Development of an ELISA to measure the humoral immune response of hybrid striped bass Morone chrysops×M. saxatilis to Streptococcus iniae

A previously developed monoclonal antibody (mAb) specific for the heavy chain of hybrid striped bass (HSB) Morone chrysops×M. saxatilis immunoglobulin was used in an assay to detect the humoral response to antigens of Streptococcus iniae. In order to validate this assay, an anti‐S. iniae antibody was produced in HSB by immunization with formalin‐killed cells of S. iniae mixed with Freund's complete adjuvant (FCA). After boosting with cells mixed with Freund's incomplete adjuvant (FIA), fish were challenged with live S. iniae by i.p. injection. This resulted in mortalities of 100%, 13% and 7% for non‐immunized, immunized and non‐challenged fish respectively. Live S. iniae were recovered only from moribund non‐immunized challenged fish, indicating that the immunization conferred protection against S. iniae. Sera were taken at 28 and 42 days post challenge and anti‐S. iniae antibody was measured by both agglutination and indirect ELISA. Titres of anti‐S. iniae antibody increased only in the immunized group as measured by agglutination and ELISA. Serum complement measured in the final bleeding was significantly higher in the immunized group. Western blotting using immune HSB serum indicated that the predominant antigen in this case was the high molecular weight polysaccharide of S. iniae.     

Extracellular products from Mycobacterium spp. in fish

Mycobacterium spp. isolated from food and ornamental fish in Thailand (TB1, TB40, TB267 and TB268), and the type strains Mycobacterium marinum (NCIMB 1298), M. fortuitum (NCIMB 1294) and M. chelonae (NCIMB 1474) were cultured in Long's medium, Eagle's minimum essential medium, Sauton's medium and modified Sauton's medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB 40, TB267, TB268 and M. marinum NCIMB 1298 in particular, whereas growth and production of ECP for all strains was limited in Long's medium. SDS-PAGE protein profiles of ECPs from 14-day culture supernatants showed major bands at 65 and <14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M. marinum averaged approximately four- to 10-fold higher than from strains cultured for 14 days at 28°C. Enzyme testing for the type strains indicated only mucinase activity for M. marinum, while lipase and RNase activities were detected for M. chelonae and M. fortuitum . Protease and DNase activities could not be detected for any of the Mycobacterium spp. tested. The medium lethal dose (LD₅₀) of ECP to rainbow trout and Nile tilapia was greater than 400 μg protein fish^–1.     

Enhancement of protective immunity in blue gourami,Trichogaster trichopterus (Pallas), against Aeromonas hydrophila and Vibrioanguillarum by A. hydrophila major adhesin

Blue gourami, Trichogaster trichopterus (Pallas), were intraperitoneally immunized with major adhesin, a 43 kDa OMP protein isolated from fish Aeromonas hydrophila, in the presence of Freund's complete adjuvant (FCA). Three weeks later, a booster injection of adhesin without FCA was administered. Control group fish were similarly treated with phosphate‐buffered saline (PBS) and FCA. Results showed that anti‐adhesin serum obtained from fish after booster immunization exhibited very strong ability in agglutinating bacterial cells. Although this antiserum had no bactericidal effect, it could significantly inhibit serologically different strains of A. hydrophila from invading EPC (Epithelioma papillosum of carp) cells in vitro. In addition, the proliferative response of head kidney leucocytes of these immune fish was significantly increased as compared to that of the control. The results also showed that the major adhesin could provide significant protective immunity to fish against the challenge by homologous and heterologous strains of A. hydrophila and one virulent strain of Vibrio anguillarum.     

