Immunological and molecular discrimination of Mycoplasma bovis from other mycoplasma species in cattle

Ghadersohi, Ali (1997) Immunological and molecular discrimination of Mycoplasma bovis from other mycoplasma species in cattle. PhD thesis, James Cook University.

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Abstract

Mycoplasma bovis is one of the most pathogenic bovine mycoplasmas causing extensive economic losses due to mastitis, pneumonia, keratoconjunctivitis, abortion and infertility. Current methodologies for detecting and identifying Mycoplasma bovis are time consuming and have poor sensitivity and specificity. The present studies were carried out to develop improved diagnostic techniques for the discrimination of Mycoplasma bovis from other Mycoplasma species and other bacterial species that cause infections in cattle. Four different detection techniques were developed and evaluated. These were isolation by conventional cultural methods, antigen and antibody detection by enzyme linked immunosorbent assay, development of a specific DNA probe and development of a polymerase chain reaction assay using primers specific for Mycoplasma bovis.

To evaluate cultural methods for the detection of M. bovis, milk samples and lung tissue specimens were obtained from the Atherton Tableland and a Townsville abattoir, respectively. A total of 500 lungs was examined for macroscopic evidence of pneumonia. Forty lungs had lesions macroscopically resembling Mycoplasma pneumonia. Fifteen were examined by histopathology and the microscopic lesions of interstitial pneumonia suggested that Mycoplasma was involved. Mycoplasma bovis was isolated from three cases suggesting a sensitivity for cultural methods of 20%. From the 92 individual quarter milk samples tested, two were positive by cultural methods. In the polymerase chain reaction developed, 35 of 92 quarters (38%) were positive for Mycoplasma bovis, again confirming the poor sensitivity of culture. Cultural methods were extremely insensitive and time consuming and commonly the cultures were overgrown by bacterial contaminants.

As an alternative to cultural methods, the detection of antibodies to Mycoplasma bovis and Mycoplasma bovis antigens in clinically diseased animals was attempted. An indirect enzyme linked immunosorbent assay was initially developed to detect the presence of Mycoplasma bovis antibody in animal and human sera that was used in the production of the Mycoplasma media used in these studies. The enzyme linked immunosorbent assay was also used to detect hybridomas secreting Mycoplasma bovis antibody during the production of monoclonal antibodies. It was found that under optimal conditions the indirect enzyme linked immunosorbent assay is fast and reproducible and a convenient and sensitive method for the detection of Mycoplasma bovis antibody in cattle sera. The limitations of the method include an extensive purification of antigen, and also some non-specific background signal. To improve antibody and antigen detection a panel of 15 monoclonal antibodies was produced using Mycoplasma bovis whole-cell antigen. Only one monoclonal antibody was specific to Mycoplasma bovis, while others were found to cross react with Mycoplasma arginini, Mycoplasma agalactiae, Mycoplasma bovirhinis, Mycoplasma group 7, Mycoplasma bovigenitalium and Mycoplasma dispar. An indirect enzyme linked immunosorbent assay using the specific monoclonal antibody was useful for the clinical identification of Mycoplasma bovis isolated using cultural methods. Using this procedure, the three Mycoplasma isolated from the lung samples and the two Mycoplasma isolates from quarter milk samples were confirmed as a Mycoplasma bovis. This method was found to be difficult to standardise for the direct detection of Mycoplasma antigen in clinical samples, due to low sensitivity and specificity. A blocking ELISA was developed for the detection of specific Mycoplasma bovis antibody in serum. Using this method the specificity and sensitivity of the assay were improved and the background reaction was eliminated. This method revealed the existence of Mycoplasma bovis antibodies in 60 out of 100 (60%) serum samples from north Queensland dairy cattle. However the use of antibody detection by enzyme linked immunosorbent assay is limited by the fact that antibody titres become detectable only 10 to 14 days after the onset of disease. Because the sensitivity of the enzyme linked immunosorbent assay was not sufficient for reliable identification of sera containing antibodies to Mycoplasma bovis, a DNA probe for detection of Mycoplasma bovis was developed.

A genomic library was prepared from Mycoplasma bovis DNA digested with Sau 3AI and cloned into pUC19. Colony hybridisation, using a probe prepared from purified Mycoplasma bovis DNA, was used to identify colonies of interest. Mycoplasma bovis DNA fragments were retrieved from recombinant plasmids by digestion with EcoRI and HindIII. This DNA was used to prepare randomly primed probes for dot blot hybridisation analysis with immobilised DNA from two strains of Mycoplasma bovis, Mycoplasma dispar, Mycoplasma agalactiae, two strains of Mycoplasma bovigenitalium, Mycoplasma ovipneumoniae, a Group 7 strain, Mycoplasma arginini, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Streptococcus uberis, Corynebacterium bovis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pasturella sp., Brucella abortus, Yersinia enterocolitica, Enterobacter cloacae, Enterobacter faecalis, Serratia marcescens, Bacillus subtilis and Escherichia coll. Four probes were found to hybridise only with M bovis and M ovipneumoniae genomic DNA, whereas one probe reacted only with genomic DNA from one of the two Mycoplasma bovis strains. Sensitivity of the dot blot hybridisation assay was 200 colony forming units per millilitre.

