Comparative Susceptibility of Channel Catfish,Ictalurus punctatus; Blue Catfish,Ictalurus furcatus; and Channel (♀) × Blue (♂) Hybrid Catfish to Edwardsiella piscicida,Edwardsiella tarda,and Edwardsiella anguillarum

Members of the genus Edwardsiella are important pathogens of cultured and wild fish globally. Recent investigations into the phenotypic and genotypic variation of Edwardsiella tarda have led to the segregation of E. tarda into three distinct taxa: E. tarda, Edwardsiella piscicida, and Edwardsiella anguillarum. In catfish aquaculture in the southeastern USA, E. piscicida has been more commonly associated with disease than E. tarda or E. anguillarum, and recent research has demonstrated E. piscicida to be more pathogenic in channel catfish than E. tarda or E. anguillarum. Anecdotal reports from industry suggest an increased prevalence of E. piscicida associated with the culture of channel (♀) × blue (♂) hybrid catfish. This work investigated the comparative susceptibility of channel catfish, blue catfish, and their hybrid cross to molecularly confirmed isolates of E. tarda, E. piscicida, and E. anguillarum. There was significantly higher mortality in hybrid catfish compared to channel catfish following intracoelomic injection of E. piscicida. To our knowledge, E. piscicida is the first bacterial pathogen to demonstrate increased pathogenicity in hybrid catfish compared to channel catfish.     

Vaccination of Full‐sib Channel Catfish Families Against Enteric Septicemia of Catfish with an Oral Live Attenuated Edwardsiella ictaluri Vaccine

This study evaluated the efficacy of an oral live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish in 20 full‐sib fingerling channel catfish families. The vaccine was delivered orally by feeding fish a diet coated with an attenuated E. ictaluri isolate. Sixty‐nine days postvaccination, control and vaccinated fish were challenged with virulent E. ictaluri and mortality was examined for 21 d postchallenge. Vaccinated fish had significantly lower mortality than nonvaccinated fish following challenge (P < 0.001). Mortality of vaccinated fish was 1.7 ± 1.4% as opposed to 47.8 ± 28.7% in nonvaccinated fish. Relative percent survival ranged from 87.7 to 100% with an average of 95.2 ± 4.0% (±SE) among the 20 families of fish. There were significant differences in mortality among families in nonvaccinated fish (P < 0.01) while there were no differences among vaccinated families of fish. Results indicate that the live attenuated E. ictaluri vaccine is effective at reducing mortality in channel catfish exposed to virulent E. ictaluri. These data demonstrate that genetic differences among healthy families of channel catfish are not major considerations in developing an effective vaccination program utilizing the oral vaccination platform described in this study.     

A rapid bioassay for bactericides against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare

The most common bacterial diseases in pond‐raised channel catfish Ictalurus punctatus (Rafinesque) are enteric septicemia of catfish and columnaris, caused by Edwardsiella ictaluri and Flavobacterium columnare respectively. Medicated feed containing antibiotics is one management approach that catfish producers use in the treatment of bacterial diseases. However, the future use of all types of medicated feed in catfish aquaculture is uncertain. To discover effective alternatives to antibiotics, a rapid 96‐well microplate bioassay utilizing E. ictaluri and F. columnare to evaluate natural compounds and extracts was developed. In this bioassay, bacterial growth is determined by absorbance measurements of microplate wells after 24 h incubation and then confirmed by detecting cell viability after the addition of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide with additional incubation for 24 h. The minimum inhibitory concentration, minimum bactericidal concentration and 50% inhibition concentration (IC50) are determined by graphing the absorbance data. The 24 h IC50 results of test compounds are compared with the 24 h IC50 results of the drug controls oxytetracycline and florfenicol. Among the antibiotics evaluated, doxycycline and tetracycline appear more effective against E. ictaluri and F. columnare than either drug control. This bioassay is rapid, reproducible and economical for evaluating a large number of compounds and extracts.     

