Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus , caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10⁷ copies μg⁻¹ DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10⁶–10⁸ copies of CyHV-2 μg⁻¹ DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22–10 °C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (±SE) of 7.3 ± 11 to 394 ± 55 μg⁻¹ DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.     

A sensitive FRET probe assay for the selective detection of Mycobacterium marinum in fish

Mycobacterium marinum is the causative agent of mycobacteriosis in wild and cultured fish and of atypical infection in humans. For the diagnosis of M. marinum , cultural and traditional polymerase chain reaction (PCR) methods are currently used. However, these protocols, although able to discriminate within Mycobacterium spp., have proved to be time-consuming or difficult to carry out. For this reason, the aim of this study was to obtain a rapid and specific diagnostic tool to quantify fish Mycobacterium spp. or to discriminate M. marinum from other mycobacteria. A primary PCR amplification with SYBR Green had a detection limit (dl) of 10² Mycobacterium DNA copies with a log-linear quantification range up to 10⁴ ( R ² = 0.99). The second PCR using FRET probes, flanking a region containing species specific nucleotide variations, was designed and validated with synthetic erp gene fragments corresponding to different mycobacterial species, different whole mycobacteria suspensions, experimentally infected fish tissues, tissues from experimentally infected fish, and samples of cultured fish. The results show that the FRET probes demonstrate a high specificity as the melting curve analysis allowed efficient discrimination of M. marinum from Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium pseudoshottsii , Mycobacterium shottsii and Mycobacterium ulcerans . The kidney is the organ with the strongest detection signal and using fish tissues the method has a mean sensitivity of 50 DNA copies/PCR.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).     

Identification of immune cells interacting with Vibrio spp. and its in vitro post-phagocytic killing mechanism of haemocytes in the penaeid shrimp, Penaeus indicus H. Milne Edwards

Immune cells were identified and their interaction towards Vibrio alginolyticus, V. parahaemolyticus and V. anguillarum was studied in vitro in the penaeid shrimp, Penaeus indicus. Haemocytes were divided into agranulocytes, semi-dense granulocytes and dense granulocytes according to their morphology. Agranulocytes (100%) and 0.3–0.7% of granulocytes were actively involved in coagulation. Granulocytes were involved in in vitro phagocytosis and encapsulation of foreign materials. Phagocytosis was enhanced by prior opsonization of bacteria with cell-free shrimp haemolymph. Semi-dense granulocytes were phagocytic towards V. alginolyticus with and without opsonization at the rate of 91.1% and 83.1%, respectively ( P < 0.05 ). Granulocyte death observed after 2 h with opsonized haemolymph was 26.1%. About 64.5% of dense granulocytes and 23.2% of semi-dense granulocytes were actively involved in encapsulation, forming capsules. A spectrophotometric nitroblue tetrazolium (NBT) reduction assay was used to demonstrate the production of superoxide anions (O₂⁻) by shrimp haemocytes. All the Vibrio spp. were able to induce superoxide anions (O₂⁻) during phagocytosis. Live Vibrio sp. induced O₂⁻ production in haemocytes in a dose-dependent manner. Significant activity was detected with a 40:1 bacteria to haemocyte ratio ( P < 0.05 ). NBT reduction assay for measuring the post-phagocytic killing mechanism in shrimp haemocytes might be a valuable tool for monitoring shrimp health and immunological studies.     

Ultrasonic immunization of sea bream, Pagrus major (Temminck & Schlegel), with a mixed vaccine against Vibrio alginolyticus and V. anguillarum

