Use of essential oils extracted from three Origanum species for disinfection of cultured rotifers (Brachionus plicatilis)

Essential oils extracted from three plant species of the genus Origanum, Origanum vulgare subsp. hirtum, Origanum onites L. and Origanum marjorana L., were assayed as potential antibacterial agents for disinfection of rotifers Brachionus plicatilis. Survival of rotifers in seawater with added oregano essential oils was significantly higher than in the control treatment (P < 0.05). When rotifers were incubated in seawater with added oregano essential oils at a final concentration of 10 mg L^?1 for 4 h, the numbers of total culturable bacteria were not significantly lower than in the control treatment (P > 0.05), while the numbers of presumptive Vibrio in rotifers were significantly reduced compared with the control treatment (P < 0.05).     

Exclusion of Vibrio spp. by an antagonistic marine actinomycete Streptomyces rubrolavendulae M56

A marine actinomycete Streptomyces rubrolavendulae M56 isolated from the sediments of Bay of Bengal and displaying biogranulation property was used for the study. The strain showed antagonistic property against vibrios, the opportunistic pathogens in aquaculture. The efficacy of the biogranules of actinomycete M56 in competitive exclusion of Vibrio spp. was tested both in vitro and in vivo. Streptomyces rubrolavendulae M56 biogranules could significantly exclude the pathogenic Vibrio spp. in co‐culture experiments (in vitro). In vivo exclusion of Vibrio spp. in a Penaeus monodon postlarval rearing system was evaluated by treatment of the rearing water with biogranules of S. rubrolavendulae M56. The experiments proved that S. rubrolavendulae M56 biogranules could reduce the pathogenic Vibrio spp., while maintaining total heterotrophic bacterial count. Therefore, the actinomycete biogranules (M56) can be used as a promising alternative to antibiotics in the shrimp larval production system which is often affected by vibriosis.     

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.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

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.     

Identification of novel sRNAs involved in oxidative stress response in the fish pathogen Vibrio alginolyticus by transcriptome analysis

Vibrio alginolyticus as an important pathogen in aquaculture can encounter the oxidative stress produced by the immune system during infection. Previous studies showed that sRNAs have important functions in response to oxidative stress in bacteria; however, less of sRNAs related to oxidative stress response were identified in V. alginolyticus. In this study, a total of 749 novel sRNAs were identified by RNA sequencing; among them, 128 sRNAs were up‐ or downregulated in response to oxidative stress. In addition, 1,870 genes exhibited variation on mRNA levels in oxidative stress response. By analysing the target genes of the sRNAs, we concluded that these sRNAs could regulate expressions of genes responsible for iron transport, catalase, GSH‐dependent defence system, electron transferred and stress response. Moreover, the functions of the sRNAs are also seemed related to the pathogenicity in V. alginolyticus. Based on the results, we constructed the oxidative stress model in V. alginolyticus. This study provides us the first outlook of sRNAs function in oxidative stress response in V. alginolyticus. Furthermore, this study can help us to prevent and control this important opportunistic pathogen in aquaculture.     

Conditioning,maturation and year‐round natural spawning of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system

The present experiment was aimed at studying the conditioning, maturation and natural spawning of orange‐spotted grouper, Epinephelus coioides, in a recirculatory aquaculture system (RAS). Thirty fish (n = 30; 3.35 ± 0.05 kg) were stocked in a circular tank of 125 m³ capacity fitted with an RAS for conditioning and broodstock development. After 15 days, 15 fish were implanted with 17 α methyl testosterone and letrozole at the rate of 5 mg and 0.2 mg/kg body weight, respectively, for conversion from female to male. The gonadal development started after 1 month, and by 90th day, 63.53 ± 3.78% and 2.07 ± 0.84% of the oocytes attained a size of 400–500 μm and 500–600 μm respectively. Natural spawning commenced in the RAS from 4th month onward after stocking and spawning continued round the year. The spawning pair showed courtship behaviour with a typical vertical burst of swimming just before release of gametes. The total number of eggs spawned during 1 year was 47.23 million with spawning frequency varying form 5 to 13 times per month. The association of spawning events with new moon day (lunar cycle) weakened as time progressed. The mean monthly fertilization and hatching rates varied from 77.80 ± 3.34% to 83.70 ± 1.76% and 82.80 ± 4.21% to 88.33 ± 1.39% respectively. The study proved that RAS is an efficient system that provides a stable, controllable and conducive environment for year‐round natural breeding of orange‐spotted grouper.     

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).     

Spat availability of commercial bivalve species recruited on artificial collectors from the northern Gulf of California. Seasonal changes in species composition

This study reports a year‐round recruitment of spat of four commercial bivalve species; Pteria sterna, Euvola vogdesi, Pinctada mazatlanica and Pinna rugosa collected in the region of Puerto Peñasco, north‐eastern coast of the Gulf of California. Bimonthly recruitment of commercial bivalve spat on netlon^® collectors was evaluated for six sites from June 2007 to August 2008. To describe spat recruitment abundances with environmental parameters, sea surface temperature (°C) and surface chlorophyll a concentration (mg m^?3) were characterized by means of monthly Aqua/MODIS satellite data. For each species a repeated measures anova was used to evaluate differences in the number of spat between months, sites and depths. Maximum sea surface temperature was recorded in August–September (~31.5°C) and the minimum in January–February (~15°C), while the minimum surface chlorophyll a was observed in June–September (mean range = 1.5–2 mg m^?3) and the maximum in January–March (mean range = 2–5 mg m^?3). Spat recruitment showed distinct patterns; P. sterna can be characterized as having a Winter–Spring pattern, E. vogdesi a winter pattern, while P. mazatlanica and P. rugosa a summer spat recruitment pattern. This information constitutes part of the fundamental data needed for the development of aquaculture and conservation initiatives in the region based on wild spat supply.     

