Equine influenza

GENERAL INTRODUCTION: ORTHOMYXOVIRIDAE

Introduction

Equine influenza is an acute and highly contagious respiratory disease of horses, donkeys, mules and zebras caused by infection with type A influenza viruses.

Epidemics of acute respiratory disease in horses have been reported for several centuries,^{36, 88} but it was only in 1956 during a widespread epidemic among horses in eastern Europe that the first isolate of equine influenza virus was recovered.⁷³ The virus, characterized as H7N7, was designated as influenza A/equine/Prague/56 and caused epidemics during the 1960s and 1970s.³⁴

In the early spring of 1963 a major epidemic occurred in the USA which was caused by an influenza A virus of a different antigenic subtype, H3N8.^{34, 86} This novel virus, designated as influenza A/equine/Miami/63, was probably introduced into the equine population of Florida with the importation of horses from Argentina.¹⁰² Isolation of an H3N8 virus was also made from South American horses in 1963.⁸⁶ Equine H3N8 was subsequently introduced into France towards the end of 1964,^{10, 11, 35} where it spread rapidly through Europe in the late winter and spring of 1965.³⁴ H3N8 was repeatedly isolated from horses involved in an epidemic that spread throughout the USA and Canada in 1965¹³ and in the same year horses in England experienced influenza, but this was caused by an H7N7 virus.³⁴

There have been numerous reports of outbreaks of equine influenza in Europe, North America and many other countries since the emergence of the H3N8 subtype.^{3, 10, 11, 18, 19, 46, 48, 55, 66, 67, 69, 70, 82, 95, 99, 104} However, equine H7N7 viruses have not been isolated since 1980 and are considered extinct.⁹⁷ In contrast, H3N8 viruses continue to cause severe respiratory disease among non-vaccinated horses. Periodically, as occurred in Europe in 1979 and 1989 and Japan in 2007, there are large epidemics even among recently vaccinated horses associated with significant antigenic changes in H3N8 viruses.^{48, 67, 104} Following the outbreak in Europe in 1989 in which there was significant failure of vaccines to prevent infection, there was a further divergence of H3N8 viruses into two antigenically distinct lineages (so-called American and Eurasian, due to their initial geographic distributions). Field and experimental studies provided increasing evidence of failure of inactivated viruses of these two lineages to cross-protect. At the time this confirmed the importance of including representative viruses of both lineages in vaccines. These studies also showed the importance of updating vaccines by replacing older outdated vaccine strains with more recent and epidemiologically relevant viral strains. This led to the establishment of an Expert Surveillance Panel (ESP) for vaccine strain selection.²³ The ESP includes representatives from OIE reference laboratories and WHO experts. The panel review surveillance data collected by both OIE reference- and collaborating laboratories from around the world on an annual basis. The panel makes recommendations on which strains should be included in vaccines, which are published by the OIE.

Eurasian lineage strains (subtypes) have not been isolated since 2005 and are no longer thought to circulate. Consequently, there is no longer a requirement to include a Eurasian lineage strain in vaccines.⁷² Meanwhile, the American lineage evolved further into three sub-lineages: Kentucky, Florida and South American.⁵³ In the early 2000s the Florida sub-lineage diverged into clades 1 and 2, which are antigenically and genetically distinct.¹⁵ Although clade 1 viruses have predominantly been isolated from North America, they have been responsible for causing large outbreaks in South Africa, Japan, Australia and South America and in recent years there have been isolated from outbreaks in Europe.^{3, 5, 14, 16, 38, 40, 48, 80, 104} In contrast, the majority of clade 2 viruses have been isolated from horses in Europe and Asia.^{15, 32, 37, 84, 95, 101, 107} Reassortment between the different lineages, sub-lineages and clades of H3N8 has also been reported.^{15, 64, 65, 101} For example, viruses encoding a clade 1 haemagglutinin (HA) and clade 2 neuraminidase (NA) have previously been isolated in the UK, revealing evidence of co-infection with the two clades.¹⁰¹ The current OIE-recommendation is to include an example of each clade in vaccines.⁷²

Aetiology

Influenza viruses are pleomorphic, spherical or filamentous virions with a diameter of 80 to 120 nm and have a segmented, single-stranded RNA genome of negative sense. The eight gene segments code for two surface glycoproteins; the haemagglutinin (HA, restricted to H when assigning subtypes e.g. H3) and neuraminidase (NA, restricted to N when assigning subtypes e.g. N8), the internal matrix protein and nucleoprotein and other structural and non-structural proteins involved in virus replication (Figure 1). The RNA segments are closely associated with the nucleoprotein (NP) and are surrounded by the matrix (M1) protein which is itself closely associated with the lipid envelope containing the two surface glycoproteins (Figure 1).⁴⁵ The haemagglutinin is the major surface glycoprotein making up approximately 25 per cent of the virus protein as compared with 5 per cent for the neuraminidase. Influenza virus is enveloped and, as such, is not infectious for long outside the host and is rapidly inactivated by sunlight and disinfectants.

