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The first reported cases of elephant endotheliotropic herpesvirus infectious haemorrhagic disease in Malaysia: case report

Background Elephant endotheliotropic herpesvirus haemorrhagic disease (EEHV HD) is the leading cause of death in captive Asian elephant calves in Asia, North America, and Europe with a mortality rate of ~ 65% in calves that are under human care. Although EEHV HD was first found in elephant camps, more recently it was identified in wild populations which poses a greater threat to the elephant population. Deaths due to EEHV HD have been seen in wild elephants, but the in-situ prevalence and mortality rate is unknown. We report the first EEHV HD cases in Malaysia from 3 wild born endangered Bornean elephant calves from Sabah with known typical clinical signs. Case presentation The first calf died within 24 h of the onset of clinical signs; the second calf died within 12 h of the onset of clinical signs. The third calf succumbed within 72 h. Necropsies revealed that all 3 calves had similar presentations of EEHV HD but in the third calf with less severity. We conducted conventional polymerase chain reaction (cPCR) assays and found EEHV DNA at all 7 loci in the 3 calves; it was identified as EEHV1A, the virus type that has been found in most other reported cases. Conclusion Typical EEHV HD clinical signs and the molecular confirmation of EEHV by cPCR and sequencing point to EEHV as the cause of death. Further genetic investigation of the strain is in progress.


Background
Elephant endotheliotropic herpesvirus (EEHV) was the first and so far, only described herpesvirus causing critical disease in elephants with visible morphological changes [1] and resulted in an 85% fatality rate in infected individuals [2,3]. Following a novel EEHV report [2], more than 100 similar cases have been identified across the globe. Captive born juvenile Asian elephants (Elephas maximus) were affected in the majority of the confirmed cases [4,5]. Over the past 20 years, this disease has had a mortality rate of 65% in young Asian elephants between 3 months to 15 years of age in Europe and North America in human care [3]. Furthermore, EEHV haemorrhagic cases are increasingly reported in the endemic wild and captive populations in Asia [3,[6][7][8][9].
Most EEHV cases linked with systemic endotheliolytic disease affecting Asian elephants belong to the EEHV1 group (EEHV1A and EEHV1B), although there has been several cases of morbidity and mortality due to EEHV4 and EEHV5 in Asian elephants [10][11][12][13][14]. Historically there had been two deaths due to EEHV2 in African elephants (Loxodonta africana) and a death due to EEHV6, [4,15] as well as a survivor of EEHV3B [16] and EEHV6 [13]. Since 2019, there have been three deaths, five survivors Open Access *Correspondence: mei-ho@conservationmedicine.org † Mei-Ho Lee and Senthilvel K. S. S. Nathan have Joint first authors 1 Conservation Medicine, 47000 Sungai Buloh, Selangor, Malaysia Diana Ramirez: Deceased 31st October 2018 Full list of author information is available at the end of the article of EEHV HD, and five cases of barely clinical/subclinical EEHV, all due to either EEHV3A or EEHV3B, as well as a death from EEHV2 and what seems to have been a wellcontrolled viraemia from EEHV6 [17,Latimer E,personal communication]. Though the transmission mechanisms of EEHV have not been fully understood, viral DNA is found in trunk secretions and other bodily fluids and is believed to be involved in transmission [18].
EEHV-infected elephants display an acute onset of lethargy, generalised oedema of head and limbs, oral ulceration, cyanosis of the tongue, tachycardia, and death after a period of 1-7 days. Lymphopenia and thrombocytopenia are commonly seen in blood evaluations. Increasingly, there are reports of elephants surviving EEHV HD with treatment including antivirals, fluids, platelets, and immunostimulants, among other therapies [4,17,[19][20][21][22][23]. Pericardial effusion, intestinal haemorrhage, and mucosal ulcerations are common necropsy findings. Target tissues for EEHV include heart, tongue, liver, and large intestine. Histological examinations usually reveal microhaemorrhages, oedema and inflammation in these organs. Lesions can be accompanied by intranuclear herpesvirus inclusions in capillary endothelial cells [1].
There is no prior report published of EEHV infection in Malaysia or from the Bornean elephant-a subspecies of Asian elephant on the island of Borneo, mainly in Sabah, a state in East Malaysia. Bornean elephants, Elephas maximus borneensis, are one of the four distinct subspecies of the Asian elephants. They are classified as endangered according to the International Union for Conservation of Nature (IUCN) Red list of threatened species due to habitat loss. More recently, the species is also threatened from illegal hunting, poisoning and attacks by those trying to protect crops. Sabah has an ex-situ population of 24 elephants in human care at the Sepilok Orangutan Rehabilitation Centre, (Sandakan, SORC), Borneo Elephant Sanctuary (Kinabatangan) and Lok Kawi Wildlife Park (Kota Kinabalu). The state's wild population is estimated at 2000 individuals [24].
There are two hypotheses regarding the origins of these elephants-they were introduced to Borneo either from Sulu [25] or Sumatera and Peninsular Malaysia or were indigenous to Borneo but derived from a Sundaic stock [26]. Genetic studies of mitochondrial DNA divergence supports the latter hypothesis [26,27]. In the wild, they can be found at the northern and north-eastern parts of Borneo in the state of Sabah in Malaysia and the upper Sembakung River in Kalimantan, Indonesia [28]. With the many rampant threats including EEHV HD, low population numbers and the uniqueness of the species in terms of its habitat range and genetic makeup, the conservation and the health of this species is tremendously important to ensure the survival of this iconic species in the wild and in human care.

