Salinity a significant environmental factor for Vibrio harveyi virulence in Fenneropenaeus indicus

The effect of ambient salinity on the haemolymph variables of Fenneropenaeus indicus and its susceptibility to Vibrio harveyi infection under salinity stress has been studied. Adult shrimps were acclimated to 5‰ (hypo osmotic), 25‰ (iso osmotic) and 35‰ (hyper osmotic) salinity levels and the animals were injected with a mid logarithmic culture of V. harveyi at sub lethal level and haemolymph parameters were analysed. Haemolymph proteins, intracellular superoxide anion production, phenoloxidase (PO), alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were found to be at elevated level both at 5‰ and 35‰ post challenge. The haematological responses showed a progressive increase (P < 0.05) up to post challenge day 5 (PCD 5) followed by a considerable decline at all salinities with the lowest being at 35‰. The alterations in the variables were higher in shrimps held at 5‰. However, the V. harveyi infection was severe in animals held at 35‰. The reduction in the parameters could be correlated with the decrease in survival rate of shrimps at 35‰ with a concurrent increase in V. harveyi at this salinity. Multiple regression analysis revealed that ACP (P < 0.001), haemocyte protein HCP (P < 0.001) and PO (P < 0.05) could explain 91% variability in the shrimp survival. These parameters may be used as effective shrimp health indicators. It is evident from the study that ambient salinity alters the haemolymph variables, modulates the virulence in V. harveyi and makes the shrimps more vulnerable to infection at higher salinity. The virulence of V. harveyi is increased at 35‰ salinity as being evidenced from the high mortality at this salinity. The study emphasizes the importance of salinity as an important environmental factor both in terms of host susceptibility and virulence of the pathogen.     

Antibacterial effect of medium-chain fatty acid: caprylic acid on gnotobiotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> nauplii against shrimp pathogens <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">V. parahaemolyticus</Emphasis>

The present study was carried out to determine the antibacterial effect of caprylic acid in the culture system of Artemia franciscana nauplii inoculated with shrimp pathogens Vibrio harveyi and V. parahaemolyticus. To begin with, the antibacterial effect of different concentrations (1, 10 and 100 mM) of caprylic acid against V. harveyi and V. parahaemolyticus was assessed through bacterial growth study. This indicated that at 100 mM concentration, the growth of the pathogens was completely inhibited within 5 h, whereas, in 1.0 and 10 mM concentrations, effective inhibition was observed with the extension of time. Subsequently, the influence of pH variation on the growth inhibitory effect of 10 mM caprylic acid against V. harveyi and V. parahaemolyticus at different pH (5–7) was also made through bacterial growth study. At pH 5, the pathogen growth was very less, compared with pH 6 and 7. The Artemia nauplii (instar II) reared in 10 mM caprylic acid supplemented medium were challenged with V. harveyi and V. parahaemolyticus, and the mortality was recorded at an interval of 6 h up to 60 h. In this study, the mortality of Artemia nauplii reduced to 20.61 and 16.30% in V. parahaemolyticus and V. harveyi challenged groups, respectively. The present results provide evidence for the potential antibacterial activity of caprylic acid in aquaculture against luminescent vibrios.     

Immunostimulating influence of herbal biomedicines on nonspecific immunity in Grouper Epinephelus tauvina juvenile against Vibrio harveyi infection

Herbals such as Cynodon dactylon, Piper longum, Phyllanthus niruri, Tridax procumbens, and Zingiber officinalis were extracted with acetone, benzene, butanol, and petroleum ether and screened against the pathogen Vibrio harveyi isolated from the infected Grouper Epinephalus tauvina. Among the different solvent extractions screening to V. harveyi, petroleum ether extracts were suppressed significantly (P < 0.05). Equal proportions of the all-plant extracts were mixed with the artificial feeds at concentrations of 100, 200, 400, and 800 mg kg⁻¹ of diet and fed to grouper juveniles of 20 ± 2 g average weight for a period of 60 days. Every 20 days, fish juveniles were challenged with V. harveyi and the immune response was studied. The herbal diets significantly (P < 0.05) increased the survival, growth, and immune responses compared to the control group. The herbal diets were significantly improved (P < 0.01) in immune parameters such as phagocytic activity and albumin–globulin (A–G) ratio. Among the different concentrations of the herbals in the diet, the 400 mg kg⁻¹ diet was the most effective in the experiment.     

