Enzootic bovine leukosis (EBL) is a malignant lymphoma of cattle that is mainly caused by bovine leukemia virus (BLV) infection. In this study, PCR-RFLP was used to investigate the frequency of the DRB3*009:02 allele in several farms with different herd management practices in Japan. A total of 742 Holsteins (384) and Japanese Blacks (230) were used as the sample size for the study, which was larger than the number of cattle in the study area with a confidence level of 95% and a margin of error of 8. PBMCs isolated from whole blood from clinically healthy cattle were used for examination. The presence of BLV provirus infection was determined by qPCR targeting the env region. BLV antibodies were detected using a commercial ELISA kit. The results showed that 35 cattle were heterozygous for DRB3*009:02. The frequency on each farm varied between farms, and PCR analysis showed that the prevalence of BLV also varied between farms. The incidence rate (4.7%) was lower than in previous studies. The BLV seroprevalence (14.4%) in this study was lower than the BLV infection rate (35%) in the study. Holstein dairy cows had low levels of BL resistance genes, confirming the spread of the virus within the farm's herd. Cattle on farms with low BLV-positive rates had a resistance gene-carrying rate of 16.9%, meanwhile, those who did not carry the resistance gene had a rate of 91.4%. BLV provirus levels vary between farmers, with herds carrying low BL-resistance genes tending to have higher levels of BLV provirus. In light of the current BLV epidemic, herd composition reform, along with aggressive breeding of BL-resistant sires, is a required component to increase the herd of BL-resistant sires and improve livestock productivity.

Bovine viral diarrhea (BVD) is caused by the BVD virus (BVDV) and has been reported worldwide in cattle. To estimate BVDV circulation among cattle where few BVD cases were reported in southern Japan, 1910 serum samples collected from 35 cattle farms without a BVD outbreak were investigated to detect antibodies against BVDV-1 and BVDV-2 using an indicator virus with a cytopathogenic effect and the luciferase gene, respectively. Neutralizing antibodies against BVDV-1 and BVDV-2 were detected more frequently in 18 vaccinated farms than in 17 nonvaccinated farms. In the nonvaccinated farms, 9.6%, 1.8%, and 13.8% of the cattle were estimated to have a history of infection with BVDV-1, BVDV-2, and both, respectively. The median rate of within-herd anti-BVDV-1 seropositivity among cattle in the nonvaccinated farms was 22.0%; however, a high within-herd seropositivity (>50%) was confirmed in the two farms. The force of infection, basic reproduction number, and annual probability of BVDV-1 infection were estimated as 0.072 (95% confidence interval [CI]: 0.062-0.084), 0.36 (95% CI: 0.31-0.42), and 0.73% (95% CI: 0.61-0.87%), respectively, using the age-specific positive rate of anti-BVDV-1 antibodies. These parameters should be further applicable for developing epidemiological models which illustrate the BVDV dynamics in the field.

Bovine viral diarrhea (BVD), caused by bovine viral diarrhea virus (BVDV), has a significant economic impact on affected farms worldwide. For effective disease control, it is crucial to select an appropriate vaccine based on the specific genotype of BVDV. Therefore, developing a rapid and reliable assay to detect and genotype BVDV is imperative for controlling the spread of disease. In this study, we developed a TaqMan assay to detect and genotype BVDV types 1 and 2 directly in bovine serum without extraction of RNA. The direct BVDV TaqMan assay effectively detected both BVDV1 and BVDV2 with confirmed specificity and showed no cross-reactivity with any of the other viruses tested, including bovine respiratory syncytial virus, bovine coronavirus, Akabane virus, bovine herpesvirus 1, bovine parainfluenza virus 3, bovine immunodeficiency virus, and bovine leukemia virus. The assay could detect the virus in serum samples with a titer as low as 102 TCID50/mL in two out of three trials for BVDV1 and all three trials for BVDV2, indicating that its sensitivity is equivalent to that of virus isolation. Our findings represent a significant advancement in BVDV detection and typing directly from bovine serum.

