Equine influenza, a contagious disease resulting from infection with influenza A viruses (subtype H7N7 or H3N8), is considered the most important viral respiratory disease in the equine industry.1 The disease is characterized in influenza immunologically naïve horses by pyrexia, signs of depression, coughing, and nasal discharge and is often complicated by secondary bacterial infections that can lead to pneumonia and death.1,2 The nature and severity of clinical signs, which develop 1 to 5 days after infection, depend on the horse's immunologic status, the infecting viral dose, the route of inoculation, and viral characteristics.3-5 The short incubation period and persistent cough contribute to the rapid spread of the disease. Morbidity can be as high as 100% in a susceptible group of horses. Within the past 20 years, all major outbreaks of equine influenza were caused by the subtype H3N8.6 This subtype has the following 2 divergent lineages: the American lineage, which is widespread, and the Eurasian lineage, which is currently thought to be confined to the European continent.7,8 However, with the expanding population of sporting horses in developed countries, where travel and population mixing occur routinely, horses in the United States are at risk of contracting the European strain as well. Results from experimental studies and field observations indicate that the viruses from the 2 lineages are sufficiently different to adversely affect vaccine efficacy,9 and as a result, the OIE currently recommends including representative viruses of both lineages in equine influenza vaccines. Researchers believe the H7N7 viruses to be extinct, and in 2000, the Expert Surveillance Panel of the OIE stated that no epidemiologic evidence exists to support inclusion of H7N7 virus in current vaccines, as was practiced historically.10
Vaccination and management regimens are critical for the prevention and control of equine influenza.11 Since the 1960s, inactivated whole-virus vaccines or surface antigen vaccines (split vaccines) have been used to control equine influenza.12 Although widely used, vaccine failures occurred, resulting in significant losses in the equine industry.13-15 For example, an outbreak in the spring of 2003 occurred among unvaccinated horses and those vaccinated with an inactivated vaccine, causing substantial morbidity with > 1,300 horses affected in the United Kingdom.16 Many infected horses in this outbreak had been vaccinated recently with an inactivated vaccine that contained representative viruses from the European and American lineages. Virus characterization revealed the 2003 outbreak viruses to be closely related to those circulating in North America in the previous year, and for the first time, the virus from the Florida sublineage of American viruses was isolated in the United Kingdom.16
To improve current equine influenza vaccines, new strategies have focused on the development of vaccines that, upon administration, closely mimic natural infection and therefore are expected to provide superior protection.17 In 2003, 2 modified-live rCP-EIV vaccinesa,b from the same manufacturer were licensed for use in Europe.
These rCP-EIV vaccines each contain 2 canarypox vectors that express the HA gene of influenza A/eq/Newmarket/2/93 (H3N8) and A/eq/Kentucky/94 (H3N8) viruses. We have recently demonstrated that vaccination with one of these productsb was effective in protecting influenza immunologically naïve horses against a challenge with the American lineage virus that caused the latest outbreak in the United Kingdom.18 The purpose of the study reported here was to assess in influenzanaïve horses the onset and duration of immunity of the modified-live rCP-EIV vaccine against challenge with a representative virus of the European lineage of influenza H3N8 viruses.
Materials and Methods
rCP-EIV vaccine—A freeze-dried vaccinea (serial Nos. 0RM 715 5B26, 5M12, and 5Q15) containing 2 modified-live canarypox virus recombinantsc that expressed the HA gene of the influenza H3N8 virus strains A/eq/Newmarket/2/93 (vCP1533) and A/eq/Kentucky/94 (vCP1529) was used. The rCP-EIV vaccines were reconstituted with 1 dose (1 mL) of tetanus toxoid diluent immediately prior to use to contain ≤ 6.7 log10 50% fluorescence assay infectious doses for vCP1529 and vCP1533.
Animals—Forty-nine male Welsh Mountain Ponies that were seronegative for equine influenza virus and were between 1 and 3 years of age were used. Ponies were housed on a designated farm. The institutional animal care and use committee approved the experimental protocol. Ponies were fed a diet of hay and proprietary horse feed, and fresh water was available ad libitum. Microchips were used to identify the ponies; additionally, a tail tag was placed prior to the animals being challenged.
