Iron supplementation in the diets of hybrid catfish (Ictalurus punctatus × I. furcatus) juveniles affected haematocrit levels and potentially decreased disease resistance to Edwardsiella ictaluri.

To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.

Influence of probiotic and prebiotic supplementation on intestinal microbiota and resistance to Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus) following florfenicol administration.

Enteric septicemia of catfish (ESC), caused by the gram-negative enteric bacteria Edwardsiella ictaluri, is a significant threat to catfish aquaculture in the southeastern United States. Antibiotic intervention can reduce mortality; however, antibiotic use results in an imbalance, or dysbiosis, of the gut microbiota, which may increase susceptibility of otherwise healthy fish to enteric infections. Herein, recovery of the intestinal microbiota and survivability of channel catfish in response to ESC challenge was evaluated following a 10-day course of florfenicol and subsequent probiotic or prebiotic supplementation. Following completion of florfenicol therapy, fish were transitioned to a basal diet or diets supplemented with a probiotic or prebiotic for the remainder of the study. Digesta was collected on Days 0, 4, 8 and 12, beginning on the first day after cessation of antibiotic treatment, and gut microbiota was characterized by Illumina sequencing of the 16S rRNA gene (V4 region). Remaining fish were challenged with E. ictaluri and monitored for 32 days post-challenge. Florfenicol administration resulted in dysbiosis characterized by inflated microbial diversity, which began to recover in terms of diversity and composition 4 days after cessation of florfenicol administration. Fish fed the probiotic diet had higher survival in response to ESC challenge than the prebiotic (p = .019) and negative control (p = .029) groups.

Changes in immune genes expression, immune response, digestive enzymes -antioxidant status, and growth of catla (Catla catla) fed with Astragalus polysaccharides against edwardsiellosis disease.

The effect of four level of Astragalus polysaccharides (APs) supplementation diets, (CD: control diet and three experiment diet (E), EA: 100 mg kg-1 APs; EB: 200 mg kg-1 APs; EC: 300 mg kg-1 APs) on growth, changes in haemato-biochemical parameters and metabolic-digestive enzymes, enhancement of antioxidant activity, innate-adaptive immune response, and cytokine gene expression were studied in catla (Catla catla) against Edwardsiella tarda. The healthy and challenged groups fed the CD displayed no mortality, while fish fed EA or EC revealed 10% mortality, but the mortality was only 5% in diet EB. Fish fed diet EB and EC revealed significantly better growth rates and high RBC count during the experimental period. Albumin and globulin levels were significant improved when fish were fed the diet EB and EC from weeks 6-8. The superoxide dismutase (SOD) was significant ameliorated by EB feeding from weeks 4-8. In contrast, serum myeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA)/lipid peroxidation (LPO), glutathione peroxidase (GPx), respiratory burst activity (RBA), bactericidal action (BCA), serum lysozyme activity (SLA), nitric oxide synthase (NOS), head kidney leukocytes response proliferation (HKLP), hemolytic action (HLA), hydrogen peroxides (H2O2), and immunoglobulin (Ig) were significantly improved from week 6-8. Groups fed the APs enriched diets had significant ameliorated interleukin (IL)-1ß and interferon (IFN)-γ mRNA expression after 6 and 8 weeks of feeding. However, IL-10 and major histocompatibility complex (MHC)-1 mRNA expressions were significant enhanced in catla fed all APs diets on week 8. APs enriched diets revealed significant improved tumor necrosis factor (TNF)-α and TNF receptor-associated factor-6 (TRAF6) mRNA expression on week 4, but toll-like receptor-2 (TLR2) and TLR4 mRNA expression were significant enhanced by diet EB and EC after weeks 6 and 8. Similarly, the lysozyme (Lyz)-C and Lyz-G mRNA levels in the head kidney (HK) increased by APs feeding on weeks 6 and 8, whereas the EB diet, the expression of nucleotide binding oligomerization domain-1 (NOD1) was significantly improved on weeks 6 and 8, but NOD2 mRNA expression was only significant enhanced after 8 weeks of diet EB. By feeding healthy catla and E. tarda challenged fish fed diet EB, resulted in significantly increased growth, haemato-biochemical indices, metabolic-digestive enzymes, antioxidant activities, innate-adaptive immune responses, and cytokine gene expression mainly between 6 and 8 weeks.

