Isolation and identification of Shewanella alga and its pathogenic effects on post-larvae of abalone Haliotis diversicolor supertexta

Abalone are gastropod molluscs that inhabit coastal reefs in tropical, subtropical and temperate areas. Their culture has become economically important in terms of job creation and generation of wealth in rural China. The herbivorous gastropod, Haliotis diversicolor supertexta, is a commercially important abalone species cultured on the southern coast of China, including Taiwan (Chen 1984, 1989). Culture of H. diversicolor supertexta has expanded greatly since 1986 due to successful artificial propagation and the development of multiple-tier basket systems for raising the abalone in land-based farms (Yang & Ting 1986; Chen & Lee 1999). However, since late 2000, farmers across southern China have experienced mass mortality of abalone reared in hatchery ponds with survival rates of post-larvae reported to be only 10%. Settlement failure of spat is also observed in nursery ponds (Lee, Liu, Chen & Huang 2001). Mass mortality is particularly prevalent in post-larvae between 10 and 30 days old. Signs of infection include whitening, shrunken muscles and a slower reaction. Post-larvae eventually detach from the diatom films on which they are cultured, however the cause of these mortalities is unclear.

The genus Shewanella is a minor genus of the family Vibrionacae (Derby & Hammer 1931). They are commonly regarded as microorganisms of aquatic products (Derby & Hammer 1931; Jorgenson & Hub 1989) and opportunistic pathogens of both aquatic animals and humans (Brink, Van Straden & Rensburg 1995; Butt, Figueroa & Mart 1997). They have also been shown to be virulent to juvenile zebra mussels (Gu & Mitchell 2002), while Shewanella alga has been identified as the causative agent of ulcer disease in the cultured marine fish Scinenops ocellata (Chen, Hu, Chen & Zhang 2003). In the present study, we describe the isolation, identification and virulence of a pathogenic strain of S. alga (strain 27) isolated from diseased abalone post-larvae found in Shanwei, Guangdong Province, China in July 2003.

To isolate the microorganisms from post-larvae during disease outbreaks, 13-day old post-larvae together with their attached diatom films were transferred from nursery ponds to the laboratory and placed in sterile Petri dishes. The post-larvae were washed five times with sterile phosphate-buffered saline (PBS), and then homogenized with a sterile glass grinder with 5–10 mL sterile saline solution. The homogenate was serially diluted in sterile saline and 100 μL of each dilution was plated onto Zobell's 2216E agar plates (Difco, Detroit, MI, USA) supplemented with 2.5% NaCl, which were then incubated at 25 °C for 5–7 days. Dominant bacterial colonies with similar morphologies were selected and repeatedly streaked onto 2216E plates until pure cultures were obtained. Stocks of purified cultures were stored in 2216E liquid medium supplemented with 10% glycerol at −70 °C.

Shewanella alga strain 27 was initially characterized using API 20E identification kits (Bio-Merieux, Marcy l'Etoile, France) by comparing the reaction profile of strain 27 with S. alga type strain ATCC55627.

Genomic DNA was extracted from strain 27 according to Sambrook, Fritsch & Maniatis (1989). Briefly, after culturing the bacteria overnight, cells were collected by centrifugation at 2640 g for 30 min and resuspended in 500 μL TE buffer (10 mm Tris-HCl, 1 mm EDTA, 0.1 m NaCl, pH 8.0) containing 1 mg mL⁻¹ lysozyme. After incubation at 37 °C for 30 min, 30 μL 10% sodium dodecylsulphate was added and the tubes were inverted gently several times until complete lysis of the pellet was observed. Five microlitres of 5 mg mL⁻¹ proteinase K were added and the tubes were incubated for 1 h at 37 °C. After incubation the solution was extracted several times with phenol to remove proteins, and once with chloroform to remove traces of phenol. DNA was precipitated using a 0.8 volume of isopropanol and washed with 70% ethanol. The extracted DNA was dried and dissolved in 10 mm Tris-HCl, pH 8.0 and stored at −20 °C. PCR amplification of the 16S rDNA from strain 27 was carried out using a pair of universal primers: 63F 5′-CAGGCCTAACACATGCAAGTC-3′ and 4R 5′-TGACTGCCAA(G)GGCATC CA-3′. The PCR mix contained 20 μm of each primer, 200 μm of 10X PCR buffer and deoxynucleoside triphosphate admixture (dATP, dCTP, dGTP and dTTP), 1.5 mm MgCl₂, 2.5 U Taq DNA polymerase and 1 μg template DNA. The PCR was performed using an initial denaturation step for 1 min at 95 °C, followed by 35 cycles of denaturation at 94 °C for 45 s, primer annealing at 55 °C for 45 s and extension at 72 °C for 1 min. In the final cycle, extension was run at 72 °C for 4 min. After cleaning the PCR products with a Qiagen spin column (Qiagen, Hilden, Germany), direct sequencing of the PCR product was carried out as reported by Thompson, Balsdon, Cai & Collins (1992). Blast was used to identify the species closest phylogenetically to the sequence obtained for strain 27.

