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

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

Ecological Factors Characterizing the Prevalence of Bacterial Tick-Borne Pathogens in Ixodes ricinus Ticks in Pastures and Woodlands

“…For Rickettsia sp., the sequences that we obtained were similar to the sequence of R. helvetica. Some of our infected ticks may also have been infected by other Rickettsia spp., such as symbionts (32). Regarding R. helvetica, the prevalences that we found were in the range of (49) or lower than (4) the prevalence found in previous work.…”

Cryptic vector divergence masks vector‐specific patterns of infection: an example from the marine cycle of Lyme borreliosis

“…Ferquel et al 2006; Halos et al 2006; Kipp et al 2006; Brisson et al 2008). Despite the improvement that these techniques represent compared with immunofluoresence methods (Kahl et al 1998), our results show that they can still have low sensitivity and result in a significant underestimate of pathogen presence.…”

Changes in soil diversity and global activities following invasions of the exotic invasive plant, Amaranthus viridis L., decrease the growth of native sahelian Acacia species

“…16S rRNA gene fingerprints of the bacterial community present in soil samples were performed by TTGE using the Dcode universal mutation detection system (Bio‐Rad Laboratories) under the conditions modified from Corgié et al (2004). This molecular method has already been proven to give sufficient and reproducible information about modifications in the soil microbial community structure (Muyzer & Smalla, 1998) and has been used in various environmental samples (Halos et al , 2006). The polyacrylamide gels [6% (w/v) acrylamide, 0.21% (w/v) bisacrylamide, 7 M urea, 1.25 × Tris‐acetate‐EDTA and 0.2% (v/v) glycerol] were allowed to polymerize for 1 h. DNA samples (10 μL) were separated by electrophoresis in 1.25 × Tris‐acetate‐EDTA at a constant voltage (100 V), with a temperature gradient from 57 to 67 °C (temperature increment of 2 °C h −1 ).…”