Elephant Endotheliotropic
Herpes Virus-Hemorrhagic
Disease (EEHV-HD)
Compiled by Willem Schaftenaar
History:
EEHV-HD is caused by a delta-herpesvirus. The virus has evolved with the elephant species and is older than the currently living elephant species. Most (if not all) adult elephants are latently infected with EEHV.
Young elephants between 1 and 9 years of age can be susceptable for an often fatal syndrom: EEHV-Hemorrhagic Disease. It is hypothesized that the long half-lifetime of maternal antibodies (EEHV maternal antibodies can circulate for up to 36 months in a calf) protects the calf against developing this syndrome. If this is true, it means that calves need to be exposed to EEHV during the phase in which antibodies are decreasing, but still protecting the calf.
Fatal cases in Asian elephants have been reported over 20 times more than in African elephants. One of the hypotheses is that African elephant are shedding the virus much more frequent than Asian elephants, which offers a better opportunity for the calf to build up a solid immunity during the period that it is still protected by maternal antibodies.
Asian elephants can carry EEHV1a, EEHV1b, EEHV4 and EEHV5, of which EEHV1a has caused the vast majority of the fatal cases.
African elephants can carry EEHV2, EEHV3a, EEHV3b, EEHV6 and EEHV7. EEHV3 has been associated with fatal cases, while the other African EEHV-subtypes are associated with lymphoid nodules in lungs and skin of African elephants. There is one report of a fatal case caused associated with EEHV3 in an Asian elphant.
Like all herpesviruses, EEHV has a latent phase in a so far unknown tissue of the elephant body. For reasons that are not yet known, the virus can be reactivated, probably due to a (temporary) weakening of the elephant's immune system. The virus migrates to the mucous membranes of the mouth, trunk, eyes and the female genital tract. Shedding in semen or mucous membranes of the penis has not yet been reported.
(Maternal) antibodies
Humoral antibodies can be demonstrated by using (still) experimental assays. A fluorescence based assay (Lips assay) has become available in the USA (Fuery, 2020) and an ELISA-based assay in the Netherlands (Hoornweg, 2021). Serological studies using these assays demonstrated that maternal antibodies remain circulating for up to 36 months in elephant calves (Fuery, 2020). These maternal antibodies are transmitted in the uterus. The long period during which they are circulating at high levels in a young elephant, suggests that this species is able to absorb antibodies excreted by the dam in her milk. However, this hypothesis still needs to be proven. EEHV-subspecies and subtype-specificity has been demonstrated for these assays (Hoornweg, 2023). Another finding was that antibodies against EEHV4 were not protective against fatal EEHV1a infections, while antibodies against EEHV1a seem to protect against illness caused by EEHV4 and EEHV5. Hoornweg et al. studied 23 fetal EEHV-HD cases in European zoos and found that all fatalities had low antibody levels against gH/gL of the EEHV (sub)species they succumbed to (Hoornweg, 2024).
During the first 12 months of life, maternal antibodies seem to remain stable at a high level, which seems to protect the calf from developing Hemorrhagic Disease when infected by EEHV. This may explain why clinical EEHV-cases have never been reported below the age of 1 year. This has lead to the hypothesis that young elephants need to be exposed to EEHV while they are still (partly) protected by maternal antibodies. Shedding of EEHV by herd mates is therefore essential for the calf to build up natural immunity. In an elephant that is permanently infected with EEHV, shedding takes place after reactivation of EEHV. In 2 elephant orphanages in Sri Lanka (31 and 93 elephants), all calves had high levels of EEHV-gB antibodies. These 2 institutions never lost a calf to EEHV-HD. This leads to the conclusion that the larger herd sized in these 2 orphanages (compared to zoos increases the likelihood of cantact between EEHV-shedders. Herpes viruses in general can become reactivated during a stressful situation, when the immune system of the host seems to become weaker, possibly under the influence of endogenous glucocorticosteroids. Specific stress inducers that result in EEHV-reactivation are not yet known for elephants. It is tempting to hypothesize that social stress could be one of those factors, as elephants are highly social animals. Zoos generally try to avoid stress situations for their animals, including elephants, especially when there is a young calf in the herd. In the light of the recent findings, the absence of stress might as well work against the development of acquired immunity against EEHV in young calves.
