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.     

Potential of Chinese chive oil as a natural antimicrobial for controlling <Emphasis Type="Italic">Flavobacterium columnare</Emphasis> infection in Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

Chinese chive Allium tuberosum oil was studied for its diallyl sulfide content and its antimicrobial activity against Flavobacterium columnare in fish both in vitro and. The oil was found to have a very low concentration of diallyl monosulfide relative to the other diallyl sulfides (diallyl disulfide, diallyl trisulfide, and diallyl tetrasulfide) identified. In the in vitro study, the Chinese chive oil had a bacteriocidal effect on all tested strains of F. columnare, with varied minimal inhibitory concentrations. The median lethal dose (LD₅₀) of FC4 for Nile tilapia Oreochromis niloticus was determined to be 3.72 × 10³ CFU/fish. In the in vivo trial, no mortality was observed in fish fed fish diets supplemented with 800 mg/kg Chinese chive oil and 100 mg/kg of oxytetracycline hydrochloride 5 days prior to infection with F. columnare strain 4 at a LD₅₀. These results indicate that Chinese chive oil has the potential to replace antibiotics for controlling fish disease caused by F. columnare.     

Occurrence of Listonella anguillarum in seed production environments of Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The present study was undertaken to investigate the distribution of Listonella anguillarum in the rearing water, fish and diets (rotifers) of Japanese flounder (Paralichthys olivaceus). A total of 793 isolates were obtained from the seed production environment of Japanese flounder and 175 out of them were identified as L. anguillarum by biochemical characterization, polymerase chain reaction (PCR) detection for VAH1 haemolysin gene and phylogenetic analysis of 16S ribosomal deoxyribonucleic acids (rDNA) sequences. These results strongly suggested that L. anguillarum is rapidly and accurately identified by the combination of incubation on thiosulphate–citrate–bile salt–sucrose agar at 35°C overnight and PCR detection for the VAH1 haemolysin gene. All flounder specimens and all rotifer samples harboured L. anguillarum at high densities of 6.9 × 10³–6.3 × 10⁵ colony forming units (CFU) g^?1 and 1.5 × 10⁴–2.3 × 10⁶ CFU g^?1, respectively, while as low as 5.0 × 10⁰–2.0 × 10¹ CFU mL^?1 of L. aguillarum were detected in only two of 11 seawater samples, even though no vibriosis occurred in larval and juvenile flounder of tanks. This fact strongly suggests that L. anguillarum is an inhabitant in the seed production environments of Japanese flounder.     

Isolation and identification of Vibrio campbellii as a bacterial pathogen for luminous vibriosis of Litopenaeus vannamei

Outbreak of luminescent disease was reported from Litopenaeus vannamei shrimp farms in Zhangpu County, Southern China during May–July 2011. The clinical signs included fluorescent, less food consumption and high mortality. Bacteria were isolated from the infected shrimps. The pathogen, a luminescent bacterium named VH1 was identified as Vibrio campbellii based on MLSA analysis (16S rDNA, rpoD and toxR). The haemolysin (hly) gene specific in V. campbellii was detected in strain VH1. Pathogenicity test using immersion infection confirmed that strain VH1 was virulent to L. vannamei postlarvae and juveniles, and the LC₅₀ value was 1.55 × 10⁶ CFU mL^?1 and 1.7 × 10⁶ CFU mL^?1 respectively.     

Haemorrhagic septicaemia in the hybrid surubim (Pseudoplatystoma corruscans×Pseudoplatystoma fasciatum) caused by Aeromonas hydrophila

Intensive culture of the hybrid surubim (Pseudoplatystoma corruscans × Pseudoplatystoma fasciatum) in Brazil is responsible for the occurrence of diseases and consequent economic losses. However, the causative agents are not well known. The objective of this study was to isolate and to characterize the pathogenic agent responsible for mortalities in cultured surubim and to demonstrate its virulence. Ten fish from a fish farm located in the Mato Grosso do Sul State (Brazil) were collected and 14 haemolytic bacteria characterized as Aeromonas hydrophila were isolated from the kidneys (eight) and brain (six). As an experimental challenge, fish weighing 98.1±23.6 g were injected with 1 mL of saline solution and 2 × 10², 2 × 10⁴, 2 × 10⁶ and 2 × 10⁸ CFU A. hydrophila mL^?1. Fish infected with 2 × 10⁸ CFU showed increased external and internal symptoms and mortality of 50±12.5% after 96 h. Increased A. hydrophila concentration was responsible for a decrease in haematocrit percentage and erythrocyte number, lymphocytes and eosinophils, as well as an increase in monocytes, neutrophils, serum agglutination titre and serum antimicrobial activity. It was concluded that A. hydrophila was responsible for characteristic symptoms of bacterial haemorrhagic septicaemia as well as important haematological and immunological alterations, which led to surubim mortality.     

