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Nucleotide binding and oligomerization domain (NOD)1 and NOD2 are important cytoplasmic pattern recognition receptors (PRRs) and key members of the NOD-like receptor (NLR) family. They sense a wide range of bacteria or their products and play a key role in inducing innate immunity. This report describes the role of NOD1 and NOD2 receptors signalling in innate immunity in the Indian major carp, mrigal (Cirrhinus mrigala). Tissue-specific expression analysis of NOD1 and NOD2 genes by quantitative real-time PCR (qRT-PCR) revealed their wide distribution in various organs/tissues. In the untreated fish, the highest expression of NOD1 and NOD2 was detected in liver and blood, respectively. Stimulation with NOD1- and NOD2-specific ligands, i.e. iE-DAP and MDP, activated NOD1 and NOD2 receptor signalling in vivo and in vitro resulting in significant (p less than 0.05) induction of downstream signalling molecule RICK, and the effector molecules IL-1 beta, IL-8 and IFN- gamma in the treated group as compared to their controls. In response to both Gram-positive and Gram-negative bacterial infections, NOD1 and NOD2 receptors signalling were activated and IL-1 beta, IL-8 and IFN- gamma were induced. These findings highlight the important role of NOD receptors in eliciting innate immune response during the pathogenic invasion to the fish.

Gut microbiota has shown tight and coordinated connection with various functions of its host such as metabolism, immunity, energy utilization, and health maintenance. To gain insight into whether gut microbes affect the metabolism of fish, we employed fast-growing transgenic common carp (Cyprinus carpio L.) to study the connections between its large body feature and gut microbes. Metagenome-based fingerprinting and high-throughput sequencing on bacterial 16S rRNA genes indicated that fish gut was dominated by Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes, which displayed significant differences between transgenic fish and wild-type controls. Analyses to study the association of gut microbes with the fish metabolism discovered three major phyla having significant relationships with the host metabolic factors. Biochemical and histological analyses indicated transgenic fish had increased carbohydrate but decreased lipid metabolisms. Additionally, transgenic fish has a significantly lower Bacteroidetes:Firmicutes ratio than that of wild-type controls, which is similar to mammals between obese and lean individuals. These findings suggest that gut microbiotas are associated with the growth of fast growing transgenic fish, and the relative abundance of Firmicutes over Bacteroidetes could be one of the factors contributing to its fast growth. Since the large body size of transgenic fish displays a proportional body growth, which is unlike obesity in human, the results together with the findings from others also suggest that the link between obesity and gut microbiota is likely more complex than a simple Bacteroidetes:Firmicutes ratio change.

Laribacter hongkongensis is a potential emerging pathogen, associated with community-acquired diarrhoea. For epidemiological purposes, different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing, have been developed for this pathogen. However, these methods require specialized equipment and costly reagents. More importantly, they are labour-intensive and time-consuming, which is not really suitable for foodborne disease outbreak investigations. In this study, we developed a rapid and reliable method using 22-mer primers specific for the enterobacterial repetitive intergenic consensus sequence (ERIC). PFGE was used for comparison, to evaluate this method. A total of 81 isolates of L. hongkongensis were examined: 79 isolates recovered from food of diverse origins and two strains derived from patients with L. hongkongensis-associated infection. Typing patterns and clustering analysis indicated that the 81 L. hongkongensis isolates were grouped into 21 and 13 genotypes by ERIC-PCR and PFGE, respectively. ERIC-PCR was found as reproducible as PFGE. A high percentage (70.4%) of isolates yielded distinguishable ERIC-PCR patterns, which were concordant with the results from PFGE. These results suggest that ERIC-PCR is valuable for use in the epidemiological investigation of L. hongkongensis.

Chemical analyses and mass spectrometry were used to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas bestiarum strain K296, serotype O18. ESI-MS revealed that the most abundant A. bestiarum LPS glycoforms have a hexa-acylated or tetra-acylated lipid A with conserved architecture of the backbone, consisting of a 1,4'-bisphosphorylated β-(1→6)-linked D-GlcN disaccharide with an AraN residue as a non-stoichiometric substituent and a core oligosaccharide composed of Kdo1Hep6Hex1HexN1P1. 1D and 2D NMR spectroscopy revealed that the O-specific polysaccharide (OPS) of A. bestiarum K296 consists of a branched tetrasaccharide repeating unit containing two 6-deoxy-l-talose (6dTalp), one Manp and one GalpNAc residues; thus, it is similar to that of the OPS of A. hydrophila AH-3 (serotype O34) in both the sugar composition and the glycosylation pattern. Moreover, 3-substituted 6dTalp was 2-O-acetylated and additional O-acetyl groups were identified at O-2 and O-4 (or O-3) positions of the terminal 6dTalp. Western blots with polyclonal rabbit sera showed that serotypes O18 and O34 share some epitopes in the LPS. The very weak reaction of the anti-O34 serum with the O-deacylated LPS of A. bestiarum K296 might have been due to the different O-acetylation pattern of the terminal 6dTalp. The latter suggestion was further confirmed by NMR.

The TNF superfamily B cell activating factor (BAFF) is a central cytokine in several diseases. A BAFF gene has been cloned from grass carp (Ctenopharyngodon idella), analyzed its structure, and investigated its expression pattern in various tissues after Aeromonas hydrophila and Aquareovirus infection. The open reading frame of grass carp BAFF (gcBAFF) consists of 804 bases encoding 267 amino acids. Phylogenetic analysis shows the gcBAFF is most closely related to other teleost BAFFs with the highest similarity to zebrafish. RT-PCR analysis shows the gcBAFF transcript is expressed in a wide range of tissues with the highest expression in skin and spleen. Upon induction by A. hydrophila and Aquareovirus, its expression is significantly up-regulated in gill, liver, kidney, spleen and skin as compared to PBS injected fish. The association of increased BAFF expression after bacterial and viral infections suggests that it plays a potentially important role in immune system of fish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: In this study, the antibacterial properties and active ingredient of plant extracts and its effect on the performance of crucian carp (Carassius auratus gibelio var. E'erqisi, Bloch) were assessed., Results: The transmission electron microscopy and flow cytometric analysis showed that the antibacterial activity of plant extracts is due to the disruption of the cell membrane and the leakage of cytoplasmic contents. The UPLC-MS/MS analysis showed that the contents of gallic acid, (-)-epigallocatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin gallate, aloe-emodin, rhein, emodin, chrysophanol, and physcion, were 5.27%, 3.30%, 1.08%, 19.32%, 5.46%, 0.23%, 0.56%, 1.28%, 0.75% and 0.39% in plant extracts, respectively. Results of feeding experiment showed that feeding crucian carp with 1.0% and 2.0% plant extracts significantly enhanced specific growth rate, serum total protein, lysozyme, catalase and superoxide dismutase activities, and decreased the feed conversion rate, malondialdehyde contents and the mortality rate (P < 0.05)., Conclusion: It can be concluded that plant extracts added to fish feed can act as natural antimicrobial and immunostimulants to prevent pathogenic infection, enhance immune response, and promote growth of the fish., (© 2012 Society of Chemical Industry.)

