Efficacy of combination of Lacticaseibacillus rhamnosus and Bifidobacterium bifidum probiotic strains against mixed infections of bovine Rotavirus A and Cryptosporidium parvum induced diarrhea by modulating host immune response in mouse model.

Neonatal calf diarrhea is a serious health issue in dairy calves with significant economic losses. The immature gut immunity and the inadequate passive transfer of immunity in neonates favour the transmission and replication of the bovine rotavirus A (RVA) and the apicomplexan Cryptosporidium spp., and others, causing significant morbidity and mortality. To prevent such losses against these major pathogens, the efficacy of two-strain probiotics, namely Lacticaseibacillus (L.) rhamnosus strain NCDC 610 and Bifidobacterium (B.) bifidum strain BbVK3, were explored in a laboratory mouse model. A total of 120 seven-day-old Swiss albino suckling mice were grouped into six: Gr I (pups gavaged with RVA, 106.5TCID50/ml @ 100 μl/pup on 0-day), Gr II (gavaged with C. parvum oocysts @ 105 on 0-day), Gr III (pups infected with RVA on 0-day followed by C. parvum oocysts @ 105 after 36 h of infection), Gr IV (pups pre-treated with10 μl of cell-free supernatant of two-strain probiotic mix (L. rhamnosus strain NCDC 610 @ 108 CFU/ml and B. bifidum strain BbVK3 @ 108 CFU/ml) from 2 days prior to mixed infection till 7 day post-infection with RVA and C. parvum), Gr V (two-strain probiotic mix from day one to day 7 post-mixed infections), and Gr VI (sterile PBS). The mice were observed daily for the clinical signs of diarrhea and 4 mice from each group were sacrificed on 3, 5, 7, 11, and 16 day post-infection for sample collection. The samples were analyzed for the counting of the C. parvum oocyst in the feces, gross and histopathological lesions in the ileum, RVA antigen in the distal ileum by immunohistochemistry, cellular apoptosis by TUNEL assay, bacterial loads in the ileocaecal and rectal contents by culturing, RVA quantification and expression levels of TLR-2, TLR-3, TNF-α, MUC-2, INF-γ, IL-12, TGF-β, and IL-10 genes in distal ileum by the Real time-PCR, and the sIgA in fecal contents and serum by ELISA. The prophylactic group (Gr IV) showed reduced severity and duration of diarrhea, reduced severity of intestinal lesions and apoptosis, reduced viral load and oocyst count, reduced levels of TNF-α, and higher Il-10, and TGF-β levels, higher levels of innate immune response (higher levels of INF-γ, TLR-3, TLR-2, and reduced levels of Il-12), and up-regulation of MUC-2 genes as compared to infected (Grs 1, II, III) and therapeutic (Gr V) groups. The study showed promising prophylactic potential of the two-strain probiotic mix of L. rhamnosus strain NCDC 610 and B. bifidum strain BbVK3 against the mixed infections of rotavirus A and C. parvum.