Characterizations of Elephant Endotheliotropic Herpesvirus Type 1A and 4 Co-Infections in Asian Elephant (Elephas maximus) Calves.

Three cases of double infection with elephant endotheliotropic herpesvirus (EEHV) types 1A and 4 in captive Asian elephants are presented. The first calf was a 4-year-old female that showed initial signs of lethargy and depression. The second calf was a 6-year-old female that displayed signs of depression and diarrhea and died within 48 h of the start of supportive treatment. The third was a 2-year-old male that died suddenly while living with the herd. Necropsies were performed in the first and second elephants, while only a tongue sample was collected from the third calf. EEHV infection was confirmed via quantitative PCR (qPCR) and gene sequencing, revealing double subtypes of EEHV1A and -4 infections. This study describes the hematological and pathological characteristics within the host following double EEHV infection.

Response of elephant peripheral blood mononuclear cells when stimulated with elephant endotheliotropic herpesvirus glycoprotein B (EEHV-gB).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the most highly fatal infectious disease among young Asian elephants. Despite the fact that antiviral therapy has been widely used, its therapeutic outcomes remain uncertain. Additionally, the virus has yet to be successfully cultivated in vitro in the process of develop viral envelope glycoproteins for vaccine design. The present study aims to investigate and evaluate EEHV1A glycoprotein B (gB) antigenic epitopes as potential candidates for further vaccine development. Epitopes of EEHV1A-gB were employed in in silico predictions and designed by using online antigenic predicting tools. Candidate genes were then constructed, transformed and expressed in the E. coli vectors prior to examine their potential for acceleration elephant immune responses in vitro. Elephant peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy juvenile Asian elephants were investigated for their proliferative capability and cytokine responses after being stimulated with EEHV1A-gB epitopes. Exposure of elephant PBMCs to 20 µg/mL of gB for 72 h resulted in a significant proliferation of CD3 + cells when compared with the control group. Furthermore, proliferation of CD3 + cells was associated with a marked up-regulation of cytokine mRNA expression, involving IL-1ß, IL-8, IL-12 and IFN-γ. It remains to be determined whether these candidate EEHV1A-gB epitopes could activate immune responses in animal models or elephants in vivo. Our potentially promising results demonstrate a degree of feasibility for the use of these gB epitopes in expanding EEHV vaccine development.

Dexmedetomidine Effectively Sedates Asian Elephants (Elephas maximus).

This study investigated the sedative effects of dexmedetomidine in Asian elephants. We hypothesized that 2 µg/kg dexmedetomidine would provide sufficient standing sedation. A crossover design study was performed in three Asian elephants. Each elephant was assigned to 1 of 3 treatment groups-1 (D1), 1.5 (D1.5) or 2 (D2) µg/kg dexmedetomidine (intramuscular injection, IM) with a two-week 'washout period' between doses. Elephants were monitored for 120 min. At 120 min (Ta), atipamezole was administered IM. Sedation and responsiveness scores were evaluated. Physiological parameters (pulse rate, respiratory rate, and %SpO2) and clinical observations were monitored during the study and for 3 days post drug administration. D2 provided the longest sedation (approximately 70 min), compared to D1 and D1.5. After Ta, each elephant's sedative stage lessened within 10-15 min without complications. No significant abnormal clinical observations were noted throughout and during the 3-days post study period. These data suggest that a single 2 µg/kg IM dexmedetomidine injection provides sufficient standing sedation for approximately 70 min in Asian elephants.

Assessing Coagulation Parameters in Healthy Asian Elephants (Elephas maximus) from European and Thai Populations.

The Asian elephant population is continuously declining due to several extrinsic reasons in their range countries, but also due to diseases in captive populations worldwide. One of these diseases, the elephant endotheliotropic herpesvirus (EEHV) hemorrhagic disease, is very impactful because it particularly affects Asian elephant calves. It is commonly fatal and presents as an acute and generalized hemorrhagic syndrome. Therefore, having reference values of coagulation parameters, and obtaining such values for diseased animals in a very short time, is of great importance. We analyzed prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentrations using a portable and fast point-of-care analyzer (VetScan Pro) in 127 Asian elephants from Thai camps and European captive herds. We found significantly different PT and aPTT coagulation times between elephants from the two regions, as well as clear differences in fibrinogen concentration. Nevertheless, these alterations were not expected to have biological or clinical implications. We have also sequenced the coagulation factor VII gene of 141 animals to assess the presence of a previously reported hereditary coagulation disorder in Asian elephants and to investigate the presence of other mutations. We did not find the previously reported mutation in our study population. Instead, we discovered the presence of several new single nucleotide polymorphisms, two of them being considered as deleterious by effect prediction software.

Clinical characteristics of elephant endotheliotropic herpesvirus (EEHV) cases in Asian elephants (Elephas maximus) in Thailand during 2006-2019.

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.

