Search

A 6-month feeding trial was conducted to compare the effects of extruded pellet (EP) and moist pellet (MP) feed on the growth performance, non-specific immunity, and intestinal microbiota of olive flounder. A total of 60,000 fish with an average weight of 70.8 ± 6.4 g were divided into two groups and fed with one of two experimental diets. At the end of a 6-month feeding trial, the weight gain and specific growth rate of the fish fed with the MP diets were significantly higher than those of fish fed with EP (P < 0.05). However, the EP group exhibited a lower feed conversion rate than the MP group, meaning that the EP diet was more cost-effective. Whole-body proximate compositions and non-specific immune responses (superoxide dismutase, myeloperoxidase, and lysozyme activity) were not significantly different between the two groups. There were no significant differences in the α-diversity of the intestinal bacterial community of the two groups. However, the composition of microorganisms at the phylum to genus level was different between the groups. The EP group was rich in Actinobacteria, Corynebacterium, Bacillus, and Lactobacillus, whereas the MP group was dominated by Proteobacteria, Vibrio, and Edwardsiella. Collectively, the MP diet increased growth performance and pathogen concentration in the gut; whereas EP improved feed conversion and beneficial Bacillus and Lactobacillus proportion in the intestinal microbial community.

Experiments were conducted to identify different ratios of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 mixtures at a concentration of 1 × 108 CFU/g diet; the effects on growth and cellular and humoral immune responses and the characteristics of disease protection in olive flounder (Paralichthys olivaceus). Flounder were divided into six groups and fed control diet D-1 (without Bacillus sp. SJ-10 and L. plantarum KCCM 11322), positive control diets D-2 (Bacillus sp. SJ-10 at 1 × 108 CFU/g feed) and D-3 (L. plantarum KCCM 11322 at 1 × 108 CFU/g feed); or treatment diets D-4 (3:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.75 + 0.25 × 108 CFU/g feed), D-5 (1:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.50 + 0.50 × 108 CFU/g feed), or D-6 (1:3 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.25 + 0.75 × 108 CFU/g feed) for 8 weeks. Group D-4 demonstrated better growth and feed utilization (P

In recent years, considerable and growing attention has been given to the application of host-associated microorganisms as a more suitable source of probiotics in aquaculture sector. Herein, we isolated and screened the olive flounder gut microbiota for beneficial bacterial strains that might serve as potential probiotics in a low fishmeal extruded aquafeed. Among the ten identified isolates, Bacillus amyloliquefaciens SK4079 and B. subtilis SK4082 were screened out based on their heat-resistant ability as well as enzymatic and non-hemolytic activities. Although both strains were well able to utilize carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) as a single carbon source in the minimal nutrient M9 medium, B. subtilis exhibited significantly higher cellulase, xylanase, and protease activities than B. amyloliquefaciens. The two selected strains were well able to degrade the undesirable anti-nutritional component of the SBM, which would limit its utilization as protein source in aquafeed industry. Significantly higher biofilm formation capacity and notably stronger adhesive interactions with the flounder’s skin mucus were detected in B. subtilis than B. amyloliquefaciens. Immobilization of the spores from the selected strains, in a SBM complex carrier, remarkably enhances their thermal resistance at 120 °C for 5 min and different drying conditions. It was also interesting to learn that the B. subtilis spores could survive and remain viable after being sprayed onto extruded low-fish meal feed pellets for as long as 6 months. Overall, the findings of the present study could help the food/feed industries achieve their goal of developing cost-effective yet efficient products.

The aim of this study was to investigate the effects of intestinal microbial manipulation by dietary probiotic supplementation on digestive enzyme activity, immune-related gene transcription, intestinal structure alteration, and viability against pathogenic challenge in olive flounder. Similar-sized flounders (14.92 ± 0.21 g) were divided into three groups and supplemented with a control (without probiotic) or 1 × 108 CFU/g diet of each of Bacillus sp. SJ-10 (ProB) and Lactobacillus plantarum (ProL) for eight weeks. At the end of the feeding trial, the estimated intestinal microbial richness (Chao1) and diversity (Shannon) demonstrated a significant (P

