189

17 

Adeno, Hendra, and Equine Rhinitis Viral Respiratory DiseasesC.J. (Kate) Savage, Deborah Middleton, and Michael J. Studdert*

C H A P T E R 

Equine Adenoviruses

Equine adenovirus type 1 (EAdV1) appears as a subclinical infection in most immunocompetent foals and horses. However, there is serologic evidence of widespread infection in equine populations in numerous different countries around the world.1,2 It causes mild, upper respiratory tract (URT) disease in normal foals but has caused acute URT disease, as well as follicular conjunctivitis in foals that have been experimentally infected. One of these foals was specific-pathogen-free (SPF) and colostrum-deprived but immunocompetent at 6 weeks of age; the other foal was younger, had received colostrum, and sus-tained decreased clinical signs, despite its age (4 days).3 Equine adenovirus type 1 is the dominant pathogen in Arabian foals with severe combined immunodeficiency disease (SCID), which is a uniformly fatal, inherited disorder. These foals have a progressive EAdV1 bronchopneumonia, as well as EAdV1-related pathology in many other organs and tissues.

Etiology

Equine adenoviruses are members of the genus Mastadenovirus, family Adenoviridae. Only a single antigenic type of EAdV1 has been isolated from horses with respiratory disease.4,5 A second serotype, equine adenovirus type 2 (EAdV2), has been isolated from the feces of foals with diarrhea.6 The biophysical proper-ties of EAdV are similar to those of adenoviruses of other species. Equine adenoviruses are nonenveloped, 70 to 80 nm in diameter, and the capsid is composed of 252 capsomers; 240 hexamers occupy the faces and edges of the 20 equilateral triangular facets of an icosahedron, and 12 pentamers occupy the corners. The inner core contains the double-stranded deoxy-ribonucleic acid (DNA) genome, which for EAdV1 is 34.4 kb in length. Restriction endonuclease maps and genome orienta-tion data were published for EAdV1,7 and genomic sequence data for both viruses were also published.8,9 Nucleotide sequence data for the EAdV2 genome corroborated at the molecular level that EAdV2 is distinct from EAdV1 and that the two viruses evolved separately.8 Cavanagh et al10 recently sequenced the EAdV1 genome. An unexpected finding was the similarity to two other recently characterized bat adenoviruses.10

Epidemiology

Equine adenovirus type 1 occurs worldwide, and seroprevalence rates vary from less than 2% to 100%, depending on the sero-logic test used and the age, breed, activity, and size of the popu-lation sample reported.5 The prevalence of antibody to EAdV1

increases with age so that, in some populations, about 70% of yearlings and 2-year-old horses have EAdV1 antibody. Serologic evidence indicates a high infection rate in the first year of life, but in some populations, 50% of horses under 1 year lack EAdV antibody and are presumably at risk for EAdV disease.4

Interestingly, research in California using polymerase chain reaction (PCR), followed by virus isolation if positive, did not demonstrate EAdV1 infection from nasopharyngeal swabs from 47 apparently healthy racehorses, which were in race training at the time. No horses were positive.11

All adenovirus infections appear to be followed by a latent carrier status and shedding of the virus; however, this is poorly characterized for EAdV1. Studies in the Pirbright, United Kingdom (UK), pony herd indicated that EAdV1 infections were often subclinical, and that virus may persist in the naso-pharyngeal mucus for up to 68 days after primary infection.12 Equine adenovirus type 1 was isolated from a foal, without clinical signs at the time of isolation, at 3 days of age.13 It seems that foals may acquire infection from their dams or other horses in their cohort during the suckling period, even in the presence of detectable levels of maternal antibody. Thus, if there is per-sistent or repeated EAdV infection, the passive immunity of the foal would be converted to an active immunity, probably without significant clinical disease. Disease may develop if primary infection occurred after maternal antibody had declined, or if the foal was unable to produce an active immune response. This overview of the early natural history of EAdV1 is supported by the natural history of EAdV1 in Arabian foals with SCID, in which the transmission pattern and consequences of infection are viewed in the absence of an active immune response.14

Equine adenovirus type 2 has been isolated in Australia and New Zealand.6,15 Sera from horses of diverse breeds and ages were collected from widely separated geographic areas in Aus-tralia; 327 horse sera were tested for EAdV2 neutralizing anti-body, and 77% of these sera were positive (maximum titer, 1 : 640). This pattern of infection is likely to occur worldwide.

Pathogenesis

Equine adenovirus type 1 presumably is acquired as a droplet or close-contact respiratory or ocular infection. The virus rep-licates in epithelial cells throughout the respiratory tract, pro-ducing lysis and sloughing of these cells and a hyperplastic response in underlying noninfected cells. It is likely that respira-tory disease in foals is more likely to be clinical, if there is total or partial failure of maternal antibody transfer. Recovery from disease caused by EAdV1 alone in immunologically competent horses presumably occurs within a week to 10 days, but mixed infections with other viruses and bacteria may cause more severe and prolonged disease.12,16 Multiple infections involving various combinations of EHV-1 and EHV-4, equine rhinitis A

*The authors acknowledge and appreciate the original contributions of these authors, whose work has been incorporated into this chapter.

190 Section 2 Viral Diseases

respiratory disease—only 50% of these (2/4) were positive on nasopharyngeal swab for EAdV1 via PCR. These two positive foals died, but there was no histologic evidence of infection with EAdV at necropsy. In this study, if PCR were positive, then virus isolation was attempted. One control foal was positive for EAdV1 isolation in cell culture, as were two hospitalized foals. None of the 47 race horses had EAdV1 detected via PCR from nasopharyngeal swabs; however, two (2/12) nonhospitalized mares were positive. Interestingly, their foals were positive control foals as well.11

However, despite evidence showing no infection in 2- to 6-year-old racehorses in training at a single point in time,11 infection can occur in equine populations and may be a cause of undiagnosed respiratory disease in young horses in work. As previously mentioned, it can cause upper respiratory disease with nasal discharge and conjunctivitis. Other clinical signs may include coughing after exercise and enlarged submandibular lymph nodes.1,5

Respiratory disease signs were described in a single, experi-mentally infected SPF foal that was cesarean delivered, colostrum-deprived, and artificially reared in an EAdV-free environment.3 The foal was healthy and 6 weeks old when infected and had been shown to be immunocompetent. After intranasal infection with EAdV1, clinical signs included muco-purulent nasal discharge, severe follicular conjunctivitis (Fig. 17-2), transient anorexia and pyrexia, and sustained tachypnea. There were no changes in blood leukocyte numbers. Equine adenovirus type 1 was readily isolated from nasal, conjunctival, and rectal swabs and from lung, trachea, bronchial lymph nodes, and small intestine tissue homogenates obtained after necropsy at 6 days after infection.

