ccdC Regulates Biofilm Dispersal in Bacillus velezensis FZB42.

Bacillus velezensis FZB42 is a plant growth-promoting rhizobacterium (PGPR) and a model microorganism for biofilm studies. Biofilms are required for the colonization and promotion of plant growth in the rhizosphere. However, little is known about how the final stage of the biofilm life cycle is regulated, when cells regain their motility and escape the mature biofilm to spread and colonize new niches. In this study, the non-annotated gene ccdC was found to be involved in the process of biofilm dispersion. We found that the ccdC-deficient strain maintained a wrinkled state at the late stage of biofilm formation in the liquid-gas interface culture, and the bottom solution showed a clear state, indicating that no bacterial cells actively escaped, which was further evidenced by the formation of a cellular ring (biofilm pellicle) located on top of the preformed biofilm. It can be concluded that dispersal, a biofilm property that relies on motility proficiency, is also positively affected by the unannotated gene ccdC. Furthermore, we found that the level of cyclic diguanylate (c-di-GMP) in the ccdC-deficient strain was significantly greater than that in the wild-type strain, suggesting that B. velezensis exhibits a similar mechanism by regulating the level of c-di-GMP, the master regulator of biofilm formation, dispersal, and cell motility, which controls the fitness of biofilms in Pseudomonas aeruginosain. In this study, we investigated the mechanism regulating biofilm dispersion in PGPR.

Biomimetic virus-like mesoporous silica nanoparticles improved cellular internalization for co-delivery of antigen and agonist to enhance Tumor immunotherapy.

Nanocarrier antigen-drug delivery system interacts specifically with immune cells and provides intelligent delivery modes to improve antigen delivery efficiency and facilitate immune progression. However, these nanoparticles often have weak adhesion to cells, followed by insufficient cell absorption, leading to a failed immune response. Inspired by the structure and function of viruses, virus-like mesoporous silica nanoparticles (VMSNs) were prepared by simulating the surface structure, centripetal-radialized spike structure and rough surface topology of the virus and co-acted with the toll-like receptor 7/8 agonist imiquimod (IMQ) and antigens oocyte albumin (OVA). Compared to the conventional spherical mesoporous silica nanoparticles (MSNs), VMSNs which was proven to be biocompatible in both cellular and in vivo level, had higher cell invasion ability and unique endocytosis pathway that was released from lysosomes and promoted antigen cross-expression. Furthermore, VMSNs effectively inhibited B16-OVA tumor growth by activating DCs maturation and increasing the proportion of CD8+ T cells. This work demonstrated that virus-like mesoporous silica nanoparticles co-supply OVA and IMQ, could induce potent tumor immune responses and inhibit tumor growth as a consequence of the surface spike structure induces a robust cellular immune response, and undoubtedly provided a good basis for further optimizing the nanovaccine delivery system.

Molecular Characterization of Nitrogen-Containing Compounds in Humic-like Substances Emitted from Biomass Burning and Coal Combustion.

N-containing organic compounds (NOCs) in humic-like substances (HULIS) emitted from biomass burning (BB) and coal combustion (CC) were characterized by ultrahigh-resolution mass spectrometry in the positive electrospray ionization mode. Our results indicate that NOCs include CHON+ and CHN+ groups, which are detected as a substantial fraction in both BB- and CC-derived HULIS, and suggest that not only BB but also CC is the potential important source of NOCs in the atmosphere. The CHON+ compounds mainly consist of reduced nitrogen compounds with other oxygenated functional groups, and straw- and coal-smoke HULIS exhibit a lower degree of oxidation than pine-smoke HULIS. In addition, the NOCs with higher N atoms (N2 and/or N3) generally bear higher modified aromaticity index (AImod) values and are mainly contained in BB HULIS, especially in straw-smoke HULIS, whereas the NOCs with a lower N atom (N1) always have relatively lower AImod values and are the dominant NOCs in CC HULIS. These findings imply that the primary emission from CC may be a significant source of N1 compounds, whereas high N number (e.g., N2-3) compounds could be associated with burning of biomass materials. Further study is warranted to distinguish the NOCs from more sources.

