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.

Microbiome variations among age classes and diets of captive Asian elephants (Elephas maximus) in Thailand using full-length 16S rRNA nanopore sequencing.

Asian elephant (Elephas maximus) is the national symbol of Thailand and linked to Thai history and culture for centuries. The elephant welfare improvement is one of the major components to achieve sustainable captive management. Microbiome inhabiting digestive tracts have been shown with symbiotic relations to host health. This work provided high-resolution microbiome profiles of 32 captive elephants at a species level by utilizing full-length 16S rRNA gene nanopore sequencing. Eleven common uncultured bacterial species were found across elephants fed with solid food including uncultured bacterium Rikenellaceae RC9 gut group, Kiritimatiellae WCHB1-41, Phascolarctobacterium, Oscillospiraceae NK4A214 group, Christensenellaceae R-7 group, Oribacterium, Oscillospirales UCG-010, Lachnospiraceae, Bacteroidales F082, uncultured rumen Rikenellaceae RC9 gut group, and Lachnospiraceae AC2044 group. We observed microbiome shifts along the age classes of baby (0-2 years), juvenile (2-10 years), and adult (> 10 years). Interestingly, we found distinct microbiome profiles among adult elephants fed with a local palm, Caryota urens, as a supplement. Potential beneficial microbes have been revealed according to the age classes and feed diets. The retrieved microbiome data could be provided as good baseline microbial profiles for monitoring elephant health, suggesting further studies towards dietary selection suitable for each age class and the use of local supplementary diets.

Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus).

Elephants are susceptible to Mycobacterium tuberculosis (M. tb) complex (MTBC) infections. Diagnosis of tuberculosis (TB) in elephants is difficult, and most approaches used for human TB diagnosis are not applicable. An interferon gamma release assay (IGRA) to diagnose TB in Asian elephants (Elephas maximus) using peripheral blood mononuclear cells (PBMCs) has been previously developed. Although the assay is shown to be valid in determining MTBC infection status, the laborious PBMC isolation process makes it difficult to use. In this study, we simplified the method by using whole blood cultures (WC) as the starting material. Using PBMC cultures for IGRA, the MTBC infection status of 15 elephants was first confirmed. Among these animals, one has been previously confirmed for M. tb infection by both TB culture and PCR and the other was confirmed for MTBC infection in this study by droplet digital PCR (ddPCR) method. WC for IGRA consisted of an unstimulated sample, a mitogen stimulated sample, and sample stimulated with recombinant M. tb antigens, ESAT6 and CFP10. Using WC for IGRA in the 15 enrolled elephants, the results showed that 7 out of 15 samples yielded MTBC infection positive status that were completely concordant with those from the results using PBMCs. To test this method, WC for IGRA were applied in another elephant cohort of 9 elephants. The results from this cohort revealed a perfect match between the results from PBMC and WC. Responses to ESAT6 or CFP10 by PBMC and WC were not completely concordant, arguing for the use of at least two M. tb antigens for stimulation. Given the ease of sample handling, smaller blood sample volumes and equivalent efficacy relative to the PBMC approach, using WC for IGRA provides a novel, rapid, and user-friendly TB diagnostic method for determining the MTBC infection in elephants.

Taxonomic status of otter species in Nakai-Nam Theun National Park, Lao PDR, based on DNA evidence.

Otter populations are threatened by habitat loss, pollution, conflicts with humans, and illegal wildlife trade to meet the demand for pets, for their fur, and for parts used in traditional medicines. Baseline information on the distribution, population genetic diversity, and connectivity is crucial to inform conservation management decisions; however, reliable data from otter populations in Southeast Asia remain scarce. In this study, we conducted baseline otter fecal DNA surveys based on mitochondrial DNA (mtDNA) to identify species, assess the occurrence, and map the spatial distribution of genetic diversity and evolutionary relationships of otter populations using 1700 bp Cytochrome B - Control Region and mitogenome from Nakai-Nam Theun National Park in the Annamite Mountains of Lao PDR. Of the total 56 samples identified to species, the majority (87.5%) was of the widely distributed Eurasian otter with three haplotypes (Lutra lutra; LLLA01-LLLA03), with a calculated haplotype diversity of 0.600 and a nucleotide diversity of 0.00141 based on mitogenome. The second species was the Asian small-clawed otter with only one haplotype detected (Aonyx cinereus; ACLA01). All Eurasian otter haplotypes were newly characterized and clustered within the strongly supported South-Southeast-North Asian clade of Lutra lutra. Compared with the European clade, the high mtDNA diversity of Lutra lutra in Nakai-Nam Theun National Park potentially reflects long-term demographic stability and lesser degree of population bottleneck during the last glacial maxima (LGM, ~21,000 years ago). The single haplotype detected in Asian small-clawed otters had not been detected in previous genetic studies. Our research is the first otter-specific noninvasive genetic study in Lao PDR and provides baseline insights into the otter population diversity in a regional priority site for biodiversity conservation.