Figure 1 Structure of the influenza A virus particle. The outer envelope of the virion is derived from the plasma membrane of the infected cell, through which the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA) protrude. The membrane ion channel protein M2 also spans the lipid bilayer. A layer of matrix protein (M1) lines the interior surface of the envelope.  The RNA gene segments are at the centre of the particle, closely associated with nucleoprotein (NP) and bound to the viral polymerase complex for replication.

There are three types of influenza viruses: A, B and C, which are all classified within the Orthomyxovirus group, the virus type being determined by the antigenic character of the nucleoprotein and matrix proteins. All equine influenza viruses are type A and the antigenic character of the HA and NA defines subtypes within a virus type. Among influenza A viruses 16 HAs and 9 NAs have been identified to date in wild aquatic birds, the natural host for influenza A viruses.^{31, 98} Two further subtypes, H17N10 and H18N11, have been detected in bats⁹¹ However, in the viruses that infect horses, only two subtypes, H7N7 and H3N8, have been recognized.

Antigenic drift occurs when mutations in the gene sequence result in amino acid substitutions, particularly in the haemagglutinin. Antigenic drift has been detected in H3N8 viruses to the extent that at least two antigenically divergent lineages were co-circulating in the 1990s, and two antigenically distinct clades of the Florida sub-lineage circulate today.^{3, 25, 72, 84}

The technique of phylogenetic characterization has demonstrated that the two clades of the Florida sub-lineage are becoming increasingly divergent.¹⁰¹ Phylogenetic relationships between viruses are established using evidence from sequencing of the HA gene and are confirmed by antigenic analysis using reciprocal HI tests and ferret anti-sera.¹⁰¹ Experts of the OIE/WHO have recommended that if equine vaccines are to be effective, they should contain representatives from each of these clades.⁷²

Major or subtype changes in the surface glycoproteins occur as a result of recombination with other influenza viruses and are called antigenic shifts. Antigenic shift gives rise to new viruses that may result in pandemics in susceptible populations. New viruses may also infect horses as a result of cross species transmission from aquatic birds, considered the major reservoir for influenza A viruses, as was seen with the emergence of equine H3N8 viruses in horses in Miami in 1963. This is thought to be the origin of the current H3N8 viruses, but also occurred in China in 1989 when a different avian H3N8 influenza virus caused an outbreak with high mortality.³⁹ Fortunately such significant cross species transmission events are a rare.

In 2004, equine influenza virus caused an outbreak of respiratory disease in greyhounds in the USA.²¹ The virus has since adapted to dogs and is termed canine influenza H3N8. Two small equine H3N8 outbreaks have been reported in foxhounds in the UK^{24, 71}  and dogs seroconverted following the Australian outbreak in 2007,²² however the H3N8 virus has not become established in canines outside the USA. It has also been demonstrated that the adapted canine virus is unable to infect horses.^{83, 106}

Epidemiology

Equine influenza is a highly infectious disease characterized by rapid spread in susceptible populations and morbidity rates are consequently high. In a study conducted during 1965 only 11 out of 634 susceptible horses resisted the disease, giving a morbidity rate of 98,2 per cent.³⁴ Equine influenza has been reported in many parts of the world, including North and South America, the West Indies, Europe, North Africa, the Middle East, India, China, Singapore, Japan, and Mongolia. The introduction of equine influenza into South Africa in 1986 and 2003, Hong Kong in 1992, Dubai in 1995, Puerto Rico and the Philippines in 1997 and Japan and Australia in 2007 were other milestones in the transmission of this virus throughout the world.^{19, 58, 99} Following the 2007 outbreak in Australia, New Zealand and Iceland are the only countries that are known to have remained entirely free from the infection The disease is considered endemic in the USA, UK and other European countries, based on the occurrence of almost annual outbreaks.

