Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity.

Rotavirus (RV) causes significant morbidity and mortality in children worldwide. The intestinal microbiota plays an important role in modulating host-pathogen interactions, but little is known about the impact of commonly used probiotics on human RV (HRV) infection. In this study, we compared the immunomodulatory effects of Gram-positive (Lactobacillus rhamnosus strain GG [LGG]) and Gram-negative (Escherichia coli Nissle [EcN]) probiotic bacteria on virulent human rotavirus (VirHRV) infection and immunity using neonatal gnotobiotic piglets. Gnotobiotic piglets were colonized with EcN, LGG, or EcN+LGG or uncolonized and challenged with VirHRV. Mean peak virus shedding titers and mean cumulative fecal scores were significantly lower in EcN-colonized compared with LGG-colonized or uncolonized piglets. Reduced viral shedding titers were correlated with significantly reduced small intestinal HRV IgA Ab responses in EcN-colonized compared with uncolonized piglets post-VirHRV challenge. However the total IgA levels post-VirHRV challenge in the intestine and pre-VirHRV challenge in serum were significantly higher in EcN-colonized than in LGG-colonized piglets. In vitro treatment of mononuclear cells with these probiotics demonstrated that EcN, but not LGG, induced IL-6, IL-10, and IgA, with the latter partially dependent on IL-10. However, addition of exogenous recombinant porcine IL-10 + IL-6 to mononuclear cells cocultured with LGG significantly enhanced IgA responses. The greater effectiveness of EcN in moderating HRV infection may also be explained by the binding of EcN but not LGG to Wa HRV particles or HRV 2/4/6 virus-like particles but not 2/6 virus-like particles. Results suggest that EcN and LGG differentially modulate RV infection and B cell responses.

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.

Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model.

Rotaviruses (RVs) are a leading cause of childhood diarrhea. Current oral vaccines are not effective in impoverished countries where the vaccine is needed most. Therefore, alternative affordable strategies are urgently needed. Probiotics can alleviate diarrhea in children and enhance specific systemic and mucosal Ab responses, but the T cell responses are undefined. In this study, we elucidated the T cell and cytokine responses to attenuated human RV (AttHRV) and virulent human RV (HRV) in gnotobiotic pigs colonized with probiotics (Lactobacillus rhamnosus strain GG [LGG] and Bifidobacterium lactis Bb12 [Bb12]), mimicking gut commensals in breastfed infants. Neonatal gnotobiotic pigs are the only animal model susceptible to HRV diarrhea. Probiotic colonized and nonvaccinated (Probiotic) pigs had lower diarrhea and reduced virus shedding postchallenge compared with noncolonized and nonvaccinated pigs (Control). Higher protection in the Probiotic group coincided with higher ileal T regulatory cells (Tregs) before and after challenge, and higher serum TGF-ß and lower serum and biliary proinflammatory cytokines postchallenge. Probiotic colonization in vaccinated pigs enhanced innate serum IFN-α, splenic and circulatory IFN-γ-producing T cells, and serum Th1 cytokines, but reduced serum Th2 cytokines compared with noncolonized vaccinated pigs (Vac). Thus, LGG+Bb12 induced systemic Th1 immunostimulatory effects on oral AttHRV vaccine that coincided with lower diarrhea severity and reduced virus shedding postchallenge in Vac+Pro compared with Vac pigs. Previously unreported intestinal CD8 Tregs were induced in vaccinated groups postchallenge. Thus, probiotics LGG+Bb12 exert divergent immunomodulating effects, with enhanced Th1 responses to oral AttHRV vaccine, whereas inducing Treg responses to virulent HRV.

Prenatally acquired vitamin A deficiency alters innate immune responses to human rotavirus in a gnotobiotic pig model.

