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1.
Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.  相似文献   

2.
Enteromyxum leei is a myxozoan histozoic parasite that infects the intestine of several teleost fish species. In gilthead sea bream (Sparus aurata), it provokes a chronic disease, entailing anorexia, delayed growth, reduced marketability and mortality. Direct fish-to-fish transmission, relevant in aquaculture conditions, has been demonstrated for E. leei via effluent, cohabitation, and oral and anal routes. However, the minimum time of exposure for infection has not been established, nor the possible effect on the fish immune response. Two effluent trials were performed at different temperatures (high: average of 25.6°C; and low: constant at 18°C), different times of exposure to the effluent (1, 3, 5 and 7 weeks) and different population densities. The results showed that 1 week was enough to infect 100% of fish at high temperature and 58.3% at low temperature. High temperature not only increased the prevalence of infection in posterior intestine, but also induced a higher production of specific antibodies, limiting the progression of the infection along the intestine. Longer time of exposure to the parasite and higher fish densities facilitated E. leei infection. These results show that effective diagnosis, lowering animal density and removal of infected fish are key aspects to manage this disease in aquaculture facilities.  相似文献   

3.
The effects of water temperature on the development of the enteric myxosporeans, Enteromyxum fugu and Enteromyxum leei, were investigated in experimentally infected tiger puffer, Takifugu rubripes. After naïve tiger puffer were fed gut tissue infected with both E. fugu and E. leei, they were divided into separate tanks and kept at different constant temperature regimes between 10 and 25 °C. Regardless of the water temperature tested, E. fugu was consistently detected with a high prevalence of infection (60–100%), although no sporulation occurred at 10 and 15 °C. Development of E. leei and the onset of disease were suppressed by low water temperatures (<15 °C). However, a temperature increase to 20 °C promoted the development of E. leei, followed by an increase of disease rate in the fish. The present study demonstrates that water temperatures below 15 °C have an inhibitory effect on the development of E. fugu and E. leei, resulting in suppression of enteromyxosis at low temperatures.  相似文献   

4.
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.  相似文献   

5.
Enteromyxoses are relevant diseases for turbot and gilthead sea bream aquaculture. The myxozoan parasites invade the intestinal mucosa, causing a cachectic syndrome associated with intestinal barrier alteration; nonetheless, their pathological impact is different. Turbot infected by Enteromyxum scophthalmi develop more severe intestinal lesions, reaching mortality rates of 100%, whereas in E. leei-infected gilthead sea bream, the disease progresses slowly, and mortality rates are lower. The mechanisms underlying the different pathogenesis are still unclear. We studied the distribution and expression changes of E-cadherin, a highly conserved protein of the adherens junctions, in the intestine of both species by immunohistochemistry and quantitative PCR, using the same immunohistochemical protocol and common primers. The regular immunostaining pattern observed in control fish turned into markedly irregular in parasitized turbot, showing an intense immunoreaction at the host–parasite interface. Nevertheless, E-cadherin gene expression was not significantly modulated in this species. On the contrary, no evident changes in the protein distribution were noticed in gilthead sea bream, whereas a significant gene downregulation occurred in advanced infection. The results contribute to the understanding of the different host–parasite interactions in enteromyxoses. Host and parasite cells appear to establish diverse relationships in these species, which could underlie the different pathological picture.  相似文献   

6.
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.  相似文献   

7.
As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 104/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.  相似文献   

8.
Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers’ specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.  相似文献   