There are many different factors that influence the epidemiology of equine influenza. Some relate to the intrinsic properties of the virus, some to the pathogenesis of the disease, the immune system of the horse and, not least, to the equine population structure and workings of the equine industry. A key factor in the spread of equine influenza in the last few decades has been the increase in transportation of horses over long distances by air. Horses incubating the disease, or those that are clinically or subclinically infected, can introduce the infection into a susceptible population if they are not quarantined adequately. Examples of this have been seen in the UK, South Africa, Hong Kong, Dubai, Puerto Rico and the Philippines.^{58, 99} The 2007 outbreak in Australia was attributed to a breakdown in quarantine procedures which led to the spread of virus to naïve domestic animals via human contact.¹⁹ In some areas of the world, quarantining has been virtually ignored in order to facilitate the international movement of horses for racing, competition and breeding purposes, particularly among Thoroughbreds. In addition, inadequate vaccination may lead to horses with partial immunity and result in the suppression of clinical signs. This may make infections difficult to recognize clinically while still allowing virus excretion.⁶² Such horses probably play an important role in the maintenance and spread of equine influenza. However, countries such as Australia, New Zealand, Dubai, Hong Kong and Japan now all have extremely stringent quarantine requirements, even for major competitions, in order to prevent the introduction of equine influenza infection to their susceptible horse populations. In most of these countries, vaccination is used in conjunction with strict quarantine and pre- and post-import testing.

Influenza viruses in horses infect the epithelium of the upper and lower respiratory tract, resulting in a frequent and harsh cough. This cough is an efficient way of spreading the virus in aerosols³⁵ and over distances of 32 metres⁵⁶ or more, although the upper limits of distances over which airborne spread occurs are poorly defined and are likely to be dependent on many local factors. This makes it difficult to provide firm recommendations regarding safe distances of separation between groups of horses in order to prevent transmission of equine influenza compared to other equine contagious pathogens. Although it is believed that influenza is transmitted almost exclusively by direct contact between horses, evidence from the South African outbreaks in 1986 and 2003, and the Australian outbreak in 2007, indicated that the virus could be transmitted indirectly by people and contaminated vehicles.^{19, 27} Virus excretion in fully susceptible horses lasts between seven and ten days, although infected animals are rarely infectious for more than seven days. Data from experimental challenge infections suggest that large amounts of virus may be shed continuously over the infectious period, with >10³ EID₅₀ influenza virus being recovered from single daily nasopharyngeal swabs.⁵⁸ Coughing may persist for longer than virus excretion, but the short incubation period also contributes to the rapid spread of infection. Although long-term carriers have been postulated as being an important element in the epidemiology of equine influenza, there is no good evidence to support this. Immunity to equine influenza is short-lived, with clinical immunity lasting little more than a year. Immunity to infection may be even more short-lived, therefore, allowing reinfection to occur without clinical signs within a matter of months after a previous infection.⁴⁴ Unlike human influenza, there is no evidence that the occurrence of equine influenza is influenced by season, however climatic conditions such as relative humidity, temperature and wind speed have been linked to an increased risk of virus spread during an outbreak.²⁹ Outbreaks of disease, particularly in the USA and some parts of Europe, are primarily related to the movement of horses and to the introduction of young stock onto racetracks with large mobile populations of horses and where the infection may be endemic.

Features of equine influenza are similar in other equine species, such as donkeys, mules and zebras,²⁷ although a high mortality rate has been reported in donkeys.⁸⁵ Indigenous equids are likely to be important reservoirs for equine influenza viruses in regions where widespread vaccination of very large populations is impractical.

The widespread epidemic that was caused by a variant of the H3N8 subtype in the USA and Europe during 1979–80⁴⁷ first raised the possibility that antigenic drift could be playing a major role in the epidemiology of equine influenza. Retrospective studies on the antigenic character of H3N8, however, demonstrated that Miami/63-like and Kentucky/81-like viruses had been co-circulating in the equine population for some years.⁴⁷

Challenge studies have shown good correlation between vaccine-induced neutralizing antibody levels directed against HA and measured by single radial haemolysis (SRH) and protective immunity against infection with antigenically similar viruses (‘homologous’ viruses).^{57, 60, 61, 63} Experimental challenge infections using three different levels of nebulized virus (10⁶ to 10^7,6 EID₅₀/ml) required levels of antibody measured by SRH of between >120 mm² and >154 mm² to protect against infection,⁵⁷ with the protective threshold increasing incrementally with the infectious viral dose. Field studies have confirmed these observations in vaccinated racehorses. Horses with pre-exposure SRH levels ≥140 mm² did not show evidence of infection by subsequent seroconversion or development of antibody measurable by nucleoprotein antigen enzyme-linked immunosorbent assay (ELISA).^{69, 92} However, population modelling has shown that there is an increased risk of infection in horses vaccinated with strains heterologous to the outbreak strain.⁷⁹ Field studies have also confirmed the need for inclusion of antigenically relevant strains in vaccines^{55, 70} due to the decrease over time of cross-protectivity between vaccine and challenge viral strains. This results from antigenic drift and means that increasingly higher levels of protective immunity are required from outdated vaccines in order to achieve minimal protection.²⁵ In the case of the 2003 outbreak in Newmarket, the mean SRH level of horses that did not seroconvert during the outbreak was 203.6 mm². This was significantly higher than the mean value of 156.2 mm² for those that did seroconvert.⁶⁷ Ultimately, when circulating field strains become sufficiently different from vaccine strains, they can cause clinical influenza irrespective of the frequency of administration and degree of response to vaccination, as was seen in Europe after 1989, in the UK in 2003 and Japan in 2007.^{48, 54, 67, 104}

