Expression, immunolocalization and serodiagnostic value of Tc38630 protein from Trypanosoma congolense.

Animal African trypanosomosis is a serious constraint to livestock sector development in sub-Saharan Africa. The disease, mainly caused by Trypanosoma congolense, has a limitation in its diagnosis and treatment. There is urgent need for a simple, rapid detection technique to replace the few available serological tests that are of variable sensitivity and specificity. Currently, there is a promising use of recombinant proteins to improve on the trypanosome lysate to detect antibodies. In this respect, we have identified a stage-specific gene that is relatively highly expressed in metacyclic and blood trypomastigotes of T. congolense. According to previously obtained differential protein expression data, the gene TcIL3000.0.38630 (1,236 bp) is by 8.5 times more expressed in metacyclic and blood trypomastigotes than in procyclic trypomastigotes and epimastigotes. The same stage specific expression pattern was shown in Western blot analysis. In addition, in confocal laser scanning microscopy the Tc38630 protein was present in the cytosol and on the cell surface of metacyclic and blood trypomastigotes. Through bioinformatics, the Tc38630 had N-terminal signal sequence, hydrophilic extracellular domain, single transmembrane alpha-helix and short cytoplasmic domain, which is characteristic of the Trypanosoma brucei invariant surface glycoprotein. However, unlike T. brucei invariant surface glycoprotein, the Tc38630 existed as a single copy gene with a probable allelic polymorphism at the Nar I restriction site. The recombinant Tc38630-based ELISA detected antibodies against Tc38630 as early as 7 days post infection in experimentally infected mouse model. Taken together, our results suggest that the Tc38630 is a novel potential diagnostic antigen of Animal African trypanosomosis.

The life cycle of Trypanosoma (Nannomonas) congolense in the tsetse fly.

BACKGROUND: The tsetse-transmitted African trypanosomes cause diseases of importance to the health of both humans and livestock. The life cycles of these trypanosomes in the fly were described in the last century, but comparatively few details are available for Trypanosoma (Nannomonas) congolense, despite the fact that it is probably the most prevalent and widespread pathogenic species for livestock in tropical Africa. When the fly takes up bloodstream form trypanosomes, the initial establishment of midgut infection and invasion of the proventriculus is much the same in T. congolense and T. brucei. However, the developmental pathways subsequently diverge, with production of infective metacyclics in the proboscis for T. congolense and in the salivary glands for T. brucei. Whereas events during migration from the proventriculus are understood for T. brucei, knowledge of the corresponding developmental pathway in T. congolense is rudimentary. The recent publication of the genome sequence makes it timely to re-investigate the life cycle of T. congolense. METHODS: Experimental tsetse flies were fed an initial bloodmeal containing T. congolense strain 1/148 and dissected 2 to 78 days later. Trypanosomes recovered from the midgut, proventriculus, proboscis and cibarium were fixed and stained for digital image analysis. Trypanosomes contained in spit samples from individually caged flies were analysed similarly. Mensural data from individual trypanosomes were subjected to principal components analysis. RESULTS: Flies were more susceptible to infection with T. congolense than T. brucei; a high proportion of flies infected with T. congolense established a midgut and subsequent proboscis infection, whereas many T. brucei infections were lost in the migration from foregut to salivary glands. In T. congolense, trypomastigotes ceased division in the proventriculus and became uniform in size. The trypanosomes retained trypomastigote morphology during migration via the foregut to the mouthparts and we confirmed that the trypomastigote-epimastigote transition occurred in the proboscis. We found no equivalent to the asymmetric division stage in T. brucei that mediates transition of proventricular trypomastigotes to epimastigotes. In T. congolense extremely long epimastigotes with remarkably elongated posterior ends were observed in both the proboscis and cibarium; no difference was found in the developmental stages in these two organs. Dividing trypomastigotes and epimastigotes were recovered from the proboscis, some of which were in transition from trypomastigote to epimastigote and vice versa. It remains uncertain whether these morphological transitions are mediated by cell division, since we also found non-dividing cells with a variously positioned, juxta-nuclear kinetoplast. CONCLUSIONS: We have presented a detailed description of the life cycle of T. congolense in its tsetse fly vector. During development in the fly T. congolense shares a common migratory pathway with its close relative T. brucei, culminating in the production of small metacyclic trypanosomes that can be inoculated with the saliva. Despite this outward similarity in life cycle, the transitional developmental stages in the foregut and mouthparts are remarkably different in the two trypanosome species.

