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A Longitudinal Study of Hematology and Stress Biomarker Profiles in Young Asian Elephants (Elephas Maximus) in Relation to Elephant Endotheliotropic Herpesvirus (EEHV) in Thailand.

ABSTRACT: 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.

SUBMITTER: Boonprasert K

PROVIDER: S-EPMC8471699 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Pathogenesis of hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV) in Asian elephants (Elephas maximus).

Project description:Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is an acute fatal disease in elephants. Despite the fact that the underlying pathogenesis of EEHV-HD has been proposed, it remains undetermined as to what mechanisms drive these hemorrhagic and edematous lesions. In the present study, we have investigated and explained the pathogenesis of acute EEHV-HD using blood profiles of EEHV-HD and EEHV-infected cases, hematoxylin and eosin (H&E) stain, special stains, immunohistochemistry, quantitative polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR). It was found that EEHV genomes were predominantly detected in various internal organs of EEHV-HD cases. Damage to endothelial cells, vasculitis and vascular thrombosis of the small blood vessels were also predominantly observed. Increases in platelet endothelial cell adhesion molecules-1 (PECAM-1)- and von Willebrand factor (vWF)-immunolabeling positive cells were significantly noticed in injured blood vessels. The expression of pro-inflammatory cytokine mRNA was significantly up-regulated in EEHV-HD cases when compared to EEHV-negative controls. We have hypothesized that this could be attributed to the systemic inflammation and disruption of small blood vessels, followed by the disseminated intravascular coagulopathy that enhanced hemorrhagic and edematous lesions in EEHV-HD cases. Our findings have brought attention to the potential application of effective preventive and therapeutic protocols to treat EEHV infection in Asian elephants.

| S-EPMC8217522 | biostudies-literature

Possible roles of monocytes/macrophages in response to elephant endotheliotropic herpesvirus (EEHV) infections in Asian elephants (Elephas maximus).

Project description:Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the primary cause of acute, highly fatal, hemorrhagic diseases in young Asian elephants. Although monocytopenia is frequently observed in EEHV-HD cases, the role monocytes play in EEHV-disease pathogenesis is unknown. This study seeks to explain the responses of monocytes/macrophages in the pathogenesis of EEHV-HD. Samples of blood, frozen tissues, and formalin-fixed, paraffin-embedded (FFPE) tissues from EEHV1A-HD, EEHV4-HD, co-infected EEHV1A and 4-HD, and EEHV-negative calves were analyzed. Peripheral blood mononuclear cells (PBMCs) from the persistent EEHV4-infected and EEHV-negative calves were also studied. The results showed increased infiltration of Iba-1-positive macrophages in the inflamed tissues of the internal organs of elephant calves with EEHV-HD. In addition, cellular apoptosis also increased in the tissues of elephants with EEHV-HD, especially in the PBMCs, compared to the EEHV-negative control. In the PBMCs of persistent EEHV4-infected elephants, cytokine mRNA expression was high, particularly up-regulation of TNF-? and IFN-?. Moreover, viral particles were observed in the cytoplasm of the persistent EEHV4-infected elephant monocytes. Our study demonstrated for the first time that apoptosis of the PBMCs increased in cases of EEHV-HD. Furthermore, this study showed that monocytes may serve as a vehicle for viral dissemination during EEHV infection in Asian elephants.

| S-EPMC6730851 | biostudies-literature

In vivo characterization of target cells for acute elephant endotheliotropic herpesvirus (EEHV) infection in Asian elephants (Elephas maximus).

Project description:Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

| S-EPMC7347588 | biostudies-literature

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

Project description: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.

| S-EPMC9312089 | biostudies-literature

Elephant endotheliotropic herpesvirus 5, a newly recognized elephant herpesvirus associated with clinical and subclinical infections in captive Asian elephants (Elephas maximus).

Project description:Elephant endotheliotropic herpesviruses (EEHVs) can cause acute hemorrhagic disease with high mortality rates in Asian elephants (Elephas maximus). Recently, a new EEHV type known as EEHV5 has been described, but its prevalence and clinical significance remain unknown. In this report, an outbreak of EEHV5 infection in a herd of captive Asian elephants in a zoo was characterized. In February 2011, a 42-yr-old wild-born female Asian elephant presented with bilaterally swollen temporal glands, oral mucosal hyperemia, vesicles on the tongue, and generalized lethargy. The elephant had a leukopenia and thrombocytopenia. She was treated with flunixin meglumine, famciclovir, and fluids. Clinical signs of illness resolved gradually over 2 wk, and the white blood cell count and platelets rebounded to higher-than-normal values. EEHV5 viremia was detectable starting 1 wk before presentation and peaked at the onset of clinical illness. EEHV5 shedding in trunk secretions peaked after viremia resolved and continued for more than 2 mo. EEHV5 trunk shedding from a female herd mate without any detectable viremia was detected prior to onset of clinical disease in the 42-yr-old elephant, indicating reactivation rather than primary infection in this elephant. Subsequent EEHV5 viremia and trunk shedding was documented in the other five elephants in the herd, who remained asymptomatic, except for 1 day of temporal gland swelling in an otherwise-healthy 1-yr-old calf. Unexpectedly, the two elephants most recently introduced into the herd 40 mo previously shed a distinctive EEHV5 strain from that seen in the other five elephants. This is the first report to document the kinetics of EEHV5 infection in captive Asian elephants and to provide evidence that this virus can cause illness in some animals.

| S-EPMC3746547 | biostudies-literature

Endotheliotropic herpesvirus infection in Asian elephants (<i>Elephas maximus</i>) of Assam, India.