A Longitudinal Study of Hematology and Stress Biomarker Profiles in Young Asian Elephants (Elephas Maximus) in Relation to Elephant Endotheliotropic Herpesvirus (EEHV) in Thailand.

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.

Impact of Weight Carriage on Joint Kinematics in Asian Elephants Used for Riding.

BACKGROUND: Elephants in Thailand have changed their roles from working in the logging industry to tourism over the past two decades. In 2020, there were approximately 2700 captive elephants participating in activities such as riding and trekking. During work hours, riding elephants carry one or two people in a saddle on the back with a mahout on the neck several hours a day and over varying terrain. A concern is that this form of riding can cause serious injuries to the musculoskeletal system, although to date there have been no empirical studies to determine the influence of weight carriage on kinematics in elephants. METHODS: Eight Asian elephants from a camp in Chiang Mai Province, Thailand, aged between 21 and 41 years with a mean body mass of 3265 ± 140.2 kg, were evaluated under two conditions: walking at a normal speed without a saddle and with a 15% body mass load (saddle and two persons plus additional weights). Gait kinematics, including the maximal angles of fore- and hindlimb joints, were determined using a novel three-dimensional inertial measurement system with wireless sensors. RESULTS: There were no statistical differences between movement angles and a range of motion of the fore- and hindlimbs, when an additional 15% of body mass was added. CONCLUSION: There is no evidence that carrying a 15% body mass load causes significant changes in elephant gait patterns. Thus, carrying two people in a saddle may have minimal effects on musculoskeletal function. More studies are needed to further test longer durations of riding on different types of terrain to develop appropriate working guidelines for captive elephants. Nevertheless, elephants appear capable of carrying significant amounts of weight on the back without showing signs of physical distress.

Pathogenesis of hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV) in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is an acute fatal disease in elephants. Despite the fact that the underlying pathogenesis of EEHV-HD has been proposed, it remains undetermined as to what mechanisms drive these hemorrhagic and edematous lesions. In the present study, we have investigated and explained the pathogenesis of acute EEHV-HD using blood profiles of EEHV-HD and EEHV-infected cases, hematoxylin and eosin (H&E) stain, special stains, immunohistochemistry, quantitative polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR). It was found that EEHV genomes were predominantly detected in various internal organs of EEHV-HD cases. Damage to endothelial cells, vasculitis and vascular thrombosis of the small blood vessels were also predominantly observed. Increases in platelet endothelial cell adhesion molecules-1 (PECAM-1)- and von Willebrand factor (vWF)-immunolabeling positive cells were significantly noticed in injured blood vessels. The expression of pro-inflammatory cytokine mRNA was significantly up-regulated in EEHV-HD cases when compared to EEHV-negative controls. We have hypothesized that this could be attributed to the systemic inflammation and disruption of small blood vessels, followed by the disseminated intravascular coagulopathy that enhanced hemorrhagic and edematous lesions in EEHV-HD cases. Our findings have brought attention to the potential application of effective preventive and therapeutic protocols to treat EEHV infection in Asian elephants.

Successful treatment of elephant endotheliotropic herpesvirus infection in an Asian elephant (Elephas maximus) calf by oral acyclovir medication: Case report.

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.

Possible roles of monocytes/macrophages in response to elephant endotheliotropic herpesvirus (EEHV) infections in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the primary cause of acute, highly fatal, hemorrhagic diseases in young Asian elephants. Although monocytopenia is frequently observed in EEHV-HD cases, the role monocytes play in EEHV-disease pathogenesis is unknown. This study seeks to explain the responses of monocytes/macrophages in the pathogenesis of EEHV-HD. Samples of blood, frozen tissues, and formalin-fixed, paraffin-embedded (FFPE) tissues from EEHV1A-HD, EEHV4-HD, co-infected EEHV1A and 4-HD, and EEHV-negative calves were analyzed. Peripheral blood mononuclear cells (PBMCs) from the persistent EEHV4-infected and EEHV-negative calves were also studied. The results showed increased infiltration of Iba-1-positive macrophages in the inflamed tissues of the internal organs of elephant calves with EEHV-HD. In addition, cellular apoptosis also increased in the tissues of elephants with EEHV-HD, especially in the PBMCs, compared to the EEHV-negative control. In the PBMCs of persistent EEHV4-infected elephants, cytokine mRNA expression was high, particularly up-regulation of TNF-α and IFN-γ. Moreover, viral particles were observed in the cytoplasm of the persistent EEHV4-infected elephant monocytes. Our study demonstrated for the first time that apoptosis of the PBMCs increased in cases of EEHV-HD. Furthermore, this study showed that monocytes may serve as a vehicle for viral dissemination during EEHV infection in Asian elephants.

Survival analysis of confirmed elephant endotheliotropic herpes virus cases in Thailand from 2006 - 2018.

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.

Effect of time and temperature on stability of progestagens, testosterone and cortisol in Asian elephant blood stored with and without anticoagulant.

