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PCR-RFLP analysis: a promising technique for host species identification of blood meals from tsetse flies (Diptera: Glossinidae)

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Abstract

A polymerase chain reaction with the restriction fragment length polymorphism (PCR-RFLP) method using universal primers complementary to the conserved region of the cytochrome b gene (cyt b) of the mitochondrion DNA (mtDNA) of vertebrates was applied to the identification of the origin of blood meals in tsetse flies. Blood samples from ten potential tsetse hosts of the family bovidae (cattle, water buffalo, red buffalo, waterbuck, springbok, goat, sheep, sable antelope, oryx and dik-dik) were included in this study. Sites for appropriate restriction endonucleases cuts were chosen by pairwise alignment of the amplified 359 bp fragments. A flow chart of endonucleases digestion using three restriction enzymes (e.g. TaqI, AluI and HindII) for the unequivocal identification of the respective bovid species was developed. A number of additional non-specific DNA fragments attributed to the co-amplification of cytochrome b pseudogenes were observed in some species (e.g. in red buffalo and dik-dik after digestion with AluI) but did not hamper assignment of bovid species. The detection rate of host DNA in tsetse by PCR-RFLP was 100, 80, 60 and 40% at 24, 48, 72 and 96 h after in vitro feeding, respectively. Identification of the last blood meal was possible even when tsetse had previously fed on different hosts.

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Acknowledgements

We wish to thank Prof. Dr. Klaus Eulenberger (Leipziger Zoo), Dr. Wolfram Rietschel (Zoologisch-Botanischer Garten Wilhelma Stuttgart), PD. Dr. Kai Fröhlich (Institute for Zoo and Wildlife Research Berlin) and Dr. Andreas Ochs (Zoologischer Garten Berlin) for providing blood samples from different bovid species used in the study. Special thanks to Mrs. Angelika Wiemann for excellent technical assistance. This work was funded by the Ständige Kommission für Forschung und wissenschaftlichen Nachwuchs (FNK) of the FU Berlin.

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Authors and Affiliations

  1. Federal Office of Consumer Protection and Food Safety, Diedersdorfer Weg 1, 12277, Berlin, Germany

    Stephan Steuber

  2. Institute for Parasitology and International Animal Health, Freie Universität Berlin, Königsweg 67, 14163, Berlin, Germany

    Ahmed Abdel-Rady & Peter-Henning Clausen

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  1. Stephan Steuber
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  2. Ahmed Abdel-Rady
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  3. Peter-Henning Clausen
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Correspondence to Stephan Steuber.

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Steuber, S., Abdel-Rady, A. & Clausen, PH. PCR-RFLP analysis: a promising technique for host species identification of blood meals from tsetse flies (Diptera: Glossinidae). Parasitol Res 97, 247–254 (2005). https://doi.org/10.1007/s00436-005-1410-y

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  • DOI: https://doi.org/10.1007/s00436-005-1410-y

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