Rheum officinale extract promotes the innate immunity of orange-spotted grouper (Epinephelus coioides) and exerts strong bactericidal activity against six aquatic pathogens.

We investigated the antimicrobial properties and the effects of Rheum officinale extract (ROE) on nonspecific immune parameters of orange-spotted grouper (Epinephelus coioides) in vitro and in vivo. The in vitro analysis was conducted by treating grouper primary head kidney leukocytes with various concentrations of ROE. The phagocytic rate of the leukocytes was elevated in a dose-dependent manner from 0.01 to 0.1 mg/ml, but decreased with higher concentrations of ROE (0.5 and 1.0 mg/ml). The production of reactive oxygen species (ROS) was strongly enhanced in a dose-dependent manner by treatment with ROE doses of 0.1-10.0 mg/ml. However, morphological changes (e.g., rounding and shrinkage of cells, chromatin condensation, fragmentation, and appearance of apoptotic bodies) were observed in the leukocytes after incubation with higher concentrations of ROE (1.0 and 10.0 mg/ml). A 28-day feeding trial was performed to assess the impact of dietary administration of ROE on grouper innate immunity parameters. Fish were fed with feed supplemented with 0, 0.1, 1.0, or 5.0 g ROE per kg of feed. The phagocytic activity of the animals' leukocytes was significantly elevated in all ROE-fed groups on day 1 and in groups fed with ROE at 0.1 or 1.0 g/kg on day 14. Production of ROS was substantially increased on day 1 in fish fed with ROE at 1.0 and 5.0 g/kg, but decreased steadily later on. The ability to generate ROS increased steadily until day 7 in fish fed the lowest concentration of ROE (0.1 mg/ml), but decreased thereafter. ROE showed excellent antibacterial activity against six pathogens of aquatic animals: Vibrio parahaemolyticus, V. vulnificus, V. alginolyticus, V. carchariae, Aeromonas hydrophila, and Edwardsiella tarda. The minimum inhibitory and bactericidal concentrations of measured ROE-derived anthraquinones were 10.57-84.53 µg/ml and 10.57-169.05 µg/ml, respectively.

High dose of dietary vitamin D3 modulated the yellow catfish (Pelteobagrus fulvidraco) splenic innate immune response after Edwardsiella ictaluri infection.

Vitamin D3 (VD3) has been shown to modulate the innate immune response in mammals but this has been rarely reported in fish. The current study found that increasing dietary VD3 content can reduce the density of yellow to dark brown pigmented macrophage aggregates (PMAs) in the spleens of yellow catfish infected with Edwardsiella ictaluri. The results of next-generation sequencing showed that a high dose of dietary VD3 (16,600 IU/kg) mainly affected the splenic immune response during Edwardsiella ictaluri infection via negative regulation of 'NF-κΒ transcription factor activity', 'NIK/NF-κΒ signaling' and the 'i-kappab kinase/NF-κΒ signaling' pathways. Follow-up qPCR showed that dietary VD3 increased the expression of NF-κΒ inhibitor iκb-α, decreased the expression of nf-κb p65, il-6, il1-ß and tnf-α, and down-regulated the expression of nik, ikks and nf-κb p52 in the NIK/NF-kappaB signaling pathway. The above results indicate that dietary VD3 can modulate the splenic innate immune response of yellow catfish after Edwardsiella ictaluri infection by inhibiting the NF-κB activation signaling pathways.

Effect of cassic acid on immunity and immune-reproductive genes transcription in Clarias gariepinus against Edwardsiella tarda.

