Equine Infectious Diseases || Equine Influenza Infection

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Equine Influenza InfectionGrabriele A. Landolt, Hugh G.G. Townsend, and D. Paul Lunn

C H A P T E R

Etiology

Influenza A viruses are members of the family Orthomyxoviri-dae, which contains enveloped viruses with segmented, single-stranded, negative-sense ribonucleic acid (RNA) genomes (Fig. 13-1). The Orthomyxoviridae comprise five genera: influenza A, B, and C viruses; Thogotoviruses; and isavirus. Influenza A viruses can be distinguished from type B and C based on the antigenic nature of their nucleoprotein (NP) and matrix (M) proteins. In contrast to influenza A viruses, which can be iso-lated from a wide variety of species (including horses), influenza B viruses appear to infect primarily humans. Influenza C has been isolated mostly from humans, although they have also been shown to infect pigs and dogs.1-3 Whereas influenza A and B contain eight separate segments of single-stranded RNA, influenza C viruses possess only seven.4

The virions of influenza A include a host cell–derived lipid envelope and are 80 to 120 nm in diameter. Embedded in the lipid envelope are the virus-encoded glycoproteins hemaggluti-nin (HA) and neuraminidase (NA)5,6 and the integral M2 protein, which functions as an ion channel. The HA serves as the viral receptor–binding protein and is responsible for fusion between the virion envelope and the host cell. The receptor-binding site is located on the globular head portion of the molecule and forms a pocket that is inaccessible to antibodies. Thus the amino acid residues creating the pocket are largely conserved among viruses.6-10

The HA is the major target of the host immune response, and there are five antigenic sites on the HA molecule, covering much of the surface of the globular head portion. Immune pressure is the driving force in the selection of mutants with amino acid substitutions in these antigenic sites, allowing the mutant virus to escape neutralizing antibodies (antigenic drift).

The NA is the second large surface glycoprotein. It is a type II integral membrane glycoprotein5,10 and is responsible for the cleavage of the α-ketosidic linkage between a sialic acid mole-cule and an adjacent sugar D-galactose or D-galactosamine.11 The NA facilitates the mobility of the influenza virus virion by removing sialic acid residues from viral glycoproteins and infected cells, therefore assisting in the release of the budding virus particles.12-14 As with the HA, the NA is a major antigenic determinant and undergoes substantial antigenic variation.15,16

The most abundant virion protein is the viral matrix protein (M1), which likely underlies the envelope, serves as the major structural protein of the virion, and associates with the ribonu-cleoprotein (RNP) complexes of the virus. The eight separate RNPs of influenza A have the appearance of flexible rods17 and are speculated to consist of a segment of RNA loosely encapsid-ated by several NP molecules. Located at the end of each RNP are the three viral polymerase proteins PB1, PB2, and PA. The segmented nature of the viral genome is a critically important feature of the influenza A virus structure. In the event that cells are infected with two (or more) different viruses, the exchange of RNA segments between the viruses allows the generation of progeny viruses containing novel combinations of genes. This phenomenon is referred to as genetic reassortment18,19 (Fig. 13-2). In theory, random incorporation of RNA segments could lead to the creation of 254 new gene combinations from two paren-tal viruses. Nonetheless, the identification of selective packaging

Figure 13-1 Schematic diagram of structural components of influenza A virus. Three integral membrane proteins—hemagglutinin (HA), neuraminidase (NA), and the ion channel protein (M2)—are embedded in the lipid envelope of the virion. The matrix protein (M1) is thought to underlie the lipid envelope. Associated with the viral ribonucleic acid (vRNA) is the viral polymerase complex, consisting of PA, PB1, and PB2. The viral nucleoprotein (NP) encapsidates the vRNA segments.

Lipid envelope

HA (hemagglutinin)

NA(neuraminidase)

M2 (ionchannel)

M1 (matrix protein)

NP (nucleoprotein)

PA, PB1, PB2(polymerasecomplex)

Figure 13-2 Schematic diagram illustrating genetic reassortment. In the event that cells are infected with two (or more) different viruses, the exchange of RNA segments between the viruses allows the generation of progeny viruses containing novel combinations of genes. In theory, random incorporation of RNA segments could lead to the creation of 254 new gene combinations from two parental viruses.

142 Section 2 Viral Diseases

centered largely on the geographic region of origin,7 the Ameri-can lineage strains since appear to predominate with only rare isolation of the Eurasian lineage strains.56,57 Continued genetic divergence has resulted in the formation of three American-like sublineages with distinct antigenic characteristics: a South American lineage, a Kentucky lineage (newly also referred to as the classic American lineage), and a Florida lineage.39 The Florida sublineage strains have spread across Europe, and the majority of viruses isolated in Europe since 2003 belong to this sublineage.56,58,59 Further genetic evolution of the Florida sub-lineage has resulted in the formation of two groups of viruses (referred to as Florida sublineage clades 1 and 2 viruses) with divergent HA sequences. The viruses isolated from the 2003 South African and the 2007 Japanese and Australian outbreaks were closely related to Florida sublineage clade 1 viruses and were most likely of North American origin.41,56 Although the genetic and antigenic evolution of H3N8 equine influenza viruses is significant in terms of immunization, when compared to human influenza viruses, equine strains have demonstrated relatively little genetic diversion.

Epidemiology

Influenza is the most frequently diagnosed and economically important cause of viral respiratory disease of the horse.36,60-62 Outbreaks of this disease have occured regularly throughout the world. New Zealand and Iceland may be the only countries where outbreaks of the disease have not occurred.

All ages and breeds of Equidae may be infected with the virus.63-65 Natural disease occurs in individual foals,63,64,66,67 but in endemic countries only one outbreak of influenza among young foals (3-6 months of age) has been reported to date.67 Longitudinal studies of North American racehorse populations, before the availability of highly efficacious vaccines, showed that the highest incidence of disease was observed in 2- and 3-year-old horses,62,68 likely caused by commingling of animals that lacked previous exposure to viral antigen.69

With the exception of occasional outbreaks in naïve popula-tions, equine influenza is of greatest importance in large popula-tions, where the disease is endemic and movement of animals among regions is routine. In addition to age and commingling of susceptible animals, influenza-specific serum antibody con-centration is a highly specific correlate of protection against infection and disease. Animals with high concentrations of homologous antibody are almost always protected against experimental challenge.70-74 During a 3-year study of a large population of racehorses, animals with high concentrations of serum antibody had 10 to 40 times lower odds of developing disease than did horses with no detectable antibody.68 Horses exposed to viral antigen within the past 6 to 12 months through natural infection75,76 or administration of a potent killed vaccine77 or an intranasal, temperature-sensitive, modified live vaccine78 may show evidence of reduced clinical signs and decreased viral shedding in the presence of little or no detect-able antibody.

