Multifocal outbreak of equine influenza in vaccinated horses in Argentina in 2018: Epidemiological aspects and molecular characterisation of the involved virus strains.

BACKGROUND: Equine influenza is an important cause of respiratory disease of horses worldwide. The equine influenza virus (EIV) undergoes antigenic drift through the accumulation of amino acid substitutions in the viral proteins, which may lead to vaccine breakdown. OBJECTIVES: To describe the epidemiological findings and the molecular characteristics of the EIV detected during the multifocal outbreak that occurred in Argentina between March and July 2018 and evidence a vaccine breakdown. STUDY DESIGN: Observational, descriptive study. METHODS: Virus was detected in nasopharyngeal swabs using real-time reverse transcriptase PCR (RT-PCR). Nucleotide and deduced amino acid sequences of the haemagglutinin (HA) and neuraminidase (NA) genes were obtained from EIV positive nasopharyngeal swabs, and phylogenetic analysis was undertaken. Amino acid sequences were compared against the current World Organisation for Animal Health (OIE)-recommended Florida clade 1 vaccine strain and strain components of vaccines used in Argentina. Serum samples were tested using haemagglutination inhibition test. RESULTS: Equine influenza virus infection was confirmed using real-time RT-PCR and serological testing. The phylogenetic analysis of the HA and NA genes revealed that all the EIV identified during the outbreak belong to the H3N8 subtype, Florida clade 1. Multiple amino acid changes, some of them at antigenic sites, were observed in the circulating virus when compared with the strains included in the most commonly used vaccine in Argentina. Seventy-six percent of the affected horses had been vaccinated with this vaccine, suggesting the occurrence of vaccine breakdown. MAIN LIMITATIONS: The study does not include antigenic characterisation and full genome sequencing of Argentinian strains, that could provide additional information. CONCLUSIONS: The occurrence of this multifocal equine influenza outbreak in regularly vaccinated horses is a field evidence of vaccine breakdown, reinforcing the necessity of keeping vaccine strains updated according to OIE recommendations. It also underlines the importance of the implementation of appropriate quarantine measures and restriction of horse movement in the face of disease.

On-site detection of equid alphaherpesvirus 3 in perineal and genital swabs of mares and stallions.

Equine coital exanthema (ECE) is an infectious, venereally transmitted muco-cutaneous disease affecting mares and stallions, caused by equid alphaherpesvirus 3 (EHV3). Diagnostic tools for rapid identification of EHV3 are of primary importance to diminish the risk of EHV3 dissemination at the time of breeding. In the last years, it has been shown that the performance of the insulated-isothermal polymerase chain reaction (iiPCR) is comparable to virus isolation, nested PCR and real-time PCR (qPCR) in detecting pathogens of various animal species. Analytical sensitivity and specificity of the iiPCR were compared with a qPCR, using a plasmid containing the target region of the EHV3 glycoprotein G gene and an Argentinian EHV3 isolate (E/9283/07 C3A). In order to evaluate the diagnostic performance of the iiPCR, nucleic acids of 85 perineal and genital swabs (PGS) of mares and stallions were extracted by tacoTM mini and tested by both techniques. EHV3 was detected in 46 and 45 of the 85 PGS by the iiPCR and qPCR, respectively. There was almost perfect agreement between the two diagnostic methods (98.82%; 95% CI: 95.03-100%; κ = 0.98). The iiPCR had a limit of detection of 95.00% at 6 genome equivalents per reaction and a detection endpoint for viral DNA comparable to that of the qPCR, and did not react with six non-targeted equine pathogens. The iiPCR represents a sensitive and specific method for the rapid on-site diagnosis of EHV3 infection. Its routinely implementation in breeding facilities, and artificial insemination and embryo transfer centers, will contribute to prevent the dissemination of this venereal, highly contagious disease in horses.

Neuropathogenic and non-neuropathogenic genotypes of Equid Herpesvirus type 1 in Argentina.

Infection with Equid Herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism (A(2254)/G(2254)) in the genome region of the open reading frame 30 (ORF30), which results in an amino acid variation (N(752)/D(752)) of the EHV-1 DNA polymerase, is significantly associated with the neuropathogenic potential of naturally occurring strains. In order to estimate the prevalence of the EHV-1 neuropathogenic genotype in our country, we analyzed the ORF30 genome region of Argentinean EHV-1 isolates. The study was carried out by real time allelic discrimination PCR in 90 equine EHV-1-positive samples, being 89 from 54 cases of abortion outbreaks (two of which were in association with neurological disease) and one from the respiratory tract of a healthy horse in training. Our results indicate that 7% (4/54) of the abortion outbreaks studied were induced by the neuropathogenic (G(2254)) genotype of EHV-1 and 50% (2/4) of them were associated with simultaneous neurological disease. This information emphasizes the necessity to extreme the hygienic and preventive measures to diminish EHV-1 infections and consequently reduce the risk of epizootic neurological disease as has been recently observed in other countries.