Enzymes from the gut bacteria of Atlantic cod,Gadus morhua and their influence on intestinal enzyme activity

Gut‐associated bacteria of fish are known to produce enzymes which aid in digestion. The presence and activities of these bacteria in cold‐water fishes like Atlantic cod are less known. Therefore, we have characterized the activities of extracellular enzymes of GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.), two bacteria isolated from the gastrointestinal tract of Atlantic cod. Additionally, we examined if these bacteria when delivered through feeds could influence the activity of selected intestinal enzymes. GP21 was able to produce amylase, chitinase, cellulase and protease, whereas GP12 could produce only chitinase and protease. These enzymes were produced extracellularly and they were found to be catalytically active at acidic conditions (pH 2–5) and at temperatures ranging from 15 to 30 °C. Orally delivered bacteria could possibly influence the activity of intestinal enzymes after 40 days, rather than after 20 days of feeding. Thus GP21 and GP12, the potential probiotic organisms, could support digestion in Atlantic cod.     

Responses of Atlantic cod <Emphasis Type="Italic">Gadus morhua</Emphasis> head kidney leukocytes to phytase produced by gastrointestinal-derived bacteria

This study identified phytase-producing bacteria that were previously isolated from the gastrointestinal tract of Atlantic cod, Gadus morhua and determined its effect on head kidney leukocytes. Out of the 216 bacterial strains tested, the two phytase producers were identified as Pseudomonas sp. and Psychrobacter sp. based on their 16S rDNA sequence. Crude phytase from these two bacterial strains was produced employing the shake flask method. Even though the total protein of the crude phytase was not significantly different for the two bacteria, the phytase activity of the crude enzyme produced by Pseudomonas sp. (97.1 ± 16.7 U) was significantly higher than that of the enzyme from Psychrobacter sp. (75.9 ± 2.4 U). When cod head kidney leukocytes were incubated with the crude phytase (50 μg ml⁻¹), it resulted in enhanced cell proliferation, higher myeloperoxidase, and acid phosphatase activities. Extracellular responses—respiratory burst activity and hydrogen peroxide production were not enhanced by the crude enzyme. As a consequence, the growth of two pathogenic bacteria Aeromonas salmonicida and Vibrio anguillarum was not suppressed by the supernatants obtained from head kidney leukocytes incubated with the crude bacterial phytase. Thus, the enzyme from phytase-producing intestinal bacteria of Atlantic cod can stimulate intracellular head kidney leukocyte activities but not the production of extracellular substances that are involved in antibacterial response. These have implications on the potential use of bacterial phytase as feed supplement to boost cellular immune response of the fish and could be employed as a health management strategy in culture systems.     

Identification of bacteria from the normal flora of perch, Perca fluviatilis L., and evaluation of their inhibitory potential towards Aeromonas species

Pathogenic Aeromonas sobria has been identified as a causative agent of ulcerative disease in farmed European perch, Perca fluviatilis L. To study the effect of the normal intestinal bacterial flora of perch against A. sobria, we sampled 193 bacterial isolates from the perch digestive tract. The isolates were identified by sequence analysis of the 16S rRNA gene and their inhibitory potential against A. sobria was evaluated in vitro. Nineteen of the strains isolated showed inhibition and were also tested against other aeromonad and non-aeromonad fish pathogens including Yersinia ruckeri and Vibrio anguillarum. Isolates showing inhibition were primarily Pseudomonas spp.; however, inhibitory Shewanella spp., and Delftia sp. were also identified. A Pseudomonas chlororaphis isolate showed inhibition against all fish pathogens tested.     

饲料添加益生菌对多病原阳性的凡纳滨对虾生长与存活的影响

采用假交替单胞菌(Pseudoalteromonas sp.) KL-3 2010和微小杆菌(Exiguobacterium sp.) KL-C2 2014作为益生菌，进行凡纳滨对虾(Litopenaeus vannamei)投喂实验，研究上述菌株对带毒对虾的生长与存活的影响。假交替单胞菌KL-3 2010对致急性肝胰腺坏死副溶血弧菌(Vibrio parahemolyticus) (VPAHPND 20130629002S01)有拮抗作用和胞外蛋白酶活性，微小杆菌KL-C2 2014有胞外蛋白酶活性。待试对虾经检测为白斑综合征病毒(WSSV)、致急性肝胰腺坏死病副溶血弧菌(VPAHPND)和虾肝肠胞虫(EHP)弱阳性。经过为期60 d的养殖实验，结果显示，与投喂普通颗粒饲料的对照组相比，投喂假交替单胞菌KL-3 2010的对虾存活率提高了213%±43% (P<0.01)；投喂微小杆菌KL-C2 2014的对虾平均生长率提高了105.5%±28.1% (P<0.05)；交替投喂2株菌的对虾存活率提高了184%±52% (P<0.05)，平均生长率提高了70.6%±32.8%。肠道可培养优势菌研究表明，2株益生菌的投喂显著影响了对虾肠道优势菌群的种类。本研究为带毒虾苗的养殖提供一种有效的病害防控和促生长的手段。     