Equine influenza has a short incubation period. Experimen-tally infected animals experience fever and begin to shed large quantities of virus in nasal secretions within 48 hours of infection.78-81 Secondary bacterial infections of the respiratory tract, largely resulting from proliferation of β-hemolytic strep-tococci, are routinely observed69,82-85 and are considered impor-tant in the pathogenesis of bacterial pneumonia of horses.86-88 Influenza morbidity rates within susceptible groups of horses may be as high as 60% to 90%.47,89,90 Mortality rates are usually less than 1%,50 although a 1998 outbreak caused by a newly emergent strain of the virus in China was associated with a

signals within the 3′ and 5′ coding regions of the influenza virus genes suggests that the incorporation of RNA segments into the virion is only partially random.20

Influenza A viruses can be divided into subtypes based on antigenic properties of their HA and NA envelope glycopro-teins. To date, seventeen HA subtypes and ten NA subtypes have been recognized. All but one of the HA and NA subtypes have been recovered from aquatic birds.3,21-24 These birds play a particularly important role in influenza epidemiology because they provide a vast global reservoir of viruses of the majority of subtypes. Phylogenetic analyses have indicated that viruses from aquatic birds were the ancestral source of the current lineages of mammalian viruses.21,25-27 In contrast, only a limited number of subtypes of influenza viruses have been associated with infec-tion of mammalian species. In humans, only viruses of H1, H2, H3, N1, and N2 subtypes have circulated widely in the popula-tion3,26,27; only H1, H3, N1, and N2 subtypes have been consis-tently isolated from pigs28-30; and apart from occasional reports of horses infected with viruses of subtypes H1N1, H2N2, and H3N2 (usually in association with human infections),31 equine influenza infections have been restricted to viruses of H7N7 (A/equine/1) and H3N8 (A/equine/2) subtypes.32-35

Outbreaks of a disease resembling influenza have been reported as early as 1751, although the etiologic agent was not isolated until 1956.32,33 The virus, designated A/Equine/1/Prague/56, was isolated during an outbreak of influenza in Czechoslovakia and was characterized as H7N7.36 H7N7 influ-enza viruses have not been detected in the horse population since the late 1970s.21,37,38

In contrast, equine H3N8 viruses, first isolated in the United States in 1963 (A/equine/2/Miami/1/63), continue to circulate in large parts of the world, except Australia, New Zealand, and Iceland.37,39,40 In August 2007, equine influenza infection was confirmed for the first time in Australia. The virus appears to have been introduced into the Australian horse population by importing horses from Japan, a country that was also experienc-ing an outbreak of the disease at the same time.41,42 However, by employing stringent quarantine procedures, movement restrictions, and vaccinations, the outbreak was contained with no new cases reported after December 2007.43 In December 2008, 12 months after identification of the last clinical case, Australia regained its equine influenza free status in accordance with the OIE Terrestrial Animal Health Code.44,45

Despite intensive vaccination programs, equine H3N8 influ-enza infections have remained a serious health and economic problem throughout most parts of the world. In the late 1980s, severe widespread influenza outbreaks were observed in horses in South Africa,46 in India,47,48 and in the People’s Republic of China,49-51 where equine influenza viruses were not known to be circulating. The H3N8 virus of the South African outbreak was most likely introduced by importation of infected horses from the United States or Europe.37,52 The outbreaks in the People’s Republic of China were caused by both conventional strains of equine H3N8 virus and viruses that were antigenically and genetically distinguishable from other circulating equine H3N8 viruses. Phylogenetic analysis of said virus showed that it had evolved independent of the existing equine lineage. Its genetic features were of avian lineage, indicating that the virus had probably spread directly to horses from the avian reservoir without genetic reassortment.49 In late 2003, a second major equine influenza outbreak occurred in South Africa and the outbreak was most likely also due to a breakdown in biosecurity measures.53

Since the early to middle 1980s, the equine H3N8 influenza viruses have diverged into two distinct evolutionary lineages, Eurasian and American.* While both lineages initially circulated

*References 7, 27, 39, 40, 54, 55.

143Chapter 13 Equine Influenza Infection

in the endemic populations in North America and Europe. Data collected during the 2007 outbreak in Australia, cases clustering around large urban centers105 with their high concen-tration of horses and premises but spreading over a total area of 300,000 km2 and infecting an estimated 9360 properties and 70,000 horses has provided new information on the epidemiol-ogy of the disease in naïve populations.105-108 The outbreak followed a classic epidemic curve, peaking in 6 and lasting 18 weeks. The initial distant spread was due to transport of infected horses within 10 days of release of virus.105-107 Local spread within 5 to 10 km of infected premises was related to direct horse to horse contact, aerosol droplets from coughing horses, and indirect contact (fomites and human assisted).63,106-109 The contribution of airborne virus in the spread of the epidemic is unknown.107,110 Commonly, 75% to 100% of horses on infected properties showed clinical signs of disease within 5 to 9 days.63,111 Mares on Thoroughbred studs were most severely affected.90 Signs in foals and yearlings were generally mild. Despite one cluster of fatalities in foals, death caused by influenza was extremely uncommon.112

Spread of disease across the entire country was prevented through restricted movement of horses, augmented by biosecu-rity and vaccination protocols.106,108,109 Strict biosecurity was associated with a significant decrease in the odds of a premise becoming infected.113 Vaccination with a highly efficacious vaccine was implemented after the peak of the epidemic. Its impact on disease occurrence in the general population has not been determined.106

Pathogenesis

Influenza A viruses replicate and induce pathologic changes throughout the entire respiratory tract, with the most signifi-cant pathology present in the lower respiratory tract.3 The primary targets of low pathogenic strains of influenza A viruses in mammalian species are the airway epithelial cells. After exposure of the upper airway mucosa to virus, infection is initi-ated by binding of the influenza virus HA to sialic acid residues on target cells located in the upper respiratory tract. However, for the virus to gain access to the cellular receptors, the virion first has to penetrate a mucus layer that forms a protective barrier over the cell surface. The viral NA promotes virus access to respiratory epithelial cells by destroying mucous glycopro-teins15 and removing decoy receptors present on mucins, cilia, and cellular glycocalix.16

After viral attachment, the virion is taken up into the cell through receptor-mediated endocytosis. After internalization and acidification of the endosomal compartment, the HA protein undergoes a conformational change,114,115 which leads to the insertion of the hydrophobic fusion peptide into the endosomal membrane and ultimately to the fusion of the viral and cellular membranes. After membrane fusion and release of the RNPs into the cell’s cytoplasm, the RNPs are actively trans-ported into the nucleus,116 where messenger RNA (mRNA) synthesis is initiated. During virus replication, the virus-encoded nonstructural protein (NS1) inhibits cellular protein synthesis by inhibiting the maturation of cellular mRNAs. The NS1 protein is multifunctional, and apart from inhibiting both poly-adenylation and splicing of cellular pre-mRNAs, it also coun-teracts interferon (IFN)-dependent and IFN-independent antiviral responses.76,117,118

In polarized epithelial cells, low pathogenic influenza viruses assemble and bud from the apical surface of the cells.76,119,120 Interaction between the cytoplasmic tails of the HA and NA proteins and the internal proteins (most likely M1) are thought to be the main driving force behind the formation of the

mortality rate of 20% in some herds.49 Morbidity rates within large groups of horses with varying degrees of previous expo-sure to influenza antigen may range from 20% to 37%.60,91 Outbreaks of equine influenza may occur at any time of the year, although seasonal outbreaks have been reported,60,74 prob-ably related to the yearly convergence of component causes (risk factors) resulting in disease at regular intervals within individual sites or geographic locations.