The clinical response to experimental infection of a 4-day-old foal that received colostrum but was artificially reared from 12 hours after birth in an EAdV1-free environment and that had a 1 : 320 SN antibody titer and a 1 : 40 HI antibody titer was similar to that of the previous foal, except fever was not as marked or as sustained, the conjunctivitis was less severe, and nasal discharge was minimal.3 Equine adenovirus type 1 was readily isolated from nasal and conjunctival swabs and from trachea and lung but not from rectal swabs, small intestine, or bronchial lymph nodes, when a necropsy was conducted 6 days after infection.

Severe or fatal bronchopneumonia has been occasionally recorded in nonimmunodeficient Thoroughbred foals.27,28 The severity of disease caused by infection with EAdV1 in Arabian SCID foals is well described, including severe bronchopneumonia.

and equine rhinitis B viruses, and equine adenoviruses were recognized in 15 of 69 outbreaks of respiratory disease of horses in the UK.1 The significance of individual viruses in the patho-genesis of disease cannot be identified in such studies.

Equine adenovirus type 1 may infect cells of the gastroin-testinal (GI) tract and may be shed in feces; consequently, it may also be transmitted through a fecal-oral cycle. Equine ade-novirus type 1 was isolated from neuritis of the cauda equina in two of three cases, but no adenovirus was detected by immu-nofluorescence.17 It was probably an incidental contaminant.17

ImmunityImmunocompetent foals develop EAdV1 antibody, recover spontaneously from the disease, and generally do not possess recoverable virus by day 10 after infection.16 In experimental infections of colostrum-deprived and colostrum-fed foals, the colostrum-fed foals had less severe changes than colostrum-deprived foals.3,16

Reinfection by EAdV1 was found to occur frequently in a group of 16 mares and foals, and many of these infections (or reinfections) occurred in the presence of high levels of circulat-ing antibody.18 Immunoglobulin A (IgA) in nasal secretions is responsible for resistance to reinfection in human infections with adenovirus or rhinoviruses. A similar situation could occur in horses, when rapidly declining levels of nasal antibody after an infection could soon render the horse susceptible to reinfec-tion, despite high serum antibody levels.

After intramuscular (IM) immunization with live EAdV1 and subsequent development of high serum antibody levels, an SPF foal proved resistant to intranasal challenge with EAdV1.3 There was a greater than twofold increase in serum-neutralizing (SN) antibody after challenge; clinical disease did not develop and virus was not isolated. Immunity was correlated with prior exposure to the virus and high circulating SN antibody levels.

An experimental inactivated EAdV1 vaccine elicited high antibody titers in rabbits, mice, and foals. Using nude mice as a model of T-cell immunodeficiency, it was shown that production of EAdV1 SN antibody and to a lesser extent, hemagglutination-inhibiting (HI) antibody was T lymphocyte dependent.19 As a measure of cell-mediated immune responses, an EAdV1-specific in vitro lymphocyte blastogenesis assay was developed and evaluated using lymphocytes from four vaccinated and two unvaccinated control horses. The four vaccinated horses showed marked increases in stimulation indices in response to EAdV1 antigen (maximum stimulation indices, 5.3-18.6).20

As mentioned, EAdV1 is associated as a dominant pathogen in the uniformly fatal, inherited disease syndrome, SCID.21,22 When first recognized in the early 1970s, SCID was estimated to cause the death of about 3% of all purebred Arabian foals. Foals are born with a total absence of T and B lymphocytes, and SCID is inherited as an autosomal recessive gene.23,24 A consis-tent and dominant feature of SCID is a progressive EAdV1 bronchopneumonia (Fig. 17-1). The virus causes pathology in a wide variety of other organs and tissues in SCID foals, includ-ing the GI tract, liver, pancreas, and bladder.

Clinical Findings

Many authors have isolated EAdV1 from nasopharyngeal swabs obtained from horses of varying ages with and without respira-tory disease.3,11,25-27 Clinical signs in immunocompetent horses are likely to be minimal, with EAdV1 infection frequently being subclinical and asymptomatic.11 In a study of hospitalized and healthy foals and horses in California, Bell et al found that EAdV1 was detected via PCR in nasopharyngeal swabs of hos-pitalized (6/15) and control (nonhospitalized foals with a normal physical examination [7/12]) foals. No difference in incidence was found (p = 0.34). Four hospitalized foals had

Figure 17-1 Lung of 63-day-old purebred Arabian foal that had severe com-bined immunodeficiency disease (SCID). There is extensive bronchopneumo-nia. The lungs have failed to collapse. On the cut surface of the lung, essentially all bronchi are plugged with thick, cream-colored exudate.

191Chapter 17 Adeno, Hendra, and Equine Rhinitis Viral Respiratory Diseases

Virus IsolationEquine adenovirus type 1 and EAdV2 are highly host cell spe-cific and have been cultivated only in cells of equine origin (e.g., fetal equine kidney)11 in which both viruses produce a cyto-pathic effect. On light microscopy and hematoxylin-eosin (HE) staining, intranuclear inclusion bodies are a prominent feature of the cytopathology of adenovirus-infected cells. On thin-section EM, virions assembled in the nucleus form crystalline aggregates.

