calves became partially immune after 4 drug-attenuated infections based on pre-defined parasitological and immunological parameters, including a significant reduction in worm burden, an increase in the percentage of larvae and a change in cytokine expression profile. During the final drugattenuated infection, three calves were drug treated and allowed to rest for 34 weeks and then orally dosed with a tap water placebo. These calves were used as controls. The remaining three calves, which also underwent 4 rounds of infection-treatment-resting procedures and allowed to rest 34 weeks between the treatments, were orally infected with a single-dose of 105 L3 infective larvae for 14 days. At the end of the experiment, calves were sacrificed, and the abomasal contents were collected. The abomasal luminal pH was measured using a standard pH meter. The sample was snap frozen in liquid nitrogen prior to storage at 280uC until DNA was extracted. Fecal egg count was monitored during the repeat infection experiment using zinc sulfate double centrifugation, and parasite burdens were determined as previously described. Roche/454 Pyrosequencing The abomasal microbiota was characterized by two sequencing approaches using the Roche/454 GS FLX Titanium chemistry, the 16S rRNA gene and the whole genome shotgun. For the first approach, unidirectional sequencing of amplicon libraries was performed according to the manufacturer’s instructions with a modification. This modification, using a specific fusion primer design, accommodates amplification using the GS FLX Titanium emPCR Kits. Five hundred ng of DNA were used to generate libraries using the GS FLX Titanium Rapid Library Preparation method ” for WGS sequencing. Therefore, emulsion PCR was carried out using a Lib-L kit for both approaches. Pyrosequencing was conducted using a GS FLX Titanium System following the manufacturer’s protocol. Sequence analysis, protein prediction and annotation 16S rDNA raw sequence reads were first decoded based on sample-specific 8-bp bar codes; their quality was checked, and artifacts “2987739 were removed. Sequence reads shorter than 200 bp were excluded. The sequence read that passed the quality filters were analyzed using the RDP classifier at both 80% and 95% confidence threshold levels for Salianic acid A chemical information taxonomic classification and phylogenetic inference. The 16S rDNA sequences were then analyzed using CD-HITOTU for the abomasal microbial composition at the species level. This algorithm uses a greedy incremental clustering process to identify OTU from 16S rDNA tags, which involves 3 major steps: raw read filtering and trimming, selection of error-free reads, and clustering selected representative reads into individual OTU at a user-specific cutoff. The program avoids over estimation of OTU, a common problem for many existing programs, and results in a rapid and more accurate estimation of microbial diversity in complex microbial ecosystems. OTU identified were then annotated using FR-HIT against the GreenGene database. The 16S rDNA sequences were further analyzed using Fast UniFrac. Briefly, the core set of the 16S GreenGene database was downloaded, and the input 16S sequences were analyzed using MegaBLAST. The resultant hit table was input into the Fast UniFrac server for Principal Coordinates Analysis. Several quality control filters were applied to WGS raw reads before analysis. First, host contaminants were removed using FR-HIT against the bovine genome Btau 4.0. The possible artifacts wer