Infectious pancreatic necrosis virus in fish by‐products is inactivated with inorganic acid (pH 1) and base (pH 12)

The aquaculture industry needs a simple, inexpensive and safe method for the treatment of fish waste without heat. Microbial inactivation by inorganic acid (HCl) or base (KOH) was determined using infectious pancreatic necrosis virus (IPNV) as a model organism for fish pathogens. Salmonella and spores of Clostridium perfringens were general hygiene indicators in supplementary examinations. IPNV, which is considered to be among the most chemical‐ and heat‐resistant fish pathogens, was reduced by more than 3 log in 4 h at pH 1.0 and pH 12.0. Salmonella was rapidly inactivated by the same treatment, whereas spores of C. perfringens were hardly affected. The results indicate that low and high pH treatment could be particularly suitable for fish waste destined for biogas production. pH treatment at aquaculture production sites could reduce the spread of fish pathogens during storage and transportation without disturbing the anaerobic digestion process. The treatment could also be an alternative to the current energy‐intensive steam pressure sterilization of fish waste to be used by the bioenergy, fertilizer and soil improver industries.     

Studies on the inactivation of selected viral and bacterial fish pathogens at high pH for waste disposal purposes

This study investigated the use of alkaline hydrolysis at ambient temperature for inactivation of selected fish pathogens in fish tissues under conditions approximating those that are likely to be found in the aquaculture industry. Infectious salmon anaemia virus (ISAV) and Lactococcus garvieae have been determined in a previous study to be the most resistant virus and bacteria to pH 12 from a wide range of viruses and bacteria tested. They were spiked at high titres into fish extracts that were then treated with 1 m sodium hydroxide (NaOH). Viable L. garvieae was not detected in the treated fish extract after 1 h, and ISAV was not detected after 24‐h exposure. Field mortalities of Atlantic salmon, Salmo salar L., caused by infectious pancreatic necrosis virus were treated by alkaline hydrolysis at ambient temperature. The macerated fish mortalities contained a high titre of virus (3.38 × 10⁸ TCID₅₀ g^?1) that was reduced to approximately 2.2 × 10³ TCID₅₀ g^?1 after 24‐h exposure to NaOH, and virus was not detected after exposure for 48 h. The results suggest that alkaline hydrolysis at ambient temperature has potential as a biosecure treatment method for fish by‐products containing fish pathogens.     

Effects of water temperature on mortality in Megalocytivirus‐infected rock bream Oplegnathus fasciatus (Temminck et Schlegel) and development of protective immunity

Rock bream iridovirus (RBIV) causes huge losses, especially in rock bream Oplegnathus fasciatus. Rock bream injected with RBIV and held at 29, 26, 23 or 20 °C had 100% mortality. Conversely, all infected fish held at 17 °C survived even after the temperature was progressively increased to 26 °C at 100 dpi. Rock bream exposed to virus and held for 2, 4 and 7 days at 23/26 °C before the temperature was reduced to 17 °C had mortality rates of 26.6/73.2%, 66.6/100% and 93.4/100%, respectively, through 100 dpi. When surviving fish had the water temperature increased from 17 to 26 °C at 100 dpi, they did not exhibit signs of disease and had low virus copy numbers (below 10³). To investigate the development of a protective immune, rock bream were infected with RBIV and held at 23 °C before shifting the water temperature to 17 °C at 4 dpi. All injected fish survived until 120 dpi. While 100% of the previously unexposed fish died, 80.2% of the previously infected fish survived. When the survivors were rechallenged again at 160 dpi, no further mortality occurred. The high survival rate of fish following rechallenge with RBIV indicates that protective immunity was established in the surviving rock bream.     

Interactions between temperature and size on the growth,size heterogeneity,mortality and cannibalism in cultured larvae and juveniles of the Asian catfish,Pangasianodon hypophthalmus (Sauvage)

Temperature effects vary throughout the ontogeny, but are proportionally more variable during the early life stages than in older fish. The larvae of a few species have been studied but contrasting trajectories were observed, thereby making it difficult to predict how temperature impacts on the growth, survival and size heterogeneity in a particular species. This study examined these interactions in young [0.9 mg–1 g wet mass (WM)] Pangasianodon hypophthalmus, one of the most extensively cultured tropical fish. Fish were raised at five temperatures from 23 to 33 °C in a recirculated water system, fed in excess with a high‐energy feed (Artemia then a formulated feed, >50% protein), and examined at 1‐day (first feeding days) or 4‐day intervals (older fish). The temperature that produced the fastest growth (T°_opt) was 31 °C at the start of exogenous feeding (0.9 mg); it increased to 32.7 °C at 8 mg then decreased by 0.7 °C for each 10‐fold increase of WM. Size heterogeneity was lower and survival was higher (70–85% from 0.9 mg to 1 g) when the temperature was close to T°_opt. Comparisons with other species suggest that the slope of the relationship between T°_opt and fish size has a strong latitudinal component, and may also reflect the fish sensitivity to oxygen.     