High mobility group box 1 (HMGB1) is a nuclear weapon in the immune arsenal and a master regulator of innate immunity, at the crossroads between innate and adaptive immunity. To clarify the immune characterizations of HMGB1 in fishes, two co-orthologs of HMGB1 (CiHMGB1a and CiHMGB1b) were identified in grass carp Ctenopharyngodon idella by local EST database searching and RACE techniques. mRNA expressions of the two HMGB1 genes are widespread in fifteen tissues investigated. The transcripts of CiHMGB1a and CiHMGB1b were significantly up-regulated and reached peak at 24h post GCRV challenge in spleen and head kidney tissues (P<0.05). The modulations are slow post-bacterial PAMP stimulations by contrast with those after viral PAMP or GCRV challenge. They are inhibited by bacterial PAMPs, but are enhanced by viral PAMP or virus. mRNA expression of CiHMGB1a is high and strongly modulated by nucleic acids and transcription of CiHMGB1b is low and mildly regulated by nucleic acids and capsids of GCRV. The over-expression vectors were constructed and transfected into C. idella kidney cell line to obtain stably expressing recombinant proteins. In HMGB1 over-expressed cells, mRNA expressions of IPS-1, MyD88 and Mx1 were down-regulated, whereas TRIF was found to be up-regulated and IFN-I showed no change in its expression. After GCRV challenge, the transcripts of IPS-1, MyD88 and Mx1 were up-regulated, while IFN-I showed down-regulation, and TRIF showed up-regulation after an initial phase of decline. The titer assay demonstrated no antiviral activity of HMGB1s. The results indicated mRNA expressions of HMGB1a and HMGB1b are enhanced by GCRV or viral PAMP, and are inhibited by bacterial PAMPs; HMGB1a and HMGB1b collaborate with each other and play important roles in modulating the innate immune responses, although without direct antiviral effect; the immune network triggered by HMGB1 work together in concert to maintain homeostasis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A total of 71 Aeromonas strains were isolated in the south of Jiangsu Province China in order to analyze the difference ofAeromonas spp. distribution between diseased fish and water environment. The sequence of 16S rDNA and gyrB demonstrated that the 71 Aeromonas isolates could be divided into 4 species, including A. veronii (55), A. hydrophila (11), A. salmonicida (3) and A. media (2). A. veronii was the most common species isolated from fish and water environment. All Aeromonas isolates were screened for three putative virulence genes, aer, hly and alt. hly was the most common gene among three virulence genes.

The formation of eight biogenic amines (BAs), total volatile base nitrogen (TVB-N), aerobic plate counts (APC), and sensory evaluation in crucian carp (Carassius auratus) during storage in ice and at 4°C were investigated. The sum of contents of all eight biogenic amines (SBA) in crucian carp increased from 49.82 to 197.09 mg/kg on day 36 when stored in ice and from 49.82 to 219.35 mg/kg on day 24 when stored at 4°C. TVB-N content increased with storage time; good relationships between TVB-N content and SBA were observed during storage in ice and at 4°C (R(2) = 0.95 and 0.93, respectively). APC of all samples significantly (P < 0.05) increased with storage time in the first 12 days and toward the end of the storage period, there were only minor changes; good relationships between APC and SBA were observed during storage in ice and at 4°C (R(2) = 0.96 and 0.58, respectively). According to sensory evaluation results, the crucian carp became unacceptable on days 24 and 16 during storage in ice and at 4°C, respectively. Putrescine and tyramine contents could be good quality evaluation indices for crucian carp during storage.

The present study was conducted to test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6.33 (SE 0.03) g, were fed with diets containing graded concentrations of arginine, namely, 9.8 (control), 12.7, 16.1, 18.5, 21.9 and 24.5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0.05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na⁺/K⁺-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0.05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18.0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.

Integrated livestock-fish aquaculture utilizes animal excreta and urine as pond fertilizers to enhance growth of plankton and other microorganisms eaten by the fish. In Vietnam, pigs are commonly integrated with fish and horticulture in household-based VAC systems (vuon = garden; ao = pond; chuong = pigsty), but the level of fecal contamination in the fish produced is unknown. This study was carried out to assess the level Escherichia coli contamination of fish meat and gut contents of grass carp (Ctenopharyngodon idellus), silver carp (Hypophthalmichthys molitrix), and rohu (Labeo rohita) cultured in randomly selected five VAC ponds (with pig manure) and five non-VAC ponds (without pig manure) at sites in periurban Hanoi, Vietnam. Fish muscle tissue samples contained E. coli at <10 or 320 or 820 CFU/g, regardless of the culture system from which they originated. In contrast, the intestinal contents of fish raised in manure-fed ponds contained E. coli at 4.75, 5.25, and 5.07 log CFU/g for silver carp, grass carp, and rohu, respectively, about 100 times higher than the contamination of fish from the control ponds. The results indicate that muscle tissue of fish raised in VAC systems has a low level of fecal contamination despite high levels of E. coli in their gut. Thus, a critical point to control food safety of such fish is the prevention of fecal cross-contamination during degutting and cleaning of the fish at markets and in the home.