Challenge virus—Influenza strain A/eq/Sussex/89 (H3N8) was used for challenge in these studies. The virus represents the Eurasian lineage of influenza H3N8 viruses. The strain was isolated at the Animal Health Trust in November 1989 from a nasopharyngeal swab taken from an infected horse in Sussex, United Kingdom (laboratory accession No. 95839). After 1 passage in horses, the recovered virus underwent 3 passages in embryonated chicken eggs to provide the challenge stock. The infectivity of the virus stock was measured by titration in embryonated chicken eggs and expressed as EID50 per milliliter.
Experimental design—Separate challenge experiments were performed to study onset and duration of immunity provided by the rCP-EIV vaccine. In trial 1, vaccinated and control ponies were challenged 2 weeks after completion of the primary vaccination program consisting of 2 doses. In trial 2, vaccinated and control ponies were challenged either 5 months after 2 doses of the rCP-EIV vaccine (trial 2a) or 1 year after the first boosting dose of the rCP-EIV vaccine administered 5 months after completion of the primary vaccination program (trial 2b). All vaccines were administered by deep IM injection in the neck.
Vaccination protocol for trial 1—Fourteen ponies were assigned to 2 groups (1 and 2) in a randomized manner with each group containing 6 and 8 ponies, respectively. Ponies from group 1 received a primary course of 2 doses of the rCP-EIV vaccine at an interval of 5 weeks on day 0 and day 35; group 2 ponies were not vaccinated and served as control animals.
Vaccination protocol for trial 2—Thirty-five ponies were randomly assigned to 4 groups (A, B, C, and D). Group A contained 14 ponies, with all the other groups containing 7 ponies each. Ponies of group A received a primary course of 2 doses of the rCP-EIV vaccine administered 5 weeks apart on day 0 and day 35; ponies in group C received a primary course of 2 doses of the rCP-EIV vaccine on day 0 and day 35 followed by a boosting dose of the vaccine 5 months later (day 175). Ponies in groups B and D acted as contemporary controls for group A and C ponies, respectively, and were vaccinated with tetanus toxoid diluent when ponies in groups A and C received rCP-EIV vaccines.
Challenge protocol—Ponies were exposed by use of a nebulizerd to an aerosol generated from approximately 20 mL of allantoic fluid containing a total of 107.3 EID50 of influenza virus A/eq/Sussex/89. After the aerosol had been generated, ponies were kept in the challenge box for ≥ 60 additional minutes. Ponies from trial 1 were challenged 2 weeks after the completion of the primary program of vaccination. Group A and B ponies from trial 2 were challenged 5 months after completion of the primary vaccination program. Group C and D ponies from trial 2 were challenged 12 months after the third dose of rCP-EIV vaccine administered 5 months after the primary vaccination series.
Serologic methods—Whole-blood samples for serologic measurements were collected from a jugular vein of each pony from trials 1 and 2 at regular intervals. Sera were prepared and stored at 20°C until analysis. Sera were assayed for antibodies by SRH19 with the following representative antigens: influenza A/eq/Newmarket/1/93 (closely related to A/eq/Kentucky/94) and A/eq/Newmarket/2/93 virus antigens. Antibody titers were expressed as the area of hemolysis (mm2). Seroconversion, indicating infection, was taken as an increase in the area of the zone of hemolysis of 25 mm2 or 50%, whichever was the smaller, between samples obtained on the day of challenge and at 2 weeks after challenge.2
Clinical examinations—Ponies were examined daily from the day of challenge until 10 (trial 2a) or 14 (trials 1 and 2b) days after the challenge for signs consistent with equine influenza, including nasal discharge, coughing, dyspnea, signs of depression, and anorexia. Signs were scored by use of a standardized protocol (Appendix). Rectal temperatures were measured on the same days during the afternoon. Rectal temperatures > 38.8°C were considered clinically relevant.
