An in vitro study of the replication, morphology and DNA base composition of Mycoplasma ovipneumoniae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand
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Mycoplasma ovipneumoniae can almost invariably be isolated from the lungs of sheep with chronic pneumonia, which is a prevalent disease in New Zealand hoggets. At Massey University, a study is in progress to establish the part, if any, played by M. ovipneumoniae in the pathogenesis of the disease. This thesis represents an in vitro investigation of some properties of M. ovipneumoniae. It was undertaken as part of the larger study, and is presented in that context. To establish a method for the production of high titre exponential phase inocula for use in disease transmission experiments, the growth of M. ovipneumoniae in FM4 broth was studied. It was found that a maximum titre of 1.0 to 3.0 x 109 CFU/ml was produced regardless of the inoculum size or degree of aeration. The organism had a minimum division time of 1.7 hr; had no stationary phase and in the late death phase was inactivated with a half-life of about 0.5 hr. The organism was stored at -70° with little loss in titre (less than two-fold) over an 18 month period. Shaking cultures became sufficiently turbid during growth to allow meaningful measurements to be made using an SP20 spectrophotometer. In defined conditions, viz. when a shaking culture is in the exponential phase and contains 2.0 to 10.0 x 108 CFU/ml, the viable cell count can be estimated from turbidity measurements. Electron microscopy of M. ovipneumoniae showed that the cells are roughly spherical, 400 to 700nm in diameter, probably replicate by binary fission, contain ribosomes and fibrils of deoxyribonucleic acid, and are bounded by a trilaminar membrane bearing projections 12nm long. No specialized structural feature such as the attachment sites found in M. pneumoniae was detected. The New Zealand isolate of M. ovipneumoniae was morphologically indistinguishable from the standard M. ovipneumoniae strain isolated in Australia. Although the above description could be applied to many mycoplasma species, it should be noted that the average cell diameter of M. ovipneumoniae (about 550nm) is larger than that found for most but not all species of mycoplasma. The base composition of the DNA of M. ovipneumoniae determined by the thermal denaturation and buoyant density studies was 28.1% GC and 28.0% GC respectively. This relatively low GC content falls within the accepted range for mycoplasma species (23 - 40% GC) and within the much narrower range (26.8 - 28.5% GC) of glycolytic mycoplasmas causing respiratory disease in domestic animals.