Development of a real‐time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 10¹ amplicon copies per assay (equivalent to 2 × 10^–9 ng/µl) using bacterial DNA and of 1.44 × 10¹ gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

Development of an Indirect ELISA to Detect Humoral Response to Flavobacterium columnare Infection of Channel Catfish,Ictalurus punctatus

ABSTRACT

The humoral antibody response of channel catfish, Ictalurus punctatus, to experimental Flavobacterium columnare infection was measured in control (non-infected) and infected (intraperitoneal- and intramuscular-injected) fish by an indirect enzyme-linked immunoassay (ELISA). The antibody response of the experimentally infected fish was significantly higher (P<0.0001) than that of the non-infected channel catfish by both indirect ELISA and agglutination. The indirect ELISA utilized goat anti-channel catfish IgM and a commercially available rabbit anti-goat IgG enzyme conjugate. The antibody response was directed against a cell-free sonicated antigen preparation derived from live whole F. columnare cells. Indirect ELISA and agglutination results correlated (r² = 0.60) for anti-F. columnare antibody response in experimentally and non-infected fish. We were also able to correlate ELISA and agglutination results of 60 fish naturally infected with F. columnare(r² = 0.76). Indirect ELISA will allow for rapid monitoring of humoral antibody, following natural exposure or vaccination against F. columnare, with a small sample of serum.     

Lack of association between Flavobacterium columnare genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.) × Oreochromis aureus (Steindachner)

Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex‐reversed hybrid tilapia, Oreochromis niloticus (L.) × O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established genomovars of F. columnare: I, II, II‐B, III and I/II. The mean per cent mortality of fish challenged with genomovar I, II and III isolates ranged from 0 to 100, 3.3–78 and 3.3–75%, respectively. The mean per cent mortality of fish challenged with genomovar II‐B ranged from 35 to 96.7%, and the only genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.     

In vitro comparisons of the inhibitory activity of florfenicol,copper sulphate and potassium permanganate towards Aeromonas hydrophila and Flavobacterium columnare

Aeromonas hydrophila and Flavobacterium columnare, the aetiological agents of motile aeromonas septicaemia (MAS) and columnaris disease respectively, have been recently causing crippling mortalities to the sunshine bass, Morone chrysops female ×Morone saxatilis male (Percichthyidae), industry in the United States. Isolates of A. hydrophila and F. columnare obtained from fish that died during farm outbreaks were subjected to in vitro evaluation of florfenicol (FFC), copper sulphate (CuSO₄) and potassium permanganate (KMnO₄). Florfenicol inhibited the growth of A. hydrophila and F. columnare more than CuSO₄ and KMnO₄. The minimum inhibition concentration (MIC) of FFC was 0.04 ± 0 and 0.2 ± 0.1 mg L^?1 for A. hydrophila and F. columnare respectively, while the 50% inhibition concentration (IC50) for A. hydrophila and F. columnare was 0.23 ± 0.01 and 0.4 ± 0.2 mg L^?1 respectively. Copper sulphate was more effective against A. hydrophila than KMnO₄; CuSO4 had a MIC of 83.2 ± 0 mg L^?1 compared to 158.0 ± 0 mg L^?1 for KMnO₄. Copper sulphate was also more effective against F. columnare than KMnO₄. The IC50 values of CuSO₄ and KMnO₄ towards F. columnare were 4.8 ± 0.3 and 8.7 ± 1.6 mg L^?1 respectively, and the minimum bactericidal concentration values of CuSO₄ and KMnO₄ towards F. columnare were 25.0 ± 0 and > 158.0 mg L^?1 respectively. In addition, F. columnare was more sensitive to CuSO₄ and KMnO₄ than A. hydrophila.     

Ultra highly sensitive method for detecting Flavobacterium psychrophilum using high‐gradient immunomagnetic separation with a polymerase chain reaction

We attempted to develop an ultrahigh sensitive method for detecting Flavobacterium psychrophilum using high‐gradient immunomagnetic separation (HGIMS) with a polymerase chain reaction (PCR). HGIMS is a magnetic separation method in which the magnetic force is strengthened by introducing a magnetic gradient between the magnetic filter and nearby column. Because immunomagnetic beads specifically react with target cells, target cells are collected efficiently. Accumulated beads are released from the filter by removing the external magnetic force. After concentrating the samples using the HGIMS system, DNA was extracted from the samples, and PCR was applied to detect F. psychrophilum. Our primers did not react with reference bacteria and reacted specifically with F. psychrophilum. The detection sensitivity using the HGIMS system was higher than that of the method without the HGIMS system, and the total assay time, including sample preparation, was <3.5 h. PCR products of the expected size were obtained from samples of concentrated 4 × 10^?1 to 4 × 10³ cfu mL^?1F. psychrophilum more than 80% of the time using the HGIMS system. Furthermore, our proposed method could be useful for the specific detection of F. psychrophilum from actual samples. Our proposed method is suitable for the highly sensitive detection of F. psychrophilum.     

