Dichelobacter nodosus is a fastidious, strictly anaerobic bacterium, an obligate parasite of the ruminant hoof, and the essential causative agent of virulent ovine footrot. The clinical disease results from a complex interplay between the pathogen, the environment, and the host. Sheep flocks diagnosed with virulent but not benign footrot in Australia may be quarantined and required to undergo a compulsory eradication program, with costs met by the farmer. Virulence of D. nodosus at least partially depends on the elaboration of a protease encoded by aprV2 and manifests as elastase activity. Laboratory virulence tests are used to assist diagnosis because clinical differentiation of virulent and benign footrot can be challenging during the early stages of disease or when the disease is not fully expressed due to unfavorable pasture conditions. Using samples collected from foot lesions from 960 sheep from 40 flocks in four different geographic regions, we evaluated the analytical characteristics of qPCR tests for the protease gene alleles aprV2 and aprB2, and compared these with results from phenotypic protease (elastase and gelatin gel) tests. There was a low level of agreement between clinical diagnosis and quantitative PCR (qPCR) test outcomes at both the flock and sample levels and poor agreement between qPCR test outcomes and the results of phenotypic virulence tests. The diagnostic specificity of the qPCR test was low at both the flock and individual swab levels (31.3% and 18.8%, respectively). By contrast, agreement between the elastase test and clinical diagnosis was high at both the flock level (diagnostic sensitivity [DSe], 100%; diagnostic specificity [DSp], 78.6%) and the isolate level (DSe, 69.5%; DSp, 80.5%).

Aims This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. Methods and Results Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P < 0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1–V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A.® Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. Conclusions Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit‐for‐purpose strategy is recommended. Significance and Impact of the Study Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.

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Virulent footrot is an economically significant disease in most sheep-rearing countries. The disease can be controlled with vaccine targeting the fimbriae of virulent strains of the essential causative agent, However, the bacterium is immunologically heterogeneous, and 10 distinct fimbrial serogroups have been identified. Ideally, in each outbreak the infecting strains would be cultured and serogrouped so that the appropriate serogroup-specific mono- or bivalent vaccine could be administered, because multivalent vaccines lack efficacy due to antigenic competition. If clinical disease expression is suspected to be incomplete, culture-based virulence tests are required to confirm the diagnosis, because control of benign footrot is economically unjustifiable. Both diagnosis and vaccination are conducted at the flock level. The aims of this study were to develop a PCR-based procedure for detecting and serogrouping directly from foot swabs and to determine whether this could be done accurately from the same cultured swab. A total of 269 swabs from the active margins of foot lesions of 261 sheep in 12 Merino sheep flocks in southeastern Australia were evaluated. DNA extracts taken from putative pure cultures of and directly from the swabs were evaluated in PCR assays for the 16S rRNA and genes of Pure cultures were tested also by the slide agglutination test. Direct PCR using extracts from swabs was more sensitive than culture for detecting and serogrouping strains. Using the most sensitive sample collection method of the use of swabs in lysis buffer, was more likely to be detected by PCR in active than in inactive lesions, and in lesions with low levels of fecal contamination, but lesion score was not a significant factor. PCR conducted on extracts from swabs in modified Stuart's transport medium that had already been used to inoculate culture plates had lower sensitivity. Therefore, if culture is required to enable virulence tests to be conducted, it is recommended that duplicate swabs be collected from each foot lesion, one in transport medium for culture and the other in lysis buffer for PCR.

We compared the use of recto-anal mucosal swab (RAMS) culture and faecal culture for the detection of E. coli O157 in a mob of Merino sheep. Fifty Merino wethers and maiden ewes housed in indoor pens were sampled on five occasions. We detected E coli O157 in 32% (16/50) of sheep, with weekly prevalence ranging from 4% (2/50) to 16% (8/50). Overall, 12·5% (2/16) were detected by RAMS culture only, and 37·5% (6/16) were detected by faecal culture only. The level of agreement between the two sampling methods was moderate [kappa statistic = 0·583, 95% confidence interval (CI) 0·460-0·707]. The relative sensitivities of RAMS and faecal culture were 67% (95% CI 41-86) and 57% (95% CI 34-77), respectively. We identified four super-shedding sheep using direct faecal culture. Although the majority of culture-positive sheep were detected at one sampling point only, 3/4 super-shedding sheep were culture-positive at two sampling points, and 1/4 was culture-positive at four sampling points. Persistent culture positivity may indicate sheep that could be considered 'super-shedders' at some point. The use of immunomagnetic separation further improved the rate of detection of E. coli O157, which was isolated from 1/34 animals that were previously negative by enrichment culture alone. A significant difference between sampling weeks was detected for both faecal (P = 0·021) and RAMS (P = 0·006), with the prevalence at the mid-point of sampling (week 4) significantly (P < 0·05) higher than at the beginning or end of the study. Study conditions (penned sheep) might have been responsible for the high prevalence and the epidemic pattern of infection observed, and could serve as a future model for studies of E. coli O157 transmission, shedding and super-shedding in sheep.