We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate immune responses and human rotavirus (HRV) vaccine efficacy in a gnotobiotic (Gn) piglet model of HRV diarrhea. The VAD and vitamin A-sufficient (VAS) Gn pigs were vaccinated with attenuated HRV (AttHRV) with or without concurrent oral vitamin A supplementation (100,000 IU) and challenged with virulent HRV (VirHRV). Regardless of vaccination status, the numbers of conventional and plasmacytoid dendritic cells (cDCs and pDCs) were higher in VAD piglets prechallenge, but decreased substantially postchallenge as compared with VAS pigs. We observed significantly higher frequency of CD103 (integrin αEß7) expressing DCs in VAS versus VAD piglets postchallenge, indicating that VAD may interfere with homing (including intestinal) phenotype acquisition. Post-VirHRV challenge, we observed longer and more pronounced diarrhea and higher VirHRV fecal titers in nonvaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFN-α levels were induced in control VAD versus VAS piglet sera at postchallenge day 2. Ex vivo HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs prechallenge also produced more IFN-α. In contrast, at postchallenge day 10, we observed reduced IFN-α levels in VAD pigs that coincided with decreased TLR3(+) MNC frequencies. Numbers of necrotic MNCs were higher in VAD pigs in spleen (coincident with splenomegaly in other VAD animals) prechallenge and intestinal tissues (coincident with higher VirHRV induced intestinal damage) postchallenge. Thus, prenatal VAD caused an imbalance in innate immune responses and exacerbated VirHRV infection, whereas vitamin A supplementation failed to compensate for these VAD effects.

Unraveling the Differences between Gram-Positive and Gram-Negative Probiotics in Modulating Protective Immunity to Enteric Infections.

The role of intestinal microbiota and probiotics in prevention and treatment of infectious diseases, including diarrheal diseases in children and animal models, is increasingly recognized. Intestinal commensals play a major role in development of the immune system in neonates and in shaping host immune responses to pathogens. Lactobacilli spp. and Escherichia coli Nissle 1917 are two probiotics that are commonly used in children to treat various medical conditions including human rotavirus diarrhea and inflammatory bowel disease. Although the health benefits of probiotics have been confirmed, the specific effects of these established Gram-positive (G+) and Gram-negative (G-) probiotics in modulating immunity against pathogens and disease are largely undefined. In this review, we discuss the differences between G+ and G- probiotics/commensals in modulating the dynamics of selected infectious diseases and host immunity. These probiotics modulate the pathogenesis of infectious diseases and protective immunity against pathogens in a species- and strain-specific manner. Collectively, it appears that the selected G- probiotic is more effective than the various tested G+ probiotics in enhancing protective immunity against rotavirus in the gnotobiotic piglet model.

Comparison of probiotic lactobacilli and bifidobacteria effects, immune responses and rotavirus vaccines and infection in different host species.

Different probiotic strains of Lactobacillus and Bifidobacterium genera possess significant and widely acknowledged health-promoting and immunomodulatory properties. They also provide an affordable means for prevention and treatment of various infectious, allergic and inflammatory conditions as demonstrated in numerous human and animal studies. Despite the ample evidence of protective effects of these probiotics against rotavirus (RV) infection and disease, the precise immune mechanisms of this protection remain largely undefined, because of limited mechanistic research possible in humans and investigated in the majority of animal models. Additionally, while most human clinical probiotic trials are well-standardized using the same strains, uniform dosages, regimens of the probiotic treatments and similar host age, animal studies often lack standardization, have variable experimental designs, and non-uniform and sometime limited selection of experimental variables or observational parameters. This review presents selected data on different probiotic strains of lactobacilli and bifidobacteria and summarizes the knowledge of their immunomodulatory properties and the associated protection against RV disease in diverse host species including neonates.

Replication of influenza A virus in swine umbilical cord epithelial stem-like cells.

