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Iranian Journal of fisheries Sciences 12(4)802-812 2013 Isolation, identification and phylogenetic analysis of a pathogen of
Haliotis diversicolor supertexta (L.) with mass mortalities
Zhou Jing*1 and Cai Junpeng2
Received: February 2013 Accepted: April 2013 Abstract
This study was conducted to determine a disease outbreak in 14 day old post-larvae of
abalone (Haliotis diversicolor supertexta) which caused mass mortality in July 2010 in
Shanwei, China. Twenty-nine bacterial strains were isolated from a sample pool of 10
diseased post-larval abalones on 2216E marine agar plates during a natural outbreak of the
disease. Among them, a dominant isolate (referred to as strain 21) was found to be highly
virulent to post-larvae in experimental challenge tests, with an LD50 value of 1.0 ×104
colony forming units (CFU) mL-1 on day 3. API 20NE kits and 16S rDNA sequence
analysis, identified strain 21 as Oceanomonas doudoroffii. It was susceptible to 10 and
moderately susceptible to 1 of the 16 antibiotics examined when antibiotic sensitivities of
the bacterium were assayed. Results of this study implicated Oceanomonas doudoroffii
strain 21 as a cause of mortalities in post-larval abalone from Shanwei, China.
Keywords: Haliotis diversicolor supertexta, massive death, challenge test, Oceanomonas
doudoroffii, 16S rDNA sequencing 1-College of Life Science, QuFu Normal University, QuFu, China 2-College of Food Science and Light Industry, South China University of Technology, Guangzhou, China Corresponding author’s email: 803 Jing & Junpeng Isolation, identification and phylogenetic analysis of a pathogen of… Introduction
Production of various species of the genus collected during an outbreak of post-larval Haliotis is a very important part of aquaculture disease in July 2010 when the post-larvae in the Orient since they have been regarded as very precious seafood. However, from 2002 Post-larvae were on average approximately onwards, mass mortalities of post-larvae of the small abalone, Haliotis diversicolor supertexta, have persisted and forced many abalone farms collected with a pipette fitted with a sterile to be closed (Cai et al., 2006a). Diseased 1-mL tip from bio-films, and placed in a post-larval abalone became pale and lethargic sterile polystyrene Petri dish containing 0.5 mL sterile phosphate buffered saline (PBS) post-larvae came off the diatom bio-films on which they grew one to two days after these symptoms appeared. Some did not come off the bio-films but their shells were left empty. glass grinder with 0.5 mL sterile PBS after Upon examination of the cause(s) of these being rinsed 3 times with PBS. A 10-fold outbreaks, Cai et al. (2006a,b,c) revealed that dilution series ranging from 10-1 to 10-5 was Vibrio parahaemolyticus, Shewanella algae, V. prepared from this homogenate. Each sample alginolyticus and Klebsiella oxytoca were all in the dilution series was plated out on associated with mass mortality of post-larval abalone in Shanwei and Fujian. However, it is not yet known if any of these pathogens were incubation at 25°C, representative colonies, different farms in the same region. We report according to their different morphologies and abundance on the culture media, were Oceanomonas doudoroffii as a cause of mass selected and purified for characterization and mortalities in the post-larval abalone, H. diversicolor supertexta in July 2010 in Abalone and virulence test
Abalone of 20 days post-fertilization with an average shell length of ca. 0.9 mm were Materials and methods
collected from an abalone farm in Shenzhen, Bacterial isolation
Guangdong Province. There was no incidence of disease outbreaks at this site during the abalone, that used for the bacterial isolation hatching season. These abalone were used for experimental challenge tests which were run according to the protocol described by Cai et al. Iranian Journal of Fisheries Sciences 12(4), 804 (2006a). Briefly, each post-larval abalone, with To verify Koch’s postulate in the case of its attached diatom film, was first cut into the most virulent strain, each of the post-larvae pieces of approximately 1 cm2 or less and in the challenge tests was picked-up under a placed in a sterile 2-L beaker and rinsed 3 magnifier, thoroughly rinsed with sterile PBS times with 500 mL autoclaved and aerated and used for re-isolation and identification of seawater containing six antibiotics (Takara, bacteria. Mortality was attributed to the bacterium isolated if it was recovered in pure norfloxacin, 10 mg/L; erythromycin, 15 mg/L; culture from dead post-larvae (Brock et al., gentamicin, 40 mg/L; penicillin G, 200,000 IU/L; and polymyxin B, 300 mg /L) and then Bacterial characterization
