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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.

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.

Illegal wildlife trade is a major threat to global biodiversity. Asian elephants (Elephas maximus) are highly valued by various cultures as religious symbols and tourist attractions, which has led to a high demand for captive elephants. Owing to the unviability of captive breeding programs, several captive elephant populations are maintained by illegally obtaining wild Asian elephants. Morbidity and mortality rates among captive populations are high, whereas reproduction is low. In this study, we examined the genetic diversity among elephants using microsatellite genotyping and mitochondrial D-loop sequences of three captive elephant populations. The study results showed very low nucleotide diversity D-loop sequences and high variations in microsatellite genotyping, with an extensive variation of the gene pool estimates from different populations. This suggests that the optimal male selection during breeding could aid in maintaining the genetic diversity among captive populations. Forward genetic simulation revealed a decreasing genetic diversity in the fixed state within 50 generations. However, largely different gene pools can be effectively used to infer original elephant sources; this would facilitate the development of an identification certificate integration with machine learning and image processing to prevent illegal legislation owing to registration fraud between wild and domestic elephants. Implementing the proposed approaches and recommendations would aid in the mitigation of the illegal capture and domestic trade of wild elephants in Thailand and contribute to the success of future conservation plans in the blueprint of sustainable development goals.

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.

The occurrence of osteoarthritis (OA) in marine mammals is still questionable. Here we investigated the prevalence of OA in marine (dolphin and dugong) and terrestrial mammals (Asian elephant, Asiatic buffalo, camel, cat, cattle, deer, dog, domestic goat, horse, human, hyena, impala, lion, Malayan tapir, Assam macaque, mule, pig, rabbit, red kangaroo, sheep, tiger and waterbuck). Skeletal remains obtained from five institutes were used as subjects; a total of 45 different parts (locations) of bones were observed for OA lesions. The prevalence of OA was reported as number of OA lesions/total number of bones. Our results revealed that the presence of OA in marine species (dolphin and dugong) was 2.44% and 3.33%, respectively. In dolphins, the highest OA occurrence was on the left and right humeral trochlea, with 13.68% and 12.63%, respectively, while the highest number of OA lesions in dugongs was on the lumbar vertebrae (8.79%). No significant difference (P > 0.05) in the prevalence of OA between sexes in dolphins and dugongs was observed, but we found a significant difference (P < 0.05) in 24 bone locations of human bones, which had the highest OA prevalence (48.93%), followed by dogs (3.94%). In conclusion, OA can occur in marine mammals, similar to terrestrial mammals, even though their natural habitat is the ocean.

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.

Elephant endotheliotropic herpesvirus (EEHV) infection is a conservation threat to the endangered Asian elephant (Elephas maximus), causing fatal hemorrhagic disease in juvenile elephants throughout the world, including Thailand. This study revealed a subclinical EEHV1 infection rate of 5.5% in healthy captive Asian elephants in Thailand (n = 362). The virus was detected in all age classes above one year old, in both sexes, and across the country – even in facilities with no history of hemorrhagic disease (EEHV HD). Subclinical EEHV infection in Thailand urgently requires proper health management.

Figure S1. Stacked bar plots with numbers of negative and positive animals per herd (ranked on herd size). Using as definitions for a positive sample: both OD ratio ≥ 3 (graph a) and OD ratio ≥ 4 (graph b). (PDF 58 kb)

Table S1. Seroprevalence based on an EEHV1A glycoprotein B protein antigen specific ELISA of elephants sampled throughout Thailand (nâ =â 994) between 2010 and 2015, and the percentage of samples testing positive1, inconclusive2 or negative3 relative to potential EEHV risk factors based on different interpretation modalities: 1:100 dilution; 1:200 dilution. (PDF 58 kb)

Table S2. Univariable regression analysis of potential risk factors for the presence of EEHV antibodies in elephants sampled throughout Thailand between 2010 and 2015 (n = 994) based on an EEHV1A glycoprotein B protein antigen specific ELISA. Seroprevalence is based on strict cutoff: positive if both OD ratio’s > 4. (PDF 52 kb)