The same hypothesis could be valid for elephants in wild situations: if social stress factors are absent in some of the wild situations (less contact with other herds due to habitat fragmentation, less contact with bulls in musth), reactivation frequency of EEHV may be reduced in (sub)adults, preventing calves younger than 12 months from building up immunity during the crucial time frame when they are still protected by maternal antibodies.
Symptoms and diagnosis:
10-14 days before the elephant shows external symptoms of EEHV-HD, the presence of the virus can be demonstrated in the blood by qPCR (EDTA blood sample). It is important to monitor the presence of EEHV in calves between 1 and 9 years of age on a weekly base. As soon as the presence of EEHV has been confirmed, the number of monocytes and platelets are indicative for the further development of the virus in the days to come. When monocytes and platelets are stable and the viral load remains below 5.000 Viral Genome Equivalents (VGE's)/ml, close observation is required. As soon as the viral load in the blood increases or monocytes or platelets drop, immediate treatment is required.
If the initial viremia has passed unnoticed, the young elephant may display one or more of the following unspecific symptoms: lethargy, reduced appetite, lameness, abnormal sleeping pattern, soft feces. In more advanced cases petechiae are seen on the tongue, edema on the head and front legs and finally cyanosis (purple tongue). Sometimes the severe symptoms are the first ones to be discovered.
Photo: courtesy of Florence Ollivet-Courtois
The most relevant tools needed for the diagnosis of EEHV-HD are: qPCR and total WBC, platelet count and blood smear (manual count of monocyte and heterophyls). The monocyte/heterophil (M/H) ratio is an important prognostic indicator for EEHV-HD. A ratio below 1 is reason for great concern and immediate treatment should be started.
Blood smears are essential for manual differentiation of the white blood cells and recognition of the morphology. Note that the presence band-heterophils in young elephants is a bad sign!
Lactate is an important serum parameter to monitor in a EEHV-HD case. Normal values are between 0--0.11 mmol/L (0-1 mg/dL). Values >0.44 mmol/L (4 mg/dL) are indicative for perfusion problems due to DIC (see below). EEHV-HD patients often have lactate value > 0.22 mmol/L (2 mg/dL) (Wiedner, pers. comm. 2022).
Disseminated Intravascular Coagulopathy (DIC)
One of the main reasons why an EEHV infection can result in severe disease or death is the development of DIC in the young calves that are not sufficiently protected by (maternal) antibodies. DIC is the result of an extreme, derailed immune response when endothelial cells are damaged by the virus. In 2 separate studies the occurrence of DIC has been clearly demonstrated to occur in fatal EEHV-HD (Guntawang, 2021; Perrin, 2021). In the treatment protocol for EEHV-HD, DIC needs to be addressed with priority.
Cytokine storm?
In recent years the question came up whether a cytokine storm (as described by some authors in human hemorrhagic fevers like Ebola and Dengue) plays a role in the development of EEHV-related DIC in elephants. No study so far has demonstrated that this could be true for EEHV as well.
Photo: courtesy African Lion Safari Park
Photo: courtesy Amersfoort Zoo
Pathological findings at necropsy
The most prominent signs of EEHV-HD at necropsy are those that resulted from DIC: hemorrhages in most of the organs, joints and muscles, ranging from petechiae to large hematomas. There may also be a hydro-or hemopericardium.
