Aquaculture 418–419 (2014) 171–176

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Aquaculture
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Reductions of Vibrio parahaemolyticus in oysters after bacteriophage

application during depuration
Rong Rong a, Hong Lin a, Jingxue Wang a,⁎, Muhammad Naseem Khan a,b, Meng Li a
a
Food Safety Laboratory, Ocean University of China, Qingdao 266003, PR China
b
Microbiological Analytical Centre, FMRRC, PCSIR Labs. Complex Karachi, 75280, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Vibrio parahaemolyticus is closely associated with oysters in China, which are normally consumed raw or lightly
Received 10 September 2013 cooked. Bacteriophages are a safe bio-controlling agent, and have been recognized in aquaculture for their path-
Accepted 17 September 2013 ogen reduction properties. This study investigated the potential application of the bacteriophage VPp1 during
Available online 25 September 2013
depuration to reduce V. parahaemolyticus in oysters at different multiplicity of infection (MOI) and temperature
levels. Oysters were infected with 105, 106, and 107 colony-forming units (CFU)/mL of V. parahaemolyticus in the
Keywords:
Vibrio parahaemolyticus
seawater and each infected group was treated with three different MOI values: 10, 1, and 0.1. Infected oysters
Bacteriophage were depurated in non-recirculating seawater at 22 °C, 20 °C, 16 °C, and 12 °C for 36 h. Results revealed that
Depuration temperatures b20 °C were safe for oyster rearing. Depuration at 16 °C with 0.1 MOI was the best condition for
Oyster reducing V. parahaemolyticus in oysters, which decreased by 2.35–2.76 log CFU/g within 36 h. This study pro-
vides the basis for the use of bacteriophages in depuration techniques to eliminate V. parahaemolyticus in oysters.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Vibrio parahaemolyticus is an enteric pathogen that is widely distrib-
uted in coastal areas, and is a causative agent of gastroenteritis in
humans after consumption of contaminated seafood (Feldhusen,
2000; Potasman et al., 2002; Sala et al., 2009; Su and Liu, 2007).
V. parahaemolyticus are the most prevalent seafood-associated patho-
genic bacteria in the world, and are frequently found in the oysters of
China (Chen et al., 2010; Drake et al., 2007). Oysters are filter feeders
that accumulate food particles and small organisms by circulating large
volumes of seawater through their system; consequently, microorgan-
isms, including human pathogens, become absorbed along with nutri-
ents and accumulate in their bodies (Lees, 2000; Meujo et al., 2010). In
China, oysters are consumed raw or lightly cooked; therefore, contami-
nated oysters are potential vectors for pathogenic V. parahaemolyticus
(Larsen et al., 2013; Lees, 2000; Ye et al., 2012).
Many methods have been applied to eliminate human pathogens, in-
cluding Vibrio spp., from oysters. Several post-harvest processes (PHP)
have been reported previously: heat/cool pasteurization (Andrews
et al., 2000; Cook, 2003), irradiation (Andrews et al., 2003; Mahmoud,
2009; Mahmoud and Burrage, 2009), high hydrostatic pressure (Kural
and Chen, 2008; Ma and Su, 2011; Prapaiwong et al., 2009), ultraviolet
(UV) exposure (Hamamoto et al., 2007; Phuvasate et al., 2012), and
rapid freezing with frozen storage (Liu et al., 2009). Recently, combined
PHP techniques were also studied, such as high hydrostatic pressure
combined with moderate heating (Ye et al., 2012). However, all of
⁎ Corresponding author. Tel./fax: +86 532 82032389.
E-mail address: snow@ouc.edu.cn (J. Wang).
these processes have shown negative effects on the consumers' culinary
experience of oysters. Furthermore, several oysters commonly die during
these processing techniques. All of these limitations of PHP influence the
consumption of oysters by consumers (Chae et al., 2009; Larsen et al.,
2013).
Shellfish depuration is a method applied to eliminate human
pathogens from live oysters. Depuration is a control process in
which shellfishes are reared in seawater treated by chlorine, ozone,
or UV light for a few hours in order to reduce pollutants in their bodies
by filtering (Croci et al., 2002; Rodrick and Schneider, 2003; Wang et al.,
2010). This method was established for reducing fecal contamination in
shellfish, such as Salmonella and Escherichia coli, but is generally not
effective against Vibrio spp. (Lee et al., 2008; Otwell et al., 1991). How-
ever, low temperature depuration was found to be effective in reducing
V. parahaemolyticus in oysters by 2–3 log as estimated by most probable
number (MPN)/g (Phuvasate et al., 2012); although this process took
several days. Additionally, the chemical treatments commonly used
for seawater disinfection result in chemical hazards in seafood. The de-
velopment and evaluation of new strategies, with no adverse effects on
the oysters, to reduce V. parahaemolyticus in raw oysters is a prospective
research area.
Feeding oysters with bacteriophages that are natural and harmless
to humans or animals is a relatively inexpensive method (Sulakvelidze
et al., 2001). Because bacteriophages are compatible with food, they
can be applied for therapeutic or bio-sanitization purposes (Hanlon,
2007; Skurnik and Strauch, 2006). The potential of bacteriophages to
control bacterial diseases has been supported by several studies demon-
strating an efficient reduction of pathogen levels in aquaculture species,
such as shrimp (Karunasagar et al., 2007; Vinod et al., 2006) and finfish

