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: [email protected]
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 Character 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 Formatted: Font: 10 pt, Italic, Complex Script Font: 10 pt, Italic similarity with Oceanomonasdoudoroffii DSM 7028T. Numbers at branching points refer to bootstrap Formatted: Font: 10 pt, Italic, Complex Script values (500 re-samplings). Formatted: Font: 10 pt, Italic, Complex Script Formatted: Font: 10 pt, Italic, Complex Script Font: 10 pt, Italic
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
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