title: Inhibition of the endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia.
abstract: BACKGROUND
  The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks.
  
  
  METHODS
  Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont "Candidatus Midichloria mitochondrii" (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM.
  
  
  RESULTS
  Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel "Candidatus Neoehrlichia" spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai.
  
  
  CONCLUSIONS
  Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel "Candidatus Neoehrlichia" spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.
authors.affiliation: Vector and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia. a.gofton@murdoch.edu.au.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Gofton
authors.affiliation: Vector and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia. c.oskam@murdoch.edu.au.
authors.forename: Charlotte L
authors.initials: CL
authors.lastname: Oskam
authors.affiliation: School of Biological Sciences, The University of Sydney, Sydney, New South Wales, Australia. nathan.lo@sydney.edu.au.
authors.forename: Nathan
authors.initials: N
authors.lastname: Lo
authors.affiliation: Faculty of Veterinary Science, The University of Sydney, Sydney, New South Wales, Australia. tiziana.beninati@gmail.com.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Beninati
authors.affiliation: School of Biological Sciences, The University of Sydney, Sydney, New South Wales, Australia. toweiheng@yahoo.com.
authors.forename: Heng
authors.initials: H
authors.lastname: Wei
authors.affiliation: School of Biological Sciences, The University of Sydney, Sydney, New South Wales, Australia. vmccarl@yahoo.com.au.
authors.forename: Victoria
authors.initials: V
authors.lastname: McCarl
authors.affiliation: Trace and Environmental DNA Laboratory, Department of Environment and Agriculture, Curtin University, Perth, Western Australia, Australia. dcgomuiri@gmail.com.
authors.forename: Dáithí C
authors.initials: DC
authors.lastname: Murray
authors.affiliation: Vector and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia. a.paparini@murdoch.edu.au.
authors.forename: Andrea
authors.initials: A
authors.lastname: Paparini
authors.affiliation: Vector and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia. telleashagreay@gmail.com.
authors.forename: Telleasha L
authors.initials: TL
authors.lastname: Greay
authors.affiliation: School of Molecular Biosciences and Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia. andrew.holmes@sydney.edu.au.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Holmes
authors.affiliation: Trace and Environmental DNA Laboratory, Department of Environment and Agriculture, Curtin University, Perth, Western Australia, Australia. michael.bunce@curtin.edu.au.
authors.forename: Michael
authors.initials: M
authors.lastname: Bunce
authors.affiliation: Vector and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia. una.ryan@murdoch.edu.au.
authors.forename: Una
authors.initials: U
authors.lastname: Ryan
authors.affiliation: Vector and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia. p.irwin@murdoch.edu.au.
authors.forename: Peter
authors.initials: P
authors.lastname: Irwin
mesh_terms: D020561:Alphaproteobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D000818:Animals; D001096:Arachnid Vectors / Q000145:classification / Q000382:microbiology*; D001315:Australia; D001898:Borrelia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D018884:Ixodes / Q000145:classification / Q000382:microbiology*; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D013559:Symbiosis
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S