Publications by authors named "Hirohisa Mekata"

37 Publications

Bovine respiratory coronavirus enhances bacterial adherence by upregulating expression of cellular receptors on bovine respiratory epithelial cells.

Vet Microbiol 2021 Apr 17;255:109017. Epub 2021 Feb 17.

Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, 889-2192, Japan; Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki, 889-2192, Japan; Center for Animal Disease Control, University of Miyazaki, Miyazaki, 889-2192, Japan. Electronic address:

Bovine coronavirus (BCoV) is one of the agents causing bovine respiratory disease complex (BRDC), with single infection tending to be mild to moderate; the probability of developing pneumonia in BRDC may be affected by viral and bacterial combinations. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection enhances adherence of Pasteurella multocida (PM) to cells derived from the bovine lower respiratory tract but that BRSV infection in cells derived from the upper respiratory tract reduces PM adherence. In this study, we sought to clarify whether the modulation of bacterial adherence to cells derived from the bovine upper and lower respiratory tract is shared by other BRDC-related viruses by infecting bovine epithelial cells from the trachea, bronchus and lung with BCoV and/or PM. The results showed that cells derived from both the upper and lower respiratory tract were susceptible to BCoV infection. Furthermore, all cells infected with BCoV exhibited increased PM adherence via upregulation of two major bacterial adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PAF-R), suggesting that compared with BRSV infection, BCoV infection differentially modulates bacterial adherence. In summary, we identified distinct interaction between bovine respiratory viruses and bacterial infections.
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http://dx.doi.org/10.1016/j.vetmic.2021.109017DOI Listing
April 2021

Pseudorabies virus infection in hunting dogs in Oita, Japan: Report from a prefecture free from Aujeszky's disease in domestic pigs.

J Vet Med Sci 2021 Apr 15;83(4):680-684. Epub 2021 Feb 15.

Centre for Animal Disease Control, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, Miyazaki 889-2192, Japan.

We isolated two pseudorabies virus (PRV) isolates (designated OT-1 and OT-2) from two hunting dogs exhibiting neurological manifestations after eating the flesh of wild boar hunted in Oita prefecture, Kyushu Island, Japan. The isolates corresponded to a previously reported PRV (MY-1 strain) isolated from a hunting dog in neighboring Miyazaki prefecture, and it clustered into genotype II based on the glycoprotein C sequence. Our results suggest that this common PRV strain may have been maintained in wild boars on Kyushu Island even though domestic pigs in this area have attained an Aujeszky's disease-free status.
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http://dx.doi.org/10.1292/jvms.20-0450DOI Listing
April 2021

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Small-Animal Veterinarians and Nurses in the Japanese Prefecture with the Highest Case Load.

Viruses 2021 02 2;13(2). Epub 2021 Feb 2.

Center for Animal Disease Control, University of Miyazaki, Miyazaki 889-2192, Japan.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative agent of SFTS, an emerging tick-borne disease in East Asia, and is maintained in enzootic cycles involving ticks and a range of wild animal hosts. Direct transmission of SFTSV from cats and dogs to humans has been identified in Japan, suggesting that veterinarians and veterinary nurses involved in small-animal practice are at occupational risk of SFTSV infection. To characterize this risk, we performed a sero-epidemiological survey in small-animal-practice workers and healthy blood donors in Miyazaki prefecture, which is the prefecture with the highest per capita number of recorded cases of SFTS in Japan. Three small-animal-practice workers were identified as seropositive by ELISA, but one had a negative neutralization-test result and so was finally determined to be seronegative, giving a seropositive rate of 2.2% (2 of 90), which was significantly higher than that in healthy blood donors (0%, 0 of 1000; < 0.05). The seroprevalence identified here in small-animal-practice workers was slightly higher than that previously reported in other high-risk workers engaged in agriculture and forestry in Japan. Thus, enhancement of small-animal-practice workers' awareness of biosafety at animal hospitals is necessary for control of SFTSV.
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http://dx.doi.org/10.3390/v13020229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912989PMC
February 2021

Isolation of Severe Fever with Thrombocytopenia Syndrome Virus from Various Tick Species in Area with Human Severe Fever with Thrombocytopenia Syndrome Cases.

