Rogéria M. Ventura, Gentilda F. Takeda and Marta M. G. Teixeira
Department of Parasitology, University of São Paulo, S.P. Brazil. E-mail: mmgteix@biomed. icb2. usp. br
Acknowledgments
References
T. evansi is a member of the subgenus Trypanozoon, which also comprises T. brucei and T. equiperdum, differing from these species by the absence of kDNA maxicircle, which renders it unable to develop cyclically in vector insects. Thus, T. evansi is transmitted only mechanically by blood-sucking insects (mainly tabanids) and bats (Hoare, 1972; Wells, 1976). The lack of maxicircle kDNA appears to facilitate the utilization of a wide range of mechanical vectors by T. evansi, and thus, the spreading of this parasite from Africa to other parts of the world (Lun, 1972). Thus, T. evansi is widespread in Africa, Asia, and South America, causing "surra" a disease of horses, camels, dogs, cattle, buffaloes and several wild animals. The disease is fatal in horses and camels, while wild animals usually appear to act as reservoirs (Hoare, 1972). Strains of T. evansi from all geographical regions and hosts present very similar zymodemes (Gibson, 1980) and kDNA minicircle sequences (Borst et al., 1987; Songa et al., 1990; Ou et al., 1991). Polymorphism of chromosome-sized DNA molecules is the more effective technique for strain discrimination (Lun et al., 1992). However, this type of analysis, besides requiring a large number of parasites, is very laborious and time consuming.
Routine identification of T. evansi is still based on morphology that may require previous isolation of parasites. T. evansi can be directly detected by microscopic analysis of samples from infected animal blood and/or from experimentally inoculated mice, whereas cultures can be obtained only in very special conditions. Nevertheless, in addition to poor sensitivity, morphological identification requires skilled technicians.
Serological diagnosis, besides not distinguishing past from present infections, can be precluded by antigenic variation. Molecular methods for T. evansi detection based on kDNA minicircles are not helpful for dyskinetoplastic strains. Therefore, available methods for detection and identification besides being of low sensitivity, are time consuming and impractical for epidemiological surveys of hosts and vectors.
Thus, diagnostic methods for T. evansi have obvious shortcomings, and we are searching for a simple, fast and sensitive method for T. evansi detection, which precludes animal inoculation and detects even dyskinetoplastic strains.
In Brazil, T. evansi has been found in many hosts and appears to be endemic in Mato Grosso States (Stevens et al., 1990; Franke et al., 1994; Nunes et al., 1993; Silva et al., 1995). However, large scale epidemiological surveys as well as isolation and characterization of many isolates have not yet been performed. Moreover, several aspects of vector and host biology, pathogenicity, and the genetic variability of Brazilian isolates are of very limited knowledge. In order to study genetic polymorphism and to define molecular markers for diagnosis and classification, we decided to compare ribosomal and spliced leader (SL) genes and RAPD markers of T. evansi with other Trypanosoma spp. We characterized 6 T. evansi strains isolated from dogs and rodents from Campo Grande, M.S., Brazil, as well as several other species of Trypanosoma.
Morphological analysis of these T. evansi strains showed high level of dyskinetoplasty. In addition, we confirmed that they displayed different behaviours in experimentally infected mice, in both parasitaemia and mortality. Virulent strains, presenting high parasitaemia and 100% of mortality in few days, and strains that showed lower parasitaemia and mortality were identified. Moreover, the avirulent strains presented relapsing infections, with waves of parasitaemia, characteristic of the antigenic variation process.
In order to develop simple methods for T. evansi characterization and identification we analyzed several DNA targets by different approaches. RFLP analysis of ribosomal (SSU and LSU) gene and amplification followed by RFLP of ribosomal ITS (Riboprinting) and spliced leader (SL) gene did not show high heterogeneity among strains with only small differences detected. The relevance of this finding needs to be investigated by analysis of other strains. We also analyzed these parasites by RAPD assays, which also did not reveal significant variability among strains. Therefore, despite the strong differences in the parasitemia and mortality observed in mice infected with different strains, small genetic polymorphism was noted in the Brazilian strains.
Genetic variability also needs to be investigated by the comparative analysis of a higher number of Brazilian isolates as well as their comparison with isolates from other countries. It is worth mentioning that similar zymodemes were previously demonstrated among Brazilian strains from dogs, capybaras and horses (Stevens et al., 1989; Franke et al., 1994; Nunes et al., 1993). In addition, enzymatic similarity was also identified among T. evansi strains from different countries (Gibson et al., 1980; Boid, 1988) Nevertheless, in addition to RFLP of rDNA and SL DNA, RAPD and ITS/Riboprinting also displayed patterns which allows the discrimination of T. evansi from other Trypanosoma species. Therefore, our results disclosed several species-specific markers for T. evansi which are being exploited for the development of rapid diagnostic assays. To define these species-specific markers, so they may be utilized as probes and/or as PCR primers, we are currently cloning potential diagnostic ribosomal and spliced-leader sequences.
In order to identify T. evansi -specific sequences we are also selecting DNA fragments amplified in RAPD-PCR assays. We selected a 8nt oligonucleotide primer that amplified a unique and species-specific DNA fragment. This PCR assay was specific and sensitive for T. evansi identification, since it detected less than one parasite and did not amplify DNA from other species of Trypanosoma, even from the closely related T. brucei and T. equiperdum. We have cloned and characterized the DNA fragment, Te664 from a dog strain of T. evansi, which was also tested as a probe in slot blot hybridization assay. Moreover, this probe proved to be very sensitive, indicating the target of this probe to be a specific repetitive DNA sequence of T. evansi genoma. Thus, our results suggest that this DNA fragment could be useful as a species-specific probe since it hybridized exclusively with T. evansi and not with any other species of Trypanosoma, including T. brucei and T. equiperdum.
In summary, analysis of genetic variability using different DNA sequences revealed only microheterogeneity between T. evansi strains. However, this analysis disclosed several molecular markers which are able to distinguish T. evansi from other Trypanosoma spp. We are aware that they need to be further evaluated on a large number of isolates as well as directly on crude preparations of blood samples. However, preliminary results suggest a simple, fast, specific and sensitive method for T. evansi identification can be achieved by these approaches.
I would like to thank all members of my laboratory for their valuable collaboration. I also wish to thank Dr. Vania L. B. Nunes (U.F. do Mato Grosso do Sul, Brazil) for supplying the T. evansi strains used in this study. This work was supported by FAPESP.
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