Rural-Urban Differences in Healthcare Use in Persons With Dementia Between 2000 and 2019: A Quebec Population-Based Study.

Background: Rural persons with dementia face medical services gaps. This study compares the health service utilization of rural and urban community-dwelling individuals with incident dementia. Methods: This study used a repeated annual cross-sectional cohort design spanning a period from 2000 to 2019 analyzing age-adjusted rates for 20 indicators of service use and mortality one year after diagnosis in Quebec administrative databases. Results: Of 237,259 persons, 20.1% were rural. Most rural persons had more emergency department visits and hospitalizations, shorter stays, less alternate level of care and fewer family physicians' and cognition specialists' visits. All groups had similar long-term care and mortality rates. Conclusion: Policy implications of these disparities are discussed.

Sex Differences in Healthcare Utilization in Persons Living with Dementia Between 2000 and 2017: A Population-Based Study in Quebec, Canada.

Objectives: Describe sex differences in healthcare utilization and mortality in persons with new dementia in Quebec, Canada. Methods: We conducted a repeated cohort study from 2000 to 2017 using health administrative databases. Community-dwelling persons aged 65+ with a new diagnosis of dementia were included. We measured 23 indicators of healthcare use across five care settings: ambulatory care, pharmacological care, acute hospital care, long-term care, and mortality. Clinically meaningful sex differences in age-standardized rates were determined graphically through expert consultations. Results: Women with dementia had higher rates of ambulatory care and pharmacological care, while men with dementia had higher acute hospital care, admission to long-term care, and mortality. There was no meaningful difference in visits to cognition specialists, antipsychotic prescriptions, and hospital death. Discussion: Men and women with dementia demonstrate differences in healthcare utilization and mortality. Addressing these differences will inform decision-makers, care providers and researchers and guide more equitable policy and interventions in dementia care.

Rural and urban differences in quality of dementia care of persons with dementia and caregivers across all domains: a systematic review.

BACKGROUND: There are challenges in healthcare service delivery in rural areas, and this may be especially true for persons with dementia, who have higher needs to access to the healthcare system, and may have difficulties to commute easily and safely to these services. There is a growing body of literature regarding geographical disparities, but there is no comprehensive systematic review of geographical differences in persons with dementia across all domains of care quality. Therefore, the objective of this study is to conduct a systematic review of the literature on rural and urban differences in quality of dementia care outcomes of persons with dementia across all quality-of-care domains. METHODS: We performed a digital search in Ovid MEDLINE on July 16, 2019, updated on May 3, 2021, for French or English records. We selected studies that reported outcome from at least one domain of quality of dementia care (Access, Integration, Effective Care, Efficient Care, Population Health, Safety, and Patient-Centered) in both rural and urban persons with dementia or caregivers. We used rigorous, systematic methods for screening, selection, data extraction and we analyzed outcomes reported by at least two studies using vote counting and appraised the certainty of evidence. Finally, we explored sources of heterogeneity. RESULTS: From the 38 included studies, we found differences in many dementia care domains. Rural persons with dementia had higher mortality rates (Population Health), lower visits to any physicians (Access), more hospitalizations but shorter stays (Integration), higher antipsychotic medications (Safety), lower use of home care services and higher use of nursing home (Patient-Centered Care) compared to urban persons with dementia. CONCLUSIONS: This comprehensive portrait of rural-urban differences in dementia care highlights possible geographically based inequities and can be used by researchers and decision makers to guide development of more equitable dementia care policies.

National Influenza Annual Report, Canada, 2021-2022: A brief, late influenza epidemic.

