Carnobacterium maltaromaticum vs. Vibrio (Listonella) anguillarum in the midgut of Atlantic cod (Gadus morhua L.): an ex vivo study

The present investigation addresses whether the midgut (MG) of Atlantic cod (Gadus morhua L.) is an infection route for Vibrio (Listonella) anguillarum serotype 02 β and if Carnobacterium maltaromaticum, a probiotic bacterium, can out‐compete the pathogen and modulate the autochthonous MG microbiota. This was investigated by using an ex vivo method the intestinal sac, utilized previously in studies on Atlantic cod and Atlantic salmon (Salmo salar L.). Exposure of the MG to V. (L.) anguillarum did not reveal any cell damage indicating that the MG does not appear to be an infection route for V. (L.) anguillarum in healthy Atlantic cod. This finding together with previous observations on Atlantic cod and Atlantic salmon indicate that intestine as an infection route might vary between these two species. When the MG was exposed to C. maltaromaticum, no cell damage or cellular disruptions were observed. As budding from the apices of microvilli was observed in all treatments exposed to bacteria, we suggest that budding might be involved in the primary barrier against bacterial infection. However, to clarify this hypothesis, further studies are needed. Exposure of the MG to the probiotics and pathogenic bacteria indicated that C. maltaromaticum, to some extent, is able to out‐compete V. (L.) anguillarum but the topic merits further investigation. Analysis of the MG microbiota after sterile saline solution and bacterial exposure indicates that bacteria related to Staphylococcus sciuri belong to the autochthonous gut microbiota in Atlantic cod.     

Lactic acid bacteria in fish: a review

Fish are continuously exposed to a wide range of microorganisms present in the environment, and the microbiota of fish have been the subject of several reviews. This review evaluates lactic acid bacteria in fish, and focuses on the several investigations that have demonstrated that Streptococcus, Leuconostoc, Lactobacillus, and Carnobacterium belong to the normal microbiota of the gastrointestinal tract in healthy fish. However, it is well known that the population level of lactic acid bacteria associated with the digestive tract is affected by nutritional and environmental factors like dietary polyunsaturated fatty acids, chromic oxide, stress and salinity. Pathogenic lactic acid bacteria such as Streptococcus, Enterococcus, Lactobacillus, Carnobacterium and Lactococcus have been detected from ascites, kidney, liver, heart and spleen. Some antibiotic treatments and vaccinations have been proposed to cure or prevent these diseases that seem, however, to spread with the development of fish culture. It has also been reported that some lactic acid bacteria isolated from the gastrointestinal tract of fish can act as probiotics. These candidates are able to colonise the gut, and act antagonistic against Gram-negative fish pathogens. These harmless bacteriocin-producing strains may reduce the need to use antibiotics in future aquaculture.     

水产养殖中使用的抗菌药物及细菌耐药性

目前，抗菌药物在水产养殖动物疾病防治中占有重要地位，然而，在水产养殖中使用抗菌药物也可产生一系列的副作用，例如细菌耐药性的产生和药物在鱼体和环境中的残留，本文就水产药物使用的历史，现状，适应症，副作用，耐药性的现状，来源，产生机制，耐药菌判定的标准，以及该领域的研究现状等问题进行综述与分析，以便为合理使用抗菌药物提供理论依据。     

Prevalence of a fish pathogen, Listonella anguillarum, in the intestinal tract of fish collected off the coast of Japan

This study evaluates the influence of temperature manipulation on circulating levels of sex steroids and gonadal maturation in F1 generation Senegalese sole Solea senegalensis breeders kept under either continuous light (LL) or natural photoperiod (NP). Under LL, progressive water temperature reduction during early winter [treatment MT (modified temperature)] significantly elevated [compared with exposure to LL and constant temperature (CT)] plasma steroid levels in females (January–February) and males (January) and proportion of females initiating maturation (January), suggesting a modulatory role of decreasing temperature in the early phases of sole gonadal recrudescence. Under NP, treatment MT did not stimulate female gonadal maturation despite the significantly higher plasma testosterone levels (February) compared with exposure to NP and CT, which may indicate that females at early‐intermediate phases of ovarian development, the predominant stages of maturation at early winter, were less responsive to the treatment. Conversely, treatment MT significantly enhanced male androgen production from January onwards, together with an increase in proportions of running individuals (March), indicating that males were also responsive to this treatment at mid‐late phases of testicular development. Our data further support the strong influence of water temperature on the overall control of gonadal maturation in Senegalese sole.     

