Polymicrobial pneumonias occur frequently in cattle, swine, and sheep, resulting in major economic losses. Individual pathogens comprising these complex infections may be mild on their own but can instead exhibit synergism or increase host susceptibility. Two examples of such pathogens, Mycoplasma ovipneumoniae (M. ovipneumoniae) and influenza D viruses (IDVs), naturally infect domestic sheep. In sheep, the role of M. ovipneumoniae in chronic nonprogressive pneumonia is well-established, but the pathogenesis of IDV infection has not previously been studied. We utilized a specific-pathogen-free sheep flock to study the clinical response to IDV infection in naïve vs. M. ovipneumoniae-exposed lambs. Lambs were inoculated intranasally with M. ovipneumoniae or mock infection, followed after four weeks by infection with IDV. Pathogen shedding was tracked, and immunological responses were evaluated by measuring acute phase response and IDV-neutralizing antibody titers. While lamb health statuses remained subclinical, M. ovipneumoniae-exposed lambs had significantly elevated body temperatures during IDV infection compared to M. ovipneumoniae-naïve, IDV-infected lambs. Moreover, we found a positive correlation between prior M. ovipneumoniae burden, early-infection IDV shedding, and IDV-neutralizing antibody response. Our findings suggest that IDV infection may not induce clinical symptoms in domestic sheep, but previous M. ovipneumoniae exposure may promote mild IDV-associated inflammation.

Mycoplasma spp. are facultative pathogens that contribute to the pathogenesis of multiple bovine diseases, including the bovine respiratory disease complex, and have been shown to form biofilms. Biofilm formation is associated with increased antibiotic resistance in many organisms, but accurate determination of antimicrobial susceptibility in biofilms is challenging. In Mycoplasma spp., antimicrobial susceptibility is routinely determined using metabolic pH-dependent color change. However, biofilm formation can lead to reduced metabolism, making interpretation of metabolic readouts difficult. Therefore, we developed and optimized a new flow cytometry-based method for antimicrobial susceptibility testing in biofilm-forming Mycoplasma, termed the live/dead antimicrobial susceptibility test (LD-AST). The LD-AST measures the proportion of live bacteria upon exposure to antibiotics, works robustly with both planktonic and biofilm cultures, and enables the determination of the minimum bactericidal concentration (MBC) for a given antibiotic. We used two strains of Mycoplasma bovis (Donetta PG45 and Madison) and two clinical Mycoplasma bovoculi isolates (MVDL1 and MVDL2) to determine the impact of biofilm growth on antimicrobial susceptibility for gentamicin, enrofloxacin, or tetracycline. All Mycoplasma strains were susceptible to all antibiotics when cultured as planktonic cells, with MBCs in the expected range. However, three out of four strains (Donetta PG45, MVDL1, and MVDL2) were completely resistant to all three antibiotics when newly adhered biofilms were analyzed, whereas M. bovis Madison gave variable results. For mature biofilms that were cultured for 4–5 days before antibiotic exposure, results also were variable, with some strains showing an increased resistance with certain antibiotics and a decreased resistance with others. Overall, these results are consistent with earlier reports that biofilms can exhibit increased antimicrobial resistance.