Project description:Background and aimElephant 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 methodsA 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).ResultsIn 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.ConclusionA 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.

| S-EPMC6925033 | biostudies-literature

Identification of shedders of elephant endotheliotropic herpesviruses among Asian elephants (Elephas maximus) in Switzerland.

Project description:Elephants, particularly Asian (Elephas maximus), are threatened by lethal elephant hemorrhagic disease (EHD) due to elephant endotheliotropic herpesviruses (EEHV). At least five of seven known EEHV types have been associated to EHD, with types 1, 4, and 5 predominantly affecting Asian elephants. In Switzerland, at least three Asian elephants have been lost due to EHD but nothing is known about the present EEHV1 circulation. Moreover, the prevalence of other EEHV types has never been assessed. Intermittent shedding of EEHV can be monitored through collecting trunk secretions and analyzing them by PCR methods that discriminate the different EEHV types. To identify EEHV shedders, seven of eight Asian elephants in a Swiss zoo were trained to provide trunk wash samples. These were collected at intervals over a period of four months and tested by PCR for presence of EEHV1 through 6. Moreover, the quality of each sample was assessed by testing for the elephant TNF-alpha gene. Overall, 57% of the samples were valid with five of seven participating elephants identified as EEHV shedders. Two of those shed virus only once, whereas the other three, all closely related among each other, shed virus on multiple occasions. One of the frequent shedders had been in very close contact to all of the three EHD victims. Therefore, we speculate that this particular animal may represent the virus source in all three cases. However, when subtyping was conducted, the presently circulating virus was identified as EEHV1B, while the virus subtype causing EHD had been 1A in all three cases. In addition to four animals excreting EEHV1, a recently introduced animal was observed to shed EEHV3/4. We suggest that the policy of trunk washing to identify and characterize EEHV-shedders is to be endorsed in zoos with ongoing or planned elephant breeding programs.

| S-EPMC5415103 | biostudies-literature

Kinetics of viral loads and genotypic analysis of elephant endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus).

Project description:Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal hemorrhagic disease in juvenile Asian elephants (Elphas maximus); however, sporadic shedding of virus in trunk washes collected from healthy elephants also has been detected. Data regarding the relationship of viral loads in blood compared with trunk washes are lacking, and questions about whether elephants can undergo multiple infections with EEHVs have not been addressed previously. Real-time quantitative polymerase chain reaction was used to determine the kinetics of EEHV1 loads, and genotypic analysis was performed on EEHV1 DNA detected in various fluid samples obtained from five Asian elephants that survived detectable EEHV1 DNAemia on at least two separate occasions. In three elephants displaying clinical signs of illness, preclinical EEHV1 DNAemia was detectable, and peak whole-blood viral loads occurred 3-8 days after the onset of clinical signs. In two elephants with EEHV1 DNAemia that persisted for 7-21 days, no clinical signs of illness were observed. Detection of EEHV1 DNA in trunk washes peaked approximately 21 days after DNAemia, and viral genotypes detected during DNAemia matched those detected in subsequent trunk washes from the same elephant. In each of the five elephants, two distinct EEHV1 genotypes were identified in whole blood and trunk washes at different time points. In each case, these genotypes represented both an EEHV1A and an EEHV1B subtype. These data suggest that knowledge of viral loads could be useful for the management of elephants before or during clinical illness. Furthermore, sequential infection with both EEHV1 subtypes occurs in Asian elephants, suggesting that they do not elicit cross-protective sterilizing immunity. These data will be useful to individuals involved in the husbandry and clinical care of Asian elephants.

| S-EPMC3746492 | biostudies-literature

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

Project description:BackgroundElephant 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.MethodsA 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.ResultsAntibody 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%).ConclusionsThis study produced baseline serological data for captive elephants throughout Thailand, and showed a significant EEHV burden likely to be maintained in the captive population.

| S-EPMC6415343 | biostudies-literature

Production of antibody against elephant endotheliotropic herpesvirus (EEHV) unveils tissue tropisms and routes of viral transmission in EEHV-infected Asian elephants.

Project description:Elephant endotheliotropic herpesvirus (EEHV) is one of the most devastating viral infectious diseases in elephants worldwide. To date, it remains unclear how elephants get infected by the virus, where the virus persists, and what mechanisms drive the pathogenesis of the disease. The present study was aimed to develop an antibody against glycoprotein B (gB) of EEHV, investigate the EEHV tissue tropisms, and provide the possible routes of EEHV transmission in Asian elephants. Samples from elephant organs that had died from EEHV1A and EEHV4 infections, peripheral blood mononuclear cells (PBMC) from EEHV4- and non-EEHV-infected calves were used in this study. The results of western immunoblotting indicated that the antibody can be used for detection of gB antigens in both EEHV1A- and EEHV4-infected samples. Immunohistochemical detection indicated that the EEHV gB antigens were distributed mainly in the epithelial cells of the salivary glands, stomach and intestines. Immunofluorescence test of PBMC for EEHV gB in the EEHV4-infected calf indicated that the virus was observed predominantly in the mononuclear phagocytic cells. The findings in the present study unveil tissue tropisms in the EEHV1A- and EEHV4-infected calves and point out that saliva and intestinal content are likely sources for virus transmission in EEHV-infected Asian elephants.

| S-EPMC5856810 | biostudies-literature

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