This study evaluated the morphology and elemental composition of Asian elephant (Elephas maximus) bones (humerus, radius, ulna, femur, tibia, fibula and rib). Computerized tomography was used to image the intraosseous structure, compact bones were processed using histological techniques, and elemental profiling of compact bone was conducted using X-ray fluorescence. There was no clear evidence of an open marrow cavity in any of the bones; rather, dense trabecular bone was found in the bone interior. Compact bone contained double osteons in the radius, tibia and fibula. The osteon structure was comparatively large and similar in all bones, although the lacuna area was greater (P < 0.05) in the femur and ulna. Another finding was that nutrient foramina were clearly present in the humerus, ulna, femur, tibia and rib. Twenty elements were identified in elephant compact bone. Of these, ten differed significantly across the seven bones: Ca, Ti, V, Mn, Fe, Zr, Ag, Cd, Sn and Sb. Of particular interest was the finding of a significantly larger proportion of Fe in the humerus, radius, fibula and ribs, all bones without an open medullary cavity, which is traditionally associated with bone marrow for blood cell production. In conclusion, elephant bones present special characteristics, some of which may be important to hematopoiesis and bone strength for supporting a heavy body weight.

Elephant endotheliotropic herpesvirus (EEHV) is an infection associated with fatal hemorrhagic disease in young Asian elephants ( Elephas maximus). This brief communication describes the postmortem evaluation of two Asian elephant calves diagnosed with EEHV4 and EEHV1A in conjunction with Clostridium perfringens infection. Case 1 was a 7-mo-old, male captive-born Asian elephant that developed diarrhea and died 2 days after clinical presentation. Examination of the heart, lungs, liver, and spleen revealed predominantly basophilic intranuclear inclusion bodies in the endothelial cells of the blood vessels. Case 2 was a 3-mo-old, female wild-born Asian elephant that showed signs of lethargy, anorexia, and convulsions and died 6 hr after clinical presentation. No intranuclear inclusion bodies were observed. The heart, lung, liver, and spleen of both calves tested positive for EEHV by polymerase chain reaction. Phylogenetic analysis identified EEHV4 and EEHV1A in Case 1 and 2, respectively. Additionally, liver, spleen, and hemorrhagic intestinal tissue samples tested positive for C. perfringens α, β, and ε toxins. This is the first reported case to describe coinfection of EEHV and C. perfringens in Asian elephant calves.

Captive-held elephants were recruited from several parts of Thailand and released as part of a reintroduction project. Wild elephants with a herd matriarch generally contain the same matrilineal line and are genetically related. However, reintroduced elephants are less likely to be related, but are known to establish social groups. The objective of this study was to investigate the genetic relatedness and behavioral relationships of elephants reintroduced into forested areas in central and northern Thailand. Blood samples were collected from 53 elephants before release into the Sublanka (SLK) and Doi Phamuang (DPM) Wildlife Sanctuaries, and DNA was extracted for microsatellite and mitochondrial analysis. Direct observations of social bonding behaviors were done weekly for 12 months after release, and an association index (AI) calculated for each individual. The results showed a low relatedness for both populations; the observed heterozygosity and number of mitochondrial haplotypes were 0.739 and 13 at SLK ( n =26), and 0.808 and 11 at DPM ( n =27), respectively. Across both locations, 33 elephants formed into 11 groups (range 2–6 individuals/group). The average AI and pairwise genetic relatedness of elephant groups were 0.517±0.039 and 0.078±0.019, respectively, and were not correlated ( r =−0.036; p =0.78). Twenty elephants were not associated with specific groups and had average AI and pairwise genetic relatedness of 0.002±0.001 and 0.047±0.013, respectively, which were not correlated ( r =−0.074; p =0.491). Several mitochondrial haplotypes were found within the same group. Thus, social bonding of the reintroduced elephants was not influenced by genetic relatedness. Rather, groups formed in association with the presence of an elephant calf. Additionally, many elephants occasionally preferred isolation. Thus, reintroduction procedures should favor introducing elephant calves, or adults with calves to increase the chance of group formation and establishment of stable elephant herds.

