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Kinetics of viral loads and genotypic analysis of elephant endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus)
Abstract
Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elphas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3-8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7-21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.
Supplementary resources (56)
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... DNA extraction on EDTA WBs was carried out as previously described. 19 qPCR DNA from each sample was screened using three independent qPCR assays: EEHV2, EEHV3-4-7, and EEHV6. 17 EEHV3, EEHV4, and EEHV7 possess significant genetic similarities and were thus combined in a single assay, EEHV3-4-7 (Ling, pers. ...
... Tumor necrosis factor-a (TNF) was used as an internal positive control to detect elephant genomic DNA and to rule out PCR inhibitors in the samples. 19 The qPCR assays were implemented in duplicate using EEHV-type specific forward and reverse primers, 5'-hydrolysis probes, and Environmental Master Mix (Life Technologies, Carlsbad, CA 92008, USA, Catalog No. 4398021), and processed on a StepOnePlus real-time PCR machine (Applied BioSystems, Waltham, MA 02451, USA). The TNF assay was implemented as a single reaction. ...
... 5 Similarly, Asian elephants had a range of À88 to 18 days (EEHV1A, EEHV1B, EEHV4, EEHV5A, and EEHV5B viremias). 1,4,6,8,19 Table 3). 2,5 The intensity and duration of shedding needed for transmission of EEHV to at-risk elephants is unknown for both species. ...
This study examined the viral shedding kinetics of elephant endotheliotropic herpesvirus (EEHV) in African elephants (Loxodonta africana) compared to viral shedding behavior in Asian elephants (Elephas maximus). Little is known about the transmission dynamics and epidemiology of this disease in African elephants. In light of recent clinical cases and mortalities, this paper aims to identify trends in viral biology. Trunk wash samples were collected from 22 African elephants from four North American zoological institutions that had recently experienced herd viremias or translocations. Processing of these samples included DNA extraction followed by qPCR to quantitate viral DNA load. The results were then compared with available literature that chronicled similar cases in Asian and African elephants. Minimal EEHV shedding was detected in response to varied herd translocations. Increased shedding was recorded in herds in which an elephant experienced an EEHV viremia when compared to baseline shedding. These index infections were followed by subsequent viremias in other elephants, although it is not known if these were recrudescence, transient controlled viremias, and/or primary infections via transmission to other elephants. When compared to historically published data, it was observed that EEHV3 cases in African elephants and EEHV1A cases in Asian elephants had consistently higher levels of viral DNA in the blood than were shed in trunk secretions, a fact that is seemingly inconsistent with such severe cases of disease and the high mortality rates associated with those respective types. The findings produced in this study highlight the need for more routine monitoring of viral shedding in African elephant herds to elucidate possible EEHV transmission and recrudescence factors for ex situ population management.
... Currently, EEHV infection is diagnosed with detection of EEHV DNA in blood, respiratory secretions, or tissues by real-time quantitative PCR (real-time qPCR; Stanton et al. 2010Stanton et al. , 2012Stanton et al. , 2013. Viremia is usually detected in symptomatic elephants, whereas trunk wash (TW) and bronchoalveolar lavage (BAL) samples are used to detect viral shedding and for EEHV surveillance in asymptomatic elephants . ...
... However, to date, no studies have reported detection of EEHV infection in blood or respiratory samples of living elephants in this free-ranging population. Our aim was to screen retrospectively blood and respiratory samples (TW and BAL) collected antemortem from free-ranging African elephants in KNP, to determine to what extent EEHV is circulating in this population and, to identify which EEHV types are present with validated real-time qPCR assays (Stanton et al. 2010(Stanton et al. , 2012(Stanton et al. , 2013. ...
... Extracted DNA (5 lL) from each sample was screened by four separate real-time qPCR assays (tumor necrosis factor [TNF], EEHV2, EEHV3-4-7, EEHV6). These assays are 100% efficient above 10 copies per reaction and use sequence detection primers, TaqMane MGB probes, and TaqMane Environmental Master Mix 2.0 (Applied Biosystems, Carlsbad, California, USA), as described (Stanton et al. 2010(Stanton et al. , 2012(Stanton et al. , 2013. The EEHV3-4-7 assay has been validated for EEHV3-4 and only partially validated for EEHV7 (P. ...
Elephant endotheliotropic herpesvirus (EEHV) infection can cause acute, often fatal, EEHV hemorrhagic disease in free-ranging and human-managed Asian elephants (Elephas maximus) and human-managed African elephants (Loxodonta africana). However, significant knowledge gaps exist pertaining to the presence of EEHV in free-ranging African elephant populations. We retrospectively screened 142 opportunistically collected samples (blood, n=98; bronchoalveolar lavage (BAL) fluid, n=21; trunk wash (TW) fluid, n=23) obtained between 2010 and 2020 from 98 free-ranging African elephants in the Kruger National Park, South Africa, for the presence of different EEHVs, as well as determining the real-time quantitative PCR positivity rate in this population. With the use of validated, previously published DNA extraction and real-time quantitative PCR protocols provided by the National Elephant Herpesvirus Laboratory (Washington, DC, USA), EEHV was detected in nine male African elephants from samples collected in 2011 (n=1), 2013 (n=1), 2018 (n=2), 2019 (n=4), and 2020 (n=1). Viral detection was more common in respiratory compared with blood samples. Six elephants tested positive for EEHV2 subtype (blood, n=2; BAL, n=3; TW, n=2), including one individual that tested positive on matched respiratory samples (BAL and TW). Four elephants tested positive for EEHV3-4-7 (blood, n=1; BAL, n=2; TW, n=1), whereas EEHV6 was not detected in any of the study animals. One elephant tested positive for both EEHV2 and EEHV3-4-7 in the same BAL sample. Even though the levels of viremia varied between 158 and 1,292 viral genome equivalents/mL blood and viral shedding of EEHV2 and EEHV3-4-7 was detected in respiratory samples, no clinical signs were observed in these apparently healthy elephants. These findings are consistent with reports of asymptomatic EEHV infection in human-managed African elephants.
... On observe ensuite la détection possible de ces mêmes virus dans les sécrétions nasales. Il y a jusqu'à un mois de délais entre la virémie et la détection dans les lavages de trompe et ainsi la possible contamination d'un autre individu (Stanton et al., 2013a). Cette présence du virus dans les sécrétions nasales peut ensuite perdurer pendant plusieurs semaines à plusieurs mois ( Figure 26). ...
... Cependant, la plupart des autres co-infections identifiées concernaient comme agents pathogènes l'EEHV luimême. En effet, de nombreux cas présentaient, soit différentes souches d'un même type(Stanton et al., 2013a), soit différents types ou sous-types viraux, la plupart des situations incriminant lesEEHV1 et 4 (Seilern-Moy et al., 2016a). ...
... On suppose cependant qu'elle peut être longue, de plusieurs semaines à plusieurs mois. En effet, chez plusieurs individus, une virémie a été détectée à basse concentration durant plusieurs mois avant apparition de signes cliniques(Stanton et al., 2013a).Concernant la dissémination du virus dans l'organisme, parmi tous les herpesvirus connus, trois types cellulaires sont les plus fréquemment infectés : les cellules neuronales, les lymphocytes et les monocytes. Pour l'EEHV, ce n'est que très récemment que des virus ont été identifiés au sein ...
... In the present study, we developed Asian elephant-specific reagents and methods, which we have used to elucidate the cellular immune response to EEHV. We have studied a herd of latently infected elephants with a normal pattern of reactivation and control (12,13) in order to identify the specificity and functional profile of protective T cells. The sequenced genome of EEHV1A has identified approximately 115 open reading frames (ORFs), 37 of which are conserved core genes, common to all herpesviruses, and 15 of which are conserved within the betaherpesvirus and gammaherpesvirus subfamilies (14,15). ...
... After primary infection EEHV establishes a lifelong latency, with occasional reactivation as demonstrated by intermittent shedding from mucosal surfaces such as the trunk and in saliva (12,13). Based on routine monitoring of trunk wash samples or prior detection of viremia, we confirmed that all but two juveniles in the Houston zoo herd were latently infected with EEHV1 (Table 2). ...
... Study population. The elephants in this study belong to a single herd of nine elephants consisting of five females (ages 5 months and 7, 27, 36, and 48 years old) and four males (3,7,12, and 52 years old). All elephants in the herd are routinely monitored for EEHV viremia and as such, blood is regularly obtained from them for the purposes of EEHV screening and further study (IACUC approval AN-5182). ...
Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, an endangered species. One hypothesis to explain this vulnerability of some juvenile elephants is that they fail to mount an effective T cell response to the virus. To our knowledge, there have been no studies of Asian elephant T cell responses to EEHV. To address this deficiency, we validated the gamma interferon (IFN-γ) enzyme-linked immunospot assay for tracking antigendirected T cell activity by monitoring rabies-specific responses in vaccinated elephants. In addition, we generated monoclonal antibodies to Asian elephant CD4 and CD8 to facilitate phenotypic T cell profiling. Using these tools, we screened healthy elephants with a history of EEHV infection for reactivity against nine EEHV proteins whose counterparts in other herpesviruses are known to induce T cell responses in their natural hosts. We identified glycoprotein B (gB) and the putative regulatory protein E40 as the most immunogenic T cell targets (IFN-γ responses in five of seven elephants), followed by the major capsid protein (IFN-γ responses in three of seven elephants). We also observed that IFN-γ responses were largely from CD4⁺ T cells. We detected no activity against the predicted major immediate early (E44) and large tegument (E34) proteins, both immunodominant T cell targets in humans latently infected with cytomegalovirus. These studies identified EEHV-specific T cells in Asian elephants for the first time, lending insight into the T cell priming that might be required to protect against EEHV disease, and will guide the design of effective vaccine strategies.
... They have all been assigned to the taxonomic genus Proboscivirus, so far the sole member of a newly proposed subfamily Deltaherpesvirinae [4,5]. Based on this newly acquired knowledge of sequences, new diagnostic PCR-assays were developed, which allowed not only detection of EEHV but also immediate type discrimination [12][13][14]. Thanks to such assays, it was also noted that various EEHV types were shed intermittently through trunk secretions [13]. Although these genome-targeting PCR assays cannot discriminate between EEHV in a latent or actively replicating state, the intermittent detection of these viruses in trunk washes has been linked to episodes of viral excretion. ...
... Each sample was subjected to 6 individual Rt-PCR assays, namely for EEHV1, EEHV2, EEHV3/4, EEHV5, EEHV6, and TNF. All real-time PCRs were done essentially as previously described [12][13][14]. Primers (Microsynth, Balgach, Switzerland) and probes (Lifetechnologies, Switzerland) as well as target genes are listed in Table 2. ...
... Primers (Microsynth, Balgach, Switzerland) and probes (Lifetechnologies, Switzerland) as well as target genes are listed in Table 2. The Asian elephant tumor necrosis factor (TNF) α gene was used as an indicator for the presence of elephant cells in the sample and as an internal amplification control for DNA under the current PCR conditions [12]. A sample was considered as "valid" only if a Ct value prior to cycle 40 was achieved with the TNF-PCR. ...
Elephants, particularly Asian (Elephas maximus), are threatened by lethal elephant hemorrhagic disease (EHD) due to elephant endotheliotropic herpesviruses (EEHV). At least five of seven known EEHV types have been associated to EHD, with types 1, 4, and 5 predominantly affecting Asian elephants. In Switzerland, at least three Asian elephants have been lost due to EHD but nothing is known about the present EEHV1 circulation. Moreover, the prevalence of other EEHV types has never been assessed. Intermittent shedding of EEHV can be monitored through collecting trunk secretions and analyzing them by PCR methods that discriminate the different EEHV types. To identify EEHV shedders, seven of eight Asian elephants in a Swiss zoo were trained to provide trunk wash samples. These were collected at intervals over a period of four months and tested by PCR for presence of EEHV1 through 6. Moreover, the quality of each sample was assessed by testing for the elephant TNF-alpha gene. Overall, 57% of the samples were valid with five of seven participating elephants identified as EEHV shedders. Two of those shed virus only once, whereas the other three, all closely related among each other, shed virus on multiple occasions. One of the frequent shedders had been in very close contact to all of the three EHD victims. Therefore, we speculate that this particular animal may represent the virus source in all three cases. However, when subtyping was conducted, the presently circulating virus was identified as EEHV1B, while the virus subtype causing EHD had been 1A in all three cases. In addition to four animals excreting EEHV1, a recently introduced animal was observed to shed EEHV3/4. We suggest that the policy of trunk washing to identify and characterize EEHV-shedders is to be endorsed in zoos with ongoing or planned elephant breeding programs.