AbstractBackgroundWhile early detection and early containment are key to controlling the African swine fever (ASF) pandemic, the lack of practical testing methods for use in the field are a major barrier to achieving this feat.ObjectivesTo describe the development of a rapid and sensitive point‐of‐care test (POCT) for ASF, and its evaluation using swine whole blood samples for field settings.MethodsIn total, 89 swine whole blood samples were collected from Vietnamese swine farms and were performed the POCT using a combination of crude DNA extraction and LAMP (loop‐mediated isothermal amplification) amplification.ResultsThe POCT enabled crude DNA to be extracted from swine whole blood samples within 10 min at extremely low cost and with relative ease. The entire POCT required a maximum of 50 min from the beginning of DNA extraction to final judgment. Compared to a conventional real‐time PCR detection, the POCT showed a 1 log reduction in detection sensitivity, but comparable diagnostic sensitivity of 100% (56/56) and diagnostic specificity of 100% (33/33). The POCT was quicker and easier to perform and did not require special equipment.ConclusionsThis POCT is expected to facilitate early diagnosis and containment of ASF invasion into both regions in which it is endemic and eradicated.

While pathogen-level quantification is an important diagnostic of disease severity and transmissibility, disease-related host biomarkers are also useful in predicting outcomes in infectious diseases. In this study, we demonstrate that combined proviral load (PVL) and host biomarker diagnostics can be used to detect bovine leukemia virus (BLV) infection, which has a negative economic impact on the cattle industry.

The bovine leukocyte antigen (BoLA) DRB3*009:02 allele is strongly associated with a low/undetectable bovine leukemia virus (BLV) proviral load. Understanding the status of cattle possessing DRB3*009:02 allele is key for BLV control by breeding. We performed a survey of DRB3*009:02-carrying cattle in two prefectures in Japan using a TaqMan assay developed previously. The allele was found in 3.8% (confidence interval (CI): 3.3-4.3) of 6020 Japanese Black female cattle. A prefecture-level difference was found: the allele was observed in 8.6% CI: 7.5-9.9) of 2242 cattle of the birth prefecture B in Kyushu/Okinawa region, and this percentage was significantly higher than those of prefecture C in Kyushu/Okinawa region (1.3% (CI: 0.4-3.4) of 319) and prefecture A in Chugoku region (0.9% (CI: 0.6-1.4) of 2741), respectively. Consideration on the difference in possession of DRB3*009:02 allele is needed to establish the more efficient control strategy of BLV infection in Japanese Black cattle.

Bovine viral diarrhea (BVD) caused by BVD virus (BVDV) leads to economic loss worldwide. Cattle that are persistently infected (PI) with BVDV are known to play an important role in viral transmission in association with the animal movement, as they shed the virus during their lifetime. In this research, the "hot spot" for BVD transmission was estimated by combining phylogenetic and epidemiological analyses for PI cattle and cattle that lived together on BVDV affected farms in Tokachi district, Hokkaido prefecture, Japan. Viral isolates were genetically categorized into BVDV-1a, 1b, and 2a, based on the nucleotide sequence of the entire E2 region. In BVDV genotype 1, subgenotype b (BVDV-1b), cluster I was identified as the majority in Tokachi district. Network analysis indicated that 12 of the 15 affected farms had cattle movements from other facilities (PI-network) and farms affected with BVDV-1b cluster I consisted of a large network. It was implied that the number of cattle movements themselves would be a risk of BVD transmission, using the PageRank algorithm. Therefore, these results demonstrate that cattle movements would contribute to disease spread and the combination of virological and epidemiological analysis methods would be beneficial in determining possible virus transmission routes.

