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Lactobacillus delbrueckii subsp. delbrueckii TUA4408L is able to differentially modulate the innate immune response of porcine intestinal epithelial cells triggered by TLR4 activation. This strain also has a remarkable ability to grow on plant substrates. These two immunological and biotechnological characteristics prompted us to evaluate whether the soymilk by-product okara fermented with the TUA4408L strain can serve as an immunobiotic feed with the ability to beneficially modulate the intestinal immunity of piglets after weaning to improve their productivity. Our in vivo studies demonstrated that the administration of immunobiotic TUA4408L-fermented okara feed significantly increased piglet growth performance and meat quality. These positive effects were associated with the ability of the TUA4408L-fermented okara feed to beneficially modulate both intestinal microbiota and immunity in pigs. The immunobiotic feed improved the abundance of the beneficial bacteria Lactobacillus and Lactococcus in the gut of pigs, reduced blood markers of inflammation, and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These findings indicate that the immunobiotic TUA4408L-fermented okara feed could be an economical and environmentally friendly option to improve the growth performance and immune health of pigs. Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Sasaki, Nana. Miyagi University; Japón Fil: Kagawa, Kyoma. Miyagi University; Japón Fil: Elean, Mariano Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Zhou, Binghui. Tohoku University; Japón Fil: Tomokiyo, Mikado. Tohoku University; Japón Fil: Islam, Md Aminul. Tohoku University; Japón. Bangladesh Agricultural University; Bangladesh Fil: Shahid Riaz Rajoka, Muhammad. Tohoku University; Japón Fil: Humayun Kober, A.K.M.. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University; Bangladesh Fil: Shimazu, Tomoyuki. Miyagi University; Japón Fil: Egusa, Shintaro. No especifíca; Fil: Terashima, Yuji. No especifíca; Fil: Aso, Hisashi. Tohoku University; Japón Fil: Ikeda Ohtsubo, Wakako. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Kitazawa, Haruki. Tohoku University; Japón

Rakkyo (Allium chinense), is a Japanese leek that is primarily used to make a popular sweet or sour pickled dish. Lactic acid bacteria are often involved in the preparation steps of fermented pickles, which helps in the effective preservation of the natural bioactive compounds of fruits and vegetable, and thereby exert several health benefits including immunomodulation and growth per-formance. This work aimed to evaluate the in vivo effects of adding Lactiplantibacillus plantarum N14 fermented rakkyo pickled juice as feed supplement on the immunocompetence and production performance of pigs. We first analyzed the nutritional composition, which revealed that the proportion of protein, lipid, and water-soluble fiber content were estimated as of 4%, 5%, and 5% in rakkyo residual liquid or juice, while 22%, 15% and 14%, respectively, were estimated in rakkyo residual powder. For the in vivo feeding trials, three groups of pigs were treated either with 5%, 20%, or 40% mixture (v/v) of fermented rakkyo pickled juice and the grinded residual liquid supplemented in the drinking water in addition to standard feed. The results of the feeding trials showed that the administration of a juice mixture of 5% or 20% (fermented pickled juice and residual liquid) had a similar trend of effects in improving the complement activity, phagocytic activity and leucocytes counts in the peripheral blood when compared to pigs fed with 40% mixture or untreated controls. Those changes were related to an improved resistance to enteric infections. Moreover, animals receiving a mixture of fermented pickled juice and fermented rakkyo residues had a higher growth rate and carcass quality than controls. The results suggested that the use of 5% mixture of fermented rakkyo pickled juice and the residual liquid through drinking water could be a cost-effective approach to promote the immune-health and production performance of pigs. This approach would contribute not only to the sustainable management of food wastes but also to the application of a value-added feed supplement for the promotion of animal health and production. Fil: Islam, Md Aminul. Tohoku University; Japón. Bangladesh Agricultural University; Bangladesh Fil: Hashiguchi, Kenji. No especifíca; Fil: Humayun Kober, A.K.M.. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University; Bangladesh Fil: Morie, Kyoko. Tohoku University; Japón Fil: Zhou, Binghui. Tohoku University; Japón Fil: Tomokiyo, Mikado. Tohoku University; Japón Fil: Shimazu, Tomoyuki. Miyagi University; Japón Fil: Aso, Hisashi. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Kitazawa, Haruki. Tohoku University; Japón

Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama. Fil: Quilodrán Vega, Sandra. Universidad de Concepción; Chile Fil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; Argentina. Tohoku University; Japón Fil: Mansilla, Flavia Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Arce, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Tohoku University; Japón Fil: Zhou, Binghui. Tohoku University; Japón Fil: Islam, Md Aminul. Tohoku University; Japón. Bangladesh Agricultural University; Bangladesh Fil: Tomokiyo, Mikado. Tohoku University; Japón Fil: Al Kassaa, Imad. Lebanese University. Faculty of Public Health; Líbano Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Kitazawa, Haruki. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón

Background Infection with bovine leukemia virus (BLV), the causative agent for enzootic bovine leukosis (EBL), is increasing in dairy farms of Japan. The tendency of tumor development following BLV infection in certain cow families and bull lines has previously been described. We therefore hypothesized the existence of a genetic component which differentiates cattle susceptibility to the disease. Results We analyzed routinely collected large-scale data including postmortem inspection data, which were combined with pedigree information and epidemiological data of BLV infection. A total of 6,022 postmortem inspection records of Holstein cattle, raised on 226 farms served by a regional abattoir over 10 years from 2004 to 2015, were analyzed for associations between sire information and EBL development. We then identified statistically the relative susceptibility to EBL development for the progeny of specific sires and paternal grandsires (PGSs). The heritability of EBL development was calculated as 0.19. Similarly, proviral loads (PVLs) of progeny from identified sires and PGSs were analyzed, but no significant differences were found. Conclusions These observations suggest that because EBL development in our Holstein population is, at least in part, influenced by genetic factors independent of PVL levels, genetic improvement for lower incidence of EBL development in cattle notwithstanding BLV infection is possible.

Mycoplasma pneumonia of swine (MPS) is caused by Mycoplasma hyopneumoniae (M.hp) and is a common chronic respiratory disease of pigs. Recently, a genetically selected variant of the Landrace pig (Miyagino L2) has a lower incidence of pulmonary MPS lesions. We investigated the pathological and immunological characteristics of MPS resistance in these pigs (n = 24) by comparing with the normal landrace pig (control: n = 24). The pathological MPS lung lesion score in MPS-selected landrace pigs was significantly lower than in the control. The gene expression of interleukin (IL)-12p40, which acts as a chemoattractant and a component of the bioactive cytokines IL-12 and IL-23, was significantly higher at the hilar lymph nodes, lung, and spleen in MPS-selected landrace pigs than in control landrace pigs, and these were negatively correlated with the macroscopic MPS lung lesion score. In summary, we demonstrate that resistance against MPS in Miyagino L2 pigs is associated with IL-12p40 up-regulation, in comparison with normal landrace pigs without the MPS vaccine. In addition, a comparative study of macroscopic MPS lung lesions and IL-12p40 gene expression in lung and hilar lymph nodes may lead to beneficial selection traits for the genetic selection for MPS resistance in pigs.

