The anamnestic serologic response to vaccination with a canarypox virus-vectored recombinant West Nile virus (WNV) vaccine in horses previously vaccinated with an inactivated WNV vaccine

A new recombinant West Nile virus (WNV) vaccine has been licensed for use in horses. Prior to the availability of the recombinant vaccine in 2004, the only equine WNV vaccine available on the market had been an inactivated vaccine. Since the recombinant vaccine only expresses selected viral genes, the question could be posed as to whether a single dose of the recombinant vaccine would be effective in producing an anamnestic serologic response in horses previously vaccinated with an inactivated WNV vaccine. In this study we demonstrate that vaccination of horses with a canarypox-vectored recombinant vaccine, under field conditions, results in a marked anamnestic response in horses previously vaccinated with an inactivated WNV vaccine.     

Detection of antibodies to West Nile virus in equine sera using microsphere immunoassay.

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

Investigation of antigen specific lymphocyte responses in healthy horses vaccinated with an inactivated West Nile virus vaccine

West Nile virus (WNV) is a single-stranded, enveloped RNA virus capable of causing encephalitic disease in horses. Unvaccinated horses are at risk for developing WNV disease in endemic geographic regions. Effective vaccination reduces disease frequency and diminishes disease severity in vaccinated individuals that become infected with WNV. Recent data indicate CD4+ lymphocytes are required for effective protection against disease; in particular, cross talk between CD4+ and CD8+ lymphocytes must be functional. The objective of this project was to investigate immune responses in horses throughout a series of three vaccinations using a commercial inactivated vaccine under natural conditions. Immune responses to vaccination were determined by neutralizing antibody titers with plaque reduction neutralization test (PRNT), IgM titer (capture ELISA), WNV specific antibody Ig subclass responses, WNV lymphocyte proliferative responses and intracellular cytokine expression. Horses were vaccinated with a series of three vaccines at 3-week intervals using an inactivated product. An initial measure of immune activation following vaccination was determined by evaluating changes in lymphocyte cytokine expression. Interferon (IFN) gamma and interleukin (IL)-4 expressing CD4+ lymphocytes significantly increased 14 days following initial vaccination compared to unvaccinated horses (P<0.05). IFN-gamma expressing CD8+ lymphocytes also increased and remained elevated for 110 days. Antigen specific lymphocyte proliferative responses were significantly increased up to 90 days following the third vaccination (P<0.05). As expected, vaccinated horses produced increased neutralizing antibody based on PRNT data and WNV antigen-specific Ig subclass responses compared with unvaccinated horses (P<0.05). Our data indicate that WNV vaccination with an inactivated product effectively induced an antigen-specific antibody responses, as well as CD4+ and CD8+ lymphocyte activation.     

Immunologic responses to West Nile virus in vaccinated and clinically affected horses

OBJECTIVE: To compare neutralizing antibody response between horses vaccinated against West Nile virus (WNV) and horses that survived naturally occurring infection. DESIGN: Cross-sectional observational study. ANIMALS: 187 horses vaccinated with a killed WNV vaccine and 37 horses with confirmed clinical WNV infection. PROCEDURE: Serum was collected from vaccinated horses prior to and 4 to 6 weeks after completion of an initial vaccination series (2 doses) and 5 to 7 months later. Serum was collected from affected horses 4 to 6 weeks after laboratory diagnosis of infection and 5 to 7 months after the first sample was obtained. The IgM capture ELISA, plaque reduction neutralization test (PRNT), and microtiter virus neutralization test were used. RESULTS: All affected horses had PRNT titers > or = 1:100 at 4 to 6 weeks after onset of disease, and 90% (18/20) maintained this titer for 5 to 7 months. After the second vaccination, 67% of vaccinated horses had PRNT titers > or = 1:100 and 14% had titers < 1:10. Five to 7 months later, 33% (28/84) of vaccinated horses had PRNT titers > or = 1:100, whereas 29% (24/84) had titers < 1:10. Vaccinated and clinically affected horses' end point titers had decreased by 5 to 7 months after vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: A portion of horses vaccinated against WNV may respond poorly. Vaccination every 6 months may be indicated in certain horses and in areas of high vector activity. Other preventative methods such as mosquito control are warranted to prevent WNV infection in horses.     

