Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red‐spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture‐based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 μg mL^?1 (3 × 10⁵ TCID50 per mL), whereas for capture‐based ELISA, the sensitivity for the antigen in solution was 17 μg mL^?1 (35 × 10⁵ TCID50 per mL). The capture‐based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10⁸ TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv‐specific IgM is required, or for use in samples where PCR is difficult to perform.     

Essential oils to control ichthyophthiriasis in pacu,Piaractus mesopotamicus (Holmberg): special emphasis on treatment with Melaleuca alternifolia

In vitro effect of the Melaleuca alternifolia, Lavandula angustifolia and Mentha piperita essential oils (EOs) against Ichthyophthirius multifiliis and in vivo effect of M. alternifolia for treating ichthyophthiriasis in one of the most important South American fish, Piaractus mesopotamicus (Holmberg), were evaluated. The in vitro test consisted of three EOs, each at concentrations of 57 μL L^?1, 114 μL L ^?1, 227 μL L^?1 and 455 μL L ^?1, which were assessed once an hour for 4 h in microtitre plates (96 wells). The in vitro results demonstrated that all tested EOs showed a cytotoxic effect against I. multifiliis compared to control groups (P < 0.05). The in vivo treatment for white spot disease was performed in a bath for 2 h day^?1 for 5 days using the M. alternifolia EO (50 μL L ^?1). In this study, 53.33% of the fish severely infected by I. multifiliis survived after the treatment with M. alternifolia (50 μL L ^?1) and the parasitological analysis has shown an efficacy of nearly 100% in the skin and gills, while all the fish in the control group died. Furthermore, the potential positive effect of M. alternifolia EO against two emergent opportunistic bacteria in South America Edwardsiella tarda and Citrobacter freundii was discussed.     

Larval feeding behaviour of angel fish Pterophyllum scalare (Cichlidae) fed copepod Eucyclops serrulatus and cladoceran Ceriodaphnia quadrangula

Three different live diets, Eucyclops serrulatus, Ceriodaphnia quadrangula and equal combination of E. serrulatus copepodid and C. quadrangula, were offered to angelfish (Pterophyllum scalare) larvae viz 1‐week, 2‐week and 3‐week old at prey densities of 2, 5 and 10 individuals mL^?1. Results showed that 1‐week‐old P. scalare larvae consumed E. serrulatus copepodid at a rate of 31.3–56.7 ind. h^?1, C. quadrangula at 8.0–12.0 ind. h^?1, and mixture of E. serrulatus and C. quadrangula at 20.7–40.7 ind. h^?1. For 2‐week‐ and 3‐week‐old larvae, consumption rate increased accordingly. The electivity indices (E) of P. scalare (1‐week‐old larvae) for E. serrulatus copepodid were +0.18, +0.23 and +0.22 at prey densities of 2, 5 and 10 ind. mL^?1 respectively. Tendency towards E. serrulatus copepodid consumption reduced by aging P. scalare as indicated by the E values for 2‐ and 3‐week‐old larvae. However, growth and survival of P. scalare larvae was greatest when fed on combination of copepod E. serrulatus and C. quadrangula.     

Isolation and identification of Vibrio campbellii as a bacterial pathogen for luminous vibriosis of Litopenaeus vannamei

Outbreak of luminescent disease was reported from Litopenaeus vannamei shrimp farms in Zhangpu County, Southern China during May–July 2011. The clinical signs included fluorescent, less food consumption and high mortality. Bacteria were isolated from the infected shrimps. The pathogen, a luminescent bacterium named VH1 was identified as Vibrio campbellii based on MLSA analysis (16S rDNA, rpoD and toxR). The haemolysin (hly) gene specific in V. campbellii was detected in strain VH1. Pathogenicity test using immersion infection confirmed that strain VH1 was virulent to L. vannamei postlarvae and juveniles, and the LC₅₀ value was 1.55 × 10⁶ CFU mL^?1 and 1.7 × 10⁶ CFU mL^?1 respectively.     

