S.K. Moloo

INTERFERENCE IN THE ESTABLISHMENT OF SUPERINFECTIONS WITH TRYPANOSOMA CONGOLENSE IN CATTLE

To examine the influence of an established infection on subsequent challenge with another unrelated trypanosome serodeme, cattle were subjected to two challenges, 5 to 6 wk apart, with unrelated isolates of Trypanosoma congolense. The primary infection inhibited the establishment of the second infection despite the initial absence of detectable antibody to the trypanosomes used for the second challenge. This was true whether the second challenge consisted of bloodstream forms of the parasite or metacyclics from infected Glossina m. morsitans. Rechallenge with bloodstream forms resulted in a slight antibody response, which was only detectable by immunofluorescence and was much less than in the challenge controls. Although animals subjected to the second challenge by tsetse flies showed no appreciable increase in parasitemia and, in most instances, no chancre reaction at the site where the tsetse bit, they developed readily detectable neutralizing antibody to the metacyclic trypanosomes. That this interference effect was not the result of specific immunity and required an active infection was confirmed by the finding that when infected animals were treated with Berenil prior to rechallenge, they were fully susceptible to the infection.

Potential value of localized skin reactions (chancres) induced by Trypanosoma congolense transmitted by Glossina morsitans centralis for the analysis of metacyclic trypanosome populations.

Summary Goats infected with Trypanosoma congolense transmitted by Glossina morsitans centralis and then treated with the trypanocidal drug diminazene aceturate are immune to tsetse‐transmitted infection with a homologous, but not heterologous trypanosome clone. Immune animals fail to develop localized skin reactions (chancres) and do not become infected, whereas on heterologous challenge chancres appear followed by parasitaemia. In this study, the feasibility of using chancre reactions to characterize several different metacyclic populations of T. congolense was evaluated. Provided goats were immunized, it was found that the chancre reaction could be used to distinguish different populations of T. congolense. However, problems were encountered when attempts were made to induce immunity against more than one trypanosome population. When goats were infected by tsetse flies with four antigenically unrelated metacyclic populations of T. congolense, either simultaneously or at 4 day intervals, adequate immunological priming was not always achieved. In fact, goats exposed to superinfection 8 to 12 days after a primary infection did not develop chancres or an immune response to the second infection (although they were immune to the organism used for primary infection). Following trypanocidal treatment these animals were subsequently completely susceptible to homologous infection with the metacyclic population used for superinfection, indicating that some form of interference phenomenon effective at the level of the skin appears to prevent or delay the development of the second trypanosome population following superinfection of infected animals.

Susceptibility of African buffalo and Boran cattle to Trypanosoma congolense transmitted by Glossina morsitans centralis

Four African buffalo (Syncerus caffer) and four Boran cattle (Bos indicus) were each exposed to the bites of 10 tsetse flies infected with Trypanosoma congolense. Although both groups of animals became infected, the buffalo showed no clinical signs of trypanosomiasis while the cattle suffered from the disease characterized by pronounced skin reactions, high parasitaemia and severe anaemia. The prepatent periods in the buffalo varied from 18 to 27 days in comparison with 11 to 14 days in the cattle. In the buffalo, skin reactions were only detectable by histological examination of skin biopsies, the peak of parasitaemia was at least a hundredfold below that in cattle and after 54 days parasites were no longer detected. In contrast, the cattle had a continuous high parasitaemia until they were treated with a trypanocidal drug 60 days after infection. Neutralizing antibody to metacyclic trypanosomes appeared in the buffalo during the prepatent period, 15-20 days after infection, whereas in cattle neutralizing antibody was not detected until 10 days after the first peak of the parasitaemia, 25-30 days after infection.

Interference between different serodemes of Trypanosoma congolense in the establishment of superinfections in goats following transmission by tsetse

