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.

Comparison of animal welfare assessment tools and methodologies: need for an effective approach for captive elephants in Asia.

Welfare is a fundamental aspect of animal management and conservation. In light of growing public awareness and welfare concerns about captive elephants, there is an urgent need for comprehensive, globally coordinated efforts for Asian elephants (Elephas maximus) that participate in religious, logging, or tourist activities in range countries where the majority reside, and where welfare issues have been identified but not addressed. This review provides a comparative analysis of available animal assessment tools. Each offers distinct features for assessment that allow institutions to select criteria for specific needs and available resources. Most are applied to general animal welfare assessments, although some are tailored to particular species, including elephants. The tools span diverse formats, from digital to primarily paper-based assessments. Assessments operate at individual and institutional levels and across multiple welfare domains. Methodologies rely on keeper ratings or expert evaluations, incorporate numerical scoring and Likert scales for welfare grading, and encompass inputs including behaviors, health, and physiological indicators. For tourist camp elephants, one challenge is that the tools were developed in zoos, which may or may not have application to non-zoological settings. Digital tools and assessment methodologies such as keeper ratings face logistical challenges when applied across tourist venues. As with any tool, reliability, validity, and repeatability are essential and must address the unique welfare challenges of diverse captive settings. We propose that a holistic, context-specific, evidence-based, and practical tool be developed to ensure high elephant welfare standards in non-zoological facilities throughout Asia.

Physiological changes in captive elephants in northern Thailand as a result of the COVID-19 tourism ban-stress biomarkers.

The international travel ban instituted by the Thai government in March 2020 in response to the COVID-19 pandemic greatly affected how tourist camp elephants were managed, with reductions in exercise opportunities, longer chaining hours, and diminished food provisioning. This study was conducted to determine how those changes affected health and welfare biomarkers in individual elephants over the 2 years of the countrywide lockdown (April 2020-April 2022). Blood and fecal samples were collected from 58 elephants at six camps (monthly in Year 1, quarterly in Year 2) and analyzed for stress biomarkers - fecal glucocorticoid metabolites (fGCM), serum oxidative stress [malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)], and stress leukograms. Overall, fGCM concentrations increased within the first few months and remained higher than pre-COVID levels, as did the H/L ratio, a measure affected by cortisol. Serum 8-OHdG, an indicator of DNA oxidative damage, also increased over time, while monocytosis and lymphopenia further suggested alterations in immune function as a result of stress. By contrast, another marker of oxidative stress, serum MDA, declined, possibly in response to reduced roughage and supplement intake. A notable finding was a seasonal pattern of fGCM that was significantly different from previous studies. Whereas higher fGCM during the rainy season were observed in this study, previously, concentrations were highest during the winter, high tourist season. Thus, ironically, both the presence and absence of tourists have been associated with increased fGCM concentrations, albeit for different reasons. Camp management factors negatively affecting stress outcomes included shorter chain lengths, longer chain hours, lack of exercise, and reduced roughage and supplements. Overall, it was clear that camps struggled to maintain adequate care for elephants during the COVID-19 pandemic, highlighting the importance of tourist income and need for contingency plans to cope with potential future disruptions to tourism.

Characteristics of gut microbiota in captive Asian elephants (Elephas maximus) from infant to elderly.

Gut microbiota play an important role in the health and disease of Asian elephants, however, its characteristics at each stage of life have not been thoroughly investigated in maintaining and regulating health of elephants. This study, therefore, aimed to characterize the profiles of the gut microbiota of captive Asian elephants from infants to the elderly. Gut microbiota were identified by 16S rRNA sequencing from the feces of captive Asian elephants with varying age groups, including infant calves, suckling calves, weaned calves, subadult and adult elephants, and geriatric elephants. The diversity of the gut microbiota was lowest in infants, stable during adulthood, and slightly decreased in the geriatric period. The gut microbiota of the infant elephants was dominated by milk-fermenting taxa including genus Bifidobacterium of family Bifidobacteriaceae together with genus Akkermansia. The fiber-fermenting taxa such as Lachnospiraceae_NK3A20_group were found to be increased in suckling elephants in differential abundance analysis by Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC). The gut microbiota profiles after weaning until the adult period has been uniform as indicated by no significant differences in beta diversity between groups. However, the composition of the gut microbiota was found to change again in geriatric elephants. Understanding of the composition of the gut microbiota of captive Asian elephants at various life stages could be beneficial for promoting good health throughout their lifespan, as well as ensuring the welfare of captive elephants.

