Impact of nutrition and rotavirus infection on the infant gut microbiota in a humanized pig model

Abstract

Background: Human rotavirus (HRV) is a major cause of viral gastroenteritis in infants; particularly in developing countries where malnutrition is prevalent. Malnutrition perturbs the infant gut microbiota leading to sub-optimal functioning of the immune system and further predisposing infants to enteric infections. Therefore, we hypothesized that malnutrition exacerbates rotavirus disease severity in infants.

Methods: In the present study, we used a neonatal germ free (GF) piglets transplanted with a two-month-old human infant's fecal microbiota (HIFM) on protein deficient and sufficient diets. We report the effects of malnourishment on the HRV infection and the HIFM pig microbiota in feces, intestinal and systemic tissues, using MiSeq 16S gene sequencing (V4-V5 region).

Results: Microbiota analysis indicated that the HIFM transplantation resulted in a microbial composition in pigs similar to that of the original infant feces. This model was then used to understand the interconnections between microbiota diversity, diet, and HRV infection. Post HRV infection, HIFM pigs on the deficient diet had lower body weights, developed more severe diarrhea and increased virus shedding compared to HIFM pigs on sufficient diet. However, HRV induced diarrhea and shedding was more pronounced in non-colonized GF pigs compared to HIFM pigs on either sufficient or deficient diet, suggesting that the microbiota alone moderated HRV infection. HRV infected pigs on sufficient diet showed increased microbiota diversity in intestinal tissues; whereas, greater diversity was observed in systemic tissues of HRV infected pigs fed with deficient diet.

Conclusions: These results suggest that proper nourishment improves the microbiota quality in the intestines, alleviates HRV disease and lower probability of systemic translocation of potential opportunistic pathogens/pathobionts. In conclusion, our findings further support the role for microbiota and proper nutrition in limiting enteric diseases.

Keywords: Humanized pig; Malnutrition; Microbiota; Protein diet; Rotavirus.

Fig. 1

Microbiota data of HIFM transplantation into GF pigs at PTD7. a Microbiota relative abundance at the phylum level. b Microbiota comparison between the HIFM original sample and HIFM pig samples. In orange is the microbiota shared between original HIFM and HIFM pig feces and intestinal tissues; In purple are the unique OTUs detected only in HIFM pigs; and in grey are the OTUs undetected for the designated sample. HIFM and HIFM F: original HIFM feces; F: HIFM pigs feces; D: HIFM pigs duodenum; J: HIFM pigs jejunum; I: HIFM pigs ileum; C: HIFM pigs colon

Fig. 2

Schematics of animal experiment indicating times of HIFM transplantation, HRV challenge, and samples collection. Pigs were transplanted at 4 days of age, challenged at 14 days with 10⁶ FFU/pig of HRV Wa(G1P [8]) human strain, and euthanized at 32 days of age (dotted arrows). Tissues sampling and measurement of clinical parameters were indicated by solid arrows. HIFM-Human infant fecal microbiota; PTD-Post transplant days; PCD-Post HRV challenge days

Fig. 3

Impact of diet and the microbiota on body weight gain, diarrhea, and HRV shedding. a Body weight gain; a, b c, sufficient diet pig groups had significantly higher body weight gain than the deficient groups for HIFM+HRV challenged, GF+HRV, or HIFM+No HRV pigs respectively (P < 0.01). Bars represent standard errors. b Severity of diarrhea after HRV challenge. In black, pigs nourished with a sufficient diet; in grey, pigs nourished with a deficient diet. Diarrhea scoring was based on the phenotypic scale from 0 to − 3, where zero was for solid feces; − 1 when feces were pasty; − 2 when feces were semi-liquid; and − 3 when feces were liquid. -1.5 was the threshold where any values below were considered as typical diarrhea symptoms. a or b: deficient diet group had significantly lower diarrhea score than the sufficient diet group for the HIFM or GF pigs respectively; c: the sufficient diet HIFM group had significantly lower diarrhea score than the deficient diet HIFM group (P < 0.05). Bars represent standard errors. c HRV shedding. The letters a, b, or c indicate the cumulative log (CCFU) of the designated group significantly higher than the Deficient+GF+HRV, Sufficient+GF+HRV, or Sufficent+HIFM+HRV group at the corresponding time point, respectively (P < 0.05). Bars represent standard errors