Emergence of Edwardsiella piscicida in Farmed Channel ♀, Ictalurus punctatus × Blue ♂, Ictalurus furcatus,Hybrid Catfish Cultured in Mississippi

There is a trend toward the increased incidence and prevalence of Edwardsiella piscicida septicemia in US catfish aquaculture, particularly in channel ♀, Ictalurus punctatus, × blue ♂, I. furcatus, hybrid catfish. From 2013 to 2017, a total of 3242 disease case submissions were made to the Aquatic Research and Diagnostic Laboratory (ARDL) at the Thad Cochran National Warmwater Aquaculture Center in Stoneville, MS. Of these, 1400 (43.2%) were hybrids. E. piscicida was suspected in 138 (4.3%) of cases, the majority of which (89.1%) were from hybrid catfish. A molecular survey of these isolates confirmed the majority (92.0%) to be E. piscicida. Furthermore, cases of E. piscicida from hybrids submitted to the ARDL and the Aquatic Diagnostic Laboratory of the Mississippi State University College of Veterinary Medicine in Starkville, MS, were documented for gross lesions and histological analysis. Grossly, E. piscicida presents with small dermal ulcerations, a raised fluid‐filled cranial midline lesion that is frequently ulcerated, hemorrhage in the gills, exophthalmia, and abdominal distension. Internally, lesions include splenomegaly, straw‐colored ascites, renomegaly, and occasionally hemorrhagic intestines. Histopathological examination is in agreement with gross observations, and infected fish repeatedly demonstrate a mononuclear meningoencephalitis, hemorrhagic branchitis, splenitis, ulcerative dermatitis, granulomatous interstitial nephritis, and hepatitis coupled with a hemorrhagic enteritis.     

Efficacy of Sarafloxacin Hydrochloride in Treating Naturally-Induced Edwardsiella ictaluri Infections in Channel Catfish,Ictalurus punctatus

Sarafin (sarafloxacin hydrochloride), a new antibacterial, was evaluated in the field on a naturally induced infection of Edwardsiella ictaluri in channel catfish, -Ictalurus punctatus. Healthy channel catfish (mean weight = 50 g) were stocked into nine cages at 200 fish per cage in a pond with an undergoing E. ictaluri infection. Seven days after stocking, dead fish were observed in the cages with clinical signs of enteric septicemia of channel catfish (ESC). After E. ictaluri was confirmed through isolative biochemical tests, medicated feed was applied for five consecutive days. During this period, fish in three control cages received a commercial 32% protein floating feed, three other cages of fish served as positive controls and were fed Romet, and three cages received the test feed with Sarafin. Both medicated feeds reduced the increase in cumulative percent mortality. In the control cages, cumulative percent mortalities continued to increase throughout the study period. Average daily mortality rates were significantly lower following both treatments of medicated feed, and treatments receiving Sarafin showed the greatest reduction in average daily mortality rates. Average daily mortality rates in the control did not change after the medicated feeding period. Toward the end of the study, temperatures reached 30°C, above the active range of ESC infections, and all mortalities ceased.     

The Relationship Between Vaccine Dose and Efficacy in Channel Catfish Ictalurus punctatus Vaccinated as Fry with a Live Attenuated Strain of Edwardsiella ictaluri (RE‐33)1

Channel catfish Ictalurus punctatus were vaccinated at 12 d of age (post‐hatch) by a 2‐min bath immersion with attenuated Edwardsiella ictaluri RE‐33 at doses of 2.5 × 10⁵, 2.5 × 10⁶, and 2.4 × 10⁷ colony‐forming units CFU/mL of water. Following vaccination, RE‐33 was recovered from a greater percentage of fry that were vaccinated at the high and intermediate doses compared to fry vaccinated at the lowest dose. Independent of dose, the greatest percentage of RE‐33 positive fry occurred between 1 and 6 d post‐vaccination with a significant decrease in positive fry observed on day 12. A significant increase in mortality occurred 6 to 12 d post‐vaccination in fry vaccinated at the highest dose. No differences in post‐vaccination mortalities occurred between the other treatments. Following virulent E. ictaluri challenge, mortalities of fish vaccinated at doses of 2.5 × 10⁶ and 2.4 × 10⁷ CFU/mL were significantly less than those of fish vaccinated at 2.5 × 10⁵ CFU/mL and sham‐vaccinated control fish. These data show that vaccination with RE‐33 can offer protection against subsequent virulent E. ictaluri infection.     

First detection of Edwardsiella ictaluri (Proteobacteria: Enterobacteriaceae) in wild Australian catfish

The bacterium Edwardsiella ictaluri is considered to be one of the most significant pathogens of farmed catfish in the United States of America and has also caused mortalities in farmed and wild fishes in many other parts of the world. E. ictaluri is not believed to be present in wild fish populations in Australia, although it has previously been detected in imported ornamental fishes held in quarantine facilities. In an attempt to confirm freedom from the bacterium in Australian native fishes, we undertook a risk‐based survey of wild catfishes from 15 sites across northern Australia. E. ictaluri was detected by selective culturing, followed by DNA testing, in Wet Tropics tandan (Tandanus tropicanus) from the Tully River, at a prevalence of 0.40 (95% CI 0.21–0.61). The bacterium was not found in fishes sampled from any of the other 14 sites. This is the first report of E. ictaluri in wild fishes in Australia.     