Vector Borne Zoonotic Dis 2021 Feb 3. Epub 2021 Feb 3.

Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan.

Severe fever with thrombocytopenia syndrome (SFTS), caused by , generally called SFTS virus (SFTSV), is an emerging zoonosis in East Asia. In Japan, 50-100 cases of SFTS have been reported each year since the first case was reported in 2013. SFTS is a tick-borne infectious disease, and SFTSV has been isolated from ticks in China and South Korea. and are considered the primary vectors in Japan. However, the other tick species seldom feeding on humans might also play an important role in maintaining the virus in nature. In this study, we collected ticks on vegetation around the location where two SFTS patients were estimated to have been infected in Miyazaki Prefecture, Japan, isolated live SFTSV, and performed a phylogenetic analysis. A total of 257 ticks were collected, and SFTSV RNA was detected in 19.5% (9/46) of tick pools. A total of 10 infectious SFTSVs were successfully isolated from , , , , and . Furthermore, the whole viral sequences isolated from ticks were highly homologous to sequences isolated from SFTS patients in the same sampling area in the past. These results suggest that SFTSVs are maintained in these tick species in the sampling area and sporadically transmitted to humans. Surveillance of SFTSV in ticks provides important information about the risk of incidental transmission to humans.
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http://dx.doi.org/10.1089/vbz.2020.2720DOI Listing
February 2021

Metagenomic identification, sequencing, and genome analysis of porcine hepe-astroviruses (bastroviruses) in porcine feces in Japan.

Infect Genet Evol 2021 Mar 14;88:104664. Epub 2020 Dec 14.

Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki, Japan; Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan; Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan. Electronic address:

Recently, hepe-astrovirus-like RNA viruses named bastroviruses (BastVs), have been found in human, pig, bat, and rat fecal samples. In this study, we determined nearly complete genome sequences of four BastVs in the feces of healthy pigs. Genetic characterization revealed that these porcine BastVs (PBastVs) and BastVs from other animals including humans, had the same genome organization, that is, they contained three predicted conserved domains of viral methyltransferase, RNA helicase, and RdRp in the nonstructural ORF1 and the astrovirus capsid domain in the structural ORF2. Phylogenetic analyses using RNA-dependent RNA polymerase and the capsid region revealed that PBastVs branched with bat and rat BastVs; however, the groups formed by each host were distantly related to human BastVs. Pairwise amino acid sequence comparison demonstrated that PBastVs shared 95.2-98.6% and 76.1-95.5% sequence identity among each other in the ORF1 and ORF2 regions, respectively; the sequence identities between PBastVs and BastVs from other animals were 21.4-42.5% and 9.1-20.6% in the ORF1 and ORF2 regions, respectively. This suggested that BastVs were derived from a common ancestor but evolved independently in each host population during a prolonged period. Putative recombination events were identified in the PBastV genome, suggesting that PBastVs gain sequence diversity and flexibility through recombination events. In an analysis of previously obtained metagenomic data, PBastV sequence reads were detected in 7.3% (23/315) of fecal samples from pigs indicating that PBastVs are distributed among pig populations in Japan.
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http://dx.doi.org/10.1016/j.meegid.2020.104664DOI Listing
March 2021

Direct Transmission of Severe Fever with Thrombocytopenia Syndrome Virus from Domestic Cat to Veterinary Personnel.

Emerg Infect Dis 2020 12;26(12):2994-2998

Two veterinary personnel in Japan were infected with severe fever with thrombocytopenia syndrome virus (SFTSV) while handling a sick cat. Whole-genome sequences of SFTSV isolated from the personnel and the cat were 100% identical. These results identified a nosocomial outbreak of SFTSV infection in an animal hospital without a tick as a vector.
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http://dx.doi.org/10.3201/eid2612.191513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706950PMC
December 2020

Bovine Respiratory Syncytial Virus Enhances the Adherence of to Bovine Lower Respiratory Tract Epithelial Cells by Upregulating the Platelet-Activating Factor Receptor.

Front Microbiol 2020 31;11:1676. Epub 2020 Jul 31.

Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki, Japan.