Safety of an attenuated West Nile virus vaccine, live Flavivirus chimera in horses

REASON FOR PERFORMING STUDY: West Nile virus (WNV) infection is endemic and able to cause disease in naive hosts. It is necessary therefore to evaluate the safety of new vaccines. OBJECTIVES: To establish: 1) the safety of a modified live Flavivirus/West Nile virus (WN-FV) chimera by administration of an overdose and testing for shed of vaccine virus and spread to uninoculated sentinel horses; 2) that this vaccine did not become pathogenic once passaged in horses; and 3) vaccine safety under field conditions. METHODS: There were 3 protocols: 1) In the overdose/shed and spread study, horses were vaccinated with a 100x immunogenicity overdose of WN-FV chimera vaccine and housed with sentinel horses. 2) A reversion to virulence study, where horses were vaccinated with a 20x immunogenicity overdose of WN-FV chimera vaccine. Horses in both studies were evaluated for abnormal health conditions and samples obtained to detect virus, seroconversion and dissemination into tissues. 3) In a field safety test 919 healthy horses of various ages, breeds and sex were used. RESULTS: Vaccination did not result in site or systemic reactions in either experimental or field-injected horses. There was no shed of vaccine virus, no detection of vaccine virus into tissue and no reversion to virulence with passage. CONCLUSIONS: WN-FV chimera vaccine is safe to use in horses with no evidence of ill effects from very high doses of vaccine. There was no evidence of reversion to virulence. In addition, administration of this vaccine to several hundred horses that may have been previously exposed to WNV or WNV vaccine resulted in no untoward reactions. POTENTIAL RELEVANCE: These studies establish that this live attenuated Flavivirus chimera is safe to use for immunoprophylaxis against WNV disease in horses.     

Efficacy, duration, and onset of immunogenicity of a West Nile virus vaccine, live Flavivirus chimera, in horses with a clinical disease challenge model

REASON FOR PERFORMING STUDY: West Nile virus (WNF) is a Flavivirus responsible for a life-threatening neurological disease in man and horses. Development of improved vaccines against Flavivirus infections is therefore important. OBJECTIVES: To establish that a single immunogenicity dose of live Flavivirus chimera (WN-FV) vaccine protects horses from the disease and it induces a protective immune response, and to determine the duration of the protective immunity. METHODS: Clinical signs were compared between vaccinated (VACC) and control (CTRL) horses after an intrathecal WNV challenge given at 10 or 28 days, or 12 months post vaccination. RESULTS: Challenge of horses in the immunogenicity study at Day 28 post vaccination resulted in severe clinical signs of WNV infection in 10/10 control (CTRL) compared to 1/20 vaccinated (VACC) horses (P<0.01). None of the VACC horses developed viraemia and minimal histopathology was noted. Duration of immunity (DPI) was established at 12 months post vaccination. Eight of 10 CTRL exhibited severe clinical signs of infection compared to 1 of 9 VACC horses (P<0.05). There was a significant reduction in the occurrence of viraemia and histopathology lesion in VACC horses relative to CTRL horses. Horses challenged at Day 10 post vaccination experienced moderate or severe clinical signs of WNV infection in 3/3 CTRL compared to 5/6 VACC horses (P<0.05). CONCLUSIONS: This novel WN-FV chimera vaccine generates a protective immune response to WNV infection in horses that is demonstrated 10 days after a single vaccination and lasts for up to one year. POTENTIAL RELEVANCE: This is the first USDA licensed equine WNV vaccine to utilise a severe challenge model that produces the same WNV disease observed under field conditions to obtain a label claim for prevention of viraemia and aid in the prevention of WNV disease and encephalitis with a duration of immunity of 12 months.     

Identification of hyperendemic foci of horses with West Nile virus disease in Texas

OBJECTIVE: To determine whether West Nile virus (WNV) disease hyperendemic foci (hot spots) exist within the horse population in Texas and, if detected, to identify the locations. SAMPLE POPULATION: Reports of 1,907 horses with WNV disease in Texas from 2002 to 2004. Procedures: Case data with spatial information from WNV epidemics occurring in 2002 (1,377 horses), 2003 (396 horses), and 2004 (134 horses) were analyzed by use of the spatial scan statistic (Poisson model) and kriging of empirical Bayes smoothed county attack rates to determine locations of horses with WNV disease in which affected horses were consistently (in each of the 3 study years) clustered (hyperendemic foci, or hot spots). RESULTS: 2 WNV hot spots in Texas, an area in northwestern Texas and an area in eastern Texas, were identified with the scan statistic. Risk maps of the WNV epidemics were qualitatively consistent with the hot spots identified. Conclusions and CLINICAL RELEVANCE: WNV hot spots existed within the horse population in Texas (2002 to 2004). Knowledge of disease hot spots allows disease control and prevention programs to be made more efficient through targeted surveillance and education.     