Previously, we evaluated the effect of the immunobiotic strain Lactobacillus rhamnosus CRL1505 on the transcriptomic response of porcine intestinal epithelial (PIE) cells triggered by the challenge with the Toll-like receptor 3 (TLR-3) agonist poly(I:C) and successfully identified a group of genes that can be used as prospective biomarkers for the screening of new antiviral immunobiotics. In this work, several strains of lactobacilli were evaluated according to their ability to modulate the expression of IFNα, IFNβ, RIG1, TLR3, OAS1, RNASEL, MX2, A20, CXCL5, CCL4, IL-15, SELL, SELE, EPCAM, PTGS2, PTEGES, and PTGER4 in PIE cells after the stimulation with poly(I:C). Comparative analysis of transcripts variations revealed that one of the studied bacteria, Lactobacillus plantarum MPL16, clustered together with the CRL1505 strain, indicating a similar immunomodulatory potential. Two sets of in vivo experiments in Balb/c mice were performed to evaluate L. plantarum MPL16 immunomodulatory activities. Orally administered MPL16 prior intraperitoneal injection of poly(I:C) significantly reduced the levels of the proinflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-15 in the intestinal mucosa. In addition, orally administered L. plantarum MPL16 prior nasal stimulation with poly(I:C) or respiratory syncytial virus infection significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced levels of the proinflammatory mediators TNF-α, IL-6, and IL-8 were found in MPL16-treated mice. Improved levels of IFN-β and IFN-γ in the respiratory mucosa were observed in mice treated with L. plantarum MPL16 when compared to control mice. The immunological changes induced by L. plantarum MPL16 were not different from those previously reported for the CRL1505 strain in in vitro and in vivo studies. The results of this work confirm that new immunobiotic strains with the ability of stimulating both local and distal antiviral immune responses can be efficiently selected by evaluating the expression of biomarkers in PIE cells. Fil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Garcia-Castillo, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad de Concepción; Chile Fil: Masumizu, Yuki. Tohoku University; Japón Fil: Indo, Yuhki. Tohoku University; Japón Fil: Islam, Md Aminul. Tohoku University; Japón Fil: Suda, Yoshihito. Miyagi University; Japón Fil: García Cancino, Apolinaria. Universidad de Concepción; Chile Fil: Aso, Hisashi. Tohoku University; Japón Fil: Takahashi, Hideki. Tohoku University; Japón Fil: Kitazawa, Haruki. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón

About 1000 species of bacteria are present in the human intestine. Some Gram-negative bacteria such as Escherichia coli or Salmonella spp. among intestinal bacteria have lipopolysaccharide (LPS), which might induce inflammation of human intestines. Actually, LPS, especially its lipid A constituent, is toxic. Small amounts of LPS in bacteria cause inflammation of mucosa and other tissues in humans. Such bacteria may be regulated by beneficial lactic acid bacteria to maintain human health. Many lactic acid bacteria show cancer prevention activity and anti-inflammatory activity in intestines. Recently, Pediococcus pentosaceus AK-23 was isolated from fermentative vegetable pickles for neutralization of LPS. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23, by ultrafiltration using a 300 kDa membrane and a 100 kDa membrane after cell wall digestion by lysozyme. Gel running blue native electrophoresis revealed the existence of a 217 kDa protein. The band of the protein having the ability to bind LPS on the gel was analyzed for amino acid homology. As the result, it is revealed as part of a subunit of heat shock protein (HSP). Furthermore, it displayed LPS binding or hydrophobic motifs. The protein neutralized LPS to release fatty acid as myristic acid and glucose from polysaccharide. These findings suggest that HSP in P. pentosaceus AK-23 neutralizes LPS to decompose it compising fatty acid and polysaccharide.

Emerging threats of antimicrobial resistance necessitate the exploration of effective alternatives for healthy livestock growth strategies. &lsquo, Immunosynbiotics&rsquo, a combination of immunoregulatory probiotics and prebiotics with synergistic effects when used together in feed, would be one of the most promising candidates. Lactobacilli are normal residents of the gastrointestinal tract of pigs, and many of them are able to exert beneficial immunoregulatory properties. On the other hand, wakame (Undaria pinnafida), an edible seaweed, has the potential to be used as an immunoregulatory prebiotic when added to livestock feed. Therefore, in order to develop a novel immunosynbiotic, we isolated and characterized immunoregulatory lactobacilli with the ability to utilize wakame. Following a month-long in vivo wakame feeding trial in 8-week-old Landrace pigs (n = 6), sections of intestinal mucous membrane were processed for bacteriological culture and followed by identification of pure colonies by 16S rRNA sequence. Each isolate was characterized in vitro in terms of their ability to assimilate to the wakame and to differentially modulate the expression of interleukin-6 (IL-6) and interferon beta (IFN-&beta, ) in the porcine intestinal epithelial (PIE) cells triggered by Toll-like receptor (TLR)-4 and TLR-3 activation, respectively. We demonstrated that feeding wakame to pigs significantly increased the lactobacilli population in the small intestine. We established a wakame-component adjusted culture media that allowed the isolation and characterization of a total of 128 Lactobacilli salivarius colonies from the gut of wakame-fed pigs. Interestingly, several L. salivarius isolates showed both high wakame assimilation ability and immunomodulatory capacities. Among the wakame assimilating isolates, L. salivarius FFIG71 showed a significantly higher capacity to upregulate the IL-6 expression, and L. salivarius FFIG131 showed significantly higher capacity to upregulate the IFN-&beta, expression, these could be used as immunobiotic strains in combination with wakame for the development of novel immunologically active feeds for pigs.

