Application of Four Genotyping Methods to Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle.

Mycoplasma bovis is a significant pathogen of feedlot cattle, responsible for chronic pneumonia and polyarthritis syndrome (CPPS). M. bovis isolates (n = 129) were used to compare four methods of phylogenetic analysis and to determine if the isolates' genotypes were associated with phenotypes. Metadata included the health status of the animal from which an isolate was derived (healthy, diseased, or dead), anatomical location (nasopharynx, lung, or joint), feedlot, and production year (2006 to 2018). Four in silico phylogenetic typing methods were used: multilocus sequence typing (MLST), core genome MLST (cgMLST), core genome single nucleotide variant (cgSNV) analysis, and whole-genome SNV (wgSNV) analysis. Using Simpson's diversity index (D) as a proxy for resolution, MLST had the lowest resolution (D = 0.932); cgSNV (D = 0.984) and cgMLST (D = 0.987) generated comparable results; and wgSNV (D = 1.000) provided the highest resolution. Visual inspection of the minimum spanning trees found that the memberships of the clonal complexes and clades had similar structural appearances. Although MLST had the lowest resolution, this methodology was intuitive and easy to apply, and the PubMLST database facilitates the comparison of sequence types across studies. The cg methods had higher resolution than MLST, and the graphical interface software was user-friendly for nonbioinformaticians, but the proprietary software is relatively expensive. The wgSNV approach was the most robust for processing poor-quality sequence data while offering the highest resolution; however, application of its software requires specialized training. None of the four methods could associate genotypes with phenotypes.

Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl. Sex- and strain-specific differences in protein abundances are identified and described, and the measured values are freely accessible via our MouseQuaPro database: http://mousequapro.proteincentre.com . Together, this large library of quantitative MRM-MS assays established in mice and the measured baseline protein abundances represent an important resource for research involving mouse models.

Comparative Microbiomes of the Respiratory Tract and Joints of Feedlot Cattle Mortalities.

A comparative study of microbiota of the respiratory tract and joints of bovine respiratory disease (BRD) cattle mortalities was undertaken. Nasopharynx, trachea, lung and joint samples were collected from 32 cattle that died of BRD, "cases", and 8 that died of other causes, "controls". Bacterial diversity was lower (p < 0.05) in the nasopharynx, trachea and lungs of cases as compared to controls. In cases, alpha-diversity (p < 0.05) was lower in the lungs and joints than the nasopharynx. Proteobacteria, Tenericutes, Bacteroidetes, Firmicutes and Actinobacteria were the most abundant phyla in all samples. Relative abundances of Mycoplasma spp. in the lung, Pasteurella spp. in the trachea and lung, and Histophilus spp. in the lung, trachea and nasopharynx of cases were higher (p < 0.001) than controls. Mycoplasma spp. comprised 20.5% of bacterial flora in the joint, 36.0% in the lung, 22.4% in the trachea and 8.8% in the nasopharynx. Mannheimia spp. (21.8%) and Histophilus spp. (10.4%) were more abundant in lungs. Cattle that died of BRD possessed less diverse respiratory microbiomes with a higher abundance of respiratory pathogens. Mycoplasma spp. were prominent members of pneumonic lungs and joints displaying septic arthritis.

Genome-Wide Association Study of Nucleotide Variants Associated with Resistance to Nine Antimicrobials in Mycoplasma bovis.

Antimicrobial resistance (AMR) studies of Mycoplasma bovis have generally focused on specific loci versus using a genome-wide association study (GWAS) approach. A GWAS approach, using two different models, was applied to 194 Mycoplasma bovis genomes. Both a fixed effects linear model (FEM) and a linear mixed model (LMM) identified associations between nucleotide variants (NVs) and antimicrobial susceptibility testing (AST) phenotypes. The AMR phenotypes represented fluoroquinolones, tetracyclines, phenicols, and macrolides. Both models identified known and novel NVs associated (Bonferroni adjusted p < 0.05) with AMR. Fluoroquinolone resistance was associated with multiple NVs, including previously identified mutations in gyrA and parC. NVs in the 30S ribosomal protein 16S were associated with tetracycline resistance, whereas NVs in 5S rRNA, 23S rRNA, and 50S ribosomal proteins were associated with phenicol and macrolide resistance. For all antimicrobial classes, resistance was associated with NVs in genes coding for ABC transporters and other membrane proteins, tRNA-ligases, peptidases, and transposases, suggesting a NV-based multifactorial model of AMR in M. bovis. This study was the largest collection of North American M. bovis isolates used with a GWAS for the sole purpose of identifying novel and non-antimicrobial-target NVs associated with AMR.

Antimicrobial Sensitivity Testing of Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle.

Mycoplasma bovis is particularly adept at evading the immune system, resulting in chronic infections of the lungs and joints of feedlot cattle. The chronicity of the lesions results in prolonged antimicrobial therapy, possibly exacerbating antimicrobial resistance. This cross-sectional study generated in vitro antimicrobial susceptibility testing (AST) data on 211 M. bovis isolates recovered from 159 healthy, diseased, and dead cattle, spanning the period of 2006-2018. Nine antimicrobials commonly administered to western Canadian feedlot cattle were assessed. The data were analyzed with non-parametric statistical tests with a level of significance of p < 0.05 (two-tailed). Minimum inhibitory concentration (MIC) values tended to increase between the isolates from healthy versus dead cattle and over time (2006-2018). Isolates from dead versus healthy cattle were more likely to be resistant to tulathromycin, gamithromycin, tylosin and enrofloxacin. There was no difference in the distributions of the MICs generated from the isolates recovered from the lungs and joints (p ≥ 0.124) and the lungs and deep nasal passages (p ≥ 0.157) of the same animals.

Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle.

Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at Δ748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (Δ748Δ2059 or Δ748Δ2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a Δ748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.