0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2013.09.028
172 R. Rong et al. / Aquaculture 418–419 (2014) 171–176
(Nakai and Park, 2002; Park and Nakai, 2003). However, the application
of phages in shellfish depuration has not yet been thoroughly studied.
This study was conducted to evaluate the effectiveness of a single bacte-
riophage (VPp1) aimed at V. parahaemolyticus, and to identify the mul-
tiplicity of infection (MOI) level along with optimum temperature to
achieve a better purification effect in live oysters infected (artificially
contaminated) with pathogenic V. parahaemolyticus (ATCC 17802).
2. Materials and methods
2.1. Phage preparation
The bacteriophage VPp1, a lytic phage which was previously isolated
from sewage samples by our laboratory (Yong et al., 2013), was proved
to be effective in lysing V. parahaemolyticus at a much lower MOI
(0.0001) and the phage titer was approximately 109 plaque-forming
units (PFU)/mL. It was replicated with V. parahaemolyticus in 2216E
broth (Qingdao Haibo Technology Co. Ltd.) at 37 °C overnight with rota-
tion (130 rpm). Double-plaque assays were performed, as described by
Adams (1959) to determine the phage titer, and phages were stored at
4 °C.
2.2. Bacterial strains
V. parahaemolyticus (ATCC 17802), the Japanese seafood outbreak
strain, was used to infect oysters. The strain was grown in 2216E
broth (Qingdao Haibo Technology Co. Ltd.) at 37 °C overnight with con-
tinuous shaking (130 rpm).
2.3. Oysters
Live oysters (Ostrea plicatula) were purchased from the NanShan
seafood market, Qingdao, China. Natural seawater was collected
from the Qingdao coastal area. Running water was used to wash
the oysters superficially. Then, the oysters were kept in a polyethyl-
ene tank at ambient water temperature for 24 h before being
infected with V. parahaemolyticus. The seawater was changed every
8 h during the pre-immersion period.
2.4. Accumulation of V. parahaemolyticus in oysters
Fresh bacterial culture was prepared (~109 colony-forming units
(CFU)/mL) and poured into a set of three aquaria with 50 L of natural sea-
water for different volumes (5 mL, 50 mL, and 500 mL). The final concen-
tration of V. parahaemolyticus in the three infected groups was 105, 106,
and 107 CFU/mL, respectively. And there was no V. parahaemolyticus
added in the negative control. Oysters were transferred to all four
aquaria and kept for 48 h. Dissolved oxygen (DO) was maintained
in the aquaria through an air pump at a rate of 120 L/min. This exper-
iment was performed during four different months at different ambient
water temperatures (August, ~22 ± 0.2 °C; September, ~20 ± 0.1 °C;
October, ~16 ± 0.1 °C; November, ~12 ± 0.2 °C). Water temperature
was measured with a thermometer every morning, noon and evening.
V. parahaemolyticus accumulated in oysters, and the mortality of oysters
was recorded regularly to optimize the appropriate seawater tempera-
ture and time of accumulation for the next step.
2.5. Oyster depuration by phage
Oysters from all three infected groups were transferred to the
depuration tank (0.3 × 0.6 × 0.2 m, without trays) separately. Oysters
were depurated in a non-flowing water system at the laboratory-
scale. Natural seawater used in depuration tanks was disinfected with
3% hydrogen peroxide solution. The depuration process with phage