This study was conducted to evaluate the effects of supplementation of Wakame seaweed stalks on the immunity and intestinal microflora of pigs. Three separate experiments were performed: Relatively young (start at 20-30 kg; Experiments 1 and 2) and fattening period (70 kg; Experiment 3). All pigs (including the control group) were fed the same commercial feed, free from antibiotic additives, but in the feed for the treatment groups, 1% seaweed powder was added. There were no group differences observed in daily weight gain and feed intake in Experiments 1 and 2 between groups; however, daily weight gain was significantly higher in the treatment group compared to the control group in Experiment 3. The percentage of peripheral blood natural killer cells of the treatment group was significantly higher than that of the control group in all experiments. Although addition of seaweed changed the gene expression of cytokine and toll-like receptors of the small intestinal Peyer's patches slightly, seaweed seems to alter intestinal microflora preferentially, for instance, there was an increase in Lactobacillus and a decrease of Escherichia coli observed. These results suggest that Wakame seaweed can be used as supplement for pig feed to improve the gut health and immunity of pigs.

The moment arm of gluteus medius proportionated to distance from femoral head tends to be decreased postoperatively in valgus-impacted femoral neck fractures treated by in situ internal fixation. The aim of this article is to introduce a new gentle technique to correct the deformity. The innovative technique of Minimally Invasive Closed Reduction for Impacted Femoral neck fractures (MICRIF) mainly focused to disimpact valgus neck fractures into anatomical position. Patients were positioned on the fracture table to fix the hip joint in abduction and internal rotation. A 2.4-mm diameter Kirschner wire was inserted a few centimetres outside the iliac crest piercing the acetabular beak to enter the femoral head, followed by repositioning of the lower extremity from abduction into neutral. This method provides satisfactory anatomical reduction. Thereafter, a surgical implant was applied to osteosynthesize the reduced fracture. This simple technique effectively provides anatomical reduction in valgus impacted femoral neck fracture.

Recently, many scholars have reported lactic acid bacteria (LAB) functions, such as anticancer activity and anti-inflammatory activity for intestines. To decrease inflammatory substances such as endotoxins, LAB consumed safely with meals were isolated from food and food ingredients. First, LAB were isolated as 168 strains of bacillus LAB (49 strain) and coccus LAB (119 strains) from food ingredients and fermented foods such as rice, rice bran, malt, grains, miso soy paste, and some pickles. Their LAB (168 strains) were cultivated in medium containing endotoxin from Escherichia coli O18 LPS at 15 and 30 °C for 64 h to identify endotoxin-eliminating LAB. Consequently, the AK-23 strain was screened as an endotoxin-eliminating LAB strain. The strain decreased endotoxin in YP medium without sugar at 30 °C for 64 h until 9% of endotoxin. The strain was identified as Pediococcus pentosaceus according to morphological characteristics such as its cell shape, physiological characteristics related to its fermentation type, assimilation of sugars, pH tolerance, optimum growth temperature, and molecular biological characteristics as its homology to 16S rRNA. To investigate the location of the endotoxin-eliminating substance, 4 fractions were separated from AK-23 cells as extracellular, cell wall digestion, cytoplasm, and cell membrane fractions. The endotoxin-decreasing substance, located on a cell wall, was identified as a 217 kDa protein.

