Quantitative and microscopic tracking of Borrelia afzelii transmission from infected Ixodes ricinus nymphs has shown a transmission cycle different from that of Borrelia burgdorferi and Ixodes scapularisBorrelia afzelii organisms are abundant in the guts of unfed I. ricinus nymphs, and their numbers continuously decrease during feeding. Borrelia afzelii spirochetes are present in murine skin within 1 day of tick attachment. In contrast, spirochetes were not detectable in salivary glands at any stage of tick feeding. Further experiments demonstrated that tick saliva is not essential for B. afzelii infectivity, the most important requirement for successful host colonization being a change in expression of outer surface proteins that occurs in the tick gut during feeding. Spirochetes in vertebrate mode are then able to survive within the host even in the absence of tick saliva. Taken together, our data suggest that the tick gut is the decisive organ that determines the competence of I. ricinus to vector B. afzelii We discuss possible transmission mechanisms of B. afzelii spirochetes that should be further tested in order to design effective preventive and therapeutic strategies against Lyme disease.

• Keywords
• Borrelia, Borrelia afzelii, Ixodes ricinus, Lyme disease, tick-borne pathogens, transmission,
• MeSH
• Arachnid Vectors microbiology   physiology   MeSH
• Borrelia burgdorferi Group physiology   MeSH
• Ixodes microbiology   physiology   MeSH
• Humans MeSH
• Lyme Disease microbiology   transmission   MeSH
• Mice, Inbred C3H MeSH
• Mice MeSH
• Nymph microbiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. RESULTS: We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98-99%), while the remaining OTUs belonged to Proteobacteria (51-81%), Actinobacteria (6-30%) and Firmicutes (2-7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. CONCLUSIONS: Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species.

• Keywords
• Borrelia, Bothriocroton undatum, Coxiella burnetii, DNA extraction, Goanna tick, Illumina, Ixodidae, MiSeq, NGS,
• MeSH
• Borrelia classification   genetics   isolation & purification   MeSH
• DNA, Bacterial chemistry   genetics   isolation & purification   MeSH
• Entomology methods   MeSH
• Phylogeny MeSH
• Ixodidae microbiology   MeSH
• Microbiological Techniques methods   MeSH
• DNA, Ribosomal chemistry   genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Sensitivity and Specificity MeSH
• Cluster Analysis MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Geographicals
• Australia MeSH
• Names of Substances
• DNA, Bacterial MeSH
• DNA, Ribosomal MeSH
• RNA, Ribosomal, 16S MeSH