9.
This study evaluated the growth and immune response of gibel carp (Carassius auratus gibelio) cultured under no feed addition biofloc technology (BFT) system at different total suspended solid (TSS) concentrations (10, 300, 600, 800 and 1,000 mg/L for group BF0‐NF, BF300‐NF, BF600‐NF, BF800‐NF and BF1000‐NF) for 30 days. The results demonstrated that bioflocs contained rich nutrients, and gibel carp eaten bioflocs showed higher weight gain, specific growth and survival. Digestive enzyme activities such as pepsin and amylase increased significantly in BF300/600/800/1000‐NF groups than those in BF0‐NF group. Antioxidant response including superoxide dismutase and total antioxidant capacity in serum and skin mucus was also enhanced significantly (p < .05). In addition, six immune‐related genes were examined by RT‐qPCR. Compared with BF0‐NF group, expression levels of immune genes intelectin (ITLN), dual specificity phosphatase 1 (DUSP 1), keratin 8 (KRT 8), myeloid‐specific‐peroxidase (MPO), c‐type lysozyme (c‐lys) and interleukin‐11 (IL‐11) were up‐regulated by 78.1‐, 23.9‐, 13.8‐, 138.8‐, 401.8‐ and 91.1‐fold, respectively. The highest expression values were observed at TSS of 600–800 mg/L. This study suggested that bioflocs can be uptaken by gibel carp as a food source, and have a potential to be used as a supplemental food for aquaculture.  相似文献   

10.
Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria‐submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti‐(active caspase‐3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi‐infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi‐infected turbot compared with non‐infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase‐3‐positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.  相似文献   

11.
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12.
Y. Wang  M. Li  K. Filer  Y. Xue  Q. Ai  K. Mai 《Aquaculture Nutrition》2017,23(5):1113-1120
This trial was conducted to evaluate the effects of replacing dietary fish oil with Schizochytrium meal for Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Six test microdiets were formulated using Schizochytrium meal to replace 0 g/kg, 250 g/kg, 500 g/kg, 750 g/kg, 1000 g/kg or 1500 g/kg fish oil DHA. No significant differences were observed in survival, growth, final body length and activities of digestive enzyme among shrimp fed different diets (p > .05). No significant differences were observed in C20:5n‐3 (EPA) in muscle samples (p > .05). C18:3n‐3 and C20:4n‐6 in muscle increased as Schizochytrium meal replacement level increased (p < .05). No significant differences were observed in C22:6n‐3 (DHA) and n‐3 fatty acids among shrimp fed diets that algae meal replaced 0 g/kg ‐ 1000 g/kg of fish oil. Shrimp fed diet R150 had higher DHA content than other groups and had higher n‐3 fatty acids than that of shrimp fed diets R50, R75 and R100 (p < .05). C18:2n‐6, PUFA and n‐6 fatty acids in muscle increased, while n‐3/n‐6 ratio decreased with increasing algae meal replacement level from 0 g/kg to 1000 g/kg (p < .05). In conclusion, Schizochytrium meal could replace 1500 g/kg fish oil DHA in the microdiets without negatively affecting shrimp larvae survival, growth and activities of digestive enzyme.  相似文献   

13.
No gold standard assay exhibiting error‐free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non‐culture assays in three matrices (phosphate‐buffered saline, ovarian fluid and kidney tissue). Non‐culture assays included polyclonal enzyme‐linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane‐filtration FAT, nested polymerase chain reaction (nested PCR) and three real‐time quantitative PCR assays. Injection challenge of specific pathogen‐free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.  相似文献   

14.
A feeding trial was conducted to assess the effects of dietary Schizochytrium meal supplementation on survival, growth performance, activities of digestive enzymes and fatty acid composition in Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Four isonitrogenous and isolipidic diets were formulated to contain graded levels of Schizochytrium meal: 0% (S0, the control diet), 2% (S2), 4% (S4) and 6% dry matter (S6). Results showed that there was no significant difference in survival of shrimps among dietary treatments (P > 0.05). Shrimps fed diets with 2% and 4% microalgae meal had significantly higher specific growth rate (SGR) than that of shrimps fed diets with 0% and 6% microalgae meal, and no significant differences were observed between shrimps fed diets with 2% and 4% microalgae meal (P > 0.05). Activity of trypsin in the pancreatic and intestinal segments, and activity of amylase in the pancreatic segments were not significantly affected by dietary microalgae meal levels (P > 0.05). Specific activities of both alkaline phosphatase and leucine‐aminopeptidase in intestine and purified brush border membrane of intestine were significantly higher in shrimps fed diet with 2% microalgae meal (P < 0.05). There were no significant differences in C18:2n‐6, n‐3 fatty acids, n‐6fatty acids, PUFA and n‐3/n‐6 in muscle samples among dietary treatments. C16:1n‐7, C18:1n‐9, MUFA, C18:3n‐3 and C20:5n‐3 decreased, however, C20:4n‐6 increased in the muscle as dietary microalgae meal level increased. In conclusion, 4% Schizochytrium meal in microdiets of shrimps can improve growth performance and may be a valuable additive in the microdiets of shrimps.  相似文献   

