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.     

Exercise alters the immune response to equine influenza virus and increases susceptibility to infection.

Equine influenza virus remains a major health concern for the equine industry in spite of ongoing vaccination programmes. Previous work has shown that the immune system of horses can be affected by strenuous exercise. The possible adverse consequence of exercise-induced alterations in lymphocyte responses measured in vitro was unknown. Here we demonstrate that subjecting vaccinated ponies to a 5 day strenuous exercise programme results in a significant suppression of their T cell-mediated immune response to equine influenza virus as measured by decreased lymphoproliferation and gamma interferon production measured in vitro. These same ponies also demonstrated increased susceptibility to influenza disease following a challenge exposure to the same strain of virus. Rested ponies that had received the same vaccine and challenge were completely protected from disease. Our results demonstrate that exercise-induced suppression of the equine immune response to influenza virus can be associated with an increased susceptibility to disease.     

Antibody responses of horses to equine influenza viruses during a postepizootic period in Japan.

The antibody responses to equine influenza viruses were investigated during a postepizootic period of the disease. Serum samples were collected from a total of 128 horses on three occasions during the years 1967-77. No significant increase of hemagglutination-inhibition antibody titers to subtypes 1 and 2 of equine influenza virus were detected in any of the sera tested. The maternal hemagglutination-inhibition antibody titers of foals decreased over a four month interval. A marked increase of the titers was recognized in only the equine influenza virus vaccinated horses. These findings suggest that equine influenza virus was not prevalent in the horse populations during the observation period. In such conditions, the dissemination of equine influenza viruses in the horses is discussed in relation to introduction of the disease from abroad. We also examined whether the doctrine of original antigenic sin, an immunological phenomenon recognized in human influenza, was applicable for equine influenza. However, no marked increase of hemagglutination-inhibition antibody titer to the primary infecting subtype in the 44 horses was observed after administration of the heterologous subtype vaccine.     

Safety, efficacy, and immunogenicity of a modified-live equine influenza virus vaccine in ponies after induction of exercise-induced immunosuppression

OBJECTIVE: To determine safety, efficacy, and immunogenicity of an intranasal cold-adapted modified-live equine influenza virus vaccine administered to ponies following induction of exercise-induced immunosuppression. DESIGN: Prospective study. ANIMALS: Fifteen 9- to 15-month old ponies that had not had influenza. PROCEDURE: Five ponies were vaccinated after 5 days of strenuous exercise on a high-speed treadmill, 5 were vaccinated without undergoing exercise, and 5 were not vaccinated or exercised and served as controls. Three months later, all ponies were challenged by nebulization of homologous equine influenza virus. Clinical and hematologic responses and viral shedding were monitored, and serum and nasal secretions were collected for determination of influenza-virus-specific antibody isotype responses. RESULTS: Exercise caused immunosuppression, as indicated by depression of lymphocyte proliferation in response to pokeweed mitogen. Vaccination did not result in adverse clinical effects, and none of the vaccinated ponies developed clinical signs of infection following challenge exposure. In contrast, challenge exposure caused marked clinical signs of respiratory tract disease in 4 control ponies. Vaccinated and control ponies shed virus after challenge exposure. Antibody responses to vaccination were restricted to serum IgGa and IgGb responses in both vaccination groups. After challenge exposure, ponies in all groups generated serum IgGa and IgGb and nasal IgA responses. Patterns of serum hemagglutination inhibition titers were similar to patterns of IgGa and IgGb responses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that administration of this MLV vaccine to ponies with exercise-induced immunosuppression was safe and that administration of a single dose to ponies provided clinical protection 3 months later.     

Studies on the antigenicity of an inactivated, aluminum hydroxide adjuvant equine influenza vaccine.

An inactivated, aluminum hydroxide adjuvant equine influenza vaccine was tested in horses and guinea pigs to determine the levels of antigen that would elicit maximum serological responses. Vaccine containing serial twofold increments of A/Equi-1/Prague and A/Equi-2/Miami strains of equine influenza virus was administered to random groupings of both types of test animals. The hemagglutination inhibition antibody response for each group was then measured. Results in horses and guinea pigs were compared to determine if the equine serological values could be related to a potency test in laboratory animals. The highest mean hemagglutination inhibition antibody response in horses occurred in groups vaccinated, respectively, with 128 or 256 hemagglutination units of A/Equi-1 and 512 or 1024 hemagglutination units of A/Equi-2 antigen. Groups vaccinated with further two- or fourfold increases in these antigens had mean hemagglutination inhibition titers that were somewhat lower than the maximum levels. When graded doses of vaccine were given to guinea pigs, their hemagglutination inhibition antibody titers reached a plateau of maximum values, similar to the serological response in vaccinated horses. Test horses remained clinically free from signs of equine influenza during the year following vaccination and no untoward post-vaccination reactions were observed.     

