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Background Starting infusion at a rate based on the Baxter formula is the global standard. However, with this infusion formula, an excessive amount of fluid may be stored inside the body (fluid creep), which may lead to severe respiratory disorders or abdominal compartment syndrome. The present study explored whether the Baxter formula or the Modified Brooke formula is more beneficial for resuscitation in extensively burned patients. Methods The study design was a randomized controlled trial. One group received conventional intervention based on the Baxter formula, and the other received intervention using the infusion method by the Modified Brooke formula. Thirty-nine patients were enrolled in this study by admission to each hospital for 3 years. Of the 39 cases, 3 dropped out according to the study protocol, so 36 cases were analyzed, and 17 patients in the Modified Brooke formula group and 19 in the Baxter formula group were used for the analyses in this study. Results There were no statistically significant differences between the 2 groups in the age, gender, body weight, burn severity, infusion volume required within 2 days after admission, serum creatinine, and acute kidney injury based on the KDIGO guideline within 48 h after arrival at the hospital, daily P/F ratio within 1 week after hospitalization, survival rate on the 28th day, and discharge survival rate. There were no cases of abdominal compartment syndrome during the observation period. The Modified Brooke formula group failed to achieve the target urine output corresponding to the infusion volume within 8 h after arrival, especially in ≥40% TBSA burn patients, but it did not become a clinical problem. Conclusion We were unable to conclusively demonstrate that the Modified Brooke formula blocked the occurrence of “fluid creep” in extensively burned patients. However, it was suggested that starting the initial resuscitation at half the conventional volume might contribute to an improved physiological condition in the acute phase of major burns.

This review briefly describes the virus classification, clinical signs, epidemiology, diagnosis, disinfection, and vaccines related equine group A rotavirus (RVA) infection. Equine RVA is one of the most important pathogens causing diarrhoea in foals. The main transmission route is faecal–oral, and the clinical signs are diarrhoea, fever, lethargy, and anorexia (decreased suckling). Some human RVA rapid antigen detection kits based on the principles of the immunochromatographic assay are useful for the diagnosis of equine RVA infection. The kits are used in daily clinical practice because of their rapidity and ease of handling. Equine RVA is a non-enveloped virus and is more resistant to disinfectants than enveloped viruses such as equine influenza virus and equine herpesvirus. Although amphoteric soaps and quaternary ammonium compounds are commonly used in veterinary hygiene, they are generally ineffective against equine RVA. Alcohol products, aldehydes, and chlorine- and iodine-based compounds are effective against equine RVA. Inactivated vaccines have been used for equine RVA infection in some countries. Pregnant mares are intramuscularly inoculated with a vaccine, and thus their colostrum has abundant antibodies against RVA at the time of birth. According to G and P classification defined in accordance with the VP7 and VP4 genes, respectively, the predominant equine RVAs circulating in horse populations globally are G3P[12] and G14P[12] equine RVAs, but the vaccines contain only the G3P[12] equine RVA strain. Ideally, a G14P[12] equine RVA should be added as a vaccine strain to obtain a better vaccine effect.

The antibody response in horses inoculated with 2 doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals (1 to 3 months) was evaluated with regard to the persistence of virus-neutralizing (VN) antibodies. The durations for which the geometric mean VN titers were maintained significantly higher than those before the first vaccination (P

Background Prognostic burn index (PBI) is a unique model utilized to predict mortality of burn patients in Japan. In contrast, other prediction models are rarely used in Japan, and their accuracy and predictive value are unknown. The present study aimed to compare commonly used burn prediction models and determine the appropriate model for mortality prediction in Japanese burn patients. Methods Japanese burn patients registered in the nationwide burn registry of Japanese Society for Burn Injury between April 1, 2011 and March 31, 2019 were reviewed retrospectively. The prognostic performance of PBI was compared with Baux score, revised Baux score, abbreviated burn severity index (ABSI), Ryan score and Belgian outcome in burn injury score (BOBI). The primary outcome was in-hospital mortality. Results The study included 7911 acute burn patients. The overall mortality rate was 10.7%, the median age was 52 (interquartile range, 26–72) years, and the median % total body surface area was 7% (interquartile range, 3%–17%). The areas under the receiver operating characteristic curve for PBI, Baux score, ABSI, revised Baux score, Ryan score, and BOBI were 0.940 (95% confidence interval [CI]: 0.931–0.948), 0.943 (95% CI: 0.934–0.951; p = 0.002), 0.945 (95% CI: 0.937–0.953; p = 0.058), 0.946 (95% CI: 0.937–0.953; p = 0.002), 0.859 (95% CI: 0.846–0.870; p Conclusion Although the performance of PBI was good, it was not superior to the Baux score, revised Baux score, and ABSI. These three scores have a high prognostic accuracy. Hence, they are considered as alternative burn prognostic scores in Japan. The Baux score was an optimal prognostic model for patients with burns in Japan.

A rare genotype G13P[18] group A rotavirus (RVA/Horse-tc/JPN/MK9/2019/G13P[18]) was isolated from a diarrhoeic foal for the first time in 28 years. The genotype constellation of the virus was assigned to G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11 and was the same as that of the first isolated strain, RVA/Horse-tc/GBR/L338/1991/G13P[18]. Phylogenetic analysis suggests that the virus is related to RVA/Horse-tc/GBR/L338/1991/G13P[18] and is distant from typical equine rotaviruses of the G3P[12] and G14P[12] genotypes.

An enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide for the E2 glycoprotein was developed for the serodiagnosis of Getah virus infection in horses. To identify an immunogenic epitope, a series of 20-mer peptides (n = 22) for the E2 protein was screened with pooled sera from horses infected with Getah virus. Peptide P11 (PTEEEIDMHTPPDIPDITLL) showed the strongest reaction. ELISA using P11 (E2-P11-ELISA) detected increased antibody levels in all seven experimentally infected horses and in five out of nine vaccinated horses. Out of 28 naturally infected horses, 25 were seronegative in their acute sera but turned seropositive in their convalescent sera. For the remaining three horses whose acute sera were seropositive, an endpoint method with serial dilutions detected a ≥ 4-fold increase in titer between paired sera. The concordance between E2-P11-ELISA and a virus-neutralization test in terms of seropositivity was assessed using a series of 220 horse sera, resulting in almost perfect agreement, with a kappa coefficient value of 0.865. E2-P11-ELISA had a sensitivity of 93.3% (95% CI 86.6-97.1%) and a specificity of 95.0% (95% CI 92.5-96.4%). This highly sensitive and specific E2-P11-ELISA should be useful for serodiagnosis of Getah virus infection in horses.

Equine influenza virus is an important pathogen for the horse industry because of its economic impact, and vaccination is a key control measure. Our previous work suggested that a mutation at position 144 in the hemagglutinin of Florida sublineage clade 2 viruses reduces the cross-neutralizing activity of antiserum against a former vaccine strain. To confirm this suggestion, here, we generated viruses by reverse genetics. Antibody titers against the mutated viruses were one-tenth to one-sixteenth of those against the former vaccine strain. Our findings confirm that this single amino acid substitution reduces the cross-reactivity of antiserum against this former Japanese vaccine.

In 2020, an outbreak of equine coronavirus (ECoV) infection occurred among 41 horses at a riding stable in Tokyo, Japan. This stable had 16 Thoroughbreds and 25 horses of other breeds, including Andalusians, ponies and miniature horses. Fifteen horses (37 %) showed mild clinical signs such as fever, lethargy, anorexia and diarrhoea, and they recovered within 3 days of onset. A virus neutralization test showed that all 41 horses were infected with ECoV, signifying that 26 horses (63 %) were subclinical. The results suggest that subclinical horses played an important role as spreaders. A genome sequence analysis revealed that the lengths from genes p4.7 to p12.7 or NS2 in ECoV differed from those of ECoVs detected previously, suggesting that this outbreak was caused by a virus different from those that caused previous outbreaks among draughthorses in Japan. Among 30 horses that tested positive by real-time RT-PCR, ECoV shedding periods of non-Thoroughbreds were significantly longer than those of Thoroughbreds. The difference in shedding periods may indicate that some breeds excrete ECoV longer than other breeds and can contribute to the spread of ECoV.

This study evaluated the effects of 12-hour transportation on immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Possible replication of EHV-1 and EHV-4 was monitored by real-time PCR of nasal swabs and peripheral blood mononuclear cells (PBMCs), and changes in systemic and mucosal antibodies were investigated. Six healthy Thoroughbreds with transport experience were transported in commercial trucks, repeating the same three-hour route four times. Blood samples for cortisol measurement were taken before departure and every three hours. Nasal swabs, PBMCs, nasal wash and serum samples were collected before departure, at unloading, two and six days after arrival. Cortisol concentration increased significantly after three and six hours of transport (P < 0.05), confirming acute transport stress. However, no evidence of viral replication or lytic infection was observed, and serum virus neutralization (VN) titers for EHV-1 and EHV-4 were unchanged, except for one horse that showed a four-fold decrease in titer against EHV-1 after transportation. Urea and total IgA concentration in nasal washes increased significantly after transportation (P < 0.05), while total IgA/protein ratio was unchanged. A transient, ≥4-fold decrease in VN titers for EHV-1 in nasal wash concentrates was observed in four out of six horses after transportation (geometric mean titer declined from 202 to 57, P < 0.05), suggesting suppression of VN capacity in the nasal mucosa may contribute to susceptibility to EHV-1 after transportation. VN antibodies against EHV-4 in nasal secretion were not detected at any timepoint.

An inactivated equine influenza virus (EIV) vaccine and a live equine herpesvirus type 1 (EHV-1) vaccine are usually administered concurrently to Thoroughbred racehorses in Japan. The objective of this study was to evaluate whether concurrent administration of an inactivated EIV vaccine and a live EHV-1 vaccine in Thoroughbred racehorses influences the antibody response against EIV. We compared the antibody response against EIV in horses administered both vaccines on the same day (Group A; n = 27) and the response in horses administered an inactivated EIV vaccine first and then a live EHV-1 vaccine 1-2 weeks later (Group B; n = 20). In both groups, geometric mean hemagglutination inhibition (HI) titers against A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010 increased significantly after EIV vaccination. However, the percentage of horses that showed a twofold increase or greater in HI titers against A/equine/Yokohama/aq13/2010 was significantly higher in Group B (75%) than in Group A (37%; P = .02). These results suggest that the concurrent use of an inactivated EIV vaccine and a live EHV-1 vaccine reduced the immune response against EIV to some extent, and it would be better to use these vaccines consecutively, especially for naive horses or horses whose vaccination history is incomplete.