After natural infection, ponies were reported to be resistant to infection for 32 weeks, with partial clinical protection per-sisting for more than 1 year.75 Similar data are not available for horses, although a longitudinal study of a large population of racehorses showed that horses present during an outbreak of respiratory disease in one season were significantly less likely to show clinical signs of disease during an outbreak in the follow-ing year.68 Efficacious vaccines against equine influenza are available, and their widespread use is having a significant impact on the epidemiology of the disease. Although vaccination does not generally provide sterile immunity or full protection against clinical disease for more than a few months, the vaccination of populations of horses is reducing the frequency of disease out-breaks and the frequency and severity of clinical signs when outbreaks occur among vaccinated animals.74,84,92-95

Outbreaks of equine influenza occur most often when sus-ceptible animals are congregated and kept in close contact with each other (e.g., racetracks, horse shows, sales yards, airplanes). Human studies show that spread is through direct-contact transmission with infected subjects, droplet transmission (con-tagious droplets greater than 10 µm and capable of being pro-jected over moderate distances by coughing), and airborne transmission (infectious droplets less than 5 µm, capable of wide dissemination in confined environments and of reaching the lower respiratory tract of susceptible individuals).96 No experimental studies have shown that transmission occurs through fomites, although human epidemiologic studies provide indirect evidence.97,98 Among horses, rapid and effective spread is enhanced by a 2-day incubation period, high concentrations of virus in nasal secretions, an explosive cough, the practice of housing horses in confined spaces, and possibly, the ability of the virus to survive in wet environments (e.g., water bowls) for 72 hours and on dry surfaces (e.g., clothing, grooming equip-ment, vehicles, feed) for 48 hours.97

Anecdotal evidence suggests that disease spreads very quickly in small groups of confined animals and that all suscep-tible horses may become infected within 2 to 3 days. However, outbreaks occurring in large groups or populations, comprised of animals with varying histories of exposure and immunity, may last for 3 to 4 weeks,60 thus providing at least some oppor-tunity to institute procedures to limit the extent and severity of such outbreaks.

Several studies show that after experimental infection, horses typically shed virus for 6 to 7 days.52,78-80 Although a carrier state does not occur, subclinical infections and viral shed-ding are probably common, particularly after infection of par-tially immune animals. Partial immunity is likely in animals that have not been recently exposed or vaccinated or may be caused by mismatching of vaccine strains and circulating field virus.84,93,99 These animals are likely important in the spread of the disease within and between groups of horses37,100,101 and, along with fomites,102-104 provide a rational explanation for disease outbreaks among horses that have not experienced direct exposure to clinically diseased animals. The international transport of subclinically affected horses has been the suspected cause of many reported outbreaks of the disease in countries or regions with large numbers of susceptible animals.103

Outbreaks of equine influenza in naïve populations, such as those occurring in recent years in South Africa, India, and Australia, present a different picture than those occurring


coughing may persist for 21 days. In severe infections, lung sounds become increased in amplitude, with adventitial sounds sometimes detected. Ultrasonographic imaging of the thorax has been used to demonstrate pulmonary consolidation; pneu-monia is a common sequela between 7 and 14 days after infec-tion.135 Persistent respiratory disease can occur beyond 14 days after infection and is thought to be the result of secondary bacterial infection.

Morbidity can approach 100% in outbreaks in susceptible populations. Mortality is typically low, although neonatal infec-tion can be fatal,66 resulting in severe bronchial and interstitial pneumonia. The effects of influenza virus infection in donkeys and mules is typically more severe than in horses and can result in mortality.136 It is also important to recognize that subclinical disease with viral shedding may be common in previously vac-cinated horses and represents an important source of conta-gion.136 The effects of influenza virus infection can be significantly exacerbated by even moderate exercise, resulting in increased weight loss and other clinical signs.135

Immunity

Equine influenza virus infection generates a broad range of adaptive immune responses in systemic and mucosal compart-ments,81 and infection also stimulates important innate immune responses.137 In the horse, many investigators have demon-strated that antibody responses are strongly associated with protection. A protective immune response, such as that follow-ing infection, is characterized by induction of influenza virus–specific immunoglobulin G isotype a and b (IgGa, IgGb) and immunoglobulin A (IgA) antibodies in both the circulation and the nasopharyngeal secretions, with the IgG isotype responses predominating in the circulation and IgA in the respiratory tract.81,138,139

Nasal IgA is an important mediator of protective immunity to influenza virus infection in other species,140,141 through neu-tralization of viral particles at the respiratory epithelium and in

budding particles.121,122 By removing carbohydrate chains from the virion and the cell surface, NA enzymatic activity is thought to be required to release the newly formed influenza virus virions completely from the cell.12-14

The virus spreads quickly throughout the respiratory tract, damaging the respiratory epithelial cells, particularly in the trachea and bronchial tree.104,123 Virus replication leads to cell death, largely through virus-induced apoptosis,124-126 and subse-quent desquamation and denudation of respiratory epithelial cells. Histologic evaluation of infected respiratory epithelium reveals vacuolization and swelling of the columnar ciliated cells, accompanied by clumping and subsequent loss of cilia.127 Con-sequently, tracheal mucociliary clearance is impaired, predispos-ing affected animals to the development of secondary bacterial infections.34,123,128,129 Within 1 day after the onset of clinical signs, focal erosions of the respiratory cells down to the basal layer are evident. Viral antigen can be demonstrated predomi-nantly in the respiratory epithelial cells and only rarely in the basal cell layer.130 The disruption of the superficial cell layers allows opportunistic bacteria to invade the respiratory epithe-lium of both the upper and the lower respiratory tract, leading to bacterial bronchopneumonia and other complications.3,104,130 Submucosal edema and hyperemia occur with peribronchial and peribronchiolar infiltration by neutrophils and mononu-clear cells.3,37,127 About 3 to 5 days after onset of illness, regen-eration of the epithelium begins, characterized by the appearance of mitotic figures in the basal cell layer.3 In uncomplicated cases, complete resolution of the epithelial damage takes a minimum of 3 weeks.129,131

Complications of equine influenza virus infections include secondary bacterial pneumonia, myositis, myocarditis, and limb edema; in rare cases, neurologic disease may be observed.35,128,130,132 Attempts at virus recovery from the heart or brains of affected horses have been unsuccessful to date, although a recent study demonstrated a mild, transient increase in cardiac troponin I in ponies experimentally inoculated with equine influenza virus and supports a direct relationship between influenza infection and myocardial damage.133 Yet, based on the lack of evidence for virus replication in these tissues, it has been speculated that myocardial damage is caused indirectly, such as through an increase in the expression of inflammatory mediators (e.g., nitric oxide).134 Finally, it has been suggested that influenza infection might predispose horses to the development of recur-rent airway obstruction (RAO) and exercise-induced pulmo-nary hemorrhage (EIPH).35,104,128

Clinical Findings

Clinical signs of influenza virus infection were extensively described after its first discovery,128 and the same clinical find-ings are still observed almost 40 years later. Signs of disease are typically seen 48 hours and sometimes as early as 24 hours, after exposure in natural or experimentally infected horses. Pyrexia is typically the first clinical sign, with temperatures sometimes exceeding 106° F (41.1° C), peaking at 48 to 96 hours after infection. The increase in rectal temperature may be biphasic with a second peak of pyrexia observed around day 7 after infection. Nasal discharge follows, initially serous (Fig. 13-3), but typically becoming mucopurulent by 72 to 96 hours after infection. Coughing, sometimes paroxysmal, typically develops during this same period. Retropharyngeal lymphadenopathy is a variable but common finding, as is tachypnea. Most affected horses become anorexic at the time of the initial pyrexia, although this typically resolves in 1 to 2 days. Weight loss is well documented after influenza virus infection. Clinical signs typically resolve in 7 to 14 days in uncomplicated cases, although

