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.

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.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS (EEHV) IN FREE-RANGING AFRICAN ELEPHANTS (LOXODONTA AFRICANA) IN THE KRUGER NATIONAL PARK, SOUTH AFRICA.

Elephant endotheliotropic herpesvirus (EEHV) infection can cause acute, often fatal, EEHV hemorrhagic disease in free-ranging and human-managed Asian elephants (Elephas maximus) and human-managed African elephants (Loxodonta africana). However, significant knowledge gaps exist pertaining to the presence of EEHV in free-ranging African elephant populations. We retrospectively screened 142 opportunistically collected samples (blood, n=98; bronchoalveolar lavage (BAL) fluid, n=21; trunk wash (TW) fluid, n=23) obtained between 2010 and 2020 from 98 free-ranging African elephants in the Kruger National Park, South Africa, for the presence of different EEHVs, as well as determining the real-time quantitative PCR positivity rate in this population. With the use of validated, previously published DNA extraction and real-time quantitative PCR protocols provided by the National Elephant Herpesvirus Laboratory (Washington, DC, USA), EEHV was detected in nine male African elephants from samples collected in 2011 (n=1), 2013 (n=1), 2018 (n=2), 2019 (n=4), and 2020 (n=1). Viral detection was more common in respiratory compared with blood samples. Six elephants tested positive for EEHV2 subtype (blood, n=2; BAL, n=3; TW, n=2), including one individual that tested positive on matched respiratory samples (BAL and TW). Four elephants tested positive for EEHV3-4-7 (blood, n=1; BAL, n=2; TW, n=1), whereas EEHV6 was not detected in any of the study animals. One elephant tested positive for both EEHV2 and EEHV3-4-7 in the same BAL sample. Even though the levels of viremia varied between 158 and 1,292 viral genome equivalents/mL blood and viral shedding of EEHV2 and EEHV3-4-7 was detected in respiratory samples, no clinical signs were observed in these apparently healthy elephants. These findings are consistent with reports of asymptomatic EEHV infection in human-managed African elephants.

EEHV1A glycoprotein B subunit vaccine elicits humoral and cell-mediated immune responses in mice.

Asian elephants are an endangered species facing many threats, including severe hemorrhagic disease (HD) caused by the elephant endotheliotropic herpesvirus (EEHV). EEHV-HD is the leading cause of death in captive juvenile Asian elephants in North America and Europe, and also affects elephants in their natural range countries. Significant challenges exist for successful treatment of EEHV-HD, which include timely recognition of disease onset and limited availability of highly effective treatment options. To address this problem, our goal is to prevent lethal disease in young elephants by developing a vaccine that elicits robust and durable humoral and cell-mediated immunity against EEHV. EEHV glycoprotein B (gB) is a major target for cellular and humoral immunity in elephants previously exposed to EEHV. Therefore, we generated a vaccine containing recombinant EEHV1A gB together with a liposome formulated TLR-4 and saponin combination adjuvant (SLA-LSQ). CD-1 mice that received one or two vaccinations with the vaccine elicited significant anti-gB antibody and polyfunctional CD4+ and CD8+ T cell responses, while no adverse effects of vaccination were observed. Overall, our findings demonstrate that an adjuvanted gB protein subunit vaccine stimulates robust humoral and cell-mediated immune responses and supports its potential use in elephants.

Elephant Endotheliotropic Herpesvirus 4 and Clostridium perfringens Type C Fatal Co-Infection in an Adult Asian Elephant (Elephas maximus).

Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is an acute, often fatal, multisystemic hemorrhagic disease and one of the most significant causes of mortality of Asian elephants in captivity. Most fatal cases of EEHV-HD are associated with EEHV1A and EEHV1B in juveniles. This case report describes the clinical and pathological features of a fatal co-infection of Clostridium perfringens type C and EEHV-HD, caused by EEHV4, in an adult female Asian elephant. Although fatal clostridial enterotoxemia has been occasionally reported in elephants, this report highlights the importance of having both EEHV-HD and clostridial enterotoxemia as potential differential diagnoses in cases of widespread tissue necrosis and internal hemorrhage in elephants, regardless of the animal age group, due to their macroscopic similarities, frequent co-occurrence and cumulative morbid potential.

