Development and evaluation of a point-of-care test with a combination of EZ-Fast DNA extraction and real-time PCR and LAMP detection: evaluation using blood samples containing the bovine leukaemia DNA.

Along with progress in globalization of society, the spread of infectious diseases has accelerated worldwide. The deployment of highly sensitive genetic tests is essential for early diagnosis and early containment of potential outbreaks and epidemics, as well as routine surveillance, although tedious and expensive nucleic acid extraction steps represent a major drawback. Here we developed a simple and rapid DNA extraction method, named as an EZ-Fast kit, applicable to the field setting. The kit does not require advanced laboratory equipment or expensive DNA extraction kits and achieves crude DNA extraction within 10 min at extremely low cost and can easily be performed in field settings. When combined with real-time PCR and LAMP analyses, the performance of the POCT, using 183 bovine blood samples, was similar to that of the existing DNA extraction method: 92·5% (135/146) (real-time PCR) and 93·7% (133/142) (LAMP) diagnostic sensitivities, and 100% diagnostic specificities. The developed POCT provides a powerful tool to facilitate on-site diagnosis in a field setting.

Molecular epidemiological survey and phylogenetic analysis of bovine influenza D virus in Japan.

The influenza D virus, a new member of the Orthomyxoviridae family, is predominantly found in cattle. Although viral pathology and clinical disease in cattle appear mild, this virus plays an important role as a trigger of bovine respiratory disease (BRD). BRD is a costly illness worldwide. Thus, epidemiological surveys of the influenza D virus are necessary. Here, we conducted a molecular epidemiological survey for the influenza D virus in healthy and respiratory-diseased cattle in Japan. We found that 2.1% (8/377) of the cattle were infected with influenza D. The cattle with and without respiratory symptoms had approximately equal amounts of the virus. A full-genome sequence analysis revealed that the influenza D virus that was isolated in Japan formed an individual cluster that was distinct from the strains found in other countries. These results suggest that this virus might have evolved uniquely in Japan over a long period of time and that the viral pathology of Japanese strains might be different from the strains found in other countries. Continuous surveillance is required to determine the importance of this virus and to characterize its evolution.

Campylobacter and Salmonella are prevalent in broiler farms in Kyushu, Japan: results of a 2-year distribution and circulation dynamics audit.

AIM: To elucidate the distribution and circulation dynamics of Campylobacter and Salmonella in Japanese chicken broiler flocks. METHODS AND RESULTS: A 2-year investigation of the distribution of Campylobacter and Salmonella was conducted in 25 broiler flocks at nine farms in Japan from 2013 to 2014. Campylobacter and Salmonella tested positive in 11 (44·0%) and 24 (96·0%) broiler flocks respectively. One hundred and ninety-five Campylobacter and 184 Salmonella isolates were characterized into 12 Campylobacter (including two novel genotypes) and three Salmonella MLST genotypes. Only Salmonella isolation between caecal and environmental samples were significantly correlated. Further, one litter sample tested positive for Salmonella before new chicks were introduced. The Campylobacter strains rapidly lost culturability within 2-18 days; in contrast, the Salmonella strains survived from 64-211 days in artificially inoculated water samples. CONCLUSION: No persistent circulation-mediated Campylobacter contamination was observed. In contrast, circulation of Salmonella in broiler houses was seen, apparently due to the litter excreted from broiler flocks, as well as Salmonella-contaminated water and feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the distribution, genotypic data and circulation dynamics of Campylobacter and Salmonella as recently observed in Japanese chicken broiler farms.

Kinetics of regulatory dendritic cells in inflammatory responses during Trypanosoma evansi infection.

Trypanosoma evansi (T. evansi) causes a wasting disease in almost all mammals. Trypanosoma evansi infection gives rise to the inflammatory responses that contribute to the development of inflammation-associated tissue injury. To determine what kinds of inflammatory molecules play roles in the pathogenicity of T. evansi infection, polymerase chain reaction array analysis was performed on samples from the infected and uninfected mice. The inflammatory cytokine and chemokine storm, caused mainly by macrophages, was observed. On the other hand, the expression levels of Ccl8 and Il10 in splenocytes were also markedly increased. These results suggested an augmentation in the number and activity of regulatory dendritic cells (DCs). Therefore, the kinetics of regulatory DCs in T. evansi-infected mice were investigated. During T. evansi infection, the regulatory DCs became prevalent, with reducing the amount of inflammatory DCs. Interestingly, when the regulatory DCs were implanted into T. evansi-infected mice, the survival was prolonged, and the expression levels of inflammatory molecules were suppressed. Taken together, these results showed that a subset of regulatory DCs acted as a potential regulator of the inflammatory responses.