Mycoplasma bovis is an economically important bacterial pathogen of cattle (Bos taurus) and bison (Bison bison) that most commonly causes pneumonia, polyarthritis, and mastitis. It is prevalent in cattle and ranched bison; however, infections in other species are rare. In early 2019, we identified M. bovis in free-ranging pronghorn (Antilocapra americana) in northeastern Wyoming. Here, we report on additional pronghorn mortalities caused by M. bovis, in the same approximately 120-km2 geographic region 1 yr later. Genetic analysis by multilocus sequence typing revealed that the mortalities were caused by the same M. bovis sequence type, which is unique among all sequence types documented thus far in North America. To explore whether pronghorn maintain chronic infections and begin assessing M. bovis status in other sympatric species, we used PCR testing of nasal swabs to opportunistically survey select free-ranging ungulates. We found no evidence of subclinical infections in 13 pronghorn sampled from the outbreak area (upper 95% binomial confidence limit [bCL], ∼24.7%) or among 217 additional pronghorn (upper 95% bCL, ∼1.7%) sampled from eight additional counties in Wyoming and 10 in Montana. All mule deer (Odocoileus hemionus; n=231; upper 95% bCL, ∼1.6%) sampled from 11 counties in Wyoming also were PCR negative. To assess the potential for environmental transmission, we examined persistence of M. bovis in various substrates and conditions. Controlled experiments revealed that M. bovis can remain viable for 6 h in shaded water and 2 h in direct sunlight. Our results indicate that environmental transmission of M. bovis from livestock to pronghorn is possible and that seasonality of infection could be due to shared resources during late winter. Further investigations to better understand transmission dynamics, to assess population level impacts to pronghorn, and to determine disease risks among pronghorn and other ungulate taxa appear warranted.

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