Acute columnaris infection in channel catfish, Ictalurus punctatus (Rafinesque): efficacy of practical treatments for warmwater aquaculture ponds

Columnaris disease was induced in channel catfish, Ictalurus punctatus (Rafinesque), by bath exposure to four highly virulent isolates of Flavobacterium columnare. In untreated controls, mortality began 20 h after exposure and reached 100% by 48 h. Mortality in channel catfish given antibiotic treatments with oxytetracycline or a combination of sulphadimethoxine and ormetoprim in feed prior to bacterial challenge was zero with all four strains of F. columnare. Diquat (Zeneca Agricultural Products, Wilmington, DE, USA) was the most effective bath treatment; mortality with all four strains was zero. With potassium permanganate, chloramine-T, hydrogen peroxide and copper sulphate, bath treatment efficacy varied significantly among strains (P = 0.0346) and among treatments (P = 0.0033). Bath treatments with chloramine-T and potassium permanganate significantly reduced (P < 0.05) mortality from 100 to 75 and 69%, respectively, but copper sulphate and hydrogen peroxide treatments were not effective. Based on our results, oral antibiotics prevented columnaris disease but, of the bath treatments, only Diquat produced a dramatic reduction in the mortality of acutely infected fish. Diquat is labelled for aquatic use as an herbicide in the USA but in large ponds it is prohibitively expensive.     

Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque)

The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.     

Evaluation of diquat against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

A trial was performed to evaluate the efficacy of diquat (6,7-dihydrodipyrido[1,2-a:2',1'-c]pyrazinediium dibromide) against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Diquat is an Environmental Protection Agency-approved herbicide and has the potential to be legally and practically used against columnaris. Channel catfish were challenged, by cutaneous abrasion, and waterborne exposure to F. columnare and treated once at 22-h post-challenge with 2.5, 5.0, 10.0 and 15 mg L⁻¹ of diquat active ingredient for 6 h. At the conclusion of the trial, 21-day post-challenge, diquat at 5.0, 10.0 and 15 mg L⁻¹ significantly ( P  < 0.05) reduced the mortality of infected fish from 95% in the challenged non-treated fish to 68%, 59% and 49%, respectively. In vitro , the minimum inhibitory concentration (MIC) of 23 isolates of F. columnare was assayed. The majority of the isolates had an MIC value of 5 μg mL⁻¹ (15 of the 23 isolates). Infected fish exhibited acute clinical signs similar to a natural infection. The skin had severe ulcerative necrotizing dermatitis and the muscles had severe necrotizing myositis. The gills had severe multifocal necrotizing branchitis. The results demonstrate that diquat would reduce mortalities caused by an acute columnaris infection.     

柱状黄杆菌强毒株和弱毒株胞外蛋白中差异蛋白的初步鉴定

柱状黄杆菌(Flavobacterium columnare)是一种世界范围的水产动物致病菌,是中国重要养殖鱼类草鱼(Ctenopharyngodon idellus)、鳜(Siniperca chuatsi)等烂鳃病的病原。本研究以1972年从患"烂鳃病"草鱼上分离的两株冻干柱状黄杆菌G4和G18菌株为研究对象,并将G4株再次分离纯化得纯化菌株,命名为G4R3。对草鱼鱼苗浸泡攻毒结果显示,G4R3的LD50至少比G18的高3个数量级,因此G4R3为"强毒株",G18为"弱毒株"。利用蛋白质组学方法分析柱状黄杆菌强毒株G4R3和弱毒株G18的胞外蛋白,经过双向电泳并结合图像分析,共发现了34个点是差异表达的蛋白。胶内酶解、肽质量指纹图谱和串联质谱分析后,鉴定出其中的7个蛋白点,代表滑动蛋白K、腺酐甲硫氨酸合成酶和一种可能的膜蛋白等3种蛋白,它们可能是柱状黄杆菌的毒力因子。     

Feed deprivation of channel catfish, Ictalurus punctatus (Rafinesque), influences organosomatic indices, chemical composition and susceptibility to Flavobacterium columnare

