Submitted to: Conference on Gastrointestinal Function
Publication Type: Abstract Only
Publication Acceptance Date: 4/6/2005
Publication Date: 4/6/2005
Citation: Whitehead, T.R., Dehority, B.A. 2005. Characterization of microbial populations of in vitro ruminal protozoal cultures by 16S rDNA sequence analyses [abstract]. Conference on Gastrointestinal Function. Paper No. 32. Interpretive Summary:
Technical Abstract: Previous reports have suggested that in vitro maintenance of ruminal protozoa requires the presence of a bacterial population. Whether the bacteria are used as a source of food or alternatively provide a nutritional factor is as yet unclear. Little is known about the bacterial populations that are present in single species or clone cultures of ruminal protozoa, and if these populations vary between different protozoal species. Antibiotic treatment has been used previously by a number of researchers to reduce or eliminate the bacterial populations of in vitro cultures to determine the effects on the protozoa. For both species used in the present study, incubation with antibiotics had no immediate effect on protozoal concentrations. However, washing the cells and transferring half the culture to fresh medium several times did not prevent death of the protozoa by 8-12 days. We have initiated a study of the bacterial flora present in cultures of Entodinium caudatum and Epidinium caudatum with and without antibiotic treatment (penicillin and streptomycin). Bacteria from cultures were recovered and total DNA prepared. DNA sequence analyses of PCR amplified 16S rDNA genes derived from eubacterial primers were carried out (40 sequences/culture). Similarity analyses of the 16S rDNA sequences indicated the presence of both Gram-negative and Gram-positive anaerobic bacteria from all cultures. Bacterial diversity, as measured by the number of Operational Taxonomic Units (OTUs), was much less in the Entodinium caudatum cultures as compared to the Epidinium caudatum cultures. Almost all of the OTUs could not be identified to known bacteria when compared to 16S rDNA sequences in the GenBank database. Antibiotic treatments resulted in a shift towards more Gram-positive OTUs with both cultures. These results indicate that the bacterial flora varies between protozoal species, and antibiotic treatment results in a shift in the bacterial populations.