Experimental challenge of horses after prime-boost immunization with a modified live equid alphaherpesvirus 1 vaccine administered by two different routes.

The immune response and protective efficacy of a modified equid alphaherpesvirus 1 (EHV-1) vaccine administered by two different routes were tested in horses. Horses that received intramuscular (IM) priming and an intranasal (IN) booster with a 28-day interval (IM-IN group [n = 6]), IN priming and IM booster (IN-IM group [n = 5]), or no vaccination (control group [n = 6]) were challenged with EHV-1 strain 10-I-224 28 days after the second vaccination. Both vaccinated groups had significantly higher serum virus-neutralizing titers than the control group, with increased levels of serum IgGa, IgGb, and IgA antibodies (p < 0.05). The nasal antibody response was dominated by the IgGa and IgGb subclasses in both vaccinated groups, with no IgA antibody response. After challenge infection, three of six control horses were pyretic for 1-4 days post-inoculation (dpi), whereas none in the vaccinated groups were pyretic during this period. The number of horses that were pyretic at 5-10 dpi was 4 out of 6 for the controls, 3 out of 6 for the IM-IN group, and 2 out of 5 for the IN-IM group. Nasal virus replication in the IN-IM group (3-4 dpi) and IM-IN group (3 dpi) was significantly lower than in the control group (p < 0.05). All of the control horses showed viremia, whereas two horses in the IM-IN group and one in the IN-IM group did not. In conclusion, although IM-IN or IN-IM vaccination did not elicit a mucosal IgA response, it provided partial protection at a level similar to that of the conventional program, likely due to systemic antibodies and mucosal IgG subclass responses.

Surveillance of Getah virus in mosquitoes and racehorses from 2016 to 2019 at a training center in Ibaraki Prefecture, Japan, a site of several previous Getah virus outbreaks.

Mosquitoes and EDTA-treated blood samples from febrile racehorses were investigated for Getah virus infection from 2016 to 2019 at the Miho Training Center, where several outbreaks of Getah virus have occurred. We collected 5557 mosquitoes and 331 blood samples from febrile horses in this study. The most frequently captured mosquito species was Culex tritaeniorhynchus (51.9%), followed by Aedes vexans nipponii (14.2%) and Anopheles sinensis (11.2%). Getah virus was detected in mosquitoes (Aedes vexans nipponii) in 2016 (strain 16-0810-26) but not in 2017-2019. Six of 74 febrile horses in 2016 and one of 69 in 2019 tested positive for Getah virus; none of the horses tested positive in 2017 or 2018. Phylogenetic and sequence analysis showed that strain 16-0810-26 was closely related to strains that had been isolated from horses and a pig around the training center in 2014-2016 but have not been detected in samples collected at the training center since 2017. In contrast, the strain isolated from the infected horse in 2019 (19-I-703) was genetically distinct from the strains isolated from horses and a pig in 2014-2016 and was more closely related to a strain isolated in 1978 at the training center. The source of strain 19-I-703 is unclear, but the virus was not detected in other horses sampled in 2019. In summary, we found that the distribution of mosquito species present at the training center had not changed significantly since 1979, and although a small outbreak of Getah virus infection occurred among horses at the training center in 2016, limited Getah virus activity was detected in mosquitoes and horses at the training center from 2017 to 2019.

Antigenic comparison of H3N8 equine influenza viruses belonging to Florida sublineage clade 1 between vaccine strains and North American strains isolated in 2021-2022.

Equine influenza virus strains of Florida sublineage clade 1 (Fc1) have been circulating in North America. In this study, virus neutralization assays were performed to evaluate antigenic differences between Fc1 vaccine strains and North American Fc1 strains isolated in 2021-2022, using equine antisera against A/equine/South Africa/4/2003 (a vaccine strain recommended by the World Organisation for Animal Health) and A/equine/Ibaraki/1/2007 (a Japanese vaccine strain). Antibody titers against four North American Fc1 strains isolated in 2021-2022 were comparable to those against the homologous vaccine strains. These results suggest that current Fc1 vaccine strains are effective against North American strains from 2021-2022.

