A field study of persistence of antibodies in California horses vaccinated against western, eastern, and Venezuelan equine encephalomyelitis.

As a result of the continuing threat of Venezuelan equine encephalomyelitis (VEE), a study was made to determine if revaccination against VEE (TC-83 vaccine) was feasible and if revaccination could be incorporated into other routine vaccination practices. Of the horses given annual vaccination with bivalent western equine encephalomyelitis (WEE) and eastern equine encephalomyelitis (EEE) vaccine, 57% retained detectable serum-neutralizing (SN) antiboyd titers for VEE 18 months after the initial VEE vaccination was given. Of horses with no record of WEE-EEE vacinnation, 100% retained detectable VEE SN antibody titers over the same period. The VEE geometric mean titer was 25 times greater for horses not previously vaccinated against WEE-EEE than for horses given annual WEE-EEE vaccination at the time of VEE vaccination. In horses vaccinated against VEE 18 months previously, the geometric mean titer increased from 4 to 70 at 48 days after the intitial WEE-EEE vaccination. This increase indicated that similar antigenic factors for VEE are possibly present in bivalent WEE-EEE vaccine. In horses previously vaccinated against WEE-EEE and VEE, the best SN antibody response to VEE revaccination occurred when VEE vaccine was given simultaneously with the bivalent WEE-EEE vaccine. Of 150 serum samples tested by both the SN and the hemagglutination-inhibiton tests, agreement between positive reactions at greater than or equal to 1:10 was 70% for VEE, 81% for EEE, and 87% for WEE.     

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.     

The involvement of mast cells and mast cell proteinases in the intestinal response to equine cyathostomin infection

Cyathostomins (Cyathostominae) are regarded as the most pathogenic equine nematode worldwide. These nematodes are difficult to control in equine populations due to emerging anthelmintic resistance and evasion of encysted larval cyathostomins to regular modern anthelmintics. Mast cells and their proteinases have been shown to play a role in the mammalian immune response to nematode infections. Involvement of mast cells and mast cell proteinases in the equine immune response to cyathostomin infection is proposed. A technique was established to perform immunohistochemical staining using polyclonal rabbit anti-equine mast cell proteinase-1 (eqMCP-1) and anti-equine tryptase on formalin-fixed large intestinal sections, from horses classified as cyathostomin positive and negative at the time of death based upon larval enumeration. Quantitative analysis of antibody labelled mast cells was used to detect mast cell proteinases in equine large intestinal sections positive and negative for cyathostomin larvae. This demonstrated an increase in equine tryptase labelled mucosal and submucosal mast cells in cyathostomin positive horses. This study has established an immunohistochemical technique to demonstrate mast cell proteinases in formalin-fixed large intestinal sections. This technique may be used to determine possible involvement of mast cells and their proteinases in the equine immune response to cyathostomin larvae. Further studies are required to define a specific role.     

The effect of age on serum antibody titers after rabies and influenza vaccination in healthy horses

BACKGROUND: The proportion of geriatric horses within the equine population has increased in the past decade, but there is limited information on the immune function of these animals. HYPOTHESIS: Aged horses will have a lesser increase in serum antibody response to vaccination. ANIMALS: Thirty-four aged healthy horses (> or = 20 years) and 29 younger adult horses (4-12 years) of various breeds. METHODS: All horses were vaccinated with vaccines of killed rabies and influenza virus. Horses in each age group were allocated to receive either rabies or influenza booster vaccine 4 weeks after the initial vaccination. Serum samples were taken at 0, 4, 8, and 24 weeks. Rabies serum neutralization titers and equine influenza virus specific antibody sub-isotypes (IgGa, IgGb, IgG(T), and IgA) as well as single radial hemolysis (SRH) titers were determined. RESULTS: Rabies antibody titers were similar in the 2 age groups at all sampling times. Aged horses had higher IgGa and IgGb influenza antibody titers before vaccination than younger horses but similar titers after vaccination (P= .004 and P= .0027, respectively). Younger horses had significantly greater increases in titer than aged horses at all sampling times for IgGa (P= .001) and at 8 and 24 weeks for IgGb (P= .041 and .01, respectively). There was no detectable serum IgG(T) at any time point. A significant booster vaccine effect was seen for both antirabies and anti-influenza titers. Anti-influenza titer before vaccination also had a significant effect on subsequent antibody response. CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy aged horses generated a primary immune response to a killed rabies vaccine similar to that of younger adult horses. Aged horses had a significantly reduced anamnestic response to influenza vaccine.     