Summary When domestic ruminants cyclically infected with Trypanosoma congolense are superinfected with a different serodeme of the same species, an interference phenomenon occurs which delays the development of the second cyclical infection. Experiments were carried out to investigate the influence of the time interval between the two infections on the degree of interference and to follow the course of the superinfection clinically, serologically and histologically. Goats infected with tsetse‐transmitted T. congolense IL 1587 were either infected simultaneously or 7, 14, 18, 28 or 35 days later with a different serodeme, T. congolense IL 1180. Skin reactions due to superinfection were either absent or smaller in size and delayed in onset compared with control animals undergoing a primary infection with T. congolense IL 1180 which had been initiated by tsetse fly bites. All animals were treated with the trypanocidal drug Berenil 21 days after superinfection and 3 weeks later challenged with T. congolense IL 1180 using tsetse flies. The goats that had been infected simultaneously with the two serodemes were immune to the homologous challenge, but 11 (85%) of the animals that had been superinfected were not. The effect of interference on the host immune response to T. congolense IL 1180 was most marked in animals superinfected at day 7; thereafter there was evidence that the ability to respond immunologically against secondary infection was gradually recovered. Histological examination of skin removed 7 days after a simultaneous infection of goats with two serodemes, showed trypanosomes and a cellular reaction similar to that following infection with a single serodeme. In skin removed 7 days after superinfection of goats that had been infected for 7 days, the cellular response was less pronounced and trypanosomes were not seen, although by 14 to 16 days numerous trypanosomes were present and there was a marked cellular infiltrate. It is postulated that the presence or absence of a factor induced shortly after initiation of a trypanosome infection in the skin of goats might delay parasite development when metacyclic trypanosomes are deposited by tsetse following superinfection.

Interference in the establishment of tsetsetransmitted Trypanosoma congolense, T. brucei or T. vivax superinfections in goats already infected with T. congolense or T. vivax

An interference phenomenon that delays superinfection with a trypanosome species different from that used for the initial infection has been found to occur in goats. Following tsetse transmission of Trypanosoma brucei to goats already infected with T. congolense, there was a delay in chancre development, as well as in the appearance of T. brucei and anti-T. brucei antibodies in the blood when compared to previously uninfected goats. However, there was no delay in the establishment of a tsetse-transmitted superinfection with T. vivax in goats already infected with either T. congolense or in animals already infected with a different serodeme of T. vivax.

Comparative sensitivity of dot-ELISA, PCR and dissection method for the detection of trypanosome infections in tsetse flies (Diptera: Glossinidae)

A visually read dot-enzyme linked immunosorbent assay (dot-ELISA) developed for the detection of trypanosomes in tsetse flies (Glossina spp.) was evaluated in the laboratory and under field conditions. In the evaluation, the fly dissection method was used as a standard technique and compared to the polymerase chain reaction (PCR). In laboratory studies, 133 and 126 tsetse flies were experimentally infected with different stocks of Trypanosoma brucei and T. congolense, respectively. Twenty-five days after infection, the flies were dissected and tested for the presence of trypanosomes using dot-ELISA and PCR. Dot-ELISA detected 98.4% of T. brucei and 94% of T. congolense infections in tsetse midguts, while PCR detected 97.6% of T. brucei and 96% of T. congolense tsetse midgut samples. For field evaluation of dot-ELISA, 700 tsetse flies were caught and screened for trypanosome infections by dissection. Seven of these (1%) had trypomastigotes in the midgut, 23 (3.3%) in the proboscis and none had trypanosomes in the salivary glands. All the flies with midgut infections also had trypanosomes in their proboscides. Five of the seven flies (71.4%) with midgut infections revealed by dissection, were also positive for T. congolense by the dot-ELISA and PCR techniques. Dot-ELISA detected T. congolense infections in an additional 86 (12.4%) of the 700 flies dissected. Of the 23 infections in the proboscis, 16 were T. vivax. Dot-ELISA detected 13 of the 16 (81%) while PCR detected 15 of 16 (94%) T. vivax infections. No T. brucei infection was detected by any of the methods in all the 700 tsetse flies examined. The results obtained from both the laboratory and field studies indicate that the dot-ELISA and PCR techniques are sensitive and species-specific in revealing trypanosome infections in tsetse flies. While dot-ELISA required a single test to detect T. congolense, several primer pairs were needed for PCR. The potential use of dot-ELISA as a tool for studying the epidemiology of trypanosomosis, while considering its field applicability and relatively lower cost is discussed.

Parasite kinetics and cellular responses in goats infected and superinfected with Trypanosoma congolense transmitted by Glossina morsitans centralis.