Characterization of serum and mucosal antibody responses and relative per cent survival in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and challenge with Flavobacterium psychrophilum

Serum and mucosal antibody responses of juvenile rainbow trout, Oncorhynchus mykiss, were characterized by enzyme‐linked immunosorbent assay (ELISA) following immunization with various preparations of formalin‐killed Flavobacterium psychrophilum cells. The protective nature of these preparations was then determined by immunizing rainbow trout fry and challenging with the bacterium. Juvenile rainbow trout immunized intraperitoneally (i.p.) with formalin‐killed F. psychrophilum emulsified with Freund's complete adjuvant (FCA), and i.p. with formalin‐killed F. psychrophilum either with or without culture supernatant generated significant serum antibody responses by 6 and 9 weeks, respectively. Significant mucosal antibody responses were detected by 9 weeks only in fish immunized i.p. with killed F. psychrophilum/FCA. Following immunization and bacterial challenge of rainbow trout fry, protective immunity was conferred in F. psychrophilum/FCA and saline/FCA groups with relative per cent survival values of up to 83 and 51, respectively. Significant protection was not observed in treatment groups immunized by immersion or i.p. without adjuvant at the challenge doses tested. Results suggest that stimulation of non‐specific immune factors enhances the ability of fish to mount a protective immune response, but specific antibody appears necessary to provide near complete protection. In this study, an ELISA was developed to monitor anti‐F. psychrophilum antibody production in trout. The relationship of such responses to protective immunity suggests that future vaccination strategies against coldwater disease may require stimulation of both the innate and adaptive arms of the immune response.     

Mixed mycobacterial infections in farmed sturgeons

Based on microbiological and histopathological examinations and DNA sequencing, several outbreaks of mycobacteriosis in the reared sturgeons, including Chinese sturgeon (Acipenser sinensis Gray) and Amur sturgeon (Acipenser schrencki), were identified during 2009 to 2010. Forty‐nine isolates of non‐tuberculous mycobacteria(NTM)were isolated from 19 diseased sturgeons. In total, seven species of Mycobacterium were identified, namely, Mycobacterium chelonae, Mycobacterium marinum, Mycobacterium gordonae, Mycobacterium fortuitum, Mycobacterium szulgai, Mycobacterium arupense and Mycobacterium porcinum. Among them, M. marinum was found to be more prevalent (89.5%) compared with the other mycobacterial species. When two molecular biological methods, PCR‐DGGE (denaturing gradient gel electrophoresis) analysis and rpoB gene library sequencing, were used to analyse the mycobacterial DNAs extracted from the diseased fish tissues, mixed infections of two or three mycobacterial species were found being the predominant infection form (94.7%) in sturgeon mycobacteriosis. M. marinum was the only one species that caused sturgeon mycobacteriosis alone. Virulence assay showed that M. marinum possessed stronger pathogenicity to zebrafish killing 100% of fish in 28 days at 10³ cfu/fish than the other species. These results suggested that M. marinum is the major pathogenic bacteria in sturgeon mycobacteriosis. To the best of our knowledge, this study is the first report on mycobacteriosis in farmed Chinese and Amur sturgeons as well as the first isolation of M. porcinum and M. arupense from fish.     

Therapeutic and prophylactic immunization against Streptococcus iniae infection in hybrid striped bass (Morone chrysops×Morone saxatilis)

Vaccination strategies have traditionally been used as preventative or prophylactic measures against disease (prophylactic immunization) in uninfected fish. Alternatively, therapeutic or remedial measures, such as antibiotic administration, are commonly employed to treat disease in infected fish. Vaccination as a therapeutic measure (therapeutic immunization), however, has not been adequately explored in sub‐clinically infected fish. Therapeutic and prophylactic immunization with three Streptococcus iniae vaccines, formalin‐killed whole S. iniae cells (FKC vaccine), concentrated S. iniae extracellular products (greater than 2 kDa) (ECP vaccine) and a combination of killed cells and extracellular products (FKC+ECP vaccine), were tested in hybrid striped bass, Morone chrysops×Morone saxatilis, previously naturally infected with S. iniae. Fish (mean weight 10.0 g) were injected intraperitoneally (IP) or intramuscularly (IM) with one of each of the vaccines, tryptic soy broth (TSB‐control) or non‐injected (non‐injected control) to evaluate therapeutic effects (Trial 1). Survivors of the natural infection and ECP and FKC+ECP vaccine immunization and another lot of non‐injected control fish were immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 at 44 days post‐immunization to evaluate vaccine efficacy (Trial 2). Hybrid striped bass (1.0 g) were also IM injected with S. iniae ECP vaccine at an aquaculture facility and immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 12 weeks post‐immunization (Trial 3). The ECP and FKC+ECP vaccines, regardless of injection route, significantly (P<0.001) increased survival in asymptomatic, sub‐clinically infected fish thereby providing therapeutic merit. Hybrid bass immunized IP or IM had mean per cent survival values ranging from 78 to 96 at 44 days post‐immunization (Trial 1) and 69–97 post challenge (Trial 2). Survival of fish injected with TSB or immunized with FKC vaccine was significantly lowered and ranged from 12 to 13 by IP injection and 40 to 50 by IM injection and thus, the FKC vaccine had no therapeutic effect. The survival of hybrid striped bass IM immunized with S. iniae ECP vaccine in field Trial 3 was 91 and the RPS was 83. These results demonstrate that therapeutic immunization using S. iniae ECP and FKC+ECP vaccines can control a natural S. iniae infection. Furthermore, S. iniae ECP or FKC+ECP vaccines can also be used prophylacticly to protect hybrid striped bass against subsequent pathogen challenge.     

