Takashi Yamanaka

Equine Research Institute, Totigi, Tochigi, Japan

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Publications (38)41.74 Total impact

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    ABSTRACT: Recently, outbreaks associated with equine coronavirus (ECoV) have occurred in Japan and the United States. While ECoV is likely to be pathogenic to horses, it has not been shown that experimental inoculation of horses with ECoV produces clinical signs of disease. In this study, we inoculated three Japanese draft horses with an ECoV-positive diarrheic fecal sample to confirm infection after inoculation and to investigate the clinical course and virus shedding patterns of ECoV. Virus neutralization tests showed that all three horses became infected with ECoV. Two of the three horses developed clinical signs similar to those observed during ECoV outbreaks, including fever, anorexia, and gastrointestinal dysfunction. All horses excreted a large amount of virus into their feces for more than 9 days after inoculation regardless of the presence or absence of clinical signs, which suggests that feces are an important source of ECoV infection. ECoV was also detected in nasal swabs from all horses, suggesting that respiratory transmission of ECoV may occur. Both symptomatic horses developed viremia, while the asymptomatic horse did not. White blood cell counts and serum amyloid A concentrations changed relative to the clinical condition of the inoculated horses; these may be useful markers for monitoring the clinical status of horses infected with ECoV. This is the first report of induction of clinical signs of ECoV infection in horses by experimental inoculation. These clinical and virological findings should aid further investigation of the pathogenesis of ECoV.
    Archives of virology. 08/2014;
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    ABSTRACT: Equine herpesviruses type 1 (EHV-1) is a major cause of winter pyrexia in racehorses in two training centers (Ritto and Miho) in Japan. Until the epizootic period of 2008-2009, a vaccination program using a killed EHV-1 vaccine targeted only those of the 3-year-old susceptible horses with low antibody levels to EHV-1 antigens. However, because protective effect was not satisfactory, in 2009-2010 the vaccination program was altered to target all 3-year-old horses. To evaluate vaccine efficacy, we investigated the number of pyretic horses due to EHV-1 or equine herpesvirus type-4 (EHV-4) infection, or both, and examined the vaccination coverage in the 3-year-old population and in the whole population before and after changing the program. The mean estimated number of horses infected with EHV-1 or EHV-4, or both, among pyretic horses from 1999-2000 to 2008-2009 was 105±47 at Ritto and 66±44 at Miho. Although the estimated number of infected horses did not change greatly in the first period of the current program, it decreased from the second period, with means of 21±12 at Ritto and 14±15 at Miho from 2010-2011 to 2012-2013. Vaccination coverage in the 3-year-old population was 99.4% at Ritto and 99.8% at Miho in the first period, and similar values were maintained thereafter. Coverage in the whole population increased more gradually than that in the 3-year-old population. The results suggested that EHV-1 epizootics can be suppressed by maintaining high vaccination coverage, not only in the 3-year-old population but also in the whole population.
    Clinical and vaccine Immunology: CVI 05/2014; · 2.60 Impact Factor
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    ABSTRACT: Reasons for performing studyThe protection induced by an equine influenza (EI) vaccine strain depends on its antigenic relatedness to the challenge virus. Although the World Organisation for Animal Health (OIE) recommend that both Florida sublineage clade 1 (Fc1) and clade 2 (Fc2) viruses should be included in EI vaccines, Japanese EI vaccines have not, thus far, been updated to include a Fc2 virus.Objectives To evaluate the efficacy of antibodies raised against Japanese EI vaccine strains in the neutralisation of recent Fc2 viruses.Study designAntigenic analysis.Methods Virus neutralisation tests were performed using antisera from experimentally infected horses and from horses that had received a primary course of the currently available vaccines.ResultsAntiserum raised against the Japanese EI vaccine strain A/equine/La Plata/1993, exhibited poor cross-neutralising activity against the Fc2 viruses isolated recently in Ireland and the UK which have the substitution alanine to valine at position 144 in antigenic site A of the haemagglutinin gene. In contrast the antiserum exhibited good cross-neutralising activity against the Fc2 viruses without the substitution. This finding was supported in experiments with antisera collected from vaccinated horses.Conclusions This suggests that the efficacy of the Japanese EI vaccine for some of the recent Fc2 viruses is suboptimal and that vaccines should be updated in accordance with the OIE recommendations.
