Non‐invasive fast real‐time PCR assay for detection of the enteric parasite Enteromyxum scophthalmi in cultured turbot (Scophthalmus maximus L.)

Enteromyxum scophthalmi is a myxozoan parasite that causes severe parasitic diseases in cultured turbot affecting mainly the intestine of the host. It is characterized by producing acute enteritis, starvation and eventually death. Current diagnosis of E. scopthalmi use traditional techniques, based on the identification of the morphology of the parasite. These techniques take extended time to be carried out and do not favour the adoption of control measure at turbot farms and require the sacrifice of fish. This study develops a fast real‐time PCR molecular tool for the detection of E. scophthalmi in infected farmed turbot. This methodology is applicable for routine controls on the farm at every stage of the parasite infection. Results of the study demonstrate the robustness, specificity, efficiency and reliability of the technique. In addition, this study also provides a non‐invasive procedure of sampling through swaps. This allows control, prevention and diagnosis of the parasite infection at turbot farms while maintaining the welfare of the cultivated fish and avoiding sacrifice of the fish sampled.     

Bromodeoxyuridine DNA labelling reveals host and parasite proliferation in a fish‐myxozoan model

Enteromyxum leei is a myxozoan parasite responsible for enteritis in gilthead sea bream (Sparus aurata). The parasite proliferates in the paracellular space of the intestinal epithelium and induces an inflammatory reaction. To assess intestinal cell turnover and parasite proliferation, fish were infected with the parasite by anal intubation; after 17 and 64 days, the cell proliferative marker bromodeoxyuridine (BrdU) was administered; and after 24 hr, tissue samples were taken for immunohistochemical detection. Parasite exposure induced increased epithelial and immune cell proliferation in all intestinal segments at all time points, even before parasite establishment. This increased turnover was triggered early after intubation and mainly at a local level, as shown by an increased proliferating cell nuclear antigen (pcna) gene expression only at the posterior intestine after 17 days (not found in lymphohaematopoietic organs). Incorporation of BrdU in parasite secondary and tertiary daughter cells indicated that parasite endogeny is not by schizogonial division, which uses de novo synthesis pathway of pyrimidines. Altogether, BrdU immunolabelling and pcna gene expression showed the rapid proliferative response of the fish intestines upon a myxozoan infection and how this response is effectively triggered even before the parasite reaches or establishes in the site.     

Apoptosis inhibition of Atlantic salmon (Salmo salar) peritoneal macrophages by Piscirickettsia salmonis

To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post‐exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post‐clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC‐1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.     

Combined antiparasitic and anti‐inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis

The histiophagous scuticociliate Philasterides dicentrarchi is the aetiological agent of scuticociliatosis, a parasitic disease of farmed turbot. Curcumin, a polyphenol from Curcuma longa (turmeric), is known to have antioxidant and anti‐inflammatory properties. We investigated the in vitro effects of curcumin on the growth of P. dicentrarchi and on the production of pro‐inflammatory cytokines in turbot leucocytes activated by parasite cysteine proteases. At 100 μm , curcumin had a cytotoxic effect and completely inhibited the growth of the parasite. At 50 μm , curcumin inhibited the protease activity of the parasite and expression of genes encoding two virulence‐associated proteases: leishmanolysin‐like peptidase and cathepsin L‐like. At concentrations between 25 and 50 μm , curcumin inhibited the expression of S‐adenosyl‐L‐homocysteine hydrolase, an enzyme involved in the biosynthesis of the amino acids methionine and cysteine. At 100 μm , curcumin inhibited the expression of the cytokines tumour necrosis factor‐alpha (TNF‐α) and interleukin‐1 beta (IL‐1β) produced in turbot leucocytes activated by parasite proteases. Results show that curcumin has a dual effect on scuticociliatosis: an antiparasitic effect on the catabolism and anabolism of ciliate proteins, and an anti‐inflammatory effect that inhibits the production of proinflammatory cytokines in the host. The present findings suggest the potential usefulness of this polyphenol in treating scuticociliatosis.     

Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone‐like DNA‐binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP‐GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co‐located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase‐3 activity and pro‐apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.     

Pro‐inflammatory caspase‐1 activation during the immune response in cells from rainbow trout Oncorhynchus mykiss (Walbaum 1792) challenged with pathogen‐associated molecular patterns

In response to pathogens, the higher vertebrate innate immune system activates pro‐inflammatory caspase‐1 which is responsible for the processing and secretion of several important cytokines involved in the host's defence against infection. To date, caspase‐1 has been described in few teleost fish, and its activity has been demonstrated through substrate cleavage and inhibition by pharmacological agents. In this study, the detection of the active form of caspase‐1 during the immune response in salmonid fish is described, where two antibodies were produced. These antibodies differentially recognize the structural epitopes of the inactive pro‐caspase‐1 and the processed active form of the caspase. Firstly, caspase‐1 activation was demonstrated in vitro by ELISA, Western blotting and immunocytochemistry in rainbow trout macrophages exposed to different pathogen‐associated molecular patterns plus the pathogen Aeromonas hydrophila. This activity was clearly abrogated by a caspase inhibitor and seems to be unrelated to IL‐1β secretion. Caspase‐1 activation was then validated in vivo in gill cells from fish challenged with Aeromonas salmonicida. These results represent the first demonstration of caspase‐1 activation in salmonids, and the first evidence of the putative regulatory role which this protease plays in inflammatory response in this fish group, as described for some other teleosts and mammals.     