Prevalence of serum and salivary virus-neutralizing antibodies against equine coronavirus in four riding stables in Japan.

To assess the prevalence of equine coronavirus infection in riding horses, virus-neutralizing tests were performed on serum and saliva samples collected at four facilities in Japan. Seropositivity rates ranged from 79.2% to 94.6%, suggesting widespread circulation of the virus in these populations. Antibody prevalence in saliva samples from two facilities that had experienced outbreaks in the previous year (67.6% and 71.4%) was significantly higher than at the other facilities without reported outbreaks (41.7% and 45.2%, P<0.05). The presence of salivary antibodies in a high proportion of horses is therefore suggestive of recent exposure to the virus.

Distribution of equine coronavirus RNA in the intestinal and respiratory tracts of experimentally infected horses.

Equine coronavirus (ECoV) causes pyrexia, anorexia, lethargy, and sometimes diarrhoea. Infected horses excrete the virus in their faeces, and ECoV is also detected in nasal samples from febrile horses. However, details about ECoV infection sites in the intestinal and respiratory tracts are lacking. To identify the ECoV infection sites in the intestinal and respiratory tracts, we performed an experimental infection study and analysed intestinal and respiratory samples collected from four infected horses at 3, 5, 7, and 14 days post-inoculation (dpi) by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and in situ hybridization (ISH). Two horses became febrile, but the other two did not. None of the horses had diarrhoea or respiratory signs, and severe cases were not observed in this study. None of the horses showed obvious abnormalities in their intestinal or respiratory tracts. Real-time RT-PCR and ISH showed that ECoV RNA was present throughout the intestinal tract, and ECoV-positive cells were mainly detected on the surface of the intestine. In one horse showing viremia at 3 dpi, ECoV RNA was detected in the lung by real-time RT-PCR, but not by ISH. This suggests that the lung cells themselves were not infected with ECoV and that real-time RT-PCR detected viremia in the lung. The other three horses were positive for ECoV RNA in nasal swabs but were negative in the trachea and lung by real-time RT-PCR and ISH. This study suggests that ECoV broadly infects the intestinal tract and is less likely to infect the respiratory tract.

Persistence of virus-neutralizing antibodies in horses inoculated with two doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals.

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<0.05) were up to 5 months in horses that received the vaccination with a 1-month interval (n=17) and 7 months for those that received it with a 2-month (n=17) or 3-month interval (n=14 or 17). The vaccination program with the 2-month interval was the most effective in maintaining VN antibodies for a long duration with the smallest gap of antibody decline between the first and second vaccinations.

Antimicrobial resistance profiles and phylogenetic groups of Escherichia coli isolated from healthy Thoroughbred racehorses in Japan.

In this study, we investigated the occurrence of antimicrobial resistance in commensal Escherichia coli isolated from healthy Thoroughbred (TB) racehorses in Japan. A total of 212 fecal samples were individually collected from TB racehorses from March 2017 to August 2018 at Japan Racing Association training centers. E. coli was isolated by using selective agar media, deoxycholate-hydrogen sulfide-lactose (DHL) and eosin methylene blue (EMB). A total of 417 E. coli isolates were examined against 10 antimicrobial agents by using the broth microdilution method. The 417 E. coli isolates were phylogenetically grouped using a multiplex polymerase chain reaction. The highest proportion of resistance was observed for streptomycin (30.9%, 129/417) followed by ampicillin (19.4%, 81/417), trimethoprim (15.8%, 66/417), tetracycline (8.4%, 35/417), chloramphenicol (2.6%, 11/417), kanamycin (1.2%, 5/417), nalidixic acid (0.5%, 2/417), cefazolin (0.2%, 1/417), colistin (0.2%, 1/417), and gentamycin (0%). Multidrug-resistant (MDR) E. coli was detected in 7.9% (33/417) of isolates. The proportions of resistance against ampicillin, streptomycin, kanamycin, and chloramphenicol and of multidrug-resistant phenotypes in E. coli belonging to phylogenetic group B2 were significantly higher than those of other groups. This study clarified the distribution of antimicrobial-resistant (AMR) E. coli in Japanese racehorses. A continuous monitoring program for antimicrobial resistance is required to control the spread of AMR bacteria in racehorses.