In order to clarify the effectiveness of ultrasonication on vaccine delivery, juvenile sea bream, Pagrus major , were treated with eight different ultrasonic methods. A mixed vaccine against Vibrio alginolyticus and V. anguillarum was used to immunize the fish . The intensity and frequency of the ultrasound were 280 mW cm^–2 and 35 kHz, respectively. The ultrasonic methods included continuous or pulsed ultrasound for 3 min, and continuous or pulsed ultrasound for 3 min before and/or after immersion for 3 min. Of all the eight ultrasonic methods tested, `pulsed ultrasound followed by immersion' and `immersion, pulsed ultrasound, and followed by immersion again' provided the best protection, which were comparable with protection of fish immunized by intraperitoneal injection. Moreover, the convenience of applying these two ultrasonic methods for immunization was comparable with the immersion method and was much better than intraperitoneal injection. If 2 × 10⁸ CFU mL^–1 of this mixed vaccine was used for vaccination repeatedly five times by ultrasonic methods, it could still produce good protection for the immunized sea bream. Therefore, the ultrasonic method is an effective and practical approach for fish vaccination on a large scale.     

Enhancement of resistance to bacterial infection in rainbow trout, Oncorhynchus mykiss (Walbaum), by oral administration of bovine lactoferrin

Abstract Bovine lactoferrin was evaluated for its ability to enhance resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Vibrio anguillarum. Oral administration of lactoferrin (100 mg kg⁻¹) to fish daily for 3 days prior to an intraperitoneal challenge with V. anguillarum resulted in increased survival rates, and enhanced resistance against Streptococcus sp., although to a lesser extent. In lactoferrin-treated fish, an increase in phagocytic and chemiluminescent (CL) activities of pronephros cells against V. anguillarum was observed. The phagocytic and CL activities of cells against Streptococcus sp. were also significantly increased. However, no in vitro bactericidal activities of lactoferrin against V. anguillarum or Streptococcus sp. were observed. This suggests that the lactoferrin enhanced the resistance of the rainbow trout against bacterial infection through the activation of phagocytes.     

The influence of different levels of dietary vitamin E on sea bass Dicentrarchus labrax flesh quality

Sea bass Dicentrarchus labrax fillet quality was investigated after feeding with four diets (A, B, C or D) containing different levels of dietary vitamin E (139 mg kg^–1, 254 mg kg^–1, 493 mg kg^–1 and 942 mg kg^–1, respectively). Six-hundred and eighty fish (mean initial weight 208 g) were equally divided into four 20 m³ tanks and fed for 87 days. Filtered seawater with a temperature ranging from 18.2 to 26.3 °C was supplied continuously. At the end of the experiment, fish were stored at 1 °C for 12 days. At one, three, six, nine and 12 days, 20 fish per group were processed for proximate composition, vitamin E and induced thiobarbituric acid reactive substances (TBARs) analyses. No significant differences in proximate composition were registered between groups. The flesh lipid content ranged from 88.0 g kg^–1 (group B) to 96.8 g kg^–1 (group A). Vitamin E fillet content was significantly different between groups, reaching levels of 98.0, 150.7, 225.2 and 302.0 μg g^–1 lipids for group A, B, C and D, respectively. Induced TBARs values were statistically different only for group A compared with the other groups. No significant variations were registered in relation to preservation time. Because of the positive influence of vitamin E on seafood quality and the correlation between its dietary level and flesh deposition, the α-tocopherol content of the diet should be well above fish minimum requirements.     

Linear models to predict the digestible lipid content of fish diets

Values for the digestible contents of nutrients in diets and feed ingredients are of utmost importance in nutritional strategies for fish. Prediction from dietary composition would eliminate lengthy, tedious and demanding digestibility experiments with fish. Apparent digestible lipid (DL) content [range 7.6–353.4 g kg⁻¹ dry matter (DM)] in compound diets can be predicted with high accuracy ( n  = 610; studies =127; fish species = 34; R ² = 0.9515; RMSE = 16.9504) from dietary crude lipid (CL) content (range 12.0–388.7 g kg⁻¹ DM) by the linear regression equation DL =−2.7303 + 0.9123 CL. Validation of this equation against 65 values from 15 independent studies presented R ² and mean prediction error (MPE) values of 0.9947 and 0.0671, respectively. The corresponding equation for 37 individual feed ingredients evaluated in 24 studies with 18 fish species ( n  = 180) was found to be DL = −1.5824 + 0.8654 CL ( R ² = 0.9717; RMSE = 8.3765). However, validation of the latter is currently hampered by a lack of independent values.     

