JSPS-6 Influenza D virus and bovine coronavirus play important roles in bovine respiratory disease in Japan

Bovine respiratory disease (BRD) is one of the most commonly diagnosed causes of morbidity and mortality in the cattle industry. Co-infection with several viruses and bacteria causes BRD. Despite the use of antibiotics and several commercial vaccines against viruses, such as bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV) 1 and 2, bovine herpesvirus 1, bovine adenovirus 7 and bovine parainfluenza virus 3, BRD remains the most common and costly problem in Japan. Therefore, we suspect that viruses not treated by the vaccines are a possible cause of BRD.Metagenomic analysis has recently allowed comprehensive viral characterization of the bovine respiratory tract. Ng et al. reported that bovine adenovirus 3, bovine rhinitis A virus and influenza D (FluD) virus were significantly associated with BRD in the USA [1]. Mitra et al. reported that FluD virus was significantly associated with respiratory disease, and viruses that are commonly associated with BRD, such as BVDV, bovine herpesvirus 1, BRSV, and bovine parainfluenza virus were detected less frequently in Mexico and the USA [2]. Although the viral pathologies and clinical diseases in sole infection appear mild, these results suggest that they play an essential role as a trigger of BRD.To identify which viruses were associated with BRD in Japan, we first performed a viral metagenomic analysis using nasal swab samples from respiratory-diseased cattle. The results suggested that bovine coronavirus (BCoV) and FluD virus played a significant role. Therefore, we conducted a molecular-epidemiological survey of nasal swab samples from respiratory-diseased and healthy cattle to assess the contribution of BCoV and FluD virus. Finally, we performed a phylogenetic analysis and assessed the antigenicity

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

Application of an Improved Micro-amount of Virion Enrichment Technique (MiVET) for the Detection of Avian Influenza A Virus in Spiked Chicken Meat Samples

Highly sensitive detection of pathogens is effective for screening meat during quarantine inspection and export. The “micro-amount of virion enrichment technique” (MiVET) was recently developed, which is a new method combining virus concentration with immunomagnetic beads and simple RNA extraction with sodium dodecyl benzenesulfonate (SDBS) for the specific and sensitive detection of avian influenza viruses (AIVs). AIV subtypes H3N2 and H4N2 were used to spike the surface of chicken breast meat samples. The modified MiVET protocol was tested by comparing it against three different homogenate preparation conditions, as well as in samples with added α-amylase and collagenase to digest inhibitors. The performance of the modified MiVET was evaluated by real-time RT-PCR assay targeting the matrix gene. Compared with conventional RNA extraction, the modified MiVET reproducibly concentrated AIVs in chicken meat samples with 100–1000-fold improvement by 60 s-hand homogenization. The 30 s- and 60 s-stomacher homogenizations resulted 100-fold and 10–100-fold improvement, respectively. The modified MiVET required < 60 min from homogenate preparation to final RNA elution. Further, use of the modified MiVET also decreased the rate of false-negative results. The modified MiVET is effective for the rapid and highly sensitive detection of AIVs in chicken meat samples, and can be applied to quarantine and export inspection at airports and seaports

Single-Nucleotide Polymorphism on Spermatogenesis Associated 16 Gene-Coding Region Affecting Bovine Leukemia Virus Proviral Load

Bovine leukemia virus (BLV) is an etiological agent of malignant lymphoma in cattle and is endemic in many cattle-breeding countries. Thus, the development of cattle genetically resistant to BLV is desirable. The purpose of this study was to identify novel single-nucleotide polymorphisms (SNPs) related to resistance to BLV. A total of 146 DNA samples from cattle with high BLV proviral loads (PVLs) and 142 samples from cattle with low PVLs were used for a genome-wide association study (GWAS). For the verification of the GWAS results, an additional 1342 and 456 DNA samples from BLV-infected Japanese Black and Holstein cattle, respectively, were used for an SNP genotyping PCR to compare the genotypes for the identified SNPs and PVLs. An SNP located on the spermatogenesis associated 16 (SPATA16)-coding region on bovine chromosome 1 was found to exceed the moderate threshold (p &lt; 1.0 &times; 10&minus;5) in the Additive and Dominant models of the GWAS. The SNP genotyping PCR revealed that the median values of the PVL were 1278 copies/50 ng of genomic DNA for the major homozygous, 843 for the heterozygous, and 621 for the minor homozygous genotypes in the Japanese Black cattle (p &lt; 0.0001). A similar tendency was also observed in the Holstein cattle. We found that cattle with the minor allele for this SNP showed 20&ndash;25% lower PVLs. Although the mechanisms through which this SNP impacts the PVL remain unknown, we found a novel SNP related to BLV resistance located on the SPATA16 gene-coding region on bovine chromosome 1

