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Equine Veterinary Journal ISSN 0425-1644 DOI: 10.1111/evj.12302
Editorials
Report of the International Equine Influenza Roundtable Expert Meeting at Le Touquet, Normandy, February 2013 Introduction Equine influenza (EI) is an important equine respiratory pathogen and a high-priority disease for the equine industry globally. Equine influenza virus (EIV) has a global distribution; it is endemic in many countries and there are occasional incursions in Japan, South Africa and Hong Kong, with only Australia, New Zealand and Iceland being considered free. In 2007, EIV was introduced into Japan and Australia, severely disrupting the equine industry, with control and eradication costing an estimated $AU1.6 billion in Australia. Equine influenza virus has subsequently been detected in recently imported, vaccinated horses in quarantine stations in Japan and Dubai. Many of the bodies responsible for regulating racing and equestrian sport in Europe and North America have required EIV vaccination since the 1980s, in order to safeguard competition and facilitate the movement of horses. The majority of horses in the racing and competition sectors are vaccinated against EIV, but outside these sectors the proportion of vaccinated horses is unknown. In most countries, too few horses are vaccinated to provide the herd protection required to prevent propagation of an epizootic, if an incursion of an EIV strain with sufficient antigenic differences to currently circulating strains were to occur. Surveillance in order to monitor outbreaks and to characterise the antigenic and genetic characteristics of circulating EIV strains, in particular changes in the haemagglutinin (HA) surface glycoprotein, is central to EI control. France, Germany, Ireland, the UK and the USA have the most extensive surveillance schemes. Haemagglutination inhibition (HI) assays and HA sequence data allow predictions of antigenic and genetic drift to be made. Antigenic mapping (cartography) is used to assist interpretation of HI data. Haemagglutinin sequence data are shared between collaborating laboratories, which facilitates phylogenetic analysis and mapping of the evolutionary changes in EIV. The expert surveillance panel (ESP) of the World Organisation for Animal Health (OIE) reviews these data and publishes recommendations for EI vaccine strain updates periodically. Most recently (2010), the ESP recommended that H7N7 viruses and Eurasian H3N8 viruses are no longer required and that strains representative of clade 1 (A/eq/South Africa/04/ 2003-like or A/eq/Ohio/2003-like viruses) and clade 2 (A/eq/Richmond/1/ 2007-like viruses) viruses of the Florida sublineage should be included. Equine H3N8 viruses change more slowly than human influenza viruses, and thus ESP recommendations have changed only twice in the last 8 years. However, at the time of writing, there are no EIV vaccines meeting the European regulatory requirements that are completely compliant with these recommendations, probably because updating EI vaccines is comparatively burdensome, time consuming and expensive. Vaccination against EIV is a highly important control measure. Vaccination has had a significant beneficial effect on the prevalence of EIV infection, but there is no room for complacency: outbreaks still occur annually, despite vaccination. In 2012, individual cases or outbreaks of EI associated with clade 1 and clade 2 viruses, closely related antigenically, but not identical, to the recommended vaccine strains were recorded in both nonvaccinated and vaccinated horses in Argentina, Chile, France, Germany, Ireland, the UK and the USA. The lack of clarity and the conflicting opinions on key aspects of EI within the European equine industry create a risk that the value of EI vaccination may be challenged, with the potential outcome that vaccination, and hence (national) herd protection, may fall to a level at which the prevalence of EI increases, equine welfare decreases and the equine industry
This is an open access article under the terms of the Creative Commons AttributionNonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
experiences significant economic losses through interference with competition, breeding, movement and trade. A meeting of international experts on EI was held in Le Touquet, France, on 6 February 2013. The goal of the meeting was to address and reach consensus on some of the most pressing questions on EI and its control, including surveillance, the spectrum of clinical disease, diagnosis, biosecurity, protective immunity, key factors in vaccine selection, vaccine efficacy, vaccine strain updates, reasons for vaccination failure and future research. Nine critical questions on current issues in EIV became apparent during the panel’s discussions and form the basis for this report.