Coinfection by bovine respiratory syncytial virus (BRSV) and (PM) frequently has been observed in cattle that develop severe pneumonia. We recently reported that BRSV infection significantly increased PM adherence to bovine lower respiratory tract epithelial cells. However, the molecular mechanisms of enhanced PM adherence are not completely understood. To investigate whether BRSV infection regulates any cellular adherence receptors on bovine bronchus- and lung-epithelial cells, we performed proteomic and functional analyses. The proteomic analysis showed that BRSV infection increased the accumulation of the platelet-activating factor receptor (PAFR) in both cell types. Molecular experiments, including specific blockade, knockdown, and overexpression of PAFR, indicated that PM adherence to these cell types depended on PAFR expression. These findings highlight the role, in cattle with severe pneumonia, of the synergistic effect of coinfection by BRSV and PM in the lower respiratory tract.
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http://dx.doi.org/10.3389/fmicb.2020.01676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411089PMC
July 2020

Bovine Respiratory Syncytial Virus Decreased Pasteurella multocida Adherence by Downregulating the Expression of Intercellular Adhesion Molecule-1 on the Surface of Upper Respiratory Epithelial Cells.

Vet Microbiol 2020 Jul 2;246:108748. Epub 2020 Jun 2.

Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki, Japan; Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan; Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan. Electronic address:

The synergistic infection of bovine respiratory syncytial virus (BRSV) and Pasteurella multocida (PM) may predispose cattle to develop severe pneumonia. Previously, we reported that BRSV infection significantly decreased PM adherence to the upper respiratory epithelial cells. It may allow bacteria to invade into the lower respiratory tract and lead to severe pneumonia. To investigate whether BRSV infection regulates the cell surface adherence receptor on bovine trachea epithelial cells (bTECs), we performed proteomic and functional analyses. BRSV infection decreased the expression of intercellular adhesion molecule-1 (ICAM1) on bTECs. Inhibition and knockdown experiments using anti-ICAM1 antibody and siRNAs targeting ICAM1 indicated that PM adherence to bTECs was dependent on ICAM1 expression. These data suggest that under normal conditions bTECs may capture PM in the upper respiratory tract, while BRSV infection reverses this mechanism. The proposed gateway function of bTECs is disrupted by BRSV infection that may facilitate bacterial invasion into the lower respiratory tract and lead to secondary or more severe respiratory infection.
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http://dx.doi.org/10.1016/j.vetmic.2020.108748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265823PMC
July 2020

Molecular epidemiological survey and phylogenetic analysis of bovine respiratory coronavirus in Japan from 2016 to 2018.

J Vet Med Sci 2020 Jun 9;82(6):726-730. Epub 2020 Apr 9.

Center for Animal Disease Control, University of Miyazaki, Miyazaki 889-2192, Japan.

Bovine coronavirus (BCoV) is an etiological agent of bovine respiratory disease (BRD). BRD is a costly illness worldwide; thus, epidemiological surveys of BCoV are important. Here, we conducted a molecular epidemiological survey of BCoV in respiratory-diseased and healthy cattle in Japan from 2016 to 2018. We found that 21.2% (58/273) of the respiratory-diseased cattle were infected with BCoV. The respiratory-diseased cattle had virus amounts 4.7 times higher than those in the asymptomatic cattle. Phylogenetic analyses showed that the BCoV identified in Japan after 2005 formed an individual lineage that was distinct from the strains found in other countries. These results suggest that BCoV is epidemic and has evolved uniquely in Japan.
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http://dx.doi.org/10.1292/jvms.19-0587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324836PMC
June 2020

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

Food Environ Virol 2020 06 19;12(2):167-173. Epub 2020 Mar 19.

Center for Southeast Asian Studies, Kyoto University, 46 Shimoadachicho, Yoshida, Sakyo-ku, Kyoto, 606-8501, Japan.

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.
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http://dx.doi.org/10.1007/s12560-020-09425-1DOI Listing
June 2020

Slaughterhouse survey for detection of bovine viral diarrhea infection among beef cattle in Kyushu, Japan.

J Vet Med Sci 2019 Oct 5;81(10):1450-1454. Epub 2019 Aug 5.

Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan.

