<i>Firmicutes</i> to <i>Bacteroidetes</i> ratio.

<p><i>Firmicutes</i> to <i>Bacteroidetes</i> ratio in the colon contents (lumen) was significantly higher (ANOVA p = 0.0021, post-tests both p<0.01) in the traditional diet fed group treated with antibiotics compared with either defined nutrient diet with antibiotics.</p

Effects of nutritional status and diets on mortality, disease severity and infection burden in mice challenged with <i>C</i>. <i>difficile</i>.

<p>Effects of nutritional status and diets on mortality, disease severity and infection burden in mice challenged with <i>C</i>. <i>difficile</i>.</p

Defined Nutrient Diets Alter Susceptibility to <i>Clostridium difficile</i> Associated Disease in a Murine Model

<div><p>Background</p><p><i>Clostridium difficile</i> is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of <i>C</i>. <i>difficile</i> infection in a murine model.</p><p>Methods</p><p>C57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with <i>C</i>. <i>difficile</i> strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and <i>C</i>. <i>difficile </i>growth and toxin production were quantified <i>ex vivo </i>in intestinal contents from untreated or antibiotic-treated mice fed with the different diets.</p><p>Results</p><p><i>C</i>. <i>difficile </i>infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. <i>C</i>. <i>difficile</i> shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. <i>Ex vivo</i> inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and <i>C</i>. <i>difficile</i> growth in both defined diets compared with a traditional diet.</p><p>Conclusions</p><p>Low protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.</p></div

Treatment schedule for mice for the <i>in vivo</i> infection model (A) and <i>ex vivo C</i>. <i>difficile</i> inoculation studies (B).

<p>Treatment schedule for mice for the <i>in vivo</i> infection model (A) and <i>ex vivo C</i>. <i>difficile</i> inoculation studies (B).</p

<i>C</i>. <i>parvum</i> priming enhances Th1-type cytokine responses to re-challenge in protein malnourished mice.

<p>(A) Ileal inflammatory mediators and chemokines and (B) cytokines measured three days after 10⁷ <i>C</i>. <i>parvum</i> challenge in previously uninfected (PBS) compared with mice primed with 10⁶ <i>C</i>. <i>parvum</i> (Cp10^<i>6</i>) 20 days prior to re-challenge. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

<i>C</i>. <i>parvum</i> priming leads to sustained changes in ileal tissue chemokine and cytokine profiles during protein malnutrition.

<p>Mice were conditioned on PD for 5 days prior to infection with 10⁶ <i>C</i>. <i>parvum</i> (Cp10⁶). Luminex was performed for measurement of chemokines and cytokines in ileal tissues at. day 3 (D3) and day 23 (D23) post challenge compared to uninfected controls (PBS) (n = 3-4/group). (A) Primary <i>C</i>. <i>parvum</i> infection led to increases in CXCL9, CXCL10, CCL-3, CCL-5, and CCL11 on D3. On D23, TNFα, IL1β, and IL-8 were diminished in infected mice relative to uninfected controls, however, CCL-5 continued to be elevated and other chemokines had returned to baseline. (B) Only IL12p40 and IL-13 were modestly elevated three days after primary <i>C</i>. <i>parvum</i> challenge. There was a relative decrease in all Th2-type cytokines through 23 days post-<i>C</i>. <i>parvum</i> compared with uninfected controls. (n = 3-4/group). *<i>P</i><0.05 for PBS vs Cp10⁶ as indicated; #<i>P</i><0.05 for Cp10⁶ D3 vs Cp10⁶ D23 as indicated.</p

Protein malnutrition alters basal immune responses to primary <i>C</i>. <i>parvum</i> exposure, but secondary responses are intact.

<p>Immunologic responses to two different recombinant <i>Cryptosporidium</i> sporozoite antigens (CApy and Cp15) were performed at 13–15 days post <i>C</i>. <i>parvum</i> challenge in mice fed either control-diet (<i>C</i>. <i>parvum</i>^CD) or protein-deficient diet (<i>C</i>. <i>parvum</i>^pd) and results were compared with naïve age and diet-matched uninfected controls (PBS^CD and PBS^pd). Mice began respective diets 12 days prior to <i>C</i>. <i>parvum</i> challenge and remained on the same diets post-challenge. (A) Cytokine secretion in splenocytes of naïve (uninfected) CD or PD-fed mic after stimulation with <i>Cryptosporidium</i> antigens. (B) Serum antibody production as anti-CApy or anti-Cp15 IgG titer (<i>*P<</i>0.05). (C) Cytokines secreted after CApy or Cp15 antigen stimulation in (C) mesenteric lymph nodes. (D) Cytokine secretion in splenocytes expressed as fold change relative to CD-fed uninfected controls. (*<i>P</i><0.05 as indicated). Data is representative of pooled individual responses from two separate tissue harvests (n = 4-5/group).</p

Severe protein malnutrition in mice selectively enhances intestinal disruption and severity of cryptosporidiosis.