    Equine Veterinary Journal 04/2014; · 2.29 Impact Factor
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    ABSTRACT: Although many disinfectants are commercially available in the veterinary field, information on the virucidal effects of disinfectants against equine group A rotavirus (RVA) is limited. We evaluated the performance of commercially available disinfectants against equine RVA. Chlorine- and iodine-based disinfectants showed virucidal effects, but these were reduced by the presence of organic matter. Glutaraldehyde had a virucidal effect regardless of the presence of organic matter, but the effect was reduced by low temperature or short reaction time, or both. Benzalkonium chloride had the greatest virucidal effect among the three quaternary ammonium compounds examined, but its effect was reduced by the presence of organic matter or by low temperature or a short reaction time. These findings will be useful for preventing the spread of equine RVA infection.
    Journal of Veterinary Medical Science 03/2014; · 0.88 Impact Factor
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    ABSTRACT: Virus-neutralizing (VN) testing is essential for evaluating virus-specific immunity in equine herpesvirus type-1 (EHV-1) infection. We developed a focus-reduction neutralization test (FRNT) for EHV-1 using 96-well plates for faster large-scale testing with sufficient sensitivity. We used an overlay medium containing Avicel (FMC Biopolymer), a microcrystalline cellulose with lower viscosity than the methylcellulose. The foci were visualized by immuno-staining with anti-EHV-1 gp14 monoclonal antibody. The FRNT successfully detected seroconversion in horses experimentally infected with EHV-1 (n=3) and in those infected naturally (n=16). The FRNT for EHV-1 was high-throughput and time saving. The FRNT can be used in large-scale seroepidemiological studies of EHV-1 and in evaluating vaccine efficacy.
    Journal of Veterinary Medical Science 04/2013; · 0.88 Impact Factor
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    ABSTRACT: Non-specific hemolysis has often been observed during complement-fixation (CF) tests for equine herpesvirus type-1 (EHV-1), even when the sera have virus-specific CF antibodies. This phenomenon has also been reported in CF tests for various infectious diseases of swine. We found that the sera from 22 of 85 field horses (25.9%) showed non-specific hemolysis during conventional CF testing for EHV-1. Because pretreatment of swine sera with potassium periodate (KIO4) improves the CF test for swine influenza, we applied this method to horse sera. As we expected, horse sera treated with KIO4 did not show non-specific hemolysis in the EHV-1 CF test, and precise determination of titers was achieved.
    Journal of Equine Science 01/2013; 24(4):71-4.
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    ABSTRACT: Disinfection is one of the most important biosecurity measures to minimize disease spread during outbreaks of equine influenza. Although many disinfectants are commercially available, information about their effects against equine influenza A virus (EIV) is limited. This report describes an evaluation of the effects of six disinfectants against EIV (∼104.7 egg infectious dose 50/200 μL) under different conditions (reaction time [10 and/or 30 minutes], temperature [4°C–25°C], and the absence and/or presence of uninfected allantoic fluid that served as our best choice of “organic matter” equivalent). Although the efficacy of didecyldimethylammonium chloride decreased with decreasing reaction temperature, the compound showed the highest efficacy of the three quaternary ammonium compounds tested in this study. The effects of sodium dichloroisocyanurate and nonoxynol iodine were not affected by reaction time or temperature, but they were affected by the presence of organic matter. Antec Virkon S containing potassium peroxymonosulfate and sodium chloride consistently inactivated EIV regardless of reaction time, temperature, and the presence of organic matter. These findings will help us to take rational biosecurity measures during outbreaks of equine influenza.
    Journal of Equine Veterinary Science 01/2013; · 0.62 Impact Factor
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    ABSTRACT: BACKGROUND: Both the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test. FINDINGS: After vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold. CONCLUSIONS: These results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.
    Acta Veterinaria Scandinavica 11/2012; 54(1):63. · 1.00 Impact Factor
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    ABSTRACT: Since equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, which is called canine influenza A virus (CIV), has become widespread in dogs. To date, it has remained unclear whether or not CIV-infected dogs could transmit CIV to horses. To address this, we tested whether or not close contact between horses and dogs experimentally infected with CIV would result in its interspecies transmission. Three pairs of animals consisting of a dog inoculated with CIV (10(8.3) egg infectious dose 50/dog) and a healthy horse were kept together in individual stalls for 15 consecutive days. During the study, all the dogs and horses were clinically observed. Virus titres in nasal swab extracts and serological responses were also evaluated. In addition, all the animals were subjected to a gross pathological examination after euthanasia. All three dogs inoculated with CIV exhibited clinical signs including, pyrexia, cough, nasal discharge, virus shedding and seroconversion. Gross pathology revealed lung consolidations in all the dogs, and Streptococcus equi subsp. zooepidemicus was isolated from the lesions. Meanwhile, none of the paired horses showed any clinical signs, virus shedding or seroconversion. Moreover, gross pathology revealed no lesions in the respiratory tracts including the lungs of the horses. These findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.
    Acta Veterinaria Scandinavica 04/2012; 54:25. · 1.00 Impact Factor
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    ABSTRACT: We have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting H7N7 equine influenza virus (EIV). The detection limit of the RT-LAMP assay was a virus dilution of 10(-4), which was 10 times more sensitive than that of Espline Influenza A&B-N and the same as that of reverse transcription polymerase chain reaction. The RT-LAMP assay specifically amplified H7N7 EIV strains but did not amplify several pathogens related to equine respiratory disease including H3N8 EIV strains. Because it provides ease of manipulation, the RT-LAMP assay is suitable for large-scale surveillance for H7N7 EIV. In addition, the combination of the H3N8 RT-LAMP assay, which was developed previously, with the H7N7 RT-LAMP assay should be useful to discriminate between H3N8 and H7N7 EIVs in clinical laboratories.
    Journal of Veterinary Medical Science 02/2012; 74(7):929-31. · 0.88 Impact Factor
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    ABSTRACT: Equine influenza A virus (EIV) of the H3N8 subtype is an important pathogen causing acute respiratory disease in horses. Peramivir is a selective inhibitor of the influenza virus neuraminidase (NA). The characteristics of peramivir are not only its capacity for parenteral administration, but also its strong affinity for NA and slow off-rate from the NA-peramivir complex, suggesting that it could lead to a prolonged inhibitory effect and thus allow a lower dosing frequency. The aims of this study were to evaluate the inhibitory efficacy of peramivir against the NA activities of EIV in vitro and the treatment efficacy of a single intravenous dose of peramivir in horses experimentally infected with EIV. Peramivir inhibited the activities of NA from the seven contemporary EIV strains in vitro, with 50% inhibitory concentrations ranging from 0.10 to 0.20nmol/L. Horses treated with a single IV dose of peramivir (3000mg/600mL/animal, 7.8-9.3mg/kg of bodyweight) showed significantly milder clinical signs (pyrexia, nasal discharge and cough) with a shorter duration than control horses injected with normal saline. Moreover, the mean duration of virus shedding for the horses treated with peramivir was significantly shorter than for the control horses. These findings suggested that a single IV administration of peramivir had good potential for the treatment of equine influenza, and may help to limit the spread of the disease in the horse population.
