Prevalence and Whole Genome Sequence Analysis of Mycoplasma bovis Isolates From Bulk Tank Milk of Dairy Farms in Tennessee, USA

Abstract

Background: Mycoplasma bovis mastitis is an important disease of dairy cows that causes substantial economic losses. However, its prevalence in different states in the United States (US), including Tennessee, is not well known. Furthermore, recent studies showed a high prevalence of bovine hemotropic mycoplasmas in US dairy farms.

Objectives: Determine the prevalence of M. bovis in bulk tank milk (BTM) of dairy farms in Tennessee and evaluate the genetic diversity, virulence factors, and antimicrobial resistance genes of the identified isolates. In addition, the prevalence of Mycoplasma wenyonii and Candidatus Mycoplasma haemobos in the bulk tank milk was determined.

Methods: Seventy-five BTM samples were collected from 59 dairy farms. Of the 59 farms, 56 are in Tennessee and the remaining 3 farms are in the neighboring states, Georgia (n = 2) and Alabama (n = 1). Milk samples were tested using bacterial culture, PCR, and qPCR. M. bovis isolates were genetically characterized by pangenome analysis.

Results: Of the 56 farms, 3 (5.3%) were positive for M. bovis by bacterial culture and 43 (76.7%) were positive by PCR. Pangenome analysis showed clustering of current isolates with mastitis isolates from the US, Israel, and Europe. Of 75 BTM samples tested by qPCR, 42 (56%) and 51 (68%) were positive for M. wenyonii and C. M. haemobos, respectively.

Conclusions: M. bovis intramammary infection is prevalent in Tennessee dairy farms.

Keywords: M. bovis; Mycoplasma wenyonii; Candidatus Mycoplasma haemobos; mastitis; virulence factors.

FIGURE 1

Spatial distribution of sampled counties. (NB: The farm in Hardin County possibly started operation after Tennessee Department of Agriculture generated the data).

FIGURE 2

Basic Local Alignment Search Tool (BLAST) analysis showed amplification of uvrC gene from one of the Mycoplasma bovis isolates. (MB41_uvrCF_C: Sanger sequence read; CP011348: GenBank Accession number for M. bovis ).

FIGURE 3

Phylogeny of 221 Mycoplasma bovis isolates developed using PPanGGOLiN. The isolates from this study belong to clade 4 and 12. The branch labels include isolate ID, country, year of isolation and disease. (A: arthritis; A^: abortion; AU: Australia; B: bovine respiratory disease; C: conjunctivitis; CH: Switzerland; CN: China; GER: Germany; HU: Hungary; IL: Israel; J: joint; L: lung; LITH: Lithuania; M: mastitis; N: nasal swab; ND: no data; P: pneumonia; PG2: Mycoplasma agalactiae, outgroup; POL: Poland; ROM: Romania; RU: Russia; S: semen; USA: United States of America; V: vulvitis, ***This study; **Positive control; *Reference strain).