Antibacterial activity of Allium sativum against multidrug-resistant Vibrioharveyi isolated from black gill–diseased Fenneropenaeus indicus

The present study evaluated the in vitro and in vivo inhibitory effects of Allium sativum extracts against the multidrug-resistant Vibrio harveyi, isolated from the black gill–diseased Fenneropenaeus indicus. Initially, the antibiogram of pathogenic V. harveyi was documented for its resistance towards selected commercial antibiotics. Further, antibacterial activity of A. sativum against Vibrio harveyi was performed by the agar well diffusion method. V. harveyi culture was seeded over the agar plates, and each of the garlic extracts (freshly squeezed garlic extract—FSGE, freeze-dried garlic extract—FDGE and methanolic garlic extract—MGE) was tested at three different concentrations. FSGE showed greater inhibitory activity against V. harveyi than FDGE and MGE. Three diets of standard meal preparation for shrimp containing 0% garlic as a control, 0.5% garlic and 1% garlic treatments were formulated to evaluate the effects of garlic on the experimental infection of shrimp with V. harveyi. The combined results of long- and short-term garlic (1%) extract treatment showed a 75% reduction in cumulative mortality. The present study discusses the advantages of using freshly squeezed garlic extract treatment in shrimp culture that could offer a promising alternative to the use of antibiotics in aquaculture to control disease.     

半滑舌鳎溃疡病病原菌的分离、鉴定及其致病性分析

采用2216E和TCBS培养基分别从患溃疡病半滑舌鳎的肠道和体表病灶分离获得菌株40株,经血平板检验分离菌株的溶血活性,体外筛选并鉴定出3株潜在致病性菌株,即哈维氏弧菌、溶藻弧菌以及副溶血性弧菌。通过将3株潜在致病菌与健康半滑舌鳎的肠道上皮细胞共培养,检测病原菌刺激后半滑舌鳎肠道上皮细胞相关免疫基因的相对表达量及共培养后肠道上皮细胞的凋亡率,对病原菌的致病性进行细胞水平的筛选。结果显示,经哈维氏弧菌刺激后肠道上皮细胞白介素10基因(IL-10)表达量极显著上调,说明哈维氏弧菌引起的共培养上皮细胞免疫反应最为剧烈,且哈维氏弧菌与肠道上皮细胞共培养后导致细胞的凋亡率高达48.3%,其次为溶藻弧菌(36%)和副溶血性弧菌(34.5%),推测哈维氏弧菌为半滑舌鳎溃疡病高致病性弧菌。通过对半滑舌鳎进行浸浴回感实验,结果发现,哈维氏弧菌、溶藻弧菌和副溶血性弧菌人工回感后第6天半滑舌鳎的累计死亡率分别为100%、95%和75%,与细胞水平检测结果相吻合。实验表明,所筛选到的3株弧菌均具有较强的毒性和致病性,尤其以哈维氏弧菌致病性最强,并由此推测半滑舌鳎溃疡病可能由多种致病菌共同感染所致。     

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.     

Effect of lytic peptides on selected fish bacterial pathogens*

Abstract. Two synthetic lytic peptides, derivatives of the naturally occurring lytic peptide cecropin B, were tested for their effect on eight pathogenic fish bacteria. All bacteria were grown on tryptic soy agar at 30°C. Four levels (dilutions) of lytic peptides were incubated with viable cells in the log phase for 1 h. Standard plate counts were made after 24±2h with control plates (no toxin) representing 100% survival. Significant differences (P<0.05) in the toxicity of the two peptides were found for six out of eight bacterial pathogens. Peptide concentration or dilution level also caused significant difference (P<0.01) in bacterial counts. Peptide type by dilution level interactions were significant (P<0.01) for only two bacterial pathogens. The LD₅₀. in molar concentrations of the lytic peptides on the bacterial pathogens, ranged from approximately 10^?6 to 10^?9. The possible role of utilizing these peptides to enhance disease resistance in aquaculture is discussed.     