Monoclonal antibodies to equine interferon-alpha (IFN-alpha): new tools to neutralize IFN-activity and to detect secreted IFN-alpha

Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generation of six monoclonal antibodies (mAbs) to equine IFN-alpha. Four of these mAbs inhibited the protective anti-viral effect of equine leukocyte IFN in bioassays. One mAb (clone 240-2) showed a high-neutralizing capacity. An ELISA was established using two anti-equine IFN-alpha mAbs (clones 29B and 240-2) and its analytical sensitivity for was found to be around 800 pg/ml and 3 U/ml for rIFN-alpha and equine leukocyte IFN, respectively. When analyzing samples with a likely dominance of IFN-alpha among type I IFNs, such as supernatants from equine peripheral blood mononuclear cells stimulated with CpG-oligodeoxyribonucleotides, the results obtained by ELISA and IFN bioassay showed a high agreement (r(2)(sp)=0.98). When analyzing samples likely containing a mixture of type I IFNs, such as serum and nasal secretions from virally infected horses, the ELISA only detected some of the IFN-activity recorded in the bioassay. Overall, the data showed that the new anti-equine IFN-alpha mAbs are valuable tools to detect native IFN-alpha for further characterization of the early innate immune response and anti-viral immunity in horses.     

Antibody Responses Against Equine Influenza Virus Induced by Concurrent and by Consecutive Use of an Inactivated Equine Influenza Virus Vaccine and a Modified Live Equine Herpesvirus Type 1 Vaccine in Thoroughbred Racehorses

An inactivated equine influenza virus (EIV) vaccine and a live equine herpesvirus type 1 (EHV-1) vaccine are usually administered concurrently to Thoroughbred racehorses in Japan. The objective of this study was to evaluate whether concurrent administration of an inactivated EIV vaccine and a live EHV-1 vaccine in Thoroughbred racehorses influences the antibody response against EIV. We compared the antibody response against EIV in horses administered both vaccines on the same day (Group A; n = 27) and the response in horses administered an inactivated EIV vaccine first and then a live EHV-1 vaccine 1–2 weeks later (Group B; n = 20). In both groups, geometric mean hemagglutination inhibition (HI) titers against A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010 increased significantly after EIV vaccination. However, the percentage of horses that showed a twofold increase or greater in HI titers against A/equine/Yokohama/aq13/2010 was significantly higher in Group B (75%) than in Group A (37%; P = .02). These results suggest that the concurrent use of an inactivated EIV vaccine and a live EHV-1 vaccine reduced the immune response against EIV to some extent, and it would be better to use these vaccines consecutively, especially for naïve horses or horses whose vaccination history is incomplete.     

Immunity to equine influenza: relationship of vaccine-induced antibody in young Thoroughbred racehorses to protection against field infection with influenza A/equine-2 viruses (H3N8)

Field outbreaks of influenza that occurred in vaccinated Thoroughbred racehorses in Newmarket in 1995 and 1996 were investigated by nucleoprotein ELISA and serology. Investigations showed that serum levels of vaccine-induced single radial haemolysis (SRH) antibody correlated closely with protective immunity against equine influenza and were consistent with observations made in previous experimental studies using nebulised aerosol challenge. In the second part of this study, antibody levels stimulated by vaccination were investigated to examine probable protection in high risk groups, such as yearlings and horses in training. Results for yearlings correlated closely with experimentally derived antibody profiles described for several equine influenza vaccines. The horses in training had levels of antibody immediately prior to revaccination, which were higher than those measured in the yearlings. In conclusion, SRH antibody, used in the investigation of outbreaks and surveillance of post vaccination responses, was shown to correlate with and validate experimental vaccination and challenge models currently used in ponies in the licensing of modern vaccines. There may be benefit from serological monitoring of horses following vaccination through identification of susceptible periods to infection and demonstration of poor vaccine responders. This would allow appropriate and timely amendment of vaccination strategies to maximise protective immunity against influenza.     

Humoral and cell-mediated immune responses of old horses following recombinant canarypox virus vaccination and subsequent challenge infection