SerologyAdenoviruses are typed on the basis of SN assays. Most adeno-viruses hemagglutinate appropriately chosen red blood cells (RBCs), and HI assays are used for antibody detection. Hemag-glutination is mediated by the knob-like tip of the penton binding to receptors on the RBC surface. Type-specific antigenic determinants defined by SN and HI assays are located on the outward-facing surface of the hexamers. Equine adenovirus type 1 possesses the common group-specific Mastadenovirus antigen.30 An HI antibody to EAdV1, by definition, is type specific.6,30 Extensive analysis of adenoviruses recovered from horses with respiratory disease, including SCID Arabian foals, indicated that on SN and HI assays, all were a single antigenic type, designated EAdV1.4,31 On SN assay, EAdV2 is unrelated to EAdV1.6

The production of soft feces in adult horses, indicative of GI infection, has been reported as a sole manifestation of EAdV infection.1 Replication of EAdV1 in cells of the GI tract was confirmed after experimental intranasal infection.3

Equine adenovirus type 1 was reported as a potential cause of abortion in mares, and abortion was reproduced experimen-tally after intrauterine inoculation of the virus.16 However, claims for the natural occurrence of EAdV abortion are unsubstantiated.

Diagnosis

Clinical LaboratoryVirus isolation from nasopharyngeal and conjunctival swabs during the acute phase of infection is possible but not fre-quently reported. Equine adenovirus type 1 may also be isolated from rectal swabs but would need to be differentiated from EAdV2. Polymerase chain reaction primers have been designed for the detection of both EAdV1 and EAdV2.29 Detection of adenovirus in negatively stained preparations from fecal samples by electron microscopy (EM) is readily achieved. Immune pre-cipitation, complement fixation, hemagglutination, HI, and SN assays have been extensively used in diagnosis and seroepide-miologic studies. Equine adenovirus type 1 hemagglutinates human blood group O and equine erythrocytes but not those of sheep or chicken.30

Figure 17-2 A, Nasal discharge in 9-week-old specific-pathogen-free (SPF) foal 6 days after experimental intranasal/intraocular infection with equine adenovi-rus type 1 (EAdV1).17 B, Conjunctivitis in the same foal 6 days after infection. C, Low-power image of bronchiolitis of the same foal. Note proliferation and dis-orientation of bronchial epithelial cells and the highly cellular bronchial exudate. Arrows indicate adenovirus inclusion bodies in nonsloughed bronchial epithelial cells.

A B

C

192 Section 2 Viral Diseases

Figure 17-3 Phylogenic relationship of Hendra virus to other members of the family Paramyxoviridae, based on analyses of predicted amino acid sequences of virus matrix proteins. (Courtesy Linfa Wang, CSIRO Livestock Industries’ Animal Health Laboratory.)

Humanparainfluenza-2

Simian

Hendra SendaiHuman parainfluenza-3

MeaslesRinderpest

Phocid distemperCanine distemper

Nipah

virus-41Simianvirus-5

Mumps

Rubulavirus

Respirovirus

Henipavirus

Morbillivirus

Family Paramyxoviridae

Newcastledisease

Pathologic Findings

After experimental infection the colostrum-deprived SPF foal showed gross and histopathologic evidence of rhinitis, conjunc-tivitis, tracheitis, and pneumonia. There was both bronchopneu-monia and interstitial pneumonia in affected areas of lung. Duodenal villous atrophy and idiopathic glomerular hyperpla-sia were also observed. Equine adenovirus antigen was detected by indirect immunofluorescence antibody staining of trachea and lung but not in frozen sections of bronchial lymph node or small intestine. In the SPF foal that received colostrum, gross and histologic evidence of EAdV1 disease was similar but less severe than that observed in the SPF colostrum-deprived foal.3

Therapy and Prevention

There are no specific therapies for equine adenovirus infections, and vaccines have not been marketed. For both prophylaxis and therapy, as for other neonatal and perinatal infectious diseases, the provision of supplemental passive antibody either through colostrum or parenterally administered EAdV1 hyperimmune sera should be considered when there is a failure or partial failure of maternal antibody transfer. An accurate diagnosis of EAdV1 would rarely be known, and in most cases, supportive treatment should be used where necessary.

Public Health Considerations

No human public health implications are associated with equine adenoviruses.

Hendra Virus: a Henipavirus

Hendra virus (HeV), formerly known as equine morbillivirus, was first recognized in 1994 as the cause of an outbreak of acute respiratory disease. It is a rare infection of horses that is also a lethal zoonosis, and it has been recognized in two states of Australia: Queensland and New South Wales. The initial out-break affected 20 Thoroughbred racehorses in Hendra, a suburb of Brisbane, Queensland, Australia. Fourteen horses died or were euthanized at the time of the outbreak. The trainer of the horses and a stable hand were infected and became seriously ill with an influenza-like illness, and the trainer died from severe interstitial pneumonia.32 In the following year, a third human patient was identified, in this case dying from encephalitis that was attributed to HeV infection. Interestingly, exposure of this individual had most likely occurred 13 months earlier when assisting with the necropsy examination of two horses that were retrospectively diagnosed with Hendra disease.33,34 Since then, numerous sporadic incidents of single or multiple deaths in horses have been reported, as well as infection and/or death of people attending to diseased horses. There is strong epidemio-logic evidence of horse-to-horse transmission of HeV in the two largest outbreaks in 1994 and 2008 (Table 17-1). All cases have occurred in northern New South Wales (NSW) or Queensland (QLD).35-37

Pteropid bats (flying foxes) are the reservoir host for Hendra virus, as well as for Nipah virus, which belongs to the same genus (Henipavirus) as Hendra but found in Malaysia, Cambo-dia, India, and Bangladesh. Nipah virus caused a major disease outbreak in Malaysia in 1999, where virus spillover from flying foxes led to respiratory disease and febrile encephalitis in domestic pigs and humans; seroconversion of horses was rarely identified. Nipah virus also regularly causes human disease in Bangladesh and nearby India, but with different epidemiologic features; thus human-to-human transmission is commonly

reported, with no strong evidence for involvement of domestic animals in the chain of transmission to humans.