Distribution and sources of DDT and its metabolites in porewater and sediment from a typical tropical bay in the South China Sea.

Dichlorodiphenyltrichloroethane (DDT) is well known for its harmful effects and has been banned around the world. However, DDT is still frequently detected in natural environments, particularly in aquaculture and harbor sediments. In this study, 15 surface sediment samples were collected from a typical tropical bay (Zhanjiang Bay) in the South China Sea, and the levels of DDT and its metabolites in sediment and porewater samples were investigated. The results showed that concentrations of DDXs (i.e., DDT and its metabolites) in bulk sediments were 1.58-51.0 ng g-1 (mean, 11.5 ng g-1). DDTs (DDT and its primary metabolites, dichlorodiphenyldichloroethane (DDD) and dichlorodiphenyldichloroethylene (DDE)) were the most prominent, accounting for 73.2%-98.3% (86.1% ± 12.8%) of the DDXs. Additionally, high-order metabolites (i.e., 1-chloro-2,2-bis(4'-chlorophenyl)ethylene (p,p'-DDMU), 2,2-bis(p-chlorophenyl)ethylene (p,p'-DDNU), 2,2-bis(p-chlorophenyl)ethanol (p,p'-DDOH), 2,2-bis(p-chlorophenyl)methane (p,p'-DDM), and 4,4'-dichlorobenzophenone (p,p'-DBP)) were also detected in most of the sediment and porewater samples, with DDMU and DBP being predominant. The DDTs concentration differed among the sampling sites, with relatively high DDTs concentrations in the samples from the aquaculture zone and an area near the shipping channel and the Haibin shipyard. The DDD/DDE ratios indicated a reductive dichlorination of DDT to DDD under anaerobic conditions at most of the sampling sites of Zhanjiang Bay. The possible DDT degradation pathway in the surface sediments of Zhanjiang Bay was p,p'-DDT/p,p'-DDD(p,p'-DDE)/p,p'-DDMU/p,p'-DDNU/ … /p,p'-DBP. The DDXs in the sediments of Zhanjiang Bay were mainly introduced via mixed sources of industrial DDT and dicofol, including fresh input and historical residue. The concentrations of DDXs in porewater samples varied from 66.3 to 250 ng L-1, exhibiting a distribution similar to that in the accompanying sediments. However, the content of high-order metabolites was relatively lower in porewater than in sediment, indicating that high-order degradation mainly occurs in particles. Overall, this study helps in understanding the distribution, source, and degradation of DDT in a typical tropical bay.

Influence of lung aeration on diaphragmatic contractility during a spontaneous breathing trial: an ultrasound study.

BACKGROUND: A spontaneous breathing trial (SBT) is a major diagnostic tool to predict successfully extubation in patients. Several factors may lead to weaning failure, including the degree of lung aeration loss and diaphragm dysfunction. The main objective was to compare the diaphragmatic contractility between patients with high lung aeration loss and low lung aeration loss during a 30-minute SBT by ultrasound. METHODS: This was a prospective single-center study. Lung ultrasound aeration score (LUS) and diaphragmatic thickening fraction (DTF) were measured during mechanical ventilation 1 h before SBT (T-1), 30 min (T1), and 120 min (T2) after the start of the SBT during quiet breathing. The right and left DTF were compared between patients with LUS ≥ 14 (high lung aeration loss), considered at high risk of post-extubation distress, and those with LUS < 14 (low lung aeration loss). The relationship between the LUS and DTF and the changes in LUS and DTF from T-1 to T2 in patients with LUS ≥ 14 were assessed. RESULTS: Forty-nine patients were analyzed; 33 had LUS ≥ 14 and 16 had LUS < 14 at T1. The DTF at T1 was significantly higher in patients with LUS ≥ 14 than in those with LUS < 14: the right median (IQR) DTF was 22.2% (17.1 to 30.9%) vs. 14.8% (10.2 to 27.0%) (p = 0.035), and the left median (IQR) DTF was 25.0% (18.4 to 35.0%) vs. 18.6% (9.7 to 24.2%) (p = 0.017), respectively. There was a moderate positive correlation between the LUS and the DTF (Rho = 0.3, p = 0.014). A significant increase in the LUS was observed from T-1 to T1, whereas no change was found between T1 and T2. The DTF remained stable from T-1 to T2. CONCLUSIONS: During a SBT, diaphragmatic contraction acts differently depending on the degree of pulmonary aeration. In patients with high lung aeration loss, increased diaphragmatic contractility indicates an additional respiratory effort to compensate lung volume loss that would contribute to successful SBT. Further studies are needed to evaluate the combined evaluation of lung aeration and diaphragmatic function to predict the successful weaning of patients from mechanical ventilation.

