised in vitro depends in part (35%) on the cytochrome electron transport system and in part (65%) on the -GPO system.. For T congolense procyclics, 25% of respiration depends on the cytochrome system and 75% on the -GPO system, while respiration of T vivax procyclics raised in vitro for 4 days depends entirely on the -GPO system.

Towards Improved Host Responses

Several studies at ILRAD focus on the complex interactions between trypanosomes and their hosts. Research concentrates on factors which affect host antibody production and on other host responses which play a role in resistance to the parasites. A better understanding of these interactions could lead to several possibilities for improved disease control.

Immune Responses

When mice are infected with T b brucei, they produce specific antibodies directed against parasite VSGs, as well as a variety of unrelated antibodies. These antibodies are produced by one type of lymphoid cell, the B-cell. B-cells are stimulated to multiply soon after infection. This occurs well before antibodies are produced, particularly in susceptible mouse strains.

In 1982, five mouse strains were infected with a T vivax stabilate derived from West Africa. All mice of the most susceptible strain died within 10 days of infection, while none of the most resistant strain died within 10 days and 50% were still alive after 40 days. Specific immune responses were analysed using the immune lysis test. The most susceptible mouse strain produced no antibodies either specific to the parasite or non-specific, while the most resistant strain produced both specific and non-specific antibodies. Further experimentation showed that the susceptible mice were in fact capable of responding to parasite antigens. Spleen cells taken from the susceptible mice were analysed using the fluorescence-activated cell sorter, and their B cells were found to be dividing. Thus these cells, while stimulated to divide, are apparently inhibited from completing the final stages of differentiation into antibody secreting cells.

Studies in livestock have shown that immune responses to metacyclic trypanosomes are highly specific for particular parasite populations. When cattle or goats are infected by tsetse with metacyclic forms of T congolense or T b brucei and treated at least 15 days later with trypanocidal drugs, they resist rechallenge with the same trypanosome stock but are fully susceptible to challenge with different stocks of the same species. In 1982, further experiments were carried out to determine whether cattle would develop a similar resistance based on infection by intradermal inoculation, using metacyclic trypanosomes propagated in vitro. Cattle were infected in this way with metacyclic forms of T congolense. They were treated 60 days later with the trypanocidal drug diminazene aceturate (Berenil-Farbwerke Hoechst AG). Ninety days after treatment, they were challenged by inoculation with the same T congolense stock and were immune to reinfection.

Research was also carried out in 1982 to isolate and characterize the specific parasite VATs responsible for recurrent peaks of host antibody production. Three steers were infected with a T congolense stock (ILNat 3.1) and two steers with a stock of T b brucei (MITat 1.2). Blood samples were taken every day and examined for the presence of infecting VATs using the IFA test. A reappearing T b brucei VAT has been identified and is now being cloned for further study.

When animals infected with trypanosomes are presented with unrelated antigens, they produce lower levels of specific antibody than uninfected animals, or none at all. In many cases, the capacity of infected animals to respond to trypanosome VSGs is also reduced. This depression of the immune response is more marked in infected animals which are susceptible to trypanosomiasis than in infected animals which are more resistant. Experiments in 1982 showed that spleen cells taken from mice infected with T vivax or T b brucei produce considerably more prostaglandin hormones than spleen cells of uninfected mice. The concentration of prostaglandins produced by spleen cells taken from infected mice reaches levels which other investigators have shown are sufficient to reduce the immune response in living animals. Among infected mice, spleen cells from more susceptible strains often produce more prostaglandins than spleen cells from resistant strains. The addition to the spleen cell cultures of trypanosomes which were coated with antibodies or killed by freezing and thawing dramatically increases the amount of prostaglandins produced, especially when the spleen cells are from infected mice. These findings suggest that prostaglandin synthesis in animals infected with trypanosomes may contribute to the depression of the immune response.

The results of this study also suggest that dead or antibody-coated trypanosomes provide the fatty acid precursor for prostaglandin synthesis or provide an enzyme, such as phospholipase A2, which makes fatty acids available. If this is the case, the degree of immunodepression in an infected animal may be related to the number of parasites which multiply and die in the bloodstream. Further work concentrates on identifying the type of spleen cell which produces prostaglandins and the role of these cells in regulating parasitaemia and the immune response.

Other host responses

Host capacity to control parasite growth rates appears to be a key factor in resistance to trypanosomiasis. Scientists at ILRAD are studying host reactions which may limit trypanosome growth and development in the skin at the site of an infected tsetse bite, in the lymphatic system, and in the bloodstream. Studies also cover responses in the central nervous system at later stages of infection.