The Indian major carp cultured in ponds in the North Eastern hilly states of India frequently suffer from fungal disease during winter months resulting in mass mortality. This study examined the pathogenic fungi isolated from farmed raised Indian major carp fingerlings and identified as Saprolegnia. For treatment, the diseased fish were exposed to 4g salt per litre of water for 2 min followed by dip treatment with 5ppm KMnO4 for 10 min, thrice every week for a period of 6 weeks. The treatment resulted in recovery from the disease after 6 weeks from the beginning of treatment. Soon after recovery, the pond management practices such as removal of pond bottom soil, application of lime and replenishment with freshwater were followed in the infected ponds. Our study concluded that rapid decrease in pond water temperature from 22 to 8 degrees C that remains low for months together coupled with increased water pH (9) and decreas dissolved oxygen (4ppm) causes saprolegniasis to the fingerlings of Indian major carps.

Toll-like receptor 2 (TLR2) is a member of TLR family. It recognizes a wide range of bacteria and their products, and is involved in inducing innate immune responses. In this article, we reported inductive expression of TLR2 and myeloid differentiation primary response gene 88 (MyD88)-dependent signaling in the Indian major carp, mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) genes by quantitative real-time PCR (qRT-PCR) revealed constitutive expression of these genes in all embryonic developmental stages, indicating their involvement in embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by qRT-PCR showed their wide distribution in various organs and tissues. Highest expression of TLR2 was in gill, MyD88 in liver and TRAF6 was in kidney. Inductive expression of TLR2, MyD88 and TRAF6 genes were observed following peptidoglycan (PGN)-treatment, and Streptococcus uberis and Aeromonas hydrophila infections. Expression of interleukin (IL)-8 and TNF-α in various organs were significantly enhanced by PGN-treatment and bacterial infections, and were closely associated with TLR2 induction. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish, and indicated it's important role in immune surveillance of various organs during pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish, and will be helpful in developing preventive measures against infectious diseases in fish.

Physiological and biochemical responses in kidneys of fishes with different trophic levels to toxic cyanobacterial blooms were studied. We sampled four fishes: the phytoplanktivorous Hypophthalmichthys molitrix and Aristichthys nobilis, the omnivorous Carassius auratus, and the carnivorous Culter ilishaeformis. Alterations of the antioxidant (GSH) and the major antioxidant enzymes (CAT, SOD, GPx, GST) were monitored monthly. Catalase and glutathione S-transferase were significantly higher during blooms than before and after blooms. All fishes showed ultrastructural alterations during blooms, which mainly are inosculation of foot processes in epithelial cell of glomeruli and mitochondria swelling in the proximal tubules. The results suggested that kidney impairment from chronic exposure of toxic cyanobacterial blooms might be the first step, and then followed by hepatic failure. Compared with livers in terms of physiological status, the weaker antioxidant ability of kidney made it more susceptible to chronic MCs exposure, besides its effective accumulation of MC metabolites., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Selection in fish for disease resistance is one of the most useful approaches to solve disease problems. Genetic variation in resistance to edwardsiellosis in fullsib families of rohu, Labeo rohita was investigated in the present study. A large variation in the susceptibility pattern (0 to 94.74 percent survival) against Edwardsiella tarda challenge was observed among 57 families. Additive genetic variation showed a heritability of 0.38 ± 0.08 across the year-class survival. The apparent resistant families showed more delayed mortality than the apparent susceptible ones. The cross-protection provided by aeromoniasis-resistant lines of rohu to edwardsiellosis was also studied to evaluate the possibility of selection for both diseases simultaneously. Challenge of F1-generation aeromoniasis-resistant and -susceptible lines with E. tarda showed significant difference in survival between the lines with higher percent survival in resistant line. This study suggests that direct selection method may be used reliably in selection programs and selection for multiple diseases simultaneously can be considered for rohu.

Silver carp fish pastes inoculated with or without a douchi starter culture containing live Aspergillus oryzae were fermented for 30 days to produce two different fermented products, designated CulF and ConF, respectively. Protein degradation and flavor compound production during the course of fermentation were monitored. Proteolytic activity, generally higher in CulF than in ConF (P < 0.05) and dominated by acidic and serine proteases, declined to an overall minimum after 30 days. Myosin in the CulF and ConF extractives was completely degraded after 1 and 5 days, respectively. The content of free amino acids and low molecular weight (<1.3 kDa) peptides rose rapidly in CulF and progressively in ConF (P < 0.05). Ethanol, silanediol, pyrazine, phenol, and formic acid were prevalent volatile compounds in CulF, whereas butanol, butanoic acid, and acetic acid were abundant in ConF. Therefore, douchi-inoculated fermentation is an attractive process to produce savory fish pastes.

Background: RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) and mediating the induction of type I interferon and inflammatory cytokines in innate immune response. Though the mechanism is well characterized in mammals, the study of the accurate function of RIG-I in teleosts is still in its infancy., Methodology/principal Findings: To clarify the functional characterizations of RIG-I in grass carp Ctenopharyngodon idella (CiRIG-I), six representative overexpression plasmids were constructed and transfected into C. idella kidney (CIK) cell lines to obtain stably expressing recombinant proteins, respectively. A virus titer test and 96-well plate staining assay showed that all constructs exhibited the antiviral activity somewhat. The quantitative real-time RT-PCR (qRT-PCR) demonstrated that mRNA expressions of CiIPS-1, CiIFN-I and CiMx2 were regulated by not only virus (GCRV) or viral PAMP (poly(IC)) challenge but also bacterial PAMPs (LPS and PGN) stimulation in the steadily transfected cells. The results showed that the full-length CiRIG-I played a key role in RLR pathway. The repressor domain (RD) exerted an inhibitory function of the signaling channel under all utilized challenges. Caspase activation and recruitment domains (CARDs) showed a positive role in GCRV and poly(I:C) challenge. Helicase motifs were crucial for the signaling pathway upon LPS and PGN stimulation. Interestingly, ΔCARDs (CARDs deleted) showed positive modulation in RIG-I signal transduction., Conclusions/significance: The results provided some novel insights into RIG-I sensing with a strikingly broad regulation in teleosts, responding not only to the dsRNA virus or synthetic dsRNA but also bacterial PAMPs.