The present study was investigated the dietary administration of cassic acid (CA) on growth, innate immunity, transcription profiles of estrogen and follicle-stimulating hormones as well as lysozyme enzyme determined in Clarias gariepinus against Edwardsiella tarda. The weight gain (WG), protein efficacy ratio (PER), and feed conversion ratio (FCR) were significantly improved in infected fish fed dietary administration with CA at 5 and 10 mg kg-1 diets. The survival is higher (96.7% and 98.3%) in the infected groups fed at 5 and 10 mg kg-1 CA diets. The red (RBC) and white (WBC) blood cells, hemoglobin (Hb), and packed cell volume (PCV) was found significantly high in the infected fish feeding at 5 and 10 mg kg-1 CA diets. Total protein and albumin were significantly increased with 5 and 10 mg kg-1 CA diets among weeks 1-4 while the globulin and albumin: globulin ratio increased of these diet only after week 2. The phagocytic and respiratory burst activities were enhanced statistically the infected fish fed at 10 mg kg-1 CA diet group whereas the production of superoxide anion (SOA) and nitric oxide (NO) were significantly increased at 5 and 10 mg kg-1 CA diets. The lymphocyte proliferation and myeloperoxidase (MPO) activity were significantly high the infected fish when fed at 5 and 10 mg kg-1 CA diets after 2nd week whereas the alternate complement activity (ACP), generation of reactive oxygen species (ROS), and lysozyme activity (Lyz) were observed at 5 and 10 mg kg-1 CA diets among weeks 1-4. The accumulative mortality was 10% in infected fish fed at 1 and 5 mg kg-1 CA diets whereas 15% mortality found with 10 mg kg-1 CA diet. The highest levels of estrogen receptor alpha (ERα) mRNA expression found in gonad while the highest levels of follicle stimulating hormone-beta subunit (FSH-ß) mRNA expression found in testis of the infected fish given at 5 and 10 mg kg-1 CA diets. The up-regulation of chick-type lysozyme (c-Lyz) mRNA was observed at 5 and 10 mg kg-1 CA diets after 2nd week while goose-type lysozyme (g-Lyz) mRNA was up-regulation amongst weeks 1-4 of these diets. The present study suggested that E. tarda challenged fish after feeding with 5 and 10 mg kg-1 CA diets did not affect growth and hemato-biochemical parameter, but it enhanced nonspecific immune system and improving ERα, FSH-ß, c-Lyz, and g-Lyz mRNA expression in C. gariepinus against E. tarda.

Novel pectin isolated from Spirulina maxima enhances the disease resistance and immune responses in zebrafish against Edwardsiella piscicida and Aeromonas hydrophila.

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 µg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 µg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1ß: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1ß) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture.

An investigation of dairy calf management practices, colostrum quality, failure of transfer of passive immunity, and occurrence of enteropathogens among Australian dairy farms.

Calf preweaning morbidity and mortality risks have been reported as high in several countries, with average values approximating 35 and 7%, respectively. However, limited data are available for calf morbidity and mortality risks on Australian dairy farms. The aims of this study were (1) to investigate current calf management practices on dairy farms in Australia and their association with herd-level morbidity and mortality using a questionnaire-based, cross-sectional study; and (2) to estimate the prevalence of common enteropathogens causing diarrhea, the failure of passive transfer of immunity, and poor colostrum quality in a sample of Australian dairy farms. We analyzed 106 completed questionnaires and samples from 23 farms (202 fecal, 253 calf serum, and 221 colostrum samples). Morbidity and mortality risks reported by farmers in preweaned heifers were 23.8 and 5.6%, respectively. These risks were above the Australian dairy industry targets in 75.5 and 66.7% of respondents. The zoonotic pathogens Cryptosporidium spp. and Salmonella spp. were the most prevalent enteropathogens, with a true prevalence of 40.9 and 25.2%, respectively. Salmonella O-group D was present in 67.9% of Salmonella-positive samples, followed by O-groups B (17.9%) and C (10.7%). Failure of transfer of passive immunity (IgG <10 g/L) was observed in 41.9% of calves (mean herd-level prevalence of 36.2%), and only 19.5% of colostrum samples met the standards for immunoglobulin content and microbiological quality. Collectively, these data indicate that there is still considerable room for improvement in calf-rearing practices on Australian dairy farms, particularly with regard to colostrum management and feeding hygiene.

Antibacterial Activity of Constituents from Mangosteen Garcinia mangostana Fruit Pericarp against Several Channel Catfish Pathogens.

Bacterial diseases cause major financial damage to the producers of Channel Catfish Ictalurus punctatus in the southeastern USA. The two most common bacterial diseases among pond-raised Channel Catfish are enteric septicemia of catfish, caused by the gram-negative bacterium Edwardsiella ictaluri, and columnaris disease, caused by the rod-shaped, gram-negative bacterium Flavobacterium columnare. Streptococcosis is another, less-common bacterial disease in catfish and is caused by the gram-positive coccus Streptococcus iniae. Catfish farmers typically rely on commercial antibiotics and other chemicals to prevent the economic damage from these diseases. Environmentally benign and efficacious alternatives to the currently used antibiotics and chemicals will tremendously help the catfish aquaculture industry. As part of our ongoing efforts in the search for such novel compounds, we investigated ethyl acetate and methanol extracts of mangosteen Garcinia mangostana fruit pericarp via bioassay-guided fractionation. Gamma-mangostin (γ-mangostin) was isolated and identified as the most promising active metabolite against F. columnare. One of the constituents in the mangosteen fruit pericarp, alpha-mangostin (α-mangostin), is the major xanthone; α-mangostin was found to be 10-fold less active than γ-mangostin when minimum inhibitory concentration values were compared.

