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A Specific DNA Hybridization Probe for Detection of Pasteurella Piscicida

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A Specific DNA Hybridization Probe for Detection of Pasteurella Piscicida DISEASES OF AQUATIC ORGANISMS Vol. 7: 203-210, 1989 Published December 7 Dis. aquat. Org. A specific DNA hybridization probe for detection of Pasteurella piscicida Jiang Zhao, Takashi Aoki* Department of Fisheries. Faculty of Agriculture, Miyazaki University, Miyazaki 889-21, Japan ABSTRACT: A 692 base pair (bp) DNA fragment from the bacterial fish pathogen Pasteurella piscicida was cloned into pUC9 and used as a hybridization probe for the identification of P, piscicida. The radiolabeled probe hybridized with itself, but did not hybridize in colony hybridization with other fish pathogens (including Aeromonas hpdrophila, A. salmonicida, Edwardsiella tarda, Pseudomonas angul- liseptica, Vibrio anguillarum and Yersinia ruckeri), or with other related organisms (Haen~ophilus influenzae, Pasteurella haemolytica, and P. multocida). Photobiotin-labeled probe hybridization detected P. piscicida chromosomal DNA by Southern and dot blot hybridizations. The radiolabeled probe directly detected P. piscicida on nitrocellulose filters smeared with infected kidney and spleen tissue of yellowtail Seriola quinqueradiata. The 3"P-labeled DNA probe was capable of detect~ng3.9 ng of purified DNA and lo5 cells of P. piscicida. The cloned specific DNA probe can be used for rapid detection and identification of P. piscicida. INTRODUCTION MATERIALS AND METHODS Pasteurella piscicida is the causative agent of Bacterial strains and plasmid. The bacterial strains pseudotuberculosis in yellowtail Serjola quin- used are listed in Table 1. Twenty-nine strains of Pas- queradiata (Kimura & Kitao 1971), one of the most teurella piscicida were collected from diseased yellow- important causes of mortality in cultured yellowtail. tail in various areas of Japan and 1 strain from white The identification of P. pisciada has been based on its perch, Morone anlericana, in the USA (Janssen & Sur- morphological and biochemical characteristics. How- galla 1968). One P, piscicida strain (KG8601),isolated ever, classification using these features is cumbersome in 1986 from Kagoshima in Japan, was used for the and time-consuming. The different illorphological and cloning of its chromosomal DNA. Other fish pathogens biochemical characteristics of the species of bacteria (4 strains of Aeromonas hydrophila, 4 of A. sal- are prescribed by their genes. Recently, randomly monicida, 4 of Edwardsiella tarda, 2 of Pseudomonas cloned chromosomal DNA fragments and cloned genes anguilliseptica, 4 of Vibl-io anguillarum, and 2 of Yer- encoding species-specific characteristics have been sinia ruckeri), were used in testing the specificity of used as specific hybridization probes for rapid identifi- the probe, as were 1 strain each of Haemophilus cation of organisms (Salyers et al. 1983, N~shibuchiet influenzae, Pasteurella haemolytica and Pasteurella al. 1985, Carter et al. 1987, Mifflin et al. 1987).By using molutocida. Escherichia coli Strain JM103 (Maniatis et a species-specific DNA probe to a specific nucleotide al. 1982) was used as the host for propagating the sequence of chromosomal DNA from Vibrio anguil- plasmid. The plasmid, pUC9 (Maniatis et al. 1982), larun~,we easily and rapidly identified V. anguillarunl was used for the cloning of P. piscicida chromosomal using colony hybridization (Aoki et al. 1989). DNA. In the present study, we describe a species-specific Media and growth conditions. Pasteurella pisclcida DNA fragment of chromosomal DNA from Pasteurella strains were cultured at 25 "C in nutrient (N) broth piscicida which, by direct hybridization, specifically (beef extract: 15 g; polypeptone: 10 g; NaC1: 5 g; distil- detects the causative agent in diseased marine fish. led water: 1 1; pH: 7.2) supplemented with 0.3 % yeast extract and 1.5 % NaC1. The other Pasteurella species and Haemophilus influenzae were grown at 37 "C in Addressee for correspondence heart infusion broth (HI, Nissui Co. Ltd.) containing 5 O/o Inter-Research/Printed in F. R. Germany 204 Dis. aquat. Org. 7: 203-210, 1989 Table l. Bacterial strains used in this study Bacterid Slrdin Hvst Source Aeromonas h ydrophila ATCC15467 Schubert (19741 Wakabayashi et al. (1981) 1;; ] Eel Nomura & Aoki (1985) A-54 Nomura & Aoki (1985) Aeron~onassalmonicida ATCC14 174 Brooktrout ltao et al. (1985) N8004 OB8101 Amago HR8101 l Edwardslella tarda SA83 18 Thls study Flounder NG8407 t M2840 1 Eel E-l 1-2 l Haemophilus influenzae NCTC8143 Kilian & Blberste~n(1984) Fasteul-ella haen~olytica rJCTC338G Carter (1984) Pasteurella multocida NCTC10322 Carter (1984) Pasteurella pisac~da El-18218 KG8206 KG8232 MEP8215 EH8306 EH8310 M28303 M28309 NG8304 EH8405 EH84 11 EHP84133 EHP84 135 SA8406 SA8407 Yellowtail This study KG84305 EHP85468 EHP85477 WKY8575 NZ8574 MZ8575 SA8514 SA85 16 KG8601 EH860 1 EH8606 EH86 10 NG8609 M286 15 Snieszko Whlte perch Janssen & Surgalla (1968) Pseudornonas angullliseptica NCMB1949 Nakajima et al. (1983) Eel ET-2 l Nakajima et al. (1983) Vibrio anguillarun~ ATCC 19264 Baumann et