Heat-killed (HK) Bacillus sp. SJ-10 (B), HK Lactobacillus plantarum (P), and their combination were dietary supplemented to olive flounder (Paralichthys olivaceus) to quantify the effects on growth, innate immunity, and disease resistance. Four test diets were supplied: a control feed free of HK probiotics, 1 × 108 CFUs g–1 single treatments of each of HK B (HKB) and HK P (HKP), and an equal proportion of (0.5 HKB + 0.5 HKP) × 108 CFUs g–1 (HKB0.5 HKP0.5). At 8 weeks of completion feeding trail, HKB0.5 HKP0.5 significantly (P

To identify and quantify the effects of a combination of dietary 1 × 108 CFU/g Lactococcus lactis subsp. lactis I2 (LI2) and 0.1% β-glucooligosaccharides (BGO) on the growth and immunity of olive flounder (Paralichthys olivaceus), a feeding experiment was conducted. Flounder (14 ± 0.5 g) were divided into two groups and fed control and synbiotic feeds for 8 weeks. Investigations were carried out on growth and feed utilization, innate immunity, serum biochemical parameters, intestinal lactic acid bacterial (LAB) viability, microvillus length, and changes in the expression levels of genes encoding pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6). Results demonstrated the synbiotic diet had significantly better (p < 0.05) responses in terms of weight gain and specific growth rate, three innate immune parameters (respiratory burst, serum lysozyme, and superoxide dismutase), intestinal LAB viability, and the relative TNF-α expression level (p < 0.05). Moreover, after challenge with Streptococcus iniae (1 × 108 CFU/ml), the synbiotically fed group exhibited significantly higher (p < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Therefore, according to the results of the present study, synbiotic feed (LI2 + BGO) increased growth, modulated innate immune parameters and protected olive flounder against streptococcosis.

The water curtain (WC) system has been applied to the greenhouse agricultural practice as an additional heat source during the cold season in South Korea. Thus, heavy groundwater extraction induces a drawdown of the groundwater level and an influx of adjacent surface water into the aquifers. Along with many reports on physicochemical transitions caused by groundwater–surface water mixing, not much knowledge exists about the resulting possible shifts and/or transitions of the subsurface and/or groundwater microbial community structures. Here, we studied a WC system's active site during a winter season to evaluate potential shifts of microbial community structures across the groundwater and surface water by next generation sequencing in combination with conventional physicochemical monitoring of groundwater. We found that there were shifts of groundwater microbial communities at groundwater (WJ-1 and WJ-2) near the adjacent stream, but there was a relatively delayed response of the community in the groundwater (WJ-3) located at some distance from surface water despite proximity to the wells of heavy groundwater extraction.

This study was conducted to investigate the long‐term effect of a low fish meal (FM) diet comprising plant‐based protein sources (PPS) on changes of gut microbial diversity in olive flounder (Paralichthys olivaceus) over the course of life. Two experimental diets were prepared to contain 74% FM (control) or 52% FM with 22% PPS (30% FM replacement, FM30). Fish were fed one of the two experimental diets for 8 months, and we collected the midgut contents to analyze the gut bacterial community by Illumina MiSeq based on the metagenomic sequences in the V3–V4 regions of 16S rRNA. We found that there were nine dominant phyla, which in turn presented Proteobacteria, Firmicutes, and Actinobacteria as the three major phyla in the gut microbiota of the flounder. At genus level, the dominant genera were Delftia, Prevotella, and Chthoniobacter at the juvenile stage (below 100 g/fish); Chthoniobacter, Bacillus, and Bradyrhizobium at the grower stage (400 g/fish); Chthoniobacter, Bacillus, and Delftia at the subadult stage (800 g/fish); and Lactobacillus and Prevotella at the adult stage (over 1,000 g/fish). The microbial diversity in olive flounders arched from the juvenile and subadult stage and reached a plateau thereafter. The fish fed the FM30 diet significantly had an increased abundance of Lactobacillus and Photobacterium and had less abundance of Prevotella and Paraprevotella than the control. However, the effect of dietary PPS was not significant on total microbial richness, indicating no negative effect as feed sources on the intestinal microbiota in olive flounder. These results indicate that the life stage of olive flounder is more important in modulating intestinal microbiota than is the diet. It could also be concluded that dietary PPS might be used as a potential fish meal alternative without any compromising effects on microbial diversity of olive flounder for long‐term feeding., This study was conducted to investigate the long‐term effect of a low fish meal diet FM30 comprising plant‐based protein sources (PPS) on changes of gut microbial diversity in olive flounder (Paralichthys olivaceus) over the course of life. The data indicate that the life stage is more important in modulating intestinal microbiota than is the diet. The dietary PPS might be used as a potential fish meal alternative without any compromising effects on microbial diversity of olive flounder for long‐term feeding.

