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Tsetse (Glossina spp.) are the primary vectors of animal and human trypanosomosis in tropical Africa, and are a continuing threat to livestock production, human health and agricultural development over much of the continent. Knowledge on the feeding behaviour of tsetse flies is of importance in improvement of vector control and disease eradication strategies. Serological techniques using host-specific antisera were developed several decades ago to identify the source of vertebrate blood from the intestinal tracts of wild-caught tsetse flies (WEITZ 1963; STAAK et al., 1986; CLAUSEN et al., 1998). Up to now, however, some problems exist with the accurate identification of phylogenetically closely related species due to interfering cross reactivity between species leading to a considerable percentage of bloodmeal hosts only classified down to the family level (CLAUSEN et al., 1998). The aim of this study was to adapt the restriction fragment length polymorphism (PCR-RFLP) analysis technique for host species identification of tsetse flies.
Blood samples from ten species of the family Bovidae were enrolled in the study. A variable 359 bp region of vertebrates cytochrome b gene (cyt b) was amplified using universal primers and subjected to PCR-RFLP analysis. To obtain sequence information on the cyt b gene of the different bovid species tested, the retrieval tool of the National Centre of Biotechnology Information (NCBI) was utilized. Search for appropriate restriction enzyme sites were supported by means of the free available programme, NEB cutter V2.0.
PCR-RFLP analysis largely yielded the predicted fingerprints and the bovid species under investigation were accurately identified. Unexpected patterns with a surplus of additional bands after digestion were noticed in some samples tested but did not readily hamper species identification in our study. Such additional bands are commonly attributed to the co-amplification of cyt b pseudogenes. It is thought that they are evolutionarily incorporated into nuclear genome during repair of chromosomal breaks by non-homologous recombination.
In tsetse flies, adequate PCR amplification of the animal host DNA for subsequent RFLP analysis amounted to 100%, 80%, 60% and 40% at 24, 48, 72 and 96 h after feeding, respectively. In case of subsequent feeding on different hosts animals 48 h apart, generally the last bloodmeal could be identified. It is concluded that the PCR-RFLP analysis is a promising method for host species identification in tsetse flies and most probably other haematophagous arthropods.