Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture.

Information obtained from fungal air samples can assist in the assessment of health hazards and can be useful in proactive indoor air quality monitoring. The objective of the present study was to evaluate the PCR-TTGE technique for the analysis of fungal diversity in the air. Eleven air samples were collected in five different sites using the bioimpactor CIP 10-M (Arelco). After a 2 hours sampling period, the collection liquid was recovered for subsequent cultivation and PCR-TTGE. A set of three fungi-specific primers (Fungcont 1, Fungcont 2+GC and Fungcont 3) was designed for the partial amplification of the 18S rRNA gene. The amplification was obtained in a single reaction tube by a semi-nested PCR. For identification, the TTGE bands were extracted and sequenced. PCR-TTGE allowed the clear separation of amplicons corresponding to distinct fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The number of fungal taxa detected after culture was systematically higher than the number of taxa found using PCR-TTGE. However, few fungal species were detected by PCR-TTGE and not by cultivation, suggesting that the combination of these approaches may provide a better analysis of fungal diversity in air samples than either method alone.

Evidence of Bartonella sp. in questing adult and nymphal Ixodes ricinus ticks from France and co-infection with Borrelia burgdorferi sensu lato and Babesia sp.

Ticks are known vectors for a wide range of pathogenic microorganisms. Their role in the transmission of some others is so far only suspected. Ticks can transmit multiple pathogens, however, little is known about the co-existence of these pathogens within questing ticks. We looked for the presence of DNA from three micro-organisms, Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. which are known or suspected tick-borne pathogens, using a cohort of 92 questing Ixodes ricinus ticks collected from pastures in northern France. DNA was extracted from each individual tick and the presence of the three pathogens was investigated using Polymerase Chain Reaction (PCR) amplification. Nine among 92 samples (9.8%) demonstrated PCR products using Bartonella specific primers, 3 among 92 (3.3%) using Borrelia burgdorferi sensu lato specific primers and 19 among 92 (20.6%) using Babesia specific primers. Seven among 92 samples (7.6%) were PCR positive for at least two of the pathogens and one sample was positive for all three. Adult ticks (12/18; 67%) showed significantly higher infection rates compared to nymphs (11/74; 15%) for all three pathogens (P < 0.001). This study is the demonstration of the simultaneous presence of Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. in questing Ixodes ricinus ticks.

Determination of an efficient and reliable method for DNA extraction from ticks.

Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P < 0.05). There was no significant difference between methods 2 and 3. Method 3 was however more adapted to cohort studies than method 2. This technique was validated for cohort tick DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.

Role of Hippoboscidae flies as potential vectors of Bartonella spp. infecting wild and domestic ruminants.

The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.