Dietary soybean β‐conglycinin suppresses growth performance and inconsistently triggers apoptosis in the intestine of juvenile grass carp (Ctenopharyngodon idella) in association with ROS‐mediated MAPK signalling

The current study aimed to investigate the effects of dietary soybean β‐conglycinin on growth performance and intestine apoptosis in juvenile grass carp (Ctenopharyngodon idella). For fish fed with the 80 g β‐conglycinin/kg diet for 7 weeks, the specific growth rate and feed intake were decreased. In the proximal intestine, dietary β‐conglycinin did not induce DNA fragmentation, tended to decrease the reactive oxygen species (ROS) content, and decreased ROS‐generating enzyme (NADPH oxidase [NOX]) activity. Subsequently, in the mid‐intestine, dietary β‐conglycinin caused DNA fragmentation, tended to increase the ROS content, increased caspase‐3, caspase‐8 and caspase‐9 activities, upregulated the mRNA levels of proapoptotic molecules (apoptotic protease‐activating factor‐1 [Apaf1] and Bcl‐2‐associated X protein [BAX]) and mitogen‐activated protein kinase (MAPK)‐related signal molecules (Jun N‐terminal kinase (JNK) and p38 MAPK) and increased the protein levels of p38 MAPK and phospho‐p38 MAPK. Moreover, in the distal intestine, dietary β‐conglycinin induced DNA fragmentation, elevated NOX activity and the ROS content and increased caspase‐3, caspase‐8 and caspase‐9 activities, death ligand (TNF‐α) mRNA expression level, and p38 MAPK and phospho‐p38 MAPK protein levels. In summary, dietary soybean β‐conglycinin suppressed fish growth and inconsistently caused apoptosis among the different intestinal segments which was partially associated with ROS‐mediated MAPK signalling.     

Immunohistochemical diagnosis of tenacibaculosis in paraffin‐embedded tissues of Senegalese sole Solea senegalensis Kaup, 1858

A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post‐inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR‐based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin‐embedded tissues.     

Immunohistochemical characterization of polyclonal antibodies against Enteromyxum leei and Enteromyxum scophthalmi (Myxozoa: Myxosporea), intestinal parasites of fish

The enteric myxozoan parasites Enteromyxum leei (Diamant, Lom et Dyková) and Enteromyxum scophthalmi Palenzuela, Redondo et Álvarez‐Pellitero are responsible for high weight loss in infected fish, which leads to subchronic disease and low mortality rates in gilthead sea bream (GSB), Sparus aurata L., and to high mortality rates in turbot, Psetta maxima (L.). The detection of initial parasite stages in histological sections is particularly difficult, but can be simplified by means of specific antibodies. Rabbit polyclonal antibodies (pAbs) were raised against E. scophthalmi and E. leei, and direct enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry were used to characterize their sensitivity and specificity. Both pAbs were adsorbed (apAb) with non‐infected intestines to avoid non‐specific labelling of fish tissues and to improve their specificity. The highest titre obtained in ELISA was 1: 32 000 for apAb‐Eleei and 1:16 000 for apAb‐Escoph. Working dilutions in immunohistochemistry were 1:1000 for apAb‐Eleei and 1:8000 for apAb‐Escoph. Both apAbs labelled proliferative and sporogonic stages with high specificity. apAb‐Escoph was very specific, whereas apAb‐Eleei cross‐reacted with Sphaerospora dicentrarchi Sitjà‐Bobadilla et Álvarez‐Pellitero and Sphaerospora testicularis Sitjà‐Bobadilla et Álvarez‐Pellitero, suggesting the presence of shared antigens. These pAbs stand as new tools for antigenic characterization and the diagnosis of both Enteromyxum species.     

Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial‐targeting secretory protein (MTSP) 3141 with an N‐terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141‐GFP fusion protein co‐localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N‐terminus but not C‐terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related gene (Bcl‐2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N‐terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.     

Tissue distribution of hepatopancreatic parvo‐like virus of shrimp in freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of hepatopancreatic parvo‐like virus (HPV) of shrimp in different organs of freshwater rice‐field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT‐PCR, ELISA, Western blot and q‐PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large‐scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT‐PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q‐PCR. The results revealed a steady decrease in C_T values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post‐larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice‐field crab could be used as an alternative host for HPV replication and also for large‐scale production of HPV.     

