Identification and pathogenicity study of emerging fish pathogens Acinetobacter junii and Acinetobacter pittii recovered from a disease outbreak in Labeo catla (Hamilton, 1822) and Hypophthalmichthys molitrix (Valenciennes, 1844) of freshwater wetland in West Bengal,India

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD₅₀ of 1.24 × 10⁵ and 1.88 × 10⁷ cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

Case definition for clinical and subclinical bacterial kidney disease (BKD) in Atlantic Salmon (Salmo salar L.) in New Brunswick,Canada

Bacterial kidney disease (BKD) is considered an important cause of loss in salmon aquaculture in Atlantic Canada. Causative agent of BKD is the Gram‐positive bacteria Renibacterium salmoninarum. Infected salmon are often asymptomatic (subclinical infection), and the disease is considered chronic. One of the challenges in quantifying information from farm production and health records is the application of a standardized case definition. Case definitions for farm‐level and cage‐level clinical and subclinical BKD were developed using retrospective longitudinal data from aquaculture practices in New Brunswick, Canada, combining (i) industry records of weekly production data including mortalities, (ii) field observations for BKD using reports of veterinarians and/or fish health technicians, (iii) diagnostic submissions and test results and (iv) treatments used to control BKD. Case definitions were evaluated using veterinarians’ expert judgements as reference standard. Eighty‐nine and 66% of sites and fish groups, respectively, were associated with BKD at least once. For BKD present (subclinical or clinical), sensitivity and specificity of the case definition were 75–100% varying between event, fish group, site cycle and level (site pen). For clinical BKD, sensitivities were 29–64% and specificities 91–100%. Industry data can be used to develop sensitive case definitions.     

Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp,Catla catla (Hamilton)

Probiotic potential of the autochthonous bacteria in catla, Catla catla has been evaluated through determination of antagonistic activity (in vitro) of the cellular components of gut bacteria against seven fish pathogens. Altogether 208 strains were isolated, inhibitory activity of the isolates was evaluated through cross‐streaking and 16 primarily selected antagonistic strains were confirmed using the double‐layer method. Four bacteria that showed antagonism against ≥4 pathogens were selected as putative probiotics. The intracellular, extracellular, whole‐cell and heat‐killed cell components exhibited bactericidal activity against the pathogens. In addition, the selected strains were capable of producing different extracellular enzymes, competent to grow in intestinal mucus and could tolerate diluted bile juice. Analysis of 16SrRNA partial gene sequence revealed that both the strains CC1FG2 and CC1FG4 were Bacillus methylotrophicus (KF559344 and KF559345), while the isolates CC1HG5 and CC2HG7 were Bacillus subtilis subsp. spizizenii (KF559346) and Enterobacter hormaechei (KF559347) respectively. Bio‐safety evaluation through intra‐peritoneal injection of the isolates did not induce any pathological signs or mortalities in C. catla. The study confirmed probiotic properties of autochthonous gut bacteria in C. catla and demonstrated potential for using them as bio‐control agents. However, in vivo studies are essential to explore their efficacy in the commercial aquaculture.     

Characterization of a megalocytivirus from cultured rock bream,Oplegnathus fasciatus (Temminck & Schlege), in China

Megalocytivirus belongs to the Iridoviridae family and is known to affect finfish. Megalocytivirus epizootics have been reported to occur in several cultured fish species in China; however, no megalocytivirus associated with rock bream, Oplegnathus fasciatus (Temminck & Schlege), has been documented. In this study, we characterized for the first time a megalocytivirus, rock bream iridovirus (RBIV)‐C1, detected in cultured rock bream in a fish farm in China that had been inflicted with a high‐mortality disease outbreak. Sequence analysis of three conserved genes showed that RBIV‐C1 shares over 90% overall identities with a number of known megalocytiviruses. Electron microscopic examination revealed RBIV‐C1 as hexagonal particles similar to those reported for megalocytiviruses. In vivo infection study indicated that, following inoculation into rock bream, RBIV‐C1 induced 100% mortality and upregulated the expression of Mx, IL‐1β and IL‐8. The infected fish exhibited pathological signs similar to those observed in naturally diseased fish. Furthermore, studies in a turbot (Scophthalmus maximus L.) model indicated that RBIV‐C1 induced acute infection in turbot that led to 100% mortality. These results indicate that RBIV‐C1 is highly virulent to rock bream as well as turbot and that RBIV‐C1 is closely related to a number of previously reported megalocytivirus and likely a genetic variant of the latter.     