Effect of Bacillus cereus as a water or feed additive on the gut microbiota and immunological parameters of Nile tilapia

We evaluated the effects of Bacillus cereus, as an additive in water and feed, on the gut microbiota and immunological parameters of Nile tilapia (Oreochromis niloticus) fingerlings. Experiments were performed in tanks and net cages respectively. Experiment 1: Tilapia were housed in tanks for 42 days, and B. cereus was added to the water at 1.0 × 10⁴ cfu mL^?1 (Treatment 1) and 1.0 ×10⁵ cfu mL^?1 (Treatment 2) weekly. For the control, no probiotic was added. Experiment 2: Tilapia were housed in cages for 42 days, and the feed was supplemented with B. cereus at 1.0 × 10⁷ cfu g^?1 (Treatment 1) and 1.0 × 10⁸ cfu g^?1 (Treatment 2) weekly. For the control, no probiotic was added. Each treatment contained three replicates, with 50 male tilapias per replicate. The fish from the probiotic treatments in both tank and cage experiments had significantly higher serum lysozyme and peroxidase activities than the control. In the cage experiment, alkaline phosphatase and total superoxide dismutase activities in tilapia were significantly higher in probiotic treatments compared with the control. The results of polymerase chain reaction‐denaturing gradient gel electrophoresis showed that B. cereus supplementation in the feed and water affected the autochthonous gut bacteria community of tilapia and stimulated various potentially beneficial bacteria. Therefore, B. cereus, as a water or feed additive, could enhance the immune status and affect the gut microbiota of tilapia. Bacillus cereus was more effective as a feed supplement rather than a water additive for enhancing the immune status of tilapia.     

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.     

A3α‐peptidoglycan extracted from Bifidobacterium sp. could enhance immunological ability of Apostichopus japonicus

To assess the effects of A3α‐peptidoglycan (A3α‐PG) extracted from Bifidobacterium sp. on the immune response and disease resistance of sea cucumber, different concentrations (0, 0.5, 5 and 50 mg mL^?1) of A3α‐PG suspensions were used to perform hypodermic injection on Apostichopus japonicus, followed by a Vibrio splendidus challenge. Total coelomocyte count (TCC), phagocytosis activity and activities of four immunological enzymes in both cell‐free coelomic fluid (extra‐cellular, EC) and coelomocyte lysate supernatant (intracellular, IC), including acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD) and peroxidase (POD), were measured at 2, 6, 14 and 24 h post injection (hpi). The TCC was not significantly affected (P > 0.05) by A3α‐PG, ranging from 1.84 × 10⁶ to 3.53 × 10⁶ cells mL^?1. The coelomocyte phagocytosis activity was significantly activated (P < 0.05) in all the A3α‐PG treatments, whereas no significant difference was observed between them except 24 hpi (P > 0.05). The EC‐ACP activity in the 5.0 mg mL^?1 treatment increased significantly (P < 0.05) at all sampling times, while the IC‐ACP activity in the 50 mg mL^?1 treatment increased significantly (P < 0.05) at 2 hpi. Also, the 5.0 mg mL^?1 treatment had significant (P < 0.05) increase in the EC‐ALP activity within 14 hpi and the EC‐POD activity at 2 hpi, respectively, while significantly (P < 0.05) enhanced IC‐ALP and IC‐POD activities were observed in the 50 mg mL^?1 treatment within 6 hpi and at 2 hpi, respectively. Only the 5.0 mg mL^?1 treatment showed significant (P < 0.05) increase in the EC‐SOD activity at 2 hpi and IC‐SOD activity within 14 hpi, respectively. The challenge test showed that the animals treated with 50 mg mL^?1 of A3α‐PG had notably lower cumulative mortality after 14 days following V. splendidus exposure. All together, these results suggest that A3α‐PG could positively enhance immune response that effectively promotes the health status of A. japonicus against V. splendidus infection.     