Figure 13-3 Serous nasal discharge typical of horse with acute influenza virus infection. (Courtesy Dr. John Barneso.)


protection.147,157,159 Studies using modified vaccinia Ankara vector vaccines have demonstrated that NP-specific equine immune responses can also result in reduced clinical disease after challenge infection, although the degree of protection was inferior to that induced by HA vaccination.157 Influenza virus NP is an internal viral structural protein, and therefore NP-specific antibodies are not capable of virus neutralization and do not control virus shedding in the horse157 or other species.160 NP typically serves as an important target antigen for cellular immune responses and can elicit cross-protective immunity to heterologous strains of influenza virus.161 In the equine vaccination studies conducted to date, NP-specific immune responses included both lymphoproliferative and IFN-γ responses, and it is possible that NP-specific immune responses could make a significant contribution to equine immunity to influenza virus infection.

Diagnosis

In a group of susceptible horses, a presumptive diagnosis of influenza virus infection can be made based on the rapid spread of an acute, febrile respiratory disease characterized by a dry, hacking cough.104 However, laboratory diagnosis is required to confirm and differentiate influenza from other respiratory pathogens. The methods presently used for diagnosis of equine influenza virus infections include virus isolation, antigen detec-tion by fluorescent antibody and ELISA testing, reverse transcriptase–polymerase chain reaction (RT-PCR)–based assays, and serologic analyses.104,162-165 However, many of these methods have one or more disadvantages, such as lack of sen-sitivity, long turnaround time, prohibitive costs, or the need for a high degree of technical expertise in the laboratory. Thus, to institute optimal control measures, it might be necessary to combine several of these diagnostic tools to identify the etio-logic agent accurately and rapidly. The World Organization for Animal Health (OIE) publishes the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals at their Web site (http://www.oie.int/), which provides an extremely useful resource for current testing methodology.

Antigen Detection

Virus IsolationVirus isolation from clinical samples is critical for epidemiologic investigation and vaccine production and is generally carried out in embryonated chicken eggs or cell culture. In the naïve horse, by the third or fourth day after infection, large amounts of virus are shed into the secretions of the respiratory tract.34,165 Therefore the best results for virus isolation can often be achieved by collecting nasal swab samples (Fig. 13-4) within the first 24 to 48 hours after onset of clinical illness.35 In par-tially immune animals the length of virus shedding is often shorter, decreasing the diagnostic sensitivity of virus isolation. Therefore it is useful to sample the more immunologically naïve horses in a group to increase the likelihood of demonstrating infectious virus.104 Nasal swab samples are best collected using polyester-tipped swabs. Cotton swabs should be avoided because influenza viruses can adhere to the cotton fibers, decreasing the likelihood of isolating virus from the sample. The swabs should be placed in sterile viral transport medium and kept on ice until further analysis.

Traditionally, embryonated chicken eggs have been the bio-logic system of choice for isolating influenza viruses. The robust yield of virus from eggs has led to their widespread use in research laboratories and for vaccine production. However, their use may be of limited value for diagnostic laboratories.

the intracellular compartment.142-144 Nasal IgA responses are a characteristic of the protective immunity that follows equine influenza infection,138,139 and influenza virus–specific IgA-producing B lymphocytes have been detected in mucosal lamina propria and lymph nodes draining the nasopharynx of the horse.81 Virus-specific IgG antibodies can also contribute to immune exclusion at the respiratory epithelium in a mouse model, although the lack of specialized mechanisms for trans-porting IgG to the respiratory surface means that its role is less important.145 In the horse, there is evidence for local production of influenza virus–specific IgGa and IgGb at respiratory mucosal surfaces after infection,81,146 and indirect evidence that virus-specific nasal IgGb antibody responses can contribute to a reduction in nasal shedding of influenza virus.147 However, IgG antibody responses tend to be more short-lived in equine respi-ratory secretions than IgA responses.148,149

In the circulation, IgGa and IgGb are thought to be the principal protective IgG subisotype responses to influenza virus,81,139 whereas IgG(T) responses are not associated with protection.139 Circulating antibody has been measured in a number of ways in horses, including conventional hemagglutina-tion inhibition (HI) assays, single radial hemolysis (SRH) assays, virus neutralization (VN) assays, and enzyme-linked immuno-sorbent assay (ELISA).139,150 Of these techniques, SRH and ELISA may have the greatest sensitivity and utility, and it appears that SRH results correlate closely with IgGb ELISA results (Lunn and Townsend, unpublished data). Much atten-tion has focused on the correlation between levels of circulating antibody measured by SRH tests and protection from influenza virus infection.74 This tool has proved very useful, and SRH responses are often used to measure vaccination effect and predict protection. However, after circulating antibody responses to a prior influenza virus infection have waned, horses can remain protected against a further challenge.75 In addition, cir-culating antibody responses measured by SRH to a cold-adapted, modified live influenza vaccine are almost undetectable, although this vaccine provides long-lasting protection from challenge infection.78,151 Taken together, these observations illus-trate that although circulating antibody responses are an impor-tant predictor of protection against influenza virus protection, a lack of antibody does not invariably predict susceptibility.

The role of cellular effectors in resistance to equine influenza virus infection is less well investigated. Virus-specific cytotoxic T lymphocytes (CTLs) are important for protection from influ-enza virus infection,152,153 and there is a single description of the measurement of major histocompatibility complex (MHC)-restricted CTL responses to equine influenza virus.154 The lack of other CTL studies reflects the difficulty in detecting this equine immune response to influenza virus using available methods. Currently, influenza virus–specific lymphoprolifera-tive responses and interferon gamma (IFN-γ) gene expression may be the best available measures of virus-specific cellular immune responses in the horse. Production of IFN-γ is an indi-cator of T-helper 1 (Th1) cell-mediated immunity and can contribute to immunologic protection of humans from influ-enza virus infection and disease.141 Furthermore, several studies indicate an association between IFN-γ production and the con-comitant generation of antigen-specific CTL responses.155,156 A number of studies have demonstrated the development of equine IFN-γ responses to influenza virus consequent to either infection or vaccination.81,157,158 Similarly, influenza virus–specific lymphoproliferative responses have also been associated with protective immunity.81,146,157

The importance of HA-specific immune responses in pro-tection from influenza virus infection is well known, and vaccination studies in horses using deoxyribonucleic acid (DNA) vaccination and recombinant vaccines expressing the HA gene all confirm the importance of the HA antigen for

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A viruses from different host species. An additional advantage of ELISA-based assays over virus isolation is that these tests are able to detect virions that have lost their infectivity during sample handling, storage, and transport to the laboratory.