2株溶藻弧菌烈性噬菌体的分离鉴定及其生物学特性

为寻找并丰富溶藻弧菌噬菌体资源,实验以溶藻弧菌VAHN1为宿主菌,采用双层平板法从海南虾塘水样及福建海产品样本中分离溶藻弧菌噬菌体。通过透射电镜、限制性内切酶及构建发育树等方法对所获溶藻弧菌噬菌体进行分类鉴定;同时分析其生理生化性能。结果显示,本研究分离获得2株溶藻弧菌噬菌体VAP9与VAP21,其噬菌斑均清晰透亮,直径约1.5～2.0 mm。2株噬菌体核酸均为双链DNA,于透射电镜下可见其头部均呈正二十面体结构,2株噬菌体均属肌尾噬菌体科。噬菌体VAP9与VAP21对理化环境具有良好的耐受性;VAP9最适pH为6～8,VAP21最适pH为7～11;2株噬菌体可耐受通用杀菌浓度的过氧乙酸,且对氯仿与乙醚不敏感,同时对紫外线具有一定耐受性。2株噬菌体的最佳感染复数均为0.001,对供试溶藻弧菌的裂解率达95.2%,可裂解部分副溶血性弧菌,但无法裂解除溶藻弧菌与副溶血性弧菌外的弧菌属、葡萄球菌属、假单胞菌属等其他种属的供试细菌。噬菌体VAP9与VAP21可高效抑制溶藻弧菌VAHN1的生长,且2株噬菌体的混合制剂对溶藻弧菌的抑制效果优于单株噬菌体。将噬菌体VAP9及VAP21保守蛋白序列于N...     

Growth performance,blood biochemical parameters,immune response,and antioxidant defense of Asian seabass (Lates calcarifer) fingerlings exposed to monovalent and bivalent vaccines against Streptococcus iniae and Vibrio harveyi

The present study investigated the effects of monovalent and bivalent vaccines against Streptococcus iniae and Vibrio harveyi on growth performance, blood biochemical parameters, immune system, and antioxidant defense of Asian seabass (Lates calcarifer) fingerlings. Fish were exposed to seven experimental groups containing phosphate buffer saline (control), S. iniae vaccine by injection, S. iniae vaccine by immersion method, V. harveyi vaccine by injection, V. harveyi vaccine by immersion method, S. iniae/V. harveyi vaccines by immersion method, and S. iniae/V. harveyi vaccines by injection in triplicate for 60 days. Vaccination resulted in a significant alteration of the final weight (FW), feed conversion ratio (FCR), daily weight gain (DWG), relative growth rate (RGR), glucose, lysozyme, and complement levels at 60 days post-vaccination. Also, serum glutathione activity significantly increased in group SVIM (S. iniae/V. harveyi vaccines by immersion method) compared to the control group at 60 days post-vaccination. The level of MDA in serum was significantly decreased in SIN (S. iniae vaccine by injection) and SVIM groups compared to the control group 60 days post-vaccination where the value of this parameter was significantly higher in VIM (V. harveyi vaccine by immersion method) and SVIN (S. iniae/V. harveyi vaccines by injection) groups. The results of this study suggest that exposure to monovalent and bivalent vaccines against S. iniae and V. harveyi could alter the growth performance, nonspecific immune response, and antioxidant defense of Asian seabass fingerlings.