... EEHVs, in particular EEHV-1, are the cause of acute haemorrhagic disease (EEHV-HD) in young Asian elephants (Elephas maximus), and clinical EEHV infections account for most of the fatalities in juvenile Asian elephants, both in their natural habitat and in captivity [7][8][9][10][11][12][13]. EEHV-1 induced acute haemorrhagic disease is manifested initially by sudden onset of non-specific clinical signs, which often develop into haemorrhagic disease and sudden death within 1 to 7 days [9,12]. It has also been documented that EEHV viraemia is detectable at least several days before the onset of clinical signs [13,14]. To enable early therapeutic intervention in response to detection of EEHV viraemia, some zoological collections have implemented regular blood sampling and laboratory testing of juvenile Asian elephants. ...
... Conferring from herpesviruses' properties, it could be assumed that once an elephant is infected it remains a latent carrier for life. Screenings of elephant herds have revealed widespread prevalence of the viruses and their periodical reactivation and shedding through biological fluids, especially trunk secretions [13][14][15][16][17][18][19]. Routes of EEHVs transmission to naïve individuals are still unclear, however, considering other herpesviruses' transmission routes, contact with infected bodily secretions is a very likely possibility and vertical transmission cannot be excluded. ...
... It is known that several elephant calves have survived laboratory confirmed EEHV infections. These elephant calves were treated with FCV at doses of 5.5-8.0 mg/ kg twice daily (BID) rectally [9,18,19,[21][22][23][24] or with GCV at 5 mg/kg BID intravenously followed by oral administration [14,25]. One elephant calf with advanced EEHV-1 clinical signs has been treated successfully with ACV at 12mg/kg BID intravenously for 15 days along with penicillin G and supportive treatment including vitamin C, physiological saline and 5 % Dextrose [26]. ...
Background
Elephant Endotheliotropic Herpesviruses (EEHVs) can cause acute haemorrhagic disease in young Asian elephants (Elephas maximus) and clinical EEHV infections account for the majority of their fatalities. The anti-herpesviral drug famciclovir (FCV) has been used routinely to treat viraemic at-risk elephants, but thus far without proven efficacy. This paper presents clinical and virological investigations of two EEHV-1A infected elephants treated with FCV, and discusses anti-herpesvirus therapies of viraemic elephants. Cases presentationsTwo 1.5 year old male Asian elephants at a zoological collection in the UK developed clinical EEHV-1A infections. Case 1 showed signs of myalgia for the duration of 24 hours before returning back to normal. EEHV-1A DNAemia was confirmed on the day of clinical signs and continued to be present for 18 days in total. Trunk shedding of the virus commenced 10 days after detection of initial DNAemia. Case 2 tested positive for EEHV-1A DNAemia in a routine blood screening sample in the absence of clinical signs. The blood viral load increased exponentially leading up to fatal clinical disease seven days after initial detection of DNAemia. Both calves were treated with 15 mg/kg FCV per rectum on detection of DNAemia and penciclovir, the FCV metabolite, could be detected in the blood at assumed therapeutic levels. The early indicators for clinical disease were a marked absolute and relative drop in white blood cells, particularly monocytes prior to the detection of viraemia. The most prognostic haematological parameter at later stages of the disease was the platelet count showing a continuous sharp decline throughout, followed by a dramatic drop at the time of death. Conclusions
The EEHV-1A viraemic animals investigated here further highlight the ongoing threat posed by these viruses to juvenile Asian elephants. The findings call into question the efficacy of rectal FCV in clinical cases and direct towards the use of alternative anti-herpesvirus drugs and complementary treatments such as plasma infusions if no improvement in either viral load or the above-mentioned blood parameters are observed in the initial days of viraemia despite anti-herpesvirus therapy.
... Asian elephant population faces serious threat due to EEHV which accounted for acute fatalities in Asian elephant calves during late 1980s [3,4,31,32]. Latent nature of herpes viruses permits to establish a carrier status in adult Asian elephants and intermittent shedding of the virus without associated clinical disease [31][32][33]. Hence, the present research was conducted to study pathoepidemiology of EEHV-HD affecting Asian elephants in India. ...
... Microscopic findings recorded in the present study were consistent with the gross lesions characterised by severe vascular changes in internal organs which was also documented by other workers [22]. Furthermore, in the present study, the vascular changes were more associated with the microvasculature which reaffirms the findings of previous studies [33,36]. Lesions in blood vessels comprised of oedema, endothelial cell swelling, denudation and mild to moderate degree of vascular leucocytosis. ...
In the present research pathology and molecular diagnosis of elephant endotheliotropic herpes virus-haemorrhagic disease (EEHV-HD) among Asian elephants was studied. Out of 76 cases, 20 were positive for EEHV infection in PANPOL and POL1 based semi-nested PCR. Out of 20 samples, 10 samples were fatal cases of EEHV-HD while 10 were of either subclinical or latent infection. Acute onset haemorrhagic disease with EEHV-HD had anorexia, facial and neck swelling, cyanotic buccal mucosa and tongue, nasal and ocular discharge, and colic. The hallmark of gross finding in all cases were severe haemorrhagic lesions in the internal organs viz. cyanosis of tongue with multifocal petechial haemorrhages, diffuse epicardial and endocardial haemorrhages, swollen liver (rounded edges) with parenchymal haemorrhages, serosal and mucosal haemorrhages in gastrointestinal tract, congested kidneys with corticomedullary haemorrhages, highly congested meninges, and brain capillaries with haemorrhages. Microscopic findings in all the cases had severe vascular changes in the visceral organs. Microthrombi was present in the vasculature of tongue, heart, lung, liver, kidney, and brain. The endothelial lining of most of the blood vessels were swollen with apoptotic changes. Amphophilic to basophilic intranuclear inclusion bodies were observed in the endothelial cells. Immunostaining using anti-EEHV DNAPOL hyperimmune sera revealed intense positive signals in the endothelium of blood vessels and their walls. Quantification of viral load in necropsy tissue samples revealed highest in the heart (7.4 × 106/μg of sample) and least in the brain (9 × 103/μg of sample). The PCR amplicons from EEHV1 specific genes (POL1(U38) and TER were subjected to partial genome sequencing which had 99.9% similarity with the EEHV1A subtype. It was concluded that Asian elephants in India are latently infected for EEHV1 and in all the fatal EEHV-HD cases, EEHV1A subtype was the causative agent with characteristic pathomorphological changes in visceral organs.
... Death can occur quickly, within 1-7 days after clinical onset [4,5], with a mortality rate of up to 85% [1,6]. Identifying changes in blood hematology in response to EEHV infection are useful in diagnosing and monitoring disease progression [7], being detectable several days before clinical signs manifest [2,8,9]. Lymphopenia, monocytopenia, and thrombocytopenia are hallmarks of EEHV-HD viremia [2,8,[10][11][12]. ...
... Whole blood samples were processed within 24 h after collection using a commercial kit (NucleoSpin ® , Macherey-Nagel GmbH & Co. KG, Dueren, Germany) in accordance with the manufacturer's recommended protocol for 200 µL of whole blood, and extracted DNA was eluted in 100 µL of elution buffer. Concentrations of DNA were calculated by use of a spectrophotometer [9]. ...
Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is a virulent disease that causes severe hemorrhage and sudden death in Asian elephant calves. A change in hematology profiles is one indicator of infection before clinical signs appear; however, to be effective, individual baselines and age-matched reference values are needed. Stress has been speculated to be a factor in clinical EEHV cases, but relationships have not been demonstrated empirically. This study evaluated blood hematology and several stress response markers—salivary cortisol, fecal glucocorticoid metabolites (FGM), salivary Immunoglobulin A (SIgA), and fecal IgA (FIgA) in samples collected for 1 year from three healthy calves with no EEHV history (non-EEHV), and six that had previously been infected, developed clinical signs and survived (prior-EEHV). Hematology values between non-EEHV and prior-EEHV elephants were not different and within published reference ranges. Concentrations of salivary cortisol, FGM, SIgA, and FIgA also were variable and showed seasonal differences, but no relationships to prior EEHV status. One of the prior EEHV calves became re-infected, developed hemorrhagic disease (HD), and died during the study period. That calf exhibited lymphocytopenia, monocytopenia, and thrombocytopenia. Additionally, all stress biomarker concentrations were lower in the 12 days before viremia was observed. Thus, as in other studies, changes in hematology occur with EEHV infection, while preliminary data in one calf suggests that stress-response measures might also be informative and should be studied further.
... In betaherpesviruses, such as HCMV, the virus is transmitted mainly by bodily secretions, including breast milk, saliva, urine, and genital excretion 13 . To date, only saliva and intestinal secretions have been demonstrated or speculated as the major routes of EEHV transmission 22,32,33 ; whether any of the other bodily secretions are involved has not been determined. Moreover, given our findings that epithelia of the trunk and tongue are permissive for EEHV replication, it is reasonable to speculate that the primary infection site of EEHV in recipient animals is the epithelia of the trunk or oral cavity; and from there the virus spreads to other target organs via circulating blood leukocytes. ...
... This seems reasonable since a significant increase of TUNEL positive cells and Iba-1 positive cells was observed in tissue parenchyma of the EEHV-infected internal organs 23 . However, based on results in the present study and that EEHV gB was found in the monocytes/macrophages 22 , and that EEHV DNA loads in the infected cases were observed in the PBMCs rather than serum 25,32 , it is strongly suggested that monocytes/macrophages are, in fact, major targets for EEHV replication in vivo. Evidence has shown that the violence of viremia in several models of viral infections, including African swine fever virus, dengue virus and HIV, is facilitated by infection of blood leukocytes 10 . ...
Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.
... All the Asian elephants were known to be positive for at least one EEHV species, previously confirmed by quantitative real-time polymerase chain reaction (qPCR) using blood; none of the African elephants had been previously tested for EEHV (Molter and Howard, pers comm.). 5,9,10 The Asian elephants (n = 5 female, n = 4 male) ranged from 1 to 48 yr old, and the African elephants (n = 6 female, n = 6 male) from 4 to 28 yr old. Due to differences in herd management at each institution, sampling was not uniform: whole blood, trunk wash, and chewed hay and browse were collected from each Asian elephant, and oral swab, trunk wash, and feces from each African elephant. ...
... 9 Parallel qPCR reactions were performed for elephant tumor necrosis factor α (TNFα) to determine quality of elephant host DNA present and as a control for qPCR conditions. 10 Samples were considered positive if >10 viral genome equivalents/reaction were detected or if the average cycle threshold was <40. 9 Using an exogenous internal positive control assay, all chewed plant samples were found to contain PCR inhibitors that prevented DNA amplification via qPCR. 4 As a result, conventional PCR assays (typically less sensitive to PCR inhibitors) designed to detect the herpesviral DNA polymerase (DPOL) gene from α, β and γ herpesvirus subfamilies and mammalian ferritin (to determine quality of elephant host DNA and as a control for PCR conditions) were used to test Asian elephant trunk wash and chewed plant samples and African elephant fecal samples. ...
Elephant endotheliotropic herpesvirus (EEHV) hemorrhagic disease (EEHV-HD) threatens Asian elephant (Elephas maximus) population sustainability in North America. Clusters of cases have also been reported in African elephants (Loxodonta africana). Risk to range country elephant populations is unknown. Currently, EEHV detection depends upon sampling elephants trained for invasive blood and trunk wash collection. To evaluate noninvasive sample collection options, paired invasively collected (blood, trunk wash and oral swabs), and noninvasively collected (chewed plant and fecal) samples were compared over 6 wk from 9 Asian elephants and 12 African elephants. EEHV shedding was detected simultaneously in a paired trunk wash and fecal sample from one African elephant. Elephant γ herpesvirus-1 shedding was identified in six chewed plant samples collected from four Asian elephants. Noninvasively collected samples can be used to detect elephant herpesvirus shedding. Longer sampling periods are needed to evaluate the clinical usefulness of noninvasive sampling for EEHV detection.
... Ennek megfelelően alakulnak ki a betegségre jellemző tünetek és kórbonctani elváltozások. Egy tanulmány szerint a klinikai tünetek megjelenése előtt már akár 28 nappal is kimutatható a vérben a vírus-DNS, ami aztán megjelenik az ormányváladékban is (35). ...
... Egy elefánt egyszerre több EEHV-törzzsel is fertőzött lehet, egy tünetmentes állat ormányváladékában lévő vírus-DNS pedig egy már lezajlott és a látenciából aktiválódott fertőzésre is utalhat (35). Hogy ezek miben befolyásolják egymást és a betegség többi részletét, az további kutatásokat igényel. ...
