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.     

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.     

Effects of dietary herbal formulae combined by Astragalus polysaccharides,chlorogenic acid and allicin in different combinations and proportions on growth performance,non‐specific immunity,antioxidant status,vibriosis resistance and damage indexes of Litopenaeus vannamei

In this study, a feeding trial followed by a challenge test was performed to evaluate effects of six herbal formulae which were different combinations of Astragalus polysaccharides (APS), chlorogenic acid (CGA) and allicin on growth performance, non‐specific immune response, antioxidant capacity, disease resistance and biomolecule damage of white shrimp Litopenaeus vannamei. Shrimp were fed seven diets, control diet (basal diet with no herbal formulae) and six herbal formula diets (G1–G6, basal diet supplemented with 0.1% APS + 0.05% allicin, 0.1% APS + 0.1% allicin, 0.1% CGA + 0.05% allicin, 0.1% CGA + 0. 1% allicin, 0.1% APS + 0.1% CGA and 0.1% APS + 0.1% CGA + 0.05% allicin respectively), for 21 days. After that, shrimp were challenged with Vibrio harveyi and then the cumulative mortality of shrimp was recorded for 7 days post challenge. The results showed that there were no significant differences in growth performance among all groups, while the non‐specific immune responses and antioxidant indexes were significantly improved (p < .05) in shrimp fed herbal formula diets when compared to the control. Meanwhile, the lowest cumulative mortality was observed in shrimp fed herbal formula diets with 0.1% APS, 0.1% CGA and 0.05% allicin supplementation after V. harveyi challenge. Additionally, herbal formulae could not cause biomolecule damage to the hepatopancreas of shrimp. In conclusion, these results indicated that synergistic effect of APS, CGA and allicin helped to boost immunity, antioxidant capacity and disease resistance of shrimp without biomolecule damage.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

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.     

Characterization of phenotype variations of luminescent and non‐luminescent variants of Vibrio harveyi wild type and quorum sensing mutants

Vibrio harveyi, a luminescent Gram‐negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non‐luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence‐related phenotypic traits of the wild‐type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non‐luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non‐luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.     

Ingestion of food pellets containing Escherichia coli overexpressing the heat‐shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection

Feeding aquatic animals with bacterial encapsulated heat‐shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK‐DnaJ‐GrpE, the prokaryotic equivalents of Hsp70‐Hsp40‐Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g^?1 pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.     

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange‐spotted grouper (Epinephelus coioides)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable Montanide^TM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.     

Antimicrobial activity of partially characterized analytes from Bacillus pumilus(B2)

This study was carried out to characterize the antimicrobial substance produced by the strain of Bacillus pumilus (B2) obtained from Novozymes Biologicals Inc. to compare its antimicrobial activity by agar well diffusion assay and bacteriocin activity assay via critical dilution method against seven different strains of Vibrio spp., specifically V. alginolyticus (A01), V. cholerae (C01), V. fluvialis (F01, F02), V herveyii (H), V. mimicus (M01), V. parahaemolyticus (P01) and V. vulnificus (V01, V02). All Vibrio spp. were isolated from the hemolymph and intestine of the white faeces disease‐infected moribund pacific white‐leg shrimp Litopenaeus vannamei (Boone 1931) and one strain (V. harveyi) from its diseased postlarva. The cell‐free neutralized supernatant (CFNS) of B2 showed moderate thermo‐stability being stable up to 70°C for 60 min with, however, reducing activity above 80°C for 20 min. B2 antimicrobials showed a stable activity within the pH ranging from 6 to 10 at room temperature and at 4°C, while residual antimicrobial activity of crude CFNS showed tolerance to salinity up to 7% of sodium chloride below 4°C. No B2 activity was obtained while exposed to proteolytic enzyme, such as proteinase k and pepsin, while its activity kept stable exposed to lipase. Initial B2 characterization for antimicrobial substance in CFNS revealed proteinaceous in nature owing to activity loss against proteolytic enzymes and no lipid moiety activity against lipase, which could be categorized as bacteriocin‐like inhibitory substance having potential application against several strains of Vibrio spp. in aquaculture.     

Bacterial population in intestines of white shrimp,Litopenaeus vannamei fed a synbiotic containing Lactobacillus plantarum and galactooligosaccharide

The aims of this study were to unravel the intestinal microbiota of Litopenaeus vannamei after being fed a diet without (control) or with the synbiotic (SYN) for 60 days using next‐generation sequencing technology to see if changes in the intestinal microbiota were involved in the improved growth performance and health status of the shrimp. Next‐generation sequencing data showed that six phyla, 11 classes, 19 orders, 30 families, 58 genera and 73 species with taxonomic names assigned were detected. The majority of the operational taxonomic units (OTUs) was shared between the SYN and control shrimp and comprised 37 OTUs. However, intestinal biodiversity analyses revealed that SYN‐fed shrimp had a higher species richness, evenness and Shannon–Weaver index than did shrimp fed the control diet, but without reaching statistical significance. Interestingly, shrimp fed the SYN diet exhibited improved colonization of Lactobacillus plantarum and reduced prevalences of Vibrio harveyi and Photobacterium damselae in the intestines. These findings indicate that the SYN was able to modulate the intestinal bacterial community of shrimp and could be used to control vibriosis in shrimp.     

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.     

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.     

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.     

