Efficacy of citric acid and sodium hypochlorite as disinfectants against Mycoplasma bovis.

Mycoplasma bovis, a cattle pathogen of major economic importance across the globe, causes a range of diseases, including pneumonia and mastitis. Because of the limited options for effective treatment of these diseases, prevention and control are preferred to diagnosis and treatment. In this study, the efficacies of citric acid and sodium hypochlorite as disinfectants against M. bovis were tested using a modification of a standardised method for assessing the efficacy of disinfectants against bacteria. A citric acid concentration of 0.5 % was found to be an effective disinfectant, reducing infectivity by close to 106 fold, while sodium hypochlorite at 1% was found to have similar efficacy to 0.5 % citric acid. A 0.04 % concentration of sodium hypochlorite was effective against M. bovis only in the absence of any organic material. Under these conditions, 0.25 % citric acid found to have similar efficacy. These findings indicate that 0.5 % citric acid or 1 % sodium hypochlorite are likely to be effective disinfectants for M. bovis under field conditions and 0.04 % sodium hypochlorite or 0.25 % citric acid are likely to be effective following removal of organic material.

iTRAQ-based proteomic analysis of Mycoplasma bovis NM-28 strain from two generations for vaccine screening.

Mycoplasma bovis is an important pathogenic bacterium affecting cows and cattle. Clinically, an inactivated vaccine of M. bovis is mainly used to prevent infection by this bacterium. The changes that occur in the antigen when M. bovis is continuously passaged in vitro remain unknown. Therefore, we performed an in vitro serial passage of the M. bovis NM-28 strain, which was isolated and identified in our laboratory. An isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics method was used to analyse the differences between generations 3 and 60. Many major membrane proteins or protective antigens reported in the literature did not exhibit changes between these generations. We found an imbalance between growth rate and nutrition in the 60th generation. The proteomics results were verified by western blotting and real-time PCR. Growth curves were also prepared based on colony-forming units (CFUs) between the 3rd and 60th generations. The number of colonies in the 60th generation in the stationary phase was 5 × 109 CFU mL-1, which was 10-fold higher than that in the 3rd generation. The 60th generation of the NM-28 strain can be used as an inactivated vaccine strain of M. bovis to lower production costs compared to use of the 3rd generation.

Bovine Epithelial in vitro Infection Models for Mycoplasma bovis.

Mycoplasma bovis causes bovine mycoplasmosis. The major clinical manifestations are pneumonia and mastitis. Recently an increase in the severity of mastitis cases was reported in Switzerland. At the molecular level, there is limited understanding of the mechanisms of pathogenicity of M. bovis. Host-pathogen interactions were primarily studied using primary bovine blood cells. Therefore, little is known about the impact of M. bovis on other cell types present in infected tissues. Clear in vitro phenotypes linked to the virulence of M. bovis strains or tissue predilection of specific M. bovis strains have not yet been described. We adapted bovine in vitro systems to investigate infection of epithelial cells with M. bovis using a cell line (MDBK: Madin-Darby bovine kidney cells) and two primary cells (PECT: bovine embryonic turbinate cells and bMec: bovine mammary gland epithelial cells). Two strains isolated before and after the emergence of severe mastitis cases were selected. Strain JF4278 isolated from a cow with mastitis and pneumonia in 2008 and strain L22/93 isolated in 1993 were used to assess the virulence of M. bovis genotypes toward epithelial cells with particular emphasis on mammary gland cells. Our findings indicate that M. bovis is able to adhere to and invade different epithelial cell types. Higher titers of JF4278 than L22/93 were observed in co-cultures with cells. The differences in titers reached between the two strains was more prominent for bMec cells than for MDBK and PECT cells. Moreover, M. bovis strain L22/93 induced apoptosis in MDBK cells and cytotoxicity in PECT cells but not in bMec cells. Dose-dependent variations in proliferation of primary epithelial cells were observed after M. bovis infection. Nevertheless, an indisputable phenotype that could be related to the increased virulence toward mammary gland cells is not obvious.

Semen as a source of Mycoplasma bovis mastitis in dairy herds.

