Serological detection of Edwardsiella ictaluri Hawke lipopolysaccharide antibody in serum of channel catfish, Ictalurus punctatus Rafinesque

Abstract. Bacterial agglutination, passive haemagglutination, complement-dependent passive haemolysis, indirect immunofluorescence, agar gel immunodiffusion and agglutination with fractions of immunized fish serum were compared for detecting humoral antibody to the lipopolysacchande (LPS) of Edwardsiella icialuri Hawke in channel catfish. Bacterial agglutination titres averaged 1: 672; passive haemagglutination titres averaged 1: 1152; and complement-dependent haemolysis titres averaged 1: 2360. Serum from non-vaccinated fish ranged from 0 to 1:32. Indirect fluorescence and immunodiffusion demonstrated positive reactions to the LPS antibody. Fractionation of immune sera produced three fractions, one of which strongly haemagglutinated E. ictaluri but the other two did not. All six serological techniques were sensitive to E. ictaluri LPS antibody.     

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.     

TaqMan real-time polymerase chain reaction assay for rapid detection of Flavobacterium columnare

Flavobacterium columnare is a ubiquitous Gram-negative bacterium that causes columnaris disease in a wide variety of fish worldwide. Timely detection of this bacterium is important to prevent its spreading and to reduce the economic loss to fish farmers. We developed a TaqMan-based real-time polymerase chain reaction (PCR) targeting a 113 bp nucleotide region of the chondroitin AC lyase gene of F. columnare G₄. Specificity of the assay evaluated with 20 isolates of F. columnare and 15 other taxonomically or ecologically related bacteria revealed that the primers and probe were 100% specific for detection of F. columnare. The sensitivity limit of detection of F. columnare in pure cultures, over a range of dilutions [3.1 × 10⁰–3.1 × 10⁶ colony-forming units (CFU) mL⁻¹], was observed to be ∼3 bacterial cells. The lowest limit of detection in nucleic acids from pure culture of F. columnare was 5.4 fg and the assay was linear with the log of amount of nucleic acid (R²=0.994) over that range (5.4 ng–5.4 fg). In tissues (blood, gills and kidney) of F. columnare experimentally infected fish, the bacterial numbers measured by TaqMan real-time PCR ranged from 3.4 × 10⁰ to 9.5 × 10⁵ CFU mL⁻¹. In both F. columnare experimentally infected and spiked samples, positive PCR results were confirmed by bacteriological culture with 100% agreement. The TaqMan real-time PCR developed in this study is specific, sensitive and reproducible for the detection and quantitation of F. columnare in infected fish.     

Oral and parenteral vaccines against <Emphasis Type="Italic">Flavobacterium columnare</Emphasis>: evaluation of humoral immune response by ELISA and in vivo efficiency in Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

Flavobacterium columnare is a bacterial pathogen for many freshwater fish species. It is responsible for outbreaks in fish farms worldwide, causing high mortality rates. Fish vaccination is a potential approach for prevention and control of disease, with oral vaccines suitable for fish because of their easier application, low cost and minimum stress to fish. Alginate microparticles have been widely used as controlled release systems, including for fish vaccination. The aim of this study was to evaluate the capacity of oral and parenteral vaccines against F. columnare to induce a humoral response, as well as the in vivo efficiency in Nile tilapia fingerlings. The fingerlings were immunized with bacterin by intraperitoneal (i.p.), intramuscular (i.m.), oral and immersion routes, as well as orally with alginate microparticles containing formalin-killed bacteria. A sandwich ELISA was developed to detect specific antibodies against F. columnare. The animals were challenged with pathogenic strain BZ-1 to determine the relative percentage of survival. A significant humoral response was induced by bacterin administered by i.p. and i.m. routes (P < 0.05). However, none of the vaccine preparations were effective in protecting fish against F. columnare infection (P < 0.05). In spite of high antibody levels, there was no relation between immunoglobulin titers and resistance to columnaris for Nile tilapia fingerlings. These data suggest that use of serological analysis as the only method to determine vaccine efficiency against F. columnare infection in Nile tilapia can lead to imprecise results for the usefulness of these products in vivo.     

