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Trypanosoma evansi is a flagellate protozoan parasite responsible for “surra”. To generate T. evansi antigens for serodiagnosis, parasites are generally propagated in laboratory animals before isolation. The alternation of animal models using axenic cultivation systems to produce trypomastigotes of various Trypanosoma species is currently available but has never been applied in Thailand. The isolation protocol for separation of live T. evansi trypomastigotes from animal blood components before in vitro cultivation has not been clearly documented. This study focused on validation of trypomastigote isolation method, in vitro cultivation of T. evansi Thai strains, and its virulence ability in vivo. In this study, two strains of T. evansi collected from Thailand were used. Trypanosoma evansi trypomastigotes were propagated in mice, and three different isolation methods, including: low-speed centrifugation, high-speed centrifugation, and ion exchange chromatography using diethylaminoethyl (DEAE) cellulose (or DE52), were compared. Four solutions of in vitro cultivation media, two different in vitro cultivation containers, and different trypomastigote densities for initiation of in vitro culture were compared. Virulence test using in vitro-adapted parasite for 100 days was conducted in vivo. The results showed that the DE52 isolation method was suitable for separation of live T. evansi trypomastigotes from animal blood components before conducting in vitro cultivation. Trypanosoma evansi Thai strains were successfully cultivated and multiplied in HMI-9 Solution I using 25 cm2 flasks and 12-well plates. The parasite was growing slowly at the initiation of in vitro culture for 15–16 days, and then rapidly increased to 10, 20, 50, 100, and 200 folds, approximately. The doubling times were varied from 11.95 ± 8 h to 41.18 ± 4.29 h in vitro. The maximum densities have reached from 0.14 × 106 to 4.63 × 106 trypomastigotes/ml. Virulence test showed that the in vitro-cultivated T. evansi was virulent in mice. In conclusion, T. evansi Thai strains were successfully isolated and cultivated in vitro for the first time. The isolation and in vitro cultivation protocols were clearly provided. The benefit of using the in vitro cultivation system helps in the production of T. evansi antigen, and replacing the use of experimental animals. It is also useful for the development of diagnostic tests in the future.

A growing number of atypical human infections due to the livestock parasite Trypanosoma evansi, or to the rat parasite Trypanosoma lewisi, are reported in humans in Asia. In some cases, clinical evolutions request treatments, however, so far, there were very few attempts to control T. lewisi using trypanocidal drugs. In a study published elsewhere, the efficacy of human trypanocides is evaluated in laboratory rats, and it concludes that none of them is able to cure rats experimentally infected with T. lewisi. Control of T. lewisi in rat would be a step for identification of drugs against this parasite. In the present study, 4 veterinary drugs: diminazene aceturate, isometamidium chloride, melarsomine hydrochloride and quinapyramine sulfate and chloride, were evaluated at low and high doses, in intra-muscular injections to normal rats experimentally infected with a stock of T. lewisi from Thailand. None of these treatments being efficient, a trial was also made using melarsomine hydrochloride in T. evansi infected rats and in mixed T. lewisi and T. evansi infected rats, in order to demonstrate the efficacy of the drugs under the present protocol. T. evansi was cleared from the rat's blood the day after the treatment, while, T. lewisi remained unaffected until the end of the experiment. These observations clearly demonstrated the efficacy of melarsomine hydrochloride against T. evansi and its inefficacy against T. lewisi. In conclusion none of the veterinary drugs was efficient against this stock of T. lewisi. Other protocols using higher doses or other drugs and T. lewisi stocks should be investigated in further studies. The control of T. lewisi infection in Wistar rats, using veterinary trypanocidal drugs, remains so far unsuccessful.