Molecular Characterization of Water- and Methanol-Soluble Organic Compounds Emitted from Residential Coal Combustion Using Ultrahigh-Resolution Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Water-soluble organic compounds (WSOC) and methanol-soluble organic compounds (MSOC) in smoke particles emitted from residential coal combustion were characterized by ultrahigh-resolution mass spectrometry. The results showed that the molecular compositions of WSOC and MSOC are different. S-containing compounds (CHOS and CHONS) are found to be the dominant components (65-87%) of the WSOC, whereas CHO and CHON compounds make a great contribution (79-96%) to the MSOC samples. It is worth noting that greater abundance of S-containing compounds was found in smoke produced from coal combustion compared to biomass burning and atmospheric samples. The molecular compositions of WSOC and MSOC also varied significantly depending on the maturity of the coal. The WSOC and MSOC derived from the combustion of low-maturity coal contained a higher proportion of oxidized functional groups but with a lower degree of aromaticity than the compounds derived from the combustion of high-maturity coal. Our findings suggest that organic molecules with a high modified aromaticity index, low O/C ratio, and low polarity showed stronger light absorption. This study also suggests that CHO and CHON compounds significantly contributed to the light absorption of WSOC and MSOC and that the contribution of CHON may be stronger.

Abundance and Light Absorption Properties of Brown Carbon Emitted from Residential Coal Combustion in China.

Brown carbon (BrC) fractions, including water-soluble organic carbon (WSOC), water-soluble humic-like substances (HULISw), alkaline soluble organic carbon (ASOC), and methanol soluble organic carbon (MSOC) were extracted from particles emitted from the residential combustion of coal with different geological maturities. The abundances and light absorption properties of these BrC fractions were comprehensively studied. The results showed that the abundances of the different constituents of the BrC fraction varied greatly with the extraction solvent, accounting for 4.3%-46%, 2.3%-23%, 3.2%-14%, and 76%-98% of the total carbon content in particles. The specific UV-vis absorbance (SUVA254) of BrC fractions followed the order of MSOC > ASOC > HULISw > WSOC. The WSOC and MSOC fractions from the combustion of low maturity coal had relatively low SUVA254 and high SR values. The mass absorption efficiencies (MAE365) for ASOC and MSOC were higher than for WSOC, and WSOC and MSOC from low maturity coal combustion had relatively low levels of light absorption. These findings indicated that coal combustion is a potential source of atmospheric BrC and the abundance and light absorption of the coal combustion-derived BrC fractions were strongly dependent on the extraction methods used and the coal maturity rather than the coal shapes.

Molecular Characterization of Water-Soluble Humic like Substances in Smoke Particles Emitted from Combustion of Biomass Materials and Coal Using Ultrahigh-Resolution Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Water-soluble humic like substances (HULIS) in smoke particles emitted from combustion of biomass materials and coal were characterized by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry. The formulas identified were classified into four main groups: CHO, CHON, CHOS, and CHONS. The average H/C and O/C ratios are 1.13-1.33, 1.01-1.13, 1.26-1.48, 1.09-1.24 and 0.21-0.41, 0.27-0.45, 0.41-0.46, 0.44-0.61 for the CHO, CHON, CHOS, and CHONS groups, respectively. The CHO compound was the predominant component (43%-72%) of the smoke HULIS from biomass burning (BB) and coal combustion, followed by the CHON group for BB-smoke HULIS and the S-containing groups (i.e., CHOS and CHONS) for coal-smoke HULIS. These results indicate that the primary HULIS emitted from biomass burning contain a high abundance of CHON species, which appear to be made up mainly of oxidized nitrogen functional groups such as nitro compounds and/or organonitrates. The coal-smoke HULIS contained more compounds with relatively low molecular weight and high aromaticity index (AImod). They were significantly enriched in S-containing compounds with high double bond equivalent (≥4), and O/S ratios suggest that they are most likely made up of aromatic organosulfates and nitrooxy organosulfates that are usually found in polluted atmospheres. These findings imply that the primary emissions from combustion of biomass and coal fuels are potential sources of water-soluble HULIS in an atmospheric matrix and that coal combustion is an especially important source of sulfate compounds.

