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.     

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus).

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.     

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus)

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.     

Molecular responses of ceruloplasmin to Edwardsiella ictaluri infection and iron overload in channel catfish (Ictalurus punctatus)

Ceruloplasmin is a serum ferroxidase that carries more than 90% of the copper in plasma and has documented roles in iron homeostasis as well as antioxidative functions. In our previous studies, it has been shown that the ceruloplasmin gene is strongly up-regulated in catfish during challenge with Edwardsiella ictaluri. However, little is known about the function of this gene in teleost fish. The objective of this study, therefore, was to characterize the ceruloplasmin gene from channel catfish, determine its genomic organization, profile its patterns of tissue expression, and establish its potential for physiological antioxidant responses in catfish after bacterial infection with E. ictaluri and iron treatment. The genomic organization suggested that the catfish ceruloplasmin gene had 20 exons and 19 introns, encoding 1074 amino acids. Exon sizes of the catfish ceruloplasmin gene were close to or identical with mammalian and zebrafish homologs. Further phylogenetic analyses suggested that the gene was highly conserved through evolution. The catfish ceruloplasmin gene was mapped to both the catfish physical map and linkage map. The catfish ceruloplasmin gene was mainly expressed in liver with limited expression in other tissues, and it was significantly up-regulated in the liver after bacterial infection alone or after co-injection with bacteria and iron-dextran, while expression was not significantly induced with iron-dextran treatment alone.     

Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri

Aims:  To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons.
Methods and results:  The Edw. ictaluri rrn operons were identified from a 5–7 kbp insert lambda library and from Edw. ictaluri fosmid clones. We present the complete sequences and analysis of all eight Edw. ictaluri rrn operons and unique regions located upstream and downstream. Two rrn operons were located in tandem with 169 bp separating them, which is apparently a conserved feature between Edw. ictaluri and Edwardsiella tarda. I- Ceu I enzyme digestion of Edw. ictaluri genomic DNA and analysis by pulsed field gel electrophoresis indicated that rrn operon number and chromosomal locations are conserved within the species Edw. ictaluri .
Conclusions:  The rrn operons of Edw. ictaluri have similar structure and flanking regions compared with other members of the family Enterobacteriaceae ; however, the presence of eight copies of the rrn operon makes Edw. ictaluri unique within the family .
Significance and impact of the study:  This research clarifies previous phylogenetic analyses of Edw. ictaluri and provides support for the Edw. ictaluri genome sequencing project. In addition, we identified a unique feature of two rrn operons that shows potential for the development of a diagnostic PCR method.     

Survivability and immune responses after challenge with Edwardsiella ictaluri in susceptible and resistant families of channel catfish, Ictalurus punctatus

Diseases in catfish farming are prevalent and costly, particularly the bacterial disease Enteric Septicemia of Catfish. Considerable research has focused on different aspects of this disease, including the biology of the causative agent, Edwardsiella ictaluri. However, no satisfactory treatment or preventive has resulted from these efforts. One solution is to increase the natural disease resistance of the fish through genetic selection. Recent research has demonstrated that genetic factors influence resistance to infection in mammals as well as fish. Selective breeding for disease resistance in channel catfish is ongoing, however differences in defence mechanisms among E. ictaluri challenged strains and families are only now being investigated. Antigen-specific as well as non-specific immune responses of full-sib families of channel catfish to laboratory challenge with E. ictaluri have been investigated. Both resistant and sensitive families produce a humoral response as specific antibody, but there were no differences found in the level of specific antibody produced. The sensitive family produced a slightly higher percentage of B lymphocytes in mononuclear cell preparations from peripheral blood, while the resistant family had a higher percentage of T lymphocytes in those preparations. The most significant observation was that the resistant family produced more macrophage aggregations in the spleen and posterior kidney throughout the infection than the sensitive family. Neither family produced stress-associated amounts of cortisol.     

Molecular characterization of three L-type lectin genes from channel catfish, Ictalurus punctatus and their responses to Edwardsiella ictaluri challenge

