Monoassociation of Preterm Germ-Free Piglets with Bifidobacterium animalis Subsp. lactis BB-12 and Its Impact on Infection with Salmonella Typhimurium

Preterm germ-free piglets were monoassociated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12) to verify its safety and to investigate possible protection against subsequent infection with Salmonella Typhimurium strain LT2 (LT2). Clinical signs of salmonellosis, bacterial colonization in the intestine, bacterial translocation to mesenteric lymph nodes (MLN), blood, liver, spleen, and lungs, histopathological changes in the ileum, claudin-1 and occludin mRNA expression in the ileum and colon, intestinal and plasma concentrations of IL-8, TNF-α, and IL-10 were evaluated. Both BB12 and LT2 colonized the intestine of the monoassociated piglets. BB12 did not translocate in the BB12-monoassociated piglets. BB12 was detected in some cases in the MLN of piglets, consequently infected with LT2, but reduced LT2 counts in the ileum and liver of these piglets. LT2 damaged the luminal structure of the ileum, but a previous association with BB12 mildly alleviated these changes. LT2 infection upregulated claudin-1 mRNA in the ileum and colon and downregulated occludin mRNA in the colon. Infection with LT2 increased levels of IL-8, TNF-α, and IL-10 in the intestine and plasma, and BB12 mildly downregulated them compared to LT2 alone. Despite reductions in bacterial translocation and inflammatory cytokines, clinical signs of LT2 infection were not significantly affected by the probiotic BB12. Thus, we hypothesize that multistrain bacterial colonization of preterm gnotobiotic piglets may be needed to enhance the protective effect against the infection with S. Typhimurium LT2.

Toll-Like Receptor 4 Signaling in the Ileum and Colon of Gnotobiotic Piglets Infected with Salmonella Typhimurium or Its Isogenic ∆rfa Mutants

Salmonella Typhimurium is a Gram-negative bacterium that causes enterocolitis in humans and pigs. Lipopolysaccharide (LPS) is a component of the outer leaflet of Gram-negative bacteria that provokes endotoxin shock. LPS can be synthesized completely or incompletely and creates S (smooth) or R (rough) chemotypes. Toll-like receptors (TLR) 2, 4, and 9 initiate an inflammatory reaction to combat bacterial infections. We associated/challenged one-week-old gnotobiotic piglets with wild-type S. Typhimurium with S chemotype or its isogenic ∆rfa mutants with R chemotype LPS. The wild-type S. Typhimurium induced TLR2 and TLR4 mRNA expression but not TLR9 mRNA expression in the ileum and colon of one-week-old gnotobiotic piglets 24 h after challenge. The TLR2 and TLR4 stimulatory effects of the S. Typhimurium ∆rfa mutants were related to the completeness of their LPS chain. The transcription of IL-12/23 p40, IFN-γ, and IL-6 in the intestine and the intestinal and plasmatic levels of IL-12/23 p40 and IL-6 but not IFN-γ were related to the activation of TLR2 and TLR4 signaling pathways. The avirulent S. Typhimurium ∆rfa mutants are potentially useful for modulation of the TLR2 and TLR4 signaling pathways to protect the immunocompromised gnotobiotic piglets against subsequent infection with the virulent S. Typhimurium.

Chlamydia suis is associated with intestinal NF-κB activation in experimentally infected gnotobiotic piglets

ABSTRACT Chlamydia suis intestinal infection of single-animal experimental groups of gnotobiotic newborn piglets was previously reported to cause severe, temporary small intestinal epithelium damage. We investigated archived intestinal samples for pro-inflammatory nuclear factor kappa B (NF-κB) activation, Interleukin (IL)-6 and IL-8 production and immune cell influx. Samples were collected 2, 4 and 7 days post-inoculation with C. suis strain S45/6 or mock inoculum (control). Increased nuclear localization of epithelial NF-κB, representative of activation, in the jejunum and ileum of C. suis-infected animals, compared to uninfected controls, began by 2 days post-infection (dpi) and persisted through 7 dpi. Infected animals showed increased production of IL-8, peaking at 2 dpi, compared to controls. Infection-mediated CD45-positive immune cell influx into the jejunal lamina propria peaked at 7 dpi, when epithelial damage was largely resolved. Activation of NF-κB appears to be a key early event in the innate response of the unprimed porcine immune system challenged with C. suis. This results in an acute phase, coinciding with the most severe clinical symptoms, diarrhea and weight loss. Immune cells recruited shortly after infection remain present in the lamina propria during the recovery phase, which is characterized by reduced chlamydial shedding and restored intestinal epithelium integrity.

Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

High Mobility Group Box 1 and TLR4 Signaling Pathway in Gnotobiotic Piglets Colonized/Infected with L. amylovorus, L. mucosae, E. coli Nissle 1917 and S. Typhimurium

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be actively secreted by immune cells after different immune stimuli or passively released from cells undergoing necrosis. HMGB1 amplifies inflammation, and its hypersecretion contributes to multiple organ dysfunction syndrome and death. We tested possible immunomodulatory effect of commensal Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM) or probiotic Escherichia coli Nissle 1917 (EcN) in infection of gnotobiotic piglets with Salmonella Typhimurium (ST). Transcription of HMGB1 and Toll-like receptors (TLR) 2, 4, and 9 and receptor for advanced glycation end products (RAGE), TLR4-related molecules (MD-2, CD14, and LBP), and adaptor proteins (MyD88 and TRIF) in the ileum and colon were measured by RT-qPCR. Expression of TLR4 and its related molecules were highly upregulated in the ST-infected intestine, which was suppressed by EcN, but not LA nor LM. In contrast, HMGB1 expression was unaffected by ST infection or commensal/probiotic administration. HMGB1 protein levels in the intestine measured by ELISA were increased in ST-infected piglets, but they were decreased by previous colonization with E. coli Nissle 1917 only. We conclude that the stability of HMGB1 mRNA expression in all piglet groups could show its importance for DNA transcription and physiological cell functions. The presence of HMGB1 protein in the intestinal lumen probably indicates cellular damage.

The Impact of Actotoxumab Treatment of Gnotobiotic Piglets Infected With Different Clostridium difficile Isogenic Mutants

Nosocomial infections with Clostridium difficile are on the rise in the Unites States, attributed to emergence of antibiotic-resistant and hypervirulent strains associated with greater likelihood of recurrent infections. In addition to antibiotics, treatment with Merck anti-toxin B (TcdB) antibody bezlotoxumab is reported to reduce recurrent infections. However, treatment with anti-toxin A (TcdA) antibody actotoxumab was associated with dramatically increased disease severity and mortality rates in humans and gnotobiotic piglets. Using isogenic mutants of C. difficile strain NAPI/BI/027 deficient in TcdA (A−B+) or TcdB (A+B−), and the wild type, we investigated how and why treatment of infected animals with anti-TcdA dramatically increased disease severity. Contrary to the hypothesis, among piglets treated with anti-TcdA, those with A+B− infection were disease free, in contrast to the disease enhancement seen in those with wild-type or A−B+ infection. It seems that the lack of TcdA, through either deletion or neutralization with anti-TcdA, reduces a competitive pressure, allowing TcdB to freely exert its profound effect, leading to increased mucosal injury and disease severity.

A sparse covarying unit that describes healthy and impaired human gut microbiota development

Characterizing the organization of the human gut microbiota is a formidable challenge given the number of possible interactions between its components. Using a statistical approach initially applied to financial markets, we measured temporally conserved covariance among bacterial taxa in the microbiota of healthy members of a Bangladeshi birth cohort sampled from 1 to 60 months of age. The results revealed an “ecogroup” of 15 covarying bacterial taxa that provide a concise description of microbiota development in healthy children from this and other low-income countries, and a means for monitoring community repair in undernourished children treated with therapeutic foods. Features of ecogroup population dynamics were recapitulated in gnotobiotic piglets as they transitioned from exclusive milk feeding to a fully weaned state consuming a representative Bangladeshi diet.

Effects of microbiota-directed foods in gnotobiotic animals and undernourished children

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.

Piperazine-Derivative MMV665917: An Effective Drug in the Diarrheic Piglet Model of Cryptosporidium hominis

Background Cryptosporidiosis, an enteric protozoon, causes substantial morbidity and mortality associated with diarrhea in children <2 years old in low- to middle-income countries. There is no vaccine and treatments are inadequate. A piperazine-based compound, MMV665917, has in vitro and in vivo efficacy against Cryptosporidium parvum. In this study, we evaluated the efficacy of MMV665917 in gnotobiotic piglets experimentally infected with Cryptosporidium hominis, the species responsible for >75% of diarrhea reported in these children. Methods Gnotobiotic piglets were orally challenged with C hominis oocysts, and oral treatment with MMV665917 was commenced 3 days after challenge. Oocyst excretion and diarrhea severity were observed daily, and mucosal colonization and lesions were recorded after necropsy. Results MMV665917 significantly reduced fecal oocyst excretion, parasite colonization and damage to the intestinal mucosa, and peak diarrheal symptoms, compared with infected untreated controls. A dose of 20 mg/kg twice daily for 7 days was more effective than 10 mg/kg. There were no signs of organ toxicity at either dose, but 20 mg/kg was associated with slightly elevated blood cholesterol and monocytes at euthanasia. Conclusions These results demonstrate the effectiveness of this drug against C hominis. Piperazine-derivative MMV665917 may potentially be used to treat human cryptosporidiosis; however, further investigations are required.