Selection of lactic acid bacteria as candidate probiotics and in vivo test on Artemia nauplii

Lactic acid bacteria (LAB) were isolated from rotifer cultures in a marine hatchery to search for potential probiotics for marine animals. Fifteen strains were first selected among a total of 55, according to antibacterial activity against Vibrio sp. Among eight strains identified as Lactobacillus casei, four were highly adhesive, suggesting some ability for surface colonization. The other strains were identified as Lactobacillus plantarum, Lactobacillus dextrinicus, and Leuconostoc sp. To validate probiotic potential, Artemia were challenged against pathogenic Vibrio alginolyticus, with or without one of six selected LAB strains. The six strains protected Artemia against the pathogen, to some extent on condition that nutrient enrichment was provided. La. casei BR51 and X2 were preferred, as they were efficient even in the absence of nutrient supply. La. casei X2 was finally selected as candidate probiotic, due to the best growth performances of Artemia, with or without the pathogen.     

Probiotic characteristics and aflatoxin B1 binding ability of Debaryomyces hansenii and Kazaschtania exigua from rainbow trout environment

The aim of this study was to evaluate probiotic properties and the aflatoxin B₁ adsorption ability of yeasts isolated from rainbow trout intestine and fish feed to assess their use in the formulation of feed additives. Growth at pH 2, bacterial pathogens inhibition, bacterial pathogens co‐aggregation, autoaggregation, homologous and heterologous inhibition against lactic acid bacteria were evaluated. Moreover, aflatoxin B₁ adsorption was tested. All strains were able to maintain viable (10⁷ cells/ml) at pH 2. All strains isolated from intestine were identified as Kazaschtania exigua, while strains isolated from feed were all identified as Debaryomyces hansenii. Kazaschtania exigua RC035 and RC037 showed the strongest antimicrobial activity while K. exigua RC037 and RC038 were the most efficient co‐aggregating bacterial pathogens. All strains exhibited strong autoaggregation. None of the tested yeast strains showed homologous inhibition towards other yeasts and heterologous inhibition towards lactic acid bacteria strains. Debaryomyces hansenii RC031 demonstrated aflatoxin B₁ adsorption capacity (21%). The results of the present study indicate that select strains of Kazaschtania exigua and D. hansenii showed potential to improve the health of rainbow trout by inhibiting pathogens and binding AFB₁ and their use as probiotics may improve the production of rainbow trout in aquaculture systems.     

鲫肠道乳酸菌的分离及益生特性

为获得鱼源益生乳酸菌,本研究从健康鲫肠道内分离鉴定得到38株乳酸菌,并选择其中8株乳酸菌进行体外益生特性分析。结果显示,8株乳酸菌均能在p H=4.5和10%鲫胆汁环境中存活,对嗜水气单胞菌、豚鼠气单胞菌、无乳链球菌和副溶血弧菌均有较强的抑菌能力,但菌体表面疏水性和自凝集能力具有菌株特异性。选择5株疏水性较高(63%~89%)、自凝集能力强(37%~45%)的乳酸乳球菌进行体外黏附能力测定,结果显示,黏附能力在菌株间具有差异性(4.5%~7.9%),但均能显著降低嗜水气单胞菌NJ-35的体外黏附率,黏附抑制率达30%~35%;最后对筛选出的3株高黏附力的乳酸乳球菌进行安全性评价,发现3株菌对鲫均无致病力。鱼源乳酸菌的筛选,为鲫养殖生产中益生菌的实际应用提供了理论依据。     

Interactions of microorganisms isolated from gilthead sea bream, Sparus aurata L., on Vibrio harveyi, a pathogen of farmed Senegalese sole, Solea senegalensis (Kaup)

Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole.     

Cross-protection after immunization of Atlantic salmon, Salmo salar L., against different strains of Vibrio salmonicida

Abstract. Atlantic salmon were immunized with one or other of two bacterins made from different strains of Vibrio salmonicida. The two strains differed both in geographic origin and plasmid profile. One of the strains had given rise to disease outbreaks in salmon and also in cod which were kept in net-pens. However, by using a virulent challenge, it appeared that salmon, immunized with any of the two strains, were also protected against infection with the other. This result indicates that the two strains are serologically similar.     