Pathogenesis

Influenza virus, after attaching to cilia, infects epithelial cells in the mucosa of both the upper and lower respiratory tracts. The haemagglutinin (HA) mediates the attachment of virus to host cells. Antigenically, the HA is of prime importance as antibodies that react with it can neutralize the virus. There are five antigenic sites on the HA molecule, and amino acid changes in all five sites produce antigenic drift of significant epidemic potential.^{55, 87, 100}

Neuraminidase (NA) has essential roles in virus entry and release.  It has sialidase activity which allows transport of virus through mucin by removing decoy receptors in the respiratory tract. It also destroys HA receptors, thus allowing elution of progeny virus from infected cells and release of virus particles. Antibodies to human influenza NA can inhibit cell to cell spread of virus and contribute to protection.⁴¹ However, the importance of equine influenza NA to immunity is not currently known.  NA has also been the target of antiviral drugs, designed to inhibit its function thereby preventing viral spread. Experimental use of NA inhibitors have shown their potential for the treatment of equine influenza virus infection.^{103, 105}

Virus replication is rapid with viral antigen appearing in cells within two hours,²⁸ and complete deciliation of the epithelium of the trachea occurs within four days of infection.⁸

Clinical signs

The clinical features of equine influenza have been previously reviewed in detail.³⁴

In controlled experimental challenge studies, the incubation period usually varies between one and three days with a range of 18 hours to five days with the length of the incubation period being inversely related to the virus dose.⁵⁹

Coughing and fever are the most common clinical signs of equine influenza, the cough being dry, harsh and initially non-productive. Coughing is frequent during the first week of infection, and in uncomplicated cases given sufficient rest, will disappear within one to three weeks. There is usually a mild rhinitis and no obvious swelling of the submaxillary lymph nodes. The nasal discharge is initially serous but subsequently becomes mucopurulent when secondary bacterial infection of the respiratory tract has occurred.

Peak temperatures have been reported as 41,2 °C³⁴ and the fever may be biphasic during the initial, purely viral phase of the disease, with the height and duration of pyrexia being related to both the subtype involved and the infectious dose received.⁵⁹ Continuous fever beyond four or five days accompanied by pronounced mucopurulent nasal discharge is invariably due to secondary bacterial infection. Other signs of influenza infection include anorexia, dyspnoea and myalgia, and there may be icterus in some cases. Cardiac damage may occur, particularly in older animals and in those that have been worked during the acute phase of the disease.³⁴

Mortality rates are usually very low in uncomplicated cases, with the exception of young foals that have not acquired maternal-derived immunity. Death in foals occurred during H3N8 outbreaks in Britain in 1965⁵⁹ and in South Africa in 1986,²⁷ particularly in those that were born at the time that their dams were suffering from the acute disease. These foals developed very high temperatures (41 to 42 °C), stopped sucking, had increased heart rates and manifested signs of severe respiratory distress, including tachypnoea (80 to 90 respirations per minute) and soft gurgling sounds on auscultation of the thorax. Pneumonia developed soon afterwards and they became weak and died seven to ten days later.

Influenza is also a cause of mortality in donkeys and mules.⁸⁵ Necropsies performed on donkeys affected in the H3N8 outbreak in Britain in 1969 revealed acute bronchopneumonia complicated by secondary bacterial infection.⁸⁵

Secondary complications and sequelae to equine influenza are common in stressed or neglected animals. Bacterial infections (including Streptococcus equisimilis, Streptococcus zooepidemicus, Escherichia coli, Staphylococcus spp., Pseudomonas spp., Pasteurella spp., Aerobacter spp., Actinobacillus equuli and Bordetella bronchiseptica) result in purulent nasal discharges, pharyngitis, conjunctivitis, and sometimes bronchopneumonia. Strangles and purpura haemorrhagica have also occurred in association with influenza infections. Chronic pulmonary disease following uncomplicated influenza or secondary bacterial infection was common after the 1965 pandemic. Horses with signs of coughing had bronchitis combined with bronchiolitis for months after the acute infection. Such sequelae have been attributed to too short a period of rest during the acute infection, early resumption of hard work, poor environmental conditions, and inadequate antibacterial therapy.

