Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.     

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 x 10(2) and 2.5 x 10(5) cells g(-1) of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the time-consuming traditional bacteriological culture techniques.     

Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare

Enteric septicemia of catfish (ESC) and columnaris disease are 2 bacterial diseases significantly affecting the aquaculture industry, and thus rapid diagnosis of disease is imperative for making judicious management decisions. A rapid indirect fluorescent antibody (IFA) test with antibody conjugated fluorochromes having 2 different spectral properties (Alexa Fluor 488-emitting green fluorescence, and Alexa Fluor 594-emitting red fluorescence) was compared with bacteriological culture (accepted standard) for simultaneous detection of Edwardsiella ictaluri (EI) and Flavobacterium columnare (FC) in 3 groups of experimentally infected channel catfish (Ictalurus punctatus Rafinesque), and a fourth group that acquired an aquarium-infection with F. columnare. A total of 303 samples (derived from kidney, brain and nares) from 101 fish were concurrently examined by both tests. Fish in the 3 experimentally infected groups (I to III) were culture positive for the bacteria with which they were infected, and fish in Group IV, (the spontaneously infected fish) revealed F. columnare only. The IFA test compared favorably in sensitivity (EI= 80.7 %; FC = 87.2%) and specificity (EI = 83.9%; FC = 88.9%) with the standard bacteriological culture. The positive predictive value (EI = 96.2% Group I, 90.8% Group II, 93.7% Groups I and II combined; FC = 95.2% Group II, 95.3% Groups II, III and IV combined) was high, while the negative predictive value (EI = 66.7% Group I, 31.3% Group II, 59.5% Groups I and II combined; FC = 73.7% Group II, 72.7% Groups II, III and IV combined) was relatively low. The IFA test will serve as an efficient tool for rapid simultaneous detection of E. ictaluri and F. columnare in outbreaks of disease.     

Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare

A 16-S rRNA gene from the chromosomal DNA of the fish-pathogenic bacterium Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set. The primer set amplified a specific 1193-bp DNA fragment from F. columnare strains but not from related bacteria, F. psychrophilum, F. aquatile, F. branchiophilum, or other bacterial pathogens of fish, Flexibacter maritimus, Cytophaga johnsonae, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, and Streptococcus iniae or from the non-fish pathogen Escherichia coli. The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if the more sensitive nested PCR is used). DNA was extracted by a boiled-extraction method or by commercial column purification. The PCR product was detected at DNA concentrations below 0.1 ng and from as few as 100 bacterial cells. Nested PCR using universal eubacterial primers increased the sensitivity five-fold, allowing detection of F. columnare strains at DNA concentrations below 0.05 ng and from as few as 10 bacterial cells in apparently healthy, asymptomatic fish. The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al. [Fish Pathol. 29 (1994) 271.] and that of Bader and Shotts [J. Aquat. Anim. Health 10 (1998) 311.]. The new primer set is as good or better than the previously published primer sets for detecting F. columnare in all samples and under all conditions tested.     

Flavobacterium columnare chemotaxis to channel catfish mucus

Flavobacterium columnare is a Gram-negative pathogen of many species of wild and cultured fish. Isolates from diseased channel catfish belong to either genomovar I or II. Genomovar II isolates were found to be more virulent than genomovar I isolates. The objective of the present study was to determine whether differences exist in the chemotactic response of these genomovars to mucus obtained from the skin, gills and intestines of healthy channel catfish using the capillary chemotaxis assay. Mucus from the skin and gill induced a greater chemotactic response by F. columnare than mucus from the intestine. Sixty percent of mucus from the skin of individual catfish yielded a positive chemotactic response from F. columnare. Finally, skin mucus induced a greater chemotactic response in genomovar II F. columnare than in genomovar I F. columnare isolates. The data indicate that mucus from channel catfish results in a chemotactic response by F. columnare. This positive chemotactic response may be an important first step for F. columnare colonization of channel catfish skin or gills. Although the role that chemotaxis plays in the virulence of F. columnare is not fully defined, the chemotactic response of genomovar ll isolates suggests that chemotaxis is associated with virulence.     