immersed in 1 L of the same mixed antibiotic solution for a day with change of the water sample pool of 10 diseased post-larval abalone every 6 h. Once pour plate technique had were checked to establish if they could grow confirmed bacteria-free status, challenge tests on thiosulfate citrate bile salt sucrose medium were carried out. A series of dilutions of 4 (TCBS; Difco, Detroid, USA) supplemented different bacterial suspensions (24 h bacterial with 2.5 % (w/v) NaCl. Further experiments culture, 103–106 CFU/mL, final concentrations) were carried out only to characterize the most was run in the tests with 2-L beakers as virulent bacterial strains identified in the containers (Trevors and Lusty, 1985). To each 2-L beaker, 1L of autoclaved, aerated 3% The most abundant and virulent bacterial salinity sand-filtered seawater, 20 bacteria-free strain (viz. strain 21) was subjected to standard post-larval abalones and the appropriate morphological, physiological, and biochemical concentration of bacteria were added. Three examination. Gram-reaction, oxidase, catalase beakers without any added bacteria were used and hydrolysis of aesculin and gelatin were tested as described by Baumann et al. (1972) All twenty-nine isolates, recovered from the and Smibert and Krieg (1994). The ability to diseased abalone homogenate, were subjected use phenol as the sole carbon source was to challenge tests, performed over a three-day determined on minimal media that contained period and in triplicate. LD50 values were 2% NaCl (w/v) and 4 mM phenol for up to 7 calculated on day 3. LD50 values of >108 CFU mL-1 were considered a-virulent, and values of between 104 and 105 CFU/mL were considered virulent, while values between 106 and 107 production from carbohydrates with 1% (w/v) CFU mL-1 were considered weakly virulent in of each compound. Other biochemical tests line with the virulence criteria of Mittal et al. were carried out using API 20NE test kits 805 Jing & Junpeng Isolation, identification and phylogenetic analysis of a pathogen of… Marcy-I’Etoile, France) according to the included three cell wall synthesis inhibitors (ampicillin, 10 µg; cefamezin, 30 µg; and In order to characterize strain 21 at the molecular level, 16S and its rDNA sequencing permeability interferer (polymyxin B, 300 µg), and phylogenetic analyses was performed. eight protein synthesis inhibitors (amikacin, 30 Strain 21 was grown overnight at 25°C in µg; chloramphenicol, 30 µg; erythromycin, 15 marine broth with shaking at 180 rpm. Cells µg; gentamicin, 10 µg; kanamycin, 30 µg; from cultures were harvested by centrifugation neomycin, 30 µg; streptomycin, 10 µg; and tetracycline, 30 µg) and four nucleic acid suspended in 1x TE buffer (10 mM Tris-HCl, synthesis inhibitors (ciprofloxacin, 5 µg; norfloxacin, 10 µg; novobiocin, 5 µg; and rDNA of strain 21 were run as reported by Cai Escherichia coli ATCC 25922 was also et al. (2006b). After confirmation of successful included in the analysis as a control bacterium. amplifications by electrophoresis of 5 µL PCR Antibiotic sensitivity to a particular antibiotic products on a 1% agarose gel, the products (i.e. sensitive, intermediately sensitive or were purified using a PCR purification kit resistant) was assessed according to the (Takara, China). PCR direct sequencing was recommended cut-off levels of the zone size. done as reported by Thompson et al. (1992). Sequence data was then deposited in GenBank. Twenty-nine representative colonies were isolated from sampled diseased post-larval phylogenetically closest to the sequence of abalone during the disease outbreak in July 2010 in Shanwei. Selection of these colonies determined using the Neighbor-Joining method was based on differences in morphology and (Saitou and Nei, 1987) with the program EGA their relative abundances on marine agar plates. version 4.0. The stability of inter-relationships One isolate, designated as strain 21, was found of bacteria in the phylogenetic tree was to be the dominant colony on the plates. assessed by performing bootstrap analysis with Bacterial challenge tests were performed on all 29 strains. Results showed that strain 21 Sensitivity of strain 21 to 16 various
was the most virulent isolate, killing 100 % of chemotherapeutic agents
the post-larvae on day 3, and had a LD50 value To investigate whether antibiotics could be of 1.0×104 CFU mL-1 (Table 1) while 3 of the used in the control of strain 21, sensitivity other isolates (strains 1, 9 and 29) were weakly experiments were performed as reported by virulent with LD50 values ranging from 2.3 Cai et al. (2006a).The discs used in the assay ×106 CFU mL-1 to 4.5 ×107 CFU mL-1, and the Iranian Journal of Fisheries Sciences 12(4), 806 remaining 25 isolates were classified as were also observed in the post-larvae of the a-virulent with LD50 values greater than 1.0 ×108 CFU mL-1. No mortality was observed in Bacteriological examinations showed that the controls. Gross symptoms observed in strain 21 was re-isolated as pure cultures from moribund post-larvae during natural outbreaks moribund post-larvae from challenge tests and thus Koch’s postulates were fulfilled. Table 1: LD50 values on day 3 post-infection, calculations based on challenge tests carried out on
the 29 isolates recovered from diseased post-larval abalone
LD50 value*
LD50 value
(CFU mL-1)
(CFU mL-1)
