Roger D. Ayling

Evaluation of MALDI-ToF as a method for the identification of bacteria in the veterinary diagnostic laboratory

Matrix-Assisted Laser Desorption Ionisation-Time of Flight (MALDI-ToF) Mass Spectrometry with Bruker MALDI Biotyper software was evaluated as a method for identifying veterinary bacteria. For 620 isolates (~100 bacterial species), identification by MALDI-ToF and non-16S rDNA methods (mainly phenotypic/biochemical) agreed to species-level (95.3%) and to species/genus-level (100%), but in the absence of 16S rDNA as a gold standard. For a further panel of 107 anaerobes and 234 aerobes (~100 bacteria species) using 16S rDNA results as the gold standard, MALDI-ToF/biochemical tests showed 97.8/96.6% species-level and 99.6/93.5% genus-level agreement for aerobes and 95.3/93.6% species-level and 100/95.3% genus-level agreement for anaerobes compared to the gold standard. Where results were obtained from direct spots, direct spots overlaid with formic acid and extracts, 89.4% of 180 aerobes and 90.1% of 152 anaerobes were identified by MALDI-ToF. MALDI-ToF was shown to be a rapid and reliable method to identify veterinary bacteria.

Mycoplasma bovis : mechanisms of resistance and trends in antimicrobial susceptibility

Mycoplasma bovis is a cell-wall-less bacterium and belongs to the class Mollicutes. It is the most important etiological agent of bovine mycoplasmoses in North America and Europe, causing respiratory disease, mastitis, otitis media, arthritis, and reproductive disease. Clinical disease associated with M. bovis is often chronic, debilitating, and poorly responsive to antimicrobial therapy, resulting in significant economic loss, the full extent of which is difficult to estimate. Until M. bovis vaccines are universally available, sanitary control measures and antimicrobial treatment are the only approaches that can be used in attempts to control M. bovis infections. However, in vitro studies show that many of the current M. bovis isolates circulating in Europe have high minimum inhibitory concentrations (MIC) for many of the commercially available antimicrobials. In this review we summarize the current MIC trends indicating the development of antimicrobial resistance in M. bovis as well as the known molecular mechanisms by which resistance is acquired.

An experimental vaccine composed of two adjuvants gives protection against Mycoplasma bovis in calves

Mycoplasma bovis is a major pathogen affecting cattle causing bronchopneumonia, mastitis, and other disorders. Only autogenous vaccines made specifically for individual farms are available in parts of Europe and the United States. A novel experimental vaccine composed of a field M. bovis isolate combined with saponin and Emulsigen(®) adjuvants was evaluated. Eighteen 3-4 week old calves were placed in three equal groups: vaccinated (Vac), positive control (PC) and negative control (NC). The Vac calves were subcutaneously injected with 8ml of the vaccine; the PC and NC calves received phosphate buffered saline (PBS). Three weeks later the Vac and PC calves were challenged with a virulent M. bovis strain, the NC group received PBS. Throughout the study clinical observations, microbiology and immunological tests were carried out. Three weeks post challenge two calves from each group were euthanased for necropsy and histopathological examination. The vaccine effectively stimulated the humoral immune response, with high titres of anti-M. bovis specific antibodies and total Ig concentration. This vaccine also intensified the IgA response. A clinically protective effect of the vaccine was demonstrated as it also reduced the gross pathological lung lesions and nasal shedding of M. bovis.

Serological survey to determine the occurrence of respiratory Mycoplasma infections in the Polish cattle population

Mycoplasma bovis and Mycoplasma mycoides subspecies mycoides small colony (MmmSC) are causes of bovine mycoplasmosis and contagious bovine pleuropneumonia (CBPP), respectively, and are responsible for serious economic losses in cattle around the world. CBPP was last reported in Poland in 1939 but bovine mycoplasmosis is believed to be endemic. A survey of 3670 serum samples for antibodies to M bovis and MmmSC from 361 herds in 16 Polish provinces Poland between 2007 and 2010 found no evidence of CBPP. The seroprevalence of M bovis, however, appeared high with 76.7 per cent of samples giving a positive reaction in the ELISA test, which did not appear to reflect the clinical disease status of the cattle. Adjusting the sensitivity of the test reduced the prevalence to 28.2 per cent and reflects the levels reported in other European countries

Immunoproteomic characterisation of Mycoplasma mycoides subspecies capri by mass spectrometry analysis of two-dimensional electrophoresis spots and western blot.