Canadian seasonal influenza circulation had been suppressed since the beginning of the coronavirus disease 2019 (COVID-19) pandemic. This suppression was reported globally and generated concern that the return of community influenza circulation could be intense and that co-circulation of influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was possible and potentially severe. Community circulation of influenza returned to Canada during the 2021-2022 influenza season. The influenza epidemic began in week 16 (mid-April 2022) and lasted only nine weeks. This epidemic was driven by influenza A(H3N2) and was exceptionally late in the season, low in intensity and short in length. Community co-circulation of influenza and SARS-CoV-2 was observed in Canada for the first time during the 2021-2022 seasonal influenza epidemic. The unusual characteristics of the 2021-2022 influenza epidemic suggest that a breadth of factors moderate transmission dynamics of the two viruses. Concerns of an intense seasonal influenza epidemic did not come to fruition during the 2021-2022 season; therefore, high influenza susceptibility remains, as does predisposition to larger influenza epidemics. Ongoing circulation of SARS-CoV-2 creates uncertainty about dynamics of future influenza epidemics, but influenza vaccination remains a key public health intervention available to protect Canadians. Public health authorities need to remain vigilant, maintain surveillance and continue to plan for both heightened seasonal influenza circulation and for the potential for endemic co-circulation of influenza and SARS-CoV-2.

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.

A Luciferase Immunoprecipitation System (LIPS) assay for profiling human norovirus antibodies.

A luciferase immunoprecipitation systems (LIPS) assay was developed to define the antigenic specificity and titer of antibodies directed against human norovirus (HuNoV). Recombinant proteins, expressed by plasmid constructs encoding Renilla luciferase (Ruc) fused to the full-length HuNoV major capsid protein (VP1) (Ruc-antigen), were generated for ten HuNoV strains. In addition, subdomain constructs Ruc-Shell (S) and Ruc-Protruding (P) were engineered for a representative GII.4 norovirus (strain GII.4/2006b). The LIPS assay measured antibody levels in a well-defined panel of HuNoV-specific sera, and the results were compared to an ELISA standard. In hyperimmune sera, the LIPS produced titers similar to or higher than those measured by the ELISA of HuNoV-specific antibodies. The specificity of antibodies in various sera was profiled by LIPS with a panel of diverse Ruc-antigens containing full-length HuNoV VP1 proteins or VP1 subdomains, and the assay detected both specific and cross-reactive antibodies. Competition assays, in which antibodies were pre-incubated with one or more intact VLPs representing different genotypes, proved useful in further assessment of the antibody specificity detected by LIPS in complex polyclonal sera. The profiling of HuNoV-specific antibodies in the high-throughput LIPS format may prove useful in defining the strength or specificity of the adaptive immune response following natural infection or vaccination.

Effects of Racecadotril on Weight Loss and Diarrhea Due to Human Rotavirus in Neonatal Gnotobiotic Pigs (Sus scrofa domesticus).

Diarrheal disease is the second leading cause of death in children younger than 5 y, and the most common cause of acute watery diarrhea in young children worldwide is rotaviral infection. Medicines to specifically reduce diarrhea would be a desirable adjunctive treatment to supportive fluid therapy to decrease the mortality rate of diarrheal diseases. In this study, we evaluated the efficacy of an antisecretory drug, racecadotril, in treating human rotavirus (HRV)-induced diarrhea in a neonatal gnotobiotic pig model. In total, 27 gnotobiotic pigs were randomly assigned (n = 9 per group) to receive either racecadotril, chlorpromazine (positive-control drug), or PBS (mock treatment) after inoculation with HRV. Pigs were weighed daily and rectal swabs were collected to determine fecal consistency scores and virus shedding. Rotaviral infection was confirmed by ELISA and cell culture immunofluorescence. Overall, the racecadotril-treated pigs had less severe illness than either the chlorpromazine- or mock-treated groups; this conclusion was supported by the lower fecal-consistency scores, shorter duration of diarrhea, and significant gain in body weight during the course of the study of the racecadotril-treated pigs. Through its influence on decreasing intestinal hypersecretion, racecadotril was better able to control the clinical signs of rotaviral infection in the gnotobiotic pigs. These results lend support for using racecadotril as a treatment for rotaviral diarrhea.

Modeling human enteric dysbiosis and rotavirus immunity in gnotobiotic pigs.

BACKGROUND: Rotavirus 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. RESULTS: Significantly 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. CONCLUSION: Alterations 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.

High Protective Efficacy of Probiotics and Rice Bran against Human Norovirus Infection and Diarrhea in Gnotobiotic Pigs.