Effects of Calcium and Magnesium Hardness of Incubation Waters on the Hatching Success of Hybrid Eggs of ♀ Channel Catfish,Ictalurus punctatus × ♂ Blue Catfish,Ictalurus furcatus

Hatching efficiency is generally lower in channel catfish, Ictalurus punctatus × blue catfish, Ictalurus furcatus, hybrid catfish hatcheries compared to hatcheries hatching pond‐spawned channel catfish eggs. Egg quality and hatching water hardness are known variables influencing the efficiency of hybrid catfish fry production in hatcheries. Previous research recommended that at least 60 mg/L hardness had to be contributed only by calcium to optimize the hatching success of hybrid catfish eggs. However, whether the effect of waterborne magnesium in hatching waters facilitates or impedes the hatching success of hybrid catfish is not known. Two trials were conducted to determine the effects of calcium alone or magnesium hardness, in combination with the hatching success of hybrid catfish eggs. Trial 1 showed that if the previously identified minimum hardness criterion of 60 mg/L was met by calcium in the hatching waters, added magnesium had no effect on the hatching success of hybrid catfish eggs. Trial 2 showed that the previously identified minimum hardness of 60 mg/L can be met by either calcium alone or magnesium replacing up to 50% of the total hardness in the hatchery waters for optimal hatching success of hybrid catfish eggs. Calcium provides a specific requirement during embryonic development of catfish. The results from this study can be used to design treatment processes for hatchery water supplies to improve efficiency of hybrid catfish fry production in hatcheries.     

Susceptibility of channel catfish, Ictalurus punctatus (Rafinesque), to Edwardsiella ictaluri challenge following copper sulphate exposure

Channel catfish, Ictalurus punctatus (Rafinesque), with or without a preliminary 24 h exposure to 2 mg copper sulphate L(-1), were challenged with 7.5 x 10(6) colony forming units L(-1) of Edwardsiella ictaluri to determine the effect of copper sulphate on disease resistance. Catfish previously exposed to copper sulphate were significantly more resistant to the bacterial challenge than those not exposed. Catfish not exposed to copper sulphate suffered 35.5% mortality while catfish exposed to copper sulphate experienced 14.1% mortality. Copper concentrations were the same in tank waters of both exposed and control fish at the time of challenge, eliminating the possibility that copper in the water may have been toxic to bacteria. Copper concentrations in freeze dried and ground tissues of unexposed, exposed, and purged channel catfish were highest in fish before copper sulphate exposures suggesting that elevated tissue levels of copper were not responsible for the increased resistance to bacterial challenge. Competition for sites of bacterial attachment to gill or epithelial cells may account for the reduction in mortality; although this is not supported by the low copper content of fish tissue after copper exposure.     

A Comparison of Immersion, Immersion/Oral Combination and Injection Methods for the Vaccination of Channel Catfish Ictalurus punctatus Against Edwardsiella ictaluri

Abstract.— Four different vaccination regimes, including non-vaccinated controls (C), immersion/oral combination (IO). late immersion (LI), and injection (IJ), were evaluated for antibody production and protection from challenge in channel catfish Ictalurus punctatus that were hatched and raised in recirculating culture systems over a 28-wk period. For IO, fry were vaccinated by immersion on day 12 post-hatch and given an oral booster during week 10 post-hatch. The LI and IJ delivered vaccine was also administered at week 10 post-hatch. No titers were detected in the IO group prior to the oral booster at week 10. Mean titers rose sharply after the week 10 vaccinations in all treatment groups, maintained a high level for about 8 wk, and slowly declined over the next 4 wk. Injected fish had significantly higher titers during the primary response period than either LI or IO fish. There was no statistical difference in titers between LI and IO fish during the primary response. After an oral booster delivered during week 22, titers in LI and IO fish increased significantly at week 25, while titers in IJ fish declined despite the booster delivery. An immersion booster delivered during week 25 resulted in significant increases in titers in all vaccinated groups. Of multiple challenges, the only one not marred by concurrent columnaris infections revealed a low, but significant level of protection in IJ fish at 12 wk post-vaccination.     