Flavobacterium columnare is a bacterial pathogen causing high mortality rates for many freshwater fish species. Fish vaccination with a safe and effective vaccine is a potential approach for prevention and control of fish disease. Here, in order to produce bacterial ghost vaccine, a specific Flavobacterium lysis plasmid pBV-E-cat was constructed by cloning PhiX174 lysis gene E and the cat gene with the promoter of F. columnare into the prokaryotic expression vector pBV220. The plasmid was successfully electroporated into the strain F. columnare G4cpN22 after curing of its endogenous plasmid. F. columnare G4cpN22 ghosts (FCGs) were generated for the first time by gene E-mediated lysis, and the vaccine potential of FCG was investigated in grass carp (Ctenopharyngodon idellus) by intraperitoneal route. Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%). These results showed that FCG could confer immune protection against F. columnare infection. As a nonliving whole cell envelope preparation, FCG may provide an ideal alternative to pathogen-based vaccines against columnaris in aquaculture.

Gut microbiota has become an integral component of the host, and received increasing attention. However, for many domestic animals, information on the microbiota is insufficient and more effort should be exerted to manage the gastrointestinal bacterial community. Understanding the factors that influence the composition of microbial community in the host alimentary canal is essential to manage or improve the microbial community composition. In the present study, 16S rRNA gene sequence-based comparisons of the bacterial communities in the grass carp (Ctenopharyngodon idellus) intestinal contents and fish culture-associated environments are performed. The results show that the fish intestinal microbiota harbors many cellulose-decomposing bacteria, including sequences related to Anoxybacillus, Leuconostoc, Clostridium, Actinomyces, and Citrobacter. The most abundant bacterial operational taxonomic units (OTUs) in the grass carp intestinal content are those related to feed digestion. In addition, the potential pathogens and probiotics are important members of the intestinal microbiota. Further analyses show that grass carp intestine holds a core microbiota composed of Proteobacteria, Firmicutes, and Actinobacteria. The comparison analyses reveal that the bacterial community in the intestinal contents is most similar to those from the culture water and sediment. However, feed also plays significant influence on the composition of gut microbiota.

Neutralizing complement C9 in grass carp Ctenopharyngodon idella sera with rabbit anti-C9 sera against fish complement C9, demonstrated that bactericidal activity against Aeromonas hydrophila of the C9-deficient fish sera was greatly impaired. These results indicated that the fish complement C9 plays a key role in pathogen killing through the lytic pathway., (© 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.)

Indian major carp (Catla catla) was subjected to study the immunostimulatory effects when the grass Cynodon dactylon(L) ethanolic extract administrated as feed supplement. C. catla was fed with 0% (Control), 0.05% (group I), 0.5% (group II) and 5% (group III) extract provided for 60 days. Blood samples were collected at every 10 days of interval up to 60 days for analyzing the non-specific humoral (lysozyme activity, antiprotease activity and haemolytic complement) and cellular (production of reactive oxygen and nitrogen species, myeloperoxidase activity) immune response study. The results indicate that C. dactylon ethanolic extract administered as feed supplement significantly (P < 0.05) enhanced most of the non-specific immune parameters tested. Among the experimental diet groups, significantly increased response of non-specific immunity was seen in group III (5%). Disease resistant analysis against Aeromonas hydrophila was performed in control group and plant extract treated fish for 7, 14, 21 and 28 days. Relative percent survival rate (RPS) was observed in treated samples, which is directly proportional to concentration of the extract. Additionally, electron microscopic studies and gelatin zymography for Matrix Metalo Proteinase (MMPs) were examined in spleen at 7th and 28th days of feeding. Administration of C. dactylon mixed diet delayed the lymphocyte destruction with positive ultrastructural changes. An induced stress (A. hydrophila infection) was observed by using MMPs expression, which was reduced in the experimental diet groups than the control. All these experimental results prove that C. dactylon ethanolic extract enhances the immunity of Catla fish., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study was performed to determine the efficacy of three immunomodulators viz., β-1,3 glucan, chitosan and raffinose on the innate immune response of koi, Cyprinus carpio koi. Kois were divided into 4 groups and each group was fed with diets supplemented with or without immunostimulant for 56 days. Total leukocyte counts (WBC), the non-specific humoral (lysozyme, alternative complement pathway and superoxide dismutase) and cellular (phagocytic capacity and respiratory burst activity) responses were determined and compared with controls (no supplement) after 7, 14, 21 and 56 days of feeding. The results of 8 weeks feeding trial showed that β-1,3 glucan supplementation significantly enhanced koi growth, whereas other immunostimulants did not. Variation in the levels of responses was evident among different supplements. Compared with chitosan or raffinose, β-1,3 glucan could maintain the immunity of kois at a higher level during the experimental period. However, continuously applying β-1,3 glucan, chitosan or raffinose into the diet caused immunity fatigue in koi. No significant change in alternative complement pathway (ACP) activity was observed for any of the three supplements over the four different periods. After feeding for 14 days, the total leukocyte count (WBC), respiratory burst activity and superoxide dismutase (SOD) activity of the kois fed with chitosan or raffinose continuously remained relatively unchanged, subsequently decreased on the 56th day, but SOD did not. Meanwhile, lysozyme activity was no longer significantly higher on the 7th day, and for phagocytic capacity on the 14th day. After 56 days, these three immunostimulants groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with Aeromonas veronii. These results indicated that dietary intake containing immunostimulants could enhance the immune responses of koi and improve its resistance to infection by A.veronii. Especially supplementation with β-1,3 glucan to the kois for 56 days showed considerable improvement in the growth, survival and immune response of the kois., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community-acquired gastroenteritis and traveller's diarrhoea, we developed a duplex species-specific PCR assay., Methods and Results: Full-length of the 16S-23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR-based sequencing. Two species-specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species-specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2.1 × 10(-2) ng μl(-1) genomic DNA which corresponds to 5000 CFU ml(-1)., Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis., Significance and Impact of the Study: This species-specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Bacteria communities living in mucus secretions of common carp Cyprinus carpio (Cyprinidae) were exposed to the organic nanomaterial fullerene (C(60)) to evaluate its potential bactericidal effects. End points analyzed were viability, growth, reactive oxygen species (ROS) concentration, and total antioxidant competence against peroxyl radicals. Viability was not affected (p > 0.05), whereas growth was arrested (p < 0.05) after 3 hours of exposure to the three concentration of C(60) assayed (0.1, 1, and 10 mg/L). Levels of RO measured at different C(60) concentration showed that some colonies were reactive (significant dose-response relation, p < 0.05) to C(60), whereas others were not. The nonreactive colonies to C(60) presented higher antioxidant competence to peroxyl radicals compared with the reactive colonies (p < 0.05). The strains isolated and identified by polymerase chain reaction (PCR) products of 16S rRNA showed a predominance of Aeromonas genus between all the isolated Gram-negative bacteria. Thus, the present results indicate that C(60) affects bacterial communities that live in mucus secretions of common carp.