Simultaneous detection of pathogens causing francisellosis,furunculosis and vibriosis in Atlantic cod by multiplex polymerase chain reaction

An assay for the simultaneous detection of three major bacterial pathogens of Atlantic cod with multiplex polymerase chain reaction (mPCR) was developed. Primers targeting the flanking regions of genes groEL, gyrB and amiB were standardized to diagnose francisellosis, furunculosis and vibriosis respectively. The detection limit of Francisella piscicida, Aeromonas salmonicida and Vibrio anguillarum in the mPCR assay ranged from 10 ng to 100 pg of bacterial DNA mL^?1, 10 μg to 50 ng of bacterial DNA mL^?1 and 1 μg to 100 ng of bacterial DNA mL^?1 respectively. These primers were found to be specific in the determination of the respective genes in the individual pathogen without cross‐reaction, thus making the assay an efficient tool to detect the presence of these pathogens simultaneously or individually.     

Production of bactericidal substances by a marine vibrio isolated from cultures of the scallop Argopecten purpuratus

A marine vibrio (strain C33) having inhibitory effects on the growth of the pathogen Vibrio anguillarum-VAR was isolated from seawater used in mass culture of the north-Chilean scallop Argopecten purpuratus. This bacterial isolate demonstrated broad inhibitory activity on several bacterial strains, including some pathogenic vibrios. Ethyl acetate extracts of extracellular products of strain C33 were separated by thin layer chromatography (TLC), and three fractions thus obtained were found to have antimicrobial activity when tested using microplate bioassays. One of the fractions (A2) having marked antimicrobial activity, was further purified using TLC and analyzed by IR spectrophotometry and NMR'H and was characterized on a preliminary basis as an aliphatic hydroxyl ether. This compound demonstrated bacteriostatic activity against the important marine pathogens Vibrio parahaemolyticus and V. splendidus, and as discussed in the paper, may be useful in developing natural strategies for the control of pathogens in mass cultures of Argopecten purpuratus and possibly other molluscs affected by these bacteria.     

Unmethylated CpG oligodeoxynucleotides activate head kidney leukocytes of Atlantic cod, <Emphasis Type="Italic">Gadus morhua</Emphasis>

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs are strong inducers of immune response in most mammalian organisms. The use of these synthetic CpG motifs in fish, particularly in salmonids and carp, resulted in the modulation of their immune system. However, much less is known in other species of fish such as gadoids including Atlantic cod, Gadus morhua. Using head kidney (HK) leukocytes of cod in an in vitro study, we determined the effects of some established CpG-ODNs on the cellular responses of the fish immunocytes. Incubation of the HK leukocytes with 2 μM concentration of the CpG-ODNs resulted in enhanced respiratory burst. There were differential effects on the activities of acid phosphatase and cellular myeloperoxidase. Only CpG-ODN 1826 triggered a significant increase in the level of both enzymes. On the other hand, the supernatants derived from the HK leukocytes after incubation with different CpG-ODNs did not possess bactericidal activity against Vibrio anguillarum and Aeromonas salmonicida. This study has shown that CpG-ODNs at low concentrations are able to stimulate respiratory burst in cod but have minimal effects on cellular enzymatic activities and antibacterial action.     

Isolation and screening of probiotic candidates from marron,Cherax cainii (Austin, 2002) gastrointestinal tract (GIT) and commercial probiotic products for the use in marron culture

Six strains of bacteria including Bacillus mycoides (A10) and Shewanella species (A12) isolated from healthy marron intestine, Bacillus species (PM1), Bacillus subtilis (PM3), Bacillus sp. (PM4) and Bacillus sp. (AQ) from commercial probiotic products were investigated for probiotic potential in marron culture. Antibiotic susceptibility tests indicated PM3 and PM4 were susceptible to all nine antibiotics evaluated. A10, A12 and AQ were resistant to class penicillins, whereas PM1 was resistant to class penicillin and macrolides. All strains were non‐pathogenic for marron. Strong inhibition against Vibrio mimicus and Vibrio cholerae non‐01 was exhibited by PM4 and PM3. A10 inhibited V. mimicus at 72 h of growth, but not V. cholerae non‐01, whereas A12 inhibited V. cholerae non‐01 but not V. mimicus, and AQ showed no inhibition activity. A wide range of enzymes were produced by A10 and AQ using the API ZYM test. Protease enzymes were produced by PM3, PM4, AQ and PM1. In order of effectiveness, the following bacteria have probiotic potential: B. subtilis (PM3), Bacillus sp. (PM4) and B. mycoides (A10). Further study is required to determine the bacterium or any combination that gives a multibeneficial effect on marron.     

Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.     

