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Borrelia burgdorferi can induce Lyme disease. Approved Lyme vaccines for horses are currently not available. In an effort to protect horses, veterinarians are using Lyme vaccines licensed for dogs. However, data to assess the response of horses to, or determine the efficacy of this off-label vaccine use are missing. Here, antibodies against outer surface protein A (OspA), OspC, and OspF were quantified in diagnostic serum submissions from horses with a history of vaccination with canine Lyme vaccines. The results suggested that many horses respond with low and often short-lasting antibody responses. Subsequently, four experimental vaccination trials were performed. First, we investigated antibody responses to three canine vaccines in B. burgdorferi-naïve horses. One killed bacterin vaccine induced antibodies against OspC. OspA antibodies were low for all three vaccines and lasted less than 16weeks. The second trial tested the impact of the vaccine dose using the OspA/OspC inducing bacterin vaccine in horses. A 2mL dose produced higher OspA and OspC antibody values than a 1mL dose. However, the antibody response again quickly declined, independent of dose. Third, the horses were vaccinated with 2 doses of a recombinant OspA vaccine. Previous vaccination and/or environmental exposure enhanced the magnitude and longevity of the OspA antibody response to about 20weeks. Last, the influence of intramuscular versus subcutaneous vaccine administration was investigated for the recombinant OspA vaccine. OspA antibody responses were not influenced by injection route. The current work highlights that commercial Lyme vaccines for dogs induce only transient antibody responses in horses which can also be of low magnitude. Protection from infection with B. burgdorferi should not be automatically assumed after vaccinating horses with Lyme vaccines for dogs.

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naive horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virus-infected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8+/IFN-γ+ and detectable from d32pi on. Peripheral blood IFN-γ+ T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.

Neonatal foals respond poorly to conventional vaccines. These vaccines typically target T-helper (Th) cell dependent B-cell activation. However, Th2-cell immunity is impaired in foals during the first three months of life. In contrast, neonatal basophils are potent interleukin-4 (IL-4) producers. The purpose of this study was to develop a novel vaccine triggering the natural capacity of neonatal basophils to secrete IL-4 and to evaluate if vaccination resulted in B-cell activation and antibody production against EHV-1 glycoprotein C (gC). Neonatal vaccination was performed by oral biotinylated IgE (IgE-bio) treatment at birth followed by intramuscular injection of a single dose of streptavidin-conjugated gC/IL-4 fusion protein (Sav-gC/IL-4) for crosslinking of receptor-bound IgE-bio (group 1). Neonates in group 2 received the intramuscular Sav-gC/IL-4 vaccine only. Group 3 remained non-vaccinated at birth. After vaccination, gC antibody production was not detectable. The ability of the vaccine to induce protection was evaluated by an EHV-1 challenge infection after weaning at 7 months of age. Groups 1 and 2 responded to EHV-1 infection with an earlier onset and overall significantly increased anti-gC serum antibody responses compared to control group 3. In addition, group 1 weanlings had a decreased initial fever peak after infection indicating partial protection from EHV-1 infection. This suggested that the neonatal vaccination induced a memory B-cell response at birth that was recalled at weanling age after EHV-1 challenge. In conclusion, early stimulation of neonatal immunity via the innate arm of the immune system can induce partial protection and increased antibody responses against EHV-1., Funding for this project was provided by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University ‘A Novel Strategy to Boost Antibody Production to EHV-1 in Neonates’ (http://vet.cornell.edu/research/Zweig/). Monoclonal antibody development for horse cell surface markers and cytokines was supported by USDA grant #2005-01812 ‘The US Veterinary Immune Reagent Network’ and #2015-67015-23072 ‘Equine Immune Reagents: Development of monoclonal antibodies to improve the analysis of immunity in horses’ (https://nifa.usda.gov/).

