Diagnosis and control of Mycoplasma bovis and Mycoplasma mycoides subspecies Mycoides small colony in cattle
Mycoplasmas are responsible for many important diseases of animals, including contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides SC and calf pneumonia, arthritis and mastitis caused by Mycoplasma bovis. However, diagnostic techniques currently available are laborious and imprecise. The work described in this thesis concentrates on the critical evaluation of existing techniques and the development of improved procedures. The role of antimicrobial resistance in limiting the options for disease control was also considered. Diagnostic methods for the detection of M. mycoides subsp. mycoides SC in clinical material were critically evaluated during a CBPP outbreak in Portugal. Immunoblotting was more sensitive and specific than CFT or ELISA. The polymerase chain reaction (PCR) was more rapid and sensitive than culture. However immunocytohistochernistry (ICC) was far the best test for detecting M. mycoides subsp. mycoides SC antigen in lungs. A rapid latex agglutination test (LAT) to detect CBPP using a carbohydrate extract of M. mycoides subsp. mycoides SC was developed. Analysis of the carbohydrate extract composition demonstrated that fucose, glucosamine and galactose are present in the ratio of 1:2:16 respectively. N-acetyl neuraminic acid was also detected. Evaluation of the LAT with sera from negative, naturally infected and experimentally infected cattle demonstrates that the test clearly differentiates positive and negative CBPP sera. The LAT compared favourably with the CFT but was not as specific as the immunoblotting; however the LAT had the advantage of being more rapid and robust and could be used in the field. Molecular methods including the polymerase chain reaction (PCR) and 16S rRNA gene sequencing were assessed for their potential use in the diagnostic laboratory. A PCR method for identifying M. bovis was adapted, evaluated and introduced as a routine laboratory test. Using a set of universal 16S rRNA gene primers, amplicons of two serologically untypable isolates, one from a peregrine falcon, and the other from an ostrich were obtained. Results imply that the isolates may be new mycoplasma species. The development of antimicrobial resistance has been seen in many microorganisms but little evidence exists for resistance in mycoplasmas. Consequently, the in vitro effect of five antimicrobials; danofloxacin, oxytetracycline, spectinomycin, florfenicol and tilmicosin on 62 isolates of M. bovis and 20 of M. mycoides subsp. mycoides SC was investigated. Nfinimum inhibitory concentrations (MICs) and mycoplasmacidal (MMC) values were determined. Evidence of antimicrobial resistance by M. bovis is shown. The potential for M. mycoides subsp. mycoides SC to develop antimicrobial resistance against spectinomycin in vitro is also demonstrated.