Seasonal patterns of oxidative stress markers in captive Asian elephants in Thailand and relationships to elephant endotheliotropic herpesvirus shedding.

Introduction: Oxidative stress refers to an imbalance between oxidant and antioxidant activity and accumulation of reactive oxygen species, which can have detrimental effects on animal health. Annual fluctuations in oxidative stress status can occur, increasing disease susceptibility during certain time periods. However, a full understanding of factors related to oxidative stress in Asian elephants and how to mitigate the negative consequences is lacking. Methods: This study measured six serum oxidative stress markers [reactive oxygen species (ROS), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), albumin, glutathione peroxidase (GPx), and catalase] and two stress markers [serum cortisol and fecal glucocorticoid metabolites (fGCM)] in 23 captive Asian elephants in Thailand over a 12 months period to examine relationships with age and season. Results: Seasonal variations were observed, with several markers exhibiting significantly higher concentrations in the summer (ROS, MDA, 8-OHdG, albumin) and lower values during the rainy/winter seasons (MDA, 8-OHdG, albumin, catalase). By contrast, GPx was the only marker to be highest during the rainy season. For the stress markers, higher fGCM concentrations were noted during the rainy season, which contrasts with earlier studies showing more activity in the winter (tourist season). Positive correlations were found between the temperature-humidity index and ROS, GPx, and fGCM, while a negative correlation was observed with serum albumin. Elephant endotheliotropic herpesvirus (EEHV) shedding events were associated with higher concentrations of ROS and MDA. A moderate negative correlation was observed between 8-OHdG and the PCR threshold cycle of EEHV shedding (Ct), indicating DNA damage may be involved in EEHV shedding in elephants. Discussion: Results revealed significant age and seasonal effects on several oxidative stress markers, indicating those factors should be considered in study design and data interpretation. There also may be physiological adaptations in oxidative stress conditions in relation to environmental changes that could impact health outcomes.

Measures of Oxidative Status Markers in Relation to Age, Sex, and Season in Sick and Healthy Captive Asian Elephants in Thailand.

Oxidative stress is a pathological condition that can have adverse effects on animal health, although little research has been conducted on wildlife species. In this study, blood was collected from captive Asian elephants for the assessment of five serum oxidative status markers (reactive oxygen species (ROS) concentrations; malondialdehyde, MDA; albumin; glutathione peroxidase, GPx; and catalase) in healthy (n = 137) and sick (n = 20) animals. Health problems consisted of weakness, puncture wounds, gastrointestinal distress, eye and musculoskeletal problems, and elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD). Fecal samples were also collected to assess glucocorticoid metabolites (fGCMs) as a measure of stress. All data were analyzed in relation to age, sex, sampling season, and their interactions using generalized linear models, and a correlation matrix was constructed. ROS and serum albumin concentrations exhibited the highest concentrations in aged elephants (>45 years). No sex differences were found for any biomarker. Interactions were observed for age groups and seasons for ROS and catalase, while GPx displayed a significant interaction between sex and season. In pairwise comparisons, significant increases in ROS and catalase were observed in summer, with higher ROS concentrations observed only in the adult female group. Lower catalase activity was exhibited in juvenile males, subadult males, adult females, and aged females compared to subadult and adult elephants (males and females) in winter and the rainy season. There was a positive association between catalase activity and fGCMs (r = 0.23, p < 0.05), and a number of red blood cell parameters were positively associated with several of these biomarkers, suggesting high oxidative and antioxidative activity covary in red cells (p < 0.05). According to health status, elephants with EEHV-HD showed the most significant changes in oxidative stress markers, with MDA, GPx, and catalase being higher and albumin being lower than in healthy elephants. This study provides an analysis of understudied health biomarkers in Asian elephants, which can be used as additional tools for assessing the health condition of this species and suggests age and season may be important factors in data interpretation.

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.

Variability of gait characteristics in lameness elephant.