The effects of Bacillus sp. supplementation of different types of cells, such as spores and vegetative cells, on fish growth performance, intestinal microbial diversity alteration and immunity in juvenile olive flounder (Paralichthys olivaceus) were investigated. The fish were divided into three groups and provided with either a basal diet (control), Bacillus sp. vegetative cell (VCS) or spore (SS) supplemented diet for 8 weeks. At the end of the experiment, significant differences (P

Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 108 CFU g−1) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P

In recent years, Bacillus spp. have garnered attention as probiotic supplements in aquafeed owing to the production of heat stable and low pH resistant spores. Herein, we isolated and characterized an autochthonous Bacillus licheniformis KCCM 43270 from the intestine of olive flounder (Paralichthys olivaceus) for supplementation in low‐fishmeal extruded aquafeeds. The KCCM 43270 was screened based on amylase, protease, cellulase, and lipase as well as non‐hemolytic activities. The isolate was able to grow in carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) when used as a single carbon source in the minimal nutrient M9 medium. The KCCM 43270 spores displayed complete survival in acid (pH 2.5) and bile (0.3%, w/v) for 3 hr, strong biofilm formation, and nearly 50% adhesion with intestinal mucus. The spores of the isolate also showed significant survival ability at 80, 90, 100°C for 60, 30, and 1 min, respectively. In addition, the spores in a blend of SBM complex carrier showed significant heat stability at 120°C for 5 min and under different drying conditions. Furthermore, the spores also survived the extrusion process during low‐fishmeal aquafeed manufacturing, implying the potential application of B. licheniformis KCCM 43270 in aquafeed industry.

In the swine industry, antibiotics or chemical therapeutics have been traditionally used as growth promoters to improve growth performance and prevent subclinical disease infection [15]. However, indiscriminate use of antibiotics has increased, and the spread of antibiotic-resistant pathogens as well as public concern with regard to their cross-transfer to humans through the consumption of pork that contains antibiotic resistance [4]. As a consequence, there is considerable attention on the development of alternative antibiotics that can be used for preventive feeding in pig nutrition and do not endanger the environment with their residues [19]. Naturally acquired compounds have been recently highlighted as promising alternative antimicrobial agents that could strengthen defense mechanisms and promote resistance to microbial infection by enhancing nonspecific systemic immunity [4, 10]. Salmonella enterica serovar Typhimurium (S. Typhimurium) is a frequently isolated Salmonella serotype in pigs and a well-known public health risk in commercial Salmonella-infected pork products [5]. The clinical signs of S. Typhimurium infection in pigs are increased body temperature accompanied by diarrhea and enterocolitis [26]. After recovering from the illness, the pigs are often stunted and grow slowly or remain as asymptomatic carriers in which the bacteria can persist without triggering any clinical signs [1, 3]. Therefore, Salmonella infection has economic concerns in swine production systems and negative implications in human public health [7]. Hence, limiting Salmonella infection in pigs could improve livestock production and human food safety [12]. Biotite and bentonite, which are silicate minerals, were originally used in various industrial applications based on their properties as catalysts, ion exchangers and absorbents [6, 18, 23]. Our previous studies have shown that dietary supplementation with biotite promotes the expression of several cytokines, such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and promotes concanavalin A (T-cell mitogen)-induced lymphocyte proliferation in pigs [11, 13]. Biotite also shows antiviral activity against several viral agents, such as porcine circovirus, porcine reproductive and respiratory syndrome virus, and bovine herpesvirus type 1 [8, 11]. Furthermore, bentonite has several beneficial biological effects that are associated with its adjuvant-like action with several antibacterial agents. Bugla-Ploskonska et al. [2] reported that a bentonite and lysozyme therapy efficiently killed Salmonella enterica spp. and Salmonella bongori spp. Bentonite also shows an enhanced bactericidal effect against Escherichia coli when combined with metal or metal ion nanoparticles, such as silver (Ag), zinc oxide (ZnO), titanium oxide (TiO2), tungsten trioxide (WO3), tin dioxide (SnO2), cupric oxide (CuO) and magnesium oxide (MgO) [14, 17, 22]. These collective observations suggest that biotite combined with bentonite could be an alternative to antibiotics as a feed additive. Therefore, we investigated the bacterial clearance effect of biotite and bentonite mixture (BBM) on experimental infection with S. Typhimurium from the perspective of fecal shedding and the patterns of Salmonella distribution in disease-relevant tissues (cecum, colon and mesenteric lymph nodes (MLN)). We also examined several immune responses under normal and challenged conditions in the present study.