Summary Mycobacterium tuberculosis (M. tb) has been shown to be the main causative agent of tuberculosis in elephants worldwide. M. tb may be transmitted from infected humans to other species including elephants and vice versa, in case of prolonged intensive contact. An accurate diagnostic approach covering all phases of the infection in elephants is required. As M. tb is an intracellular pathogen and cellmediated immune (CMI) responses are elicited early after infection, the skin test is the CMI assay of choice in humans and cattle. However, this test is not applicable in elephants. The interferon gamma (IFN-c) assay is considered a good alternative for the skin test in general, validated for use in cattle and humans. This study was aimed at development of an IFN-c assay applicable for diagnosis of tuberculosis in elephants. Recombinant elephant IFN-c (rEpIFN-c) produced in eukaryotic cells was used to immunize mice and generate the monoclonal antibodies. Hybridomas were screened for IFN-c-specific monoclonal antibody production and subcloned, and antibodies were isotyped and affinity purified. Western blot confirmed recognition of the rEpIFN-c. The optimal combination of capture and detection antibodies selected was able to detect rEpIFN-c in concentrations as low as 1 pg/ml. The assay was shown to be able to detect the native elephant IFN-c, elicited in positive-control cultures (pokeweed mitogen (PWM), phorbol myristate acetate plus ionomycin (PMA/I)) of both Asian and African elephant whole-blood cultures (WBC). Preliminary data were generated using WBC from non-infected elephants, a M. tb infection-suspected elephant and a cultureconfirmed M. tb-infected elephant. The latter showed measurable production of IFN-c after stimulation with ESAT6/CFP10 PPDB and PPDA in concentration ranges as elicited in WBC by Mycobacterium tuberculosis complex (MTBC)-specific antigens in other species. Hence, the IFN-c assay presented potential as a diagnostic tool for the detection of elephant tuberculosis. Validation of the assay will require its application in large populations of non-infected and infected elephants.

Background Lesions related to working conditions and improper saddle design are a concern for a variety of working animals including elephants. The objectives of the present study were to determine the prevalence of cutaneous lesions in anatomic regions (i.e., neck, girth, back, tail) in contact with saddle-related equipment among elephants in Thailand working in the tourism industry, and to identify potential risk factors associated with these lesions. Data for this cross-sectional study were collected between May 2007 and July 2007 on 194 elephants from 18 tourism camps across Thailand. Results There was a high prevalence (64.4 %; 95 % CI 57.3 – 71.2) of active lesions, most often located on the back region. Using multilevel multivariable logistic regression modelling containing a random intercept for camp we identified the following risk factors: increasing elephant age, the use of rice sacks as padding material in contact with the skin, and the provision of a break for the elephants. Working hours had a quadratic relationship with the log odds of an active lesion where the probability of an active lesion initially increased with the number of working hours per day and then declined possibly reflecting a “healthy worker” bias where only animals without lesions continue to be able to work these longer hours. Conclusions While we recognize that the cross-sectional nature of the study posed some inferential limitations, our results offer several potential intervention points for the prevention of these lesions. Specifically, we recommend the following until longitudinal studies can be conducted: increased monitoring of older elephants and the back region of all elephants, working less than 6 hours per day, and the avoidance of rice sacks as padding material in contact with skin. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0438-1) contains supplementary material, which is available to authorized users.

Intact plasma and acrosome membranes and functional mitochondria following cryopreservation are important attributes for the fertilizing ability of spermatozoa. In the present study, functional and ultrastructural changes of Asian elephant spermatozoa after cryopreservation either in TEST + glycerol or HEPT + dimethyl sulphoxide (DMSO) were evaluated by fluorescent techniques and electron microscopy. Sperm frozen in TEST + glycerol had higher proportion of sperm with intact plasma (49.1 +/- 9.2% vs. 30.9 +/- 3.9%) and acrosomal (53.7 +/- 4.9% vs. 35.8 +/- 6.1%) membranes, as well as active mitochondria (57.0 +/- 7.2% vs. 42.0 +/- 5.0%) than those cryopreserved in HEPT + DMSO. The results obtained from electron microscopy were similar to those obtained by fluorescence microscopy. The percentage of normal spermatozoa was higher when spermatozoa were frozen in TEST + glycerol than those frozen in HEPT + DMSO (31.8 +/- 5.6 vs. 28.5 +/- 6.4). The ultrastructural alterations revealed by transmission electron microscopy could be classified as (i) distension of plasma membrane, while the acrosome was swollen; (ii) disruption or loss of plasma membrane, while acrosome was swollen with distended outer acrosomal membrane; (iii) disruption or loss of plasma and outer acrosomal membrane with leakage of acrosome content; (iv) extensive vesiculation of plasma and outer acrosomal membrane and leakage of acrosome content; (v) a complete loss of both plasma membrane and outer acrosomal membrane; and (vi) swelling of mitochondria. These findings suggest that the freezing and thawing procedure caused structural damage to elephant spermatozoa, especially in the plasma membrane, acrosome and mitochondria. Fluorescence and electron microscopic evaluations are potentially a powerful tool in the analysis of elephant spermatozoa after freezing and thawing.