Originally intended for the diagnosis of human influenza infections, an antigen-capture ELISA has been adapted for the detection of equine H3N8 viral antigen.174,175 Briefly, to detect viral antigen, a “capture” antibody, directed against the influenza NP, is linked to a 96-well plastic plate. The clinical sample is added to the wells, and if viral antigens are present, they will be bound to the immobilized antibody. Subsequently, bound viral antigen is detected by use of a second enzyme-linked antibody. The antigen-capture ELISA was evaluated during an outbreak of equine influenza in the United Kingdom in 1989 and, when used in combination with virus isolation, was found to enhance the virus detection rate by 44%.176 Moreover, the test was shown to be particularly useful for detection of viral antigen in samples heavily contaminated with bacteria.104,165

Commercial development of optical immunoassay (OIA)-based test kits (e.g., Flu OIA assay [Biostar, Boulder, CO], Directigen Flu-A assay [Becton Dickinson Microbiology Systems, Cockeysville, MD), designed to detect the NP of human influenza A viruses, has facilitated the widespread use of this procedure. Many investigators found such commercial diagnostic kits to be useful, and frequently, these assays were considerably more sensitive than traditional virus isolation.177-181 Furthermore, these OIAs proved to be highly specific and rapid, whereas virus isolation in embryonated chicken eggs required up to three passages before hemagglutination became evident.177-179,181 Yet, while these test kits were able to identify infected horses consistently at the peak of virus shedding, they may not be sensitive enough to detect low levels of virus shed-ding reliably.7 Therefore, whenever possible, horses with severe clinical signs are preferable for testing during a suspected influ-enza virus outbreak to increase the likelihood of obtaining positive results.179

ImmunofluorescenceEmploying influenza virus–specific fluorochrome-labeled anti-bodies, immunofluorescence (IF) is based on the immunodetec-tion of virus-infected cells obtained from nasal scrapings or tracheal washes. Although the assay was reported to be highly sensitive and rapid,165,182 IF requires substantial sample prepara-tion and handling. Briefly, samples are centrifuged to separate the cells from respiratory mucus. The cells are washed, spotted onto glass slides, acetone fixed, and incubated with influenza-specific antibodies. Antigen-positive cells are detected by use of a secondary fluorochrome-labeled antibody.104,165,182 A study comparing the detection of influenza viruses by IF to a com-mercially available OIA for diagnosis of influenza virus infec-tions in humans found that the IF had a significantly higher sensitivity than the OIA.182

Reverse Transcriptase–Polymerase Chain ReactionDuring the past decade, advances in PCR technology and other DNA amplification techniques have resulted in these methods becoming key tools in diagnostic laboratories.183-185 By choosing appropriate oligonucleotide primers, a selected region of the viral genome can be amplified. In vitro DNA synthesis is cata-lyzed by a special DNA polymerase isolated from thermophilic bacteria that is stable at high temperatures. PCR is extremely sensitive and can theoretically detect a single-copy of DNA in a sample. Trace amounts of RNA can be detected in the same way by first transcribing them into DNA with RT.

RT-PCR–based assays have been used successfully for the detection of a broad range of influenza A virus subtypes from clinical samples.164,172,182,186,187 In contrast to virus isolation, RT-PCR does not require the presence of viable virus,188 and

Depending on the virus strain, amount of virus present in the sample, sample quality, and handling, detection of the presence of infectious virus by egg inoculation can take a minimum of 2 or 3 days. In horses with mild or subclinical infections, viral titers in nasal secretions are often low, sometimes requiring several passages before sufficiently high viral titers are produced to allow detection using conventional HA assays.165

Alternatively, equine influenza virus can be propagated in cell culture. Although the virus can infect a variety of primary and continuous cell lines, many of these do not support produc-tive viral replication.166-169 The most widely used cells are Madin-Darby canine kidney (MDCK) epithelial cells. Unfortu-nately, the use of cell culture for influenza virus isolation also has several limitations. For example, depending on the protocol and cell lines used, substantial differences in influenza recovery rates from clinical samples can occur.170 In addition, MDCK cells are generally considered less permissive than embryonated chicken eggs for equine influenza viruses.171 Other, less fre-quently used cell lines include mink lung epithelial cells (Mv1Lu) and chick embryo fibroblasts. Mv1Lu cells in particu-lar might provide a useful alternative system for the isolation of influenza A viruses from clinical samples. Recent studies found that Mv1Lu cells and mixtures of MDCK and Mv1Lu cells supported the replication of a wider range of influenza A virus than MDCK cells alone.172,173

ImmunoassaysA number of ELISAs using monoclonal antibodies to detect the viral NP in nasal swab samples have been developed as a more rapid alternative to virus isolation. Although influenza A viruses can differ substantially in their HA and NA genes, the sequences of the internal genes, such as the M, NP, and the NS genes, are highly conserved among all subtypes and strains of influenza A viruses.4 As such, a diagnostic test aimed at the detection of NP is likely to be capable of identifying a wide variety of influenza

Figure 13-4 Technique to obtain nasal mucosal swab for antigen detection or virus isolation. Short, polyestertipped (noncottontipped) swab is most appropriate for sample collection. Virus isolation from a nasal mucosal swab is most sensitive for detection of equine influenza virus if obtained during the first 24 to 48 hours of fever.


course of infection. The tests commonly used to detect influenza-specific antibodies include HI, VN, complement fixa-tion (CF), SRH, and ELISA-based testing.

Hemagglutination InhibitionHemagglutination inhibition tests are simple, sensitive, inex-pensive, and rapid and therefore are often the method of choice for assaying antibodies to influenza A virus. The test relies on the hemagglutination activity of the influenza HA and the ability of HA-specific antibodies to inhibit the virus from agglu-tinating erythrocytes (Fig. 13-5). Briefly, dilutions of serum are incubated with virus, and erythrocytes are added. After incuba-tion, the HI titer is read as the highest dilution of serum that inhibits hemagglutination. A fourfold or greater increase in HI antibody titer is regarded as evidence of infection.165,202 Hemag-glutination inhibition antibodies define subtype-specific anti-gens on the virus particle, thus allowing the differentiation of equine influenza H3N8 and H7N7 subtypes.203 In addition, HI assays have found wide application in the analysis of antigenic differences between strains in equine and human influenza surveillance. One of the main shortcomings of the HI test is interlaboratory variation. Although a study comparing the sen-sitivity of HI and SRH tests on human sera found that both tests had similar sensitivity, the interlaboratory reproducibility of HI was significantly lower.202

Single Radial HemolysisFor SRH tests, sheep erythrocytes, which have previously been incubated with influenza virus, are mixed with guinea pig com-plement and incorporated in agarose gels (Fig. 13-6). Heat-inactivated serum samples are then added to wells cut into the gel, and the antibody titer is determined based on the zone of hemolysis induced by diffusion of the antibody-positive sample

therefore the sensitivity of RT-PCR methods is often substan-tially higher than for virus isolation. Despite these advantages, the technique can also have a number of shortcomings. Because of the assay’s high sensitivity, the greatest problem facing the diagnostic application of PCR is the production of false-positive results. These are often attributable to contamination by nucleic acids, particularly from previously amplified mate-rial (carryover). Any contaminant, even the smallest airborne remnant, may be multiplied and produce a false-positive result. In fact, a recent study reported that even administration of an inactivated whole-virus influenza vaccine at time of nasal swab samples collection resulted in the contamination of the sample with the vaccine virus and in the creation of a false-positive RT-PCR result.189 Contamination of the nasal swab sample most likely occurred through aerosolized vaccine, contami-nated fomites, or inadvertent sample handling.189 False-negative results might also be encountered when employing RT-PCR–based testing and can result from the nature of the sample tested. Ribonucleases (RNases) are present in various quan-tities in specimens collected from the respiratory tract, and these enzymes can gradually digest viral RNA.190,191 This may reduce the sensitivity of RT-PCR–based tests in clinical samples that contain large amounts of RNases and a low concentration of viral RNA. In addition, PCR assay inhibitors (e.g., lactofer-rin, hemoglobin) can represent a substantial problem in diag-nostic PCR-based assays.192,193 The presence of PCR inhibitors have been reported in about 2% of samples from the respira-tory tract.194