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.     

斑鳠烂鳃病病原菌的研究

从池塘患烂鳃病的斑鳠(Mystus guttatus)亲鱼病灶中分离出一株毒力较强的致病菌-Mg2,经形态学观察、生理生化特性和16S rRNA基因序列分析鉴定,其主要特征为：菌体细长,无鞭毛和荚膜,革兰氏阴性,大小0.5×(6.5－11)μm,菌落黄色,边缘不整齐呈假树根状。氧化酶、过氧化氢酶阳性,分解酪素和明胶,硝酸盐还原阳性;不分解纤维素、几丁质、酪氨酸、七叶灵和淀粉,吲哚和葡萄糖产气阴性。对恩诺沙星、诺氟沙星、萘啶酸、红霉素、洁霉素敏感,菌株的16S rRNA基因序列分析结果表明：Mg2菌株与柱状黄杆菌聚类,基因序列的同源性在97.5%以上,综合生理生化、分子生物学两方面的分类鉴定结果,Mg2菌株应归类鉴定为柱状黄杆菌（Flavobacterium Columnare）。     

Catfish hybrid Ictalurus punctatus × I. furcatus exhibits higher resistance to columnaris disease than the parental species

We present experimental data on susceptibility to columnaris disease, caused by the bacterium Flavobacterium columnare, in hybrid catfish (female channel catfish Ictalurus punctatus × male blue catfish I. furcatus) (C×B). Under our experimental conditions, C×B hybrids were significantly more resistant to columnaris disease caused by the highly virulent strain of F. columnare BGFS-27 (genomovar II) than channel catfish and blue catfish. Channel and blue catfish cumulative mortalities after immersion challenge were 74 and 87%, respectively, whereas mortality in the C×B hybrid was 31%. Susceptibility to the strain ARS-1 (genomovar I) was lower among all catfishes, although channel catfish was the least resistant species at 32% cumulative mortality. By contrast, C×B hybrid and blue catfishes were strongly resistant to the ARS-1 strain, with <10% mortality. Our data suggest enhanced disease resistance of the C×B hybrid to columnaris disease.     

Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish

AIMS: To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare. METHODS AND RESULTS: Genetic variability among Fl. columnare isolates was characterized using restriction fragment length polymorphism analysis of the 16S rDNA gene, intergenic spacer region (ISR) sequencing, and amplified fragment length polymorphism (AFLP) fingerprinting. Thirty Fl. columnare cultures isolated from different fish species and geographical origins as well as reference strains were included in the study. Fifteen isolates belonged to genomovar I while eleven were ascribed to genomovar II. Analysis of the ISR sequence confirmed the genetic differences between both genomovars but revealed a higher diversity among genomovar I isolates. The maximum resolution was provided by AFLP fingerprinting, as up to 22 AFLP profiles could be defined within the species. CONCLUSIONS: We confirmed the division of Fl. columnare isolates from cultured fish into different genogroups. We showed that both genomovars I and II are present in channel catfish from the US. We described a unique genetic group represented by four Fl. columnare isolates from tilapia in Brazil which appears to be related to both genomovars. We were able to further subdivide the species by analysing the ISR. Finally, the use of AFLP allowed us to fingerprint the species at clone level without losing the higher genetic hierarchy of genomovar division. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the fish pathogen Fl. columnare.     

Passive immunization of channel catfish Ictalurus punctatus with anti-Flavobacterium columnare sera

Passive immunization of channel catfish Ictalurus punctatus (Rafinesque) was conducted to determine if anti-Flavobacterium columnare serum was protective when injected intraperitoneally (i.p.) into channel catfish. The anti-F. columnare serum was produced by actively immunizing (i.p. injection) channel catfish with sonicated whole cells or purified lipopolysaccharide (LPS) of F. columnare in Freund's adjuvant. Serum anti-F. columnare activity was verified by Western blotting and ELISA of serum. Normal serum and sterile culture broth were used as controls. Complement was inactivated in all sera by heating. After 48 h, passively immunized fish were challenged with virulent F. columnare by i.p. injection. A group of unchallenged fish served as controls. The immune response of catfish to the antigenic fractions was different when examined by Western blotting. Antibody produced with whole-cell antigen responded to a broad range of molecular weight components, while LPS antigens were restricted to a pair of bands near 20 kDa. Control fish injected with culture medium experienced 100% mortality 14 d post-challenge. Relative percent survival was 77 and 73 for catfish passively immunized with anti-LPS and anti-whole-cell serum, respectively. Results suggest that antibodies in the serum are involved in the protective immune response against columnaris disease in channel catfish.     