The local skin reaction

The chancre which develops in the skin at the site of an infected tsetse bite represents the host's first response to the parasite. When resistant wild animals are bitten by infected tsetse flies, they develop much smaller chancres than susceptible livestock. The prepatent period is also much longer before parasites can be detected in the blood. This suggests that the resistance of wild species is related to a capacity to control the parasites in the skin before they reach the bloodstream. Investigations at ILRAD concentrate on the possible role of the local skin reaction in conferring resistance, in particular on the structural changes and the distribution of parasites in the chancre and on the specific parasite material which induces the skin response. This work may indicate how parasite proliferation is controlled in the skin and may provide markers for genetic resistance.

In one study, cattle were challenged by tsetse flies infected with T congolense. Skin biopsies and blood were collected at regular intervals and anaemia and parasitaemia were monitored. The biochemical analysis of tissue taken from the chancres revealed a variety of proteins and peptides (see Figure 25). Some of these have been shown to be aminopeptidases, while others are as yet only partially characterized. It is not yet clear whether these chancre-associated proteins and peptides are of host or parasite origin. The next step will be to characterize chancre-associated proteins, peptides and other molecules from animals which are resistant to trypanosomiasis.

Figure 25. Reverse phase high performance liquid chromatographic (HPLC) analysis of bovine chancre-associated peptides. This chromatogram shows the resolution of 80% ethanol-soluble peptides and other small molecules obtained from chancre extracts taken on days 0, 7, 10 and 14 after cattle were challenged by tsetse infected with T congolense. The study reveals a progressive appearance of molecules with retention times (Rt) between 3.8 and 5.33 minutes, reaching a 50-fold increase on day 10. Two other major chancre-associated peptides with approximate Rts of 7.0 and 9.6 minutes show increases of 14- and 6-fold respectively.

In a related study carried out in collaboration with the Swiss Tropical Institute, goats and cattle were challenged by tsetse flies infected with the three major trypanosome species, and biopsies were taken of chancres at intervals after infection. Chancre formation in these animals will be compared with skin reactions produced in infected buffalo.

Goats were also inoculated intradermally with bloodstream forms of T vivax, T b brucei and T congolense. Using this method, it was possible to measure how many trypanosomes were injected in the skin and to rule out the effects of tsetse saliva. With T vivax and T b brucei, a skin reaction was produced which appeared identical to a chancre. The onset, size and duration of the skin reaction depended on the number of trypanosomes inoculated: as few as 102 T b brucei parasites were sufficient to produce a chancre, whereas 104 T vivax were required. With T congolense, no chancre developed even when 107 bloodstream parasites were inoculated. .

When an animal is bitten by an infected tsetse fly, uncoated procyclic trypanosomes may be injected into the skin along with the metacyclic forms. To test the possible role of procyclic trypanosomes in stimulating chancre formation, goats were inoculated intradermal with procyclic parasites. When 108 T b brucei or T congolense procyclics were inoculated, an intense inflammatory reaction was detectable during the first 3 to 4 days. Smaller numbers of parasites did not produce a reaction.

Another study is in progress to determine what parasite material stimulates the skin reaction. T congolense metacyclics have been collected from tsetse flies and placed in millipore diffusion chambers. Chambers with a pore size of 0.45. µm have been implanted under the skin of goats. A definite skin reaction occurs, similar to, but smaller than a chancre, despite the fact that the trypanosomes are unable to exit from the chamber and initiate infection.

The role of the lymphatic system

Trypanosome have been observed in lymphatic vessels in the region of the chancre and in enlarged lymph nodes draining the site of infected tsetse bites. In 1982, investigations continued on the role of the lymphatic system in the development of trypanosome infections.

Calves were challenged by tsetse infected with T congolense and the lymph nodes draining the resulting chancres were cannulated. Lymph flow was measured and the cellular and parasite content of the lymph was monitored. Trypanosomes were detected in efferent lymph 3 to 5 days before they were observed in the bloodstream, as shown in Figure 26. Antibodies specific to the infecting parasite clone were detected in both lymph and plasma 2 weeks after infection. Further work will concentrate on typing lymphatic cells in the chancre, the efferent lymph and the bloodstream and identifying which cell types respond to trypanosome antigen.

Figure 26. Parasitaemia in the lymph and blood of calves infected with T congolense. Trypanosomes were always detected earlier in lymph than in blood, but the peak levels of parasitaemia were 1 to 2 logs higher in the bloodstream.

Control of parasites in the bloodstream

For T b brucei and possibly for other trypanosome species, the level and duration of parasitaemic waves in the bloodstream appear to be related to parasite differentiation from an actively dividing to a non-dividing form. Earlier research has shown that some factor in the host plays a role in the timing of parasite differentiation and thus in the level of parasitaemia, because differentiation occurs at different times in different host species and strains, even when the parasite population is genetically identical.