mine, maltose, gluconate, and adipate. Growth analysis (Table 2) showed that strain 21 was a was detected when cultivated in minimal salts Gram-negative rod and an obligate aerobic. It medium that contained 2% NaCl and phenol as the sole carbon and energy source. Based on requirement for growth, nitrate reduction, these features and comparisons with those of caprate, malate and citrate utilization. It was (=ATCC27123T) in API 20NE tests, strain 21 acidification, arginine dihydrolase, gelatin and conformed to the description of O. doudoroffii aesculin hydrolysis and utilization of glucose, arabinose, mannose, mannitol, N-acetylglucosa- 807 Jing & Junpeng Isolation, identification and phylogenetic analysis of a pathogen of… Table2: Physiological characteristics of strain 21 as revealed by API 20NE test
Strain 21
DSM 7028T
To further characterize strain 21 at the 2004). A similarity search done by using the molecular level, 16S rDNA PCR sequencing BLAST program showed that strain 21 had the analysis was performed. PCR amplification of closest relationship with O. doudoroffii DSM 16S rDNA and the it’s region yielded an 7028T (99% similarity, AB019390) (Fig. 1) amplicon of approximately 1.8 kp in size. PCR similarities with O. baumannii strain GB6 sequences of 16S rDNA and its region of strain (AF168367) and Oceanisphaera litoralis DSM 15406 (AJ550470), respectively. Thompson et Formatted: Font: (Default) Times New Roman,
11 pt, Complex Script Font: B Nazanin, 11 pt
al. (2004) pointed out that 16S sequence similarities of ≥ 97% could be considered as (1331 bases) of strain 21 was manually aligned the same species, and thus strain 21 was using the clustal method in the megalign identified at the molecular level as O. program (DNAStar) with its related bacteria in the Order Aeromonadales (Martin and Joseph, Iranian Journal of Fisheries Sciences 12(4), 808 Table 3: Sensitivity of strain 21 to various chemotherapeutic agents
Chemotherapeutic agents
Disc content (µg)
Sensitivity a
a R: resistance; S: sensitive; MS: moderately sensitive
challenge tests also revealed that only strain 21 was highly virulent to post-larvae as judged by chemotherapeutic agents examined, as shown in Table 3, indicating that strain 21 exhibited virulent and the rest 25 were a-virulent. This suggests that post-larval abalone is more examined and was resistant to norfloxacin, susceptible to strain 21 than its counterparts Strain 21 was Gram-negative rods, obligate Discussion
aerobic, oxidase-positive and could utilize In the current study, even though there were malate, succinate, citrate and galactose for 29 representative colonies isolated on 2216E growth. On the basis of these biochemical agar plates from the diseased post-larval characteristics (Table 2) and comparison with abalone sampled during the disease outbreak in the API 20E results of the reference strain July 2010 in Shanwei, only strain 21 was predominant on the agar plates. Live bacterial 809 Jing & Junpeng Isolation, identification and phylogenetic analysis of a pathogen of… (Geoffrey et al., 2001) were its closest confirmed to be O. doudoroffii. The results of the 16S rDNA sequencing revealed that strain litoralis DSM 15406T(Ivanova et al., 2005), 21 shared 99% similarity with the type strain Tolumonas auensis DSM 9187T(Fischer et al., of O. doudoroffii viz. strain Bry (ATCC 1996) and the genus Aeromonas were more 27123T, accession number AB019390). Strain distantly related. This relationship is displayed 21 may therefore be considered as O. in the 16S rRNA gene sequence dendrogram doudoroffii. Phylogenetic analysis based on the based on the additive treeing algorithm of nearly complete 16S rRNA gene sequence of strain 21 indicated that O. doudoroffii ATCC 27123T and O. baumannii ATCC 700832T 100 Aeromonas enteropelogenes X71121 Oceanisphaera litoralis AJ550470 Fig. 1 Dendrogram of 16S rRNA gene sequence relatedness, showing that strain 21 is phylogenetically
closely related to members of the genus Oceanomonas, and shares 99% 16S rRNA gene sequence
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similarity with Oceanomonas doudoroffii DSM 7028T. Numbers at branching points refer to bootstrap
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values (500 re-samplings).
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Iranian Journal of Fisheries Sciences 12(4), 810 the prevent disease outbreaks on abalone farms. experiments, a dose of 1 ×106 CFU/mL of A possible alternative to the use of antibiotics strain 21 was able to cause 100 % mortalities may be the application of probiotics, especially within 3 days, and had an LD50 value as low since Macey and Coyne (2005) demonstrated as 1.0 ×104 CFU/mL, while the other 3 strains were classified as weak virulent with LD50 resistance in Haliotis midae, after treating the values ranging from 1.0 ×106 CFU mL-1 to 1.0 ×107 CFU mL-1, and the remaining 25 isolates were classified as a-virulent with proteomics of these extracellular products as LD50 values greater than 1.0 ×108 CFU mL-1. targets for therapy or prophylaxis of this This is well correlated with the severity of the deadly infection in post-larvae of abalone H. actual outbreak of post-larval disease as only about 90% of the mortality occurred in that particular outbreak and about 50 post-larvae Acknowledgements
were still retained on nearly every bio-film after the administrations of antibiotics. Furthermore, the same bacterium could be post-larvae after bacterial challenge and References
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