Photobacterium damselae subsp. damselae isolated from juvenile black seabream Acanthopagrus schlegeli

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1 Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae isolated from juvenile black seabream Acanthopagrus schlegeli Masayuki Nakanishi, Takashi Kamaishi*, Ikunari Kiryu*, and Naoki Sobajima In August 2007, an infectious disease was observed in cultured juvenile black seabream Acanthopagrus schlegeli in Kunda bay. The maximum motality per day was 1.3% of the stock. The average size of diseased fish was 73 mm in fork length and 6.3 g in body weight. All moribund fish examined showed severe anemia, especially pale gill, as the main clinical sign. Bacteria were isolated from the kidneys of moribund fish. Isolates were identified as Photobacterium damselae subsp. damselae based on physiological and biochemical tests, including API20E, the oligonucleotide DNA array, and multiplex PCR assay for the investigation of partial genes of 16S rrna and urec. Photobacterium damselae subsp. damselae Acanthopagrus schlegeli 1.3% 1Photobacterium damselae subsp. damselae P. damselae subsp. piscicida (, 2004) (Muroga et al.,1977) P. damselae subsp. damselae ( (L) 73 mm (BW) 6.3 g) 2 1% NaCl BHI (Difco) Table 1 API20E 2 AsK0701 AsK0702 Multiplex PCR 2 P. damselae subsp. piscicida (OT51443 ) P. damselae Table 1 List of the bacterial isolates studied Isolate Date of isolation Host Age of host ork length Body weight Organ Ask0701 Aug Black seabream (Acanthopagrus schlegeli) 0 69 mm 4.5 g Kidney Ask mm 7.2 g OT51443 Jun Yellowtail (Seriola quinqueradiata) 32 g PK9705 Jun Japanese flounder (Paralichthys olivaceus) 38 mm*1 0.5 g Kidney *1, Total length. * National Research Institute of Aquaculture, Minamiise, Mie Japan

2 Vibrio damsela Vibrio (Listonella) anguillarum (PK9705 ) PK 9705 ( V.anguillarum A type) V (Listonella) anguillarum A type 4 (L mm BW g) m HematoxylinEosin (HE) May Grunwald (L mm, BW g), 20 3 DNA 16S l (Matsuyama et al., 2006) 15 2 API20E (V4.1) (Biomerieux) AsK0701 AsK0702 (1988) % NaCl BHI 25 1 (OT ) 25 O/129 1% NaCl % 1% (BBL) 5 ( ) ( ) AsK0701 AsK0702 OA CP NB OM CL SiX NA ST NT OTC ABPC P. damselae subsp. piscicida subsp. Table 2 Oligonucleotide sequences of primers used for multiplex PCR (Osorio et al. (1999) and Osorio et al. (2000)) R R Primer Car1 Car2 Ure5' Ure3' Nucleotide sequence (5' 3') GCT TGA AGA GAT TCG AGT CAC CTC GCG GTC TTG CTG TCC GGA ATA GGT AAA GCG GG CTT GAA TAT CCA TCT CAT CTG C damselae Osorio et al. (2000) Car1 Car2 ( 267 bp) Ure5' Ure3' ( 448 bp) 2 Multiplex PCR Car1 Car2 subsp. piscicida subsp. damselae 16S rrna (Osorio et al., 1999) Ure5' Ure3' subsp. damselae (urec) Table 2 Multiplex PCR Table 3 DNA 4 TrisEDTA buffer (ph 8.0) 100 L ,000 rpm 3 DNA PCR PrimeSTAR Max (Takara) Table 3 Components of the multiplex PCR mixture PrimeSTAR Max (Takara)*1 2 Primer Car1 R Car2 Ure5' R Ure3' 100 pmol/ L 100 pmol/ L 100 pmol/ L 100 pmol/ L Deionized, distilled water DNA template Total *1, Including polymerase, buffer, and dntps L L * l,,, , p. 49 * 2,,,,,2007 DNA 19, p P. damselae subsp. damselae

3 ig. 1 Light micrograph of the hepatopancreas of an affected black seabream, Acanthopagrus schlegeli. Note hemorrhage ( ) at the margin of the pancreas. hpv, hepatic portal vein. HE stain. ig. 2 Light micrograph showing hemorrhage ( ) in the spinal ganglion of an affected black seabream, Acanthopagrus schlegeli. HE stain. DNA % (ABPC) % (L 69 mm BW 4.5 g L 75 mm BW 7.2 g) AsK 0701AsK0702 ( ) 4 1 (ig.1) 1 (ig.2) 16S P. damselae ig. 3 Spots of Photobacterium damselae were positive in the 16S rrnabased microarray (A). Vibrio harveyi (B1) and P. damselae (B2) were detected in the DNA tip for the discrimination of fish pathogens Vibrio and Photobacterium species (B). No signal was obtained from the array for fish pathogenic virus (C)

4 Table 4 Biochemical characteristics of isolates from diseased black seabream (Acanthopagrus schlegeli) and reference strains Characteristics Gram stain Cell form Motility Cytochrome oxidase Catalase H2S production MR test VP test 2,3Butanediol production Indole production Nitrate reduction Urease Malonate utilization Gelatin liquefaction Casein digestion Starch hydrolysis Esculin hydrolysis Tween 80 ONPG Phenylalanine deaminase O/129 sensitivity Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization Hugh & Leifson test DNase Growth on TCBS Chitin hydrolysis Lecithinase Growth in 0% NaCl 0.5% 1.0% 1.5% 3.0% 7.0% Isolates References AsK0701 AsK0702 OT51443*1 PK9705*2 Short rod Short rod Short rod Short rod w Gr Gr Y, positive; w, weakly positive;, negative;, fermentative; Gr, green; Y, yellow. *1, Photobacterium damselae subsp. piscicida. *2, Vibrio (Listonella) anguillarum. (ig.3a) Vibrio harveyi P. damselae (ig.3b) (ig.3c) API20EAsK0701 AsK0702 profile number P. damselae ( 99.9%ID T Table 4 Table 5 VP O/129 DNase TCBS % NaCl 2,3 P. damselae subsp. piscicida (OT51443 ) O/129 DNase TCBS 0.5% NaCl V. (Listonella) anguillarum (PK9705 ) VP OA CP NB OM CL NA ST NT OTC ABPC SiX Multiplex PCR ig bp 448 bp P. damselae subsp. damselae P. damselae 58 P. damselae subsp. damselae

