Concentration and molecular detection of bovine adenoviruses for fecal source tracking

To assess the utility of bovine adenoviruses (BAdV) as fecal source indicators, we determined their prevalence in bovine manure and examined techniques for their concentration and detection in environmental samples. We compared procedures to recover adenovirus DNA and remove polymerase chain reaction (PCR) inhibitors from fecal and manure samples spiked with human adenovirus type 41 (HAdV-41). The Zymo Research Soil Microbe (ZRsm) DNA extraction kit(TM) demonstrated the greatest sensitivity and inhibitor removal of the methods evaluated and facilitated the PCR detection of exogenous HAdV-41 in bovine, canine, cervid, and porcine fecal samples and in bovine urine, bedding and manure. Unamended animal waste samples were extracted with the ZRsm kit and interrogated for indigenous atadenoviruses and mastadenoviruses using novel, genus-specific semi-nested PCR assays targeting the AdV hexon gene; genetic similarity among human and bovine mastadenoviruses, which has confounded the design of BAdV-specific PCR primers, was overcome by the design of PCR assays based on allele-specific PCR. Eight of the ten prototype BAdV, plus two BAdV that diverged from previously reported genotypes, were amplified from samples having a suspected urine component; all bovine fecal samples examined were BAdV-negative. Phylogenetic analysis of partial mastadenovirus hexon sequences demonstrated unambiguous discrimination of human vs. animal viruses and implied the discovery of three novel Mastadenovirus species.;The detection of viruses in water samples commonly requires the concentration of 10- to >100-L samples. Electropositive NanoCeramRTM syringe filters were evaluated for the retention and recovery of two bacteriophages (MS2 and PRD1) and two adenoviruses (BAdV-1 and HAdV-41) from water. We demonstrated ≥ 99% removal and 0--90% recovery of bacteriophages from feed water, depending on bacteriophage identity and the composition of filter eluents evaluated. Electrostatic and hydrophobic mechanisms contributed to bacteriophage-filter binding. However, optimized eluents were ineffective at recovering adenoviruses: we observed ≥98.6% retention of both adenoviruses on the NanoCeram filters, but less than 5% recovery of these targets. Considering these findings, the recovery of specific viral targets must be verified and optimized before NanoCeram filters are adopted generally for the concentration of viruses in water samples.