Gn Pigs Research Articles

Virus Shedding and Diarrhea: A Review of Human Norovirus Genogroup II Infection in Gnotobiotic Pigs.

For nearly twenty years, gnotobiotic (Gn) pigs have been used as a model of human norovirus (HuNoV) infection and disease. Unique in their ability to develop diarrhea and shed virus post oral challenge, Gn pigs have since been used to evaluate the infectivity of several genogroup II HuNoV strains. Nearly all major pandemic GII.4 variants have been tested in Gn pigs, with varying rates of infectivity. Some induce an asymptomatic state despite being shed in large quantities in stool, and others induce high incidence of both diarrhea and virus shedding. Non-GII.4 strains, including GII.12 and GII.6, have also been evaluated in Gn pigs. Again, rates of diarrhea and virus shedding tend to vary between studies. Several factors may influence these findings, including age, dosage, biological host factors, or bacterial presence. The impact of these factors is nuanced and requires further evaluation to elucidate the exact mechanisms behind increases or decreases in infection rates. Regardless, the value of Gn pig models in HuNoV research cannot be understated, and the model will surely continue to contribute to the field in years to come.

MRNA-Based Vaccines Are Highly Immunogenic and Confer Protection in the Gnotobiotic Pig Model of Human Rotavirus Diarrhea.

Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated the immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6], or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T-cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as the positive and negative control, respectively. Upon challenge with virulent Wa HRV, a significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG antibody-secreting cells in the ileum, spleen, and blood seven days post-challenge, as well as VP8*-specific IFN-γ-producing T-cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to the P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and the significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for the further development of mRNA-based rotavirus vaccines.

Intestinal colonization with Escherichia fergusonii enhances infectivity of GII.12 human norovirus in gnotobiotic pigs

The role of gut microbiota [especially, histo-blood group antigen (HBGA)-expressing bacteria] in influencing human norovirus (HuNoV) infections is unclear. We investigated if infectivity of GII.12 HuNoV in gnotobiotic (Gn) pigs is altered by intestinal colonization with Escherichia fergusonii known to express HBGA A and H on their cell surface. Fifteen piglets were randomly grouped: (1) E. fergusonii + HuNoV (n = 6), (2) HuNoV alone (n = 6), and (3) Mock-inoculated (n = 3). Pigs (8-11-day-old) were inoculated orally with GII.12 HuNoV strain HS206 (9.5 log10 genomic equivalents/pig) or mock. For 2 days prior to viral inoculation, pigs were inoculated orally with E. fergusonii [8 log10 colony forming units/pig/day]. Daily fecal consistency, fecal viral RNA or E. fergusonii shedding, and histopathology (at euthanasia) were evaluated. Unlike the reduced infectivity of GII.4 HuNoV observed previously in Gn pigs colonized with Enterobacter cloacae known to express HBGA A, B, and H on the surface, E. fergusonii + HuNoV pigs exhibited significantly higher cumulative fecal HuNoV RNA shedding at PIDs 6–14 and 1–21 compared with HuNoV alone pigs. Mean days of fecal HuNoV RNA shedding were also significantly greater in E. fergusonii + HuNoV pigs (11.8 ± 1.6 days) compared with HuNoV alone pigs (7.0 ± 1.0 days). By immunofluorescent staining, HuNoV antigen-positive bacteria were detected on the surface of the intestinal epithelium, possibly enhancing attachment of HuNoV to enterocytes, suggesting a potential mechanism by which intestinal colonization with E. fergusonii promoted infectivity of GII.12 HuNoV in Gn pigs.

Human Rotavirus Replicates in Salivary Glands and Primes Immune Responses in Facial and Intestinal Lymphoid Tissues of Gnotobiotic Pigs.

Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs.