Simultaneous detection of Aeromonas salmonicida,Flavobacterium psychrophilum,and Yersinia ruckeri,three major fish pathogens,by multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

Freezing induces biased results in the molecular detection of Flavobacterium columnare

Specific PCR detection and electron microscopy of Flavobacterium columnare revealed the risk of false-negative results in molecular detection of this fish pathogen. Freezing and thawing destroyed the cells so that DNA was for the most part undetectable by PCR. The detection of bacteria was also weakened after prolonged enrichment cultivation of samples from infected fish.     

Use of suppressive subtractive hybridization to identify Flavobacterium columnare DNA sequences not shared with Flavobacterium johnsoniae

Aims:  To identify specific sequences in the fish pathogen Flavobacterium columnare not shared by Flavobacterium johnsoniae .
Methods and Results:  Suppressive subtractive hybridization (SSH) was used to selectively amplify and clone F. columnare -specific sequences. A highly virulent strain of F. columnare was used as tester and the type strain of F. johnsoniae was used as driver. After library construction, 192 clones were selected and sequenced. From those, 110 clones contained unique F. columnare -specific sequences that were verified using dot blot hybridization. Sequence sizes ranged from 55 to 872 bp with 45 363 bp sequenced in total.
Conclusions:  Specific F. columnare sequences representing all but one (motility related) functional categories were annotated. Several putative virulence factors were identified in F. columnare such as a collagenase, a chondroitinase, proteases, as well as drug resistance and iron transport-related genes.
Significance and Impact of the Study:  Suppressive subtractive hybridization is a cost-effective method for identifying genetic differences between Flavobacterium spp. The number of sequences available from F. columnare has been doubled.     

First identification of Flavobacterium columnare infection in farmed freshwater striped catfish Pangasianodon hypophthalmus

The bacterium Flavobacterium columnare was recovered and identified as the aetiological agent causing freshwater columnaris infection in farmed striped catfish Pangasianodon hypophthalmus (Sauvage) fingerlings that had suffered high mortality rates within commercial hatchery ponds in Vietnam. The gross clinical signs were typical of columnaris-infected fish. Histological examination found numerous Gram-negative, filamentous bacteria present on the skin, muscle and gill tissues of affected fish. The yellow-pigmented bacteria were isolated and identified as F. columnare using primary, biochemical and PCR methods. An experimental immersion-challenge study with 2 strains was also performed. It fulfilled Koch's postulates and showed a median lethal concentration (LC50) of 4.27 × 105 and 1.66 × 106 cfu ml-1 for the F. columnare strains FC-HN and FC-CT, respectively. To the best of our knowledge this is the first report of freshwater columnaris infection in P. hypophthalmus.     

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.     

Effect of Pseudomonas sp. MT5 baths on Flavobacterium columnare infection of rainbow trout and on microbial diversity on fish skin and gills