Distribution and biomass of an underfished vendace, Coregonus albula, population in a mesotrophic German reservoir

Abstract  The distribution and overall biomass of an underfished vendace, Coregonus albula L., population in the mesotrophic Henne Reservoir (Germany) was studied using hydroacoustics and gill nets. Additionally, midwater trawling was carried out. Overall fish biomass, based on five hydroacoustic surveys (June to September 2002), ranged from 188 kg ha⁻¹ in early August to 302 kg ha⁻¹ in September 2002. The overall mean fish biomass was 256 kg ha⁻¹ (±48 kg ha⁻¹ SD). Biomass of fish smaller than 25 cm total length (mostly vendace) varied from 56 kg ha⁻¹ in August to 99 kg ha⁻¹ in September, with an overall mean fish biomass of 74 kg ha⁻¹ (±17 kg ha⁻¹ SD). The echograms showed temporal variation in fish distribution in Henne Reservoir. In June, fish were fairly evenly distributed over the whole reservoir but in September a dense aggregation of fish (mostly vendace) was found in the deeper water layers near the dam. The distribution of vendace stock, its impact on water quality and fisheries management, biomanipulation and effort for mass removal are discussed.     

Fish community composition of a tropical nonestuarine embayment in Zanzibar,Tanzania

ABSTRACT:   By using a seine net, fish samples were taken from the nonestuarine Chwaka Bay (Zanzibar, Tanzania) from the mangroves, mud/sand flats and seagrass beds. Sampling was done twice per month between November 2001 and October 2002. In total, 150 fish species belonging to 55 families were identified. Diversity ( H' ) ranged from 1.9 in mud/sand flats to 3.4 within the Chwaka seagrass beds. Mean density of fishes was significantly higher in the mangrove creeks than in any other habitat (mean = 238.7 ind./1000 m²). Highest, but non-significantly different mean biomasses were recorded in the mangrove creeks (1.7 kg/1000 m²) and in the Marumbi seagrass beds (1.6 kg/1000 m²). The mangrove channel had the lowest biomass (0.6 kg/1000 m²). A high overlap in species composition (as high as 93.4% similarity) was found for adjoining habitats (i.e. mangrove creeks and mangrove channel), while habitats that were far apart showed low overlap (6.6% similarity for the Marumbi seagrass beds and mangrove creeks). On average, 58.4 and 63.2% in terms of abundance and biomass, respectively, of the fish assemblage of Chwaka Bay were of commercial fishery importance. Thus, Chwaka Bay appears to be an important juvenile habitat for various commercially important fish species.     

病原性鳗弧菌(Vibrio anguillarum)双重PCR与LAMP检测方法的建立

本研究检测了分离自发病大菱鲆、半滑舌鳎及鲤鱼的22株病原鳗弧菌(Vibrio anguillarum)毒力相关基因的携带情况,并建立了病原鳗弧菌的分子生物学检测方法。以PCR方法检测8个毒力相关基因的分布,结果显示,22株病原鳗弧菌均可扩增出6个基因(empA、vah1、vah4、flaA、rtxA和tonB)目的条带,未扩增出virA和angM基因;针对vah4和rtxA设计引物进行双重PCR扩增,同一PCR反应体系可扩增出两条目的条带,灵敏度为2.4×103 CFU/ml,对照菌无任何扩增条带;以vah4设计引物进行LAMP扩增,病原鳗弧菌可扩增出阶梯状条带,呈现阳性反应,6株对照菌无阶梯状扩增条带且呈现阴性反应,LAMP扩增灵敏度为2.4×101 CFU/ml。LAMP检测灵敏度是双重PCR的100倍,LAMP技术与PCR比较,操作简便、快速、灵敏度高且不需昂贵仪器,LAMP检测鳗弧菌的方法更适合于养殖生产实际应用。     

Growth of juvenile tilapia, Oreochromis niloticus L. from Lakes Zwai, Langeno and Chamo (Ethiopian rift valley) based on otolith microincrement analysis