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.
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http://dx.doi.org/10.1292/jvms.19-0045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863731PMC
October 2019

Co-infection of epithelial cells established from the upper and lower bovine respiratory tract with bovine respiratory syncytial virus and bacteria.

Vet Microbiol 2019 Aug 12;235:80-85. Epub 2019 Jun 12.

Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan; Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan. Electronic address:

Bovine respiratory disease complex is a major disease affecting the global cattle industry. Multiple infections by viruses and bacteria increase disease severity. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection increases adherence of Pasteurella multocida to human respiratory and bovine kidney epithelial cells. To examine the interaction between the virus and bacteria in bovine respiratory cells, we generated respiratory epithelial cell lines from bovine trachea (bTEC), bronchus (bBEC), and lung (bLEC). Although all established cell lines were infected by BRSV and P. multocida susceptibility differed according to site of origin. The cells derived from the lower respiratory tract (bBEC and bLEC) were significantly more susceptible to BRSV than those derived from the upper respiratory tract (bTEC). Pre-infection of bBEC and bLEC with BRSV increased adherence of P. multocida; this was not the case for bTEC. These results indicate that BRSV may reproduce better in the lower respiratory tract and encourage adherence of bacteria. Thus, we identify one possible mechanism underlying severe pneumonia.
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http://dx.doi.org/10.1016/j.vetmic.2019.06.010DOI Listing
August 2019

Development of a fluorescent loop-mediated isothermal amplification assay for rapid and simple diagnosis of bovine leukemia virus infection.

J Vet Med Sci 2019 May 27;81(5):787-792. Epub 2019 Mar 27.

Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, Miyazaki, Miyazaki 889-2192, Japan.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a condition that threatens the sustainability of the livestock industry. A fluorescent loop-mediated isothermal amplification (fLAMP) assay targeting BLV env sequences was developed and used to evaluate 100 bovine blood samples. Compared with a conventional real-time PCR (rPCR) assay, the fLAMP assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. The rPCR assay took 65 min, while the fLAMP assay took 8 min to 30 min from the beginning of DNA amplification to final judgement with a comparable limit of detection. The fLAMP is a potential tool for the rapid and simple diagnosis of BLV infection to supplement ELISA testing and can be used by local laboratories and slaughterhouses without special equipment.
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http://dx.doi.org/10.1292/jvms.19-0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541838PMC
May 2019

Evaluation of the natural vertical transmission of Theileria orientalis.

Vet Parasitol 2018 Nov 1;263:1-4. Epub 2018 Oct 1.

Center for Animal Disease Control, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 8892192, Japan; Divisions of Research & Education for Livestock and Veterinary Clinic, Honkawa Ranch, Takase, Hita 8770056, Japan.

Bovine theileriosis, caused by Theileria orientalis, is endemic from East Asia to Oceania. Even though the disease is mainly transmitted by Haemaphysalis ticks, the T. orientalis parasite can also be transmitted vertically. To develop proper control measures, the frequency of each transmission route must be elucidated. However, the frequency of vertical transmission, including transplacental transmission, of T. orientalis in naturally infected cattle is still controversial. This study aimed to clarify the frequency of the vertical transmission of T. orientalis in naturally infected cattle. Blood samples were collected from 204 T. orientalis-infected dams and their 211 newborn calves (including 7 sets of twins) within the first 24 h as well as 30 days after birth. Furthermore, 31 and 24 calves born to T. orientalis-infected and uninfected dams, respectively, were continuously surveyed for infection until 5 months of age. A total of 5 (2.4%) dams were diagnosed with mild anemia, whereas most of the dams were asymptomatic based on hematological examination and clinical signs. PCR analysis was performed on whole blood to determine the presence of T. orientalis in calves, and no calves were PCR positive 0 and 30 days after birth. However, 9.6% and 0% of the calves born to T. orientalis-infected and uninfected dams, respectively, tested positive at 3 and 5 months of age. The sampled calves were fed in-house, and the survey was conducted during the cold season; thus, horizontal transmission through blood-sucking insects rarely occurred. Therefore, the vertical transmission of T. orientalis took as long as 3 months to become detectable by PCR and occurred in approximately 10% of field cattle.
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http://dx.doi.org/10.1016/j.vetpar.2018.09.017DOI Listing
November 2018

New Micro-amount of Virion Enrichment Technique (MiVET) to detect influenza A virus in the duck faeces.

