Production and evaluation of specific antisera against sera of various vertebrate species for identification of bloodmeals of Glossina morsitans centralis.

Specific antisera against sera of 46 species of vertebrates were prepared. The antisera to 21 Bovidae species were raised in goats except the antiserum to goat serum which was raised in sheep. The antisera to 3 Suidae species were produced either in domestic pigs or warthogs, while antisera to most of the other vertebrate species were raised in rabbits. The antisera were used in an enzyme-linked immunosorbent assay (ELISA) to identify the source of bloodmeals ingested by teneral and non-teneral tsetse at different time intervals after feeding. The bloodmeal donors were identifiable in 100% of the teneral tsetse up to 40 h post-feeding and in 87.5% in those tested up to 74 h post-feeding. Non-teneral tsetse digested the species-distinguishing bloodmeal components faster than the tenerals. Bloodmeals could be identified in 100% non-tenerals at 20 h post-feeding but only 67.5% and 50% of the bloodmeals could be identified 40 h and 74 h post-feeding, respectively. The antisera were also able to identify mixed bloodmeals from closely related species.

Study on the sequential tsetse-transmitted Trypanosoma congolense, T. brucei brucei and T. vivax infections to African buffalo, eland, waterbuck, N'Dama and Boran cattle.

Susceptibility of African buffalo, eland, waterbuck, N'Dama and Boran cattle to sequential Glossina morsitans centralis-transmitted infections of Trypanosoma congolense, T. brucei brucei and T. vivax was compared, and their possible role as reservoirs of these parasites for G. moristans centralis, G. pallidipes, G. austeni, G. brevipalpis and G. longipennis determined. The buffalo, eland, waterbuck and N'Dama controlled T. congolense parasitaemias and were able to prevent anaemia. By contrast, one Boran became severely anaemic whilst the other controlled parasitaemia and anaemia. When the above five species of Bovidae were rechallenged with T. brucei brucei they showed persistent parasitaemias but did not develop anaemia. The buffalo died of other causes. When the remaining four bovids were rechallenged with T. vivax they became infected with mixed T. vivax/T. b. brucei parasites. Eland, waterbuck and N'Dama controlled parasitaemias and anaemia whereas the Boran became anaemic. Cyclical development of T. congolense occurred in G. moristans centralis when fed on the bovid hosts, with buffalo being infective for tsetse flies for a much longer period. There was no relationship between the levels of T. congolense parasitaemia in the bovid hosts and the resultant infection rates in tsetse flies. Glossina m. centralis was more susceptible than G. pallidipes to T. brucei brucei whilst G. austeni the least; G. brevipalpis and G. longipennis were refractory to the mature infection. Again there was no relationship between T. brucei brucei parasitaemia levels in the hosts and infection rates in the flies. Glossina m. centralis and G. pallidipes showed mixed T. brucei brucei/T. vivax infections whilst G. austeni, G. brevipalpis and G. longipennis became infected with T. vivax alone. Tsetse flies showed higher T. vivax infection rates when fed on the hosts with high parasitaemias than thosewith low parasitaemias. Thus trypanotolerant African buffalo, eland, waterbuck, N'Dama as well as some trypanosusceptible Boran cattle can serve as reservoirs of single or mixed trypanosome infections for tsetse flies. This study has also shown that the Ag-ELISA on the sera from the five bovid hosts had low sensitivity and species-specificity. Examinations of thin wet blood films and buffy coats with a phase-contrast microscope were not sensitive enough to detect the parasites on all occasions. Xenodiagnosis using mice for T. brucei brucei and T. congolense infections, and tsetse flies for all the three trypanosome species were most sensitive for the detection of trypanosome infections in the bovid hosts.

Comparative sensitivity of antigen-detection enzyme immunosorbent assay and the microhaematocrit centrifugation technique in the diagnosis of Trypanosoma brucei infections in cattle.

Four Boran cattle were infected with Trypanosoma brucei using Glossina morsitans centralis and were left untreated throughout the experimental period of 18 months. During this period, sequential blood samples were collected and examined for the presence of antitrypanosome antibodies and their antigens. Using the buffy coat technique (BCT), trypanosomes were detected in 38 (16.3%) of the 233 blood samples. Unlike the BCT, antigen-detection enzyme-linked immunosorbent assay (Ag-ELISA) diagnosed infections in 189 (81.1%) of the blood samples. These results were supported by the presence of antitrypanosome antibodies in the same samples. Thus Ag-ELISA was 5.5 times more sensitive than the BCT. Towards the end of the observation period, G.m. centralis tsetse were fed on the aparasitaemic cattle to determine whether they still harboured the infection as the persistent antigenaemia seemed to suggest. Bloodmeals from the four cattle were infective to tsetse, thus emphasising the importance of Ag-ELISA in diagnosis of sub-patent infections.

Monoclonal antibodies that distinguish Trypanosoma congolense, T. vivax and T. brucei.

Monoclonal antibodies (MoAbs) were derived against in-vitro-propagated procyclic forms of Trypanosoma congolense, T. vivax, T. brucei brucei and T.b. rhodesiense in order to identify antigens for use in immunodiagnosis of African trypanosomiasis. The antibodies have been tested against procyclic and bloodstream form trypanosomes of 13 T. congolense, six T. vivax six T.b. brucei, four T.b. rhodesiense, five T.b. gambiense and three T. simiae isolates from different geographical areas by indirect immunofluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The MoAbs raised against T.b. brucei reacted with all the brucei group of trypanosomes but not with T. congolense, T. simiae or T. vivax. Likewise, MoAbs against T. congolense reacted with T. congolense and T. simiae but not with any of the other species, while those against T. vivax reacted with T. vivax only. The antigens recognized by these MoAbs were present in lysates of bloodstream trypanosomes as well as midgut (for T. congolense and T. brucei) and epimastigote forms from infected Glossina morsitans centralis. There was no reactivity of the MoAbs with Theileria parva, Anaplasma marginale, Babesia bigemina or Plasmodium falciparum. These antibodies and the antigens they recognize should, therefore, prove useful in the development of assay systems for immunodiagnosis of African trypanosomiasis.

Immunological characterization and expression in Escherichia coli and baculovirus systems of a Trypanosoma vivax antigen detected in the blood of infected animals.

A monoclonal antibody (MAb)Tv27 employed in an antigen-detection enzyme immunosorbent assay (Ag-ELISA) for diagnosis of Trypanosoma vivax infection was shown to react with a T. vivax-specific protein of an approximate molecular weight of 10 kDa. This protein is diffusely distributed throughout the cytosol and nucleus of metacyclic forms, bloodstream forms, and procyclic-like elongated trypomastigotes, but is not detectable in epimastigotes of T. vivax. The T. vivax-specific antigen prepared from parasite lysates appeared to be of lower molecular mass than the form expressed in either Escherichia coli or in baculovirus-infected silkworm insect cells. In the recombinant baculovirus-infected cells, the protein was expressed mostly as an 18-kDa peptide with less abundant forms of 13 and 12 kDa, while the protein expressed in E. coli was approximately 14 kDa. Both the low- and higher-molecular-weight proteins are recognized by the MAb Tv27 in Western blots and in Ag-ELISA. Although the crude preparations of the protein produced by the insect cells are labile when kept for more than 2 hr at 24 degrees C, they retained reactivity at temperatures below 4 degrees C for several weeks. The proteins expressed in both the insect cells and E. coli captured anti-T. vivax antibodies in sera prepared from trypanosome-infected animals. Since the recombinant protein expressed in the baculovirus-infected cells is available in large homogeneous quantities, it would serve as a positive control in Ag-ELISA and is also usable for antibody detection assays.