The design and evaluation of a genetic probe for Megasphaera elsdenii

M.Sc.The remarkable adaptability of the ruminant with respect to the variety of feeds of different composition which it can utilize is attributable to the presence, in the rumen, of a complex microbial population with diverse nutritional requirements and enzymatic activities. For economic reasons it is essential in feedlot practice to meet, as closely as possible, the energy requirements of ruminants being rounded off for slaughter purposes. This necessitates adapting the animals in the shortest possible time from predominantly pasture to high-concentrate diets, i.e. diets containing a high proportion of readily fermentable carbohydrates. However, a very rapid transition between these diets frequently results in a perturbation of the microbial population of the rumen. In particular, the sudden increase in the concentration of readily fermentable carbohydrates favours the proliferation of lactic acid-producing species, whereas the numbers of lactic acid-utilizing species increase more slowly. The resulting accumulation of lactic acid in the rumen causes a marked drop in pH which has an adverse effect on digestive processes in general. In mild cases, this may merely have a temporary negative effect on food intake and growth of the animal, but in the worst cases it may lead to ruminal stasis, cause permanent damage to the lining of the rumen wall and even result in death. Even though the incidence and severity of acute lactic acidosis may be restricted through careful diet formulation and inclusion of buffers and ionophore antibiotics in the diet, a small proportion of the animals brought into feedlots do not adapt readily to the highconcentrate diets and the performance of a larger proportion is probably negatively affected by sub-acute acidosis. The Nutritional Microbiology Section of the ARC Animal Nutrition and Animal Products Institute at Irene therefore embarked on the development of an alternative preventative and therapeutic treatment against lactic acidosis, viz. the administration of live, laboratory-grown cultures of highly-active lactate utilizers, specially selected for their potential for survival and multiplication in the rumen of animals on feedlot diets. By selective enrichment and extensive screening procedures a collection of phenotypically similar isolates of anaerobic, large, Gram-negative, acid-tolerant and ionophore-resistant cocci was obtained, which were presumptively identified as strains of Megasphaera elsdenii. These organisms utilized both L(+) and D(-) lactate and showed high specific growth rates on relatively inexpensive media even at low pH values, and they appeared to hold good promise for the intended application. In order to test the effectiveness of treatment of acidosis-compromised animals with cultures of these bacteria, information on their persistence in the rumen was required. Conventional microbiological counting methods were unsuitable for this purpose because of slowness and unsatisfactory specificity and precision. The present investigation deals primarily with the design and evaluation of a speciesspecific 16S rRNA probe for M elsdenii. It was intended for following the contribution of this species to the total microbial population of the rumen, as affected by the sudden exposure to high-concentrate diets. Furthermore, the phylogenetic relationship between the recent isolates, the type strain and previously sequenced strains of M elsdenii and M cerevisiae, as well as representatives of other rumen bacteria was investigated. In conclusion, the method for measuring the relative abundance of M elsdenii in rumen microbial populations with the aid of a custom-designed rRNA probe satisfactorily met the requirements of the proposed application