Patho-epidemiology of certain enteric viral pathogens in dairy calves and immunopathology of Rotavirus A infection in experimental mouse model

Diarrhoea in calves is a multifactorial disease, several infectious and noninfectious factors responsible for calf diarrhoea. The rotavirus diarrhoea occurs either single or in combination with other pathogens - like Coronavirus, Cryptosporidium spp., Escherichia coli pathotypes. In the present study, a total of 816 samples, comprising of 771 faecal and 45 intestinal tissue of ≤3- month -old bovine calves belonging to 5 districts of MP, one district of CG and 4 districts of UP were analyzed for the presence of microbial pathogens. Forty one (5.02%) cases (diarrhoeal and non-diarrhoeal) were tested positive for the group A rotavirus (RVA) and 7 (0.85%) cases positive for coronavirus by antigen ELISA test. Highest prevalence of RVA was noted in Pratapgarh district (10%) of UP, Durg district (9.67 %) of CG and Rewa district (5.35%) of MP. Coronavirus prevalence was higher in Indore district of MP. Out of total 438 diarrhoeal samples (393 faecal samples and 45 intestinal contents), 33 (7.53%) cases were positive for rotavirus, 32 (7.30 %) cases positive for the cryptosporidium oocysts (by MZN staining), and 6 (1.36%) cases positive for coronavirus. Rotavirus occurrence was maximum in 21–30 days -old calves, thereafter it decreased with age. Occurrence of coronavirus was found increasing with advancement of age. Age-wise occurrence of cryptosporidium was higher in 0–20 days of age and declined with increase in the age of calves. Parity-wise occurrence of rotavirus, coronavirus and cryptosporidium was high in first parity, than it decreased correspondingly with parity. Rotavirus occurrence was high in pasty yellow faecal samples, but cryptosporidium and coronavirus occurrence was high in watery diarrhoea. Occurrence of rotavirus was highest in winter season, but for cryptosporidium and coronavirus it was highest in monsoon season. In addition, cryptosporidium was also concurrently associated in two cases of rotavirus positive samples and in one case of coronavirus positive sample. Interestingly, no co-infection of rotavirus and coronavirus was found in the present study. In 45 screened carcasses of bovine calves, enteritis due to RVA was detected in 2 calves and enteritis due to coronavirus in 2 calves. Enteritis was recorded due to E. coli patho-types (eae, bfp and fimA gene by PCR) in 12 calve, enteritis due to Staphylococcus spp in 5 calves, enteritis due to both E.coli and P. multocida mixed infectionin 10 calves, parasitic enteritis in 3 calves and enteritis due to miscellaneous reasons in 11 calves. In the experimental study, 30 Swiss albino suckling mice inoculated with dose of 100μl (TCID₅₀/ml, 5x10⁶.⁵) of RVA/mouse were kept as the Infected Group. The similar treatment was given to 15 suckling mice with sterile PBS and kept as the Control Group. The pups were sacrificed at 1, 2, 4, 7 and 14 DPI. No clinical sing of diarrhoea during the entire period of the experiment was observed in mice however, these micewere found dull/depressed at 1ˢᵗ, 2ⁿᵈ and 4ᵗʰ DPI. The faeces were found positive for RVA at 1ˢᵗ DPI to 7ᵗʰ DPI in the infected group. The sIgA and IgG was quantified in the rectal contents by using mouse immunoglobulin A (IgA) sandwich ELISA kit. The level of sIgA and IgG in rectal contents showed significantly decreasing trends from 2ⁿᵈ DPI to 7ᵗʰ DPI in the Infected Group of mice as compared to that of the Control Group. However, the levels of IgG in sera of suckling mice did not show any significant difference between the Infected Group and the Control Group of mice at all time points of sacrifice. On morphometrical analysis of the jejunum and the ileum showed, decreasing trends of VH (Villous height), VCR (Villi Crypt ratio) and VW (Villous width) from 2ⁿᵈ dpi to 14ᵗʰ dpi in the Infected Group of mice as compared with the Control Group. However, CD (Crypt depth) in the jejunum and the ileum showed increasing trends from 2ⁿᵈ dpi to 14ᵗʰ dpi in the Infected Group of mice. The H & E stained sections of the small intestines were scored for the presence of lesions viz., vacuolization of enterocytes, cellular infiltration in the lamina propria and the cryptic glands hyperplasia. The HPS (Histo-pathological score) of the jejunum and the ileum showed increasing trends from 1ˢᵗ DPI to 7ᵗʰ DPI in the Infected Group of mice, after that HPS became normal as in the Control Group of mice. The lesion was more sever in the jejunum at initial stage after that it became more severe in the ileum. In the present study RVA, antigen was demonstrated in the jejunum as well as in the ileum, at the tip of villi, base of villi, crypts as well as in the Peyer‘s patches by IHC. Relative expression (By Real-Time PCR) of TNF-α in the ileum of the Infected Group showed trends of up- regulation till 7ᵗʰ DPI. The IL-12 expression in the Infected Group of mice rose upto 7ᵗʰ DPI. The expression of IL- 10mRNA in the Infected Group of mice showed rising trend from 1ˢᵗ DPI to 14ᵗʰ DPI. Overall, the expression of IFN-ymRNA in the Infected Group of mice remained lower from day 1ˢᵗ DPI to 14ᵗʰ DPI.