Lameness has a significant impact not only on the economy but also on elephant welfare. Several gait characteristics are altered to compensate for the discomfort. The traditional approach to detecting lameness has relied on mahout and veterinarian observation. The study aimed to determine how lameness affected the variability of an elephant's gait by using a three-dimensional inertial measurement unit (IMU) with Wi-Fi sensors. Five elephants with lameness, as determined by an experienced veterinarian and two, non-lamed elephants, aged between 58-80 years were included in the study. Gait biomechanics including limb segment motion, obtained from individually gyrometric- and accelero-based parameters and demonstrated as a graphical pattern showing changes in absolute rotation angle over time. The result revealed some character changes in gait kinematics parameters, but it was heterogeneity with an inconclusive pattern. The interlimb coordination could be a part of maintaining the actual locomotion pattern, or it could be a result of the mild degree of lameness for which all of the clients have compensated. This study introduces a new objective method for quantifying gait changes caused by lameness, additional research is required to make this objective more clinically applicable.

Development of an immunochromatographic strip test for antigen detection of elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus (EEHV) is the causative agent of EEHV-hemorrhagic disease (EEHV-HD) in elephants worldwide. This disease is highly virulent and a predominant cause of fatalities in young Asian elephants. Rapid diagnosis and aggressive therapies have been determined to be a key strategy in the successful treatment of this disease. Herein, we have developed the immunochromatographic strip test for EEHV detection. Accordingly, 31.2 kDa of partial EEHV DNA polymerase (DNApol) protein was expressed in Escherichia coli and used to generate rabbit polyclonal anti-EEHV DNApol antibodies. These were then used to develop an ICS test for EEHV antigen detection using the double-antibody sandwich colloidal gold method. Anti-EEHV DNApol antibodies conjugated with 40 nm colloidal gold solution were used as a detector, while rabbit anti-EEHV DNApol and goat anti-rabbit IgG antibodies immobilized on the nitrocellulose membrane were used as the test and control lines, respectively. The test had a detection limit of 1.25 × 105 viral genome copies (vgc)/mL of EEHV obtained from blood samples. Moreover, no specialized equipment or laboratory infrastructure was required in the administration of this test. This developed ICS test for EEHV antigen detection can be used in field application for the rapid detection of EEHV in resource-limited environments.

Physiological changes in captive elephants in Northern Thailand as a result of the COVID-19 tourism ban - muscle, liver, metabolic function, and body condition.

The international travel ban initiated in March 2020 due to the COVID-19 pandemic greatly affected how captive elephants were managed in Thailand. A lack of tourists and associated income meant elephants were chained longer with reduced food provisions, had fewer mahouts, and limited exercise like riding, which likely affected health and welfare. Fifty-eight elephants from six tourist camps were assigned a body condition score (BCS) and blood samples were collected monthly for 2 years during the travel ban to measure: (1) muscle enzymes [creatine kinase (CK), aspartate aminotransferase (AST)]; (2) liver enzymes [aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT)]; (3) lipids [total cholesterol (TC), triglycerides (TG), low (LDL) and high (HDL) density lipoproteins]; and metabolic function [glucose, insulin, fructosamine]. Serum CK concentrations were lower at the end of the study, possibly due to no tourist activities like riding. Changes in liver function included increased AST and ALP, also possibly due to physical inactivity. Feeding less bananas and sugar cane was associated with fewer elephants in the obese category and lower TG concentrations. However, increases in glucose, insulin and fructosamine were observed as local people returned to feed elephants after lifting travel restrictions. In sum, changes in several health biomarkers were observed in association with restricted activities and food resources. Camps need better plans to meet the health and welfare needs of elephants during any future disruptions to the tourism industry, although reduced feeding of sweet treats appeared to have positive effects on body condition and metabolic function.

Effect of the COVID-19 pandemic and international travel ban on elephant tourist camp management in northern Thailand.

The COVID-19 pandemic has had a significant impact on the tourism industry, especially in Thailand. Starting in April 2020, the Thai government banned international travel and all elephant tourist camps closed. A wide variety of management changes were implemented because of the lack of income from tourists. This study surveyed 30 camps that cared for >400 elephants in northern Thailand to obtain information on camp, elephant, and mahout management during the COVID-19 pandemic from April 2020 to 2022 compared to the year before. The survey consisted of questionnaires that interviewed elephant camp owners, managers, veterinarians, and mahouts, and captured information on changes in camp operations, including numbers of tourists, elephants and mahouts, elephant and mahout activities, and veterinary care. Results revealed significant changes in camp structure, elephant work activities and general care. Staff layoffs led to a decrease in the ratio of mahouts to elephants from 1:1 to 1:2. Elephant activities, distance walked, and amounts of food were reduced when compared to pre-COVID-19, while chain hours were increased due to reduced activity. Overall, the COVID-19 crisis altered elephant management significantly, potentially affecting animal welfare resulting from changes in nutrition, health, exercise, and numbers of mahouts. We hope to use these data to develop better management plans and guidelines for elephant camps in Thailand so they can cope with the current and potential imminent pandemics that result in decreased tourism income. A follow-up study will measure health and welfare markers in relation to COVID-19 induced changes to determine if any camps adapted management to still meet elephant health and welfare needs, and could serve as models for responding to future pandemics.