L-type lectins have a leguminous lectin domain and can bind to high-mannose type oligosaccharides. In the secretory pathway, L-type lectins play crucial roles in selective protein trafficking, sorting and targeting. Three L-type lectins were cloned in the channel catfish, Ictalurus punctatus, the 53 kDa endoplasmic reticulum ER-Golgi intermediate compartment protein (ERGIC-53), the vesicular integral protein of 36 kDa (VIP36) and VIP36-like. Phylogenetic analysis indicated that the catfish genes are orthologous to their counterparts in other species. Southern blot analysis demonstrated that all three L-type lectin genes are likely single-copy genes in the catfish genome. Analysis of expression in healthy tissues using quantitative real time RT-PCR indicated that all three genes are expressed widely in all tested tissues, but with strong tissue preference of expression: ERGIC-53 was found to be abundantly expressed in the liver, VIP36 was found to be abundantly expressed in the head-kidney, whereas VIP36-like was found to be abundantly expressed in the brain. Upon infection with Edwardsiella ictaluri, expressions of the three genes all had significant up-regulation in the head-kidney, but had distinct expression patterns: ERGIC-53 was gradually induced with the highest expression 7 days after challenge in the head-kidney, but was down-regulated in the liver, spleen, and brain. VIP36 was highly induced in the head-kidney, and 3 days after challenge in the brain, but was not up-regulated in any other tissues or timepoints after challenge. Expression levels of the catfish VIP36-like gene appeared to also respond to infection, albeit with differing patterns among the tested tissues. Taken together, our results indicate that all three L-type lectin genes may be involved in the immune responses of catfish after infection with E. ictaluri.     

Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection

An Edwardsiella ictaluri expression library was screened for clones expressing antigenic E. ictaluri proteins using anti-E. ictaluri serum, which resulted in the isolation of 32 clones. The clones were partially characterized and 4 were selected for complete analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimensional PAGE, Western blotting, and DNA sequencing were used to analyze expressed antigenic proteins and encoded genes. Sequence analysis identified 4 putative open reading frames (ORFs) in the insert of Clone 4d6, which corresponded to antigenic acidic proteins of 55, 20 and 18 kDa expressed by both the clone and E. ictaluri cells. The predicted gene products of these ORFs were similar to several products of the imp locus of Rhizobium leguminosarum bv. trifolii. The imp locus of R. leguminosarum contains 14 genes that encode proteins involved in a putative temperature-dependent protein secretion system. In addition there was significant amino acid identity for a variety of hypothetical proteins from R. solanacearum, Ps. aeruginosa, A. tumefaciens, Y. pestis, and Salmonella typhimurium. Overlapping inserts of Clones 1.4, 5d2, and 5d3 encoded ORFs similar to Escherichia coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda. These genes encode D-3-phosphoglycerate dehydrogenase (serA), ribose 5-phosphate isomerase (rpiA), a specific inhibitor of chromosomal initiation of replication (iciA), a hypothetical protein (yggE), a protein involved in responses to osmotic stress (yggB), fructose 1,6-bisphosphate aldolase (fda), and phosphoglycerate kinase (pgk). Cloned antigenic E. ictaluri proteins of 33, 27, 35 and 45 kDa appeared to be products of the ORFs similar to yggE, rpiA, iciA, and fda respectively. All the cloned antigenic proteins were recognized by antiserum from catfish that had recovered from enteric septicemia of catfish (ESC), indicating that these antigens are expressed during the infectious process. The cloned antigenic proteins were subsequently evaluated as subunit vaccines for protection against wild-type E. ictaluri. All vaccine treatments were protective against E. ictaluri in catfish, but results were inconclusive due to high levels of cross-reactive protection afforded by the E. coli host strain of the cloning vector.     

In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri

Macrophages from catfish vaccinated with an Edwardsiella ictaluri vaccine and macrophages from non-vaccinated catfish were used in in vitro and in vivo studies with red-fluorescent E. ictaluri to assess phagocytic ability, reactive oxygen and nitric oxide production and bactericidal activity. In the in vitro experiment, macrophages were harvested from vaccinated and non-vaccinated fish and then exposed to red-fluorescent E. ictaluri. Results of this study showed that E. ictaluri can survive and replicate in macrophages from non-vaccinated catfish (relative percent killing, RPK, from 0.011 to 0.620 and from ?0.904 to 0.042 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) even in the presence of reactive oxygen and nitrogen products. Macrophages from vaccinated fish were significantly (p < 0.05) more efficient in killing E. ictaluri (RPK from 0.656 to 0.978 and from 0.011 to 0.620 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) and produced significantly (p < 0.05) higher amounts of ROS (10-fold increase) and nitrogen oxide (about 10-fold increase) than macrophages from non-vaccinated fish. In the in vivo experiment, vaccinated and non-vaccinated catfish were injected with red-fluorescent E. ictaluri to allow the interaction between macrophages and other components of the immune system. After 6 h, macrophages were harvested from the fish and seeded in glass chamber slides and bactericidal activity was measured in vitro. Results showed in vivo interaction of other components of the immune system enhanced bactericidal activity of macrophages from vaccinated fish. In another set of experiments, catfish were intraperitoneally injected with fluorescent bacteria opsonized with immune serum or non-opsonized and necropsied in the first 48 h after bacterial challenge to observe localization of E. ictaluri between vaccinated and non-vaccinated catfish. Vaccinated fish were able to control the dispersion of E. ictaluri in the body and red-fluorescent bacteria were observed only in the spleen, anterior and trunk kidney. In non-vaccinated fish E. ictaluri was able to replicate and invade all organs with the exception of the brain. We further determined that macrophages seeded with E. ictaluri could cause infection in non-vaccinated fish upon reinoculation with in vitro infected-macrophages. Overall, the results indicated that macrophages from vaccinated fish are activated and responsible for rapid clearance of infection upon re-exposure to virulent E. ictaluri.     