Roundtable discussion report Question 1. What are the best detection tools for equine influenza? The selection of appropriate diagnostic tests is fundamental to biosecurity and infection control, whether dealing with an EI outbreak or formulating a screening programme. Clinical signs are the first warning of an EI outbreak but vary tremendously in severity in both vaccinated and nonvaccinated horses. This appears to be influenced by a combination of factors, including age, ability to mount an immune response, virus strain, time since previous vaccination, and perhaps also co-infection with equine herpes virus. Fever, inappetance and a frequent dry, hacking cough in one or more horses is highly suggestive of EIV infection. However, mild or subclinical disease may not raise the suspicion of EIV infection, meaning that early diagnosis and intervention are not facilitated and that outbreaks propagate or are allowed to pass undetected. Virological findings are poorly correlated with clinical signs: horses with mild or subclinical disease may be responsible for significant virus shedding and represent a source of contagion, complicating biosecurity protocols and infection control measures, where identification of infected horses relies on clinical signs. Virus shedding differs significantly between vaccinated and nonvaccinated horses. Vaccinated horses may shed no or little EIV if infected at or close to the peak of immunity (i.e. shortly after immunisation) [1–8]. However, the duration and amount of virus shed tends to increase with the time since the last immunisation [5,9–11]. Appropriate diagnostic testing is critical, whether managing outbreaks or constructing screening programmes to reduce the risk of EIV entry into competitions. Equine influenza virus is shed intermittently from the nasopharynx and may be detectable for 2–8 days after infection in naïve animals and for a significantly shorter period in vaccinated horses. Case selection and sample timing are very important. Cases should be selected on the basis of initial clinical signs, such as pyrexia within the last 24–48 h, with twice-daily rectal temperature monitoring of all at-risk horses. Tailor-made nasopharyngeal swabs provide better sensitivity than nasal swabs [5]. Rapid transport in suitable conditions in the correct transport medium to an appropriate diagnostic laboratory is essential. The panel reviewed the current diagnostic tests (Table 1). Virus-specific nucleic acid detection by real-time reverse transcription-polymerase chain reaction (rtRT-PCR) is the most sensitive assay currently available [12–17]. This rapid test is highly suitable for situations where quick decision making is required, such as the mass screening of horses in Australia in 2007 [13]. The assay is widely available and is recommended as the test of choice for outbreak control (to confirm disease in individuals or in groups of horses) and for biosecurity protocols (at entry into equestrian competitions or prior to movement). Test results need to be interpreted carefully. In screening programmes, all positive horses without clinical signs should be regarded as potentially
Equine Veterinary Journal 46 (2014) 645–650 © 2014 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of British Equine Veterinary Association.
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Report of the international equine influenza roundtable expert meeting
TABLE 1: Summary of currently available tests and their key features Test
Samples required and comments
Virus isolation
Polyester-tipped nasopharyngeal swabs are ideal; (short) nasal swabs are adequate; collect into virus transport medium; positive results are most likely in the first 24–48 h after infection. Culture in eggs or cell culture. Gold standard, but slow and prone to false negatives. Successful virus isolation is an essential component of surveillance programmes Polyester-tipped nasopharyngeal swabs are ideal; (short) nasal swabs are adequate; collect into virus transport medium. Nucleoprotein enzyme-linked immunosorbent assay is used widely. Patient-side enzyme-linked immunosorbent assays for detection of human influenza A and B viruses are useful for rapid screening of horses Polyester-tipped nasopharyngeal swabs are ideal; (short) nasal swabs are adequate; collect into virus transport medium. The RT-PCR is rapid, highly sensitive, specific and widely available; first choice in outbreak control and screening programmes for equine influenza Serum. Haemagglutination inhibition is the classical assay; subject to considerable interlaboratory variation. Single radial haemolysis is more reliable; titres correlate with susceptibility to infection (>75 mm2 suggests clinical protection, >150 mm2 suggests virological protection)
Antigen detection
Nucleic acid detection
Antibody detection (serology)
infected and managed accordingly. All positive samples above the validated cut-off for the assay should be regarded as evidence of exposure to EIV. However, positive samples with low levels of RNA are difficult to interpret because RT-PCR (and enzyme-linked immunosorbent assay) can detect nonviable and degraded virus even after horses are no longer infectious or constitute a risk of infection to other horses. For example, during the 2007 outbreak in Australia, RNA was detected up to 34 days after infection [18]. Assessment of the risk posed by horses with weakly positive RT-PCR tests should be interpreted based on sampling on consecutive days and in light of clinical and epidemiological data.
Question 2. How can contamination be prevented during an outbreak? All equine businesses need to be prepared for when, and not if, an EIV outbreak occurs. Biosecurity (even simple hygiene measures) is an unfamiliar concept that is not accepted by all parts of the equine industry. Simple measures, such as hand washing, minimising horse-to-horse contact and controlling fomites, significantly reduce the risk of disease transmission. Such measures can be implemented easily by all equine businesses, race meetings and competitions. Biosecurity protocols were developed for the Olympic Games in London 2012 and by the Federation Equestre Internationale. Some countries have established epidemiological surveillance centres for equine disease, such as Reseau d’EpidemioSurveillance en Pathologie Equine (RESPE) in France, RESPE in Ireland (iRESPE) and the International Collating Centre (ICC) in the UK. During an outbreak, a crisis management centre, consisting of individuals from the equine industries in Europe, is established to propose and coordinate an emergency response plan. Simple biosecurity measures combined with quarantine and suspension of movement of horses should be implemented immediately, and early veterinary involvement is essential. Horses should be regarded as contagious for at least 2 weeks, although 3 weeks of quarantine may be more prudent. Rectal temperatures should be monitored twice daily and recorded. The spread of EIV in vaccinated and/or mixed populations may not be as explosive as in naïve populations because of slower disease transmission in vaccinated horses. It is important that all of those managing outbreaks in older, vaccinated horses should be aware of these differences. The high level of biosecurity required by, for example, the pig and poultry industries, is unachievable across much of the equine industry, owing to the frequent movement and mixing of horses. Tighter control of
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the transportation of horses, such as accurate registration procedures and compliance with basic sanitary conditions, is of the utmost importance because of the major role that movement of horses plays in virus spread. The ability to implement control measures and the attitude to risk determine what control measures would be appropriate. The measures that can be achieved realistically need to be taken into account when constructing control programmes. In the face of an outbreak, it would appear logical to vaccinate primed horses, because they are likely to develop a rapid, anamnestic response. Vaccination should be recommended for in-contact horses that have shown no clinical signs in the previous 24 h. Booster vaccination appears to be effective even when the vaccine does not contain the circulating virus, because the high titres of antibody generated by booster vaccination are likely to be cross-protective. Likewise, the booster vaccine does not appear to need to match previously administered vaccines [19,20], although there are few published data on this.