Field Efficacy of Florfenicol for Control of Mortality in Channel Catfish, Ictalurus punctatus (Rafinesque), Caused by Infection With Edwardsiella ictaluri

A field study to assess the efficacy of florfenicol (FFC) against enteric septicemia of catfish (ESC) was conducted with pond‐reared channel catfish fingerlings held in 0.1‐acre earthen ponds. Fish were challenged with Edwardsiella ictaluri in a natural pond outbreak or by cohabitation with E. ictaluri‐infected fish held in netpens. Fourteen ponds were assigned in equal number to two treatment groups, that is, either treated (with 10 mg FFC/kg body weight in medicated feed) or not treated (control) for 10 consecutive d. The threshold for enrollment into the study was 0.3% cumulative mortality attributed to ESC. Treatment was initiated on different dates for each pond because each pond was enrolled when 33 fish/pond were diagnosed with ESC based on clinical signs, lesions, or positive cultures. Mortality was monitored during the 10‐d treatment period and during a 14‐d posttreatment observation period. At the end of the 14‐d posttreatment observation period, all fish were euthanized, and 20 fish from each pond were examined by gross necropsy and evaluated for the presence of E. ictaluri by bacterial culture. The odds of a mortality in the control group were 2.20 times the odds of a mortality in the FFC‐treated group. Significantly fewer (P≤ 0.05) FFC‐medicated catfish died in comparison to unmedicated catfish. The minimum inhibitory concentration of FFC for this strain of E. ictaluri was 0.25 μg/mL in all fish that were assayed. The mean zone of inhibition (Kirby Bauer) was 36.8 mm from E. ictaluri isolates of test fish. There were no FFC treatment‐related lesions seen on gross pathology. FFC was efficacious and safe for control of mortality from E. ictaluri infection in catfish.     

Uptake and Clearance of Edwardsiella ictaluri in the Peripheral Blood of Channel Catfish Ictalurus punctatus Fingerlings during Immersion Challenge

Uptake and clearance of Edwardsiella ictaluri in the peripheral blood of channel catfish Ictalurus punctatus fingerlings were monitored for 216 h after exposure to E. ictaluri for 4 h and 8 h under static conditions. Most fish exposed to E. ictaluri developed bacteriemia 24 h post-exposure, and by 72 h post-exposure E. ictaluri was recovered from all the blood of all exposed fish. The number of E. ictaluri colony forming units (CFU) in the blood of moribund fish ranged between 1.7 × 10³ to 1.6 × 10⁵ CFU/50 μL whole blood. Clearance of bacteria from the blood was observed by 216 h post-exposure and all fish surviving bacterial exposure developed agglutinating antibody against E. ictaluri . The pathogenesis of the infection was accompanied by the shedding of viable E. ictaluri into the water which may serve as a mechanism by which fish to fish transmission occurs.     

斑点叉尾鮰病毒“自杀性”ＤＮＡ疫苗的构建及其体外表达检测

根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到"自杀性"DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)"自杀性"DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但"自杀性"DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。     

斑点叉尾鮰病毒“自杀性”DNA疫苗的构建及其体外表达检测

根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到自杀性DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)自杀性DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但自杀性DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。     

Replacing Soybean Meal with Alternative Protein Sources in Diets for Pond‐raised Hybrid Catfish, ♀ Ictalurus punctatus × ♂ Ictalurus furcatus

The present study investigated the replacement of soybean meal with combinations of two or three alternative protein sources in diets for pond‐raised hybrid catfish, ♀ Ictalurus punctatus × ♂ Ictalurus furcatus. Alternative protein sources evaluated included cottonseed meal, distillers dried grains with solubles (DDGS), peanut meal, and porcine meat and bone meal (PMBM). Hybrid catfish fingerlings with a mean initial weight of 35 g/fish were stocked into 25 earthen ponds (0.04 ha) at a density of 14,826 fish/ha. Fish were fed once daily to apparent satiation for 166 d. No significant differences were observed for total diet fed, net yield, weight gain, survival, carcass yield, fillet yield, or fillet proximate composition among dietary treatments. Results show soybean meal may be completely replaced by combinations of cottonseed meal and one or two other alternative protein sources including DDGS, peanut meal, and PMBM in the diet without markedly affecting production and processing characteristics and fillet proximate composition of pond‐raised hybrid catfish. These alternative diets may be used during foodfish production when prices are favorable.     

