Abstract

Encephalomyocarditis virus (EMCV) is in the genus Cardiovirus and family Picornaviridae. It has a worldwide distribution and infects a wide range of species including mammals, birds, and arthropods. In the majority of species infection is incidental without causing disease.^2,5,8 Infection in humans is relatively common; however, disease is rare.^6,14 However, the virus EVCV is pathogenic in certain species. The domestic species most commonly affected are pigs (Sus scrofa). EMCV has also caused disease in numerous species of both free-ranging and captive wildlife.^{1,2,5,8,10,13} Non-human primates are particularly susceptible. Species affected include chimpanzees (Pan troglodytes), orangutans (Pongo pygmaeus), gibbons (Hylobates spp.), baboons (Papio spp.), mandrills (Mandrillus sphinx), De Brazza’s guenons (Cercopithecus neglectus), African green moneys (Cercopithecus aethiops), black and white colobus monkeys (Colobus guereza), squirrel monkeys (Saimiri sciureus), golden-lion tamarins (Leontopithecus rosalia rosalia), owl monkeys (Aotus spp.), ring-tailed lemurs (Lemur catta), and ruffed lemurs (Varecia variegata). Non-primate species affected include addax (Addax nasomaculatus), scimitar-horned oryx (Oryx dammah), lowland nyala (Tragelaphus angasii), gerenuk (Litocranius walleri), Thompson’s gazelle (Gazella thompsoni), llama (Lama glama), dromedary camel (Camelus dromedaries), African elephant (Loxodonta africana), black rhinoceros (Diceros bicornis), pygmy hippopotamus (Choeropsis liberiensis), two-toed sloths (Choloepus didactylus), lions (Panthera leo), Goodfellow’s tree-kangaroo (Dendrolagus goodfellowi), Lumholtz’s tree-kangaroo (Daphnia lumholtzi),¹ quokka (Setonix brachyurus), common wombat (Vombatus ursinus), and numerous rodent species.⁷ Interestingly there have been no reports of EMCV infection (no seroconversion in EMCV endemic areas) or disease in gorillas (Gorilla gorilla). EMCV is highly pathogenic in African elephants, while Asian elephants (Elephas maximus) seroconvert without developing disease.²

The epidemiology of EMCV is poorly understood.^2,5 Rodents are thought to be the primary reservoir of the virus. However, virus is rarely isolated from rodents during epizootics of EMCV, so infection in rodents is thought to be transient. Virus is most likely excreted in feces and urine. There is evidence of a correlation between increased rodent numbers and incidence of disease.^4,11,13 Anecdotally, this has been our experience at Taronga Zoo. Other species including arthropods may also act as reservoirs or transport hosts for the virus. Animals may be infected by consuming contaminated food or water (the source of infection may, therefore, be remote from the facility where disease occurs), direct contact with contaminated environments, or consumption of rodent carcasses.¹⁰ The primary route of infection is oral. Vertical transmission has been demonstrated both experimentally and in natural infections in swine and has been suspected in some non-human primate species.² Whether an infected animal remains subclinical or develops clinical disease depends on many factors including viral dose, virus strain, species of animal, age, health, and immune status of the animal. EMCV can survive in the environment for extended periods and fomite transmission is possible. The virus does not survive desiccation and is readily killed by heating to 60°C for 30 minutes and most disinfectants. A gender-related susceptibility to the pathogenic effects of EMCV has been observed, with females being more resistant than males. All EMCV related chimpanzee deaths (n=7) at Taronga Zoo have been males. Numerous unvaccinated female chimpanzees have had elevated antibody titers indicating exposure without disease. This has also been observed in mice³ and in an EMCV epizootic in African elephants where 83% of fatalities were adult bulls, despite there being a slightly higher infection rate in female elephants.⁴

The most common clinical presentation of EMCV infection in zoo animals is sudden death due to acute, non-suppurative, necrotizing myocarditis in an otherwise healthy animal. Occasional signs such as lethargy, anorexia, weakness, dyspnea, incoordination, salivation, and frothing at the nares have been reported 12–24 hours prior to death. Antemortem diagnosis is difficult due to the peracute nature of the disease. Animals rarely survive long enough to seroconvert.^2,5,8 Gross pathologic findings can be subtle and include pale, streaked, or mottled myocardium; epicardial or myocardial hemorrhage; cardiac dilation; pale myocardium; pericardial effusion; hydrothorax; ascites and pulmonary edema; and congestion. Histologically there is focal to diffuse, interstitial, non-suppurative myocarditis. Encephalitis is rarely seen in zoo and wild animals. Pancreatic necrosis may be seen in some cases. A definitive diagnosis can be made by virus isolation. The preferred tissues for virus isolation are heart and spleen.^2,5,8 Immunohistochemistry can also be used to confirm diagnosis.⁸

