Endomicrobia in termite guts: symbionts within a symbiont (Phylogeny, cospeciation with host flagellates, and preliminary genome analysis)

"Endomicrobia" are intracellular symbionts of termite gut flagellates that represent a distinct lineage in the novel bacterial phylum Termite Group I (TG-1). The evolutionary history of "Endomicrobia" with respect to their symbiosis with host flagellates was investigated using phylogenetic analyses and in situ identification based on small-subunit ribosomal RNA (SSU rRNA) sequences. By analyzing SSU rRNA sequences extracted from manually separated flagellate cells, "Endomicrobia" were shown to be widely distributed among termite gut flagellates. Symbionts originating from the same genus of flagellates invariably formed a host-specific monophyletic cluster in the phylogenetic tree. Their intracellular location in the cytoplasm was confirmed by fluorescent in situ hybridization (FISH) using oligonucleotide probes designed specifically for each symbiont and for the host. The phylogeny of "Endomicrobia" and host flagellates belonging to the parabasalid genus Trichonympha was investigated further in detail. SSU rRNA trees of the symbiont and the host exhibited overall congruence, which suggested cospeciation. Pairwise distance analysis and FISH corroborated the phylogenetic evidence, and these results allowed the construction of evolutionary scenarios for the divergence of "Endomicrobia" and their acquisition by flagellate hosts. "Endomicrobia" share their intracellular habitat with other bacterial symbionts. Bacterial SSU rRNA sequences originating from Trichonympha flagellates of Incisitermes marginipennis and Zootermopsis nevadensis revealed the occurrence of several distinct phylogenetic groups, representing Treponema spp., "Endomicrobia", δ-Proteobacteria, Bacteroidetes, and Mycoplasmatales. The Proteobacteria symbionts were shown to densely colonize the surface and the cytoplasm of the flagellates in high abundance. Since no pure cultures of "Endomicrobia" or their host flagellates are available, a method for the physical enrichment of "Endomicrobia" was established to gain more insights into the nature of these symbionts. "Candidatus Endomicrobium trichonymphae" (CET), the symbiont of Trichonympha flagellates, was selected as representative and enriched from gut contents of Z. nevadensis. High-molecular-weight DNA extracted from the enrichment is currently used for genome sequencing at the DOE Joint Genome Institute. A recently assembled 80-kb contig of CET revealed first insights into its metabolism, including hexuronate metabolism and the possible formation of H2. "Endomicrobia" are also present in the gut of the wood-feeding cockroach Cryptocercus punctulatus, which is considered to share a common ancestor with termites. Analysis of SSU rRNA sequences obtained from whole-gut DNA of this cockroach revealed the phylogenetic positions of six lineages (morphotypes) of parabasalid flagellates. Sequences obtained from manually isolated flagellates, which have long been assigned to the genus Trichonympha, turned out to be a previously undescribed lineage of Parabasalia. Since this new lineage may represent one of the earliest branches of parabasalid flagellates, the recovery of “Endomicrobia" sequences also from this flagellate underscores the presence of these endosymbionts already in the flagellates of the hypothetical dictyopteran ancestor of termites and cockroaches. The results of this study collectively document that "Endomicrobia" are prevalent and persistent endosymbionts of termite gut flagellates. This study also provides a better understanding of the phylogenetic properties of their biotic environment, i.e., the host flagellates and the cohabiting bacteria, which may help to explain the functional roles of "Endomicrobia" and their symbiotic interactions

Comparative Study of Bacterial Populations in the Feces of Pasture-kept and Stabled Horses and Ponies

Poster Session

Draft genome sequence of Weissella viridescens UCO-SMC3, isolated from the slime of helix aspersa Müller snails