Mycoplasma bovis infections are responsible for substantial economic losses in the cattle industry, have significant welfare effects and increase antibiotic use. The pathogen is often introduced into naive herds through healthy carrier animals. In countries with a low prevalence of M. bovis, transmission from less common sources can be better explored as the pathogen has limited circulation compared to high prevalence populations. In this study, we describe how M. bovis was introduced into two closed and adequately biosecure dairy herds through the use of contaminated semen during artificial insemination (AI), leading to mastitis outbreak in both herds. Epidemiological analysis did not reveal an infection source other than semen. In both farms the primary clinical cases were M. bovis mastitis in cows inseminated with the semen of the same bull four weeks before the onset of the disease. One semen straw derived from the semen tank on the farm and other semen lots of this bull were positive for M. bovis. In contrast, semen samples were negative from other bulls that had been used for insemination in previous or later oestrus to those cows with M. bovis mastitis. Furthermore, cgMLST of M. bovis isolates supported the epidemiological results. To our knowledge this is the first study describing the introduction of M. bovis infection into a naive dairy herd via processed semen. The antibiotics used in semen extenders should be re-evaluated in order to provide farms with M. bovis-free semen or tested M. bovis-free semen should be available.

Effect of atmospheric carbon dioxide concentration on the cultivation of bovine Mycoplasma species.

Recommendations for bovine mycoplasma culture CO2 concentrations are varied and were not empirically derived. The objective of this study was to determine whether the growth measures of bovine mycoplasma isolates differed when incubated in CO2 concentrations of 10 or 5% or in candle jars (2.7 ± 0.2% CO2). Growth of Mycoplasma bovis (n = 22), Mycoplasma californicum (n = 18), and other Mycoplasma spp. (n = 10) laboratory isolates was evaluated. Isolate suspensions were standardized to approximately 108 cfu/mL and serially diluted in pasteurized whole milk to achieve test suspensions of 102 and 106 cfu/mL. One hundred microliters of each test dilution was spread in duplicate onto the surface of a modified Hayflick's agar plate. Colony growth was enumerated on d 3, 5, and 7 of incubation. A mixed linear model included the fixed effects of CO2 treatment (2.7, 5, or 10%), species, day (3, 5, or 7), and their interactions, with total colony counts as the dependent variable. Carbon dioxide concentration did not significantly affect overall mycoplasma growth differences, but differences between species and day were present. Colony counts (log10 cfu/mL) of M. bovis were 2.6- and 1.6-fold greater than M. californicum and other Mycoplasma spp., respectively. Growth at 7 d of incubation was greater than d 3 and 5 for all species. These findings were confirmed using field isolates (n = 98) from a commercial veterinary diagnostic laboratory. Binary growth responses (yes/no) of the field isolates were not different between CO2 treatments but did differ between species and day of incubation. On average, 57% of all field isolates were detected by 3 d of incubation compared with 93% on d 7. These results suggest that the range of suitable CO2 culture conditions and incubation times for the common mastitis-causing Mycoplasma spp. may be broader than currently recommended.

Milk acidification to control the growth of Mycoplasma bovis and Salmonella Dublin in contaminated milk.

