African buffalo serum contains novel trypanocidal protein.

The high ability of African buffalo, as compared to domestic cattle, to control infections with Trypanosoma brucei brucei ILTat 1.4 organisms did not correlate with the timing or magnitude of parasite surface coat-specific antibody responses and may have resulted from the constitutive presence in buffalo blood of a novel trypanocidal factor. Buffalo plasma and serum contained material that killed bloodstream stage T. b. brucei, T. b. rhodesiense, T. b. gambiense, T. evansi, T. congolense, and T. vivax organisms during four h of incubation at 37 degrees C in vitro. Serum from eland was also trypanocidal whereas serum from oryx, waterbuck, yellow-back duiker, cattle, horse, sheep, goat, mouse, rat, and rabbit was not trypanocidal. The buffalo serum trypanocidal material was not lipoprotein, or IgG, and had the following properties: 1) a density of > 1.24 g/ml determined by flotation ultracentrifugation; 2) insolubility in 50% saturated ammonium sulphate; 3) non-reactivity with anti-bovine IgM, and anti-bovine IgG; 4) non-reactivity with protein G, and protein A; 5) a relative molecular mass of 152 kDa determined by chromatography on Sephacryl S 300, and of 133 kDa determined by chromatography of the 50% SAS cut of IgG-depleted buffalo serum on Superose 12; 6) no associated cholesterol; and 7) inactivation by digestion with proteinase K that was immobilized on agarose.

Isolation of Trypanosoma spp. from wild tsetse flies through procyclic expansion in Glossina morsitans centralis.

Procyclic trypanosomes from wild tsetse flies were membrane-fed to Glossina morsitans centralis in order to develop an optimal technique for propagating field isolates. A 70% success rate was achieved in isolating Trypanosoma simiae and a variety of genotypes of T. congolense originating from G. pallidipes, G. brevipalpis and G. swynnertoni. Parasites matured into forms infective for mammals, and could be maintained by passage of gut forms to new groups of flies. In experiments with laboratory stocks, we also passaged immature gut infections of T. congolense and T. brucei from various tsetse species to G. m. centralis. The optimal technique was investigated for procyclic T. congolense through addition of various compounds to goat blood using G. m. centralis and G. m. morsitans as recipients. From these experiments, many approaches to procyclic expansion appeared possible. However, a simple and practical method based on the use of fresh goat blood for rapid feeding of G. m. centralis is recommended. Application of this technique should aid in the resolution of questions relating to the cryptic diversity of Nannomonas trypanosomes in diverse host and vector communities.

Dynamics of host blood effects in Glossina morsitans sspp. infected with Trypanosoma congolense and T. brucei.

The pattern of infection in Glossina morsitans morsitans and G. m. centralis membrane-fed on eland, buffalo or goat blood mixed with Trypanosoma congolense or T. brucei was studied from day 1 to day 10. Tsetse were initially permissive vectors, with most flies harbouring infections of 10(4)-10(5) parasites on day 3. However, after a second blood meal on day 3, flies cleared many infections, with G. m. morsitans clearing more infections than G.m. centralis. Infective feeds of goat blood consistently increased final infection rates by limiting the number of infections lost between days 3 and 6. In further experiments with G. m. morsitans only, this effect was replicated by feeding flies on erythrocytes but not on serum. These results suggest that compounds from some mammalian erythrocytes match the target specificity of G. m. morsitans midgut lectins and, hence, have a protective effect on trypanosome establishment in the fly.

Relationships between protease activity, host blood and infection rates in Glossina morsitans sspp. infected with Trypanosoma congolense, T. brucei and T. simiae.

Midgut protease activity in Glossina morsitans centralis and G.m. morsitans, at 48 h post bloodmeal averaged 1.8IU of trypsin-like activity. These two tsetse subspecies differ in their susceptibility to trypanosome infection. Except for low levels in flies fed on waterbuck blood (0.7 IU), activity did not differ in flies fed a variety of host bloods (goat, pig, cow, buffalo, eland) and trypanosome species (Trypanosoma congolense, T. brucei, T. simiae). Protease activity was also not correlated with infection rates, despite large differences in infection rates among experiments. Nevertheless, addition of 0.06 M D(+)-glucosamine to parasitaemic blood resulted in a three-fold reduction in protease activity, coincident with a large increase in infection rate. This effect did not occur when parasites or D(+)-glucosamine were added alone to the bloodmeal, suggesting that the effect was due to metabolism of D(+)-glucosamine by parasites.

Cross-reactivity of workshop antibodies with cells from domestic and wild ruminants.

Reactivities of the monoclonal antibodies (mAbs) from the workshop panel with cells from cattle, sheep, goats, Cape buffalo (Syncerus caffer) and waterbuck (Kobus defassa) were tested. One hundred and sixty-nine mAbs reacted with bovine cells and 111 with sheep cells; 86 were shown to react with goat cells, 71 with buffalo cells and 70 with waterbuck cells. Some mAbs cross-reacted with all five ruminants tested, and are likely to react with epitopes that are conserved in other ruminant species. Such mAbs will obviate the need to produce mAbs panels to leukocyte antigens of other ruminants.