al. (1984) PT834 1 Aoki et al. (1989) PT-24 } AYU Kitao et al. (1983) ET- l Eel htao et al. (1983) Yersinla rucker) 114 Stave et al. (1 987) I Rainbow trout 11-29 I Stave et al. (1987) fildes extract (Oxoid Ltd.). Vibrio anyr~illarumstrains ruckeri were cultured at 25 "C in N broth. Escherichia were cultured at 25 "C in HI broth containing l '?L NaC1, col; was grown at 37 "C in Luria-Bertani broth (LB) Pseudomonas anguilliseptjca and Aeromonas salmon- (polypeptone: 10 g; yeast extract: 5 g; NaCI. 5 g; distil- icida were cultured at 20 'C in HI and N broth, respec- led water: l l; pH 7.0) (Maniatis et al. 1982). Each tively. A. hydrophila, Edwardsiella tarda, and Yersinja medium also contained 1.5 O/O agar to isolate organisms. Zhao & Aoki: A DNA probe for PasteureUa pksacida 205 Isolation of DNA. The chromosomal DNA of Pas- 7.8, and 3.9 ng of DNA were blotted on nitrocellulose teurella piscjcida, Strain KG8601, and the fish-patho- filters. gens Aeromonas hydrophila A-10, A. saln~onicjda Hybridization with 32P labeled DNA probe was car- HTCC14174, and Vibrio anguillarum PT8341 were iso- ried out on filters at 65 "C for 36 h in 4 x SSC containing lated by previously reported methods (Silhavy 1984). 10 X Denhardt's solution (l'10 Ficoll, 1 % polyvinylpyr- Plasmid DNA was prepared by the alkaline lysis rolidone, 1 % bovine serum albumin), 0.5 % sodium method of Birnboim & Doly (1979). Both isolated dodecyl sulfate (SDS), and 1.5 mg ml-' heat-denatured chromosomal DNA and plasmid DNA were purified by salmon-sperm DNA. Filters were washed with 2 X SSC centrifugation to equilibrium in cesium chloride- + 0.1 % SDS, dried, and subjected to autoradiography. ethidium bromide gradients. Hybridization with the photobiotin-labeled probe was Molecular cloning of DNA probe. The chromosomal carried out at 42 "C for 12 to 18 h in a hybridization DNA of Pasteurella piscicida was digested with Hind solution containing 45 % formamide, 5 X SSC, l x 111. The vector plasmid, pUC9, was also digested with Denhardt's solution, 25 mM sodium phosphate (pH 6.5), the same enzyme and then dephosphorylated with 5 YO dextran sulfate, and 0.2 mg ml-' heat-denatured bacterial phosphatase (Takara Shuzo Co. Ltd, Kyoto, salmon-sperm. After hybridization, the probe-target Japan). The digested DNA was mixed together and DNA hybrid was detected with conjugate dyes of the ligated with T4 ligase (Nippon Gene Co. Ltd, Osaka, B1.u GENE (Bethesda Research Laboratories) strep- Japan). The recombinant plasmids were transformed to ta.uidin-alkaline phosphatase. Escherichia coli JM103, and transformants selected on Colony hybridization. The organisms were inocu- L-agar (Maniatis et al. 1982) supplemented with 50 yg lated onto autoclaved, gridded nitrocellulose filters ml- ' ampicillin and X-gal (5-bromo-4-chloro-3-indolyl- (Toyo Roshi Co. Ltd, Japan) overlayed on an agar plate. P-D-galactopyranoside). After incubation, the filter was removed from the plate Labeling of DNA probe. A cloned DNA fragment and treated with NaOH, neutralized, baked, and hy- with a molecular weight of about 0.7 kilobase (kb) was bridized with probe DNA. Hybridization reactions selected, at random, for use as the probe, and was were carried out by the method of Grunstein & Hog- isotopically labeled with (CV-~*P)~CTP(New England ness (1975). Nuclear Corp., Boston, USA) by nick translation (Rigby The minimum number of Pasteurella piscicida (Strain et al. 1977). KG8601) cells detected was determined by hybridi.za- The DNA probe was labeled with photobiotin tion with the same probe. Bacterial cells diluted 10-fold (Biotechnology Research Enterprises S.A. Pty Ltd), and from 10"o 10' were spotted on a nitrocellulose filter. the Blu GENE nonradioactive DNA detection system DNA hybridization was thcn performed as described (Bethesda Research Laboratories, USA) was used. by Grunstein & Hogness (1975). Probe DNA was mixed with equal volumes of photo- Detection of Pasteurella piscicida in fish. Nineteen biotin acetate in an Eppendorf tube, irradiated for 20 diseased and 3 healthy Seriola quinqueradiata were min with a 400W lamp from a distance of 10 cm, and obtained from an experimental fishery station. These diluted to 100 p1 with TE buffer (10 mM Tris HCl, 1mM fish were dissected and examined to determine EDTA, pH 8.0) followed by 100 111 2-butanol. The mix- whether nodules of kidney and spleen, characteristic of ture was centrifuged and the 2-butanol phase dis- the pseudotuberculosis, were present in the kidney and carded. Photobiotin-labeled DNA was purified by 3 spleen of fish. Kidney and spleen tissue from each fish successive ethanol precipitations, and finally resus- was sampled using a platinum loop and smeared onto pended in TE buffer and stored at -20 'C. nitrocellulose filters. Hybridization was then carried Southern and dot blot hybridization. For Southern out with the same probe and by the same method as blot hybridization, chromosomal DNAs of Pasteurella used in the colony hybridization procedure. At the pjscicida, Aeromonas hydrophila, A, salmonicida, and same time, the causative agent was isolated on agar Vibrio anguillarum were cleaved by Hind 111, and elec- media and identified.
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