Enzootic bovine leukosis is a lethal neoplastic disease caused by bovine leukemia virus (BLV), belongs to family Retroviridae. The BLV proviral load (PVL) represents the quantity of BLV genome that has integrated into the host’s genome in BLV-infected cells. Bovine leukocyte antigen (BoLA) class II allelic polymorphisms are associated with PVLs in BLV-infected cattle. We sought to identify relationships between BoLA-DRB3 allelic heterozygosity and BLV PVLs among different cattle breeds. Blood samples from 598 BLV-infected cattle were quantified to determine their PVLs by real-time polymerase chain reaction. The results were confirmed by a BLV-enzyme-linked immunosorbent assay. Restriction fragment length polymorphism-polymerase chain reaction identified 22 BoLA-DRB3 alleles. Multivariate negative binomial regression modeling was used to test for associations between BLV PVLs and BoLA-DRB3 alleles. BoLA-DRB3.2*3, *7, *8, *11, *22, *24, and *28 alleles were significantly associated with low PVLs. BoLA-DRB3.2*10 was significantly associated with high PVLs. Some heterozygous allele combinations were associated with low PVLs (*3/*28, *7/*8, *8/*11, *10/*11, and *11/*16); others were associated with high PVLs (*1/*41, *10/*16, *10/*41, *16/*27, and *22/*27). Interestingly, the BoLA-DRB3.2*11 heterozygous allele was always strongly and independently associated with low PVLs. This is the first reported evidence of an association between heterozygous allelic combinations and BLV PVLs.

As genetically resistant individuals, the "elite controllers" (ECs) of human immunodeficiency virus infection have been focused on as the keys to developing further functional treatments in medicine. In the livestock production field, identifying the ECs of bovine leukemia virus (BLV) infection in cattle is desired to stop BLV transmission chains on farms. Cattle carrying the bovine leukocyte antigen (BoLA)-DRB3*009:02 allele (DRB3*009:02) have a strong possibility of being BLV ECs. Most of cattle carrying this allele maintain undetectable BLV proviral loads and do not shed virus even when infected. BLV ECs can act as transmission barriers when placed between uninfected and infected cattle in a barn. To identify cattle carrying DRB3*009:02 in large populations more easily, we developed a pooled testing system. It employs a highly sensitive, specific real-time PCR assay and TaqMan MGB probes (DRB3*009:02-TaqMan assay). Using this system, we determined the percentage of DRB3*009:02-carrying cattle on Kyushu Island, Japan. Our pooled testing system detected cattle carrying the DRB3*009:02 allele from a DNA pool containing one DRB3*009:02-positive animal and 29 cattle with other alleles. Its capacity is sufficient for herd-level screening for DRB3*009:02-carrying cattle. The DRB3*009:02-TaqMan assay showed high-discriminative sensitivity and specificity toward DRB3*009:02, making it suitable for identifying DRB3*009:02-carrying cattle in post-screening tests on individuals. We determined that the percentage of DRB3*009:02-carrying cattle in Kyushu Island was 10.56%. With its ease of use and reliable detection, this new method strengthens the laboratory typing for DRB3*009:02-carrying cattle. Thus, our findings support the use of BLV ECs in the field.

The cattle industry is suffering economic losses caused by bovine leukemia virus (BLV)and enzootic bovine leukosis (EBL), the clinical condition associated with BLV infection. Thispathogen spreads easily without detection by farmers and veterinarians due to the lack of obviousclinical signs. Cattle movement strongly contributes to the inter-farm transmission of BLV. Thisstudy quantified the farm-level risk of BLV introduction using a cattle movement analysis. Ageneralized linear mixed model predicting the proportion of BLV-infected cattle was constructedbased on weighted in-degree centrality. Our results suggest a positive association between weightedin-degree centrality and the estimated number of introduced BLV-infected cattle. Remarkably, theintroduction of approximately six cattle allowed at least one BLV-infected animal to be added to thefarm in the worst-case scenario. These data suggest a high risk of BLV infection on farms with ahigh number of cattle being introduced. Our findings indicate the need to strengthen BLV controlstrategies, especially along the chain of cattle movement.