Immunization of rainbow trout Oncorhynchus mykiss (Walbaum) with a crude lipopolysaccharide extract from Flavobacterium psychrophilum

Control methods for Flavobacterium psychrophilum are limited and oftentimes ineffective; hence, research efforts have focused on vaccine development. This study tested the hypothesis that a crude lipopolysaccharide (LPS) extract from F. psychrophilum will elicit a protective immune response in rainbow trout Oncorhynchus mykiss (Walbaum) against F. psychrophilum challenge. Rainbow trout (mean weight, 3 g) were immunized intraperitoneally with the following treatment and control preparations: 10 μg of crude LPS with or without Freund's complete adjuvant (FCA), 25 μg of crude LPS with or without FCA and saline with or without FCA. Immunization of fish with 10 or 25 μg of crude LPS/FCA resulted in significant antibody responses against F. psychrophilum using ELISA with a whole‐cell lysate as the coating antigen, but only minimal levels of protection were conferred following F. psychrophilum challenge at 14 weeks post immunization. Western blot analyses demonstrated that fish exhibited antibodies specific for low‐molecular mass proteins present in the crude LPS extract, but did not exhibit antibodies specific for F. psychrophilum LPS. The results indicate that higher immunization doses and/or the use of an alternative extraction method that yields larger LPS molecules (23–70 kDa) may be necessary to elicit specific antibody responses against F. psychrophilum LPS.     

Renal threshold for urinary glucose excretion by tilapia in response to orally administered carbohydrates and injected glucose

Two experiments, oral carbohydrate administration (Experiment 1) and vein glucose injection (Experiment 2), were conducted to gain more insight into the ability of hybrid tilapia, Oreochromis niloticus × O. aureus, to utilize different carbohydrates and to establish the kidney threshold for urinary glucose excretion. In Experiment 1, both glucose and starch were administered orally after the tilapia were fasted for 24 h. Plasma and urine were sampled from the fish at selected time intervals from 1 to 24 h thereafter. Higher (p<0.05) plasma and urine glucose concentrations were found in fish fed on glucose than in fish fed on starch. The concentration of plasma glucose of tilapia peaked at 3 h (25.45 mM for glucose; 8.24 mM for starch) after the oral ingestion of both carbohydrates. Maximum urinary glucose concentrations (48 mM for glucose; 10 mM for starch) in fish fed glucose and starch were at 3 and 4 h post administration. In Experiment 2, five concentrations (0, 0.08, 0.12, 0.16 and 0.24 g glucose ml^–1) of glucose solution were injected into the caudal vein of the tilapia. Urine were sampled from the fish at 30-min time intervals from 0 to 6 h after the injection. Blood was sampled at 1 h after the injection. Higher urinary glucose concentrations were observed in fish injected with 0.12 g glucose ml^–1. When the urinary glucose concentrations in fish injected with the various glucose concentrations were plotted against the plasma glucose concentrations of the fish 1 h after injection, the kidney threshold for urinary glucose excretion in tilapia appeared to be about 6 mM.     