Case presentation
The first occurrence of EEHV infection at SORC occurred in a 24-month-old E. m. borneensis calf (Table 1). Symptomatic treatment (prednisolone 1 mg/kg, papase, oxytetracycline 20 mg/kg, intravenous fluid therapy) was administered. The calf died within 24 h of the onset of clinical signs. Over 1 L of serosanguinous epicardial fluid  1D) was retrieved from the pericardial sac. The calf had facial swelling (Fig. 1A). Over 6-12 h the swelling increased, and discolouration of the tongue apex was observed (Fig. 1B). The necropsy revealed prominent facial and truncal oedema with cyanotic swollen tongue. Generalised subcutaneous petechiation ( Fig. 1C) with moderate subcutaneous oedema was observed. Severe haemorrhaging was evident in the heart extending from the epicardium through the myocardium and papillary musculatures (Fig. 1E). Generalised haemorrhage and oedema along the gastrointestinal tract including the mesentery was observed (Fig. 1F), and the liver was enlarged with mild petechiation. Following the first calf 's death, all other elephants at the SORC were administered acyclovir 19 mg/kg, oxytetracycline 20 mg/kg, vitamin C and virgin coconut oil supplements. The second EEHV case occurred 8 days later in another 24-month-old calf (Table 1). Intravenous fluid therapy was administered immediately, but the calf died less than 12 h later. The third death occurred 3 days later in a 36-month-old calf (Table 1). Treatment as described above was given. The calf regained appetite for milk and solid food the next day following intensive fluid therapy (rectal and intravenous). Nevertheless, on the next morning, the calf stopped eating and died several hours later.
The necropsy of the second dead calf revealed similar findings to the first one. The necropsy of the third calf showed similar findings to the first two calves but with less severity. Dullness, inappetence and lethargy were observed in all cases. While tongue cyanosis and swelling, and facial and trunk oedema were only observed in the first two calves. Vesicles and oral ulceration were not observed in all three calves.
Organ samples were collected from all three dead calves for Escherichia coli culture and histopathological examination. Blood samples from the last two dead calves and from five apparently healthy elephants from the SORC (Table 1)   were conducted on the extracted DNA for herpesviruses using consensus [29,30] and EEHV PCRs [4,13] ( Table 2). For further genetic investigations on the variabilities on these loci, all blood and organ samples were rescreened at WHGFL with PCRs on conserved gene loci-U60(TERex3), U71(gM), and U77(HEL) and on hypervariable gene loci-U48.5(TK), U48(gH-TK), U51(vGPCR1), and E54(vOX2-1) [Hayward G, unpublished data, 31] (Table 3). Since the serum and whole blood from the first dead calf were unavailable, initial consensus and EEHV PCRs were not performed for this animal. Instead, the organ samples were used for the subsequent EEHV PCRs.
Correct sized PCR products on electrophoresis agarose were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Purified products were sequenced using their corresponding specific primers. The nucleotide sequences were analysed with the Geneious 10.1.3 software (Auckland, New Zealand) and compared to sequences in GenBank for homology analysis using the Basic Local Alignment Search Tool (BLAST; National Center for Biotechnology Information). Cleaned sequences from the initial EEHV PCR were aligned with 17 published EEHV DNA polymerase sequences using the Geneious Prime 2021.1.1 software (Auckland, New Zealand) Geneious Alignment (Alignment type: Global alignment with free end gaps; Cost matrix: Identity, Table 4). Aligned sequences were used to build a phylogenetic tree using the Geneious Prime 2021.1.1 software (Auckland, New Zealand) Geneious Tree Builder, with the Jukes-Cantor genetic distance model and 1000 bootstrap replicates (Fig. 2).
Histopathological examination showed that inclusion bodies were insignificant in the organs of the three calves, but the histopathological lesions were suggestive of enteritis in the first calf and viral infection in the second and third. Using the consensus terminase and polymerase herpesvirus, the polymerase specific EEHV 3/4 and the EEHV terminase PCR assays, herpesvirus was not detected in all tested samples. Of the seven individuals from the SORC screened using the polymerase pan EEHV PCR assays, three individuals (second and third dead calves and one of the five healthy animals) were sequence-confirmed for EEHV, with 91-93% identical sequences to EEHV 1A and 1B (North American isolates) and elephantid herpesvirus 1 DNA polymerase. Phylogenetic analysis showed that there was 100% sequence agreement within the three calves with the initial EEHV PCR detection and they were more related to EEHV 1A from Lao (Accession no. KJ400033.1) than the other sequences used for the comparison. The EEHV detection for the first dead calf was confirmed with sequences from the followup PCRs. The detailed analysis of the results from the follow-up genetic investigations will be presented in a subsequent manuscript.