Probiotics have been recognized as vaccine adjuvants and therapeutic agents to treat acute gastroenteritis in children. We previously showed that rice bran (RB) reduced human rotavirus diarrhea in gnotobiotic pigs. Human noroviruses (HuNoVs) are the major pathogens causing non-bacterial acute gastroenteritis worldwide. In this study, Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) were first screened for their ability to bind HuNoV P particles and virions derived from clinical samples containing HuNoV genotype GII.3 and GII.4, then the effects of LGG+EcN and RB on HuNoV infection and diarrhea were investigated using the gnotobiotic pig model. While LGG+EcN colonization inhibited HuNoV shedding, probiotic cocktail regimens in which RB feeding started 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized gnotobiotic pigs exhibited high protection against HuNoV diarrhea and shedding, characterized by significantly reduced incidence (89 versus 20%) and shorter mean duration of diarrhea (2.2 versus 0.2 days), as well as shorter mean duration of virus shedding (3.2 versus 1.0 days). In both probiotic cocktail groups, the diarrhea reduction rates were 78% compared with the control group, and diarrhea severity was reduced as demonstrated by the significantly lower cumulative fecal scores. The high protective efficacy of the probiotic cocktail regimens was attributed to stimulation of IFN-γ+ T cell responses, increased production of intestinal IgA and IgG, and maintenance of healthy intestinal morphology (manifested as longer villi compared with the control group). Therefore, probiotic cocktail regimens containing LGG+EcN and RB may represent highly efficacious strategies to prevent and treat HuNoV gastroenteritis, and potentially other human enteric pathogens.

Enterobacter cloacae inhibits human norovirus infectivity in gnotobiotic pigs.

Human noroviruses (HuNoVs) are the leading cause of epidemic gastroenteritis worldwide. Study of HuNoV biology has been hampered by the lack of an efficient cell culture system. Recently, enteric commensal bacteria Enterobacter cloacae has been recognized as a helper in HuNoV infection of B cells in vitro. To test the influences of E. cloacae on HuNoV infectivity and to determine whether HuNoV infects B cells in vivo, we colonized gnotobiotic pigs with E. cloacae and inoculated pigs with 2.74 × 10(4) genome copies of HuNoV. Compared to control pigs, reduced HuNoV shedding was observed in E. cloacae colonized pigs, characterized by significantly shorter duration of shedding in post-inoculation day 10 subgroup and lower cumulative shedding and peak shedding in individual pigs. Colonization of E. cloacae also reduced HuNoV titers in intestinal tissues and in blood. In both control and E. cloacae colonized pigs, HuNoV infection of enterocytes was confirmed, however infection of B cells was not observed in ileum, and the entire lamina propria in sections of duodenum, jejunum, and ileum were HuNoV-negative. In summary, E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs, with or without E. cloacae colonization.

Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency.

Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.

B-Cell-Deficient and CD8 T-Cell-Depleted Gnotobiotic Pigs for the Study of Human Rotavirus Vaccine-Induced Protective Immune Responses.

Genetically modified pigs have become available recently. In this study, we established the gnotobiotic pig model of human rotavirus (HRV) infection using cloned pigs with homozygous disruption in the gene encoding immunoglobulin heavy chain (HCKO), which totally impairs B-cell development. To clarify importance of B cells and cytotoxic T cells in rotavirus immunity, CD8 cells in a subset of the pigs were depleted by injecting antipig CD8 antibodies and the immune phenotypes of all pigs were examined. HCKO pigs, CD8 cell-depleted HCKO pigs, and wild-type (WT) pigs were vaccinated with an attenuated HRV vaccine and challenged with virulent HRV. Protection against HRV infection and diarrhea was assessed postchallenge and detailed T-cell subset responses were determined pre- and postchallenge. Significantly longer duration of virus shedding was seen in vaccinated HCKO pigs than in WT pigs, indicating the importance of B cells in vaccine-induced protective immunity. Vaccinated HCKO/CD8(-) pigs shed significantly higher number of infectious virus than WT pigs and non-CD8-depleted HCKO pigs, indicating the importance of CD8 T cells in controlling virus replication. Therefore, both B cells and CD8 T cells play an important role in the protection against rotavirus infection. HCKO and HCKO/CD8(-) pigs did not differ significantly in diarrhea and virus shedding postchallenge; increased CD4 and CD8(-) γδ T-cell responses probably compensated partially for the lack of CD8 T cells. This study demonstrated that HCKO pigs can serve as a valuable model for dissection of protective immune responses against viral infections and diseases.