Extraction, isolation and characterization of bioactive compounds from chloroform extract of Carica papaya seed and it's in vivo antibacterial potentiality in Channa punctatus against Klebsiella PKBSG14.

The relative efficacy of the isolated pure compound, extracted from Carica papaya seed has been tested against live fish, Channa punctatus infected with pathogenic strains of KlebsiellaPKBSG14 (gene bank accession no.KJ162158) at a dose of 0.75 CFU/ml in vivo. The isolated compound has been characterized by chromatography and mass spectroscopy studies using FTIR, 1HNMR and 13c NMR to identify as well as to determine the nature of the pure compound. This study revealed the extracted biological molecule is oleic acid, a long chained saturated fatty acid (LFAs) with a molecular formula C18H34O2. Later this compound was analyzed for its efficacy as an antibacterial agent in vivo through cytotoxicological and genotoxicological assays. A dose of 0.5 mg/kg and 1 mg/kg b.w of isolated pure oleic acid has been tested and it showed effective result in regard to DNA fragmentation, comet tail length and toxicity biomarkers like ROS generation. The results of in vivo studies showed similar effects on spleen cells with regard to cell viability by PI staining, cell cycle analysis and also Annexin-FITC assay. Thus, the overall results suggest that oleic acid increases drug bioavailability and thereby has a better chemo-preventive action against bacterial infection in vivo.

Effects of dietary Tenebrio molitor meal on the growth performance, immune response and disease resistance of yellow catfish (Pelteobagrus fulvidraco).

This study evaluated the influence of diets containing mealworm (Tenebrio molitor) meal in partial substitution of fishmeal on growth performance and immune responses of juvenile yellow catfish (Pelteobagrus fulvidraco). Four diets were formulated to contain 0 (the control diet), 9, 18 and 27 g mealworm meal per 100 g diet with 0%, 25%, 50% and 75% of fishmeal replacement, respectively. Yellow catfish were randomly divided into 4 groups with 3 replicates in each group. The fish in each group were fed with one of the four experimental diets for 5 weeks. Growth performance, plasma parameters (SOD, MDA, IgM, C3, lysozyme) and immune related genes (MHC II, IL-1, CypA, IgM, HE) of yellow catfish were determined at the end of the feeding trial, as well as 24 h post bacterial (Edwardsiella ictaluri) challenge. The present results showed that dietary inclusion of mealworm meal (MW) had no negative effects on the growth performance of the juvenile yellow catfish, compared to the control group. At the end of the feeding trial, plasma MDA contents of MW supplemented groups were significant lower than the control group. Plasma SOD activities increased significantly with the increasing dietary MW contents at the end of feeding trial (pre-challenge) and 24 h post challenge with E. ictaluri. Significant increase of plasma lysozyme activity was found in MW supplemented groups compared to the control group 24 h post bacterial challenge. Plasma IgM levels increased significantly with the increasing dietary MW contents at the end of feeding trial. Compared with the control group, the immune related genes of MHC II, IL-1, IgM and HE of the fish in the MW supplemented groups significantly upregulated pre-challenge or 24 h post bacterial challenge. Finally, it was observed that the survival rate of the 27% MW group was significant higher (P < 0.05) than the control group but was not significantly differed from the 18% MW group. The present results indicated that dietary inclusion level of at least 18% MW could improve the immune response and the bacterial resistance of yellow catfish without any negative growth effects.

Antimicrobial Use for and Resistance of Zoonotic Bacteria Recovered from Nonhuman Primates.