In vitro and in vivo antifungal activity of Satureja cuneifolia ten essential oil on Saprolegnia parasitica strains isolated from rainbow trout (Oncorhynchus mykiss,Walbaum) eggs

In the present study, the chemical composition and the antifungal properties against Saprolegnia parasitica (in vitro and in vivo) of the essential oils of thyme (Satureja cuneifolia) from Mediterranean region of Turkey were evaluated for the first time. The composition of oils was analysed using gas chromatography/mass spectrometry (GC/MS). The major constituents of oil of S. cuneifolia were cavracrol (46,84%) and cymene (16.90%). Antifungal effects of S. cuneifolia essential oil against S. parasitica strains (A1 and E1) were detected by disc diffusion and tube dilution assays. The antifungal effect of S. cuneifolia was determined to be stronger against S. parasitica E1 isolate (MIC 50 μL mL^?1, MLC 250 μL mL^?1) compared with S. parasitica A1 isolate (MIC 50 μL mL^?1, MLC 500 μL mL^?1). Following in vitro assays, effective doses of S. cuneifolia for disease control in rainbow trout eggs experimentally infected with S. parasitica were investigated. For this aim, infected eggs were treated with the essential oil (0, 5, 10, 20 and 50 ppm) during incubation period (21 days) after fertilization. Formalin (5 mL L^?1) was used as positive control. Hatching rate of eggs at the end of incubation period were calculated. The highest hatching rates were recorded in S. parasitica E1 strain at 5 and 10 ppm concentrations of S. cuneifolia and in S. parasitica A1 strain at 10 and 20 ppm (P < 0.05).     

A comparison between marine and terrestrial invertebrate meals for mirror carp (Cyprinus carpio) diets: Impact on growth,haematology and health

Invertebrate meals (e.g. polychaetes and insects) present novel and sustainable high‐quality nutrient sources for use in fish feed formulations. To test this innovative source, an eleven‐week feeding trial was conducted evaluating the effects of replacing the fishmeal (FM) component as an example of a superior protein source (FM CTRL) with ragworm meal (RW, Nereis virens) and/or silkworm pupae (SWP, Bombyx mori) in mirror carp (Cyprinus carpio) diets. Three experimental diets with partial replacement of FM (diets: RW + FM, SWP + FM and RW + SWP + FM) were formulated. All diets were formulated to be iso‐nitrogenous, iso‐lipidic and iso‐energetic. Growth performance and feed utilization indices were assessed, and the feeding trial concluded with the analysis of haematological parameters to provide an indication of carp physiological and health status. Mean weight gain was greatest in mirror carp fed RW + FM (60.83 fish^?1 day^?1; P < 0.05 vs. all other diets) followed by SWP + FM (40.62 g fish^?1 day^?1; P < 0.05 vs. all other diets). The least weight gain was achieved in fish fed FM + SWP + RW+ and FM CTRL (34.34 and 33.96 g fish^?1 day^?1, respectively; not significantly different from each other). Fish fed on RW + FM diet had significantly lower plasma ammonia concentrations than any other dietary groups (P = 0.04). Mirror carp fed on SWP + FM diet (111.52 units mL^?1) were observed to have a marked enhancement in alternative complement activity than FM CTRL (79.21 units mL^?1, P = 0.041). Both ragworm and silkworm pupae meal present attractive sustainable functional feed component in carp diets, with benefits on enhancing growth performance and specific physiological parameters.     

Estimation of infectious dose and viral shedding rates for infectious pancreatic necrosis virus in Atlantic salmon,Salmo salar L., post‐smolts

Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post‐smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 10² to 10^?4 50% end‐point tissue culture infectious dose (TCID₅₀) mL^?1 sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post‐smolts was <10^?1 TCID₅₀ mL^?1 by bath immersion (4 h at 10 °C). The peak shedding rate for IPNV following intraperitoneal challenge using post‐smolts was estimated to be 6.8 × 10³ TCID₅₀ h^?1 kg^?1 and occurred 11 days post‐challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.     

Differential pathogenicity of five Streptococcus agalactiae isolates of diverse geographic origin in Nile tilapia (Oreochromis niloticus L.)

