Evaluation of zebrafish Danio rerio as a model for enteric septicemia of catfish (ESC)

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 x 10(3), 6 x 10(4), or 6 x 10(5) colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 x 10(4) CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 x 10(2) CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 x 10(5), 1.16 x 10(6), and 1.16 x 10(7) CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.     

Evidence that Enteric Septicemia of Catfish (ESC) was Present in Arkansas by the Late 1960s: New Insights into the Epidemiology of ESC

Abstract

Enteric septicemia of catfish (ESC), a disease of channel catfish Ictalurus punctatus, was first reported in 1979 based on isolates obtained from 1976 through 1978. Channel catfish that had been preserved in 1970, labeled “nutritional cranial spot,” and stored at the Stuttgart National Aquaculture Research Center were tested with Gram stains, histology, and immunohistochemistry to demonstrate that the specimens were actually infected by Edwardsiella ictaluri (the causative agent of ESC). A reexamination of catfish disease case records has indicated that ESC might have been present in Arkansas in 1969. Investigation of these old records and specimens has led to insights on the discovery and epidemiology of the disease.     

Risk Factors Associated with Enteric Septicemia of Catfish on Mississippi Commercial Catfish Farms

Abstract

A gram-negative bacterium, Edwardsiella ictaluri, is the cause of enteric septicemia of catfish (ESC), which is one of the most prevalent bacterial diseases in farm-raised catfish. The objective of this study was to identify risk factors associated with ESC mortalities and are reported by farm personnel. To identify risk factors a catfish management database was developed. The odds ratios (OR) of the final multivariable logistic regression model were: (1) volume of the pond (OR, 0.56), (2) interval from harvest until a mortality event (OR, 1.49), (3) interval from stocking until a mortality event (OR, 0.52), (4) nitrite measured within 14 d of a mortality (OR, 3.49), (5) total ammonia measured within 14 d of a mortality (OR, 20.48), and (6) sum of feed fed for 14 d prior to the disease outbreak (OR, 1.02), all of which were significantly (P ≤ 0.05) associated with ESC occurrence. This study showed that some commonly recorded production variables were associated with ESC outbreaks and if monitored could help identify “at risk” ponds prior to disease outbreaks.

Received September 18, 2013; accepted December 17, 2013     

Oral Vaccination of Channel Catfish against Enteric Septicemia of Catfish Using a Live Attenuated Edwardsiella ictaluri Isolate

Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are typically administered by immersion when fry are transferred from the hatchery to rearing ponds. While this approach is a practical method of mass delivery, this strategy administers vaccines to very young fish, which lack a fully developed immune system. To circumvent this limitation, an oral vaccination strategy was evaluated as a means of immunizing catfish at the fingerling stage of production, when fish possess a more complete immune arsenal. A virulent E. ictaluri isolate (S97-773) was attenuated by successive passage on media containing increasing concentrations of rifamycin. In laboratory trials, cultured vaccine was diluted and mixed with feed (100 mL diluted vaccine/454 g feed). This mixture was then fed to Channel Catfish Ictalurus punctatus fingerlings. Two separate dilutions of cultured vaccine (1:10 and 1:100) were used to create the vaccine–feed mixture, equating to estimated doses of 5 × 10⁷ and 5 × 10⁶ CFU/g of feed, respectively. After 30 d, catfish were exposed by immersion (1 × 10⁶ CFU/mL) to the virulent parental strain of E. ictaluri. The target dose (1:100 dilution, ～5 × 10⁶ CFU/g of feed) offered exceptional protection (relative percent survival = 82.6–100%). In addition, negligible deaths occurred in fish vaccinated at 10 times the target dose (1:10 dilution, ～5 × 10⁷ CFU/g of feed). In pond trials, antibody production increased 18-fold in orally vaccinated fish. When compared with nonvaccinated controls, vaccination significantly improved survival, feed fed, feed conversion, biomass produced, and total harvest. This research demonstrates Channel Catfish can be successfully immunized in a commercial setting against E. ictaluri with a single dose of an orally delivered, live attenuated, E. ictaluri vaccine.

Received July 31, 2014; accepted March 2, 2015     

Impact of Endocrine‐Disrupting Chemicals on Reproductive Function in Zebrafish (Danio rerio)

The prevalence of endocrine‐disrupting chemicals (EDCs) in the aquatic environment has been associated with the wide detection of alterations in the development and physiology of vertebrates. Zebrafish, as a model species, has been extensively used in toxicological research. In this review, we focus on recent published evidence of the harmful effects of EDCs on reproductive function in zebrafish, including skewed sex ratio, immature gonads, diminished sexual behaviour, decreased sperm count, reduced spawning and fertilization. These impairments mostly result from disruption to sex‐steroid hormones induced by endocrine disruptors. We also discuss other effects of exposure to EDCs. In EDC exposure research, despite incomplete assessments of altered gonad histopathology and sexual behaviour, these present potential effective biomarkers or pathways for evaluating the reproductive function in zebrafish on EDC exposure. To date, the pernicious effects of some EDCs on the reproductive performance in laboratory zebrafish are well understood; however, similar alterations remain for further determination in wild‐type fish and more kinds of EDCs. More studies should be performed under established scientific regulatory criteria to investigate the impact of EDCs on reproduction in zebrafish. Moreover, further research is required to explain the definite mechanism of sexual differentiation, which helps in understanding the shift of sexual phenotype with EDC exposure.     

