DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS INFECTION AMONG HEALTHY ASIAN ELEPHANTS (ELEPHAS MAXIMUS) IN SOUTH INDIA
Systemic infections with Elephant Endotheliotropic Herpesviruses (EEHV) cause a rapid onset acute hemorrhagic disease with an 85% mortality rate. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. An ultrasonographic assessment and two biopsies were performed on 39 Asian elephants, and these lymph nodes were classified ultrasonographically as active, inactive or chronically active. Although fatal EEHV1-associated hemorrhagic disease has been reported in range countries, data are lacking regarding the prevalence of subclinical EEHV infections among in situ Asian elephants. In fact, heart attacks and circulatory problems are estimated to cause between 11.4 percent and 20 percent of deaths in non-infant captive elephants. During her 37 years, she was artificially inseminated at least 112 times. These virus types likely initially diverged close to 100 million years ago when the ancestors of modern elephants split from all other placental mammals and then evolved into two major branches with high- or low-G+C content about 35 million years ago.
EEHV was first diagnosed in 1995 (Richman and others 1999). While many strains of EEHV have been identified through PCR, none have been cultured. Angela Fuery , Ph.D., Jie Tan , B.S., RongSheng Peng , B.S., Joseph P. 1, pp. This article describes the first case of EEHV infection in Lao People’s Democratic Republic of a 2.5-yr-old domestic male Asian elephant. Although fatal EEHV1-associated hemorrhagic disease has been reported in range countries, data are lacking regarding the prevalence of subclinical EEHV infections among in situ Asian elephants. S.
Using DNA prepared from trunk washes, we detected EEHV1, EEHV3/4, and EEHV5 at frequencies of 7, 9, and 20% respectively. Molewaterplein 50, 3015 GE Rotterdam, Netherlands (Martina and Osterhaus). In addition, the immune response of Asian elephants to EEHV1 infection has not been described. None of the trunk washes was positive for EEHV2 or 6. This project recognizes that conservation often imposes a significant opportunity cost on local communities, and that these same communities often have skills and experience that can be harnessed to promote conservation. It was used to detect EEHV1 in trunk secretions of 3 of the 5 elephants surveyed during the 15-week period. 1999; Ehlers et al.
2001; Fickel et al. 2001; Garner et al. 2009; Latimer et al. The Rotterdam Zoo in association with the International Elephant Foundation (IEF) would like to invite you to participate in the 2011 Elephant and Rhino Research and Conservation Symposium scheduled for October 10-14, 2011 in Rotterdam, The Netherlands. The probosciviruses most commonly associated with morbidity and mortality in captive Asian (Elephas maximus) elephants are EEHV1A and EEHV1B (Richman et al. 1999; Fickel et al. 2001; Stanton et al.
2013), which account for the majority of fatal cases of herpesvirus-associated hemorrhagic disease examined in detail (Richman and Hayward 2011). Death due to EEHV1 infection is associated with widespread capillary endothelial-cell necrosis resulting in diffuse hemorrhagic disease, subcutaneous edema of the head and proboscis, lameness, and ultimately myocardial failure (Richman et al. 1999, 2000a, b). Death attributable to EEHV1 infection most commonly occurs in prereproductive, subadult Asian elephants (Richman and Hayward 2011). Although EEHV1 is the proboscivirus most commonly associated with pathology, other probosciviruses produce morbidity and mortality in elephants. At least two African elephant calf deaths have been associated with EEHV2 infection (Richman et al. 1999) and EEHV3 and EEHV4 have each been associated with the death of an Asian elephant calf (Latimer et al.
2011). Blood samples collected from an apparently healthy adult Asian elephant gave rise to the discovery of EEHV5 (Latimer et al. Elephant hierarchical ranking was reported by the keepers, and was based on observations of dominant and submissive behaviours seen in interactions between the animals over time. 2013). HCMV pUL69 is a nuclear phosphoprotein that consists of 744-amino-acid residues and has a molecular mass of ∼105 to 116 kDa. 2013). A transient detection of EEHV6 was found in the blood of a 1-yr-old African elephant calf with mild symptoms (Latimer et al.
PMID 17884307. Although the vast majority of reports describing EEHV1-associated deaths have originated from institutions holding captive Asian elephants in North America and Europe, EEHV1 is not exclusive to captive elephant populations. Case reports have emerged from range countries, including nine cases of EEHV1-associated deaths involving camp and free-ranging elephants in South India, as well as the death of a wild-born orphan elephant calf in Cambodia (Reid et al. 2006; Zachariah et al. 2013). These reports described Asian elephants with typical pathologic lesions associated with acute EEHV1 infection and were confirmed by PCR assays specific for EEHV1 DNA. Evidence of EEHV1 infection in South India is particularly concerning because the region holds the largest population of Asian elephants.