Background Regarding blast injuries, fatal damage leading to immediate death is considered due to shock-lung, respiratory arrest, or circulatory failure induced by a neurological reflex, such as the severe vagal nerve reflex. It has not yet been determined whether a bulletproof vest protects against or aggravates shock-lung or the neurological reflexes considered to be fatal in the super-acute phase after an explosion. Purpose The purpose of this study was to clarify whether a bulletproof vest would protect the body in a pig blast model using a blast tube built at National Defense Medical College, which is the first such blast tube in Japan. Methods Seventeen pigs were divided into two groups: the body armor group (n = 6) and the non-body armor group (n = 11). Pigs underwent tracheal intubation with IV and A lines were secured to measure vital signs were checked and collect blood samples. Then, under intravenous anesthesia, the pigs were tightly fixed in the left lateral position on a table and exposed from the back neck to the upper lumbar back to the blast wave and wind with or without body armor, with the driving pressure of the blast tube set to 3.0 MPa. We checked the vital signs, collected blood samples, and observed the pigs for 3 hours after injury. Results When the surviving and dead pigs were compared, blood gas analyses revealed significant differences in PaO2, PaCO2, and pH in the super-early phase. In addition, all pigs injured by the blast wave and wind had lung hemorrhage, and 14 of 17 pigs had intra-abdominal hemorrhage with splenic injury. All 6 animals in the body armor group and 6 of the 11 animals in the control group survived for 3 hours after injury. Respiratory arrest immediately after exposure to the blast wave and wind was considered to influence the outcome in our pig model. Conclusions Respiratory arrest within several minutes after injury influenced the mortality of pigs. Body armor may have the beneficial effect in protecting against respiratory arrest immediately after an explosion.

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

In order to establish an efficient system for serological diagnosis of equine viral arteritis in Japan, we compared enzyme-linked immunosorbent assays (ELISAs) provided by two manufacturers (Nisseiken Co., Ltd., Tokyo, Japan, and VMRD Inc., Pullman, WA, U.S.A.) by testing a series of horse sera. The results revealed that 159 of 160 virus-neutralizing (VN) antibody-positive serum samples were positive in both the Nisseiken-ELISA and VMRD-ELISA. Of the VN-negative sera (n=157), 134 and 154 samples were negative in the Nisseiken-ELISA and VMRD-ELISA, respectively. Sensitivity was 99.4% for both the Nisseiken-ELISA and VMRD-ELISA. The specificity of the VMRD-ELISA (98.1%) was significantly higher than that of the Nisseiken-ELISA (85.4%, P

Background We studied a recent epizootic of Getah virus infection among pigs in the southern part of Ibaraki Prefecture and the northern part of Chiba Prefecture, Japan, focusing on its possible association with outbreaks in racehorses in 2014 and 2015. The genomic sequence of a Getah virus strain from an infected pig was analyzed to evaluate the degree of identity with the strains from horses. Results Sera were collected from pigs from September to December 2012 to 2015 in south Ibaraki (380 pigs in 29 batches), and from September to December 2010 to 2015 in north Chiba (538 pigs in 104 batches). They were examined by using a virus-neutralizing test for Getah virus. Seropositivity rates in 2012–2013 in south Ibaraki and 2010–2012 in north Chiba ranged from 0% to 1.6%. In south Ibaraki, seropositivity rates in 2014 (28.8%) and 2015 (65.0%) were significantly higher than those in the previous years (P

Baloxavir marboxil (BXM), an inhibitor of the cap-dependent endonuclease of the influenza virus polymerase acidic protein (PA), exerts an antiviral effect against influenza A virus. It has been available in Japan since March 2018. This study evaluated the antiviral efficacy of BXM against equine influenza A virus (EIV) by an experimental challenge study using horses. Six horses were experimentally inoculated with EIV, and BXM was administered to the three horses at 2 days post inoculation. Horses treated with BXM showed milder clinical signs than horses without treatment and shed less virus. These results suggest that BXM is effective against EIV. The PA gene of viruses present in the nasopharyngeal swabs collected from horses treated with BXM was sequenced. Two mutations have been detected in viruses recovered from horses treated with BXM. These mutations were the substitution of isoleucine with threonine at position 38 (PA-I38T) and that of asparagine with aspartic acid at position 675 in PA (PA-N675D). A mutated virus with PA-I38T was less susceptible to BXM than viruses with PA-N675D or without mutation. A PA-I38T mutation has also been detected in viruses recovered from humans treated with BXM and is responsible for the reduction in susceptibility to BXM. This suggests that we should not unthinkingly use BXM for the treatment of EI. BXM is likely to easily induce resistance in influenza A viruses, not only in humans but also in horses.