Fig. 4

Beta diversity analysis of fecal samples before and after HRV challenge from HIFM pigs on a deficient or sufficient diets. a Relative abundance at the phylum level. b Microbiota diversity over time between sufficient and deficient diets based on the OTU assignment after open OTU picking with the Greengene database. PTD- Post HIFM transplant day; PCD- Post HRV challenge day

Fig. 5

Impact of the diets on fecal microbiota of HIFM+HRV pigs. The relative abundance between deficient and sufficient diets for a given time point is shown. Results are represented via a phylogenetic tree (Graphlan), combined with relative abundance data. Labeled in red are the bacteria detected in higher abundance in deficient diet; while in green are the bacteria detected in higher abundance in sufficient diet. The labeling of the taxonomic levels from the outside (phylum) to the inside (genus), while the tree start (root) from the center and goes outside. Nodes are indicated by a circle. Bacteria (node) more abundant in one of the diets is shown in red or green, no change is shown in gold. Bacteria are designated with alphabet in red or green corresponding to the node

Fig. 6

Beta diversity analysis of intestinal samples from HIFM+HRV and HIFM+No HRV pigs nourished with deficient or sufficient diet. Comparison of relative abundances at the phylum level between diets for the HIFM+HRV (a) and HIFM+No HRV (b) pigs. Microbiota diversity in intestinal tissue samples based on diet (c) or HRV challenge status (d). OTUs were assigned by open OTU picking with the Greengene database. D: Duodenum; J: Jejunum; I: Ileum; C: Colon; HRV: HRV challenged tissue; Non-HRV: Non-HRV challenged tissue

Fig. 7

Impact of the diets on the intestinal microbiota in HIFM+HRV pigs. The relative abundance between deficient and sufficient diets for a given tissue is shown. Results are represented via a phylogenetic tree (Graphlan), combined with relative abundance data. Labeled in red are the bacteria detected in higher abundance in deficient diet; while in green are the bacteria detected in higher abundance in sufficient diet. The labeling of the taxonomic levels from the outside (phylum) to the inside (genus), while the tree start (root) from the center and goes outside. Nodes are indicated by a circle. Bacteria (node) more abundant in one of the diets is shown in red or green, no change is shown in gold. Bacteria are designated with alphabet in red or green corresponding to the node

Fig. 8

Beta diversity analysis of systemic tissues samples from HIFM+HRV and HIFM+No HRV pigs nourished with a deficient or sufficient diet. Comparison of relative abundances at the phylum level between diets for the HIFM+HRV (a) and HIFM+No HRV (b) pigs. Microbiota diversity in systemic tissues samples based on diet (c) or HRV challenge status (d). OTUs were assigned by open OTU picking with the Greengene database. HRV: HRV challenged tissue; Non-HRV: Non-HRV challenged tissue

Fig. 9

Impact of the diets on the systemic tissues microbiota of HIFM+HRV pigs. The relative abundance between deficient and sufficient diets for a given tissues is shown. Results are represented via a phylogenetic tree (Graphlan), combined with relative abundance data. Labeled in red are the bacteria detected in higher abundance in deficient diet; while in green are the bacteria detected in higher abundance in sufficient diet. The labeling of the taxonomic levels from the outside (phylum) to the inside (genus), while the tree start (root) from the center and goes outside. Nodes are indicated by a circle. Bacteria (node) more abundant in one of the diets is shown in red or green, no change is shown in gold. Bacteria are designated with alphabet in red or green corresponding to the node