Kaolinitic clay protects against Flavobacterium columnare infection in channel catfish Ictalurus punctatus (Rafinesque)

Columnaris disease, caused by the bacterial pathogen Flavobacterium columnare, continues to be a major problem worldwide in both wild and cultured freshwater finfish. Despite the far-reaching negative impacts of columnaris disease, safe and efficacious preventatives and curatives for this disease remain limited. In this study, we evaluated the potential of kaolin (Al₂Si₂0₅(OH)₄), a type of clay, for the prevention of columnaris disease. Channel catfish, Ictalurus punctatus (Rafinesque), fingerlings were experimentally challenged with Flavobacterium columnare in untreated water or with water containing kaolin (1 g L⁻¹). Over the 7-day course of study, kaolin treatment led to significantly (P < 0.001) improved survival (96%) as compared to untreated fish (78% survival). Histological examination of the gills revealed that kaolin-treated fish had substantially less gill damage than untreated controls. Quantitative PCR analysis of gill tissue revealed that kaolin significantly reduced F. columnare adhesion (measured at 1 h post-challenge) and colonization (24 h post-challenge). Incubation of kaolin with F. columnare in vitro demonstrated that kaolin reduced the number of F. columnare cells in culture supernatants, presumably through the formation of physical complexes through adsorption. In summary, kaolin can improve survival, reduce gill pathologies and reduce bacterial attachment to key tissues associated with columnaris disease in channel catfish by binding to F. columnare.     

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

Evaluation of a 4‐h static copper sulphate treatment against experimental infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus)

The efficacy of copper sulphate (CuSO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus) was evaluated. Fish were challenged, by waterborne exposure to F. columnare in an ultra‐low flow‐through water delivery system, and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 at 5.5 h post challenge for 4 h. Bacterial challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged with F. columnare and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 post challenge had mortalities of 6.4% and 18.3%, respectively, compared with the positive control, which had 90.2% mortality. The mortality of challenged fish treated with CuSO₄ at 4.2 and 2.1 mg L^?1 was significantly different from the positive control. There was no significant difference between the mortalities of the 4.2 and 2.1 mg L^?1 treatments. The results suggest that CuSO₄ has clear therapeutic value against columnaris infections.     

Protective efficacy of Nigella sativa seeds and oil against columnaris disease in fishes

Columnaris disease, caused by the bacterium Flavobacterium columnare, is currently the most frequently reported bacterial disease affecting farm‐raised channel catfish in the USA. Common treatments against the disease include the use of medicated feed that has led to emergent antibiotic resistant strains of F. columnare. Nigella sativa (Black cumin) is a medicinal herb commonly used by many cultures as a natural remedy for numerous disorders. Recently, we have discovered the antibacterial activity of N. sativa and its oil extract against F. columnare. In this study, we showed N. sativa oil (NSO) strongly inhibited the growth of all of the strains of F. columnare tested and yielded significantly larger zones of inhibition than those produced by oxytetracyclin. We tested the protective effect against columnaris disease in vivo by incorporating NSO (5%) or N. sativa seeds (NSS) (5%) into fish feeds. Fishes (Ictalurus punctatus and Danio rerio) fed amended diets displayed significantly lower mortality than those fed control diets. Per cent mortalities in control groups ranged from 77% to 44% and from 70% to 18% in zebrafish and channel catfish, respectively. A dose study using different NSS concentrations showed that 5% NSS offered the most protection against columnaris disease in channel catfish.     

Evaluation of the therapeutic effect of potassium permanganate at early stages of an experimental acute infection of Flavobacterium columnare in channel catfish,Ictalurus punctatus (Rafinesque)

The efficacy of potassium permanganate (KMnO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus, was evaluated. Fish were experimentally challenged by waterborne exposure for 2 h to F. columnare after cutaneous abrasion, and treated with KMnO₄ at 2.0 mg L^?1 above the KMnO₄ demand at 0, 1, 2 or 4 h postchallenge for 24 h. Challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged and treated with KMnO₄ at 0, 1, 2 or 4 h postchallenge had mortalities of 26%, 63%, 64% and 83% respectively. The mortality of challenged fish treated with KMnO₄ at 0 h postchallenge (26%) was significantly less than the positive control (77%). The mortalities of challenged fish treated at 1, 2 or 4 h postchallenge were not significantly different from the positive control fish. The results suggest that KMnO₄ has a clear therapeutic value in early stages of columnaris infection but limited therapeutic value once the infection has progressed.     

