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-mediatedisothermal 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 detectionwere optimized for the detection of E. tarda in 45 min at 65°C.No amplification of the target hemolysin gene was detected inother related bacteria. When the LAMP primers were used, detectionof edwardsiellosis in infected Japanese flounder kidney, andspleen and seawater cultures was possible. We have developeda rapid and sensitive diagnostic protocol for edwardsiellosisdetection in fish. This is the first report of the applicationof LAMP for the diagnosis of a fish pathogen.

INTRODUCTION

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Introduction

Edwardsiellosis is a common bacterial disease of fish causedby gram-negative, motile-flagellate, rod-shaped bacteria Edwardsiellatarda. Infection by E. tarda in epidemic proportions has beenreported to have a disastrous effect on fish culture. E. tardahas also been recognized as a serious human pathogen causinga variety of clinical syndromes (13). Among several virulencefactors studied with E. tarda (5, 12), hemolysis by hemolysinsis considered to be a major cause of death in the infected fish.Hemolysin produced by E. tarda has been cloned and extensivelycharacterized (2, 3). Although hemolysis and necrosis of organsare very clear indications of edwardsiellosis, it is difficultto diagnose the infection in its initial stages or during mildinfection.

Outbreaks of edwardsiellosis are first seen in the form of infectionsaffecting only a few fish; these fish are able to survive onlyfor a few days. When early detection and proper chemotherapyare not undertaken, the infection spreads to the surroundingareas, creating an edwardsiellosis epidemic. Control of edwardsiellosisis attempted through monitoring and stocking specific-pathogen-freestocks. However, diagnosis of E. tarda is made only on the basisof isolation and biochemical identification and of detection(using enzyme-linked immunosorbent assays) of antigens in infectedorgans like spleen, kidney, and liver. However, a rapid andsensitive detection technique is required to detect edwardsiellosisin fish at early stages to control the disease at the source.

The loop-mediated isothermal amplification (LAMP) reaction isan autocycling strand displacement DNA synthesis performed usinga DNA polymerase with a high level of strand displacement activityand a set of specially designed inner and outer primers (11).First, according to the principles of the procedure, the innerprimers (the forward inner primers [FIP]) anneal to hybridizethe target DNA and the first strand is synthesized by Bst polymerase;the outer primers (F3), at a lower concentration, hybridizeand displace the synthesized first strand linked with the FIP-linkedcomplementary strand, which forms the looped structure at oneend. This strand initiates a process in which a backward innerprimer (BIP) hybridizes the complementary strand and initiatesstrand synthesis and, later, strand displacement by B3 innerprimers to form a dumbbell shape. This stem-loop structure actsas a template for the mixture of cauliflower-structured productsin a LAMP reaction, as thoroughly described by Notomi and coworkers(11). Four highly specific designed primers are known to hybridizewith six distinct sequence sites of the template DNA; therefore,the amplification of the target DNA is highly specific. LAMPis a sensitive method which can amplify a few copies of DNAto a magnitude of 10⁹ CFU in less than an hour under isothermalconditions (7-11). This method is promising for rapid diagnosisin cases of infections. LAMP has a wide-ranging applicabilityin the detection of microorganisms. Recently, Maruyama and coworkers(6) applied a LAMP technique for the in situ detection of anstx₂ gene in Escherichia coli. Here we have developed a rapidand sensitive method using LAMP to detect edwardsiellosis infish and in their environment. This is the first report of theuse of a LAMP method for the detection of an environmental isolate.

Development of primers.

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Development of primers.

A highly specific set of primers was designed, and these wereefficient in amplifying an E. tarda hemolysin gene (Fig. 1).Primers for LAMP were designed to target the hemolysin gene(ethA) from E. tarda (accession no. BAA21097), by using primersoftware at the Net Laboratory website (http://www.venus.netlaboratory.com/partner/lamp/index.html),as the gene had low homology to the same gene from other bacteria.For efficient stem-loop formation, the size of the target DNAshould be 130 to 200 bp. The BIP for the E. tarda hemolysingene consisted of B1 (20 nucleotides [nt]), a TTTT linker, andB2 (19 nt) (5'-TTGGTACCATCGGCAAGCCG-TTTT-GGTATCGCTGCTGCTCTGC-3');the FIP consisted of F1c (20 nt), a TTTT linker, and a complementarysequence of F2c (19 nt) (5'-GCCTTTCTTCACCGCCCCTT-TTTT-GGCGTTAGCGTCGACTACAG-3').Primers B3 and F3 were 5'-TGGATCTGGGTGGTCGTC-3' and 5'-AGCCAACGTACCCAGGTC-3'(Fig. 1A). The PCR primers (Fw, 5'-AAGTCGAGCGGTAGCAGG-3'; Rv,5'-GGTGAGCCATTACCTCACCT-3') targeted 290 bp of 16S rRNA fromE. tarda.

