Immunohistochemical and molecular detection of natural cases of bovine rotavirus and coronavirus infection causing enteritis in dairy calves

Abstract

Bovine rotavirus (BRoV) and bovine coronavirus (BCoV) are major enteric viral pathogens responsible for calve diarrhoea. They are widespread both in dairy and beef cattle throughout the world and causing huge economic losses. The diagnosis of these agents is very difficult due to non-specific nature of lesions and the involvement of some intrinsic and extrinsic risk factors. We performed postmortem of 45 calves, which was below three months of age. Out of 45 necropscid calves, three (6.66%) cases were positive for BRoV and four (8.88%) cases were found positive for BCoV, screened by reverse transcriptase polymerase chain reaction (RT-PCR). Further RT-PCR positive cases were confirmed by immunohistochemistry (IHC) in paraffin-embedded intestinal tissue sections. Three cases of enteritis caused by BRoV showed the hallmark lesions of the shortening and fusion of villi, denudation and infiltration of mononuclear cells in the lamina propria. The BRoV antigen distribution was prominent within the lining epithelium of the villi, peyer's patches in the ileum and strong immunoreactions in the lymphocytes and some macrophages of the mesenteric lymph nodes. Four cases in which BCoV was detected, grossly lesions characterized by colonic mucosa covered with thick, fibrinous and diphtheritic membrane. Histopathologically, jejunum showed skipping lesion of micro-abscesses in crypts. The BCoV antigen distribution was prominent within the necrotic crypts in the jejunum and cryptic micro-abscesses in the colon and ileum. It is the first report of BRoV and BCoV antigen demonstration in the jejunum, colon, ileum, Peyer's patches and mesenteric lymph nodes of naturally infected calves from India by using IHC.

Keywords: Coronavirus; Immunohistochemistory; RT-PCR; Rotavirus.

Fig. 1

PCR amplification of VP6 gene of RVA showing 227 bp size amplicon in 1.5% agrose gel. Lane 1 loaded 1 kb plus DNA ladder; samples in lane 2, 3 and 4 are positive samples, lane 5 as non template control.

Fig. 2

PCR amplification of N gene of bovine coronavirus showing 172 bp size amplicon in 1.5% agrose gel. Lane 2 loaded 1 kb plus DNA ladder, samples in lane 3, 4, 5 and 6 are positive samples and lane 1 as positive control plasmid 172 bp.

Fig. 3

Histopathology of rotavirus infected calves: Paraffin sections of a calf (41A/17, 537A/16) stained with H & E stain showing exfoliation of villi, capillary plexus engorgement, moderate infiltration in the lamina propria (a) and Peyer's patches depletion (b) in the ileum. The section of mesenteric lymph node showing edema/congestion, medullary cords and sinuses infiltrated with MNCs (c). H&E.

Fig. 4

Immunohistochemistry of bovine rotavirus infection in bovine calves:a) Antibody control; b)The RVA antigen distribution in calves (41A/17, 537A/16) is seen as reddish brown signals (DAB as chromogen, arrows) in paraffin sections of mesenteric lymph nodes(b) (MLN) (Inset, 400X) and c) Peyer's patches (PP), Mayer's haematoxylin used as counter stain.

Fig. 5

Gross and histopathological lesions of corona virus infected calf: Gross image of the colon (40A/18) showing thick necrotic bran -like diphtheritic deposits over the mucosa (a, arrow). Microscopically, thick homogeneous necrotic material is seen over the ulcerated mucosa of the colon (b, arrows). Crypt lining cells are necrotic (c, arrows) and the lamina propria infiltrated with MNCs. Microabscesses in the crypt (c, inset). Mesenteric lymph nodes showed moderate depletion of many cortical follicles with thick capsule (d). H & E.

Fig. 6

Immunohistochemistry of bovine coronavirus in tissues section of calves: a) antibody control). The bovine coronavirus (BCoV) antigen distribution is seen as dark brown (DAB as chromogen) positive signals in lining epithelial cells of villi and necrotic crypts in the jejunum (thin arrows, b) and microabscesses (thick arrow, c) respectively and d) In MNCs of mesenteric lymph nodes (MLN) also showed positive signals of BCoV antigen (arrows) (inset, antibody control no signals).