was conducted at two ambient water temperatures, 16 °C and 12 °C,
for 36 h in two different months, October and November, respectively.
In each trial, oysters were depurated with the bacteriophage treat-
ment at MOI 10, 1, and 0.1.
2.5.1. Depuration at 16 °C
For each infected group (105, 106, and 107 CFU/mL), oysters
(n = 120) infected with V. parahaemolyticus were divided into four
treatment groups (n = 30 each): a) phage treatment with an MOI
of 10, b) phage treatment with an MOI of 1, c) phage treatment
with an MOI of 0.1, and d) no phage treatment (negative control
group).
2.5.2. Depuration at 12 °C
For each infected group (105, 106, and 107 CFU/mL), oysters (n = 60)
infected with V. parahaemolyticus were divided into two treatment
groups (n = 30 each): 1) phage treatment with an MOI of 0.1 and 2)
no phage treatment (negative control group). Oysters were not fed dur-
ing the whole experimental process.
2.6. Microbiological analysis
To determine the V. parahaemolyticus counts, oysters were sampled
from treatment groups at different intervals during the accumulation
and depuration process. For each bacterial count, three oysters were
randomly selected from every treatment group; oysters were homoge-
nized, and 10 g was blended in 90 mL (1:10 w/v) sterile physiological
saline in a sterile laboratory blender. The homogenized samples were
then ten-fold serially diluted, and 0.1 mL from each dilution was spread
on thiosulfate citrate bile salts sucrose (TCBS) agar plates. All plates
were incubated at 37 °C for 18 to 24 h (Qingdao Luqiao Technology
Co. Ltd.). All experiments were performed in triplicate.
2.7. Data analysis
Results are presented as mean values, and standard deviations of the
mean are indicated with error bars. The counts of V. parahaemolyticus in
different groups were converted to base 10 logarithms before being an-
alyzed. Then the data were analyzed with SPSS 17.0 software (Chicago,
IL, USA). One-way analysis of variance (ANOVA) was used to compare
significant differences (P b 0.05) among various data sets. Figures were
constructed with OriginPro 8.5 software (OriginLab, Northampton, MA,
USA).
3. Results
3.1. Mortality–temperature relationships
The survivals of oysters associated with different levels of
V. parahaemolyticus accumulation at different temperatures, ranging
from 12 to 22 °C, are shown in Table 1. In the negative control (without
V. parahaemolyticus), the highest morality rates observed were 36% at
22 °C and 5% at 20 °C. No deaths occurred at 16 °C or 12 °C after 48 h
of V. parahaemolyticus accumulation, both in the negative control and
infected groups. The overall mortality rate was higher in infected oys-
ters (N 50%) compared with the negative control. Then in the following
experiment, 16 °C and 12 °C were selected.
3.2. Accumulation of V. parahaemolyticus in oysters
Live oysters were assessed after 48 h of pathogen accumulation at
16 °C (Fig. 1). Accumulation of V. parahaemolyticus in oysters increased
rapidly during the first 6 h, and then counts decreased in all three infected
groups and remained comparatively steady after 12 h and onward. Ac-
cumulation of V. parahaemolyticus in oysters at 12 °C showed the same
tendency (data not shown). This suggests that V. parahaemolyticus
could colonize the oysters up to a certain level after accumulation for
 R. Rong et al. / Aquaculture 418–419 (2014) 171–176 173