It is increasingly recognized that among vaccinated horses that have some, but incomplete, immunity, outbreaks of clinically mild influenza do occur and these are often also characterised by rapid spread of signs, such as nasal discharge and occasional cough, where they do occur. In such outbreaks there is frequently mild signs that may not be recognized or diagnosed as influenza and often the first sign noted is poor training and racing performance.^{49, 66, 70} Outbreaks have been described in which the infection circulated sub clinically for 18 days before recognizable clinical signs were observed.⁸²

Pathology

Haematological changes during equine influenza infections are non-specific and variable and hence are not diagnostic for the disease. Anaemia, leukopenia and lymphopenia are often observed in the first three to seven days, with an increased neutrophil to lymphocyte ratio in the first two or three days after infection.^{2, 34} Monocytosis may also be observed during early convalescence from about day seven after infection.

The most important pathological changes associated with infection of horses with influenza viruses occur in the lower respiratory tract and include bronchitis, bronchiolitis and interstitial pneumonia accompanied by congestion, oedema and neutrophil infiltration.

Secondary bacterial infections and other complications may result in conjunctivitis, pharyngitis, purulent bronchopneumonia, chronic pulmonary disease, guttural pouch infections, purpura haemorrhagica and strangles.

Diagnosis

The clinical features of equine influenza in susceptible animals (rapidly spreading disease manifested by a harsh, dry cough, high temperature and nasal discharge) are sufficiently characteristic to permit a tentative diagnosis. However, in animals that have previously experienced the infection or that have waning vaccinal immunity, it is difficult to differentiate influenza from other respiratory infections. In such situations, or where equine influenza has not occurred in the locality in the recent past, laboratory diagnosis is required, involving virus isolation, agent detection (RNA or protein) or serology.

Specimens for virus detection should be collected as soon as possible after the onset of pyrexia and coughing, as the period of virus excretion may be as short as one to two days in primed animals. Virus may be detected from nasopharyngeal secretions collected into virus transport medium, usually by swabbing or lavage of the nasopharynx or by lavage of the trachea through an endoscope. The transport medium should not contain fetal calf serum for reasons explained below. The samples should be submitted to the laboratory as quickly as possible.

The adoption of more widespread vaccination has made the diagnosis of influenza infection less straightforward,with clinical signs being less severe, blood samples from acute cases already possessing moderate levels of serum antibody, and the quantities of live virus retrievable from the respiratory tract being greatly reduced. The sensitivity of quantitative (or real-time) reverse-transcription polymerase chain reaction (qRT-PCR) enables the detection of viral RNA, even at low levels, in extracts from nasopharyngeal swabs. qRT-PCR assays have been developed targetting the more conserved gene segments such as matrix (M) and nucleoprotein (NP). These assays are more sensitive than virus isolation in eggs or detection of NP protein using rapid antigen detection (RAD) kits.⁷² A qRT-PCR assay targetting the M gene, originally developed for the detection of a wide range of influenza A viruses,  was effectively used during the eradication programme in Australia in 2007³⁰  and is used for the diagnosis and surveillance of equine influenza by OIE reference laboratories.^{37, 84} Following initial diagnosis, sequencing of the HA and NA genes is performed to confirm subtype.

Viral antigen may also be detected on site (sometimes referred to as ‘horse-side’ or similar) using a variety of different RAD tests developed for use in detection of human influenza A infection; however, samples should whenever possible be submitted to an OIE reference laboratory for confirmation and further analysis, including virus isolation and RNA sequence determination. Influenza virus can be cultured in embryonated hens’ eggs or in susceptible mammalian cells such as Madin- Darby canine kidney (MDCK) cells. Embryonated hens’ eggs are inoculated via the allantoic cavity of 9- to 11-day-old embryos. Amniotic and allantoic fluids are harvested after incubation for 48 to 72 hours at 34 °C and examined for haemagglutinating activity using chick erythrocytes. Virus isolation in mammalian cell cultures, e.g. MDCK cells, is less satisfactory than isolation in eggs because this process increases antigenic heterogeneity and can be less sensitive than embryonated hens’ eggs. The virus samples need to be inoculated into the cell culture in the presence of trypsin and the absence of serum. The presence of virus, which may not cause obvious cytopathic effect, is detected by assay of tissue culture fluid for haemagglutinating activity after four to seven days’ incubation at 34 °C. Two or three passages may be required in order to isolate infectious virus. Isolated viruses should be submitted to OIE reference laboratories for detailed sequence analysis of HA and NA and antigenic characterisation.

