Hematological parameters and phagocytic activity in fat snook (Centropomus parallelus) bred in captivity

The objective of this work was to determine the hematological parameters and the phagocytic capacity of peritoneal macrophages of fat snook related to sex, stage of gonadal maturation and seasonal cycle. Blood was collected from 135 animals (78 females and 57 males) and used for determinations of: erythrocyte number, hematocrit, hemoglobin, erythrocyte indices mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), total and differential leukocyte counts, and thrombocyte count. The phagocytic capacity and phagocytic index were determined after Saccharomyces cerevisiae inoculation in the peritoneal cavity of the animals. The hematological results according to sex showed that the erythrocyte, total leukocyte and thrombocyte counts were statistically higher in males than females, with the latter showing a higher MCV. Concerning to erythrocyte count, hematocrit and hemoglobin concentration analyzed separately by sex and stage of gonadal maturation, males were found to have significantly elevated values in the mature stage and decreased levels in the resting stage. The results of the erythrocyte and leukocyte series, thrombocytes and phagocytic activity related to seasonal cycle showed significant differences in both sexes, where hematocrit and hemoglobin concentration were lower in winter and higher in the other seasons, mean corpuscular volume was higher in the summer and lower in the winter and fall, total leukocytes and thrombocytes lower in the spring and higher in the fall, lymphocytes low in the winter and summer and high in the spring and phagocytic capacity and phagocytic index high in the summer and low in the winter and fall. The results showed that the hematological values in males are statistically higher than those in females, the erythrocyte values in males increase with the progression of gonadal maturation and that winter is the season of the year least favorable for hematological and phagocytic responses for survival of fat snook kept in captivity. The parameters studied could be utilized in the evaluation of the health status of this species in captivity. (C) 2012 Elsevier Ltd. All rights reserved.

Hemolytic activity of alternative complement pathway as an indicator of innate immunity in pacu (Piaractus mesopotamicus)

The objective of this study was to evaluate the methodology to establish the hemolytic activity of alternative complement pathway as an indicator of the innate immunity in Brazilian fish pacu (Piaractus mesopotamicus), in addition to verifying the influence of beta-glucan as an immunostimulant. Fish were fed with diets containing 0, 0.1 and 1% beta-glucan, during seven days, and then inoculated with Aeromonas hydrophila. Seven days after the challenge, they were bled for serum extraction. The methodology consisted of a kinetic assay that allows calculating the required time for serum proteins of the complement to promote 50% lysis of a rabbit red blood cell suspension. The method developed in mammals was successfully applied for pacu and determined that the hemolytic activity of the proteins of the complement system (alternative pathway) increased after the pathogen challenge, but was not influenced by the beta-glucan treatment.

Gonadal steroids levels and vitellogenesis in the formation of oocytes in Prochilodus lineatus (Valenciennes) (Teleostei: Characiformes)

The objective of this study was to obtain information about the possible mechanisms related to poor reproductive performance in tropical rheophilic fish. To that effect, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively, for curimbata (Prochilodus lineatus) breeders. Fish were maintained under experimental conditions for 18 months, and during this period females were randomly sampled every two months for biometric analysis (n=30), blood (n=5/sampling) and ovary (n=5/sampling). After this period EPs females (EPFs) and Cs females (CFs) were submitted to the induced breeding experiments. The results showed that rearing curimbata for such long time in a cage at this stocking density, reduces its growth, plasma E2 levels and vitellogenesis. During vitellogenesis, the mean plasma estradiol levels of CFs were three times lower than those of EPFs (P<0.01). CFs presented poorer results than EPFs for all the examined parameters of reproductive performance. Taken together these data showed that the reduced estradiol levels during vitellogenesis (and the consequently less intense transition from the previtellogenic to vitellogenic phase) and reduced amounts of yolk are mechanisms associated with the formation of low quality oocytes and shortened and delayed breeding season in this species. Moreover, our data showed that the onset of vitellogenesis (six months before the spawning season) must be considered as a key period related to the formation of oocytes of good quality, and adequate management should be provided throughout the year.