The pattern of host resistance to T b brucei has been studied in detail in mouse strains which are relatively resistant or susceptible to trypanosomiasis. Trypanosome populations rise to a peak in the bloodstream as long-slender parasites divide. The level and duration of these parasitaemic peaks appears to depend on the timing of differentiation from rapidly dividing, long-slender parasites to the senescent short-stumpy forms. Studies in 1982 confirmed that trypanosome differentiation is slower in susceptible mouse strains. This slower differentiation is accompanied by a delayed host antibody response, but rapid parasite differentiation in resistant mice does not appear to be due to more efficient antibody production. Rather, host antibody responses seem only to be effective against the parasites after a number of short stumpy forms have appeared and the expansion of the trypanosome population has already slowed down substantially.

Clearance studies with 75Se-methionine labeled trypanosomes and adoptive transfer experiments have shown that antibodies produced against specific parasite VSGs do not destroy either long-slender or short-stumpy T b brucei parasites selectively, nor do they stimulate the parasites to differentiate to the short-stumpy form. Furthermore, parasite growth and differentiation rates were found to be the same in irradiated mice with or without reconstituted spleen cells, although antibody was detected in the spleen-cell-reconstituted mice and not in the others. This indicates that control of parasitaemia is not entirely dependent on the production of antibodies.

The development of T b brucei infections has also been compared in relatively susceptible 1-week-old calves and more resistant yearlings. As in mice, this study showed that resistance in cattle is apparently related to the ability to control parasite growth rates in the bloodstream and this in turn is related to the timing of parasite differentiation.

When mice were treated with Corynebacterium parvum, an adjuvant which enhances resistance to trypanosomiasis, and infected 4 days later with T b brucei, parasite differentiation occurred earlier than in controls. During the first 9 days after infection, peaks of parasitaemia were lower and of shorter duration in the treated mice, as illustrated in Figure 27. No difference was found in antibody production between the treated mice and the controls during the first 7 days after infection, which indicates that some other response was involved in the control of parasitaemia. The factors which regulated parasite growth and differentiation were partly radio-insensitive.

Figure 27. When C57B1/6 mice were treated with C parvum 4 days before challenge with T b brucei, the subsequent level of parasitaemia was significantly reduced.

Resistance to trypanosomiasis, as measured by survival, appears to be an inherited trait involving several genes. Specific host responses which contribute to resistance may be under less complex genetic control. To investigate this possibility, the genetic bases of factors regulating the growth of T vivax and T b brucei in the bloodstream were compared in susceptible and resistant mice. Random matings were established between a susceptible and resistant strain, and offspring were tested for their capacity to control parasitaemia. F1 crosses were then crossed with Fl s and bred back to both the parental strains. The results indicate that control of parasitaemia is based on a co-dominant gene system which is possibly fully as complex as the genetic basis of resistance measured in terms of survival.

To test the effects of host products on trypanosome survival, growth and differentiation rates, T b brucei parasites at different stages of development were maintained in a cell- and serum-free culture medium and exposed to a variety of substances they might encounter in the mammalian bloodstream. Without any additions to the medium, long-slender forms survived for 40 hours: during the first 5 hours they retained 100% infectivity for mice, from the 5th to the 20th hour they showed progressively less infectivity, and from the 20th to the 40th hour they developed into stumpy-like forms which were not infective for mice but were viable and had a surface coat. When moved to appropriate culture conditions, they differentiated to procyclic-like forms, showing that they were not irreversibly committed to degeneration. Intermediate parasite forms survived for 20 hours in culture and underwent changes similar to those occurring in the long-slender forms in their last 20 hours of culture. Short-stumpy parasites survived in culture for 4 to 5 hours, undergoing changes similar to those occurring during the last 10 hours of culture of the long-slender and intermediate forms.

The addition of transferrin, bovine serum albumin, foetal bovine serum, normal mouse plasma or normal mouse serum to the cultures all extended the life-span of the parasites, but had no effect on declining infectivity. Thirteen other substances were tested, including prostaglandins, insulin and epidermal growth factor, but these had no effect on parasite viability or infectivity for mice. Spleen cells from normal or trypanosome-infected mice extended the parasite life-span, stimulated the parasites to multiply and slowed down their loss of infectivity. These effects were more pronounced when spleen cells were taken from relatively susceptible mouse strains than from resistant strains, while spleen cells from mice of intermediate susceptibility were intermediate in terms of their capacity to extend parasite survival and infectivity.

In a related study, actively dividing T congolense parasites were cultured in vitro with cells taken from resistant and susceptible strains of mice. Subpopulations of mouse spleen cells and peritoneal exudate cells were used. As in the previous study, the trypanosomes survived less well when cultured with cells from relatively resistant mice. Trypanosome viability was also lower when the parasites were cultured with cells taken from mice previously injected with C parvum, as shown for the peritoneal exudate population in Figure 28. When trypanosomes were cultured with cells taken from cattle or buffalo, the results also reflected relative susceptibilities in vivo: T b brucei parasites survived equally well when cultured with cattle or buffalo cells, but T congolense cultured with buffalo cells did not survive well. These systems are now being used for a detailed investigation of the mechanisms involved in host resistance to trypanosomiasis and may be useful in screening to identify relatively resistant animals.