Background Equine herpesvirus type 1 (EHV-1) infection is a major cause of pyrexias in winter among Japanese racehorses. In 2014–2015, the Japan Racing Association (JRA) changed the EHV-1 vaccine from an inactivated vaccine to a live vaccine (both produced by Nisseiken). To evaluate the effect of changing the vaccines, the capacities of these vaccines to induce virus-neutralizing (VN) antibodies were compared, and an epizootiological investigation of EHV-1 was performed at the JRA Ritto Training Center during epizootic periods from 2010–2011 to 2016–2017. Results Three-year-old horses that received the first dose of live vaccine showed higher geometric mean (GM) VN titers (205 and 220) than those that received inactivated vaccine (83, P

Blunt vertebral artery injury (BVAI) is a well-known potentially fatal complication of cervical spine injury. The condition is reported to be associated with vertebral fractures and cervical hyperextension. However, appropriate patient screening methods remain to be elucidated. This study aimed to identify the risk factors associated with BVAI in patients with cervical spine injury. We conducted a retrospective, observational, single-centered study, including 137 patients with cervical spine injury transferred to our center from April 2007 to December 2016. Evaluation for BVAI was available in 62 patients based on magnetic resonance angiography or multi-detector computed tomography angiography. BVAI was classified using the Biffl grade. Among the 62 patients evaluated, 13 (21%) were diagnosed with BVAI. All injuries were classified as Biffl grade 2 (50%) or 4 (50%). Univariate analysis of patients with and without BVAI showed that cervical dislocation (p = 0.041) and low average hemoglobin level (p = 0.032) were associated with BVAI. On multivariate logistic regression analysis, cervical dislocation (odds ratio 1.189; 95% confidence interval 1.011–1.399, p = 0.036) remained a significant predictor of BVAI. Based on receiver operating characteristic (ROC) analysis, a dislocation > 6.7 mm was selected as the optimal cutoff value for prediction of BVAI (sensitivity and specificity, 87.5% and 71.4%, respectively). BVAI frequently occurred in combination with cervical spine dislocation, and the distance of the cervical dislocation was identified as a useful predictor of BVAI.

Antibody response in horses after accelerated-schedule Getah virus vaccination was evaluated for its potential adoption during outbreaks. One-year-old Thoroughbred horses received two doses of priming vaccinations following an accelerated schedule (accelerated group: 14-day interval, n = 30) or the conventional schedule (control group: 28-day interval, n = 30). At Day 14, both groups showed similar seropositive rates (66.7% in control group and 73.3% in accelerated group) and geometric mean (GM) virus-neutralizing titers (5.2 [95% confidence interval (CI), 3.0-8.8] in control group and 5.3 [95% CI, 3.1-8.9]). At Day 28, the controls showed a lower seropositive rate (40.0%) and GM titer (2.2 [95% CI, 1.5-3.3]), whereas these figures were significantly higher in the accelerated group, at 80.0% and 7.0 (95%CI, 4.2-11.6, P.05). The control group's antibody response peaked on Day 42, with a seropositive rate of 80.0% and GM titer of 11.3 (95% CI, 5.6-24.0). From Day 42, the accelerated group showed a faster decline in seropositive rate and GM titer than the control group. Despite the relatively short persistence of antibodies after a second vaccination, the accelerated vaccination schedule proved effective in bridging the detrimental immunity gap that is observed in conventionally vaccinated horses, suggesting the potential usefulness of this accelerated vaccination schedule as an emergency control measure.

Background: Patient-reported outcomes (PROs) of quality of life (QOL) during chemotherapy involving lung cancer are very important for the medical staffs. Patients’ satisfaction and healthy changes were evaluated by the patient-self assessment. Materials and Methods: From July 2007 to April 2008, a total of 19 patients received chemotherapy. The QOL data were collected by using the QOL questionnaire for cancer patients treated with anticancer drugs (QOL-ACD) and the anti-aging QOL assessment (AA-QOL). The AA-QOL contained 51 items: 30 of physical and 21 of mental symptoms of the elderly and the aging population. The patients replied to the questions at two different times, i.e., at pre-chemotherapy (baseline) and at post-chemotherapy (2 weeks after the chemotherapy). Results: Regarding the hematological toxicities, for the grade 3/4 toxicities, there were 12 neutropenia (12/19, 63.2%) and 3 thrombocytopenia (3/19, 15.8%). For the grade 3 febrile neutropenia, there were 5 cases (5/19, 26.3%). Regarding the non-hematological toxicities, there was no grade 3 and grade 4 toxicities. Based on the outcomes of the QOL-ACD, the three items (“physical condition”, “social attitude”, and “overall QOL”) at post-chemotherapy became significantly worse compared to the baseline. Regarding the outcomes of the AA-QOL, 4 items of physical symptoms (“thirst”, “anorexia”, “early satiety”, and “diarrhea”) became significantly worse compared to the baseline. Regarding the mental symptoms, 2 items (“nothing to look forward in life” and “a sense of uselessness”) became significantly worse compared to the baseline. Conclusion: Regarding the PROs of the QOL during the chemotherapy term, both the physical and mental symptoms had become worse. To clarify the changes in the QOL during chemotherapy is very important for multidisciplinary teamwork, which should play the role of providing the appropriate cares and treatment as patient-support.

We previously reported that horse antiserum against the Japanese equine influenza vaccine virus, A/equine/La Plata/1993 (LP93) exhibited reduced cross-neutralization against some Florida sublineage Clade (Fc) 2 viruses, for example, A/equine/Carlow/2011 (CL11). As a result, Japanese vaccine manufacturers will replace LP93 with A/equine/Yokohama/aq13/2010 (Y10, Fc2). To assess the benefit of updating the vaccine, five horses vaccinated with inactivated Y10 vaccine and five vaccinated with inactivated LP93 were challenged by exposure to a nebulized aerosol of CL11. The durations of pyrexia (≥38.5°C) and other adverse clinical symptoms experienced by the Y10 group were significantly shorter than those of the LP93 group.

The molecular epidemiology of equine group A rotaviruses (RVAs) in Ireland from 2011 to 2015 was investigated. Of 438 diagnostic specimens submitted from foals with enteric disease, 102 (23.3%) were positive for RVA using an immunochromatographic assay. G genotypes were determined for 76 equine RVAs, of which 68 (89.5%) were G3 and eight (10.5%) were G14. Of 18 RVAs (12 G3 and six G14) characterised by P genotyping, all were P[12]. G3P[12] and G14P[12] were the most prevalent genotypes of RVA in foals in Ireland, similar to other countries and consistent with previous studies in Ireland from 1999 to 2005. Phylogenetic analysis showed that G3P[12] and G14P[12] RVAs were related to equine RVAs recently detected in Europe, Brazil and South Africa, and to the vaccine strain H-2.

A rapid and sensitive diagnostic method is needed to help prevent the spread of equine influenza virus. The cobas Influenza A/B & RSV test for the cobas Liat system (Roche Diagnostics) is based on real-time reverse transcription polymerase chain reaction and is designed to broadly detect influenza A virus RNA within 20 minutes. It detected a broad range of equine influenza virus strains, and detected equine influenza virus RNA from nasal swabs of infected horses at the same level as real-time reverse transcription polymerase chain reaction, although it returned some invalid results (7.7%). This suggests that cobas Influenza A/B & RSV test is highly sensitive to equine influenza virus, but should be improved to reduce the rate of invalid results.

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.

We sequenced the eight segments of influenza A virus strains A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010, which are strains of the Florida sublineage clades 1 and 2 of the H3N8 subtype equine influenza virus. These strains have been used as vaccine strains in Japan since 2016 in accordance with World Organization for Animal Health (OIE) recommendations.

Equine coronavirus has been responsible for several outbreaks of disease in the United States and Japan. Only one complete genome sequence (NC99 isolated in the US) had been reported for this pathogenic RNA virus. Here, we report the complete genome sequences of three equine coronaviruses isolated in 2009 and 2012 in Japan. The genome sequences of Tokachi09, Obihiro12-1 and Obihiro12-2 were 30,782, 30,916 and 30,916 nucleotides in length, respectively, excluding the 3’-poly (A) tails. All three isolates were genetically similar to NC99 (98.2–98.7 %), but deletions and insertions were observed in the genes nsp3 of ORF1a, NS2 and p4.7. Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2565-1) contains supplementary material, which is available to authorized users.

Equine group A rotavirus (RVA) G3P[12] and G14P[12] strains cause gastroenteritis in foals worldwide. Both of these strains have been co-circulating in Japan since G14P[12] strains emerged in the late 1990s. Although it is important to comprehensively understand the evolution of RVA strains, whole-genome sequence data on recent equine RVA strains in Japan are lacking. Therefore, in this study, whole-genome analysis of 23 equine RVA isolates from the late 1990s and 2009-2010 and the vaccine strain RVA/Horse-tc/JPN/HO-5/1982/G3P[12] (HO-5) was performed. The G3 strains, including strain HO-5, shared a G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 genotype constellation, and all of their 11 gene segments were highly conserved, regardless of the year of isolation. G14 strains also exhibited an identical genotype constellation (G14-P[12]-I2-R2-C2-M3-A10-N2-T3-E2-H7), but, phylogenetically, segregated into two lineages within the VP7-G14 and NSP4-E2 genotypes. G14 strains were closely related to G3 strains in their VP4, VP1-3, NSP1-3 and NSP5 gene segments. Interestingly, the NSP4 gene of all G3 and G14 strains isolated in the late 1990s branched into a bovine-RVA-like NSP4 gene cluster. These results indicate that, apart from VP7, VP6, and NSP4 genes, the Japanese equine RVA strains share a highly conserved genetic backbone, and that strains possessing a bovine-RVA-like NSP4 gene were predominant in the late 1990s in Japan.

Equine influenza (EI) is a respiratory disease caused by equine influenza A virus (EIV, H3N8) infection. Rapid diagnosis is essential to limit the disease spread. We previously reported that some rapid antigen detection (RAD) tests are fit for diagnosing EI although their sensitivity is not optimal. Here, we evaluated the performance of the newly developed RAD test using silver amplification immunochromatography (Quick Chaser Auto Flu A, B: QCA) to diagnose EI. The detection limits of QCA for EIVs were five-fold lower than the conventional RAD tests. The duration of virus antigen detection in the infected horses was longer than the conventional RAD tests. We conclude that QCA could be a valuable diagnostic method for EI.