Detection and phylogenetic analysis of Dabieshan tick virus and Okutama tick virus in ticks collected from Cape Toi, Japan

New technologies have led to the discovery of novel tick-borne and tick-associated viruses. Dabieshan tick virus (DaTV) and Okutama tick virus (OkTV), which belong to the family Phenuiviridae, were discovered in ticks in China and Japan, respectively, in the 2010s. Although it is unknown whether these viruses cause disease in animals or humans, all tick-associated viruses have the potential to become etiological agents of infectious diseases through gene reassortment. Therefore, it is important to elucidate the ecology of these viruses, regardless of their pathogenicity. In this study, ticks were collected year-round in Cape Toi, Miyazaki Prefecture, Japan, and an epidemiological survey of tick-associated phenuiviruses was performed. A total of 516 ticks collected from the vegetation by dragging flannel sheets were used for analysis. Pan-phenuivirus reverse transcription PCR was performed on the tick samples, and DaTV and OkTV were detected. We found that 37.0% (85/230) and 23% (16/71) of nymphal and adult Haemaphysalis longicornis were infected with DaTV, respectively, and 10% (6/62) and 13% (1/8) of nymphal and adult Haemaphysalis flava were infected with OkTV, respectively. Phylogenetic analysis indicated that the DaTV identified in this study formed a unique clade that was distinct from the strains identified in China. The survey revealed that DaTV is distributed not only in China, but also in Japan. We believe that this study contributes to our understanding of the prevalence of tick-associated viruses

Avoidance of Natural Suckling from Dams with Bovine Leukemia Virus Is a Low Priority Countermeasure against Postnatal Transmission

Although natural suckling from dams with bovine leukemia virus (BLV) has not been recommended in Japan, the frequency of BLV transmission through natural suckling under natural conditions is still unclear. The purpose of this study was to elucidate the risk of BLV transmission through natural suckling. Dams with BLV were classified into three groups (high, middle, low) based on the proviral loads (PVLs). PCR positivity of their colostrum and the correlations between the ratios of calves with BLV and types of feeding milk were analyzed. In dams with low PVLs, no colostrum or calves were confirmed to have BLV. In dams with middle and high PVLs, 17 out of 25 (68.0%) colostrum were PCR positive, and 10 out of 23 (43.4%) and 13 out of 29 (44.8%) calves with natural suckling and artificial rearing were infected with BLV, respectively. No difference was confirmed between the infection rates of natural-suckled and artificially reared calves. Thus, we concluded that the avoidance of natural suckling from dams with BLV and the introduction of artificial rearing were low priority countermeasures against BLV transmission

Avoidance of Natural Suckling from Dams with Bovine Leukemia Virus Is a Low Priority Countermeasure against Postnatal Transmission

Although natural suckling from dams with bovine leukemia virus (BLV) has not been recommended in Japan, the frequency of BLV transmission through natural suckling under natural conditions is still unclear. The purpose of this study was to elucidate the risk of BLV transmission through natural suckling. Dams with BLV were classified into three groups (high, middle, low) based on the proviral loads (PVLs). PCR positivity of their colostrum and the correlations between the ratios of calves with BLV and types of feeding milk were analyzed. In dams with low PVLs, no colostrum or calves were confirmed to have BLV. In dams with middle and high PVLs, 17 out of 25 (68.0%) colostrum were PCR positive, and 10 out of 23 (43.4%) and 13 out of 29 (44.8%) calves with natural suckling and artificial rearing were infected with BLV, respectively. No difference was confirmed between the infection rates of natural-suckled and artificially reared calves. Thus, we concluded that the avoidance of natural suckling from dams with BLV and the introduction of artificial rearing were low priority countermeasures against BLV transmission