Literature review about the endotheliotropic herpesvirus of elephants
... Since then, five additional EEHV species have been identified in other Asian and African elephants with mild or lethal acute systemic disease [9][10][11][12][13][14] and in lung nodules, as well as at low levels in some random necropsy tissue samples from healthy adult African elephants [15]. Specific DNA-based PCR blood and trunk wash tests that were developed to detect all seven known EEHV species [10,[15][16][17][18] have revealed that these viruses are natural and largely benign infections endemic to both Asian and African elephants in the wild, as well as in zoos. However, primary infection with predominantly just the one highly pathogenic type EEHV1A leads to lethal HD in up to 80% of Asian calves that develop characteristic acute clinical symptoms, although many others survive with lower grade or even asymptomatic mild disease [4,17,19]. ...
... Specific DNA-based PCR blood and trunk wash tests that were developed to detect all seven known EEHV species [10,[15][16][17][18] have revealed that these viruses are natural and largely benign infections endemic to both Asian and African elephants in the wild, as well as in zoos. However, primary infection with predominantly just the one highly pathogenic type EEHV1A leads to lethal HD in up to 80% of Asian calves that develop characteristic acute clinical symptoms, although many others survive with lower grade or even asymptomatic mild disease [4,17,19]. Overall, as reviewed by Long et al [5], EEHV HD has affected more than 20% of all Asian elephant calves born in North American and European zoos between 1995 and 2015, and has been the cause of 65% of the deaths that occurred amongst North American zoo or circus reared calves during that time period. ...
Thirteen new lethal cases of acute hemorrhagic disease (HD) with typical histopathogical features were identified in young Asian elephants (Elephas maximus indicus) in India between 2013 and 2017. Eight occurred amongst free-ranging wild herds, with three more in camp-raised orphans and two in captive-born calves. All were confirmed to have high levels of Elephant Endotheliotropic Herpesvirus type 1A (EEHV1A) DNA detected within gross pathological lesions from necropsy tissue by multi-locus PCR DNA sequencing. The strains involved were all significantly different from one another and from nine previously described cases from Southern India (which included one example of EEHV1B). Overall, eight selected dispersed PCR loci totaling up to 6.1-kb in size were analyzed for most of the 22 cases, with extensive subtype clustering data being obtained at four hypervariable gene loci. In addition to the previously identified U48(gH-TK) and U51(vGPCR1) gene loci, these included two newly identified E5(vGPCR5) and E54(vOX2-1) loci mapping far outside of the classic EEHV1A versus EEHV1B subtype chimeric domains and towards the novel end segments of the genome that had not been evaluated previously. The high levels of genetic divergence and mosaic scrambling observed between adjacent loci match closely to the overall range of divergence found within 45 analyzed North American and European cases, but include some common relatively unique polymorphic features and preferred subtypes that appear to distinguish most but not all Indian strains from both those in Thailand and those outside range countries. Furthermore, more than half of the Indian cases studied here involved calves living within wild herds, whereas nearly all other cases identified in Asia so far represent rescued camp orphans or captive-born calves.
... Nevertheless, since the present study demonstrated that salivary glands harbor EEHV gB in the EEHV1A-infected and EEHV4-infected calves, it is reasonable to hypothesize that the primary replication site for EEHV1A and EEHV4 may be in these glands and that the infectious virions are shaded in the saliva. This hypothesis could be further explained by the fact that EEHV1A viral nucleic acid has been observed in the saliva and elephant trunk washes of the acute fatal EEHV1A-infected calves 35 . It has been shown that dissemination of EEHV viral nucleic acid in blood (DNAemia) was detected several days prior to the observation of the clinical signs 7,35 , and shedding of EEHV1A in trunk washes has been observed to commence 10 days after the initial detection of DNAemia 7 . ...
... This hypothesis could be further explained by the fact that EEHV1A viral nucleic acid has been observed in the saliva and elephant trunk washes of the acute fatal EEHV1A-infected calves 35 . It has been shown that dissemination of EEHV viral nucleic acid in blood (DNAemia) was detected several days prior to the observation of the clinical signs 7,35 , and shedding of EEHV1A in trunk washes has been observed to commence 10 days after the initial detection of DNAemia 7 . Moreover, since elephant trunk washes from healthy and asymptomatic elephants in the EEHV-infected herd were also found positive for viral nucleic acid 13 , it is suggested that tissues, such as salivary glands, may also serve as a site of latency for EEHV during persistent infection. ...
Elephant endotheliotropic herpesvirus (EEHV) is one of the most devastating viral infectious diseases in elephants worldwide. To date, it remains unclear how elephants get infected by the virus, where the virus persists, and what mechanisms drive the pathogenesis of the disease. The present study was aimed to develop an antibody against glycoprotein B (gB) of EEHV, investigate the EEHV tissue tropisms, and provide the possible routes of EEHV transmission in Asian elephants. Samples from elephant organs that had died from EEHV1A and EEHV4 infections, peripheral blood mononuclear cells (PBMC) from EEHV4- and non-EEHV-infected calves were used in this study. The results of western immunoblotting indicated that the antibody can be used for detection of gB antigens in both EEHV1A- and EEHV4-infected samples. Immunohistochemical detection indicated that the EEHV gB antigens were distributed mainly in the epithelial cells of the salivary glands, stomach and intestines. Immunofluorescence test of PBMC for EEHV gB in the EEHV4-infected calf indicated that the virus was observed predominantly in the mononuclear phagocytic cells. The findings in the present study unveil tissue tropisms in the EEHV1A- and EEHV4-infected calves and point out that saliva and intestinal content are likely sources for virus transmission in EEHV-infected Asian elephants.
... Although close monitoring and early detection of EEHV infection together with good medical care have helped to save several acutely infected juvenile Asian elephants, the use of antiviral drugs such as famciclovir (FCV) or ganciclovir (GCV) for treatment is still controversial [2,16]. ...
... The young elephant AZ_2016 was treated with ganciclovir (GCV) 5 mg/kg [2,16] by oral administration twice daily for 3 days (Source: Houston Zoo Asian Elephant EEHV Treatment Protocol, April 2015). GCV was injected into bread pieces, mixed with smashed beets, and given by hand to ensure complete uptake. ...
Elephant endotheliotropic herpesviruses (EEHVs) are a continuous threat for young Asian elephants. We report a laboratory-confirmed infection of a 5-year-old female Asian elephant (AZ_2016) in the Berlin Zoologischer Garten. Initially, high EEHV-1 loads were detected in trunk swabs obtained from the young elephant during routine screening. The animal showed no clinical signs except for slight irritability. EEHV-1 was continuously shed for almost one year, with fluctuations in viral load from time to time. Our investigations highlight the continuous threat of EEHV-1 to young captive Asian elephants and stress the importance of routine monitoring of captive elephants to allow early detection of infection.
... It is now known that viremia is detectable days to weeks prior to the development of clinical signs, and therefore routine monitoring through weekly blood testing is recommended for all institutions holding Asian elephant calves. 2, 8,31 Serial blood collection is also necessary to measure the level of viremia and relevant hematologic and biochemical blood variables during the course of a clinical case to assess efficacy of therapies and progression of the disease. ...
... Primers and TaqMan probe for EEHV-1 qPCR used in this study were as described previously. 18 Primers and TaqMan probe for the Asian elephant TNFa qPCR were as described previously, 31 substituting the VIC fluorophore with a HEX fluorophore for the hydrolysis probe. Primers and TaqMan probe for Asian elephant b-actin qPCR were as described previously. ...
Lancing a finger elicits minimal pain in humans and is applied routinely to obtain small volumes of blood for clinical diagnostics. A modified lancet bleeding method and several blood sampling matrices were evaluated in this study for the purpose of routine elephant endotheliotropic herpesvirus (EEHV) surveillance in Asian elephants (Elephas maximus). The procedure enabled weekly sampling from elephants as young as 9 mo of age. The blood sampling matrices were evaluated for their sensitivity measuring β-actin, tumor necrosis factor α, and/or EEHV-1 by quantitative polymerase chain reaction assays. Foam and flocked swabs produced significantly (P < 0.05) lower quantitation cycles, ie, increased analytical sensitivity, than filter papers, Whatman® FTA cards, or conventional cotton-tipped swabs. The two swab types also demonstrated comparable analytical sensitivity to that of a similar volume of EDTA whole blood for the detection of EEHV-1 DNA. This lancet-and-swab technique proved satisfactory for the detection of EEHV-1 viremia in two Asian elephant calves, and in one instance viremia could be detected 5 days prior to the development of clinical signs. Low blood yield from the lancet application may reduce sensitivity and compromise early detection of viremia. Therefore, standard venipuncture remains the recommended blood sampling method, and training for consistent and regular vein access should continue to be the priority for collections holding elephants. However, if appropriate measures are taken to collect an optimum blood volume, this lancet-and-swab technique offers a suitable alternative for EEHV surveillance in situations where venipuncture may not be practical.
... Facilities that care for either species of elephants should be and are increasingly well prepared for such cases, and the increased awareness of this disease has likely contributed to the successful treatment of several individuals in recent years. 1,14,17 The availability of PCR testing for diagnosis and monitoring has greatly enhanced the surveillance and aided in decision-making for clinical cases. ...
... Clinical signs have been shown with EEHV1 to begin at 10 4 VGC/ml, and fatal episodes typically are accompanied by over 10 6 VGC/ml in whole blood. 17 Trunk wash fluid in this case contained mild to moderate levels of viral particles compared to more classic EEHV1 cases, peaking at day 78 (2,403 VGC/reaction); EEHV1 clinical cases have shown 10 3 -10 6 VGC/reaction. It is interesting to note that this elephant calf was shedding low levels of virus through the entire period of analysis (6 mo after initial signs of infection). ...
This epidemiologic study follows a 5-yr-old male African elephant (Loxodonta africana) during an episode of hemorrhagic disease (HD) due to elephant endotheliotropic herpesvirus 3B (EEHV3B) utilizing data from complete blood counts, electrophoresis and acute phase protein analysis, and polymerase chain reaction (PCR) of multiple body fluids during and after the clinical episode. The elephant presented with sudden onset of marked lethargy and inappetence followed by hypersalivation, hyperemia of the conjunctivae and focally on the tongue, and swellings on the head and ventrum. A moderate leukocytopenia with band neutrophilia, lymphopenia, monocytopenia, and thrombocytophilia was followed by a rise in all three cell types by day 10. Moderate increases in serum amyloid A and C-reactive protein were noted in the first weeks of illness. Conventional PCR of whole blood yielded a strong positive result for EEHV3B. Quantitative PCR revealed moderate viremia, which slowly returned to undetectable levels by day 35 of treatment. EEHV3B was shed in trunk wash samples starting at day 22 for 10 days at moderate levels, and then at low levels for up to 8.5 mo. All three female herd mates shed low levels of EEHV3B in trunk washes intermittently starting from day 28 of the calf's illness until over 7 mo afterward. The majority of saliva samples from the calf over the 8.5-mo period were also positive for EEHV3B. A subfraction of saliva samples from a female herdmate was positive from days 127-190 following disease onset in the calf. Four elephant gammaherpesviruses were detected sporadically from the calf and female herdmates during this same time period. Treatment was started at the onset of clinical signs and consisted of rectal and oral fluids and oral famciclovir. This is the first case of EEHV3B HD in an elephant species and the first thorough epidemiologic evaluation of EEHV HD in an African elephant.
... However, EEHV1 and EEHV5 have been reported in healthy Asian elephants (Atkins et al., 2013;Sariya et al., 2012;Stanton et al., 2014), and EEHV2, EEHV3, EEHV6, and EEHV7 have been detected in healthy African elephants (Zong et al., 2015). Multiple EEHV infections have been reported in both Asian and African individual elephants, suggesting that infection with one subtype of EEHV does not prevent them from infection with other subtypes of EEHVs (Stanton et al., 2013;Zong et al., 2015). Because EEHVs cannot be cultured, polymerase chain reaction (PCR) or real-time PCRbased methods have been used to detect the virus in whole-blood samples, samples of necropsied tissue, conjunctiva swabs, trunk-wash fluid, trunk swabs, or palate samples (Hardman et al., 2012;Latimer et al., 2011;Sariya et al., 2012;Stanton et al., 2014). ...
... Previous reports have shown that EEHV1 viremia can be found in both clinical and subclinical EEHV1 infection. However, DNA of EEHV1 has been detected up to 28 days before the onset of clinical signs (Stanton et al., 2013). Thus, the detection of EEHVs in whole blood may help veterinarians during the initial treatment of infected elephants before the onset of irreversible disease progression. ...
Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in elephants, especially young captive Asian elephants (Elephas maximus). Currently, seven EEHV types have been reported. In this study, EEHVs were examined in whole-blood samples derived from 56 captive Asian elephants from eight provinces in Thailand by nested PCR using primers specific to the viral DNA polymerase gene in an attempt to monitor EEHV elephant cases. After EEHV testing, one sample (1.78%) was positive and found to be closely related to EEHV4 with 99% amino acid identity. This sample was from a three-year-old female Asian elephant with no clinical signs. These data suggest that asymptomatic EEHV4 infection can occur in Asian elephants.
... This study suggested that EEHV1 was most likely to belong to a group of endogenous viruses that co-evolved with Asian elephants, rather than being transmitted from African elephants (Zachariah et al., 2013). This theory was also supported by the fact that EEHV1 was found to be shed occasionally from adult Asian elephants which showed no clinical signs (Hardman et al., 2012;Stanton et al., 2013), thus adult elephants could potentially play a role as a viral reservoir. ...
... This is suggested that a single locus analysis may not be sufficient to draw a conclusion concerning epidemiological relatedness and multiple gene analysis is needed. EEHV is shed in elephant secretions including those from trunk and conjunctiva (Hardman et al., 2012;Atkins et al., 2013;Stanton et al., 2013), with evidence this shedding pattern is intermittent (Hardman et al., 2012), thus it is likely there is ongoing exposure within a herd. The findings from our study also suggested that disease occurrence is sporadic despite the high likelihood of all animals encountering the virus, and therefore the development of disease following EEHV infection likely depends on the host immune response. ...
... Two days after beginning intravenous administration, the condition was improved drastically, for instance, body temperature returned to normal, and there was a reduction of tongue cyanosis. In addition, the improvement was also followed by a decrease in viral load, which proves the theory of study conducted by Stanton et al. (2013), which states that the dynamics of viral loads in the blood can affect the clinical condition of an elephant with EEHV infection. ...
Elephants are charismatic exotic animals. As the largest land animal on the earth, their maximum weight can reach more than 7 tons and 4 meters in height. Apart from their exoticness, however, elephants, particularly Asian elephants are now losing more than 70% of their habitat. As a result, the International Union for Conservation of Nature (IUCN) has red-listed Asian elephants as the critically endangered animal. Various conservation efforts have been implemented, such as translocation of the elephants, control of poaching, educational campaigns, and research. Nonetheless, over the past two decades, Elephant Endotheliotropic Herpesvirus (EEHV), a newly emerging disease has caused a serious threat to Asian elephants’ health worldwide. The virus causes a rapid, acute, and fatal haemorrhagic disease as the major clinical manifestation in adult Asian elephants and particularly in juvenile elephants in both wild and captive populations. This article provides a literature review regarding the EEHV infection in Asian elephants.
... Low-level viremia observed on qPCR could represent subclinical primary infection, a transient recrudescence of viremia that will not progress to EEHV-HD, or it could represent the early subclinical phase of EEHV-HD that can occur up to 30 d before development of clinical signs. 25 Methods of determining subclinical EEHV-related disease may lead to early intervention with supportive care and antiviral therapy, thus increasing survivorship. 20 The potential for a multiday low-level viremia phase prior to the onset of clinical signs allows for evaluation of the heart for myocardial damage early in the disease process. ...
Cardiac troponin I (cTnI) is specific to myocardial tissue, highly conserved across taxa, and a reliable indicator of myocardial disease in human and veterinary medicine. Biomarkers, like cTnI, may be useful for cardiac evaluation of elephants because the application of other modalities is complicated by the size of the animal. The goal of this study was to establish observed ranges for plasma cTnI in Asian elephants (Elephas maximus) measured by two point-of-care analyzers. Blood was collected from captive juvenile (≤15 yr; n = 9), adult (16-50 yr; n = 42), and geriatric (>50 yr; n = 16) elephants. Following centrifugation, heparinized plasma was stored at 5°C prior to and in between analyses on iSTAT (Abbott Point of Care Inc, Princeton, NJ 08540, USA) and HUBI-QUANpro (Humiasis Co, Ltd, Anyang-si 14042, South Korea) analyzers. With the exception of two results, plasma concentrations of cTnI were below the limit of quantification (LOQ < 0.05 ng/ml) for the HUBI-QUANpro (n = 64), which prohibited comparison between the two analyzers. Observed ranges were determined for plasma cTnI concentrations reported by the iSTAT for the entire population sampled (n = 58; mean 0.011 ng/ml; SD ± 0.013 ng/ml; range 0.00-0.07 ng/ml; 95% CI 0.008-0.015 ng/ml; median 0.01 ng/ml) and with outliers excluded (n = 50; mean 0.007 ng/ml; SD ± 0.007 ng/ml; range 0.00-0.02 ng/ml; 95% CI 0.005-0.009 ng/ml; median 0.01 ng/ml). No significant differences were observed between age classes (P = 0.70) or sexes (P = 0.34). Higher cTnI concentrations were significantly correlated with increasing age (Pearson's r = 0.426; P = 0.002). Future studies are warranted to investigate the diagnostic potential of plasma cTnI in Asian elephants.
... Elephant endotheliotropic herpesvirus (EEHV) is a causative agent of EEHV-hemorrhagic diseases (EEHV-HD) in elephants worldwide (Richman et al., 1999). Several previous reports have determined that the EEHV viral load was found to be higher in the peripheral blood of EEHV-infected cases than in other clinical fluid samples including saliva, feces, trunk wash or urine samples (Common et al., 2021;Stanton et al., 2013). Furthermore, elephant monocytes have been hypothesized that they serve as a vehicle for dissemination of the virus during the EEHV viremic phase (Srivorakul et al., 2019). ...
Elephant endotheliotropic herpesvirus (EEHV) is the causative agent of EEHV-hemorrhagic disease (EEHV-HD) in elephants worldwide. This disease is highly virulent and a predominant cause of fatalities in young Asian elephants. Rapid diagnosis and aggressive therapies have been determined to be a key strategy in the successful treatment of this disease. Herein, we have developed the immunochromatographic strip test for EEHV detection. Accordingly, 31.2 kDa of partial EEHV DNA polymerase (DNApol) protein was expressed in Escherichia coli and used to generate rabbit polyclonal anti-EEHV DNApol antibodies. These were then used to develop an ICS test for EEHV antigen detection using the double-antibody sandwich colloidal gold method. Anti-EEHV DNApol antibodies conjugated with 40 nm colloidal gold solution were used as a detector, while rabbit anti-EEHV DNApol and goat anti-rabbit IgG antibodies immobilized on the nitrocellulose membrane were used as the test and control lines, respectively. The test had a detection limit of 1.25 ×10⁵ viral genome copies (vgc)/mL of EEHV obtained from blood samples. Moreover, no specialized equipment or laboratory infrastructure was required in the administration of this test. This developed ICS test for EEHV antigen detection can be used in field application for the rapid detection of EEHV in resource-limited environments.
... Low-level viremia observed on qPCR could represent subclinical primary infection, a transient recrudescence of viremia that will not progress to EEHV-HD, or it could represent the early subclinical phase of EEHV-HD that can occur up to 30 d before development of clinical signs. 25 Methods of determining subclinical EEHV-related disease may lead to early intervention with supportive care and antiviral therapy, thus increasing survivorship. 20 The potential for a multiday low-level viremia phase prior to the onset of clinical signs allows for evaluation of the heart for myocardial damage early in the disease process. ...
Elephant endotheliotropic herpesvirus (EEHV) is one of the most important causes of mortality in Asian elephants (Elephas maximus). The unusual tropism of EEHV for endothelial cells of capillaries can lead to catastrophic vascular dysfunction, hemorrhage, cardiac damage, and death. Cardiac troponin I (cTnI) is an intracellular protein of cardiomyocytes that is released into circulation in levels directly correlated to the severity of cardiomyocyte damage. The purpose of this study was to assess if cTnI could be used to distinguish when EEHV viremia leads to clinical disease versus subclinical infection. Thirty-seven individual Asian elephants contributed 53 blood samples that were evaluated for EEHV viremia using quantitative polymerase chain reaction and analyzed for cTnI using a high-sensitivity assay. Viremia was categorized as none (24/53), low (< 20,000 vge/ml, 12/53) and high (20,000 vge/ml, 17/53). Seven of the nonviremic samples had detectable cTnI. Nine low-viremia samples were positive for EEHV1 (1A and 1B combined) and lacked a detectable cTnI. Fourteen high-viremia samples were positive for EEHV1 and had detectable cTnI. There was statistical significance between having viremia and having a detectable cTnI value (P = 0.0001), and animals with EEHV1 viremia were more likely to have a positive cTnI value (P = 0.04). The presence of cTnI was associated with the presence of clinical signs, with higher values of cTnI in the presence of clinical signs versus subclinical viremia (P = 0.0001). In addition, four elephants contributed multiple samples from a single viremic event and results displayed a trend of elevation in troponin values with progression of EEHV viremia. The association of EEHV viremia with cTnI suggests these markers might be used in conjunction to help predict when EEHV viremia is likely to progress to EEHV-HD for an individual.
... To ensure adequate expression of gBΔfur731 from recombinant MVA, DF-1 cells and elephant primary endothelial cells were infected with MVA-gBΔfur731 or wtMVA. Immunoblotting using sera from an adult elephant with prior EEHV1 infection [55,56] and known gB immunoreactivity [16] detected a protein with a molecular weight expected for gBΔfur731 in both chicken and elephant cells (Fig 1C). The predicted molecular weight of native unmodified gBΔfur731 is 83.3kDa. ...
Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease (EEHV-HD) in Asian elephants and is the largest cause of death in captive juvenile Asian elephants in North America and Europe. EEHV-HD also has been documented in captive and wild elephants in their natural range countries. A safe and effective vaccine to prevent lethal EEHV infection would significantly improve conservation efforts for this endangered species. Recent studies from our laboratory suggest that EEHV morbidity and mortality are often associated with primary infection. Therefore, we aim to generate a vaccine, particularly for EEHV1 naïve animals, with the goal of preventing lethal EEHV-HD. To address this goal, we generated a Modified Vaccinia Ankara (MVA) recombinant virus expressing a truncated form of glycoprotein B (gBΔfur731) from EEHV1A, the strain associated with the majority of lethal EEHV cases. Vaccination of CD-1 mice with this recombinant virus induced robust antibody and polyfunctional T cell responses significantly above mice inoculated with wild-type MVA. Although the vaccine-induced T cell response was mainly observed in CD8⁺ T cell populations, the CD4⁺ T cell response was also polyfunctional. No adverse responses to vaccination were observed. Overall, our data demonstrates that MVA-gBΔfur731 stimulates robust humoral and cell-mediated responses, supporting its potential translation for use in elephants.
... For example, a latent case in Napoli reactivated during a period of Salmonella septicaemia (F Molenaar, 2021). As it is not yet possible to distinguish between 1a and 1b using qPCR, co-infections may have been concealed or consecutive excretion events may have encompassed sequential infections (Fuery et al., 2020;Stanton et al., 2013Stanton et al., , 2010 ...
Background: Elephant endotheliotropic herpesvirus (EEHV) infections can lead to a clinical haemorrhagic disease (HD) process, which is the primary cause of death in captive Asian elephant (Elephas maximus) calves jeopardising the sustainability of captive populations. Intense monitoring remains the most efficient strategy in disease control since antiviral treatment is lacking and EEHV exhibits a 70% mortality rate within seven days of initial clinical signs. Six known variants infect Asian elephants (1a/b, 4a/b, 5a/b), which can become latent and recrudesce during immunosuppression. Considering the majority (if not all) of adults are seropositive, it is crucial to understand which management strategies promote reactivation to reduce the probability of clinical HD in naïve elephants. This study aimed to assess the effect of Asian elephant herd management on EEHV 1a/b and 4a/b excretion, as evidence of latent infection recrudescence following management events. The objectives were to: (1) describe EEHV excretion among two facilities experiencing varying degrees of management-derived stress (mild/acute versus social/none perceived); (2) determine whether social stress influences EEHV recrudescence odds or relative excretion and, if so, the isolated effect of between and within herd moves; and (3) evaluate recrudescent virological behaviour.
Methods: Two European zoos provided 176 samples coinciding with discrete management contexts: 11 trunk washes and oral swabs were collected amid health exams (mild stress), sedations and euthanasia (acute stress), while 165 trunk and conjunctival swabs were obtained during between and within herd moves (social stress). Between herd moves represents moving elephants away and introducing new individuals, while within describes novel mixing for breeding. RT-qPCR was employed to detect EEHV 1a/b and 4a/b, alongside the housekeeping gene EF-1-ɑ for data normalisation (via ΔCT). Only descriptive statistics were possible for the inter-facility comparison. For objectives 2 and 3, generalised estimating equations (GEEs) were used to determine the influence of management on odds ratios (OR) of EEHV recrudescence and relative viral load. Recrudescent virological behaviour parameters included load volume, excretion onset and duration.
