Abstract
Elephant endotheliotropic herpes virus (EEHV), a fatal herpes viral infection in young Asian elephant calves has become a concern in elephant camps and breeding centers. EEHV has a very rapid onset and progression and often leads to a fatal outcome. It was first reported from North American zoos; later many cases were reported throughout the world.6 In Nepal, five sporadic cases have been reported since 2002, from which four of the affected calves have died. Only clinical signs and postmortem lesions were the basis for diagnosing herpes in the past. For the EEHV-affected female elephant calf presented in 2012 at the breeding center, recent diagnostic molecular tests were performed. The calf died despite the early supportive treatment with aciclovir (Ovir-800, XL Laboratories Pvt. Ltd., Rajasthan, India; 10 mg/kg PO q 8 h) because famciclovir or ganciclovir were not available. Typical signs and symptoms of classical EEHV infection (lethargy, facial edema, cyanosis of the tongue) were observed before her death. Blood samples collected before death revealed a severe lymphocytopenia, monocytopenia and thrombocytopenia. Histopathologic examination showed presence of basophilic intranuclear inclusion bodies in capillary endothelial cells of various tissues. DNA extracted from tissue samples was analyzed by conventional PCR methods, and DNA sequencing confirmed EEHV type 1A. This is the first confirmed herpesvirus case in Nepal.
Introduction
Elephant endotheliotropic herpesvirus (EEHV) has been a problem in young elephants throughout the world. This disease is highly pathogenic to juveniles from 1–4 y of age and lethal in most of the cases.5 The number of reports of this disease has crossed over 60 with mortality of 85% and death within 24–48 h after the onset of first clinical symptoms of lethargy, facial edema and tongue cyanosis.9 Thus, EEHV poses one of the biggest threats in captive elephant breeding programs.4,9,14,20
Herpesvirus in elephants was first reported in the early 1970s in an African elephant (Loxodonta africana) as inclusion bodies in ultrastructural findings from pulmonary nodules.1,10 Later, the virus was also detected as intranuclear inclusion bodies from cutaneous skin lesions,7 and the virus was also identified in the tissue samples from the dead Asian elephant (Elephas maximus).11 The first index case for the herpesvirus infection was from the National Zoo, Washington, D.C. in 1995 which was studied extensively and then reported in 1996.13 After that, several cases from all over the world were investigated to understand this novel disease. In 1999, this virus was suggested to be specific for elephants, different from other B-herpesvirus and then termed as elephant endotheliotropic herpesvirus (EEHV).14
Phylogenetically, EEHV is classified into family Herpesviridae under the genus Proboscivirus.2 To date, eight different strains of EEHV are known. These include two chimeric variants of EEHV-1 (i.e., EEHV-1A and EEHV-1B), EEHV-2, EEHV-3, EEHV-4, EEHV-5, EEHV-6 and EEHV-7. Their range of pathogenicity and distribution is also variable. For example, the wild African elephants frequently carry four viruses (EEHV-2, EEHV-3, EEHV-6 and EEHV-7) in benign lung nodules and cutaneous nodules, and these elephants are less susceptible to disease severity. On the other hand, EEHV-1 is the most commonly isolated strain in the Asian elephant and most pathogenic both in the wild and captivity.4,5,19
Understanding disease dynamics in EEHV has been a challenge. Laboratory findings of this virus in asymptomatic elephants even for the fatal strain4,16,18 suggests the latency and reactivation characteristics as described for Herpesviridae family viruses,8 but EEHV mechanisms of latency and pathogenesis are expected to be different from other herpesviruses like cytomegaloviruses and roseoloviruses.5 Continued inability of the virus to grow in the cell culture has limited the understanding of its pathophysiology.15 Sporadic detection of EEHV-1 gene in nasal secretions, conjunctival swabs and blood from clinically healthy Asian elephants,4,16,17 wide range of genetic diversity among Indian EEHV-1 cases19 and identification of the first EEHV-4 case in Thailand15 corroborate the ideas of the Asian elephant as a natural host species and the virus could be commonly distributed in host species causing sporadic clinical infection in the young calves. Recent advances in molecular methods have led to a better detection and understanding of EEHV strains. Still the important epidemiological feature like prevalence, exposure and pathogenesis are complex to understand. A study on EEHV-1 viral kinetics and genetics16 has also provided new information on further understanding this disease. A single elephant can be infected with multiple EEHV strains though simultaneous infection with both strains at the same time has not been found. Sterilizing immunity against one subtype does not confer sterilizing immunity against subsequent infection with other strains.