Hemorrhages in the heart, intestines, brain and liver of a yound elephant that died of EEHV1a-HD. Photos by Arun Zacharia
Treatment of EEHV-HD
Early treatment of EEHV-HD is essential for the survival of the elephant. The list of recommended drugs is shown below. The clinician should not hesitate to administer all these drugs and should even sedate the sick elephant if needed for its treatment. Repetitive sedations have been given to sick calves without negative effects. If butorphanol is used to obtain sedation, it should not be antagonized as it will help to relieve the pain in the patient.
Circulatory support:
Rectal fluids:
Luke-warm water 10-20 ml/kg BW TID or QID, up to every 2 hours
NB: As repeated rectal fluid administrations may be needed, the anus of the elephant may become sensitive to these procedures. Rx: mix 15 ml lidocaine 2% with some lubricant and carefully apply on the anus. Wait for 10 minutes before placing the tube in the rectum for the administration of fluids.
Crystalloids:
IV as a bolus of 0.3-4 ml/kg BW
Whole blood transfusion:
Indicated in case of anemia or severely delayed coagulation. The advantage of whole blood administration lays in the rapid availability: no waiting time for preparing plasma. A practical strategy is to collect sufficient blood from a donor elephant to make it available for whole blood (1-2 L) and save the rest to prepare it for plasma transfusions.
Dosage whole blood transfusion: 1-2 L.
Cross matching needs to be done prior to the transfusion.
Plasma transfusion:
IV bolus of 0.5-2 ml/kg BW (after minor cross matching of donor and recipient blood)
For plasma transfusion in elephants see: Emergency care for elephants clinically ill from Elephant Endotheliotropic Herpes Virus–hemorrhagic disease (EEHV-HD, EAZA Elephant TAG, compiled by Fieke Molenaar (ZSL-Whipsnade zoo), Mads Bertelsen and Kathryn Perrin (Copenhagen zoo), Imke Lueders (GEOLifes), Lauren Howard (Houston zoo), Willem Schaftenaar (vet adv. EAZA Elephant TAG, 9 February 2021)
Plasma is currently considered one of the best supportive therapies to provide, as platelets, clotting factors and potentially protective antibodies can thus be provided. Note that the freezing process activates platelets, which may render them useless at the time of transfusion. Therefore - where possible - freshly collected plasma is preferred. The following should be considered for plasma transfusions:
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If frozen plasma is available, this can be given in an early stage of the disease to save time (despite the activated and spent platelets).
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Blood collection from an adult elephant (plasma donor) should be initiated to provide fresh plasma as soon as possible.
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Cross-matching the donor animals with the recipients, especially if one donor will be used on multiple occasions.
For more information about plasma transfusion: click here
Antiviral treatment:
Several antiviral drugs are routinely used, although none of these have proven to be effective; preliminary studies are suggesting that the TK-gene of EEHV does not make the virus sensitive for the group of “ciclovirs” that is currently used. Famciclovir has been used most frequently, followed by ganciclovir. In the absence of the former antivirals, aciclovir has been given in several cases.
Famciclovir: 15 mg/kg orally or rectally, TID
Aciclovir: 15 mg/kg BID orally, rectally or IV
(Ganciclovir: 5 mg/kg BW BID 5 mg/kg IV, BID, each dose given slowly diluted in 1 liter of NaCl. NB Ganciclovir is not preferred, as it is considered a potential human carcinogen, teratogen, and mutagen)
Antibiotic treatment:
A broad-spectrum antibiotic is recommended as the integrity of the intestinal wall may be disrupted and gut bacteria may leak into the abdominal cavity.
Pain management:
Pain management (opioids, NSAIDs) is recommended if there are clear signs of pain or discomfort
Butorphanol (first choice): 0.008-0.014 mg/kg IM Q 4 hrs
Flunixin: 0.25 to 0.5 mg/kg IM SID
Omeprazole: 0.7 to 1.4 mg/kg PO SID
Anti-inflammatory treatment:
Gluco-corticosteroid drugs are indicated in case of suspicion of DIC.