Results: One facility exhibited 16 EEHV 1a/b excretion events, otherwise all samples were negative, limiting an inter-facility comparison. Social stress was associated with a non-significant increase in EEHV detection odds (social stress OR=2.88, 95% CI=0.363-22.8, p=0.317; between herd moves OR=1.5, 95% CI=0.167-13.5, p=0.717). Within herd moves significantly elevated EEHV recrudescence odds (OR=6.86, 95% CI=0.823-57.1, p=0.0751), if a one-week excretion onset delay is incorporated. Viral loads were lower during social stress, however this was comparative to a positive case that may have been excreting unusually high volumes due to a split toenail. Excretion onset ranged from six to 54 days and from 3.59 to 11.09 ΔCTs.
Conclusions: Social stress appeared to promote recrudescence, where within herd moves posed the strongest and only significant influence (p=0.0425). EEHV was not detected at a facility where the elephants are known carriers, demonstrating the importance of longitudinal sampling. This study indicated recrudescent cases should be considered an exposure risk to naïve individuals and show comparable virological behaviour to primary infections. Further research is needed to establish at which threshold EEHV is infectious and quantify the degree of risk imposed.
... There are several limitations associated with this study. As EEHV 1a and 1b are indistinguishable by this qPCR, co-infections may have been concealed or consecutive shedding events may have encompassed sequential infections [24,30,52]. Four of five sampled elephants had previously resided elsewhere; relocation can expose elephants to novel strains [12,13], increasing the odds of co-infection. ...
Haemorrhagic disease associated with elephant endotheliotropic herpesvirus (Elephantid herpesvirus, EEHV) infections is the leading cause of death for Asian elephant (Elephas maximus) calves. This study assessed the effect of captive herd management on EEHV shedding, as evidence of latent infection reactivation, focusing on: (1) the influence of social change on the odds of recrudescence; (2) the respective effects of between and within herd moves; and (3) characteristics of recrudescent viral shedding. Trunk and conjunctival swabs (n = 165) were obtained from six elephants at an EAZA-accredited zoo, collected during a period of social stability, and at times of social change. Longitudinal sampling took place at times of moving two bulls out of the collection and one new bull into an adjacent enclosure to the cow herd (between herd moves), and during a period of mixing this new bull with the cow herd to facilitate mating (within herd moves). Quantitative PCR was employed to detect EEHV 1a/b, 4a/b, and EF–1–α (housekeeping gene). Generalised estimating equations determined EEHV recrudescence odds ratios (OR) and relative viral DNA load. Sixteen EEHV 1a/b shedding events occurred, but no EEHV 4a/b was detected. All management-derived social changes promoted recrudescence (social change OR = 3.27, 95% CI = 0.412–26, p = 0.262; and between herd moves OR = 1.6, 95% CI = 0.178−14.4, p = 0.675), though within herd movements posed the most significant increase of EEHV reactivation odds (OR = 6.86, 95% CI = 0.823−57.1, p = 0.075) and demonstrated the strongest relative influence (post hoc Tukey test p = 0.0425). Shedding onset and magnitude ranged from six to 54 days and from 3.59 to 11.09 ΔCts. Differing challenges are associated with between and within herd movements, which can promote recrudescence and should be considered an exposure risk to naïve elephants.
... Because of the similarity between EEHV3A and EEHV3B E34 proteins, it is unlikely that this biomarker will be sufficient to distinguish antibody responses generated after infection with these individual virus species. In Asian elephants, it has been documented that EEHV-HD caused by EEHV1A is not always sufficient to prevent illness caused by a related species, EEHV1B, and infection with EEHV1B is not sufficient to provide protection from illness caused by EEHV1A (18)(19)(20). EEHV1A and EEHV1B have an average divergence across their genomes of approximately 4.5%. However, the average divergence between EEHV3A and EEHV3B is somewhat greater and approaches 9% (G. ...
Distinct but related species of elephant endotheliotropic herpesviruses (EEHVs) circulate within Asian and African elephant populations. Primary infection with EEHVs endemic among Asian elephants can cause clinical illness and lethal EEHV hemorrhagic disease (EEHV-HD). The degree to which this occurs among African elephants has not been fully established. Recent cases of EEHV-HD caused by the EEHV3 species in African elephants housed in North American zoos has heightened concern about the susceptibility of this elephant species to EEHV-HD. In this study, we utilize the luciferase immunoprecipitation system (LIPS) to generate a serological assay specific for EEHV3 in African elephants by detecting antibodies against the EEHV3 E34 protein. The results showed that the majority of tested elephants from four separate and genetically unrelated herds, including five elephants that survived clinical illness associated with EEHV3, were positive for prior infection with EEHV3. However, African elephants who succumbed to EEHV3-HD were seronegative for EEHV3 prior to lethal infection. This supports the hypothesis that fatal EEHV-HD caused by EEHV3 is associated with primary infection rather than reactivation of latent virus. Lastly, we observed that African elephants, like Asian elephants, acquire abundant anti-EEHV antibodies prenatally and that anti-EEHV3 specific antibodies were either never detected or declined to undetectable levels in those animals that died from lethal disease following EEHV3 infection. IMPORTANCE Prior to 2019, only five cases of clinical disease from EEHV infection among African elephants had been documented. Since 2019, there have been at least seven EEHV-HD cases in North American zoos, resulting in three fatalities, all associated with EEHV3. Evidence is accumulating to suggest that EEHV-associated clinical illness and death among Asian elephants is due to primary infection and may be associated with waning anti-EEHV antibody levels in young elephants. The development of the EEHV3 serological test described in this study enabled us to confirm that similar dynamics may be contributing to EEHV-HD in African elephants. The ability to screen for EEHV immune status in African elephant calves will have a major impact on managing captive African elephant herds and will provide new tools for investigating and understanding EEHV in wild populations.
... 5,14,15 Currently, detection of excretion relies on polymerase chain reaction (PCR)-based methods of detection on samples such as trunk-washes, conjunctival swabs and saliva swabs. 12,13,16,17 Insight into excretion patterns, from an epidemiological viewpoint, identifies strains of circulating virus within a herd and 'at-risk periods' for juveniles. To be able to achieve this non-invasively would increase animal welfare in captive settings. ...
Background: Elephant endotheliotropic herpesvirus (EEHV)-associated haemorrhagic disease (EEHV-HD) is a leading cause of death in Asian elephant calves across the world. Cases of EEHV-HD have been detected in free-living calves through post-mortem examination (PME) indicating the presence of the virus in the wild. In the absence of a non-invasive sampling method, little research into free-living populations has been possible. This study aimed to provide evidence that faeces can be used as a non-invasive sampling method for the detection of EEHV excretion using quantitative poly-merase chain reaction.
Methods: Serial saliva swabs and faecal samples were taken from five captive Asian elephants in Thailand over 12 weeks. To ensure the presence of detectable elephant DNA within the sample, qPCR was run for amplification of the Asian elephant tumour necrosis factor (TNF-α) gene, EEHV1 and EEHV4.
Results: Of 28 sample pairs, seven saliva samples were positive for EEHV, of which two had paired positive faecal samples.
Conclusions: This study presents the first evidence that EEHV is excreted in faeces at detectable levels. This method may in future be used for improved understanding of the epidemiology of EEHV in free-living elephant populations , as well as detection of EEHV excretion in captive herds.
... One notable difference in case 4 was that no hematologic changes or clinical signs were seen until day 3 of the viremia, and the peak whole blood viral count (1.1 3 10 6 VGE/ml on day 5) was substantially higher overall and consistent with levels often associated with fatality in Asian elephants. 1,18 The viral load in all surviving elephants gradually decreased to undetectable levels within 2-3 wk of the onset of illness; however, transient low levels of EEHV2, EEHV3, and EEHV6 DNA are detected regularly in whole blood from these individuals and other subclinically infected elephants within this herd. ...
Acute hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV) infection is well recognized as a major threat to young Asian elephants (Elephas maximus) but has been less frequently documented in African elephants (Loxodonta africana). This report describes five sequential cases of EEHV3A infection in African elephants in managed care at one institution. All elephants developed disease within a 4-mo period. The first two cases were 6.5- and 7.5-yr-old females that presented with depressed mentation, anorexia, hematuria, and diarrhea. Both elephants died within 48-72 hr of the onset of illness despite treatment. Postmortem findings included widespread edema, ascites, and extensive petechiae and ecchymoses on the heart, liver, and spleen and within the gastrointestinal and urogenital tracts. Histologic examination identified disseminated vascular necrosis with edema, hemorrhage, and rare endothelial cell intranuclear inclusions typical of herpesvirus in multiple organs. The third and fourth cases were a 13-yr-old male and a 12-yr-old female that presented with minimal to no clinical signs, but with marked changes in hematologic parameters and high viremia detected by quantitative polymerase chain reaction (qPCR). Both elephants survived the infection with early and aggressive treatment. The fifth case was a 37-yr-old female that presented with lethargy and a decreased appetite. Low viremia was detected by qPCR, and mild to moderate hematologic changes were noted. Early treatment resulted in a successful outcome. This case series documents the first known reports of clinical disease and fatality associated with EEHV3A in African elephants.
... Plusieurs éléphanteaux de zoo asiatiques présentant des symptômes précoces de la maladie, à savoir une langue violette, une léthargie ou des oedèmes au niveau du visage et du cou semblent avoir été sauvés suite un traitement systémique de FCV(Schmitt et al., 2000). Huit éléphants du zoo de Houston ont également été traités au FCV et/ou GCV durant une épidémie à EEHV-1(Stanton et al., 2013). ...
Neuf herpèsvirus sont connus pour infecter les équidés. Parmi eux, l’herpèsvirus équin 1 (HVE-1) induit les formes de la maladie les plus graves. En effet, ce virus provoque des troubles respiratoires, des avortements, des morts néonatales et des troubles nerveux qui mènent souvent à l’euthanasie de l’animal. La prophylaxie, reposant sur les bonnes pratiques sanitaires et la vaccination, demeure le meilleur moyen de lutte contre l’ensemble des herpèsvirus équins. Des vaccins qui réduisent efficacement les troubles respiratoires et la dissémination de l’HVE-1 ont été développés. Cependant, ces derniers ne préviennent pas les avortements et n’ont aucun effet démontré contre la forme nerveuse. De plus, la couverture vaccinale demeure insuffisante en France. Les traitements aux antiviraux constituent donc une approche complémentaire dans la lutte contre l’HVE-1. Cependant, trop peu d’études ont évalué l’effet de molécules contre ce virus, limitant les perspectives d’utilisation. Pour répondre à cette problématique, nous avons développé un protocole de criblage à moyen/haut débit à l’aide de la technologie RTCA xCELLigence®, basée sur la mesure d’impédance cellulaire. Suite au criblage de 2891 molécules, 21 candidats ont été identifiés pour leur efficacité contre l’HVE-1. Parmi ceux-ci, l’aphidicoline, la décitabine, le ganciclovir, l’idoxuridine, le pritelivir et le valganciclovir ont présenté les meilleurs effets. L’activité de ces molécules a été confirmée sur différents modèles cellulaires en présence de différentes souches d’HVE-1. Cette étude a conduit à l’identification et à l’étude du mode d’action d’une nouvelle molécule efficace contre l’HVE-1, la décitabine. Cet analogue de la déoxycitidine a également montré un effet synergique in vitro lorsqu’elle est associée au valganciclovir. Lors de la seconde phase de ce travail, nous avons testé l’efficacité d’un traitement au valganciclovir lors d’un challenge expérimental avec infection par nébulisation d’une souche d’HVE-1 (C2254) récemment isolée au cours de l’épizootie de 2018. Cette étude a permis de démontrer qu’une dose de 6,5 mg/kg de valganciclovir, administrée 2 fois par jour, permettait de maintenir un bon niveau de protection avant la mise en place de la réponse immunitaire humorale. En effet, ce traitement permet de réduire les signes cliniques, l’excrétion virale et la virémie cellulaire induits par l’HVE-1. Ces travaux réalisés in vivo démontrent l’efficacité du valganciclovir contre l’HVE-1 et le criblage réalisé in vitro ouvre de nouvelles perspectives de traitement, en particulier avec des associations de molécules.
