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

Description

Mycoplasma bovis mastitis is responsible for an annual economic loss of more than $100 million in the United States (U.S.) mainly due to production loss and culling. There are no effective control measures and antimicrobial drugs are less effective due to increased resistance among field isolates and intrinsic resistance of Mycoplasma to commonly used beta-lactams. Despite the significant effects of M. bovis mastitis on productivity, its prevalence in different States in the U.S. including Tennessee is not well known. The main objectives of this study were to determine the prevalence of M. bovis in bulk tank milk of dairy farms in Tennessee and determine the genetic diversity and virulence factors of the identified isolates. Overall, 75 bulk tank milk samples were collected from 59 dairy farms. Out of 59 farms, 56 are in Tennessee and the remaining 3 farms were in the neighboring states, Georgia (n=2) and Alabama (n=1). From the 56 farms, 3 (5.3%) were positive for M. bovis by bacterial culture and 43 (76.7%) were positive by PCR. We conducted whole genome sequencing of M. bovis isolates (three field isolates from Tennessee, one from Viriginia and one from American Type Culture Collection (ATCC 25523)). Pangenome analysis showed clustering of current isolates with isolates from U.S., Israel, and Europe as well as clustering with isolates that cause mastitis rather than strains isolated from cases of other M. bovis diseases. To our knowledge, this is the first study that focused on prevalence of M. bovis mastitis in Tennessee and is the first report of M. bovis detection in bulk tank milk in East Tennessee. The widespread presence of M. bovis in BTM, detected by PCR, is concerning suggesting the potential spread of the disease. Urgent action is needed to implement enhanced testing of individual cows on farms using repeated sampling and parallel testing with different methods.

Steps to reproduce

Bulk tank milk samples were collected from dairy farms; enriched in Friis broth DNA and plated on mycoplasma agar. DNA was extracted using MagAttract HMW kit and tested for M. bovis uvrc gene. DNA-Seq library was prepared using MagicPrep™ NGS system. Sequencing was performed and Mi-Seq platform and raw reads were trimmed and quality filtered using fastp. PPanGGOLiN (v2.0.3) was used for the construction and analysis of M. bovis pangenome. Phylogenetic tree reconstruction was performed with IQ-TREE 2 (v2.2.6). MLST analysis was performed using PubMLST (https://pubmlst.org/) with the DNA sequences of assembled draft genomes in FASTA format. To identify virulence factors, DNA sequences of the draft genomes assembled in this study were used as a query in the virulence factors database (VFDB). Similarly, the genomes were used as a query in the comprehensive antibiotic resistance database (CARD).

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