In this study, we describe the isolation and characterization of epithelial stem-like cells from the swine umbilical cord and their susceptibility to influenza virus infection. Swine umbilical cord epithelial stem cells (SUCECs) expressed stem cell and pluripotency associated markers such as SSEA-1, SSEA-4, TRA 1-60 and TRA 1-81 and Oct4. Morphologically, cells displayed polygonal morphology and were found to express epithelial markers; pancytokeratin, cytokeratin-18 and occludin; mesenchymal cell markers CD44, CD90 and haematopoietic cell marker CD45 were not detected on these cells. The cells had extensive proliferation and self- renewal properties. The cells also possessed immunomodulatory activity and inhibited the proliferation of T cells. Also, higher levels of anti-inflammatory cytokine IL-10 were detected in SUCEC-T cell co-cultures. The cells were multipotent and differentiated into lung epithelial cells when cultured in epithelial differentiation media. We also examined if SUCECs are susceptible to infection with influenza virus. SUCECs expressed sialic acid receptors, used by influenza virus for binding to cells. The 2009 pandemic influenza virus and swine influenza virus replicated in these cells. SUCECs due to their differentiation and immunoregulatory properties will be useful as cellular therapy in a pig model for human diseases. Additionally, our data indicate that influenza virus can infect SUCECs and may transmit influenza virus from mother to fetus through umbilical cord and transplantation of influenza virus-infected stem cells may transmit infection to recipients. Therefore, we propose that umbilical cord cells, in addition to other agents, should also be tested for influenza virus before cryopreservation for future use as a cell therapy for disease conditions.

Strategies for design and application of enteric viral vaccines.

Enteric viral infections in domestic animals cause significant economic losses. The recent emergence of virulent enteric coronaviruses [porcine epidemic diarrhea virus (PEDV)] in North America and Asia, for which no vaccines are available, remains a challenge for the global swine industry. Vaccination strategies against rotavirus and coronavirus (transmissible gastroenteritis virus) infections are reviewed. These vaccination principles are applicable against emerging enteric infections such as PEDV. Maternal vaccines to induce lactogenic immunity, and their transmission to suckling neonates via colostrum and milk, are critical for early passive protection. Subsequently, in weaned animals, oral vaccines incorporating novel mucosal adjuvants (e.g., vitamin A, probiotics) may provide active protection when maternal immunity wanes. Understanding intestinal and systemic immune responses to experimental rotavirus and transmissible gastroenteritis virus vaccines and infection in pigs provides a basis and model for the development of safe and effective vaccines for young animals and children against established and emerging enteric infections.

Porcine lung mesenchymal stromal cells possess differentiation and immunoregulatory properties.

INTRODUCTION: Mesenchymal stem (stromal) cells (MSCs) possess self-renewal, differentiation and immunoregulatory properties, and therefore are being evaluated as cellular therapy for inflammatory and autoimmune diseases, and for tissue repair. MSCs isolated from bone marrow are extensively studied. Besides bone marrow, MSCs have been identified in almost all organs of the body including the lungs. Lung-derived MSCs may be more effective as therapy for lung diseases as compared to bone marrow-derived MSCs. Pigs are similar to humans in anatomy, physiology and immunological responses, and thus may serve as a useful large animal preclinical model to study potential cellular therapy for human diseases. METHODS: We isolated MSCs from the lungs (L-MSCs) of 4-6-week-old germ-free pigs. We determined the self-renewal, proliferation and differentiation potential of L-MSCs. We also examined the mechanisms of immunoregulation by porcine L-MSCs. RESULTS: MSCs isolated from porcine lungs showed spindle-shaped morphology and proliferated actively in culture. Porcine L-MSCs expressed mesenchymal markers CD29, CD44, CD90 and CD105 and lacked the expression of hematopoietic markers CD34 and CD45. These cells were multipotent and differentiated into adipocytes, osteocytes and epithelial cells. Like human MSCs, L-MSCs possessed immunoregulatory properties and inhibited proliferation of T cells and interferon-γ and tumor necrosis factor-α production by T cells and dendritic cells, respectively, and increased the production of T-helper 2 cytokines interleukin (IL)-4 and IL-13 by T cells. L-MSCs induced the production of prostaglandin E2 (PGE2) in MSC-T cell co-cultures and inhibition of PGE2 significantly restored (not completely) the immune modulatory effects of L-MSCs. CONCLUSIONS: Here, we demonstrate that MSCs can be isolated from porcine lung and that these cells, similar to human lung MSCs, possess in vitro proliferation, differentiation and immunomodulatory functions. Thus, these cells may serve as a model system to evaluate the contribution of lung MSCs in modulating the immune response, interactions with resident epithelial cells and tissue repair in a pig model of human lung diseases.