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

Changes in ELISA serology are frequently used to determine antibiotic treatment success for Lyme disease in horses. This concept was based upon a previous report showing a marked decline in ELISA values in experimentally infected and antibiotic-treated ponies. Changes in Lyme serology following antibiotic treatment in naturally infected horses have not been reported. The objective of this study was to compareBorreliaELISA antibody concentrations in naturally exposed horses both before and following antibiotic treatment for Lyme disease. A retrospective study was performed comparing oxytetracycline- or doxycyclinetreated (n = 68) and untreated (n = 183) horses from a single equine practice and their change inBorreliaELISA values over a similar time period. Antibiotictreated horses had a decline in ELISA values in comparison to control horses (P ≤ 0.05) and untreated horses were twice as likely to have their ELISA values increase (OR = 0.5; 95% C.I. = 0.3–0.9) compared to treated horses. The magnitude of the decline in ELISA units following treatments was small compared to that previously reported in experimentally infected and treated ponies. Field-exposed horses with highBorrelia burgdorferiELISA values who are treated with either oxytetracycline or doxycycline can be expected to have only a small decline in ELISA values following treatment. Persistently high ELISA titres following appropriate treatments for Lyme disease may not, without appropriate clinical signs, be a reason for more prolonged treatment.

OBJECTIVE To compare antibody responses of horses naturally infected with West Nile virus (WNV) and those vaccinated against WNV, to identify whether vaccination interferes with the ability to diagnose WNV infection, and to determine the duration of antibody responses after vaccination. SAMPLE Sera from horses naturally infected with WNV (n = 10) and adult WNV-naïve horses before and after vaccination with a live canarypox virus–vectored vaccine (7) or a killed virus vaccine (8). PROCEDURES An established WNV IgM capture ELISA was used to measure IgM responses. Newly developed capture ELISAs were used to measure responses of 8 other WNV-specific immunoglobulin isotypes. A serum neutralization assay was used to determine anti-WNV antibody titers. RESULTS WNV-specific IgM responses were typically detected in the sera of WNV-infected horses but not in sera of horses vaccinated against WNV. Natural infection with and vaccination against WNV induced an immunoglobulin response that was primarily composed of IgG1. West Nile virus–specific IgG1 was detected in the sera of most horses 14 days after vaccination. Serum anti-WNV IgG1 and neutralizing antibody responses induced by the killed-virus vaccines were higher and lasted longer than did those induced by the live canarypox virus–vectored vaccine. CONCLUSIONS AND CLINICAL RELEVANCE On the basis of these findings, we recommend that horses be vaccinated against WNV annually near the beginning of mosquito season, that both IgM and IgG1 responses against WNV be measured to distinguish between natural infection and vaccination, and that a WNV IgG1 ELISA be used to monitor anti-WNV antibodies titers in vaccinated horses.

Phylogenetic and antigenic analyses indicate that recent circulating equine-2 influenza viruses in the United States have been alternating between two genetic and antigenic distinct lineages since 1996. The evolution rates for these two lineages, the Kentucky and the Florida lineage, are very similar. For the earlier isolates in the Kentucky lineage, there are multiple and sequential nonsynonymous substitutions at antigenic sites B and D. However, there are no changes at any of these antigenic sites for KY98 and OK00. In the Florida lineage, except for NY99 with one amino acid substitution at antigenic site B, viruses in this lineage do not have nonsynonymous substitutions at any of the antigenic sites. The lack of amino acid substitutions at these antigenic sites suggests a mechanism other than immune selection is responsible for the maintenance of these viral lineages. Serological analysis indicates that these two lineages are antigenic distinct, and the pattern of reactivity of horse sera towards these two lineages alternates in consecutive years, parallel to the "switching" of virus lineage seen in the phylogenetic tree. This alternate circulation may play a role in the maintenance of these two lineages of equine-2 influenza virus.