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.

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.

Development of Nonstructural Protein-Based Indirect ELISA to Identify Elephant Endotheliotropic Herpesvirus (EEHV) Infection in Asian Elephants (Elephas maximus).

Disease caused by elephant endotheliotropic herpesvirus (EEHV) infection is the most highly fatal hemorrhagic disease in Asian elephant calves worldwide. To date, adult elephants that have been infected with EEHV have predominantly displayed mild clinical signs, while they are believed to serve as EEHV shedders to other elephants. Hence, the diagnostic tools employed for monitoring EEHV-active infection are considered vitally important. In this study, partial EEHV-DNA polymerase (DNApol) nonstructural proteins (NSPs) were used to detect anti-EEHV antibodies through the use of an in-house indirect enzyme-linked immunosorbent assay (ELISA). The results were then compared to those obtained from a PCR test. In this study, a total of 175 serum samples were collected from Asian elephants living in elephant camps located in Chiang Mai and Lampang Provinces, Thailand. The elephants were aged between 2 and 80 years old. The overall percentages of positive samples by the PCR and EEHV-DNApol ELISA tests were 4% (21/175) and 12% (21/175), respectively. The ELISAs demonstrated values of 77.9% (95% posterior probability interval (PPI) = 52.5-95%) sensitivity and 87.7% (PPI = 82.5-91.9%) specificity, respectively. Accordingly, the sera obtained from the elephants exhibiting no clinical signs of EEHV infection, and those who were negative according to PCR tests, revealed a value of 14% seropositivity for EEHV-DNApol. Our results indicate that these asymptomatic, active EEHV-infected elephants could likely serve as a source of EEHV shedding within elephant herds. Consequently, the developed EEHV-DNApol NSPs-based ELISA test employed in the present study may be of use for routine monitoring and identification of EEHV shedders in elephant herds, and could be an alternative diagnostic tool for EEHV detection in Asian elephants.

Relationship among Serum Progestagens, Cortisol, and Prolactin in Pregnant and Cycling Asian Elephants in Thailand.

The aim of this study was to examine relationships among serum progestagens, cortisol, and prolactin in pregnant and normal cycling Asian elephants living in tourist camps in northern Thailand. Samples were collected twice a month for 22 months from nine elephants. Of those, four were pregnant (24.3 ± 2.9 years of age; range 21−28 years) and five (20.2 ± 9.6 years; range 8−34 years) exhibited normal ovarian cycles based on serum progestagen analyses. Gestation was divided into three periods: 1st (week 1−31), 2nd (week 32−62), and 3rd (week 63 to parturition), while the estrous cycle was divided into the follicular and luteal phases. Serum progestagens were higher during the luteal phase of the cycle (p < 0.003), whereas cortisol and prolactin were similar. In pregnant elephants, there were no differences in serum progestagens or cortisol concentrations across the three gestational periods, whereas prolactin concentrations increased significantly during the 2nd and 3rd periods (p < 0.0001). By contrast, prolactin concentrations in nonpregnant elephants were consistently low throughout the ovarian cycle. In one cycling female, prolactin concentrations were similar to pregnant elephants, perhaps because she was an allomother to two calves. Another cycling female exhibited consistently elevated cortisol concentrations, 5 to 10 times higher than the other elephants. There were no correlations between serum progestagens, cortisol, and prolactin throughout gestation; however, serum progestagens and cortisol were positively related in cycling elephants (r = 0.386, p < 0.001). From our results, there were a number of individual differences in reproductive hormonal patterns, so it is important to develop personalized monitoring programs for each elephant to enhance breeding success and create sustaining captive populations of elephants in Asia.

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.

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.

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.

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.