Intranasal P particle vaccine provided partial cross-variant protection against human GII.4 norovirus diarrhea in gnotobiotic pigs.

UNLABELLED: Noroviruses (NoVs) are the leading cause of nonbacterial acute gastroenteritis worldwide in people of all ages. The P particle is a novel vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. This study utilized the neonatal gnotobiotic pig model to evaluate the protective efficacies of primary infection, P particles, and virus-like particles (VLPs) against NoV infection and disease and the T cell responses to these treatments. Pigs either were vaccinated intranasally with GII.4/1997 NoV (VA387)-derived P particles or VLPs or were inoculated orally with a GII.4/2006b NoV variant. At postinoculation day (PID) 28, pigs either were euthanized or were challenged with the GII.4/2006b variant and monitored for diarrhea and virus shedding for 7 days. The T cell responses in intestinal and systemic lymphoid tissues were examined. Primary NoV infection provided 83% homologous protection against diarrhea and 49% homologous protection against virus shedding, while the P particle and VLP vaccines provided cross-variant protection (47% and 60%, respectively) against diarrhea. The protection rates against diarrhea are significantly inversely correlated with T cell expansion in the duodenum and are positively correlated with T cell expansion in the ileum and spleen. The P particle vaccine primed for stronger immune responses than VLPs, including significantly higher numbers of activated CD4+ T cells in all tissues, gamma interferon-producing (IFN-γ+) CD8+ T cells in the duodenum, regulatory T cells (Tregs) in the blood, and transforming growth factor ß (TGF-ß)-producing CD4+ CD25- FoxP3+ Tregs in the spleen postchallenge, indicating that P particles are more immunogenic than VLPs at the same dose. In conclusion, the P particle vaccine is a promising vaccine candidate worthy of further development. IMPORTANCE: The norovirus (NoV) P particle is a vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. P particles can be easily produced in Escherichia coli at high yields and thus may be more economically viable than the virus-like particle (VLP) vaccine. This study demonstrated, for the first time, the cross-variant protection (46.7%) of the intranasal P particle vaccine against human NoV diarrhea and revealed in detail the intestinal and systemic T cell responses by using the gnotobiotic pig model. The cross-variant protective efficacy of the P particle vaccine was comparable to that of the VLP vaccine in pigs (60%) and to the homologous protective efficacy of the VLP vaccine in humans (47%). NoV is now the leading cause of pediatric dehydrating diarrhea, responsible for approximately 1 million hospital visits for U.S. children and 218,000 deaths in developing countries. The P particle vaccine holds promise for reducing the disease burden and mortality.

Probiotic Lactobacillus rhamnosus GG enhanced Th1 cellular immunity but did not affect antibody responses in a human gut microbiota transplanted neonatal gnotobiotic pig model.

This study aims to establish a human gut microbiota (HGM) transplanted gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV) vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG) feeding (14 doses, up to 109 colony-forming-unit [CFU]/dose) effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 106 CFU/dose) was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics) that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.

Dual functions of Lactobacillus acidophilus NCFM as protection against rotavirus diarrhea.