The influence of host blood on infection rates in Glossina morsitans sspp. infected with Trypanosoma congolense, T. brucei and T. simiae.

Trypanosoma congolense, T. brucei and T. simiae isolated from wild-caught Glossina pallidipes were fed to laboratory-reared G. morsitans centralis and G.m. morsitans to determine the effect of host blood at the time of the infective feed on infection rates. Bloodstream forms of trypanosomes were membrane-fed to flies either neat, or mixed with blood from cows, goats, pigs, buffalo, eland, waterbuck and oryx. The use of different bloods for the infective feed resulted in differences in infection rates that were repeatable for both tsetse subspecies and most parasite stocks. Goat, and to a lesser extent, pig blood facilitated infection, producing high infection rates at low parasitaemias. Blood from cows and the wildlife species produced low infection rates, with eland blood producing the lowest. Addition of D(+)-glucosamine (an inhibitor of tsetse midgut lectin) increased infection rates in most cases. These results indicate the presence of species-specific factors in blood that affect trypanosome survival in tsetse. In certain hosts, factors actually appear to promote infection. The nature of these factors and how they might interact with midgut lectins and proteases are discussed.

Disease research in the wildlife-livestock interface in Kenya.

Selected results of wildlife disease research in Kenya are given against the background of the socio-economic conflict in the wildlife/livestock interface. An attempt is made to rank the three areas of conflict between wildlife and livestock: feeding competition, disease control and predation. Disease survey results reveal the lack of wildlife reservoirs, with the exception of some important problem areas. Research on trypanosomiasis identifies a variety of adaptations evolved in wild Bovidae. The most striking result is the isolation of serum proteins from buffalo with trypanocidal activity against all common species of trypanosomes. The importance of wild Bovidae as reservoir hosts for theileriosis of livestock is discussed. The African buffalo presents the only known reservoir host of economic importance. The use of parasite stocks derived from buffalo has been effective to immunize cattle under field conditions in spite of the presence of an unknown number of antigenic types. The occurrence of common antigens indicated by successful immunization in the field was also confirmed by the recognition of common antigenic epitopes by cloned cytotoxic T cells. These results are encouraging for the plans afoot for large scale immunizations in Kenya. The co-existence of livestock and wildlife is threatened by declining profits and increasing costs for wildlife production and the absence of a general policy to encourage the full economic use of wildlife in areas where it competes with livestock.

A comparison of African buffalo, N'Dama and Boran cattle as reservoirs of Trypanosoma vivax for different Glossina species.

Teneral Glossina morsitans centralis were fed on the flanks of African buffalo, N'Dama or Boran cattle infected with Trypanosoma vivax IL 2337. The infected tsetse were maintained on goats and on day 25 after the infected feed, the surviving tsetse were dissected to determine the infection rates. The mean mature infection rates (% +/- S.E.) in the tsetse fed on buffalo, N'Dama and Boran cattle were 34.3 +/- 9.9, 33.7 +/- 13.4 and 58.9 +/- 7.1, respectively. Logistic regression analysis indicated that infection rates in the labrum and hypopharynx of the tsetse were significantly lower when fed on the infected buffalo or N'Dama than Boran cattle. Similarly, the risk of infection was significantly lower in male than female tsetse. When teneral G. m. centralis, G. pallidipes, G. p. gambiensis, G. brevipalpis and G. longipennis were fed simultaneously on either the buffalo cow, the N'Dama bull or the Boran steer infected with T. vivax IL 2337, the mature infection rates were higher in the two morsitans group than the two fusca group tsetse, whilst G. p. gambiensis was relatively refractory to the infection, irrespective of the host species on which they fed. Logistic regression analysis indicated that the infection rates in the labrum and hypopharynx were significantly different amongst the five tsetse species for each of the three infected host animals. Nevertheless, the trypanotolerant African buffalo and N'Dama cattle may serve as reservoirs of T. vivax infection as can trypanosusceptible Boran cattle.

Trypanosomiasis in the black rhinoceros (Diceros bicornis Linnaeus, 1758).

A black rhinoceros (Diceros bicornis) moved from a tsetse-free to a tsetse-infested area in Kenya was monitored for two months following translocation. The animal acquired a Trypanosoma vivax infection from natural tsetse challenge, but survived without requiring treatment with trypanocides. The infection was characterised by moderately high parasitaemia, with symptoms of anaemia, leukopaenia and thrombocytopaenia. Although confirmed to be T. vivax through deoxyribonucleic acid hybridisation and parasite development in tsetse in the proboscis only, the parasite had unusual morphology and motility. It also failed to infect normally susceptible hosts such as cows and goats, and produced unusually low infection rates in Glossina morsitans centralis and G. brevipalpis.

A comparison of African buffalo, N'Dama and Boran cattle as reservoirs of Trypanosoma congolense for different Glossina species.

