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.

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-α and IFN-γ. 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.

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.

An episode of serial mortality occurred in a population of wild-caught, aquarium-reared sharphead flyingfish Hirundichthys oxycephalus. Most diseased fish had ulcerative hemorrhagic lesions on the skin, especially on the head, and displayed multiple, fine, whitish nodules scattered on the internal organs, including the kidney, spleen and liver. Histopathological examination revealed the nodules to be granulomatous lesions consisting of epithelioid cells with a thin outermost rim of fibroblasts and connective tissue, and the central caseous core of the granulomas was associated with acid-fast bacilli. Though the granulomatous lesions were systemically present in the major internal organs, the most severe lesions were in the kidney and spleen. In addition, melanized fungal hyphae were also found in the kidney and spleen. Acid-fast bacterial strains were isolated from affected fish and most were identified as Mycobacterium marinum by molecular examination. A few dematiaceous fungal strains were also isolated from some fish and identified as Exophiala aquamarina by morphological and molecular examination. This is a novel description of a case of co-infection of a nontuberculous mycobacterium (M. marinum) and a dematiaceous fungus (E. aquamarina) in a flyingfish species.

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.

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.

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Running head: HISTIOCYTIC SARCOMA IN CIVET This case study had focused on a male, 7-year-old Asian palm civet (Paradoxurus hermaphroditus) with a history of biting its tail and the development of skin masses around its inguinal area, prior to its death. Macroscopically, multiple firm white nodular masses of 0.5-5 cm in diameter were found in the subcutis of the inguinal area, and in the lungs, spleen and liver. Microscopically, masses in the skin, lungs and spleen were composed of neoplastic spindle cells admixed with mononuclear cells and multinucleated giant cells. The neoplastic cells were arranged in a sheet pattern. Immunohistochemically, the neoplastic cells were immunohistochemically positive for vimentin, Iba-1, CD 204 and Human leukocyte antigen (HLA)-DR, while the cells were negative for cytokeratin and smooth muscle actin. Based on the histopathological and immunohistochemical results, disseminated histiocytic sarcoma was diagnosed.