A strain was isolated from a soil sample collected from Weihe river in Shaanxi province (108°03'E 34°14'N), which was identified as Alcaligenes faecalis by 16S rRNA analysis. A compound M showing potent immune activity was isolated from secondary metabolites of the strain through bioassay-guided isolation techniques. The structure of the compound M was elucidated using FT-IR, EI-MS, 1H NMR and 13C NMR spectra and identified as cyclo-(L-Pro-Gly)5 which was first time reported as a natural product. We evaluated the immune effects of the cyclo-(L-Pro-Gly)5 on the basis of serum lysozyme activity, bacterial agglutination titre assay, superoxide anion production and phagocytic activity assay, and they were found to be significantly increased by cyclo-(L-Pro-Gly)5. The effects of cyclo-(L-Pro-Gly)5 on immune-related gene expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 6β (IL-6β) and inducible nitric oxide synthase 1β (iNOS-1β) mRNA in the blood leucocytes have been augmented by cyclo-(L-Pro-Gly)5. The challenge experiment showed that crucian carp injected the cyclo-(L-Pro-Gly)5 had significantly (P < 0.05) lower cumulative mortality (13.0%) compared with the control (45.4%) after infection with live Aeromonas hydrophila. These results suggested that cyclo-(L-Pro-Gly)5 is a possible immunostimulant and may strengthen the immune response and protect the heath status of crucian carp against A. hydrophila., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

A bacterial strain with putative immunomodulatory properties was isolated from Xi'an hot springs in China. Comparison of 16S rRNA gene revealed a 97% similarity between the tested strain (designated XA-1) and Anoxybacillus kamchatkensis. Two compounds isolated from the secondary metabolites of XA-1 were identified by spectral data (infrared, nuclear magnetic resonance and mass spectrometry) as: (1) cyclo (Gly-L-Pro) and (2) cyclo (L-Ala-4-hydroxyl-L-Pro). Two cyclic dipeptides showed stimulatory properties towards a range of parameters when a dose of 20mg kg(-1) body weight was intraperitoneally injected in naive common carp, Cyprinus carpio. Innate immune parameters (serum SOD, lysozyme and bactericidal activity, and phagocytic activity by peripheral blood leucocytes) along with the expression of two immune-related genes (IL-1β and iNOS) in blood were examined after 7, 14, 21, and 28 days of injection. In the absence of infection, immunomodulators should ideally not affect normal physiology and immunity of the host; possible negative outcomes of activated immune responses in the naive state are discussed. Protection by two bacterial dipeptides was assessed in an intraperitoneal injection challenge trial with live Aeromonas hydrophila. Both compounds reduced mortality, with the highest survival rate observed in the group that received compound 2 (80%) followed by the group that received compound 1 (65%) while control group scored the worse (15%). Elucidation of the involved protective mechanisms in carp requires future studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tissue inhibitors of metalloproteinases (TIMPs) are the primary inhibitors of matrix metalloproteinases (MMPs) in tissues. In this study, a TIMP-2 gene was isolated from grass carp Ctenopharyngodon idella (CiTIMP-2b) and characterized. This cDNA sequence encoded a signal peptide, N- and C-terminal domains. CiTIMP-2b is highly homologous to the orthologous in zebrafish and other teleosts, suggesting TIMP-2b is highly conserved during teleost evolution. CiTIMP-2b gene is expressed in a wide range of tissues including blood, brain, muscle, trunk kidney, liver, head kidney, skin, spleen, heart, gill, intestine and fin, with the highest level of transcripts in spleen. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in all tissues. The CiTIMP-2b transcript is present at unfertilized eggs, which suggests that CiTIMP-2b transcript is maternally inherited. These results suggest that the CiTIMP-2b would play an important role in the A. hydrophila-related diseases and early embryonic development stages in grass carp., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Context: Infectious diseases are one of the major factors affecting the production of fish worldwide. The pathogens (especially bacteria) affect the immune system of fish and the administration of immunostimulants can increase resistance to infectious diseases by enhancing both specific and nonspecific defense mechanisms., Objective: In the present study, we have conducted an experiment on the pathogen-infected Cyprinus carpio Linn. (Cyprinidae), using Euphorbia hirta Linn. (Euphorbiaceae) plant leaves as immunostimulants., Materials and Methods: The aqueous extract of the leaves was prepared and the immunostimulant action was recorded by giving different concentrations of plant extract supplemented diet., Results and Discussion: The results obtained from the studies show that the higher concentration of the extract (50 g/kg diet) provided significant immune response (specific and nonspecific) on the fish. The 50 g/kg leaf extract of E. hirta enhanced the phagocytic ratio on 10th and 15th day after the infection. The results of the specific and nonspecific immunostimulation studies are statistically significant., Conclusion: This work will guide the researchers for the discovery of significant aquaculture nutrients to improve the immunostimulant action on fish.

This study evaluated the influence of toxic cyanobacterial water blooms on the blood indices of the common carp, Cyprinus carpio L. Experimental fish were exposed to a natural population of cyanobacterial water blooms (mainly Microcystis aeruginosa and M. ichthyoblabe), which contained microcystins [total concentration 133-284 μg g⁻¹ (DW), concentration in water 2.8-7.4 μg L⁻¹]. Haematological indices showed marked changes in fish exposed to the cyanobacterial population in comparison with the control group. Statistical evaluation of the influence of cyanobacterial water blooms on biochemical indices of the juvenile carp showed a distinct decrease in albumin, alanine aminotransferase, total bilirubin, calcium, cholesterol, glucose, phosphorus and iron when compared to controls. Values of red blood counts [haemoglobin, haematocrit (PCV), mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration] and lactate were significantly increased compared to controls. After exposure to cyanobacterial water bloom, the carp were kept in clean water to monitor the persistence of biochemical indices. The influence of cyanobacterial populations on calcium, cholesterol, glucose, lactate, phosphorus and PCV persisted up to 28 days after conclusion of the experiment. Duration of exposure, toxicity and density of cyanobacterial water blooms had an important impact on individual haematological indices., (© 2010 Blackwell Publishing Ltd.)