海水鱼类细菌性疾病病原及其检测、疫苗研究概况

概述了海水鱼类常见细菌性疾病包括弧菌病、冬季溃疡病、巴斯德氏菌病、疖病、海水屈挠杆菌病、假单胞菌病、链球菌病、细菌性肾病的病原研究及其检测、疫苗研究概况,为及时了解当前细菌性疾病的研究进展及发展趋势以采取更有效的措施防治疾病提供参考依据。     

剑尾鱼在检测细菌毒力方面的应用

用１８株从鱼类所分离的细菌攻毒剑尾鱼,结果显示细菌对剑尾鱼的毒力与回归感染或攻毒敏感鱼的毒力结果较为一致,另外,用剑尾鱼感染病原菌的适宜途径为背肌注射,与原代比较,近交高代剑尾鱼个体之间对病原菌的反应较为一致,感染后死亡时间相对集中。水生实验动物剑尾鱼在检测鱼类细菌的毒力方面有较好的应用前景。     

In vitro antibacterial effect of berberine hydrochloride and enrofloxacin to fish pathogenic bacteria

Berberine the main antibacterial substance in the rhizome of Coptis chinensis Franch, is used as an antimicrobial agent for a long time. This study was carried out to investigate the potential of berberine alone or in combination with enrofloxacin against six common fish pathogens for use in fish disease management in aquaculture. The minimum inhibitory concentrations (MICs) of berberine hydrochloride against Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio vulnificus, Edwardsiella ictaluri, Escherichia coli and Streptococcus agalactiae were determined and to be >500, >500, >500, 300, 400 and 100 μg mL^?1 respectively. The minimal bactericidal concentrations (MBCs) of berberine hydrochloride to E. coli, E. ictaluri and S. agalactiae were 300–500 μg mL^?1. In combination with enrofloxacin, the MICs and MBCs of berberine hydrochloride significantly decreased against E. coli and E. ictaluri but not against Streptococcus dysgalactiae. These results demonstrated that the berberine hydrochloride enhanced the bactericidal effect of enrofloxacin and vice versa. The synergistic bactericidal effect of berberine hydrochloride and enrofloxacin suggest its potential use in fish disease management in aquaculture. This is the first study on the inhibitory effect of berberine hydrochloride against fish pathogenic bacteria.     

Enzyme‐producing bacteria isolated from fish gut: a review

Digestion of food depends on three main factors: (i) the ingested food and the extent to which the food is susceptible to the effects of digestive enzymes, (ii) the activity of the digestive enzymes and (iii) the length of time the food is exposed to the action of the digestive enzymes. Each of these factors is affected by a multitude of secondary factors. The present review highlights the experimental results on the secondary factor, enzymatic activity and possible contribution of the fish gut microbiota in nutrition. It has been suggested that fish gut microbiota might have positive effects to the digestive processes of fish, and these studies have isolated and identified the enzyme‐producing microbiota. In addition to Bacillus genera, Enterobacteriaceae and Acinetobacter, Aeromonas, Flavobacterium, Photobacterium, Pseudomonas, Vibrio, Microbacterium, Micrococcus, Staphylococcus, unidentified anaerobes and yeast are also suggested to be possible contributors. However, in contrast to endothermic animals, it is difficult to conclude the exact contribution of the gastrointestinal microbiota because of the complexity and variable ecology of the digestive tract of different fish species, the presence of stomach and pyloric caeca and the relative intestinal length. The present review will critically evaluate the results to establish whether or not intestinal microbiota do contribute to fish nutrition.     