Coincidence of low genetic diversity and increasing population size in wild gaur populations in the Khao Phaeng Ma Non-Hunting Area, Thailand: A challenge for conservation management under human-wildlife conflict.

The gaur (Bos gaurus) is found throughout mainland South and Southeast Asia but is listed as an endangered species in Thailand with a decreasing population size and a reduction in suitable habitat. While gaur have shown a population recovery from 35 to 300 individuals within 30 years in the Khao Phaeng Ma (KPM) Non-Hunting Area, this has caused conflict with villagers along the border of the protected area. At the same time, the ecotourism potential of watching gaurs has boosted the local economy. In this study, 13 mitochondrial displacement-loop sequence samples taken from gaur with GPS collars were analyzed. Three haplotypes identified in the population were defined by only two parsimony informative sites (from 9 mutational steps of nucleotide difference). One haplotype was shared among eleven individuals located in different subpopulations/herds, suggesting very low genetic diversity with few maternal lineages in the founder population. Based on the current small number of sequences, neutrality and demographic expansion test results also showed that the population was likely to contract in the near future. These findings provide insight into the genetic diversity and demography of the wild gaur population in the KPM protected area that can inform long-term sustainable management action plans.

The first report on internal transcribed spacer region-based characterization of microfilaria in Asian elephants (Elephas maximus) in Thailand.

BACKGROUND AND AIM: Filarial infections can significantly impact the health of both humans and animals. In elephants, filariasis has been associated with cutaneous dermatitis and skin nodules. However, molecular evidence for such infections is limited in Thailand. This study aimed to identify the morphological and molecular characteristics of microfilaria in captive Asian elephants in Thailand. MATERIALS AND METHODS: Whole blood collected from the ear vein of 129 captive Asian elephants was hematologically analyzed, and the blood parasites were evaluated using three standard techniques: The microcapillary test, thin blood smears, and polymerase chain reaction (PCR). RESULTS: Conventional PCR revealed that approximately 17% (22/129) of the sampled elephants were positive for microfilaria. Microscopy revealed that microfilariae are large, unsheathed, with extended nuclei, a short headspace, and a curved tail tapering at the end. Results of internal transcribed spacer region analysis show that the elephant microfilariae are closely related to Onchocerca spp. All of the elephants positive for microfilaria presented with neither skin lesion nor anemic signs. Microfilaria infection was not associated with age; however, microfilariae were more likely to be detected in male elephants due to differences in management systems. CONCLUSION: This is the first study to provide both morphological and molecular evidence of microfilaria in Thai elephants. There is an urgent need to investigate the long-term and large-scale effects of microfilaria on the health of elephants.

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.

The occurrence of elephant endotheliotropic herpesvirus infection in wild and captive Asian elephants in Thailand: Investigation based on viral DNA and host antibody.