In diagnostic laboratory settings the use of RT-PCR can be limited by cost and the availability of adequate test sample volume. To overcome these shortcomings, multiplex PCR assays have been developed. In multiplex PCR, more than one target sequence can be amplified by including more than one pair of primers in the reaction. Multiplex PCR has been shown to be a valuable and cost-effective tool for monitoring the emergence of new variants and subtypes of influenza A viruses.195,196 In addition, multiplex PCR has also been used successfully to screen clinical samples simultaneously for multiple equine respiratory pathogens.197 In contrast to conventional PCR methods, real-time PCR does not require post-PCR processing steps (e.g., gel electrophoresis and determination of fragment size) and can therefore generate results within 4 to 5 hours. By employing a target-specific fluorescent probe, real-time PCR is a highly sensitive and specific method for the detection of equine influenza virus in clinical samples and can also be used for quantification of virus.198-200 Based on these advantages, real-time RT-PCR–based assays are now widely used in routine diagnostic settings.

Antibody Detection

In the past, serologic tests have been the key tool by which influenza infections were diagnosed. Most serologic assays are fairly easy to perform and cost-effective. In addition, a large number of samples can be collected and tested simultaneously, facilitating large-scale herd surveillance. Because many horses have been vaccinated, however, diagnosis of influenza infection can often be made only by testing paired samples (acute and convalescent titers) collected 10 to 21 days apart and demon-strating at least a fourfold increase in antibody titer over a period of several weeks (seroconversion). Therefore serologic testing often provides only retrospective information, and sub-clinical infections, which may not be accompanied by serocon-version, might not be detected.201 In addition, because of the inherent variability of immunoassays, paired samples always should be run by the same laboratory. To diagnose influenza infection, seroconversion should be demonstrated, and there-fore baseline (acute) samples must be collected early in the

Figure 13-5 Schematic diagram of hemagglutination inhibition (HI) assay. Chicken red blood cells (RBCs) agglutinate in the presence of influenza virus, but this can be inhibited by the addition of influenza virus–specific antibody (Ab). HI results in cells coating the bottom of a roundbottomed test plate, whereas lack of antibody allows for clumping of cells into a pellet.

I) Prepared test antigen: RBCs with adsorbed virus

2) Add patient serum

Antibody blocks hemagglutinin:no RBC agglutination

Hemagglutinin not blocked:RBC agglutination

+ (anti-influenza Ab) – (no specific Ab)

+ –


process of selecting a vaccine to limit the spread of infection, an important consideration was that the vaccine, in combination with an appropriate serologic test, would provide DIVA capacity.211,212 Consequently, the canarypox-vectored vaccine (Recombitek, Merial, Duluth, GA) was chosen, because it induces an antibody response only to the influenza HA protein. Coupled with a blocking ELISA (bELISA) detecting serum antibodies to the influenza NP, this strategy allowed vaccinated horses to be distinguished from infected horses with high diag-nostic sensitivity (approximately 97%), and this testing strategy was used extensively during the Australian equine influenza control and eradication effort.213,214

Pathologic Findings

Given the limited mortality associated with equine influenza infection, there are few reports of gross pathologic findings, although an early publication summarizes the original studies performed when equine influenza virus infection was first rec-ognized.128 Subacute inflammatory disease of the nasal mucosa, pharynx, larynx, and trachea is observed. Pulmonary changes include bronchitis, peribronchitis, and perivasculitis, with sub-acute interstitial pneumonia, edema, and focal bronchopneu-monia. Myocarditis is a variable finding. Neonatal infection with equine influenza virus can result in severe fatal bronchointer-stitial pneumonia accompanied by pulmonary congestions, tracheitis, bronchiolar and alveolar necrosis, and squamous metaplasia,215-217 although such occurrences are rare.

Hemogram findings in equine influenza virus infection include a moderate normocytic, normochromic anemia.128 The leukogram typically shows a leukopenia, which results from both neutropenia and lymphopenia of 3 to 5 days’ duration128,218;

from the well.138,204,205 An increase of 50% or 25 mm2 is con-sidered evidence of recent infection.202 Although more labor intensive than HI assays, SRH tests have been shown to be more reproducible than HI tests.202 Since it was found that the level of antibody measured by SRH after vaccination correlates well with the level of protection, SRH may also be used to predict the level of antibody-mediated immunity and determine the need for revaccination.72,73,165

ELISA-Based AssaysELISAs detecting antibodies to equine influenza H3 HA have been developed as an alternative method to traditional HI tests and were found to be sensitive, rapid, and reproducible.206 For example, an assay employing an HA protein produced in a baculovirus expression system demonstrated broad reactivity with serum antibodies generated after infection with heterolo-gous H3N8 influenza virus strains.207 Because conventional serologic tests do not provide information as to whether exist-ing antibodies were produced in response to infection or vac-cination, an ELISA aimed at the detection of antibodies to the NS1 protein has been developed.208 Because antibodies to NS1 can be demonstrated only in influenza virus–infected horses,209 and NS1 is antigenically and genetically highly conserved across influenza A viruses,210 NS1 is a good candidate for a differential diagnostic marker, capable of differentiating between infected and vaccinated animals (DIVA). Subsequent testing of horses that were either experimentally infected with A/equine/Kentucky/1/81 (H3N8) and A/equine/La Plata/1/93 (H3N8) or vaccinated with inactivated influenza H3N8 and H7N7 virus demonstrated that the NS1-based ELISA was a useful tool to distinguish postvaccination antibody titers from those gener-ated by recent infection.208 A slightly different approach to differentiate between infected and vaccinated horses was taken in the 2007 equine influenza outbreak in Australia. During the

Figure 13-6 Left, Schematic diagram of single radial hemolysis (SRH) assay. Sheep red blood cells (RBCs) coated with influenza virus and guinea pig complement are mixed with molten agar and poured in a plate. Test equine serum and control serum can be added to individual wells cut in the agar, and the zone of hemolysis corresponds to the amount of influenza virus–specific complementfixing antibody (Ab) present in the test equine serum. Right, Typical result.