Withholding feed has been suggested as a strategy to manage infectious disease of channel catfish, Ictalurus punctatus (Rafinesque). In a previous study, we demonstrated that deprivation of feed for as little as 7 days reduced innate resistance of catfish to Flavobacterium columnare. This study was conducted to evaluate the effect of feeding regimens [no feeding (NF), fed once every other day to satiation (FEOD) and fed once daily to satiation (FD)] on organosomatic indices, physiological changes and susceptibility of channel catfish to F. columnare. Fish that were not fed for 2 and 4 weeks had a significant increase (P < 0.05) in gutted weight:-wet weight ratio and decrease in other organosomatic indices [gut index (GI), mesenteric fat index (MFI) and hepatosomatic index (HSI)]. Haematology was not effected by feeding regimen except at week 4, when a significantly higher haemoglobin level was observed in the NF fish. Serum protein did not differ at week 2, but the level at week 4 of the NF fish (35.91 mg mL(-1)) was significantly lower than that of the FD fish (41.77 mg mL(-1)). Significantly lower (P < 0.05) blood glucose (39.5 and 40.3 mg dL(-1)) and liver glycogen (1.7 and 1.8 mg g(-1)) were seen in the NF fish at weeks 2 and 4, respectively, as compared with blood glucose and liver glycogen levels of FD fish (67.5 and 92.8 mg dL(-1) and 46.5 and 52.6 mg g(-1) at weeks 2 and 4, respectively) and FEOD (82.8 and 85.5 mg dL(-1) and 45.1 and 51.4 mg g(-1) at weeks 2 and 4, respectively). Mortality in the NF fish caused by F. columnare (78%) was significantly higher (P < 0.05) than mortality in the FD and FEOD treatments (0.0 and 1.7%, respectively). Blood glucose and liver glycogen showed the same trend of low values for NF fish following challenge (week 6). Blood glucose, liver glycogen, GI and HSI are sensitive indicators for channel catfish deprived of feed (NF) for 4 weeks. Blood glucose and liver glycogen levels around 40 mg dL(-1) and 2 mg g(-1), respectively, are indicative of starvation in juvenile channel catfish. Moreover, NF fish were susceptible to F. columnare infection. Thus, it is suggested that in the absence of natural food, juvenile channel catfish should be fed at least once every other day to apparent satiation to maintain normal physiological function and improve resistance to F. columnare.     

Effects of low salinities on Flavobacterium columnare infection of euryhaline and freshwater stenohaline fish

Channel catfish, Ictalurus punctatus (Rafinesque), goldfish, Carassius auratus (L.), striped bass, Morone saxatilis (Walbaum), and Gulf sturgeon, Acipenser oxyrinchus desotoi Vladykov, were acclimatized to fresh water or salinities of 9.0‰ or less and then exposed to Flavobacterium columnare (formerly known as Flexibacter columnaris ), the bacterial pathogen that causes columnaris disease. None of the fish acclimatized to 3.0 or 9.0‰ salinity died, and all deaths in lower salinities occurred between 1 and 5 days after exposure to F. columnare . Mortality was 97.7% in fresh water and 67.1% in 1.0‰ salinity for channel catfish (model SE, 1.8) and 66.5% in fresh water and 40.8% in 1.0‰ salinity for goldfish (model SE, 1.2); and 96.9% in fresh water and 61.7% in 1.0‰ salinity for striped bass (model SE, 1.8). After exposure to F. columnare , none of the Gulf sturgeon died. Flavobacterium columnare was isolated from the skin and gills of all fish dying during the experiments, but was not isolated from survivors in fresh water and 1.0‰ salinity 21 days after bacterial exposure. In vitro growth of bacteria was significantly higher in 1.0 or 3.0‰ salinity than in control medium (0.3‰ salinity). However, in vitro adhesion of bacteria was reduced with increasing salinity, which could explain the lower mortality of fish at higher salinities.     

Isolation and characterization of strains of Flavobacterium columnare from Brazil

Flavobacterium columnare is an important pathogen of freshwater fish, implicated in skin and gill disease, often causing high mortality. An outbreak of skin disease in fingerling and adult Nile tilapia, Oreochromis niloticus (L.), cultivated in a recirculation system, was investigated. Four strains were isolated and characterized by biochemical reactions, enzyme production, fatty acid profile and analysis of the 16S-23S rDNA intergenic spacer region. All strains were identified as F. columnare. Experimental infection assays with one of these strains (BZ-5-02) were conducted and pathogenicity (by intramuscular route) was demonstrated in Nile tilapia and channel catfish, Ictalurus punctatus (Rafinesque). This is the first report of characterization of Brazilian strains of F. columnare.     