Role of gastrointestinal microbiota in fish

The gastrointestinal (GI) tract of an animal consists of a very complex and dynamic microbial ecosystem that is very important from a nutritional, physiological and pathological point of view. A wide range of microbes derived from the surrounding aquatic environment, soil/sediment and feed are found to colonize in the GI tract of fish. Among the microbial groups, bacteria (aerobic, facultative anaerobic and obligate aneraobic forms) are the principal colonizers in the GI tract of fish, and in some fish, yeasts are also reported. The common bacterial colonizers in the GI tract of freshwater and marine fish include Vibrio, Aeromonas, Flavobacterium, Plesiomonas, Pseudomonas, Enterobacteriaceae, Micrococcus, Acinetobacter, Clostridium, Fusarium and Bacteroides, which may vary from species to species as well as environmental conditions. Besides, several unknown bacteria belonging to Mycoplasma, Arthrobacter, Brochothrix, Jeotgailbacillus, Ochrobactrum, Psychrobacter and Sejongia species in the GI tract of different fish species have now been identified successfully using culture‐independent techniques. Gnotobiotic and conventional studies indicate the involvement of GI microbiota in fish nutrition, epithelial development, immunity as well as disease outbreak. This review also highlights the need for manipulating the gut microbiota with useful beneficial microbes through probiotic, prebiotic and synbiotic concepts for better fish health management.     

Phytase-producing bacteria in the digestive tracts of some freshwater fish

Isolation and enumeration of phytase‐producing bacterial flora in the foregut and hindgut regions of the gastrointestinal tracts of 10 culturable freshwater teleosts of different feeding habits, namely rohu (Labeo rohita), catla (Catla catla), mrigal (Cirrhinus mrigala), bata (Labeo bata), kalbasu (Labeo calbasu), Nile tilapia (Oreochromis niloticus), climbing perch (Anabas testudineus), common carp (Cyprinus carpio), silver carp (Hypophthalmichthys molitrix) and grass carp (Ctenopharyngodon idella), have been carried out. Microbial culture of the gut mucosa on selected nutrient media following the enrichment culture technique was performed for bacterial isolation. The bacterial isolates were screened on the basis of their enzyme‐producing ability. The bacterial population on the tryptone soya agar (TSA) plate was maximum in the hindgut region of bata, followed by mrigal and minimum in the foregut region of Nile tilapia. In modified phytase screening medium (MPSM), phytase‐producing strains were recorded at higher densities in the foregut region of mrigal and grass carp and minimum in the foregut region of bata. In case of the hindgut, maximum phytase‐producing strains were present in grass carp and mrigal and minimum in rohu. In general, in MPSM, the bacterial population was lower in the hindgut region of all the 10 species of fish examined. The phytase‐producing ability of the selected 31 strains (16 from the foregut and 15 from the hindgut region) was determined by clearing zones on phytate‐containing plates. Among these isolates, 22 strains (12 from the foregut and 10 from the hindgut region) were selected as potent phytase producers according to a quantitative enzyme assay. The highest phytase activity was observed in the bacterial strains LF1 and LH1 isolated from the fore and the hindgut regions of rohu respectively. Both the strains were identified as Bacillus licheniformis on the basis of phenotypic characteristics as well as 16S rDNA sequence analysis.     

益生菌对幼鱼生长发育的影响及调控机制

近年来,高密度、集约化的水产养殖模式导致养殖鱼类疾病频发,其中幼鱼发病率和死亡率的上升严重影响水产养殖业的健康快速发展。如何保障幼鱼的健康是当前水产养殖业亟需解决的问题。益生菌能够通过多种方式提高幼鱼的抗病力、增强幼鱼对营养素的摄取、促进幼鱼的生长发育。已有大量的研究阐述了益生菌在幼鱼生长发育及免疫调节中的作用,但相关机制还需深入探究。本文综述了近年来益生菌在幼鱼生长发育及抗病方面发挥的有益作用及其可能的机制,讨论了益生菌在幼鱼养殖应用时可能存在的问题以及发展方向,为益生菌在水产动物幼体养殖中的应用提供了理论参考。     