This report describes the draft genome sequence of Weissella viridescens UCO-SMC3, isolated from Helix aspersa Müller slime. The reads were generated by a whole-genome sequencing (WGS) strategy on an Illumina MiSeq sequencer and were assembled into contigs with a total estimated size of 1,612,814 bp. A total of 2,455 genes were predicted, including 2,301 protein-coding sequences. The draft genome sequence of W. viridescens UCO-SMC3 will be useful for further studies of specific genetic features and for understanding the mechanisms of its beneficial properties in the skin.Fil: Garcia Cancino, Apolinaria. Universidad de Concepción; ChileFil: 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; Argentina. Tohoku University; JapónFil: Espinoza Monje, Marcela. Universidad de Concepción; ChileFil: Campos Martin, Jorge. Universidad de Concepción; ChileFil: Garcia Castillo,Valeria. Tohoku University; Japón. Universidad de Concepción; ChileFil: Nakano, Yukha. Tohoku University; JapónFil: Ikeda Ohtsubo, Wakako. Tohoku University; JapónFil: Gutierrez, Cristian. Universidad de Concepción; ChileFil: Morita, Hidetoshi. Okayama University; JapónFil: Kitazawa, Haruki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japó

Feed, Microbiota, and Gut Immunity: Using the Zebrafish Model to Understand Fish Health

Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for in vivo imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions.</p

Evaluation of the immunomodulatory ability of lactic acid bacteria isolated from feedlot cattle against mastitis using a bovine mammary epithelial cells In vitro assay

Bovine mastitis, the inflammation of the mammary gland, affects the quality and quantity of milk yield. Mastitis control relies on single or multiple combinations of antibiotic therapy. Due to increasing antibiotic resistance in pathogens, the intramammary infusion of lactic acid bacteria (LAB) has been considered as a potential alternative to antibiotics for treating and preventing bovine mastitis through the improvement of the host immunity. Probiotic effects are a strain-dependent characteristic; therefore, candidate LAB strains have to be evaluated efficiently to find out the ones with the best potential. Here, we investigated LAB strains originally isolated from feedlot cattle’s environment regarding their ability in inducing the Toll-like receptor (TLR)-triggered inflammatory responses in bovine mammary epithelial (BME) cells in vitro. The BME cells were pre-stimulated with the LAB strains individually for 12, 24, and 48 h and then challenged with Escherichia coli-derived lipopolysaccharide (LPS) for 12 h. The mRNA expression of selected immune genes—interleukin 1 alpha (IL-1α), IL-1β, monocyte chemotactic protein 1 (MCP-1), IL-8, chemokine (C-X-C motif) ligand 2 (CXCL2), and CXCL3 were quantified by real-time quantitative PCR (RT-qPCR). Results indicated that pretreatment with some Lactobacillus strains were able to differentially regulate the LPS inflammatory response in BME cells; however, strain-dependent differences were found. The most remarkable effects were found for Lactobacillus acidophilus CRL2074, which reduced the expression of IL-1α, IL-1β, MCP-1, IL-8, and CXCL3, whereas Lactobacillus rhamnosus CRL2084 diminished IL-1β, MCP-1, and IL-8 expression. The pre-stimulation of BME cells with the CRL2074 strain resulted in the upregulated expression of three negative regulators of the TLRs, including the ubiquitin-editing enzyme A20 (also called tumor necrosis factor alpha-induced protein 3, TNFAIP3), single immunoglobin IL-1 single receptor (SIGIRR), and Toll interacting protein (Tollip) after the LPS challenge. The CRL2084 pre-stimulation upregulated only Tollip expression. Our results demonstrated that the L. acidophilus CRL2074 strain possess remarkable immunomodulatory abilities against LPS-induced inflammation in BME cells. This Lactobacillus strain could be used as candidate for in vivo testing due to its beneficial effects in bovine mastitis through intramammary infusion. Our findings also suggest that the BME cells immunoassay system could be of value for the in vitro evaluation of the immunomodulatory abilities of LAB against the inflammation resulting from the intramammary infection with mastitis-related pathogens.Fil: Fukuyama, Kohtaro. Tohoku University; JapónFil: Islam, Md. Aminul. Tohoku University; Japón. Bangladesh Agricultural University; BangladeshFil: Takagi, Michihiro. Tohoku University; JapónFil: Ikeda Ohtsubo, Wakako. Tohoku University; JapónFil: Kurata, Shoichiro. Tohoku University; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Comparative geochemical evaluation of toxic metals pollution and bacterial communities of industrial effluent tributary and a receiving estuary in Nigeria