    The Veterinary Journal 02/2012; 193(2):358-62. · 2.42 Impact Factor
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    ABSTRACT: The immunoglobulin G (IgG) subclass response was investigated in horses with or without pyrexia after natural infection with equine herpesvirus type 1 (EHV-1) in the field. All horses were kept at the training centers of the Japan Racing Association and were immunized with an inactivated EHV-1 vaccine before EHV-1 infection. An IgG subclass response dominated by IgGa and IgGb was induced in horses without pyrexia after EHV-1 infection. In contrast, horses that developed pyrexia showed increased IgGc and IgG (T) subclass production in addition to IgGa and IgGb. Although inactivated EHV-1 vaccines are considered to induce a mainly Th-2-biased response, these results indicated that the responses in horses inoculated with inactivated EHV-1 vaccine were not uniform, and that horses with a Th-1-biased response were likely to be protected from pyrexia.
    Journal of Veterinary Medical Science 01/2012; 74(6):791-5. · 0.88 Impact Factor
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    ABSTRACT: To investigate the pathology of equine influenza, necropsy of 7 horses experimentally infected with equine influenza A virus (EIV) subtype H3N8 was conducted on post-infection days (PID) 2, 3, 7, and 14. Histopathologically, rhinitis or tracheitis including epithelial degeneration or necrosis with loss of ciliated epithelia and a reduction in goblet cell numbers, was observed in the respiratory tracts on PIDs 2 and 3. Epithelial hyperplasia or squamous metaplasia and suppurative bronchopneumonia with proliferation of type II pneumocytes were observed on PIDs 7 and 14. Viral antigen was detected immunohistochemically in the epithelia of the nasal mucosa, trachea, and bronchi on PIDs 2 and 3. The sodA gene of Streptococcus equi subsp. zooepidemicus, a suspected cause of suppurative bronchopneumonia, was detected in paraffin-embedded lung tissue sections, but only on PIDs 7 and 14. These findings suggest that damage caused to ciliated epithelia and goblet cells by EIV infection results in secondary bacterial bronchopneumonia due to a reduction in mucociliary clearance.
    Journal of Equine Science 01/2012; 23(2):17-26.
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    ABSTRACT: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to the detection of equine influenza virus (EIV). Because equine influenza is caused currently by EIV of the H3H8 subtype, the RT-LAMP primer set was designed to target the hemagglutinin gene of this subtype. The detection limit of the RT-LAMP assay was a virus dilution of 10(-5); which was 10(3) times more sensitive than the Espline Influenza A&B-N test and 10 times more sensitive than a reverse transcription polymerase chain reaction (RT-PCR) assay. The specificity of the RT-LAMP assay was examined by using several equine pathogens and nasal swabs collected from horses with fever in 2010 after EIV was eradicated in Japan. No cross-reactions were observed. Using 100 nasal swabs collected from horses with fever during an EIV outbreak in 2007, the RT-LAMP assay detected EIV in 52 samples, whereas the Espline test and the RT-PCR assay detected EIV in only 17 and 41 samples, respectively. These results indicate that the RT-LAMP assay is specific for EIV and more sensitive than the Espline test and the RT-PCR assay. Because it provides high sensitivity and ease of manipulation without the need for a thermal cycler or gel electrophoresis, the RT-LAMP assay should be applicable for laboratory diagnosis of EIV.
    Journal of virological methods 08/2011; 178(1-2):239-42. · 2.13 Impact Factor
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    ABSTRACT: A single non-synonymous nucleotide substitution of guanine (G) for adenine (A) at position 2254 in the viral DNA polymerase gene (encoded by open reading frame [ORF] 30) of equine herpesvirus type 1 (EHV-1) has been significantly associated with neuropathogenic potential in strains of this virus. To estimate the prevalence of EHV-1 strains with the neuropathogenic genotype (ORF30 G(2254)) in the Hidaka district--a major horse breeding area in Japan--we analyzed the ORF30 genomic region in cases of EHV-1 infection in this area during the years 2001-2010. Of the 113 cases analyzed, 3 (2.7%) were induced by ORF30 G(2254) strains. This prevalence is lower than those observed in the U.S.A. (10.8-19.4%), Argentina (7.4%), France (24%), and Germany (10.6%).