Evaluation of an outbreak of West Nile virus infection in horses: 569 cases (2002)

OBJECTIVE: To characterize an outbreak of West Nile virus (WNV) infection in horses in North Dakota in 2002, evaluate vaccine effectiveness, and determine horse characteristics and clinical signs associated with infection. DESIGN: Retrospective study. ANIMALS: 569 horses. PROCEDURE: Data were obtained from veterinary laboratory records, and a questionnaire was mailed to veterinarians of affected horses. RESULTS: Affected horses were defined as horses with typical clinical signs and seroconversion or positive results of virus isolation; affected horses were detected in 52 of the 53 counties and concentrated in the eastern and northeastern regions of the state. Among affected horses, 27% (n = 152) were vaccinated against WNV, 54% (309) were not, and 19% (108) had unknown vaccination status; 61 % (345) recovered, 22% (126) died, and 17% (98) had unknown outcome. The odds of death among nonvaccinated horses were 3 and 16 times the odds among horses that received only 1 or 2 doses of vaccine and horses that were vaccinated according to manufacturer's recommendations, respectively. Horses with recumbency, caudal paresis, and age > 5 years had higher odds of death, whereas horses with incoordination had lower odds of death, compared with affected horses without these characteristics. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination appears to have beneficial effects regarding infection and death caused by WNV.     

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.     

Clinical West Nile virus infection in 2 horses in western Canada

Two horses had a history of ataxia and weakness or recumbency. One recovered and was diagnosed with West Nile virus (WNV) infection by serologic testing. The other was euthanized; it had meningoencephalomyelitis, WNV was detected by polymerase chain reaction. West Nile virus infection is an emerging disease. Year 2002 is the first year in which cases have been seen in Saskatchewan.     

Surveillance of West Nile virus in horses in Canada: A retrospective study of cases reported to the Canadian Food Inspection Agency from 2003 to 2019

The objectives of the study were to describe the regional and provincial incidence rates and the weekly distribution of 842 reported West Nile virus (WNV) cases in horses in Canada between 2003 and 2019. This study also investigated characteristics of cases reported to the Canadian Food Inspection Agency (CFIA) between 2015 and 2019. The western region (British Columbia, Alberta, Saskatchewan, and Manitoba) had higher incidence rates than the eastern region (Ontario, Quebec, and Atlantic provinces) and overall, Saskatchewan registered the highest incidence. Over the study period, an earlier weekly preliminary onset of WNV cases was observed in the western region. The vast majority of cases were unvaccinated (96%), most cases were Quarter Horses (68%) and the risk of mortality was 31.9%. The findings of this study may be useful in informing veterinary equine practitioners about measures to prevent WNV disease in horses in Canada.     

Evaluation of the efficacy provided by a Recombinant Canarypox-Vectored Equine West Nile Virus vaccine against an experimental West Nile Virus intrathecal challenge in horses

Efficacy of the Recombitek Equine West Nile Virus (WNV) vaccine was evaluated against a WNV intrathecal challenge model that results in WNV-induced clinical disease. Ten vaccinated (twice at days 0 and 35) and 10 control horses were challenged 2 weeks after administration of the second vaccine with a virulent WNV by intrathecal administration. After the challenge, eight of 10 controls developed clinical signs of encephalomyelitis whereas one vaccinate exhibited muscle fasciculation only once. Nine controls and one vaccinate developed a fever. Histopathology revealed mild to moderate nonsuppurative encephalitis in eight controls and one vaccinate. None of the vaccinates and all of the controls developed WNV viremia after challenge. All vaccinated horses developed antibodies to WNV after vaccination. These and results of previous studies demonstrate efficacy of the Recombitek WNV vaccine against WNV-induced clinical disease and natural challenge with WNV-infected mosquitoes.     

Serologic Responses of West Nile Virus Seronegative Mature Horses to West Nile Virus Vaccines

A 42-day study was conducted to assess the impact of three West Nile virus vaccines given either as separate injections or incorporated with their counterpart equine encephalitis and tetanus vaccines on serological responses under field use conditions. Two hundred forty mature, West Nile virus seronegative (<4) horses were followed serologically pre- and postprimary and secondary vaccination with six different vaccination programs, all including West Nile virus antigens. Forty horses were unvaccinated sentinel horses. All vaccines stimulated both a primary and secondary (booster) response to vaccination that was significantly higher than that of seronegative controls. However, inclusion of West Nile virus with equine encephalitis viruses and tetanus toxoid in vaccines had a significant detrimental impact on West Nile virus serum neutralization antibody production to both the primary and secondary vaccinations.     

2009 West Nile disease epidemic in Italy: first evidence of overwintering in Western Europe?

For the second consecutive year a West Nile disease (WND) epidemic has affected Italy causing disease in horses and humans. The infection re-occurred in the same places of the 2008 and moved westerly and southerly involving new areas and regions. The whole genome sequence of the Italian 2009 West Nile disease isolate (WNDV) was compared with those responsible for the 2008 WND outbreaks. The epidemiological findings of the two years of epidemic were compared as well. The high identity between 2008 and 2009 WNV strains (>99%), the earlier virus circulation in 2009 and the re-occurrence of the disease starting from the bordering infected areas reached by the infection in the previous year, strongly support the hypothesis of the overwintering of the virus and the endemisation to local host populations.     

Evaluation of factors associated with positive IgM capture ELISA results in equids with clinical signs compatible with West Nile virus infection: 1,017 cases (2003)

OBJECTIVE: To describe the prevalence of West Nile virus (WNV) infection and evaluate factors associated with positive IgM capture ELISA results in equids with clinical signs compatible with WNV infection. DESIGN: Retrospective case series. SAMPLE POPULATION: Laboratory submission forms from 1,104 equids tested for WNV in Colorado in 2003. PROCEDURES: Submission forms accompanying samples submitted for detection of WNV via IgM capture ELISA were obtained from the Colorado state veterinarian and diagnostic laboratories performing the tests. Data on signalment, clinical signs, history of vaccination against WNV, and assay results were collected from laboratory submission forms. Equids with clinical signs compatible with WNV infection in which IgM capture ELISA results were positive were considered as case equids. RESULTS: 1,104 equids were tested for WNV; 1,017 (92.1%) had clinical signs compatible with WNV infection. Among equids with clinical signs compatible with WNV infection, the odds of testing positive for WNV via IgM capture ELISA were lower in males and in vaccinated equids and higher in equids with moderate and severe illness, compared with females, unvaccinated equids, and equids with mild illness. CONCLUSIONS AND CLINICAL RELEVANCE: Among equids with clinical signs compatible with WNV infection, vaccination against WNV, severity of clinical signs, duration of illness, and region in Colorado were associated with increased risk of having a positive IgM capture ELISA result.     

Seroprevalence and risk factors for infection with West Nile virus in Saskatchewan horses, 2003

The primary objectives of this study were to determine the seroprevalence of West Nile virus (WNV) infection of horses in Saskatchewan in 2003 and to identify risk factors for the infection. Blood samples were collected in August and October from 212 horses in 20 herds in 5 geographic zones. After accounting for within-herd clustering, the proportion of horses that had been infected with WNV, as determined by IgG and IgM antibody response, was 55.7% (95% confidence interval, 44.9% to 65.8%). The proportion of antibody-positive horses differed among herds (0% to 100%) and across ecoregions (20% to 76%). Horses in southern ecoregions were more likely to have either IgM antibodies or IgG concentrations suggesting infection than were horses in northern ecoregions. The use of mosquito-control measures was associated with decreased risk. After accounting for ecoregion, there was no difference between recipients of an inactivated WNV vaccine and nonrecipients in the occurrence of antibodies reflecting natural infection.     

A case-control study of factors associated with development of clinical disease due to West Nile virus, Saskatchewan 2003

REASON FOR PERFORMING STUDY: West Nile virus (WNV) was first diagnosed in Saskatchewan equids in 2002. AWNV epidemic was considered highly likely for 2003, which would provide a unique opportunity to study all aspects of WNV subclinical infection and clinical disease development in a relatively naive population. HYPOTHESIS: There are individual equid attributes and management risk factors associated with development of clinical disease. Specifically, this study could address the question of vaccine efficacy for the prevention of development of clinical disease. METHODS: A case-control study was conducted in the summer of 2003 during a province-wide outbreak of WNV. Between 5 and 10 equids were sampled from each of 23 case premises with clinical disease and 23 control premises with no apparent or confirmed clinical disease. Data were analysed to identify risk factors for the development of clinical disease. RESULTS: The proportion of equids serologically positive for natural exposure to West Nile virus was 64% (193/300). Nonvaccinated equids were 23 times (95%CI limits 3.0, 168.5, P = 0.002) more likely to develop clinical disease than those vaccinated. The estimate of vaccine efficacy in this field study was 96% (95%CI limits 67%, 99%). CONCLUSIONS: The study demonstrated that vaccination was strongly associated with the prevention of clinical disease. POTENTIAL RELEVANCE: Vaccination is an effective, practical method of prevention of clinical disease.     

Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)

West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.     

Investigation of an outbreak of encephalomyelitis caused by West Nile virus in 136 horses

OBJECTIVE: To describe an outbreak of encephalomyelitis caused by West Nile virus (WNV) in horses in northern Indiana. DESIGN: Case series. ANIMALS: 170 horses. PROCEDURES: Horses with clinical signs suggestive of encephalomyelitis caused by WNV were examined. Date, age, sex, breed, and survival status were recorded. Serum samples were tested for anti-WNV antibodies, and virus isolation was attempted from samples of brain tissue. Climate data from local weather recording stations were collected. An epidemic curve was constructed, and case fatality rate was calculated. RESULTS: The most common clinical signs were ataxia, hind limb paresis, and muscle tremors and fasciculations. Eight horses had been vaccinated against WNV from 2 to 21 days prior to the appearance of clinical signs. West Nile virus was isolated from brain tissue of 2 nonvaccinated horses, and anti-WNV IgM antibodies were detected in 132 nonvaccinated horses; in 2 other nonvaccinated horses, anti-WNV antibodies were detected and WNV was also isolated from brain tissue. Thirty-one (22.8%) horses died or were euthanatized. The peak of the outbreak occurred on September 6, 2002. Ambient temperatures were significantly lower after the peak of the outbreak, compared with prior to the peak. CONCLUSIONS AND CLINICAL RELEVANCE: The peak risk period for encephalomyelitis caused by WNV in northern Indiana was mid-August to mid-September. Reduction in cases coincided with decreasing ambient temperatures. Because of a substantial case fatality rate, owners of horses in northern Indiana should have their horses fully protected by vaccination against WNV before June. In other regions of the United States with a defined mosquito breeding season, vaccination of previously nonvaccinated horses should commence at least 4 months before the anticipated peak in seasonal mosquito numbers, and for previously vaccinated horses, vaccine should be administered no later than 2 months before this time.     

Detection and molecular analysis of West Nile virus infections in birds of prey in the eastern part of Austria in 2008 and 2009

The emergence of West Nile virus (WNV) was expected in Austria since the initial discovery of the infection in neighbouring Hungary in 2003/2004. In 2008 six cases of West Nile disease were diagnosed at the Institute for Veterinary Disease Control M?dling, Austrian Agency for Health and Food Safety (AGES), involving five goshawks (Accipiter gentilis) and one gyrfalcon (Falco rusticolus), which were found dead in the eastern Austrian federal states of Lower Austria, Vienna and Styria, respectively. Pathomorphological and immunohistochemical findings suggested a WNV infection. Virus was isolated in embryonated specific pathogen free chicken eggs and propagated in mouse neuroblastoma cells (NA), in which a cytopathic effect occurred. The virus was identified and characterised by electron microscopic examination and molecular detection using RT-PCR, sequencing, and phylogenetic analysis. The Austrian WNV sequences exhibited nucleotide identities of 99.9% to the lineage 2 WNV sequences described in Hungary since 2004. In addition, 71 sera of 14 different bird species were screened for the presence of WNV antibodies using a commercial ELISA: 43.7% of the tested samples showed antibody titers. Selected positive sera were also subjected to WNV neutralisation tests, in which the ELISA results were verified in 66%. The results of this study confirm unambiguously the presence of a lineage 2 WNV infection in birds of prey in the eastern part of Austria.