Prenatal vitamin A deficiency impairs adaptive immune responses to pentavalent rotavirus vaccine (RotaTeq®) in a neonatal gnotobiotic pig model.

Vitamin A deficiency (VAD) is associated with increased childhood mortality and morbidity in impoverished Asian and African countries, but the impact of VAD on rotavirus (RV) vaccine or infection is poorly understood. We assessed effects of gestational and dietary induced pre- and post-natal VAD and vitamin A supplementation on immune responses to a pentavalent rotavirus vaccine, RotaTeq(®) in a neonatal gnotobiotic pig model. Vaccine efficacy was assessed against virulent G1P[8] human rotavirus (HRV) challenge. VAD and vitamin A sufficient (VAS) piglets were derived from dietary VAD and VAS sows, respectively. VAD piglets had significantly lower levels of hepatic vitamin A compared to that of VAS piglets. RotaTeq(®)-vaccinated VAD piglets had 350-fold higher fecal virus shedding titers compared to vaccinated VAS piglets post-challenge. Only 25% of vaccinated non-vitamin A supplemented VAD piglets were protected against diarrhea compared with 100% protection rate in vaccinated non-supplemented VAS piglets post-challenge. Intestinal HRV specific immune responses were compromised in VAD piglets. Vaccinated VAD piglets had significantly lower ileal HRV IgG antibody secreting cell (ASC) responses (pre-challenge) and duodenal HRV IgA ASC responses (post-challenge) compared to vaccinated VAS piglets. Also, intestinal HRV IgA antibody titers were 11-fold lower in vaccinated VAD compared to vaccinated VAS piglets post-challenge. Persistently elevated levels of IL-8, a pro-inflammatory mediator, and lower IL-10 responses (anti-inflammatory) in vaccinated VAD compared to VAS piglets suggest more severe inflammatory responses in VAD piglets post-challenge. Moreover higher IFN-γ responses pre-challenge were observed in VAD compared to VAS piglets. The impaired vaccine-specific intestinal antibody responses and decreased immunoregulatory cytokine responses coincided with reduced protective efficacy of the RV vaccine against virulent HRV challenge in VAD piglets. In conclusion, VAD impaired antibody responses to RotaTeq(®) and vaccine efficacy. Oral supplementation of 100,000 IU vitamin A concurrent with RV vaccine failed to increase the vaccine efficacy in VAD piglets.

Lactobacilli and Bifidobacteria enhance mucosal B cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model.

B cells play a key role in generation of protective immunity against rotavirus infection, a major cause of gastroenteritis in children. Current RV vaccines are less effective in developing countries compared to developed countries. Commensals/probiotics influence mucosal immunity, but the role of early gut colonizing bacteria in modulating intestinal B cell responses to RV vaccines is largely unknown. We co-colonized neonatal gnotobiotic pigs, the only animal model susceptible to HRV diarrhea, with 2 dominant bacterial species present in the gut of breastfed infants, Lactobacillus rhamnosus strain GG and Bifidobacterium animalis lactis Bb12 to evaluate their impact on B cell responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine. Following HRV challenge, probiotic-colonized, AttHRV vaccinated piglets had significantly lower fecal scores and reduced HRV shedding titers compared to uncolonized, AttHRV vaccinated pigs. The reduction in HRV diarrhea was significantly correlated with higher intestinal IgA HRV antibody titers and intestinal HRV-specific IgA antibody secreting cell (ASC) numbers in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs. The significantly higher small intestinal HRV IgA antibody responses coincided with higher IL-6, IL-10 and APRIL responses of ileal mononuclear cells (MNCs) and the immunomodulatory effects of probiotics genomic DNA on TGF-ß and IL-10 responses. However, serum RV IgG antibody titers and total IgG titers were significantly lower in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs, both pre- and post-challenge. In summary, LGG and Bb12 beneficially modulated intestinal B cell responses to HRV vaccine.

In vivo gut transcriptome responses to Lactobacillus rhamnosus GG and Lactobacillus acidophilus in neonatal gnotobiotic piglets.

