Use of species-specific DNA probes for detection and identification of trypanosome infection in tsetse flies

Abstract

Species- and subspecies-specific trypanosome DNA hybridization probes have been employed in the detection and identification of trypanosome infections in Glossina morsitans centralis. Several ways of sample preparation including the use of tsetse organ suspensions, proboscides and dissected midguts, as well as tsetse abdominal content touch-blots were explored. The results of hybridization of radio-isotope-labelled species-specific DNA probes to tsetse samples indicated that it was possible to detect trypanosomes in the organs where parasite development is known to characteristically occur for each subgenus. Duplicate slot-blots of samples prepared from midguts of tsetse infected with 2 strains of T. congolense and from non-infected fly controls show that it is not only possible to detect infection in tsetse but also to identify the strain of parasite present in a sample after hybridization with the DNA probes specific for each strain. The results, obtained after hybridization of sequential abdominal touch-blots from the same fly with the DNA probe specific for one strain of T. congolense, indicated that at least 8 positive signals can be observed after an overnight exposure. Because of their simplicity and potentially low cost, the techniques described here would be appealing for screening large numbers of tsetse samples from the field for the presence of any trypanosome residing in the guts or proboscis of the vector. In addition, the possibility of doing multiple touch-blots from the same fly gives the opportunity of detecting mixed trypanosome infections in the vector.

Barcoding in trypanosomes

“…The rise of molecular methods DNA probes based on non-coding satellite repeats were the first molecular methods sensitive enough for the direct identification of trypanosomes in both host and vector samples without requiring cell cultures (Kukla et al 1987;Gibson et al 1988;McNamara et al 1989). The development of the polymerase chain reaction (PCR) heralded a major advance in the sensitivity of diagnostic techniques; PCR-based methods can identify trypanosomes at the subspecies level, they are suitable for analysis of mixed infections and can be applied to samples where parasite numbers are vanishingly low Gibson, 2009;Matovu et al 2010).…”

Molecular diagnosis of infections and resistance in veterinary and human parasites

“…During the 1980s and early 1990s, probe tests were improved by a number of technological advances including alternative substrates for example nylon membranes, and safer and more effective labels such as biotin-streptavidin and fluorescent chemicals. These DNA probe tests can be quite useful, and the literature contains many examples of DNA-probe based tests detecting organisms responsible for disease in mammalian organisms including viruses (Tabares, 1987;Venter et al, 1995), bacteria (Visser and Ambrosio, 1987;Santha et al, 1987;Thomas et al, 1989;Thaker et al, 1990), Amoebozoan (Samuelson et al, 1989), Apicomplexan (Franzen et al, 1984;Posnett and Ambrosio, 1989;Kajiwara et al, 1990;Savva and Holliman, 1990;Ndiritu et al, 1996), Diplomonad (Lu et al, 1994), Kinetoplastid (Eys et al, 1987;Greig and Ashall, 1987;Kukla et al, 1987) and Schizopyrenidan (Sparagano, 1993) protozoan parasites, cestodes (Harrison et al, 1990;Chapman et al, 1995, trematodes (Heussler et al, 1993Kaplan et al, 1995), filarial nematodes (Williams et al, 1988;Poole and Williams, 1990;Dissanayake et al, 1991), arthropods (Cooper et al, 1991;Johnson et al, 1992) and fungal pathogens (Bowman, 1992;Sandhu et al, 1995).…”

The detection and quantification of a digenean infection in the snail host with special emphasis on Fasciola sp.

“…DNA-based blotting DNA probes for the identification and detection of parasites, either in their definitive hosts or in different intermediate hosts, have been shown to be useful diagnostic and epidemiologic tools (Kukla et al 1987;Delves et al 1989;Webster et al 1989). The first DNA-based technique that aimed to detect specifically F. hepatica in snails was described in 1993 (Heussler et al 1993).…”