Immunogenic properties and mycoplasmal pneumonia of swine (MPS) lung lesions were compared between the immunity-selected Large White line and the non-selected Large White line. The selected Large White line showed a higher level of pulmonary MPS lesions compared with the non-selected Large White line. Subsequent to vaccination, the percentage of natural killer cells and T cells (CD3(+) CD4(+) CD8(-) and CD3(+) CD4(-) CD8(+) T cells) were significantly increased in the non-selected line but remained unchanged in the immunity-selected Large White line. Secretion of Mycoplasma hyopneumoniae vaccine-specific immunoblogulin G and phagocyte activity in peripheral blood were significantly higher in the immunity-selected Large White line than in the non-selected line. Expression of interleukin (IL)-4 and IL-6 messenger RNA in hilar lymph nodes was significantly lower in the immunity-selected Large White line than in the non-selected line. However, expression of IL-10 in all immune tissues was significantly higher in the immunity-selected Large White line. These results suggest that the selection for high immunity was not effective in increasing resistance to MPS lung lesions.

Mycoplasma pneumonia of swine (MPS) lung lesions and immunogenic properties were compared between a Landrace line that was genetically selected for reduced incidence of pulmonary MPS lesions, and a non-selected Landrace line. The MPS-selected Landrace line showed significantly lower degrees of pulmonary MPS lesions compared with the non-selected Landrace line. When changes in immunity before and after vaccination were compared, the percentage of B cells in the peripheral blood of the MPS-selected Landrace line was significantly lower than that of the non-selected line. Furthermore, the concentration of growth hormone and the mitogen activity of peripheral blood mononuclear cells in the MPS-selected Landrace line showed significantly (P

The genome of the immunomodulatory Lactobacillus plantarum MPL16, a strain able to ferment wakame (Undaria pinnatifida), is described here. The reads were assembled into contigs with a total size 3,278,495 bp. The genome information will be useful for further specific genetic studies of this strain that evaluate its immunomodulatory and biotechnological properties. Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Suda, Yoshihito. Miyagi University; Japón. Tohoku University; Japón Fil: Masumizu, Yuki. Miyagi University; Japón. Tohoku University; Japón Fil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Clua, Maria Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Ikeda Ohtsubo, Wakako. Tohoku University; Japón. Miyagi University; Japón Fil: Kitazawa, Haruki. Miyagi University; Japón. Tohoku University; Japón

Purpose In this study, we aimed to characterize toll-like receptor (TLR)-3-mediated inflammatory immune response in porcine intestinal epithelial (PIE) cells and in PIE-immune cell co-cultures and, to evaluate if these in vitro systems are useful for selecting immunomodulatory lactic acid bacteria. Results We demonstrated that these systems are valuable tools for the in vitro study of the inflammatory response triggered by TLR3 in intestinal epithelial cells (IECs) and of the interaction between IECs and immune cells. In addition, we showed that PIE cells could be used for the selection of immunobiotic lactobacilli strains with anti-inflammatory activities. We found that Lactobacillus casei MEP221114 is an immunobiotic candidate for modulation of TLR3-mediated inflammatory responses. Conclusion The present study deepened our understanding of the mechanisms of immunobiotic action by demonstrating that the interaction between some lactobacilli strains and IECs can up-regulate the mRNA expression of TLR negative regulators and that this effect could help to regulate the production of inflammatory mediators during the generation of a TLR3-mediated immune response.