A retrospective comparison of induction with thiopental/guaifenesin and propofol/ketamine in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine during arthroscopic surgery.

This study compares clinical characteristics between induction with thiopental/guaifenesin and propofol/ketamine in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine. Clinical records of 214 horses that underwent arthroscopic surgery between 2015 and 2016 were retrospectively retrieved. Horses were premedicated with medetomidine and midazolam to sedate at the adequate level for smooth induction, and then induced with either thiopental (4.0 mg/kg) and guaifenesin (100 mg/kg) in Group TG (n=91) or propofol (1.0 mg/kg) and ketamine (1.0 mg/kg) in Group PK (n=123). Anesthesia was maintained using sevoflurane with constant rate infusion of medetomidine. Quality of induction/recovery, sevoflurane requirement, cardiovascular function and recovery characteristics were evaluated. Anesthetic induction scores (median, range) for Group TG (5, 2-5) and Group PK (5, 2-5) were not significantly different. There were no significant differences in end-tidal sevoflurane concentration (mean ± standard deviation) between Group TG and Group PK (both 2.4 ± 0.2%). Dobutamine infusion rate (µg/kg/min) required for keeping mean arterial blood pressure (MAP) above 70 mmHg in Group PK (0.43, 0.10-1.40) was significantly lower than in Group TG (0.67, 0.08-1.56). Recovery score in Group PK (5, 2-5) was significantly higher than in Group TG (4, 2-5). Both propofol/ketamine and thiopental/guaifenesin provided a smooth induction of anesthesia. Moreover, induction with propofol/ketamine resulted in lower dobutamine requirements for keeping MAP above 70 mmHg during maintenance, and better quality of recovery. Induction with propofol/ketamine would be preferable to thiopental/guaifenesin in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine during arthroscopic surgery.

Extended-spectrum ß-lactamase-producing Escherichia coli isolated from healthy Thoroughbred racehorses in Japan.

Extended-spectrum ß-lactamase-producing Escherichia coli (ESBLEC) have become a major health concern in both human and veterinary medicine. These bacteria could become a critical problem in equine medicine due to the limited number of antimicrobial drugs available. However, there are no previous reports of ESBLEC isolated from horses in Japan. The objectives of this study were to investigate the occurrence of ESBLEC isolated from feces in healthy Thoroughbred racehorses in Japan. Feces samples were collected from 147 healthy Thoroughbred racehorses by equine veterinarians at the Japan Racing Association (103 from Miho Training Center and 44 from Ritto Training Center) between March 2017 and April 2018. Samples were screened for ESBLECs using MacConkey agar supplemented with 1 µg/ml cefotaxime. Detection of ESBL genes was performed by PCR and confirmed by DNA sequencing. Horizontal transmission was demonstrated by conjugation assay. In this study, 24 ESBLECs were isolated from twelve horse feces samples (8.2%). All ESBLECs harbored blaCTX-M-2, and both blaTEM-1 and blaCTX-M-2 were detected in nine isolates (37.5%). ESBLECs showed resistance to all ß-lactam antibiotics (100%) tested, followed by trimethoprim (66.7%), streptomycin (62.5%), tetracycline (25.0%), and oxytetracycline (25.0%). Horizontal transmission was successfully demonstrated by conjugation assay in eight of 13 isolates, and blaCTX-M-2 was detected by PCR in all transconjugants. This study showed that racehorses in Japan are potential reservoirs of ESBLECs.

Comparison of oxidative stress under different propofol administration protocols in Thoroughbred racehorses by bOS and bAP assessment.

It is desirable to reduce surgery-induced oxidative stress (OS) because it can cause immune suppression and delayed wound healing. Propofol is known to have antioxidant potential and to reduce OS in humans, but there have been no studies of this issue in horses. This study was conducted to evaluate OS under three different propofol administration protocols in Thoroughbred racehorses undergoing arthroscopic surgery with sevoflurane anesthesia. Blood oxidative stress (bOS) and blood antioxidant power (bAP) were used as OS biomarkers. Both bOS and bAP significantly decreased after surgery in all groups, but no differences in these reductions were found among them. Different propofol administration protocols with sevoflurane anesthesia did not cause a difference in OS in Thoroughbred racehorses that underwent arthroscopic surgery.

Serosurveillance of equine coronavirus infection among Thoroughbreds in Japan.

BACKGROUND: Equine coronavirus (ECoV) causes fever, lethargy, anorexia and gastrointestinal signs in horses. There has been limited information about the prevalence and seasonality of ECoV among Thoroughbreds in Japan. OBJECTIVES: To understand the epidemiology and to evaluate the potential risk of ECoV infection to the horse industry in Japan. STUDY DESIGN: Longitudinal. METHODS: The virus-neutralisation (VN) test was performed using sera collected three times a year at 4 months intervals from 161 yearlings and at 6-7 months intervals from 181 active racehorses in Japan in 2017-2018, 2018-2019 and 2019-2020. VN titre ≥1:8 was defined as seropositive, and ≥4-fold increase in titres between paired sera was regarded as indicative of infection. RESULTS: The VN test showed that 44.1% (71/161) of yearlings were seropositive in August, when they first entered the yearling farm. The infection rate was significantly higher between August and December (60.9%, 98/161) than between December and the following April (5.6%, 9/161; p = 0.002). Among the racehorses, it was significantly higher between November and the following May (15.5%, 28/181) than between the preceding April/May and November (0%; p = 0.02). The morbidity rates during the estimated periods of viral exposure were 39.2% in the yearlings and 4% in the racehorses. No horses showed any severe clinical signs. MAIN LIMITATIONS: Clinical records did not cover the period during horses' absence from the training centre. CONCLUSIONS: ECoV was substantially prevalent in Thoroughbred yearlings and racehorses in Japan, and there was a difference in epizootic pattern between these populations in terms of predominant periods of infection. ECoV infection was considered to be responsible for some of the pyretic cases in the yearlings. However, no diseased horses were severely affected in either population, suggesting that the potential risk of ECoV infection to the horse industry in Japan is low.

Comparison of 4 agar gel immunodiffusion kits for serologic detection of equine infectious anemia virus antibodies.

Using 85 sera collected from horses that had been experimentally infected with equine infectious anemia virus (EIAV) and 200 field sera collected from racehorses in Japan, we compared 4 agar gel immunodiffusion (AGID) kits for serologic detection of EIAV antibodies from Idexx, VMRD, IDvet, and the National Engineering Research Center of Veterinary Biologics, China (NECVB). The positive control lines were sufficiently clear in all kits for evaluation to be made, with slight differences in sharpness: NECVB was the sharpest, followed by VMRD, IDvet, and Idexx. The test results for all 285 samples agreed among the 4 kits, with 62 positives and 223 negatives. The sensitivities and specificities of VMRD, IDvet, and NECVB compared with the Idexx kit were 100%, and the kappa coefficient values between the kits were 1.0 for all combinations. We concluded that the testing capacity of these 4 kits was virtually identical.

Performance of a microfluidic immunofluorescence assay kit for equine influenza virus antigen detection.

Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription polymerase chain reaction as a reference assay, the microfluidic immunofluorescence assay kit showed a sensitivity of 60.7% when evaluating nasopharyngeal swab samples of three horses experimentally infected with EIV. Comparing with the other two rapid antigen detection kits based on immunochromatography and silver amplification immunochromatography, the microfluidic immunofluorescence assay kit exhibited higher sensitivity than the former assay (53.6%) and the same sensitivity as the latter (60.7%). The microfluidic immunofluorescence assay kit did not detect nine non-EIV viruses including one equine coronavirus strain and seven bacteria, suggesting a high specificity for EIV antigens. Similar to other rapid antigen detection kits, the microfluidic immunofluorescence assay kit could be an effective diagnostic tool to detect EIV in the field.

Growth properties and immunogenicity of a virus generated by reverse genetics for an inactivated equine influenza vaccine.

BACKGROUND: Keeping vaccine strains up to date is the key to controlling equine influenza (EI). Viruses generated by reverse genetics (RG) are likely to be effective for quickly updating a vaccine strain. OBJECTIVES: To evaluate the growth properties of an RG virus in embryonated chicken eggs, and to evaluate antibody responses to a formalin-inactivated vaccine derived from the RG virus in Thoroughbred horses. STUDY DESIGN: In vitro and in vivo experiments. METHODS: Wild-type (WT) viruses (A/equine/Ibaraki/1/2007) or RG viruses (consisting of haemagglutinin [HA] and neuraminidase genes derived from A/equine/Ibaraki/1/2007 and the six other genes derived from high-growth A/Puerto Rico/8/34) were inoculated into embryonated chicken eggs, and the allantoic fluids were harvested at every 24 hours after inoculation. WT and RG viruses were inactivated by formalin for vaccine use. Ten unvaccinated yearlings (five each for WT or RG vaccine) received the first two doses of a primary vaccination course 4 weeks apart followed by their third dose 12 weeks later. Twenty vaccinated adult horses (10 each for WT or RG vaccine) received a single dose of a booster vaccination. RESULTS: The RG virus had high growth properties in embryonated chicken eggs. Unvaccinated yearlings responded poorly to the first vaccination, especially those that received the RG vaccine, but mounted better responses to the second and the third vaccinations, and maintained relatively high haemagglutination inhibition (HI) titres up to 28 weeks after the first vaccination. Vaccinated adult horses did not respond remarkably to the booster vaccination, but no horses showed titres below their pre-booster values even at 12 weeks after vaccination. The RG virus elicited immunogenicity in horses adequate for vaccine use. MAIN LIMITATIONS: No virus challenge study was performed. CONCLUSIONS: The RG viruses are useful as an EI vaccine strain, and quick updates of an EI vaccine strain can be achieved by using RG techniques.

Antibody Responses to a Reverse Genetics-Derived Bivalent Inactivated Equine Influenza Vaccine in Thoroughbred Horses.

Updating vaccine strains is important to control equine influenza (EI). Previously, we reported that a monovalent inactivated EI vaccine derived from a virus generated by reverse genetics (RG) elicited immunogenicity in horses. In the present study, we compared antibody responses to a bivalent inactivated EI vaccine generated by RG and a commercially available bivalent inactivated EI (CO) vaccine derived from wild-type equine influenza viruses in Thoroughbred horses. The CO vaccine contained A/equine/Ibaraki/1/2007 (Florida sub-lineage clade 1) and A/equine/Yokohama/aq13/2010 (Florida sub-lineage clade 2) as vaccine strains. We generated two RG viruses possessing the hemagglutinin and neuraminidase genes from A/equine/Ibaraki/1/2007 or A/equine/Yokohama/aq13/2010. These viruses were inactivated by formalin, and the hemagglutinin titer of the RG vaccine was adjusted to be the same as that of the CO vaccine. Sixteen unvaccinated yearlings (7 for the RG vaccine group and 9 for the CO vaccine group) received two doses of a primary vaccination course four weeks apart. Thirty-two vaccinated adult horses (18 in the RG-vaccinated group and 14 in the CO vaccine group) received a single dose of a booster vaccination. The patterns of hemagglutination inhibition antibody response to the primary and booster vaccinations were similar for the RG and CO groups in unvaccinated yearlings and vaccinated adult horses. These results suggest that a bivalent vaccine derived from RG viruses elicits equivalent immunogenicity to that elicited by a CO vaccine derived from wild-type viruses. RG viruses can, therefore, be used in multivalent as well as monovalent vaccines for horses.

