The toxicity of chlorine dioxide to clownfish and its bactericidal capability to pathogenic strains of vibrio spp.

Global ornamental fish transportation ranging from hours to days can produce multiple stress factors impact fish health and cause mortality. Clownfish, particularly Amphiprion ocellaris, are among the most traded saltwater ornamental fish. Vibrio includes several pathogenic strains that affect aquatic animals. Consequently, prophylactic treatment of the water or fish is recommended. In this study, six Vibrio strains including V. alginolyticus, V. parahaemolyticus and V. harveyi isolated from sick A. ocellaris and one V. harveyi strain from a sick East Asian fourfinger threadfin (Eleutheronema rhadinum) were tested for their sensitivity to a popular disinfectant, chlorine dioxide (ClO2). The results showed that 0.25 ppm ClO2 effectively suppressed five of the seven tested Vibrio strains for 24 h; however, 0.1 ppm ClO2 is safer for A. ocellaris. Meanwhile, ClO2 2.5 ppm reduced the bacterial counts to below 3.3 × 105 CFU/mL for 24 hours. The LC50 of ClO2 for A. ocellaris was 0.87 ppm at 10 min and 0.72 ppm at 24 h post treatment. Mild changes in water quality, including dissolved oxygen (DO), temperature and pH, were recorded during the trial. More research is necessary to understand the sensitivity of various aquatic animal pathogens to ClO2 and its toxicity to different aquatic animals.

The efficacy of new oral vaccine feeds against Salmonid novirhabdovirus in rainbow trout.

Salmonid novirhabdovirus (IHNV) causes infectious haematopoietic necrosis (IHN) in salmonid species. Despite an injectable plasmid-based DNA vaccine of the glycoprotein (G) gene is effective, there are no oral vaccines for mass vaccination of rainbow trout (Oncorhynchus mykiss) fry. Recombinant baculoviruses were generated, used in cabbage looper (Trichoplusia ni) insect larvae to produce IHNV G and IHNV G-C5a proteins. Western blotting and chemiluminescence assays confirmed the expression of recombinant proteins, which were added to the fish feeding and top-coated with unflavored gelatin binder. Commercial rainbow trout were fed with experimental diets containing either IHNV G or IHNV G-C5a proteins for 2 weeks, and boosted 4 weeks after. Four weeks post-booster, fish were challenged with IHNV by immersion. Survival upon the infection challenge was evaluated. Spleen were sampled at 7 and 14 days post infection (dpi). Non-vaccinated and IHNV G fed trout reached a mortality of 91.7 and 97.6%, and 70.9 and 88.4%, respectively at 8 and 15 dpi. The IHNV G-C5a fed group exhibited a reduced mortality of 51.2% at 8 dpi, reaching 81.7% at 15 dpi, suggesting some level of antiviral protection. The individual viral load was measured by RT-qPCR detection of IHNV N gene, showing no significant difference across experimental groups. The transcription modulation of selected immune response markers was evaluated across experimental groups, including Type I IFN-a, Mx-1, CD4, and IgM. Further study is needed to assess how new oral vaccines may become effective to mitigate IHNV pathogenesis in juvenile trout by modulating the host immune response to protect towards IHNV exposure.

Autophagy modulation in rainbow trout Oncorhynchus mykiss L. and resistance to experimental infection with Flavobacterium psychrophilum.

Previously, rainbow trout fed deoxynivalenol (DON) or partially fed (pair-fed) for 4 weeks before and during experimental infection with Flavobacterium psychrophilum had significantly decreased mortality rates. Similar results were obtained in the present study after 12 days, but not after 6 days, feeding 5 ppm DON or pair-fed before infection. Furthermore, feeding 250 ppm chloroquine (CQ) also reduced mortality (p = .052) compared with controls and may have promise for treatment of some fish disease. Parallel groups of fish were maintained on the respective treatments for 15 days, with an additional group that was fasted, but were not infected to monitor autophagy. Fish that were fasted or fed DON had significantly increased LC3II in the liver and fasted fish had significantly decreased LC3II in muscle compared with controls using western blot. There was no difference in LC3II signal in the spleen of any treatment group. Fish that were fasted or pair-fed had significant up-regulation of the Atg genes atg4, atg7, lc3, gabarap and atg12 in muscle using quantitative PCR. Less alteration of Atg expression was seen in liver. Fish treated with CQ had significantly increased expression of atg4, becn1, lc3 and atg12 in the liver. Fish fed DON for 15 days had few alterations of Atg genes in either the liver or muscle. It is still not clear if autophagy is responsible for the resistance of rainbow trout fed DON, CQ or pair-fed before F. psychrophilum infection.

