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Yanase T.,Japanese National Institute of Animal Health | Matsumoto Y.,Japan National Institute of Agrobiological Science | Matsumori Y.,Goto Livestock Hygiene Service Center | Aizawa M.,Okinawa Prefectural Institute of Animal Health | And 6 more authors.
Journal of Medical Entomology | Year: 2013

Although Culicoides biting midges act as a vector of important human and domestic animal diseases, their ecology is poorly understood. The lack of proper identification systems of Culicoides larvae is one of the main obstacles to progress in research. Based on mitochondrial sequences of 19 Japanese Culicoides species, we designed a universal primer set to amplify the partial sequence of the mitochondrial cytochrome c oxidase I (cox 1). The polymerase chain reaction product amplified from extracted DNA of Culicoides larvae using the primer set was directly sequenced, and species identification based on the variation at cox1 was conducted. Using the molecular identification system, we sorted 243 specimens of field-collected larvae from the southern part of Japan into 10 species including Culicoides arakawae (Arakawa), Culicoides oxystoma Kieffer, and Culicoides brevitarsis Kieffer, which are regarded as vectors of important livestock animal diseases. Eight species of Culicoides larvae, including C. arakawae and C. oxystoma, were recovered from active paddy fields and an abandoned paddy field. The result suggests that paddy fields contribute to breeding a variety of Culicoides species and maintenance and spread of Culicoides-borne pathogens. In contrast, larvae of C. brevitarsis were collected from cattle dung in pastures. The molecular identification system described herein using nucleotide sequences successfully achieved larval identification and will be useful for a better understanding of larval habitats of Culicoides biting midges. © 2013 Entomological Society of America.


PubMed | Domestic Livestock Disease Identification Office, Kusu Livestock Hygiene Service Center, Okinawa Prefectural Institute of Animal Health, Kagoshima Central Livestock Hygiene Service Center and 2 more.
Type: | Journal: Journal of virological methods | Year: 2015

TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine -actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.


Yokoyama N.,Obihiro University of Agriculture and Veterinary Medicine | Sivakumar T.,Obihiro University of Agriculture and Veterinary Medicine | Ota N.,Obihiro University of Agriculture and Veterinary Medicine | Igarashi I.,Obihiro University of Agriculture and Veterinary Medicine | And 12 more authors.
Infection, Genetics and Evolution | Year: 2012

In the present study, we investigated the possible tick vectors that can transmit Theileria orientalis in eastern Hokkaido, Japan. Questing ticks collected from three different districts, Taiki, Otofuke, and Shin-Hidaka, of Hokkaido included Ixodes persulcatus, Haemaphysalis megaspinosa, Haemaphysalis douglasi, and Ixodes ovatus, while all the ticks collected from Yonaguni island of Okinawa were identified as Haemaphysalis longicornis. When the ticks were screened by polymerase chain reaction (PCR) for T. orientalis, the parasite was commonly detected among all tick species. Genotype-specific PCR assays revealed that all tick species in Hokkaido were predominantly detected with type 2, while ticks collected from Okinawa (H. longicornis) were predominantly detected with type 1. Consistent with the genetic diversity of T. orientalis in ticks, genotyping PCR assays from cattle grazed in the same Hokkaido sampling locations identified type 2 as the most prevalent genotype. This study provides the first identification of I. persulcatus, H. megaspinosa, H. douglasi, and I. ovatus as possible tick vectors of T. orientalis, and finds that the variety of vectors apparently capable of transmitting T. orientalis is wider in Japan than expected. The authors suggest that tick control strategies should be modified in Hokkaido based on the seasonal activities of ticks identified in the present study. © 2012 Elsevier B.V.


PubMed | Japanese National Institute of Animal Health, Yaeyama Livestock Hygiene Service Center and Okinawa Prefectural Institute of Animal Health
Type: Journal Article | Journal: BMC veterinary research | Year: 2016

In Japan, epizootic arboviral infections have severely impacted the livestock industry for a long period. Akabane, Aino, Chuzan, bovine ephemeral fever and Ibaraki viruses have repeatedly caused epizootic abnormal births and febrile illness in the cattle population. In addition, Peaton, Sathuperi, Shamonda and DAguilar viruses and epizootic hemorrhagic virus serotype 7 have recently emerged in Japan and are also considered to be involved in abnormal births in cattle. The above-mentioned viruses are hypothesized to circulate in tropical and subtropical Asia year round and to be introduced to temperate East Asia by long-distance aerial dispersal of infected vectors. To watch for arbovirus incursion and assess the possibility of its early warning, monitoring for arboviruses was conducted in the Yaeyama Islands, located at the most southwestern area of Japan, between 1994 and 2014.Blood sampling was conducted once a year, in the autumn, in 40 to 60 healthy cattle from the Yaeyama Islands. Blood samples were tested for arboviruses. A total of 33 arboviruses including Akabane, Peaton, Chuzan, D Aguilar, Bunyip Creek, Batai and epizootic hemorrhagic viruses were isolated from bovine blood samples. Serological surveillance for the bovine arboviruses associated with cattle diseases in young cattle (ages 6-12 months: had only been alive for one summer) clearly showed their frequent incursion into the Yaeyama Islands. In some cases, the arbovirus incursions could be detected in the Yaeyama Islands prior to their spread to mainland Japan.We showed that long-term surveillance in the Yaeyama Islands could estimate the activity of bovine arboviruses in neighboring regions and may provide a useful early warning for likely arbovirus infections in Japan. The findings in this study could contribute to the planning of prevention and control for bovine arbovirus infections in Japan and cooperative efforts among neighboring countries in East Asia.