Mycoplasma mycoides subspecies capri is one of the causative agents of contagious agalactia in goats. The disease is characterised by mastitis, pneumonia, arthritis, keratitis and in acute cases septicaemia. No vaccine is currently available that has been demonstrated to prevent disease.

Mycoplasma bovis infections and co-infections with other Mycoplasma spp. with different clinical manifestations in affected cattle herds in eastern region of Poland

Abstract The aim of the study was to evaluate the presence of Mycoplasma bovis infection and co-infections with other Mycoplasma spp. infections in cattle. The tested population was one in the eastern region of Poland containing 66 dairy cows and 23 calves showing different clinical signs and suffering from pneumonia, mastitis, and arthritis. The incidence of M. bovis in co-infections with other Mycoplasma spp. was examined using serological traditional mycoplasma culture methods, and the molecular methods - PCR and polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE). The PCR/DGGE method for detecting Mycoplasma spp. in cattle was used for the first time in Poland. The seroprevalence of M. bovis in the affected cattle herds in the eastern region of Poland was 47.8% in calves and 19.7% in dairy cows. The direct detection and identification of M. bovis from nasopharyngeal swabs by PCR revealed that 56.5% of calves were positive, but all of the dairy cows were negative. The PCR/DGGE identified eight (34.8%) instances of M. arginini and eight (26.1%) instances of M. bovirhinis co-infecting with M. bovis in ten calves. The seroprevalence of M. bovis in the tested population was 33.7%. Any future attempts to control mycoplasma infections require an insight into the current epidemiological situation of M. bovis infection and its relationship to other mycoplasmas in causing clinical disease in cattle. Using these diagnostic methods we have demonstrated that mycoplasmal infections are often caused by multiple species of Mycoplasma and not just the primary M. bovis pathogen.

Mycoplasma in cattle

IT was good to hear on Radio 4′s Farming Today programme recently that a Cumbrian farmer, who had sustained disastrous losses two years ago, which resulted in him culling his entire herd, was in the process of restocking his dairy farm. He had reported huge milk production losses, arthritis, poor fertility and illthrift caused by ‘multiple strains of three mycoplasmas: Mycoplasma bovis , Mycoplasma wenyonii and Candidatus Mycoplasma haemobos’. Whether the last two organisms can cause disease is unproven but M bovis is well recognised as …

Investigations into the seasonal presence of Mycoplasma species in fattening lambs.

The presence of infection with Mycoplasma species in association with lung consolidation, environmental temperature and relative humidity was investigated in 410 clinically healthy fattening lambs from five different feedlots in Extremadura (southwestern Spain). Isolates of Mycoplasma species were obtained (n= 117), including Mycoplasma ovipneumoniae (n = 18) and Mycoplasma arginini (n = 99). Two seasonal periods were identified. The first period, which included February, March, September, October, and November, had an average temperature of 17.5 ± 4.7 °C and a relative humidity of 61.3 ± 15.8%. The second seasonal period, which included the months from April to August, had an average temperature of 22.9 ± 5.5 °C and a relative humidity of 48.4 ± 10.7%. Most Mycoplasma species were isolated from the second seasonal period, indicating that higher temperatures and lower relative humidity favour the presence of Mycoplasma species. M. arginini was also associated with lung consolidation.

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

ABSTRACT 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.

Analysis of the immune response of calves to various saponin-based adjuvants for an experimental Mycoplasma bovis vaccine

Mycoplasma bovis is a primary infectious agent of many disorders in cattle including bovine respiratory disease. No commercial vaccines against M. bovis are available in Europe. The immune response of calves to three saponin-based adjuvants combined with a field Polish M. bovis strain was evaluated. Four groups of six calves each were injected subcutaneously with the M. bovis strain combined with either saponin, saponin + Emulsigen®, saponin + Emulsigen® + alphatocopherol acetate, or with phosphate-buffered saline as control group. Blood and nasal swab samples were collected up to day 84 post injection. All formulations effectively stimulated the humoral and the cellular immune response of the calves, but the course of the response depended on the adjuvant formulation. These immunological data provide additional information supporting the findings of previous M. bovis saponin and Emulsigen® vaccine challenge studies to facilitate the development of successful M. bovis vaccines.