Comparative stability of two salmonid viruses and poliovirus in fresh, estuarine and marine waters

Abstract. The inactivation rates in the aquatic environment of two fish pathogens, infectious pancieatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV), were compared with that of poliovirus type 1 as a representative of the human enterovirus group. The survival studies were performed using untreated fresh, estuarine and sea water samples held at 15 and 20°C. The results indicated that the salmonid viruses survived longer than poliovirus in the saline waters, whereas in fresh water poliovirus was the most stable of the three viruses. IPN virus proved to be most stable in estuarine water at 15°C, whereas the survival of IHN virus was favoured in fresh water. We also observed that at 20°C the inactivation rate for each virus was independent of salt concentration in estuarine and sea water. Although temperature exhibited a marked effect on virus stability in fresh water, the salmonid viruses presented similar survival patterns at both temperatures in sea water. In general the period of greatest viral inactivation correlated with higher bacterial numbers in the waters.     

Comparative Properties of Bluefish (Pomatomus saltatrix) Gels Formulated by High Hydrostatic Pressure and Heat

High pressure was applied at levels of 300 to 3,742 atm for 30 min to formulate gels from bluefish meat paste, and the properties of the resulting gels were compared with those of heat-induced gels formulated at 90°C for 20 min, or at 60°C for 60 min. The moisture content of the pressure-induced gels was similar to that of the heat-induced gels, while protein contents and pH values of pressurized gels were slightly lower than those formulated by heat. Gels formed by pressure were more translucent as compared with those formulated by heat. Texture measurement indicated that there were no significant differences between the elasticities of the gels obtained under various pressures, although gel firmness increased with pressure. Overall, the heat-induced gels formulated at 90°C for 20 min were firmer but had similar elasticities to pressure-induced gels, while gels formulated at 60°C for 60 min were comparatively softer and had lower elasticities. The salt-extractable protein and protein digestibility studies indicated that pressure treatment formed gels with less protein denaturation and which were more digestible than the fish gels formulated by heat. The results from proteolytic activity studies showed that the pressure range used in this study was less effective in inactivating the endogenous proteases in the fish flesh than heat.     

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C‐E3‐E2‐6K‐E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10 weeks post‐vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus‐neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7 days and scoring of pathological changes after 21 days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus‐neutralizing antibodies.     

Influences of population density,temperature and latitude on the growth of invasive topmouth gudgeon Pseudorasbora parva

Plasticity in life‐history traits provides advantages for introduced fish in overcoming demographic bottlenecks that would otherwise inhibit establishment. Here, the influence of population density, temperature and latitude was tested on the growth increments and growth rates of invasive populations of topmouth gudgeon Pseudorasbora parva, a small Asian cyprinid fish that is invasive across Europe. Aquaria experiments tested the roles of fish number and temperature on growth increments under a fixed food supply, pond experiments tested the role of density on growth increments, and a field study completed in England and Wales tested the influence of density and latitude on growth rates. In aquaria experiments, whilst growth increments were higher at 21 and 23 °C than at 19 and 25 °C, fish number had a greater influence on growth than temperature. Higher growth increments were produced at lower densities. In experimental ponds, growth increments were significantly higher in ponds with low densities of P. parva compared with those at elevated densities. In the field study comprising 10 wild populations across a latitudinal gradient of 4.0°N, a difference in mean air temperatures of approximately 3 °C, and estimated densities between 0.5 and 65.0 m^?2, population density was the only significant predictor of growth rates. Whilst populations at very low densities comprised of significantly faster growing individuals, there were no significant differences when densities were between 15 and 65 m^?2. Thus, invasive P. parva populations have considerable growth plasticity, especially at low densities, with this likely to be important in their ability to colonise new environments.     

