"Molecular Characterization and Epidemiology of Mycoplasma bovis"
Open Access
- Author:
- Soehnlen, Marty Katherine
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 28, 2011
- Committee Members:
- Bhushan M Jayarao, Dissertation Advisor/Co-Advisor
Bhushan M Jayarao, Committee Chair/Co-Chair
Subhashinie Kariyawasam, Committee Member
Arthur Hattel, Committee Member
Eugene Joseph Lengerich, Committee Member
Jeanne Ann Lumadue, Committee Member
David R Wolfgang, Committee Member - Keywords:
- vaccination
epidemiology
cattle
Mycoplasma bovis
veterinary medicine
antimicrobial - Abstract:
- Mycoplasma bovis is an important pathogen causing pneumonia, mastitis, arthritis, conjunctivitis, otitis, genital infections, and septicemia in cattle. A molecular epidemiological analysis of M. bovis strains submitted to the Pennsylvania Animal Diagnostic Laboratory (PA-ADL) between December 1, 2007 and November, 30 2008 was conducted. During this period, M. bovis represented 63.6% of Mycoplasma species isolated from samples submitted to PA-ADL. The ability to separate isolates into clonally distinct groups showing strong genetic heterogeneity was demonstrated through the utilization of a novel technology, amplified fragment length polymorphism (AFLP). The results were consistent with the existence of at least two clonally distinct groups, although there was no clear geographical, month of isolation, or source origination relationship, indicating a currently unclassified characteristic is responsible for the strain heterogeneity. AFLP may serve as a valuable tool for molecular characterization of M. bovis strains from the United States. Mycoplasma bovis isolates submitted to the PA-ADL (n=192) were tested for antimicrobial susceptibility to enrofloxacin, erythromycin, florfenicol, spectinomycin, ceftiofur, tetracycline, and oxytetracycline using a broth micodilution testing method which incorporated a redox color change reagent. The antimicrobials which were most effective for M. bovis using the broth microdilution method were florfenicol, enrofloxacin, and tetracycline with a minimum inhibitory concentration (MIC) range of 2 - 32 µg/mL, 0.1 - 3.2 µg/mL, and 0.05 - >12.8 µg/mL, respectively. A significant difference in the susceptibility levels between quarter milk and lung M. bovis isolates was found for spectinomycin. Using CLSI-approved breakpoints for other bovine respiratory disease pathogens it was determined that enrofloxacin, spectinomycin, ceftiofur, erythromycin, tetracycline, oxytetracycline, and florfenicol demonstrated resistance in 2.1%, 58.3%, 100%, 100%, 12.5%, 22.4%, and 11.4% of isolates, respectively. MIC values of a subset of the M. bovis isolates (n=12) were tested using a novel side-scatter flow cytometric technique with concurrent staining using SYBR green I and propidium iodide. The most effective antimicrobials for the 12 isolates were determined to be enrofloxacin and florfenicol. Flow cytometry offers potential in clinical applications due to high throughput-capability, quick turn-around time, and the objective nature of interpreting results. Based upon the increasing resistance patterns of M. bovis to several currently administered veterinary antimicrobials, novel small molecule compounds were screened for potential inhibition of Mycoplasma bovis growth and dose-dependent response was evaluated. The determination of inhibition of M. bovis growth in a milk environment using novel small molecule natural compounds was also studied. The data suggest that 32 of the 483 compounds tested were able to inhibit growth of M. bovis using a tetrazolium salt assay. Methanesulfonic acid, 3-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyloxy](1S,3R,4R,5R)-1,4,5-trihydroxycyclohexane carboxylic acid, S-carboxymethyl-L-cysteine, L-aspartic acid, dihydrotachysterol, eriodictyol, and (+)-alpha-tocopherol acid succinate demonstrated a dose response in the tetrazolium broth culture assay and at 3 hours and 24 hours in fresh quarter milk. Small molecule natural compounds offer the potential for prophylactic or therapeutic use on organic and natural farms as a viable alternative to traditional antimicrobial agents. A longitudinal study was conducted to determine the cumulative incidence and prevalence of M. bovis in special-fed veal calves from 4 Pennsylvania herds. The period prevalence of M. bovis colonization was 90.5% for the nasal swabs and 38% for swabs collected at the bronchial bifurcation. A total of 90 lung lesions were identified from the 252 calves in the study. A total of 42% of the lung lesions collected were culture positive for M. bovis, while 37% of bronchi swabs were culture positive. Evidence suggests that M. bovis, in concert with other respiratory pathogens, is responsible for pneumonic lesions in veal calves. It was determined that calves with a member of the Pasteurellaceae family cultured from a lung lesion was 10.8 (OR=10.7885; 95% CI 2.2245-52.3231) times more likely to also have M. bovis present in the lesion than Pasteurellaceae alone. The data suggest that nasal swab testing on special-fed veal farms can be used to screen calves for the presence of M. bovis, but are unlikely to be successful at determining exactly which calves are at increased risk of developing lung lesions. A blinded, controlled trial of two commercially available M. bovis bacterin vaccines for the prevention of respiratory disease associated with M. bovis infection in special-fed veal calves was conducted. Vaccine A efficacy was compared to a placebo and vaccine B efficacy was compared to 0.9% sterile saline solution. Upper-respiratory tract colonization was not impacted by vaccination status. The presence of lung lesions was significantly reduced by treatment of vaccine A (p=0.0325), however lung lesions from tissues positive for M. bovis was not significantly reduced. Vaccine B was not shown to significantly reduce total lung lesions or M. bovis-specific lung lesions. There was no association between specific antibody concentrations and M. bovis-associated morbidity in the veal calves. Approximately 30% of all calves in the study were found to be seropositive for M. bovis upon acclimation to the barn, indicating a potential problem of maternal M. bovis in Pennsylvania. A detectable difference in the IL-1â and TNF-á level for vaccinated calves (vaccine A and vaccine B) was seen as compared to control calves. Under the field conditions of this study, vaccine efficacy for special-fed veal calves was determined to be 44% and less than 1% for vaccine A and vaccine B, respectively. In summary, the findings presented indicate that M. bovis is an important public health concern in Pennsylvania. Although virulence specific factors or environmental factors contributing to the severity of M. bovis-associated disease have yet to be discovered, this work has provided a basis for continued investigation. Further understanding of the changing epidemiology of M. bovis in Pennsylvania will provide valuable information for the design and implementation of M. bovis prevention and management strategies in the United States.