Response of Pond‐raised Fingerling Hybrid Catfish, ♀ Ictalurus punctatus × ♂ Ictalurus furcatus,to Dietary Protein Concentrations and Sources

This study examined four experimental diets with different protein concentrations and sources for pond‐raised fingerling hybrid catfish, ♀ Ictalurus punctatus × ♂ Ictalurus furcatus. A 35% protein diet with fishmeal was used as the control diet. Test diets were 32 and 28% all‐plant‐protein diets and a 28% protein diet with porcine meat, bone, and blood meal. Small fingerlings with a mean initial weight of 2.9 g/fish were stocked into 20 earthen ponds (0.04 ha) at a density of 172,970 fish/ha. They were fed once daily to apparent satiation for 107 d. No significant differences were observed for total diet fed, gross yield, final weight, survival, or condition factor among dietary treatments. However, fish fed the 28 and 32% all‐plant‐protein diets had a significantly higher feed conversion ratio than fish fed the 35% protein diet with fishmeal. There were no significant differences in chlorophyll a and nitrite concentrations in the pond water, but ponds receiving the 35% protein diet had significantly higher ammonia than those receiving 28% protein diets. Economic analysis suggested potential cost savings by using low‐protein and all‐plant‐protein diets for hybrid catfish fingerling production.     

Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33)

These studies were conducted to evaluate the efficacy of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In one study channel catfish fingerlings (72 d of age post hatch) were immersed for 30 min in water containing E. ictaluri RE-33 at dosages of 1 × 10⁶, 1 × 10⁷ and 2 × 10⁷ CFU/ML of water. No mortalities were observed following vaccination. Following exposure to virulent Edwardsiella ictaluri the cumulative mortality of fish vaccinated with dosages of at least 1 × 10⁷ CFU/mL were significantly lower than that of non-vacccinated fish in both laboratory and field challenges. Vaccination with 1 × 10⁶ CFU RE-33mL provided some protection during the laboratory challenge but failed to protect fish under field conditions. In a second study, vaccination of 6 full-sib families of channel catfish at a vaccine dosage of 1 × 10⁷ CFU/mL resulted in a relative percent survival among families ranging from 67.1 to 100%. Significant differences in mortality were found among families and between vaccinated and unvaccinated groups, but there was no family by vaccine interaction. Families with the highest mortality after vaccination were also shown to have the highest mortality without vaccination (r = 0.82; P = 0.04).     

Cross-protection elicited in channel catfish (Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri strains

Cross-protection of channel catfish ( Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri and challenged with six E. ictaluri strains was examined in four trials. The relative per cent survival among low-dose immunized and then challenged fish ranged from 27.7% to 100%. Significant protection ( P <0.05), with the exception of strain ATCC-33202, was conferred by immunization with a given E. ictaluri strain challenged either with a homologous or a heterologous strain. Antibody titres of pooled serum collected on day 22 from surviving fish examined by enzyme-linked immunosorbent assay (ELISA) ranged from 1:40 to 1:320, but no differences were apparent among different vaccinated groups. The protein profiles of six E. ictaluri strains examined by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a relatively homogeneous pattern. Immuno-blots probed with pooled serum from immunized and challenged fish showed a pattern similar to LPS-reaction patterns observed with E. ictaluri in other studies. Since the present studies further corroborate that E. ictaluri is a clonal bacterial species with no apparent antigenic differences, it is possible that immunization with a single E. ictaluri field strain should confer protection against any other strain.     

Growth Response and Resistance to Edwardsiella ictaluri of Channel Catfish,Ictalurus punctatus,Fed Diets Containing Distiller’s Dried Grains with Solubles

A study was conducted to examine the effect of dietary levels of distiller’s dried grains with solubles (DDGS) on growth, body composition, hematology, immune response, and resistance of channel catfish, Ictalurus punctatus, to Edwardsiella ictaluri challenge. Five diets containing 0, 10, 20, 30, and 40% DDGS with supplemental lysine (Diets 1–5) as partial replacements of a combination of soybean meal and cornmeal on an equal protein basis were fed to juvenile catfish (13.33 ± 0.25 g) for 12 wk. Growth performance and feed utilization efficiency were similar for fish in all treatments. Body lipid and moisture increased and decreased, respectively, in fish feed DDGS‐containing diets relative to the control group. Dietary treatment had no effect on red and white blood cell counts. Hemoglobin and hematocrit were significantly higher in fish fed diets containing DDGS than in those fed the control diet. Fish fed 20–40% DDGS diets had increased serum total immunoglobulin, and those fed the 30% DDGS diet had significantly increased antibody titers 21 d following E. ictaluri challenge. Other immune variables evaluated were not affected by dietary treatments. Preliminary results on bacterial challenge showed an increased resistance against E. ictaluri in fish fed DDGS‐containing diets (Diets 2–5).