Genomic analysis of the 2017 Aotearoa New Zealand Outbreak of Mycoplasma bovis and its position within the global population structure

Barbara Mary Binney^1*Edna Gias¹Jonathan Foxwell^2,3Alvey Little⁴Patrick Jon Biggs^5,6Nigel French⁷Callum Lambert⁸Hye Jeong Ha⁴Glen Carter⁹Miklós Gyuranecz¹⁰Bart Pardon¹¹Sarne De Vliegher¹²Filip Boyen¹³Volker Krömker¹⁴Nicole Wente¹⁵Timothy John Mahony¹⁶Justine Gibson¹⁷Tamsin Sarah Barnes¹⁷Nadeeka Wawegama¹⁸Alistair Legione¹⁸Martin Heller¹⁹Christiane Schnee¹⁹Sinikka Pelkonen²⁰Tiina Autio²⁰Hidetoshi Higuchi²¹Satoshi Gondaira²¹Michelle Mcculley^1*

• ¹Genomics Team, National Animal Health Laboratory, Biosecurity New Zealand, Upper Hutt, New Zealand
• ²Ministry for Primary Industries, Wellington, New Zealand
• ³Bacteriology & Mycology, National Animal Health Laboratory, Biosecurity New Zealand, Upper Hutt, New Zealand
• ⁴Virology Mammalian, National Animal Health Laboratory, Biosecurity New Zealand, Upper Hutt, New Zealand
• ⁵Infectious Disease Research Center, Massey University, Palmerston North, Manawatu-Wanganui, New Zealand
• ⁶School of Food Technology and Natural Sciences, Massey University, Palmerston North, New Zealand
• ⁷Tāwharau Ora, School of Veterinary Sciences, College of Sciences, Massey University, Palmerston North, Manawatu-Wanganui, New Zealand
• ⁸Bioprocessing and Fermentation, Biotechnologies, Callaghan Innovation,, Lower Hutt,, New Zealand
• ⁹Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
• ¹⁰HUN-REN Veterinary Medical Research Institute, Budapest, Hungary
• ¹¹Department of Internal Medicine, Reproduction and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
• ¹²M‑team & Mastitis and Milk Quality Research Unit, Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke,, Belgium
• ¹³Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Flemish Brabant, Belgium
• ¹⁴Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Capital Region of Denmark, Denmark
• ¹⁵Department of Bioprocess Engineering and Microbiology, Faculty II, Hannover University of Applied Sciences and Arts, Hanover, Germany
• ¹⁶The University of Queensland, Queensland Alliance for Agriculture and Food Innovation,, Gatton QLD,4343, Australia
• ¹⁷The University of Queensland, School of Veterinary Science, Gatton, QLD,4343, Australia
• ¹⁸Asia Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria, Australia
• ¹⁹Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Germany
• ²⁰Animal Health Diagnostic Unit, Laboratory and Research Division, Finnish Food Authority, Kuopio, Finland
• ²¹Animial Health Laboratory, Department of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Japan

In 2017 an outbreak of Mycoplasma bovis (M. bovis), an infectious agent of cattle, was identified in Aotearoa New Zealand. This study characterizes the genomic population structure of the outbreak in New Zealand and compares it with the known global population structure. The New Zealand outbreak strain was genotyped as ST21 using a multilocus sequence type (MLST) protocol from pubMLST. A comprehensive collection of 840 genomes from the New Zealand outbreak, showed a pattern of clonal expansion when characterized by MLST, core genome MLST (cgMLST) and whole genome MLST (wgMLST). A lineage of genomes was found with no in silico identifiable pta2 locus, a housekeeping gene used in the MLST scheme. Our results support the need for Whole Genome Sequencing (WGS) as well as MLST genotyping in M. bovis outbreaks. We also compared a sample set of 40 New Zealand genomes to 47 genomes from other countries. This group had 79 ST21 genomes and eight genomes that were single nucleotide polymorphism (SNP) variants within the MLST loci of ST21. Two of the 47 international genomes showed signs of extensive unique recombination. Unique alleles in six genes were identified as present only in the New Zealand genomes. These novel variants were in the genes; haeIIIM encoding for cytosine-specific methyltransferase, cysC encoding for cysteinyl tRNA synthetase, era encoding for GTPase Era, metK encoding for S-adenosylmethionine synthase, parE encoding for DNA topoisomerase, and hisS encoding for histidine-tRNA ligase. This finding could be due to a population bottleneck, genetic drift, or positive selection. The same sample set of 40 New Zealand genomes were compared using MLST to 404 genomes from 15 other countries and 11 genomes without a known country. A FastBAPS analysis of 455 genomes showed a global population structure with 11 clusters. Some countries, such as Canada, Denmark and Australia contained both internally closely related genomes and some genomes that were more closely related to genomes found in other countries. These results support the importance of understanding the national and international movement patterns of cattle and their genetic material, as possible routes of transmission, when managing the spread of M. bovis.