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

Abstract

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.

Figure 1

Gross findings of elephant carcasses case 1 and case 4. Representative images of calves infected by EEHV1A (A–C) and EEHV4 (D–F). Infection of EEHV1A in the calves revealed petechial and ecchymotic hemorrhages with edema throughout the internal organs, including the subcutaneous tissue of the sublingual area (A), heart (B), and lymph node (C). EEHV4 infection in the calf resulted in mild hemorrhages of the heart (D), but caused severe hemorrhages and edema (arrowhead) in the submucosal and the subseroal areas of the stomach (E) and rectum (F).

Figure 2

SDS-PAGE of tissue lysates from EEHV1A-infected, EEHV4-infected, and non-EEHV-infected calves. SDS-PAGE gel of the elephant tissue lysates stained with Coomassie Blue (A) and immunostained with polyclonal anti-EEHV gB antibodies (B). The anti-EEHV gB antibodies demonstrated a positive signal against EEHV1A gB (lane 1 and lane 2) and EEHV4 gB (lane 3 and lane 4) at molecular weights of ~97 kDa and ~98 kDa, respectively, compared to the protein molecular weight marker (lane M), while they showed a negative signal in the elephant sample that died from non-EEHV-infection (lane 5). Lane M = protein molecular weight marker; lane 1 = total protein from case 1; lane 2 = total protein from case 2; lane 3 = total protein from case 3; lane 4 = total protein from case 4; and lane 5 = total protein from case 5.

Figure 3

Representative images of histopathological and immunohistochemical findings of the EEHV1A-infected calf. Sections of the heart (A–C), colon (D–F), spleen (G–I), and lymph nodes (J–L) of the EEHV1A-infected calf showed mild-to-moderate degree of hemorrhages with infiltration of lymphohistiocytic inflammatory cells. Immunohistochemical labeling with anti-EEHV gB antibodies revealed positive signals in the cytoplasm of the mononuclear phagocytic cells within the blood vessels of the heart (B,C) and spleen (H,I). The colon showed positive signals (arrowhead) in the cytoplasm of intact villi (E,F) and necrotic cells within the intestinal lumen. The lymph nodes showed rarely immunolabeling positive cells (arrowhead) of the lymphoid follicle (K,L). The negative controls were sections of EEHV1A-infected calves that were incubated with normal rabbit serum instead of rabbit anti-EEHV gB antibodies, or incubation of non-EEHV-infected tissue sections with rabbit anti-EEHV gB antibodies.

Figure 4

Histopathological and immunohistochemical findings of the salivary glands of EEHV1A-infected and EEHV4-infected calves. Representative images of sections from the salivary gland of the EEHV1A-infected calf (A,C,E,G) and the EEHV4-infected calf (B,D,F,H). Infection with EEHV1A revealed moderate-to-severe, multifocal, lymphohistiocytic inflammation of the serous and the mucous acini, while less severity was observed in the EEHV4-infected calf (A,B). Immunohistochemical labeling of EEHV gB in the EEHV1A-infected calf indicated strong positive signals in the cytoplasm of the epitheliums of serous and mucous acini (C,E). EEHV gB immunolabeling positive cells were also shown to be observed in the cytoplasm of the cuboidal and columnar epithelial cells of the striated and the interlobular ducts (arrowhead, G,H). Some EEHV gB immunolabeling positive cells (arrow) were found within the lumen of the salivary ducts (G). The negative controls were sections of EEHV1A- and EEHV4-infected calves that were incubated with normal rabbit serum instead of rabbit anti-EEHV gB antibodies, or incubation of non-EEHV-infected tissue sections with rabbit anti-EEHV gB antibodies.

Figure 5

Representative images of histopathological and immunohistochemical findings of the EEHV4-infected calf. Sections of the stomach (A–C), cecum (D–F), colon (G–I), and rectum (J–L) of the EEHV4-infected calf showed severe hemorrhage and edema of the laminar propria and submucosa layer, with moderate degree of lymphohistiocytic cell infiltration. Immunohistochemical labeling with rabbit anti-EEHV gB revealed strong and diffused positive signals in the cytoplasm of the gastric mucosa (B,C), while less positive cells were observed in the cecum (E,F) and colon (H,I). The strong intensity of the positive signals in the basal and/or crypts of the intestinal epithelium of the rectum were noticeable (K,L). The negative controls were sections of EEHV4-infected calves that were incubated with normal rabbit serum instead of rabbit anti-EEHV gB antibodies, or incubation of non-EEHV-infected tissue sections with rabbit anti-EEHV gB antibodies.

Figure 6

Double immunofluorescence test and quantitative assessment of PBMCs from non-EEHV-infected and EEHV4-infected calves. PBMCs obtained from healthy, non-EEHV-infected calf and fresh carcass of an elephant that had died from EEHV4 infection were immunolabeled with mouse anti-Iba-1 (green in A) and rabbit anti-EEHV gB antibodies (red in A). The nuclei were detected using bisbensimide fluorescence stain (blue in A). The percentage of Iba-1 immunolabeling positive cells of PBMCs in the EEHV4-infected calf was significantly lower than that in the non-EEHV-infected calf (B). More than 50% of the Iba-1-positive cells were immunolabeling positive for anti-EEHV gB antibodies (C). Negative controls were PBMCs from non-EEHV-infected calf incubated with the anti-EEHV gB antibodies, or PBMCs from the EEHV4-infected calf incubated with normal rabbit serum. Asterisks indicate statistically significant difference between EEHV4-infected and non-EEHV-infected calves (p < 0.05).