Evaluation of a novel multiplex PCR amplicon sequencing assay for detection of human pathogens in Ixodes ticks

Hojgaard, Andrias; Osikowicz, Lynn M.; Eisen, Lars; Eisen, Rebecca J.

doi:10.1016/j.ttbdis.2020.101504

Cited by 11 publications

(29 citation statements)

References 24 publications

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Section: Methodsmentioning

confidence: 99%

“…We tested for genera of parasites or bacteria previously described as pathogenic in humans and spread by tick vectors (Eisen et al 2017, Hojgaard et al 2020). We used a previously described multiplex PCR amplicon sequencing assay where PCR primers were designed to amplify genera of both parasite and bacterial DNA (Hojgaard et al 2020). In addition to the PCR primers detecting the microbes, the PCR multiplex also has PCR primers that will amplify tick DNA (actin) and therefore serve as a positive control for both the presence and quality of DNA.…”

Section: Methodsmentioning

confidence: 99%

“…The genera specific multiplex PCR reactions were performed in 25 µl, which included 12.5 µl 2× Sso Advanced (BioRad, Hercules, CA, USA), 10 µl tick nucleic acids extract, and 2.5 µl PCR primers resuspended in PCR grade water. Following the multiplex PCR reaction NGS sequencing libraries were generated as previously described (Hojgaard et al 2020, 2021), and sequencing was performed using the MiSeq system (Illumina, San Diego, CA) with the MiSeq reagent kit Nano 500V2 according to the manufacturer's protocol (Illumina). All sequences were analyzed using CLC Genomic Workbench (Qiagen) software, and reference sequences as previously described (Hojgaard et al 2020).…”

Section: Methodsmentioning

confidence: 99%

“…Following the multiplex PCR reaction NGS sequencing libraries were generated as previously described (Hojgaard et al 2020, 2021), and sequencing was performed using the MiSeq system (Illumina, San Diego, CA) with the MiSeq reagent kit Nano 500V2 according to the manufacturer's protocol (Illumina). All sequences were analyzed using CLC Genomic Workbench (Qiagen) software, and reference sequences as previously described (Hojgaard et al 2020).…”

Section: Methodsmentioning

confidence: 99%

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Ticks and tick-borne microbes identified through passive and active surveillance in Alaska

Hahn

Hojgaard

Disler

et al. 2023

Journal of Medical Entomology

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Rapid environmental change in Alaska and other regions of the Arctic and sub-Arctic has raised concerns about increasing human exposure to ticks and the pathogens they carry. We tested a sample of ticks collected through a combination of passive and active surveillance from humans, domestic animals, and wildlife hosts in Alaska for a panel of the most common tick-borne pathogens in the contiguous United States to characterize the diversity of microbes present in this region. We tested 189 pooled tick samples collected in 2019-2020 for Borrelia spp., Anaplasma spp., Ehrlichia spp., and Babesia spp. using a multiplex PCR amplicon sequencing assay. We found established populations of Ixodes angustus Neumann (Acari: Ixodidae), Ixodes uriae White (Acari: Ixodidae), and Haemaphysalis leporispalustris Packard (Acari: Ixodidae) in Alaska, with I. angustus found on a variety of hosts including domestic companion animals (dogs and cats), small wild mammals, and humans. Ixodes angustus were active from April through October with peaks in adult and nymphal activity observed in summer months (mainly July). Although no known human pathogens were detected, Babesia microti-like parasites and candidatus Ehrlichia khabarensis were identified in ticks and small mammals. The only human pathogen detected (B. burgdorferi s.s.) was found in a tick associated with a dog that had recently traveled to New York, where Lyme disease is endemic. This study highlights the value of a combined passive and active tick surveillance system to detect introduced tick species and pathogens and to assess which tick species and microbes are locally established.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Ticks and tick-borne microbes identified through passive and active surveillance in Alaska

Hahn

Hojgaard

Disler

et al. 2023

Journal of Medical Entomology

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“…Amplicon DNA was also sequenced by Illumina. Amplicon libraries from the PCR reactions were generated as previously described [30,31] and sequencing was performed on the MiSeq system (Illumina, San Diego, CA) using MiSeq Reagent Kits Nano 500V2 according to the manufacturer's protocol (Illumina). For sequence analysis, we used CLC Genomic Workbench (Qiagen) software, using reference generated from GenBank sequences of B. miyamotoi strain CT13-2396 (GCA_001767415.1) and LB-2001 (GCF_017748095.1).…”

Section: Nanopore and Illumina Sequencing (Ngs) And Analysismentioning

confidence: 99%

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure

et al. 2023

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Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations.

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When to Think About Other Borreliae:

Rodino

Pritt

2022

Infectious Disease Clinics of North America

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Evaluation of a novel multiplex PCR amplicon sequencing assay for detection of human pathogens in Ixodes ticks

Cited by 11 publications

References 24 publications

Ticks and tick-borne microbes identified through passive and active surveillance in Alaska

Ticks and tick-borne microbes identified through passive and active surveillance in Alaska

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure

When to Think About Other Borreliae:

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