Dexamethasone: 0.05-0.1 mg/kg IV or IM SID for 1-3 days. Please note that in human medicine DIC (e.g. in Covid-19 cases) is treated with Dexamethasone 0.1mg/kg SID for 7-10 days (https://www.who.int/news-room/q-a-detail/coronavirus-disease-covid-19-dexamethasone#:~:text=Recommendation%201%3AWHO%20strongly,medication%20for%20another%20condition.)
Triamcinolon: 0.067 mg/kg IV SID for 1-3 days
Immunostimulating drugs:
Immunostimulants have been used in one case of EEHV1a-HD: Interferon alpha 2a or 2b (25 mIU/2.5 ml Intron A, Merck or 4.5 mIU/0.5 ml Roferon A, Roche) were administered at 27–33 mIU intramuscularly once a day on days 1–12 then every 48 hours to day 20, administered by dart on days with no treatment session, incomplete delivery on days 8 and 14. Bacterial plasmid DNA in a liposome carrier (Zelnate DNA immunostimulant, Bayer HealthCare LLC) was given to the same elephant (2 ml intramuscularly on days 0, 4, 7 and 12).
It should be noted that the same elephant was also given anti-inflammatory treatment (dexamethasone).
Adjunctive drugs:
Oxygen should always be standby and administered as soon as signs of hypoxemia are seen.
Furosemide (1 mg/kg IM ) has been given occasionally.
Vitamin C, routine used in Asia (dosage depends on product; use equine dose).
Vitamin E (dosage depends on product; use equine dose).
Monitoring the course of the disease:
The serum lactate level gives an indication of the organ perfusion. In EEHV-HD patients, the lactate level is often higher than 2 mmol/L (normal value: 0-1 mmol/L). Rehydration by the fluid administrations will help to decrease an elevated lactate.
Platelet counts during the treatment course are helpfull in evaluating the success of the treatment. The administration of whole blood and plasma will compensate partly the loss of platelets and also provide antibodies if the donor is an adult elephant. It is advisable to make sure that the donor does have antibodies.
Blood pressure: in severe EEHV-case, the blood pressure may decrease or decrease. Fluid administration may help to stabilize the blood pressure. When the patient has a vascular shock, the blood pressure may be low. A fast administered bolus of rectal fluids (0.5-5 ml/kg BW) within 15-30 minutes may help to increase the blood pressure.
Treatment of EEHV-HD
Cross-matching procedure
Based on design elaborated by Houston Zoo, Inc.
Step one: Prepare a 3-5% red cell suspension.
1. Collect blood from both donor and recipient in EDTA.
2. Centrifuge the tube and separate the plasma from the red cells. Save both.
3. Place 1 drop of recipient red cells into a small (2-5 ml) clean test tube.
4. Add approx. 1-2 ml of normal saline to the tube with the red cells (or 1 drop RBC to 40 drops saline)
5. Centrifuge at 2500 RPM for 20 seconds.
6. Remove the supernatant, leaving the red cell button on the bottom.
7. Repeat steps 4-6 three times (for a total of 4 washes).
8. Add 1 drop of newly washed recipient red cells to a new test tube.
9. Add approximately 20-40 drops of saline and mix to suspend the red cells. This should be an approximate 3-5% cell suspension to work with.
Step two: Minor cross-match (for plasma transfusion).
1. Add 1 drop of the recipient’s 3-5% red cell suspension to a labeled test tube. Add 1 drop of the recipient’s 3-5% red cell suspension to another labeled test tube to be used as a control.
2. Add 2 drops of donor plasma or serum to the test tube.
3. Add 2 drops of saline to the control tube.
4. Incubate these tubes at 37oC for 15 minutes.
5. Centrifuge the tubes for 20 seconds at 2500 RPM.
6. Observe the supernatant for signs of haemolysis. If present in the cross-match tube and not the control tube, the match is not compatible. If present in both, start again with a new cell suspension.