Twenty-day-old abalone post-larvae (average shell length, 0.9 mm) were collected from an abalone farm in Shenzhen, Guangdong Province where no mass mortalities occurred in that hatching season. They were rinsed three times with 500 mL of autoclaved and aerated sea water containing six antibiotics (chloramphenicol, 20 mg L⁻¹; norfloxacin, 10 mg L⁻¹; erythromycin, 15 mg L⁻¹; gentamicin, 40 mg L⁻¹; penicillin G, 200 000 IU L⁻¹ and polymyxin B, 300 mg L⁻¹) (Cai, Zhou & Cai 2004) and then bathed in 1 L of the same mixed antibiotic solution for 24 h with a change of water every 6 h. After confirmation of bacteria-free status by using the pour plate technique with ground post-larvae, LD₅₀ tests were run.

Post-larvae placed in a beaker (1 L) supplied with autoclaved and aerated sand-filtered sea water of 30 g L⁻¹ salinity were acclimatized to 25 °C before exposure to a bacterial challenge. An LD₅₀ test at 25 °C was conducted by adding suspensions of bacteria [24 h cultures diluted to give final concentrations of 10³ to 10⁶ colony forming units (CFU) mL⁻¹] to 20 abalone post-larvae per dose. Sterile PBS was added to parallel beakers as controls (Control 1). In addition, a strain of experimentally confirmed non-pathogenic but unidentified bacterium was used as a second control (Control 2) using dilutions between 10³ and 10⁶ CFU mL⁻¹. Mortalities were recorded daily for 3 days post-challenge. Re-isolation and identification of the bacteria from moribund post-larvae was conducted using 2216E agar plates.

Stock cultures of strain 27 were grown in 2216E broth for 24 h at 25 °C. Bacteria were harvested by centrifugation at 10 000 g for 15 min and resuspended in sterile saline to a dilution with a turbidity equivalent to a Macfarland No. 0.5 standard (0.5 mL BaSO₄ + 99.5 mL 0.36 n HCl). The bacterial suspension (0.1 mL) was spread onto Mueller-Hinton agar (Difco) and antibiotic discs added to the cultures (Koneman, Allen, Janda, Paul & Winn 1992). The discs used in the assay included ampicillin (10 μg), chloramphenicol (30 μg), polymyxin B (300 μg), ciprofloxacin (5 μg), penicillin G (10 IU), erythromycin (15 μg), cefamezin (30 μg), kanamycin (30 μg), gentamicin (10 μg), neomycin (30 μg), norfloxacin (10 μg), amikacin (30 μg), novobiocin (5 μg), streptomycin (10 μg), SMZ-TMP (trimethoprim/Sulfamethoxazole) (25 μg) and tetracycline (30 μg). The plates were incubated at 35 °C for 18 h and the inhibition of the bacteria by the chemotherapeutic agents scored (Koneman et al. 1992).

The bacterial strain (27) isolated from diseased abalone post-larvae was a Gram-negative, motile, straight rod, which was oxidase positive, catalase positive and nonfermentative. On the basis of these biochemical characteristics (Table 1) and comparison with the API 20E results of a S. alga type strain (ATCC 55627), strain 27 was confirmed as belonging to the genus Shewanella with 99.9% confidence. The results of the 16S rDNA sequencing revealed strain 27 shared 99% similarity with the type strain of S. alga, viz. strain Bry (ATCC 55627, accession number X81621). Strain 27 may therefore be considered as S. alga. The 16S rDNA sequence was deposited in GenBank under accession number DQ026023.

The LD₅₀ of strain 27 for 20-day-old abalone post-larvae was 1.8 × 10⁴ CFU mL⁻¹ (Table 2). Pure homogeneous colonies of bacteria were re-isolated from moribund and whitened post-larvae infected during the bacterial challenge, and these were confirmed to be the same as the challenge strain. No mortality was observed in the two control groups. The same gross signs as those observed in moribund post-larvae during natural outbreaks were observed in the moribund/dead abalone post-larvae following bacterial challenge.

Strain 27 was susceptible to 11 and moderately susceptible to two of the 16 chemotherapeutic agents tested (Table 3).

Bacterial infection has been established to be one of the major threats in wild and farmed abalones (Lafferty & Kuris 1993; Gardner, Harshbarger, Lake, Sawyer, Price, Stephenson, Haaker & Togstad 1995). Shewanella alga strain 27 was found to be virulent to abalone post-larvae and may be an important pathogen in abalone culture. Strain 27 is susceptible to 13 of the 16 chemotherapeutic agents tested suggesting the possibility of using antibiotics to prevent disease-related losses in abalone culture.