Antioxidant defenses and biochemical changes in the neotropical fish pacu, Piaractus mesopotamicus: Responses to single and combined copper and hypercarbia exposure

This study investigated the potentially detrimental effects of copper and elevated aquatic CO2 (hypercarbia), alone or in combination, on pacu, Piaractus mesopotamicus. Fish were exposed for 48 h to control (no copper addition in normocarbia), to 400 mu g Cu2+L-1, to hypercarbic (1% CO2; PCO2=6.9 mm Hg) water and to 400 mu g Cu2+L-1+ hypercarbia. In liver the single factors caused an increase in lipid hydroperoxide concentration that was not observed when the factors were combined. Copper exposure elicited increased hepatic superoxide dismutase activity, irrespective of aquatic CO2 level. On the other hand, the effects of copper on hepatic glutathione peroxidase activity were dependent on water CO2 levels. The two stressors combined did not affect hepatic catalase activity. Hypercarbic water caused a decline in plasma glucose concentration, but this was not observed when hypercarbia was combined with copper exposure. Copper caused a decrease in branchial Na+/K+-ATPase activity that was independent of water CO2 level. Copper caused an increase in branchial metallothionein concentration that was independent of water CO2 level. Thus, branchial metallothionein and Na+/K+-ATPase were effective biomarkers of copper exposure that were not affected by water CO2 level. (C) 2012 Elsevier Inc. All rights reserved.

Growth, body composition and hematology of juvenile pacu (Piaractus mesopotamicus) fed increasing levels of ractopamine

The effects of four levels of dietary ractopamine (RAC) on growth, body composition and hematology of pacu, Piaractus mesopotamicus juveniles (103.6+/-3.3g) were studied. Fish were housed into 12 circular tanks of 1 m(3) - 15 fish per tank - and fed for 60 days with practical diets supplemented with 0; 10; 20 or 40mg RAC per kg of diet, in a totally randomized design trial (n=3). Fish fed diets containing up to 40mg RAC/kg diet for 60 days did not have improved growth or body composition parameters. There were no significant differences in hematocrit, hemoglobin and mean corpuscular hemoglobin. Plasma glucose and triglycerides were significantly smaller in fish fed with RAC; however no significant differences between RAC levels were detected. The inclusion of up to 40mg RAC/kg of diet did not improve growth and body composition, but influenced some hematological and biochemical parameters of juvenile pacu.

The fatty acid profiles and energetic substrates of two Nile tilapia (Oreochromis niloticus, Linnaeus) strains, Red-Stirling and Chitralada, and their hybrid

Protein and lipid content as well as the fatty acid (FA) composition of storage tissues were analysed in two varieties of Oreochromis niloticus (Red-Stirling and Chitralada) and their hybrid. The animals were maintained in cages for 11 months. The samples were taken when the animals weighed 10, 50, 100, 250 and 500 g. The results showed that changes in the metabolic processes occur during an increase in body mass in both varieties of tilapia and also their hybrid, but that these differences are not found in animals collected at the commercial weight. The protein content of the fillet and liver decreased with growth and the same protein content associated with growth was found for fillet lipid content. The genetic variety did not influence the FA profile of the fillet, but different genotypes had different hepatic FA compositions. Even with the same lipid content, the hepatocytes of Chitralada accumulated higher levels of polyunsaturated fatty acids (PUFA) n6 in triglycerides and increased C22:6n3 in the hepatocyte membranes. The higher n6PUFA content was compensated by a lower fraction of saturated FA in the hepatocyte triglycerides. The skin of Chitralada also had higher n6PUFA and C22:6n3 contents, suggesting a higher ability to deposit PUFA in the skin due to alterations in the liver synthetic pathway.

Development of new molecular methods for the diagnosis and the study of viral diseases of fish

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

Appearance of red mark syndrome / cold water strawberry disease in Switzerland and Austria

Red mark syndrome (RMS) or cold water strawberry disease (CWSD) is a non-lethal skin disease of rainbow trout Oncorhynchus mykiss that is of high economic importance in the UK. The disease is temperature-dependent, with up to 60% morbidity at water temperatures below 15 degrees C. Although CWSD is horizontally transmissible, the aetiology is still unknown. Here we describe the first cases of RMS on the European mainland in the alpine regions of Switzerland and Austria. In Switzerland, morbidity remained around 1% after the first outbreak, whereas in Austria no further cases were diagnosed.

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.