Plasmatic levels of cortisol in the response to acute stress in Nile tilapia, Oreochromis niloticus (L.), previously exposed to chronic stress

Many studies have been made about the physio-logical effects of isolated chronic or acute stress. However, few studies have been made to assess the combination of both responses. The fish submitted to chronic stress may be subjected to an additional acute stressor. The aim of the present work was to evaluate the acute stress response in Nile tilapia Oreochromis niloticus (L.) previously subjected to chronic stress. For this, two experiments were performed. In the first experiment, the fish were subjected to chronic stress followed by an additional acute stress. In the second experiment, the fish were submitted only to an acute stress. The data showed that Nile tilapia fingerlings can adapt to chronic stress situations, and this decreases, but does not eliminate, their capacity to respond to an additional acute stressor. In both experiments, plasma cortisol levels reached a peak 1 h after administration of the acute stressor. In fish previously submitted to chronic stress, the highest concentration of plasma cortisol measured was 196 ng mL^–1. This value was significantly different from the cortisol concentration obtained in the second experiment (267 ng mL^–1) with non-chronically stressed fish. The data also suggest that the chronic stress response can provoke a reduction in performance and growth rates compared with non-stressed fish.     

Immunohistochemical and Taqman real-time PCR detection of mycobacterial infections in fish

Real‐time PCR and immunohistochemistry (IHC) assays were developed to detect fish mycobacterial infections at the genus level, based on the RNA polymerase β subunit (rpoB) gene and polyclonal anti‐Mycobacterium rabbit serum, respectively. The PCR assay positively identified a number of pathogenic mycobacteria including Mycobacterium abscessus, M. avium ssp. avium, M. bohemicum, M. chelonae ssp. chelonae, M. farcinogenes, M. flavescens, M. fortuitum ssp. fortuitum, M. gastri, M. gordonae, M. immunogenicum, M. malmoense, M. marinum, M. montefiorense, M. phlei, M. phocaicum, M. pseudoshottsii, M. salmoniphilum, M. senegalense, M. shottsii, M. smegmatis, M. szulgi and M. wolinskyi. A detection limit equivalent to 10² cfu g^?1 was registered for M. salmoniphilum‐infected fish tissue. The IHC precisely localized both free and intracellular mycobacteria in tissues and detected mycobacterial infections down to 10² cfu g^?1 tissue. Both assays were found to be more sensitive than Ziehl–Neelsen (ZN) staining, where the detection limit was below 8 × 10³ cfu g^?1 tissue. Although specificity testing of the real‐time PCR against a panel of non‐Mycobacterium spp. revealed a degree of cross‐reaction against pure DNA extracted from Nocardia seriolae and Rhodococcus erythropolis, no cross‐reactions were identified (by either real‐time PCR or IHC) on testing of formalin‐fixed paraffin‐embedded (FFPE) tissues confirmed to be infected with these bacteria. The broad applicability of both assays was confirmed by analysis of FFPE tissues from a range of fish species infected with diverse Mycobacterium spp. The results indicate that both assays, alone or in combination, constitute sensitive tools for initial, rapid diagnosis of mycobacteriosis in fish. This should in turn allow rapid application of more specific studies, i.e. culture based, to identify the specific Mycobacterium sp. involved.     

Transmission of Mycobacterium chelonae and Mycobacterium marinum in laboratory zebrafish through live feeds

The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is particularly concerning because it is a common disease associated with chronic infections in these fish. Infections are also a source of uncontrolled experimental variance that may influence research results. Live feeds for zebrafish are common and include paramecia (Paramecium caudatum), brine shrimp (Artemia franciscana) and rotifers (Branchionus spp.). Although nutritionally beneficial, live feeds may pose a biosecurity risk. In this study, we investigate transmission of Mycobacterium chelonae and Mycobacterium marinum through these three live feeds. We show that all three live feeds ingest both M. marinum and M. chelonae and can transmit mycobacterial infections to zebrafish. This observation emphasizes the need for live feeds to be included in the consideration of potential biosecurity risks. This study is of importance to other beyond the zebrafish community, including those of additional aquatic models and those using live feeds for other types of aquaculture.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