Using Diagnostic Laboratory Records to Monitor Occurrence of Enteric Septicemia of Channel Catfish in Mississippi

Abstract

Diagnostic laboratory information was used to monitor the temporal and spatial occurrence of enteric septicemia of catfish (ESC) in Mississippi. From 1980 through 1988, the Mississippi Cooperative Extension Service (MCES) entered information on 15,649 fish-kill investigations into a computerized data base. Analysis of the records revealed the following trends and patterns. From 1980 through 1985, there was an increase in laboratory submissions and in the ESC proportional morbidity rate (PMR; proportion of diagnosed epizootics that were specific for ESC. However, from 1986 to 1988, although the total yearly number of laboratory submissions had stabilized, there was a decrease in the PMR of ESC cases. During the 9-year interval, 427 producers from 56 counties had ESC diagnosed in their ponds. Twenty-eight producers from four counties were responsible for about 40% of all ESC cases.     

Comparison of two formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish (Danio rerio)

ObjectiveTo compare two commercial formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish.Study designProspective, blinded, randomized study.AnimalsA total of 20 adult Danio rerio (Tuebingen strain).MethodsZebrafish were divided into two groups of 10 (five female, five male) and placed in individual immersion baths containing 10 mg L^–1 of unpreserved alfaxalone (group 1) or preserved alfaxalone (group 2). Anaesthetists blinded to treatment used a composite score scale (CSS) (range 0–12) to assess fish every 30 seconds until induction of anaesthesia. Anaesthetic induction occurred when equilibrium and response to stimulus were lost. Fish were then placed in a clean water bath and scored every 60 seconds. Recovery from anaesthesia was defined as a CSS of ≤ 1. Time variables recorded were anaesthetic induction time (AIT), anaesthetic recovery time (ART) and total procedure time (TPT). Fish were observed for evidence of roupgross external pathology during the procedure. Following anaesthesia, four fish from each group were randomly chosen and euthanized for gill histopathology analysis immediately after recovery criteria were met. Data are presented as mean ± standard deviation. An independent t test was used to compare the difference in average anaesthetic time variables between groups (α = 0.05).ResultsThere were no statistical differences between groups in reported variables. TPT, AIT and ART were 10.2 ± 1.2, 1.9 ± 0.9 and 8.3 ± 1.2 minutes for group 1 and 10.8 ± 2.9, 2.4 ± 1.2 and 8.4 ± 2.7 minutes for group 2. No gross external pathology was evident, and no fish died during the experimental period. Histopathology showed normal gill pathology and no difference between the groups.Conclusions and clinical relevanceImmersion anaesthesia using 10 mg L^–1 of either formulation of alfaxalone resulted in anaesthesia of similar quality and duration.     

Pathogenesis of Enteric Septicemia of Channel Catfish,Caused by Edwardsiella ictaluri: Bacteriologic and Light and Electron Microscopic Findings

Abstract

To clarify early events in the pathogenesis of enteric septicemia of catfish, 140 channel catfish Ictalurus punctatus (8–10 months old) were each infected with approximately 1.0 × 10⁹ colony-forming units of Edwardsiella ictaluri by intragastric intubation. Fish were sacrificed at 0, 0.25, 0.5, 1, 3, 6, 12, 24, 48, 72, 96, and 120 h postinfection (PI). Multiple tissue samples at all scheduled sampling times were evaluated by gross observation, light and electron microscopy, and immunohistochemical methods. In addition, at each sampling time, stomach, intestine, trunk kidney, and liver were cultured to quantitate bacteria. Trunk kidney cultures were positive by 0.25 h PI, indicating rapid transmucosal passage. In the intestine, E. ictaluri was seen in contact with the brush border at 0.5 h PI. Also at 0.5 h PI, dilated basilar cells with large intracytoplasmic inclusions were observed adjacent to the basement membrane. From 1 to 3 h PI, occasional necrotic enterocytes were seen on tips of intestinal folds. Proprial leukocytes were rare before 24 h PI but common thereafter. Immunoelectron microscopy showed E. ictaluri in vacuoles within phagocytes as early as 24 h PI in the intestinal mucosa. In other tissues, earliest observed microscopic lesions (48 h PI) consisted of bacteria within vacuoles of phagocytic cells contained within blood vessels. Bacteria were also seen within degenerate vacuoles in enterocytes and hepatocytes at 72, 96, and 120 h PI. This study confirms that E. ictaluri can invade channel catfish within 0.25 h PI by crossing the intestinal mucosa and suggests that the bacterium may have invasion and survival strategies similar to those of other enteroinvasive members of the Enterobacteriaceae.     