Laribacter hongkongensis is a recently discovered bacterium associated with gastroenteritis. In this study, a total of 199 isolates of this species obtained from aquatic products (n=462) in Guangzhou City, China, were examined for their susceptibility to 19 antimicrobial agents and the presence of antimicrobial resistance integrons. The genetic relatedness of the isolates with integrons was also evaluated. A PCR-based method was used to screen integrons and found that 13 (6.5%) of the isolates harbored class 1 integrons. The antimicrobial resistance rates of integron-positive isolates were significantly higher than integron-negative ones for cefepime, ciprofloxacin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole and rifampicin. Genetic sequence analysis revealed that these integrons contained various antimicrobial-resistance genes (dfrA1, dfrA14, dfrA17, dfrA32, aadA1, aadA2, aadA5, cmlA5, arr2, ereA and orfC) organized into different gene cassettes arrangements including a novel array of dfrA14-arr2-cmlA5. Pulsed-field gel electrophoresis (PFGE) yielded 13 different patterns among 13 integron-positive isolates, which could be grouped into four clusters. These indicate the dispersal of multi-resistant integrons among different molecular types. To the best of our knowledge, this is the first report showing distribution and characterization of class 1 integrons among L. hongkongensis isolates., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We measured the adhesion of candidate probiotic lactic acid bacteria (LAB) to carp intestinal mucus. The percentage of adherent bacteria varied among strains. Four strains, two with high adhesion and two with low adhesion in vitro, were tested for in vivo colonization ability. Carp were fed LAB-containing feed for 12 d, and then unsupplemented feed until day 33, and the numbers and compositions of intestinal LAB were analyzed during the entire period. LAB with lower in vitro adhesion disappeared quickly from the intestine after LAB feeding stopped. LAB with higher in vitro adhesion remained in the intestine 3 weeks after LAB feeding stopped, indicating a strong correlation between mucus adhesion in vitro and colonization ability in vivo. Next we isolated nine candidate probiotic LAB with high in vitro mucus-binding ability. Three of them were fed to carp, and all three were stably maintained in the intestine.

Calcium phosphate nanoparticles as an antigen/protein delivery was explored in a fish model Labeo rohita H. S-layer protein (of Aeromonas hydrophila) adsorbed on nano sized calcium phosphate particles elicited both innate and adaptive immune parameters which persisted up to 63 days of post immunization through parenteral immunization and gave cross protections., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A bacterial strain, designated DR-834 and producing immunostimulatory activities to carp (Cyprinus carpio), was isolated from Qinghai-Tibetan Plateau permafrost soil. Cultural characteristic studies suggested that this strain belongs to the genus Bacillus. The nucleotide sequence of the 16S rRNA gene of strain DR-834 exhibited close similarity (99%) with the 16S rRNA gene of Bacillus simplex. Two compounds showing potent activity were isolated from secondary metabolites of the strain through bioassay-guided isolation techniques and identified by spectral data (infrared, nuclear magnetic resonance and mass spectrometry) as: (1) 4-trans-hydroxy-l-proline and (2) cyclo-(l-Pro-Gly)(2). They were found to be significantly increased the selected innate immune function parameters, serum SOD activity, serum lysozyme activity, serum bactericidal activity, superoxide anion production and phagocytic activity by isolated blood leucocytes. The effects of two compounds on immune-related genes expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 1beta (IL-1beta) and inducible nitric oxide synthase (iNOS) mRNA in the blood have been augmented by 4-trans-hydroxy-l-proline and cyclo-(l-Pro-Gly)(2). Compounds 1 and 2 administration the challenge with live Aeromonas hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in relative percent survival (RPS) values. Compound 2 produced the highest protection with the RPS values of 87.50, 77.78, 55.56 and 55.56 after 1, 2, 3 and 4 weeks, respectively. The study indicates that the isolated compounds could be positively influence the immune response and protect the heath status of carp against A. hydrophila infection., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

Expression of four reference genes of grass carp, including beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1alpha), was studied in tissues of normal individuals and bacteria-infected individuals. EF1alpha had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1alpha in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1alpha is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp.

Aeromonas hydrophila is a Gram-negative bacteria associated with many disease conditions of aquatic animals. Two rough attenuated variants were derived from two smooth virulent types of this bacterium by continuous passage separately in laboratory over a period of 8 years in brain-heart infusion agar. These variants were checked to be rough lipopolysaccharides (LPS) types and attenuated since they neither produced any disease nor mortality in the injected fishes. The immunogenic potential of these variants remained unaltered as compared to parent smooth types and fish immunized with these variants resisted the infection and mortality upon cross challenge. Because of their intact immunogenic potential and loss of virulence, they may be used as candidates for fish immunization., ((c) 2010 Elsevier Ltd. All rights reserved.)

The present study was performed to investigate the effect of dietary conjugated linoleic acid (CLA) on the growth performance, immune parameters and disease resistance to Aeromonas hydrophila of juvenile Jian carp. A total of 900 juvenile Jian carp (10.71 +/- 0.02 g) were randomly allocated into six groups of each three replicates, feeding isonitrogenous (35% crude protein) and isolipidic (7.8% crude fat) diets containing graded amounts of CLA (0, 0.5, 0.8, 1.4, 1.7, and 2.0%) for 60 days. After a 60-day growth test, a challenge trial was conducted by intraperitoneal injection of Aeromonas hydrophila to determine the effect of CLA on disease resistance of juvenile Jian carp. The results showed that diets incorporated with a proportion of up to 2.0% CLA without effect on weight gain, feed intake, and feed conversion ratio, but improved immune parameters and disease resistance of juvenile Jian carp. The dietary CLA level of optimal disease resistance to Aeromonas hydrophila was estimated to be 1.7% in juvenile Jian carp using broken-line analysis.