A 10-week feeding trial was conducted to examine the effects of dietary spirulina and quercetin on growth, innate immunity, disease resistance and dietary antioxidant capacity in the juvenile olive flounder. Triplicate groups of fish (initial body weight, 2.9 ± 0.01 g) were fed one of isonitrogenous (48% crude protein) and isocaloric (17.4 MJ/kg DM) experimental diets containing 0% spirulina (as a control), 3.4% spirulina, or 6.8% spirulina with or without supplementation of 0.5% quercetin (designated as CON, SP3.4, SP6.8, and SP6.8 + Q, respectively) at a rate of 3% body mass twice daily. Higher dietary antioxidant capacity was found with spirulina supplementation, and the highest value (P < 0.05) was obtained with SP6.8 + Q diet. At the end of the feeding trial, no significant effects were observed on growth performance, body composition and disease resistance against Edwardsiella tarda. Lysozyme activity was significantly increased by spirulina supplementation (P < 0.05), and the highest value was observed in the group fed SP6.8 + Q diet. Also, significantly higher respiratory burst activity (P < 0.05) was found in SP3.4 group. According to the results of this study, dietary supplementation of 3.4% spirulina may enhance innate immunity of olive flounder.

Antibiotics continue to be used as growth promoters in the poultry industry. Honeybee (Apis melifera) venom (HBV) possesses a number of beneficial biological activities, particularly for regulating the immune system. The aim of the present study was to evaluate the immunoprophylactic effects of HBV against Salmonella Gallinarum in broiler chicks as an initial step towards developing eco-friendly alternatives to reduce antibiotic use. HBV was administered using a spray technique. HBV improved body weight gain, particularly in the presence of infection. Moreover, HBV enhanced antibody production activity against formalin-killed S. Gallinarum. The CD4(+):CD8(+) T lymphocyte ratio, relative mRNA expression levels of interleukin-18 and interferon-γ, and serum lysozyme activity also increased following HBV administration before the infection period as well as during infection. HBV reinforced bacterial clearance and increased survivability against S. Gallinarum. Corresponding pathological analyses demonstrated that the HBV-sprayed group displayed mild and less severe abnormal changes compared with those in the control group. It was presumed that the prophylactic effects of HBV against S. Gallinarum were associated with its non-specific immune response stimulating activity. Thus, HBV may provide an alternative to reduce antibiotic use in the poultry industry.

Antimicrobials are used as feed additives to improve growth performance and to prevent subclinical disease challenge in industrial animals. However, these drugs can lead to the development of resistant strains of bacteria. Shiitake mushrooms (SM) (Lentinula edodes) have long been popular as a health food in East Asia. Moreover, SM-derived polysaccharides are well-known as immunostimulants that possess antimicrobial properties. The aim of the present study was to evaluate the immunoprophylactic effects of SM against Bordetella bronchiseptica infection in mice as an initial step towards the development of eco-friendly feed additives to reduce the use of antimicrobials. Although SM had no effect on body weight gain under the un-infected conditions, SM alleviated progressive weight loss and helped in the recovery of body weight in B. bronchiseptica infected mice. Dietary supplementation with SM reinforced bacterial clearance in the infected mice. Of note, SM markedly increased the percentage of various T lymphocytes and the relative mRNA expression levels of tumor necrosis factor-α and interferon-γ in the bronchial lymph node early in the infection. Taken together, these findings suggest that SM could help in the improvement of body weight gain during B. bronchiseptica infection and may enhance the protective immune activity against a subclinical disease challenge, such as B. bronchiseptica infection in mice, probably by a strong stimulation of non-specific immune responses. Hence, SM may provide an alternative to reduce use of antimicrobials. Confirmation of the beneficial effects of SM as a feed additive is now required in industrial animals.