Diversity of siderophore‐producing bacteria isolated from the intestinal tracts of fish along the Japanese coast

This study examined the diversity of siderophore‐producing bacteria in the intestinal tracts of coastal fish in Japan and screened candidate bacterial strains for probiotic use in aquaculture. Of the 2637 bacteria isolated from the 27 fish specimens (13 species) and six environmental samples collected in this study, 266 isolates exhibited the ability to produce siderophores. Siderophore producers were detected in the intestines of 18 of the fish specimens caught (68%) at densities of 2.3 × 10⁴–2.3 × 10⁸ CFU g^?1, in all three seawater samples at 2.0 × 10²–1.3 × 10³ CFU mL^?1 and in all three sand samples at 2.6 × 10¹–2.8 × 10⁴ CFU g^?1. These findings suggest that siderophore‐producing bacteria are widely distributed in the intestinal tracts of coastal fish and their environments. Analysis of 16S rRNA gene sequences revealed that the siderophore producers belonged to 38 species, of which Vibrio splendidus, Vibrio ichthyoenteri and Vibrio crassostreae accounted for 32.7%, 19.5% and 11.3% of the 266 isolates, respectively, suggesting that these bacteria are indigenous to the intestinal tract of coastal fish. Six bacterial species, Enterovibri norvegicus, Photobacterium leiognathi, Photobacterium phosphoreum, Photobacterium rosenbergii, V. crassostrea and Vibrio scophthalmi were identified as possible candidates for use as probionts in fish aquaculture.     

Identification and Detection of the Pathogenic Bacteria Responsible for Swollen Abdomen Disease in Cultured Turbot,Scophthalmus maximus,and Flounder,Paralichthys olivaceus

Swollen abdomen disease (SAD) seriously threatens the aquaculture of turbots and flounders. Two dominant bacterial strains, FS1 and FS2, were isolated from the livers and kidneys of fish with diagnosed SAD. Applications of biochemical analyses, sequence analyses of 16S ribosomal RNA gene and heat shock protein 60 gene revealed two distinct pathogenic bacterial species, Edwardsiella tarda and Vibrio alginolyticus. These two hypothesized SAD‐causing pathogens were validated by challenge trials on flounder, Paralichthys olivaceus. Challenges with E. tarda and V. alginolyticus demonstrated lethal dose 50 (LD₅₀) values at 1.51 × 10⁵ colony‐forming units (CFU) and 1.05 × 10⁵ CFU, respectively. To develop a rapid SAD diagnosis method in flounders and turbots, a multiplex polymerase chain reaction (PCR) assay method was developed to simultaneously detect E. tarda and V. alginolyticus. Our multiplex PCR assay successfully detected as low as 10⁵ CFU/mL E. tarda and V. alginolyticus in flounders and turbots. No other common fish pathogens were detected with the multiplex PCR, suggesting a high specificity of this assay. The multiplex PCR assay developed in this study showed a great reliability in detecting SAD‐causing bacterial pathogens. Further optimization of this assay may contribute to the development of a novel SAD diagnosis tool in aquaculture.     

Application of real-time PCR for early diagnosis of diseases caused by Aeromonas salmonicida,Vibrio anguillarum,and Tenacibaculum maritimum in turbot: A field study

In the present study a multiplex real-time PCR method was developed for early detection of diseased fish infected by Aeromonas salmonicida, Vibrio anguillarum, and/or Tenacibaculum maritimum. The method consisted of the detection of three species-specific genes after DNA extraction with a commercial kit. Three types of samples were tested, and the results were compared with those of traditional diagnosis. The method obtained a limit of detection of 10⁴ cfu/mL (2 x 10² cfu/tube). Additionally, 27 samples from fish showing signs of disease were correctly diagnosed by the developed methodology, demonstrating its suitability for implementation in aquaculture.     

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.     

Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida

A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10⁴ ± 1 × 10⁴ CFU g^?1 (without enrichment) and 40 ± 10 CFU g^?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.     

Efficacy of an experimentally inactivated Streptococcus agalactiae vaccine in Nile tilapia (Oreochromis niloticus) reared in Brazil

Tilapia aquaculture is one of the fastest‐growing segments of fish production in Brazil. Nile tilapia (Oreochromis niloticus) is largely cultivated in the state of Parana, where Streptococcus agalactiae is the cause of severe disease outbreaks. The objective of this paper was to evaluate an inactivated S. agalactiae vaccine in tilapia for the control of streptococcal disease outbreaks. Tilapia, weighing approximately 20 g each, were intraperitoneally (i.p.) inoculated with 0.1 mL of the vaccine at a dose of 2.0 × 10⁸ colony‐forming unit (CFU) mL^?1. One group of tilapia (treatment 1) received one vaccine dose, and the other group of tilapia (treatment 2) received two doses, with an interval of 21 days. The control group was i.p. inoculated with 0.1 mL tryptic soy broth fish^?1. Immunized and control tilapia were i.p. challenged with 0.1 mL of 3.0 × 10⁷ CFU mL^?1 at 30 days post vaccination. The fish were monitored daily for disease signs and for mortality for 16 days post challenge. A statistically significant difference (P=0.0045) was found between the mortality of treatments 1 and 2. The value of relative per cent of survival of 83.6% and 96.4%, respectively, indicate that this vaccine was efficient in Nile tilapia.     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.