Transbound Emerg Dis 2019 Jan 16;66(1):341-348. Epub 2018 Oct 16.

Faculty of Veterinary Medicine, Azabu University, Sagamihara, Japan.

Transboundary animal diseases, including highly pathogenic avian influenza, cause vast economic losses throughout the world. While it is important to identify the sources and propagation routes of the spread, such strategies are often hindered by incomplete epidemiological evidence. Isolation/detection of micro-amounts of pathogens from environmental samples is rarely successful due to the very low contamination level. This paper describes the development of the micro-amount of virion enrichment technique (MiVET), a simple and highly sensitive method that combines the use of a complex comprising a polyclonal antibody and protein G-coated magnetic beads for virion capture, and simple sodium dodecyl benzenesulfonate (SDBS) elution for low volume samples. The performance of the MiVET was evaluated using avian influenza A viruses (AIVs) in artificially spiked samples by real-time reverse transcription polymerase chain reaction (rRT-PCR). Four AIVs, H3N2, H4N2, H5N2 and H7N7, were used to artificially spike 50 ml of phosphate-buffered saline (PBS) and 1 ml of 10%-25% duck faecal supernatants. The MiVET system successfully concentrated AIVs in both PBS and faecal samples with at least 2 and 1 log greater efficacy, respectively, than conventional RNA extraction methods. The MiVET could be completed in <30 min from the beginning of sample preparation to final RNA extraction. The MiVET effectively prevented the effects of inhibitors in faecal samples, and did not require special equipment. This is the first report of this novel type of system, which is expected to be useful for the detection of micro-amounts of various veterinary and human viruses to elucidate their circulation dynamics in the environment, and for rapid and sensitive diagnosis with greater detection power.
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http://dx.doi.org/10.1111/tbed.13027DOI Listing
January 2019

Comparison of two agar gel immunodiffusion protocols for diagnosing equine infectious anemia.

J Vet Med Sci 2018 Aug 12;80(8):1245-1247. Epub 2018 Jun 12.

Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan.

This study compared agar gel immunodiffusion (AGID) protocols for diagnosing equine infectious anemia. Two commercial testing kits were used: one following the Japanese Act on Domestic Animal Infectious Diseases Control and one following the World Organisation for Animal Health (OIE) manual. From 651 samples tested, both protocols gave identical results for 647 samples (23 samples tested positive; 624 tested negative). Non-specific reactions were observed in 21 samples testing negative by the Japanese protocol, but none were observed with the OIE protocol. The kappa coefficient value was 0.962, indicating almost perfect agreement between the two protocols. This study found no difference in diagnostic agreement between the two protocols, but the OIE protocol produced non-specific reactions less frequently than the Japanese protocol.
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http://dx.doi.org/10.1292/jvms.18-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115256PMC
August 2018

Bovine respiratory syncytial virus infection enhances Pasteurella multocida adherence on respiratory epithelial cells.

Vet Microbiol 2018 Jul 30;220:33-38. Epub 2018 Apr 30.

Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan; Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan. Electronic address:

Primary infection with bovine respiratory syncytial virus (BRSV) predisposes cattle to secondary infection with bacteria that cause bovine respiratory disease complex (BRDC). However, the interaction between BRSV and bacteria is unclear. This in vitro study examined the adherence of Pasteurella multocida (PM) to BRSV-infected cells was assessed in colony forming unit assays, by flow cytometry analysis, and by indirect immunofluorescence analysis (IFA) of epithelial cells (A549, HEp-2, and MDBK). An in vitro model based on infection of BRSV-infected epithelial cells revealed that PM adherence to BRSV-infected cells was 2- to 8-fold higher than uninfected cells. This was confirmed by flow cytometry analysis and IFA. Epithelial cell expression of mRNA encoding cytokines and chemokines increased after exposure to PM, but increased further after co-infection with BRSV and PM. BRSV-mediated adherence of PM to epithelial cells may underlie the serious symptoms of BRDC.
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http://dx.doi.org/10.1016/j.vetmic.2018.04.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117154PMC
July 2018

New hematological key for bovine leukemia virus-infected Japanese Black cattle.

