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.

Development of a Protocol for Biomechanical Gait Analysis in Asian Elephants Using the Triaxial Inertial Measurement Unit (IMU).

Gait analysis is a method of gathering quantitative information to assist in determining the cause of abnormal gait for the purpose of making treatment decisions in veterinary medicine. Recent technology has offered the wearable wireless sensor of an inertial measurement unit (IMU) for determining gait parameters. This study proposed the use of a triaxial IMU, comprising an accelerometer, a gyroscope, and a magnetometer, for detecting three-dimensional limb segment motion (XYZ axis) during the gait cycle in Asian elephants. A new algorithm was developed to estimate the kinematic parameter that represents each limb segment of the forelimbs and hindlimbs while walking at a comfortable speed. For future use, this study aimed to create a new prototype of the IMU with a configuration that is tailored to the elephant and apply machine learning in an effort to achieve greater precision.

Pharmacokinetics and analytical determination of acyclovir in Asian elephant calves (Elephas maximus).

A therapeutic regimen that includes antiviral drugs is critical for the survival of Asian elephant (Elephas maximus) calves infected with elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD), with acyclovir showing considerable promise. The purpose of this study was to determine the pharmacokinetics and bioavailability of acyclovir following intravenous (IV) and oral (PO) administration in Asian elephants. A single dose of acyclovir (15 mg/kg, IV or 45 mg/kg, PO) was administered to four healthy elephant calves, with a minimum 2-week washout period between treatments. Serial plasma samples were collected after each injection for acyclovir analysis using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Maximum plasma acyclovir concentrations were 27.02 ± 6.79 µg/mL at 0.94 ± 0.31 h after IV administration, and 1.45 ± 0.20 µg/mL at 3.00 ± 0.70 h after PO administration. The half-life of the elimination phase (T1/2) was 5.84 ± 0.74 and 8.74 ± 2.47 h after IV and PO administration, respectively. After IV administration, acyclovir concentrations were higher than the half-maximal inhibitory concentration (IC50) of those found for herpes simplex virus (HSV) 1 and 2 in humans, and equid alpha herpesvirus-1 (EHV-1) for at least 12 h. By contrast, the bioavailability of oral administration was low, only 6.03 ± 0.87%, so higher doses by that route likely are needed to be effective. Due to the high concentration of plasma acyclovir after IV administration, the dose may need to be adjusted to prevent any negative side effects.

Developing a non-invasive method of detecting elephant endotheliotropic herpesvirus infections using faecal samples.

BACKGROUND: Elephant endotheliotropic herpesvirus (EEHV)-associated haemorrhagic disease (EEHV-HD) is a leading cause of death in Asian elephant calves across the world. Cases of EEHV-HD have been detected in free-living calves through post-mortem examination (PME) indicating the presence of the virus in the wild. In the absence of a non-invasive sampling method, little research into free-living populations has been possible. This study aimed to provide evidence that faeces can be used as a non-invasive sampling method for the detection of EEHV excretion using quantitative polymerase chain reaction. METHODS: Serial saliva swabs and faecal samples were taken from five captive Asian elephants in Thailand over 12 weeks. To ensure the presence of detectable elephant DNA within the sample, qPCR was run for amplification of the Asian elephant tumour necrosis factor (TNF-α) gene, EEHV1 and EEHV4. RESULTS: Of 28 sample pairs, seven saliva samples were positive for EEHV, of which two had paired positive faecal samples. CONCLUSIONS: This study presents the first evidence that EEHV is excreted in faeces at detectable levels. This method may in future be used for improved understanding of the epidemiology of EEHV in free-living elephant populations, as well as detection of EEHV excretion in captive herds.

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.

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.