There is no treatment for the latent herpesvirus infection in animals or even in humans. Famciclovir and ganciclovir are used in clinical cases, believing that these can suppress viral replication and cellular damage while the virus is circulating and thus an early detection of EEHV following early treatment could lead to treatment success.6 Recent study on EEHV-1 cases16 showed that viral DNA (i.e., viremia) is detected in elephants before the onset of clinical illness. The viral DNA in blood precedes the detection in trunk washes. Regular monitoring of the herd for viremia and virus in trunk washes could help in making the decision for early treatment before the clinical symptoms appear, especially in young elephants in which the disease is often fatal. Even though these drugs might suppress viremia and widespread organ infection, it probably could not prevent the viral localization in specific organs without any clinical manifestation. So, it was found in these studies16,18 that even after viremia was detected in negligible amounts after successful treatment, viral DNA remained detected in trunk washes in higher levels for a much longer period, and there were also cases where DNA was detected in trunk washes at the point where no viremia was detected in the blood.
Various studies have been done in captive elephants in North American and European zoos, but with fewer studies being done on these viruses in the Asian elephant range countries this limits the understanding about these viruses in these areas. The purpose of this article is to report a case of EEHV infection in a captive-born Asian elephant, which is the first confirmed case of EEHV in Nepal.
Case Study
The following case describes EEHV in a juvenile Asian elephant in Nepal. This female calf was born from a captive dam and wild sire on 9 May 2011 and was a herd member (herd consisted of mature cycling females, gravid females and juveniles with their mother) in the elephant breeding center. She was healthy and playful and had normal body condition and health before she showed herpesvirus symptoms. Mahouts reported her sluggish behavior in the evening on 21 November 2012. The next day in the morning she was accessed for a health examination in which she was inactive, sluggish and reluctant to move and had developed cyanotic spots on the tongue. She was highly suspicious for EEHV infection. An attempt to measure her body weight was not successful so she was estimated by her age at 500 kg. Since Famciclovir and ganciclovir were not available, treatment was initiated with aciclovir as the only alternative, the therapeutic efficiency of which is questionable.6
Treatment was started on 22 November 2012 with aciclovir (Ovir-800, XL Laboratories Pvt. Ltd., Rajasthan, India; 10 mg/kg PO q 8 h), meloxicam (Melonex Power, Intas Pharmaceuticals Ltd; 0.4 mg/kg IM once daily) and ampicillin-dicloxacillin (Moxel-D, Alembic; 5 mg/kg IM once daily) and was continued until she died. Acyclovir was administered per os, putting the tablets inside bananas, three times a day; 10:00 am, 6:00 pm and 2:00 am and she was accepting the treatments. On 23 November, facial, trunk and limb edema was pronounced and she was hypothermic (95.9°F). Heart sounds were not easily audible, and pulse was very feeble. The day before she died, 24 November, severe body trembling and hypothermia (94.2°F) were pronounced at the time she was examined (10:00 a.m.). She was given intravenous fluids after warming them in lukewarm water. 500 ml Rintose (Wockhardt Ltd., India), 500 ml normal saline and 500 ml Ringers lactate were given by placing a 21-gauge IV catheter in her aural vein, glued to the skin. In the evening, she was weak and lying in sternal and lateral recumbency. After some struggling, she could get up and walk, but there was trunk trembling and she moved with difficulty. From 6:00 pm onwards, she was very weak and unable to accept the aciclovir. She died on 25 November at 4:00 a.m.