Short-term handling stress affects the humoral immune responses of juvenile Atlantic cod,Gadus morhua

Juveniles of Atlantic cod, Gadus morhua L., weighing 30–50 g were subjected to short-term handling stress by exposing them to air for 3 min and immediately placing in the rearing tank for recovery. Indices for the primary stress response as well as selected immune- and metabolism-related parameters were measured in the plasma before the application of the stressor and at 1-h, 24-h and 7-day post-stress. Plasma cortisol significantly increased at 1-h post-stress then returned to the prestress levels at 24-h post-exposure. Plasma glucose and total antioxidant capacity had two peaks at 1 h and 7 days after handling stress. Alkaline phosphatase significantly increased at 24-h post-exposure, while no significant changes in myeloperoxidase activity was observed. Lysozyme and antiprotease activities were significantly downregulated at 1- and 24-h post-exposure, respectively. These findings showed that short-term handling stress in Atlantic cod juveniles resulted in temporal elevation of the primary stress response and metabolism with a corresponding decrease in its defense against bacterial pathogens.     

Colonization in the fish intestinal tract and production of inhibitory substances in intestinal mucus and faecal extracts by Carnobacterium sp. strain K1

A non-virulent Carnobacterium sp., designated strain K1, isolated from the gastrointestinal tract of Atlantic salmon, Salmo salar L., which produced inhibitory substances against bacterial fish pathogens, was examined in vitro for characteristics important for the colonization of the fish gastrointestinal tract and in vivo for persistence in the tract after oral dosing. In vitro growth experiments showed that the cells of this strain were metabolically active in both the intestinal mucus and faeces from salmonids. The production of growth inhibitors against the two common fish pathogens Vibrio anguillarum and Aeromonas salmonicida by Carnobacterium sp. strain K1 was demonstrated in vitro in mucus and faecal extracts. Furthermore, it was demonstrated that the Carnobacterium cells remained viable in the gastrointestinal tract for several days and that no detrimental effect to the fish was observed as a result of the presence of the bacterium.     

Isolation and inhibitory effect of anti-<Emphasis Type="Italic">Vibrio</Emphasis> substances from <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. A1-J11 isolated from the coastal sea water of Kagoshima Bay

ABSTRACT:   Among several marine antagonistic bacteria isolated from the sea water of Kagoshima Bay, Pseudoalteromonas sp. A1-J11 was found to produce anti- Vibrio substances. The anti- Vibrio substances were extracted from the culture supernatant of the strain with chloroform and isolated using reverse-phase (Cosmosil 75C₁₈-OPN) column chromatography followed by high-pressure liquid chromatography (Mightysil RP-18 GP Aqua). Purified substances, designated as AVS-03 a , c and d showed similar ultraviolet absorption spectra with λ_max at 215, 235, 315 and 327 nm in methanol. AVS-03 d , the major anti- Vibrio substance, was thermostable up to 100°C and pH stable over a pH range higher than 4.0, and also showed strong inhibitory activities, specifically against Vibrio harveyi strains.     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

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.     

Isolation and characterization of Bacillus spp. M001 for potential application in turbot (Scophthalmus maximus L.) against Vibrio anguillarum

The aim of this study was to screen Bacillus strains from the guts of Scophthalmus maximus, Paralichthys olivacues, Epinephelus coioides and Clupanodon punctatus, for use as probiotics in aquaculture. Eight Bacillus strains were screened, and strain M001 was selected for probiotic study based on its antagonistic activity against multiple aquatic bacterial pathogens including Vibrio anguillarum, V. campbellii, V. vulnificus, V. parahamolyticus, Streptococcus sp. and Edwardsiella tarda. M001 was identified as B. amyloliquefaciens based on the biochemical tests and 16S rRNA gene analysis. In vitro experiments revealed that M001 was able to grow at a wider range of temperature, pH and salinity and was capable to use turbot mucus as nutrient for growth. Additionally, M001 isolate greatly inhibited the growth of V. anguillarum by producing antibacterial substances and was acid tolerance, non‐antibiotic resistance and non‐harmful. Thereafter, the potential probiotic effect of M001 was tested in turbot by dietary administration of M001 at a dose of 10⁸ CFU g^?1 diet for 42 days. No significant differences of weight gain, specific growth rate and feed ratio were found in the M001‐diet group of fish compared with control fish, but which increased, respectively, by 5.5%, 4.7% and 7.0% after 42 days of feeding. Several digestive enzyme activities were found to increase significantly in the M001‐diet group, including protease and amylase activities in hepatopancreas, protease activity in intestine and lipase activity in stomach (P < 0.05). Sera superoxide dismutase activity and total protein content (P < 0.05) were also increased significantly (P < 0.05) in the M001‐diet group. The challenge experiment showed that the M001‐diet group of fish showed a relative per cent of survival of 62.7% against V. anguillarim infection. The Bacillus M001 identified from this study has good potential to provide vibriosis control as probiotic feed additive for turbot aquaculture.