Development of Chimeric Hepatitis B (HBV) – Norovirus (NoV) P particle as candidate vaccine against Hepatitis B and norovirus infection

IntroductionHepatitis B remains a global problem with no effective treatment. Here, a mucosal vaccine candidate was developed with HBsAg and HBcAg, to provide both prophylactic and therapeutic protection against hepatitis B. The antigens were presented using the P particle of human norovirus (HuNov). As a result, the chimeric HBV – HuNoV P particle can act as a dual vaccine for hepatitis B and HuNoV. MethodsThe vaccine candidate was expressed and purified from Escherichia coli BL21 (DE3) cells. HBV-HuNoV chimeric P particles were successfully expressed and isolated, with sizes of approximately 25.64 nm. Then, the HBV-HuNoV chimeric P particles were evaluated for safety and immunogenicity in mice and gnotobiotic (Gn) pigs. After three doses (5 µg/dose in mice and 200 µg/dose in Gn pigs) of intranasal immunization, humoral and cellular immune responses, as well as toxicity, were evaluated. ResultsThe vaccine candidate induced strong HBV-HuNoV specific IFN-γ producing T-cell responses in the ileum, spleen, and blood of Gn pigs. Serum IgG and IgA antibodies against HBV-HuNoV chimeric P particles also increased significantly in Gn pigs. Increased HBsAg- and HuNoV-specific serum IgG responses were observed in mice and Gn pigs, although not statistically significant. The vaccine candidate did not show any toxicity in mice. ConclusionsIn summary, the chimeric HBV-HuNoV P particle vaccine given intranasally was safe and induced strong cellular and humoral immune responses in Gn pig. Modifications to the vaccine structure and dosage need to be evaluated in future studies to further enhance immunogenicity and induce more balanced humoral and cellular responses.

A New Gnotobiotic Pig Model of P[6] Human Rotavirus Infection and Disease for Preclinical Evaluation of Rotavirus Vaccines.

Human rotavirus (HRV) is a leading cause of gastroenteritis in children under 5 years of age. Licensed vaccines containing G1P[8] and G1-4P[8] strains are less efficacious against newly emerging P[6] strains, indicating an urgent need for better cross protective vaccines. Here, we report our development of a new gnotobiotic (Gn) pig model of P[6] HRV infection and disease as a tool for evaluating potential vaccine candidates. The Arg HRV (G4P[6]) strain was derived from a diarrheic human infant stool sample and determined to be free of other viruses by metagenomic sequencing. Neonatal Gn pigs were orally inoculated with the stool suspension containing 5.6 × 105 fluorescent focus units (FFU) of the virus. Small and large intestinal contents were collected at post inoculation day 2 or 3. The virus was passaged 6 times in neonatal Gn pigs to generate a large inoculum pool. Next, 33-34 day old Gn pigs were orally inoculated with 10-2, 103, 104, and 105 FFU of Arg HRV to determine the optimal challenge dose. All pigs developed clinical signs of infection, regardless of the inoculum dose. The optimal challenge dose was determined to be 105 FFU. This new Gn pig model is ready to be used to assess the protective efficacy of candidate monovalent and multivalent vaccines against P[6] HRV.

Probiotic as Adjuvant Significantly Improves Protection of the Lanzhou Trivalent Rotavirus Vaccine against Heterologous Challenge in a Gnotobiotic Pig Model of Human Rotavirus Infection and Disease.

This preclinical study in the gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and disease evaluates the effect of probiotic Lactobacillus rhamnosus GG (LGG) as a mucosal adjuvant on the immunogenicity and cross-protective efficacy of the Lanzhou live oral trivalent (G2, G3, G4) vaccine (TLV, aka LLR3). Gn pigs were immunized with three doses of TLV with or without concurrent administration of nine doses of LGG around the time of the first dose of the TLV vaccination, and were challenged orally with the virulent heterotypic Wa G1P[8] HRV. Three doses of TLV were highly immunogenic and conferred partial protection against the heterotypic HRV infection. LGG significantly enhanced the intestinal and systemic immune responses and improved the effectiveness of protection against the heterotypic HRV challenge-induced diarrhea and virus shedding. In conclusion, we demonstrated the immune-stimulating effects of probiotic LGG as a vaccine adjuvant and generated detailed knowledge regarding the cross-reactive and type-specific antibody and effector B and T cell immune responses induced by the TLV. Due to the low cost, ease of distribution and administration, and favorable safety profiles, LGG as an adjuvant has the potential to play a critical role in improving rotavirus vaccine efficacy and making the vaccines more cost-effective.

Gut Microbiota of Obese Children Influences Inflammatory Mucosal Immune Pathways in the Respiratory Tract to Influenza Virus Infection: Optimization of an Ideal Duration of Microbial Colonization in a Gnotobiotic Pig Model.