The aim of this study was to evaluate antimicrobial susceptibility of Aeromonas spp. isolates from common carp and koi carp coming from randomly chosen farms. The isolates were tested for susceptibility to 8 antimicrobial agents using the standard agar dilution susceptibility test. In all isolates, PCR was used to detect the presence of tet(A-E) genes, integrase genes, and gene cassettes. From the total 72 isolates of motile aeromonads sampled from koi carp, 36 isolates (50%) were resistant to oxytetracycline, 18 (25%) to ciprofloxacin, 5 (7%) to chloramphenicol, 5 (7%) to florfenicol, and 11 (15%) to trimethoprim. Among 49 isolates of motile aeromonads collected from common carp, 20 (41%) were resistant to oxytetracycline, 3 (6%) to chloramphenicol, and 3 (6%) to florfenicol. The resistance of aeromonads isolated from koi carp was significantly higher to ciprofloxacin (P=0.00024). The presence of class 1 integrons was detected in these isolates only (P=0.00024). Tet genes were detected in 40% (48/121) of isolates, with tet(E) being the most dominant. Our results demonstrated a significant difference in the incidence of resistant isolates collected from koi carp and common carp (P=0.00042). This difference can be ascribed to a distinct antibiotic policy established on consumer fish farms versus ornamental fish farms. The potential risk for resistant bacteria to spread and transmit infection to humans should be considered in cases of technological crossover between the two types of fish farms., (Copyright 2009 Elsevier B.V. All rights reserved.)

A recombinant protein for the S-layer protein of Aeromonas hydrophila was produced and its ability to protect common carp Cyprinus carpio L. against six virulent isolates of A. hydrophila was assessed. A group of 120 carp (30-40 g) were vaccinated intra-peritoneally with 0.1 ml of adjuvanted vaccine (30 microg protein per fish). Another group of 120 carp were injected with 0.1 ml of PBS-adjuvant mixture to serve as controls. Twenty fish from each group were challenged with each one of six virulent isolates of A. hydrophila 35 days post-vaccination. The fish were maintained in 12 separate tanks before terminating the experiment at 16 days post-challenge. The relative percentage survival (RPS) for the six isolates of A. hydrophila ranged from 56 to 87%. The difference in survival rate of fish challenged with four of the isolates was statistically significant in vaccinated fish compared to control fish, when analysed using a Chi-square test. The results of the study suggest that the recombinant S-layer protein of A. hydrophila could be useful as a vaccine antigen to protect fish against different isolates of this pathogenic bacterium., ((c) 2010 Elsevier Ltd. All rights reserved.)

Edwardsiella tarda is an important Gram-negative bacterium that causes systemic infections in a wide range of hosts including fish. The pathogenic mechanisms in this disease are still poorly understood in fish. Indian major carp, Labeo rohita were intraperitoneally challenged with a pathogenic isolate of E. tarda to measure sequential changes in immunity level. A significant decrease in the superoxide production, myeloperoxidase, alternative complement activity, total protein levels and antiprotease activity of serum was marked in the infected fish. However, the serum lysozyme activity and haemagglutination titre were raised in the infected fish. Similarly, a significant rise in specific antibody titre was noticed on and after 10 days post-challenge. This study also elucidates the changes in the relative expression of some immune-related genes viz., interleukin 1-beta (IL-1beta), inducible nitric oxide synthase (iNOS), complement component C3, beta(2)-microglobulin, CXCa, tumor necrosis factor-alpha (TNFalpha), and C-type and G-type lysozymes during the infection. Significant up-regulation of IL-1beta, iNOS, C3, CXCa and expression of both types of lysozyme genes was noticed at 6-12 h post-challenge (h.p.c.) whereas down-regulation of beta(2)-microglobulin and TNFalpha genes was observed after 48 h p.c. The results obtained here strengthen the understanding on molecular pathogenesis of edwardsiellosis in L. rohita., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Background: To improve the quality of processed grass carp (Ctenopharyngodon idellus) products and control the accumulation of hazardous substances therein, minced grass carp slices were salted for 6 h at room temperature and then inoculated with mixed starter cultures of Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen and Monascus anka and fermented for 12 h at 30 degrees C. The changes in some characteristics and biogenic amine contents of the fermented muscles were investigated., Results: During the 12 h fermentation at 30 degrees C, muscles inoculated with mixed starter cultures showed a rapid decrease in pH from 6.0 to 5.1 and suppression of the growth of enterobacteria and pseudomonads. The fermented muscles exhibited better colour, appearance, flavour and overall acceptability than the control (P < 0.05). The changes in non-protein nitrogen and free amino acid contents of the fermented muscles and in their sodium dodecyl sulfate polyacrylamide gel electrophoresis profiles indicated that severe hydrolysis of muscle proteins occurred during fermentation. The accumulation of biogenic amines in the muscles was efficiently reduced by fermentation with mixed starter cultures., Conclusion: Fermentation with mixed starter cultures of L. casei, S. lactis, S. cerevisiae Hansen and M. anka significantly improved the characteristics of grass carp muscles and controlled the accumulation of biogenic amines.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon-tagged A. hydrophila strains using mini Tn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10(-3) and 10(-6) and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 x 10(4) CFU mL(-1). Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.

Transferrin partial complementary DNAs were cloned from the livers of five species in four genera of Indian carps (Indian major carp species: Labeo rohita, Catla catla and Cirrhinus mrigala; medium carp: Puntius sarana; minor carp: Labeo bata) subsequent to polymerase chain reaction amplification with published heterologous primers or self-designed primers derived from conserved regions of transferrin cDNA sequences. The partial transferrin cDNAs of the five species of carps had sizes from 624 to 633bp (487bp for L. rohita) and encoded an open reading frame consisting of 206-211 (162 for L. rohita) amino acids. The alignments of carp cDNA sequences showed 85-97% homology and 71-93% homology in deduced amino acid sequences. A phylogenetic tree of amino acid sequences of transferrin cDNAs from carps showed that the relationship among the four genera of Indian carps is well correlated with that derived from classic morphologic analyses. The hypothesized cleavage site and interdomain bridge of transferrin molecule were predicted for the above carp species and interestingly the cleavage site amino acid sequence was found to be conserved among all the carps. To study the tissue-specific expression of the transferrin gene, various tissues (liver, kidney, spleen, brain, muscle, testis, heart, intestine, gill and fin) from apparently healthy (control), moribund and survived C. mrigala experimentally infected with Aeromonas hydrophila infection were analyzed. Transferrin mRNA was detected only in liver RNA and to lesser extent in brain tissue out of the 10 tissues analyzed irrespective of bacterial infection.