Equine influenza (EI) vaccine has been widely used. However, the causative EI virus (H3N8) undergoes continuous antigenic drift, and the vaccine strains must be periodically reviewed and if necessary, updated to maintain vaccine efficacy against circulating viruses. In 2016, the Japanese vaccine was updated by replacing the old viruses with the Florida sub-lineage Clade (Fc) 2 virus, A/equine/Yokohama/aq13/2010 (Y10). We investigated the virus neutralization (VN) antibody response to Fc2 viruses currently circulating in Europe, after booster or primary immunization with the new vaccine. These European viruses have the amino acid substitution A144V or I179V of the hemagglutinin. In horses that had previously received a primary course and bi-annual boosters with the old vaccine booster, immunization with the updated vaccine increased the VN antibody levels against the European Fc2 viruses as well as Y10. There were no significant differences in the VN titers against Y10 and the Fc2 viruses with A144V or I179V substitution in horses that had received a primary course of the updated vaccine. However, a mixed primary course where the first dose was the old vaccine and the second dose was the updated vaccine, reduced VN titers against the European viruses compared to that against Y10. In summary, the new vaccine affords horses protective level of VN titers against the Fc2 viruses carrying A144V or I179V substitution, but our results suggest that the combination of the old and new vaccines for primary immunization would not be optimum.

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.

Objective Automated external defibrillators (AEDs) have been rented in various places in Japan. When rental AEDs are placed in locations where the probability of sudden cardiac arrest is high and permanent placement of AEDs is difficult, the possibility of improving survival rates might increase. In this preliminary study, we investigated how, when, and where rental AEDs have been used in Japan to clarify their characteristics when used in actual situations and to facilitate better usage in the future. Methods We investigated the total number of AEDs rented, the duration of rental of each AED, the total number of AEDs rented monthly, the rental sites, the frequency and location of use, the number of defibrillations, and the time to defibrillation success for devices rented between January 2008 and December 2010 by a single company in Japan. Results The number of AEDs rented annually was 590 at 391 sites in 2008, 767 at 465 sites in 2009, and 847 at 477 sites in 2010. More AEDs were rented during the summer. The devices were actually used on 17 individuals, of whom 2 individuals (at a beach and a marathon) underwent defibrillation, and 1 individual (at a marathon) survived. Conclusion Rental AEDs can play an important role in emergency cases occurring during seasonal and temporary outdoor events. The provision of rental AEDs in locations where permanent AEDs would be unfeasible may offer a useful strategy for efficiently improving survival rates in the future.

Equine herpesvirus 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 susceptible 3-year-old horses with low antibody levels to EHV-1 antigens. However, because the protective effect was not satisfactory, in 2009-2010 the vaccination program was altered to target all 3-year-old horses. To evaluate the vaccine's efficacy, we investigated the number of horses with pyrexia 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 changes in the program. The mean (± standard deviation [SD]) estimated numbers of horses infected with EHV-1 or EHV-4 or both, among pyretic horses from 1999-2000 to 2008-2009 were 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 (±SD) 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 suggest 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.

Getah virus causes fever, skin eruptions, and limb edema in horses. For a high-throughput and time-saving method for serodiagnosis, we explored immunogenic antigens of Getah virus, and established an enzyme-linked immunosorbent assay (ELISA) using a recombinant protein. Western blot analysis using sera from infected horses showed strong reaction with viral antigens around 46 kDa corresponding to E1 or E2 glycoproteins. Recombinant E2 (rE2) protein reacted more strongly with infected horse sera than did rE1 protein in both Western blotting and ELISA. In ELISA using rE2 protein (rE2-ELISA), for all horses experimentally infected with Getah virus (n = 7), optical density (OD) exceeded the cutoff value at 14 days post-infection. ODs in five of nine vaccinated horses also slightly exceeded the cutoff value after vaccination. Among naturally infected horses (n = 28), 24 were seronegative in the acute sera, which turned seropositive in the convalescent sera. For the four horses seropositive in the acute sera, an endpoint method with serial dilutions of paired sera detected a ≥4-fold increase in titer. In conclusion, we established rE2-ELISA that could detect horse antibodies against Getah virus after experimental and natural infections; this should be useful in the diagnosis and surveillance of Getah virus infection.