Replacement of fish oil with a DHA‐rich Schizochytrium meal on growth performance,activities of digestive enzyme and fatty acid profile of Pacific white shrimp (Litopenaeus vannamei) larvae

This trial was conducted to evaluate the effects of replacing dietary fish oil with Schizochytrium meal for Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Six test microdiets were formulated using Schizochytrium meal to replace 0 g/kg, 250 g/kg, 500 g/kg, 750 g/kg, 1000 g/kg or 1500 g/kg fish oil DHA. No significant differences were observed in survival, growth, final body length and activities of digestive enzyme among shrimp fed different diets (p > .05). No significant differences were observed in C20:5n‐3 (EPA) in muscle samples (p > .05). C18:3n‐3 and C20:4n‐6 in muscle increased as Schizochytrium meal replacement level increased (p < .05). No significant differences were observed in C22:6n‐3 (DHA) and n‐3 fatty acids among shrimp fed diets that algae meal replaced 0 g/kg ‐ 1000 g/kg of fish oil. Shrimp fed diet R150 had higher DHA content than other groups and had higher n‐3 fatty acids than that of shrimp fed diets R50, R75 and R100 (p < .05). C18:2n‐6, PUFA and n‐6 fatty acids in muscle increased, while n‐3/n‐6 ratio decreased with increasing algae meal replacement level from 0 g/kg to 1000 g/kg (p < .05). In conclusion, Schizochytrium meal could replace 1500 g/kg fish oil DHA in the microdiets without negatively affecting shrimp larvae survival, growth and activities of digestive enzyme.     

Suitable dietary chitosan improves the growth performance,survival and immune function of tiger shrimp,Penaeus monodon

Different levels of dietary chitosan on growth performance, survival and stress tolerance to air exposure was studied in tiger shrimp, Penaeus monodon. Shrimp (mean initial wet weight about 1.16 g) were fed with six different diets (C0, C0.05, C0.1, C0.2, C0.3 and C0.4) containing six level of chitosan (0%, 0.05%, 0.1%, 0.2%, 0.3% and 0.4% respectively) in triplicate for 60 days. Growth performance [final body wet weight (FBW); weight gain (WG); biomass gain (BG)] of shrimp fed chitosan‐containing diets were higher (P < 0.05) than that of shrimp fed the basal diet, shrimp fed C0.1 diet showed the highest value of growth performance. Survival of shrimp in C0.1 and C0.2 diet groups were higher (P < 0.05) than that of shrimp in C0, C0.05 and C0.4 diet groups but without statistical difference (P > 0.05) in shrimp fed C0.3 diet group. Whole body and muscle lipid contents decreased with increasing dietary chitosan levels. Plasma total cholesterol and triglyceride contents of shrimp fed C0 diet was significantly higher (P < 0.05) than that of shrimp fed chitosan‐containing diets. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of shrimp fed C0 diet were higher than those of shrimp fed chitosan‐containing diets. Digestive gland malondialdehyde (MDA) and carbonyl protein contents of shrimp fed chitosan‐containing diets were lower (P < 0.05) than that of shrimp fed C0 diet. Total haemocyte count of shrimp fed C0 diet was lower (P < 0.05) than that of shrimp fed chitosan‐containing diets. On the contrary, the haemolymph clotting time of shrimp fed C0 diet was higher (P < 0.05) than that of shrimp fed chitosan‐containing diets. In conclusion, all results suggested that dietary intake containing 0.1% and 0.2% chitosan enhanced the growth of shrimp, whereas a higher level than 0.3% and 0.4% decreased growth of shrimp. Second‐degree polynomial regression analysis of WG and BG indicated that the optimum supplement of dietary chitosan level should be 0.19–0.21%.     

Immunostimulatory effect of mangrove‐derived marine yeasts in Penaeus monodon

This study attempted to assess the efficacy of mangrove‐derived marine yeasts as a source of immuno‐stimulant in the tiger shrimp, Penaeus monodon. The shrimp was fed on the diets in supplementation with yeast biomass at 10% for a period of 15 days and then challenged with the pathogenic Vibrio cholerae. The animals were assessed for immune responses in terms of total haemocyte counts, phenol oxidase and endobiotics. Among the yeasts tested, Rhodotorula minuta was found to have high immunostimulatory effect in the shrimps especially after challenge with the pathogenic vibrio.     

Production of specific dsRNA against white spot syndrome virus in the yeast Yarrowia lipolytica

White spot syndrome virus (WSSV) is the most aggressive disease affecting cultured shrimp. One possibility to tackle it is by means of RNA interference (RNAi) induced by the presence of double‐stranded RNA (dsRNA). Normally, dsRNA is a product of the cellular machinery to gene regulation, but it can be produced synthetically and introduced into specific tissues or cells and thereby induce RNAi. Although in vitro production of dsRNA is possible, this is high cost. An alternative is to produce dsRNA in vivo using biological systems such as bacteria or yeasts. In this regard, Yarrowia lipolytica offers distinctive advantages for dsRNA production. The objective was to develop a Y. lipolytica strain able to produce dsRNA‐specific against WSSV and to evaluate its antiviral activity in the white leg shrimp Litopenaeus vannamei. From the 0.4 and 0.6 Kb fragments of the ORF89 gene, a dsRNA‐ORF89‐producing construct was built in the plasmid pJC410; the resulting construct (pARY410) was used to transform Y. lipolytica to drive the specific expression of dsRNA‐ORF89. Yeast colonies positive to the WSSV‐ORF89 gene were selected. The expression of dsRNA‐ORF89 and RNAse III was measured being detected at 32 and 48 hr. Subsequently, the antiviral activity of dsRNA‐ORF89 was tested in a WSSV challenge bioassay. The results showed survival in dsRNA‐ORF89 shrimp (25%) compared to control organisms treated with total RNA from the yeast P01‐AS harvested at 32 hr. In conclusion, Y. lipolytica is a convenient host to produce and deliver dsRNA‐ORF89 able to protect WSSV‐challenged shrimp.