Screening of potential probiotic LAB for aquaculture from adult common carp intestine was performed seasonally. Lactococcus lactis h2 and Lactococcus raffinolactis h47, which show cholic acid resistance and strong antibacterial activity against fish pathogens, were selected from predominant LAB in summer and winter respectively. Enterococcus pseudoavium h50, with the strongest antimicrobial activity among the strains isolated through 1 year, was also selected. Streptococcus iniae I1, with strong antimicrobial activity, was selected from predominant LAB in young common carp intestine. Direct screening of LAB with cholic acid resistance was also carried out seasonally. The antibacterial activity of the isolates was tested, and Lactobacillus fuchuensis K11 was selected from the summer isolates. In addition, five candidate strains were selected from the winter samples. The candidates' levels of cholic acid resistance and antibacterial activity were better than or at the least matched those of their corresponding type strains. All the candidates grew over a wide range of temperatures.

Phytate is the most abundant organic phosphorus compound in nature, and microbial mineralization of phytate by phytase is a key process for phosphorus recycling in the biosphere. In the present study, beta-propeller phytase (BPP) gene fragments were readily amplified from the intestinal contents of grass carp (Ctenopharyngodon idellus) directly or from phytate-degrading isolates from the same source, confirming the widespread occurrence of BPP in aquatic communities. The amounts of sequences collected using these two methods differed (88 distinct genes versus 10 isolates), but the sequences showed the same general topology based on phylogenetic analysis. All of the sequences fell in five clusters and were distinct from those of Anabaena, Gloeobacter, Streptomyces, Flavobacterium, Prosthecochloris, and Desulfuromonas, which have never been found in the grass carp intestine. Analysis of the microbial diversity by denaturing gradient gel electrophoresis demonstrated that unculturable bacteria were dominant bacteria in the grass carp intestine and thus the predominant phytate-degrading organisms. The predominant cultured species corresponding to the phytate-degrading isolates, Pseudomonas, Bacillus and Shewanella species, might be the main source of known BPPs. A phytase from Brevundimonas was first obtained from cultured species. Combining our results with Lim et al.'s inference that phytate-mineralizing bacteria are widely distributed and highly diverse in nature (B. L. Lim, P. Yeung, C. Cheng, and J. E. Hill, ISME J. 1:321-330, 2007), we concluded that BPP is the major phytate-degrading enzyme in nature, that most of this enzyme might originate from unculturable bacteria, and that the distribution of BPP may be related to the type of niche. To our knowledge, this is the first study to experimentally estimate BPP diversity in situ.

Aims: In the present study, we focused on one of the Aeromonas veronii isolates that exhibited marked adhesion onto carp intestine and studied its membrane-associated proteins for their possible involvement in mucosal adhesion., Methods and Results: We isolated a strain of Aer. veronii (CWP11) that exhibited a high degree of temperature-dependent adhesion activity onto carp intestinal tract and studied its adhesion factor. A proteomic analysis of the membrane-associated fraction showed the presence of multiple proteins that were specifically expressed in CWP11 cells cultured at 25 degrees C. Of these, a 30 kDa protein was identified to be OmpA by a mass fingerprint analysis. Cloning and nucleotide sequencing of the ompA region of CWP11 revealed the presence of two tandem ompA homologues (ompAI-ompAII). Escherichia coli that expressed either OmpAI or OmpAII exhibited marked adhesion onto carp intestinal surface. Disruption of ompAI by a homologous recombination technique resulted in marked reduction of the adhesion activity in CWP11., Conclusion: The OmpA homologue plays an important role in the adhesion of the Aer. veronii strain onto the surface of intestinal tract., Significance and Impact of the Study: We successfully identified an OmpA homologue to be an adhesion factor of Aer. veronii, an optimistic pathogen that habituates in carp intestinal tract.

Four hundred and twenty intestinal content samples (not including intestinal tissues) of freshwater fishes (60 silver carps, 100 carps, 100 crucian carps, 60 catfishes and 100 zaieuws) caught from one water reservoir were examined bacteriologically for the occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. C. perfringens could be isolated in 75 intestinal contents samples (17.9%) from freshwater fish including: 13 silver carps, 2 carps, 12 crucian carps, 40 zaieuws, and 8 catfishes. In 75 isolates, 58 strains (77.3%) were C. perfringens toxin type C (alpha and beta toxin positive), 13 strains (17.3%) were toxin type A (alpha toxin positive) and 4 strains (5.3%) were toxin type B (alpha, beta and epsilon toxin positive). In addition, the gene encoding for beta2 toxin was found in 47 strains (62.7%) of all the isolates, seven from type A, two from type B, and 38 from type C. The gene encoding for enterotoxin was not found in any isolate. These amplified toxin gene fragment were cloned and sequenced and compared with reference strains, the identity varied from 98.15% to 99.29%. This is the first report of C. perfringens alpha, beta, epsilon, beta2 toxins in freshwater fish and of beta, epsilon toxins in fish in general, and is the first discovery that the beta2 toxin could be detected in strains of type B. The origin of this bacterium and its importance to human food poisoning in freshwater fish is discussed.

The potential of a Bacillus cereus isolate (NRRL 100132) as a biological agent for aquaculture has been demonstrated in vitro and in vivo. The functionality of this isolate across a range of physiological conditions, including salinity, pH and temperature, based on rearing of high-value ornamental Cyprinus carpio, was investigated. Temperature had a significant influence on germination, specific growth rate and increase in cell number of B. cereus in shake-flask cultures, whilst salinity and pH did not have a measurable effect on growth. Controlled studies in bioreactors and modelling of the data to the Arrhenius function indicated the existence of high and low growth temperature domains. The rates of pathogenic Aeromonas hydrophila suppression and decrease in waste ion concentrations (ammonium, nitrite, nitrate and phosphate) were translated into a linear predictive indicator of efficacy of the B. cereus isolate at different temperatures. The present study confirmed the robustness of the B. cereus isolate (NRRL 100132) as a putative biological agent for aquaculture and further demonstrated a novel method for the assessment of in vitro biological efficacy as a function of temperature.