Nasal swab specimens and virus isolation—Nasal swab specimens were taken daily from each pony for 10 (trial 2a) or 14 (trials 1 and 2b) days starting on the first day after the challenge, and the swab extracts were titrated for infectious virus as described.20 The virus titers were expressed as EID50 per milliliter of swab extract.
Data analysis—Serologic responses of the vaccinated groups at various times after they had been vaccinated were reported descriptively. Rectal temperature responses from days 1 to 10 after challenge were analyzed by use of a mixed model with repeated measurements. Rectal temperature on the day of challenge was included in the model as a covariate. If a significant group × day interaction was found, day-to-day pairwise comparisons were made with an F test.
The severity of clinical signs (sickness score) was compared among the groups by assigning ponies to 1 of 2 of the following disease categories: no or mild clinical disease and moderate-to-severe clinical disease as described previously.18 Briefly, the sickness score was calculated by use of the daily scores for each clinical sign on the basis of an algorithm, which gave a double weighting to the scores for dyspnea, signs of depression, and anorexia. Thus, sickness score = (daily score for nasal discharge) + (daily score for cough) + 2 × (daily score for anorexia) + 2 × (daily score for signs of depression) + 2 × (daily score for dyspnea). Each pony was classified according to the most severe daily score recorded during the after-challenge observation period with a score of 0 for no disease, 1 for mild disease, 2 to 4 for moderate disease, and > 4 for severe disease. Differences in the incidence of moderate to severe disease (scores ≥ 2) among the groups were analyzed by use of a Fisher exact test. For data with a normal distribution (Shapiro-Wilks test), the number of days with any clinical sign (daily clinical score > 0) was compared between groups by use of a Student t test. Otherwise, a nonparametric test was performed.
The total number of days during which ponies excreted virus was recorded, and the total amount of virus excreted by ponies was calculated from the area under time-titer curves. Virus excretion between groups was compared by use of the Student t test or Wilcoxon rank sum test. Differences between the groups in the proportion of ponies shedding virus during the period after challenge were analyzed by use of the Fisher exact test.
All data were analyzed by use of computer software programs.e,f Significance was based on the 2-tailed tests of the null hypothesis that resulted in a value of P ≤ 0.05.
Results
Serologic responses to vaccination—None of the ponies had detectable serum antibodies against influenza H3N8 virus at the start of the trial, and none of the unvaccinated ponies seroconverted to influenza virus prior to the time of challenge, indicating that no field infection with influenza H3N8 virus occurred. The antibody responses to vaccination in terms of peak titers and kinetics were similar for both influenza A/eq/Newmarket/2/93 and A/eq/Kentucky/94 viruses, and only the results for the first are presented. In both trials, all ponies responded to vaccination with high serum antibody titers 2 weeks after the first vaccination. Two weeks after the second vaccination, mean serum antibody titers peaked at 167 mm2 (range, 147 to 186 mm2) and 221 mm2 (range, 172 to 273 mm2) for ponies in trials 1 and 2 (groups A and C combined), respectively. Detectable serum antibody titers were present 5 months after the second vaccination and before the third rCP-EIV vaccine dose in all ponies from trial 2, with a mean titer of 72 mm2 (range, 20 to 124 mm2). All ponies from group C (trial 2b) developed a strong booster response to the third dose of rCP-EIV vaccine (5 months after the second vaccine dose) with mean SRH serum antibody titers of 182 mm2 (range, 151 to 233 mm2) at 4 weeks after vaccination. The titer of 182 mm2 was comparable to the mean titer found at 4 weeks after second vaccination (176 mm2; range, 113 to 232 mm2). In the 12 months following the third dose of the rCP-EIV vaccine, serum antibody titers declined slowly so that by the time of challenge, the mean antibody titer had decreased to 110 mm2 (range, 75 to 172 mm2; Figure 1).
Antibody responses of group C ponies (trial 2b) against influenza A/eq/Newmarket/2/93 virus after vaccination with rCP-EIV vaccine (V) and challenge (ch).