7. If no haemolysis, then gently rock the test tube back and forth to re-suspend the cell button. Observe the cell button while rocking the tube and grade for the presence of agglutination. Grade on a 0-4 scale where 0 is no agglutination and 4 is heavy clumping. Record your results.
Step three: Major cross-match (for whole blood transfusion).
1. Add 1 drop of the donor’s 3-5% red cell suspension to a labeled test tube. Add 1 drop of the donor’s 3-5% red cell suspension to another labeled test tube to be used as a control.
2. Add 2 drops of recipient’s plasma or serum to the test tube.
3. Add 2 drops of saline to the control tube.
4. Incubate these tubes at 35-37oC for 15 minutes.
5. Centrifuge the tubes for 20 seconds at 2500 RPM.
6. Observe the supernatant for signs of haemolysis. If present in the cross-match tube and not the control tube, the match is not compatible. If present in both, start again with a new cell suspension.
7. If no haemolysis, then gently rock the test tube back and forth to re-suspend the cell button. Observe the cell button while rocking the tube and grade for the presence of agglutination. Grade on a 0-4 scale where 0 is no agglutination and 4 is heavy clumping. Record your results.
References:
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Fuery, A, Pursell,T., Tan, J, Peng, R, Burbelo, P.D., Hayward, G.S., Ling, P.D.2020. Lethal Hemorrhagic Disease and Clinical Illness Associatedcwith Elephant Endotheliotropic Herpesvirus 1 Are Caused by Primary Infection: Implications for the Detection of Diagnostic Proteins. J. Vir. Volume 94 Issue 3.
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Guntawang T, Sittisak T, Kochagul V. ,Srivorakul S., Photichai K., Boonsri K., Janyamethakul T.,
Boonprasert K., Langkaphin W.5, Chatchote Thitaram C. and Pringproa K. 2021. Pathogenesis of hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV) in Asian elephants (Elephas maximus). Scientific Reports (2021). 11:12998. https://doi.org/10.1038/s41598-021-92393-8 -
Hoornweg TE, Schaftenaar W, Maurer G, van der Doel PB, Molenaar F, Chamour-Galante A, Vercammen F, Rutten V and de Haan CAM. 2021. Elephant Endotheliotropic Herpes Virus is omnipresent in elephants in European zoos and an Asian elephant range country. Viruses 2021, 13, 283. https://doi.org/10.3390/v13020283.
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Hoornweg TE, Perere VP, Karunarathne NS, Schaftenaar W, Mahakapuge AN, Kalupahana AN, Rutten VPMG, de Haan CAM. 2022 . Young elephants in a large herd maintain high levels of elephant endotheliotropic herpesvirus-specific antibodies and do not succumb to fatal haemorrhagic disease. Transboundery and Emerging Diseases 69-5. https://doi.org/10.1111/tbed.14644.
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Hoornweg TE, Schaftenaar W, Rutten VPMG, de Haan CAM. 2024. Low gH/gL (Sub)Species-Specific Antibody Levels Indicate Elephants at Risk of Fatal Elephant Endotheliotropic Herpesvirus Hemorrhagic Disease. Viruses. 2024; 16(2):268. https://doi.org/10.3390/v16020268.
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Howard L.L. & Schaftenaar W. 2017. Elephant Endotheliotropic Herpes Virus. In: Fowler’s Zoo and Wild Animal Medicine Current Therapy, Volume 9.
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Perrin KL, Kristensen AT, Bertelsen MF, Denk D. 2021. Retrospective review of 27 European cases of fatal elephant endotheliotropic herpesvirus‑haemorrhagic disease reveals evidence of disseminated intravascular coagulation. Scientific Reports (2021) 11:14173, https://doi.org/10.1038/s41598-021-93478-0.
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Wissink N. et al. 2018.Using in-house hematology to direct decisionmaking in the successful treatment and monitoring of a clinical and susequently subclinical case of Elephant Endotheliotropic Her Vitus 1B. J. of Zoo and Wildlife Med., 50(2): 498-502
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