Effects of between and within Herd Moves on Elephant Endotheliotropic Herpesvirus (EEHV) Recrudescence and Shedding in Captive Asian Elephants (Elephas maximus).

Haemorrhagic disease associated with elephant endotheliotropic herpesvirus (Elephantid herpesvirus, EEHV) infections is the leading cause of death for Asian elephant (Elephas maximus) calves. This study assessed the effect of captive herd management on EEHV shedding, as evidence of latent infection reactivation, focusing on: (1) the influence of social change on the odds of recrudescence; (2) the respective effects of between and within herd moves; and (3) characteristics of recrudescent viral shedding. Trunk and conjunctival swabs (n = 165) were obtained from six elephants at an EAZA-accredited zoo, collected during a period of social stability, and at times of social change. Longitudinal sampling took place at times of moving two bulls out of the collection and one new bull into an adjacent enclosure to the cow herd (between herd moves), and during a period of mixing this new bull with the cow herd to facilitate mating (within herd moves). Quantitative PCR was employed to detect EEHV 1a/b, 4a/b, and EF-1-α (housekeeping gene). Generalised estimating equations determined EEHV recrudescence odds ratios (OR) and relative viral DNA load. Sixteen EEHV 1a/b shedding events occurred, but no EEHV 4a/b was detected. All management-derived social changes promoted recrudescence (social change OR = 3.27, 95% CI = 0.412-26, p = 0.262; and between herd moves OR = 1.6, 95% CI = 0.178-14.4, p = 0.675), though within herd movements posed the most significant increase of EEHV reactivation odds (OR = 6.86, 95% CI = 0.823-57.1, p = 0.075) and demonstrated the strongest relative influence (post hoc Tukey test p = 0.0425). Shedding onset and magnitude ranged from six to 54 days and from 3.59 to 11.09 ΔCts. Differing challenges are associated with between and within herd movements, which can promote recrudescence and should be considered an exposure risk to naïve elephants.

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.

The first reported cases of elephant endotheliotropic herpesvirus infectious haemorrhagic disease in Malaysia: case report.

BACKGROUND: Elephant endotheliotropic herpesvirus haemorrhagic disease (EEHV HD) is the leading cause of death in captive Asian elephant calves in Asia, North America, and Europe with a mortality rate of ~ 65% in calves that are under human care. Although EEHV HD was first found in elephant camps, more recently it was identified in wild populations which poses a greater threat to the elephant population. Deaths due to EEHV HD have been seen in wild elephants, but the in-situ prevalence and mortality rate is unknown. We report the first EEHV HD cases in Malaysia from 3 wild born endangered Bornean elephant calves from Sabah with known typical clinical signs. CASE PRESENTATION: The first calf died within 24 h of the onset of clinical signs; the second calf died within 12 h of the onset of clinical signs. The third calf succumbed within 72 h. Necropsies revealed that all 3 calves had similar presentations of EEHV HD but in the third calf with less severity. We conducted conventional polymerase chain reaction (cPCR) assays and found EEHV DNA at all 7 loci in the 3 calves; it was identified as EEHV1A, the virus type that has been found in most other reported cases. CONCLUSION: Typical EEHV HD clinical signs and the molecular confirmation of EEHV by cPCR and sequencing point to EEHV as the cause of death. Further genetic investigation of the strain is in progress.

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.

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.

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.

Attempt to Isolate Elephant Endotheliotropic Herpesvirus (EEHV) Using a Continuous Cell Culture System.