Analysis of erythropoietin and erythropoietin receptor genes expression in cattle during acute infection with Trypanosoma congolense.

Acute Trypanosoma congolense infection induced moderate, transient anemia in N'Dama cattle (trypanotolerant) and severe anemia in Boran cattle (trypanosusceptible). Erythropoietin receptor (EpoR) was cloned and sequenced from the two breeds of cattle. A single position mutation of Tyr in the Boran to His in the N'Dama predicted amino acid sequence was revealed. The mRNA transcription of erythropoietin (Epo) in kidneys and EpoR in the bone marrow of infected cattle was determined by competitive reverse transcription and the polymerase chain reaction (RT-PCR). Though Epo mRNA transcription increased in the kidneys during infection, the increase was not significantly different (p>0.05) between the two breeds of infected cattle. The level of EpoR transcripts in the bone marrow of infected N'Damas was significantly higher (p<0.05) than that detected in the marrows from infected Boran cattle. While infection seem to increase levels of transcription of IL-1alpha and beta, and TNFalpha in kidneys from both Boran and N'Dama cattle, no significant difference was detected in the level of mRNAs of these cytokines in the kidney from the two breed of cattle. The amount of IFNgamma mRNA transcripts were not changed with infection in N'Dama cattle, while on the contrary a significant higher levels of IFNgamma was found in kidneys from infected Boran cattle as compared to the other groups. A significant (p<0.05) increase in the levels of IL-1alpha and beta, and IFNgamma mRNA transcripts were detected in the marrows of infected Borans as compared to the infected N'Dama cattle. In this study the increase in the level of TNFalpha mRNA in the marrows of the two infected breeds was not different. This implies there is no negative effect of TNFalpha on hematopoiesis during acute infection. These findings suggest that the levels of Epo and EpoR in the infected Boran cattle were inadequate for their degree of anemia, which might be due in part to high expression of IFNgamma during acute infection with T. congolense.

Effects of vasopressor drugs on number of Trypanosoma congolense in ruminant blood.

The effects of intravenous administration of vasopressor drugs on parasite concentration was studied in cows and sheep infected with Trypanosoma congolense. Ephedrine (USP), adrenaline (BP) and insulin increased jugular concentration of the parasite. Maximum effects were reached between 1 1/2 and 2 h with ephedrine and at 2 1/2-3 h with adrenaline. Increases in parasitaemia resulting from epinephrine were sustained only in sheep and adrenaline gave similar effects in both ruminants. Only epinephrine significantly increased parasitaemia within 45 min of administration. Ephedrine, hydralazine and guanethidine had no significant effects on parasitaemia. It is suggested that one of the mechanisms by which trypanocides increase jugular concentration of T. congolense may be by vasoconstriction of the capillary endothelium.

In vitro cultivation of Trypanosoma congolense: the production of infective metacyclic trypanosomes in cultures initiated from cloned stocks.

Glossina morsitans were infected with two cloned stocks of T. congolense. The proboscides, foreguts and midguts of infected flies were then used as sources of trypanosomes in vitro at 28 degrees C in the presence of bovine dermal collagen explants. Cultures were established in which trypanosomes differentiated into adhering colonies of epimastigote forms which could then be maintained and subcultured in Eagle's Minimum Essential Medium supplemented with foetal calf serum for over 40 weeks. Within 2-4 weeks of establishment of each culture or subculture the epimastigote trypanosomes differentiated into metacyclic trypanosomes which could be harvested from supernatant medium at concentrations of 1 X 10(5)-3 X 10(6) parasites/ml. These organisms were used to induce the formation of local skin reactions in rabbits. Successful cultivation of infective trypanosomes appeared to depend on the initial adhesion of the parasites to the surface of the flask where they subsequently differentiated first into epimastigote and then to metacyclic forms.