    Journal of Veterinary Medical Science 08/2011; 73(12):1663-7. · 0.88 Impact Factor
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    ABSTRACT: We evaluated loop-mediated isothermal amplification (LAMP) as a means of detecting equine herpesvirus type 1 (EHV-1) DNA directly from nasal swabs. To increase the sensitivity, we added a step in which the samples were heat-treated to the original LAMP procedure. The detection limit of the LAMP assay with heat treatment was 10 times more sensitive than the original LAMP assay even when the DNA extraction step was omitted. In addition, the LAMP assay with heat treatment was more sensitive than the original LAMP assay and the polymerase chain reaction using clinical samples. The LAMP assay with heat treatment is easy to perform and so should be applicable to the diagnosis of EHV-1 infections in clinical laboratories.
    Journal of Veterinary Medical Science 05/2011; 73(9):1225-7. · 0.88 Impact Factor
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    ABSTRACT: Using a total of 2018 fecal samples collected between 2003 and 2008 from foals with diarrhea, the molecular epidemiology of group A equine rotaviruses circulating in Japan was investigated by the reverse transcription-polymerase chain reaction (RT-PCR) typing and sequence analysis of the VP4 (P type) and VP7 (G type) genes. A total of 1149 samples showed positive reactions with RT-PCR, of which 462 samples (40.2%) were positive for G3 type, 502 samples (43.7%) were positive for G14 type, and 185 samples (16.1%) were positive for both G3 and G14 types. To examine P types, 59 G3 and 56 G14 positive samples were used. The majority of the samples (96.5%) were characterized as P[12] type. In a phylogenetic analysis, the VP4 gene of the P[12] type in Japan was found to be conserved for a long time. The VP7 sequences of the G3 type were found to be clustered in the same group as the HO-5 strain, which is a G3 strain that was isolated in 1982 in Japan. In contrast, the VP7 sequences of the G14 type, which were in circulation between 2003 and 2008, were clustered differently from those of the G14 type strains isolated in Japan in the late 1990 s. These results suggest that the VP7 gene of the G3 type has been conserved over 25 years, while the VP7 gene of the G14 type circulating between 2003 and 2008 appears to have re-emerged in or invaded Japan around 2000.
    Veterinary Microbiology 04/2011; 152(1-2):67-73. · 3.13 Impact Factor
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    ABSTRACT: It is strongly suspected that equine influenza virus (EIV) is the origin of canine influenza virus (CIV, H3N8), which was first isolated in U.S.A. in 2004, on the basis of phylogenetic analyses. Although the distribution of influenza virus sialoreceptors seems to be associated with this interspecies transmission, there have been scant data of comparison about distributions of sialoreceptors on the whole respiratory tract between horses and dogs. We examined the histological distribution of influenza virus sialoreceptors on the upper and lower respiratory tract in detail in both animals using double lectin staining with Maackia amurensis (specific for SAα2,3Gal) and Sambucus sieboldiana (specific for SAα2,6Gal). SAα2,3Gal was observed on the surface of ciliated epithelial cells in the nasal mucosa, trachea and bronchus in both animals. The results may indicate that dogs are susceptible to EIV without alteration of receptor binding specificity.
    Journal of Veterinary Medical Science 01/2011; 73(1):125-7. · 0.88 Impact Factor
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    ABSTRACT: In the past 25 years, there has been only one case of Japanese encephalitis in horses in Japan. We determined the full genome sequence of the Japanese encephalitis virus (JEV) strain JEV/eq/Tottori/2003 isolated from an afflicted horse and also analyzed its virulence in mice. The sequence analysis showed that the genome of JEV/eq/Tottori/2003 is similar to that of genotype I, a dominant genotype of JEV presently circulating in Japan. Its neurovirulence, but not neuroinvasiveness, was still as high as it was for genotype III, thus indicating the necessity for continuation of a vaccination program of horses against JEV.
    Journal of Veterinary Medical Science 01/2011; 73(6):813-6. · 0.88 Impact Factor
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    ABSTRACT: An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were naïve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses naïve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.
    Journal of Veterinary Medical Science 12/2010; 73(4):535-9. · 0.88 Impact Factor