Antimicrobial activity in bacteria isolated from Senegalese sole, Solea senegalensis, fed with natural prey

Intestinal bacteria in marine fish may produce antimicrobial substances which inhibit pathogenic bacteria. The aim of this study was to determine the influence of a change of fish diet on the antimicrobial activity of the culturable aerobic gut microflora of Senegalese sole, Solea senegalensis. Pre‐adult 15‐month‐old fish previously fed on an artificial diet, were fed polychaetes (Hediste diversicolor), which form part of the natural diet of Senegalese sole. Samples were taken0, 3 and 6 weeks after start of the experiment from the stomach, small and large intestine of the fish. The bacterial strains isolated from these samples were sub‐cultured to pure cultures and stored at ?80°C. Several biochemical tests were run to obtain some basic phenotypic characteristics of the isolated strains. Amplification and sequencing of 16S rDNA fragments were used to identify the majority of the bacterial strains isolated. The identification by use of this molecular approach gave results in agreement to the phenotypic characterization. Feeding with polychaetes significantly increased (P<0.05) the numbers of presumptive Vibrio isolates in the gut. The number of bacterial strains with antimicrobial activity, as determined by two in vitro approaches, was significantly (P<0.05) increased by feeding with polychaetes.     

Calcium alginate capsules for oral administration of fish probiotic bacteria: assessment of optimal conditions for encapsulation

The application of probiotics in aquaculture represents a promising alternative for disease prevention. These microorganisms must survive adverse conditions during handling and storage to be administered in high concentrations to the host. This is especially important when stressful conditions, such as low water contents, are present in animal feed. Bacterial encapsulation has been used to maintain probiotic viability in human food; however, the usefulness of the encapsulation techniques in animal feed needs to be evaluated. In the present study, optimal conditions for encapsulation in alginate of a fish probiotic Shewanella putrefaciens strain (Pdp11) have been determined. Different alginate (1%, 2%, 3% and 4% w/v) and calcium chloride (0.5%, 1% 2% and 3% w/v)concentrations were used to elaborate alginate beads. The results obtained indicate that S. putrefaciens Pdp11 can be encapsulated successfully in calcium alginate beads. The percentages of encapsulated cells were above 80%, the lowest efficiency rates corresponding to high calcium availability (3%). In addition, capsules containing viable S. putrefaciens Pdp11 can be stored at 4 °C for at least 1 month, survival rates being above 90%. On the contrary, storage of the capsules at 22 °C resulted in 40% viability loss within 30 days. Finally, the survival of encapsulated probiotics through fish gastrointestinal tract has been demonstrated.     

Screening of gastrointestinal bacteria of Indian major carps for a candidate probiotic species for aquaculture practices

Probotic selection could be detrimental for the success of aquaculture practices. While, in most of the cases emphasis is given to the production of antibacterial compounds, and in vitro inhibitory activities of a bacterium for choosing a probiotic strain, however, other criteria like non‐antibiotic resistant, non‐haemolytic nature and non‐pathogenic potentiality of probiotic strain are often overlooked. We have screened gastrointestinal bacteria isolated from all the three species of Indian major carps viz., Labeo rohita, Catla catla and Cirrhinus mrigala for a suitable probiotic. Bacteria like Aeromonas (Aeromonas species, A. hydrophila), Micrococcus, Corynebacterium, Plesiomonas, Bacillus (Bacillus species, B. subtilis) and Pseudomonas (Pseudomonas species, P. aeruginosa) exhibited antibacterial activities against different pathogens. Among these, B. subtilis was found to grow at a wider range of temperatures, pH and salt concentrations and was non‐haemolytic, non‐antibiotic resistant, non‐pathogenic and inhibited all the tested primary and secondary pathogens. Dietary supplementation of the bacteria at 10⁸ CFU g^?1 feed also enhanced the growth of Indian major carp, L. rohita. Based upon these characteristics, we aimed to develop a suitable probiotic for aquaculture practices.     

Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria

Because of the evolving resistance of microorganisms to existing antibiotics, there is an increasing need for new antibiotics not only in human but also in veterinary medicine. Competition for space and nutrients led to the evolution of antimicrobial defence strategies in the aquatic environment. Therefore, aquatic organisms, e.g., seaweeds, offer a particularly rich source of potential new drugs. The aim of our studies was to identify seaweeds, which possess activities against fish pathogenic bacteria and could be an alternative to the commonly used antibiotics in aquaculture.Dichloromethane, methanole and water extracts of 26 species of cultivated seaweeds were screened for their antibacterial activities against five fish pathogenic bacteria strains (Aeromonas salmonicida ssp. salmonicida, Aeromonas hydrophila ssp. hydrophila, Pseudomonas anguilliseptica, Vibrio anguillarum, Yersinia ruckeri). The dichloromethane extracts of Asparagopsis armata, Ceramium rubrum, Drachiella minuta, Falkenbergia rufolanosa, Gracilaria cornea and Halopitys incurvus showed strong antibacterial activities. V. anguillarum and P. anguilliseptica were the two most susceptible bacteria strains. The screening results confirm the possible use of seaweeds as a source of antimicrobial compounds or as a health-promoting food for aquaculture.     

A historical review of the key bacterial and viral pathogens of Scottish wild fish

Thousands of Scottish wild fish were screened for pathogens by Marine Scotland Science. A systematic review of published and unpublished data on six key pathogens (Renibacterium salmoninarum, Aeromonas salmonicida, IPNV, ISAV, SAV and VHSV) found in Scottish wild and farmed fish was undertaken. Despite many reported cases in farmed fish, there was a limited number of positive samples from Scottish wild fish, however, there was evidence for interactions between wild and farmed fish. A slightly elevated IPNV prevalence was reported in wild marine fish caught close to Atlantic salmon, Salmo salar L., farms that had undergone clinical IPN. Salmonid alphavirus was isolated from wild marine fish caught near Atlantic salmon farms with a SAV infection history. Isolations of VHSV were made from cleaner wrasse (Labridae) used on Scottish Atlantic salmon farms and VHSV was detected in local wild marine fish. However, these pathogens have been detected in wild marine fish caught remotely from aquaculture sites. These data suggest that despite the large number of samples taken, there is limited evidence for clinical disease in wild fish due to these pathogens (although BKD and furunculosis historically occurred) and they are likely to have had a minimal impact on Scottish wild fish.     

Bacteriocins and their applications for the treatment of bacterial diseases in aquaculture: a review

Aquaculture is one of the fastest growing food producing sectors in the world. The increase in the world population requires faster growth in aquaculture for better food availability and to overcome malnutrition. But this worldwide growth of aquaculture is overwhelmed by catastrophic fish diseases and spoilage during cultivation and preservation problems caused by pathogenic bacteria. Various remedies are available for food preservation and also from the bacterial diseases in the fish production. Due to the emergence of antibiotic resistance and adverse effects, an alternative to antibiotic is the need of the hour. The proteins such as bacteriocins, i.e. ribosomally synthesized antimicrobial peptides (AMPs) and possess antagonistic against closely related and other bacteria. These proteins are produced by most lineages of bacteria which are playing key roles in recognition and possess a cognate immunity system for self‐protection as well as host protection from infections. These proteins are potent immunomodulators with broad spectrum inhibition properties which are further used as novel therapeutic agents. In this review, we have tried to summarize the bacteriocins on the basis of their classifications, structural and functional attributes, mode of actions, bacteriocins isolated from fish and gut microbiota and presence of beneficiary bacteria in the fish gut. Further, this study highlighted where further research is a prerequisite to increase our basic understanding and search for novel bacteriocins to elucidate the proteins/peptides having antimicrobial properties for disease control in aquaculture as an alternative to antibiotics.     

Bioassay‐guided isolation and identification of active compounds from Macleaya microcarpa (Maxim) Fedde against fish pathogenic bacteria

Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L^?1 for A. hydrophila and 50 mg L^?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L^?1 for A. hydrophila, 100 mg L^?1 for V. harveyi, and 125 mg L^?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L^?1 for A. hydrophila and 200 mg L^?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.