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FIG. 1. (A) Nucleotide sequence of partial hemolysin gene used to design inner and outer primers for LAMP. The nucleotide sequences and the positions used to design the primers are represented by dashed lines and labels. (B) A schematic diagram showing the positions at which the primers attach for amplification of the target gene. The picture has been adapted from data provided by Eiken Chemical Co. Ltd.

DNA extraction and LAMP.

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Dna extraction and lamp.

The DNA template was prepared by growing a 1-ml culture of E.tarda (FPC 498; isolated from a Japanese flounder, Nagasaki,Japan) in Luria-Bertani broth to late logarithmic phase (12h postinoculation) and by centrifugation at 5,000 x g for 3min to form a pellet. The pellet was then resuspended in 500µl of Tris-EDTA buffer, boiled for 5 min at 95°C,and centrifuged to collect the supernatant. This supernatantwas used for LAMP and PCRs. LAMP reactions were carried outusing a Loopamp kit (Eiken Chemical Co. Ltd., Tokyo, Japan).In brief, LAMP was carried out in a total of 25 µl ofreaction mixture containing 40 pmol of FIP and BIP, 5 pmol ofF3 and B3, 12.5 µl of 2x reaction mixture (40 mM Tris-HCl[pH 8.8], 20 mM KCl, 16 mM MgSO₄, 20 mM [NH]₄SO₄, 0.2% Tween20, 1.6 M betaine, deoxynucleotide triphosphates [dNTPs] [2.8mM each]), Bst DNA polymerase, and template DNA. The reactionmixture was incubated at 65°C for 45 min and then at 80°Cfor 2 min to terminate the reaction. A total of 2 µl ofthe product was analyzed in 2.0% agarose gel by electrophoresis.The reaction produced many bands of various sizes, ranging fromapproximately 100 bp to the capacity of the loading well (Fig.1).

Optimization of LAMP for E. tarda detection.

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Optimization of lamp for...

Detection (using LAMP) of E. tarda in diseased fish was standardizedin this study. To optimize the conditions needed for the specificamplification of the hemolysin gene of E. tarda, the DNA templatefrom strain FPC 498 (Table 1) was used. The product was formedat 65°C with an amplification time of 60 min. No amplificationwas detected at 60 and 63°C (Fig. 2A). When time-specificamplification for 15, 30, 45, and 60 min was carried out, amplificationof the E. tarda DNA template at 65°C was detected at asearly as 45 min (Fig. 2B). On the basis of the above analyses,the conditions of amplification were optimized as 45 min at65°C.

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TABLE 1. E. tarda strains used in this study

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FIG. 2. Determination of LAMP conditions for E. tarda detection. (A) Effect of temperature on the amplification of a LAMP product. Amplification (using FPC 498 as the DNA template) at 60, 63, and 65°C was carried out for 60 min. (B) Effect of time on amplification of E. tarda by LAMP. Amplification using FPC 498 was carried out at 65°C for 15, 30, 45, and 60 min. A 100-bp molecular mass marker is shown in the extreme right lane, and the results of a control reaction with no template (NT) are shown in the first lane.

To determine the specificity of detection, LAMP reactions werecarried out (using E. tarda hemolysin-specific LAMP primers)in experiments with E. tarda, Salmonella enterica serovar Typhi,E. coli, Shigella sonnei, Proteus mirabilis (kindly providedby Yoshitaka Goto, Miyazaki University, Miyazaki, Japan), Vibriosp., Aeromonas hydrophila, Enterococcus sp., and Photobacteriumsp. After 45 min at 65°C, using templates of DNA from otherenteric bacteria did not produce any amplification. Some ofthese isolates are known to produce enterotoxins and have beenisolated from fish-rearing facilities. Five strains of E. tarda(Table 1) obtained from farms in Japan and tested using E. tardaLAMP primers gave specific amplification products (Fig. 3).