It has been considered that drinking oxygenated water improves oxygen availability, which may increase vitality and improve immune functions. The present study evaluated the effects of oxygenated drinking water on immune function in pigs. Continuous drink- ing of oxygenated water markedly increased peripheral blood mononuclear cell proliferation, interleukin-1β expression level and the CD4 + :CD8 + cell ratio in pigs. During Salmonella Typhimurium infection, total leukocytes and relative cytokines expression levels were significantly increased in pigs consuming oxygenated water compared with pigs consuming tap water. These findings suggest that oxygen- ated drinking water enhances immune activity in pigs and increases immune responses of pigs during S. Typhimurium Infection.

Aluminosilicate is the major component of clay minerals such as zeolite, bentonite and clinoptilolite. The minerals possess a number of beneficial activities, especially in regulating the immune system. The aims of the present study were to evaluate immune enhancing effects of dietary aluminosilicate supplement (DAS) in mice, and to demonstrate clearance effects of DAS against porcine circovirus type 2 (PCV2) in experimentally infected pigs as an initial step towards the development of an antibiotic substitute for use in pigs. Relative messenger RNA expression levels of interferon-gamma, interleukin-4 and tumor necrosis factor-alpha, phagocytic activities of polymorphonuclear leucocytes, serum antibody production level and spleen B cell ratio were significantly increased in the DAS groups of mice compared with the control group (each feeding group had three replications with 5 mice each). The results indicated that general immune activity including cellular and humoral immunity could be enhanced by DAS in mice. In experimentally PCV2-infected pigs, the load of viral genome in nasal swab, serum and lung of the DAS group of pigs was significantly decreased compared with the control group at 28 days post-infection (each group three pigs). Corresponding histopathological analyses demonstrated that pigs in the DAS group displayed mild and less severe abnormal changes compared with the control group, indicating that DAS reinforces clearance of PCV2 in experimentally infected pigs. This may relate to general immune enhancing effects of DAS in mice. Therefore DAS will help the health of animal, especially in swine.

Porcine circovirus type 2 (PCV2) infections cause important respiratory diseases in the pig industry and are associated with many bacterial, mycoplasmal, and viral complications. In this study, a heterologous PCV2 major capsid protein (MCP) was expressed in the Bordetella bronchiseptica aroA mutant strain (BBS-MCP) and used as a live vaccine vehicle. Mice and pigs were immunized with live BBS-MCP via the intranasal route. The antibodies against MCP were induced successfully in the serum as determined by ELISA. In the PCV2 challenge experiment, viral DNA was removed successfully from the lymph nodes of pigs vaccinated with live BBS-MCP. Overall, BBS-MCP is believed to be a good candidate for the development of a live-attenuated vaccine against PCV2 infections.

The chemokine IP-10 (CXCL10) and its cellular receptor CXCR3 are upregulated in the lung during murine gammaherpesvirus 68 (MHV-68) infection. In order to determine the role of the CXCR3 chemokine receptor in the immune response to MHV-68, CXCR3 −/− mice were infected with the virus. CXCR3 −/− mice showed delayed clearance of replicating MHV-68 from the lungs. This correlated with delayed T-cell recruitment to the lungs and reduced cytolytic activity prior to viral clearance. Splenomegaly and the numbers of latently infected cells per spleen were transiently increased. Ηowever, CXCR3 −/− mice showed normal virus-specific antibody titers and effective long-term control of MHV-68 infection.

Honeybee (Apis melifera) venom (HBV), which includes melittin and lipid-soluble ingredients (chrysin and pinocembrin), elicited increases in the CD4(+)/CD8(+) T lymphocyte ratio, relative mRNA expression levels of the T helper type 1 (Th 1) cytokines (interferon-γ and IL-12) and reinforced viral clearance of an experimental porcine reproductive and respiratory syndrome (PRRS) virus infection in our previous study. On the basis of that previous study, we have now developed poly-d,l-lactide-co-glycolide (PLGA)-encapsulated HBV nanoparticles (P-HBV) for longer sustained release of HBV. We administered P-HBV to pigs via the rectal route, and then evaluated the potential immune-enhancing and bacterial clearance effects of P-HBV against Salmonella enterica serovar Typhimurium. The CD4(+)/CD8(+) lymphocyte ratio, proliferative capacity of peripheral blood lymphocytes and relative mRNA expression levels of IFN-γ and IL-12 (produced mainly by Th1 lymphocytes) were significantly increased in the P-HBV group up to 2 weeks post-administration of P-HBV. After S. Typhimurium infection, the P-HBV group showed a marked reduction in microbial burden in feces and all tissue samples (including the ileum, cecum, colon, and mesenteric lymph node (MLN)), a significant increase in Th 1 cytokines (IFN-γ, IL-2, and IL-12) and a marked decrease in a Th 2 cytokine (IL-4) in all tissue samples and peripheral blood lymphocytes. Thus, P-HBV may be a promising strategy for immune enhancement and prevention of S. Typhimurium or other bacterial infections.