Hematology examination was performed from a blood sample collected before death. Manual blood cell differentiation and platelet count was performed. The tests revealed low hemoglobin levels (10.7 g/dl), lymphocytopenia (2.88 x 103/µl), monocytopenia (0.45 x 103/µl) and severe thrombocytopenia (53 x 103/µl). Postmortem examination was performed on the day of her death. The tongue had raised, white nodules over the surface and was cyanotic. Extensive hemorrhages were observed in the subcutaneous blood vessels. Generalized serosal hemorrhages were also observed over the peritoneum and muscles. Kidneys were enlarged and severe hemorrhage were observed in intestinal serosa, and uterus. The heart had a burnt appearance with severe involvement of the epicardium. Endocardium was also affected and there was severe edema over the papillary muscles. Histopathologic examination was performed on the formalin-fixed tissue with identification of inclusion bodies in the heart, liver and tongue. Fresh tissue samples were collected in cold box and then deep frozen until further analyzed.
Molecular tests were performed with DNA extracted from frozen whole blood and minced liver tissue using DNeasy Blood & Tissue Kit (Qiagen) as per the manufacturer’s recommended protocol. Conventional PCR was performed and Platinum Blue SuperMix (Invitrogen) was used to provide necessary reagents for PCR. Pan EEHV POL and EEHV1-specific U38 POL primers used in this study were given by Dr. Gary Hayward (Johns Hopkins School of Medicine, Baltimore, MD) for the herpes project of Emily Picciotto (student, Tufts Cummings School of Veterinary Medicine). Pan EEHV POL PCR was performed with forward primer (L1 LGH6710) and reverse primer (R1 LGH6711) while EEHV-1 Specific U38 Pol PCR was performed with forward primer (L1 LGH7446) and reverse primer (R1 LGH7445). The thermocycler program used was as follows: 95°C for 2 minutes, then 45 cycles of 95°C for 40 seconds, 50°C for 45 seconds, 73°C for 1 minute, then final 72°C for 7 minutes.
Laboratory tests showed positive results from both PCR with PAN POL and EEHV-1 specific U38 POL primers. Specific DNA bands could be visualized after gel electrophoresis on 1.5% agarose gel. No second round nested PCR were performed since there were adequate DNA levels for visualization in the first round PCR. Bands detected of the appropriate length were excised using a clean scalpel and DNA fragments were extracted using a Qiaex II Gel Extraction Kit (Qiagen). DNA sequencing confirmed that EEHV-1A was responsible for the death of the calf.
Discussion
Nepal has a captive elephant population of more than 215 individuals distributed in five conservation areas of terai belt. They are involved in tourism activities as well as in the management of protected areas. In addition to elephant tuberculosis as the most problematic disease in captive populations, herpesvirus has now been identified causing fatal infection in juveniles.
EEHV was first confirmed in 2006 to be present in Asia12 and since then, other herpesvirus cases from the Asian elephant range countries have also been reported. Still, studies in these range countries have not been done in as much detail as in the North American and European zoos. Thus, the exact status of Herpesvirus in these range countries is still largely unexplored. The recent finding of the EEHV-1 vast genetic diversity in South Indian EEHV cases encompasses almost all of the overall genetic range of EEHV-1 variants from North America and Europe.19 Still, the North-West population of elephants (captive as well as wild that are closely related to the elephants of Nepal) are not accessed for herpesvirus diagnostics. When considering wild tamed native elephants, elephants gifted from other range countries (e.g., Thailand, Burma, India) and wild bulls involved in captive breeding, these conditions suggest that there could be a vast EEHV diversity with the possibility of EEHV strains in other range countries that enter and get established in the Nepalese elephant population. This situation could be further complicated with superinfection16 of EEHV strains.
Wild, as well as wild-born but captive-raised, juveniles were confirmed with the EEHV in South India19 which suggests that this virus could affect the survivability of juveniles in the wild population. EEHV could be one of the contributing factors for decreasing the range of endangered Asian elephants in most of its range countries and for the crossing of the critical endangered threshold level for this species in Sumatra and Vietnam,3 though the correlation between these two has not yet been made. EEHV spread in range countries could also be aggravated by the trans-boundary migration of elephants; more in countries like Nepal, Bhutan, Bangladesh and Indonesia where the trans-boundary migration population consists of a major fraction of the wild population.
