N. Rash

Outbreak of upper respiratory disease in horses caused by Streptococcus equi subsp. zooepidemicus ST-24.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is generally considered a commensal and an opportunistic pathogen of the upper airways in horses. Establishing whether certain strains of S. zooepidemicus can cause upper respiratory disease as a host-specific pathogen of horses, and if there are certain genogroups of S. zooepidemicus that are more virulent than others is of major clinical importance. In this study, we describe an outbreak of upper respiratory disease in horses that was associated with S. zooepidemicus. Upper respiratory samples were cultured, analyzed by real-time PCR for S. zooepidemicus and S. equi, and genetically differentiated by sequencing of the SzP protein gene and multi-locus sequence typing (MLST). Serum samples were analyzed for antibodies against S. equi and common viral respiratory pathogens. The ST-24 strain of S. zooepidemicus was isolated from all horses with clinical signs of disease, while the healthy horses carried other strains of S. zooepidemicus. Bacteriological, molecular and serological analyses strongly suggest that a single strain (ST-24) was responsible for the disease outbreak, and that certain strains of this presumed commensal may be more virulent than others.

Equine mesenchymal stromal cells and embryo-derived stem cells are immune privileged in vitro

IntroductionAutologous mesenchymal stem cells (MSCs) are an attractive concept in regenerative medicine, but their mechanism of action remains poorly defined. No immune response is reported after in vivo injection of allogeneic equine MSCs or embryo-derived stem cells (ESCs) into the equine tendon, which may be due to the cells’ immune-privileged properties. This study further investigates these properties to determine their potential for clinical application in other tissues.MethodsMitomycin C-treated MSCs, ESCs, or differentiated ESCs (dESCs) were cultured with allogeneic equine peripheral blood mononuclear cells (PBMCs), and their effect on PBMC proliferation, in the presence or absence of interferon-gamma (IFN-γ) was determined. MSCs and super-antigen (sAg)-stimulated PBMCs were co-cultured directly or indirectly in transwells, and PBMC proliferation examined. Media from MSC culture were harvested and used for PBMC culture; subsequent PBMC proliferation and gene expression were evaluated and media assayed for IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 and IL-6 proteins with enzyme-linked immunosorbent assay (ELISA).ResultsCo-culture of PBMCs with ESCs or dESCs did not affect baseline proliferation, whereas co-culture with MSCs significantly suppressed baseline proliferation. Stimulation of PBMC proliferation by using super-antigens (sAgs) was also suppressed by co-culture with MSCs. Inhibition was greatest with direct contact, but significant inhibition was produced in transwell culture and by using MSC-conditioned media, suggesting that soluble factors play a role in MSC-mediated immune suppression. The MSCs constitutively secrete IL-6, even in the absence of co-culture with PBMCs. MSC-conditioned media also brought about a change in the cytokine-expression profile of sAg-stimulated PBMCs, significantly reducing PBMC expression of IL-6, IFN-γ, and TNF-α.ConclusionsEquine MSCs and ESCs possess a degree of innate immune privilege, and MSCs secrete soluble factors that suppress PBMC proliferation and alter cytokine expression. These properties may make possible the future clinical use of allogeneic stem cells to help standardize and broaden the scope of treatment of tissue injuries.

How to Meet the Last OIE Expert Surveillance Panel Recommendations on Equine Influenza (EI) Vaccine Composition: A Review of the Process Required for the Recombinant Canarypox-Based EI Vaccine

Vaccination is highly effective to prevent, control, and limit the impact of equine influenza (EI), a major respiratory disease of horses. However, EI vaccines should contain relevant equine influenza virus (EIV) strains for optimal protection. The OIE expert surveillance panel annually reviews EIV evolution and, since 2010, the use of Florida clade 1 and 2 sub-lineages representative vaccine strains is recommended. This report summarises the development process of a fully- updated recombinant canarypox-based EI vaccine in order to meet the last OIE recommendations, including the vaccine mode of action, production steps and schedule. The EI vaccine ProteqFlu contains 2 recombinant canarypox viruses expressing the haemagglutinin of the A/equine/Ohio/03 and A/equine/Richmond/1/07 isolates (Florida clade 1 and 2 sub-lineages, respectively). The updated EI vaccine was tested for efficacy against the representative Florida clade 2 EIV strain A/equine/Richmond/1/07 in the Welsh mountain pony model. Protective antibody response, clinical signs of disease and virus shedding were compared with unvaccinated control ponies. Significant protection was measured in vaccinated ponies, which supports the vaccine registration. The recombinant canarypox-based EI vaccine was the first fully updated EI vaccine available in the EU, which will help to minimise the increasing risk of vaccine breakdown due to constant EIV evolution through antigenic drift.

Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution

BackgroundEquine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective.ResultsThe group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2.ConclusionsVarious lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs.

Localised mitogenic activity in horses following infection with Streptococcus equi

Streptococcus equi subspecies equi (S. equi) is the causative agent of strangles, a highly contagious upper respiratory disease of equids. Streptococcus equi produces superantigens (sAgs), which are thought to contribute to strangles pathogenicity through non-specific T-cell activation and pro-inflammatory response. Streptococcus equi infection induces abscesses in the lymph nodes of the head and neck. In some individuals, some abscess material remains into the guttural pouch and inspissates over time to form chondroids which can harbour live S. equi. The aim of this study was to determine the sites of sAg production during infection and therefore improve our understanding of their role. Abscess material, chondroids and serum collected from Equidae with signs of strangles were tested in mitogenic assays. Mitogenic sAg activity was only detected in abscess material and chondroids. Our data support the localised in vivo activity of sAg during both acute and carrier phases of S. equi infection.

Polarisation of equine pregnancy outcome associated with a maternal MHC class I allele: Preliminary evidence.

Identification of risk factors which are associated with severe clinical signs can assist in the management of disease outbreaks and indicate future research areas. Pregnancy loss during late gestation in the mare compromises welfare, reduces fecundity and has financial implications for horse owners. This retrospective study focussed on the identification of risk factors associated with pregnancy loss among 46 Thoroughbred mares on a single British stud farm, with some but not all losses involving equid herpesvirus-1 (EHV-1) infection. In a sub-group of 30 mares, association between pregnancy loss and the presence of five common Thoroughbred horse haplotypes of the equine Major Histocompatibility Complex (MHC) was assessed. This involved development of sequence specific, reverse transcriptase polymerase chain reactions and in several mares, measurement of cytotoxic T lymphocyte activity. Of the 46 mares, 10 suffered late gestation pregnancy loss or neonatal foal death, five of which were EHV-1 positive. Maternal factors including age, parity, number of EHV-1 specific vaccinations and the number of days between final vaccination and foaling or abortion were not significantly associated with pregnancy loss. In contrast, a statistically significant association between the presence of the MHC class I B2 allele and pregnancy loss was identified, regardless of the fetus/foals EHV-1 status (p=0.002). In conclusion, this study demonstrated a significantly positive association between pregnancy loss in Thoroughbred mares and a specific MHC class I allele in the mother. This association requires independent validation and further investigation of the mechanism by which the mares genetic background contributes to pregnancy outcome.

Mitogenic Equine Isolates of Streptococcus zooepidemicus From North America Host Superantigen Genes Similar to Those Hosted in Europe

Abstract Streptococcus equi subsp. zooepidemicus (Sz) is an important opportunist pathogen of the equine respiratory and reproductive tracts. It is highly variable with respect to sequence type and virulence factors including SzP and SzM. Recent studies in the UK have shown that many Sz strains host genes for mitogenic superantigens including szeF, szeN, szeP, seeI, seeL, and seeM. The aims of the present study were to estimate the prevalence of mitogenicity in equine Sz in North America and establish whether mitogenicity is more likely to be associated with isolates from a specific site of infection. Twenty‐six percent (23/90) of strains randomly selected from over 600 Sz isolated in the United States from 1969 to 1994 were mitogenic for equine peripheral blood mononuclear cells. All but five of these mitogenic Sz encoded one or more of the superantigen genes szeF, szeN, szeP, seeI, seeL, and seeM. Homologues of seeH in S. equi were not detected in any Sz strain. Polymerase chain reaction analysis revealed the presence of seeI, szeF, szeN, or szeP in 91% (10/11) of isolates from lymph node abscesses confirming earlier reports of a significant association of superantigens with lymph node abscessation in the UK. In contrast, only 24% (4/17) of Sz isolates from the reproductive tract were mitogenic and/or hosted seeL, seeM, szeF, szeP, or szeN. HighlightsTwenty‐six percent of randomly selected Streptococcus zooepidemicus strains from equines in North America were mitogenic.Superantigen genes of these mitogenic strains are similar to those in Europe.Isolates from lymph node abscesses were very likely to host superantigen genes.Isolates from the reproductive tract were unlikely to host these genes.