Use of direct LAMP screening of broiler fecal samples for Campylobacter jejuni and Campylobacter coli in the positive flock identification strategy

Rapid identification of Campylobacter-positive flocks before slaughter, following freezing and heat treatment for the Campylobacter-positive carcasses at the slaughterhouses is an effective control strategy against foodborne campylobacteriosis. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the direct screening of naturally contaminated chicken cloacal swabs for Campylobacter jejuni/Campylobacter coli to compare this assay with conventional quantitative culture methods. In a comparison study of 165 broilers, the LAMP assay showed 82.8% (48/58 by conventional culture) sensitivity, 100% (107/107) specificity, 100% (48/48) positive predictive value (PPV), and 91.5% (107/117) negative predictive value (NPV). In a comparison of 55 flocks, LAMP showed 90.5% (19/21) sensitivity, 100% (34/34) specificity, 100% (19/19) PPV, and 94.4% (34/36) NPV. In the cumulative total of 28 farm-level comparisons, LAMP showed 100% (12/12) sensitivity, 100% (16/16) specificity, 100% (12/12) PPV, and 100% (16/16) NPV. The LAMP assay required less than 90 min from the arrival of the fecal samples to final results in the laboratory. This suggests that the LAMP assay will facilitate the identification of C. jejuni/C. coli-positive broiler flocks at the farm level or in slaughterhouses before slaughtering, which would make it an effective tool in preventing the spread of Campylobacter contamination

Molecular Epidemiology and Whole-Genome Analysis of Bovine Foamy Virus in Japan

Bovine foamy virus (BFV) is a member of the foamy virus family in cattle. Information on the epidemiology, transmission routes, and whole-genome sequences of BFV is still limited. To understand the characteristics of BFV, this study included a molecular survey in Japan and the determination of the whole-genome sequences of 30 BFV isolates. A total of 30 (3.4%, 30/884) cattle were infected with BFV according to PCR analysis. Cattle less than 48 months old were scarcely infected with this virus, and older animals had a significantly higher rate of infection. To reveal the possibility of vertical transmission, we additionally surveyed 77 pairs of dams and 3-month-old calves in a farm already confirmed to have BFV. We confirmed that one of the calves born from a dam with BFV was infected. Phylogenetic analyses revealed that a novel genotype was spread in Japan. In conclusion, the prevalence of BFV in Japan is relatively low and three genotypes, including a novel genotype, are spread in Japan

Cattle with a low bovine leukemia virus proviral load are rarely an infectious source

Bovine leukemia virus (BLV) is an etiological agent of fatal B-cell leukemia and malignant lymphoma in cattle. Cattle with higher BLV proviral loads represent a higher risk of both horizontal and vertical transmission. Therefore, quantifying the proviral load of BLV is important in identifying major infectious sources and protecting BLV-free cattle from exposure to infected cattle. In this study, we confirmed that cattle with very low BLV proviral loads did not transmit the virus to virus-free cattle under conventional conditions. We observed a total of 7 tests in which a BLV-infected bull was allowed to cohabit with 57 to 92 BLV-free cattle for 12 or 22 months. We then evaluated the frequency of viral transmission. A BLV-infected bull with a “very low proviral load” (i.e., fewer than 100 proviral copies/50 ng of genomic DNA) did not transmit the virus to any virus-free cattle in 2 out of 2 tests. However, a BLV-infected bull with a “low proviral load” (i.e., 100 to 500 copies/50 ng) transmitted the virus to a total of 3 virus-free cattle in 2 out of 5 tests. These results suggest that BLV-infected cattle with “very low proviral loads” do not transmit the virus under conventional conditions, while cattle with “low proviral loads” can transmit the virus, although at low rates. We believe that the results of this study will promote the construction of effective measures to prevent BLV infection and control the spread of BLV