Trypanosoma congolense infected tsetse were fed on the flanks of goats at sites drained by the prefemoral lymph node. The efferent lymphatic of this lymph node was surgically cannulated and the lymph was collected daily and examined for appearance of parasites, lymph flow and cells. Trypanosomes were detected in the lymph 4 days after infection, which was 2 days prior to the appearance of the local skin reaction or the presence of parasites in the blood. Once the animal became parasitaemic, trypanosomes were found to recirculate in the lymphatic system, appearing in the lymph of the contralateral lymph node 11 days after infection. In goats infected with T. congolense and superinfected 12 or 13 days later with a different tsetse-transmitted T. congolense serodeme, parasites belonging to the second serodeme were apparently delayed in their development in the skin and appeared up to 7 days later in the efferent lymph when compared to control animals. This delay in development might have implications for field situations where superinfections frequently occur; it might result in limiting the number of serodemes of T. congolense an animal can be infected with at any one time.

Detection and differentiation between trypanosome species in experimentally infected tsetse flies (Glossina spp.) using dot-ELISA

A modified NC membrane-based dot-ELISA was used to detect and differentiate between Trypanosoma brucei, T. congolense and T. simiae procyclics in the midguts of experimentally infected tsetse flies. The modification of the assay consisted of (a) the lysis of T. congolense or T. simiae in NC membrane applied sample dots using Triton X-114, and (b) treatment of sample applied NC membrane strips with hydrogen peroxide to remove non-specific stains. Also, T. brucei was detected in the salivary glands, and T. congolense and T. vivax were detected in the mouthparts, however, in dot-ELISA without modification. In all the assays, T. brucei and T. congolense parasites were detected directly using MoAbs specific to each of them, whereas T. simiae parasites were detected by exclusion using a T. congolense specific and Nannomonas subgenus-specific MoAbs. The sensitivity of the assay for detecting midgut infections was 90.5%, 84.6% and 94.4% in detecting T. brucei, T. congolense and T. simiae, respectively. Sample dots stored at room temperature (19-26 degrees C) under desiccated conditions did not show any loss in activity in 90 days. However, after 7 days of storage, a ring-pattern reaction appeared on some sample dots that were tested with T. brucei specific MoAb, irrespective of whether T. brucei antigens were present or not. These ring reactions, however, did not interfere with correct interpretation of the assay results. The specificity of the assay for detection of T. brucei in the salivary glands was 100% and the sensitivity was 90%. Also, T. vivax and T. congolense organisms were each detected in the mouthparts of infected tsetse flies, with 100% specificity. The sensitivity was, however, lower, 43.8% for T. vivax and 55.6% for T. congolense.

Skin hypersensitivity to bites of Glossina morsitans centralis in goats

Abstract—Four goats, which had been previously bitten on two different occasions by Glossina morsitans centralis infected with Trypanosoma congolense and subsequently treated on each occasion with the trypanocidal drug diminazene aceturate were each fed upon by six uninfected tsetse. Hypersensitivity reactions developed in the skin of two of the goats within 1 hr after the bite, and reached a maximum intensity over the next 24 hr. In order to determine whether hypersensitive skin reactions could be induced by tsetse saliva alone, six different goats were bitten by uninfected tsetse up to eight times. Only a few small nodular skin reactions were noticed. However, following infection and treatment and another sequence of uninfected tsetse bites, hypersensitive skin reactions did occur in two animals, although the reactions were not as severe as anticipated. The hypersensitivity reactions usually precede but sometimes overlap the chancre reaction elicited by metacyclic trypanosomes transmitted by tsetse.Résumé—Quatre chèvres sont préalablement infectées en deux occasions par Glossina morsitans centralis porteuse de Trypanosoma congolense et traitées consécutivement avec un trypanocide, le diminazène acéturate. Chaque chèvre sert ensuite à nourrir 6 mouches tsétsé non infectées. On observe des réactions d’hypersensibilité au niveau cutané sur deux des chèvres dans l’heure qui suit la piqûre. Cette réaction atteint son intensité maximale dans les 24 hr suivantes.De manière à determiner si cette réaction d’hypersensibilité cutanée est uniquement induite par la salive de la tsétsé, 6 autres chèvres sont piquées jusqu’ à 8 fois de suite par des tsétsé non infectées. Seules quelques petites réactions cutanées nodulaires sont observées. Ces six animaux sont ensuite infectés par des tsétsé porteuses de T. congolense et traitées consécutivement avec le diminazène acéturate. Une autre séquence de piqûres par des tsétsé non infectées est réalisée. Deux animaux développent une réaction d’hypersensibilité cutanée moins sévère que celle observée précédemment. Ces réactions d’hypersensibilité cutanée précèdent habituellement l’apparition du chancre cutané. Elles peuvent parfois se superposer au chancre provoqué par les formes métacycliques de T. congolense injectées par la tsétsé.