Efficacy of a commercial vaccine for preventing disease caused by influenza virus infection in horses.

OBJECTIVE: To evaluate efficacy of a commercial vaccine for prevention of infectious upper respiratory tract disease (IURD) caused by equine influenza virus. DESIGN: Double-masked, randomized, controlled field trial. ANIMALS: 462 horses stabled at a Thoroughbred racetrack. PROCEDURE: Vaccine or saline solution placebo was administered 4 times in the population at 6-week intervals. The vaccine contained 3 strains of inactivated influenza virus, and inactivated equine herpesvirus type 4. Horses received 1 or 2 doses of vaccine or placebo prior to onset of a natural influenza epidemic, and were examined 5 d/wk to identify and monitor horses with IURD. Serum antibody concentrations were determined, and virus isolation was performed. RESULTS: Vaccination of horses prior to the influenza epidemic did not result in significant decrease in risk of developing respiratory tract disease. Severity of clinical disease was not different between affected vaccinated horses with IURD and controls with IURD, but median duration of clinical disease was 3 days shorter in vaccinated horses. Serum concentrations of antibodies to H3N8 influenza viruses were lower prior to initial vaccination in horses that were sick during the epidemic, and did not increase in these horses in response to vaccination. On arrival at the racetrack, young horses had lower antibody concentrations than older horses, and did not respond to vaccination as well. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination was of questionable benefit. A greater degree of protection must be obtained for influenza vaccines to be effective in protecting horses from IURD. Objective field evaluations of commercial vaccines are needed to adequately document their efficacy.     

Effect of influenza A/equine/H3N8 virus isolate variation on the measurement of equine antibody responses.

This study has tested the effect of using homologous or heterologous equine influenza A virus isolates to evaluate serum antibody levels to influenza A virus in vaccinated and naturally-infected horses. In addition, the potential effect of antigenic selection of virus variants in egg versus tissue culture propagation systems was studied. Serum antibody levels in samples from horses recently infected with a local influenza A virus isolate (A/equine 2/Saskatoon/1/90) or recently vaccinated with a prototype isolate (A/equine 2/Miami/1/63) were assessed by hemagglutination inhibition and by single radial hemolysis using cell or egg-propagated A/equine 2/Saskatoon/1/90, A/equine 2/Miami/1/63 or A/equine 2/Fontainebleau/1/79. There were no significant differences in hemagglutination inhibition or single radial hemolysis antibody levels obtained with homologous or heterologous isolates or between viruses propagated in either eggs or cell culture. However there was a trend to higher titers in the hemagglutination inhibition assay when cell-propagated virus was used. These results suggest that antigenic variation in equine influenza A virus isolates and host-cell selection of antigenic variants during virus propagation may not be of sufficient magnitude to influence serological evaluation of antibody responses by hemagglutination inhibition or single radial hemolysis.     

Effect of chronic clenbuterol administration and exercise training on immune function in horses

Effects of longitudinal exercise training and acute intensive exercise (simulated race test) on immune function have not been reported in horses. Clenbuterol, a beta2-adrenergic agonist, is used to manage inflammatory airway disease in horses. This study investigated the interaction of 8 wk of exercise training with or without 12 wk of clenbuterol administration in horses. Twenty-three untrained standardbred mares (10 +/- 3 yr, Mean +/- SE) were used and divided into four experimental groups. Horses given clenbuterol plus exercise (CLENEX; n = 6) and clenbuterol alone (CLEN; n = 6) received 2.4 microg/kg BW of clenbuterol twice daily (in an average volume of 20 mL) on a schedule of 5 d on and 2 d off for 12 wk. The CLENEX group was also aerobically trained 3 d/wk. Mares given exercise alone (EX; n = 5) were aerobically trained for 3 d/wk, and the control group (CON; n = 6) remained sedentary. Both EX and CON horses were administered similar volumes (approximately 20 mL) of molasses twice daily. A simulated race test (SRT) resulted in an elevation in lymphocyte number postexercise (P < 0.05). There was no significant difference after acute exercise in either monocyte or granulocyte number. Acute exercise resulted in a decrease (P < 0.05) in the percentage of CD4+ and an increase (P < 0.05) in the percentage of CD8+ cells. The SRT resulted in a decreased lymphoproliferative response to pokeweed mitogen (P < 0.05). A SRT had no effect on antibody production in response to equine influenza vaccine. The EX group demonstrated greater cortisol concentrations at rest and at all other time points postexercise after completing the training regimen compared with CLENEX horses (P < 0.05). Preexercise (SRT) peripheral blood monocyte number was lower in CLENEX horses than in other treatment groups (P < 0.05). Clenbuterol and exercise training did not significantly affect post-SRT changes in leukocyte numbers. Exercise training resulted in a decrease (P < 0.05) in the percentage of CD8+ cells post-SRT compared with other groups, but the percentage of CD4+ cells was not altered by either clenbuterol or exercise conditioning. Lymphocyte proliferative response was not affected by clenbuterol or exercise treatment. Horses demonstrated responses to bouts of acute exercise as noted with other species, namely humans and rodents.     