Summary Clinical, pathological, immunohistochemical, serological and microbiological findings are described for 2 geographically and temporally distinct equine arteritis virus (EAV) epidemics in newborn foals. Outbreak A occurred at a commercial Standardbred breeding facility; Outbreak B began in a group of research animals. Clinical signs were severe and primarily referrable to the respiratory tract. Fever and leucopenia and/or thrombocytopenia were observed in foals surviving for more than 24 h. The most common gross pathological findings were limited to the respiratory tract. Common histopathological findings included interstitial pneumonia, lymphocytic arteritis and periarteritis with fibrinoid necrosis of the tunica media. Renal tubular necrosis was noted in 2 foals. Immunoperoxidase histochemistry combined with virus isolation was diagnostic in all cases.

Borrelia burgdorferi, the etiologic agent of Lyme disease is a tick born spirochetal infection. Clinical signs of Lyme borreliosis are uncommon in horses, but when present they are often vague and nonspecific. In horses, Lyme borreliosis has been implicated in musculoskeletal, neurological, reproductive, and ocular disorders, including uveitis, but definitive diagnosis can be challenging as the causative agent is rarely isolated and serologic tests can be unreliable and do not confirm active disease. Here, we report two cases of equine uveitis associated with B. burgdorferi based on the identification of spirochetes within ocular fluids and confirmed with PCR testing. The two cases illustrate some of the challenges encountered in the recognition and diagnosis of equine Lyme borreliosis. Although only one of many possible causes of equine uveitis, Lyme disease should be considered a differential diagnosis, especially in endemic areas. Given the possibility for false negative results of serum tests during uveitis associated with B. burgdorferi and the failure of such tests to confirm active infection, a combination of cytologic assessment, antibody, and/or PCR testing of ocular fluids may be worthwhile if the clinical suspicion for Lyme uveitis is high.

Horses naturally exposed to West Nile Virus (WNV) or vaccinated against WNV develop humoral immunity thought to be protective against development of clinical disease in exposed or infected animals. No reports evaluate the efficacy of passive transfer of naturally acquired specific WNV humoral immunity from dam to foal. The purpose of this study was to investigate passive transfer of naturally acquired immunity to WNV to foals born in a herd of semi-feral ponies, not vaccinated against WNV, in an endemic area, with many dams having seroconverted because of natural exposure. Microwell serum neutralization titers against WNV were determined in all mares and foals. Serum IgG concentration was determined in foals by serial radial immunodiffusion. Differences in IgG concentration between seropositive and seronegative foals were examined by means of the Mann-Whitney U-test. Linear regression was used to evaluate the association between mare and foal titers. Seventeen mare-foal pairs were studied; 1 foal had inadequate IgG concentration. IgG concentration was not different between seronegative and seropositive foals (P = .24). Mare and foal titers were significantly correlated in foals with adequate passive transfer of immunity (Spearman's rho = .84; P.001);90% of the foal's titer was explained by the mare's titer (R2 = 0.91; P.001). Passive transfer of specific immunity to WNV is present in pony foals with adequate passive transfer of immunity born to seroconverted mares.

One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein–based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0–48.0), although the rNS1 MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine ( P < 0.0001) or naturally infected with WNV ( P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.

Salem virus (SalV) is a recently identified equine virus belonging to the family Paramyxoviridae. The only known isolate was obtained from a horse that was involved in a disease outbreak of undetermined nature and the circumstances of its isolation suggested an etiologic role. However, the experimental infection of a colostrum-deprived foal failed to reproduce the disease; only mild neutropenia and temperature elevation were recorded. An additional attempt to establish an etiological relationship with the disease was made by conducting a retrospective evaluation of the serological profiles of animals involved in the outbreak. Animals reported as being affected by the disease according to a comprehensive United Sates Department of Agriculture (USDA) database were found to be 48% (n=27) positive for antibodies to SalV, but the percent positive for all horses, affected and unaffected, was actually higher at 56% (n=62). For 15 affected horses for which paired acute and convalescent serum specimens were available, no unequivocal seroconversions to SalV were identified. Furthermore, the horse from which SalV was isolated was not listed as one of the animals affected by the disease. In total, the evidence suggests that SalV was not the etiological agent of the disease and that its isolation was fortuitous.