Citation: American Journal of Veterinary Research 68, 2; 10.2460/ajvr.68.2.213
Serologic responses to challenge—All control ponies from trials 1 and 2 seroconverted to influenza A/eq/Newmarket/2/93 virus after challenge, indicating infection and virus replication. Four of 6 vaccinated ponies from trial 1, all vaccinated ponies from trial 2a, and 6 of 7 vaccinated ponies from trial 2b seroconverted to influenza A/eq/Newmarket/2/93 virus. The 3 vaccinated ponies (2 from trial 1 and 1 from trial 2b) that did not seroconvert had prechallenge SRH antibody titers of 174, 187, and 172 mm2, respectively.
Rectal temperatures—Control ponies from trials 1 and 2a had a biphasic increase in rectal temperature that peaked on day 2 and again between days 5 and 6 (Figure 2). The peak temperature response in control ponies from trial 2b occurred on day 4 after challenge. Seven of 8 control ponies from trial 1 developed fever for a mean duration of 4.0 days with temperatures as high as 40.5°C. None of the vaccinated ponies from trial 1 developed rectal temperatures of > 38.8°C. In trial 2a, 6 of 7 control ponies became pyrexic (maximum, 40.9°C), whereas 9 of 14 vaccinated ponies developed transient fever (maximum, 40.4°C) when challenged 5 months after the vaccinated ponies completed the primary vaccination course. Fever lasted for a mean of 5.4 and 0.9 days in the control and vaccinated ponies, respectively. All control ponies in trial 2b developed febrile responses after challenge (maximum, 41.1°C) at 1 year after the third vaccination of the treatment group that lasted for 4 to 9 days with a mean duration of 5.3 days. Only 3 of 7 vaccinated ponies developed mild transient fever (maximum, 39.1°C) that lasted for 1 or 2 days. A significant (P < 0.001) group × day interaction was found for all 3 challenges as a result of increased rectal temperatures in control ponies. Overall, the mean rectal temperatures between days 2 and 7 after challenge in the vaccinated ponies were significantly (P < 0.001) lower than those in control ponies for all 3 trials.
Mean rectal temperatures from days 0 to 10 after challenge in vaccinated and control ponies at 2 weeks (A) and 5 months (B) after 2 doses of rCP-EIV vaccine and at 12 months (C) after the third dose of rCP-EIV vaccine.
Citation: American Journal of Veterinary Research 68, 2; 10.2460/ajvr.68.2.213
Clinical signs—All control ponies from trial 1 developed clinical signs typical of influenza; coughing and nasal discharge were observed in all 8 control ponies for a mean duration of 5.0 days (range, 1 to 8 days) and 5.4 days (range, 2 to 10 days), respectively (Table 1). Two ponies had signs of depression, and 3 ponies developed anorexia. The only clinical sign observed in the vaccinated ponies was serous nasal discharge (maximum score of 1) in 3 ponies lasting 1 day only.
Incidence and duration of clinical disease in vaccinated and unvaccinated (control) ponies after challenge with influenza A/eq/Sussex/89 virus.
| Trial | Group | Challenge | Duration | P value* | Incidence (No. of ponies) | P value* | |
|---|---|---|---|---|---|---|---|
| Mean No. of days (range) | None to mild | Moderate to severe | |||||
| 1 | V (n = 6) | 2 WPV-2 | 0.5 (0–1) | 0.002 | 6 | 0 | < 0.001 |
| C (8) | 7.1 (3–11) | 0 | 8 | ||||
| 2a | V (14) | 5 MPV-2 | 1.0 (0–4) | 0.003 | 12 | 2 | 0.003 |
| C (7) | 6.4 (0–9) | 1 | 6 | ||||
| 2b | V (7) | 12 MPV-3 | 1.1 (0–3) | 0.002 | 7 | 0 | 0.005 |
| C (7) | 6.7 (4–12) | 1 | 6 | ||||
Values of P < 0.05 are significant.