Antibody responses after vaccination against equine influenza in the Republic of Korea in 2013

In this study, antibody responses after equine influenza vaccination were investigated among 1,098 horses in Korea using the hemagglutination inhibition (HI) assay. The equine influenza viruses, A/equine/South Africa/4/03 (H3N8) and A/equine/Wildeshausen/1/08 (H3N8), were used as antigens in the HI assay. The mean seropositive rates were 91.7% (geometric mean antibody levels (GMT), 56.8) and 93.6% (GMT, 105.2) for A/equine/South Africa/4/03 and A/equine/Wildeshausen/1/08, respectively. Yearlings and two-year-olds in training exhibited lower positive rates (68.1% (GMT, 14) and 61.7% (GMT, 11.9), respectively, with different antigens) than average. Horses two years old or younger may require more attention in vaccination against equine influenza according to the vaccination regime, because they could be a target of the equine influenza virus.     

Risk factors for disease associated with influenza virus infections during three epidemics in horses

OBJECTIVE: To identify risk factors associated with respiratory tract disease in horses during 3 epidemics caused by influenza virus infections. DESIGN: Cross-sectional and prospective longitudinal observational studies. ANIMALS: 1,163 horses stabled at a Thoroughbred racetrack. PROCEDURES: Investigations were conducted during a 3-year period. An epidemic of respiratory tract disease caused by influenza virus infections was identified in each year. Routine observations and physical examinations were used to classify horses' disease status. Data were analyzed to identify factors associated with development of disease. RESULTS: Results were quite similar among the epidemics. Concentrations of serum antibodies against influenza virus and age were strongly associated with risk of disease; young horses and those with low antibody concentrations had the highest risk of disease. Calculation of population attributable fractions suggested that respiratory tract disease would have been prevented in 25% of affected horses of all horses had high serum antibody concentrations prior to exposure. However, recent history of vaccination was not associated with reduction in disease risk. Exercise ponies had greater risk of disease than racehorses, which was likely attributable to frequent horse-to-horse contact. CONCLUSIONS AND CLINICAL RELEVANCE: Particular attention should be paid to young horses, those with low serum antibody concentrations, and horses that have frequent contact with other horses when designing and implementing control programs for respiratory tract disease caused by influenza virus infections. It appears that control programs should not rely on the efficacy of commercial vaccines to substantially reduce the risk of disease caused by influenza virus infections.     

Occurrence of infectious upper respiratory tract disease and response to vaccination in horses on six sentinel premises in northern Colorado

REASONS FOR PERFORMING STUDY: Horses vaccinated against common agents of infectious upper respiratory disease (IURD) may not have detectable serum antibody and may not be protected from clinical disease. OBJECTIVES: The objectives of this study were to 1) investigate the serological response of horses to vaccination against influenza virus (H3N8 and H7N7) and equine herpesviruses (EHV) in a field setting and 2) evaluate associations among vaccination status, serum antibody concentrations, and occurrences of IURD in monitored horses. METHODS: In this study, horses on 6 Colorado premises were vaccinated parenterally against influenza virus and EHV, and serological response evaluated. Horses were monitored, and biological samples collected from individuals with clinical IURD and control horses. RESULTS: Of 173 horses, 61 (35.3%), 21 (12.1%) and 4 (2.3%) seroconverted in response to vaccination against EHV, influenza virus H7N7 and influenza virus H3N8, respectively. CONCLUSIONS: Outbreaks of IURD in study horses were associated with influenza virus H3N8 and Streptococcus equi infection, and serological response to vaccination with conventional products was poor. POTENTIAL RELEVANCE: These results confirm that horses may not respond with detectable serological responses to conventional vaccination against common respiratory viruses and, therefore, suggest that alternate methods of protecting horses against common respiratory viruses should be sought.     

Serological observations on an epidemic of equine influenza in India

During the epidemic of equine influenza which occurred in India in 1987, serum samples were collected at late acute/early convalescent phase (7–9 days), at 5 weeks and at 18–23 weeks after onset of illness, from six affected horses from the Union Territory of Changigarh, and Nawanshahr, Punjab State, India, and were examined for antibodies to A/eq-1 and A/eq-2 influenza viruses by hemagglutination inhibition (HI) tests. It was found that the antibody response to A/eq-1 virus strains, Ludhiana/87 and Prague/56, was stronger and antibodies persisted at high levels in four animals. The fifth animal showed a diagnostic decrease in HI titers while the sixth animal seroconverted. The corresponding HI titers to A/eq-2/Ludhiana virus showed a 4-fold decrease in all six animals.Another nine equine animals in the single convalescent serum samples had detectable or high HI titers against A/eq-1 and A/eq-2 viruses.In serum samples from horses and ponies, taken 5 weeks to 9 months after onset of illness, little or no difference in antibody titers to A/eq-2/Miami/63 and A/eq-2/Fontainebleau/79 strains was found.It seems clear that the antibody titers that ensued were indicative of recent influenza infections. Apparently, two distinct equine influenza viruses, A/eq-1 and A/eq-2, were involved during the epidemic, infecting the equine animals simultaneously in the region.     