Limited data are available regarding the nutrition and feeding of captive Asian elephants in range countries. In this study, feeding regimens of two collections in northern Thailand and their actual diets shaped by availability of forage and mahout preferences were assessed for nutritional quality. The composition of dietary intake, fecal output, and the dietary regimen were individually recorded for 5 days in 10 elephants. The proportion of forage in the diet represented 41 to 62% of the dry matter intake (DMI) in one collection whereas in the other collections it varied between 68 and 72%. Between 8.5 and 24% of the diet consisted of commercial pellets, and hulled rice represented up to 25% of the DMI in one collection. Sugar cane, corn cobs, and fruits such as bananas were eaten in smaller amounts. Body condition scores and weights were measured, which revealed that nine animals were in good condition. Representative samples of each food as well as fecal samples were analyzed for dry matter, crude protein, fat, crude fiber, gross energy, ash, calcium, and phosphorus. Diet adequacy was assessed by calculating the digestible nutrients in the rations and by comparing them to the recommendations from literature. The digestible energy (DE) intake varied between 0.6 and 1.4 megajoules (MJ) per kg(0.75) per day; therefore, higher than the estimated recommendations of 0.65 MJ per kg(0.75) per day for nine of the elephants. In all elephants the crude protein intake was less than the maintenance recommendations and ranged between 6.01 and 7.56% of the DMI. Calcium intake was low in one collection and there was an inverse calcium: phosphorus ratio, which was inadequate. The present study adds to the knowledge of captive elephant diets in Asia and is a starting point for further research, which is necessary to design optimum diet plans for captive Asian elephants in Thailand.

Bone turnover markers could offer a potential alternative means for the early diagnosis of metabolic bone disease in young growing elephants although the baseline of bone turnover markers in elephant is not well established. The aim of this study was to determine any relationship between the age of captive Asian elephants (Elephas maximus) and markers of bone formation. Serum samples from 24 female Asian elephants were collected to evaluate levels of two bone formation markers, namely, osteocalcin (OC) and bone-specific alkaline phosphatase (BAP). Both intact and N-terminal midfragment OC and BAP were negatively correlated with age. The findings demonstrate that younger elephants have a higher rate of bone turnover than older elephants. Use of these and additional bone markers could lead to the establishment of validated protocols for the monitoring of bone disease in elephants.

Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S–23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential.

Background Artificial insemination (AI) using frozen-thawed semen is well established and routinely used for breeding in various mammalian species. However, there is no report of the birth of elephant calves following AI with frozen-thawed semen. The objective of the present study was to investigate the fertilizing ability of chilled and frozen-thawed semen in the Asian elephant following artificial insemination (AI). Methods Semen samples were collected by from 8 bulls (age range, 12-to 42-years) by manual stimulation. Semen with high quality were either cooled to 4°C or frozen in liquid nitrogen (-196°C) before being used for AI. Blood samples collected from ten elephant females (age range, 12-to 52-years) were assessed for estrus cycle and elephants with normal cycling were used for AI. Artificial insemination series were conducted during 2003 to 2008; 55 and 2 AI trials were conducted using frozen-thawed and chilled semen, respectively. Pregnancy was detected using transrectal ultrasonography and serum progestagen measurement. Results One female (Khod) inseminated with chilled semen became pregnant and gave birth in 2007. The gestation length was 663 days and the sex of the elephant calf was male. One female (Sao) inseminated with frozen-thawed semen showed signs of pregnancy by increasing progestagen levels and a fetus was observed for 5 months by transrectal ultrasonography. Conclusion This is the first report showing pregnancy following AI with frozen-thawed semen in the Asian elephant. Successful AI in the Asian elephant using either chilled or frozen-thawed semen is a stepping stone towards applying this technology for genetic improvement of the elephant population.