Although EEHV1 mortalities have been reported in range countries, the prevalence and impact of proboscivirus infection among camp or free-ranging populations is unknown. Cracknell, Jonathan (2008). Vet J. Journal of General Virology 87 (Pt 10): 2781-2789. Care of geriatric specimens is becoming increasingly common, including such disorders as diabetes, heart failure, chronic arthritis, and neoplasia. 2012). The novel gammaherpesvirus EGHV5 #NAG6 was found in a biopsy DNA sample taken from a papillomatous nodule present inside the trunk of a healthy 27-year-old wild-born male Asian elephant in Ohio in 2007.
The following oligonucleotides were used to PCR amplify the gene: forward primer 5’-ATGATCACAAATGTAAATTTGATGTACGGT-3’; and reverse primer, 5’-CCCACCGGGTTGAGATACT-3’. Although the ability to detect subclinical EEHV infection has improved greatly, the prevalence of subclinical proboscivirus infection among in situ or ex situ elephants has not been fully determined. On the basis of recent reports of subclinical EEHV1 infection of captive Asian elephants in North America and Europe, as well as previous documentation of EEHV1-associated deaths in South India, we hypothesized that EEHV1, and possibly other EEHV species, would be detectable in trunk secretions or conjunctival swabs of Asian elephants in South India. To test this hypothesis, we traveled to the Wildlife Disease Research Laboratory in Kerala, India and used previously published methodology and qPCR assays to perform a cross-sectional study in which DNA prepared from trunk washes and conjunctival swabs, collected from three geographically distinct cohorts of Asian elephants in South India, were screened for the presence or absence of EEHV1, EEHV2, EEHV3/4, EEHV5, and EEHV6 DNA. These studies were conducted during October and November 2011 with the approval and participation of the Department of Forest and Wildlife in Kerala and Tamil Nadu, India. In the preceding accompanying paper (10), we reported an analysis of the results of extensive Sanger DNA sequencing that generated a total of 378 kb of EEHV genomic DNA sequence derived directly from pathological necropsy tissue samples from eight different elephants that suffered from fatal acute EEHV-associated hemorrhagic disease. 1).
Figure 1.Map of South India showing the locations of the three elephant cohorts included in this study: Mudumalai National Park; Guruvayur (Guruvayur Sri Krishna Temple); and Kodanad (Kodanad Elephant Orphanage). The Wildlife Disease Research Laboratory is located in Sultan Bathery. Forty-six elephants were included in the study. The age range of the group was 3 mo to 72 yr and the median age was 35 yr. The cohort includes elephants that lived primarily in captive situations, such as the Guruvayur Temple elephants and juvenile orphan calves in Kodanad, to elephants that interacted extensively with free-ranging elephants such as the Kumki elephants at the elephant camp at Mudumalai National Park. All the elephants included in this study were routinely under human care at some point. Trunk washes were collected and processed as described by Stanton et al.
(2010). Trunk-wash samples were stored at 4 C until processed for DNA purification. Conjunctival swabs were collected using sterile cotton-tipped applicators placed in the conjunctival sac of one eye of an elephant, then stored in 1 mL of sterile saline solution at 4 C until processed for DNA using a commercially available DNA purification kit and the manufacturer’s recommended protocol (DNeasy Blood and Tissue Kit, Qiagen Inc., Valencia, California, USA). Following trunk-wash DNA preparation, 5 of 60 µL from each DNA preparation (n = 46) were screened using five independent qPCR assays: Asian elephant tumor necrosis factor-alpha (TNF); EEHV1; EEHV2; EEHV3/4; EEHV5; and EEHV6 (Stanton et al. 2012, 2013). The TNF assay detects Asian elephant genomic DNA and is included as an internal PCR amplification control to determine if samples contain amplifiable elephant genomic DNA (Stanton et al. 2013).
As previously described, a single qPCR assay is used to detect a sequence of the EEHV3 and EEHV4 terminase gene that is 100% identical (Stanton et al. 2012). This assay will be referred to as the EEHV3/4 assay. All qPCR assays were performed using PCR primers, 5′-hydrolysis probes, and qPCR reaction reagents as previously described (Stanton et al. 2010, 2012). All qPCR assays were performed in duplex format.