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly three decades ago ^1,2, so far, to the best of these authors'' knowledge, no method has been developed to allow for the in situ visualization of the bacteria in histological sections.While bacterial localization has been determined in vivo in previous studies using plate counts ³, radiometric labeled ⁴, or bioluminescent bacteria ⁵, most of these studies have only been performed at the gross organ level, with one exception ⁶. This limitation is of particular concern because E. ictaluri has a complex infection cycle ^1,7, and it has a variety of virulence factors ^8,9. The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi ¹⁰, which is in the same taxonomic family.Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.¹¹.Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium.This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.Download video file.^{(33M, mov)}     

Sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65 degrees C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.     

Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea)

In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.     

Transmission and detection of Flavobacterium columnare in channel catfish Ictalurus punctatus

A specific and rapid PCR detection method for Flavobacterium columnare based on the 16S-23S rDNA intergenic spacer region (ISR) of the ribosomal RNA operon has been developed. The ISR of 30 F. columnare strains and other Flavobacterium species was amplified using universal primers and sequenced. Once F. columnare specific sequences within the ISR were recognized, specific PCR primers were designed against them (FCISRFL and FCISRR1). The primers were sensitive and able to detect as low as 7 colony forming units from pure culture by PCR. The new PCR detection method was applied to experimentally infected channel catfish. Two different experiments in which channel catfish fingerlings were infected by intramuscular injection or by immersion bath showed the advantage of the PCR method over standard culture techniques. F. columnare was detected by PCR in both tank water and catfish tissue samples with a higher frequency and in less time than standard microbiological methods. Furthermore, PCR detection confirmed that F. columnare can be transmitted horizontally indirectly through the water column without fish-to-fish contact. The newly developed PCR detection method for F. columnare was more sensitive and rapid than standard culture on bacteriological media for detection of F. columnare in channel catfish tissues and in tank water.     

Environmental stress and bacterial infection in channel catfish, Ictalurus punctatus Rafinesque

Channel catfish, Ictalurus punctatus , were injected intraperitoneally with a sublethal dose of Aerornonas hydrophila and then stressed for 144 h by being maintained either in a dissolved oxygen concentration of 1·5 mg/1, 1·2 mg/1 total ammonia, and/or 6·5 mg/1 free CO₂ with a continuous inflow of water. A significant difference in percentage of mortality was noted between treatments ( P < 0·05). The trunk kidneys of surviving stressed fish had significantly higher total bacterial counts than non-stressed controls. A. hydrophila was isolated from 67% of the stressed fish and 9% of the control fish. Edwardsiella tarda , apparently endemic in the population, was isolated from 43% of the stressed fish and 7% of the control fish. Histopathological lesions were in the gills, liver, spleen, trunk kidney, and head kidney of stressed fish, but not control fish.     

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 degrees C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10(7) cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.     

Development and application of a loop-mediated isothermal amplification method on rapid detection of Pseudomonas aeruginosa strains

We developed and evaluated the specificity and sensitivity of a simple loop-mediated isothermal amplification (LAMP) method for rapid detection of P. aeruginosa strains. The optimal reaction condition was found to be 65°C for 45 min, with the detection limit as 100 fg DNA/tube and 10 CFU/reaction. Application of LAMP assays were performed 426 clinical samples (including 252 P. aeruginosa and 174 non- P. aeruginosa isolates) using a rapid procedure and easy result confirmation. Sensitivity of LAMP and PCR assays was found to be 97.6% (246/252) and 90.5% (228/252), respectively; with a 100% specificity for both assays.     

Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method

Here we report a loop-mediated isothermal amplification (LAMP) method for detecting Shigella and enteroinvasive Escherichia coli. The target for this LAMP method is the ipaH gene which is carried by both of the pathogens. The LAMP method efficiently detected the gene within 2 h at a minimal amount of bacteria (8 CFU) per reaction.