Bioavailability of pharmaceuticals in waters close to wastewater treatment plants: use of fish bile for exposure assessment

Pharmaceuticals are ubiquitous in surface waters as a consequence of discharges from municipal wastewater treatment plants. However, few studies have assessed the bioavailability of pharmaceuticals to fish in natural waters. In the present study, passive samplers and rainbow trout were experimentally deployed next to three municipal wastewater treatment plants in Finland to evaluate the degree of animal exposure. Pharmaceuticals from several therapeutic classes (in total 15) were analyzed by liquid chromatography-tandem mass spectrometry in extracts of passive samplers and in bile and blood plasma of rainbow trout held at polluted sites for 10 d. Each approach indicated the highest exposure near wastewater treatment plant A and the lowest near that of plant C. Diclofenac, naproxen, and ibuprofen were found in rainbow trout, and their concentrations in bile were 10 to 400 times higher than in plasma. The phase I metabolite hydroxydiclofenac was also detected in bile. Hence, bile proved to be an excellent sample matrix for the exposure assessment of fish. Most of the monitored pharmaceuticals were found in passive samplers, implying that they may overestimate the actual exposure of fish in receiving waters. Two biomarkers, hepatic vitellogenin and cytochrome P4501A, did not reveal clear effects on fish, although a small induction of vitellogenin mRNA was observed in trout caged near wastewater treatment plants B and C.

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium ("Pan-Flavo") and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.

Molecular epidemiology of Flavobacterium psychrophilum from Swiss fish farms

Bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) caused by Flavobacterium psychrophilum are 2 of the major diseases causing high fish mortality in salmonid fish farms. The molecular epidemiology of F. psychrophilum is still largely unknown. Multilocus sequence typing (MLST) has been previously used for this pathogen and underscored a correlation between clonal complexes and host fish species. Here we used MLST to study the relationships among 112 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta fario and S. t. lacustris in Swiss fish farms between 1993 and 2012. The isolates belonged to 27 different sequence types (STs). Most of the Swiss outbreaks were associated with strains belonging to clonal complexes CC-ST2 and CC-ST90, found in both rainbow trout and brown trout and represented by several STs. Eight ST singletons could not be connected to any known clonal complex. Already reported from other parts of Europe and North America, CC-ST2 was the most frequent clonal complex observed, and it caused the majority of outbreaks in Switzerland, with CC-ST90 being the second most important type. In the tightly interconnected Swiss fish farms, no association between clonal complex and host fish was detected, but a temporal evolution of the frequency of some STs was observed. The occurrence of sporadic STs suggests high F. psychrophilum diversity and may reflect the presence of different sequence types in the environment.

First outbreak of sleeping disease in Switzerland: disease signs and virus characterization.

Sleeping disease is a contagious disease mainly of freshwater farmed rainbow trout, caused by salmonid alphavirus (SAV) Subtype 2. Here we describe the first case in Switzerland. Pathological changes ranged from acute pancreas necrosis to more chronic lesions with complete loss of exocrine pancreas and simultaneous degenerative, inflammatory and regenerative heart and muscle lesions. The partial sequencing of SAV E2 and nsp3 genes placed the Swiss SAV variant within the Subtype 2 clustering together with freshwater isolates from UK and continental Europe. Although mortality stayed low, growth rates were significantly reduced, making the disease economically relevant.

Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR.

BACKGROUND Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. RESULTS We describe a qPCR technique based on the single copy gene β' DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. CONCLUSIONS This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak.

Model for ranking freshwater fish farms according to their risk of infection and illustration for viral haemorrhagic septicaemia

We developed a model to calculate a quantitative risk score for individual aquaculture sites. The score indicates the risk of the site being infected with a specific fish pathogen (viral haemorrhagic septicaemia virus (VHSV); infectious haematopoietic necrosis virus, Koi herpes virus), and is intended to be used for risk ranking sites to support surveillance for demonstration of zone or member state freedom from these pathogens. The inputs to the model include a range of quantitative and qualitative estimates of risk factors organised into five risk themes (1) Live fish and egg movements; (2) Exposure via water; (3) On-site processing; (4) Short-distance mechanical transmission; (5) Distance-independent mechanical transmission. The calculated risk score for an individual aquaculture site is a value between zero and one and is intended to indicate the risk of a site relative to the risk of other sites (thereby allowing ranking). The model was applied to evaluate 76 rainbow trout farms in 3 countries (42 from England, 32 from Italy and 2 from Switzerland) with the aim to establish their risk of being infected with VHSV. Risk scores for farms in England and Italy showed great variation, clearly enabling ranking. Scores ranged from 0.002 to 0.254 (mean score 0.080) in England and 0.011 to 0.778 (mean of 0.130) for Italy, reflecting the diversity of infection status of farms in these countries. Requirements for broader application of the model are discussed. Cost efficient farm data collection is important to realise the benefits from a risk-based approach.