Etiology

Hendra virus is a member of the genus Henipavirus in the order Mononegavirales, in the family Paramyxoviridae, subfamily Paramyxovirinae. Nipah virus and HeV form a distinct clade within the family Paramyxoviridae38 (Fig. 17-3). Virions are enveloped with a nonsegmented, single-stranded, negative-sense ribonucleic acid (RNA) genome that is 18,234 nucleo-tides in length.39 In negatively stained electron micrographs, the virus is pleomorphic and approximately 180 nm in diameter. Unusually, the HeV envelope is covered with two kinds of spikes that are 10 nm and 18 nm long and give the particle a “double-fringed” appearance, which is not a feature of previ-ously described paramyxoviruses. These spikes contain the F (fusion) glycoprotein trimers and G (attachment) glycoprotein tetramers. The nucleocapsid is 18 nm wide and has a periodicity of 5 nm.40 Hendra virus does not agglutinate RBCs from a range of species, including human type O, monkey, equine, porcine, bovine, guinea pig, chicken, and goose, when tested at 4° C, 22° C, and 37° C, or possess detectable neuraminidase activity.

Epidemiology

Flying foxes of the genus Pteropus, order Chiroptera, are the natural hosts for HeV, and antibodies have been found in all four Australian mainland species, with up to 47% of the bats testing positive.41,42 However, transmission of HeV from bats to horses is a rare event, and it is not known precisely how this takes place. There is epidemiologic evidence of horse-to-horse transmission, particularly where the outbreak has been centered on a stable complex or veterinary clinic, with transmission opportunity attributed to contamination of surfaces or equip-ment by infectious fluids from the index case.43 Transmission by aerosol does not appear to be an important means of spread under natural circumstances, and in general, the infection in horses is not considered to be highly transmissible, with direct droplet contact exposure required. Similarly, the attack rate for humans exposed to potentially infectious equine bodily fluids

193Chapter 17 Adeno, Hendra, and Equine Rhinitis Viral Respiratory Diseases

secretions, urine, feces, and blood, suggesting that the greatest transmission risk is posed at this time.45 This is in accordance with field observations around human infection with HeV.

At necropsy examination the most prominent gross lesion is firm, dark red lungs with dilated lymphatic vessels and hemor-rhage and froth in the trachea, bronchi, and bronchioles.47,48 Fibrin tags may be seen on the pleura (Fig. 17-4). The pericar-dial sac may contain serous fluid. In some horses with HeV infection, extensive subcutaneous hemorrhages were observed, but these may have been agonal.

The characteristic histologic lesion in both naturally occur-ring and experimentally induced acute HeV infection is vascu-litis with fibrinoid degeneration and necrosis of the vascular wall. This lesion typically involves smaller vessels in lung, brain (including meninges), spleen, lymph nodes, and kidney. However, lesions may also be detected in nasal mucosa, liver,

is estimated at 10%.44 The presence of antibody to HeV in asymptomatic horses has been confined to disease outbreaks suggesting that the virus is not maintained within the horse population.

Pathogenesis and Pathology

In horses exposed to HeV by the oronasal route and under con-trolled laboratory conditions, HeV RNA may be recovered from nasal secretions 48 hours after contact with the virus and may persist throughout the incubation period.45 This suggests that systemic spread of virus is preceded by local replication in the nasal cavity or nasopharynx, as has also been proposed for Nipah virus.46 Increasing gene copy number is observed in the immedi-ate presymptomatic and symptomatic stages of infection, and at this point, viral RNA is also recoverable from oral cavity

Table 17-1 Hendra Virus Outbreak in Australia*

Date/Town StateMortality†: Horses

Mortality/Morbidity: Humans Dogs

August 1994 Mackay QLD n = 2 n = 1 (died) —September 1994 Hendra QLD n = 21 n = 1 (died)

n = 1 (ill)—

January 1999 Cairns QLD n = 1 — —October 2004 Cairns QLD n = 1 n = 1 (ill) —December 2004 Townsville QLD n = 1 — —June 2006 Peachester QLD n = 1 — —October 2006 Murwillumbah NSW n = 1 — —June 2007 Peachester QLD n = 1 — —July 2007 Cairns QLD n = 1 — —July 2008 Redlands QLD n = 5 n = 2 (1 died/1 ill) —July 2008 Proserpine QLD n = 3 — —Aug 2009 Cawarral QLD n = 4 n = 1 (died) —September 2009 Bowen QLD n = 2 —May 2010 Tewantin QLD n = 1 —June to July 2011 Boonah QLD n = 3 — n = 1 (seroconverted; dog was euthanized after a second positive

antibody test, in line with current government policy)June 2011 Beaudesert QLD n = 1 — —June 2011 Logan Reserve QLD n = 1 — —June 2011 Wollongbar NSW n = 2 — —July 2011 Macksville NSW n = 1 — —July 2011 Park Ridge QLD n = 1 — —July 2011 Kuranda QLD n = 1 — —July 2011 Hervey Bay QLD n = 1 — —July 2011 Lismore NSW n = 1 — —July 2011 Boondall QLD n = 1 — —July 2011 Chinchilla QLD n = 1 — —July 2011 Mullumbimby NSW n = 1 — —August 2011 Ballina NSW n = 1 — —August 2011 South Ballina NSW n = 1 — —August 2011 Mullumbimby NSW n = 2 — —August 2011 Gold Coast Hinterland QLD n = 1 — —August 2011 Ballina NSW n = 1 — —September to October 2011 Beachmere QLD n = 3January 2012 Townsville QLD n = 1 — —May 2012 Rockhampton QLD n = 3 — —May 2012 Ingham QLD n = 1 — n = 1 (initial weak positive, clear on subsequent tests, deemed

not positive, so euthanasia not performed)June 2012 Mackay QLD n = 1 — —July 2012 Rockhampton QLD n = 1 — —July 2012 Cairns QLD n = 1 — —September 2012 Port Douglas QLD n = 1 — —October 2012 Ingham QLD n = 1 — —January 2013 near Mackay QLD n = 1 — —

QLD, Queensland; NSW, New South Wales.

*The number of horses, humans, and dogs known to be infected with or have seroconverted to Hendra virus in Australia in NSW and QLD, in chronologic order. Town names are given for clarity. All outbreaks were in QLD or NSW.†Mortality refers to animals that died or were euthanized for humane reasons or because they were seropositive or virus—isolation positive.