Bovine viral diarrhea virus (BVDV) footprint has spread across the globe and is responsible for one of the most economically important diseases in cattle. In Japan, some regional surveillance and preventive measures to control bovine viral diarrhea (BVD) have been implemented. However, BVDV infection is poorly understood in cattle industries, and there is no systematic BVD surveillance system and control program. Kyushu is the center for raising beef cattle in Japan. Therefore, this study aimed to determine the BVDV infection using a slaughterhouse survey among beef cattle in Kyushu, Japan. A total of 1,075 blood samples were collected at two regional slaughterhouses in Miyazaki prefecture from December 2015 to June 2016. Antigen ELISA was used for detection of BVDV antigen in blood samples. Two samples showed positive results (2/1,075; 0.18%). BVDV RNA was extracted from positive blood samples; the sequence was determined and analyzed by the neighbor-joining method for construction of the phylogenetic tree. Phylogenetic analysis based on the 5'-UTR revealed that the two positive samples were grouped into the same subtype BVDV-1b in the BVDV-1 genotype, but the infected cattle belonged to two different farms. In conclusion, this is the first study to identify the presence of BVDV in a slaughterhouse survey in Kyushu. These findings suggest that a slaughterhouse survey is a useful tool for developing a surveillance system for monitoring infectious diseases in cattle.

Highly pathogenic avian influenza (HPAI) outbreaks engender a severe economic impact on the poultry industry and public health. Migratory waterfowl are considered the natural hosts of HPAI virus, and HPAI viruses are known to be transmitted over long distances during seasonal bird migration. Bird migration is greatly affected by the weather. Many studies have shown the relationship between either autumn or spring bird migration and climate. However, few studies have shown the relationship between annual bird migration and annual weather. This study aimed to establish a model for the number of migratory waterfowl involved in HPAI virus transmission based on meteorological data. From 136 species of waterfowl that were observed at Futatsudate in Miyazaki, Japan, from 2008 to 2016, we selected potential high-risk species that could introduce the HPAI virus into Miyazaki and defined them as 'risky birds'. We also performed cluster analysis to select meteorological factors. We then analysed the meteorological data and the total number of risky birds using a generalised linear mixed model. We selected 10 species as risky birds: Mallard (Anas platyrhynchos), Northern pintail (Anas acuta), Eurasian wigeon (Anas penelope), Eurasian teal (Anas crecca), Common pochard (Aythya ferina), Eurasian coot (Fulica atra), Northern shoveler (Anas clypeata), Common shelduck (Tadorna tadorna), Tufted duck (Aythya fuligula) and Herring gull (Larus argentatus). We succeeded in clustering 35 meteorological factors into four clusters and identified three meteorological factors associated with their migration: (1) the average daily maximum temperature; (2) the mean value of global solar radiation and (3) the maximum daily precipitation. We thus demonstrated the relationship between the number of risky birds and meteorological data. The dynamics of migratory waterfowl was relevant to the risk of an HPAI outbreak, and our data could contribute to cost and time savings in strengthening preventive measures against epidemics.

The purpose of this study was to detect porcine epidemic diarrhea virus (PEDV) subclinically infected pigs shipped from non-case farms to slaughterhouses. Systematic sampling was conducted at two slaughterhouses. A total of 1,556 blood samples were collected from 80 case and non-case farms from pigs over 6 months old. Blood samples were centrifuged to obtain sera. Serial serum dilutions were subjected to serological examination for PEDV presence using Neutralization test (NT). The cut-off titer was set at titer of 1:2 dilution and farms with at least one positive sample in duplicate were classified as PED-positive farms. Several non-case farms (9.4%, 6/64) and 100% (16/16) of the case farms were indeed positive for PEDV. The proportion of seropositive animals from case farms was 63.7%, significantly different from that of non-case farms (4.3%, P<0.05). In both case and non-case farms, the proportion of seropositive animals in farrow-to-finish farms was significantly higher than in wean-to-finish farms (P<0.05). Seropositive animals in non-case farms were detected by NT in a sero-survey by sampling at slaughterhouses. Therefore, subclinically infected pigs should be considered prior to shipment.

BACKGROUND:Porcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd.RESULTS:In this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed.CONCLUSIONS:The developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.