Streptococcus agalactiae is an emerging pathogen of fish and has caused significant morbidity and mortality worldwide. The main objective of this study is to assess whether pathogenic differences exist among isolates from different geographic locations. Nile tilapia (Oreochromis niloticus L.) were administered an intraperitoneal injection of suspension containing USA, Brazil, Honduras, Israel, or Kuwait S. agalactiae isolates at concentrations ranging from 10² to 10⁷ cfu mL^?1. The LD₅₀ values 7 days after challenge were as follows: USA (1.0 × 10² cfu mL^?1), Brazil (1.5 × 10³ cfu mL^?1), Honduras (6.8 × 10³ cfu mL^?1), Israel (1.0 × 10⁴ cfu mL^?1) and Kuwait (7.2 × 10⁵ cfu mL^?1). Fish from all groups exhibited lethargy, anorexia, exophthalmia, corneal opacity, erratic swimming, petechiae and mortality. Opercular clearing and ascites were only found after infection with certain geographic isolates. The findings in this study indicate that S. agalactiae isolates of different geographic origin can cause significant mortalities after experimental challenge and can have different pathogenic capacities. Isolates from the Americas (USA, Brazil and Honduras) were more pathogenic to Nile tilapia than isolates from the Middle East/Asia (Israel and Kuwait).     

Effect of dietary supplementation of periphyton on growth,immune response and metabolic enzyme activities in Penaeus monodon

A 60‐day indoor trial was conducted to study the effect of periphyton supplementation on metabolic and immune responses in tiger shrimp, Penaeus monodon. Periphyton developed over bamboo substrate in outdoor tanks (15 m²) was used as dietary supplement for P. monodon (2.02 ± 0.04 g) reared in 1000 L FRP tanks. Graded levels of periphyton were included in shrimp basal diets: 0% (P0), 3% (P3), 6% (P6), 9% (P9) and P0 diet with natural periphyton (NP) over bamboo substrate. At the end of the trial, P6 and NP showed significantly higher (P < 0.01) growth rate, 23.9% and 20%, respectively, compared with control, P0. Comparatively, lower level of metabolic enzymes, such as lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and alanine aminotransferase, was recorded in treatments P3, P6 and NP compared with control, P0. The periphyton‐supplemented group, P3 had significantly higher (P < 0.05) superoxide dismutase (15.83 ± 0.96) and catalase activity (15.73 ± 0.69) compared to 6.88 ± 2.84 and 9.15 ± 0.67 unit mg^?1 protein min^?1_, respectively, in P0. Similarly, higher total haemocyte counts, 32.58 ± 1.30, 28.51 ± 3.12 and 27.26 ± 4.43 × 10⁶ cells mL^?1_, were recorded in P6, NP and P3, respectively, compared to P0, 23.57 ± 1.80 × 10⁶ cells mL^?1. After challenge with Vibrio harveyi, P3 recorded the highest relative percentage survival 67% followed by NP (58%) and P6 (42%) compared with control. However, treatment with highest periphyton inclusion (P9) did not differ significantly with P0 on growth and immunological parameters. This study indicates that periphyton supplementation at 3–6% level improves growth, immune response and metabolic activities in P. monodon.     

Hexamitiasis leads to lower metabolic rates in rainbow trout Oncorhynchus mykiss (Walbaum) juveniles

This study assessed the effects of Hexamita salmonis (Moore) on metabolism of rainbow trout Oncorhynchus mykiss (Walbaum) and its effect on the host's susceptibility to infectious pancreatic necrosis virus (IPNV) after antiparasitic treatment. Rainbow trout naturally infected with H. salmonis were treated with 10 mg metronidazole kg fish^?1 per day, and their physiological recovery was assessed through measuring resting metabolism on the 7th, 14th, 21st and 28th day after treatment. In addition, we exposed the naïve fish to H. salmonis and measured the resting metabolism (oxygen consumption as mg O₂ kg^?1 per hour) on the 10th, 20th and 30th day after the exposure to assess the variation in metabolic rates after infection. Significantly lower rates of metabolic activity (P < 0.05) were anticipated 20 days after infection with H. salmonis compared with the fish infected with H. salmonis for 10 days or with the parasite‐free fish. Similarly, the treated fish needed about 20 days to fully recover from hexamitiasis. The susceptibility of rainbow trout to IPNV remained unchanged in the presence of H. salmonis. Weight loss was significantly higher (P < 0.05) in infected than that in the parasite‐free fish. Fish should be examined regularly for H. salmonis and treated immediately whether found to prevent economic losses and excessive size variation.     