Elephant endotheliotropic herpesvirus (EEHV) infection is known to cause acute fatal hemorrhagic disease, which has killed many young Asian elephants (Elephas maximus). Until recently, in vitro isolation and propagation of the virus have not been successful. This study aimed to isolate and propagate EEHV using continuous cell lines derived from human and/or animal origins. Human cell lines, including EA. hy926, A549, U937, RKO, SW620, HCT-116 and HT-29, and animal cell lines, including CT26.CL25 and sp2/0-Ag14, were investigated in this study. Mixed frozen tissue samples of the heart, lung, liver, spleen and kidney obtained from fatal EEHV1A- or EEHV4-infected cases were homogenized and used for cell inoculation. At 6, 24, 48 and 72 h post infection (hpi), EEHV-inoculated cells were observed for cytopathic effects (CPEs) or were assessed for EEHV infection by immunoperoxidase monolayer assay (IPMA) or quantitative PCR. The results were then compared to those of the mock-infected controls. Replication of EEHV in the tested cells was further determined by immunohistochemistry of cell pellets using anti-EEHV DNA polymerase antibodies or re-inoculated cells with supernatants obtained from passages 2 or 3 of the culture medium. The results reveal that no CPEs were observed in the tested cells, while immunolabeling for EEHV gB was observed in only U937 human myeloid leukemia cells. However, quantitation values of the EEHV terminase gene, as well as those of the EEHV gB or EEHV DNA polymerase proteins in U937 cells, gradually declined from passage 1 to passage 3. The findings of this study indicate that despite poor adaptation in U937 cells, this cell line displays promise and potential to be used for the isolation of EEHV1 and EEHV4 in vitro.

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.

EVALUATION OF THE EFFICACY OF TWO DIFFERENT SAMPLING SITES FOR THE DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS (EEHV) IN THREE ASIAN ELEPHANTS (ELEPHAS MAXIMUS) IN IRELAND.

Elephant endotheliotropic herpesvirus (EEHV) causes a disease that primarily affects juvenile Asian (Elephas maximus) elephants, causing acute hemorrhage and death. Due to the severity of the disease, many zoos have developed EEHV active surveillance programs. Currently, trunk washes are the standard for testing elephants for shedding of EEHV, but it has also been detected from other mucosal surfaces. This study compared the efficacy of oral swabs and trunk washes for the detection of EEHV shedding using previously validated quantitative polymerase chain reaction (qPCR) methods. Oral swab and trunk wash samples from three juvenile elephants at the Dublin Zoo in Ireland were collected in tandem and tested from April to September 2017. Of the 51 paired samples, 21 trunk wash samples were positive for EEHV1, while only 2 of the oral swab samples were positive for EEHV1, suggesting that trunk wash samples are more effective for detecting shedding of EEHV in Asian elephants compared with oral swabs.

NONINVASIVE SAMPLING FOR DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS AND GENOMIC DNA IN ASIAN (ELEPHAS MAXIMUS) AND AFRICAN (LOXODONTA AFRICANA) ELEPHANTS.

Elephant endotheliotropic herpesvirus (EEHV) hemorrhagic disease (EEHV-HD) threatens Asian elephant (Elephas maximus) population sustainability in North America. Clusters of cases have also been reported in African elephants (Loxodonta africana). Risk to range country elephant populations is unknown. Currently, EEHV detection depends upon sampling elephants trained for invasive blood and trunk wash collection. To evaluate noninvasive sample collection options, paired invasively collected (blood, trunk wash and oral swabs), and noninvasively collected (chewed plant and fecal) samples were compared over 6 wk from 9 Asian elephants and 12 African elephants. EEHV shedding was detected simultaneously in a paired trunk wash and fecal sample from one African elephant. Elephant γ herpesvirus-1 shedding was identified in six chewed plant samples collected from four Asian elephants. Noninvasively collected samples can be used to detect elephant herpesvirus shedding. Longer sampling periods are needed to evaluate the clinical usefulness of noninvasive sampling for EEHV detection.

Elephant Endotheliotropic Herpesvirus Hemorrhagic Disease in Asian Elephant Calves in Logging Camps, Myanmar.

In recent years, an alarming number of cases of lethal acute hemorrhagic disease have occurred in Asian elephant calves raised in logging camps in Myanmar. To determine whether these deaths were associated with infection by elephant endotheliotropic herpesvirus (EEHV), we conducted diagnostic PCR subtype DNA sequencing analysis on necropsy tissue samples collected from 3 locations. We found that EEHV DNA from 7 PCR loci was present at high levels in all 3 calves and was the same EEHV1A virus type that has been described in North America, Europe, and other parts of Asia. However, when analyzed over 5,610 bp, the strains showed major differences from each other and from all previously characterized EEHV1A strains. We conclude that these 3 elephant calves in Myanmar died from the same herpesvirus disease that has afflicted young Asian elephants in other countries over the past 20 years.

