Voluntary Surveillance Program for Equine Influenza Virus in the United States during 2008-2021.

A voluntary upper respiratory biosurveillance program in the USA received 9740 nasal swab submissions during the years 2008-2021 from 333 veterinarians and veterinary clinics. The nasal swabs were submitted for qPCR testing for six common upper respiratory pathogens:equine influenza virus (EIV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), equine rhinitis A virus (ERAV), and equine rhinitis B virus (ERBV). Additional testing was performed for equine gamma herpesvirus-2 (EHV-2) and equine gamma herpesvirus-5 (EHV-5) and the results are reported. Basic frequency statistics and multivariate logistic regression models were utilized to determine the associations between risk factors and EIV positivity. The EIV qPCR-positivity rate was 9.9%. Equids less than 9 years of age with a recent history of travel and seasonal occurrence in winter and spring were the most common population that were qPCR positive for EIV. This ongoing biosurveillance program emphasizes the need for molecular testing for pathogen identification, which is critical for decisions associated with therapeutics and biosecurity intervention for health management and vaccine evaluations and development.

Investigation of cross-regional spread and evolution of equine influenza H3N8 at US and global scales using Bayesian phylogeography based on balanced subsampling.

Equine influenza virus (EIV) is a highly contagious pathogen of equids, and a well-known burden in global equine health. EIV H3N8 variants seasonally emerged and resulted in EIV outbreaks in the United States and worldwide. The present study evaluated the pattern of cross-regional EIV H3N8 spread and evolutionary characteristics at US and global scales using Bayesian phylogeography with balanced subsampling based on regional horse population size. A total of 297 haemagglutinin (HA) sequences of global EIV H3N8 were collected from 1963 to 2019 and subsampled to global subset (n = 67), raw US sequences (n = 100) and US subset (n = 44) datasets. Discrete trait phylogeography analysis was used to estimate the transmission history of EIV using four global and US genome datasets. The North American lineage was the major source of globally dominant EIV variants and spread to other global regions. The US EIV strains generally spread from the southern and midwestern regions to other regions. The EIV H3N8 accumulated approximately three nucleotide substitutions per year in the HA gene under heterogeneous local positive selection. Our findings will guide better decision making of target intervention strategies of EIV H3N8 infection and provide the better scheme of genomic surveillance in the United States and global equine health.

Genome-informed characterisation of antigenic drift in the haemagglutinin gene of equine influenza strains circulating in the United States from 2012 to 2017.

Equine influenza virus (EIV) is a major infectious pathogen causing significant respiratory signs in equids worldwide. Voluntary surveillances in the United States recently reported EIV detection in horses with respiratory signs even with adequate vaccine protocols and biosecurity programs and posed a concern about suboptimal effectiveness of EIV vaccine in the United States. This study aims to determine the genetic characteristics of 58 field EIV H3N8 strains in the United States from 2012 to 2017 using the phylogenetic analysis based on the haemagglutinin (HA) gene. Amino acid substitution and acquisition of N-glycosylation of the HA gene were also evaluated. Phylogenetic analysis identified that almost all US field strains belonged to the Florida clade 1 (FC1) except one Florida clade 2 strain from a horse imported in 2014. US EIV strains in 2017 shared 11 fixed amino acid substitutions in the HA gene, compared to the vaccine strain (A/equine/Ohio/2003), and two additional amino acid substitutions were detected in 2019. The introduction of foreign EIV strains into the United States was not detected, but antigenic drift without acquisition of N-glycosylation in the HA gene was observed in US field strains until 2017. Considering the global dominance of FC1 strains, subsequent antigenic drift of US EIV strains should be monitored for better effectiveness of the EIV vaccine in the United States and global equine industries.

Investigation of a 24-Hour Culture Step to Determine the Viability of Streptococcus equi Subspecies equi Via Quantitative Polymerase Chain Reaction in Nasal Secretions From Horses With Suspected Strangles.