Figure 28. T congolense cultured with peritoneal exudate adherent cells from two mouse strains: A/J mice which are relatively susceptible to the parasite in vivo and C57BI which are relatively resistant. The cells of the A/J mice maintained T congolense in culture much better than the C57BI cells. This difference became more marked when cells were used from mice previously infected with the immunostimulator C parvum.

Effects of trypanosomiasis on the central nervous system

Cattle and goats infected with T b brucei, may develop a fatal illness characterized by clinical abnormalities in the central nervous system. This may occur during the initial infection or after treatment with trypanocidal drugs. Two projects were initiated in 1982 to investigate central nervous system involvement in T b brucei infecitions.

In one study, 12 cattle were infected with T b brucei or a mixture of T b brucei and T congolense. Sera and fluid were collected and analysed for the presence of antibodies specific to the trypanosome clones used. A slight rise in IgM antibodies was observed in the cerebrospinal fluid 2 months after infection.

The cattle given mixed infections developed abnormalities in the central nervous system and died. In 6 out of 8 of the cattle which died, a mild to moderate meningoencephalitis was apparent. Brain tissue from these animals is now being analysed for the presence of immune complexes.

In the second study, eight cattle were infected with T b brucei. Four of these animals died 4 to 6 months after infection. Despite mild anaemia and intermittent, low-level parasitaemia, they became very ill and emaciated and showed typical signs of central nervous system derangement as a result of severe meningo-encephalitis. The other four cattle were treated with the trypanocidal drug diminazene aceturate. They developed clinical signs after treatment and similar lesions were observed in their brains.

To investigate this syndrome further, 14 East African-Galla crossbred goats were infected with T b brucei. Unlike the cattle, they showed persistently high parasitaemia and developed severe anaemia. Three animals died 6 weeks after infection. The others were treated and all but one appeared to recover, but in the 3rd month after treatment half of the remaining goats developed anaemia with signs of central nervous system involvement, as shown in Figure 29. In all cases, trypanosomes were present in the cerebrospinal fluid, which also showed increases in protein and cell content. Brain tissue from these animals is being examined by electron microscopy to determine how the parasites invade the central nervous system.

Figure 29. Development of anaemia in goats infected with T b brucei and treated after 7 wks with diminazene aceturate. Fourteen goats were infected intravenously with 106 T b brucei (ILRAD 1797) and the course of anaemia measured by changes in packed cell volume (PCV). The figure shows mean PCVs up to 24 weeks after infection. Four goats died within 8 weeks, but after treatment the PCVs of the others returned rapidly to normal. However, beginning 9 weeks after treatment the infections in half the remaining goats relapsed. The average PCV of the relapsed group is shown by the broken line in the figure, while the continuation of the solid line shows the average PCV of the others.

Both studies confirm that T b brucei can be pathogenic for domestic livestock. In the second study, brain lesions were observed both after primary infections and after treatment with trypanocidal drugs. Parasites entering the central nervous system are resistant to the effects of many of the trypanocides commonly used to treat livestock because the drugs cannot cross the bloodbrain barrier. Thus relapsing infections which occur occasionally after treatment may not necessarily be the result of drug resistance or underdosage, but rather may be due to the transfer of parasites to the central nervous system and subsequent systemic reinfection.

Resistance to trypanosomiasis in livestock and wildlife

Comparative studies on host resistance to trypanosomiasis comprise an important research area at ILRAD. One goal is to find genetic markers which can be used to identify relatively resistant animals for livestock improvement programs. A better understanding of how some animals resist trypanosomiasis may also suggest promising interventions for increasing the resistance of others.

Previous work has suggested that indigenous East African livestock breeds may possess some genetic resistance to trypanosomiasis, though not to the same degree as the recognized West African trypanotolerant breeds. Studies are in progress to assess levels of resistance and determine the mechanisms involved.

In 1982, five groups of cattle of different breeds were infected with T congolense by intravenous injection. These were Boran and Ayrshire from tsetse-free areas of Kenya and three groups of Zebu from different tsetse-endemic zones. The Ayrshires were significantly more susceptible to trypanosomiasis than the other groups, followed by the Boran and the two Zebu groups from low tsetse-challenge areas, all with similar levels of susceptibility. The Zebu cattle from a high challenge area in western Kenya were generally most resistant, but this group showed considerable heterogeneity. Resistant animals controlled parasitaemia more effectively and developed less severe anaemia. The Ayrshire cattle produced less anti-trypanosome antibody than the other four groups. The other groups did not differ significantly in terms of antibody production, but antibody activity persisted longer in the more resistant animals. As the three Zebu groups had been exposed to trypanosomiasis previously, it was not possible to assess whether the resistance they demonstrated was due to innate or acquired factors.