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEΔ), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295–310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEΔ and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.

Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78T was created in lambda zap express and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 285 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastroinestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) alpha submit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identify in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).

A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae, the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non-S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae, and should prove to be a useful reagent for identification of S. hyodysenteriae.

Herbs including Curcuma longa, Houttuynia cordata, Prunus mume and Rubus coreanus have potential immune enhancing and antimicrobial effects. Probiotics also have antibacterial effects, and some are important in regulating the immune system. The aims of the present study were to evaluate the immune enhancing effects of a probiotic fermented four-herb combination (PFH) in broiler chicks and to demonstrate the prophylactic effect of PFH against Salmonella Gallinarum in experimentally infected broiler chicks as an initial step towards the development of feed supplements for promotion of immune activity and disease prevention. Continuous ingestion of PFH markedly increased lysozyme activity in serum and the spleen, peripheral blood mononuclear cell (PBMC) proliferation, the CD4(+):CD8(+) T lymphocyte ratio in the spleen and antibody production level in broiler chicks. Conversely, prostaglandin E(2) synthesis in serum and PBMC culture medium was significantly decreased in the PFH-fed chicks compared with the control group in a dose-dependent manner. In the chicks experimentally infected with S. Gallinarum, mortality was delayed in the 2% PFH-fed chicks. Moreover, the survival rates in the 2% PFH-fed group remained the highest among all the trial groups throughout the experimental period. Taken together, these findings suggest that PFH enhances immune activity in broiler chicks and increases survivability against Salmonella Gallinarum in experimentally infected broiler chicks, likely because of potent stimulation of nonspecific immune responses.

Maesil (Prunus mume) has long been used as a traditional drug and healthy food in East Asian countries. It possesses a number of beneficial biological activities including potential antimicrobial effects against pathogens. Probiotics also have antibacterial effects. Moreover, some probiotics have an important role in regulating the immune system. The present study evaluated the immune enhancing effects of fermented Maesil with probiotics (Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus acidophilus) in mice, especially against Bordetella bronchiseptica, as an initial step towards the development of feed supplements for the promotion of immune activity and prevention of disease, especially in pigs. Continuous ingestion of fermented Maesil with probiotics markedly increased the macrophage ratio in peripheral blood and the T lymphocyte ratio in the spleen. In addition, antibody production against formalin-killed B. bronchiseptica significantly increased in the mice fed fermented Maesil compared with the control group. The number of leukocytes was significantly higher in the bronchio-alveolar lavage obtained from the fermented Maesil-fed animals compared to it in the control group at day 3 (maximal peak time) after experimental B. bronchiseptica infection. Moreover, at 7 day post-infection, relative messenger RNA expression levels of tumor necrosis factor- α and interferon-γ were significantly increased in splenocytes of mice fed fermented Maesil compared with those in the control group. Taken together, these findings suggest that feed containing fermented Maesil with probiotics enhances immune activity in mice, especially against B. bronchiseptica, via the potent stimulation of non-specific immune responses.

The role of CD28-dependent costimulatory interactions in the development and maintenance of antiviral immune responses was investigated in a mouse model of gammaherpesvirus infection. CD28−/−mice could clear a productive infection with murine gammaherpesvirus 68 (MHV-68), although early lung viral titers were significantly increased. Both CD28−/−and CD28+/+mice maintained effective long-term control of MHV-68. Gamma interferon responses appeared to develop more slowly in CD28−/−mice, while cytotoxic T-cell activity was similar to that in wild-type mice. Splenomegaly developed normally in CD28−/−mice, whereas virus-specific antibody responses were significantly reduced and aberrant class switching was observed. This work demonstrates that costimulatory interactions involving CD28 are not an absolute requirement for the control of infection with MHV-68.

A gene encoding a 30 kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12 bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735 bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30 kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B. hyodysenteriae also reacted with recombinant BmpB expressed in E. coli.