OBJECTIVE: The aim of the study was to examine the dose effects of Lactobacillus acidophilus (LA) NCFM strain on rotavirus-specific antibody and B-cell responses in gnotobiotic pigs vaccinated with an oral attenuated human rotavirus (AttHRV). METHODS: Pigs were inoculated with AttHRV vaccine in conjunction with high-dose LA (14 doses, total 2.2 × 10(6) colony-forming units [CFU]), intermediate-dose LA (MidLA) (9 doses, total 3.2 × 10(9) CFU), low-dose LA (LoLA) (5 doses, total 2.1 × 10(6) CFU), or without LA feeding. Protection against rotavirus shedding and diarrhea was assessed upon challenge with a virulent HRV. Rotavirus-specific immunoglobulin A (IgA) and IgG antibodies in serum and rotavirus-specific IgA and IgG antibody-secreting cells (ASCs) and memory B cells in ileum, spleen, and blood of the pigs were measured and compared among treatment groups. RESULTS: The MidLA, but not high-dose LA or LoLA, significantly reduced rotavirus diarrhea (MidLA-only group) and significantly improved the protection conferred by AttHRV vaccine (MidLA + AttHRV group). Associated with the increased protection, MidLA significantly enhanced rotavirus-specific antibody, ASCs, and memory B-cell responses to AttHRV vaccine. High-dose LA or LoLA did not enhance virus-specific antibody and ASC responses, and hence did not improve the vaccine efficacy. CONCLUSIONS: These findings highlight the importance of dose selection and indicate that certain specific lactobacilli strains at the appropriate dose have the dual function of reducing rotavirus diarrhea and enhancing the immunogenicity and protective efficacy of rotavirus vaccines.

A neonatal gnotobiotic pig model of human enterovirus 71 infection and associated immune responses.

Vaccine development and pathogenesis studies for human enterovirus 71 are limited by a lack of suitable animal models. Here, we report the development of a novel neonatal gnotobiotic pig model using the non-pig-adapted neurovirulent human enterovirus 71 strain BJ110, which has a C4 genotype. Porcine small intestinal epithelial cells, peripheral blood mononuclear cells and neural cells were infected in vitro. Oral and combined oral-nasal infection of 5-day-old neonatal gnotobiotic pigs with 5×10(8) fluorescence forming units (FFU) resulted in shedding up to 18 days post-infection, with viral titers in rectal swab samples peaking at 2.22×10(8) viral RNA copies/mL. Viral capsid proteins were detected in enterocytes within the small intestines on post-infection days (PIDs) 7 and 14. Additionally, viral RNA was detected in intestinal and extra-intestinal tissues, including the central nervous system, the lung and cardiac muscle. The infected neonatal gnotobiotic pigs developed fever, forelimb weakness, rapid breathing and some hand, foot and mouth disease symptoms. Flow cytometry analysis revealed increased frequencies of both CD4(+) and CD8(+) IFN-γ-producing T cells in the brain and the blood on PID 14, but reduced frequencies were observed in the lung. Furthermore, high titers of serum virus-neutralizing antibodies were generated in both orally and combined oral-nasally infected pigs on PIDs 7, 14, 21 and 28. Together, these results demonstrate that neonatal gnotobiotic pigs represent a novel animal model for evaluating vaccines for human enterovirus 71 and for understanding the pathogenesis of this virus and the associated immune responses.

Lactobacillus rhamnosus GG on rotavirus-induced injury of ileal epithelium in gnotobiotic pigs.

OBJECTIVE: The aim of this study was to study the effect of continued Lactobacillus rhamnosus GG strain (LGG) feeding on rotavirus gastroenteritis in the gnotobiotic (Gn) pig model of virulent human rotavirus (HRV) infection. METHODS: Gn pigs were assigned to treatment groups: mock control, LGG only, HRV only, or LGG plus HRV. Nine days before HRV inoculation (3 days of age), pigs were fed LGG with a daily dose increase of 10-fold from 10³ to 10¹² colony-forming units (CFU). The 10¹² CFU/dose of LGG feeding continued until post-HRV inoculation day (PID) 6. Clinical sign (diarrhea), rotavirus fecal shedding, histopathology of the ileum, adherent junction and tight junction protein expression in the ileal epithelial cells, mucin production in the large and small intestinal contents, and serum cytokine responses from PID 2 to 6 were examined and compared among the treatment groups. RESULTS: Clinically, the percentage of pigs developing diarrhea, the mean duration of diarrhea, and the mean cumulative fecal scores were lower in the LGG fed pigs compared to the nonfed pigs after HRV inoculation. LGG partially protected ileal epithelium against HRV-induced compensatory increases of the adherent junction protein α-catenin and ß-catenin, tight junction protein occludin, claudin-3 and claudin-4, and leak protein claudin-2. LGG promoted mucin production because the mucin levels in the large intestinal contents of the LGG+HRV pigs were significantly higher than the HRV-only pigs on PID 2. Additionally, LGG maintained the anti-inflammatory cytokine transforming growth factor-ß level in serum after HRV infection. CONCLUSIONS: LGG is moderately effective for ameliorating rotavirus diarrhea by partially preventing injuries to the epithelium.