Question 3. What lessons can be learned from the French EIV outbreak in 2012? The epidemiology and virology of a 2012 EIV outbreak that occurred in France and potentially affected horses that were intended to compete at the London Olympic Games were reviewed. The outbreak occurred mainly in a mixed population of vaccinated and nonvaccinated horses on 3 stud farms in Normandy. Studies of the HA1 portion of the H3 gene [21] revealed a clade 2 virus, similar to strains circulating previously in Europe [1,21–27]. Equine influenza virus was confirmed on a farm with about 150 horses (eventers, broodmares, young horses and foals) in the Calvados region on 4 May 2012. Eventers were vaccinated twice annually, and all other horses were vaccinated annually. The vast majority of horses developed fever, which was accompanied, in some, by cough. Clinical signs were recorded for 11 days. Three days later, EIV was confirmed on another farm, with 70 horses (eventers, broodmares, young horses and foals), in the same region. Adult horses were vaccinated as at the first location. Young horses were not vaccinated. The vast majority of horses developed fever and cough. Clinical signs were recorded for 10 days. Two days later (9 May), EIV was confirmed in 3 horses on a 70 horse farm where competition horses were vaccinated twice annually, some horses were vaccinated annually and some horses were not vaccinated. Fifteen horses developed fever, cough and, in some cases, nasal discharge. Competition horses, if affected, had fever but no other clinical signs. Clinical signs were recorded for 10 days. A 10-day-old foal from a nonvaccinated mare had to be subjected to euthanasia owing to the severity of disease. Two asymptomatic horses, vaccinated twice annually, were diagnosed positive for EI following a routine veterinary examination prior to an international showjumping competition (CSIO 5*) in La Baule, LoireAtlantique, on 11 May. Although these horses were immediately removed from the holding stables and were not permitted to compete, this does not preclude dissemination of virus prior to the positive test result. A competition mare in Oise, Picardy, was confirmed EIV positive on 15 May. Several horses at this large riding establishment, where all 100 horses were vaccinated twice annually, were noted to be coughing. The first 3 farms are linked epidemiologically. Spread was facilitated by mingling of nonvaccinated and vaccinated horses on farms and at equestrian events (Saint Gatien, Calvados; le Touquet, Pas-de-Calais; Haras du Pin, Orne). The horses that tested positive at La Baule were at risk owing to both their geographical proximity to the first 3 farms and contact at equestrian events in Normandy. The horses on the Picardy farm were in contact via equestrian events in Normandy. Reseau d’Epidemio-Surveillance en Pathologie Equine set up a management crisis centre that brought together many equine industry stakeholders. This unit meets on a weekly basis to organise improvements in operational health measures and to maintain and disseminate timely communiqués and other seminal information. Following this outbreak, it was recommended that all competing and in-contact horses should be vaccinated twice annually. This is particularly important in young horses (1–5 years old) at the greatest risk of EIV infection. In addition, direct and indirect contact between vaccinated and nonvaccinated horses housed at the same site should be minimised and, ideally, avoided.
Equine Veterinary Journal 46 (2014) 645–650 © 2014 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of British Equine Veterinary Association.
J. Slater et al.
Question 4. Interindividual variation in equine influenza: why do some horses develop severe disease or die? Equine influenza virus infection produces a considerable spectrum of clinical disease. Characteristic clinical signs are seen in naïve animals, but less severe disease, with mild and transient or subclinical clinical signs, is generally seen in vaccinated horses. Deaths occur mainly in foals from nonvaccinated mares; thus, inadequate passive transfer of antibodies, due to poor-quality colostrum or inadequate intake, is likely to be a major factor. All mares should be vaccinated adequately to ensure that there are sufficient maternally derived antibodies in colostrum. There is evidence that infection with different EIV strains (genotypes) results in different clinical outcomes (disease-causing phenotypes) [28,29]. Certain strains appear to be more likely to produce primary viral pneumonia; others are more likely to be followed by secondary bacterial infection, whereas the remainder produce less severe disease. There is also likely to be a strong host effect, with genotype and previous immune experience playing roles.