ABSTRACTThe impact of obesity on the human microbiota, immune maturation, and influenza virus infection has not been yet established in natural host animal models of influenza. In this study, gnotobiotic (Gn) pigs were colonized with human fecal microbiota (HFM) of obese (oHFM) or healthy lean (hHFM) children and infected at different periods (2-, 3-, and 5-weeks post-transplantation) using a zoonotic influenza virus strain. The infected oHFM pigs were characterized by lower levels of Firmicutes (Lactococcus, Lactobacillus, Turicibacter, and Streptococcus) and Actinobacteria (Bifidobacterium), which was associated with higher levels of Proteobacteria (Klebsiella), Bacteroidetes, and Verrucomicrobia (Akkermansia) compared with the infected hHFM group (P < 0.01). Furthermore, these genera significantly correlated with the expression of immune effectors, immune regulators, and inflammatory mediators, and displayed opposite trends between oHFM and hHFM groups (P < 0.01). The lymphoid and myeloid immune cell frequencies were differently modulated by the oHFM and hHFM colonization, especially apparent in the 5-weeks HFM colonized piglets. In addition, oHFM group had higher pro-inflammatory cytokines (IL-6, IL-12, TNF-α, and IFNγ) gene expression in the respiratory tract compared with the hHFM colonized pigs was detected. In conclusion, pigs colonized for longer duration, established oHFM increased the immune maturation favoring the activation of inflammatory mediators, however, the influenza virus load remained comparable with the hHFM group. Further, a longer duration of microbial colonization (5 weeks) may be required to reveal the impact of microbiome on the host immune maturation and susceptibility to influenza virus infection in the humanized Gn pig model.IMPORTANCE The diversity of gut microbiome of obese people differs markedly from that of lean healthy individuals which, in turn, influences the severity of inflammatory diseases because of differential maturation of immune system. The mouse model provides crucial insights into the mechanism(s) regulating the immune systems mediated by the gut microbiota but its applicability to humans is questionable because immune cells in mice are poorly activated in microbiota humanized mice. Several important strains of Bifidobacterium, Lactobacillus, and Clostridium fails to colonize the murine gut. Thus, understanding the role of certain important commensal gut bacterial species influences upon health and disease, a suitable large animal model like pig that supports the growth and colonization of most of the important human gut bacteria and possess comparable immunology and physiology to humans is beneficial to improve health.

Escherichia coli Nissle 1917 Enhances Efficacy of Oral Attenuated Human Rotavirus Vaccine in a Gnotobiotic Piglet Model.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

Reduced rotavirus vaccine efficacy in protein malnourished human-faecal-microbiota-transplanted gnotobiotic pig model is in part attributed to the gut microbiota.

The low efficacy of human rotavirus (HRV) vaccines in low- and middle-income countries (LMIC) remains a major challenge for global health. Protein-calorie malnutrition (kwashiorkor) affects the gut microbiota and compromises immune development, leading to environmental enteropathy, vaccine failures, and increased susceptibility to enteric diseases in young children. Relationship between diet and reduced vaccine efficacy in developing countries is not well established; therefore, we investigated the interconnections between the host-microbiota-nutrition-HRV vaccine using HRV-vaccinated, human infant faecal microbiota (HIFM)-transplanted neonatal gnotobiotic pigs fed with a protein deficient or sufficient diet. The microbiota from faecal, intestinal (duodenum, ileum, jejunum, and colon), and systemic tissue (liver, spleen, and mesenteric lymph node [MLN]) samples was analysed before and after HRV challenge using MiSeq 16S rRNA sequencing. Overall, microbiota from deficient fed HIFM pigs displayed, compared to the sufficient group, significantly higher Shannon index, especially in the faeces and lower intestines; higher level of Proteus and Enterococcus, and lower level of Bifidobacterium, Clostridium, and Streptococcus in the three types of samples collected (P<0.05); and higher unique operational taxonomic units (OTUs), especially in the systemic tissues. Further, the multivariate analysis between microbiota and immunologic data showed that 38 OTUs at the genus level correlated (r2≤0.5 or ≥-0.5; P<0.05) with at least one host immune response parameter (regulatory [Tregs and transforming growth factor-β], effectors [interferon (IFN)-γ+ CD4+ and CD8+ T cells, IFN-γ and interleukin (IL)-12], and inflammatory [tumour necrosis factor-α, IL-17 and IL-22]) and with opposite trends between diet groups. Differences described above were increased after HRV challenge. We demonstrated that a protein deficient diet affects the composition of the gut microbiota and those changes may further correlate with immune responses induced by HRV and perturbed by the deficient diet. Thus, our findings suggest that the reduced efficacy of HRV vaccine observed in Gn pig model is in part attributed to the altered microbiota composition.