I) Prepared test antigen: RBCs with adsorbed virus plus complement ( ) and agarose gel

2) Add patient serum to well

Ab induces complementfixation and RBC lysis

RBC with absorbedequine influenza virus

No Ab to fix complement

+ (anti-influenza Ab)

Zone of lysis

- (no specific Ab)

Pour into plate

2 mm well


excreted primarily unmetabolized in urine, and therefore the dosage must be adjusted in patients with renal insufficiency to reduce the risk of adverse effects.229 The most common adverse effects observed as a result of amantadine therapy are central nervous system (CNS) effects.229,230 At high doses (>15 mg/kg), the drug was reported to produce acute seizures and even death in horses.228 In humans the concurrent use of antihistamines, anticholinergics, and psychotropic drugs is thought to enhance the neurotoxic effect of amantadine.226 When administered orally to healthy humans and horses, rimantadine was much less frequently associated with adverse CNS effects than amanta-dine.229,231 Moreover, the bioavailability of rimantadine after oral administration in horses appeared considerably more uniform than the oral bioavailability of amantadine. The administration of 30 mg/kg of rimantadine every 12 hours (q12h) resulted in sufficiently high plasma concentrations without causing observ-able signs of adverse effects.231

Clinical trials conducted with influenza virus–infected humans and horses suggest that amantadine and rimantadine are equally effective in reducing the severity and duration of illness.231-234 In addition, oral administration of rimantadine reduced virus load in nasal secretions, although the duration of nasal virus shedding was similar to that in the untreated con-trols.231 The potential benefits associated with the administra-tion of M2 ion channel blockers during an influenza virus outbreak may be limited by the rapid development of drug resistance. Amantadine and rimantadine resistance can develop as early as one day after start of treatment.235 Such resistant viruses may then spread to susceptible contacts and cause disease, indicating that acquisition of drug resistance is not associated with attenuation of the virus.236,237 Resistance can pose a major problem when these drugs are used in close-contact environments.238,239 In recent years, the incidence of naturally occurring amantadine-resistant seasonal human-influenza virus has increased significantly around the world. Amantadine-resistant strains were found to have spread exten-sively around the world, including to some countries where the use of the M2 channel inhibitors was minimal.240

The NA inhibitors zanamivir and oseltamivir block the activity of the viral NA protein, therefore inhibiting the release of virions from the infected cells.241,242 Both zanamivir and oseltamivir have demonstrated antiviral activity against a number of equine influenza viruses in vitro, with inhibitory concentrations generally ranging from 0.015 to 0.09 µM.243 Because of its low oral bioavailability, zanamivir is administered in nasal sprays or drops, a nebulized mist, or a dry-powder aerosol.241 To date, the drug’s clinical effectiveness has not been tested in horses. In contrast, oseltamivir phosphate (OP) is well absorbed from the equine gastrointestinal tract and rapidly metabolized to oseltamivir carboxylate (OC).244 Oral adminis-tration of OP at a dose of 2 mg/kg body weight resulted in OC plasma concentration well above the equine influenza virus inhibitory concentrations found in vitro.244 In humans, oselta-mivir has a long duration of action, thus dosing is typically recommended at twice daily for treatment and once daily for prophylaxis.241 In contrast, OC is rapidly eliminated from horse plasma. Therefore, to maintain effective plasma concentrations, it was suggested that oseltamivir dosing intervals should be less than 10 hours in the horse.244 In humans, oseltamivir is generally well tolerated. The most frequent adverse effect is nausea and less frequently, vomiting.241 In a recent study, no clinical or serum biochemistry abnormalities were observed in horses receiving 6 mg/kg of oseltamivir every 12 hours for 5 days.244 While oseltamivir has been shown to be effective for prevention and early treatment of influenza infection in humans, there are limited data on the drug’s clinical efficacy in horses. Oral admin-istration of 2 mg/kg of OP prior to (prophylaxis) and soon after experimental inoculation (treatment) was found to reduce the

neutropenia is not a consistent finding, and the neutrophil/lymphocyte ratio is often increased during this period.16,128 Monocytosis during early convalescence is a variable finding.127,128

Therapy

Medical Therapy

Symptomatic treatment is the primary form of therapy for equine influenza virus infection and should include rest in a nonstressful environment. It is important to ensure adequate hydration, and nonsteroidal antiinflammatory drugs (NSAIDs) may reduce morbidity caused by pyrexia and myalgia. Affected animals should be monitored for development of complications such as pneumonia or myocarditis, and any horse exhibiting signs of respiratory disease beyond 10 days postinfection should be considered at high risk of secondary bacterial infection. The duration of adequate convalescence is difficult to gauge but is typically much longer than owner’s expectations. Estimates for the period before horses should be returned to athletic activity vary from 50 to 100 days,219 to a week for each day of fever.136

Antiviral Therapy

Vaccination is the best option for the control of equine influ-enza, and limitations inherent to influenza immunoprophylaxis (e.g., lack of vaccine coverage or protective immunogenicity) have stimulated an interest in the use of antiviral therapy in the horse. Moreover, in contrast to vaccines, antivirals can be rapidly employed to combat an influenza virus outbreak. Despite this, use of antiviral drugs to combat equine influenza infection should be carefully deliberated. As a large body of evidence indicates that the use of antiviral drugs can result in the devel-opment of antiviral resistance among influenza A viruses, indiscriminate use of antivirals could potentially reduce the effectiveness of treatment during an outbreak.220-222 Two classes of influenza antiviral drugs are currently licensed for the pro-phylactic and therapeutic use against influenza A virus in humans: the M2 ion channel blockers and the NA inhibitors.

Amantadine (1-aminoadamantane hydrochloride) and rimantadine (methyl-1-adamantanemethylamine hydrochlo-ride) target the transmembrane domain of the M2 ion channel protein. Both drugs inhibit virus replication by blocking ion channel activity of the M2 protein through allosteric inhibition. Amantadine and rimantadine have antiviral properties against all subtypes of influenza A virus.222 Their primary antiviral action results from blocking the flow of H+ ions from the acidi-fied endosomal compartment into the interior of the virion, a process necessary for release of the viral RNPs. A second effect of both drugs is to block maturation of the HA during transport from the endoplasmic reticulum to the plasma membrane.223,224

Influenza virus inhibitory concentrations of amantadine and rimantadine range from 0.03 to 1.0 µg/mL,225 with rimantadine about fourfold to tenfold more active than amantadine.226 In humans, amantadine and rimantadine have good oral bioavail-ability and very large volumes of distribution.161,227 In horses, oral administration of amantadine was associated with substan-tial interanimal variation in bioavailability, ranging from very low (~10%) to a maximum of 70%, and was not substantially affected by prior fasting of the horses.228 Therefore oral admin-istration of amantadine in horses, although effective in some animals, might fail to produce effective plasma concentration in others. In contrast, intravenous (IV) administration of aman-tadine at doses ranging from 10 mg/kg every 8 hours (q8h) to 5 mg/kg every 4 hours (q4h) was suggested to be sufficient to maintain effective plasma concentrations.228 Amantadine is


reports of alternative successful vaccination strategies, including the use of DNA vaccination,81,146,147,255,256 and the development of a modified vaccinia Ankara (MVA) vaccine vector.157,158 The use of the MVA vector had the particular advantage of generat-ing nasal mucosal influenza virus–specific IgA. More recently, commercial vaccines using ISCOM-matrix formulations have been described.257,258