Question 5. Update on strains and immunity: what paths should be explored? Veterinarians play a critical role in EIV surveillance, but are not always aware of the importance of investigation, sample collection and reporting of suspected EIV outbreaks, vaccination breakdowns and suspected adverse reactions. In addition, gathering of data is frequently hampered by the lack of a standardised approach and economic constraints. The equine industry needs to continue to support surveillance schemes, and professional associations should promote awareness of the veterinarian’s role in disease surveillance. Equine industry- and government-supported surveillance, especially in the UK and Ireland, has allowed effective monitoring of the evolution of H3N8 EIV strains and has tracked the initial divergence into European and American lineages and the continued evolution of the American lineages, such that current circulating strains are from clades 1 and 2 of the Florida sublineage. The utility of the HI assay used to characterise HA could be improved by using standardised equine antisera in addition to ferret antisera, although the former are more difficult to generate and more cross-reactive than the latter. Some horses exhibit clinical and virological protection in the absence of (single radial haemolysis) antibody titres [1,5,30]. There is a paucity of data on the role of and targets for cell-mediated immunity in EIV, which should be a target of further research. Finally, the panel discussed the current difficulties faced by vaccine manufacturers in updating EI vaccine strains in Europe. Updates require a variation in the marketing authorisation for a vaccine regulated by EC/1234/2008. The panel considered the revision of the current guidance to be timely and agreed that it provided an opportunity further to streamline EI vaccine updates by animal health companies.
Question 6. What is the best way to assess vaccine efficacy? The regulatory framework sets out the safety, efficacy and quality data required to obtain market authorisation, but the panel agreed that efficacy in field situations is a more demanding and diverse concept than in the laboratory setting. Experimental challenge is designed to provide a controlled environment, with fewer confounding factors than in the field, to allow the evaluation of vaccine efficacy and safety in comparison to nonvaccinated control horses. Typically, relatively small groups of animals of similar ages, types and previous immune experience of EIV are used. Most studies follow a similar study design, in which animals are vaccinated in accordance with datasheet recommendations and then challenged at the onset of immunity of the vaccine. This provides a standardised approach to comparing vaccine performance and measuring ability to protect, but may not be completely representative of the vaccination protocols used in the field. It is important that immunisation in the field
Report of the international equine influenza roundtable expert meeting
should follow, as far as possible, the datasheet recommendations. Testing protocols should be standardised between laboratories so that critical points, such as challenge method (i.e. nebulisation or instillation of virus suspension, horse-to-horse transmission), sampling times, method of sampling (e.g. nasopharyngeal or nasal swabs), time of challenge and duration of follow-up, are harmonised, thus allowing comparison between studies. In Europe, vaccines for intramuscular administration contain whole inactivated virus, subunit (purified proteins), either alone or in a live vector (canary pox), vaccines containing either carbomer, carbomer and aluminium hydroxide, immune-stimulating complexes (ISCOM) or ISCOM-matrix adjuvants [31]. In North America, vaccines of different antigen and adjuvant composition are available for intramuscular administration, and a live, attenuated, intranasal vaccine is also available [32,33]. The panel agreed that all EI vaccines currently available in Europe would be expected to perform well, even against heterologous challenge, when tested at peak immunity. However, extrapolation to the field situation would be incomplete, because horses may be challenged at any time. In the field, vaccine efficacy can be monitored serologically, because there is a well-established correlation between single radial haemolysis antibody titres and protective immunity following vaccination with inactivated whole virus and subunit vaccines [34,35]. This approach has been employed in a few comparative field studies [20,36–38]. The duration of immunity of EI vaccines needs also to be evaluated alongside the constantly evolving virus. The efficacy of EI vaccines may be limited if antibody levels fall below protective levels between vaccinations [37,38] and challenge occurs during this so-called immunity gap [5], between the second and third EI immunisation [39]. The vaccination protocols that are currently accepted by racing and equestrian sport regulatory bodies do not take this into account. Ideally, there should be a more holistic assessment of vaccine efficacy that also takes data from appropriately designed and controlled studies into account. As a minimum, assessment of vaccine efficacy should include consideration of in vitro immunological data, experimental challenge data, longitudinal immunological data from different age groups of horses and surveillance data from the field.