Effect of calf starter feeding on gut microbial diversity and expression of genes involved in host immune responses and tight junctions in dairy calves during weaning transition.

Calf starters are usually offered to dairy calves to facilitate the weaning process, however, the effect of solid feed consumption on gut health has not been well studied. This study aimed to investigate the effect of calf starter feeding on the gut bacterial community and mucosal immune functions in dairy calves during weaning transition. Mucosal tissue and digesta samples were collected from rumen, jejunum, ileum, cecum, and colon upon slaughtering of calves (n=8) after feeding the experimental diets [milk replacer (MR) or milk replacer + calf starter (MR+S)] for 6 wk. Expression of toll-like receptor (TLR) 10 was downregulated along the gut, whereas TLR2 in colon and TLR6 along the gut were upregulated in MR+S-fed calves compared with MR-fed calves. Ileal TLR9 and TLR10 showed higher expression compared with the other regions regardless of the diet. Peptidoglycan recognition protein 1 demonstrated a diet- and gut-regional dependent expression pattern, whereas ß-defensin did not. The diet and gut region also affected the expression of tight junction-regulating genes claudin 4 and occludin. Bacterial diversity tended to be different between the 2 diets, whereas the bacterial density was different among gut regions and sample type. The present study revealed that changes in bacterial diversity, expression of genes encoding host mucosal immune responses, and barrier functions were associated with the MR+S diet, and suggests that solid feed consumption may alter gut microbiome and host mucosal functions during weaning transition.

Regional and age dependent changes in gene expression of Toll-like receptors and key antimicrobial defence molecules throughout the gastrointestinal tract of dairy calves.

The primary aim of this study was to determine regional and age-dependent expression patterns of Toll-like receptors (TLRs), peptidoglycan recognition protein 1 (PGLYRP1), and ß-defensin in rumen, jejunum, ileum, cecum and colon of 3 week (n=8) and 6 month old (n=8) calves. The expression of most TLRs was significantly down-regulated throughout the gastrointestinal tract (GIT) with increasing age. TLR10 expression was significantly higher in ileum than all other gut regions, irrespective of age. TLR2 and TLR4 expression were significantly higher in the cecum and colon of 6 month old calves. Furthermore, expression of ß-defensin, and PGLYRP1 was only detectable in 6 month old calves. The expression of TLRs was positively or negatively correlated with population of total bacteria and/or lactic acid bacteria depending on the GIT region. These observations indicate that innate immune responses to commensal microflora may vary significantly throughout the GIT and with age changes.

Distinct commensal bacteria associated with ingesta and mucosal epithelium in the gastrointestinal tracts of calves and chickens.

The primary aim of this study was to determine whether distinct gastrointestinal tract (GIT) microbial communities are established within ingesta and on mucosal surfaces of dairy calves and chickens to evaluate whether the principle of microbial segregation is of broad biological significance. Multivariate analysis of the predominant bacterial PCR-denaturing gradient gel electrophoresis profiles and estimated bacterial populations were compared in rumen, jejunum, ileum, cecum, and colon ingesta and matching mucosal tissues. Samples collected from 3-week old (n = 8) and 6-month old (n = 8) calves revealed that the predominant mucosa-associated bacteria were distinct from those inhabiting ingesta, and bacterial diversity varied significantly among the GIT regions. The estimated bacterial populations displayed significant regional differences for bovine mucosal (P = 0.05) and for ingesta (P = 0.03) only at 6 months of age. This indicates an established segregation of the enteric bacterial population throughout the GIT in weaned calves. Analysis of ileal and cecal bacterial profiles in chickens confirmed that the segregation of commensal bacteria between ingesta and the mucosal tissue was a common biological phenomenon. Our study provides some fundamental understanding of the impact of sample type (mucosa vs. ingesta), region, and host age on commensal bacterial establishment and segregation throughout the GIT.