Probiotics facilitate mucosal repair and maintain gut homeostasis. They are often used in adjunct with rehydration or antibiotic therapy in enteric infections. Lactobacillus spp have been tested in infants for the prevention or treatment of various enteric conditions. However, to aid in rational strain selection for specific treatments, comprehensive studies are required to delineate and compare the specific molecules and pathways involved in a less complex but biologically relevant model (gnotobiotic pigs). Here we elucidated Lactobacillus rhamnosus (LGG) and L. acidophilus (LA) specific effects on gut transcriptome responses in a neonatal gnotobiotic (Gn) pig model to simulate responses in newly colonized infants. Whole genome microarray, followed by biological pathway reconstruction, was used to investigate the host-microbe interactions in duodenum and ileum at early (day 1) and later stages (day 7) of colonization. Both LA and LGG modulated common responses related to host metabolism, gut integrity, and immunity, as well as responses unique to each strain in Gn pigs. Our data indicated that probiotic establishment and beneficial effects in the host are guided by: (1) down-regulation or upregulation of immune function-related genes in the early and later stages of colonization, respectively, and (2) alternations in metabolism of small molecules (vitamins and/or minerals) and macromolecules (carbohydrates, proteins, and lipids). Pathways related to immune modulation and carbohydrate metabolism were more affected by LGG, whereas energy and lipid metabolism-related transcriptome responses were prominently modulated by LA. These findings imply that identification of probiotic strain-specific gut responses could facilitate the rational design of probiotic-based interventions to moderate specific enteric conditions.

Vitamin A deficiency impairs adaptive B and T cell responses to a prototype monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model.

Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.

Probiotics and colostrum/milk differentially affect neonatal humoral immune responses to oral rotavirus vaccine.

Breast milk (colostrum [col]/milk) components and gut commensals play important roles in neonatal immune maturation, establishment of gut homeostasis and immune responses to enteric pathogens and oral vaccines. We investigated the impact of colonization by probiotics, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) with/without col/milk (mimicking breast/formula fed infants) on B lymphocyte responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine in a neonatal gnotobiotic pig model. Col/milk did not affect probiotic colonization in AttHRV vaccinated pigs. However, unvaccinated pigs fed col/milk shed higher numbers of probiotic bacteria in feces than non-col/milk fed colonized controls. In AttHRV vaccinated pigs, col/milk feeding with probiotic treatment resulted in higher mean serum IgA HRV antibody titers and intestinal IgA antibody secreting cell (ASC) numbers compared to col/milk fed, non-colonized vaccinated pigs. In vaccinated pigs without col/milk, probiotic colonization did not affect IgA HRV antibody titers, but serum IgG HRV antibody titers and gut IgG ASC numbers were lower, suggesting that certain probiotics differentially impact HRV vaccine responses. Our findings suggest that col/milk components (soluble mediators) affect initial probiotic colonization, and together, they modulate neonatal antibody responses to oral AttHRV vaccine in complex ways.

Lactobacilli and bifidobacteria promote immune homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs.

The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.

IgY antibodies protect against human Rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model.

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization -VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV-specific IgA ASC in the duodenum. We further analyzed the pigs immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.

Expression of complement receptors 1 (CR1/CD35) and 2 (CR2/CD21), and co-signaling molecule CD19 in cattle.

C3d is a sub-fragment of the C3 component of the complement system. Covalent binding of multiple C3ds to antigen reduces the activation threshold of cognate B lymphocytes by one thousand fold through co-ligation of the B cell antigen receptor (BCR) and complement receptor 2 (CR2/CD21). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that, in cattle, four distinct complement receptors are produced from the Cr2 gene by alternative splicing. Cattle express two major variants of the Cr2 gene representing homologues of murine CR1 and CR2, each of which is expressed in both a long and a short form. Expression of CR1 and CR2 was detected in IgM(+) cells from both the spleen and peripheral blood. Additionally, the coding sequence of CD19, the CR2 co-signaling molecule, was determined. CD19 was confirmed to be expressed by IgM(+) cells from the spleen and peripheral blood.