<p>(A) Experimental timeline. 3-week-old C57Bl/6 female mice were initiated on experimental malnutrition diets (RBD or PD) or control diet (CD) immediately upon receipt from supplier. Challenge with 10⁶ or 10⁷ <i>Cryptosporidium parvum</i> oocysts, heat-inactivated <i>C</i>. <i>parvum</i> oocysts (Δ<i>C</i>. <i>parvum</i>) or PBS occurred via oral gavage 5 days after initiating diet. Serial weights were collected daily post-challenge, and fecal parasite shedding was determined by RT-PCR. On day 3–4 post-challenge, tissue parasite burden and mucosal injury was assessed by measuring ileum villus:crypt ratios and alterations in epithelial tight-junction proteins. (B) Impact of diet on growth (**<i>P</i><0.01, ***<i>P</i><0.001 <i>C</i>. <i>parvum</i>^PD vs. <i>C</i>. <i>parvum</i> or <i>C</i>. <i>parvum</i>^RBD), fecal parasite shedding (**<i>P</i><0.01 PD or RBD vs. CD day 2, *<i>P</i><0.05 PD vs. RBD or CD day 3), tissue parasite burden (*<i>P</i><0.05 PD vs CD or RBD ileum, *<i>P</i><0.05 PD vs CD colon), and ileum villus:crypt ratios (**<i>P</i><0.01 PBS^CD vs PBS^RBD, ***<i>P</i><0.001 PBS^CD vs PBS^PD, ****<i>P</i><0.0001 <i>C</i>. <i>parvum</i>^CD or <i>C</i>. <i>parvum</i>^RBD vs <i>C</i>. <i>parvum</i>^PD, ^<i>P</i><0.05 PBS^CD vs. <i>C</i>. <i>parvum</i>^CD, ^##<i>P</i><0.01 PBS^PD vs. <i>C</i>. <i>parvum</i>^PD (n = 3-7/group). (C) Growth through three days post-challenge with either <i>C</i>. <i>parvum</i> or Δ<i>C</i>. <i>parvum</i>. (N = 5-10/group, **<i>P</i><0.01, ***<i>P</i><0.001 (<i>C</i>. <i>parvum</i> vs. either PBS or Δ<i>C</i>. <i>Parvum</i>) and fecal parasite shedding (n = 5-10/group, *<i>P</i><0.05, **<i>P</i><0.01). (D) Immunofluorescence staining of epithelial cell tight-junction proteins (ZO-1, occludin, claudin-2) in ileum of CD and PD-fed infected mice and uninfected controls (n = 4/group). (E) Dose dependent persistent growth faltering (n = 10/group, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 <i>C</i>. <i>parvum</i> 10⁷ vs <i>C</i>. <i>parvum</i> 10⁶; and ^####<i>P</i><0.0001 <i>C</i>. <i>parvum</i> vs PBS) and (F) fecal parasite shedding (**<i>P</i><0.01, ****<i>P</i><0.0001) through 21 days post-challenge. Data is representative of 2 replicate experiments.</p

Viable <i>C</i>. <i>parvum</i> priming provides greater protection against re-challenge than either CpG-ODN or <i>S</i>. Typhi.

<p>(A, B) Comparison of protective immunity following priming with either viable and heat-inactivated (Δ) <i>C</i>. <i>parvum</i> 10⁶. (A) Growth of PD-fed mice through 23 days post-priming with either 4x10⁶ viable (<i>C</i>. <i>parvum</i>) or 4x10⁶ heat-inactivated (Δ<i>C</i>. <i>parvum</i>). Mice were challenged with viable 4<i>x</i>10⁷ <i>C</i>. <i>parvum</i> oocysts on day 20 post-priming. *<i>P</i><0.05 for Δ<i>C</i>. <i>parvum-C</i>. <i>parvum</i> vs. <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> (d3 and d23); ^^<i>P</i><0.01 for <i>C</i>. <i>parvum</i>-PBS vs PBS-<i>C</i>. <i>parvum</i> (d23); ^###<i>P</i><0.001 for <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> vs. PBS-<i>C</i>. <i>parvum</i> (d23). (B) RT-PCR of <i>Cryptosporidium</i> stool shedding on experimental days 21 and 23 (day 1 and day 3 after <i>C</i>. <i>parvum</i> 10⁷ challenge, respectively). *<i>P</i><0.05 and *<i>P</i><0.01 for <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> vs either PBS-<i>C</i>. <i>parvum</i> or Δ<i>C</i>. <i>parvum-C</i>. <i>parvum</i>. (C,D) 3-week-old C57Bl/6 mice were conditioned on PD for 7 days prior to orogastric inoculation with 10⁶ <i>C</i>. <i>parvum</i>, intranasal (i.n.) 10⁹ <i>S</i>. Typhi 908<i>htr</i>, i.n. CpG-ODN 1668 (100 mcg), or PBS (100 mcl) as indicated (n = 10/group). On day 21, mice were re-challenged with either PBS or <i>C</i>. <i>parvum</i> 10⁷. (C) Growth as percentage of initial weight, normalized to the day of 10⁷ <i>C</i>. <i>parvum</i> challenge (Day 0). The group labeled “All uninfected” includes animals that received either PBS during both inoculations, CpG followed by PBS, or <i>S</i>. Typhi followed by PBS (n = 5/group x 3 = 15) given all three groups grew similarly and were never exposed to <i>C</i>. <i>parvum</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004820#pntd.0004820.s004" target="_blank">S4 Fig</a>). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 for PBS–<i>C</i>.<i>p</i>.10⁷ (red) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, ^<i>P</i><0.05 for CpG-<i>C</i>.<i>p</i>.10⁷ (yellow) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, and ^#<i>P</i><0.05 for <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ (green) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷). Horizontal lines designate significant differences at <i>P</i><0.05 between CpG-<i>C</i>.<i>p</i>.10⁷ (yellow), <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ (green), and PBS-<i>C</i>.<i>p</i>.10⁷ (red) vs. All uninfected controls, respectively. (D) Parasite fecal shedding in serial fecal pellets collected on indicated experimental days post <i>C</i>. <i>parvum</i> 10⁷ challenge. *<i>P</i><0.05 for PBS–<i>C</i>.<i>p</i>.10⁷ vs. <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, ^<i>P</i><0.05 for CpG-<i>C</i>.<i>p</i>.10⁷ vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, and ^#<i>P</i><0.05 for <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷. Data is representative of two replicate experiments.</p