Question 7. What do we expect from vaccines and why does vaccination fail? The panel discussed how the expectations of the equine industry contrasted with those of the livestock sector; with the exception of the racing and competition sectors, herd protection is not widely accepted. Much of the sector is unaware that vaccination helps to reduce the prevalence and severity of disease. The majority of owners are pleasure horse-owners with individual animals, who are not used to thinking of their animals as part of a herd, even in livery yards. These owners generally expect vaccines to produce long-lasting, sterile immunity, protecting each horse from both infection and disease. There is a significant need for veterinary practices to communicate realistic and effective messages about the appropriate use of vaccination and disease control. There are subpopulations of horses that probably never receive influenza vaccination because of economic constraints, lack of motivation, lack of awareness or cultural attitudes to preventive medicine and disease control. Population (herd) vaccination coverage is needed to control EI. A small percentage of the population fails to mount and/or maintain an adequate immune response after immunisation and therefore remains susceptible to EI despite having been vaccinated appropriately [15,20,37,38]. Although the reasons for this remain unclear, age appears to be a significant factor, with the majority of poor responders being
Equine Veterinary Journal ISSN 0425-1644 DOI: 10.1111/evj.12302
Editorials
Report of the International Equine Influenza Roundtable Expert Meeting at Le Touquet, Normandy, February 2013 Introduction Equine influenza (EI) is an important equine respiratory pathogen and a high-priority disease for the equine industry globally. Equine influenza virus (EIV) has a global distribution; it is endemic in many countries and there are occasional incursions in Japan, South Africa and Hong Kong, with only Australia, New Zealand and Iceland being considered free. In 2007, EIV was introduced into Japan and Australia, severely disrupting the equine industry, with control and eradication costing an estimated $AU1.6 billion in Australia. Equine influenza virus has subsequently been detected in recently imported, vaccinated horses in quarantine stations in Japan and Dubai. Many of the bodies responsible for regulating racing and equestrian sport in Europe and North America have required EIV vaccination since the 1980s, in order to safeguard competition and facilitate the movement of horses. The majority of horses in the racing and competition sectors are vaccinated against EIV, but outside these sectors the proportion of vaccinated horses is unknown. In most countries, too few horses are vaccinated to provide the herd protection required to prevent propagation of an epizootic, if an incursion of an EIV strain with sufficient antigenic differences to currently circulating strains were to occur. Surveillance in order to monitor outbreaks and to characterise the antigenic and genetic characteristics of circulating EIV strains, in particular changes in the haemagglutinin (HA) surface glycoprotein, is central to EI control. France, Germany, Ireland, the UK and the USA have the most extensive surveillance schemes. Haemagglutination inhibition (HI) assays and HA sequence data allow predictions of antigenic and genetic drift to be made. Antigenic mapping (cartography) is used to assist interpretation of HI data. Haemagglutinin sequence data are shared between collaborating laboratories, which facilitates phylogenetic analysis and mapping of the evolutionary changes in EIV. The expert surveillance panel (ESP) of the World Organisation for Animal Health (OIE) reviews these data and publishes recommendations for EI vaccine strain updates periodically. Most recently (2010), the ESP recommended that H7N7 viruses and Eurasian H3N8 viruses are no longer required and that strains representative of clade 1 (A/eq/South Africa/04/ 2003-like or A/eq/Ohio/2003-like viruses) and clade 2 (A/eq/Richmond/1/ 2007-like viruses) viruses of the Florida sublineage should be included. Equine H3N8 viruses change more slowly than human influenza viruses, and thus ESP recommendations have changed only twice in the last 8 years. However, at the time of writing, there are no EIV vaccines meeting the European regulatory requirements that are completely compliant with these recommendations, probably because updating EI vaccines is comparatively burdensome, time consuming and expensive. Vaccination against EIV is a highly important control measure. Vaccination has had a significant beneficial effect on the prevalence of EIV infection, but there is no room for complacency: outbreaks still occur annually, despite vaccination. In 2012, individual cases or outbreaks of EI associated with clade 1 and clade 2 viruses, closely related antigenically, but not identical, to the recommended vaccine strains were recorded in both nonvaccinated and vaccinated horses in Argentina, Chile, France, Germany, Ireland, the UK and the USA. The lack of clarity and the conflicting opinions on key aspects of EI within the European equine industry create a risk that the value of EI vaccination may be challenged, with the potential outcome that vaccination, and hence (national) herd protection, may fall to a level at which the prevalence of EI increases, equine welfare decreases and the equine industry
This is an open access article under the terms of the Creative Commons AttributionNonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
experiences significant economic losses through interference with competition, breeding, movement and trade. A meeting of international experts on EI was held in Le Touquet, France, on 6 February 2013. The goal of the meeting was to address and reach consensus on some of the most pressing questions on EI and its control, including surveillance, the spectrum of clinical disease, diagnosis, biosecurity, protective immunity, key factors in vaccine selection, vaccine efficacy, vaccine strain updates, reasons for vaccination failure and future research. Nine critical questions on current issues in EIV became apparent during the panel’s discussions and form the basis for this report.