Characterization of bovine ruminal epithelial bacterial communities using 16S rRNA sequencing, PCR-DGGE, and qRT-PCR analysis.

Currently, knowledge regarding the ecology and function of bacteria attached to the epithelial tissue of the rumen wall is limited. In this study, the diversity of the bacterial community attached to the rumen epithelial tissue was compared to the rumen content bacterial community using 16S rRNA gene sequencing, PCR-DGGE, and qRT-PCR analysis. Sequence analysis of 2785 randomly selected clones from six 16S rDNA (∼1.4kb) libraries showed that the community structures of three rumen content libraries clustered together and were separated from the rumen tissue libraries. The diversity index of each library revealed that ruminal content bacterial communities (4.12/4.42/4.88) were higher than ruminal tissue communities (2.90/2.73/3.23), based on 97% similarity. The phylum Firmicutes was predominant in the ruminal tissue communities, while the phylum Bacteroidetes was predominant in the ruminal content communities. The phyla Fibrobacteres, Planctomycetes, and Verrucomicrobia were only detected in the ruminal content communities. PCR-DGGE analysis of the bacterial profiles of the rumen content and ruminal epithelial tissue samples from 22 steers further confirmed that there is a distinct bacterial community that inhibits the rumen epithelium. The distinctive epimural bacterial communities suggest that Firmicutes, together with other epithelial-specific species, may have additional functions other than food digestion.

Changes in bacterial diversity associated with epithelial tissue in the beef cow rumen during the transition to a high-grain diet.

Our understanding of the ruminal epithelial tissue-associated bacterial (defined as epimural bacteria in this study) community is limited. In this study, we aimed to determine whether diet influences the diversity of the epimural bacterial community in the bovine rumen. Twenty-four beef heifers were randomly assigned to either a rapid grain adaptation (RGA) treatment (n = 18) in which the heifers were allowed to adapt from a diet containing 97% hay to a diet containing 8% hay over 29 days or to the control group (n = 6), which was fed 97% hay. Rumen papillae were collected when the heifers were fed 97%, 25%, and 8% hay diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis were used to characterize rumen epimural bacterial diversity and to estimate the total epimural bacterial population (copy numbers of the 16S rRNA gene). The epimural bacterial diversity from RGA heifers changed (P = 0.01) in response to the rapid dietary transition, whereas it was not affected in control heifers. A total of 88 PCR-DGGE bands were detected, and 44 were identified from phyla including Firmicutes, Bacteroidetes, and Proteobacteria. The bacteria Treponema sp., Ruminobacter sp., and Lachnospiraceae sp. were detected only when heifers were fed 25% and 8% hay diets, suggesting the presence of these bacteria is the result of adaptation to the high-grain diets. In addition, the total estimated population of rumen epimural bacteria was positively correlated with molar proportions of acetate, isobutyrate, and isovalerate, suggesting that they may play a role in volatile fatty acid metabolism in the rumen.

Correlation of particular bacterial PCR-denaturing gradient gel electrophoresis patterns with bovine ruminal fermentation parameters and feed efficiency traits.

The influence of rumen microbial structure and functions on host physiology remains poorly understood. This study aimed to investigate the interaction between the ruminal microflora and the host by correlating bacterial diversity with fermentation measurements and feed efficiency traits, including dry matter intake, feed conversion ratio, average daily gain, and residual feed intake, using culture-independent methods. Universal bacterial partial 16S rRNA gene products were amplified from ruminal fluid collected from 58 steers raised under a low-energy diet and were subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Multivariate statistical analysis was used to relate specific PCR-DGGE bands to various feed efficiency traits and metabolites. Analysis of volatile fatty acid profiles showed that butyrate was positively correlated with daily dry matter intake (P < 0.05) and tended to have higher concentration in inefficient animals (P = 0.10), while isovalerate was associated with residual feed intake (P < 0.05). Our results suggest that particular bacteria and their metabolism in the rumen may contribute to differences in host feed efficiency under a low-energy diet. This is the first study correlating PCR-DGGE bands representing specific bacteria to metabolites in the bovine rumen and to host feed efficiency traits.