Respiratory challenge with murine gammaherpesvirus 68 (MHV-68) leads to an acute productive infection of the lung and a persistent latent infection in B lymphocytes, epithelia, and macrophages. The virus also induces splenomegaly and an increase in the number of activated CD8 T cells in the circulation. Lymphotoxin- α-deficient (LTα−/−) mice have no lymph nodes and have disrupted splenic architecture. Surprisingly, in spite of the severe defect in secondary lymphoid tissue, LTα−/−mice could clear a productive MHV-68 infection, although with delayed kinetics compared to wild-type mice, and could control latent infection. Cytotoxic T-cell activity was comparable in the lungs and spleens of LTα−/−and wild-type mice. However, splenic gamma interferon responses were substantially reduced in LTα−/−mice. Furthermore, LTα−/−mice failed to develop splenomegaly or lymphocytosis. Although germinal centers were absent, LTα−/−mice were able to class switch and showed significant virus-specific antibody titers. This work demonstrates that organized secondary lymphoid tissue is not an absolute requirement for the generation of immune responses to viral infections.

The purpose of this study was to determine the presence of lipopolysaccharide-like material in the intestinal spirochaete Serpulina pilosicoli and the extent of antigenic cross-reactivity of this material in different strains of the species. Hot water-phenol, aqueous-phase extracts from five porcine and three human strains of S. pilosicoli, and from seven strains of four other Serpulina spp., were separated by SDS-PAGE and silver-stained profiles were obtained. All S. pilosicoli strains had a predominant band at c. 16 kDa. Some also had a partial ladder-like profile, consistent with the presence of semi-rough lipo-oligosaccharide (LOS); this was more obvious in Western immunoblot analysis. LOS from each S. pilosicoli strain was serologically distinct in immunoblot analysis and there was no cross-reactivity with other Serpulina spp. The serological diversity found amongst the LOS of S. pilosicoli strains may help to explain why individual people and animals can suffer repeated infections with different strains of the organism.

Group A rotaviruses are a major cause of acute gastroenteritis in young children worldwide. For the proper management of rotavirus infections, knowledge of the distribution of G and P genotypes including detection of emerging genotype is crucial. Therefore, the aim of this study is to describe epidemiological changes in rotavirus gastroenteritis in Gwangju metropolitan city, South Korea. Stool samples were collected from 14,314 patients with diarrhea, who visited hospitals in Gwangju from 2008 to 2012. Samples were screened for rotavirus with Enzyme-Linked Immunosorbent Assay(ELISA) method and rotavirus P (VP4), G (VP7) genotypes were determined by reverse-transcription polymerase chain reaction. And we performed nucleotide sequencing analysis. Among a total of 14,314 samples investigated 1,982 samples (13.8%) were ELISA positive. Genotyping of Rotavirus was performed using 526 rotavirus samples. The most prevalent circulating G genotype was G1 (40.5%), followed by G2 (27.6%), G3 (19.4%), G9 (9.7%), G4 (2.5%) and G12 (0.4%). The predominant type of P genotypes was P[8] (69.6%), followed by P[4] (27.8%) and P[6] (2.3%). In this study, 13 G-P combinations were detected. From 2008 to 2010, G1P[8] was the most prevalent, followed by G3P[8]. Whereas, 2011 and 2012, G2P[4] was the most common, followed by G1P[8]. Rotavirus gastroenteritis is a common disease associated with significant morbidity, mortality and economic burden. Ongoing rotavirus surveillance to understand the distribution of G and P genotypes will be critical for the development of effective prevention measurements.

SUMMARYMultilocus enzyme electrophoresis was used to divide 124 Australian isolates ofStreptococcus suistype 2 into 17 electrophoretic types (ETs). Isolates in ET 1 were the most frequent cause of disease amongst Western Australian pigs, but isolates of ET 8 were more commonly associated with disease in other Australian states. Multiple isolates from 10 of 19 farms all belonged to the same ET, whilst isolates from the other farms belonged to between 2 and 4 different ETs. Some isolates could be differentiated further by DNA restriction endonuclease analysis, whilst others with the same restriction pattern were located in different, but closely-related ETs. Fourteen isolates were tested for their virulence in mice. Most caused disease if given in high numbers, but isolates in ET 1 were virulent at lower dose rates. This virulent clone also was distinguished by the fact that 80% of isolates produced extracellular factor (EF).