Roundtable discussion report Question 1. What are the best detection tools for equine influenza? The selection of appropriate diagnostic tests is fundamental to biosecurity and infection control, whether dealing with an EI outbreak or formulating a screening programme. Clinical signs are the first warning of an EI outbreak but vary tremendously in severity in both vaccinated and nonvaccinated horses. This appears to be influenced by a combination of factors, including age, ability to mount an immune response, virus strain, time since previous vaccination, and perhaps also co-infection with equine herpes virus. Fever, inappetance and a frequent dry, hacking cough in one or more horses is highly suggestive of EIV infection. However, mild or subclinical disease may not raise the suspicion of EIV infection, meaning that early diagnosis and intervention are not facilitated and that outbreaks propagate or are allowed to pass undetected. Virological findings are poorly correlated with clinical signs: horses with mild or subclinical disease may be responsible for significant virus shedding and represent a source of contagion, complicating biosecurity protocols and infection control measures, where identification of infected horses relies on clinical signs. Virus shedding differs significantly between vaccinated and nonvaccinated horses. Vaccinated horses may shed no or little EIV if infected at or close to the peak of immunity (i.e. shortly after immunisation) [1–8]. However, the duration and amount of virus shed tends to increase with the time since the last immunisation [5,9–11]. Appropriate diagnostic testing is critical, whether managing outbreaks or constructing screening programmes to reduce the risk of EIV entry into competitions. Equine influenza virus is shed intermittently from the nasopharynx and may be detectable for 2–8 days after infection in naïve animals and for a significantly shorter period in vaccinated horses. Case selection and sample timing are very important. Cases should be selected on the basis of initial clinical signs, such as pyrexia within the last 24–48 h, with twice-daily rectal temperature monitoring of all at-risk horses. Tailor-made nasopharyngeal swabs provide better sensitivity than nasal swabs [5]. Rapid transport in suitable conditions in the correct transport medium to an appropriate diagnostic laboratory is essential. The panel reviewed the current diagnostic tests (Table 1). Virus-specific nucleic acid detection by real-time reverse transcription-polymerase chain reaction (rtRT-PCR) is the most sensitive assay currently available [12–17]. This rapid test is highly suitable for situations where quick decision making is required, such as the mass screening of horses in Australia in 2007 [13]. The assay is widely available and is recommended as the test of choice for outbreak control (to confirm disease in individuals or in groups of horses) and for biosecurity protocols (at entry into equestrian competitions or prior to movement). Test results need to be interpreted carefully. In screening programmes, all positive horses without clinical signs should be regarded as potentially
Equine Veterinary Journal 46 (2014) 645–650 © 2014 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of British Equine Veterinary Association.
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Report of the international equine influenza roundtable expert meeting
TABLE 1: Summary of currently available tests and their key features Test
Samples required and comments
Virus isolation
Polyester-tipped nasopharyngeal swabs are ideal; (short) nasal swabs are adequate; collect into virus transport medium; positive results are most likely in the first 24–48 h after infection. Culture in eggs or cell culture. Gold standard, but slow and prone to false negatives. Successful virus isolation is an essential component of surveillance programmes Polyester-tipped nasopharyngeal swabs are ideal; (short) nasal swabs are adequate; collect into virus transport medium. Nucleoprotein enzyme-linked immunosorbent assay is used widely. Patient-side enzyme-linked immunosorbent assays for detection of human influenza A and B viruses are useful for rapid screening of horses Polyester-tipped nasopharyngeal swabs are ideal; (short) nasal swabs are adequate; collect into virus transport medium. The RT-PCR is rapid, highly sensitive, specific and widely available; first choice in outbreak control and screening programmes for equine influenza Serum. Haemagglutination inhibition is the classical assay; subject to considerable interlaboratory variation. Single radial haemolysis is more reliable; titres correlate with susceptibility to infection (>75 mm2 suggests clinical protection, >150 mm2 suggests virological protection)
Antigen detection
Nucleic acid detection
Antibody detection (serology)
infected and managed accordingly. All positive samples above the validated cut-off for the assay should be regarded as evidence of exposure to EIV. However, positive samples with low levels of RNA are difficult to interpret because RT-PCR (and enzyme-linked immunosorbent assay) can detect nonviable and degraded virus even after horses are no longer infectious or constitute a risk of infection to other horses. For example, during the 2007 outbreak in Australia, RNA was detected up to 34 days after infection [18]. Assessment of the risk posed by horses with weakly positive RT-PCR tests should be interpreted based on sampling on consecutive days and in light of clinical and epidemiological data.
Question 2. How can contamination be prevented during an outbreak? All equine businesses need to be prepared for when, and not if, an EIV outbreak occurs. Biosecurity (even simple hygiene measures) is an unfamiliar concept that is not accepted by all parts of the equine industry. Simple measures, such as hand washing, minimising horse-to-horse contact and controlling fomites, significantly reduce the risk of disease transmission. Such measures can be implemented easily by all equine businesses, race meetings and competitions. Biosecurity protocols were developed for the Olympic Games in London 2012 and by the Federation Equestre Internationale. Some countries have established epidemiological surveillance centres for equine disease, such as Reseau d’EpidemioSurveillance en Pathologie Equine (RESPE) in France, RESPE in Ireland (iRESPE) and the International Collating Centre (ICC) in the UK. During an outbreak, a crisis management centre, consisting of individuals from the equine industries in Europe, is established to propose and coordinate an emergency response plan. Simple biosecurity measures combined with quarantine and suspension of movement of horses should be implemented immediately, and early veterinary involvement is essential. Horses should be regarded as contagious for at least 2 weeks, although 3 weeks of quarantine may be more prudent. Rectal temperatures should be monitored twice daily and recorded. The spread of EIV in vaccinated and/or mixed populations may not be as explosive as in naïve populations because of slower disease transmission in vaccinated horses. It is important that all of those managing outbreaks in older, vaccinated horses should be aware of these differences. The high level of biosecurity required by, for example, the pig and poultry industries, is unachievable across much of the equine industry, owing to the frequent movement and mixing of horses. Tighter control of
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the transportation of horses, such as accurate registration procedures and compliance with basic sanitary conditions, is of the utmost importance because of the major role that movement of horses plays in virus spread. The ability to implement control measures and the attitude to risk determine what control measures would be appropriate. The measures that can be achieved realistically need to be taken into account when constructing control programmes. In the face of an outbreak, it would appear logical to vaccinate primed horses, because they are likely to develop a rapid, anamnestic response. Vaccination should be recommended for in-contact horses that have shown no clinical signs in the previous 24 h. Booster vaccination appears to be effective even when the vaccine does not contain the circulating virus, because the high titres of antibody generated by booster vaccination are likely to be cross-protective. Likewise, the booster vaccine does not appear to need to match previously administered vaccines [19,20], although there are few published data on this.