Protective efficacy of a reverse genetics-derived inactivated vaccine against equine influenza virus in horses.

Updating vaccine strains is essential to control equine influenza. We evaluated the protective efficacy of an inactivated equine influenza vaccine derived from viruses generated by reverse genetics (RG) in horses in an experimental viral challenge study. Wild-type (WT) virus (A/equine/Tipperary/1/2019) and virus generated by RG (consisting of hemagglutinin and neuraminidase genes from A/equine/Tipperary/1/2019 and six other genes from high-growth A/Puerto Rico/8/34) were inactivated by formalin for vaccine use. Twelve 1-year-old naïve horses with no antibodies against equine influenza virus were assigned to three groups (each n = 4): control, WT, and RG. They were vaccinated twice, 4 weeks apart, and were challenged with A/equine/Tipperary/1/2019 2 weeks after the second vaccination. All four horses in the control group and one horse in the WT group had pyrexia for multiple days and respiratory illness, and one horse in the RG group had pyrexia for 2 days without respiratory illness. The mean rectal temperatures and the mean concentrations of serum amyloid A in the WT and RG groups were significantly lower than those in the control group, with no significant differences between them. The WT and RG vaccines significantly reduced viral shedding relative to the control. The protective efficacy of the RG-derived inactivated vaccine against equine influenza virus is comparable to that of the vaccine derived from WT viruses in horses. The RG technique can make it easy to update equine influenza vaccine strains.

Outbreak of equine coronavirus infection among riding horses in Tokyo, Japan.

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.

Evaluation of Antibody Response in Horses After Vaccination With an Inactivated Getah Virus Vaccine Using an Accelerated Immunization Schedule.

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.

Decreased Virus-Neutralizing Antibodies Against Equine Herpesvirus type 1 In Nasal Secretions of Horses After 12-hour Transportation.

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.

Antigenic differences between equine influenza virus vaccine strains and Florida sublineage clade 1 strains isolated in Europe in 2019.

From late 2018 to 2019, equine influenza virus (EIV) strains of Florida sublineage clade 1 (Fc1), which had until then been circulating mainly in the United States, suddenly spread across Europe causing many outbreaks, and Florida sublineage clade 2 (Fc2) strains, which had been circulating mainly in Europe, have not been detected in Europe since 2018. Since 2010, the World Organisation for Animal Health (OIE) has recommended that EIV vaccines contain an Fc1 strain that is like A/equine/South Africa/4/2003 or A/equine/Ohio/2003. Accordingly, Japanese vaccines contain A/equine/Ibaraki/1/2007 as the Fc1 strain. To evaluate the effectiveness of these vaccines against the Fc1 strains detected in Europe in 2019, we performed virus neutralization tests using horse antisera. Challenge viruses used were Irish strain A/equine/Tipperary/1/2019 and two recombinant viruses generated by reverse genetics. Recombinant viruses possessing hemagglutinin (HA) and neuraminidase (NA) derived from A/equine/Tipperary/1/2019 (rA/equine/Tipperary/1/2019) or British strain A/equine/Essex/1/2019 (rA/equine/Essex/1/2019) were generated. Equine antisera against A/equine/South Africa/2003 and A/equine/Ibaraki/2007 were produced by experimental infection. Antibody titers against A/equine/Tipperary/1/2019, rA/equine/Tipperary/1/2019, and rA/equine/Essex/1/2019 were 2.5- to 6.3-fold lower than those against the homologous vaccine strains A/equine/South Africa/4/2003 or A/equine/Ibaraki/2007. These results suggest that the ongoing evolution of the Fc1 viruses may impact on antigenicity and although antibodies against current vaccine strains neutralize the 2019 strains, ongoing surveillance is essential for optimum choice of candidate vaccine strains.