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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Publications at this Location » Publication #185353


item Scupham, Alexandra
item Baldwin, John
item Rasmussen, Mark

Submitted to: American Society for Microbiology Branch Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 9/22/2005
Publication Date: 9/22/2005
Citation: Scupham, A.J., Baldwin, J., Rasmussen, M.A. 2005. Fingerprint analysis of bacterial communities associated with single protozoa [abstract]. North Central Branch-American Society for Microbiology. p. 61.

Interpretive Summary:

Technical Abstract: GOAL: To describe bacterial communities consumed by and associated with individual protozoa, and to correlate protozoa species with the bacterial communities. METHODS: Individual rumen protozoa were isolated from washed rumen contents using drawn-out glass capillaries. Proteinase K cell lysis followed by centrifugation liberated protozoal and bacterial DNA simultaneously. Universal primers for protozoa small-subunit (SSU) ribosomal gene amplification were designed from an alignment of 262 protozoa 18S sequences retrieved from NCBI. Primers were used in nested reactions and include: 1 degree amplification, PZF2: YAA AGA YTA AGC CAT GCA TG and PZR1: AGC GAC GGG CGG TGT GTA CA; 2 degrees amplification, PZF1: CCA GCA GCC GCG GTA ATT CC and PZR2: ATC ACS GAC CTG TTA TTG CC. SSU genes were cloned into pGEM-T and sequenced. Automated Ribosomal Intergenic Spacer Analysis (ARISA) using nested primer pairs was performed to generate community fingerprints of protozoa-associated bacteria. Primers include: 1degree amplification, L-D-Bact-132-a-A-18 and ITSF; 2 degrees amplification S-D-Bact-1522-b-S-20 and ITSReub. RESULTS: SSU protozoa genes were identified by sequence as Dasytricha ruminantium (8 isolates, 96-100% sequence similarity) Isotricha prostoma (1 isolate, 96% sequence similarity), Epidinium caudatum (1 isolate, 98% sequence similarity) and two isolates without similarity to known protozoa sequences. Fingerprints between isolates were not identical, although some ARISA fragments were common to most Dasytricha. CONCLUSIONS: Lack of bacterial fingerprint identity between individuals of a protozoa species implies discrimination of consumable bacteria is likely based on bacterial size rather than bacterial species. Bacterial species represented in multiple Dasytricha ARISA fingerprints may represent associated rather than ingested bacterial species, or very abundant bacterial species. Extended SSU analysis of the rumen protozoa will allow phylogenetic analysis of animals previously identified solely by physical characteristics.