Question 3. What lessons can be learned from the French EIV outbreak in 2012? The epidemiology and virology of a 2012 EIV outbreak that occurred in France and potentially affected horses that were intended to compete at the London Olympic Games were reviewed. The outbreak occurred mainly in a mixed population of vaccinated and nonvaccinated horses on 3 stud farms in Normandy. Studies of the HA1 portion of the H3 gene [21] revealed a clade 2 virus, similar to strains circulating previously in Europe [1,21–27]. Equine influenza virus was confirmed on a farm with about 150 horses (eventers, broodmares, young horses and foals) in the Calvados region on 4 May 2012. Eventers were vaccinated twice annually, and all other horses were vaccinated annually. The vast majority of horses developed fever, which was accompanied, in some, by cough. Clinical signs were recorded for 11 days. Three days later, EIV was confirmed on another farm, with 70 horses (eventers, broodmares, young horses and foals), in the same region. Adult horses were vaccinated as at the first location. Young horses were not vaccinated. The vast majority of horses developed fever and cough. Clinical signs were recorded for 10 days. Two days later (9 May), EIV was confirmed in 3 horses on a 70 horse farm where competition horses were vaccinated twice annually, some horses were vaccinated annually and some horses were not vaccinated. Fifteen horses developed fever, cough and, in some cases, nasal discharge. Competition horses, if affected, had fever but no other clinical signs. Clinical signs were recorded for 10 days. A 10-day-old foal from a nonvaccinated mare had to be subjected to euthanasia owing to the severity of disease. Two asymptomatic horses, vaccinated twice annually, were diagnosed positive for EI following a routine veterinary examination prior to an international showjumping competition (CSIO 5*) in La Baule, LoireAtlantique, on 11 May. Although these horses were immediately removed from the holding stables and were not permitted to compete, this does not preclude dissemination of virus prior to the positive test result. A competition mare in Oise, Picardy, was confirmed EIV positive on 15 May. Several horses at this large riding establishment, where all 100 horses were vaccinated twice annually, were noted to be coughing. The first 3 farms are linked epidemiologically. Spread was facilitated by mingling of nonvaccinated and vaccinated horses on farms and at equestrian events (Saint Gatien, Calvados; le Touquet, Pas-de-Calais; Haras du Pin, Orne). The horses that tested positive at La Baule were at risk owing to both their geographical proximity to the first 3 farms and contact at equestrian events in Normandy. The horses on the Picardy farm were in contact via equestrian events in Normandy. Reseau d’Epidemio-Surveillance en Pathologie Equine set up a management crisis centre that brought together many equine industry stakeholders. This unit meets on a weekly basis to organise improvements in operational health measures and to maintain and disseminate timely communiqués and other seminal information. Following this outbreak, it was recommended that all competing and in-contact horses should be vaccinated twice annually. This is particularly important in young horses (1–5 years old) at the greatest risk of EIV infection. In addition, direct and indirect contact between vaccinated and nonvaccinated horses housed at the same site should be minimised and, ideally, avoided.
Equine Veterinary Journal 46 (2014) 645–650 © 2014 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of British Equine Veterinary Association.
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Question 4. Interindividual variation in equine influenza: why do some horses develop severe disease or die? Equine influenza virus infection produces a considerable spectrum of clinical disease. Characteristic clinical signs are seen in naïve animals, but less severe disease, with mild and transient or subclinical clinical signs, is generally seen in vaccinated horses. Deaths occur mainly in foals from nonvaccinated mares; thus, inadequate passive transfer of antibodies, due to poor-quality colostrum or inadequate intake, is likely to be a major factor. All mares should be vaccinated adequately to ensure that there are sufficient maternally derived antibodies in colostrum. There is evidence that infection with different EIV strains (genotypes) results in different clinical outcomes (disease-causing phenotypes) [28,29]. Certain strains appear to be more likely to produce primary viral pneumonia; others are more likely to be followed by secondary bacterial infection, whereas the remainder produce less severe disease. There is also likely to be a strong host effect, with genotype and previous immune experience playing roles.