A Non-Nutritive,Ambient Temperature-Processed Floating Pellet as a Carrier of Antibiotics for Channel Catfish

A non-nutritive, ambient temperature-processed floating pellet (ATFP) was used as a medium to deliver apramycin (Eli Lilly and Co., Greenfield, Indiana), 52% active, and oxytetracycline hydrochloride (Pfizer, Inc., Lee's Summit, Missouri) (91% active) to channel catfish, Ictalurus punctatus, experimentally infected with Edwardsiella ictaluri. The drugs were fed at 50 mg of active antibiotic per kilogram of fish per day. Two medicated feeding trials were run for 10 consecutive days in troughs with channel catfish, beginning 24 hours after exposure to E. ictaluri. Mortality in the non-infected control population in Trial 1 was 7.6%, significantly (P < 0.05) lower than the 88.3% of the infected, non-medicated fish. Fish fed diets containing apramycin experienced 62.5% mortality, while fish fed diets containing oxytetracycline hydrochloride had 63.3% mortality. Mortality of the two medicated groups were significantly (P < 0.05) different from either control group. In Trial II, the 23% mortality in the non-infected group was significantly lower than the 48% mortality in the infected, non-medicated group and the 41% mortality in the oxytetricycline hydrochloride-medicated group, but not different than fish fed diets containing apramycin (28%). Mortality of the two medicated groups in Trial II were significantly different from each other. The presence of thc apramycin and oxytetracycline hydrochloride in the ATFP was demonstrated by the antibiotic diffusion method on agar plates swabbed with E. ictaluri.     

Development of an ELISA to measure the humoral immune response of hybrid striped bass Morone chrysops×M. saxatilis to Streptococcus iniae

A previously developed monoclonal antibody (mAb) specific for the heavy chain of hybrid striped bass (HSB) Morone chrysops×M. saxatilis immunoglobulin was used in an assay to detect the humoral response to antigens of Streptococcus iniae. In order to validate this assay, an anti‐S. iniae antibody was produced in HSB by immunization with formalin‐killed cells of S. iniae mixed with Freund's complete adjuvant (FCA). After boosting with cells mixed with Freund's incomplete adjuvant (FIA), fish were challenged with live S. iniae by i.p. injection. This resulted in mortalities of 100%, 13% and 7% for non‐immunized, immunized and non‐challenged fish respectively. Live S. iniae were recovered only from moribund non‐immunized challenged fish, indicating that the immunization conferred protection against S. iniae. Sera were taken at 28 and 42 days post challenge and anti‐S. iniae antibody was measured by both agglutination and indirect ELISA. Titres of anti‐S. iniae antibody increased only in the immunized group as measured by agglutination and ELISA. Serum complement measured in the final bleeding was significantly higher in the immunized group. Western blotting using immune HSB serum indicated that the predominant antigen in this case was the high molecular weight polysaccharide of S. iniae.     

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.     

Identification,annotation of Mucin genes in channel catfish (Ictalurus punctatus) and their expression after bacterial infections revealed by RNA‐Seq analysis

Mucins are large glycoproteins that cover epithelial surfaces of the body and play important roles in prevention of inflammatory and various infectious diseases. In this study, five membrane‐bound and seven secreted mucin genes in the channel catfish were identified. All these identified mucin genes possess at least one PTS, von Willebrand D (VWD) or SEA domains. The expression of the 12 mucin genes in channel catfish was first studied with infection of Edwardsiella ictaluri and Flavobacterium columnare. Expression difference in MUC13a, MUC13, MUC2 and MUC5b was found in the intestine after E. ictaluri infection. Eight mucin gene expressions (except MUC3a, MUC2, MUC4 and MUC5f) were up‐regulated at 4 hr and down‐regulated after 24 hr in the gill with F. columnare infection. Expression level of MUC2 gene was up‐regulated in the intestine with E. ictaluri infection without no significant change in the gill under the F. columnare infection, which indicate that MUC2 is tissue‐specific gene expression and has different immune respond to two bacterial challenge. Taken together, the study showed mucin from the gill by F. columnare challenge induced an obvious response than mucin from the intestine with E. ictaluri infection.     