Toxic metals/metalloid contaminations of estuarine sediments due to compromised tributaries arouse significant interest in studying bacterial community that triggers natural attenuation processes. Geo-accumulation index (Igeo), contamination factor (CF), pollution load index (PLI), and Hakanson potential ecological risk index (RI) as a sum of risk factors (Er) were used to quantify toxic metal/metalloid-pollution status of Lagos Lagoon (2W) and ‘Iya-Alaro’ tributary (4W) sediments in comparison with pristine ‘Lekki Conservation Centre’ sediment (L1-B). Bacteriology of the ecosystems was based on culture-independent analyses using pyrosequencing. 2W and 4W were extremely contaminated with mercury (Igeo > 7), whereas, cadmium contamination was only observed in 4W. The two ecosystems were polluted with toxic metal based on PLI, where mercury (Er = 2900 and 1900 for 4W and 2W, respectively) posed very high ecological risks. Molecular fingerprinting revealed that Proteobacteria, Firmicutes, and Acidobacteria predominately contributed the 20 most abundant genera in the two ecosystems. The 240 and 310 species present in 2W and 4W, respectively, but absent in L1-B, thrive under the metal concentrations in the polluted hydrosphere. Whereas, the 58,000 species missing in 2W and 4W but found in L1-B would serve as indicators for systems impacted with metal eco-toxicity. Despite toxic metal pollution of the ecosystems understudied, bacterial communities play vital roles in self-recovery processes occurring in the hydrosphere

Isolation and immunocharacterization of lactobacillus salivarius from the intestine of wakame-fed pigs to develop novel "Immunosynbiotics"

Emerging threats of antimicrobial resistance necessitate the exploration of effective alternatives for healthy livestock growth strategies. ?Immunosynbiotics?, 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-β) 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-β expression; these could be used as immunobiotic strains in combination with wakame for the development of novel immunologically active feeds for pigs.Fil: Masumizu, Yuki. Tohoku University; JapónFil: Zhou, Binghui. Tohoku University; JapónFil: Humayun Kober, AKM. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University; BangladeshFil: Islam, M. Aminul. Agricultural University; Bangladesh. Tohoku University; JapónFil: Iida, Hikaru. Tohoku University; JapónFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Suda, Yoshihito. Department Of Food Agriculture, Miyagi University; JapónFil: Albarracín, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón. Universidad Nacional de Tucumán; ArgentinaFil: Nochi, Tomonori. Tohoku University; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Suzuki, Keiichi. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Transcriptome Modifications in Porcine Adipocytes via Toll-Like Receptors Activation

Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10, and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2, and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.Fil: Igata, Manami. Tohoku University; JapónFil: Islam, M. Aminul. Tohoku University; Japón. Bangladesh Agricultural University; BangladeshFil: Tada, Asuka. Tohoku University; JapónFil: Takagi, Michihiro. Tohoku University; JapónFil: Humayun Kober, AKM. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University; BangladeshFil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Ciencias de la Computación; ArgentinaFil: Aso, Hisashi. Tohoku University; JapónFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Miyazawa, Kenji. Takanashi Milk Products Co.; JapónFil: Yoda, Kazutoyo. Takanashi Milk Products Co.; JapónFil: He, Fang. Takanashi Milk Products Co.; JapónFil: Takahashi, Hideki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Modulation of porcine intestinal epitheliocytes immunetranscriptome response by Lactobacillus jensenii TL2937

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; JapónFil: Albarracín, Leonardo Miguel. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Sato, Nana. Tohoku University; JapónFil: Kanmani, Paulraj. Tohoku University; JapónFil: Kober, Humayun A.K.M.. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University. Department of Dairy and Poultry Science; BangladeshFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Suda, Yoshihito. Miyagi University; JapónFil: Nochi, Tomonori. Tohoku University; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Makino, Seiya. Meiji Co., Ltd. Food Science Research Labs.; JapónFil: Kano, Hiroshi. Meiji Co., Ltd. Food Science Research Labs.; JapónFil: Ohkawara, Sou. Meiji Seika Pharma Co., Ltd. Agricultural and Veterinary Division; JapónFil: Saito, Tadao. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó