Vet. Res. 37 (2006) 245-253
DOI: 10.1051/vetres:2005055

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Lénaïg Halos^a, Maria Mavris^a, Gwenaël Vourc'h^b, Renaud Maillard^c, Jacques Barnouin^b, Henri-Jean Boulouis^a and Muriel Vayssier-Taussat<sup>a

a</sup>  UMR 956 INRA/AFSSA/ENVA/UPVM, Microbiologie, École Nationale Vétérinaire d'Alfort, 7 avenue du Général de Gaulle, 94700 Maisons-Alfort, France
^b  Unité d'Épidémiologie Animale, INRA, 63122 Saint-Genès-Champanelle, France
^c  Unité de Pathologie du Bétail, École Nationale Vétérinaire d'Alfort, Maisons-Alfort, France

(Received 8 July 2005; accepted 10 November 2005; published online 11 February 2006)

Abstract - Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.

Key words: Ixodes ricinus / Borrelia burgdorferi sensu lato / Anaplasma phagocytophilum / Rickettsia spp. / bacterial diversity

Corresponding author: Muriel Vayssier-Taussat mvayssier@vet-alfort.fr

© INRA, EDP Sciences 2006