Investigations on the role of the salmon louse,Lepeophtheirus salmonis (Caligidae), as a vector in the transmission of Aeromonas salmonicida subsp. salmonicida

A bacteria–parasite challenge model was used to study the role of sea lice, Lepeophtheirus salmonis (Copepoda), as a vector of Aeromonas salmonicida subsp. salmonicida. Three hypotheses were tested: (i) L. salmonis can acquire A. salmonicida subsp. salmonicida via water bath exposure; (ii) L. salmonis can acquire the bacteria via parasitizing infected Atlantic salmon, Salmo salar; and (iii) L. salmonis can transmit the bacteria to naïve Atlantic salmon via parasitism. Adult L. salmonis exposed to varying A. salmonicida subsp. salmonicida suspensions (10¹–10⁷ cells mL^?1) for 1.0, 3.0 or 6.0 h acquired the bacteria externally (12.5–100%) and internally (10.0–100%), with higher prevalences associated with the highest concentrations and exposures. After exposure to 10⁷ cells mL^?1, viable A. salmonicida subsp. salmonicida could be isolated from the external carapace of L. salmonis for 120 h. Lepeophtheirus salmonis also acquired the bacteria externally and internally from parasitizing infected fish. Bacterial transmission was observed only when L. salmonis had acquired the pathogen internally via feeding on ‘donor fish’ and then by parasitizing smaller (<50 g) ‘naive’ fish. Under specific experimental conditions, L. salmonis can transfer A. salmonicida subsp. salmonicida via parasitism; however, its role as a mechanical or biological vector was not defined.     

Retracted: Effect of concentration of the microalga Dunaliella tertiolecta on survival and growth of fairy shrimp,Phallocryptus spinosa Milne Edwards, 1840 (Crustacea: Anostraca)

We investigated the effects of concentration of the microalga Dunaliella tertiolecta on the growth and survival of fairy shrimp, Phallocryptus spinosa. Newly hatched nauplii were stocked into containers, maintained at different concentrations of D. tertiolecta (at 18, 36, 54, 72 and 90 × 10⁶ cells mL^?1). All treatments were in quadruplicate and each replicate was stocked with 100 larvae in a 2‐L cylindrical bowl. We studied the survival and growth of the fairy shrimp after 3, 6, 9, 12 and 15 days of culture. The results indicated significant differences, in terms of growth and survival, of fairy shrimps fed at different algal densities. The highest and lowest growth and survival among the treatments were observed on Day 15, the highest in animals fed at a concentration of 90 × 10⁶ cells mL^?1 and the lowest in animals fed at a concentration of 18 × 10⁶ cells mL^?1. We conclude that the growth and survival of the P. spinosa increased with increasing density of algae, to a threshold level. Within certain concentration limits, the addition of D. tertiolecta substantially improved the performance of larval culture of P. spinosa, suggesting that this fairy shrimp has potential in terms of aquaculture development.     

In vivo evaluation of the effects of commercial Bacillus probiotics on survival and development of Litopenaeus vannamei larvae during the early hatchery period

Three in vivo experiments were conducted to measure the effectiveness of commercial Bacillus probiotics on survival and development of Litopenaeus vannamei larvae from nauplii 3–4 (N_3–4) to zoea 3 (Z₃) stages. Experiment I: Nine commercial Bacillus probiotics were individually added to larvae twice at N₆ and Z₁ at levels of 2, 20, and 100 mg L^?1. Only six of nine products at 20 mg L^?1 exhibited higher or significantly higher larval survival (P < 0.05) than the control. Experiment II: Two superior products from the first experiment were administered to larvae with six dose frequencies at 20 mg L^?1. For both products, three doses, once at each of N_3–4, N₆ and Z₁ stages, yielded the best larval survival and development rates (P < 0.05), and these were confirmed by enhanced activities of tryptase and amylase. Experiment III: The isolates of these two products, identified as Bacillus subtilis and Bacillus licheniformis, were delivered to larvae singly at concentrations of 1.0 × 10⁸ and 1.0 × 10⁹ CFU L^?1, or at the same concentrations by mixing the two equally. At 1.0 × 10⁹ CFU L^?1, B. subtilis exerted more beneficial effects on larvae than B. licheniformis or the mixture. Therefore, the optimal dosage and dose frequency of commercial Bacillus products should be evaluated prior to large‐scale application in shrimp hatcheries to avoid futility or even adverse effects, as spore counts and non‐bacterial ingredients are key factors influencing the efficacy of Bacillus probiotics.     