Protective effects of phlorotannins against ultraviolet B radiation in zebrafish (Danio rerio)

Exposure to ultraviolet B (UV-B) radiation has been associated with a variety of adverse effects in all forms of life, including micro-organisms, plants, animals and humans. Ultraviolet B induces cell damage at the molecular level and consequently organisms must employ strategies to protect themselves from sunlight and to repair UV-B-induced cellular damage. In this study, the UV-B protective effects of four different phlorotannins isolated from a brown alga (Ecklonia cava) were determined using zebrafish (Danio rerio) as an in vivo model. Zebrafish embryos were pretreated with phlorotannins and exposed to UV-B (50 mJ/cm(2)). The heart rate, generation of reactive oxygen species and nitric oxide, cell death and hyperpigmentation were assessed in order to evaluate UV-B-induced photo-damage. Treatment of the embryos with the algal phorotannins reduced UV-B-induced reactive oxygen species and nitric oxide levels, protected against UV-B-induced cell death and significantly reduced hyperpigmentation. We therefore suggest that phlorotannins isolated from E. cava can protect against UV-B radiation. Editor Note. Readers of the journal may be unfamiliar with the use of zebrafish embryos in research studies. There is no indication in this article of an ethical review of the study. This is because the use of fish embryos in research, at least in the UK, is not subject to a licensing procedure if they are less than 5 days post fertilization (dpf). In this study the embryos were 2 dpf.     

Assessment of diclofenac LC50 reference values in juvenile and embryonic stages of the zebrafish (Danio rerio)

The aim of the study was to compare the acute toxicity of diclofenac to juvenile and embryonic stages of the zebrafish (Danio rerio). Acute toxicity tests were performed on the aquarium fish Danio rerio, which is one of the model organisms most commonly used in toxicity testing. The tests were performed using a semi-static method according to OECD guideline No. 203 (Fish, acute toxicity test). Embryo toxicity tests were performed in zebrafish embryos (Danio rerio) in compliance with OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results were subjected to a probit analysis using the EKO-TOX 5.2 programme to determine 96hLC50 and 144hLC50 (median lethal concentration, 50% mortality after a 96 h or 144 h interval, respectively) values of diclofenac. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of Danio rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. The LC50 mean value of diclofenac was 166.6 +/- 9.8 mg/L in juvenile Danio rerio, and 6.11 +/- 2.48 mg/L in embryonic stages of Danio rerio. The study demonstrated a statistically higher sensitivity to diclofenac (P < 0.05) in embryonic stages compared to the juvenile fish.     

Exogenous expression of rat aquaporin-3 enhances permeability to water and cryoprotectants of immature oocytes in the zebrafish (Danio rerio)

Movement of water and cryoprotectants through the plasma membrane needs to be accelerated for successful cryopreservation of zebrafish oocytes/embryos, which are much larger than their mammalian counterparts. Aquaporin-3 is a water/solute channel that can transport not only water but also various cryoprotectants. In this study, we attempted to increase the permeability of immature zebrafish oocytes at stage III to water and cryoprotectants by exogenous expression of rat aquaporin-3. Immature zebrafish oocytes were injected with rat aquaporin-3 cRNA and cultured for 5-12 h. Permeability to water and cryoprotectants was then determined based on changes in the volumes of the oocytes in a hypertonic sucrose solution and various cryoprotectant solutions at 25 C. The permeability to water of the aquaporin-3 cRNA-injected oocytes was three times higher than that of intact and water-injected oocytes. The permeability of the aquaporin-3 cRNA-injected oocytes to ethylene glycol, glycerol, propylene glycol, and DMSO was also 2-4 times higher than that of intact oocytes. Thus, the permeability of immature zebrafish oocytes to water and cryoprotectants was enhanced by exogenous expression of aquaporin-3. Cryopreservation of teleost oocytes may be realized through a further increase in permeability.     