Impact of feed restriction, chloroquine and deoxynivalenol on viral haemorrhagic septicaemia virus IVb in fathead minnow Pimephales promelas Rafinesque.

Autophagy can markedly alter host response to infectious disease, and several studies have demonstrated that a restricted diet or deoxynivalenol modulates autophagy and reduces mortality of fish due to bacterial disease. The picture is less clear for viral diseases of fish. Duplicate tanks of fathead minnow, Pimephales promelas Rafinesque, were fed a replete diet (control), 100 µM chloroquine, 5 µM deoxynivalenol, 10% (fasted) or 40% of a replete diet (pair-fed) for 2 weeks and then experimentally infected by intraperitoneal injection with 2 × 105 viral haemorrhagic septicaemia virus IVb. Survival from highest to lowest for the different treatments was as follows: deoxynivalenol (average 43.3%); control (40.0%); pair-fed (35.0%); fasted (33.3%); and chloroquine (21.7%). No treatment significantly altered the survival rate of fathead minnow after VHSV IVb infection when compared to controls; however, the fish fed with chloroquine had significantly lower survival rate than the fish fed deoxynivalenol (p < .05).

Pharmacological and nutritional modulation of autophagy in a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1.

Autophagy is involved in the modulation of nutrition, immunity, and disease in humans and animals. To understand the impact of autophagy modulation on a rainbow trout gill cell line, RTgill-W1, treatments including reduced nutrition (2% fetal bovine serum compared with 10% control), rapamycin, 3-methyladenine, deoxynivalenol, and chloroquine were tested. Western blot and immunofluorescence were used to detect microtubule-associated protein 1A/1B-light chain protein and quantitative polymerase chain reaction was used to detect the expression of 10 autophagy-related genes. At 3-d post-treatment, reduced nutrition significantly (p < 0.05) increased autophagy while deoxynivalenol significantly (p < 0.01) suppressed it compared to controls. These phenomena were confirmed by using immunofluorescence to detect the number of autophagosomes in RTgill-W1. Chloroquine is critical to allow observation of microtubule-associated protein 1A/1B-light chain protein in this model. The commonly used autophagy-modulating chemicals rapamycin and 3-methyladenine either activated or suppressed microtubule-associated protein 1A/1B-light chain protein, respectively, as expected from the literature, but did not act in a consistently significant manner. Expression of five of the 10 Atg genes, including lc3, gabarap, atg4, atg7, and atg12, were altered in a similar pattern to microtubule-associated protein 1A/1B-light chain protein. The consistent trend of autophagy-related gene upregulation including becn1, lc3, gabarap, and atg9 following treatment with 3-methyladenine and chloroquine is suggestive of a novel feedback regulation in the autophagy machinery.

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1.

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p < .05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.

Autophagy-related genes in rainbow trout Oncorhynchus mykiss (Walbaum) gill epithelial cells and their role in nutrient restriction.

Autophagy is primarily an adaptive response to provide nutrients and energy following exposure to stress and starvation but can also regulate muscle mass and impact infectious disease susceptibility. Expression of 10 autophagy-related (Atg) genes in rainbow trout was monitored throughout the autophagosome formation cycle. The Atg gene sequences of rainbow trout were compared to other species to identify highly conserved regions and to generate primers. Phylogeny trees created with rainbow trout and 14 other species demonstrate that rainbow trout Atg gene sequences have greatest similarity to Atlantic salmon and other fish species. RTgill-W1 cells were subjected to nutrient restriction and compared to cells in normal nutrient conditions using quantitative reverse transcriptase polymerase chain reaction to assess changes in Atg gene expression. Nutrient restriction had a direct impact on Atg gene expression, with atg4, atg9, atg12, lc3, gabarap and becn1 undergoing the greatest differential expression (p < 0.05), most dramatically on Day 3. This was corroborated by Western blot detection of LC3, which also showed a peak of autophagy activity at Day 3 post-nutrient restriction. Atg gene expression revealed autophagy flux in RTgill-W1, as well as, those genes that were most significantly altered by nutrient restriction.