J Vet Med Sci 2018 Feb 22;80(2):316-319. Epub 2018 Jan 22.

Center for Animal Disease Control, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-2192, Japan.

The European Community's (EC) Key, which is also called Bendixen's Key, is a well-established bovine leukemia virus (BLV) diagnostic method that classifies cattle according to the absolute lymphocyte count and age. The EC Key was originally designed for dairy cattle and is not necessarily suitable for Japanese Black (JB) beef cattle. This study revealed the lymphocyte counts in the BLV-free and -infected JB cattle were significantly lower than those in the Holstein cattle. Therefore, applying the EC Key to JB cattle could result in a large number of undetected BLV-infected cattle. Our proposed hematological key, which was designed for JB cattle, improves the detection of BLV-infected cattle by approximately 20%. We believe that this study could help promote BLV control.
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http://dx.doi.org/10.1292/jvms.17-0455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836770PMC
February 2018

Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs.

J Vet Sci 2018 May;19(3):350-357

Research and Education Center for Prevention of Global Infectious Diseases of Animals, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-0045, Japan.

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, ser. Dublin, ser. Typhimurium, and ; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.
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http://dx.doi.org/10.4142/jvs.2018.19.3.350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974516PMC
May 2018

Intrauterine infection with bovine leukemia virus in pregnant dam with high viral load.

J Vet Med Sci 2017 Dec 6;79(12):2036-2039. Epub 2017 Nov 6.

Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan.

Enzootic bovine leukemia is caused by the bovine leukemia virus (BLV). BLV is transmitted vertically or horizontally through the transfer of infected cells via direct contact, through milk, insect bites and contaminated iatrogenic procedures. However, we lacked direct evidence of intrauterine infection. The purpose of this study was to confirm intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery. BLV was detected in cord and placental blood, and the BLV in the newborns showed 100% nucleotide identity with the BLV-env sequence from the dams. Notably, a newborn was seropositive for BLV but had no colostral antibodies. In this study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load.
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http://dx.doi.org/10.1292/jvms.17-0391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745186PMC
December 2017

Detection and molecular characterization of equine infectious anemia virus in Mongolian horses.

J Vet Med Sci 2017 Nov 11;79(11):1884-1888. Epub 2017 Oct 11.

Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido 060-0818, Japan.

The genetic characterization and actual prevalence of EIAV in Mongolian horse in the disease endemic region is currently unknown. Here, 11 of 776 horse serum samples from four Mongolian provinces tested positive on agar gel immunodiffusion test. Genomic DNA extracted from all seropositive samples was subjected to nested PCR assay. Among these, three samples tested positive with nested PCR assay and were identified by sequencing analysis based on long termination repeat and tat gene of the virus. Two of the three sequences were identical, with 94.0% identity with the third. These two independent Mongolian EIAV sequences were retained functional motifs, with no dramatic changes but some variability in the U5 region; they were clustered with genotypes from European countries but not with those from China, U.S.A., or Japan.
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http://dx.doi.org/10.1292/jvms.17-0202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709569PMC
November 2017

Cattle with the BoLA class II DRB3*0902 allele have significantly lower bovine leukemia proviral loads.

J Vet Med Sci 2017 Sep 28;79(9):1552-1555. Epub 2017 Jul 28.

Laboratory of Animal Infectious Disease and Prevention, Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-2192, Japan.

The bovine MHC (BoLA) class II DRB3 alleles are associated with polyclonal expansion of lymphocytes caused by bovine leukemia virus (BLV) infection in cattle. To examine whether the DRB3*0902 allele, one of the resistance-associated alleles, is associated with the proviral load, we measured BLV proviral load of BLV-infected cattle and clarified their DRB3 alleles. Fifty-seven animals with DRB3*0902 were identified out of 835 BLV-infected cattle and had significantly lower proviral load (P<0.000001) compared with the rest of the infected animals, in both Japanese Black and Holstein cattle. This result strongly indicates that the BoLA class II DRA/DRB3*0902 molecule plays an important immunological role in suppressing viral replication, resulting in resistance to the disease progression.
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http://dx.doi.org/10.1292/jvms.16-0601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627326PMC
September 2017

Phylogenetic analysis of env gene of bovine leukemia virus strains spread in Miyazaki prefecture, Japan.