Equine influenza virus is a leading cause of respiratory disease in the horse population; however, the susceptibility of old horses to EIV infection remains unknown. While advanced age in horses (>20 years) is associated with age-related changes in immune function, there are no specific recommendations regarding the vaccination of older horses even though a well-characterized effect of aging is a reduced antibody response to standard vaccination. Therefore, we evaluated the immunological and physiological response of aged horses to a live non-replicating canarypox-vectored EIV vaccine and subsequent challenge infection. Vaccination of the aged horses induced EIV-specific IgGb and HI antibodies. No specific increase in cell-mediated immune (CMI) response was induced by the vaccine as determined by EIV-specific lymphoproliferation and the detection of EIV-specific IFNγ⁺ CD5⁺T cells, IFNγ, IL-2, IL-4 and IL-13 mRNA expression. Non-vaccinated aged horses exhibited clinical signs of the disease (coughing, nasal discharge, dyspnea, depression, anorexia) as well as increased rectal temperature and viral shedding following challenge. Concomitant with the febrile episodes, we also observed increased production of pro-inflammatory cytokine mRNA production in vivo using RT-PCR. Naïve horses were included in this study for vaccine and challenge controls only. As expected, the canarypox-vectored EIV vaccine stimulated significant CMI and humoral immune responses and provided significant protection against clinical signs of disease and reduced virus shedding in naive horses. Here, we show that aged horses remain susceptible to infection with equine influenza virus despite the presence of circulating antibodies and CMI responses to EIV and vaccination with a canarypox-vectored EIV vaccine provides protection from clinical disease.     

Duration of serologic response to three viral antigens in cats

OBJECTIVE: To determine whether vaccinated cats either remained seropositive or responded serologically to revaccination against 3 key viral antigens after extended periods since their last vaccination. DESIGN: Serologic survey. ANIMALS: 272 healthy client-owned cats. PROCEDURE: Cats were > or = 2 years old and vaccinated for feline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus (FHV). On day 0, cats were revaccinated with a vaccine from the same line of vaccines as they had historically received. Antibody titers were measured in sera collected on day 0 (prevaccination titer) and 5 to 7 days later (postvaccination titer). Cats were considered to have responded serologically if they had a day-0 hemagglutination inhibition titer to FPV > or = 1:40, serum neutralization (SN) titer to FCV > or = 1:32, SN titer to FHV > or = 1:16, or > or = 4-fold increase in antibody titer after revaccination. RESULTS: The percentage of cats that had titers at or above the threshold values or responded to revaccination with a > or = 4-fold increase in titer was 96.7% for FPV, 97.8% for FCV, and 88.2% for FHV. CONCLUSIONS AND CLINICAL RELEVANCE: In most cats, vaccination induced a response that lasted up to and beyond 48 months for all 3 antigens. Although not equivalent to challenge-of-immunity studies as a demonstration of efficacy, results suggest that revaccination with the vaccine used in our study provides adequate protection even when given less frequently than the traditional 1-year interval. The study provides valuable information for clinicians to determine appropriate revaccination intervals.     

Vaccination of pregnant ponies against equine rhinopneumonitis

Bovine herpesvirus 1247 (one dose) was given subcutaneously to five pregnant pony mares between 227 and 319 days of their gestations. There were no adverse clinical reactions, and the virus was not recovered from nasal swabs collected during a 2-week period after vaccination. Four ponies foaled full-term, live, healthy foals. The foal of the fifth mare (No. 1) was found dead, but on the basis of the pathologic and virologic examinations, the virus was not considered to be the cause of the death. At 3 weeks after vaccination, the pregnant pony mares had a 13- to 250-fold increase in serum antibody titer to equine herpesvirus-1. A virulent-virus challenge exposure of all pony mares at 208 days after vaccination resulted in antibody titers greater than those just before this exposure. Virus was recovered from nasal swabs from vaccinated mares only on postexposure day 1, whereas the one control (nonvaccinated) pony shed virus for at least 3 days after challenge exposure. The immunogenic and the nonabortifacient characteristics of the herpesvirus 1247 in pregnant pony mares indicate that it may be useful to vaccinate horses against equine herpesvirus-1.     

Pathogenicity of a new neurotropic equine herpesvirus 9 (gazelle herpesvirus 1) in horses

Pathogenicity of equine herpesvirus 9 (EHV-9), a new type of equine herpesvirus isolated from Gazella thomsoni, in horses was investigated by intranasal inoculation of EHV-9 (10(7) pfu) to two conventionally reared 8-months old half-bred weanling horses. Fever higher than 39 degrees C was recorded. Virus was recovered from nasal swabs and peripheral blood mononuclear cells. Both horses developed neutralizing antibody to EHV-9. Perivascular infiltration of mononuclear cells and glial reaction were found in the olfactory and limbic systems. The results suggested that EHV-9 has a pathogenicity in horses.     

Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals

The envelope glycoprotein D of equine herpesvirus 1 (EHV-1 gD) has been shown in laboratory animal models to elicit protective immune responses against EHV-1 challenge, and hence is a potential vaccine antigen. Here we report that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant Iscomatrix elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses. The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/4 whole virus vaccine. Intramuscular inoculation of EHV-1 gD DNA encoded in a mammalian expression vector was less effective in inducing antibody responses when administered as the sole immunogen, but inoculation with EHV-1 gD DNA followed by recombinant EHV-1 gD induced increased gD ELISA and virus-neutralizing antibody titres in six out of seven horses. However, these titres were not higher than those induced by either EHV-1 gD or the whole virus vaccine. Isotype analysis revealed elevated gD-specific equine IgGa and IgGb relative to IgGc, IgG(T) and IgA in horses inoculated with EHV-1 gD or with the whole virus vaccine. Following inoculation of pregnant mares with EHV-1 gD, their foals had significantly higher levels of colostrally derived anti-gD antibody than foals out of uninoculated mares. The EHV-1 gD preparation did not induce a significant mean antibody response in neonatal foals following inoculation at 12 h post-partum and at 30 days of age, irrespective of the antibody status of the mare. The ability of EHV-1 gD to evoke comparable neutralizing antibody responses in horses to those of a whole virus vaccine confirms EHV-1 gD as a promising candidate for inclusion in subunit vaccines against EHV-1.     

Low-dose ginseng (Panax quinquefolium) modulates the course and magnitude of the antibody response to vaccination against Equid herpesvirus 1 in horses

The purpose of this study was to determine if ginseng fed at low levels enhances a horse's antibody response to vaccination against Equid herpesvirus 1 (EHV-1). For 28 d, 5 horses received ground, powdered ginseng (35 mg/kg body weight, 1.7 mg/kg total ginsenosides) in molasses as a carrier, and 5 received molasses only. On day 14, each horse was vaccinated against EHV-1. The time course of the antibody response to vaccination was significantly altered in the horses receiving ginseng, a clinically relevant increase in antibody titer being observed by postvaccination day 2 compared with day 6 in the control horses. The horses receiving ginseng also had a significant decrease in serum levels of sodium and a significant increase in serum levels of potassium. No adverse effects of ginseng treatment were identified by hematologic and blood biochemistry profiles. Thus, low-dose dietary supplementation with ginseng in healthy horses may be a useful adjunct to vaccination.     

Antibodies against equine herpesvirus 1 in the cerebrospinal fluid in the horse

Neutralizing antibodies against equine herpesvirus 1 were measured in serum and cerebrospinal fluid of 16 horses and ponies from a closed herd both before and after vaccination with modified live equine herpesvirus 1. These titers were also measured in 22 neurologically normal and 15 neurologically abnormal horses at a teaching hospital. Animals from the closed herd had prevaccination serum titers up to 1:8 and postvaccination serum titers up to 1:128. Horses from the teaching hospital had serum titers up to 1:64. Cerebrospinal fluid titers were not detected in the vaccinated horses or the neurologically normal horses but a low titer (1:8) was noted in one neurologically abnormal horse. This titer probably resulted from hemorrhage into the cerebrospinal fluid following trauma.     

Equine haptoglobin: isolation, characterization, and the effects of ageing, delivery and inflammation on its serum concentration.

Haptoglobin (Hp) was isolated from equine serum by ammonium sulphate precipitation, anion-exchange chromatography and gel filtration. Equine Hp which migrated to the alpha 2-globulin region in electrophoresis, contained 2 fractions with molecular weights (NW) of 108,000 and 105,000, and each fraction consisting of 2 subunits. Quantitative measurement of Hp in equine serum was performed by the single radial immunodiffusion method using anti-equine Hp serum. In clinically normal horses, the highest concentration of serum Hp was found in newborn foals and a high value was maintained until 12 months of age. The concentration then decreased with age. Normal Hp values were 5.25 +/- 2.36 mg/ml in foals (less than or equal to 12 months old), 2.19 +/- 1.54 mg/ml in adult horses (greater than or equal to 18 months old) and 3.62 +/- 0.81 mg/ml in all horses. Serum Hp concentration in mares during the perinatal period in comparison with the normal adult female was high for 4 months pre-partum, a passing increase at delivery, and then decreased at 2 weeks post-partum returning to normal within 1 month of delivery. In horses with experimentally-induced inflammation, serum Hp concentration began to increase immediately after treatment and reached the highest value, 1.5 to 9 times higher than those of pre-treatment at 2 to 5 days, then decreased within 4 weeks. It was also elevated in most cases of horses with clinically inflammatory signs.     

