Clinical and Pathological Findings of a Newly Recognized Disease of Elephants
Caused by Endotheliotropic Herpesviruses

Between 1983 and 2000 eight different zoos in North America had eleven different elephants (2 African, 9 Asian) get sick from a Herpesvirus (HV) with most dying. 5 cases were studied initially with 6 cases identified as being related to the HV with similarities in their cases. 2 Asian elephants survived their infection after a 3-4 week therapy with the anti-HV medication famciclovir. New diagnostic methods were developed through the course of these studies to recognize this disease, such as Polymerase Chain Reaction (PCR) testing as an early diagnostic tool. The study also found 2 distinct but similar HV affecting both African elephants (Loxodonta africana), and Asian elephants (Elephas maximus), these two viruses were confirmed as being similar with a PCR test. Materials and Methods
Samples were collected from all the elephants with the HV infection. Evaluation of the most recent cases (1995-1998) included elephants 1-4 and their disease status was evaluated. They usually had physical exams including samples of blood taken for hemograms, bacterial and viral cultures, serological testing, Vitamin E levels, and to determine the famciclovir levels. Elephants 3 and 4 were treated with famciclovir orally or rectally at 500mg/70kg three times a day for 3 to 4 weeks and survived the HV infection Elephants 1 and 2 had autopsies performed with samples were taken from all organs; all samples were stored in paraffin, and studied by light microscopes. Elephant 1 had liver, spleen, and heart samples and fresh serum for tests and other samples, and were frozen at -70C. Pathology material was requested using a survey sent to the participating zoos through the American Zoo and Aquarium Association, also by reviewing the mortality records contained in the Asian and African elephant studbook. No deaths associated with HV infections were found with these records early in the 20th century in North America. Records from over 20 elephants that had similar descriptions to Elephant 1, which was the index case, were obtained. Archival paraffin blocks of lung tissue from healthy wild African elephants reported to have HV inclusion bodies (IB) were also obtained. These tissues were processed similarly to elephants 1 and 2 for light and electron microscopy. All slides were reviewed, all major organs and tissues were examined and data collated PCR test was done on DNA from blood from elephants 3 and 4 with signs of Endotheliotropic HV, and on DNA found in lung tissue from the 3 wild African elephants. Primer sets for the elephant HV terminase and DNA polymerase gene regions were constructed from elephants 1 and 2. PCR testing was done with the product visualized Serological tests from 3 elephants (2 Asian, 1 African) as well as frozen sera from herd mates. The following viruses were tested for bluetongue virus, Epizootic hemorrhagic disease virus of deer, Bovine HV 1, 2, & 4, Malignant catarrhal fever virus, Equine HV, Equine viral arteritis virus, Encephalomyocarditis virus, Equine adenovirus , Pseudorabies virus, Porcine reproductive and respiratory syndrome virus. The elephants Penciclovir level was tested, (Penciclovir is what famciclovir is converted to), from samples drawn several times a day and compared to banked serum samples from prior to treatment. Evaluations for elephants 1-4 onset of disease was very sudden with the course lasting 1-7 days. Symptoms included lethargy, anorexia, mild colic, and edema in the head, neck and limbs. Elephant 1 had a cyanotic (blue) tongue and oral ulcers and died on day 5. Elephant 3 had bluing of the tongue which started at the tip and moved back on the 3rd-4th day. Elephant 4 had swelling and bluing of the tongue on the 4th day. Elephant 2 died on the 3rd day. Elephant 3’s tongue bluing and head/neck edema resolved after 9 days of starting treatment, the elephant also regained strength and resumed eating, Viral levels were checked daily showing levels dropped to a undetectable level by 8 weeks. Pathological findings of elephants 1 and 2 as well as seven of the 20 elephants reviewed had similar lesions attributed to the Endotheliotropic HV; six cases were Asian elephants and one African elephant. Findings include peripheral cardial effusion with hemorrages, bluing, and multiple ulcers found in the mouth, throat and large intestine. Microscopic findings included microhemorrhages throughout the heart and tongue associated with edema, small abnormal appearance of lymphocytes, monocytes and a few neutrophils between muscle cells, mild/moderate hepatic sinusoidal expansion with multifocal subacute inflammation, mild hepatocellular vacuolar degenerative changes. Ulcers found in the laryngeal mucous membranes in elephant 1 were acute, endothelial cells of capillaries in the heart muscle and tongue muscle and within the hepatic sinusoids of the liver had intranuclear viral IB. The endothelial cells with the viral IB were in close association with the microhemorrhages in the heart and tongue. Elephant 1 had almost all bacterial cultures come back negative. Cultures on multiple areas of the gastrointestinal (GI) tract had no excessive bacteria population. Tests of liver and heart also were negative for any known viruses. PCR sequencing of DNA from Elephants 3 and 4 had nearly identical Endotheliotropic HV sequences to some of the deceased Asian elephants but all changes were silent mutations. DNA from wild African elephants contained viral sequences that had 100% protein identity with the HV that was fatal for the two African elephants in the study. Serological studies showed one herd mate of elephant 1 had low antibody titer to bovine HV 4 and some others tested had low titers to malignant catarrhal fever virus and to equine adenovirus. Serosurveys from remaining viruses were negative Levels of penciclovir from elephant 3 varied from low to high depending on the time between Discussion
