ARS | Publication request: PHYLOGENETIC IDENTIFICATION OF BACTERIAL MICROFLORA OF IN VITRO RUMINAL PROTOZOAL CULTURES BY 16S RDNA SEQUENCE ANALYSES

Submitted to: International Union of Microbiological Societies Proceedings/Abstracts
Publication Type: Abstract
Publication Acceptance Date: July 28, 2005
Publication Date: June 1, 2005
Citation: Whitehead, T.R., Dehority, B.A. 2005. Phylogenetic identification of bacterial microflora of in vitro ruminal protozoal cultures by 16S rDNA sequence analyses [abstract]. International Union of Microbiological Societies. Paper No. B-1020.

Technical Abstract: Previous reports have suggested that maintenance of anaerobic ruminal protozoa in vitro requires the presence of a bacterial population. Whether the bacteria are used as a source of food and/or provide a nutritional factor is as yet unclear. However, 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 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 antibiotics (penicillin and streptomycin). Bacteria from cultures of Entodinium caudatum and Epidinium caudatum incubated in the presence and absence of penicillin and streptomycin were recovered by centrifugation. Total DNA was prepared from the bacterial pellets. 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. Diversity of the Operational Taxonomic Units (OTUs) was much less in the Entodinium caudatum cultures as compared to the Epidinium caudatum cultures. OTU sequences also varied between protozoal species. Almost all of the OTUs could not be identified with 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. Results of this study indicate that the bacterial flora varies between protozoal species, based on the different OTU sequences. Antibiotic treatment results in an apparent shift in the bacterial populations towards more Gram-positive OTUs. Such changes in bacterial flora may influence the ability of the protozoa to survive during in vitro cultivation.