Probiotic preparation VSL#3 induces remission in children with mild to moderate acute ulcerative colitis: a pilot study.

BACKGROUND: Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) that has periods of exacerbated symptoms and periods that are symptom-free. The treatment of active UC with probiotic bacteria could possibly induce remission. We evaluated the clinical efficacy and safety profile of probiotic preparation VSL#3 in the treatment of mild to moderate acute UC in the pediatric population. METHODS: Eighteen eligible patients between the ages of 3-17 with mild to moderate acute UC received open-label VSL#3 daily in 2 divided doses for 8 weeks. The disease activity pre- and post-VSL#3 therapy was assessed by the simple clinical colitis activity index (SCCAI); Mayo ulcerative colitis endoscopic score; inflammatory markers: erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP); serum cytokine profiling; and rectal tissue microbial profiling done at baseline and at week 8. RESULTS: Thirteen patients completed 8 weeks of VSL#3 treatment and 5 patients were withdrawn due to lack of improvement. Remission (defined as SCCAI or=2, but final score

Across genus plasmid transformation between Bacillus subtilis and Escherichia coli and the effect of Escherichia coli on the transforming ability of free plasmid DNA.

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.

A marine killer yeast against the pathogenic yeast strain in crab (Portunus trituberculatus) and an optimization of the toxin production.

A pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus was identified to be Metschnikowia bicuspidate according to the results of routine yeast identification and 18S rDNA and ITS sequences. After screening of more than 300 yeast strains from different sources in marine environments, it was found that strain YF07b had the highest ability to produce killer toxin against the pathogenic yeast. Strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The optimal conditions for killer toxin production by strain YF07b were the production medium with 2.0% NaCl, pH 4.5, cultivation temperature of 20 degrees C and the optimal conditions for action of the crude killer toxin against the pathogenic yeast were the assay medium with 6.0% NaCl, pH 4.5 and temperature 15 degrees C.

[Intergenus natural genetic transformation between Escherichia coli and Bacillus subtilis at different growth phase].

The culture fluids of Escheriachia coli with shuttle plasmid and Bacillus subtilis strains were mixed and coincubated for 40 minutes after culturing respectively in LB and minimal media. The steadily plasmid transfer by natural genetic transformation between these two gram-negative and gram-positive bacteria has been demonstrated by the methods of selective medium screening, DNase I sensitivity test and plasmid detection. In contrast to MM culture B. subtilis LB culture can be competent and has equivalent transformation frequency. Furthermore, the maximal transformation frequency was obtained when cells in exponential phase served as donors or recipients. It is suggested that B. subtilis solid transformation is different from liquid plasmid transformation including the whole process of DNA plasmid competence producing. Understanding the mechanisms of gene transfer between bacteria may aid in assessing the potential risk associated with the release of recombinant organisms into the environment.

The surface display of haemolysin from Vibrio harveyi on yeast cells and their potential applications as live vaccine in marine fish.

HL1 gene encoding haemolysin from Vibrio harveyi SF-1 was expressed in yeast cells and the expressed haemolysin was displayed on the cell surface. After induction for 36h in galactose-containing medium, one-third of the cells contained the displayed protein and the displayed cells had haemolytic activity on erythrocytes from flounder. The double diffusion agar analysis showed that the sera from the flounder immunized with the displayed yeast cells having the haemolytic activity could form precipitate with the purified haemolysin. ELISA analysis indicated that immunization times had great influence on increased production of the specific antibody against haemolysin in turbot immunized with the displayed yeast cells having the haemolytic activity. After the challenge with V. harveyi SF-1, it was found that earlier protection in flounder and significant protection in turbot, both of which were immunized with the displayed yeast cells having the haemolytic activity, were achieved. These results suggested that the displayed yeast cells with the haemolytic activity could be used as potential live vaccine in marine fish.