Bacterial contamination of milk fed to calves compromises calf health. Several bacterial pathogens that infect cows, including Mycoplasma bovis and Salmonella enterica ssp. enterica serovar Dublin, are shed in milk, providing a possible route of transmission to calves. Milk acidification lowers the milk pH so that it is unsuitable for bacterial growth and survival. The objectives of this study were to (1) determine the growth of M. bovis and Salmonella Dublin in milk, and (2) evaluate the efficacy of milk acidification using a commercially available acidification agent (Salstop, Impextraco, Heist-op-den-Berg, Belgium) to control M. bovis and Salmonella Dublin survival in milk. For the first objective, 3 treatments and a positive control were prepared in 10 mL of milk and broth, respectively, and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 104 cfu/mL. Each treatment was retained at 5, 23, or 37°C with the positive control at 37°C. Aliquots were taken at 4, 8, 24, 28, 32, 48, 52, and 56 h after inoculation and transferred onto agar medium in triplicate following a 10-fold dilution series in sterile phosphate-buffered saline. All plates were incubated and colonies counted. For the second objective, 4 treatments and a positive control were prepared with 100 mL of milk and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 106 cfu/mL. With the use of Salstop, treatments were adjusted to an approximate pH of 6, 5, 4, or 3.5. The positive control was left untreated. At 1, 2, 4, 6, 8, and 24 h after treatment, triplicate aliquots were taken, the pH measured, and then the aliquots were transferred onto agar medium and into broth for enrichment. Following incubation, agar colonies were counted, while broths were plated and incubated prior to colonies being counted. All trials were repeated. Mycoplasma bovis did not grow in milk, but Salmonella Dublin proliferated. The pH of all acidification treatments remained stable for 24 h. No viable M. bovis organisms were detected at 1 h of exposure to pH 3.5 and 4 or at 8 h of exposure to pH 5. Following 24 h of exposure to pH 6 M. bovis remained viable. No viable Salmonella Dublin organisms were detected at 2 and 6 h of exposure to pH 3.5 and 4, respectively. Salmonella Dublin remained viable following 24 h of exposure to pH 5 and 6. These results demonstrate that milk acidification using Salstop is effective at eliminating viable M. bovis and Salmonella Dublin organisms in milk if the appropriate pH and exposure time are maintained.

High Prevalence of Diverse Insertion Sequences within the rRNA Operons of Mycoplasma bovis.

Insertion sequences (ISs) are widespread in the genome of Mycoplasma bovis strain PG45, but no ISs were identified within its two tandemly positioned rRNA operons (rrn1 and rrn2). However, characterization of the rrn locus in 70 M. bovis isolates revealed the presence of ISs related to the ISMbov1 (IS30 family) and ISMbov4 (IS4 family) isomers in 35 isolates. ISs were inserted into intergenic region 1 (IGR-1) or IGR-3, which are the putative promoter regions of rrn1 and rrn2, respectively, and into IGR-5, located downstream of the rrl2 gene. Seven different configurations (A to G) of the rrn locus with respect to ISs were identified, including those in five annotated genomes. The transcriptional start site for the single rrn operon in M. bovis strain 88127 was mapped within IGR-1, 60 bp upstream of the rrs gene. Notably, only 1 nucleotide separated the direct repeat (DR) for ISMbov1 and the promoter -35 element in configuration D, while in configuration F, the -35 motif was a part of the ISMbov1 DR. Relative quantitative real-time (qRT) PCR analysis and growth rate comparisons detected a significant increase (P < 0.05) in the expression of the rrs genes and in the number of viable cells during log phase growth (8, 12, and 16 h) in the strains with configuration F in comparison to strains with one or two rrn operons that did not have ISs. A high prevalence of IS elements within or close to the M. bovis rrn operon-promoter region may reflect their important role in regulation of both ribosome synthesis and function. IMPORTANCE: Data presented in this study show a high prevalence of diverse ISs within the M. bovis rrn locus resulting in intraspecies variability and diversity. Such abundance of IS elements near or within the rrn locus may offer a selective advantage to M. bovis Moreover, the fact that expression of the rrs genes as well as the number of viable cells increased in the group of strains with IS element insertion within a putative promoter -35 sequence (configuration F) in comparison to that in strains with one or two rrn operons that do not have ISs may serve as a basis for understanding the possible role of M. bovis IS elements in fundamental biological processes such as regulation of ribosome synthesis and function.

Prevalence and genotype of Mycoplasma bovis in beef cattle after arrival at a feedlot.