黄颡鱼幼鱼的赖氨酸需要量

为评估饲料中赖氨酸水平对黄颡鱼幼鱼生长性能、营养成分和血液指标的影响,进而确定黄颡鱼幼鱼的赖氨酸需要量,试验设计6种等氮等脂的饲料(粗蛋白质45.00%,粗脂肪7.00%),饲料中晶体赖氨酸的添加水平分别为0、0.30%、0.60%、0.90%、1.20%和1.50%,实测各饲料中赖氨酸水平分别为1.58%、1.89%、2.15%、2.41%、2.67%和2.90%。试验选取初始体重为2.00 g左右的黄颡鱼幼鱼360尾,随机分为6组,每组3个重复,每个重复20尾,进行为期12周的养殖试验。结果表明:1.58%组黄颡鱼幼鱼的成活率显著低于其他各组(P0.05)。黄颡鱼幼鱼的增重率和特定生长率随着赖氨酸水平的增加呈先升高后降低趋势,且在赖氨酸水平为2.41%时具有最大的增重率和特定生长率。饲料效率和蛋白质效率均以1.58%组最低,显著低于除2.15%组外的其他各组(P0.05)。饲料中赖氨酸水平对黄颡鱼幼鱼的肥满度、肝体比和内脏比无显著影响(P0.05)。饲料中赖氨酸水平对全鱼和肌肉干物质、粗脂肪含量以及肌肉粗蛋白质含量均无显著影响(P0.05),但显著影响全鱼和肌肉粗灰分含量以及全鱼粗蛋白质含量(P0.05),全鱼和肌肉粗灰分含量均以2.41%组最低,全鱼粗蛋白质含量以2.41%组最高。饲料中赖氨酸水平对黄颡鱼幼鱼全血中红细胞数、白细胞数、血红蛋白含量、红细胞压积无显著影响(P0.05),对血清中总胆固醇、葡萄糖含量及谷草转氨酶活性亦无显著影响(P0.05),但对血清中谷丙转氨酶活性和甘油三酯含量有显著影响(P0.05)。以增重率为评价指标,通过线性模型分析得出黄颡鱼幼鱼对饲料中赖氨酸的需要量为2.61%(相当于饲料蛋白质的5.80%)。     

Histochemical Analysis of Glycoconjugates in the Skin of a Catfish (Arius Tenuispinis, Day)

A histochemical study using conventional carbohydrate histochemistry (periodic‐acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)‐labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose‐binding lectins (Con A, LCA and PSA), galactose‐binding lectins (PNA, RCA), N‐acetylgalactosamine‐binding lectins (DBA, SBA, SJA and GSL I), N‐acetylglucosamine‐binding lectins (WGA and WGAs), fucose‐binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC‐labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose‐binding lectins LCA and PSA; the galactosamine‐binding lectins DBA, SBA and GLS I; the glucosamine‐binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose‐binding lectin UEA and the sialic acid‐specific lectin SNA. In addition, the galactose‐binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N‐acetylgalactosamine and N‐acetylglucosamine residues.     

Olfactory Organ of Channel Catfish as a Site of Experimental Edwardsiella ictaluri Infection

Abstract

Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), is one of the most important pathogens to infect channel catfish Ictalurus punctatus. Although the full pathogenesis of E. ictaluri is unclear, the olfactory organ is thought to be a site of entry. We have examined the effects of applying E. ictaluri directly into the olfactory capsule of channel catfish. Olfactory organs of 30 experimental fish were exposed to E. ictaluri for 1 h (1 mL, 1 × 10⁶ colony-forming units/mL). Live fish were sampled at 1, 24, 48, and 72 h, and days 5 and 14 postinfection, and their olfactory organs were examined by light and electron microscopy. Damage, including loss of sensory cilia and microvilli from the olfactory mucosal surface, was observed at 1 h postinfection. Degeneration of olfactory receptors and supporting cells was evident by 24 h postinfection. The nonsensory region also showed signs of degeneration, such as columnar cells lacking cilia. Electron microscopic immunocytochemistry confirmed the presence of E. ictaluri on the mucosal surface and within the epithelium. Host leukocytes responded to bacteria by migrating through the olfactory epithelium into the interlamellar lumen and phagocytosing organisms, but phagocytosed E. ictaluri did not appear to be destroyed. Our results indicate that during initial stages of infection channel catfish olfactory epithelium is vulnerable, and E. ictaluri can enter the host through the olfactory organ. It is also possible that host phagocytic cells serve as a vehicle for the systemic dissemination of E. ictaluri     

Evaluation of brine shrimp (Artemia salina) larvae as a bioassay for mycotoxins in animal feedstuffs.

Brine shrimp larvae was tested as a possible simple biological screening system to identify specimens of animal feedstuffs that should be examined further by chemical analytical procedures for mycotoxins. All extracts of the control, nonmouldy feedstuffs increased larval mortality, this being most marked in the case of silage. Chemical and biological testing of diagnostic specimens indicated that the bioassay identified two of four chemically positive specimens and 59 of 135 chemically negative specimens and 59 identified larvicidal compounds present in normal feedstuffs gave a high percentage (56%) of false-positive bioassay results when compared to the results of chemical analyses for three mycotoxins. The use of brine shrimp larvae did not materially reduce the necessity of conducting chemical analyses for mycotoxins.