In order to evaluate probiotic strains applicable for the beneficial immunomodulation of the porcine gut (immunobiotics), we previously developed a porcine intestinal epitheliocyte cell line (PIE cells). Here, transcriptomic studies using PIE cells were performed considering that this information would be valuable for understanding the mechanisms involved in the protective activity of the immunobiotic strain Lactobacillus jensenii TL2937 against intestinal inflammatory damage in pigs. In addition, those studies would provide criteria for selecting biomarkers for the screening of new immunobiotic strains. We performed microarray analysis to investigate the transcriptomic response of PIE cells to the challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs) and, the changes induced by L. jensenii TL2937 in that response. The approach allowed us to obtain a global overview of the immune genes involved in the response of PIE cells to heat-stable ETEC PAMPs. We observed that L. jensenii TL2937 differently modulated gene expression in ETEC PAMPs-challenged PIE cells. Microarray and RT-PCR analysis indicated that the most remarkable changes in PIE cells transcriptomic profile after heat-stable ETEC PAMPs challenge were observed in chemokines, adhesion molecules, complement and coagulation cascades factors. In addition, an anti-inflammatory effect triggered by TL2937 strain in PIE cells was clearly demonstrated. The decrease in the expression of chemokines (CCL8, CXCL5, CXCL9, CXCL10, and CXCL11), complement (C1R, C1S, C3, and CFB), and coagulation factors (F3) by L. jensenii TL2937 supports our previous reports on the immunoregulatory effect of this strain. These results provided clues for the better understanding of the mechanism underlying host-immunobiotic interaction in the porcine host. The comprehensive transcriptomic profiles of PIE cells provided by our analyses successfully identified a group of genes, which could be used as prospective biomarkers for the screening and evaluation of new anti-inflammatory immunobiotics for the prevention of inflammatory intestinal disorders in pigs. Fil: Kobayashi, Hisakazu. Tohoku University; Japón Fil: Albarracín, Leonardo Miguel. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sato, Nana. Tohoku University; Japón Fil: Kanmani, Paulraj. Tohoku University; Japón Fil: Kober, Humayun A.K.M.. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University. Department of Dairy and Poultry Science; Bangladesh Fil: Ikeda-Ohtsubo, Wakako. Tohoku University; Japón Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Nochi, Tomonori. Tohoku University; Japón Fil: Aso, Hisashi. Tohoku University; Japón Fil: Makino, Seiya. Meiji Co., Ltd. Food Science Research Labs.; Japón Fil: Kano, Hiroshi. Meiji Co., Ltd. Food Science Research Labs.; Japón Fil: Ohkawara, Sou. Meiji Seika Pharma Co., Ltd. Agricultural and Veterinary Division; Japón Fil: Saito, Tadao. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Kitazawa, Haruki. Tohoku University; Japón

Background: Immunobiotic Lactobacillus jensenii TL2937 modulates porcine mononuclear phagocytes from Peyer?s patches (PPMPs) and induces a differential production of pro- and anti-inflammatory cytokines in response to Toll-like receptor (TLR)-4 activation. Objective: In view of the important role played by phagocytosis in the activation of antigen presenting cells (APCs), the aim of the present work was to examine the interaction of TL2937 with porcine PPMPs focusing on phagocytosis. In addition, this study aimed to investigate whether the effects of L. jensenii TL2937 in porcine blood monocyte-derived dendritic cells (MoDCs) are similar to those found in PPMPs considering that MoDCs do not recapitulate all functions of mucosal APCs. Results: studies showed a high ability of porcine CD172a+ PPMPs to phagocytose L. jensenii TL2937. Interestingly, our results also revealed a reduced capacity of the non-immunomodulatory L. plantarum TL2766 to be phagocytosed by those immune cells. Phagocytosis of L. jensenii TL2937 by porcine PPMPs was partially dependent on TLR2. In addition, we demonstrated that TL2937 strain was able to improve the expression of IL-1, IL-12 and IL-10 in immature MoDCs resembling the effect of this immunobiotic bacterium on PPMPs. Moreover, similarly to PPMPs those immunomodulatory effects were related to the higher capacity of TL2937 to be phagocytosed by immature MoDCs. Conclusions: Microbial recognition in APCs could be effectively mediated through ligand-receptor interactions that then mediate phagocytosis and signaling. For the immunobiotic strain TL2937, TLR2 has a partial role for its interaction with porcine APCs and it is necessary to investigate the role of other receptors. A challenge for future research will be advance in the full understanding of the molecular interactions of immunobiotic L. jensenii TL2937 with porcine APCs that will be crucial for the successful development of functional feeds for the porcine host. This study is a step in that direction. Fil: Tsukida, Kohichiro. Tohoku University; Japón Fil: Takahashi, Takuya. Tohoku University; Japón Fil: Iida, Hikaru. Tohoku University; Japón Fil: Kanmani, Paulraj. Tohoku University; Japón Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Nochi, Tomonori. Tohoku University; Japón Fil: Ohwada, Shuichi. Tohoku University; Japón Fil: Aso, Hisashi. Tohoku University; Japón Fil: Ohkawara, Sou. Meiji Seika Pharma Co., Ltd. Agricultural & Veterinary Division; Japón Fil: Makino, Seiya. Meiji Co., Ltd. Division of Research and Development; Japón Fil: Kano, Hiroshi. Meiji Co., Ltd. Division of Research and Development; Japón Fil: Saito, Tadao. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Kitazawa, Haruki. Tohoku University; Japón