... This is the first report to confirm oral acyclovir treatment reduced a high EEHV1A viral load to concentrations below the cut-off level by Day 5, although it increased again on Day 7, which may have been related to detection of dead viruses or viral gene fragments [15], and so should be investigated more in the future. The viral load pattern in our study was similar to that of Stanton [30], where clinical signs were in accordance with the pattern reflecting a true infection. In a previous case [27], successful treatment of EEHV1A was achieved using oral acyclovir (24 mg/kg) for 2 days and then intravenous acyclovir (12 mg/kg) twice a day, with supportive antibiotics (penicillin G), vitamin C, and fluid therapy. ...
Elephant endotheliotropic herpesvirus (EEHV) is a major cause of death in Asian elephant (Elephas maximus) calves. A 2-year, 11-month-old female, captive Asian elephant presented with facial edema and a mild fever. Blood samples were collected and showed EEHV1A positivity with a high viral load by real time PCR. Heterophil toxicity also was reported for the first time in this case. The calf was treated orally with acyclovir, 45 mg/kg tid for 28 days, which reduced the EEHV1A viral load to undetectable levels within 9 days and the calf survived. A successful outcome with oral acyclovir administration provides another and affordable option to treat EEHV hemorrhagic disease in Asian elephants, and one that is easier to administer in untrained calves.
... DNA was extracted from 200 µl whole blood or swab solution using FavorPrep™ Viral Nucleic Acid Extraction Kit (Favorgen Biotech Corp.; Ping-Tung, Taiwan), following the manufacturer instructions. To evaluate the quality of extracted DNA, elephant tumor necrosis factor (TNF) real-time PCR assay was used to determine the presence of elephant genomic DNA, as previously described [29]. DNA extraction was repeated once if the sample was TNF-negative. ...
Elephant endotheliotropic herpesvirus (EEHV) infection is one of the most common diseases in young elephants, causing severe fatal hemorrhagic disease. Subclinical infection was previously described; however, information about the factors associated with virus shedding and reactivation were scarce. To identify the biological and environmental factors related with EEHV detection, blood and oral swab samples were collected from nine captive Asian elephants in Thailand for one year and tested for EEHV presence using real-time PCR. Data including hematological values, management, environmental temperature, and serum cortisol levels were also recorded and analyzed. Results showed that the viral detection frequency ranged from 0–25%. The highest detection frequency was found in the two youngest elephants, aged less than 15 years. Three types of viruses, EEHV1, EEHV4, and EEHV5, were found in this study, which also detected mixed infection in five elephants. Additionally, the study found that sample type, changes in hematological values, management and health issues, and serum cortisol levels were not associated with herpesvirus detection in the elephants. However, EEHV detection percentage was significantly increased in the summer (mid-Feb to mid-May), possibly due to body fitness reduction from food source limitation and low nutrient content. To obtain a broad aspect of EEHV management, long-term EEHV monitoring is highly recommended in every captive elephant herd.
... 6 Circulating viral DNA is detectable at least several days before the appearance of clinical signs, and routine monitoring of blood samples with quantitative polymerase chain reaction (qPCR) of all at-risk calves has been advocated. 1,12 Distinct changes in white blood cell counts (WBC), with a drop in the monocyte : neutrophil ratio and a reduction in the platelet count, have been suggested as useful prognostic indicators and as a way to monitor response to treatment. 2,3 These suggestions have been applied in this case with remarkable results, and findings may provide new insights into the changes associated with EEHV infection. ...
A 3.5-yr-old asymptomatic female Asian elephant (Elephas maximus) with a high load of circulating EEHV1B DNA on qPCR on a routine blood sample, showed progressive depletion of monocytes, lymphocytes, and platelets. Twice daily IV ganciclovir, plasma transfusions, and fluid therapy coincided with a decreasing viral load, which may support potential efficacy of this antiviral drug. An increase in lymphocytes followed initial treatment and preceded the onset of clinical signs. Administration of short-acting glucocorticosteroids for two consecutive days preceded a reduction of lymphocytes, recovery and maturation of monocytes, and gradually decreasing clinical signs, illustrating the potential value of glucocorticosteroids in treatment of clinical EEHV. Three subsequent subclinical episodes with high monocyte and platelet counts did not require intervention. Decision-making was led not just by quantification of viral load and clinical signs, but more specifically by interpretation of the hematological changes using easily accessible, in-house blood smear analysis.
... The LIPS assay is sensitive and specific for EEHV infection through detection of antibodies specific for U39, U28, and U14. To establish the LIPS assay for use in Asian elephants, we assessed responses in two distinct cohorts: (i) adult elephants from a single herd with known exposure to one or more EEHVs (positive trunk washes or prior viremia), making them more likely to be chronically infected and to have an anti-EEHV antibody response (Table 1) (17)(18)(19)(20)(21)(22), and (ii) elephants that died from EEHV-HD (Table 2). Of note, 7 of the 8 EEHV-HD cases investigated in this study were confirmed to have been associated with EEHV1A infection, while the remaining case was caused by EEHV1B (Table 2). ...
Whether clinical illness and deaths associated with elephant endotheliotropic herpesvirus (EEHV) infection result from primary infection or reactivation of latent virus is a longstanding question in the field. By applying a relatively new assay, the luciferase immunoprecipitation system (LIPS), combined with the genomic sequences of the viruses, we gained the insights and tools needed to resolve this issue. Our EEHV1-specific LIPS assay should be useful for assessing the vulnerability of elephant calves to infection with different EEHVs and evaluating antibody responses to anti-EEHV vaccines. A significant proportion of the Asian elephant population is under some form of human care. Hence, the ability to screen for EEHV immune status in elephant calves should have a major impact on the management of these animals worldwide.
... The singleplex EEHV-1, ¾ and 5 real-time polymerase chain reaction (RT-PCR) assays were then performed to test each sample, and all the samples were also tested in a singleplex tumour necrosis factor a (TNFa) as an internal amplification control. The primers and the TaqMan probe for EEHV1, ¾ and 5 used were as described before (Stanton et al., 2012(Stanton et al., , 2013. Each PCR reaction was in 20 ll which contained 4 ll nuclease-free water, 10 ll real-time PCR reaction Master Mix, 1 ll pre-mixed 209 primer/ probe mix and 5 ll sample DNA. ...
A number of methods for measuring the welfare of
elephants in human care have been used within zoological
associations and rescue centres worldwide. The
measurement of glucocorticoids in relation to stress
has been particularly well validated. Measuring stress
is especially important for Asian elephant Elephas
maximus calves between one and 8 years of age which
are highly susceptible to developing elephant endotheliotropic
herpesvirus (EEHV) haemorrhagic disease.
Sleep monitoring has been used as a possible means of
assessing the welfare state of animals, although the
efficacy of this method has not been validated. Our
aim was to test whether sleep time or cortisol provided
the most significant predictor for the onset of positive
EEHV blood viraemia in a 2 year-old calf at Night
Safari, Singapore. Faecal samples were collected twice
per week and assayed for glucocorticoids. Using
closed-circuit television, the time the calf slept each
night was measured between December 2017 and
September 2018. Sleep was not a predictor of viraemia
nor of cortisol concentration in this study. However,
cortisol appeared to be related to the occurrence of viraemia.
... Among recent case reports, viral load of EEHV in Asian elephant whole blood samples during the clinical period was more than 10 3 copies/µl [2,3,15,17]. The detection limit of the LAMP assay using spiked blood samples was 10 1.2 copies/µl and was enough to detect EEHV1 viremia during the clinical period. ...
Elephant endotheliotropic herpesvirus type 1 (EEHV1) is the most important causative agent of an acute fatal hemorrhagic disease in Asian elephants (Elephas maximus). We employed loop-mediated isothermal amplification (LAMP) to develop a rapid and simple detection method for EEHV1 in blood. When used to test 21 clinical samples collected in Japan, the EEHV1 assay correctly identified one positive and 20 negative clinical samples. It was observed that when samples were spiked with synthetic DNA plasmids including EEHV1 polymerase gene, the detection limit of the LAMP assay was 101.2 copies/μl and 100-fold higher than that of conventional PCR. These advantages of the LAMP assay for EEHV1 detection may facilitate better veterinary practices for treating elephants suffering from the acute disease.
... Clinical signs are usually not seen with viremias <10 4 virus genomic equivalents (vge)/mL. 12 The highest level seen in this animal was ~2 × 10 3 vge/mL. The presence of a bar on the lower graph indicates a positive conventional PCR (cPCR) result corresponding to that date. ...
A 2-y-old male Asian elephant (Elephas maximus), with an elevated platelet count (1,100 × 10⁹/L [1,100 × 10³/mm³]), tested positive for elephant endotheliotropic herpesvirus 1A (EEHV-1A) on conventional PCR (cPCR) of EDTA whole blood. No clinical signs were ever reported and no treatment was administered, but low-level viremia persisted for 2.5 y based on results of cPCR and/or real-time PCR (rtPCR). Sequencing confirmed that the EEHV-1A detected was identical at the beginning through the end of the time period. No other elephants in the herd tested positive for EEHV-1 during this time period. Platelet counts remained elevated throughout the viremia and throughout the animal’s life, and direct correlation between the elevated platelet counts and EEHV-1A viremia could not be confirmed. We document long-term, intermittent, low-level viremia of EEHV-1A and provide additional information to consider when determining if treatment is warranted in a case of EEHV infection.
... 6,11 Viral levels in blood were measured using an EEHV1-specific TaqMan real-time PCR, as previously described. 10 The highest viral load was presented in the first collected sample on day 4, with 2.2 3 10 6 VGCs/ml blood, and decreased steadily after that (Fig. 2). A low viremia (2 3 10 2 -2 3 10 3 VGCs/ml) was detectable in blood for 2 wk and dropped to zero by day 33. ...
This article describes the treatment of clinical elephant endotheliotropic herpesvirus (EEHV) infection in a male Asian elephant ( Elephas maximus; approximately 3 yr old), the dynamics of viral load during the active infection, and genetic analysis of the virus. Treatment included injectable acyclovir (12 mg/kg iv, bid), antibiotic, vitamin, and fluids. Quantitative polymerase chain reaction was used to measure the viral levels in blood, which decreased continuously after initiation of intravenous acyclovir. Low levels of virus were detected in the blood for 2 wk, and the virus was undetectable after 1 mo. No complication was observed during the treatment period. This case report suggests that acyclovir, given parenterally, could potentially enhance survival of clinical EEHV-infected individuals.
... Close monitoring of blood, trunk washes, and saliva from apparently healthy adults in several zoo herds has also demonstrated asymptomatic subclinical primary infections with one or more EEHV species in many zoo elephants (11)(12)(13)(17)(18)(19). Characteristic features of these infections involve first a peak of low-to moderate-level viremia detectable in the blood followed, after clearing, by transient shedding in trunk wash secretions for several weeks. ...
Nearly 100 cases of lethal acute hemorrhagic disease in young Asian elephants have been reported worldwide. All tested cases contained high levels of elephant endotheliotropic herpesvirus (EEHV) DNA in pathological blood or tissue samples. Seven known major types of EEHVs have been partially characterized and shown to all belong to the novel Proboscivirus genus. However, the recently determined 206-kb EEHV4 genome proved to represent the prototype of a GC-rich branch virus that is very distinct from the previously published 180-kb EEHV1A, EEHV1B, and EEHV5A genomes, which all fall within an alternative AT-rich branch. Although EEHV4 retains the large family of 7xTM and vGPCR-like genes, six are unique to either just one or the other branch. While both branches display a highly enriched distribution of A and T tracts in intergenic domains, they are generally much larger within the GC-rich branch. Both branches retain the vGCNT1 acetylglucosamine transferase and at least one vOX-2 gene, but the two branches differ by 25 genes overall, with the AT-rich branch encoding a fucosyl transferase (vFUT9) plus two or three more vOX2 proteins and an immunoglobulin-like gene family that are all absent from the GC-rich branch. Several envelope glycoproteins retain only 15 to 20% protein identity or less across the two branches. Finally, the two plausible predicted transcriptional regulatory proteins display no homology at all to those in the alpha-, beta-, or gammaherpesvirus subfamilies. These results reinforce our previous proposal that the probosciviruses should be designated a new subfamily of mammalian herpesviruses.
... However, routine testing between 2011 and 2015 within the most closely monitored U.S. zoo herd (which consists of just eight individual Asian adults and calves) has now also detected episodes in which multiple herdmates underwent sequential mild primary viremic infections with subsequent trunk wash shedding by first EEHV5 (19) and then later EEHV4 (20). Because at least four different EEHV1A strains had been documented in lethal cases at this same facility within the previous 15 years and most of this herd had already been observed to undergo subclinical infections by strains of EEHV1A or EEHV1B or both either several years earlier or later (21)(22)(23), we conclude that it is likely that most Asian elephants eventually become infected with multiple EEHV species and subtypes. The relative timing and order of these primary EEHV infections are expected to have major impacts on the levels of immune protection to disease caused by the others. ...