The effect of the introduction of Nile tilapia (Oreochromis niloticus,L.) on small indigenous fish species (mola,Amblypharyngodon mola,Hamilton; chela,Chela cachius,Hamilton; punti,Puntius sophore,Hamilton)

This is the first controlled experiment to quantify the effect of introduced tilapia on indigenous species. This experiment was conducted in small earthen ponds (100 m²) to assess the impact of mixed‐sex or all‐male Nile tilapia (Oreochromis niloticus) on small indigenous species (SIS) commonly found in south Asia, mola (Amblypharyngodon mola), chela (Chela cachius) and punti (Puntius sophore). Ponds were fertilized, then stocked with 0.56 fish m^?2 of water surface area in the mixed‐sex and all‐male tilapia treatments and 0.42 fish m^?2 in the treatment without tilapia. No additional nutritional inputs were applied after stocking. Treatments were: mixed‐sex tilapia with SIS, mono‐sex male tilapia with SIS and SIS without tilapia (control). All treatments were stocked with 14 fish per species. All species reproduced during the 21‐month culture duration. The number of recruits varied by species, Tilapia reproduced in greater numbers than SIS. Tilapia numbers at harvest were the highest (451 ± 25/100 m²) in the mixed‐sex treatment compared with mola (221 ± 22/100 m²), chela (94 ± 8/100 m²) and punti (100 ± 7/100 m²). The number of mola was higher (399 ± 33/100 m²) in the all‐male tilapia treatment. There was reduction in the number of mola and chela in the treatment containing mixed‐sex tilapia. Gut content analysis combined with water sampling revealed that all fish species fed selectively. Significant interspecies dietary overlap was found between Nile tilapia and SIS and among SIS. Thus, there is potential for tilapia to compete with indigenous fish species when space and other resources are limiting, but a longer duration study with varying level of management is needed to determine how successfully tilapia competes with locally adapted SIS.     

Systemic and mucosal antibody response in tilapia, Oreochromis niloticus (L.), following immunization with Flavobacterium columnare

Specific antibody responses to Flavobacterium columnare (isolate ATCC 23463T) were characterized in plasma and mucus of tilapia following intraperitoneal (i.p.) injection or immersion immunization with formalin-killed sonicated or whole cell preparations. Fish (30 per treatment) received a primary immunization and were booster immunized 4 weeks later. An enzyme-linked immunosorbent assay was developed for detection and quantification of specific anti-F. columnare antibody, and it was found that formalin-killed sonicated cells in Freund's complete adjuvant (FCA) injected i.p. stimulated a significant systemic antibody response within 2 weeks (mean titre 11,200) which increased to 30,600 following secondary immunization. At 10 weeks post-immunization, the mean titre remained significantly elevated above the controls. Antibodies were also observed in cutaneous mucus of fish immunized i.p. with formalin-killed sonicated cells in FCA at 6 and 8 weeks post-immunization (mean titres 67 and 33, respectively). Although some individual fish responded, mean plasma and cutaneous mucus antibody titres were not significantly greater than controls in any of the other treatment groups. The results of this study demonstrate that tilapia can mount a significant humoral response in plasma and cutaneous mucus to F. columnare, but i.p. immunization with FCA is required to elicit this response.     