Abstract – Age and growth of juvenile tilapia, Oreochromis niloticus , from Lakes Zwai, Langeno and Chamo (Ethiopia) were studied from microincrements in otoliths. Growth in length was best described by the Gompertz model. Average growth rate of the fish was most rapid in Lake Chamo (0.39 mm  ·  day⁻¹, 1.14%  ·  day⁻¹), intermediate in Lake Zwai (0.20 mm  ·  day⁻¹, 0.72%  ·  day⁻¹) and slowest in Lake Langeno (0.16 mm  ·  day⁻¹, 0.62%  ·  day⁻¹). Similarly, back-calculation from otolith increment widths gave growth rates of 0.28 to 0.43 mm  ·  day⁻¹, 0.15 to 0.32 mm  ·  day⁻¹ and 0.11 to 0.28 mm  ·  day⁻¹ for Chamo, Zwai and Langeno fish, respectively. In addition, Fulton's condition factor was largest for Chamo tilapia and smallest for Langeno tilapia; the difference between fish from Langeno and Zwai was small. Rapid growth of juvenile O. niloticus in Lake Chamo was attributed to warm temperature and better food quality. ^Note     

Supplemental citric acid and particle size of fish bone-meal influence the availability of minerals in rainbow trout Oncorhynchus mykiss (Walbaum)

Juvenile rainbow trout Oncorhynchus mykiss (Walbaum) were fed six low-phosphorus (P) diets supplemented with two different sizes of ground fish bone-meals (fine, 68 μm or less; coarse, 250–425 μm) and a coarse bone-meal diet containing four levels of citric acid (0, 4, 8 or 16 g kg⁻¹ diet) to investigate the effects of pH and bone particle size on P bioavailability. The basal diet provided 3.4 g P   kg⁻¹ and bone-meal increased P contents to 5.4–6.0 g P   kg⁻¹. Coarse bone-meal diets supplemented with 0, 4, 8 or 16 g kg⁻¹ of citric acid had pH values of 6.0, 5.7, 5.4 and 5.0, respectively. Weight gain and whole-body water, protein and lipid contents were not influenced by bone-meal supplementation. Supplementing the basal diet with both coarse and fine bone-meal significantly increased whole-body ash content. Fish fed no bone-meal were hypophosphataemic compared with fish fed with either fine or coarse bone-meals. Phosphorus in fine bone-meal had higher availability than P in coarse bone-meal. Bone-meal supplementation significantly decreased whole-body manganese content from 8.9 μg g⁻¹ in fish fed no bone-meal to 2.3 and 4.5 μg g⁻¹ in fish fed with fine and coarse bone-meals, respectively. The concentration of magnesium increased but zinc concentration was not affected by bone-meal supplements. Citric acid increased whole-body ash content but the influence of citric acid on the body P content was not significant ( P  = 0.07). Dietary acidification by citric acid significantly increased whole-body iron in a linear fashion. The bioavailability of dietary P can be improved by fine grinding the bone in fish meals.     

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 °C for 60 days, to compare LCD-incidence. In the fish reared at 20 °C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10⁶ PCR-U mg⁻¹ tissue, were detected in the fins and skin of LCD-affected fish at 20 °C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 °C and 30 °C; however, a low level of LCDV (10³ PCR-U mg⁻¹ tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 °C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 °C.     

Carbohydrate level in the diet of silver barb, Puntius gonionotus (Bleeker) fingerlings: effect on growth, nutrient utilization and whole body composition

