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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

Alignment of partial 16S rDNA sequences and the 27F-Y and MidBlocker primers. Alignment includes partial 16S sequences of seven tick-borne bacterial pathogens and “Candidatus M. mitochondrii” with the 27F-Y and MidBlocker primers showing mismatches that allow specific blocking of “Candidatus M. mitochondrii”
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Fig1: Alignment of partial 16S rDNA sequences and the 27F-Y and MidBlocker primers. Alignment includes partial 16S sequences of seven tick-borne bacterial pathogens and “Candidatus M. mitochondrii” with the 27F-Y and MidBlocker primers showing mismatches that allow specific blocking of “Candidatus M. mitochondrii”

Mentions: In pilot 16S community profiling experiments, over 95 % of the sequences generated from each sample, from both I. holocyclus and I. ricinus ticks, belonged to CMM regardless of the sequencing depth, PCR primers, or sequencing platform used (data not shown). To inhibit amplification of these overabundant sequences during PCR, we developed a CMM-specific blocking primer (MidBlocker) [70] to be used in conjunction with the 16S universal primers 27F-Y (Fig. 1) and 338R (5’-TGCTGCCTCCCGTAGGAGT-3’) that amplify the V1-V2 16S region [71]. The MidBlocker primer was designed from an alignment of 107 partial 16S sequences including known tick-borne pathogens and endosymbionts, ubiquitous environmental bacteria, and CMM (Fig. 1). The 5’ end of the MidBlocker primer has a 7 bp overlap with the 3’ end of the 27F-Y primer, extends 15 bp downstream of the 27F-Y primer-binding site, and terminates polymerase elongation due to a C3 spacer at the 3’ end of the primer (Fig. 1). In silico analysis (not shown) suggests that the MidBlocker primer is specific to CMM and will not modulate the binding of the 27F-Y primer to other closely related Rickettsiaceae and Anaplasmataceae.Fig. 1


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)

Alignment of partial 16S rDNA sequences and the 27F-Y and MidBlocker primers. Alignment includes partial 16S sequences of seven tick-borne bacterial pathogens and “Candidatus M. mitochondrii” with the 27F-Y and MidBlocker primers showing mismatches that allow specific blocking of “Candidatus M. mitochondrii”
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Alignment of partial 16S rDNA sequences and the 27F-Y and MidBlocker primers. Alignment includes partial 16S sequences of seven tick-borne bacterial pathogens and “Candidatus M. mitochondrii” with the 27F-Y and MidBlocker primers showing mismatches that allow specific blocking of “Candidatus M. mitochondrii”
Mentions: In pilot 16S community profiling experiments, over 95 % of the sequences generated from each sample, from both I. holocyclus and I. ricinus ticks, belonged to CMM regardless of the sequencing depth, PCR primers, or sequencing platform used (data not shown). To inhibit amplification of these overabundant sequences during PCR, we developed a CMM-specific blocking primer (MidBlocker) [70] to be used in conjunction with the 16S universal primers 27F-Y (Fig. 1) and 338R (5’-TGCTGCCTCCCGTAGGAGT-3’) that amplify the V1-V2 16S region [71]. The MidBlocker primer was designed from an alignment of 107 partial 16S sequences including known tick-borne pathogens and endosymbionts, ubiquitous environmental bacteria, and CMM (Fig. 1). The 5’ end of the MidBlocker primer has a 7 bp overlap with the 3’ end of the 27F-Y primer, extends 15 bp downstream of the 27F-Y primer-binding site, and terminates polymerase elongation due to a C3 spacer at the 3’ end of the primer (Fig. 1). In silico analysis (not shown) suggests that the MidBlocker primer is specific to CMM and will not modulate the binding of the 27F-Y primer to other closely related Rickettsiaceae and Anaplasmataceae.Fig. 1

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