To increase the sensitivity of detection further, an Mycoplasma bovis-specific polymerase chain reaction assay was developed. The primers were designed using sequences obtained from the DNA probe which discriminated Mycoplasma bovis from all other Mycoplasma DNA tested. The minimum amount of target DNA that could be detected by the polymerase chain reaction assay was that isolated from 10-20 colony forming units per millilitre. The Mycoplasma bovis-specific polymerase chain reaction assay was therefore 10 times more sensitive than dot blot hybridisation. The identity of the three Mycoplasma spp. isolated from the cattle lung samples and the two isolates from the quarter milk samples (that showed evidence of clinical mastitis) were reconfirmed by using the polymerase chain reaction assay. The assay was highly sensitive and specific for the detection of Mycoplasma bovis in milk samples, lungs and also nasal secretions.

The assay was applied for the first time to estimate the prevalence of Mycoplasma bovis infection in dairy cattle in Australia. In the first study, 186 and 165 bulk milk samples from north Queensland and Victoria respectively, were supplied from milk factories following routine somatic cell counting. Based on published guidelines for the interpretation of somatic cell count in bulk milk as an indicator of mastitis in a herd, five groups were determined: 1) slight <250 x 103, 2) average 250-500 x 103, 3) above average 500-750 x 103, 4) bad 750-1000 x 103, 5) severe >1000. The association of Mycoplasma bovis infection in north Queensland milk samples with level of mastitis was: 32% of cases recognised as slight, 50% as average and in 65% of cases recognised as having above average level of mastitis. For milk samples from Victoria, Mycoplasma bovis was associated with 32% of cases recognised as having a slight level of mastitis, 71% average and 82% as having an above average level of mastitis.

In a study of 92 composite milk samples from individual cows in a single herd from Queensland, Mycoplasma bovis was detected in 55.5 % of cases recognised as having a slight level of mastitis, 70.5 % as average, 66.6% above average, 50% bad and 95% as having a severe level of mastitis.

A total of 52 quarter milk samples from cows with persistently high somatic cell counts in their composite milk were tested by Mycoplasma bovis-specific polymerase chain reaction, and culture and the association with major mastitis pathogens was investigated. Mycoplasma bovis was detected by polymerase chain reaction in 77% of samples tested of which 19% were infected with M bovis alone, without any other bacteria detected, 17% had Mycoplasma bovis in combination with a major mastitis pathogen, and 40% had Mycoplasma bovis in combination with a non-major mastitis pathogen. A high association was found between positive reactors detected by indirect or blocking enzyme linked immunosorbent assay and animals found positive for Mycoplasma bovis by Mycoplasma bovis-specific polymerase chain reaction. The prevalence of Mycoplasma bovis in the respiratory tract of cattle and calves was also investigated using Mycoplasma bovis-specific polymerase chain ration and a close correlation between Mycoplasma bovis infection of the respiratory tract and the udder of cattle was found, suggesting the probable route in the infection cycle.

It is believed that Mycoplasma bovis infections are widespread in dairy cattle and has the potential to produce disease alone or to predispose the udder to disease caused by major mastitis pathogen and other environmental pathogens. These initial studies have revealed a previously unrecognised high prevalence of Mycoplasma bovis in dairy cattle in north Queensland and Victoria. These studies also give a clear association between Mycoplasma bovis and elevated somatic cell counts. The role of Mycoplasma bovis as a cause of, or predisposing factor for bovine mastitis, is an area that requires further study.

Item ID: 28062
Item Type: Thesis (PhD)
Keywords: Mycoplasma bovis; diagnostic techniques; polymerase chain reaction assay; mastitis pathogens; bovine mastitis
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Ghadersohi,A., Coelen, R.J., Hirst, R.G. (1997) Development of a specific DNA probe and PCR for the detection of Mycoplasma bovis. Veterinary Microbiology. 56(1-2). pp. 87-98.

Ghadersohi, Ali, Fayazi, Zahra, and Hirst, Robert G. (2005) Development of a monoclonal blocking ELISA for the detection of antibody to Mycoplasma bovis in dairy cattle and comparison to detection by PCR. Veterinary Immunology & Immunopathology, 104 (3-4). pp. 183-193.

Date Deposited: 08 Aug 2013 07:24
FoR Codes: 07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070703 Veterinary Diagnosis and Diagnostics @ 100%
SEO Codes: 83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8303 Livestock Raising > 830302 Dairy Cattle @ 51%
97 EXPANDING KNOWLEDGE > 970107 Expanding Knowledge in the Agricultural and Veterinary Sciences @ 49%
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