194 Section 2 Viral Diseases

Virus IsolationHendra virus may be reisolated from clinical specimens using several cell lines—Vero cells are most commonly used.53 Pre-ferred tissue samples for this purpose are lung, spleen, kidney, and brain.

Quantitative Real-Time Polymerase Chain ReactionQuantitative real-time PCR has been used extensively to iden-tify acutely infected animals in outbreak investigations, as well as in experimental studies54,55; blood and nasal swabs are the specimens of choice for diagnostic confirmation in the live animal.

SerologySerum neutralization (SNT) and enzyme-linked immunosor-bent assay (ELISA) tests have been developed to detect anti-bodies against HeV, with application primarily to epidemiologic investigations associated with the management of outbreaks. As SNT must be carried out at BSL4, ELISA is usually employed as the first-line screening test.

Antigen DetectionImmunohistochemistry may be used for detection of HeV antigen in clinical specimens and is particularly helpful where formalin-fixed tissues only are available for examination.

Therapy

No specific therapy exists for horses with HeV infection. Because of the serious zoonotic potential of HeV, even sup-portive therapy should not be undertaken once a diagnosis is confirmed. While exclusion diagnostic testing is being carried out, any interventions necessary for reasons of animal welfare should be undertaken with due consideration of the zoonotic risk.

Prevention

VaccinationA key strategy for reducing HeV infection risk to people and other susceptible in-contact horses is to remove the virus-shedding horse from the chain of transmission. Conditions that favour spill-over of HeV from bats to horses are not well understood, and this aspect of disease emergence may not be amenable to control in the foreseeable future. Accordingly, immunization of horses holds promise both for protecting the health of horses exposed to HeV-infected bats and, in the event that an immunized horse does become infected, for reducing any viral replication that may occur to a level that prevents on-going transmission.

Efficacy testing of one candidate HeV vaccine has been carried out in laboratory mammals,56 and in horses. The vaccine is based on recombinantly expressed soluble versions of the G glycoprotein (sG) from HeV57 that has been formulated for use in horses with a proprietary adjuvant as an inactivated subunit vaccine. Preliminary data gathered after using a prime-boost immunization regime confirmed seroconversion of vac-cinated horses, and immunized horses were protected from disease following exposure to an otherwise lethal HeV chal-lenge. In addition, no viral shedding was identified and no viral genome was recovered from tissues (D. Middleton, unpub-lished observations). Clearly, vaccination of horses against HeV has the potential both to protect the health of horses and to break the chain of transmission of HeV from bats to people. Other vaccine platforms, such as canarypox virus-based recom-binant vaccine carrying the glycoprotein (G) gene,58 may in time provide an alternative product that is also fit for this

heart, stomach, intestine, uterus, and ovary. Severe involvement of the lung is common, with diffuse areas of necrotizing alveo-litis and marked fibrinous alveolar exudates accounting for the respiratory signs that are often noted at clinical examination. Syncytial cells may also develop in vascular endothelium and endothelium of lymphatic vessels, as well as in respiratory epi-thelial and lymphoid cells. Extensive deposits of viral antigen may be seen on immunohistochemical examination of affected tissues.

The few known field cases of HeV that have recovered from acute disease have been euthanized in line with current national policy. Mild to moderately severe, focal, nonsuppurative menin-goencephalitis with gliosis and perivascular cuffing has been identified in these animals, and HeV genome has been recov-ered from the brain. The pathogenesis and significance of these findings is not yet understood.

Experimentally, cats, ferrets, and guinea pigs are susceptible to HeV, and although some pathologic differences exist in these species, the diseases are similar to that observed in horses.49,50 Rats, mice, and chickens are not susceptible to experimental infection, and only one of two dogs exposed under laboratory conditions developed neutralizing antibody to the virus.47 However, during 2011 a dog on an outbreak property was identified to have antibody to HeV without having shown signs of disease (see Table 17-1).

Clinical Findings

The incubation period of HeV in horses has been proposed to be 4 to 16 days,32,51,52 after which horses show severe, acute, febrile respiratory disease sometimes accompanied by facial swelling, ataxia, and terminally, copious frothy nasal discharge as the result of pulmonary edema. More recently, neurologic signs have predominated in some outbreak events, with clinical features comprising hypersensitivity, ataxia, disorientation, facial paralysis, head tilt, circling, head pressing, and strangu-ria,43 although there is no evidence that significant viral strain mutation has occurred. The clinical course of disease is gener-ally short, with most horses dying within 48 hours of the onset of signs. Affected horses may also be found dead. Laboratory testing is essential to confirm a diagnosis of HeV infection.

Diagnosis

Hendra virus is classified as a biosafety level 4 (BSL4) agent, and laboratory diagnostic testing should be carried out under the appropriate conditions of biosafety and biocontainment.

Figure 17-4 Gross lesions in lung of horse experimentally infected with Hendra virus, showing pneumonia and dilation of lymphatics. (Courtesy CSIRO Livestock Industries’ Australian Animal Health Laboratory.)

195Chapter 17 Adeno, Hendra, and Equine Rhinitis Viral Respiratory Diseases

Equine Rhinitis a Virus and Equine Rhinitis B Viruses

Picornaviruses are recognized causes of acute upper respiratory and systemic disease in horses. When first isolated in the 1960s, the biophysical properties of these viruses indicated that they were members of the family Picornaviridae. As the majority of the isolates were acid labile (infectivity destroyed at pH 3), they were classified in the genus Rhinovirus, which includes the common cold viruses of humans, and named equine rhinoviruses. Both the naming and the classification implied that the biology, including clinical diseases and pathogenesis, would parallel human common cold viruses, which are the prototypic members of the genus Rhinovirus.

Despite seroprevalence rates of 20% to 80% for equine rhi-nitis A virus (ERAV), equine rhinitis B virus type 1 (ERBV1), and equine rhinitis B virus type 2 (ERBV2), these viruses are seldom specifically diagnosed as causes of respiratory disease in horses. Their relative “neglect” may be related to the dominant position of influenza viruses and equine herpesvirus types 1 and 4 (EHV-1, EHV-4) as causes of acute upper respiratory disease in horses but is also related to a lack of sensitive, widely avail-able, and adopted diagnostic tests.