Five iso-nitrogenous (300 g crude protein kg⁻¹ diet) semi-purified diets with graded levels of carbohydrate at 220 (D-1), 260 (D-2), 300 (D-3), 340 (D-4) and 380 (D-5) g kg⁻¹ diet were fed ad libitum to Puntius gonionotus fingerlings (average weight 0.59±0.01 g) in triplicate groups (20 fish replicate⁻¹) for a period of 90 days to determine the effect of the dietary carbohydrate level on the growth, nutrient utilization, digestibility, gut enzyme activity and whole-body composition of fish. Fifteen flow-through cement tanks of 100 L capacity with a flow rate of 0.5 L min⁻¹ were used for rearing the fish. The maximum weight gain, specific growth rate, protein efficiency ratio, RNA:DNA ratio, whole-body protein content, protease activity, protein and energy digestibility and minimum feed conversion ratio (FCR) were found in the D-2 group fed with 260 g carbohydrate kg⁻¹ diet. The highest protein and energy retention was also recorded in the same group. However, from the second-order polynomial regression analysis, the maximum growth and nutrient utilization of P. gonionotus fingerlings was 291.3–298.3 g carbohydrate kg⁻¹ diet at a dietary protein level of 300 g kg⁻¹ with a protein/energy (P/E) ratio of 20.58 −20.75 g protein MJ⁻¹.     

Effects of dietary protein to metabolizable energy ratios and total protein concentrations on the performance of yellow perch Perca flavescens

Juvenile yellow perch Perca flavescens were fed semipurified diets with varying protein to metabolizable energy ratios (PME, g protein MJ⁻¹ metabolizable energy) and nutrient densities in three experiments to determine recommended dietary protein and energy concentrations. Experiment 1 fish (18.6 g) were fed diets containing 450 g crude protein kg⁻¹ dry diet and 14.5–18.8 MJ ME kg⁻¹ dry diet for 10 weeks. No differences were found in the growth of experiment 1 fish fed the different diets. Experiment 2 fish (21.9 g) were fed diets containing 15.7 MJ ME kg⁻¹ dry diet and 210–420 g crude protein kg⁻¹ dry diet for 8 weeks. Fish fed the diet containing 340 g kg⁻¹ protein (diet PME = 22) exhibited the greatest weight gain. Experiment 3 fish (27.1 g) were fed diets with a PME of 22 and varying nutrient density (yielding 205–380 g crude protein kg⁻¹ dry diet) for 8 weeks. No differences were found in the growth of experiment 3 fish. Yellow perch fed the semipurified diets exhibited increased liver fat content, liver size and degree of liver discoloration compared with fish fed a commercial fish meal-based diet. Liver changes may have resulted from high dietary carbohydrate levels. We conclude that a protein level of 210–270 g kg⁻¹ dry diet is suitable for juvenile yellow perch provided that the dietary amino acid profile and carbohydrate content are appropriate for yellow perch.     

The relationship between stock and catch and the effect of bait on catch as determined for a UK recreational catch and release fishery

Abstract Twelve anglers fishing in a UK navigation canal for a total of 42 h caught 567 fish, mainly gudgeon, Gobio gobio (L.) ( n =306) and roach, Rutilus rutilus (L.) ( n =253) at an average catch rate of 13.4 fish angler-h⁻¹ or 128.5 g angler-h⁻¹. The species and size of fish caught were compared with the numbers determined by depletion estimates at six sections of canal using micromesh seine netting. Fifty four percent of fish caught in the net were < 60 mm FL. Gudgeon (60–99 mm) were over-represented in the anglers' catches whilst roach (60–99 mm) were under-represented. The size distribution of roach and gudgeon caught by anglers using two types of bait (small maggots and chironomid larvae) was examined and smaller fish were found to be caught using the latter.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

A pharmacokinetic study of flumequine in sea bass, Dicentrarchus labrax (L.), after a single intravascular injection

The pharmacokinetic properties of flumequine following a single intravascular injection (10 mg kg^–1 fish) were studied in sea bass, Dicentrarchus labrax (L.), 120 g held at 18 °C. The absorption half life ( t _1/2α) and the elimination half life ( t _1/2β) of the drug were calculated to be 1.05 and 10.71 h, respectively. Tissue penetration of flumequine seemed to be moderate because both the apparent volume of distribution of the drug at steady-state ( V _d(ss)) and the extensive apparent volume of the central compartment ( V _c) were found to be small (1.51 and 0.626 L kg^–1). The mean residence time ( MRT ) was short (09.73 h) and the total clearance (CL_T) of the drug was rapid (0.156 L kg^–1 h^–1).