The history of herpesvirus in captive elephants in Nepal dates only a few years back with the first report of herpesvirus symptoms on 6 November 2002. To date, there are five reports of herpesvirus infection in juvenile elephants and only one could be saved by the aggressive use of famciclovir. No such information on sudden death of elephant calves with herpesvirus symptoms was available previously and so was thought to be a novel disease in Asian elephants. The finding of Indian EEHV-1 cases as native to the Asian elephant population19 was in contrast to what was assumed earlier. This is further supported by the report of EEHV-4 in Thailand.15 It is uncertain whether modern diagnostic capability improvements have increased disease identification or if several interacting environmental agents and various other co-factors could be responsible for increasing the susceptibility to infection and the number of cases in juveniles. The first EEHV-1A confirmed case in Nepal showed that this strain could be prevalent, but still it is not known whether the calf had acquired herpesvirus from her mother or from other herd members. It is also uncertain whether the same strains were responsible for the previous herpesvirus case or that completely different strains may be involved. The impact of EEHV infection on the wild population is also unknown. Research in the future should be directed toward exploring these questions. Routine evaluation for herpes viremia in captive herd populations and further investigation in the wild will help to explore the diversity of EEHV in Nepal.
Acknowledgments
The authors would like to acknowledge the support of Mr. Kiran Rijal and Mr Chitra Bahadur Khadka of Chitwan National Park for assisting in the postmortem examination; the help of elephant caretakers and handlers are equally appreciated. National Trust for Nature Conservation contribution and help is also equally appreciated. The molecular dynamics team of CMDN are acknowledged for lab work. Emily Picciotto of Tufts University Cummings School of Veterinary Medicine is specially acknowledged, for initiation of herpesvirus research in Nepal. Simon Long and Dr. Gary Hayward of John Hopkins University are acknowledged for technical advices in carrying out the PCR. Authors would also like to thank Dr. Susan Mikota and Ms. Barbara Vincent of Elephant Care International.
Literature Cited
1. Basson, P.A., R.M. McCully, V. De Vos, E. Young, and S.P. Kruger. 1971. Some parasitic and other natural diseases of the African elephant in the Kruger National Park. Onderstepoort J. Vet. Res. 38: 239–254.
2. Davidson, A.J., R. Eberle, B. Ehlers, G.S. Hayward, D.J. McGeoch, A.C. Minson, P.E. Pellett, B. Roizman, M.J. Studdert, and E. Thiry. 2009. The order Herpesvirales. Arch. Virol. 154: 171–177.
3. Fernando, P., and Pastorini, J. 2011. Range-wise status of Asian elephants. Gajah. 35: 15–20.
4. Hardman, K., A. Dastjerdi, R. Gurrala, A. Routh, M. Banks, F. Steinbach, and T. Bouts. 2012. Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR. Vet. Rec. 170 (8), 205.
5. Hayward, G.S. 2012. Conservation: Clarifying the risk from herpesvirus to captive Asian elephants. Vet. Rec. 170: 202–203.
6. Houston Zoo. 2010. Asian Elephant EEHV Protocol.
7. Jacobson, E.R., J.P. Sundberg, J.M. Gaskin, G.V. Lollias, and M.K. O’Banion. 1986. Cutaneous papillomas associated with a herpesvirus-like infection in a herd of captive African elephants. J. Am. Vet. Med. Assoc. 189: 1075–1078.
8. Knipe, D.M., P.M. Howley, D.E. Griffin, and R.A. Lamb. 2006. Herpesviruses. In: Knipe, D., P. Howley, and W. Kluwer (eds). Field Virology. Vol II. Lippincott Williams & Wilkins. Pp: 2479–2499.