Most bovine leukemia virus (BLV)-infected cattle do not have clinical signs (aleukemic AL), but some develop persistent lymphocytosis (PL) and B-cell lymphosarcoma (enzootic bovine leucosis [EBL]). BLV infection is a well-known cause of chronic wasting disease, which is associated with a reduction in milk productivity and immunity in dairy cattle. However, the effect of BLV infection on beef cattle is not clear. The objective of this study was to investigate the effect of BLV infection on the productivity of slaughtered beef cattle. A total of 997 blood samples were collected from cattle in 2 slaughterhouses in Miyazaki prefecture, Japan. BLV-antibodies were tested in these cattle’s blood samples using enzyme-linked immunosorbent assay (ELISA), to identify BLV-infected cattle. We compared blood parameters and carcass weight between BLV ELISA-positive and ELISA-negative cattle in two age groups : young (≤ 60 months) and elder (> 60 months) groups. The results showed that the proportion of ELISA-positive cattle in the young and elder groups were 22.8% and 24.9%, respectively. The number of white blood cells (WBCs) and lymphocytes in ELISA-positive cattle was significantly higher than that in ELISA-negative cattle in the young group. In addition to the number of lymphocytes, the number of monocytes and neutrophils were also significantly higher in BLV ELISA-positive cattle than in ELISA-negative cattle in the elder group. There was no significant difference in the carcass weight between ELISA-positive and ELISA-negative cattle in both groups. The results of this study suggest that BLV infection has an effect on the host immune response in beef cattle.

Aim of our study is to establish models for predicting the number of migratory wild birds based on the meteorological data. From 136 species of wild birds, which have been observed at Futatsudate in Miyazaki, Japan, from 2008 to 2016, we selected the potential high-risk species, which can introduce highly pathogenic avian influenza (HPAI) virus into Miyazaki; we defined them as “risky birds”. We then performed regression analysis to model the relationship between the number of risky birds and meteorological data. We selected 10 wild bird species as risky birds: Mallard ( Anas platyrhynchos ), Northern pintail ( Anas acuta ), Eurasian wigeon ( Anas penelope ), Eurasian teal ( Anas crecca ), Common pochard ( Aythya ferina ), Eurasian coot ( Fulica atra ), Northern shoveler ( Anas clypeata ), Common shelduck ( Tadorna tadorna ), Tufted duck ( Aythya fuligula ), and Herring gull ( Larus argentatus ). We succeeded in identifying five meteorological factors associated with their migration: station pressure, mean value of global solar radiation, minimum of daily maximum temperature, days with thundering, and days with daily hours of daylight under 0.1 h. We could establish some models for predicting the number of risky birds based only on the published meteorological data, without manual counting. Dynamics of migratory wild birds has relevance to the risk of HPAI outbreak, so our data could contribute to save the cost and time in strengthening preventive measures against the epidemics.

[Background] In January 2014, a suspect case of highly pathogenic avian influenza (HPAI), with increasing mortality and decreasing egg production in a flock, was notified at a breeder duck farm in Gochang, Jeollabuk-do province. An infection of HPAI virus type H5N8 was confirmed in next day, and a total of 212 outbreaks were confirmed during the subsequent 193 days. This study investigated the clinical characteristics of H5N8 HPAI observed in Korean poultry farms during the 2014 epidemic. [Materials and Methods] History of manifestation of clinical signs was investigated by time of hour. Data were collected via 1) Survey by veterinary officers in local veterinary service, and 2) Clinical observation of investigators from the Animal and Plant Quarantine Agency (QIA). [Results] Clinical signs of fever, increased water consumption, and white diarrhea were observed on day 3 of post infection, progressed to loss of appetite (day 4), respiratory symptoms (day 5), drowsiness (day 6), hypotrophy and loss of vitality (day 7), and neurological signs including ataxia and wryneck (day 8). Timeline of occurrence of clinical signs was similar in chicken flock, however, increased mortality with distinctive clinical signs was observed in chicken. On the fortnight after the post infection, it was suggested that farmer can recognize an abnormality in duck flock. [Conclusion] Evolution of clinical signs of H5N8 seems slower than previous H5N1 HPAI epidemics. Finding of this study was the first guideline of H5N8 HPAI infection. It will be useful for the clinical inspection in poultry farms.