BACKGROUND AND AIM: Elephant endotheliotropic herpesvirus (EEHV) is a serious disease, threatening the life of young elephants. Many elephants have been infected with no clinical signs and may serve as carriers spreading this disease. It is important to monitor the disease through clinical signs and molecular diagnosis. In this study we investigated the occurrence of EEHV and the efficiency of different techniques used to monitor EEHV infection in various samples and populations of Asian elephants. MATERIALS AND METHODS: Blood and trunk swabs were collected from live elephants, while visceral organs (lung, digestive tract, spleen, lymph nodes, and kidney) were collected from dead elephants. EEHV was detected by polymerase chain reaction (PCR) in whole blood, trunk swabs, and visceral organs as samples, while elephant anti-EEHV immunoglobulin G (IgG) in serum was detected by enzyme-linked immunosorbent assay (ELISA). A total of 162 samples were analyzed in this study: 129 from healthy, 26 from dead, and 7 from sick elephants. RESULTS: The present study showed that the overall incidence of EEHV was 40.1% (n=65/162). Approximately 46.2% (n=12/26) and 85.7% (n=6/7) of dead and sick elephants were positive for EEHV by PCR, respectively. All sick elephants that were young and affected by EEHV clinical disease tested negative for the IgG antibody ELISA, suggesting primary EEHV infection in this group. In addition, 2.3% (n=3/129) of subclinical infections were detected using PCR, and trunk swab samples showed slightly higher sensitivity (5.3%, n=2/38) to detect EEHV than whole blood (1.2%, n=1/84). As many as, 48.4% (n=44/91) of healthy elephants were EEHV seropositive (ELISA-positive), suggesting that many elephants in Thailand had previously been infected. Overall, 30% of dead wild elephants had been infected with EEHV (n=3/10). Moreover, statistical analysis revealed no significant differences in the EEHV detection rate between different age groups or sexes (p>0.05). CONCLUSION: PCR is better than ELISA to detect EEHV active infection in dead/sick elephants and to monitor EEHV in young elephants. ELISA is suitable for detecting previous EEHV infection and carriers, particularly adults. Theoretically, we could use both PCR and ELISA to increase the sensitivity of testing, along with observing abnormal behavior to efficiently monitor this disease. Identification of EEHV carriers within elephant populations is important to prevent transmission to healthy individuals, especially young elephants with high mortality from EEHV. This is the first report from Thailand regarding EEHV infection in wild elephants, showing the importance of preventing disease transmission between captive and wild elephants.

ASIAN ELEPHANT (ELEPHAS MAXIMUS) SUFFERING FROM LIGHTNING STRIKE SUCCESSFULLY TREATED BY INTEGRATIVE VETERINARY MEDICINE.

A captive female Asian elephant (Elephas maximus), approximately 30 yr old, was struck by lightning and displayed obtundation, dysphagia, drooling, and muscle weakness. A physical examination, hematologic test, blood gas analysis, and electrocardiogram were performed. Treatments included the administration of mannitol, dexamethasone, antibiotics, analgesics, vitamins, and intravenous fluid. Conventional therapy resulted in improvement but not complete resolution of clinical signs. Therefore, acupuncture was integrated into the treatment protocol. Electroacupuncture and laser acupuncture were administered every other day; aqua acupuncture was administered once a week. No complications were observed during acupuncture treatment. Acupuncture, a form of traditional Chinese veterinary medicine (TCVM), contributed to the resolution of clinical signs in this case and may be an effective adjunctive treatment for other neurologic disorders in elephants.

Biological and environmental factors associated with the detection of elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in Thailand.

Elephant endotheliotropic herpesvirus (EEHV) infection is one of the most common diseases in young elephants, causing severe fatal hemorrhagic disease. Subclinical infection was previously described; however, information about the factors associated with virus shedding and reactivation were scarce. To identify the biological and environmental factors related with EEHV detection, blood and oral swab samples were collected from nine captive Asian elephants in Thailand for one year and tested for EEHV presence using real-time PCR. Data including hematological values, management, environmental temperature, and serum cortisol levels were also recorded and analyzed. Results showed that the viral detection frequency ranged from 0-25%. The highest detection frequency was found in the two youngest elephants, aged less than 15 years. Three types of viruses, EEHV1, EEHV4, and EEHV5, were found in this study, which also detected mixed infection in five elephants. Additionally, the study found that sample type, changes in hematological values, management and health issues, and serum cortisol levels were not associated with herpesvirus detection in the elephants. However, EEHV detection percentage was significantly increased in the summer (mid-Feb to mid-May), possibly due to body fitness reduction from food source limitation and low nutrient content. To obtain a broad aspect of EEHV management, long-term EEHV monitoring is highly recommended in every captive elephant herd.

16S rRNA Gene Amplicon Profiling of Baby and Adult Captive Elephants in Thailand.

Here, we present a 16S rRNA gene amplicon sequence data set and profiles demonstrating the bacterial diversity of baby and adult elephants from four different geographical locations in Thailand. The dominant phyla among baby and adult elephants were Bacteroidetes, Firmicutes, Proteobacteria, Kiritimatiellaeota, Euryarchaeota, and Tenericutes.