Antibody to influenza virus may be detected by its ability to inhibit the agglutination of chick erythrocytes mediated by the viral haemagglutinin in the haemagglutination inhibition or HI test. The HI test is subtype specific, and is much more sensitive for antibody to H7 viruses than it is to H3 virus antibody when whole virus is used in the assay. The sensitivity of the test for antibody to the H3 viruses may be enhanced by disruption of the virus with detergent.⁵⁰ In HI tests a four-fold or greater increase in antibody titre against H3N8 between acute and convalescent-phase sera is regarded as significant and indicative of infection, in the absence of recent vaccination. Where vaccination history is not known care may need to be taken in serologically confirming equine influenza infection. Although an ELISA has been developed for detection of serum antibodies to influenza NP, careful consideration is required when interpreting results from vaccinated horses as NP is only present in some vaccines.^{26, 52} However, this approach of detecting serum antibodies to NP was used successfully in the eradication of and declaration of freedom from equine influenza virus from Australia in 2007 as the assay, in combination with the HA only based canary-pox vectored vaccine that was being exclusively used, facilitated a DIVA strategy that differentiated infected from vaccinated animals.^{51, 74}

Subclinical infections may occur in vaccinated horses but they do not necessarily stimulate a four-fold increase in HI antibody, and such animals may be a source of infection to other horses. Significant increases in antibody may be detected using the SRH test. In this test, influenza virus is coupled to sheep erythrocytes with chromium chloride, and agarose gels prepared containing these sensitized cells and guinea pig complement. Equine serum, inactivated by heating at 56 °C for 30 minutes, is introduced into wells in the gels and incubated for 18 to 20 hours at 34 °C.⁷² The diameters of the resultant zones of haemolysis are measured with a calibrated viewer and digital recording apparatus. Seroconversion indicating infection is demonstrated by an increase in the SRH area of 25 mm² or 50 per cent, whichever is the smaller, between the acute and convalescent serum samples. The reproducibility of the assay allows the identification of infections that stimulate no more than a two-fold increase in antibody. The SRH assay has also been widely used in both experimental and field studies of vaccine efficacy, and correlation between vaccine-induced SRH antibody and protective immunity from killed vaccines is now well established.^{57, 69, 92}

Serological diagnosis of infection in a vaccinated population is complicated by the presence of vaccine-induced antibody. Some vaccines are trivalent and contain one representative strain of the H7N7 virus and two strains of the H3N8 virus, however OIE recommendations since 2010 have no longer included an H7N7 representative. Vaccination usually stimulates significant increases in antibody to both subtypes, whereas antibody to infection is subtype-specific.

Differential diagnosis

Equine influenza must be differentiated from acute respiratory diseases caused by both infectious (including other viruses and bacteria) and non-infectious agents.

Viral infections that must be considered as differential diagnoses are equid herpes virus-1 and -4 (EHV-1 and EHV-4), equine rhinitis viruses (ERV), and equine viral arteritis (EVA). Bacterial infections caused by Streptococcus equi (strangles), Streptococcus zooepidemicus, Pasteurella spp., Actinobacillus spp., Rhodococcus equi, Mycoplasma felis and others may produce similar clinical disease syndromes.

Control

Multivalent, inactivated, adjuvanted influenza vaccines for horses were developed in response to the worldwide pandemic of 1963–64.^{12, 17, 33, 81} This early work (based on experience from human vaccines) led to the development of the now broadly standard schedules for equine influenza vaccination. In these schedules it was recommended that a primary course of two doses of injections be given approximately four to six weeks apart, followed by a booster vaccination six months after the end of the primary course, and that annual boosters be administered thereafter. The same schedules are still adopted today in product data-sheet recommendations for vaccines and are the basis for the regulatory rules (including International Equestrian Federation (FEI) and British Horseracing Authority rules) for most international equine competitions.

Direct transmission from acutely infected to susceptible horses is the usual mode of transmission and the most effective means of control in the face of an outbreak are vaccination and restriction of the movement of horses. Vaccination programmes are most successful when the majority of horses that are travelling and mixing with others for competition or breeding purposes are vaccinated. This can best be achieved by instituting mandatory vaccination policies, and such systems operate in many European countries. It may not be possible to impose or monitor vaccination of indigenous equine species that do not take an active part in competition or travel nationally or internationally. Nevertheless, mandatory vaccination of high-risk groups has reduced the prevalence of equine influenza in Europe.

Currently there are at least four types of vaccines against equine influenza: whole inactivated virus vaccines, immune-stimulating complex (ISCOM) adjuvanted subunit vaccines, a live attenuated vaccine and a recombinant poxvirus-vectored vaccine.⁷⁶ Influenza vaccines may be provided in combination with vaccines for tetanus and equid herpes-virus 1 infection. Despite there being different types of vaccines against equine influenza, the same vaccination schedules currently apply to all of them. This is possibly driven by the regulations set out by international equine competition authorities.