15.
The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 101 amplicon copies per assay (equivalent to 2 × 10–9 ng/µl) using bacterial DNA and of 1.44 × 101 gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.  相似文献   

16.
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17.
A feeding experiment was conducted in a closed recirculating system to evaluate the effects of freeze‐dried spheroplasts prepared from Pyropia yezoensis (Ueda) on feed intake, growth and biochemical composition of sea cucumber, Apostichopus japonicus (Selenka). Pyropea spheroplasts (PS) were prepared through enzymatic treatment to break down the complex mixture of polysaccharides cell walls that might be easier for growth energy partitioning. Sea cucumbers were fed‐formulated diets with 10 (Diet 1), 30 (Diet 2) and 50 g/kg (Diet 3) inclusion level of PS. A diet without PS was used as a control (Diet 4). The experiment was conducted for 6 weeks maintaining water temperature 15 ± 1°C, photoperiod 18:06 hours (D:L). Feed was supplied ad‐libitum at 16.00 h once in a day, and the remaining feed and faeces were removed in the next day. Results showed that the highest growth was observed in the 50 g/kg PS diet compared to other treatments. Total weight gain, mean weight gain, net yield, protein efficiency ratio (PER) and protein gain (%) were significantly higher in the 50 g/kg PS diet (p < .05). A significantly higher percentage of energy was allocated for growth in the 50 g/kg PS diet. The highest specific growth rate and feed conversion efficiency (p < .05) were observed in the higher percentage of PS diet. Both the growth performance and biochemical analysis showed that superior growth was observed with increasing levels of PS in the diet. We infer that PS can be used as a new, cheaper feed ingredient in the formulated diet of A. japonicus.  相似文献   

18.
This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 100 amplicon copies per assay (equivalent to 2 × 10?11 ng/µl, Cq value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 100 gene copies in tissues artificially infected and 0.02 × 100 in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.  相似文献   

19.
The individual and combinational effects of probiotics on Persian sturgeon (Acipenser persicus) were evaluated through assessing the growth performance, proximate body composition, digestive enzymes and intestinal morphology. On the basis of feeding with experimental diets, the fish were assigned into four groups denoted as follows: control (basal diet), D‐Pro (basal diet + Bacillus subtilis and Bacillus licheniformis), D‐Pro/Yeast (basal diet + B. subtilis and B. licheniformis + Saccharomyces cerevisiae), Yeast (basal diet + S. cerevisiae). Maximum mean weight and length were measured for the fish fed with D‐pro regime. weight gain (WG), body weight gain (BWI%),  feed efficiency (FE%), protein efficiency rate (PER), lipid efficiency rate (LER), and condition factor (CF) demonstrated the highest values for the fish fed with the diets containing D‐Pro and/or D‐Pro/yeast. LER and food conversion ratio (FCR) showed the highest performance for the fish received the D‐Pro diet. The diets containing D‐Pro exhibited higher performance in enhancing the activity of protease and lipase than the one supplemented only with the yeast, whereas the regimes treated with the yeast exhibited higher amylase activity. The fish received D‐Pro exhibited the highest protein and fat contents, yet did the lowest contents of moisture and total ash. Taken together, the findings revealed that the dietary administration of the D‐Pro and D‐Pro/Yeast could improve the growth performance of A. persicus.  相似文献   

20.
Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal‐to‐noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species‐specific oligonucleotide probes utilizing the sequences of 16S‐23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.  相似文献   

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