Comparison of lipopolysaccharide and protein profiles between Flavobacterium columnare strains from different genomovars

Lipopolysaccharide (LPS) and total protein profiles from four Flavobacterium columnare isolates were compar. These strains belonged to genetically different groups and/or presented distinct virulence properties. Flavobacterium columnare isolates ALG-00-530 and ARS-1 are highly virulent strains that belong to different genomovars while F. columnare FC-RR is an attenuated mutant used as a live vaccine against F. columnare. Strain ALG-03-063 is included in the same genomovar group as FC-RR and presents a similar genomic fingerprint. Electrophoresis of LPS showed qualitative differences among the four strains. Further analysis of LPS by immunoblotting revealed that the avirulent mutant lacks the higher molecular bands in the LPS. Total protein analysis displayed by immunoblotting showed differences between the strains analysed although common bands were present in all the isolates. FC-RR lacked two distinct common bands (34 and 33 kDa) shared by the other three isolates. Based on the difference of LPS and total protein profiles, it is possible to discriminate the attenuated mutant FC-RR from other F. columnare strains.     

Rapid detection and identification of viral and bacterial fish pathogens using a DNA array-based multiplex assay

Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study.     

Development of an indirect enzyme-linked immunoabsorbent assay using a monoclonal antibody to identify Ictalurus sp. fillets

The deceptive marketing of imported basa, Pangasius bocourti (Sauvage), fillets as catfish has resulted in serious economic losses to the channel catfish, Ictalurus punctatus (Rafinesque), industry in the US. The similar appearance of channel catfish and basa fillets created a need for a rapid method to differentiate uncooked, cooked and/or marinated channel catfish fillets from basa fillets and other fish products. A monoclonal antibody (MAb) specific for a 36.8 kDa channel catfish fillet protein was produced and characterized by an indirect enzyme‐linked immunoabsorbent assay (ELISA) and Western blotting. This MAb was used to develop an indirect ELISA specific for a fillet protein unique to fish of the genus Ictalurus. Using this ELISA, 100% of raw and cooked channel catfish fillets were correctly identified and differentiated from other fish in a single‐blind study. These results show that the indirect ELISA using MAbs specific for unique Ictalurus sp. fillet proteins is a rapid and sensitive method for the identification of raw and cooked catfish fillets.     

Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim‐sulfadimethoxine and trimethoprim‐sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards – amongst others – ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.     

Detection of channel catfish virus in asymptomatic adult channel catfish,Ictalurus punctatus (Rafinesque)*

Abstract. Two populations of channel catfish were examined for the presence of channel catfish virus (CCV) by use of a nucleic acid probe. In one population of 22 fish with no history of CCV, viral DNA was found in every liver. These fish had previously been examined by a technique involving co-cultivation of their leucocytes with catfish tissue culture cells. The co-cultivation method had identified virus in 10 of these fish. The second fish population consisted of 14 adults that had survived a CCV outbreak in 1980. Of the 14 fish, 11 showed positive indication of CCV DNA. The tissue distribution of the CCV differed from fish to fish. All fish from the first group and one fish from the second group showed some alterations in the DNA banding patterns expected from pure CCV DNA. This might be indicative of modifications in the genomic structure of the CCV DNA when the virus is latent in a fish.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.     

Virulence of different isolates of Aeromonas hydrophila in channel catfish

The virulence of nine Aeromonas hydrophila isolates from diseased fish, diseased Macrobrachium (freshwater shrimp) and from pond water was determined in channel catfish (Ictalurus punctatus) fingerlings. According to the lethal dose ? 50% end point (LD₅₀) of each isolate, the water organisms were significantly (P < 0.05) less virulent than the isolates from diseased fish. All isolates were biochemically similar.     

Multi-Locus DNA Fingerprinting of Channel Catfish Ictalurus punctatus

Abstract.— Similarities among multi-locus DNA fingerprints of five channel catfish Ictalurus punctatus strains and the ability to identify the strain of a fish based on its fingerprint pattern were investigated. Five restriction enzymes and 13 multi-locus DNA probes were screened to identify enzyme-probe combination useful for DNA fingerprinting channel catfish. Restriction enzymes Hinf I and Dpn II, in combination with probes (CAC)n, (CGC)n, (CTC)n, (ATCC)n, and (GATA)n, produced useful fingerprints (20–30 resolvable bands for each enzyme-probe combination). Thirty individuals (3 pools of 10 individuals each) from each of five channel catfish strains (albino, Mississippi normal, USDA-102, USDA-102 select, and USDA-103) were fingerprinted with all useful enzyme-probe combinations. Band sharing among samples was higher within strains than among strains and band sharing among strains was higher for strains whose breeding history indicated a high degree of relatedness. Individual fingerprints of 18 fish from each of the USDA-102 select and USDA-103 strains revealed no strain-specific bands, but several diagnostic bands (present at high frequencies in either USDA-102 select or USDA-103 strains and at a low frequencies in other strains) were identified. Band sharing at diagnostic bands was used to correctly identify fish as USDA-102 select or USDA-103 strains with 82% accuracy from fingerprints of 17 USDA-102 select strain fish, 18 USDA-103 strain fish, and 38 fish collected from three commercial farms.     

