Skip to main content

ORIGINAL RESEARCH article

Front. Vet. Sci.
Sec. Veterinary Infectious Diseases
Volume 11 - 2024 | doi: 10.3389/fvets.2024.1443855

High throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived clinical DNA samples

Provisionally accepted
Isaac Framst Isaac Framst 1Rebecca M. Wolking Rebecca M. Wolking 2Justin Schonfeld Justin Schonfeld 3Nicole Ricker Nicole Ricker 1Janet Beeler-Marfisi Janet Beeler-Marfisi 1Gabhan Chalmers Gabhan Chalmers 1Pauline Kamath Pauline Kamath 4Grazieli Maboni Grazieli Maboni 5*
  • 1 University of Guelph, Guelph, Ontario, Canada
  • 2 Washington State University, Pullman, Washington, United States
  • 3 Public Health Agency of Canada (PHAC), Ottawa, Ontario, Canada
  • 4 University of Maine, Orono, Maine, United States
  • 5 University of Georgia, Athens, United States

The final, formatted version of the article will be published soon.

    Spillover events of Mycoplasma ovipneumoniae have devastating effects on wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of M. ovipneumoniae between sheep populations. Most work involving M. ovipneumoniae typing has used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing. Our study aimed to develop and validate an MLST workflow for typing of M. ovipneumoniae using Nanopore Rapid Barcoding sequencing and multiplex PCR. We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes of M. ovipneumoniae using archived DNA samples (N = 68) from nasal swabs. Sequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared to Nanopore Native Barcoding and Illumina MiSeq methods. Our proposed workflow is a convenient and effective method for strain typing of M. ovipneumoniae and could be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings where reduced hands-on time, cost, and multiplexing capabilities are important.

    Keywords: Long-read sequencing, short-read sequencing, Bacterial Typing Techniques/methods, Sheep respiratory disease, Mycoplasma ovineumoniae

    Received: 04 Jun 2024; Accepted: 10 Jul 2024.

    Copyright: © 2024 Framst, Wolking, Schonfeld, Ricker, Beeler-Marfisi, Chalmers, Kamath and Maboni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Grazieli Maboni, University of Georgia, Athens, United States

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.