Expression of complement receptor 2 (CD21), membrane IgM and the inhibitory receptor CD32 (FcgammaRIIb) in the lymphoid tissues of neonatal calves.

Limited active antibody responses in neonates following vaccination have been attributed to immaturity of the immune system and to the suppressive effects of maternal antibodies. The activating receptor CD21 (CR2), when co-ligated with membrane IgM (mIgM) by complement-bound antigen lowers the threshold for activation of B lymphocytes. The inhibitory receptor CD32 (FcgammaRII) when co-ligated with mIgM by antigen-antibody complexes raises the threshold for activation. Expression of these receptors, which potentially play roles in regulation of B cell responses in the presence of maternal antibodies in neonates, has been recently characterized in blood lymphocytes in neonatal calves. Little is known however about expression of these receptors in the lymphoid tissues, where immune responses are initiated. In this study, expression of CD21, mIgM and CD32 receptors by B lymphocytes was studied in a range of lymphoid tissues including spleen, lymph nodes and bone marrow from newborn and 7-week-old calves using flow cytometry. The proportion of naïve B lymphocytes in the lymphocyte gate was significantly lower in blood and spleen of newborn calves compared to 7-week-old calves. Over 90% of B lymphocytes expressed CD21 in the lymphoid tissues. In the lymph nodes and spleen, a lower proportion of mIgM(+) B lymphocytes expressed CD32 compared to blood. In addition, intensity of expression of CD32 on B cells in lymph nodes was significantly lower compared to that in blood, suggesting a lower potential for inhibitory signalling in B cells in the lymphoid microenvironment. Investigation of the CD5(+) B cell population (as an indicator of B1 B cells) suggested an increase in the proportion of IgM(+)CD5(+) cells with age in calves, in both blood and lymphoid tissue, in contrast to the situation in humans and mice. Overall, the majority of naïve B lymphocytes in lymphoid tissues in neonatal calves expressed both activating (CD21, mIgM) and inhibitory (CD32) receptors. These receptors may provide targets for novel adjuvants, to lower the threshold for activation of B cells in neonates, and enhance antibody responses.

Multiple bovine FcgammaRIIb sub-isoforms generated by alternative splicing.

Receptors for the Fc portion of immunoglobulin molecules (FcR) provide an important and vital link between circulating antibody and cellular effector functions. These receptors have been well characterized in human and murine species, however few of these receptors have been investigated in livestock. FcgammaRII (CD32) is an FcR previously shown in mice and humans to exist in multiple isoforms, both activating (FcgammaRIIa, FcgammaRIIc) and inhibitory (FcgammaRIIb), on a wide variety of cells including B cells, T cells, dendritic cells, monocytes, macrophages and platelets. On B cells, FcgammaRIIb acts to suppress cell activation and immunoglobulin production by means of an intracellular immunoreceptor tyrosine-based inhibitory motif signaling domain. Two sub-isoforms of FcgammaRIIb, designated b1 and b2, distinguished by the inclusion of an additional cytoplasmic exon in the b1 form, have been demonstrated in humans and mice, whereas only one sequence corresponding to the human and mouse b2 isoform has been identified in cattle. In this study, the expression profile of FcgammaRIIb in bovine blood mononuclear cells was characterized by collecting blood samples from mature cattle of dairy and beef breeds, and determining their FcgammaRIIb mRNA expression profile by RT-PCR. Analysis revealed the presence of two uncharacterized bovine FcgammaRIIb transcripts in addition to the single previously published transcript. Analysis of the first unknown transcript revealed high homology with published human and murine FcgammaRIIb1 sequences. This transcript was present in all cell types examined, with little variation in primary sequence between individuals or among breeds. The second unknown sequence was found to be homologous to the murine FcgammaRIIb3 (IgG-binding protein or soluble FcgammaR in humans) sequence. This transcript appears to have a much more limited expression profile, which may indicate that expression varies with the cellular activation-state of the cell. These results indicate that cattle, like humans and mice, express multiple sub-isoforms of FcgammaRIIb. These findings add further complexity to the regulation of IgG-mediated immunity and provide new insight into the role Fc receptors play in antigen acquisition and presentation in cattle.