OBJECTIVE: To determine the prevalence of Mycoplasma bovis infection in the lungs of cattle at various times after arrival at a feedlot, to measure the relationship between clinical disease status and the concentration and genotype of M bovis within the lungs, and to investigate changes in the genotype of M bovis over time. SAMPLE: Bronchoalveolar lavage fluid (BALF) from 328 healthy or pneumonic beef cattle and 20 M bovis isolates obtained from postmortem samples. PROCEDURES: The concentration of M bovis in BALF was determined via real-time PCR assays, and M bovis isolates from BALF were genotyped via amplified fragment length polymorphism (AFLP) analysis. RESULTS: Prevalence of M bovis in BALF was 1 of 60 (1.7%) at arrival to a feedlot and 26 of 36 (72.2%) and 36 of 42 (85.7%) at ≤ 15 days and 55 days after arrival, respectively. Neither the concentration nor the AFLP type of M bovis in BALF was correlated with clinical disease status. The M bovis AFLP type differed between early and later sampling periods in 14 of 17 cattle. CONCLUSIONS AND CLINICAL RELEVANCE: The findings implied spread of M bovis among calves and suggested that host factors and copathogens may determine disease outcomes in infected calves. Chronic pulmonary infection with M bovis may represent a dynamic situation of bacterial clearance and reinfection with strains of different AFLP type, rather than continuous infection with a single clone. These findings impact our understanding of why cattle with chronic pneumonia and polyarthritis syndrome inadequately respond to antimicrobial treatment.

Oral inoculation of young dairy calves with Mycoplasma bovis results in colonization of tonsils, development of otitis media and local immunity.

Because M. bovis otitis media is an economically important problem, there is a need to understand the pathogenesis of disease, not only to improve our understanding of the factors contributing to the development of this disease but also to inform the development of improved diagnostic tests and therapy. Oral ingestion of M. bovis-contaminated milk is linked, but not definitively proven, to development of otitis media. In the current study, we demonstrate that oral ingestion of M. bovis infected colostrum can result in an ascending infection and development of otitis media. Importantly, M. bovis was found to have a previously unrecognized tendency for colonization of the tonsils of calves, which most likely contributed to the subsequent development of otitis media. In contrast, transtracheal inoculation failed to produce clinically significant upper respiratory tract disease, although did induce lower respiratory tract disease. The upper respiratory tract was the major site of M. bovis-specific B cell and mucosal IgA responses in calves inoculated by the oral route. The oral inoculation route of infection presented here is particularly suited to the study of host-pathogen interactions during initial colonization of the tonsils, expansion of infection and dissemination to the lower respiratory tract and middle ear. In addition, it could be used to investigate potential new preventative or control strategies, especially those aimed at limiting colonization of the tonsils and/or spread to the middle ear.

Identification of novel small molecule antimicrobials targeting Mycoplasma bovis.

OBJECTIVES: To screen novel small molecule compounds for inhibition of Mycoplasma bovis growth and to characterize their activity in terms of dose-dependency and ability to function in milk. METHODS: Using a tetrazolium salt cytotoxicity assay, 480 natural compounds were screened to determine which of the small molecules have the potential to become therapeutic options for M. bovis prevention and treatment. The dose response was determined in broth culture and in fresh quarter milk for a subset of compounds shown to be capable of inhibiting M. bovis growth. RESULTS: Data suggest that 32 of the 480 compounds tested were able to inhibit growth of M. bovis using a tetrazolium salt assay. Methanesulphonic 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 (+)-α-tocopherol acid succinate were selected for further concentration-dependent studies and testing in fresh quarter milk. Each compound demonstrated a dose response in broth culture and at 3 h and 24 h in fresh quarter milk. CONCLUSIONS: Small molecule natural compounds are capable of inhibiting the growth of M. bovis in both a pleuropneumonia-like organism (PPLO) medium and in fresh quarter milk. Results suggest that the compounds are mycoplasmastatic in a dose-dependent manner. By inhibiting M. bovis, 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.

Mycoplasma bovis Infections in young calves.

Mycoplasma bovis has emerged as an important pathogen of young intensively reared calves in North America. A variety of clinical diseases are associated with M bovis infections of calves, including respiratory disease, otitis media, arthritis, and some less common presentations. Clinical disease associated with M bovis often is chronic, debilitating, and poorly responsive to antimicrobial therapy. Current control measures are centered on reducing exposure to M bovis through contaminated milk or other sources, and nonspecific control measures to maximize respiratory defenses of the calf. This article focuses on the clinical and epidemiologic aspects of M bovis infections in young calves.