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

Isolation,molecular characterization and antimicrobial susceptibility of Aeromonas spp. obtained from Tiger Grouper (Epinephelus fuscoguttatus) and Marble Goby (Oxyeleotris marmoratus) fish in Sabah,Malaysia

Aeromonads are ubiquitous in aquatic environments and have been implicated in fish and human infections. In this study, we isolated, studied antimicrobial susceptibility patterns and screened the existence of 15 virulence genes in aeromonads from two famously consumed fish species—seven marine Tiger Grouper (Epinephelus fuscoguttatus) and eight freshwater Marble Goby (Oxyeleotris marmoratus) from the aquaculture hatchery in Sabah, Malaysia. A total of 30 aeromonads (17 A. caviae, 9 A. rivuli, 4 A. dhakensis) were identified using PCR targeting GCAT gene, rpoD‐restriction fragment length polymorphism and multi‐locus phylogenetic analysis. All 30 strains were resistant to amoxicillin and cephalothin and five strains were multidrug‐resistant. Nine virulence genes (lip, ela, eno, fla, aerA, hylA, dam, alt and ser) present in A. dhakensis, suggesting the virulence potential of this species as a fish pathogen. This study offers as a baseline for future studies in monitoring and managing these two fish in aquaculture industry.     

Iron bioavailability of different sources in juvenile grouper Epinephelus coioides

A 70‐day experiment was implemented to study the iron (Fe) bioavailability of three Fe sources in Epinephelus coioides (initial weight, 12.19 ± 0.14 g). Treatments consisted of 0, 50, 90, 130, 170 and 210 mg supplemental Fe/kg from iron sulphate (FeSO₄), ferric citrate (Fe‐citrate) and hydroxy methionine analogue iron (Fe‐MHA). The results showed that the growth performance was not affected by Fe level regardless of Fe sources. Hepatic catalase activity (CAT), the haematocrit value (Hct), haemoglobin (Hb), mean corpuscular volume (MCV) and Fe content in whole body or liver firstly increased significantly and then plateaued as Fe level increased regardless of the Fe source, except significant decreasing of Hct in 210 mg Fe/kg group from FeSO₄. Manganese (Mn) content decreased significantly and then kept stable in the whole body and liver whatever the source was. The slope‐ratio method using FeSO₄ as the reference and Hb content, CAT activity, body and hepatic Fe concentration as the response parameter showed the average relative bioavailability of Fe‐citrate and Fe‐MHA was 103.25 ± 4.35 (n = 4) and 134.5 ± 15.67 (n = 4), respectively, with the maximum value of Fe‐MHA in any case. In conclusion, Fe‐MHA was the most effective to meet preferable haematological index, CAT activity and mineral deposition, with little difference between Fe‐citrate and FeSO₄, athough Fe levels with different sources failed to influence the growth in grouper.     

Growth performance and disease resistance against Vibrio vulnificus infection of novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus)

Groupers are economically important for aquaculture in Thailand. A novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) has been successful cross‐bred; therefore, the present work aimed to assess the hybrid's traits. The growth performance, strength and tolerance to a pathogenic bacterial infection of this hybrid were compared with its parent species, tiger grouper and giant grouper. The results of all measured growth parameters indicated that the hybrid strain grew fastest followed by giant and tiger grouper respectively. The expressions of the growth‐related genes, insulin‐like growth factor (IGF) I and II, were also analysed in fish muscle and liver which are the main target organs in fish growth regulation. Among tested species, similar expression patterns of IGF‐I and IGF‐II were detected in both organs. The levels of these genes in liver and muscle of hybrid and giant grouper were higher than those of tiger grouper comparable with the growth manner. After challenge with Vibrio vulnificus, the immunological parameters, clearance time of Vibrio in haemolymph and survival was measured to verify the fish immunity. Leucocyte number, lysozyme activity and the ability to eliminate the pathogen were very high in hybrid and giant grouper while these parameters were lower in tiger grouper. Correspondingly, the mortality rate of tiger grouper was higher than others and % survival at the end of observation time (15 days post challenge) was lowest in infected tiger grouper. Altogether, the results suggested that the hybrid grouper has desirable traits that will improve cultured grouper.     

Experimental infection of brown‐marbled grouper,Epinephelus fuscoguttatus (Forskal), with Vibrio parahaemolyticus identifies parvalbumin beta‐2 subunit I,alpha‐2‐macroglobulin,nattectin and immunoglobulin light chain,differentially expressed in resistant grouper

The mechanisms through which brown‐marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown‐marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two‐dimensional gel electrophoresis (2‐DE). The results showed that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.