In a related study, five groups of goats were challenged with T congolense by infected tsetse flies-East African goats, Galla and crossbreds of East African with Galla, Nubian and Toggenburg. No important differences were observed between these breed groups, either in the development of the chancre or in the timing, level or duration of the first parasitaemic wave. All groups developed severe anaemia by the 6th week after infection. Immune responses to the parasite, measured by immune lysis of bloodstream forms, were also similar for all groups.

Red Masai sheep from East Africa have been shown to resist trypanosomiasis better than imported Merinos. Parasite populations increase just as quickly in the Red Masai, but the resulting anaemia is milder. Studies are in progress to determine the reason for this relatively mild anaemia, but initial results have been inconclusive.

It has been observed for some time that wild animals resist trypanosome infection, but little is known about the mechanisms involved. ILRAD scientists have carried out several studies on resistance in East African wildlife species in collaboration with the Kenya Government's National Veterinary Laboratories, supported by the Government of the Netherlands.

To study the role of the local skin reaction and other responses to trypanosome infection, buffalo, oryx, waterbuck, eland, taurine cattle and East African goats were exposed to tsetse flies infected with T congolense, T b brucei or T vivax. Among animals exposed to T congolense, chancres appeared at 80 to 90% of infected bite sites in the domestic species and at 20 to 60% of bite sites in the wild species. The chancres which developed were also larger in the domestic animals. The prepatent period before parasites appeared in the bloodstream was generally much longer in the buffalo, oryx and waterbuck than in the goats and cattle, and the level of parasitaemia was lower in the wild species. All the animals showed some anaemia, as measured by reductions in packed cell volume (PCV), but only the domestic animals suffered from severe or chronic anaemia. These results, shown in Figure 30, suggest that the greater resistance to T congolense shown by the wild species may be related to better control of parasites in the skin and better control of anaemia.

 

% Skin reaction at bite sites

% Skin thickness increase

Prepatent period (days)

Maximum parasitaemia (parasites/ml)

% Drop in PCV

Buffalo

20

0

32

102-103

5

Oryx

20

0

27

104

20

Waterbuck

60

70

23

104

24

Eland

40

100

13

106

24

Cattle

80

200

13

106

37

Goats

90

300

9

106

50

Figure 30. Comparison of resistance to trypanosomiasis infection in wild and domestic animals, as shown by differences in local skin reaction, development of parasitaemia and level of anaemia. Animals were challenged by tsetse infected with T congolense.

The pattern of reactions was similar among animals exposed to T b brucei. Eland and waterbuck developed fewer chancres and less severe anaemia, but the differences between the wild and domestic species were less marked than for T congolense. It was not possible to infect the eland or water buck with T vivax using tsetse flies, though the animals were fully susceptible to intravenous challenge with bloodstream parasites. Further experimentation will determine whether this resistance to tsetse challenge is due to responses in the skin or to a resistance to metacyclic, as opposed to bloodstream, forms of the parasite.

When eland, buffalo, waterbuck and cattle were infected intravenously with bloodstream forms of T b brucei, the first wave of parasitaemia was similar in all the animals. However, after the first wave, parasite levels were considerably lower in the eland and buffalo than in the cattle and decreased gradually until parasites could no longer be detected. In the waterbuck, several parasitaemic waves occurred, similar to those observed in the cattle. However, anaemia was severe in the cattle, but very mild in all three wild species. Clearly, some factor limits parasite growth in the bloodstream of eland and buffalo, perhaps responses similar to those detected in resistant mouse strains.

In another study, wildebeest and Boran cattle were infected intravenously with bloodstream forms of T b brucei and their antibody responses were compared. Both the wildebeest and the cattle produced IgM, IgG1and IgG2 antibodies directed specifically against trypanosome antigens, but the cattle produced much lower levels of IgM than the wildebeest. The wildebeest produced antibodies in a pattern of recurrent peaks, while the cattle did not. The cattle were able to control parasitaemia within 2 months after infection, whereas the wildebeest were still slightly parasitaemic after 7 months, but the cattle became anaemic and the wildebeest did not.

A culture system has been developed which should make it possible to study these reactions in more detail. Cell lines were established from embryonic tissue taken from eland, impala, Grant's gazelle and goats. These cells lines have been tested in combination with different sera for their capacity to support the growth of bloodstream forms of T b brucei and T b rhodesiense. Parasites were maintained successfully with feeder layers of eland, impala and goat cells combined with sera from the same species, but not with cells of Grant's gazelle. Further experiments are in progress to investigate why the parasites could not be maintained with the gazelle cells. The success of the other culture systems indicates that tissue and sera from eland and impala have no innate factors which inhibit parasite growth. These results support the observation that the mechanism which enables eland to control trypanosome growth in the bloodstream is only stimulated after an initial parasitaemic wave.