Since the 1979 H3N8 epidemic in Europe, in which there was clear evidence of vaccine breakdown, there have been extensive studies on the efficacy of equine influenza vaccines, particularly with respect to their HA content, ability to stimulate antibody,⁶² and protective efficacy.⁶³ The failure of inactivated vaccines to perform adequately in the 1979 influenza epidemic was largely attributed to low potency and unrealistic vaccination schedules, although antigenic differences between the Miami/63 prototype virus contained in the vaccines and the Fontainebleau/79 variant may have contributed. A clear relationship has been demonstrated between the HA content of vaccines, the antibody level to HA stimulated in horses measured by the SRH test, and protection against infection in experimental and field challenges^{62, 63} (see Epidemiology).

Review of the equine influenza epidemic that occurred in the UK in 1989, showed this occurred largely irrespective of whether horses had high levels of vaccine-induced antibody. That finding indicated that divergence of the field strain away from vaccine strains by antigenic drift was an important cause of vaccine failure and that careful consideration should therefore be paid to the epidemiological relevance of vaccine strains. This has been substantiated by the subsequent demonstration of divergent, non-cross-reacting H3N8 lineages/sub-lineages^{25, 101} and recommendation that both Florida clades need to be included in vaccines if they are to remain effective.⁷²

The vaccine manufacturer’s dosing recommendations should be regarded as a minimum, with additional vaccination recommended in high-risk situations, such as in the face of an outbreak or heightened risk of infection. Antibody stimulated after the second dose of vaccine may persist for less than eight weeks after vaccination with aqueous products and less than four months after immunization with adjuvanted vaccines resulting in a possible immunity gap. A challenge study using an H3N8 virus demonstrated that vaccinated horses could be infected 2-3 months after the primary course of two doses and transmitted live virus to naïve animals.⁷⁸ The rapid decline in circulating antibody has been shown in experimental studies to be less marked after the third (six month booster) dose for the most recently licensed vaccines in the UK. In controlled challenge studies these vaccines provided clinical protection 12 months after the third dose.^{60, 61}

Serological surveillance of vaccinated animals indicates that repeated vaccination with inactivated vaccines eventually results in durable antibody responses to the extent that annual revaccination is adequate for the provision of a solid immunity. For example, brood mares vaccinated in the month prior to parturition usually maintain high levels of HA antibody throughout the year. However, antibody responses to equine influenza are highly variable among individual horses. More than 5 per cent of horses may fail to produce an adequate antibody response after two doses of vaccine, and even after three doses a small number of individuals fail to respond. Thus protection of individual animals in high-risk situations may require vaccination based on the results of antibody determinations. In addition, young horses, especially groups of racehorses, have for a long time been known to be particularly susceptible to influenza infection⁹⁶ and that it is harder to stimulate vaccine-induced immunity in these young horses than in older animals.⁸¹ Vaccine failure is still most commonly reported in young racehorses, with factors relating to the vaccine potency, the vaccination schedule, the horse’s immunological response and differences between the infecting and vaccine viruses probably all contributing to this phenomenon.⁶⁹ A field study conducted in the UK confirmed that some important factors related to vaccination history affected levels of SRH antibody to influenza in young Thoroughbred racehorses entering training.⁶⁸ The significant factors were found to be (i) time since last vaccination (negative association), (ii) total number of previous vaccinations (positive), (iii) age at first vaccination (positive), and (iv) the types of vaccines previously administered (negative). The study showed that horses that had received a mixture of types of UK-licensed vaccines had the highest mean titres, followed by those that had received only carbomer-adjuvanted vaccines. Horses that had received all of another type of vaccine, which included those not licensed for use in the UK, had lower mean titres. Whilst horses that had no record of previous vaccination predicably had the lowest mean titres.

An anamnestic response can be detected in primed animals four days after vaccination,⁴⁴ with significant levels of antibody developing seven to ten days later. However, the mechanisms involved in immunity induced by vaccination and by previous infection are apparently different. While parenteral inoculation with inactivated vaccines stimulates locally produced antibody in the respiratory tract⁴³ and cytotoxic T cells,⁴² protection correlates most closely with SRH antibody in serum. In vaccinated animals which do become infected the suppression of clinical signs (duration of pyrexia and coughing), and the suppression of virus excretion in terms of titre and duration, is inversely proportional to the level of circulating SRH antibody at the time of infection.

In horses that have been previously exposed to infection, immunity to reinfection only occurs in the presence of high levels of SRH antibody. However, clinical protection (suppression of pyrexia and coughing) has been observed in the absence of circulating antibody,⁴⁴ and this suggests that infection is more effective than vaccination in stimulating other mechanisms of immunity, such as local antibody or cell-mediated responses. In vaccinated horses, immunity capable of resisting infection may persist for only three to six months, whereas immunity to clinical disease persists for 12 to 18 months following natural infection.