Use of Pseudomonas sp. strain MT5 to prevent and treat Flavobacterium columnare infection was studied in 2 experiments with fingerling rainbow trout Oncorhynchus mykiss. In the first experiment, length heterogeneity analysis of PCR-amplified DNA fragments (LH-PCR) was used to assess the effect of antagonistic baths on the microbial diversity of healthy and experimentally infected fish. In the 148 samples studied, no difference was found between bathed and unbathed fish, and 3 fragment lengths were detected most frequently: 500 (in 75.7% of the samples), 523 (62.2%) and 517 bp (40.5%). The species contributing to these fragment sizes were Pseudomonas sp., Rhodococcus sp. and F. columnare, respectively. A specific PCR for detection of Pseudomonas sp. MT5 was designed, but none of the tissue samples were found to be positive, most likely indicating poor adhesion of the strain during bathing. LH-PCR was found to be a more powerful tool for detecting F. columnare in fish tissue than traditional culture methods (chi2 = 3.9, df = 1, p < 0.05). Antagonistic baths had no effect on the outbreak of infection or on fish mortality. F. columnare was also detected in healthy fish prior to and after experimental infection, indicating that these fish were carriers of the disease. In the second experiment, intensive Pseudomonas sp. MT5 antagonistic baths were given daily to rainbow trout suffering from a natural columnaris infection. Again, the antagonistic bacteria had no effect on fish mortality, which reached 95 % in both control and antagonist-treated groups in 7 d.     

Detection of the fish pathogen Flavobacterium psychrophilum in water from fish farms

Rainbow trout fry syndrome and cold-water disease are serious diseases in farmed salmonid fish. In the present study, three methods were compared, for the detection of the causative pathogen, Flavobacterium psychrophilum in water. The methods included traditional agar plate cultivation on tryptone yeast extract salts (TYES) agar, immunofluorescence antibody technique (IFAT) and nested PCR. The three methods were subsequently used for the detection of F. psychrophilum from fish farm environments. The nested PCR was the most sensitive method used for a detection of F. psychrophilum. As low as 3 CFU estimated by agar plate cultivation or 41 cells estimated by IFAT of F. psychrophilum per ml of non-sterile well water were needed for a detection using the nested PCR method. The obtained detection limits for the agar plate cultivation and the IFAT was 32 CFU/ml and 410 cells/ml, respectively. Using IFAT and nested PCR F. psychrophilum was detected most frequently in water samples from fish farms, but the pathogen was isolated from only a few samples using agar plate cultivation. In the present study IFAT and nested PCR proved to be rapid, specific and sensitive methods compared to traditional agar plate cultivation for the detection of F. psychrophilum from environmental samples. It is suggested that IFAT and nested PCR provide effective tools for the examination of F. psychrophilum in the environment.     

Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

A link between ectoparasite infection and susceptibility to bacterial disease in rainbow trout

Rainbow trout, Oncorhynchus mykiss, were infected concomitantly with Argulus coregoni and Flavobacterium columnare and their survival was compared with that of fish infected with either the parasite or the bacterium alone. The mortality of fish challenged with A. coregoni was negligible while infection with F. columnare alone led to significantly lower survival. However, compared with single infections, the mortality was significantly higher and the onset of disease condition was earlier among fish, which were concomitantly infected by A. coregoni and F. columnare. This data presents, for the first time, experimental support for the hypothesis that an ectoparasite infection increases susceptibility of fish to a bacterial pathogen.     

Treatment of columnaris disease of rainbow trout: low pH and salt as possible tools?

The impact of salt and low pH on columnaris disease of fish was studied. Survival of Flavobacterium columnare after exposure to either 4% NaCl (pH 7.2) or pH 5.0, pH 4.86 or pH 4.6 for 15 min or 1 h was studied in vitro. All conditions significantly reduced the numbers of viable bacterial cells. The effects of salt (4 and 2%) and acidic baths (pH 4.6) were studied in 2 experiments in vivo with rainbow trout Oncorhynchus mykiss infected with F. columnare. Both salt and acidic baths failed to prevent fish mortality; the overall mortality reached 100% in all groups. However, according to survival analysis, the mortality rate was lower in fish treated with 4% salt baths compared to a control group. The buffering capacity of fish skin mucus against low water pH was also studied. Fish skin mucus was an efficient buffer against decreased water pH and the pH of the skin could be remarkably higher than that of the mucus. This may explain the failure of bath treatments to prevent mortality providing that attached F. columnare are located below the mucus surface. We suggest, however, that salt and acidic bath treatments can be used to disinfect water containing F. columnare cells shed by infected fish and thus prevent the transmission of the disease.