In vitro studies have demonstrated that the enzyme polyamine oxidase, present in the bloodstream of wild and domestic ruminants, suppresses the growth of African trypanosomes in the presence of either spermine or spermidine. High levels of polyamine oxidase activity have been detected in sera from adult buffalo, cattle, eland, sheep and waterbuck, while none was detected in non-ruminant sera. Much lower levels of activity were observed in fetal bovine sera than in sera from adult cattle, but activity increased rapidly in newborn calves, reaching adult levels on the 21st day after birth.

Spermidine and putrescine were detected in bloodstream forms of T vivax and T b brucei and in procyclic forms of all three major trypanosome species. No spermine was detected. When bloodstream and procyclic forms of T b brucei were cultured with bovine serum, the spermidine they released was broken down by polyamine oxidase, forming aldehyde which gradually decomposed to putrescine. The amount of spermidine in the culture increased for 42 hours and then dropped off: at the same time, the population of trypanosome bloodstream forms increased for 42 hours and then decreased slightly (see Figure 31). This suggests that the toxic aldehyde produced from the breakdown of spermidine may suppress the growth of trypanosomes in culture.

Figure 31. Bloodstream forms of T b brucei were cultured in HEPES (25mM)-buffered RPMI 1640 medium with 20% fetal bovine serum at 28°C for 72 h. The number of trypanosomes reached a maximum concentration after 48 h and decreased slightly during the next 24 h. Likewise, the amount of spermidine released in the culture medium increased up to 48 h and decreased considerably during the next 24 h. The amount of putrescine, produced by the poiyamine oxidase activity of the serum, increased steadily throughout the 72 h period.

Epidemiology

In many field situations in Africa, livestock are exposed to more than one trypanosome species and to a large number of genetically distinct parasite populations, called serodemes. The development of techniques to identify trypanosome species and serodemes is an important component of ILRAD's epidemiology program. The epidemiology program also includes studies on the role of tsetse flies as trypanosomiasis vectors. In addition, a field project is being conducted at the Kenya coast, and ILRAD scientists are participating in a collaborative research project on livestock productivity and trypanotolerance covering sites in several African countries.

Characterization of trypanosome populations

Scientists are developing techniques to identify trypanosome species based on the analysis of antibodies present in the blood of infected animals. Monoclonal antibodies have been raised against T congolense and T b brucei parasites and the antigens recognized by these antibodies have been isolated using immunoadsorbent columns. Antibodies against these purified antigens were readily detected by enzyme-linked immunosorbent assay (ELISA) in sera taken from cattle infected with the same trypanosome species, but not in sera from cattle infected with different species. Purified T vivax antigens are now being prepared using the same technique and antigens from all three species are being characterized biochemically. By detecting antibodies produced against these antigens, it is now possible to determine how quickly antibodies are produced after trypanosome infection and how long these antibodies persist in the bloodstream after an animal recovers. This approach could be extended to identify parasites in tsetse flies.

Within a trypanosome species, serodemes can also be identified by analyzing antibodies present in the blood of infected animals. Experiments using monoclonal antibodies and sera from infected rabbits indicate that metacyclic parasites of specific T congolense and T b brucei serodemes display a limited number of VSGs about 10 to 12 in the T b brucei serodemes studied and 4 to 6 in the T congolense serodemes. These metacyclic VSGs are characteristic and constant for each serodeme.

Cattle chronically infected with bloodstream forms of T congolense or T b brucei produce antibodies against metacyclic VSGs of the infecting serodeme, but not against metacyclic VSGs of other serodemes. This indicates that bloodstream forms produce VSGs which are the same or very similar to metacyclic VSGs of the same serodeme. This study is now being extended to T vivax. The long-term objective is to develop techniques which can be used to type trypanosome isolates and determine the number of serodemes present in any given field situation. The detection of specific antibodies against trypanosome metacyclics may also indicate whether animals under field challenge conditions have developed any immunity to the local serodemes.

Another system is being developed on an experimental basis to identify trypanosome species and serodemes by analysing parasite DNA. Two genes which code for specific VSGs have been cloned from two well characterized T congolense stocks. Double stranded DNA copies (cDNA) have been synthesized from these genes and cloned in bacteria. One of the cDNA clones is being used to probe T congolense isolates from various regions of East Africa, primarily by Southern blot hybridization, to identify specific serodemes.

A third approach involves priming animals by infection with one serodeme and treatment, and then infecting them with various field isolates and monitoring the appearance of chancres. Goats develop chancres when they are primed with one serodeme of T congolense or T b brucei and then challenged with isolates of different serodemes, but no chancre develops when they are challenged with an isolate of the same serodeme as the original infection. This approach has not yet been successful in identifying T vivax serodemes.

It has also proven difficult to prime experimental animals with more than one serodeme. When goats were infected with four T congolense serodemes at 4- or 12-day intervals, the initial infection blocked subsequent infections so the animals developed full resistance to the first serodeme only. This interference may be due to a nonspecific response to the first infection which subsequently limits parasite growth in the skin. Another finding was that goats or cattle which were bitten by tsetse flies three or more times developed striking immediate hypersensitivity reactions in the skin, as shown in Figure 32, whether the flies were infected or not. Scientists are investigating this reaction in more detail to evaluate whether hypersensitivity to tsetse saliva affects the transmission of trypanosomes.

