Antibody and cellular immune responses of naïve mares to repeated vaccination with an inactivated equine herpesvirus vaccine.

Equine herpesvirus type 1 (EHV-1) continues to cause severe outbreaks of abortions or myeloencephalopathy in horses despite widely used vaccination. The aim of this work was to determine the effects of frequent vaccination with an inactivated EHV vaccine on immune development in horses. Fifteen EHV-1 naïve mares were vaccinated a total of 5 times over a period of 8 months with intervals of 20, 60, 90 and 60 days between vaccine administrations. Total antibody and antibody isotype responses were evaluated with a new sensitive EHV-1 Multiplex assay to glycoprotein C (gC) and gD for up to 14 months after initial vaccination. Antibodies peaked after the first two vaccine doses and then declined despite a third administration of the vaccine. The fourth vaccine dose was given at 6 months and the gC and gD antibody titers increased again. Mixed responses with increasing gC but decreasing gD antibody values were observed after the fifth vaccination at 8 months. IgG4/7 isotype responses mimicked the total Ig antibody production to vaccination most closely. Vaccination also induced short-lasting IgG1 antibodies to gC, but not to gD. EHV-1-specific cellular immunity induced by vaccination developed slower than antibodies, was dominated by IFN-γ producing T-helper 1 (Th1) cells, and was significantly increased compared to pre-vaccination values after administration of 3 vaccine doses. Decreased IFN-γ production and reduced Th1-cell induction were also observed after the second and fourth vaccination. Overall, repeated EHV vaccine administration did not always result in increasing immunity. The adverse effects on antibody and cellular immunity that were observed here when the EHV vaccine was given in short intervals might in part explain why EHV-1 outbreaks are observed worldwide despite widely used vaccination. The findings warrant further evaluation of immune responses to EHV vaccines to optimize vaccination protocols for different vaccines and horse groups at risk.

Analysis of soluble CD14 and its use as a biomarker in neonatal foals with septicemia and horses with recurrent airway obstruction.

Soluble CD14 (sCD14) binds bacterial lipopolysaccharide (LPS) and acts as an anti-inflammatory LPS-inhibitor in vivo. In humans, sCD14 is one of the soluble biomarkers used for various inflammatory diseases and conditions, however, sCD14 assays have not yet been evaluated in horses. Here, we developed and optimized a bead-based assay for the quantification of sCD14 in horses. The assay was then used to determine native sCD14 concentrations in serum from healthy and septic foals, in the colostrum of healthy mares and in plasma from adult horses with recurrent airway obstruction (RAO) and control horses. Healthy foals and adult horses had sCD14 concentrations in serum or plasma in the high ng/ml range. The concentration of sCD14 in colostrum samples from healthy mares was in the µg/ml range. Foals with septicemia and adult horses with RAO had significantly higher sCD14 concentrations in their circulation than the respective control groups. The findings suggest that sCD14 can become a valuable biomarker for neonatal septicemia, RAO and possibly also for other inflammatory diseases in horses. Further studies and larger samples numbers are required to determine normal sCD14 concentration ranges and those that are indicative of disease progression, severity or prognosis.

Antibodies to OspC, OspF and C6 antigens as indicators for infection with Borrelia burgdorferi in horses.

REASONS FOR PERFORMING STUDY: Lyme disease is caused by Borrelia burgdorferi, which is transmitted by infected ticks (Ixodes spp.). Reports on Lyme disease in horses have increased in recent years. Nevertheless, the diagnosis of Lyme disease in horses is still challenging owing to its vague clinical presentation and the limitations of diagnostic tests. OBJECTIVES: This study used a new serological Lyme multiplex assay to examine antibody responses to 3 antigens of B. burgdorferi, outer surface protein (Osp) C, OspF and C6, and to verify their use as markers for early and late infection stages in horses. METHODS: Multiplex analysis of antibodies to OspC, OspF and C6 in equine patient sera (n = 191) was performed. A subset of the sera (n = 90) was also tested using a commercial C6-based Lyme test. RESULTS: Antibodies to OspF and C6 highly correlate as reliable markers of infection with B. burgdorferi in horses. Antibodies to OspC, which have been confirmed as early infection markers in man and dogs, were only detected in some patient sera, suggesting that OspC antibodies are indicators of early infection in horses. Commercial C6 testing identified most infected horses but also resulted in false positive and false negative interpretations. CONCLUSIONS: Serological multiplex testing is a rapid and quantitative diagnostic method to confirm infection with B. burgdorferi and to identify the stage of infection. In horses with risk of exposure and clinical signs, multiplex testing supports the diagnosis of Lyme disease. POTENTIAL RELEVANCE: Antimicrobial treatment of B. burgdorferi is time sensitive. Treatment success decreases with time of persistent infection, while the risk of developing chronic disease increases. The ability to identify early infection with B. burgdorferi provides practitioners and clinicians with a tool to improve the diagnosis of equine Lyme disease and make treatment decisions.

A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum.

Lyme disease is a zoonotic, vector-borne disease affecting humans, dogs, horses and other species. It is caused by infection with spirochetes of the Borrelia burgdorferi sensu lato group which are transmitted to the mammalian host by infected ticks (Ixodes). Exposure to B. burgdorferi is commonly diagnosed by serological testing. The gold standard for the detection of antibodies to B. burgdorferi is a two-step procedure of an ELISA followed by confirmatory Western blotting (WB). Here, we developed and validated a new bead-based multiplex assay for the detection of antibodies to B. burgdorferi in canine serum which combined the testing by ELISA and WB in a single quantitative test. B. burgdorferi outer surface protein A (OspA), OspC and OspF were expressed in E. coli. The recombinant proteins were coupled to fluorescent beads providing the matrix of the assay. Two sets of canine sera were used for validation of the multiplex assay. First, sera from 79 dogs with known ELISA and WB results were used to establish the conditions of the assay. These samples were selected to provide similar numbers of pre-tested sera ranging from negative to high positive results and included sera from vaccinated and/or naturally infected dogs. A high correlation was observed for detection of antibodies to B. burgdorferi in the single and multiplex assays (n=79). Spearman's rank correlations were 0.93, 0.88 and 0.96 for OspA, OspC and OspF, respectively. Second, a total of 188 canine serum samples that were not tested previously were used for further multiplex assay validation. All samples were also blindly analyzed for antibodies to B. burgdorferi antigens by WB. The WB results provided a 'relative gold standard' for each antigen and were used to perform a receiver operating curve analysis. The areas under the curves were 0.93 for OspA, 0.82 for OspC, and 0.89 for OspF. Multiplex assay interpretation ranges for antibodies to all three B. burgdorferi antigens in canine serum were established by likelihood analysis. The diagnostic sensitivities of the individual OspA, OspC and OspF bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic specificities were 90%, 89% and 86%, respectively. The new multiplex assay provides a sensitive and fully quantitative platform for the simultaneous evaluation of antibodies to B. burgdorferi OspA, OspC and OspF antigens and distinguishes between antibodies that originated from vaccination or natural exposure to B. burgdorferi.