Etiology

Three acid-labile serotypes, called equine rhinoviruses 1, 2, and 3, were originally identified. A fourth serotype, termed acid-stable picornavirus (ASP), was also isolated. Sequence analysis of the genome of two strains of equine rhinovirus 1 indicated that this virus was most closely related to foot-and-mouth disease virus (FMDV),59,60 and accordingly, the virus was reclas-sified in the genus Aphthovirus, although as a separate cluster, and renamed equine rhinitis A virus (ERAV).61 Until that time, FMDV had been the sole member of the genus Aphthovirus. Analysis of the sequence of the genome of equine rhinovirus 2 indicated that, although most closely related to “encephalomyo-carditis virus,”60 which is a member of the Cardiovirus genus, it was sufficiently distinct from all other picornaviruses to be assigned as the sole member of a new genus Erbovirus (erb[o] for equine rhinitis B) and renamed equine rhinitis B virus (ERBV).61 Some ERBV1 isolates have been shown to be stable at acid pH, and these can also be distinguished from acid-labile ERBV1 by genomic sequence analysis.62 Sequence analysis of the antigenically distinct equine rhinovirus 3 indicated that this virus was a second serotype (ERBV2) within the genus Erbovirus.63 Acid-stable picornaviruses have been isolated from the respiratory tract (prototype 4442/75) and oral cavity of horses,64,65 and it has been shown that they are a third serotype (ERBV3) within the erbovirus genus.

ERAV was first isolated and characterized in the United Kingdom by Plummer.66-68 Subsequently, ERAV was isolated in many parts of the world.69 Most isolations of so-called equine rhinoviruses were antigenically similar to the virus (PERV/62) originally isolated by Plummer66 in 1962 and now designated ERAV. Hofer et al70 confirmed that there were at least two distinct serotypes of equine rhinovirus. The identity of a second serotype (prototype 1436/71) was confirmed by Newman et al,71 and after sequencing of the genome60 and renaming,61 this virus is now designated ERBV1. Steck et al72 indicated the existence of a third serotype, and genomic sequencing of the prototype strain of this virus (313/75) confirmed that it was a second member of the genus Erbovirus, proposed to be desig-nated ERBV2.64 Only one other isolation of ERBV2 from a horse with febrile respiratory disease has been reported.73,74 The so-called acid-stable equine picornaviruses were identified as a distinct serotype64,65 and as noted, are now designated ERBV3.

purpose. In November 2012, Equivac® HeV was released by Pfizer under permit issued by the Australian Pesticides and Veterinary Medicines Authority (APVMA). This vaccine is supplied and administered to horses by veterinarians under strict conditions.

Other MethodsGeneral recommendations have been made to horse owners with the aim of reducing the risk of contact of their animals with potential sources of infection such as flying fox secretions, urine, feces, spats, or aborted fetuses.41 These include providing feeding and watering locations that are under cover, avoiding feedstuffs (such as fruit) that may be attractive to flying foxes, and relocating horses away from paddocks containing flower-ing trees at times of flying fox abundance. Outbreaks of the disease currently require immediate quarantine of the prem-ises, controls on the movement of horses within a defined disease control zone, and serologic surveillance to determine the extent of infection. A greater understanding of the virus and the disease, as well as the vaccination of “at risk” horses in the future, may allow less onerous disease control programs to be used. Information on the persistence of the virus in the environment and the possibility that recovered animals may act as carriers and potential sources of infection needs further exploration.

Public Health Considerations

Although transmission of HeV to humans is a rare event, the serious consequences of infection must be considered when examining horses that may be infected with HeV, collecting clinical samples, or undertaking in vitro or in vivo laboratory tests. The known human infections have occurred after contact with the secretions or bodily fluids of infected horses at the time of terminal illness or during necropsy examination rather than directly from bats. Human-to-human transmission has not yet been reported. However, there are many difficulties inher-ent in making reliable recommendations on reducing risk of transmission from infected horses and in particular, relying on Personal Protective Equipment (PPE) for this purpose. Difficul-ties include: (1) the propensity for outbreaks to occur in a warm climate where compliance with certain items of PPE may be poor; (2) the emotional attachment of humans to their horses that leads to regular close contact, but where use of PPE is perceived as impractical; and (3) specific knowledge gaps, including lack of definition of occupational exposure limits to HeV, unknown infectious dose (especially by inhalation), and unknown viral load in the air.

Members of the horse industry involved with the care of horses that are in or have travelled from the northern NSW region and from QLD are increasingly developing protocols and procedures for managing the HeV transmission risk posed by routine activities.

In other parts of Australia, a challenge for veterinarians and horse handlers is to remain vigilant for the possibility of HeV, despite the low likelihood of its occurring. However, a horse exhibiting signs consistent with HeV that has recently arrived from QLD or northern NSW must be considered a possible or probable case, depending on the clinical signs. The fact that early signs of HeV resemble many other equine diseases, includ-ing colic, pneumonia and pleuropneumonia (travel sickness), makes this problematic. The more distinctive features of HeV, including severe respiratory distress, cardiovascular collapse, and neurologic signs, are manifest after HeV is detectable in secretions. Consequently, if the horse is demonstrating these signs and although they are not pathognomonic for HeV, it is imperative that in-contact people use PPE, biosecurity methods, and test the horse for HeV.