The most effective method of preventing EMCV infection in a zoological collection is an intensive, integrated pest management program. In collections where EMCV infections occur it is strongly recommended that qualified and trained pest-management personnel are employed to run the program. In addition, a vaccination program for susceptible species, particularly males, is recommended. Various live-attenuated and inactivated EMCV vaccines have been developed using several different adjuvant systems.^5,7,13 The safety and efficacy in terms of maintaining sustained protective antibody titres against infection have not been fully evaluated for many of these vaccines. In many cases antibody responses were variable and short-lived.^2,8 An effective inactivated vaccine was developed in response to an epizootic in African elephants in South Africa. The vaccine was shown to produce good antibody titers 7–10 days post-vaccination and protected 100% of vaccinated animals from challenge with virulent virus.⁹ There is currently no commercially available EMCV vaccine.

Taronga Zoo has had a long history of EMCV infections causing mortality in numerous species.^8,10,12 EMCV infection was also likely to have been the cause of severe dilated cardiomyopathy in a female orangutan that survived for approximately one year after onset of clinical signs. The animal had high EMCV antibody titres (1:6,240). EMCV was also the cause of death of an African elephant at Western Plains Zoo, Dubbo, Australia.¹¹

In 2000 Taronga Zoo, in conjunction with the NSW Department of Primary Industries, developed an inactivated EMCV vaccine using the highly effective adjuvant Montanide™ ISA 206. The vaccine was administered to Barbary sheep (Ammotragus lervia), Indian antelope (Antilope cervicapra), eastern wallaroos (Macropus robustus), and chimpanzees.⁸ All vaccinated animals developed significant antibody titers. The vaccine has since been used on a variety of susceptible species within the Taronga Zoo collection. Chimpanzees and orangutan are vaccinated annually, while other species such as western lowland gorilla, silvery gibbon (Hylobates moloch), quokka, and Goodfellow’s tree kangaroo are vaccinated opportunistically. All animals are vaccinated with a 2 ml dose regardless of body mass and are anesthetized for vaccination to ensure deep intramuscular injection is achieved. In smaller species the dose may be divided and injected in two sites. The great apes are vaccinated in the deltoid or triceps muscle, while most other species are vaccinated in large muscles of the thigh. Only one vaccination reaction has been observed: a quokka receiving 1 ml of vaccine into each hind leg developed a mild cellulitis at the vaccination sites, which resolved without intervention after a few days. Species that are relatively easy to restrain and anesthetize receive a primary vaccination and a booster four weeks later. Most other species are vaccinated just once and then annually or opportunistically. At the time of every vaccination a blood sample is obtained to determine EMCV antibody titers. This is done using a virus neutralization test (VNT), in which a set amount of serum from the animal is passaged in wells, until a dilution titre is obtained in which the EMCV virus in the well can be detected again. Antibody titres ≥1:64 are positive, ≤1:32 are inconclusive and <1:4 are negative. The greater the dilution titer observed before virus is detected, the greater the immune response to the vaccine.⁸

Eighteen chimpanzees have been vaccinated since 2000. Eight animals had positive titers at the time of first vaccination indicating previous exposure to the virus. Antibody titers were maintained at a high level post-vaccination and there was no evidence of waning titers over the 12-month inter-vaccination interval in the majority of animals. Since antibody titers appear to be maintained at a high level for up to and possibly longer than 12 months, the inter-vaccination interval has now been increased to 18 months. A Goodfellow’s tree-kangaroo had a negative antibody titer at the time of first vaccination and an antibody titre of 1:512 at the time of second vaccination one month later. Two quokkas were vaccinated twice, four weeks apart. Antibody titers at the time of first vaccination were negative and were 1:32 and 1:64 by the second vaccination. A silvery gibbon had a negative titer 15 months after a single primary vaccination. A male orangutan had a positive titer at the time of first vaccination and maintained high antibody titers post-vaccination after three subsequent annual vaccinations. An aged female orangutan has maintained high antibody titers despite having never been vaccinated.

Although live-virus challenge has not been conducted there has been no case of EMCV infection causing disease in any vaccinated animal at Taronga Zoo. Pest management has been rigorous since the vaccination program commenced; however, there have been periods of increased rodent numbers. It is, therefore, presumed that the vaccine induced antibody titres are protective.

Acknowledgments

The authors thank Kaye Humphreys, Peter Kirkland, Karrie Rose, and the veterinary nurses and keepers at Taronga Zoo for their assistance with this work.

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