Optimal Conditions for Long-distance Transportation of Live Black Rockfish (Sebastes schlegeli) and Changes in their Characteristics during Transport

Optimal transport conditions for the live black rockfish and changes in their characteristics during long-distance transport were investigated. Oxygen consumption by black rockfish was lowest at 4°C and increased as the water temperature increased up to a maximum at 10°C. At a water temperature of 8°C and 10°C, the plasma glucose concentration of the fish was 36.2 and 38.2 mg/dL, respectively. The mean weight of the fish reduced to approximately 2.8% of initial weight after 16 days of storage at 8°C. The fish did not feed, and the survival rate of fish stored for 16 days at 8°C was 98.4%. The longer the live fish were stored in the container, the quicker the onset of rigor mortis after the slaughter was reached. Compared with fillets from control fish, the chewiness of fillets was somewhat lower (17.3%) after storage of fish for 16 days, but the softness of the fillets was slightly higher (21.6%). From these results, it was suggested that the optimum temperature for long-distance transport of the live fish was 8°C and that the long transport period of live fishes decreased their fillet quality more rapidly after fish death.     

Tolerance response of tambaqui Colossoma macropomum (Cuvier) to water pH

Tambaqui Colossoma macropomum (Cuvier) is a fish of primary importance in Amazon aquaculture. It has been described as an acid‐resistant species that moves seasonally between white (muddy) water and black water rivers and enters the extremely dilute acidic areas of flooded jungle to feed during the rainy season. To analyse the pH tolerance of this species, tambaqui were exposed to three water pH levels for 40 days (pH 4.0, 6.0 and 8.0). The water was acidified slowly over 3 h, allowing the fish to acclimate. A similar protocol was used to adjust water pH to 8.0. No mortality was observed during the exposure period. Several haematological parameters were significantly changed in alkaline‐exposed animals, with significant decreases in haematocrit (20%), haemoglobin concentration (8%) and red blood cells (12%). Tambaqui showed severe blood variations when exposed to alkaline pH. Fish final weight, condition factor and specific growth rate (SGR) was inversely proportional to a pH increase, and SGR were higher for fish reared in acidic water. The relative insensitivity of tambaqui to low pH confirms its acid tolerance and is in accordance with its natural occurrence in black water habitats.     

The effects of stress and storage temperature on the colour and texture of pre-rigor filleted farmed cod (Gadus morhua L.)

This experiment deals with the effects of pre‐slaughter stress and storage temperature on muscle pH, fillet contraction, colour and texture in pre‐rigor filleted farmed cod fillets. The fish were either sedated with a low dose of MS‐222 (14.3 mg L^?1) (unstressed groups) or exposed to the air for 3 min (stressed groups) before being submerged in a benzocaine bath (150 g L^?1). The fish were then killed by a blow to the head, their gills cut, filleted and finally stored at either 4 or 20°C. The stressed groups had significantly lower pH values after slaughter (pH=7.0) than the unstressed groups (pH=7.3). This difference was maintained until post rigor for the fish stored at 4°C, but at 20°C it was immediately overshadowed by a decrease in pH caused by temperature‐dependent processes. The length contraction and changes in registered colour values were more pronounced at both the higher temperature and the higher level of pre‐slaughter stress. Again temperature dominated, but significant and consistent effects were registered from stress. No significant effects of stress on texture post rigor were observed. It is concluded that high storage temperature masks the majority of effects caused by pre‐slaughter stress on the measured variables. Stress management protocols, however, are important when the fillets are kept at the common storage temperature of 4°C.     

Low pathogenicity of flounder iridovirus (FLIV) and the absence of cross‐protection between FLIV and rock bream iridovirus

The genus Megalocytivirus is known to infect a wide range of cultured marine fish. In this study, we examined the pathogenicity of FLIV (Megalocytivirus from olive flounder, genotype III) and RBIV (Megalocytivirus from rock bream, genotype I) to their homologous and heterologous host species. Olive flounder (7.5 ± 1.3 cm) injected with FLIV [major capsid protein (MCP) gene copies, 6.8 × 10³–6.5 × 10⁶/fish] at 24 °C did not die until 90 days post‐infection (dpi). The average virus replication in the spleen peaked (1.27 × 10⁶/fish) at 20 dpi. Rock bream (6.5 ± 1.5 cm) injected with FLIV (8.8 × 10⁵ and 6.5 × 10⁶/fish of MCP copies) showed no mortality until 50 dpi. The rock bream that survived after FLIV infection were rechallenged with RBIV at 50 dpi had 100% mortality, showing that there is no cross‐protection between FLIV and RBIV. Temperature shifting (26 °C and 20 °C at 12 h intervals) did not cause FLIV‐specific mortality into olive flounder, but higher virus copies were observed in the fish exposed to higher stocking density. This study demonstrates that FLIV and RBIV have different antigenic and pathogenic characteristics and that FLIV has low pathogenicity to olive flounder.     