Table 1
Mortality of oysters at different water temperatures.

Temperature Vibrio parahaemolyticus Mortality of oyster during

(study month) infected group (CFU/mL) the processing period

6h 12 h 24 h 48 h

22 °C Control 4% 12% 24% 36%

(August) IG-1 (105) 8% 12% 36% 52%
IG-2(106) 8% 24% 40% 56%
IG-3(107) 8% 16% 36% 80%
20 °C Control – – – 5%
(September) IG-1(105) – – 5% 20%
IG-2(106) – – 10% 30%
IG-3(107) – – 20% 50%
16 °C Control/IG- – – – –
(October) 1/IG-2/IG-3
12 °C Control/IG- – – – –
(November) 1/IG-2/IG-3

IG; infected group (CFU/mL).

Control; without V. parahaemolyticus.
“–”; no mortality.

Fig. 2. Changes of V. parahaemolyticus counts (log CFU/g) in infected group: 105 CFU/mL in
12 h. Therefore, phage treatments were performed after 12 h of immer- the seawater during depuration with different bacteriophage VPp1 treatment (MOI = 10;
sion with V. parahaemolyticus in the seawater. MOI = 1; MOI = 0.1) and negative treatment control at 16 °C. Means with the same let-
ter are not significantly different (P N 0.05).

3.3. The efficiency of depuration at 16 °C

According to Results in Section 3.2, oysters were immersed with
V. parahaemolyticus for 12 h before depuration. After V. parahaemolyticus
accumulation at 16 °C, the initial mean values of bacteria in oysters
were 4.51, 4.98, and 6.08 log CFU/g in infected groups of 105, 106,
and 107 CFU/mL, respectively (Figs. 2, 3 and 4).
According to Fig. 2 (the infected concentration of V. parahaemolyticus
was 105 CFU/mL), after 36 h of depuration, oysters treated with phage
VPp1 (MOI = 10 and MOI = 0.1) both achieved significantly reductions
of 1.99 and 2.35 log CFU/g based on the data analysis (P b 0.05). The
reduction of oysters treated with phage VPp1 (MOI = 1) was
1.74 log CFU/g. The reduction of V. parahaemolyticus in the negative
control group was only 1.24 log CFU/g.
According to Fig. 3 (the infected concentration of V. parahaemolyticus
was 106 CFU/mL), after 36 h of depuration, compared with the reduction
in the negative control group (1.98 log CFU/g), the significant reduction
(P b 0.05) in V. parahaemolyticus count with bacteriophage treatment of
MOI = 1 and MOI = 0.1 was 3.00, and 2.73 log CFU/g, respectively. In
contrast, the bacteriophage treatment of MOI = 10 showed a slight re-
duction of 2.22 log CFU/g.
Significant reductions in V. parahaemolyticus counts were also ob-
served in Fig. 4 (the infected concentration of V. parahaemolyticus was
107 CFU/mL). The significant reduction (P b 0.05) was observed in the
treatment of MOI = 0.1, with approximately 2.76 log CFU/g reduction,
which was statistically different from the 1.82 log CFU/g reduction ob-
served in controls (untreated group). Reduction of the other two treat-
ment groups (MOI = 10; MOI = 1), was 2.33 and 2.32 log CFU/g,
respectively.
Figs. 2, 3 and 4 further showed that bacteriophage treatment of
MOI = 0.1 could achieve significant reduction in all infected groups.
Thus MOI = 0.1 was used in the following study (depuration at 12 °C).