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-β and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-β production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities. Fil: Ishizuka, Takamasa. Tohoku University; Japón Fil: Kanmani, Paulraj. Tohoku University; Japón Fil: Kobayashi, Hisakazu. Tohoku University; Japón Fil: Miyazaki, Ayako. National Institute of Animal Health. Viral Diseases and Epidemiology Research Division; Japón Fil: Soma, Junichi. National Federation of Agricultural Cooperative Associations. Institute of Animal Health. Research and Development Section; Japón Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Aso, Hisashi. Tohoku University; Japón Fil: Nochi, Tomonori. Tohoku University; Japón Fil: Iwabuchi, Noriyuki. Morinaga Milk Industry Co. Ltd. Food Science and Technology Institute; Japón Fil: Xiao, Jin-zhong. Morinaga Milk Industry Co. Ltd. Food Science and Technology Institute; Japón Fil: Saito, Tadao. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Haruki Kitazawa. Tohoku University; Japón

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.

Background: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. Methods: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). Results: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1 alpha and IL-6 in the colon compared to a vector control strain. Conclusions: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD., Article, MICROBIAL CELL FACTORIES. 14:189 (2015)

This study was conducted to evaluate immunological changes in peripheral blood leukocytes in pigs that were genetically selected for their improved resistance to mycoplasmal pneumonia of swine (MPS), using MPS vaccine as an antigen. Twelve castrated MPS-selected Landrace pigs were compared with the same number of pigs from a nonselected line by using a time-course analysis at the hematological level. After the second sensitization with MPS vaccine, the percentages of B cells, CD4(+) T cells, and natural killer (NK) cells in total leukocytes were lower in the selected line than in the nonselected line, whereas the percentage of granulocytes in total leukocytes increased in the MPS-selected line. We also assessed the proliferative ability of peripheral blood mononuclear cells (PBMCs) stimulated with Mycoplasma hyopneumoniae, lipopolysaccharide or concanavalin A, and found that although the proliferative ability of the PBMC was not different between the two lines at a steady state, the nonselected line showed a significantly higher proliferative ability after sensitization with MPS vaccine than the selected line regardless of antigens used. These results thus indicate that the selection of pigs on the basis of MPS resistance changes their immunophenotype, and would give us beneficial information for the prevention of MPS infection.

This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a +CD11R1 high and CD4 + cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response. Fil: Hosoya, Soichi. Tohoku University. Food Immunology Group; Japón Fil: Villena, Julio Cesar. Tohoku University. Food Immunology Group; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Shimazu, Tomoyuki. Tohoku University. Food Immunology Group; Japón Fil: Tohno, Masanori. Tohoku University. Food Immunology Group; Japón Fil: Fujie, Hitomi. Tohoku University. Food Immunology Group; Japón Fil: Chiba, Eriko. Tohoku University. Food Immunology Group; Japón Fil: Shimosato, Takeshi. Tohoku University. Food Immunology Group; Japón Fil: Aso, Hisashi. Tohoku University. Food Immunology Group; Japón Fil: Suda, Yoshihito. Miyagi University. Department of Food Agriculture; Japón Fil: Kawai, Yasushi. Tohoku University. Food Immunology Group; Japón Fil: Saito, Tadao. Tohoku University. Food Immunology Group; Japón Fil: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Bioquímica Aplicada; Argentina Fil: Ikegami, Shuji. Meiji Dairies Corporation Japan; Japón Fil: Itoh, Hiroyuki. Meiji Dairies Corporation Japan; Japón Fil: Kitazawa, Haruki. Tohoku University. Food Immunology Group; Japón

Background Previously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB). Results All toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1α and MCP-1 in BIE cells by activating both NF-κB and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-κB and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3). Conclusions BIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host.