Detection and Distribution of Flavobacterium columnare in Experimentally-Infected Channel Catfish,Ictalurus punctatus Using Culture and Polymerase Chain Reaction

Abstract

The authors examined the use of culture and polymerase chain reaction (PCR) to detect Flavobacterium columnare in experimentally-infected channel catfish, Ictalurus punctatus. Five treatments were utilized which included immersion exposure to 10⁶, 10⁷, 10⁸ colony forming units (CFU)/mL for 30 minutes, intramuscular injection of 10⁸ CFU/fish and a negative control (i.e., immersion in Cytophaga broth). Flavobacterium columnare was isolated and detected in mucus 30 minutes following exposure by microbiological culture and PCR in all treatments except the negative controls. Gills were positive by culture and by PCR in all treatments at 30 minutes post treatment except the 10⁶ CFU/mL immersion treatment which did not yield positive culture and PCR results until 1 hour. Culture positive samples were observed in the internal organs (anterior and posterior kidney) and blood of the 10⁷⁸ CFU/mL treatments although at low numbers (≤10 CFU). Results of PCR paralleled that of culture for the mucus and gill samples when analyzing all treatments together over time suggesting either method is useful in determination of the presence ofF. columnare. Polymerase chain reaction was significantly (P < 0.001) better at detection ofF. columnare from skin/muscle than was the use of microbiological culture. These results suggest that PCR may be useful for rapid detection of F. columnare in the mucus.     

Sickeningly Sweet: L‐rhamnose stimulates Flavobacterium columnare biofilm formation and virulence

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L‐rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L‐rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L‐rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L‐rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.     

Vertebrate mucus stimulates biofilm development and upregulates iron acquisition genes in Flavobacterium columnare

Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5–60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.     

Assessment of Aquaflor(®) , copper sulphate and potassium permanganate for control of Aeromonas hydrophila and Flavobacterium columnare infection in sunshine bass, Morone chrysops female × Morone saxatilis male

Two experiments were conducted to test the effectiveness of different therapeutants against a mixed infection of Aeromonas hydrophila and Flavobacterium columnare in sunshine bass. Experiment 1 evaluated copper sulphate, florfenicol‐medicated feed and potassium permanganate (KMnO₄) against a natural mixed infection. Experiment 2 further evaluated copper sulphate as a treatment to control an experimental mixed infection. In experiment 1, naturally infected untreated fish had the lowest final survival per cent, at 71%, while florfenicol‐medicated feed at 15 mg kg^?1 body weight for 10 days or copper sulphate at 2.1 mg L^?1 (1% of the total alkalinity) for 24 h produced the highest final survivals, at 90% and 88%, respectively. The final survival of the naturally infected fish administered florfenicol‐medicated feed was significantly different (P < 0.1) from the untreated fish. The survival curves for the florfenicol and the copper sulphate at 2.1 mg L^?1 were significantly improved from the untreated fish. In experiment 2, fish were challenged by waterborne exposure to A. hydrophila and F. columnare and either not treated or treated with copper sulphate at 2.1 mg L^?1. At the end of experiment 2, the per cent survival of the challenged fish treated with copper sulphate (99%) was significantly higher (P < 0.05) than the non‐treated (61%). The results illustrate clear benefit of florfenicol and copper sulphate against a mixed infection of A. hydrophila and F. columnare.     

Epizootics in farm-raised channel catfish, Ictalurus punctatus (Rafinesque), caused by the enteric redmouth bacterium Yersinia ruckeri

In the winters of 1995 and 1996, unusual disease outbreaks occurred on two separate channel catfish farms in Arkansas, USA. Affected fish exhibited extraordinary haemorrhaged rings around the eyes and raised haemorrhaged areas overlying the frontal foramens. Other signs included abnormal swimming, lethargy, loss of equilibrium, and exophthalmia. Bacterial isolates from the moribund fish were identified as Yersinia ruckeri by biochemical tests, no lysis by the Hafnia-specific bacteriophage 1672, and Y. ruckeri-specific growth patterns on Shotts-Waltman media. Fingerling catfish injected intraperitoneally with the bacterial isolate at 7.8 × 10⁶ bacteria fish^?1 developed lesions characteristic of the epizootics at 13, 18, and 22 °C and a biochemically identical isolate was recovered. Fingerling rainbow trout injected with the channel catfish isolate at 1 × 10⁵ bacteria fish^?1 and held at 20 °C developed signs typical of enteric redmouth by 4 days post-inoculation and were moribund by 5 days post-inoculation. Some differences of clinical signs occurred between experimentally infected rainbow trout and channel catfish. Clinical and biochemical similarities between infections of Y. ruckeri and many warmwater pathogens in affected catfish may lead to incorrect diagnosis of ERM infections.