The extreme nature and high fatality rate in the HV infected elephants is odd for infections compared to HV infections of humans and other animals. A major factor in the epidemiology of the elephant HV is the HV being almost harmless in one species (African elephants) and extremely deadly the other (Asian elephants). This is similar to HV-B which is inactive in Macaca but can be deadly in humans and like malignant catarrhal fever HV which again is inactive in sheep and wildebeest but deadly in cattle and acelaphine antelope. Findings show a probable cross-species transmission of HV from benign lesions in African elephants to Asian elephants. The finding of nearly identical HV sequences in these two species also supports this information The pathogenicity and unusual nature of the lesions in elephant HV infections is the endotheliotropism of these viruses most animal HV are epitheliotropic or have a predilection for nervous tissues, with the target organs in mammals being usually the liver, adrenal glands and brain. The finding of intranuclear IB in the endothelial cells of capillaries in the elephant lesions provides histomorphological evidence of their tropism for these vascular cells. These endothelial IB found in all nine elephants that died of the HV infection are histological hallmarks and may be pathognomonic for this disease syndrome. It was rumored the elephants that had contact with the Swiss HV case had evidence of exposure to bovine HV 2 (BHV2). Due to this report tests were done on the convalescent serum samples from elephant 3 for exposure to or cross reactivity with BHV1, BHV2 and BHV4, all test results were negative though Vitamin E level in elephant 1 was found to be within the normal range prior to death, In earlier cases low vitamin E was considered to be cause death in several of the elephants in the study group due to ecchymotic hemorrages seen at autopsy. The endotheliotropism of the elephant HV may explain the organ tropism of these viruses for the heart, liver, and the tongue. Other instances of endotheliotropism for HV have been reported with varicella-zoster virus (VZV) and human HV-8 (HHV8) Based on these findings the proposed pathogenesis is once the elephant becomes viremic, viral replication in the heart leads to endothelial cell damage resulting in capillary leakage and severe hemorrhage and edema. The magnitude of this damage leads to heart failure either by interruption of the conducting system, intracardiac swelling, or myocardial ischemia. Heart insufficiency may also lead to tongue bluing. The regression of tongue bluing in elephant 3 after treatment shows a process of capillary injury and leaking within the tongue followed by healing as the elephant recovered. The hemorrhage and congestion was shown as the bluing discoloration in elephant 3 and the bluing of the entire tongue of elephant 1. In support of the proposed disease development elephant 3 and 4 were found to be viremic early in the disease, they had all the classic clinical signs and out of the eleven elephants they’re the only two who survived. After several days of treatment when other elephants died, appetite returned, tongue bluing reversed and edema was no longer visible for elephant 3. In elephant 3, its PCR showed decreasing viral load that matched with famciclovir treatment. Elephant 4’s survival after being treated with famciclovir also shows the effectiveness of this medication in the treatment of the elephant HV. Before the identification and description of this HV in captive elephants there were a few reports of HV in some lesions in African elephants but no references to HV in Asian elephants. At the time these lesions were considered with no illness associated with them and no documentation of HV. The virus found in the African elephant lesions is similar with the virus that is deadly for Asian elephants. The same virus was also identified by PCR in biopsies from the vaginal tract of wild African elephants. This HV is now thought to be indigenous HV of African elephants. The patches from time to time will become hyperplastic which can spread the virus to other elephants. The report also indicates that there is a second related HV found in a sample from healthy wild African elephants that has 100% protein identity with the virus that was lethal for the two African elephants in the study. Results show that African elephants can harbor 2 similar HV, one that can cause fatal HV disease in Asian elephants and the other in African elephants. Intensive studies are being down to these two HV to see if they are carried latently or as mild constant infection in African elephants. The virus in the Asian elephant from Switzerland was found to be very similar to the virus for the other 9 Asian elephants from North America and was confirmed to be similar to HV cases for Asian elephants from Germany, Netherlands, and Israel, the effect of the HV on wild African elephants is unknown but no HV illnesses have been found. The data seems to blame the African elephant as the carrier of the viruses that can cause HV in the 2 species; proof of transmission has not been found. More work is required to clarify the carrier status of the African elephant for either of these viruses. Some of the work will depend on cultivating the viruses or by the development of tests so that elephants previously exposed to the HV can be found. Limiting contact between the two may also be an effective step in preventing the spread of the disease.


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