Comparative Sequence Analysis of Historic and Current Porcine Rotavirus C Strains and Their Pathogenesis in 3-Day-Old and 3-Week-Old Piglets.

The increased prevalence of porcine group C rotavirus (PRVC) in suckling piglets and the emergence of new genetically distinct PRVC strains are concerning due to the associated significant economic losses they cause to the swine industry. We sequenced and analyzed two new PRVC strains, RV0104 (G3), and RV0143 (G6) and compared their pathogenesis with that of the historic strain Cowden (G1) in gnotobiotic (Gn) pigs. Near complete genome sequence analysis confirmed that these two strains were distinct from one another and the Cowden strain. VP1, VP2, VP6, NSP1-NSP3, and NSP5 genes were more similar between Cowden and RV0143, whereas VP3, VP7, and NSP4 shared higher nucleotide identity between Cowden and RV0104. Three-day-old and 3-week-old Gn piglets were inoculated with 105 FFU/piglet of Cowden, RV0104 or RV0143, or mock. All 3-day-old piglets developed severe diarrhea, anorexia, and lethargy, with mean PRVC fecal shedding titers peaking and numerically higher in RV0104 and RV0143 piglets on post infection day (PID) 2. Histopathological examination of the small intestine revealed that the 3-day-old Cowden and RV0104 inoculated piglets were mildly affected, while significant destruction of small intestinal villi was observed in the RV0143 inoculated piglets. Consistent with the highest degree of pathological changes in the small intestines, the RV0143 inoculated piglets had numerically higher levels of serum IL-17 and IFN-α cytokines and numerically lower PRVC IgA geometric mean antibody titers. Milder pathological changes and overall higher titers of PRVC IgA antibodies were observed in 3-week-old vs. 3-day-old piglets. Additionally, diarrhea was only observed in RV0104 and RV0143 (but not Cowden) inoculated 3-week-old piglets, while levels of serum IL-10 and PRVC IgA antibodies were higher in Cowden inoculated pigs, consistent with the lack of diarrhea. Thus, we confirmed that these current, genetically heterogeneous PRVC strains possess distinct pathobiological characteristics that may contribute to the increased prevalence of PRVC diarrhea in neonatal suckling piglets.

Malnutrition Decreases Antibody Secreting Cell Numbers Induced by an Oral Attenuated Human Rotavirus Vaccine in a Human Infant Fecal Microbiota Transplanted Gnotobiotic Pig Model.

Human rotavirus (HRV) is a leading cause of morbidity and mortality in children, especially in developing countries. Malnutrition is prevalent in these countries, which may contribute to the decreased oral vaccine efficacy, posing a concern for global health. Neonatal gnotobiotic (Gn) pigs closely resemble human infants in their anatomy, physiology, and outbred status and are a unique model to investigate malnutrition, oral live attenuated HRV (AttHRV) vaccination, and subsequent virulent HRV (VirHRV) challenge. We evaluated the impact of malnutrition on AttHRV vaccine efficacy and B cell immune responses in neonatal germfree (GF) or Gn pigs transplanted with human infant fecal microbiota (HIFM). Pigs were fed either deficient or sufficient bovine milk diets. Malnutrition did not significantly affect the serum and intestinal contents total or HRV-specific IgG and IgA antibody titers pre VirHRV challenge. However, HRV-specific IgG and IgA antibody secreting cells (ASCs) were reduced in blood or intestinal tissues following AttHRV vaccination and pre VirHRV challenge in deficient HIFM transplanted pigs. Furthermore, post-VirHRV challenge, deficient HIFM pigs had decreased total Ig and HRV-specific IgG and IgA antibody titers in serum or intestinal contents, in addition to decreased HRV-specific IgG and IgA ASCs in blood and ileum, compared with sufficient HIFM pigs. Our results indicate that deficient diet impairs B cell mucosal, and systemic immune responses following HRV vaccination, and challenge. The impaired immune responses contributed to the decreased protective efficacy of the AttHRV vaccine, suggesting that malnutrition may significantly reduce the effectiveness of oral HRV vaccines in children in developing countries.