Background This study investigated the relationship between CHADS2 scores and the rate of antithrombotic drug use and clinical outcomes in patients with an initial cardioembolic stroke who had nonvalvular atrial fibrillation (NVAF). Methods In 234 patients (135 men and 99 women; mean age [±SD] 76 ± 11 years) with initial cardiogenic cerebral embolism with NVAF who were admitted to our hospital between April 2007 and March 2011, the CHADS2 score, use of warfarin, and clinical outcomes were retrospectively investigated. Results CHADS2 scores were as follows: 0 points, n = 21 (9%); 1 point, n = 72 (31%); 2 points, n = 92 (39%); 3 points, n = 47 (20%); and 4 points, n = 2 (1%). The overall warfarin use rate was low (14.1%; n = 33), and it was significantly (P = .023) lower for paroxysmal atrial fibrillation (8%) than for chronic atrial fibrillation (18.5%). The clinical outcomes evaluated by the modified Rankin Scale (mRS) score after 3 months were: CHADS2 score 0 points, mRS 0 to 2 (81%) and 3 to 6 (19%); 1 point, mRS 0 to 2 (46%) and 3 to 6 (54%); 2 points, mRS 0 to 2 (46%) and 3 to 6 (54%); and ≥3 points, mRS 0 to 2 (29%) and 3 to 6 (71%). The clinical outcome worsened as the CHADS2 score increased (P = .002). Logistic regression analysis revealed that being ≥75 years of age and having a high National Institutes of Health Stroke Scale (NIHSS) score on admission were related to a poor outcome (P < .001). Conclusions The overall warfarin use rate was low in initial cardioembolic stroke patients with NVAF. Clinical outcomes deteriorated with increases in the CHADS2 score, age ≥75 years, and NIHSS score on admission were related to a poor clinical outcome.

Arbekacin (ABK) is an aminoglycoside and widely used in Japan for treatment of patients infected with methicillin-resistant Staphylococcus aureus (MRSA). Although, ABK has concentration-dependent antibacterial activity, the peak serum concentration (C (peak)) of ABK has not yet been fully investigated as an indicator of the efficacy of ABK. The present study was conducted in patients admitted to hospitals affiliated with the ABK Dose Finding Study Group, between October 2008 and June 2011, who had pneumonia or sepsis, the cause of which was identified or suspected to be MRSA. The initial target C (peak) was set at 15-20 μg/mL and therapeutic drug monitoring was conducted. Then the relationship between serum concentration and efficacy/safety of ABK was prospectively examined to obtain sufficient clinical efficacy. In total, 89 patients from 11 clinical sites in Japan were enrolled and 29 of these patients were subjected to efficacy analysis. The mean initial dose and C (peak) were 306.9 mg/day and 16.2 μg/mL, respectively. The efficacy rate was 95 % (19/20 patients) at 5-6 mg/kg or higher, 87.5 % (7/8) for sepsis and 90.5 % (19/21) for pneumonia, and the overall efficacy rate was 89.7 % (26/29). There was no increase in the incidence of adverse events. In conclusion, we recommend the initial dose of ABK at 5-6 mg/kg or higher and the dosage regimen should be adjusted to achieve C (peak) at 10-15 μg/mL or higher in the treatment of patients with pneumonia or sepsis caused by MRSA. This strategy would surely achieve low incidence of adverse events while obtaining high clinical efficacy.

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.

Background As we reported previously, Getah virus infection occurred in horses at the Miho training center of the Japan Racing Association in 2014. This was the first outbreak after a 31-year absence in Japan. Here, we report a recurrent outbreak of Getah virus infection in 2015, sequential to the 2014 one at the same site, and we summarize its epizootiological aspects to estimate the risk of further outbreaks in upcoming years. Results The outbreak occurred from mid-August to late October 2015, affecting 30 racehorses with a prevalence of 1.5 % of the whole population (1992 horses). Twenty-seven (90.0 %) of the 30 affected horses were 2-year-olds, and the prevalence in 2-year-olds (27/613 [4.4 %]) was significantly higher than that in horses aged 3 years or older (3/1379 [0.2 %], P

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 0.20 nmol/L. Horses treated with a single IV dose of peramivir (3,000 mg/600 mL/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.

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.

It has been suggested that soil-thawing and snow-melting are critical triggers for vigorous emissions of nitrous oxide (N 2 O) from soils in cold regions. However, because soil freezing is affected by air temperature and snow cover, accurate predictions that estimate subsequent emissions of this important greenhouse gas are difficult to make. In this study, we measured in situ soil gas N 2 O and oxygen (O 2 ) concentrations at two experimental sites in northern Japan over the period of a year, from November 2008 to October 2009, to clarify the factors stimulating N 2 O production in soil at low temperatures. The sites were N-fertilized bare arable lands with different soil frost depths and snowmelt rates, according to the snow cover management imposed. Winter-to-spring net N 2 O fluxes, ranging from −0.10 to 1.95 kg N 2 O–N ha −1 , were positively correlated with the annual maximum soil frost depth (ranging from 0.03 to 0.41 m; r = 0.951***). In the plots with deeper maximum soil frost, winter-to-spring N 2 O fluxes represented 58% to 85% of the annual values. Soil N 2 O production was stimulated when the soil frost depth was greater than 0.15 m or the daily mean soil temperature at 0.05-m depth was below −2.0 °C. In the soil with the greatest frost depth, soil gas N 2 O concentrations at the depth of 0.10 m peaked at 46 ppm when soil gas O 2 concentrations fell down to 0.12 m 3 m −3 under soil temperature below 0.0 °C. Snowmelt acceleration had no stimulating effect on N 2 O production in the soil during the winter-to-spring period.

