Fachbereich Veterinärmedizin



    African trypanosomosis:
    Identification of vertebrate hosts for tsetse (Diptera, Glossinidae) by PCR-RFLP analysis (2004)

    Steuber, S.
    Abd El-Rady, A
    Clausen, P. H.
    14th Japanese-German Cooperative Symposium on Protozoan Diseases
    Düsseldorf, 20. – 24.09.2004
    Institut für Parasitologie und Tropenveterinärmedizin

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35, 22, 23
    14163 Berlin
    Tel.+49 30 838 62310 Fax.+49 30 838 62323

    Abstract / Zusammenfassung

    Knowledge on the feeding behaviour of arthropods responsible for the transmission of vector-borne diseases is of importance in improvement of vector control and disease eradication strategies. Recent models proposed to describe the epidemiology of African trypanosomosis include, as key parameters, the biting rate of tsetse and the probability of tsetse feeding on different hosts.
    Information on host-vector interactions can be obtained from studies on the natural feeding habits of different species of tsetse. For this purpose serological techniques have been developed using host-specific antisera to identify the source of vertebrate blood from the intestinal tract of wild-caught flies (Weitz 1963; Staak et al., 1981; Clausen et al., 1998). Repeated absorption of antisera with the most cross-reacting antigens will yield highly host specific antisera. Cross-reactivity between members of different groups of animal families can be eliminated whereas even after repeated absorptions some cross-reactivity will remain between phylogenetically closely related species e.g. cross reactivity between domestic pigs, bush-pigs, and warthogs, which results in a high percentage identified as suidae of unknown species.
    Recently, a combined polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) has been established for the differentiation of material from closely related animal species in food products (Meyer et al., 1995, Bellagamba et al., 2001). The aim of this study was to adapt this technique for the identification of blood meals from tsetse.
    The primers used in this study are complementary to the conserved region of the cytochrome b gene (cyt b) of the vertebrates mitochondrion DNA (mt DNA) resulting in an interspecifically different 359 bp-PCR amplicon. The selection of appropriate restriction endonucleases sites is based on the comparison of mtDNA sequence data of vertebrates drawn from the search tool of the National Centre of Biotechnology Information (NCBI) available on the web (http://www.ncbi.nlm.nib.gov). Sites for all restriction enzymes that cut the 359 bp sequence were identified by means of the programme NEB cutter V2.0 (New England Biolab Incorporation). Based on these data a theoretical flowchart of PCR-RFLP has been established. Practical examples of endonuclease digest with selected restriction enzymes will be presented to demonstrate the accurate identification of important mammalian bovid species.