Growth response and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection

This study determined the growth performance and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection. Tilapia were challenged with three doses of S. iniae (8.8 × 10³, 8.8 × 10⁴ and 8.8 × 10⁵ CFU fish^?1 for low, medium and high challenges respectively). Groups of non‐injected and tryptic soy broth‐injected fish were maintained as controls. Significantly (P<0.05) higher mortality (45.0%) occurred in the high challenge treatment than in the low challenge treatment group (29.6%). The medium challenge group had mortality (36.3%) that did not differ significantly from the high or low treatment. Few fish died in the non‐injected and broth‐injected treatments (3.4% and 0.8% respectively). The tilapia that survived S. iniae infection used to assess growth performance were selected from survivors without gross clinical signs of disease. These fish were randomly stocked at a rate of 30 fish into each 57 L aquarium in triplicate and fed to apparent satiation for 8 weeks. No significant differences were detected in weight gain, feed intake, feed efficiency ratio or survival between S. iniae‐survived tilapia and the control treatments following the 8‐week growth performance trial. Following the 8‐week feeding study, tilapia were challenged with 1 × 10⁶ CFU fish^?1 of S. iniae to assess acquired immunity. Mean cumulative mortality was significantly higher (P<0.05) in the control treatments (41.7% for the non‐injected and 43.3% for the broth‐injected fish) than in the low, medium and high challenge treatments (7.4%, 3.3% and 8.3% respectively). Serum protein was significantly (P<0.05) elevated in the S. iniae‐survived tilapia that were subsequently challenged when compared with controls challenged for the first time. Agglutinating antibody titre was significantly higher in the fish in the medium and high challenge treatments, compared with the control fish challenged for the first time. The results suggest tilapia that survive S. iniae challenge without showing overt disease signs performed as well as non‐infected tilapia. Further, the S. iniae‐survived tilapia challenged following the 8‐week growth performance trial gained acquired resistance to homologous S. iniae challenge.     

Application of multiplex quantitative polymerase chain reaction methods to detect common bacterial fish pathogens in Nile tilapia,Oreochromis niloticus,hatcheries in Costa Rica

Edwardsiella spp., Streptococcus spp., and Francisella noatunensis subsp. orientalis are some of the most important fish pathogens affecting global tilapia, Oreochromis spp., aquaculture. In Costa Rica, the aquaculture industry is dominated by freshwater‐cultured Nile tilapia, Oreochromis niloticus, which are raised in all seven national provinces. At present, little is known regarding the diversity of pathogens present in these facilities, and definitive identification of agents associated with disease outbreaks are rare. To evaluate the prevalence of common bacterial pathogens in these systems, this study used multiplex quantitative polymerase chain reaction (qPCR) assays targeting Edwardsiella, Streptococcus, and Francisella species as a diagnostic and surveillance tool. In 2017, seven different tilapia hatcheries were visited, and 350 fingerlings were subjected to necropsy and molecular diagnostic evaluation. Fish exhibiting gross signs of disease were subjected to histological and microbiological analysis. For the first time, Edwardsiella anguillarum was recovered and molecularly confirmed from diseased tilapia in Costa Rica. In addition, F. noatunensis subsp. orientalis was identified in a region of Costa Rica where it had not been previously reported.     

Leukocyte response and phagocytic activity in Nile tilapia experimentally infected with <Emphasis Type="Italic">Enterococcus</Emphasis> sp.

This study evaluated the total and differential leukocyte counting and the phagocytic activity in Nile tilapia Oreochromis niloticus experimentally injected with Enterococcus sp. in the swim bladder. Fish were distributed in four treatments in triplicates of non-injected fish, fish injected with 1 ml of sterile saline solution 0.65%, and fish injected with 1 × 10³ and 1 × 10⁶ colony-forming units (CFU) of Enterococcus diluted in 1 ml sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected for white blood cell (WBC) counts, differential counting of WBC, and phagocytic activity of blood leukocytes. The increased numbers of WBC and lymphocytes were followed by decreased number of monocyte after infection. The percentages of phagocytic activities in the blood were 55.3 and 55.9%, respectively, in tilapia injected with 1 × 10³ and 1 × 10⁶ CFU/ml.     

A comparison of the antigenicity of the extracellular products and whole-cell sonicates from Mycobacterium spp. in rabbits, mice and fish by immunoblotting and enzyme-linked immunosorbent assay