V = Vaccinated. C = Control. 2 WPV-2 = Two weeks after the second vaccination. 5 MPV-2 = Five months after the second vaccination. 12 MPV-3 = Twelve months after the third vaccination.
After challenge 5 months following the second vaccination of the treatment group (trial 2a), 6 of 7 control ponies coughed for a mean duration of 6.2 days (range, 5 to 8 days) and had nasal discharge for a mean duration of 5.2 days (range, 4 to 6 days). Five ponies in the control group had signs of depression, and 1 pony died on day 9 after challenge. Necropsy examination revealed evidence of rhinitis, necrotizing and exudative bronchiolitis, and alveolitis consistent with mixed viral and bacterial infection. Although 1 control pony did not have any clinical signs following challenge, it was fully susceptible to infection as was shown by the virus isolation data. Vaccinated ponies had only mild transient disease. Coughing was observed in only 2 of 12 vaccinated ponies lasting for 1 and 4 days, respectively. Serous nasal discharge appeared only sporadically in 5 ponies. One pony had signs of depression on day 3 after challenge.
All control ponies challenged 12 months after the third vaccination of the treatment group (trial 2b) had nasal discharge for a mean duration of 4.0 days (range, 2 to 8 days) and coughed for a mean duration of 5.4 days (range, 2 to 11 days). Three ponies became dyspneic on days 5 to 7 after challenge, 1 pony was anorexic on day 6, and another pony had signs of depression on day 13 after challenge. The only clinical sign observed in the vaccinated ponies was sporadic serous nasal discharge that lasted for 1 to 3 days. The incidence of moderate to severe disease was significantly lower in the vaccinated groups than in the control groups (P < 0.001, P = 0.003, and P = 0.005 at 2 weeks and 5 months after 2 doses and 12 months after the third dose of vaccine, respectively).
Mean number of days with any clinical sign (daily clinical score > 0) was significantly lower in the vaccinated groups, compared with the control groups (P = 0.002, P = 0.003, and P = 0.002 at 2 weeks and 5 months after 2 doses and 12 months after the third dose of vaccine, respectively; Table 1).
Virus excretion—All control ponies from trials 1, 2a, and 2b shed virus following challenge for a mean duration of 5.9 (range, 5 to 7 days), 5.1 (range, 4 to 6 days), and 4.7 days (range, 2 to 6 days), respectively (Table 2). Virus was isolated on significantly fewer days among vaccinated ponies, compared with control ponies, following challenge at 2 weeks and 5 months after the second vaccination and at 12 months after the third vaccination (P = 0.002, P < 0.001, and P = 0.002, respectively). After each challenge, the total amount of virus isolated from swab specimens obtained from vaccinated ponies was significantly less than that obtained from the control samples (P = 0.002, P = 0.001, and P = 0.002 at 2 weeks and 5 months after 2 doses and 12 months after the third dose of vaccine, respectively). The proportion of ponies shedding virus after challenge was significantly lower in the vaccinated ponies from trials 1 and 2b than in control ponies (P = 0.003 and P = 0.005, respectively). No statistical analysis was performed on the incidence of shedding in trial 2a ponies because all of those ponies shed virus.
Virus isolation from swab specimens taken from vaccinated and unvaccinated ponies after challenge with influenza A/eq/Sussex/89 virus.
| Trial | Group | Challenge | No. of ponies with positive results | Mean No. of days of virus secretion (range) | Mean amount of virus excreted (AUC [EID50 log10]) |
|---|---|---|---|---|---|
| 1 | V (n = 6) | 2 WPV-2 | 1 | 0.5 (0–3) | 1.4 (0–8.5) |
| C (8) | 8 | 5.9 (5–7) | 17.1 (15.5–19.5) | ||
| P value* | 0.003 | 0.002 | 0.002 | ||
| 2a | V (14) | 5 MPV-2 | 14 | 3.2 (2–5) | 9.0 (4.5–17.5) |
| C (7) | 7 | 5.1 (4–6) | 16.4 (14.5–17.8) | ||
| P value* | ND | < 0.001 | 0.001 | ||
| 2b | V (7) | 12 MPV-3 | 1 | 0.4 (0–3) | 1.1 (0–8) |
| C (7) | 7 | 4.7 (2–6) | 13.9 (3.5–19.5) | ||
| P value* | 0.005 | 0.002 | 0.002 |
AUC = Area under the time-titer curves. ND = Not done.
See Table 1 for remainder of key.
Discussion
Until now, conventional killed vaccines against influenza for horses have dominated the market. The protection provided by these vaccines is strongly correlated with the concentrations of circulating antibodies against HA, as long as the vaccine and challenge strains are genetically and antigenically similar.21 Historically, conventional inactivated vaccines on the market in the United States have been licensed as a 2-dose regimen with a recommendation for annual revaccination. To our knowledge, no peer-reviewed published experimental challenge data exist supporting this booster claim, and the poor durability of the immune response of unprimed horses to 2 doses of killed vaccine suggests that it would be unwise to wait 12 months before administering a booster vaccination.22 This conclusion is further supported by field experience showing that killed vaccines do not confer protection beyond 6 months after administration of the last dose,23 while in 1 study,14 a commercial killed vaccine did not protect horses with a history of recent vaccination. Although published evidence20,24 exists demonstrating long-term protection against experimental challenges following a series of 3 vaccinations with killed vaccines produced and commercialized in Europe, a critical lag in immunity (immunity gap) has been observed during the period between the second and third vaccination.25 Serum antibody titers may decrease to a substantially low enough concentration prior to the third revaccination to leave the horse or pony potentially open for infection during this time. Therefore, vaccination protocols, particularly vaccination intervals, should not be generalized but rather be based on the durability of immune response demonstrated in experimental challenge trials20,24,26,27 or in controlled field trials.14
We have constructed 2 recombinant canarypox viruses expressing the HA of 2 epidemiologically relevant equine influenza H3N8 strains.g The HA gene product is essential for viral attachment and entry into cells and therefore is an ideal candidate for use in equine influenza vaccination strategies, as was previously demonstrated with recombinant vaccinia,21 DNA,28 and recombinant modified vaccinia Ankara vaccines29 coding for the HA gene of equine influenza H3. Results of our study indicate that the rCP-EIV vaccine is effective in protecting immunologically naïve ponies against influenza virus. The applied vaccination protocol provided clinical and virologic protection against a severe challenge with influenza virus A/eq/Sussex/89 at 2 weeks and 5 months after completion of the primary vaccination course and at 12 months after the first boosting dose. The Sussex/89 virus was used for challenge because of its established virulence characteristicsh and its antigenic similarity to the current OIE-recommended Eurasian vaccine strain (A/eq/Newmarket/2/93), as was shown by results of nucleotide and amino acid analysis of the HA1 portion of the HA gene.7 Our results from the 3 independent challenge studies justify this choice and demonstrate that the Sussex/89 challenge strain consistently produces severe clinical disease in unvaccinated ponies. The rCP-EIV vaccine provided the greatest protection 2 weeks after the second dose of vaccine. Sporadic serous nasal discharge was the only clinical sign observed in the vaccinated ponies, whereas virus shedding was almost completely suppressed. Our results support those of a previous study18 in which it was concluded that the rCP-EIV vaccine provides early protection in vaccinated ponies challenged 2 weeks after either 1 or 2 doses. The modified-live rCP-EIV vaccine induced protective immunity that remained present at 5 months after the second dose, the time at which it is normally recommended that a booster dose be administered. Under normal vaccination practices, the 5-month time point represents a period of waning immunity. Importantly, in some training centers, this time frame coincides with the start of equine training programs, a recognized risk period for transmitting influenza virus.30 Similar results have been obtained in a study27 on a cold-adapted modified-live influenza vaccine. In that study, horses that were vaccinated with only 1 dose of vaccine had significantly lower clinical scores, had smaller increases in rectal temperature, and shed significantly less virus over fewer days when challenged 6 months later. It is apparent from the data of our study and that of Townsend et al27 that the timing of booster vaccination is of crucial importance for horses being prepared for sales or training and preferably should be administered no later than 2 weeks prior to the event. Protection data obtained after the third dose of the rCP-EIV vaccine support yearly boosting intervals with this vaccine, although more frequent vaccination may be required by some organizations. Although vaccination clearly reduced the duration and severity of clinical signs and the amount of virus shed following each challenge, the vaccine did not provide sterile immunity to most ponies and therefore will not allow complete control of disease spread. Because the recovered virus titers in nasal secretions from vaccinated ponies were higher than those reported from naturally infected horses,20 it is probable that these ponies would represent an infection risk to other naïve horses. However, estimates of vaccine efficacy in these experimental infection models are probably conservative; it is likely that ponies were exposed to a virus challenge dose at least 1,000 times as great as that found in the field.20,27 Therefore, we feel comfortable in stating that the rCP-EIV vaccine would stimulate long-lasting immunity under field conditions and aid in reducing transmission of virus to allow better control of the disease.