Figure 32. The skin of a goat showing a severe hypersensitivity reaction 48 h after being bitten by an uninfected tsetse fly. This animal had been bitten on two previous occasions. There is extensive superficial necrosis of the epidermis and marked cellular infiltration of the dermis accompanied by severe oedema.

A special problem in trypanosomiasis epidemiology relates to the distinction between T b brucei which infects several species of domestic livestock, and T b rhodesiense, which infects humans. These two trypanosome subspecies are morphologically indistinguishable. Experiments have been carried out at ILRAD to identify T b brucei and T b rhodesiense infections by analysing parasite DNA with restriction enzymes. This work has been successful, but DNA analysis requires long and complex purification procedures which limits its usefulness under field conditions or for large-scale studies. For this reason, a simplified laboratory technique is being developed which can be used to identify different trypanosome species from samples of blood, tsetse saliva or isolated cells, as illustrated in Figure 33. Work is now in progress to develop a similar test which will distinguish the subspecies T b brucei and T b rhodesiense, and also T b gambiense, the other trypanosome subspecies which infects humans.

Figure 33. Simplified illustration of a molecular hybridization technique for parasite identification. Suspensions of whole trypanosomes of four different stocks—T b brucei ILTat 1.3, T b brucei ILTat 1.2, T b brucei LUMP 427 and T b rhodesiense ETat 1.10—were diluted to different levels and applied to nitrocellulose filters. The filters were processed by the Southern blot method and the trypanosomes were hybridized with a probe for T b brucei ILTat 1.3 DNA. The figure shows that hybridization occurs only with parasites related to the specific probe. The degree of hybridization increases (darker spot) as the number of parasites increases. The method should be useful to distinguish the presence or absence of trypanosomes of a specific genotype, provided there is a minimum of 104 parasites in the sample.

The infectivity of different parasites for different host species may be linked to survival in the host bloodstream. Successive generations of T b rhodesiense populations have been observed which vary in terms of their sensitivity or resistance to the cytotoxic effects of human serum in vitro. This could indicate a variation in their potential viability in the human bloodstream. The reversibility of this sensitivity suggests that it is mediated by some protein or proteins which are synthesized in response to changing genetic information. ILRAD scientists have searched for differences in protein configurations between serum-sensitive and insensitive T b rhodesiense preparations using two-dimensional gel electrophoresis. However, no consistent differences have been detected yet.

Scientists have also observed a reversible pattern of sensitivity to human serum in T b brucei parasites isolated from laboratory animals and livestock. A cow was infected with a stock of T b brucei (TREU 667) which had previously been sensitive to the cytotoxic effects of human serum. Parasites appeared in the animal's bloodstream and cerebrospinal fluid. In vitro tests showed that parasites from the bloodstream were sensitive to human serum, while parasites from the cerebrospinal fluid were not. This suggests that T b brucei parasites which resist cytotoxicity in human serum may have an affinity for the central nervous system. Goats were infected with another derivative of this stock and parasites isolated from the bloodstream, the cerebrospinal fluid and the brain tissue all resisted the cytotoxic effects of human serum. Material has been collected which will be used to investigate whether changes in parasite sensitivity might be related to the expression of different surface antigens.

Studies on tsetse flies as trypanosome vectors

Tsetse studies at ILRAD concentrate on exploring improved methods for trypanosomiasis control. For instance, the release of sterile male tsetse flies has aroused considerable interest as a potential method of tsetse eradication. The success of this approach is based on the fact that female tsetse normally mate only once in their lifetime, so if most or all of the female flies in an area mate with sterile males the population will be eradicated. However, the possible impact on trypanosomiasis epidemiology of releasing large numbers of sterile males has not been fully explored.

Investigations in 1982 focused on the infection rates and transmission characteristics of male tsetse flies sterilized by gamma irradiation. Sterile and fertile males of three species-Glossina morsitans centralis, G austeni and G tachinoides—were fed on a goat infected with T b brucei and their survival rates, mating performance and transmission characteristics were compared. Ninety percent or more of the flies exposed to gamma irradiation were found to be sterile, but their mating performance was similar to that of fertile males. Differences in trypanosome infection rates between sterile and fertile males were marginal, and there were no differences in transmission characteristics between the two groups. These findings indicate that the release of sterile males as part of a tsetse control program could increase the immediate risk of trypanosomiasis. Similar studies are now in progress to examine the transmission of T congolense and T vivax by sterile male tsetse.

