From late February until mid-March 2020 a property in the Lower Hunter Valley of NSW suffered the loss of five camels and a llama by sudden death. Several of these camels were subject to necropsy. Heart lesions in the first camel necropsied prompted pathologists at EMAI to suggest testing for Encephalomyocarditis virus.
Encephalomyocarditis virus (EMCV) belongs to the family Picornaviridae, genus Enterovirus, species Cardiovirus. Picornaviruses are non-enveloped, single-stranded ribonucleic acid (RNA) viruses1. EMCV has been found worldwide and causes disease in a wide variety of mammals including non-human primates, elephants and pigs. There have been disease outbreaks in piggeries and zoos throughout Australia3.
Rodents are considered to be the likely source for EMCV infection, with outbreaks of disease usually associated with a severe infestation of mice and rats1. Rodents shed the virus in urine and faeces, which can contaminate food and water supplies. Ingestion of rodents that are dead or dying from EMCV is another possible means of infection. The virus spreads between rats asymptomatically but does cause disease in mice.
EMCV was named due to the disease observed in non-human primates (myocarditis) and mice (encephalitis). In the wide range of susceptible hosts, EMCV disease can vary in severity and location of lesions. Disease may present as fever, anorexia, listlessness, trembling, staggering, dyspnea, paralysis, or sudden death due to acute cardiac insufficiency and pulmonary oedema.
Zoonotic infections with EMCV are quite common in some parts of the world, but most human infections are asymptomatic. EMC viruses have rarely been recognised as a cause of human illness2,4.
On 22 February 2020 a camel and a llama were found dead on a property in the Lower Hunter. Although the llama and camels occupied different paddocks, they were being fed the same hay. This fact became relevant as the investigation progressed. These first two deaths were not reported, and consequently not investigated.
This 600-acre property housed 60 camels, two alpacas, one llama, 30 horses, 150 cattle, 10 sheep, 16 donkeys, seven red deer, two Asian buffalo and chickens.
On 25 February 2020 a second camel died on the same property and was referred to Hunter Local Land Services (LLS) for investigation. This second camel to die (Camel No. 2) was a 15-year-old female camel in good health and excellent condition. She had been seen the previous afternoon with no obvious clinical signs. She was running in a clean kikuyu paddock with no obvious weeds and no access to a farm dump. The camels on the property were unvaccinated, so enterotoxaemia and other clostridial diseases were considered to be differential diagnoses. In addition to pasture, the camels were being fed oaten hay and a grain-based supplement. However, there had been no change to the components or quantity of the ration.
Post-mortem examination of Camel No. 2 was unspectacular, although it was noted that areas of myocardium were pale and were consequently sampled. An anthrax ICT test was negative, and nitrate/nitrite testing of aqueous humour was negative. The lack of gaseous decomposition suggested that enterotoxaemia was not to blame. A range of fresh and fixed tissues were submitted to EMAI. EMAI commented that "the lesions in the heart are severe and responsible for the sudden death observed." Cardiac glycosides, ionophors and EMC virus were offered as possible causes. PCR testing for EMCV was attempted on blood from this camel but returned negative results.
While we were waiting for the laboratory results for Camel No. 2, a third camel had died but was not reported. A fourth camel (Camel No. 4), another 15-year-old female in good condition, subsequently died on 6 March 2020 without showing preceding signs of disease and was reported to Hunter LLS for necropsy. As with Camel No. 2, necropsy of Camel No. 4 identified heart lesions: white streaks throughout the myocardium with a dry, white appearance on cut surface. There was fibrin in the pericardial fluid, and the lungs were congested. At this stage we still hadn’t received a positive result for EMCV from Camel No. 2, so were still investigating other diagnoses. To that end, testing for arsenic, copper and chromium were requested because the camels had been observed to be licking copper, chromium and arsenic-treated posts in their shelter shed.
PCR testing of fresh lung and heart from Camel No. 4 quickly returned a positive result for EMCV. The histopathological description of the heart samples was similar to Camel No. 2 and consistent with EMCV infection (myocardial necrosis, acute, monophasic, diffuse, marked with moderate lymphohistiocytic myocarditis).
Incidentally, liver samples from Camel No. 4 did return elevated levels for copper.
Further testing of fresh lung and liver samples from Camel No. 2 subsequently identified EMCV by virus isolation.
On 16 March 2020 the fifth and final camel to die was a castrated male aged 8-10 years old. As with the other four camels to die during this outbreak, Camel No. 5 had not been observed to show signs of illness prior to death. It was just found dead in its paddock on Monday 16 March 2020. The level of decomposition of this camel suggested that he had been dead for a couple of days before being discovered. The necropsy performed on this animal was consequently limited due to the level of decomposition. Sampling was confined to fresh and fixed samples of heart as we had a presumptive diagnosis. Despite the advanced autolysis of the carcase, the heart was in sufficiently good condition that areas of pale myocardium were discernible, consistent with the appearance of the heart lesions seen during the two preceding necropsies.
Despite significant autolysis in Camel No. 5, the histopathology report noted cardiomyocyte degeneration and necrosis. The fresh heart sample was PCR positive for EMCV.
As soon as we received indication that EMCV was the cause of the deaths, the owners were advised to embark on rapid, thorough rodent control measures. EMCV outbreaks are associated with rodent carriers contaminating feed with their urine and faeces. Due to the likelihood that the existing feed stocks (primarily hay and grain-based supplement) were the source of the virus (having been contaminated by rodents), the owners were advised to use an alternative source of feed.
Recommendations were also made to disinfect food and water bowls, all affected enclosures and surrounding area. A list of disinfectants was provided that are suitable for cleaning EMCV-contaminated surfaces1. Where possible it was recommended to empty food bowls at the end of the day to discourage rodents eating and defaecating in the feed.
Since these control measures were put in place (i.e. change in the food, cleaning the facility and rodent control) there have been no further deaths on the property.
During this investigation, three camels that died suddenly tested positive for EMCV and had heart lesions consistent with EMCV infection. In an effort to demonstrate to the owner the role of rodents in spreading the virus, a captured rat was also submitted for EMCV PCR testing. Unfortunately, it returned negative results. This finding is unsurprising because only a few rodents may be carrying EMCV. The virus is more easily detected and isolated from larger animals affected by EMCV infection than from captured live rodents3.
The cornerstone to prevention of EMCV infection is rodent control and hygienic feeding practices. Research and zoologic facilities have reported lessening or a cessation of EMC outbreaks when rodent control measures are undertaken1.
Previous studies of EMCV have shown that regardless of species, the most common clinical presentation is sudden death, where the gross pathologic changes had diffuse or focal pallor of the myocardium and often a marked pulmonary congestion. Necrotising non-suppurative myocarditis was consistent on histopathology3.
EMCV is a relatively easy virus to isolate and the preferred tissues for virus isolation and PCR are fresh chilled or frozen heart, brain and spleen3. Fixed tissues should also be submitted for histopathology (heart, lungs, brain, spinal cord, liver, kidney and spleen) as well as 10ml of blood collected in a red-top tube4.
Research on EMCV has demonstrated that the virus is resistant to many environmental conditions, remaining stable at pH 3-8. EMCV can be inactivated in low humidity (<50%) and at temperature of 60⁰C after 30 minutes. Water containing 0.5ppm chlorine, iodine-based disinfectants, and mercuric chloride have been used as disinfectants for EMCV5.
Tiffany O’Connor, Veterinary Virologist DPI, Elizabeth Macarthur Agricultural Institute (EMAI).
Pedro Pinczowski, Veterinary Pathologist DPI, EMAI.