Effective prophylaxis against rotavirus diarrhea using a combination of Lactobacillus rhamnosus GG and antibodies

Abstract

Background: Rotavirus is a worldwide cause of infectious infantile diarrhea that claims over 600,000 lives annually. Recently, two new vaccine candidates have been developed but their efficacy in developing countries, still remains to be proven. Oral delivery of specific immunoglobulins provides passive immunity and is a fast acting treatment for rotavirus diarrhea. Probiotic bacteria have also gained considerable attention lately as treatment for rotavirus diarrhea. Here we report an evaluation of the therapeutic potential of different probiotics and their combination with anti - rotavirus antibodies in a mouse model of rotavirus diarrhea.

Results: Of the six probiotic bacteria tested, Lactobacillus rhamnosus strain GG had the strongest influence in reducing prevalence, duration and severity of diarrhea and was therefore chosen for combination treatment with immunoglobulins. The combination treatment reduced the diarrhea outcome measures significantly, prevented histopathological changes and reduced the virus load in the intestines.

Conclusion: The advantages associated with immunoglobulins and probiotics based therapy is that the treatment provides a rapid therapeutic effect and is cost efficient. These components do not require special storage conditions and could potentially complement the rehydration therapy that is currently used.

Figures

Figure 1

Reactivity of Hyperimmune bovine colostrums (HBC) against RRV. HBC preparation is highly reactive against RRV as assessed by ELISA. ELISA plates were coated with RRV and HBC was added in different dilutions. The reaction was developed using anti-bovine AP conjugated secondary antibody. Control colostrums preparation (Imulin®) does not show any cross-reactivity with RRV even at high concentrations.

Figure 2

Optimization of the dose for oral treatment with probiotic bacteria using L. paracasei [15] as a reference strain. L. paracasei [15] was fed in different doses and the pups challenged with RRV on day 0. 108 CFU of bacteria was selected as the optimal dose for subsequent treatment with probiotic bacteria.

Figure 3

Anti-rotavirus HBC combination treatment with different species of Lactobacilli. Mice were fed lactobacilli daily, either alone or supplemented with 10 μg anti-rotavirus HBC and challenged with RRV on day 0. Diarrhea prevalence was recorded every day and is presented as percentage prevalence. Test of significance was performed using Fischer's exact test. (A) L. paracasei [15], in combination with HBC, was able to achieve a 58% better protection than that imparted by bacteria alone on day 2. On day 3 the combination treatment achieved statistically significant reduction in diarrhea prevalence in comparison to infected but untreated mice (p = 0.001) and 53% better protection than L. paracasei [15] alone. (B) L. rhamnosus GG alone caused a significant reduction in diarrhea prevalence in comparison to untreated mice on day 3 (p = 0.009) and in combination with HBC this effect was further enhanced (p = 0.003). (C) On day 3, protection conferred by combined therapy of L. reuteri strain SD2112 with 10 HBC μg was statistically significant in comparison to untreated mice (p = 0.035). (D) Administration of either L. paracasei strain NCC 2461 alone or supplemented with anti-rotavirus HBC did not change the diarrhea profile which resembled that of the untreated mice.

Figure 4

Hematoxylin/Eosin stained sections of jejunum from mice treated with different formulations. Tissue sections were excised and embedded in paraffin. HE staining was performed by standard protocols and the samples assessed blindly for signs of rotavirus infection. (A) infected and untreated mice shows typical signs of grave rotavirus infection with swollen and vacuolized villus tips. (B) L. rhamnosus GG treated mice show moderately resolved histopathology similar to (C) 10 μg/dose HBC treated mice. (D) L. rhamnosus GG combined with HBC is able to resolve the histopathology to normalcy. (E) no histopathology associated with treatment with 100 μg/dose HBC. (F) uninfected control mice.

Figure 5

Real time PCR of intestinal tissue sections for RRV vp7 gene. Tissue samples were excised from small intestines and total cellular RNA was extracted. Real time PCR was performed for rotavirus vp7 gene. The bars represent geometric mean of the virus load after normalization with housekeeping gapdh gene. The combination of 10 μg HBC and L. rhamnosus GG was able to achieve a statistically significant reduction of virus load (as tested by Fischer's test). *** p < 0.0001, ** p = 0.0005, * p = 0.0016