9. Latimer, E., J.C. Zong, S.Y. Heaggans, L.K. Richman, and G.S. Hayward. 2011. Detection and evaluation of novel herpesviruses in routine and pathological samples from Asian and African elephants: identification of two new probosciviruses (EEHV5 and EEHV6) and two new gammaherpesviruses (EGHV3B and EGHV5). Vet. Microbiol. 147: 28–41.
10. McCully, R.M., A.P. Basson, J.G. Pienaar, B.J. Erasmus, and E. Young. 1971. Herpes nodules in the lung of the African elephant (Loxodonta africana). Onderstepoort J. Vet. Res. 38: 225–235.
11. Ossent, P., F. Guscetti, A. E. Metzler, E. M. Lang, A. Rubel, and B. Hauser. 1990. Acute and fetal herpesvirus infection in a young Asian elephant (Elephas maximus). Vet. Pathol. 27: 131–133.
12. Reid, C.E., T.B. Hildebrandt , N. Marx , M. Hunt , N. Thy , J.M. Reynes , W. Schaftenaar, and J. Fickel. 2006. Endotheliotropic elephant herpes virus (EEHV) infection. The first PCR-confirmed fatal case in Asia, Vet. Q. 28: 61–64.
13. Richman, L.K., R.J. Montali, R.C. Cambre, J. Lehnhardt, M.A. Kennedy, S. Kania, and L. Potgieter. 1996. Endothelial inclusion body disease: A newly recognized fatal herpes-like infection in Asian elephants. Proceedings of American Association of Zoo Veterinarians. Mexico. Pp 483–485.
14. Richman, L.K., R.J. Montali, R.L. Garber, M.A. Kennedy, J. Lehnhardt, T. Hildebrandt, D. Schmitt, D. Hardy, D.J. Alcendor, and G.S. Hayward. 1999. Novel endotheliotropic herpesviruses fatal for Asian and African elephants. Science. 283: 1171–1176.
15. Sripiboon, S., P. Tankaew, G. Lungka, and C. Thitaram. 2013. The occurrence of elephant endotheliotropic herpesvirus in captive Asian elephants (Elephas maximus): First case of EEHV4 in Asia. J. Zoo. Wildl. Med. 44:100–104.
16. Stanton, J.J., J.C. Zong, C. Eng, L. Howard, J. Flanagan, M. Stevens, D. Schmitt, E. Weidner, D. Graham, R.E. Junge, M.A. Weber, M. Fischer, A. Mejia, J. Tan, E. Latimer, A. Herron, G.S. Hayward, and P.D. Ling. 2013. Kinetics of viral loads and genotypic analysis of elephant Endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus). J. Zoo. Wildl. Med. 44: 42–54.
17. Stanton, J.J., S.A. Nofs, R. Peng, G.S. Hayward, and P.D. Lings. 2012. Development and validation of quantitative real-time polymerase chain reaction assays to detect elephant endotheliotropic herpesviruses-2, 3, 4, 5, and 6. J. Virol. Methods. 186: 73–77.
18. Stanton, J.J., J.C. Zong, E.Latimer, J.Tan, A.Herron, G.S. Hayward, and P.D. Ling. 2010.
19. Detection of pathogenic elephant endotheliotropic herpesvirus in routine trunk washes from healthy adult Asian elephants (Elephas maximus) by use of a real-time quantitative polymerase chain reaction assay. Am. J. Vet. Res. 71: 925–933.
20. Zachariah, A., J. Zong, S.Y. Long, E.M. Latimer, S.Y. Heaggans, L.K. Richman, and G. Hayward. 2013. Fatal herpesvirus hemorrhagic disease in wild and orphan Asian elephants in Southern India. J. Wildl. Dis. 49: 381–393.
21. Zong, J.C., E. Latimer, S.Y. Heaggans, L.K. Richman, and G.S. Hayward. 2008. Viral gene subtyping of eighteen North American cases of EEHV hemorrhagic disease. Proceedings of the International Elephant Conservation and Research Symposium. Pattaya, Thailand; International Elephant Foundation, Azle, Texas. Pp: 63.