A novel group of mammalian DNA viruses called elephant endotheliotropic herpesviruses (EEHVs) belonging to the Proboscivirus genus has been associated with nearly 100 cases of highly lethal acute hemorrhagic disease in young Asian elephants worldwide. The complete 180-kb genomes of prototype strains from three AT-rich branch viruses, EEHV1A, EEHV1B, and EEHV5, have been published. However, less than 6 kb of DNA sequence each from EEHV3, EEHV4, and EEHV7 showed them to be a hugely diverged second major branch with GC-rich characteristics. Here, we determined the complete 206-kb genome of EEHV4(Baylor) directly from trunk wash DNA by nextgeneration sequencing and de novo assembly procedures. Among a total of 119 genes with an overall colinear organization similar to those of the AT-rich EEHVs, major features of EEHV4 include a family of 26 paralogous 7xTM and vGPCR-like genes plus 25 novel or missing genes. The genome also contains an unusual distribution of tracts of 5 to 11 successive A or T nucleotides in intergenic domains between the mostly much higher GC content protein coding regions. Furthermore, an extremely high GC-rich bias in the third wobble position of codons clearly delineates the coding regions for many but not all proteins. There are also two novel captured cellular genes, including a C-type lectin (vECTL) and an O-linked acetylglucosamine transferase (vOGT), as well as an unusually large and complex Ori-Lyt dyad symmetry domain. Finally, 30 kb from a second strain proved to include three small chimeric domains, indicating the existence of distinct EEHV4A and EEHV4B subtypes.
Elephant hemorrhagic disease (EHD), caused by several Elephant endotheliotropic herpesviruses (EEHV), represents a frequently lethal syndrome, affecting both captive and free-living elephants. In summer 2022, three young Asian elephants ( Elephas maximus ) succumbed to EHD in a zoo in Switzerland, despite of considerable preventive efforts and early detection of EEHV1A viremia. In this communication, we describe the extent of preventive measures in terms of prior virus detection, active survey of viremia, and antibody status. In the course of the outbreak, the causative virus was concomitantly analyzed and eventually fully sequenced and compared to other EEHV types and strains.
The conclusions from these analyses may be summarized in three points: (1) A previously undetected EEHV1A strain had remained unrecognized among these elephants. Probably, the new virus re-emerged after almost 40 years of latency from one of the oldest elephants in the zoo. (2) While two of the three affected animals had prior immune responses against EEHV1, their strain-specific immunity proved insufficient to prevent EHD. (3) There is an urgent need to develop efficient antiviral drugs and protective vaccines. In particular, ways need to be found to circumvent the present unavailability of appropriate cell cultures and animal models.
Elephant endotheliotropic herpesvirus (EEHV) causes a fatal hemorrhagic disease and is a significant cause of mortality in juvenile Asian elephants. A loop-mediated isothermal amplification (LAMP) method was developed to rapidly diagnose EEHV viremia. However, extracting DNA from whole blood samples to perform LAMP hampers diagnosis in a field setting. Here, we established the Direct-LAMP method, using heparinized plasma without extracting the DNA to speed up and simplify the test. EEHV-positive specimens were tested using the Direct-LAMP. The detection limit was calculated to be 101.3 copies/μL using the mimetic samples, which was almost identical to the value determined in LAMP in which DNA was extracted. Hence, the Direct-LAMP provided a more rapid diagnosis to save, which could prevent elephant deaths.
Hemorrhagic disease (HD) caused by a group of elephant endotheliotropic herpesviruses (EEHV) is one of the leading causes of death for young elephants in human care. These viruses are widespread and typically persist latently in adult elephants with no negative effects; however, in juvenile Asian and more recently young African elephants, the onset of disease can be rapid and the mortality rate high. Measuring biomarkers associated with the immune response could be beneficial to understanding underlying disease processes, as well as the management of infection and HD. The goal of this study was to measure acute phase proteins and cytokines in serum collected from elephants infected with EEHV (13 Asian and 1 African) and compare concentrations according to presence, severity and outcome of disease. Serum amyloid A (SAA) and haptoglobin (HP) were higher in elephants with EEHV viremia than those without; concentrations increased with increasing viral load, and were higher in fatal cases compared to those that survived. In Asian elephants, SAA was also higher during EEHV1 viremia compared to EEHV5. Cytokine concentrations were typically low, and no statistical differences existed between groups. However, in individuals with detectable levels, longitudinal profiles indicated changes in tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) that may reflect an immune response to EEHV infection. However, the overall low concentrations detected using previously validated assays do not support the presence of a ‘cytokine storm’ and suggest more work is needed to understand if sub-optimal immune responses could be involved in disease progression. These results highlight the potential benefit of measuring circulating biomarker concentrations, such as APPs and cytokines, to improve our understanding of EEHV viremia and HD, assist with monitoring the progression of disease and determining the impact of interventions.
Background: Elephant endotheliotropic herpesvirus causes a hemorrhagic disease (EEHV-HD) that is a major cause of death in juvenile Asian elephants with EEHV1 and EEHV4 being the most prevalent.
Aim: To perform a retrospective clinical data analysis.
Methods: Records of a total of 103 cases in Thailand confirmed by polymerase chain reaction (PCR) on blood and/or tissue samples.
Results: The severity of clinical signs varied among EEHV subtypes. EEHV1A was the most prevalent with 58%, followed by EEHV4 with 34%, EEHV1B with 5.8% and EEHV1&4 co-infection with 1.9%. Overall case fatality rate was 66%. When compared among subtypes, 100% case fatality rate was associated with EEHV1&4 co-infection, 83% with EEHV1B, 75% with EEHV1A, and the lowest at 40% for EEHV4. Calves 2-4 years old were in the highest age risk group and exhibited more severe clinical signs with the highest mortality. Majority of cases were found in weaned or trained claves and higher number of cases were observed in rainy season. A gender predilection could not be demonstrated. Severely affected elephants presented with thrombocytopenia, depletion of monocytes, lymphocytes and heterophils, a monocyte:heterophil (M:H) ratio lower than 2.37, hypoproteinemia (both albumin and globulin), severe grade of heterophil toxicity, and low red blood cell (RBC) counts and pack cell volumes (PCV). Survival was not affected by antiviral drug treatment in the severely compromised animals.
Conclusion: Early detection by laboratory testing and aggressive application of therapies comprising of supportive and anti-viral treatment can improve survival outcomes of this disease.
Background
Elephant endotheliotropic herpesvirus causes a hemorrhagic disease (EEHV-HD) that is a major cause of death in juvenile Asian elephants with EEHV1 and EEHV4 being the most prevalent.
Aim
To perform a retrospective clinical data analysis.
Methods
Records of a total of 103 cases in Thailand confirmed by polymerase chain reaction (PCR) on blood and/or tissue samples.
Results
The severity of clinical signs varied among EEHV subtypes. EEHV1A was the most prevalent with 58%, followed by EEHV4 with 34%, EEHV1B with 5.8% and EEHV1&4 co-infection with 1.9%. Overall case fatality rate was 66%. When compared among subtypes, 100% case fatality rate was associated with EEHV1&4 co-infection, 83% with EEHV1B, 75% with EEHV1A, and the lowest at 40% for EEHV4. Calves 2- to 4-year old were in the highest age risk group and exhibited more severe clinical signs with the highest mortality. Majority of cases were found in weaned or trained claves and higher number of cases were observed in rainy season. A gender predilection could not be demonstrated. Severely affected elephants presented with thrombocytopenia, depletion of monocytes, lymphocytes and heterophils, a monocyte:heterophil (M:H) ratio lower than 2.37, hypoproteinemia (both albumin and globulin), severe grade of heterophil toxicity, and low red blood cell counts and pack cell volumes. Survival was not affected by antiviral drug treatment in the severely compromised animals.
Conclusion
Early detection by laboratory testing and aggressive application of therapies comprising of supportive and anti-viral treatment can improve survival outcomes of this disease.
As many as a dozen cases of lethal acute hemorrhagic disease (HD) in young captive-born Sumatran sub-species Asian elephant (Elephas maximus sumatranus roman) calves raised naturally in camps in Sumatra have been observed in recent years. To address whether these deaths, like many others documented worldwide, might be associated with acute systemic infection by elephant endotheliotropic herpesvirus (EEHV), diagnostic polymerase chain reaction (PCR) tests followed by subtype deoxyribonucleic acid (DNA) sequencing analysis were carried out on pathologic tissue samples from two lethal HD cases that occurred within 6 days of one another in calves at the same camp. Viral DNA from five selected PCR loci was found to be present at high levels in both calves and proved to be the same EEHV1A virus species that has been described most commonly previously in numerous lethal or surviving symptomatic cases in North America, Europe, India, and Thailand. Furthermore, the two cases were identical at all five PCR loci tested (covering a total of 3,050 base pairs) and were therefore likely to have been infected from the same epidemiologic source herdmate. However, the strain involved (which was subtype-D2 in the vGPCR1 locus) differed from all previously characterized EEHV1A strains. In conclusion, these two calves are the first two confirmed HD cases in Sumatra alongside several other suspected HD cases in Sumatra that have succumbed to the same devastating EEHV1A-HD that has afflicted young Asian elephants worldwide over the past 25 yr. Because the progeny of some of the 1,500 remaining red-listed critically endangered Sumatra subspecies elephants are bred naturally in camps from wild parents it seems very likely that the EEHV1A herpesvirus identified here in these HD camp cases is also present in the free-ranging Sumatran elephant population, and this will have to be taken into account in future wildlife management policies and decisions.
The elephant endotheliotropic herpesvirus (EEHV) has been a known cause of death of young elephants in Thailand for over a decade. In this study, we report on the demography, disease characteristics and mortality of 58 elephants with confirmed EEHV hemorrhagic disease between January 2006 and August 2018 using retrospective data subjected to survival analysis. Median age of EEHV presentation was 29 months, and the mortality rate was 68.97% with a median survival time of 36 h. Most EEHV cases occurred in the north of Thailand, the region where most of the country’s captive elephants reside. The hazard ratio analysis identified application of medical procedures and antiviral medications as being significant factors correlated to the risk of death. Our results indicate a need to focus EEHV monitoring efforts on young elephants and to follow current protocols that advise starting treatments before clinical signs appear.
Herpesviruses are ubiquitous and are found worldwide, most animal species can be infected with multiple herpesviruses. Some cause clinical disease and others remain symptomatic throughout life. Herpesviruses are found in both captive and wild animals including Asian elephants (Elephas maximus). Elephant Endothelioltropic Herpesvirus (EEHV) has been reported in both captive and wild Asian elephants, with a number of cases being reported in North America, Europe and Asia. It has been suggested that EEHV is associated with haemorrhagic disease, which has been attributed to a number of Asian elephant deaths, affecting mostly juveniles and calves. Clinical signs can vary from weight loss, lethargy, depression, cyanosis of the tongue and sudden death. Molecular testing using qPCR has enabled the detection of individual variants of EEHV, this thesis investigates the EEHV1 variant. EEHV1 has been highlighted as the variant that is more frequently associated with deaths. This thesis includes five studies investigating different aspects of EEHV. Including, the relationship between pregnancy and EEHV viral shedding, the use of an amended human protocol for culturing endothelial cells, EEHV tissue tropism, a potential genetic or familial link between EEHV associated deaths and the detection of potential co-pathogens. The main findings from this thesis include: 1) the use of a longitudinal study investigating a potential link between the physiological stress of pregnancy and EEHV viral shedding. This study suggested there was no link between pregnancy and EEHV viral shedding however other stressors may be involved. 2) Using an amended human umbilical vein endothelial cell protocol, the culture of Asian elephant endothelial cells was successful. The cells from this study may be used in subsequent drug testing and vaccine development. 3) Quantitative PCR was used to determine EEHV1 tropism in tissues from two deaths associated with the virus. Tropism appeared to be for the heart and liver. 4) This thesis provides results from a preliminary study into a potential link between EEHV associated deaths. The data from an Asian elephant genogram shows there is the possibility of a genetic or familial link, which requires further investigation. 5) A number of tissues from deaths associated with EEHV and or death from other causes were investigated for the presence of potential co-pathogens, including the presence of encephalomyocarditis virus (EMCV), using microarray technology. The results indicated there were no co-pathogens present in the tissues. This thesis adds to the current published data, and includes the first known preliminary study investigating a potential genetic link between elephant deaths due to EEHV.