     

Pathogenicity of halophilic Vibrio harveyi in giant freshwater prawns (Macrobrachium rosenbergii De Man)

In this study, the phenotypic and pathogenic properties in freshwater host were characterized in 14 strains of halophilic Vibrio harveyi isolated from infected marine black tiger shrimp, Penaeus monodon. The lysogenic phenotype was assayed via prophage excision and mitomycin C induction. The bacteria were grouped into two types, corresponding to lysogenic and non‐lysogenic strains. The pathogenicity was determined via direct injection of bacterial cultures into post‐larval juvenile giant freshwater prawns, Macrobrachium rosenbergii De Man. All of the infected prawns showed similar symptoms and inflamed hepatopancreas. The V. harveyi isolates derived from the first‐injected infected prawns were re‐isolated and re‐injected into healthy giant freshwater prawns, in which they retained similar infectivity. Both lysogenic and non‐lysogenic Vibrio spp. showed identical virulence associated with 100% mortality within one day post‐injection. TEM micrographs showed hepatopancreatic nuclear deformation and lipid breakdown caused by lysogenic γ‐hemolytic VL19 and non‐lysogenic β‐hemolytic V33. However, the V33 strain was associated with severely disrupted mitochondria. None of the V. harveyi strains was able to produce a biofilm. Together, our findings indicate that the lysogenic and non‐lysogenic halophilic V. harveyi isolated from marine shrimps may use different virulence factors that are responsible for their pathogenicity in freshwater prawns.     

The high prevalence of pathogenic Vibrio harveyi with multiple antibiotic resistance in scale drop and muscle necrosis disease of the hybrid grouper,Epinephelus fuscoguttatus (♀) × E. lanceolatus (♂), in China

Scale drop and muscle necrosis disease with high mortality widely occurred recently in the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a crucial cultured marine fish species in China. In this study, 30 Harveyi clade isolates of 27 Vibrio harveyi strains were isolated from diseased hybrid groupers in the south‐east and north‐east coastal areas of China. A total of 22 V. harveyi strains were determined to be pathogenic, and most challenged fish died within 2 days of infection; surviving individuals exhibited scale drop and deep dermal lesions as naturally diseased fish. Although five typical virulence genes, including luxR, toxR_Vh, chiA, serine protease and vhh widely existed in V. harveyi, no obvious correlation was established between virulent strains and virulence genes harboured in them. Furthermore, multiple antibiotic resistance was widely exhibited in Harveyi clade strains, particularly for penicillins, polypeptides, lincomycins, acetylspiramycin, streptomycin, metronidazole and bacitracin. And the multiple antibiotic resistance indices were gradually decreased from southern to northern areas of China. This study demonstrated that the pathogenic V. harveyi with multiple antibiotic resistance is highly prevalent in hybrid grouper in China, which requires particular attention.     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.     

Differences in uptake and killing of pathogenic and non‐pathogenic bacteria by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei, (Boone)

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non‐pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi‐granulocytes) have the main function in phagocytosis of both pathogenic and non‐pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non‐virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post‐inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non‐virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.     

Improvement of growth performance,water quality and disease resistance against Vibrio harveyi of postlarval whiteleg shrimp (Litopenaeus vannamei) by administration of mixed microencapsulated Bacillus probiotics

The objective of this study was to investigate the effects of mixed Bacillus on growth, water quality and disease resistance against Vibrio harveyi in whiteleg shrimp (Litopenaeus vannamei). Postlarval shrimp (PL₃₀) were fed with (a) a basal diet (the control), (b) a diet containing mixed freeze‐dried Bacillus probiotics (FB) and (c) addition of mixed microencapsulated Bacillus probiotics (MB) in culture water. Addition of FB and MB probiotics improved (p < .05) growth, feed efficiency, survival and culture water quality (ammonia and nitrite) compared to the control group although there was no difference (p > .05) between the two treated groups. Bacillus numbers in gastrointestinal tracts and culture water of FB‐ and MB‐administrated shrimp were higher (p < .05) than in the control. After a 30‐day culture, shrimp were infected with V. harveyi and monitored for 10 days. A significant reduction (p < .05) in cumulative mortality was observed in FB‐ and MB‐supplemented shrimp (43.24% and 45.05%, respectively), compared to the control (63.06%). This finding demonstrated that administration of microencapsulated probiotics was as effective as freeze‐dried probiotics for improving growth, feed efficiency, survival, Bacillus in gastrointestinal tracts, water quality (ammonia and nitrite) and conferring disease resistance to V. harveyi.     