Median infectious dose of human norovirus GII.4 in gnotobiotic pigs is decreased by simvastatin treatment and increased by age.

Human noroviruses (NoVs), a major cause of viral gastroenteritis, are difficult to study due to the lack of a cell-culture and a small-animal model. Pigs share with humans the types A and H histo-blood group antigens on the intestinal epithelium and have been suggested as a potential model for studies of NoV pathogenesis, immunity and vaccines. In this study, the effects of age and a cholesterol-lowering drug, simvastatin, on the susceptibility of pigs to NoV infection were evaluated. The median infectious dose (ID50) of a genogroup II, genotype 4 (GII.4) 2006b variant was determined. The ID50 in neonatal (4-5 days of age) pigs was ≤2.74×10(3) viral RNA copies. In older pigs (33-34 days of age), the ID50 was 6.43×10(4) but decreased to <2.74×10(3) in simvastatin-fed older pigs. Evidence of NoV infection was obtained by increased virus load in the intestinal contents, cytopathological changes in the small intestine, including irregular microvilli, necrosis and apoptosis, and detection of viral antigen in the tip of villi in duodenum. This GII.4 variant was isolated in 2008 from a patient from whom a large volume of stool was collected. GII.4 NoVs are continuously subjected to selective pressure by human immunity, and antigenically different GII.4 NoV variants emerge every 1-2 years. The determination of the ID50 of this challenge virus is valuable for evaluation of protection against different GII.4 variants conferred by NoV vaccines in concurrence with other GII.4 variants in the gnotobiotic pig model.

CD4+ CD25- FoxP3+ regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs.

The distribution and dynamic changes of CD4(+) CD25(+) FoxP3(+) and CD4(+) CD25(-) FoxP3(+) regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post-inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue-residing Treg cells, and decreased the frequencies of interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) producing CD4(+) CD25(-) Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL-10 and TGF-ß producing CD4(+) CD25(-) Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV-caused diarrhoea (r = -1, P < 0.0001). Hence, higher frequencies of functional CD4(+) CD25(-) Treg cells can be an indicator for poorer protective immunity against rotavirus. Our results highlighted the importance of CD4(+) CD25(-) Treg cells over CD4(+) CD25(+) Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4(+) CD25(-) Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF-ß producing Treg cells post-challenge. The reduced suppressive effect of Treg cells in AttHRV-vaccinated pigs would unleash effector/memory T-cell activation upon challenge. Preserving TGF-ß producing CD4(+) CD25(-) Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T-cell responses after primary rotavirus infection/vaccination and challenge.

Characterization of immune modulating functions of γδ T cell subsets in a gnotobiotic pig model of human rotavirus infection.

We characterized immune modulating functions of porcine γδ T cell subsets in rotavirus infection using a gnotobiotic pig model of human rotavirus infection and sort-purified lymphocyte autologous co-cultures. We demonstrated that CD2+CD8- and CD2-CD8- γδ T cells have mainly pro-inflammatory function as evident by directly secreting IFN-γ or promoting CD4+ αß T cell proliferation and IFN-γ production, whereas CD2+CD8+ γδ T cells mainly exert regulatory T cell function by expressing FoxP3, secreting IL-10 and TGF-ß or increasing IL-10 and TGF-ß production by CD4+ αß T cells. γδ T cells responded to rotavirus infection by increasing TLR2, TLR3, TLR9 expression and IFN-γ and/or TGF-ß production. The CD8- subsets likely differentiate into CD8+ subset by acquiring CD8 expression, explaining in part the apparently dual functions of CD2+CD8+ and CD2+CD8- subsets. Thus, both CD8+ and CD8- γδ T cell subsets can contribute to anti-rotavirus immunity and to the maintenance and restoration of intestinal and systemic homeostasis.