We investigated the pharmacokinetics of penciclovir after oral administration of its prodrug famciclovir to horses. Following an oral dose of famciclovir at 20 mg/kg, maximum plasma concentrations of penciclovir occurred between 0.75 and 1.5 hr (mean 0.94 + or - 0.38 hr) after dosing and were in the range 2.22 to 3.56 microg/ml (mean 2.87 + or - 0.61 microg/ml). The concentrations of penciclovir declined in a biphasic manner after the peak concentration was attained. The mean half-life of the rapid elimination phase was 1.73 + or - 0.34 hr whereas that of the slow elimination phase was 34.34 + or - 13.93 hr. These pharmacokinetic profiles observed were similar to those of another antiherpesvirus drug, acyclovir, previously reported in horses following oral dosing of its prodrug valacyclovir.

An equine herpesvirus type 1 (EHV-1) mutant, DeltagE, defective in glycoprotein E (gE) was evaluated as a modified live virus (MLV) vaccine. Colostrum-deprived Thoroughbred foals inoculated intranasally (i.n.) or intramuscularly (i.m.) with DeltagE did not exhibit any clinical signs of respiratory disease except for a mild nasal discharge in 1 i.n. inoculated foal on Days 1 and 3 post-infection. In contrast, the intranasal inoculation of foals with the revertant of DeltagE resulted in biphasic pyrexia, mucopurulent nasal discharge and swelling of submandibular lymph nodes. These results indicated that gE plays an important role as regards EHV-1 virulence in horses. The ability of DeltagE to protect against wild type EHV-1 challenge infection was assessed using i.m. vaccinated foals. Foals inoculated twice i.m. with 10(5) or 10(6) plaque-forming units (pfu) of DeltagE at an interval of 3 weeks exhibited no clinical evidence of local inflammation, respiratory disease or deleterious systemic responses. Remarkable increases in SN antibody titer to EHV-1 were observed in all vaccinated foals after the 2nd inoculation with DeltagE. Following a wild type EHV-1 challenge infection, vaccinated foals showed milder clinical symptoms than foals vaccinated with a placebo. Specifically, 1 of 3 foals vaccinated with 10(6) pfu of DeltagE exhibited no clinical symptoms other than a mild nasal discharge for 1 day. Additionally, the virus load of nasal shedding and viremia were reduced by vaccination. These results suggest that DeltagE would be a good candidate as an MLV vaccine.

Background Equine influenza (EI) is a highly contagious disease caused by viruses of the H3N8 subtype. The rapid diagnosis of EI is essential to reduce the disease spread. Many rapid antigen detection (RAD) tests for diagnosing human influenza are available, but their ability to diagnose EI has not been systematically evaluated. Objectives The aim of this study was to compare the performance of 22 RAD tests in the diagnosis of EI. Methods The 22 RAD tests were performed on fivefold serial dilutions of EI virus to determine their detection limits. The four most sensitive RAD tests (ImmunoAce Flu, BD Flu examan, Quick chaser Flu A, B and ESPLINE Influenza A&B-N) were further evaluated using nasopharyngeal samples collected from experimentally infected and naturally infected horses. The results were compared to those obtained using molecular tests. Results The detection limits of the 22 RAD tests varied hugely. Even the four RAD tests showing the best sensitivity were 125-fold less sensitive than the molecular techniques. The duration of virus detection in the experimentally infected horses was shorter using the RAD tests than using the molecular techniques. The RAD tests detected between 27% and 73% of real-time RT-PCR-positive samples from naturally infected horses. Conclusions The study demonstrated the importance of choosing the right RAD tests as only three of 22 were fit for diagnosing EI. It was also indicated that even RAD tests with the highest sensitivity serve only as an adjunct to molecular tests because of the potential for false-negative results.

Background Equine coronavirus (ECoV) is considered to be a diarrheic pathogen in foals. In central Kentucky in the United States, it has been shown that approximately 30 % of thoroughbred foals are infected with ECoV and thus it is considered widely prevalent. In contrast, the epidemiology of ECoV and its relationship to diarrhea in foals are poorly understood in Japan. We investigated ECoV in rectal swabs collected from thoroughbred foals in Japan. Results We collected 337 rectal swabs from 307 diarrheic foals in the Hidaka district of Hokkaido, the largest thoroughbred horse breeding region in Japan, between 2012 and 2014. In addition, 120 rectal swabs were collected from 120 healthy foals in 2012. These samples were tested by reverse transcription loop-mediated isothermal amplification and a real-time reverse transcription-polymerase chain reaction. All samples collected from diarrheic foals were negative, and only three samples (2.5 %) collected from healthy foals were positive for ECoV. Compared with central Kentucky, ECoV is not prevalent among thoroughbred foals in the Hidaka district of Hokkaido. Conclusion ECoV is not prevalent and was not related to diarrhea in thoroughbred foals in the Hidaka district of Hokkaido between 2012 and 2014.

A Getah virus strain isolated during an outbreak in racehorses in Japan in 2014 (14-I-605) was compared with the vaccine strain isolated in 1978 (MI-110). A comparison of the genome sequences of these strains revealed seven amino acid substitutions in non-structural protein 3, and one or two substitutions in each of other non-structural proteins. In contrast, the structural proteins were highly conserved (99.8–99.9 % amino acid sequence identity). Horse antisera raised against the MI-110 strain showed similar virus-neutralization titers against both MI-110 and 14-I-605 strains (512 and 256, respectively). Therefore, antigenic mutation was probably not a direct cause of the outbreak that occurred in 2014.