As a growing threat to human and animal health, antimicrobial resistance (AMR) has become a central public-health topic. Largescale surveillance systems, such as the National Antimicrobial Resistance Monitoring System (NARMS), are now established to monitor and provide guidance regarding AMR, but comprehensive literature on AMR among NHP is sparse. This study provides data regarding current antimicrobial use strategies and the prevalence of AMR in zoonotic bacteria recovered from NHP within biomedical research institutions. We focused on 4 enteric bacteria: Shigella flexneri, Yersinia enterocolitica, Y. pseudotuberculosis, and Campylobacter jejuni. Fifteen veterinarians, 7 biomedical research institutions, and 4 diagnostic laboratories participated, providing susceptibility test results from January 2012 through April 2015. Veterinarians primarily treated cases caused by S. flexneri, Y. enterocolitica, and Y. pseudotuberculosis with enrofloxacin but treated C. jejuni cases with azithromycin and tylosin. All isolates were susceptible to the associated primary antimicrobial but often showed resistance to others. Specifically, S. flexneri isolates frequently were resistant to erythromycin (87.5%), doxycycline (73.7%), and tetracycline (38.3%); Y. enterocolitica isolates to ampicillin (100%) and cefazolin (93.6%); and C. jejuni isolates to methicillin (99.5%) and cephalothin (97.5%). None of the 58 Y. pseudotuber-culosis isolates was resistant to any tested antimicrobial. Notably, resistance patterns were not shared between this study's NHP isolates and human isolates presented by NARMS. Our findings indicate that zoonotic bacteria from NHP diagnostic samples are broadly susceptible to the antimicrobials used to treat the clinical infections. These results can help veterinarians ensure effective antimicrobial therapy and protect staff by minimizing occupational risk.

Feeding of nano scale oats ß-glucan enhances the host resistance against Edwardsiella tarda and protective immune modulation in zebrafish larvae.

In this study, we prepared and characterized the oats origin of nano scale ß-glucan (NBG) and investigated the immunomodulatory properties in zebrafish larvae. Newly prepared NBG (average particle size of 465 nm) was fully soluble in water. Zebrafish larvae survival rate was increased against pathogenic bacteria Edwardsiella tarda, when NBG was added to the water (500 µg/mL) compared to NBG non-exposed controls. Moreover, quantitative real time PCR (qRT-PCR) results showed up-regulation of immune functional genes including TNF-α, IL-1ß, ß-defensin, lysozyme, IL 10, IL 12 and C-Rel indicating higher survival rate could be due to stronger immunomodulatory function of NBG (500 µg/mL). Thus, non-toxic, water soluble and biodegradable NBG from oats could be considered as the potential immunostimulant for larval aquaculture.

Comparison of the effects of dietary single and multi-probiotics on growth, non-specific immune responses and disease resistance in starry flounder, Platichthys stellatus.

An 8-week feeding trial was conducted to evaluate the effects of dietary probiotics on growth performance and non-specific immune responses in starry flounder, Platichthys stellatus. Fish averaging 46.5 ± 0.65 g (mean ± SD) were fed one of the six experimental diets; one control (Cont), and five other diets were prepared by supplementing single-probiotics 1 (Bacillus subtilis; SP1, 2 × 109 CFU kg-1 diet), single-probiotics 2 (Bacillus licheniformis; SP2, 2 × 109 CFU kg-1 diet), multi-probiotics 1 (Bacillus subtilis + Bacillus licheniformis; MP1, 2 × 109 CFU kg-1 diet), multi-probiotics 2 (commercial probiotics; Bacillus subtills + Bacillus licheniformis + Paenibacillus polymyxa + Aspergillus oryzae + Saccharomyces cerevisiae; MP2, 2 × 109 CFU kg-1 diet) and oxytetracycline (OTC) at 5 g OTC kg-1 diet. At the end of 8 weeks feeding trial, weight gain (WG) and specific growth rate (SGR) of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). Superoxide dismutase (SOD) activity of fish fed MP2 diet was significantly higher than those of fish fed OTC diet (P < 0.05). Nitro blue tetrazolium (NBT) activity and lysozyme activity of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed OTC diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1 and MP2 diets. During the Edwardsiella tarda challenge test, the first mortality occurred on day 2. After the 14 days challenge test, cumulative survival rate of fish fed MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1, MP2 and OTC diets in survival rate at the termination of the challenge test. Although there was little advantage in immunological parameters with fish fed MP diets, single and multi-probiotics were equally effective statistically. These results demonstrated that single or multi-probiotics had equal beneficial effect as an antibiotic replacer in terms of growth performance, non-specific immune responses and disease resistance in starry flounder.

Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP release in fish.

Dietary Bacillus subtilis FPTB13 and chitin, single or combined, modulate systemic and cutaneous mucosal immunity and resistance of catla, Catla catla (Hamilton) against edwardsiellosis.

Effects of dietary administration of Bacillus subtilis FPTB13 and chitin, single or combined, on the systemic immunity, mucosal immunity and resistance of catla (Catla catla) against Edwardsiella tarda infection were investigated. The probiotic attributes of B. subtilis was tested by conducting antagonism study, safety in catla, in vitro immunomodulation and dietary immunomodulation. Results of these studies indicated the probiotic potential of the strain. From the preliminary dietary immunomodulation study, a dose of 10(9) B. subtilis cells g(-1) was selected for inclusion into diets for subsequent experiments. Experimental diets were prepared by adding B. subtilis (10(9) cells g(-1)), chitin (2%) and their combination to the basal diet. Different systemic and mucosal immunological parameters viz. oxygen radical production, myeloperoxidase content, lysozyme activity, total protein content and alkaline phosphatase activity showed significant enhancement (p<0.05) after 2 weeks of feeding with the combined diet. B. subtilis and chitin alone also significantly elevated most of the immune responses. All the diets significantly increased the resistance of catla against E. tarda challenge. The highest post-challenge survival was observed in combined group (i.e. 63.33%). In conclusion, B. subtilis and chitin, alone or combined, had a health ameliorating effect in catla. The results also collectively suggest the usefulness of applying a combined probiotic and immunostimulant supplemented diet to achieve greater benefits.

The effects of feeding ß-glucan to Pangasianodon hypophthalmus on immune gene expression and resistance to Edwardsiella ictaluri.

Pangasianodon hypophthalmus (striped catfish) is an important aquaculture species and intensification of farming has increased disease problems, particularly Edwardsiella ictaluri. The effects of feeding ß-glucans on immune gene expression and resistance to E. ictaluri in P. hypophthalmus were explored. Fish were fed 0.1% fungal-derived ß-glucan or 0.1% commercial yeast-derived ß-glucan or a basal control diet without glucan. After 14 days of feeding, the mRNA expression of immune genes (transferrin, C-reactive protein, precerebellin-like protein, Complement C3 and factor B, 2a MHC class II and interleukin-1 beta) in liver, kidney and spleen were determined. Following this fish from each of the three diet treatment groups were infected with E. ictaluri and further gene expression measured 24 h post-infection (h.p.i.), while the remaining fish were monitored over 2 weeks for mortalities. Cumulative percentage mortality at 14 days post-infection (d.p.i.) was less in ß-glucan fed fish compared to controls. There was no difference in gene expression between dietary groups after feeding for 14 days, but there was a clear difference between infected and uninfected fish at 24 h.p.i., and based on principal component analysis ß-glucans stimulated the overall expression of immune genes in the liver, kidney and spleen at 24 h.p.i.

Enhanced immune response and resistance to edwardsiellosis following dietary chitooligosaccharide supplementation in the olive flounder (Paralichthys olivaceus).

This study was conducted to evaluate the effects of dietary chitooligosaccharide (COS) supplementation on peripheral leukocyte count, head kidney leukocyte phagocytic rate, phagocytic index, respiratory burst activity, serum lysozyme activity, and immune protection in Paralichthys olivaceus. A total of 300 flounder with an average body weight of 80-100 g were randomly assigned into four dietary groups: (I) basic diet (control), basic diet containing (II) 0.5% COS, and (III) 1% COS, fed continuously for 28 d, and (IV) basic diet containing 1% COS fed in 14 d intervals. Continuous feeding of 0.5% and 1% COS diets for 28 d significantly increased the number of peripheral leukocytes, head kidney leukocyte phagocytic rate, phagocytic index, respiratory burst activity, and serum lysozyme activity (P < 0.05 or P < 0.01). After a 10 d Edwardsiella tarda challenge, the immune protection rates in the 0.5% and 1% COS groups were 30% and 60%, respectively. No control fish survived the E. tarda challenge treatment. Most immune indices were slightly lower after removal of COS from the diet for 14 d, but all immune indices were observed to recover after another 14 d of COS supplementation. This study demonstrates that supplementation of a basic diet with COS enhances the non-specific immune response and improves survival rates following infection with E. tarda in P. olivaceus. An optimized interval feeding strategy with diets containing 1% COS may have potential applications in the prevention of disease in aquacultured P. olivaceus.