Evidence of high EEHV antibody seroprevalence and spatial variation among captive Asian elephants (Elephas maximus) in Thailand.

BACKGROUND: Elephant endotheliotropic herpesviruses (EEHV) can cause an acute highly fatal hemorrhagic disease in young Asian elephants (Elephas maximus), both ex situ and in situ. Amongst eight EEHV types described so far, type 1 (subtype 1A and 1B) is the predominant disease-associated type. Little is known about routes of infection and pathogenesis of EEHV, and knowledge of disease prevalence, especially in range countries, is limited. METHODS: A large cross-sectional serological survey was conducted in captive elephants (n = 994) throughout Thailand using an EEHV-1A glycoprotein B protein antigen specific antibody ELISA. RESULTS: Antibody seroprevalence was 42.3%, with 420 of 994 elephants testing positive. Associations between seropositivity and potential risk factors for EEHV infection were assessed and included: elephant age, sex, camp cluster size, management type (extensive versus intensive), sampling period (wet vs. dry season) and location of camp (region). Univariable regression analysis identified management system and region as risk factors for the presence of EEHV antibodies in elephants, with region being significant in the final multivariable regression model. Prevalence was highest in the North region of the country (49.4%). CONCLUSIONS: This study produced baseline serological data for captive elephants throughout Thailand, and showed a significant EEHV burden likely to be maintained in the captive population.

Rapid and sensitive detection of elephant endotheliotropic herpesvirus 1 (EEHV1) in blood by loop-mediated isothermal amplification (LAMP).

Elephant endotheliotropic herpesvirus type 1 (EEHV1) is the most important causative agent of an acute fatal hemorrhagic disease in Asian elephants (Elephas maximus). We employed loop-mediated isothermal amplification (LAMP) to develop a rapid and simple detection method for EEHV1 in blood. When used to test 21 clinical samples collected in Japan, the EEHV1 assay correctly identified one positive and 20 negative clinical samples. It was observed that when samples were spiked with synthetic DNA plasmids including EEHV1 polymerase gene, the detection limit of the LAMP assay was 101.2 copies/µl and 100-fold higher than that of conventional PCR. These advantages of the LAMP assay for EEHV1 detection may facilitate better veterinary practices for treating elephants suffering from the acute disease.

Endotheliotropic herpesvirus infection in Asian elephants (Elephas maximus) of Assam, India.

BACKGROUND AND AIM: Elephant endotheliotropic herpesvirus (EEHV) is an emerging disease of elephant. Therefore, a study was conducted to know the actual status of the disease in Assam State of India. MATERIALS AND METHODS: A total of 289 Asian elephants of Assam were screened during 2 years of study from April 2017 to March 2019. The clinical symptoms of diseased as well as gross and histopathological changes of dead elephants were recorded for the diagnosis of the disease. Virus involved in the occurrence of the disease was confirmed by polymerase chain reaction (PCR). RESULTS: In the present study, a total of three elephant calves out of 22 were found positive to EEHV1A. On the other hand, three adult asymptomatic elephants were also found positive for EEHV1 on screening 267 captive Asian elephants of Assam. The amplified PCR product showed band size of 520, 600, and 930 bp. The PCR amplified product with size 600 bp had shown the gene sequence for EEHV1U77/HEL. Gross lesions include congested blood vessels of the liver and intestinal mucosa, foci of petechiae in the spleen, and heart and focal ulceration in the dorsal surface of the tongue. Microscopically, the kidneys showed intertubular edema and focal areas of degeneration associated with coagulative necrosis of the tubular epithelium. The liver showed hydropic degeneration and fatty changes of the hepatocytes. There was a massive proliferation of fibroblasts in the interlobular spaces which penetrated the necrosed areas of the hepatic lobules. CONCLUSION: A total of three wild rescued elephant calves and three asymptomatic adults were found positive for EEHV1A during the 2 years of study. The PCR amplified product with size 600 bp had shown the gene sequence for EEHV1U77/HEL.