Recommendations for equine vaccination are available from a variety of sources.259,260 General recommendations are not to vaccinate in the presence of maternal immunity in foals before at least 6 months of age.261 Vaccination of mares with inacti-vated vaccines that are known to generate high-titer antibody responses 2 to 6 weeks before parturition is likely to provide protection of foals through passive transfer of immunity. Initial vaccination series for inactivated vaccines should include three doses, and this approach is recommended even when data sheets only recommend two initial doses. The timing of these initial vaccinations is important. An interval of 3 to 4 weeks between the first and second dose is recommended, but a longer interval of 3 to 4 months between the second and third dose is preferred. This results in the third dose of the initial series being administered when the antibody response to the second vaccine dose has waned, and the amplitude of the antibody response to the third dose is consequently much greater.77 This regimen of three initial doses of vaccine, with intervals of 1 month between the first and second doses and with 3 to 4 months between the second and third vaccine doses, is recommended independent of the recommendations present on data sheets. Contemporary inactivated vaccines with established protective efficacy are likely to perform well if this approach is taken, and subsequent booster vaccinations should be given at 6-month intervals in high-risk populations. The modified live cold-adapted vaccine only requires a single initial dose and is recommended for booster doses at 6-month intervals. If a known period of high risk of infection is anticipated, a booster vaccination should be given even to well-vaccinated horses 1 to 2 weeks before the risk period.

Husbandry

Control of equine influenza virus infection can be substantially addressed by adequate husbandry procedures. It is informative to review the OIE qualifications for influenza-free countries.262 If the same criteria are met for horses entering an equine estab-lishment, it is likely that influenza virus infection can be excluded. Specifically, horses should be isolated for 4 weeks before introduction into the horse population, horses should be fully vaccinated before admission to the isolation facility, no clinical signs of influenza should be detected during the isola-tion period, and no new animals should be introduced into the isolation facility during the isolation period. Some countries also perform influenza virus diagnostic testing at the beginning of the isolation period, using tests such as the Directigen ELISA (Becton, Dickinson, Franklin Lakes, NJ). The recent outbreak of equine influenza virus in Australia, a country previously free of infection, was a result of a breakdown in these quarantine procedures.43 During this outbreak and the subsequent eradica-tion of equine influenza virus to reestablish Australia’s equine influenza virus free status, new experience was gained in the use of a variety of diagnostic techniques for managing and controlling the spread of major outbreaks.263

Although the OIE criteria may be too demanding for most horse owners, adequate vaccination and 2 weeks of full isolation on the facility could be regarded as an excellent compromise. Frequently, even this standard is difficult to achieve, and we remain heavily dependent on vaccination for protection. Nev-ertheless, other husbandry procedures, including segregating equine populations on horse facilities, can be valuable. This

magnitude of nasal virus shedding and severity of clinical signs.245 In addition, researchers found that bacterial counts of Streptococcus equi subsp. zooepidemicus in bronchoalveolar lavage fluids collected 7 days after inoculation with influenza were significantly lower in horses receiving OP compared to the placebo-treated control horses.245

As with the M2 ion channel inhibitors, emergence of NA inhibitor-resistant variants is a major concern. While oseltamivir resistance has not been documented for equine influenza strains, the prevalence of drug-resistant seasonal human H1N1 viruses has rapidly increased since the late 1990s and exceeded 90% in the 2008-2009 influenza season.221,246-248

Prevention

Vaccination

Vaccination against equine influenza virus infection is a common and important practice for the control of disease, and a wide variety of vaccine formulations are available. Inactivated vac-cines remain the most common type of vaccine in use around the world, although both modified live and recombinant vac-cines are commercially available. Many publications have docu-mented the ability of inactivated equine influenza vaccines to protect against homologous viral challenge, which also demon-strates a correlation between protection and prechallenge anti-body level.71,72,249,250

The value of inactivated equine influenza virus vaccines critically depends on the quality and quantity of viral antigen and the choice of adjuvant.201,251 Some of the most successful inactivated vaccines have used adjuvants such as ISCOMS or carboxypolymer-based compounds (carbomer, carbopol),71,249 and a recent North American study showed a clear advantage to carbomer-based products.77 The use of some adjuvants, such as alum, have been associated with induction of nonprotective immune responses.139

Another critically important consideration is which strains of virus are used for preparation of the vaccine. The inclusion of the equine H7N7 virus is no longer considered necessary for equine influenza vaccines. Moreover, as no Eurasian H3N8 viruses have been isolated in recent years, the OIE expert panel on equine influenza vaccine composition no longer deems the inclusion of these viruses in vaccines necessary. However, further evolution of the American lineage H3N8 virus contin-ued to result in failure of killed vaccines in outbreaks in 2003,252 and ongoing evolution of this lineage into two new clades with distinct antigenic identity poses new challenges to vaccine for-mulations.57 Ongoing surveillance and inclusion of viral antigens representative of contemporary circulating viruses will remain a priority.

An intranasal, cold-adapted, modified live equine influenza virus vaccine based on a Kentucky 1991 H3N8 virus is available in North America and provides protection for up to 12 months after a single administration, although only a 6-month claim is made on the product data sheet.78,151 Intranasal vaccine admin-istration is generally well tolerated in horses.253 At the time of its introduction, this vaccine was found to protect against both European and American lineages, despite including only an example of the latter.254 It is now some years since there were reports of the efficacy of this vaccine against contemporary circulating viruses.77 A recombinant canarypox vector-based equine influenza vaccine has shown excellent performance against even the most recent circulating viral lineages, including those that have overcome modern inactivated vaccines.159,200 These examples illustrate the increasing role and potency of novel equine influenza vaccines not based on conventional inac-tivated formulations. Experimentally, there are a number of

between horses and other species is limited. This has led to the hypothesis that horses may be “dead-end” hosts for influenza A viruses. However, this notion has been challenged by at least four separate natural transmission events of equine H3N8 influ-enza virus to dogs that occurred in the United States,267 the United Kingdom,268,269 and in Australia.270 In addition to these naturally occurring cross-species transmission events, equine influenza virus has also been found to spread from an experi-mentally infected horse to a dog that was housed in the same stall.271 However, despite this seeming susceptibility of dogs to infection with equine H3N8 influenza virus, with the exception of the transmission event in the United States where the equine origin virus has become established in the canine population, there is no evidence of widespread circulation of H3N8 influ-enza in dogs in the United Kingdom57 or Australia.270 Moreover, recent experimental inoculations of horses and ponies with two different canine H3N8 influenza isolates did result in only mild clinical signs and minimal nasal virus shedding, suggesting that the canine isolates may have reduced infectivity for horses.272,273

The complete reference list is available online at www. expertconsult.com.

allows for potential containment of disease outbreaks because infection moves slowly through large facilities where horse populations are segregated.60 Shared grooming equipment and tack may increase the risk of contagion.68

Monitoring of vaccine response by serology has been reported to help in prevention and control of influenza virus outbreaks, and the SRH test can be used for this purpose.74,264 Surveillance for detection of influenza virus is routinely practiced in some areas with large equine populations and offers the opportunity for early detection of outbreaks and for detection of new virus strains that may not be controlled by vaccine.265

Public Health Considerations

Influenza A viruses have partial host range restriction, meaning that viruses from one species can occasionally transmit to infect another species. However, biologic barriers exist that limit such spread. Human volunteers were found to be susceptible to infection with H3 equine-lineage viruses,266 although phylo-genetic analyses suggest that exchange of influenza virus genes

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