Effects of fish oil with difference oxidation degree on growth performance and expression abundance of antioxidant and fat metabolism genes in orange spotted grouper,Epinephelus coioides

The present study was designed to assess the effects of fish oil with different oxidation degree on growth performance, serum biochemistry parameters and expressive abundance of oxidative stress and fat metabolism genes of orange spotted grouper Epinephelus coioides. The oxidized fish oil was conducted as follows: storage temperature: 4°C, ambient temperature (AT, [31.5 ± 3.5]°C); storage time: 45, 90, 135 days; antioxidant contents: 30 mg/kg (ethoxyquin [EQ]), 300 mg/kg Higher EQ (HEQ). According to the different treated conditions, 14 kinds of fish oil with different oxidation degree were obtained: T_F+EQ [positive control (fresh oil + EQ)], T_F (negative control [fresh oil]), T_4°C+45d+EQ, T_4°C+45d+HEQ, T_4°C+90d+EQ, T_4°C+90d+HEQ, T_4°C+135d+EQ, T_{4°C+135d+HEQ}, T_AT+45d+EQ, T_AT+45d+HEQ, T_AT+90d+EQ, T_AT+90d+HEQ, T_AT+135d+EQ, T_AT+135d+HEQ. Groupers were fed isonitrogenous and isolipidic diets containing 14 kinds of fish oil for 8 weeks, respectively. The results showed that survival, weight gain rate and thermal growth coefficient decreased as oxidation degree of dietary fish oil increased (p < 0.05). Higher serum total protein, triglyceride and glucose were observed with ascending oxidation degree of fish oil (p < 0.05). The genes expression levels of catalase, superoxide dismutase and glutathione peroxidase were up‐regulated with dietary oxidized level increasing (p < 0.05). In addition, the similar status also appeared in expression of peroxisome proliferator‐activated receptor gamma (PPARγ), hormone‐sensitive lipase (HSL) and fatty acid synthase (FAS) genes. In conclusion, the fish oil would show negative influence on the fish health until peroxide value and p‐anisidine value in oil exceed 12.96 meq/kg and 20.89. The best storage condition for fish oil is 4°C, 45 days and 30 mg/kg EQ which could keep fish oil available property to grouper.     

Roles of water quality and disinfectant application on inactivation of fish pathogenic Streptococcus agalactiae with povidone iodine,quaternary ammonium compounds and glutaraldehyde

Streptococcosis is an important bacterial disease in Nile tilapia causing severe economic losses to tilapia aquaculture worldwide. The effects of water quality (low‐ [LS] and high‐level [HS] soiling, to mimic clean or dirty surface conditions and temperatures) and disinfectant application (diluted concentrations and exposure time) were characterized on the inactivation of Streptococcus agalactiae isolated from diseased tilapia. Five isolates were tested against three commercial disinfectant products with the main ingredients being povidone iodine (Anidine 100?; AD), benzalkonium chloride (Better BKC 80%?; BKC 80), and a mixture of quaternary ammonium compounds and glutaraldehyde (Chloraldehyde?; CR). CR demonstrated highest efficacy to S. agalactiae inactivation, followed by BKC 80 and AD, respectively. Higher‐level soiling, low temperature, diluted concentrations and short exposure time all decreased the disinfectant efficacy. CR and BKC 80 provided more than 5‐log inactivation at 1‐min exposure at 20°C under HS conditions, and also with ten‐fold‐diluted concentrations at 60‐min exposure time at 30°C. However, AD required 10‐min exposure to effectively remove bacteria under LS conditions at 30°C. The results could facilitate aquaculture management planning that leads to operating cost reductions and improvements in biosecurity.     

Ammonia nitrogen excretion in Mandarin Fish (Siniperca chuatsi) and Grass Carp (Ctenopharyngodon idellus) fed practical diets: the effects of water temperature

To understand the actual production of fish culture about the utilization of dietary protein and excreta impact on the environment between mandarin fish (Siniperca chuatsi) and grass carp (Ctenopharyngodon idellus), the study to investigate the effect of temperatures (19 ± 0.5°C, 24 ± 0.5°C and 29 ± 0.5°C) on ammonia‐N excretion in mandarin fish and grass carp under fed and fasted states was conducted. These two species were fed a practical diet containing 325.2 g kg^?1 crude protein at 3% body weight per day. The ammonia‐N excretion rate was significantly increased when temperature increased from 19 to 29°C, and a linear relationship between ammonia‐N excretion rate and temperature. The maximum ammonia‐N excretion levels of mandarin fish and grass carp were observed at 4–8 h and 2–4 h after feeding, respectively, and the minimum values for both species were observed at 24 h after feeding. Under the feeding condition, mandarin fish had a lower ammonia‐N excretion level compared to grass carp at 24°C and 29°C. The average amount of ammonia‐N excreted by mandarin fish at 24 h is significantly higher than grass carp under fasting conditions, except 19 ± 0.5°C. These results indicated that mandarin fish might make better use of protein at higher temperature than grass carp when fed practical diets in commercial production. These results of this study suggested that mandarin fish had a lower ammonia‐N excretion level compared with grass carp, making a less contribution to environmental loading in an intensive fish culture.     

