The isolation and characterisation of ruminal mycoplasmas and their interactions with ruminal cellulolytic microorganisms : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

Abstract

Six ruminal mycoplasmas (RM10, RM11, RM12, RM13, RM14 and RM15) were isolated from the ruminal digesta of Friesian cows using anaerobic techniques. The isolates were characterised using a range of culture media and molecular techniques to provide phenotypic data. Cellular and colonial morphology were elucidated using light microscopy, scanning electron microscopy and transmission electron microscopy. It was very apparent the isolates fell into two distinct groups. Isolates RM10, RM11 and RM12 had a growth requirement for sterol, were strictly anaerobic, were able to lyse the cell wall of the Gram-negative bacterium E. coli, were proteolytic, were non-motile, had trilaminar cell membranes, had no distinguishable cell wall, were pleomorphic, were able to pass through 0.45 μm filters, were Gram-negative and produced H2. Therefore, they were classified as members of the genus Anaeroplasma, within the class Mollicutes. In the absence of 16S rRNA sequence data, it was not possible to determine the phylogeny of the isolates. Analysis of cellular proteins by SDS-PAGE demonstrated differences among the three isolates. Random Amplified Polymorphic DNA (RAPD) profiles showed RM11 and RM12 were more closely related to each other than to RM10. Isolates RM13, RM14 and RM15 had the same characteristics as isolates RM10, RM11 and RM12 except they were not able to lyse E. coli cell walls, were not proteolytic and did not produce H2. These phenotypic characteristics identified them as Anaeroplasma abactoclasticum. Analysis of cellular proteins by SDS-PAGE showed variation in high molecular weight bands which suggested RM14 and RM15 were more closely related to each other than to RM13. Evidence based on RAPD profiles of DNA confirmed these relationships. The range of carbohydrates used for growth was small and varied among the isolates. Antibiotics to which both groups were sensitive were those which inhibit protein synthesis and included, chloramphenicol, lincomycin-HCl and tetracycline. All isolates had an optimum growth pH in the range pH 6.0 to 6.8 and an optimum growth temperature in the range 42°C to 45°C. The population density of ruminal mycoplasmas in hay-fed Friesian cows was between 107-108 g-1 of ruminal digesta. A similar population density was observed in grass-fed Friesian cows. The population density of Asteroleplasma species in both sets of animals was between 105-106 g-1. Therefore, ruminal mycoplasmas represent between 0.1-1.0% of the total bacterial population in the bovine rumen. Experimental evidence showed, that when grown in coculture with ruminal cellulolytic fungi, some isolates reduced the extent of cellulose digestion by the fungus as follows; Caecomyces communis (80%), Neocallimastix frontalis (60%) and Piromyces communis (70%). Inhibition of fungal cellulolysis was most marked when the fungi were grown in coculture with An. abactoclasticum (isolates RM13, RM14 and RM15). The isolates were also examined in coculture with ruminal cellulolytic bacteria. Cellulolysis by Ruminococcus species, in coculture with ruminal mycoplasmas, was inhibited by 30-70% when growing on paper. Cellulolysis by Fibrobacter succinogenes and Clostridium chartatabidum was not inhibited, and may have been slightly stimulated. The mechanisms for the observed effects are not known.