Chelonid herpesvirus 5 (ChHV5), the likely etiologic agent of sea turtle fibropapillomatosis (FP), is predicted to be unevenly distributed within an infected turtle, with productive virus replication and virion shedding occurring in cutaneous tumor keratinocytes. In this study, we measured and compared ChHV5 DNA quantities in tumors, skin, urine, major organs, and nervous tissue samples. These samples were taken from the carcasses of 10 juvenile green turtles (Chelonia mydas) with and without clinical signs of FP that stranded in Florida, USA during 2014. Quantitative polymerase chain reaction (qPCR) for ChHV5 UL30 was used to identify ChHV5 DNA in tumors, skin, heart, kidney, nerves, and urine sampled from 5/5 FP+ and 3/5 FP-free turtles. The most frequently co-occurring sites were cutaneous tumor and kidney (n = 4). Novel data presented here include the identification of ChHV5 DNA in kidney, heart, and nerve samples from three FP-free turtles. These data support candidate nontumored anatomic sites of ChHV5 DNA localization and mobilization during two different disease states that may be involved in the ChHV5 infection cycle.
Received 08 Sep 2016 accepted 17 Apr 2017 revised 27 Mar 2017
Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in Asian and African elephants. There are quantitative real time PCR (qPCR) tests that can detect seven known EEHVs (1A, 1B, 2, 3, 4, 5, and 6) in mucosal secretions, tissue isolates, and blood samples. However, current qPCR tests are unable to distinguish between EEHV 1A and 1B or 3 and 4. To address these inadequacies, new qPCR assays were generated and validated to specifically detect EEHV 1A, 1B, and 4. Each assay demonstrated robust efficiency, a broad linear range, and low intra- and inter-assay variability. Each also proved to be specific for its EEHV target when tested against known banked samples from past EEHV cases. The EEHV1A and 1B assays were then used to characterize an eight-week, low level EEHV1 viremic event in a young Asian elephant. These new tests will allow veterinarians and researchers to pinpoint the specific species causing infection more rapidly. They will also allow veterinarians and elephant keepers to better characterize the EEHV status of each animal within their herd leading to more informed management strategies.
Several different strains of elephant endotheliotropic herpes virus-1 (EEHV-1) have been identified via polymerase chain reaction (PCR) techniques in both African and Asian elephants. EEHV-1 has been identified in both cutaneous lesions in healthy African elephants and fatal cases of hemorrhagic syndrome in Asian elephants. However, until now, no EEHV-1 strain has been identified or associated with otherwise healthy Asian elephants. This article describes recurrent nonendothelial lesions associated with EEHV-1 infection in a herd of Asian elephants not exhibiting fatal hemorrhagic syndrome. Genotypes of EEHV-1 strains, based on viral DNA polymerase and glycoprotein B, associated with fatal hemorrhagic syndrome, were compared to those identified in nonendothelial lesions. The same EEHV-1 genotypes were identified in fatal cases and mucosal lesions in otherwise healthy Asian elephants in this herd. Further studies of the Asian elephant immune system and virologic studies to determine the triggers of tissue tropism are needed before any conclusion can be reached. Elephant endotheliotropic herpes virus, EEHV, herpetic lesions, tropism.
The first herpesviruses described in association with serious elephant disease were referred to as endotheliotropic herpesviruses (EEHV) because of their ability to infect capillary endothelial cells and cause potentially fatal disease. Two related viruses, EEHV1 and EEHV2, have been described based on genetic composition. This report describes the similarities and differences in clinicopathologic features of 2 cases of fatal endotheliotropic herpesvirus infections in Asian elephants caused by a previously unrecognized virus within the betaherpesvirus subfamily. EEHV3 is markedly divergent from the 2 previously studied fatal probosciviruses, based on polymerase chain reaction sequence analysis of 2 segments of the viral genome. In addition to ascites, widespread visceral edema, petechiae, and capillary damage previously reported, important findings with EEHV3 infection were the presence of grossly visible renal medullary hemorrhage, a tropism for larger veins and arteries in various tissues, relatively high density of renal herpetic inclusions, and involvement of the retinal vessels. These findings indicate a less selective organ tropism, and this may confer a higher degree of virulence for EEHV3.
The taxonomy of herpesviruses has been updated by the International Committee on Taxonomy of Viruses (ICTV). The former family Herpesviridae has been split into three families, which have been incorporated into the new order Herpesvirales. The revised family Herpesviridae retains the mammal, bird and reptile viruses, the new family Alloherpesviridae incorporates the fish and frog viruses, and the new family Malacoherpesviridae contains a bivalve virus. Three new genera have been created in the family Herpesviridae, namely Proboscivirus in the subfamily Betaherpesvirinae and Macavirus and Percavirus in the subfamily Gammaherpesvirinae. These genera have been formed by the transfer of species from established genera and the erection of new species, and other new species have been added to some of the established genera. In addition, the names of some nonhuman primate virus species have been changed. The family Alloherpesviridae has been populated by transfer of the genus Ictalurivirus and addition of the new species Cyprinid herpesvirus 3. The family Malacoherpesviridae incorporates the new genus Ostreavirus containing the new species Ostreid herpesvirus 1.
Proliferative cutaneous lesions developed in a herd of captive African elephants (33 from an animal importer in Texas [group 1], and 63 young elephants collected in Zimbabwe [group 2]). Group-1 elephants were purchased 8 months before the arrival of the group-2 elephants. On arrival, 7 group-1 elephants had raised nodular fibrous growths, located predominantly on their trunks. Lesions were not observed in the group-2 elephants until approximately 3 months after they were acquired. Lesions on group-2 elephants began as small focal proliferative growths that regressed or that progressed into large nodular fibrous growths that were similar in appearance to those seen in the group-1 elephants. Lesions at various stages of development were biopsied and examined. Histologically, early lesions were inverted papillomas, with hyperplastic and hypertrophic epithelial cells containing amphoteric intranuclear inclusions in the lesion center. Older, large, nodular fibrous growths were ulcerated and were composed predominantly of a thickened dermis containing fibroblasts, collagen, and a mixed inflammatory cell infiltrate; inclusions were not observed in adjacent epidermal cells. Using a peroxidase-antiperoxidase technique, we did not detect group-specific papillomavirus antigens. Southern blot hybridization analysis of DNA from lesion specimens did not indicate papillomavirus-specific genomes. Electron-microscopically, inclusions consisted of aggregates of virus particles. The particles had electron-dense and electron-lucent cores and were 95 to 103 nm in diameter. Virions developed envelopes from nuclear membranes. Mature particles were seen within the cytoplasm and filled the intercellular spaces. On the basis of size, location, conformation, and envelopment, the particles most closely resembled those of herpesviruses.
A highly fatal hemorrhagic disease has been identified in 10 young Asian and African elephants at North American zoos. In the affected animals there was ultrastructural evidence for herpesvirus-like particles in endothelial cells of the heart, liver, and tongue. Consensus primer polymerase chain reaction combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and another in African elephants. Otherwise healthy African elephants with external herpetic lesions yielded herpesvirus sequences identical to that found in Asian elephants with endothelial disease. This finding suggests that the Asian elephant deaths were caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants. A reciprocal relationship may exist for the African elephant disease.
A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.
Newly discovered, lethal elephant endotheliotropic herpesviruses (EEHV) have been identified in both Asian (Elephas maximus) and African (Loxodonta africana) elephants. Carried by otherwise healthy African elephants they can be fatal mainly for young Asian elephants. Since zoos often harbour both elephant species, we conducted a survey on the presence of EEHV in Asian elephants from 12 European zoos, 3 circuses and 1 Israeli zoo. Here, we demonstrate that all EEHV that have affected Asian elephants so far belong to the EEHV1 group. We also describe the detection and the partial sequencing of an endotheliotropic herpesvirus variant (named EEHV1b) in Asian elephants, being either an EEHV endogenous to Asian elephants or indicating different sources (African elephants) of infection.
Since 1995, 4 suspected cases of Endotheliotropic Elephant Herpes Virus (EEHV) infection, i.e. based on clinical presentation, have occurred in Asia without resulting in epidemic outbreaks as expected. In order to confirm the presence of EEHV on the continent of Asia, viral DNA particles from liver samples of a wild-caught 3-year-old elephant found dead at a Cambodian elephant sanctuary and clinically diagnosed with EEHV, were PCR processed using known EEHV strain primers. The presence of EEHV viral nucleic acids was confirmed and the nucleic acids had a 99% sequence similarity to the U.S.A strain (gene bank locus: AF117265) and 97% sequence similarity to the European strain (gene bank locus: AF354746) assigning this case to the EEHV-1 cluster. More than the confirmation of EEHV on the continent of Asia, is the phylogenic relationship to the USA and European strains with no corresponding contact or transport of USA or European elephants to Asia. Thus, this brings many of the traditional theories into question. Although almost forgotten, this disease is still ramped in captive elephant populations worldwide and continues to devastate particularly the neonatal and weaning-age population. Special attention and continued research are needed specifically in the area of basic virology and epidemiology.
Demographic analysis of the captive Asian elephants in North America indicates that the population is not self-sustaining. First year mortality is nearly 30%, but perhaps more important, the fecundity is extremely low (Mx = 0.01–0.02) throughout the prime reproductive years. Without continued importation or a drastic increase in birth rates, the Asian elephant population in North America will drop to approximately 10 elephants in 50 years and be demographically extinct. Model mortality and fecundity curves needed to establish a self-sustaining Asian elephant population in North America show that fecundity must increase four to eight times the historical rates. Emerging techniques such as artificial insemination may assist in making the goal of a self-sustaining population more realizable by allowing reproduction by the numerous females that do not have access to a male, but other obstacles exist as well. A self-sustaining population will present challenges such as maintaining the significant number of male offspring that will be produced. Importation of young females from documented self-sustaining populations overseas is one option that would alleviate the need for a self-sustaining Asian elephant population in North America and the number of imports per year would be minimal. Zoo Biol 19:299–309, 2000. © 2000 Wiley-Liss, Inc.
To investigate the pathogenesis and transmission of elephant endotheliotropic herpesvirus (EEHV1) by analyzing various elephant fluid samples with a novel EEHV1-specific real-time PCR assay.
5 apparently healthy captive Asian elephants (Elephas maximus) from the same herd.
A real-time PCR assay was developed that specifically detects EEHV1. The assay was used to evaluate paired whole blood and trunk-wash samples obtained from the 5 elephants during a 15-week period. Deoxyribonucleic acid sequencing and viral gene subtyping analysis were performed on trunk-wash DNA preparations that had positive results for EEHV1. Viral gene subtypes were compared with those associated with past fatal cases of herpesvirus-associated disease within the herd.
The PCR assay detected viral DNA to a level of 1,200 copies/mL of whole blood. It was used to detect EEHV1 in trunk secretions of 3 of the 5 elephants surveyed during the 15-week period. Viral gene subtyping analysis identified 2 distinct elephant herpesviruses, 1 of which was identical to the virus associated with a previous fatal case of herpesvirus-associated disease within the herd.
EEHV1 was shed in the trunk secretions of healthy Asian elephants. Trunk secretions may provide a mode of transmission for this virus. Results of this study may be useful for the diagnosis, treatment, and management of EEHV1-associated disease and the overall management of captive elephant populations.
Systemic infections with elephant endotheliotropic herpesviruses (EEHV) cause a rapid onset acute hemorrhagic disease with an 85% mortality rate. More than 60 cases have been confirmed worldwide occurring predominantly in juvenile Asian elephants. Originally, three virus types EEHV1A, EEHV1B and EEHV2 were identified, all members of the Proboscivirus genus within the Betaherpesvirinae. However, four elephant gammaherpesviruses (EGHV) have also been found by DNA PCR approaches in eye and genital secretions of asymptomatic animals, and two more versions of the probosciviruses, EEHV3 and EEHV4, were recently detected in acute hemorrhagic disease cases. To ask whether even more species of elephant herpesviruses may exist, we have developed several new diagnostic DNA PCR assays using multiple round primers in the DNA POL region. These have been used routinely for nearly three years to screen samples submitted to the Elephant Herpesvirus Laboratory for diagnosis of possible cases of EEHV disease in blood and necropsy tissue, as well as in biopsies of other suspicious lesions or growths. Several more cases of EEHV1-associated hemorrhagic disease were confirmed, but in addition, we describe here eleven examples of other known and novel herpesviruses detected and evaluated with these reagents. They include the prototypes of four new elephant herpesviruses, two more within the proboscivirus group EEHV5 and EEHV6, plus two more gammaherpesviruses EGHV3B and EGHV5. We also report initial semi-quantitative PCR assays demonstrating very high viral loads in the blood of the EEHV3 and EEHV4-associated hemorrhagic disease cases.