Another study concentrated on the possible impact on tsetse infection rates of treating livestock with trypanocidal drugs. A population of G m morsitans was fed on a goat infected with T vivax.. The flies were then divided into three groups and each group was maintained on a different goat, one treated with Berenil, one treated with isometamidium (Samorin-May and Baker) and one untreated control. The experiment was repeated using goats infected with T congolense and T b brucei, and the survival, fecundity and infection rates of all the flies were compared.

No significant differences were detected in survival rates or reproductive performance, but infection rates were significantly reduced for all groups of tsetse maintained on goats treated with trypanocidal drugs, as shown in Figure 34. This suggests that a carefully managed drug treatment program could significantly reduce the cyclical transmission of trypanosomiasis in situations where tsetse are largely feeding on livestock.

Original infection

Number infected

Maintained on goats treated with:

Number survived

Infection rates in surviving tsetse (%)

T vivax

200

Berenil

142

2.1

T vivax

200

Samorin

82

0.0

T vivax

200

Control

131

97.7

T congolense

200

Berenil

79

19.0

T congolense

200

Samorin

50

14.0

T congolense

200

Control

28

46.4

T b brucei

200

Berenil

89

0.0

T b brucei

200

Samorin

102

0.0

T b brucei

200

control

109

22.0

Figure 34. Comparison of T vivax, T congolense and T b brucei infection rates in male and female G m morsitans infected as tenerals and then maintained on goats treated with Berenil or Samorin or on untreated control animals.

To support epidemiological research in the field, a serological system has been developed to identify the animal species on which tsetse flies have fed. Such a system has to be specific enough to differentiate closely related host species and sensitive enough to analyse even the minute quantities of blood which can be obtained from the flies. Antisera were prepared against 42 species of mammals and 4 species of birds. To test their specificity, bloodmeals were analysed from tsetse flies raised at ILRAD. Further work will concentrate on the production of species-specific monoclonal antibodies which could provide a steady source of standard antisera.

In collaboration with the Swiss Tropical Institute, scientists completed a study on changes in the ultrastructure and function of secretory cells in the tsetse uterine gland at different stages of pregnancy. Material taken from G m morsitans raised at ILRAD was examined in Basel by electron microscopy. This study demonstrated a temporal correlation between cellular dynamics and physiological events during pregnancy. The goal is to identify possible approaches to the control of tsetse populations by interfering with the reproductive process.

Epidemiological survey at the Kenya coast

An epidemiological survey was conducted in 1982 at a large dairy ranch near the Kenya coast. This location was chosen for an intensive study because the trypanosomiasis situation appeared fairly simple, with a low level of disease risk and little movement of livestock or wild animals.

A preliminary tsetse survey was carried out using bait oxen, biconical tsetse traps and searches for tsetse pupae and pupal cases. This survey revealed a small population of G austeni on the ranch in an area of dense riverine bush. Of 28 tsetse captured, 26 were dissected and 5 were found to be infected with trypanosomes: 2 with T vivax and 3 with T congolense. Four tsetse bloodmeals were analysed using antisera prepared at ILRAD. These were found to originate from a sheep, a cow, a warthog and a suni (Neotragus moschatus).

Blood samples were taken from a total of 5909 cattle on the farm. Of these, 1890 (32%) were anaemic, with a PCV of 30% or less. Twenty-five animals were found with trypanosome infections: 22 with T congolense, 2 with T b brucei and 1 with T vivax. Nearly all the infected cattle were kept near the area where the tsetse flies had been captured.

Twenty tracer cattle were brought to the tsetse-infested area and kept in an open pen. Fourteen of these became infected with T congolense about 22 days after exposure and were treated with Berenil. About 60 days after treatment, 16 animals became infected, including 13 reinfections and 3 first infections, and these were treated again. One animal became infected 3 times.

Collaborative epidemiology network

ILRAD scientists are collaborating with colleagues at ILCA and ICIPE in a research and training network which includes projects in 11 countries of West, Central and East Africa, as shown in Figure 35. ILRAD's role is to provide training and supervision for the animal health and tsetse survey components of the program.

Figure 35. Eleven countries in Africa where ILRAD is collaborating with ILCA and ICIPE in evaluating the impact of trypanosomiasis on livestock production. Major research sites are located in countries where trypanotolerant cattle are raised.

The main focus of the program is on the indigenous taurine cattle breeds of West and Central Africa, the N'Dama (shown on the cover) and West African Shorthorn, which are significantly more resistant to trypanosomiasis than Bos indicus or European Bos taurus breeds. This trait, termed trypanotolerance, has been shown to be an innate characteristic. Comparative studies on these and other breeds are in progress in Ivory Coast, Nigeria, Zaire and Gabon and will be extended in 1983 to The Gambia, Senegal, Togo, Congo and Benin. Carefully selected production situations are being monitored where different breeds of cattle, sheep and goats are being raised under well defined management regimes and levels of trypanosomiasis risk. Similar studies are under way in Kenya and Tanzania focusing on indigenous breeds of cattle, sheep and goats.

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