Teneral Glossina morsitans centralis Machado were fed on the flanks of the African buffalo (Syncerus caffer Sparrman), N'Dama (Bos taurus L.) or Boran (Bos indicus L.) cattle infected with Trypanosoma congolense Broden. The infected tsetse were maintained on rabbits and on day 30 after the infected feed, the surviving tsetse were dissected to determine the infection rates. The mean infection rates (% +/- SE) in the midgut of tsetse fed on buffalo, N'Damas and Borans were 23.5 +/- 3.3, 31.6 +/- 2.7 and 33.7 +/- 4.6, respectively. The differences were not significant. However, the mean mature infection rate in tsetse fed on the buffalo (13.2 +/- 2.1%) was significantly lower compared to the rates in tsetse fed on the N'Dama (20.4 +/- 1.4) or the Boran cattle (21.4 +/- 1.1). When groups of teneral G.m.centralis, G.pallidipes Austen, G.p.gambiensis Vanderplank, G.f.fuscipes Newstead, G.brevipalpis Newstead and G.longipennis Corti were fed simultaneously on either an infected buffalo, an N'Dama or a Boran steer, the mature infection rates ranged from 0 to 16.1%. Irrespective of the host species used, the T.congolense infection rate was highest in G.m.centralis, lowest in the palpalis and fusca group tsetse, with G.pallidipes being intermediate. Nevertheless, the trypanoresistant African buffalo and N'Dama may serve as reservoirs of T.congolense as can trypanosusceptible Boran cattle.

The influence of buffalo and bovine serum on transformation of Trypanosoma congolense from metacyclic forms to bloodstream forms in vitro.

The transformation of Trypanosoma congolense metacyclics into bloodstream forms was studied in vitro using light and electron microscopy. The trypanosomes were maintained in culture at 28 degrees C using a medium supplemented with foetal bovine serum. The metacyclics were harvested and separated from the procyclic and epimastigote forms by using anion exchange chromatography. Isolated metacyclics were incubated with different concentrations of buffalo or bovine serum at 35 degrees C for periods ranging from 3 hours to 72 hours. It was noted that the metacyclics transformed at about the same time regardless of the animal serum used. However, most of the buffalo serum tended to kill trypanosomes whereas bovine serum tended to support trypanosome growth. The killing of the trypanosomes may be due to natural toxins present in buffalo serum.

Pathogenicity of tsetse-transmitted Trypanosoma congolense for waterbuck (Kobus defassa) and Boran cattle (Bos indicus).

Five waterbuck (Kobus defassa) and four Boran cattle (Bos indicus) were infected with Trypanosoma congolense IL2895 using Glossina morsitans morsitans. At the same time, two waterbuck and two cattle were inoculated intravenously with bloodstream forms. With both methods of challenge, cattle had short prepatent periods followed by a continuous high parasitaemia. All cattle became severely anaemic and had to be treated with trypanocidal drugs to prevent death. In contrast, tsetse and intravenous challenge of waterbuck resulted in a long prepatent period, followed by brief, intermittent levels of low parasitaemia, and eventual selfcure. Waterbuck did not become anaemic, even during short bouts of parasitaemia which in general were very low. Both cattle and waterbuck developed parasite-specific antibodies, but some waterbuck failed to develop neutralizing antibodies. These results suggest that the ability of the waterbuck to resist trypanosome infection may not be mediated entirely by antibody-dependent immune processes.

Infection rates in Glossina morsitans morsitans fed on waterbuck and Boran cattle infected with Trypanosoma congolense.

Teneral Glossina morsitans morsitans were fed on waterbuck (Kobus defassa) and Boran cattle (Bos indicus) infected experimentally with Trypanosoma congolense clone IL2895. Infection rates in tsetse varied from 9 to 31% when fed on cattle, and from 2 to 59% when fed on waterbuck. In waterbuck, infections were often detected through the development of parasites in tsetse at times when parasitaemia could not be detected through microscopic examination of blood. Male and female, and 1- and 2-day-old flies were equally susceptible to infection on both hosts. Infection in tsetse was associated with a 14% absolute reduction in survival during the month following the infective feed.

Susceptibility of African buffalo and Boran cattle to intravenous inoculation with Trypanosoma congolense bloodstream forms.

This study compares the susceptibility of African buffalo (Syncerus caffer) and Boran cattle (Bos indicus) to intravenous infection with T. congolense blood stream forms. The trypanosomes multiplied in the buffaloes and the Boran and reached levels of detectable parasitaemia 4 days after infection in the Boran and 10 days after infection in the buffalo. The cattle developed severe anaemia and had to be treated 60 days after infection to save them from dying whereas the buffaloes did not develop any signs of anaemia and did not require treatment. The Boran cattle showed high levels of parasitaemia persisting throughout the experimental period with some fluctuations. The parasitaemia in the buffaloes reached a peak of 5 x 10(3)/ml, 100 fold below the maximum level in cattle, it was intermittent and by the end of the experimental period (60 days), 3 out of 4 buffaloes had eliminated the parasites from circulation. Neutralizing antibodies were detected at the time of peak parasitaemia or soon after the 1st peak parasitaemia in buffaloes whereas in the Boran cattle neutralizing antibody could not be detected until after several peaks of parasitaemia. Neutralizing antibody persisted both in the Boran and buffaloes until the end of the experimental period.