Evaluating the most suitable nonlinear growth model for turbot (Scophthalmus maximus) in aquaculture 2 (weight application): Multi‐criteria model selection and growth prediction

Seeking the most suitable model to describe the growth of turbot, we analysed growth data of two different turbot (Scophthalmus maximus) strains reared communally in a recirculating aquaculture system. We fitted 10 different nonlinear growth models to individual weight gain data (n = 2,010) during the grow‐out phase. Analyses were carried out for each strain, for sexes within strains and for a pooled data set containing both strains and sexes. To assess the model performance, three different criteria are used. Further, a growth‐simulation was performed to evaluate the shape of the generated curve. This way we could assess the capability of the models to predict future growth. The 3‐parametric Gompertz model achieved the best fit in 42.9% of all cases tested and the lowest Bayesian information criterion in 100% of cases. The model produced realistically shaped curves and asymptotic values matching the biological attributes of the species. In contrast, 5‐parametric functions projected unrealistically shaped curves and predicted improbable mature sizes. Our results show that increasing number of parameters do not lead to increasing goodness of fit, but tend to result in overfitting, and demonstrate the advantages of the 3‐parametric Gompertz model for describing the growth of turbot.     

Outbreak of mass mortality of yearling groupers of Epinephelus (Perciformes,Serranidae) associated with the infection of a suspected new enteric Sphaerospora (Myxozoa: Myxosporea) species in South China Sea

A suspected new enteric Sphaerospora species was believed to be directly associated with the mass mortality of yearling groupers of Epinephelus spp. in South China. The epizootic generally emerged from late September to late April of the following year. The infection prevalence and mortality rate were significantly negatively correlated with fish size. Clinical signs included anorexia, cachexia and extrusion of white pulp‐like substance from anus after gentle pressure on the abdomen. Upon necropsy, severe intestinal oedema, thin and transparent intestinal wall, swollen spleen, kidney and gall bladder could be observed. Wet preparation of the infected samples showed large amount of typical disporous plasmodia of the genus Sphaerospora, but no mature spores were observed. Epidemiological investigation showed that this parasite exclusively infected Epinephelus groupers. Histopathologically, this species mainly infected the epithelium of intestine and kidney tubules and caused severe epithelia sloughing and the collapse of intestinal villus. Interestingly, this enteric myxosporidiosis did not cause severe emaciation of infected fish for mass mortality usually emerged within 2–3 days after appearance of clinical signs. The species was most genetically related to Sphaerospora fugu (89% sequence identity) and phylogenetically positioned within marine Sphaerospora lineage. This is the first report of enteric sphaerosporosis of groupers.     

Yeast extract on growth,nutrient utilization and haemato‐immunological responses of Nile tilapia

Dietary supplementation (0, 10, 20, 40, 60, 80 g kg^?1) of a nucleotide source (NuPro^®) was evaluated on growth, nutrient utilization, haemato‐immunological responses and resistance to experimental infection of Nile tilapia. Fish (2.63 ± 0.63 g) were fed experimental diets to satiation three times a day for 75 days. Feed intake increased linearly with increasing levels of NuPro^® promoting higher weight gain (P < 0.05). Survival, feed conversion and protein retention were not affected. Following the growth trial, fish were challenged with Aeromonas hydrophila by intraperitoneal injection. Survival and innate immune responses (phagocytosis and lysozyme activity) were not significantly affected. However, among the haematological variables, thrombocyte, leucocyte and monocyte counts increased linearly (P < 0.05). The indirect measurement of digestibility in juvenile Nile tilapia (122.32 ± 11 g) indicated a linear decrease in protein (1.41%), dry matter (5.72%) and energy (4.10%) digestibility coefficients as dietary supplementation increased (P < 0.05). NuPro^® showed to be a beneficial additive to be supplemented in diets for juvenile Nile tilapia, providing 32.8% increase in feed intake and 28.8% in weight gain, as well as in thrombocyte, leucocyte and monocyte counts in blood, possibly indicating a more efficient defence response in the highest dietary supplementation levels tested.     

Influence of 5′‐inosine monophosphate and 5′‐guanosine monophosphate on growth,feed digestibility and activity of digestive enzymes in turbot Scophthalmus maximus

We evaluated the influence of different proportions of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on growth, feed digestibility and activity of digestive enzymes of turbot Scophthalmus maximus. Weight gain and daily feed intake were significantly higher in S. maximus fed with IMP or GMP, in comparison with fish fed with neither IMP nor GMP. The growth of 0.05% IMP + 0.05% GMP group was the best, and the intestinal digestive function was improved. The addition of IMP and GMP to fish diets significantly increased the apparent feed digestibility coefficient of dry matter and protein, as well as intestinal protease activity. The highest intestinal protease activity was observed in fish fed with 1 g/kg IMP. However, the lipase activity in hepatopancreas decreased significantly after addition of nucleic acid. According to our results, the optimal level of dietary IMP is 1 g/kg, which is in line with most of the growth performance and feed digestibility.