Equine herpesvirus 1 (EHV-1) glycoprotein D DNA inoculation in horses with pre-existing EHV-1/EHV-4 antibody

We have shown previously that equine herpesvirus 1 (EHV-1) glycoprotein D (gD) DNA elicited protective immune responses against EHV-1 challenge in murine respiratory and abortion models of EHV-1 disease. In this study, 20 horses, all with pre-existing antibody to EHV-4 and two with pre-existing antibody to EHV-1, were inoculated intramuscularly with three doses each of 50, 200 or 500microg EHV-1 gD DNA or with 500microg vector DNA. In 8 of 15 horses, inoculation with EHV-1 gD DNA led to elevated gD-specific antibody and nine horses exhibited increased virus neutralising (VN) antibody titres compared to those present when first inoculated. A lack of increase in gC-specific antibody during the 66 weeks of the experiment showed that the increase in gD-specific antibodies was not due to a natural infection with either EHV-1 or EHV-4. The increase in EHV-1 gD-specific antibodies was predominantly an IgGa and IgGb antibody response, similar to the isotype profile reported following natural EHV-1 infection.     

Serologic responses to eastern and western equine encephalomyelitis vaccination in previously vaccinated horses

A prospective study was performed to determine the serologic response of previously vaccinated horses to revaccination against eastern and western equine encephalomyelitis (EEE and WEE). Horses responded variably to each antigen, and some horses had low or undetectable antibodies 6 months after vaccination. Some horses did not develop increasing titers to EEE or WEE despite recent vaccination. Geometric mean titers peaked 2 weeks after revaccination and were significantly increased from before revaccination. Except for one horse, EEE:WEE titer ratios ranged from 0.25 to 2.0. Regular vaccination against EEE and WEE did not interfere with testing for Saint Louis encephalitis.     

The Response of Ponies to Myxovirus influenzae A-equi 2 I. Serum and Antibody Titres Following Exposure

The antibody response in serum and nasal secretions of groups of ponies vaccinated or infected with Myxovirus influenzae A-equi 2 was examined. Following infection by aerosol with live virus, a weak antibody response was recorded in both serum and secretions. Antibody levels were undetectable in secretions at 31 days after infection.

After primary intramuscular vaccination with killed virus, using sodium alginate as an adjuvant, antibody was detected only in the serum. However, following revaccination, a pronounced antibody response was demonstrated in both serum and secretions. Antibody was still detectable in all four ponies when tested 135 days later.

Only a serum antibody response was detected in ponies after primary intramuscular vaccination with a commercial vaccine. Upon revaccination nasal antibody occurred in all ponies but this only persisted for about 30 days.

Neither serum nor nasal antibody response occurred following intranasal vaccination and revaccination with a killed virus vaccine.

     

Surveillance of equine respiratory viruses in Ontario

The objective of this project was to develop and implement an active surveillance program for the early and rapid detection of equine influenza viruses in Ontario. For this purpose, from October 2003 to October 2005, nasopharyngeal swabs and acute and convalescent serum samples were collected from 115 client-owned horses in 23 outbreaks of respiratory disease in Ontario. Sera were paired and tested for antibody to equine influenza 1 (AE1-H7N7), equine influenza 2 (AE2-H3N8), equine herpesvirus 1 and 4 (EHV1 and EHV4), and equine rhinitis A and B (ERAV and ERBV). Overall, the cause-specific morbidity rate of equine influenza virus in the respiratory outbreaks was 56.5% as determined by the single radial hemolysis (SRH) test. The AE2-H3N8 was isolated from 15 horses in 5 outbreaks. A 4-fold increase in antibody levels or the presence of a high titer against ERAV or ERBV was observed in 10 out of 13 outbreaks in which AE2-H3N8 was diagnosed as the primary cause of disease. In conclusion, AE2-H3N8 was found to be an important contributor to equine respiratory viral disease. Equine rhinitis A and B (ERAV and ERBV) represented an important component in the equine respiratory disease of performing horses.     

Ataxia and paresis with equine herpesvirus type 1 infection in a herd of riding school horses

An outbreak of neurologic disease associated with serologic evidence of equine herpesvirus type 1 (EHV-1) infection occurred in a herd of 46 riding school horses. Ataxia and paresis were observed in 14 geldings and 5 barren mares. Eight affected horses had distal limb edema, 1 horse had a head tilt, and 3 others had urinary incontinence. Other clinical signs included fever, depression, and inappetance in 30 horses. Seven horses with neurologic signs were treated with acyclovir. Serum neutralizing antibody titers against EHV-1 increased 4-fold between acute and convalescent samples or exceeded 1: 256 in 19 of 44 horses, confirming recent infection. A significantly greater proportion of horses that seroconverted were mares ( P = .014). Of the 19 horses exhibiting ataxia and paresis, 17 made a complete recovery, 1 made a partial recovery, and 1 was euthanized.