Discussion and conclusions
These are the first reported cases anywhere of EEHV HD in Bornean elephants (Elephas maximus borneensis). However, the lack of previous reports in Malaysia does not necessarily indicate a low EEHV HD morbidity.

U60(TERex3)
LGH6640  Table 4 Cleaned sequences of the initial PCR of pan EEHV DNA polymerase, Round 2A using 6710/6712 [13] WB2 Whole blood from second collection, S serum, S2 serum from second collection Initially, EEHV was hypothesised to be a novel virus for Asian elephants spread via exposure to African elephants in captivity [2]. However, in 2012, Asian elephants were shown to be the host with detection in wild Indian elephants [3] as well as other subsequent detections of EEHV in wild and camp elephants in Asia [9,31]. Our cases support the latter, as the affected animals had no prior contact with African elephants.
Shedding by herd members could be the source of infection in vulnerable calves; the source of the SORC outbreak is not known. The EEHV may have been shed by the two youngest calves, aged less than 12 months old rescued four months prior to the outbreak, that may have still been protected by maternal antibodies. The calves that died were older than 24 months; none of the younger calves exhibited any clinical signs, although EEHV was detected in one of them. This is consistent with other fatal cases in individuals above 24 months old [4,10], with maternal antibodies generally waning by 24 months old, explaining the higher fatality in slightly older calves [32].
Most fatal EEHV HD cases are of juvenile Asian elephants, ranging from 12 to 84 months old and usually dying within 24 h of the first detectable clinical signs [2,10]. The course of the disease in the first two calves was peracute, both dying in less than 24 h from the onset of clinical signs, the third calf died after 72 h. Viraemia can occur several days prior to the onset of clinical signs and by this time severe vascular lesions would have developed [10]. Nevertheless, keepers should still be trained to identify the signs of the disease. Immediate administration of antiviral drugs might prevent further vascular damage, but its efficacy remains unclear and needs more investigation [23,33]. A symptomatic treatment regime, including intensive fluid therapy, anti-inflammatories, parenteral antibiotics, and vitamins, remains crucial to reduce or stop the effects of initial vascular damages [33]. The efficacy of lauric acid found in the virgin coconut oil given to the elephants for its supposedly antiviral properties [34] in the treatment of EEHV HD is inconclusive because we were not able to distinguish between the effects from the drug treatments and from the supplements. At the time of the outbreak, there was limited information available on successful treatments, new treatment guidelines published subsequently [19][20][21][22][23] provide useful guidance for future outbreaks.
Since this was the first time we found EEHV in our elephants, it is important to determine the prevalence, patterns of shedding and the duration of the disease. In addition, early detection by PCR is vital for preclinical viraemia detection; routine screening serves as an early warning system to better prepare elephant centres for managing imminent EEHV outbreaks. PCR monitoring after an outbreak event is also crucial. These approaches can help us to understand EEHV HD to reduce morbidity and mortality in wild and captive elephants that are already endangered through habitat loss and increasing conflict with humans.