5 Table 5 Carbohydrate utilization of present isolates and reference strains Characteristics Acid from: Glucose ructose Galactose Mannose Maltose Sucrose Dextrin Glycerin Arabinose Rhamnose Xylose Threhalose Lactose Cellobiose Melibiose Starch Raffinose Salicin Mannitol Adonitol Sorbitol Dulcitol Inositol Ribose Isolates References AsK0701 AsK0702 OT51443*1 PK9705*2 w w w w, positive; w, weakly positive;, negative. *1, Photobacterium damselae subsp. piscicida. *2, Vibrio (Listonella) anguillarum. et al. (1992) VP DNA P.damselae ( V.damsela ) V. harveyi P. damselae subsp. piscicida subsp. damselae (Muroga et al., 1977) O/129 P. damselae subsp. piscicida P. damselae subsp. piscicida subsp. damselae Osorio et al. (2000) Multiplex PCR Osorio et al.(2000) Car1 Car2 Ure5' Ure3' 2 Multiplex PCR P.damselae subsp. piscicida 25 subsp. damselae bp 267 bp 448 bp subsp. piscicida 25 subsp. piscicida (OT51443 ) Osorio et al. (2000) 267 bp 448 bp V. (Listonella) anguillarum (PK9705 ) 5 2 P. damselae P. damselae Vibrio damsela Seriola quinqueradiata (Sakata et al., 1989) turbot Scophthalmus maximus (ouz et al., 1992) Sakata et al. (1989) VP (Table 6, 7) ouz ig. 4 Profile of the multiplex PCRamplified DNA fragments obtained from four strains studied. Lanes: M, DNA marker (Takara 100 bp DNA marker); 1, negative control; 2, V. (Listonella) anguillarum (PK9705); 3, P.damselae subsp. piscicida (OT51443); 4 and 5, AsK0701 and AsK0702 isolated from diseased black seabream, respectively

6 Table 6 Comparison of biochemical characteristics between present isolates and Vibrio damsela (Sakata et al. (1989) and ouz et al. (1992)) Characteristics Gram stain Cell form Motility Hugh & Leifson test Cytochrome oxidase Catalase O/129 sensitivity Nitrate reduction Urease Gelatinase Indole production VP test MR test H2S production Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization Starch hydrolysis Casein hydrolysis Chitin hydrolysis Lecithinase DNase ONPG Growth on TCBS Growth in 0% NaCl 0.5% 1.0% 1.5% 3.0% 7.0% Hemolysis Present isolates Vibrio damsela AsK0701/Ask0702 Sakata et al. (1989) ouz et al. (1992) Short rod Gr NT Short rod Gr, positive;, negative;, fermentative; Gr, green; NT, not tested. Rod Gr subsp. damselae 16ureC DNA subsp. piscicida Multiplex PCR 267 bp 448 bp 2 Osorio et al. (2000) P. damselae subsp. damselae V. damsela ( P.damselae) Sakata et al. (1989) ouz et al. (1992) API20E API20E DNA Multiplex PCR P. damselae subsp. damselae Sakata et al. (1989) ouz et al. (1992) V. damsela ( P.damselae) 60 P. damselae subsp. damselae

7 Table 7 Comparison of carbohydrate utilization between present isolates and Vibrio damsela (Sakata et al. (1989) and ouz et al. (1992)) Carbohydrates Present isolates Vibrio damsela AsK0701/Ask0702 Sakata et al. (1989) ouz et al. (1992) Cellobiose w/ g Dextrin g Dructose g DGalactose g DGlucose g Maltose g DMannose g Starch g Dulcitol DRibose Glycerol w O DMannitol Melibiose Salicin Sucrose Threhalose LArabinose DSorbitol minositol Lactose Raffinose Rhamnose DXylose /w, positive; w, weakly positive;, negative; g, fermentative with gas production;, fermentative; O, oxidative. P. damselae subsp. damselae Photobacterium damselae subsp. piscicida (OT51443 ) ouz B., Larsen J.L., Nielsen B., Barja J.L., Toranzo A.E Characterization of Vibrio damsela strains isolated from turbot Scophthalmus maximus in Spain. Dis.Aquat.Org., 12: Matsuyama T., Kamaishi T., Oseko N Rapid discrimination of fish pathogenic Vibrio and Photobacterium species by oligonucleotide DNA array. ish Pahol., 41: () Muroga K., Sugiyama T., Ueki N Pasteurellosis in cultured black seabream. J.ac.ish.Anim.Husb., Hiroshima Univ., 16: Osorio C.R., Collins M.D., Toranzo A.E., Barja J.L., Romalde J.L S rrna gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis. Appl. Environ. Microbiol., 65: Osorio C.R., Toranzo A.E., Romalde J.L., Barja J.L Multiplex PCR assay for urec and 16S rrna genes clearly discriminates between both subspecies of Photobacterium damselae. Dis.Aquat.Org., 40: Sakata T., Matsuura M., Shimokawa Y Characteristics of Vibrio damsela isolated from dis

8 eased yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi, 55: P. damselae subsp. damselae