J Vet Med Sci 2017 May 23;79(5):912-916. Epub 2017 Mar 23.

Laboratory of Animal Infectious Disease and Prevention, Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-2192, Japan.

To understand how the latest dominant bovine leukemia virus (BLV) strains were introduced and spread in the Miyazaki prefecture, we collected blood samples from 3 geographic areas (north, central and south) and carried out sequence analysis of the BLV env gene. Two genotypes, genotype I, and III, were identified and the majority of the strains belonged to genotype I (71/74). To clarify a route of BLV introduction, we divided the strains into 20 subgenotypes based on their nucleotide sequences and performed phylogenetic analysis. Our study indicated that common BLV strains were comparatively evenly distributed even in the area, where the farmers have not introduced cattle from other areas and the cattle have limited exposure to BLV infection in grazing fields.
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http://dx.doi.org/10.1292/jvms.17-0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447981PMC
May 2017

Use of Direct LAMP Screening of Broiler Fecal Samples for and in the Positive Flock Identification Strategy.

Front Microbiol 2016 30;7:1582. Epub 2016 Sep 30.

Department of Veterinary Sciences, Faculty of Agriculture, University of MiyazakiMiyazaki, Japan; Center for Animal Disease Control, University of MiyazakiMiyazaki, Japan.

Rapid identification of -positive flocks before slaughter, following freezing and heat treatment for the -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 / 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 /-positive broiler flocks at the farm level or in slaughterhouses before slaughtering, which would make it an effective tool in preventing the spread of contamination.
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http://dx.doi.org/10.3389/fmicb.2016.01582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043150PMC
September 2016

Nationwide Distribution of Bovine Influenza D Virus Infection in Japan.

PLoS One 2016;11(9):e0163828. Epub 2016 Sep 28.

Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

Cattle are major reservoirs of the provisionally named influenza D virus, which is potentially involved in the bovine respiratory disease complex. Here, we conducted a serological survey for the influenza D virus in Japan, using archived bovine serum samples collected during 2010-2016 from several herds of apparently healthy cattle in various regions of the country. We found sero-positive cattle across all years and in all the prefectural regions tested, with a total positivity rate of 30.5%, although the positivity rates varied among regions (13.5-50.0%). There was no significant difference in positivity rates for Holstein and Japanese Black cattle. Positivity rates tended to increase with cattle age. The herds were clearly divided into two groups: those with a high positive rate and those with a low (or no) positive rate, indicating that horizontal transmission of the virus occurs readily within a herd. These data demonstrate that bovine influenza D viruses have been in circulation for at least 5 years countrywide, emphasizing its ubiquitous distribution in the cattle population of Japan.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040247PMC
September 2016

Six Novel O Genotypes from Shiga Toxin-Producing Escherichia coli.

Front Microbiol 2016 20;7:765. Epub 2016 May 20.

Department of Bacteriology, Faculty of Medical Sciences, Kyushu University Fukuoka, Japan.

Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny.
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http://dx.doi.org/10.3389/fmicb.2016.00765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873512PMC
May 2016

Horizontal transmission and phylogenetic analysis of bovine leukemia virus in two districts of Miyazaki, Japan.

J Vet Med Sci 2015 Sep 19;77(9):1115-20. Epub 2015 Apr 19.

Project for Zoonoses Education and Research, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-2192, Japan.

Horizontal transmission is recognized as a major infection route for bovine leukemia virus (BLV), and cattle with high viral loads are considered to be a major infectious source in a herd. However, a correlation between viral loads and the risk of infection has been insufficient to use as a foundation for BLV control strategies. In this report, we examined the epidemiology of BLV infection and the infectious source in a local area. In 2013-2014, BLV infection was investigated in 1,823 cattle from 117 farms in two adjacent districts, Miyazaki, Japan. Seropositive samples for BLV were detected with 88 cattle and in 14 farms. Phylogenetic analysis revealed that 94% of the isolates clustered into genotype I and the remaining isolate into genotype III. Among genotype I, genetically distinct strains were spread at each farm, and cattle infected with less than 3 copies/100 cells did not transmit BLV to other cattle for more than thirty months. This is the first report of concrete data of viral load in relation to viral horizontal transmission under the field condition. The data facilitate farmers and veterinarians understanding the status of BLV infected cattle. This research contributes to BLV infection control and the development of effective BLV eradication programs.
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http://dx.doi.org/10.1292/jvms.14-0624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591153PMC
September 2015

Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi.