196 Section 2 Viral Diseases

respectively. One part of this study included serum from 44 Thoroughbred horses obtained when they were newly intro-duced into a training center and their average age was 23 months, with a second sample obtained approximately 7 months later. Equine rhinitis A virus, ERBV1, and ERBV2 SN antibody was present in 8 (18%), 34 (77%), and 39 (89%) of horses, respectively, when first bled, and in 27 (61%), 34 (77%), and 38 (86%) of horses, respectively, when tested 7 months later. In this study, 19 of the 44 horses (43%) seroconverted to ERAV within 7 months of entering the training stable. For ERBV1 and ERBV2, the percentage of seropositivity between the first and second samples was about the same. Notably, however, 5 (12%) and 4 (9%) of the 44 horses, respectively, seroconverted to ERBV1 and ERBV2, although this rate of seroconversion was offset by the observation that 5 (12%) and 6 (14%) of the horses, respectively, seroreverted (became anti-body negative). Among all the horses, the average ERAV SN antibody titer was relatively high (3796), and in contrast, ERBV1 and ERBV2 titers were relatively low (average of 84 and 45, respectively) and often fell to below detectable levels (seroreverted) over time at a rate comparable to seroconver-sion. In general, Thoroughbred horses 6 to 24 months of age are serologically negative to ERAV and do not seroconvert until after entering training stables. This suggests that most horses are infected with ERAV during the period of training and racing.76,80,83

Pathogenesis

After aerosol or indirect transmission, virus replicates in nasal epithelial cells. Viremia is a regular feature of ERAV infection. Virus disappears from the blood after onset of antibody produc-tion, although it persists in the pharynx and may be isolated from feces in small amounts (<10 TCID50/g) for at least 1 month after infection (samples were not tested beyond this time).68 Isolation of the virus from feces was considered curious because virus was not isolated at any time from gut, indicating that it must either infect and persist in lower gut cells or be transported from the pharynx through the bloodstream in a manner not detectable by standard virus isolation procedures. The demonstration that ERAV is shed in urine for prolonged periods, up to 146 days in one study but almost certainly longer,77 led to a view that persistent infection may be estab-lished in the urinary tract, possibly in the bladder.

A temporary suppression of cell-mediated immunity in Standardbred horses with decreased athletic performance, in association with symptoms such as intermittent fever and mild pharyngitis, was linked to ERAV infections.84 In this study, lymphocyte proliferation assays to evaluate cell-mediated immunity and a bioassay for equine type 1 interferon were used as markers for detection of viral infection.

Clinical Findings

Equine rhinitis A virus is generally considered to cause mild to moderate respiratory disease,69,70,76,79 although clinical signs may be quite variable,70 and nasal discharge is not invariably present.76 In many cases, infection is subclinical. The incubation period is 3 to 8 days. Clinical signs in natural outbreaks of disease may include fever (41° C ± 0.5° C), anorexia, and serous nasal dis-charge that becomes mucopurulent.85 Of course, secondary bacterial infection may be involved in the production of the latter. There may be coughing and pharyngitis. Recovery in uncomplicated cases occurs within 7 days. Where pharyngitis persists, coughing may continue for 2 to 3 weeks. Although persistent shedding of ERAV in urine and feces is presumably a consequence of prolonged infection, no disease has been linked to nonrespiratory sites of infection.77

Virions measure 24 to 30 nm in diameter. The genome is a single-stranded, positive-sense RNA. Virions are not inacti-vated by lipid solvents (ether, chloroform) or by nonionic detergents.

Epidemiology

Equine rhinitis A virus, ERBV1, and ERBV2 are spread by contact through nasal secretions and aerosol inhalation.75,76 Equine rhinitis A virus is also shed in urine for prolonged periods, and urine aerosol is considered to be an important mode of transmission.77,78 As a coincidental part of a study of the carrier status of equine arteritis virus in male horses, McCollum and Timoney77 showed that 432 of 2523 (17%) postrace urine samples were positive for ERAV by virus isola-tion. The frequency of urine shedding was highest in 2-year-old horses (26%) and appeared to decline steadily to 5% in 8-year-old horses, although horses up to 10 years of age shed virus. There were no differences in urine shedding of ERAV between stallions and geldings. Persistent shedding in urine for up to 146 days was demonstrated in individual horses. In a study of 20 young stallions in which both nasopharyngeal and urine samples were examined for ERAV, only 1 of 11 stallions from which virus was isolated was double positive; 5 of the 20 stallions yielded virus from nasopharyngeal swabs and 7 from urine samples. A possible interpretation of these data is that virus shedding from urine is more prolonged than shedding from the nasal cavity. Although the role of fecal and urine shedding in the transmission of ERAV has not been fully elucidated, it appears that urine is an important mode of transmission,77 presumably by inhalation of a urine aerosol, as also proposed by Powell.78 McCollum and Timoney77 did not recover ERBV1 or ERBV2 from urine.

Although labile at pH less than 6.5, ERAV may remain infectious in the environment under favorable conditions for extended periods, perhaps months.76 Generally, ERAV is most frequently isolated, whereas ERBV1, ERBV2, and ERBV3 appear less frequently isolated. A notable exception to this view was a study of 92 horses with acute respiratory disease in Canada,79 in which ERBV was isolated from 28 of 64 (44%) nasopharyngeal swabs from horses with acute febrile respiratory disease; of the 28 virus-positive horses, 6 (21%) demonstrated a fourfold increase in ERBV2 antibody titer.

Equine rhinitis A virus, ERBV1, and ERBV2 maternal anti-body was present 12 hours after suckling in foals born to antibody-positive dams. By 10 to 12 months, however, ERAV SN antibody was not detected in any of the progeny horses, whereas ERBV1 and ERBV2 SN antibodies were common (83% and 100%, respectively).80 In a U.S. study, the overall percentage of horses less than 3 years old with SN antibodies to ERAV and ERBV was 73%, whereas 90% of horses older than 4 years were positive.81 The prevalence of ERAV antibody in Australia indicated a maximum infection rate of 47.9% (170 of 355 serums), and accordingly, at any one time, 50% of the population was susceptible.75,80 In a study in Japan, paired serum samples were collected from 3012 racehorses that devel-oped pyrexia at two training centers between 1980 and 1986.82 Seroconversion to ERAV was demonstrated in 102 (3.4%), and the mean age of horses seroconverting was 2.44 years. In a study by Carman et al,79 9 of 92 (10%) horses that presented with respiratory disease had a significant rise in ERAV SN antibody, suggesting that ERAV was the cause of respiratory disease in these horses; the corresponding figure as previously noted for ERBV was 6 of 28 (21%) horses.

