Effectiveness of stabilization methods for the immediate and short-term preservation of bovine fecal and upper respiratory tract genomic DNA.

Previous research on stabilization methods for microbiome investigations has largely focused on human fecal samples. There are a few studies using feces from other species, but no published studies investigating preservation of samples collected from cattle. Given that microbial taxa are differentially impacted during storage it is warranted to study impacts of preservation methods on microbial communities found in samples outside of human fecal samples. Here we tested methods of preserving bovine fecal respiratory specimens for up to 2 weeks at four temperatures (room temperature, 4°C, -20°C, and -80°C) by comparing microbial diversity and community composition to samples extracted immediately after collection. Importantly, fecal specimens preserved and analyzed were technical replicates, providing a look at the effects of preservation method in the absence of biological variation. We found that preservation with the OMNIgene®â€¢GUT kit resulted in community structure most like that of fresh samples extracted immediately, even when stored at room temperature (~20°C). Samples that were flash-frozen without added preservation solution were the next most representative of original communities, while samples preserved with ethanol were the least representative. These results contradict previous reports that ethanol is effective in preserving fecal communities and suggest for studies investigating cattle either flash-freezing of samples without preservative or preservation with OMNIgene®â€¢GUT will yield more representative microbial communities.

Does swab type matter? Comparing methods for Mannheimia haemolytica recovery and upper respiratory microbiome characterization in feedlot cattle.

BACKGROUND: Bovine respiratory disease (BRD) is caused by interactions among host, environment, and pathogens. One standard method for antemortem pathogen identification in cattle with BRD is deep-guarded nasopharyngeal swabbing, which is challenging, costly, and waste generating. The objective was to compare the ability to recover Mannheimia haemolytica and compare microbial community structure using 29.5 inch (74.9 cm) deep-guarded nasopharyngeal swabs, 16 inch (40.6 cm) unguarded proctology swabs, or 6 inch (15.2 cm) unguarded nasal swabs when characterized using culture, real time-qPCR, and 16S rRNA gene sequencing. Samples for aerobic culture, qPCR, and 16S rRNA gene sequencing were collected from the upper respiratory tract of cattle 2 weeks after feedlot arrival. RESULTS: There was high concordance of culture and qPCR results for all swab types (results for 77% and 81% of sampled animals completely across all 3 swab types for culture and qPCR respectively). Microbial communities were highly similar among samples collected with different swab types, and differences identified relative to treatment for BRD were also similar. Positive qPCR results for M. haemolytica were highly concordant (81% agreed completely), but samples collected by deep-guarded swabbing had lower amounts of Mh DNA identified (Kruskal-Wallis analysis of variance on ranks, P < 0.05; Dunn-test for pairwise comparison with Benjamini-Hochberg correction, P < 0.05) and lower frequency of positive compared to nasal and proctology swabs (McNemar's Chi-square test, P < 0.05). CONCLUSIONS: Though differences existed among different types of swabs collected from individual cattle, nasal swabs and proctology swabs offer comparable results to deep-guarded nasopharyngeal swabs when identifying and characterizing M. haemolytica by culture, 16S rRNA gene sequencing, and qPCR.

Recombinant Myxoma Virus Expressing Walleye Dermal Sarcoma Virus orfC Is Attenuated in Rabbits.

The poxvirus, myxoma virus (MYXV) has shown efficacy as an oncolytic virus (OV) in some cancer models. However, MYXV replication within murine cancer models and spontaneous canine sarcomas is short-lived. In mice, successful treatment of tumors requires frequent injections with MYXV. We hypothesize that treatment of cancer with a recombinant MYXV that promotes apoptosis could improve the efficacy of MYXV. The orfC gene of walleye dermal sarcoma virus (WDSV), which induces apoptosis, was recombined into the MYXV genome (MYXVorfC). A marked increase in apoptosis was observed in cells infected with MYXVorfC. To ensure that expression of WDSV orfC by MYXV does not potentiate the pathogenesis of MYXV, we evaluated the effects of MYXVorfC inoculation in the only known host of MYXV, New Zealand white rabbits. Virus dissemination in rabbit tissues was similar for MYXVorfC and MYXV. Virus titers recovered from tissues were lower in MYXVorfC-infected rabbits as compared to MYXV-infected rabbits. Importantly, rabbits infected with MYXVorfC had a delayed onset of clinical signs and a longer median survival time than rabbits infected with MYXV. This study indicates that MYXVorfC is attenuated and suggests that MYXVorfC will be safe to use as an OV therapy in future studies.