Development and Testing of Selective and Differential Media for the Recovery of Bacterial Bovine Respiratory Disease Isolates from Feedlot water Bowls

Bovine respiratory disease (BRD) is a significant disease of both beef and dairy cattle production, influenced by various factors including stressful conditions, viral and bacterial pathogens, and management practices. The four main bacterial species associated with BRD, Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, and Mycoplasmopsis bovis, have predominantly been studied using isolates obtained from sick and dead cattle. Antimicrobial usage (AMU) has been used for both metaphylactic and prophylactic control of BRD, contributing to the rise in antimicrobial resistance (AMR) within these production systems. Traditionally, AMR evaluation has focused on antimicrobial susceptibility testing (AST), but there has been a shift towards screening via molecular methods. Exploring alternative methods for AMR screening utilizing environmental fomite isolates could inform timely management decisions. Water bowls serve as promising targets for isolating these bacterial pathogens because bacteria appear to be shed into the water from the animal’s upper respiratory tract (URT). Shared water bowls have also been identified as a significant risk factor for BRD in feedlot cattle. The isolation of BRD pathogens from water bowls is challenging due to their fastidious nature and overgrowth by more competitive environmental microbes. The overarching objective was to develop culture media to aid in the recovery of P. multocida, M. haemolytica, and H. somni from feedlot water bowls. These isolates could then be used to monitor AMR at the feedlot pen level. To achieve this goal, the project was divided into four separate objectives: 1) characterize the metabolic profiles of the three Pasteurellaceae pathogens for identification of potential nutritional requirements and differentiation targets; 2) develop and test both chemically defined (CDM) and differential media (DM) using Pasteurellaceae isolates; 3) assess CDM and DM for the isolation of P. multocida and M. haemolytica from feedlot water bowl samples; and 4) characterize the antimicrobial resistance (AMR) phenotypes and genotypes of M. bovis isolates obtained from feedlot water bowls and cattle that died of BRD. Initially, biochemical characterization was performed with P. multocida, M. haemolytica and H. somni, using 189 organic compounds, 96 solute concentration conditions, and 96 pH conditions. All three BRD pathogens effectively metabolized L-cysteine. P. multocida uniquely metabolized L-glutamic acid and mannose, while M. haemolytica metabolized L-aspartic acid and maltose. Whereas H. somni metabolized L-arabinose and D-glucosamine, the fastidious nature of the organism required additional nutritional supplements. These metabolization studies helped to inform the development of a CDM and DM for P. multocida and M. haemolytica. Next, a CDM for P. multocida was developed from a M9 mineral salt base, L-cysteine, L-glutamic acid, and mannose. The CDM for M. haemolytica was comprised of L-cysteine, L-leucine, and maltose. A DM was also developed, with the differentiation factor being the fermentation of mannose for P. multocida, and maltose for M. haemolytica along with a pH indicator. Both the CDM and DM were able to sustain the growth of their respective bacterial species. All CDM required a high initial bacterial inoculum for sustained growth, while the DM supported bacterial growth at low initial concentrations. The morphology of the colonies in CDM was not visually discernible from other bacteria. In contrast, colonies in the DM were dark yellow, small, regular, and glistening, and allowed visual discernment from other bacteria. The CDM and DM were then used for isolating P. multocida and M. haemolytica from feedlot water bowls. Both DM were tested with feedlot water bowl samples collected between September and November 2023, using a total of 120 samples collected over 9 weeks. Putative colonies of P. multocida (300) and M. haemolytica (256) were screened by PCR, but all were negative for P. multocida and M. haemolytica. However, by using a previously described and well documented selective medium for Mycoplasmataceae organisms, 67 M. bovis isolates, confirmed by PCR, were recovered and underwent AST with 10 antimicrobials. All (100%) of the isolates were resistant to macrolides (MIC >64 ug/mL) and florfenicol (MIC > 8 ug/mL); 87% to oxytetracycline (MIC > 4 ug/mL); and 77% to gentamycin (MIC > 8 ug/mL). Approximately 30% of isolates had MIC > 2 ug/mL for enrofloxacin. Genomic analysis identified known resistance-associated mutations in 25 isolates, indicating common resistance mechanisms between water bowl-borne isolates and lung sample isolates. These results suggest the possibility of using environmentally sourced isolates as an alternative for AMR screening in feedlot operations.

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