Depletion study and estimation of withdrawal periods for florfenicol and florfenicol amine in pacu (Piaractus mesopotamicus)

The intensive production of farmed fish is at a global all‐time high, and the control of bacteria proliferation in fish farms requires the frequent use of antimicrobials. This practice raises important environmental concerns related to the emergence of antimicrobial‐resistant bacteria strains. Only a few antimicrobial drugs have been approved for use in aquaculture, one of which is florfenicol. This work studies the depletion and withdrawal period of florfenicol and its main metabolite, florfenicol amine, in pacu (Piaractus mesopotamicus), a neotropical characin widely farmed in the southern hemisphere. Juvenile pacu (average weight of 724 g) were stocked in a closed‐loop laboratory system with controlled water temperature (25.8°C), and were fed for 10 consecutive days with a diet containing an intended dose of 10 mg/florfenicol per kg bw. Muscle and skin tissues were collected at 1, 3, 6, 8, 10, 12 and 16 days post‐treatment, and florfenicol and florfenicol amine were quantified using a validated ultra‐high performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method. The limits of quantitation for florfenicol and florfenicol amine were 10 ng/g in muscle and 50 ng/g in skin. Considering a maximum residue limit of 1000 ng/g for the sum of florfenicol and florfenicol amine in muscle with skin in natural proportions a withdrawal period of 5 days (water temperature 25.8°C) or 129 degree days was calculated on the basis of the upper limit of the one‐sided 95% confidence interval for the 99^th percentile derived from the residue depletion study.     

Influence of feeding habits of fish on the characteristics of their visceral alkaline proteases with special reference to detergent compatibility

Technical characteristics and detergent compatibility of visceral alkaline proteases of three freshwater fish, namely Labeo rohita, Pangasianodon hypophthalmus and Cyprinus carpio of different feeding habits, were studied. Crude enzyme extract was partially purified by (NH₄)₂SO₄ precipitation and dialysis. The molecular weight was in the range of 20–63 kDa. The enzyme purification folds post‐dialysis were found to be 1.55, 1.81 and 2.17 in case of Rohu, Pangas and Common carp respectively. The alkaline protease from Rohu, Pangas and Common carp exhibited maximum activity at pH 10.0, 9.0 and 11.0 respectively. The enzyme temperature optima observed were 60°C (Rohu and Pangas) and 70°C (Common carp). SBTI and EDTA inhibited more than 90% of the activity at conc. of 50 mM. Exposure of the proteases to non‐ionic surfactants like Tween 20–80 retained about 92%–100% and 76%–100% of their activity at conc. (v/v) of 1% and 5% respectively. Proteases were found less stable in the presence of SDS. There was moderate to lesser influence of H₂O₂ and sodium perborate on the proteolytic activity. The alkaline protease from omnivorous fish was found superior compared to the herbivore and carnivore in respect of pH and temperature optima and stability with detergents and oxidizing agents.     

Effects of changes in environmental factors on the non-specific immune response of Nile tilapia, Oreochromis niloticus L.

Lysozyme acts as a non‐specific defence substance and is found in the peripheral blood, cutaneous mucus and certain tissues of marine and freshwater fishes. In the present study, we examined the effect of various environmental factors (water temperature, salinity, pH and suspended sediments) on plasma lysozyme activity in the Nile tilapia, Oreochromis niloticus L. When the fish were reared at different water temperatures (18.4, 23, 28 and 33°C), plasma lysozyme activity increased at 28°C after 2 and 4 weeks. A significant decrease in lysozyme activity was found in the fish reared at 33°C for 4 weeks. These results suggest that there is a water temperature range that affects the amount of plasma lysozyme activity that can be detected. Fish cultured at 24 g L^?1 of salinity for 2 and 4 weeks and 12 g L^?1 for 4 weeks resulted in significantly increased plasma lysozyme activity, suggesting that environmental salinity also affects the amount of plasma lysozyme that can be detected. Lysozyme activity also significantly increased when the fish were held in acidic water at pH 4.0 and in suspended sediments at 2000 mg L^?1 for 2 weeks. It was concluded that changes in some aquatic environmental factors affect the non‐specific immune responses of Nile tilapia.