It is important, when considering protection of horses against equine influenza through the use of conventional vaccination, to ensure that the most effective and potent vaccines containing all relevant viral strains, are administered at strategic times with respect to expected challenge and at adequate intervals. Additional measures should include sensible management procedures to minimize the likelihood of introducing infection. In this respect, identification and re-boosting of ‘poor responders’ to vaccination may be particularly important, as these horses are most likely to introduce the infection into groups. In fact the source of equine influenza virus that was transmitted out of the Eastern Creek quarantine station to the highly susceptible resident population and led to the 2007 Australian outbreak was suggested to be via poor vaccine responders in the quarantined population that amplified the amount of virus in the environment.¹⁹ The results of the monitoring of SRH antibody in the investigation of field outbreaks and the surveillance of post-vaccination responses correlates with and validates the experimental vaccination and challenge models currently used in ponies in the licensing of modern equine influenza vaccines. Newer vaccine technologies have led to improved protective immunity from vaccination, attempting to reproduce the superior immunity produced by natural infection.  One example of this improved vaccine-induced immunity is the cold-adapted, modified-live, intranasal vaccine, which, in controlled blind challenge studies, was shown to provide clinical protection several months after a single dose administered to naive horses. However, this vaccine is currently licensed for use in North America only. In addition to the live-attenuated vaccine, the canarypox-vectored and ISCOM based vaccines have also been shown to induce cell-mediated immune responses.^{1, 75, 77}

In recent years, two experimental vaccines have been under development and pilot studies carried out to assess their safety and efficacy. The first, another modified-live virus vaccine, was generated using reverse genetics and encoded truncated forms of the NS1 protein.²⁰ Horses inoculated with these viruses did not develop fever or other clinical signs but were shown to seroconvert. Following challenge with wild-type virus, these horses displayed reduced clinical signs and virus shedding compared to naïve controls.²⁰ One benefit of using a reverse genetics approach, such as this one, is the ease in which the vaccine can be updated by changing the glycoproteins on the viral surface. The second approach has been to develop DNA vaccines where mammalian expression plasmids incorporating the HA gene from equine influenza viruses were synthesised and used as immunogens.⁴ A pilot challenge study showed that this type of vaccine protected against clinical signs and virus replication following challenge with the homologous virus.⁴ As with the poxvirus-vectored vaccine, one downside to this type of vaccine is that an immune response is mounted only against the HA of the virus. However as with the reverse genetics approach, vaccines can be easily updated by synthesising a plasmid with the relevant HA gene.

The frequency of vaccination of mares also affects the level and duration of maternal immunity provided to their foals. This in turn affects the response of foals to primary immunization since maternally derived antibody may inhibit their antibody response^{93, 94} and has also been shown to still contribute to increased susceptibility to infection during outbreaks.⁷ For this reason vaccine manufacturers recommend initiating influenza vaccination programmes in foals at about five or six months of age. However, much younger foals are capable of mounting an immune response, and in cases where mares have little or no influenza antibody, earlier vaccination may be indicated. Foals born out of seronegative mares during an influenza outbreak may be protected by the administration of immune plasma with antibiotic cover to minimize the risk of secondary bacterial infection.

During the febrile stage of influenza, horses may be treated with non-steroidal anti-inflammatory drugs while antibiotics, such as penicillin, may be useful to combat secondary infections. Horses recovering from equine influenza, even after mild or subclinical disease, should be given adequate rest before training is resumed in order to avoid subsequent chronic respiratory disease. As a guide, the number of weeks of rest that is recommended is the same as the number of days that the animal suffered from pyrexia, with a minimum of two weeks’ rest and slow resumption of work thereafter.

Although antiviral therapy in horses with influenza infection has been tried experimentally,^{12, 103, 105} the risk of fatal side-effects, impracticality and prohibitive expense all mean that it is not used routinely. Because of the availability of rapid diagnosis and longer duration of outbreaks in large groups of horses, it has become common practice for mass vaccination to be implemented in the face of an outbreak, particularly in racing centres with large numbers of young, valuable horses. Anecdotal reports suggest that this is effective in reducing adverse clinical effects of influenza infection, probably by both stimulating immunity prior to infection and reducing the amount of virus shed. Analysis of field data from the equine influenza outbreak among vaccinated racehorses in Newmarket in 2003, provided evidence to support these previous anecdotal observations.⁷ Subsequent mathematical modelling of data derived from the same outbreak, provide more detailed evidence to further support the strategic use of widespread vaccination at the earliest opportunity after confirmation of the disease among large groups of horses at risk of suffering extensive outbreaks.⁶

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