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Inhibition of the endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia.

Gofton AW, Oskam CL, Lo N, Beninati T, Wei H, McCarl V, Murray DC, Paparini A, Greay TL, Holmes AJ, Bunce M, Ryan U, Irwin P - Parasit Vectors (2015)

Bottom Line: 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.Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation.Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Neighbour-joining tree of 16S V1-2 “Candidatus Neoehrlichia” sequences from I. holocyclus and I. ricinus ticks. Branch labels are bootstrap values inferred from 1000 replicated. Parenthesises after node labels refers to the GenBank accession number. * Indicates sequences from this study
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Fig4: Neighbour-joining tree of 16S V1-2 “Candidatus Neoehrlichia” sequences from I. holocyclus and I. ricinus ticks. Branch labels are bootstrap values inferred from 1000 replicated. Parenthesises after node labels refers to the GenBank accession number. * Indicates sequences from this study

Mentions: Three I. ricinus ticks and 15 I. holocyclus ticks contained sequences from the genus “Candidatus Neoehrlichia” and all I. ricinus-derived sequences had > 98 % sequence similarity, and clustered with “Candidatus Neoehrlichia mikurensis” reference sequences (Fig. 4). Ixodes holocyclus-derived “Candidatus Neoehrlichia” sequences formed two distinct novel clades with high bootstrap confidence (94.2 % and 97.2 %) that did not group with any “Candidatus Neoehrlichia” sequences in GenBank (Fig. 4). Sequences within each of these novel “Candidatus Neoehrlichia” clades were less than 1 % dissimilar to each other but more than 6 % dissimilar to any known “Candidatus Neoehrlichia mikurensis” or “Candidatus Neoehrlichia lotoris” 16S sequences. One I. holocyclus tick also contained sequences that grouped with relatively high bootstrap confidence (75.1 %) with Anaplasma bovis within a clade that also includes the pathogens A. platys, A. Phagocytophilum and A. odocoilei (Fig. 4).Fig. 4


Inhibition of the endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia.

Gofton AW, Oskam CL, Lo N, Beninati T, Wei H, McCarl V, Murray DC, Paparini A, Greay TL, Holmes AJ, Bunce M, Ryan U, Irwin P - Parasit Vectors (2015)

Neighbour-joining tree of 16S V1-2 “Candidatus Neoehrlichia” sequences from I. holocyclus and I. ricinus ticks. Branch labels are bootstrap values inferred from 1000 replicated. Parenthesises after node labels refers to the GenBank accession number. * Indicates sequences from this study
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493822&req=5

Fig4: Neighbour-joining tree of 16S V1-2 “Candidatus Neoehrlichia” sequences from I. holocyclus and I. ricinus ticks. Branch labels are bootstrap values inferred from 1000 replicated. Parenthesises after node labels refers to the GenBank accession number. * Indicates sequences from this study
Mentions: Three I. ricinus ticks and 15 I. holocyclus ticks contained sequences from the genus “Candidatus Neoehrlichia” and all I. ricinus-derived sequences had > 98 % sequence similarity, and clustered with “Candidatus Neoehrlichia mikurensis” reference sequences (Fig. 4). Ixodes holocyclus-derived “Candidatus Neoehrlichia” sequences formed two distinct novel clades with high bootstrap confidence (94.2 % and 97.2 %) that did not group with any “Candidatus Neoehrlichia” sequences in GenBank (Fig. 4). Sequences within each of these novel “Candidatus Neoehrlichia” clades were less than 1 % dissimilar to each other but more than 6 % dissimilar to any known “Candidatus Neoehrlichia mikurensis” or “Candidatus Neoehrlichia lotoris” 16S sequences. One I. holocyclus tick also contained sequences that grouped with relatively high bootstrap confidence (75.1 %) with Anaplasma bovis within a clade that also includes the pathogens A. platys, A. Phagocytophilum and A. odocoilei (Fig. 4).Fig. 4

Bottom Line: 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.Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation.Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus