- Top of page
- Materials and methods
- Supporting Information
Equine influenza virus (EIV) a highly contagious respiratory pathogen of horses and other equidae is endemic in Europe and North America, and major outbreaks have occurred in Asia,[1-5] Africa,[6, 7] Australia and South America (http://www.oie.int/wahis/public.php). Two virus subtypes H7N7 and H3N8 have been isolated in horses, and for nearly two decades, both virus subtypes co-circulated. It is now considered, however, that the H7N7 virus first identified in horses in 1956 is no longer in circulation. Since 1979, all outbreaks of equine influenza (EI) for which virus has been isolated have been caused by the H3N8 subtype. Both virus subtypes are included in the majority of commercially available vaccines despite the fact that the Office International des Epizooties (OIE) stipulate it is no longer a requirement for vaccines to include a H7N7 virus.
Outbreaks of EI occur predominately among naïve and partially immune populations. During the 2-year period 2010–2012, outbreaks were reported in Argentina, Brazil, Canada, Chile, China, Dominican Republic, France, Germany, India, Mongolia, Sweden, UAE, United Kingdom, USA and Uruguay (http://www.aht.org.uk/icc/linksicc.html; http://www.oie.int/wahis/public.php) in addition to those identified in Ireland during this study. At a minimum, such outbreaks result in suboptimal performance, severe disruption to training schedules and significant economic losses. The virus infects the ciliated epithelium and impairs mucociliary clearance which may take several weeks to recover, even if uncomplicated by secondary bacterial infection. Rest and restriction of horse movement are essential to the management of clinical cases. This can result in individual racehorses missing important racing fixtures which may impact on their potential value and that of their progeny. Where large populations are affected, betting revenue may decline as a result of the uncertainty relating to racing form. Continuing to exercise horses following infection has been demonstrated to exacerbate the severity of clinical disease and increase weight loss. In a study carried out in the UK, examinations of the network of contacts between racehorses suggest that the spread of infectious diseases such as EI may occur rapidly. This is largely due to the close proximity of horses within yards and horses from different yards on the training gallops. During an EI outbreak, which infiltrated the racing population in Newmarket in 2003, 21 training yards and over 1300 horses were affected during a 2-month period. Equestrian events may also be affected and sometimes cancelled. During the outbreak in Uruguay in 2012, which affected over 2000 horses, race meetings were cancelled for several weeks and movement of horses out of the country was prohibited (http://www.oie.int/wahis/public.php). Equine influenza outbreaks also resulted in the cancellation of equestrian events in Brazil (http://www.oie.int/wahis/public.php). While mortality is very rarely associated with EI, more than 40 horses died during an outbreak affecting over 74 000 horses in Mongolia in 2011. Disconcertingly, several foal deaths were also reported during EI outbreaks in France during the same year (http://www.oie.int/wahis/public.php).
Vaccination against EI is an effective method of disease control and following the introduction of mandatory vaccination of racehorses in Ireland and the UK in 1981, no major equestrian event has been cancelled as a result of the disease. The racing authorities in Ireland and the UK stipulate that the first two doses of primary vaccination be administered between 21 and 92 days apart followed by a third vaccine dose 150–215 days after the second vaccination. Thereafter, annual booster vaccination is required (http://www.tuftclub.ie; http://www.thejockeyclub.co.uk). Outbreaks have occurred, however, when the virus circulating in the field was significantly different from that contained in the vaccines.[16, 18, 19] The horse industry requires EI vaccines that prevent both clinical disease (clinical protection) and virus shedding. The role of subclinically infected vaccinated horses (horses that are not virologically protected) in the global spread of EIV is of major concern to the industry. The importation of such horses in conjunction with inadequate quarantine procedures has resulted in several major outbreaks with significant economic consequences. It is estimated that the eradication of EI from Australia following the importation of a subclinically affected horse cost in excess of one billion Australian dollars. Therefore, in order to maintain the effectiveness of vaccination, surveillance of EI viruses and prompt updating of vaccine strains is of fundamental importance. Ireland has an active EI surveillance programme as does the UK, but in many other countries, surveillance is inadequate predominately due to a lack of funding. The aims of this study were to identify: the factors involved in the spread of EI in a country where the virus is endemic, the virus strains responsible for the outbreaks, the single radial haemolysis (SRH) antibody levels correlating with protection against current virus strains circulating in the field and any evidence of vaccination breakdown as defined by disease and/or virus shedding.
- Top of page
- Materials and methods
- Supporting Information
Equine influenza outbreaks identified during this study were sporadic and self-limiting and occurred primarily among equidae which were unvaccinated or did not have up to date vaccination records. This study included the first-confirmed case of EI affecting donkeys in Ireland (premises 4). In all cases, outbreaks occurred following the movement of horses. On the majority of premises, horses were clinically affected following the introduction of a new arrival of unknown vaccination status. The risk associated with failing to isolate new arrivals and establish their antibody status was highlighted previously.[20, 31, 32] The seasonality of the outbreaks and the contribution of stabling and fomites/personnel to virus spread were also observed previously as was the influence of age on the clinical severity of disease.[2, 6, 7, 19, 20, 35, 36] However, the rate of virus isolation was greater than that reported in a similar study from 2007 to 2010. In the current study, virus was isolated from 18 of the 45 (40%) RT-PCR-positive nasal swabs which were collected from 14 of the 135 horses. In the previous study, a delay in veterinary intervention was identified as a possible contributing factor to the low rate of virus isolation. As a similar delay was observed in the present study, it is likely that the increased rate of virus isolation was due to the fact that 10 of the 14 horses were seronegative on initial sampling. Seronegative horses rapidly amplify and shed large quantities of virus and are frequently the index case during outbreaks.[37-39]
A definite correlation between SRH antibody levels (H3N8) and protective immunity has been established following experimental challenge studies and observations in the field. Horses with antibody levels >85 mm2 are clinically protected, that is, protected against clinical disease, and horses with antibody levels >150 mm2 are virologically protected, that is, protected against infection, provided the vaccine strains are closely related to those circulating in the field.[24, 38, 40] It is not possible to differentiate between H3N8 antibodies due to vaccination and natural infection. Other studies have defined pre-infection antibody titres as those of blood samples taken the day after influenza was first diagnosed.[16, 19] However, delayed veterinary intervention as observed in previous outbreaks affects interpretation of serological data, as at the time of sample collection, horses have often mounted an antibody response to the virus that is circulating on the premises. Thus, it is often not possible to determine the pre-infection SRH titre that correlates with protection. In horses vaccinated with the majority of vaccines which contain the H7N7 subtype, the measurement of antibodies against H7N7 is a useful aid to the identification of those horses that have vaccinal rather than post-infection titres as a higher H3N8 antibody level in comparison with H7N7 suggests exposure by natural infection. In this study, horses sampled pre-infection were defined as those that were seronegative or had similar H7N7 and H3N8 antibody titres. These horses exhibited a strong correlation between their pre-existing SRH antibody level and susceptibility to influenza as defined by clinical signs and confirmatory laboratory diagnosis. Seventy-eight per cent of the seronegative horses, 44% of horses with antibody levels of between 85 and 150 mm2 and 5% of horses with antibody levels >150 mm2 developed influenza. No horse with a pre-existing antibody level >151 mm2 exhibited clinical signs that were confirmed as EI. No horse with a pre-existing antibody level >210 mm2 tested positive for EI, that is, they were virologically protected. Subclinical infection was identified in 12 horses on four different premises. Of the 12 horses, six had antibody titres consistent with vaccination. The mean H3N8 antibody titre of these six horses was 175 ± 14·7 mm2 SE. Virus was isolated from one of these horses on two occasions 72 hours apart. This horse which had a H3N8 SRH antibody level of 127 mm2 had received its third vaccine dose (Equilis Prequenza Te) 9 months prior to the outbreak.
Over 80% of the horses included in this study were unvaccinated, of unknown/unavailable vaccination status or their vaccination record was out of date. Furthermore, over 70% of horses that tested positive in this study were seronegative for H7N7, suggesting no vaccinal antibodies at the time of exposure. On three premises, it was reported that the majority of horses were vaccinated; however, vaccination records were only available for between 23% and 44% of horses. Nevertheless, examination of the H7N7 and H3N8 antibody level indicated that horses whose vaccination record was unknown or unavailable were not necessarily unvaccinated and highly susceptible to infection. Some had antibody levels in excess of 150 mm2.
Of the 25 horses with up to date vaccination records, a confirmed diagnosis of EI was made in 5 (20%) of 8 (32%) clinically affected horses. The average time since last vaccination for the eight clinically affected horses was 4·8 ± 0·49 SE months. These horses had been vaccinated with all four commercially available products, Duvaxyn IE-T Plus (2), Equip FT (1), Equilis Prequenza Te (3) and ProteqFlu Te (2). Two of the horses had received their third vaccine dose of ProteqFlu Te on average 6 months prior to developing clinical signs. These horses had a mean H3N8 antibody level of 79 ± 47·3 mm2 SE on initial sampling. A third horse which had received its second dose of Equilis Prequenza Te 5 months prior to the outbreak had comparable H7N7 (106 mm2) and H3N8 (118 mm2) antibody levels, thus suggesting this horse had responded well to vaccination but was also inadequately protected. The five remaining horses were all vaccinated with products containing both virus subtypes. They had received on average 3·6 doses of vaccine and had last been vaccinated between 2 and 5 months prior to developing clinical signs. All were H7N7 seronegative and one was also H3N8 seronegative, suggesting these horses had responded poorly to vaccination. Poor response to vaccination is seen in some horses and is believed to play an important role in the transmission of virus among vaccinated horses. Two recent comparative vaccine studies have demonstrated that between 39% and 43% of Thoroughbred weanlings failed to seroconvert following first vaccination and that the incidence of poor responders was shown to be vaccine related Poor durability of antibody response between second and third vaccination and approximately 6 months following third vaccination have also previously been reported.[37, 38, 41-44]
All virus strain identified as being responsible for outbreaks in Ireland during this study were from the Florida sublineage clade 2 and were similar to those identified during previous outbreaks in Ireland in 2007 and 2008 in Europe[31, 32] and in Asia.[4, 33] Experimental challenge studies and field data have demonstrated that protection against virus shedding correlates with the degree of antigenic relatedness between the vaccine and challenge strain.[16, 19, 45] In this study, evidence of vaccine breakdown occurred in eight horses, six of which had been vaccinated with products not updated in line with the OIE recommendations of 2004 or 2010. These vaccines contained A/eq/Newmarket/1/93 (Duvaxyn IE-T Plus, Equilis Prequenza Te) and A/eq/Kentucky/98 (Equip FT) as a representative of the American lineage. Evidence of vaccine breakdown occurred in a further two horses vaccinated with ProteqFlu Te. This vaccine has been updated in line with OIE recommendations from 2004 to include A/eq/Ohio/03 as a representative of clade 1 of the Florida sublineage; however, it does not include a representative of clade 2 as recommended by the OIE since 2010.
Cross-protection is the ability of an antigen to elicit a protective immune response against infection with another antigen, for example a heterologous strain of virus. The findings of this study suggest that cross-protectivity between the viruses included in the current vaccines and the circulating (field) strains has decreased. Forty-four per cent and 18% of 68 horses that had pre-infection antibody levels that correlate with clinical protection and virological protection with homologous virus developed clinical signs. Subclinical infection was detected in horses which had a mean H3N8 SRH antibody level of 175 ± 14·7 mm2 SE, and one horse had an SRH level of 210 mm2, that is, a level higher than that usually elicited by booster vaccination. The results of this study therefore suggest that vaccinal antibody levels indicative of clinical protection against homologous virus did not correlate with protection against the clade 2 viruses responsible for these outbreaks.
Equine influenza vaccines are updated to effect qualitative rather than quantitative improvements in the immune response. Comparative vaccine studies suggest that the level of antibodies elicited by current vaccines is related to adjuvant composition rather than antigenic load or virus strains.[41, 42, 46] When there is mismatch between the circulating field strains and the vaccine strains, infection may result in clinical influenza and/or virus shedding regardless of a high SRH response to vaccination. Replacing an out-of-date virus in a vaccine with an epidemiologically relevant strain is unlikely to elicit a greater antibody response but should maximise protective immunity against EI. In order to ensure that mandatory vaccination programmes provide optimum protection, vaccines must contain strains that are antigenically similar to those circulating in the field. The findings of epidemiological investigations such as this should not be overlooked, and vaccine strains must be updated in a timely manner as recommended by the OIE.