Polymerase chain reaction (PCR)-based detection assays for Streptococcus equi subspecies equi often overestimate the prevalence of samples containing viable organisms. The objective of this study was to determine if viability could be determined using genome quantitation and detection of messenger RNA (mRNA) transcripts for the SeM gene of S. equi in pre- and post-cultured samples. Nasal secretions collected from 42 horses with suspected strangles were tested by culture and by quantitative PCR (qPCR) before and 24 hours after a culture step. Viable S. equi was determined based on the detection of S. equi via culture, the detection of mRNA transcripts for the SeM gene of S. equi by qPCR, and/or an increase in absolute number of SeM target genes of S. equi between pre- and post-cultured samples. Viability was determined in 28/42 samples based on isolation of S. equi (11 samples), the presence of mRNA transcripts for the SeM gene of S. equi (25), and/or an increase in absolute quantitation of the SeM gene of S. equi between pre- and post-culture (17). The overall agreement between culture alone and the three criteria to determine viability was 59%. The overall agreement for the detection of mRNA transcripts and increase in absolute target genes was 88% and 74%, respectively. The combination of mRNA transcripts and increase in absolute target genes was able to determine the viability status in all 42 samples. In the absence of a culture-positive result for S. equi, the determination of viability can be achieved by using molecular strategies applied to samples undergoing a 24-hour culture step.

Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates.

We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 ( HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012-2017 to determine their clade affiliation. All 14 EIV prototype strains were identified correctly as either Florida sublineage clade 1 or clade 2 using the 2 SNP target positions. Of 341 EIV rtPCR-positive samples, 337 (98.8%) and 4 (1.2%) isolates were classified as belonging to clade 1 and 2 Florida sublineage EIV, respectively. All clade 1 Florida sublineage EIV strains were detected in domestic horses, three clade 2 Florida sublineage EIV strains originated from horses recently imported into the United States, and one clade 2 Florida sublineage EIV strain originated from a healthy horse recently vaccinated with a modified-live intranasal EIV vaccine containing the American lineage strain A/eq/Kentucky/1991. EIV Florida sublineage clade differentiation using a fast and reliable multiplex rtPCR platform will help monitor the introduction of clade 2 Florida sublineage EIV strains into North America via international transportation.

Distribution and Prevalence of Myxobolus cerebralis in Postfire Areas of Plumas National Forest: Utility of Environmental DNA Sampling.

Myxobolus cerebralis is a myxozoan parasite and the etiological agent of whirling disease in salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and the benthic freshwater oligochaete worm Tubifex tubifex (Oligochaeta: Tubificidae). Wildfires can lead to the erosion of fine sediments into stream channels and have been implicated as promoting environmental conditions that are suitable for the survival and success of T. tubifex, whose presence in turn can affect the prevalence of M. cerebralis. Analysis of environmental DNA (eDNA) has the potential to be a powerful tool for evaluating the presence of microorganisms, for which direct observation is impossible. We investigated the presence of M. cerebraliseDNA in river water and river sediment samples collected from areas affected by recent fire activity in Plumas National Forest, California. We compared eDNA loads in the environment to M. cerebralis infection in T. tubifex and sentinel-exposed Rainbow Trout Oncorhynchus mykiss and the presence of T. tubifex lineages in the same environment. For the latter, we developed a multiplex quantitative PCR assay for detection of T. tubifex lineages I, III, and V. Lineage IIIT. tubifex and M. cerebralis (eDNA as well as DNA extracted from fish and worm tissues) were detected only in samples obtained from areas affected by the Moonlight wildfire. The association between M. cerebralis infection in sentinel-exposed fish and eDNA detection in environmental samples only approached significance at a P-value of 0.056. However, given the difference in relative effort between the two sampling methods (host versus nonhost environment), our data suggest that eDNA sampling of water and substrate is a promising approach for surveillance of myxozoan fish parasites.