Effects of protein hydrolysates supplementation in low fish meal diets on growth performance, innate immunity and disease resistance of red sea bream Pagrus major.

This study was conducted to evaluate the supplemental effects of three different types of protein hydrolysates in a low fish meal (FM) diet on growth performance, feed utilization, intestinal morphology, innate immunity and disease resistance of juvenile red sea bream. A FM-based diet was used as a high fish meal diet (HFM) and a low fish meal (LFM) diet was prepared by replacing 50% of FM by soy protein concentrate. Three other diets were prepared by supplementing shrimp, tilapia or krill hydrolysate to the LFM diet (designated as SH, TH and KH, respectively). Triplicate groups of fish (4.9 ± 0.1 g) were fed one of the test diets to apparent satiation twice daily for 13 weeks and then challenged by Edwardsiella tarda. At the end of the feeding trial, significantly (P < 0.05) higher growth performance was obtained in fish fed HFM and hydrolysate treated groups compared to those fed the LFM diet. Significant improvements in feed conversion and protein efficiency ratios were obtained in fish fed the hydrolysates compared to those fed the LFM diet. Significant enhancement in digestibility of protein was found in fish fed SH and KH diets and dry matter digestibility was increased in the group fed SH diet in comparison to LFM group. Fish fed the LFM diet showed significantly higher glucose level than all the other treatments. Whole-body and dorsal muscle compositions were not significantly influenced by dietary treatments. Histological analysis revealed significant reductions in goblet cell numbers and enterocyte length in the proximal intestine of fish fed the LFM diet. Superoxide dismutase activity and total immunoglobulin level were significantly increased in fish fed the diets containing protein hydrolysates compared to the LFM group. Also, significantly higher lysozyme and antiprotease activities were found in fish fed the hydrolysates and HFM diets compared to those offered LFM diet. Fish fed the LFM diet exhibited the lowest disease resistance against E. tarda and dietary inclusion of the hydrolysates resulted in significant enhancement of survival rate. The results of the current study indicated that the inclusion of the tested protein hydrolysates, particularly SH, in a LFM diet can improve growth performance, feed utilization, digestibility, innate immunity and disease resistance of juvenile red sea bream.

The effects of feeding immunostimulant ß-glucan on the immune response of Pangasianodon hypophthalmus.

Immunostimulants are food additives used by the aquaculture industry to enhance the immune response of fish, and although ß-glucans are now commonly used for this purpose in aquaculture, little is known about their effects on the immune response of Pangasianodon hypophthalmus. Thus, a variety of immune parameters (e.g. phagocytosis, respiratory burst, lysozyme, complement, peroxidase, total protein, total anti-protease, total IgM, natural antibody titres, and specific IgM titres) was examined in this species after feeding fish with a basal control diet or diets supplemented with 0.05, 0.1, or 0.2 g/kg fungal-derived ß-glucan or 0.1% commercial yeast-derived ß-glucan, as a positive control diet, for a period of four weeks. The effect of the glucans on disease resistance was then evaluated by experimentally infecting the fish with Edwardsiella ictaluri by immersion and mortalities monitored for 14 days. Samples were collected from fish for analysis at 0, 1, 3, 7, 14, 21 and 28 days post-feeding (dpf), and also at 14 days post infection (dpi). The lowest dose of fungal-derived ß-glucan (0.05%) appeared insufficient to effectively stimulate the immune response of the fish, while those fed with the two highest levels of fungal-derived ß-glucan had enhanced immune responses compared to the control group. Significantly elevated levels of respiratory burst activity on all days examined (P < 0.05) and lysozyme activity on 7 dpf were found in the group fed 0.2% fungal-derived ß-glucan, while plasma anti-protease activity was significantly enhanced (P < 0.05) by 21 dpf, natural antibody titres by 3 dpf and complement activity by 7 dpf and also at 14 dpi in the group fed 0.1% fungal-derived ß-glucan. No statistical differences was seen in the level of mortalities between the dietary groups, although the group fed with the control diet had the highest level of mortalities and the groups fed with commercial yeast-derived ß-glucan and 0.2% fungal-derived ß-glucan the lowest.