The antigenicity of extracellular products (ECPs) derived from Mycobacterium spp. isolated from snakehead, Channa striata (Bloch), and Siamese fighting fish, Betta splendens (Regan), were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera collected from immunized rabbits, mice and fish (rainbow trout). All three species responded to a 65-kDa protein present in both the ECPs and whole cell sonicates (WCSs) from a variety of Mycobacterium spp. Cross-reactivity of anti- M. tuberculosis and anti-human heat-shock protein monoclonal antibodies (MAbs), and the presence of fibronectin binding proteins secreted into ECPs of mycobacteria were also examined. The MAbs against human 60-kDa heat-shock protein cross- reacted with the band at 65 kDa in the ECPs of TB1 (isolated from snakehead fish) and the type strain M. marinum, while the anti- M. tuberculosis MAb F29–47 elicited a strong reaction with a band at 21 kDa with most of the ECPs from mycobacterial strains examined. The major fibronectin-binding proteins were located between 21 and 25 kDa. The 65-kDa protein from ECPs of Mycobacterium spp. proved strongly immunogenic to rabbits, mice and fish. Rabbit antiserum against the 65-kDa protein from strain TB267 reacted with many non- Mycobacterium WCSs, and therefore, the 65-kDa protein from Mycobacterium spp. is believed to be a common protein found in many fish bacterial pathogens.     

Comparative study of growth performance of three strains of Nile tilapia, Oreochromis niloticus, L. at two stocking densities

This study was conducted to investigate the effect of stocking density (125 or 200 fish m^?3) on the growth performance of three strains of the Nile tilapia, Oreochromis niloticus: the non‐improved strain (NS), the genetically improved farmed tilapia (GIFT) and the Freshwater Aquaculture Center selected tilapia known as the FaST selected line (SL). Each strain and density combination was triplicated in 0.42 m³ fibreglass tanks within a re‐circulating water system. Water temperature was maintained at 29.0±1.0°C. Large Nile tilapia having a mean body weight of 100–110 g were stocked in each tank and hand‐fed four times daily with commercial tilapia pellets (35% protein) for 104 days. Results showed that at the two stocking densities, the GIFT and SL strains showed a significantly higher (P<0.05) mean weight (MWT), daily growth rate (DGR), specific growth rate (SGR), feed conversion ratio (FCR) and gross yield (GY) than the NS. In all three strains, growth performance was negatively affected by stocking density. The lower density (125 fish m^?3) treatments had significantly higher MWT, DGR and SGR than the higher density one (200 fish m^?3). However, higher FCR and GY were observed at the higher density. Survival rates were high in all treatments and were not affected by strain or density. In general, the SL strain had better growth parameters than the GIFT strain. The findings of this study demonstrated the superior growth performance of the improved strains at both densities compared with the NS. The higher density (200 fish m^?3) could be more profitable for the tilapia farms in Kuwait than the lower density of (125 fish m^?3) in terms of reduced land cost and facilities, demand on the limited low‐salinity underground water and manpower.     

Evaluation of the INNO‐LiPA mycobacteria v2 assay for identification of aquatic mycobacteria

Fifty‐seven isolates of mycobacteria comprising 10 reference strains, 47 field isolates and one non‐Mycobacterium isolate were screened using commercial INNO‐LiPA v2 assay kits. All mycobacteria isolates tested hybridized with the Mycobacterium genus probe on the LiPA strip. All M. marinum, M. fortuitum and M. chelonae reference and field strains and three out of the four M. gordonae isolates hybridized to their corresponding species‐ or complex‐specific probes. Two cultures (a type strain and a field isolate) yielded mixed growth of two mycobacterial species, i.e. M. chelonae and M. fortuitum. A Mycobacterium isolate from one of these cultures was subsequently purified and correctly identified with the kit. However, sequence analysis of the 16S–23S rRNA internal transcribed spacer (ITS) region of various mycobacteria isolates revealed a misidentification of M. shottsii and M. pseudoshottsii with the kit because these isolates reacted with the M. marinum/M. ulcerans probe. Moreover, nine of the 13 field isolates presumed to be M. fortuitum from the results of the kit had closer ITS sequence homology with M. conceptionense, a species which, to our knowledge, has never been reported in fish. These findings highlight the need to redesign the M. fortuitum–M. peregrinum probe included in the INNO‐LiPA assay and to introduce additional complex‐specific probes into the kit. Nevertheless, the kit proved to be a rapid and reliable method for identifying mycobacteria in the aquatic environment and would be particularly useful in laboratories without sequencing facilities.