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FIG. 3. Specificity for hemolysin gene amplification using a LAMP reaction and different strains of E. tarda. Lanes 1 to 5, strains E 381, FPC 498, SU 226, E 22, and 158. A 100-bp molecular-mass marker (M) is shown in the extreme left lane. C, control reaction with distilled water.

Previous reports have shown that LAMP can detect target DNAat levels as low as 6 copies (10). In this study, using a LAMPmethod we detected the hemolysin gene of E. tarda from DNA obtainedfrom 10 to 10⁹ CFU of E. tarda. This was achieved by a 10-foldserial dilution of 2.9 x10⁹ CFU E. tarda in phosphate-bufferedsaline. The colonies were enumerated on a Casamino Acids (0.5%)-supplementedTrypticase soy agar plate. E. tarda detection (targeting 16SrRNA) by PCR was carried out to compare the levels of sensitivityof LAMP. All PCRs were performed according to the followingprotocol. A total of 1 µl of cDNA was mixed with 5 µlof dNTPs (10 µM concentrations of each dNTP), 0.5 µlof Taq polymerase (5 U/µl), 5 µl of each gene-specificprimer (5 pmol/µl), and 27.5 µl of water. The PCRwas performed using a PCR apparatus (MJ Research Inc., Waltham,Mass.) under conditions of 0.5 min at 94°C, 0.5 min at 60°C,and 1 min at 72°C for 30 cycles. However, PCR amplifiedthe target gene at a dilution of bacterial DNA of up to 10³CFU. The sensitivity of the PCR detection also depends (amongseveral other factors) upon the efficiency of the primers, andthe use of nested PCR can increase the sensitivity of detection.The need for a specialized machine, and the time required forthe detection by PCR, makes LAMP-mediated detection advantageous.However, until recently, a fluorescent antibody technique (4)and PCR of the hemolysin gene (1) were used to diagnose edwardsiellosis.Thus, these observations show that E. tarda detection usingLAMP is highly specific and sensitive.

Diagnosis of edwardsiellosis by LAMP.

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Diagnosis of edwardsiellosis by...

Fish diseases are a major problem in the aquaculture industrybecause of the economic losses incurred. The rapidity and sensitivityof LAMP make it a suitable technique for the detection of pathogensin aquaculture facilities and for management of disease. Healthyand E. tarda-infected samples of Japanese flounder (Paralichthysolivaceus) were obtained from Ehime Prefectural Chuyo FisheriesExperimental Station, Iyo, Japan. The kidney and spleen wereremoved aseptically, and DNA was extracted using a DNA extractionkit (ISOHAIR; Nippon Gene, Toyama, Japan). E. tarda was detectedin the infected kidney (n = 5) and spleen (n = 5) samples. Healthyspleen and kidney samples did not produce amplification whenspecific primers for E. tarda detection by LAMP were used (Fig.4). Seawater samples from E. tarda-infected Japanese flounderculture ponds contained 3.8 x 10² CFU. A total of 1 ml of theseawater was aliquoted and centrifuged at 16,000 x g to obtaina pellet, which was dissolved in 10 µl of distilled waterand tested (using LAMP) for the presence of E. tarda. Seawatersamples were positive for the presence of E. tarda.

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FIG. 4. Detection of E. tarda hemolysin gene in samples from healthy and infected Japanese flounder and in samples of seawater from an infected culture facility. K1 and K2, E. tarda-infected kidney samples; S1 and S2, E. tarda-infected spleen samples; K3 and S3, healthy kidney and spleen samples. SW1 and SW2 are seawater samples from E. tarda-infected ponds. Molecular mass marker 5 (M5) ( ${phi}$ /X174/HincII digest) is shown in the last lane on the right. All the products were electrophoresed on 2.0% agarose gels and stained with ethidium bromide. NT, no template; DW, distilled water.

In conclusion, LAMP, a rapid and highly sensitive system fordetecting edwardsiellosis, has been designed. This is the firstreport of the use of a LAMP technique, which has applicationsfor the detection of environmental isolate. This method canbe effectively used for diagnosis of edwardsiellosis in fishand in a culture environment.

FOOTNOTES

* Corresponding author. Mailing address: Masahiro Sakai Faculty of Agriculture, Miyazaki University, Gakuen kibanadai nishi 1-1, Miyazaki 889-2192, Japan. Phone and fax: 81-985-587219. E-mail: m.sakai{at}cc.miyazaki-u.ac.jp.

REFERENCES

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