Enhancement of growth performance, digestive enzyme activities and disease resistance in black tiger shrimp (Penaeus monodon) postlarvae by potential probiotics

Outbreaks of multidrug-resistant pathogens have been constraint on aquaculture production in Thailand, thereby controlling shrimp pathogens by preventive probiotics being importance to sustain the aquaculture system. In this study, the effect of potential probiotics, Bacillus subtilis and Enterococcus sp. was related to growth, digestive enzyme activities (trypsin and chymotrypsin) and pathogenic resistance by postlarval black tiger shrimp (Penaeus monodon). The experiment was divided into two treatments, in triplicate, with and without supplementation of probiotics as a food additive. Shrimp fed with probiotics for a culture period of 84 days showed a significant increase (P < 0.05) in weight (2.03 ± 0.29 g) and survival (71.91 ± 3.15 %) in comparison with non-treated shrimp (1.53 ± 0.28 g and 65.20 ± 5.68 %, respectively). Trypsin activity of treated shrimp was significantly higher (P < 0.05) than that of control shrimp whereas chymotrypsin activities of the two treatments were not significantly different (P > 0.05). After challenging P. monodon postlarvae with a shrimp pathogen, Vibrio harveyi for 10 days, percentage of mortality of P. monodon postlarvae fed with probiotics was 46.67 ± 1.44 %, significantly lower (P < 0.05) than that of non-treated shrimp (61.67 ± 6.29 %). This study showed that potential probiotics was appropriate for application in P. monodon postlarvae cultivation under laboratory condition due to improvement of shrimp weight and survival, enhancement of trypsin activity and reduced mortality causing by pathogenic V. harveyi. This is the first publication reported the effect of probiotics on trypsin and chymotrypsin activities of P. monodon postlarvae.     

Effect of Customized Probiotics on the Physiological and Immunological Responses of Juvenile Western King Prawns (Penaeus latisulcatus Kishinouye, 1896) Challenged with Vibrio harveyi

Juvenile western king prawn P. latisulcatus were fed 10⁵ colony-forming units (CFU)/mL of two probiotics Pseudomonas synxantha and P. aeruginosa for 28 days. P. latisulcatus were then challenged with V. harveyi at 0 (control), 10³, 10⁵, and 10⁷ CFU/mL. During the seven days of challenge, disease resistance of the probiotic-fed prawns was compared with that of prawns not fed probiotics. The immunological responses of the prawns did not improve during the challenge period in terms of total haemocyte count, hyalinocyte, semi-ganulocyte, granulocyte, clotting time, bacteraemia, and intestinal bacterial load. Overall, when prawns were challenged with V. harveyi, the LT₅₀ values got shorter as V. harveyi concentration increased. LT₅₀ values of prawns fed probiotics were significantly longer (P < 0.05) than those not fed probiotics. At a V. harveyi concentration of 10³ CFU/mL, the 100% survival of the prawns fed probiotics was three times more likely than those of the prawns not fed probiotics.     

Detection of V. harveyi in shrimp postlarvae and hatchery tank water by the Most Probable Number technique with PCR

V. harveyi is the cause of serious disease in the shrimp industry in Thailand during cultivation. In this study, the gyrB gene of V. harveyi NICA, isolated from shrimp in Thailand, was sequenced. A pair of specific primers (A2B3) was designed that allowed amplification of a 363 bp gene fragment of V. harveyi. No cross reaction was detected in 17 other Vibrio species tested except for V. carchariae which is a synonym for V. harveyi. The possibility of using A2B3 for confirmation and enumeration of V. harveyi by PCR was demonstrated. Of 40 possible V. harveyi strains isolated from seafood on the basis of their growth on TCBS plates and biochemical reactions, 36 gave a reaction with the specific primers. The primers could detect V. harveyi at a level of as few as 15 cells/ml. The Most Probable Number (MPN) technique was applied to enumerate V. harveyi. We have demonstrated that when PCR was applied directly to the enrichment broth of shrimp artificially inoculated with V. harveyi, the MPN value was no different from the MPN value obtained using the standard technique with selective agar. This technique was employed to enumerate V. harveyi in postlarvae and hatchery tank water. V. harveyi were detected in 18 out of 21 postlarval samples and in 14 out of 21 tank water samples. The numbers of V. harveyi detected in postlarvae and water were 150-1.1 × 10⁸/g postlarvae and 7-4.6 × 10⁴/ml of water samples, respectively. Screening of postlarvae to reduce the high risk of V. harveyi contamination in cultivation ponds is suggested as a measure to prevent the catastrophic losses caused by V. harveyi disease.     