Effective influenza vaccines will likely need to stimulate mucosal IgA on one hand to prevent virus infection and humoral IgGa and IgGb responses and cell-mediated responses on the other hand to prevent an infection developing and eliminate virus-infected cells.28 Our study focused on the protection conferred against challenge infection, and no attempt was made to assess the underlying mechanisms of protection. We have recently provided evidence that the rCP-EIV vaccine stimulates cell-mediated immunity, as it efficiently improved the T-cell response to equine influenza virus after challenge.31 Further studies are required to assess whether the rCP-EIV vaccine is capable of inducing secretory IgA at the mucosal respiratory surface.
In summary, results of our study reveal that comprehensive protection against virus challenge develops following vaccination with parenteral administration of the rCP-EIV vaccine. Of particular interest was the protection at 5 months after the second dose, indicating that vaccination essentially closed the immunity gap between the second and third doses. The rCP-EIV vaccine also provided at least a 1-year duration of immunity after third vaccination, further supporting the recommended vaccination schedule. On the basis of the results of our study and those of Edlund Toulemonde et al,18 the modified-live rCP-EIV vaccinesa,b will provide veterinarians with a powerful tool in controlling influenza infections.
ABBREVIATIONS
| OIE | Office International des Épizooties |
| rCP-EIV vaccine | Canarypox-vectored recombinant vaccine for equine influenza virus |
| HA | Hemagglutinin |
| EID50 | 50% egg infectious dose |
| SRH | Single radial hemolysis |
PROTEQFLU-Te, Merial, Lyon, France.
PROTEQFLU, Merial, Lyon, France.
ALVAC, Connaught Technology Corp, Greenville, Del.
ULTRA-NEB 2000, Devilbiss Health Care Inc, Somerset, Pa.
SAS-PC2, version 8.2, SAS Institute Inc, Cary, NC.
STATGRAPHICS Plus for Windows, Manugistics Group Inc, Rockville, Md.
Minke JM, Audonnet JC, Jessett D. Canarypox as a vector for influenza and EHV-1 genes: challenges and rewards (abstr), in Proceedings. 2nd Int Vet Vaccines Diagn Conf 2000;36.
Daly JM, Newton JR, Park A. Current epidemiology and evolution of influenza A H3N8 strains (abstr), in Proceedings. 9th Cong World Equine Vet Assoc 2006;4–9.
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Appendix
Scoring system for equine influenza.
| Clinical sign | Degree | Score |
|---|---|---|
| Coughing | No coughing | 0 |
| Coughing 1 time in 20 minutes | 1 | |
| Coughing ≥ 2 times in 20 minutes | 2 | |
| Nasal discharge | No discharge | 0 |
| Slight serous discharge | 1 | |
| Moderate discharge | 2 | |
| Severe copious discharge | 3 | |
| Dyspnea | Normal respiration | 0 |
| Abnormal respiration | 1 | |
| Depression | No signs of depression | 0 |
| Signs of depression (lethargy) | 1 | |
| Anorexia | Absent | 0 |
| Present | 1 |