Isolation and characterization of Edwardsiella ictaluri strains as pathogens from diseased yellow catfish Pelteobagrus fulvidraco (Richardson) cultured in China

Yellow catfish Pelteobagrus fulvidraco (Richardson) is a commercially important fish generally distributed in Southeast Asian countries. The well‐known aetiological agent of enteric septicaemia of catfish, Edwardsiella ictaluri, was isolated from diseased yellow catfish P. fulvidraco (Richardson) reared at two commercial fisheries in China. The economic losses due to the high mortalities (about 50%) caused by this bacterium have been increasing annually. The affected fish presented two different, typical symptoms: pale gills, slight exophthalmia and a ‘hole in the head’, and haemorrhage on the opercula, in the skin under the jaw, creating a ‘hole under the jaw’. These diseases were found frequently in cultured yellow catfish throughout China. The isolates from both outbreaks were all Gram negative, facultatively anaerobic and short rod. Morphological and biochemical tests and phylogenetic analysis based on the 16S rDNA sequences all strongly indicated that these yellow catfish isolates were highly identical to the known E. ictaluri. In addition, the isolates possessed the typical plasmid profile of E. ictaluri. Experimental infection assays were conducted and pathogenicity (by an intraperitoneal injection) was demonstrated in yellow catfish and channel catfish Ictalurus punctatus. The results showed that yellow catfish isolates were quite conservative phenotypically and genetically, and were able to cause two different, typical symptoms in this fish under unknown conditions and mechanism.     

Occurrence of trypanosomiasis in net‐cage cultured groupers (Cromileptes altivelis and Epinephelus fuscoguttatus) in Nanshan port of Sanya,Hainan province,China

In the present study, reported was an unidentified trypanosome that caused massive mortalities of cultured humpback grouper, Cromileptes altivelis (Valenciennes, 1828) and brown‐marbled grouper, Epinephelus fuscoguttatus (Forsskål, 1775) in the net‐cages in Sanya, China, and some general pathological features of E. fuscoguttatus after experimental infection of the trypanosome. Before dying in the net cages of diseased fish, listlessness and anorexia were the observable symptoms. The blood of the diseased fish was light‐coloured in comparison with that of healthy one. E. fuscoguttatus experimentally infected with this trypanosome are with different degrees of engorgement in gill lamella, liver, spleen and kidney. Tissue lesion and necrosis were observed in liver and spleen of diseased fish.     

Recovery of Hafnia alvei from diseased brown trout,Salmo trutta L., and healthy noble crayfish,Astacus astacus (L.), in Bulgaria

Hafnia alvei was isolated in Bulgaria from healthy noble crayfish, Astacus astacus (L.), and then from farmed diseased brown trout, Salmo trutta L., with signs of haemorrhagic septicaemia. The isolates were identified initially with conventional phenotyping and commercial Merlin Micronaut and API 20E rapid identification systems, followed by sequencing of the 16S rRNA gene. Hafnia alvei Bt1, Bt2 and Aa4 were of low virulence to rainbow trout and brown trout, although cytotoxicity was demonstrated by Bt1 and Bt2, but not by Aa4.     

Effects of rearing density on growth,fatty acid profile and bioremediation ability of polychaete Nereis diversicolor in an integrated aquaculture system with rainbow trout (Oncorhynchus mykiss)

The objective of this study was to assess the effects of rearing density on the growth and fatty acid profile of Nereis diversicolor and on its capability to bioremediate wastewater in an integrated culture system with rainbow trout (Oncorhynchus mykiss). To this end, a batch of juvenile N. diversicolor (0.03 ± 0.01 mg) was assigned into four different densities (T₁ = 250, T₂ = 500, T₃ = 1,000, T₄ = 2,000, besides T₀ = with no worm) in three replicates. The worm groups were fed with solid waste that was supplied from tanks containing rainbow trout (107.17 ± 13.5 g; 1.39 ± 0.18 kg/m³). During the experiment (60 days), the water temperature was 17.71 ± 0.6°C. The results revealed that SR%, SGR% and WG% of N. diversicolor in T₁ were significantly higher than those of T₂, T₃ and T₄. Both FER rate and biomass gain in T₄ were significantly higher than those in the other groups. The highest removal rate of NO₂‐N (73.72%), NH₃‐N (65.70%), PO₄‐P (59.32%), BOD₅ (69.60%) and TSS (82.33%) were observed in T₄. The major fatty acids presents were palmitic acid, oleic acid, linoleic acid, stearic acid and alpha‐linolenic acid in all worm‐treated groups, with no difference observed in the concentration of these fatty acids among them. Taken together, these findings suggest that organic wastes from rainbow trout farms could be recycled to achieve a sustainable aquaculture goal, and demonstrate that a high percentage of fatty acids in fish feed is not absorbed by rainbow trout but is done by N. diversicolor.     