Subclinical infection of captive Asian elephants (Elephas maximus) in Thailand with elephant endotheliotropic herpesvirus.

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.

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.

Molecular characterisation and genetic variation of Elephant Endotheliotropic Herpesvirus infection in captive young Asian elephants in Thailand.

Elephant Endotheliotropic Herpesvirus (EEHV) is emerging as a new threat for elephant conservation, since being identified as the cause of severe, often fatal, haemorrhagic disease in young Asian elephants. To describe positive cases and the molecular relatedness of virus detected in elephants in Thailand, we re-examined all available of EEHV samples occurring in young elephants in Thailand between 2006 and 2014 (n=24). Results indicated 75% (18/24) of suspected cases were positive for EEHV by semi-nested PCR. Further gene analysis identified these positive cases as EEHV1A (72%, 13/18 cases), EEHV1B (11%, 2/18) and EEHV4 (17%, 3/18). This study is the first to phylogenetically analyse and provide an overview of most of the known EEHV cases that have occurred in Thailand. Positive individuals ranged in age from one to nine years, with no sex association detected, and occurred across geographical locations throughout the country. All individuals, except one, were captive-born. No history of direct contact among the cases was recorded, and this together with the fact that various subtype clusters of virus were found, implied that none of the positive cases were epidemiologically related. These results concur with the hypothesis that EEHV1 is likely to be an ancient endogenous pathogen in Asian elephants. It is recommended that active surveillance and routine monitoring for EEHV should be undertaken in all elephant range countries, to gain a better understanding of the epidemiology, transmission and prevention of this disease.

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.

Potential of a gonadotropin-releasing hormone vaccine to suppress musth in captive male Asian elephants (Elephas maximus).

Musth in adult bull elephants is a period of increased androgen concentrations ranging from a few weeks to several months. For captive elephant bull management, musth presents a serious challenge because of the aggressive behavior of musth bulls toward people and other elephants. Commercially available GnRH vaccines have been shown to suppress testicular function by interrupting the hypothalamo-pituitary-gonadal (HPG) axis in many species. The aim of this study was to test the efficacy of a GnRH vaccine in elephant bulls for suppressing the HPG axis and mitigating musth-related aggressive behavior. Five adult Asian elephant bulls (22-55 years old) were immunized with a GnRH vaccine starting with an initial injection 2-4 months before the predicted musth period, and followed by three boosters at approximately 4-week intervals. Blood samples were collected twice weekly for hormone and antibody titer analysis. An increase in GnRH antibody titers was observed in all bulls after the second or third booster, and titers remained elevated for 2-3 months after the final booster. Musth was attenuated and shortened in three bulls and postponed completely in two. We conclude that GnRH vaccination is capable of suppressing symptoms of musth in adult bull elephants. With appropriate timing, GnRH vaccination could be used to control or manage musth and aggressive behavior in captive elephant bulls. However, more work is needed to identify an optimal dose, booster interval, and vaccination schedule for complete suppression of testicular steroidogenesis.

The occurrence of elephant endotheliotropic herpesvirus in captive Asian elephants (Elephas maximus): first case of EEHV4 in Asia.

Elephant endotheliotropic herpesvirus (EEHV) is a type of herpesvirus that causes acute hemorrhagic disease in Asian elephants (Elephas maximus) and is often fatal, especially in calves. This study describes the postmortem evaluation of two captive-born Asian elephants (2 and 3 yr of age, respectively) diagnosed with EEHV in Thailand. Both elephants presented only mild depression, lethargy, and anorexia before death within 24 hr of symptom onset. Necropsies were performed, and tissue samples were tested for EEHV viral presence using polymerase chain reaction. Molecular and phylogenetic evidence illustrated two types of EEHV, which were closely related to EEHV1A in Case 1 and EEHV4 in Case 2. Pathologic findings differed between the cases. More specific organ tropism was found in Case 1, where mainly the cardiovascular system was affected. In contrast, in Case 2, hemorrhages were noted in most organs, including in the gastrointestinal, respiratory, and cardiovascular systems. This report is the first to document EEHV4 in Asia and the second case of this strain to be identified in an elephant worldwide.