The trypanosomes pathogenic to livestock in Africa (Trypanosoma congolense, Trypanosoma vivax, and Trypanosoma brucei) are mainly cyclically transmitted by tsetse (Glossina). However, T. vivax, can also be mechanically transmitted by haematophagous insects. Laboratory studies have demonstrated the mechanical transmission of T. congolense, but confirmation of this under natural conditions was necessary. An experiment was therefore carried out in Lahirasso, Burkina Faso, in a corral completely covered by mosquito net, to avoid exposure to tsetse. Eight receiver heifers, free of trypanosome infection, were kept together with two donor heifers, experimentally infected with local stocks of T. congolense. On average, 291 Atylotus agrestis, freshly captured in Nzi traps, were introduced into the mosquito net daily for a period of 20 days to initiate mechanical transmission among cattle. Daily microscopical observation of their blood indicated that two of the eight receiver heifers became infected with T. congolense from days 42 and 53. Mechanical transmission of T. congolense by A. agrestis was demonstrated unequivocally with a 25% incidence over a 20-day period of exposure under a mean challenge of 29 insects/animal/day. These results, in addition to previous reports, demonstrate the ability of A. agrestis to transmit T. vivax and T. congolense to cattle in Africa by mechanical means. Efforts to eliminate cattle trypanosomosis should therefore consider the eventual persistence of disease as a result of mechanical transmission of trypanosomes by tabanids. Index descriptor and abbreviations: Trypanosoma congolense (Trypanosomatidae) is a pathogenic trypanosome found in wild and domestic herbivores, principally in cattle (Bos taurus, Bos indicus, and cross-breds), in Africa. It is cyclically transmitted by tsetse (Glossina, Diptera); however, mechanical transmission by biting insects may also occur. The present study demonstrates unequivocally the mechanical transmission of T. congolense to cattle by one of the most common African tabanids, A. agrestis. The main conclusion is that tabanids are able to transmit T. congolense; however, the incidence of transmission was lower than in studies carried out under the same conditions with T. vivax. Better models of mechanical transmission are required to understand why, on the one hand, epidemiological studies support the mechanical transmission of T. vivax but not T. congolense, and, on the other hand, experimental studies confirm that both species can be mechanically transmitted. Our studies suggest that the epidemiology of trypanosomosis in cattle involves tabanids, and hence, the eradication of tsetse-flies in Africa will not necessarily lead to the eradication of trypanosomosis in domestic livestock. ADT, apparent density of insects per trap per day (mean number of insects caught in one type of trap per 24h of trapping); D, day; NS, not statistically significant

The role of mechanical vectors in the transmission of African livestock trypanosomes has always been controversial relative to tsetse flies, their cyclical vectors. An experiment was carried out in Burkina Faso to demonstrate mechanical transmission of Trypanosoma vivax by one of the most common tabanids in Africa: Atylotus agrestis. Eight heifers (crossbred zebu × Baoule), free of trypanosome infection, were kept in a corral covered by a mosquito net, together with two heifers infected experimentally with a local stock of T. vivax. On average, 324 A. agrestis, freshly captured with Nzi traps, were introduced daily over 20 days. Parasitological, PCR and serological examinations were carried out regularly to assess infections and levels of parasitaemia. Microscopic examination of buffy-coats indicated that five of the eight receiver-heifers were infected on days 8, 13, 32, 41, and 48. PCR results indicated that these five heifers were already infected by day 13. Mechanical transmission of T. vivax by A. agrestis was demonstrated unequivocally, at a high rate (63% in 13–20 days). Conditions of transmission in this experiment are discussed in terms of natural rates of challenge. The importance of tabanids as mechanical vectors of T. vivax should be re-considered, in light of these results. Creation of tsetse free zones in Africa will generally lead to the disappearance of T. congolense, T. brucei, and most often T. vivax as well; however, in areas where T. vivax can be mechanically transmitted, clearance of tsetse may not be sufficient to eradicate livestock trypanosomosis. Index Descriptor and Abbreviations: Trypanosoma vivax (Trypanosomatidae) is a pathogenic trypanosome found in wild and domestic herbivores, principally in cattle (Bos taurus, Bos indicus, and crossbreeds), in South America and Africa. In Africa it is principally cyclically transmitted by tsetse flies (Diptera). In America it is only mechanically transmitted by tabanids (Tabanidae). The present paper describes the mechanical transmission of T. vivax in cattle by one of the most common African tabanids: A. agrestis. The main conclusion is that not only tsetse can transmit T. vivax in Africa, consequently the epidemiology of trypanosomosis in cattle is also tabanid dependant, and, the eradication of tsetse flies will not necessarily lead to eradication of T. vivax. ADT, Apparent density of insects per trap per day (mean number of insects caught in one type of trap per 24 h of trapping); PATTEC, Pan African Tsetse and Trypanosomiasis Eradication Campaign; RHD, rate of hematocrit decrease; D, day; ELISA, enzyme linked immunosorbent assay; NS, not significant (in terms of statistics); PCR, polymerase chain reaction