Formulation with CpG ODN enhances antibody responses to an equine influenza virus vaccine

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty na?ve horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.     

Mycoplasma felis pleuritis in two show-jumper horses.

Mycoplasma felis was identified as the cause of acute pleuritis in 2 show-jumping horses. The pleural exudate was proteinaceous, contained large numbers of neutrophils, and had a markedly increased lactate concentration. M. felis was isolated in pure culture from pleural fluid. Rising serum antibody titers to M. felis as well as a precipitous decline in titers to equine influenza virus were demonstrated in both horses. Pleural effusion in both horses and a pneumothorax detected in one of the horses resolved following a single drainage of pleural fluid and intravenous fluid, antibiotic, and analgesic therapy.     

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.     

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.     

Effect of age and pregnancy on the antibody and cell-mediated immune responses of horses to equine herpesvirus 1.

The cell-mediated immune response and antibody response of horses of varying ages and of pregnant horses to equine herpesvirus 1 antigen were examined. Six to eight month old horses showed either no increase or slight increases in anti-equine herpesvirus 1 serum neutralizing antibody following vaccination and revaccination with a modified live equine herpesvirus 1 vaccine. However, these same horses showed a marked increase in the cell-mediated immune response to equine herpesvirus 1 as measured by the lymphocyte transformation test. Eighteen to 21 month old horses showed four to 64-fold increases in anti-equine herpesvirus 1 serum neutralizing antibody titer following vaccination, but the cell-mediated immune response to equine herpesvirus 1 was low or absent. Only after revaccination did they show an increased cell-mediated immune response to equine herpesvirus 1. The cell-mediated immune response of mares in the latter stages of pregnancy to equine herpesivurs 1 was suppressed although antibody titers increased as much as 16-fold following exposure to virulent equine herpesvirus 1.     

Use of recombinant modified vaccinia Ankara viral vectors for equine influenza vaccination

Recombinant modified vaccinia Ankara (MVA) vectors expressing equine influenza virus genes were constructed and evaluated for use in equine vaccination. Two strains of recombinant MVA, expressing either hemagglutinin (HA) or nucleoprotein (NP) genes were constructed. Each influenza virus gene was cloned from A/equine/Kentucky/1/81 (Eq/Ky) into an MVA construction plasmid, and was introduced to the deletion III locus of the wild type MVA genome by homologous recombination. Recombinant viruses were plaque purified, and antigen expression was confirmed by immunostaining. Two ponies were primed by vaccination with 50 microg HA-DNA and two ponies were vaccinated with 50 microg NP-DNA using the PowderJect XR research device. Six and 10 weeks later, ponies were immunized with 2 x 10(9) infectious units of recombinant MVA encoding the homologous influenza antigen, equally divided between intramuscular and intradermal sites in the neck. A marked rise in influenza virus-specific IgGa and IgGb serum antibody titers was detected following administration of MVA boosters with both HA and NP antigens. Influenza virus-specific lymphoproliferative responses and IFN-gamma mRNA production were also strongly elicited by both antigens. This study demonstrates the facility with which recombinant MVA viruses expressing defined pathogen genes can be constructed, and provides preliminary evidence of the immunogenicity and safety of these vectors in the horse.     

Antibody isotype responses in the serum and respiratory tract to primary and secondary infections with equine influenza virus (H3N8)

Serum antibody (IgGab, IgM and IgA) responses to primary and secondary infection with influenza A/equine/Newmarket/79 (H3N8) by nebulised aerosol were compared with local (nasopharyngeal and tracheal) antibody responses in ponies. Circulating IgGab antibody was of long duration after primary infection, whereas IgM responses were short-lived after both primary and secondary infections. The antigenic stimulation of secondary infection with equine influenza was sufficient to induce elevations of serum IgM and IgA in the presence of high levels of circulating IgGab. These results support the potential of virus-specific IgM measurement for the detection of recent exposure to virus in horses which have high levels of circulating IgGab. Unlike serum IgGab, nasal and tracheal wash antibody of this isotype did not show long duration after primary infection, but local antibody memory was demonstrated by anamnestic responses on rechallenge. Nasopharyngeal IgA developed later than IgGab and IgM, and was more durable.