Influence of social factors on the nocturnal activity pattern of the noble crayfish,Astacus astacus (Crustacea,Decapoda) in recirculating aquaculture systems

The social environment plays a major role in the control of growth in many crustaceans, but little is known on the mechanisms involved. Two‐ and three‐year‐old noble crayfish, Astacus astacus are strictly nocturnally active. In the present article, we studied social effects on the pattern of nocturnal activity, represented by the time which the animals spent outside shelters. For this purpose, the activity of groups of uniformly large (‘dominant’) and uniformly small (‘subordinate’) individuals were recorded at different densities both separately and in mixed set‐ups. In crowded laboratory cultures, the presence of larger (dominant) conspecifics was found to reduce the time which smaller (subordinate) individuals spent outside shelters in search of food and feeding by about one‐third. It is suggested that this asymmetric inhibitory effect is an element of the functional chain which ultimately results in an extremely wide variation in individual growth rates of communally reared A. astacus. The relevance of the findings to crayfish aquaculture is discussed.     

The effect of density in larval rearing of the pullet carpet shell Venerupis corrugata (Gmelin, 1791) in a recirculating aquaculture system

The pullet carpet shell Venerupis corrugata is an economically valuable species in several European countries, however, nowadays stocks are under high fishing pressure. Hatchery production of juveniles for release is a major contributor to strengthen the stock and consequently improve the sustainability of the natural stocks. This study aimed to determine the feasibility of rearing V. corrugata larvae with different larval densities (10, 40 and 200 larvae per mL) in a recirculating aquaculture system (RAS), compared with the traditional larval rearing methodology (Batch). The mean survival, growth and metamorphic rate of V. corrugata larvae in RAS was higher (11.1%; 71.3 μm; 21.6% respectively) than in the Batch system, in all tested densities. The larval growth was not affected by the initial density until 40 larvae per mL, however, 200 larvae per mL decreased the larval growth in length nearly 54 μm. The larval rearing time was shortened in 2 days in the RAS system. The physical, chemical and microbiologic parameters suggested that the tested densities were not excessive to disturb the biofilter stability of RAS. The V. corrugata larval rearing performed at high larval stocking densities in RAS system present a reduction in the operating costs to produce this species.     

Use of Nannochloropsis sp. isolated from the East China Sea in larval rearing of sea cucumber (Apostichopus japonicus)

The supply of microalgae to hatcheries is a limiting factor for the mass larval production of sea cucumber (Apostichopus japonicus) in Fujian Province, China. In this study, Nannochloropsis sp. isolated from the East China Sea was tested as food for A. japonicus larvae. The first trial compared the effect of mono‐, bi‐ and trialgal diets comprising three microalgae (Chaetoceros muelleri, Dunaliella tertiolecta and Nannochloropsis sp.) on A. japonicus larval growth, survival, settlement and juvenile growth. The results showed that there were no significant differences in survival and settlement between larvae fed with Nannochloropsis sp. and other diets. All diet treatments yielded similar juvenile sea cucumber output. In the second trial, A. japonicus larvae were fed equally four times daily at three different rations (5000, 20 000 and 40 000 cells mL^?1 day^?1). Larvae fed 20 000 cells mL^?1 day^?1 were significantly larger than larvae in other groups and experienced the highest survival rate. In the third trial, A. japonicus larvae were fed 20 000 cells mL^?1 day^?1 in three different frequency (2, 3 and 4 meals day^?1). The greatest body length was observed in larvae that received 3 meals day^?1. Survival and settlement of larvae fed 2 meals day^?1 were significantly lower than other two groups. These results suggest that Nannochloropsis sp. can be used as a diet for the large‐scale production of A. japonicus seed, and larvae fed three times daily at a ration of 20 000 cell mL^?1 day^?1 are recommended for hatchery production of A. japonicus.     