In a more comprehensive seroprevalence study, 388 serums from 291 horses were tested for SN antibody to ERAV, ERBV1, and ERBV2.80 The prevalence of ERAV, ERBV1, and ERBV2 SN antibodies was approximately 37%, 83%, and 66%,

197Chapter 17 Adeno, Hendra, and Equine Rhinitis Viral Respiratory Diseases

ERBV1.1436/71 was 0.1 50% tissue culture infectious doses. Using this RT-PCR, DNA was amplified from 6 of 17 nasopha-ryngeal swab samples from horses that had clinical signs of acute febrile respiratory disease, but from which ERBV was not initially isolated in cell culture. The sequences of these six ERBV-positive samples had 93% to 96% nucleotide identity, with six other partially sequenced ERBV1 isolates and one ERBV2. Equine rhinitis B virus was isolated from one of the six samples at fourth cell culture passage when it was shown that the addition of 20 mg/mL MgCl2 to the cell culture medium enhanced the growth of the virus. The study highlights the utility of PCR for the identification of viruses in clinical samples that may initially be considered negative with conventional cell culture isolation.

Pathologic Findings

No detailed studies on the pathology of ERAV and ERBV infec-tions have been done other than in explant organ cultures infected with ERAV.91,92 ERAV replicated in cell and organ cultures but was released almost exclusively from nasal turbi-nate epithelium. On thin-section EM, organ cultures inoculated with ERAV appeared normal, with the exception of rare, island-like lesions in infected nasal turbinate, and virus particles were not seen.

Therapy

There is no specific antiviral therapy for rhinitis virus infection in horses. Therapy should be symptomatic and supportive.

Prevention

Vaccines for ERAV or ERBV have not been developed com-mercially until the 2012 release of an adjuvanted, inactivated vaccine to protect against ERAV. This new vaccine has a con-ditional license in the United States; efficacy and potency tests are in progress. An experimental inactivated ERAV vaccine produced primary immune responses in horses, mice (including athymic nu/nu mice), and rabbits.93 The problem of multiple serotypes recognized for FMDV does not occur because ERAV is antigenically and genomically remarkably stable over time and geographic location.90 The occurrence of three serotypes of ERBV would need to be considered in any vaccine development.

Public Health Considerations

Evidence indicates that ERAV infects humans after both natural and experimental infection. A human volunteer infected with ERAV developed severe pharyngitis and swelling of the pha-ryngeal lymph nodes accompanied by fever, and virus was iso-lated from his blood.66,67 Based on serologic data, humans may be infected with ERAV by contact with infected horses, but clinical disease or subsequent human-to-human transmission of virus was not recognized in such naturally occurring infec-tions.66,67,94 Kriegshauser et al94 tested 137 sera from veterinar-ians for the presence of ERAV and ERBV1 SN antibody. Four (2.7%) and five (3.6%) human sera had low levels of neutral-izing “activity” to ERAV and ERBV1, respectively. The authors concluded that the risk of acquiring ERAV and ERBV1 as zoo-notic infections among veterinarians appears low.

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

Plummer and Kerry68 showed that after experimental infec-tion of horses with ERAV, the incubation period averaged 4.25 days (range: 2-8 days). Illness was characterized by fever (up to 40.6° C), anorexia, nasal discharge, pharyngitis, and lymphad-enitis involving at least the submaxillary and pharyngeal lymph nodes. Bronchitis was also recognized. Based on serologic con-versions, most authors recognize that subclinical infections occur. Viremia consistently developed on average at 5.4 days (range: 3-7 days) and lasted 4.5 days (range: 4-5 days). The disappearance of virus from the blood coincided with the onset of antibody production.

Equine rhinitis B virus infection of horses may also result in an acute febrile respiratory disease characterized by coughing and lymphadenitis and recovery, although there is persistent infection and virus shedding from the respiratory tract.64,77,79,83,86 In contrast to ERAV viremia, urine and fecal shedding have not been recognized in association with ERBV infection.

Diagnosis

Clinical Laboratory and Virus IsolationEquine rhinitis A virus replicates in cultured equine cells, as well as in cells from several heterologous animal species.76 The cytopathic effect (CPE) was produced in primary cell cultures from horse, dog, rabbit, hamster, and monkey; in a diploid cell line of equine origin; in cell lines that included HeLa and Hep 2 from humans, rhesus monkey, and African green monkey kidney (LLC-MK2 and Vero cells); and in a rabbit kidney cell line (RK13). Of these, RK13 and Vero cell cultures were found to be efficient host systems for some ERAV strains.67,73,77 The CPE produced occurs at 37° C (98.6° F) and resembles that produced by other picornaviruses in which infected cells round up, shrink, and show marked nuclear pyknosis, becoming refrac-tive and eventually degenerating and detaching from the surface.81 However, primary isolation and propagation of ERAV in cultured cells have proved difficult in some cases.67,75 ERAV may replicate in primary equine fetal kidney (EFK), Vero, and RK13 cells without causing obvious CPE, and switching cell lines was necessary to maintain cytopathic ERAV.87 Similarly, primary isolation of ERBV also seems to be difficult; isolation rates achieved in some studies70,80 were not matched in other studies.73,75,88

These variable success rates correlating acute respiratory disease with rhinitis viruses may simply reflect the real situation at the time the samples are taken. An alternative view is that the noncultivability of the viruses from particular outbreaks may be an important factor. Variation in the susceptibility of the cell lines used for isolation or genetic variation (quasispe-cies) in the viruses themselves could account for higher success rates of virus isolation in some studies compared with others.

SerologyThe demonstration of rising SN antibody titer in paired sera collected about 2 weeks apart will confirm a diagnosis for ERAV or ERBV infection.

Virus DetectionReverse transcriptase–polymerase chain reaction (RT-PCR) has been developed for the detection of ERAV in nasopharyngeal swabs and other samples collected from horses with acute respi-ratory disease.87,89,90 An ERBV-specific nested RT-PCR that amplified a product within the 3Dpol and 3′ nontranslated region of the viral genome was developed.73 This RT-PCR detected all 24 available ERBV1 isolates and one available ERBV2 isolate. The limit of detection for the prototype strain

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197.e3Chapter 17 Adeno, Hendra, and Equine Rhinitis Viral Respiratory Diseases