Kyan H.,Okinawa Prefectural Institute of Health and Environment | Taira M.,Okinawa Prefectural Institute of Health and Environment | Yamamoto A.,Okinawa Prefectural Institute of Health and Environment | Inaba C.,Okinawa Prefectural Institute of Health and Environment | Zakimi S.,Okinawa Prefectural Institute of Animal Health
Japanese Journal of Infectious Diseases | Year: 2012

Toxoplasma gondii genotypes were isolated and characterized from cephalic muscle samples collected from 24 goats slaughtered at an abattoir in Okinawa between 2008 and 2009. Of the 24 samples assayed using latex agglutination, 18 were seropositive, 2 were pseudo-positive, and 4 were seronegative against T. gondii antibodies. The samples were then inoculated into laboratory mice to isolate the parasite. Among the isolated samples, 13 (72.2% of the 18 seropositive strains in the latex agglutination assay) were seropositive, 1 (50%) was pseudo-positive, and none were seronegative. However, after being frozen and stored at -20°C, all samples were found to be T. gondii-free. Of the 14 isolates of the GRA6 genotype, 6 were of type I, 7 were of type II, and 1 was of type III; the genotype distribution ratio was similar to that of T. gondii strains isolated from locally raised pigs. Moreover, no sulfonamide-tolerant dhps gene mutant of T. gondii was detected.


Sivakumar T.,Obihiro University of Agriculture and Veterinary Medicine | Tattiyapong M.,Obihiro University of Agriculture and Veterinary Medicine | Okubo K.,Obihiro University of Agriculture and Veterinary Medicine | Suganuma K.,Obihiro University of Agriculture and Veterinary Medicine | And 6 more authors.
Ticks and Tick-borne Diseases | Year: 2014

Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n= 1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100. fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata. © 2014 Elsevier GmbH.


Yanase T.,Japanese National Institute of Animal Health | Aizawa M.,Okinawa Prefectural Institute of Animal Health | Kato T.,Japanese National Institute of Animal Health | Yamakawa M.,Japanese National Institute of Animal Health | And 2 more authors.
Virus Research | Year: 2010

Sequence determination and phylogenetic analysis were conducted using the S, M and L RNA segments of the 10 Aino, 6 Peaton and 1 Sango virus (AINOV, PEAV and SANV) field isolates of the genus Orthobunyavirus in the family Bunyaviridae, respectively. The Japanese AINOV strains were genetically stable, but the sequence differences between the Japanese and Australian AINOV strains were considerably larger than those among the Japanese AINOV strains. A similar result was found in the genetic relationship among Japanese and Australian PEAVs, and SANV which was isolated in Nigeria and was thought as a synonym of PEAV, suggesting that geographic separation contributed significantly to the evolution of those viruses. The Australian AINOV strain B7974 is more closely related to the Australian PEAV strain CSIRO110 than to the Japanese AINOV strains in the S and L RNA segments, while the phylogenetic position of the M RNA segment of the B7974 strain was clustered with those of the Japanese AINOV strains. Our findings indicate that the B7974 strain is a reassortment with the M RNA segment derived from AINOV and the S and L RNA segments derived from an Australian PEAV. © 2010 Elsevier B.V.


PubMed | Okinawa Prefectural Institute of Animal Health
Type: Journal Article | Journal: The Journal of veterinary medical science | Year: 2015

In September 2012, several cows and a calf showed decreased activity, anorexia and fever on Ishigaki Island, Okinawa Prefecture, Japan, and the cases were diagnosed as bovine ephemeral fever (BEF). We isolated BEF virus (BEFV) from one of the affected cows and then determined the complete genome sequence of the G gene, which encodes a class I transmembrane glycoprotein of BEFV. The BEFV isolate in this case, ON-3/E/12, was sorted into the same cluster as other BEFV isolates in Japan, Taiwan and China obtained in 1996-2004 and was most closely related to a 2002 Chinese isolate, JT02L, according to the phylogenetic analysis of the complete G gene. Since inactivated vaccines for BEF available in Japan are considered effective against the ON-3/E/12 isolate as well as other isolates in East Asia from 1996-2004, annual vaccination should be conducted to prevent BEF in Okinawa. Additionally, in this study, we developed an RT-PCR assay to detect the BEFV gene in Japan and neighboring countries. Our assay was able to amplify target sequences in all of the tested BEFV isolates, including 18 isolates in Japan and another isolate in Australia. The assay was found to be useful also for testing RNA samples extracted from bovine peripheral blood mononuclear cells, and the detection limit of the assay was 10 copies per tube. We believe that our assay would be an important tool for the screening of BEFV infection and the diagnosis of BEF.


PubMed | Obihiro University of Agriculture and Veterinary Medicine and Okinawa Prefectural Institute of Animal Health
Type: Journal Article | Journal: Ticks and tick-borne diseases | Year: 2014

Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described -tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.

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