J Vet Med Sci 2015 Aug 27;77(8):1017-9. Epub 2015 Mar 27.

Laboratory of Infectious Diseases, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Sapporo 060-0818, Japan.

Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle.
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http://dx.doi.org/10.1292/jvms.15-0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565805PMC
August 2015

Evaluation of the natural perinatal transmission of bovine leukaemia virus.

Vet Rec 2015 Mar 15;176(10):254. Epub 2014 Dec 15.

Laboratory of Animal Infectious Disease and Prevention, Department of Veterinary Medicine, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-2192, Japan Center for Animal Disease Control, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-2192, Japan.

The perinatal transmission of bovine leukaemia virus (BLV) plays a critical role in the spread and persistence of BLV infection in cattle herds. The purpose of this study was to examine the frequency of perinatal infections in an area in Japan and investigate some risk factors associated with infection. Altogether, 129 calves born to BLV-infected cows in a herd in Japan were tested for infection immediately after birth and again at one month of age using nested PCR. Twenty-four calves (18.6 per cent) were infected with BLV, of which 14 (10.8 per cent) and 10 (7.7 per cent) calves were infected via the transplacental and the birth canal routes, respectively. Maternal viral loads, breed, the presence or absence of assistance during parturition and the number of births per dam were evaluated to investigate risk factors associated with infection. Maternal viral load was significantly correlated with the frequency of perinatal infection, and more than 40 per cent of newborn calves born to dams with high viral loads were infected with BLV. The results of this study could contribute towards developing effective eradication programmes by providing necessary data for replacement of breeding cow in the field.
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http://dx.doi.org/10.1136/vr.102464DOI Listing
March 2015

Identification of O serotypes, genotypes, and virulotypes of Shiga toxin-producing Escherichia coli isolates, including non-O157 from beef cattle in Japan.

J Food Prot 2014 Aug;77(8):1269-74

Center for Animal Disease Control, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki, 889-2192, Japan.

Bovines are recognized as an important reservoir of Shiga toxin-producing Escherichia coli (STEC). Although STEC strains are significant foodborne pathogens, not all of the STEC held by cattle are pathogenic, and which type of STEC that will become epidemic in humans is unpredictable. Information about the prevalence of serotype and virulence gene distribution in beef cattle is insufficient to develop monitoring and controlling activities for a food safety and security program. Thus, this study investigated the prevalence of O157 and non-O157 STEC in Japanese beef cattle and characterized the isolates by the type of O antigen and several virulence markers to help predict the pathogenicity. In this study, 64.2% (176 of 274) of enrichment cultures of fecal samples collected from an abattoir and farms were stx1 and/or stx2 positive by PCR. STEC strains were isolated from 22.1% (39 of 176) of the positive fecal samples, and these isolates represented 17 types of O antigen (O1, O2 or O50, O5, O8, O55, O84, O91, O109, O113, O136, O150, O156, O157, O163, O168, O174, and O177). Two selective media targeting major STEC groups, cefixime-tellurite sorbitol MacConkey agar and CHROMagar O26/O157, allowed isolation of a variety of STEC strains. The most frequently isolated STEC was O113 (8 of 39), which has previously been reported as a cause of foodborne infections. Although most of the O113 STEC isolated from infected patients possessed the enterohemolysin (hlyA) gene, none of the O113 STEC cattle isolates possessed the hlyA gene. The second most common isolate was O157 (6 of 39), and all these isolates contained common virulence factors, including eae, tir, lpf1, lpf2, and hlyA. This study shows the prevalence of O157 and non-O157 STEC in Japanese beef cattle and the relationship of O antigen and virulotypes of the isolates. This information may improve identification of the source of infection, developing surveillance programs or the current understanding of virulence factors of STEC infections.
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http://dx.doi.org/10.4315/0362-028X.JFP-13-506DOI Listing
August 2014