Parenterally Administered P24-VP8* Nanoparticle Vaccine Conferred Strong Protection against Rotavirus Diarrhea and Virus Shedding in Gnotobiotic Pigs.

Current live rotavirus vaccines are costly with increased risk of intussusception due to vaccine replication in the gut of vaccinated children. New vaccines with improved safety and cost-effectiveness are needed. In this study, we assessed the immunogenicity and protective efficacy of a novel P24-VP8* nanoparticle vaccine using the gnotobiotic (Gn) pig model of human rotavirus infection and disease. Three doses of P24-VP8* (200 μg/dose) intramuscular vaccine with Al(OH)3 adjuvant (600 μg) conferred significant protection against infection and diarrhea after challenge with virulent Wa strain rotavirus. This was indicated by the significant reduction in the mean duration of diarrhea, virus shedding in feces, and significantly lower fecal cumulative consistency scores in post-challenge day (PCD) 1–7 among vaccinated pigs compared to the mock immunized controls. The P24-VP8* vaccine was highly immunogenic in Gn pigs. It induced strong VP8*-specific serum IgG and Wa-specific virus-neutralizing antibody responses from post-inoculation day 21 to PCD 7, but did not induce serum or intestinal IgA antibody responses or a strong effector T cell response, which are consistent with the immunization route, the adjuvant used, and the nature of the non-replicating vaccine. The findings are highly translatable and thus will facilitate clinical trials of the P24-VP8* nanoparticle vaccine.

Enhanced GII.4 human norovirus infection in gnotobiotic pigs transplanted with a human gut microbiota.

The role of commensal microbiota in enteric viral infections has been explored extensively, but the interaction between human gut microbiota (HGM) and human norovirus (HuNoV) is poorly understood. In this study, we established an HGM-Transplanted gnotobiotic (Gn) pig model of HuNoV infection and disease, using an infant stool as HGM transplant and a HuNoV GII.4/2006b strain for virus inoculation. Compared to germ-free Gn pigs, HuNoV inoculation in HGMT Gn pigs resulted in increased HuNoV shedding, characterized by significantly higher shedding titres on post inoculation day (PID) 3, 4, 6, 8 and 9, and significantly longer mean duration of virus shedding. In addition, virus titres were significantly higher in duodenum and distal ileum of HGMT Gn pigs on PID10, while comparable and transient HuNoV viremia was detected in both groups. 16S rRNA gene sequencing demonstrated that HuNoV infection dramatically altered intestinal microbiota in HGMT Gn pigs at the phylum (Proteobacteria, Firmicutes and Bacteroidetes) and genus (Enterococcus, Bifidobacterium, Clostridium, Ruminococcus, Anaerococcus, Bacteroides and Lactobacillus) levels. In summary, enhanced GII.4 HuNoV infection was observed in the presence of HGM, and host microbiota was susceptible to disruption upon HuNoV infection.

Protein deficiency reduces efficacy of oral attenuated human rotavirus vaccine in a human infant fecal microbiota transplanted gnotobiotic pig model

BackgroundLow efficacy of rotavirus (RV) vaccines in developing African and Asian countries, where malnutrition is prevalent, remains a major concern and a challenge for global health. MethodsTo understand the effects of protein malnutrition on RV vaccine efficacy, we elucidated the innate, T cell and cytokine immune responses to attenuated human RV (AttHRV) vaccine and virulent human RV (VirHRV) challenge in germ-free (GF) pigs or human infant fecal microbiota (HIFM) transplanted gnotobiotic (Gn) pigs fed protein-deficient or -sufficient bovine milk diets. We also analyzed serum levels of tryptophan (TRP), a predictor of malnutrition, and kynurenine (KYN). ResultsProtein-deficient pigs vaccinated with oral AttHRV vaccine had lower protection rates against diarrhea post-VirHRV challenge and significantly increased fecal virus shedding titers (HIFM transplanted but not GF pigs) compared with their protein-sufficient counterparts. Reduced vaccine efficacy in protein-deficient pigs coincided with altered serum IFN-α, TNF-α, IL-12 and IFN-γ responses to oral AttHRV vaccine and the suppression of multiple innate immune parameters and HRV-specific IFN-γ producing T cells post-challenge. In protein-deficient HIFM transplanted pigs, decreased serum KYN, but not TRP levels were observed throughout the experiment, suggesting an association between the altered TRP metabolism and immune responses. ConclusionCollectively, our findings confirm the negative effects of protein deficiency, which were exacerbated in the HIFM transplanted pigs, on innate, T cell and cytokine immune responses to HRV and on vaccine efficacy, as well as on TRP-KYN metabolism.

Immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (Gn) pigs infected with porcine epidemic diarrhea virus (PEDV) and serum cytokine responses of Gn pigs to acute PEDV infection

Serotonin is a critical monoamine neurotransmitter molecule stored and released from enterochromaffin (EC) cells into the gut submucosa, transmitting the vomiting signal to the brain. We studied one mechanism by which vomiting is induced in pigs infected with porcine epidemic diarrhea virus (PEDV) by characterization of swine EC cells by immunohistochemistry. Conventional or gnotobiotic (Gn) 9-day-old pigs [PEDV-inoculated (n = 12); Mock (n = 14)] were inoculated orally (8.9–9.2 log10 genomic equivalents/pig) with PEDV PC21A strain or mock. This is the first identification of serotonin-positive EC cells in swine by immunohistochemistry and mainly in intestinal crypts, regardless of infection status. They were morphologically triangular-shaped or round cells with or without apical cytoplasmic extensions, respectively. At post-inoculation hour (PIH) 16 or 24, when vomiting was first or frequently observed, respectively, PEDV infection resulted in significantly reduced numbers of serotonin-positive EC cells in duodenum, mid-jejunum, ileum, or colon. However, two of three PEDV-inoculated Gn pigs that did not yet show vomiting at PIH 16 had numbers of serotonin-positive EC cells in duodenum, ileum and colon similar to those in the negative controls. These findings suggest that serotonin release from EC cells (increased serotonin levels) into the gut submucosa might occur early PEDV post-infection to stimulate the vagal afferent neurons, followed by vomiting. Serotonin might be involved in the mechanisms related to vomiting in PEDV-infected piglets. We also found that mid-jejunum was the primary site of acute PEDV infection, and that systemic innate and pro-inflammatory cytokine responses were induced during the acute stage of PEDV infection.

Effect of antibiotic, probiotic, and human rotavirus infection on colonisation dynamics of defined commensal microbiota in a gnotobiotic pig model.

We developed a gnotobiotic (Gn) pig model colonised with defined commensal microbiota (DMF) to provide a simplified and controlled system to study the interactions between intestinal commensals, antibiotics (ciprofloxacin, CIP), probiotics (Escherichia coli Nissle 1917, EcN) and virulent human rotavirus (VirHRV). The DMF included seven gut commensal species of porcine origin that mimic the predominant species in the infant gut. Gn piglets were divided into four groups: DMF control (non-treated), DMF+CIP (CIP treated), DMF+CIP+EcN (CIP/EcN treated), DMF+EcN (EcN treated) and inoculated orally with 105 cfu of each DMF strain. The pig gut was successfully colonised by all DMF species and established a simplified bacterial community by post-bacteria colonisation day (PBCD) 14/post-VirHRV challenge day (PCD) 0. Overall, Bifidobacterium adolescentis was commonly observed in faeces in all groups and time points. At PCD0, after six days of CIP treatment (DMF+CIP), we observed significantly decreased aerobic and anaerobic bacteria counts especially in jejunum (P<0.001), where no DMF species were detected in jejunum by T-RFLP. Following HRV challenge, 100% of pigs in DMF+CIP group developed diarrhoea with higher diarrhoea scores and duration as compared to all other groups. However, only 33% of pigs treated with EcN plus CIP developed diarrhoea. EcN treatment also enhanced the bacterial diversity and all seven DMF species were detected with a higher proportion of Bifidobacterium longum in jejunum in the DMF+CIP+EcN group on PBCD14/PCD0. Our results suggest that EcN increased the proportion of B. longum especially in jejunum and mitigated adverse impacts of antibiotic use during acute-infectious diarrhoea. The DMF model with a simplified gut commensal community can further our knowledge of how commensals and probiotics promote intestinal homeostasis and contribute to host health.