Experimental Lactococcus garvieae infection in rainbow trout,Oncorhynchus mykiss,Walbaum 1792: a comparative histopathological and immunohistochemical study

The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young‐Adult IP groups and Young‐Adult IM groups, each contain 30 fish) and four control groups (Young‐Adult IP Control groups and Young‐Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976 . Fish in the IP groups were intraperitoneally administered an inoculate containing 10⁶ cfu mL^?1 bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10⁸ cfu mL^?1 bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin‐biotin‐peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.     

Dynamics of glucose in the haemolymph of female giant freshwater prawn,Macrobrachium rosenbergii,influences reproductive and non‐reproductive moulting cycles

Glucose, when measured in haemolymph, has been found to reflect a useful predictor of energetic investment. This study evaluated the pattern of glucose in the haemolymph, with an attempt to gain a better insight into the role of glucose as nutritional source of ovarian development and energy reserves during reproductive and non‐reproductive moulting cycles. The haemolymph of female giant freshwater prawn, Macrobrachium rosenbergii, was obtained at eight different moulting stages, and levels of glucose were determined using an enzymatic colorimetric glucose‐oxidase method in parallel with a histological examination of ovarian development. Glucose levels were relatively low (0.15 ± 0.02 mg mL^?1) at D₀ stage, an abrupt increase (0.52 ± 0.13 mg mL^?1) during premoult D₁ stage and declined (0.32 ± 0.10 and 0.31 ± 0.09 mg mL^?1) during premoult D₂ and D₃ stages, respectively; thereafter, a slight increase (0.43 ± 0.09 mg mL^?1) occurred at post‐moult A stage. The progression of ovarian growth, marked by an increasing gonadosomatic index (GSI) pattern during the reproductive moulting cycle (C₀–D₃ stages), was directly proportionate to fluctuations in glucose levels. GSI was significantly positively correlated with glucose (R = 0.40; P < 0.05). In contrast, glucose was notably higher at post‐moult A and premoult D₂ stages during non‐reproductive moulting cycle, the period during which glucose is crucial for exoskeletal chitin synthesis. At this particular stage, a negative correlation between body weight and glucose (R = ?0.36; P < 0.05) was observed. The dynamics of glucose in the haemolymph of female M. rosenbergii correlated with ovarian growth, which signify that glucose as nutritional source for vitellogenesis, and affects the body weight of this species.     

The in‐vitro antibacterial effects of organic salts,chemical disinfectants and antibiotics against pathogens of black disease in fairy shrimp of Thailand

The antibacterial effects of organic salts, chemical disinfectants and antibiotics were evaluated on cultures of Aeromonas hydrophila C03, Aeromonas sobria C26, A. sobria C29, Aeromonas caviae C24 and Acinetobacter sp. SH‐94B, the pathogens that cause black disease found in fairy shrimps (Streptocephalus sirindhornae Sanoamuang et al. (2000) and Branchinella thailandensis Sanoamuang, Saengphan & Murugan) of Thailand. The minimal inhibitory concentrations (MICs) of organic salts (sodium chloride and potassium chloride) and antibiotics (oxytetracycline dihydrate, streptomycin sulphate, kanamycin monosulphate, chloramphenicol and ampicillin) were determined using the agar‐dilution method. The effect of chemical disinfectants (sodium hypochlorite and chlorine dioxide) was evaluated by exposing bacteria to different concentrations of these chemicals for different periods of time. Interestingly, all strains were intrinsically resistant to 0.25–3% sodium chloride and potassium chloride. The effect of sodium hypochlorite was greater than that of chlorine dioxide, and 5–20 μg mL^?1 of sodium hypochlorite was sufficient to inhibit the growth of these bacteria, but the exposure time varied, depending on the bacterial species. Of the antibiotics tested, chloramphenicol and oxytetracycline dihydrate completely inhibited the selected strains. Chloramphenicol showed the highest antibacterial effect against all pathogenic species – the MIC and minimal bactericidal concentration (MBC) ranged from 0.8 to 3.1 μg mL^?1 from 3.1 to 6.25 μg mL^?1, respectively. To achieve control of black disease during cultivation of fairy shrimp, data derived from this study can be used as a basis for further toxicity tests in vivo.