Phosphorus Availability of Common Feedstuffs to Channel Catfish Ictalurus punctatus as Measured by Weight Gain and Bone Mineralization1

The efficacy of using weight gain and bone mineralization to estimate phosphorus availability from feed ingredients for channel catfish was investigated at the conclusion of a 12-wk feeding trial. Juvenile channel catfish (initial weight = 7.8 g/fish) were fed one of seven test diets each containing phosphorus from a single source. All diets were isocaloric, isonitrogenous, and met all nutrient requirements of channel catfish except for phosphorus, which was assumed to be the factor limiting growth. Phosphorus was considered to be 90% available to fish fed the diet containing monosodium phosphate, but a relative value of 100 was assigned to this treatment for purposes of comparison. All other availability values were calculated relative to this value. Phosphorus availabilities (based on weight gain) for wheat middlings, cottonseed meal, and soybean meal were 38%, 43%, and 49%, respectively, which are in the range previously reported for channel catfish. Phosphorus availability values (based on weight gain) for dicalcium phosphate, menhaden fish meal, and meat and bone/blood meal were 82%, 75%, and 84%, respectively. These values were considerably higher (93–96%) than previously reported for catfish when based on bone ash or bone phosphorus. However, availability data based on weight gain for feedstuffs of animal origin generally agreed with phosphorus availability data reported for rainbow trout. Based on our data, mineral utilization by animals in general, and on known physiology of channel catfish, we suggest that weight gain may be a reliable indicator of phosphorus availability and that phosphorus availability values may be overestimated when base on bone mineralization.     

An assessment of the antigenic homogeneity of Edwardsiella ictaluri using monoclonal antibody

Abstract. Monoclonal antibodies were made against the reference strain of Edwardsiella ictaluri (ATCC 3320). Antibody produced by one of seven anti- E. ictaluri hybridomas reacted positively by the immunofluorescent antibody technique against 17 other E. ictaluri isolates. All hybridoma antibodies failed to react with six other bacterial species pathogenic to fish including E. tarda . Ouchterlony tests indicated that four anti- E. ictaluri clones produced only one kind of immunoglobulin. Electrophoresis of 14 different E. ictaluri isolates indicated identical protein bands at 36 and 60 kilodaltons (KD) in all isolates except an isolate from Thailand. Using the immunoblot method, channel catfish anti- E. ictaluri serum reacted with protein bands at 34 and 60 KD, which indicates that this molecular weight protein in the bacterium may be the dominant immunoprotein.     

Comparative growth and yield of channel catfish and channel × blue hybrid catfish fed a full or restricted ration

Growth and yield (kg ha^?1) of the channel catfish (Ictalurus punctatus, Rafinesque, 1818) and the channel × blue hybrid catfish [I. punctatus female ×I. furcatus (Lesueur, 1840) male], which shared the Jubilee strain of channel catfish as the maternal parent, were compared in sixteen 0.1 ha earthen ponds (14 852 fish ha^?1) during the April to November growing season. Each fish genetic group was fed a commercially formulated 32% protein feed daily to apparent satiation or at 80% of the mean daily satiation ration. Net yield and individual weight were higher for channel × blue hybrid catfish compared with channel catfish and for fish fed a full ration compared with a restricted ration. When fed a full ration, the channel × blue hybrid catfish grew faster from May to September than did the purebred channel catfish because the hybrid catfish consumed a greater percentage of its body weight at each feeding. Net yield within each fish genetic group was lower when feed ration was restricted. The per cent reduction in net yield in response to feed restriction was similar for each fish genetic group.     

Detection of channel catfish virus in channel catfish,Ictalurus punctatus (Rafinesque): use of a nucleic acid probe*

A nucleic acid probe for channel catfish virus (CCV) was constructed using recombinant DNA techniques. This probe consisted of a specific viral DNA fragment generated by digestion of CCV DNA with the restriction enzyme EcoRI. The probe was used to examine DNA isolated from tissues of fish that had been injected with CCV. Viral DNA was detected in some tissues of various injected fish. The sensitivity limit of detection was determined to be one viral DNA per cell.