Question 5. Update on strains and immunity: what paths should be explored? Veterinarians play a critical role in EIV surveillance, but are not always aware of the importance of investigation, sample collection and reporting of suspected EIV outbreaks, vaccination breakdowns and suspected adverse reactions. In addition, gathering of data is frequently hampered by the lack of a standardised approach and economic constraints. The equine industry needs to continue to support surveillance schemes, and professional associations should promote awareness of the veterinarian’s role in disease surveillance. Equine industry- and government-supported surveillance, especially in the UK and Ireland, has allowed effective monitoring of the evolution of H3N8 EIV strains and has tracked the initial divergence into European and American lineages and the continued evolution of the American lineages, such that current circulating strains are from clades 1 and 2 of the Florida sublineage. The utility of the HI assay used to characterise HA could be improved by using standardised equine antisera in addition to ferret antisera, although the former are more difficult to generate and more cross-reactive than the latter. Some horses exhibit clinical and virological protection in the absence of (single radial haemolysis) antibody titres [1,5,30]. There is a paucity of data on the role of and targets for cell-mediated immunity in EIV, which should be a target of further research. Finally, the panel discussed the current difficulties faced by vaccine manufacturers in updating EI vaccine strains in Europe. Updates require a variation in the marketing authorisation for a vaccine regulated by EC/1234/2008. The panel considered the revision of the current guidance to be timely and agreed that it provided an opportunity further to streamline EI vaccine updates by animal health companies.
Question 6. What is the best way to assess vaccine efficacy? The regulatory framework sets out the safety, efficacy and quality data required to obtain market authorisation, but the panel agreed that efficacy in field situations is a more demanding and diverse concept than in the laboratory setting. Experimental challenge is designed to provide a controlled environment, with fewer confounding factors than in the field, to allow the evaluation of vaccine efficacy and safety in comparison to nonvaccinated control horses. Typically, relatively small groups of animals of similar ages, types and previous immune experience of EIV are used. Most studies follow a similar study design, in which animals are vaccinated in accordance with datasheet recommendations and then challenged at the onset of immunity of the vaccine. This provides a standardised approach to comparing vaccine performance and measuring ability to protect, but may not be completely representative of the vaccination protocols used in the field. It is important that immunisation in the field
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should follow, as far as possible, the datasheet recommendations. Testing protocols should be standardised between laboratories so that critical points, such as challenge method (i.e. nebulisation or instillation of virus suspension, horse-to-horse transmission), sampling times, method of sampling (e.g. nasopharyngeal or nasal swabs), time of challenge and duration of follow-up, are harmonised, thus allowing comparison between studies. In Europe, vaccines for intramuscular administration contain whole inactivated virus, subunit (purified proteins), either alone or in a live vector (canary pox), vaccines containing either carbomer, carbomer and aluminium hydroxide, immune-stimulating complexes (ISCOM) or ISCOM-matrix adjuvants [31]. In North America, vaccines of different antigen and adjuvant composition are available for intramuscular administration, and a live, attenuated, intranasal vaccine is also available [32,33]. The panel agreed that all EI vaccines currently available in Europe would be expected to perform well, even against heterologous challenge, when tested at peak immunity. However, extrapolation to the field situation would be incomplete, because horses may be challenged at any time. In the field, vaccine efficacy can be monitored serologically, because there is a well-established correlation between single radial haemolysis antibody titres and protective immunity following vaccination with inactivated whole virus and subunit vaccines [34,35]. This approach has been employed in a few comparative field studies [20,36–38]. The duration of immunity of EI vaccines needs also to be evaluated alongside the constantly evolving virus. The efficacy of EI vaccines may be limited if antibody levels fall below protective levels between vaccinations [37,38] and challenge occurs during this so-called immunity gap [5], between the second and third EI immunisation [39]. The vaccination protocols that are currently accepted by racing and equestrian sport regulatory bodies do not take this into account. Ideally, there should be a more holistic assessment of vaccine efficacy that also takes data from appropriately designed and controlled studies into account. As a minimum, assessment of vaccine efficacy should include consideration of in vitro immunological data, experimental challenge data, longitudinal immunological data from different age groups of horses and surveillance data from the field.
Question 7. What do we expect from vaccines and why does vaccination fail? The panel discussed how the expectations of the equine industry contrasted with those of the livestock sector; with the exception of the racing and competition sectors, herd protection is not widely accepted. Much of the sector is unaware that vaccination helps to reduce the prevalence and severity of disease. The majority of owners are pleasure horse-owners with individual animals, who are not used to thinking of their animals as part of a herd, even in livery yards. These owners generally expect vaccines to produce long-lasting, sterile immunity, protecting each horse from both infection and disease. There is a significant need for veterinary practices to communicate realistic and effective messages about the appropriate use of vaccination and disease control. There are subpopulations of horses that probably never receive influenza vaccination because of economic constraints, lack of motivation, lack of awareness or cultural attitudes to preventive medicine and disease control. Population (herd) vaccination coverage is needed to control EI. A small percentage of the population fails to mount and/or maintain an adequate immune response after immunisation and therefore remains susceptible to EI despite having been vaccinated appropriately [15,20,37,38]. Although the reasons for this remain unclear, age appears to be a significant factor, with the majority of poor responders being