The effect of several microalgae isolated from East China Sea on growth and survival rate of postset juveniles of razor clam,Sinonovacula constricta (Lamarck, 1818)

Four species of microalgae (Thalassiosira weissflogii, Thalassiosira pseudonana, Nannochloropsis sp. and an unidentified species) were isolated from natural seawater of East China Sea. By studying the effect on growth and survival rates of two sizes of postset juveniles of razor clam, Sinonovacula constricta (Lamarck, 1818) (shell length 0.37 ± 0.05 mm and 1.07 ± 0.08 mm, respectively), the food value of these microalgae and four other commonly used microalgal species in China (Chaetoceros calcitrans, Isochrysis galbana, Platymonas subcordiformis and Nannochloropsis oculata) in single and two algal species diets were evaluated and compared. For smaller juvenile, when used as a monospecies diet, C. calcitrans and I. galbana supported the greatest shell growth and the highest survival rate, and P. subcordiformis, N. oculata and T. weissflogii produced the smallest shell growth and the lowest survival rate. Meanwhile, for the larger juvenile, single‐species algal diet had the greatest and weakest food value on T. weissflogii and N. oculata, respectively. Full‐mixed algal diets exhibited the strongest food value and most of the two species diets supported more food value than corresponding individual single microalgae diet. Moreover, shell growths and the survival rates of larger juvenile fed most of the two species diets were lower than fed single species of T. weissflogii.     

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout,Onchorynchus mykiss (Walbaum), in Spain

Flavobacterium psychrophilum is the causative agent of bacterial cold‐water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed‐field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 μg mL^?1, and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.     

An evaluation of fish health‐management practices and occupational health hazards associated with Pangasius catfish (Pangasianodon hypophthalmus) aquaculture in the Mekong Delta,Vietnam

This study aimed to evaluate the current status on the use of probiotics, disinfectants and antimicrobials in hatcheries, nurseries and grow‐out farms producing Pangasius catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam. Semi‐structured interviews were conducted with 83 aquaculture enterprises (15 hatcheries, 32 nurseries and 36 grow‐out farms). Farmers reported use of a total of 24 different antimicrobials, e.g. for treatment of bacillary necrosis and motile aeromonad septicaemia, and a variety of disinfectants, probiotics and nutritional supplements. In contrast to small‐scale farmers, all large‐scale grow‐out farmers studied were certified and therefore had higher levels of formal education and specialized aquaculture training to diagnose and treat diseases. All farmers prepared their own medicated feed with a high risk of treatment failure, negative environmental impact from released antimicrobials and resistance development. Small‐scale farmers were at particular occupational health risks when handling antimicrobials and other chemicals, e.g. mixing medicated feed with bare hands. There is an urgent need to improve knowledge and use innovative approaches, e.g. private‐public partnerships, to assure a prudent use of chemicals, to improve capacity and access to disease diagnosis, particularly for small‐scale grow‐out farmers and nurseries. Efforts to control use of antimicrobials in aquaculture should be coordinated with the livestock and human health sectors taking an One‐Health approach.     

Growth,food intake,protein retention and fatty acid profile in Octopus vulgaris (Cuvier, 1797) fed agglutinated moist diets containing fresh and dry raw materials based on aquaculture by‐products

The lack of specific compound diets for cephalopods is limiting the industrial development of some species. In this study, four agglutinated moist diets were tested in individually reared Octopus vulgaris (979 ± 151 g) for 8 weeks. All diets were based on bogue Boops boops, accidentally reared in fish farms (aquaculture by‐product), and agglutinated with alginate and calcium. One diet was based exclusively on bogue fillets, two on bogue fillets complemented with meat from two crab species (Portunus pelagicus, Grapsus grapsus) and the last one on bogue and G. grapsus meals. As a control diet, bogue and P. pelagicus were supplied fresh on alternate days. All diets induced similar feed intake (2.1–2.6% day^?1). However, the meal‐based diet induced negative growth in comparison with the control and the other agglutinated diets (0.80–0.85% day^?1). Higher lipid content in agglutinated diets (28–30% dw) in comparison with the control diet (16% dw) led to higher protein retention in muscle. These results underline the inadequacy of traditional meals in diets for cephalopods and that the inclusion of crab meat did not increase feeding rates and growth in O. vulgaris. The dietary fatty acid, with high levels of oleic and linoleic acid and low levels of ARA, clearly reflected in digestive gland, while only decreasing ARA and increasing EPA levels were observed in muscle, with no apparent negative effect on growth.