The value of biological samples collected in the field is compromised if storage conditions result in analyte degradation, especially in warmer climates like Thailand. We evaluated the effects of time and temperature on immunoactive steroid hormone stability in Asian elephant (Elephas maximus) blood stored with and without an anti-coagulant before centrifugation. For each elephant (5 male, 5 female), whole blood was aliquoted (n = 2 ml each) into 13 red top (without anticoagulant) or purple top (with anticoagulant) tubes. One tube from each treatment was centrifuged immediately and the serum or plasma frozen at -20°C (Time 0, T0). The remaining 12 aliquots were divided into stored temperature groups: 4°C, room temperature (RT, ~22°C), and 37°C, and centrifuged after 6, 24, 48 and 62 h of storage. Serum and plasma concentrations of progestagens in females, testosterone in males and cortisol in both sexes were quantified by validated enzyme immunoassays. Steroid concentration differences from T0 were determined by a randomized complete block ANOVA and Dunnett's tests. The only evidence of hormone degradation was cortisol and testosterone concentrations in serum stored at 37°C. Testosterone concentrations declined by 34% at 48 h and 52% at 62 h, cortisol was decreased by 19% after 48 h and 27% after 62 h at 37°C, respectively. None of the other aliquots displayed significant changes over time at any temperature. In conclusion, steroids appear to be stable in blood for nearly 3 days at room or refrigeration temperatures before centrifugation; steroids in samples with ethylenediaminetetraacetic acid were particularly stable. However, warmer temperatures may negatively affect steroids stored without anti-coagulant, perhaps due to red blood cell metabolism. Thus, under field conditions with no access to cold or freezer temperatures, collection of plasma is a better choice for elephants up to at least 62 h before centrifugation.

SUCCESSFUL TREATMENT OF A CLINICAL ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS INFECTION: THE DYNAMICS OF VIRAL LOAD, GENOTYPE ANALYSIS, AND TREATMENT WITH ACYCLOVIR.

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.

Differential testosterone response to GnRH-induced LH release before and after musth in adult Asian elephant (Elephas maximus) bulls.

Bull elephants exhibit marked increases in testosterone secretion during musth, and studies have shown a heightened sensitivity of the testis to GnRH-stimulated testosterone production in musth compared to nonmusth males. However, activity of the hypothalamo-pituitary-gonadal axis before or soon after musth has not been studied in detail. The aim of this study was to evaluate LH and testosterone responses to GnRH challenge in nine adult Asian elephant (Elephas maximus) bulls during three periods relative to musth: premusth, postmusth, and nonmusth. Bulls were administered 80 µg of a GnRH agonist, and blood was collected before and after injection to monitor serum hormone concentrations. The same bulls were injected with saline 2 weeks before each GnRH challenge and monitored using the same blood collection protocol. All bulls responded to GnRH, but not saline, with an increase in LH and testosterone during all three periods. The mean peak LH (1.76 ± 0.19 ng/mL; P < 0.001) and testosterone (6.71 ± 1.62 ng/mL; P = 0.019) concentrations after GnRH were higher than the respective baselines (0.57 ± 0.07 ng/mL, 3.05 ± 0.60 ng/mL). Although basal- and GnRH-induced LH secretion were similar across the stages, evaluation of the area under the curve in GnRH-treated bulls indicated that the testosterone response was greatest during premusth (2.84 ± 0.76 area units; P = 0.019) compared to postmusth (2.02 ± 0.63 area units), and nonmusth (2.01 ± 0.46 area units). This confirms earlier reports that GnRH stimulates LH release and subsequent testosterone production in bull elephants. Furthermore, although the hypothalamo-pituitary-gonadal axis is active throughout the year, the testis appears to be more responsive to LH in terms of testosterone production in the period leading up to musth, compared to the nonmusth and postmusth periods. This heightened sensitivity, perhaps as a result of LH receptor up-regulation, may prime the testis for maximal testosterone production, leading to the physiological and behavioral changes associated with musth.

Anterior ocular abnormalities of captive Asian elephants (Elephas maximus indicus) in Thailand.

OBJECTIVE: To survey and classify anterior ocular abnormalities in 1478 captive Asian elephants (Elephas maximus indicus) in six regions of Thailand. METHODS: Anterior ocular examination was performed in both eyes (n = 2956) of 1478 elephants selected from the annual health check program involving 2958 animals within six regions of Thailand from January to November 2013. Lesions were described and compared between age and gender. RESULTS: A total of 17.83% (527/2956) of examined eyes from 24.97% (369/1478) of examined elephants had anterior ocular abnormalities. The most common lesions in these examined eyes were frothy ocular discharge (5.85%), corneal edema (5.31%), and conjunctivitis (5.18%). In addition, epiphora, phthisis bulbi, other corneal abnormalities, anterior uveitis, and lens abnormalities were noted. Almost all lesions increased in frequency with age (P < 0.01). CONCLUSIONS: Regular ophthalmic examination in elephants should be included in their annual health check program. Early detection and treatment of any ocular abnormality may avoid the development of subsequent irreversible ocular pathology.