Modeling human enteric dysbiosis and rotavirus immunity in gnotobiotic pigs.

BackgroundRotavirus vaccines have poor efficacy in infants from low- and middle-income countries. Gut microbiota is thought to influence the immune response to oral vaccines. Thus, we developed a gnotobiotic (Gn) pig model of enteric dysbiosis to study the effects of human gut microbiota (HGM) on immune responses to rotavirus vaccination, and the effects of rotavirus challenge on the HGM by colonizing Gn pigs with healthy HGM (HHGM) or unhealthy HGM (UHGM). The UHGM was from a Nicaraguan infant with a high enteropathy score (ES) and no seroconversion following administration of oral rotavirus vaccine, while the converse was characteristic of the HHGM. Pigs were vaccinated, a subset was challenged, and immune responses and gut microbiota were evaluated.ResultsSignificantly more rotavirus-specific IFN-γ producing T cells were in the ileum, spleen, and blood of HHGM than those in UHGM pigs after three vaccine doses, suggesting HHGM induces stronger cell-mediated immunity than UHGM. There were significant correlations between multiple Operational Taxonomic Units (OTUs) and frequencies of IFN-γ producing T cells at the time of challenge. There were significant positive correlations between Collinsella and CD8+ T cells in blood and ileum, as well as CD4+ T cells in blood, whereas significant negative correlations between Clostridium and Anaerococcus, and ileal CD8+ and CD4+ T cells. Differences in alpha diversity and relative abundances of OTUs were detected between the groups both before and after rotavirus challenge.ConclusionAlterations in microbiome diversity and composition along with correlations between certain microbial taxa and T cell responses warrant further investigation into the role of the gut microbiota and certain microbial species on enteric immunity. Our results support the use of HGM transplanted Gn pigs as a model of human dysbiosis during enteric infection, and oral vaccine responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13099-016-0136-y) contains supplementary material, which is available to authorized users.

Experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain OH-FD22.

Porcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus in pigs. We have isolated and passaged the PDCoV strain OH-FD22 in an LLC porcine kidney (LLC-PK) cell line. Our study investigated the pathogenicity of the tissue-culture-grown PDCoV (TC-PDCoV) OH-FD22 at cell passages 5, 20 and 40 in LLC-PK cells, in eight 14-day-old gnotobiotic (Gn) pigs. Pigs (n = 3) were euthanized for pathologic examination at post-inoculation day (PID) 3, and the remainder were monitored for clinical signs, virus shedding, and serum antibody responses until PID 28. All inoculated pigs developed watery diarrhea and/or vomiting at PID 1-2 and shed the highest amount of viral RNA in feces at PID 3-5, accompanied by severe atrophic enteritis. They developed high titers of PDCoV-specific IgG/IgA and virus-neutralizing antibodies in serum at PID 23-24. Histologic lesions were limited to the villous epithelium of the jejunum and ileum at PID 3. Two inoculated pigs tested at PID 23-24 had small to moderate numbers of PDCoV antigen-positive cells in the intestinal lamina propria and mesenteric lymph nodes, but not in enterocytes. An analysis of full-length S and N genes of TC- and Gn-pig-passaged OH-FD22 revealed a high genetic stability in cell culture and pigs. TC-PDCoV OH-FD22 (cell passages 5, 20 and 40) was enteropathogenic, and the pathogenicity was similar to that of the original field virus. The TC-PDCoV OH-FD22 will be useful for further pathogenesis studies and for evaluating if higher-level cell-culture passaged virus becomes attenuated for vaccine development.

Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN-, LGG-, and EcN + LGG-colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK-cell function and decreased frequencies of apoptotic and TLR4+ mononuclear cells (MNCs). Consistent with the highest NK-cell activity, splenic CD172+ MNCs (DC enriched fraction) of EcN-colonized pigs produced the highest levels of IL-12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG-colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN-treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN-α, IL-12, and IL-10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN-mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC-IL-12-NK immune axis.