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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

Rank abundance plots of bacterial genera identified with and without blocking. The ranked relative abundance of bacterial genera identified in 10 I. holocyclus and 10 I. ricinus ticks when amplified with (blue lines) and without (red lines) the MidBlocker primer. X-axis represents the number of bacterial genera identified
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Fig2: Rank abundance plots of bacterial genera identified with and without blocking. The ranked relative abundance of bacterial genera identified in 10 I. holocyclus and 10 I. ricinus ticks when amplified with (blue lines) and without (red lines) the MidBlocker primer. X-axis represents the number of bacterial genera identified

Mentions: Consequent to the blocking step, all samples had a significantly higher taxonomic diversity when amplified with the MidBlocker primer than when amplified without the MidBlocker primer (p < 0.05; Mann-Whitney U-Test). Amplification without the MidBlocker primer resulted in the detection of 32 and 14 bacterial genera in I. holocyclus and I. ricinus samples respectively, while inhibition of CMM 16S sequences resulted in the detection of 103 and 89 additional bacterial genera in I. holocyclus and I. ricinus samples respectively (Fig. 2). Furthermore, the MidBlocker primer did not appear to inhibit the amplification of other Rickettsiales closely related to CMM, as confirmed by the identification of members of the closely related Rickettsia and “Candidatus Neoehrlichia” genus in I. holocyclus and I. ricinus samples amplified with the MidBlocker primer.Fig. 2


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)

Rank abundance plots of bacterial genera identified with and without blocking. The ranked relative abundance of bacterial genera identified in 10 I. holocyclus and 10 I. ricinus ticks when amplified with (blue lines) and without (red lines) the MidBlocker primer. X-axis represents the number of bacterial genera identified
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Rank abundance plots of bacterial genera identified with and without blocking. The ranked relative abundance of bacterial genera identified in 10 I. holocyclus and 10 I. ricinus ticks when amplified with (blue lines) and without (red lines) the MidBlocker primer. X-axis represents the number of bacterial genera identified
Mentions: Consequent to the blocking step, all samples had a significantly higher taxonomic diversity when amplified with the MidBlocker primer than when amplified without the MidBlocker primer (p < 0.05; Mann-Whitney U-Test). Amplification without the MidBlocker primer resulted in the detection of 32 and 14 bacterial genera in I. holocyclus and I. ricinus samples respectively, while inhibition of CMM 16S sequences resulted in the detection of 103 and 89 additional bacterial genera in I. holocyclus and I. ricinus samples respectively (Fig. 2). Furthermore, the MidBlocker primer did not appear to inhibit the amplification of other Rickettsiales closely related to CMM, as confirmed by the identification of members of the closely related Rickettsia and “Candidatus Neoehrlichia” genus in I. holocyclus and I. ricinus samples amplified with the MidBlocker primer.Fig. 2

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