Effects of dietary immunostimulant combination on the growth performance,non‐specific immunity and disease resistance of cobia,Rachycentron canadum (Linnaeus)

The aim of this study was to examine the effects of the immunostimulant combination (IC) containing β‐glucan, A3α‐peptidoglycan, vitamin C and vitamin E on the growth performance, non‐specific immunity and protection against Vibrio harveyi infection in cobia (Rachycentron canadum). Fish were fed diets containing six graded levels of IC (0, 1, 2, 3, 4 and 5 g kg^?1 diet) for 8 weeks. The results showed that the survival rate ranged from 81.1 to 84.4% with no significant difference among all the groups (P > 0.05) after the feeding experiment. Dietary IC significantly increased the specific growth rate (SGR), serum lysozyme, alternative complement pathway (ACH50) activity, phagocytosis percentage (PP) and respiratory burst activity of head kidney macrophages of cobia. Moreover, feeding of supplemented diets containing 3.0 g kg^?1 IC resulted in significantly lower mortality against the pathogens, V. harveyi compared with the control group. To elevate the growth and immune resistance ability of cobia, the optimal dose of dietary IC administration, determined by second‐order polynomial regression analysis was 3.43 and 2.71 g kg^?1 diet, respectively, on the basis of the SGR and mortality after challenge with V. harveyi.     

Multilocus sequence analysis of Vibrionaceae isolated from farmed amberjack and the development of a multiplex PCR assay for the detection of pathogenic species

Since 2011, high mortality rates and symptoms consistent with vibriosis have been observed in farmed amberjack (Seriola dumerili) in Japan. To identify 41 strains isolated from diseased amberjack, a multilocus sequence analysis using nine concatenated genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) was conducted. Twenty‐seven strains were identified as Vibrio harveyi, suggesting an epidemic of V. harveyi infection in amberjack farms. Other strains were identified as Vibrio anguillarum, Vibrio owensii and Photobacterium damselae subsp. damselae. To develop an efficient diagnostic method for vibriosis in amberjack, a multiplex PCR system was developed to identify V. anguillarum, V. harveyi and P. damselae subsp. damselae. The method successfully discriminated between these three bacterial species, with amplification products of 350 bp for V. anguillarum, 545 bp for V. harveyi and 887 bp for P. damselae subsp. damselae and can be used for diagnosis in aquaculture farms.     

A study on the influence of selected Indian herbal active principles on enhancing the immune system in Fenneropenaeus indicus against Vibrio harveyi infection

Five herbs such as Acalypha indica, Hygrophila spinosa, Picrorhiza kurooa, Tinospora cordifolia and Zingiber officinale were selected to screen for the in vitro immunostimulant activity against the shrimp pathogen Vibrio harveyi using different organic polar and non-polar solvents. After initial screening, the best extract of the five herbs was mixed thoroughly in equal proportion and made it an artificial immunostimulant diets (ISD) such as ISD-1(500), ISD-2 (1,000), ISD-3 (1,500) and ISD-4 (2,000) mg?kg^?1. The control diet was prepared without adding immunostimulant herbal extracts. The Indian white shrimp, Fenneropenaeus indicus having the weight of 8.0?±?1.0?g, were fed with the above diets for 60?days. After the completion of feeding trail, they were challenged with V. harveyi at the rate of 10⁷CFU/ml and found out the cumulative mortality, growth parameters, haematological and immunological parameters. The weight gain and specific growth rate were significantly (P?<?0.05) increased from the control group to experimental group. The immunostimulant active principles helped to decrease the coagulation time and improved the total haemocyte count (THC), phagocytosis, phenol oxidase (PO) activity, haemagglutinin activity and bacterial clearance.