7 a
V. parahaemolyticus in oysters (lg CFU/g)

b
6 c

0 10 20 30 40 50
Time (h)
Fig. 3. Changes of V. parahaemolyticus counts (log CFU/g) in infected group: 106 CFU/mL in
Fig. 1. Accumulation of V. parahaemolyticus in oysters in different infected groups after the seawater during depuration with different bacteriophage VPp1 treatment (MOI = 10;
48 h. Final concentrations of V. parahaemolyticus (log CFU/g) in infected group MOI = 1; MOI = 0.1) and negative treatment control at 16 °C. Means with the same let-
(a):105 CFU/mL; infected group (b): 106 CFU/mL and infected group (c):107 CFU/mL. ter are not significantly different (P N 0.05).
174 R. Rong et al. / Aquaculture 418–419 (2014) 171–176
3.4. The efficiency of depuration at 12 °C
According to Results in Section 3.2, oysters were immersed with
V. parahaemolyticus for 12 h before depuration. After V. parahaemolyticus
accumulation at 12 °C, the initial mean value of bacteria in oysters was
4.61, 4.90, and 6.09 log CFU/g in infected groups of 105, 106, and
107 CFU/mL, respectively.
According to Fig. 5 (MOI = 0.1 was used), the counts of V.
parahaemolyticus in oysters were reduced by 1.02, 1.78, and
1.29 log CFU/g after 36 h depuration of infected group A (105 CFU/mL),
B (106 CFU/mL), and C (107 CFU/mL), respectively.
Though reductions of accumulated V. parahaemolyticus in oysters
were significant at 12 °C after 36 h of depuration, the final counts at
12 °C were nonetheless higher in all infected groups compared to
those depurated at 16 °C under the same infected concentration of
V. parahaemolyticus. Thus, the lower depuration temperature had a
negative impact on depuration efficiency for the same infected con-
centration of V. parahaemolyticus.
4. Discussion
Bacteriophages, as specific pathogen-killers, are effective agents for
controlling bacterial infections in aquaculture, and do not affect normal
flora (Hawkins et al., 2010; Park and Nakai, 2003). Protective effects of
bacteriophages have been reported across different fields by many re-
searchers (Bueno et al., 2012; Lim et al., 2012; Oliveira et al., 2010;
Stelios et al., 2011; Steven et al., 2011).
Oysters are always sold live, and depuration is often the preferred
method for controlling microbiological contamination throughout
the world (Lees et al., 2010; Rodrick and Schneider, 2003). Elimina-
tion of V. parahaemolyticus in oysters is important for public health,
as this pathogen could easily accumulate in raw oysters (Cliver,
1995; Lees, 2000). Bacterial elimination mostly depends on the
self-purification (depuration) process that is achieved by filtering
oysters (de Abreu Corrêa et al., 2007); however, this process could not
effectively purge V. parahaemolyticus from their tissues (Lee et al.,
2008). Therefore, bacteriophage was applied as a new depuration
method for eliminating V. parahaemolyticus from live oysters.
Fig. 4. Changes of V. parahaemolyticus counts (log CFU/g) in infected group: 107 CFU/mL in
the seawater during depuration with different bacteriophage VPp1 treatment (MOI = 10;
MOI = 1; MOI = 0.1) and negative treatment control at 16 °C. Means with the same let-
ter are not significantly different (P N 0.05).
6.5
a
v.parahaemolyticus in oysters (lg CFU/g)
a b
6.0
c
bc b
5.5
c d
5.0
a
a
a
4.5 a
4.0 b c
b
3.5 c
b
3.0
d
0 5 10 15 20 25 30 35 40
Time (h)
Fig. 5. Changes of V. parahaemolyticus counts (log CFU/g) in infected group (a): 105 CFU/mL,
infected group (b): 106 CFU/mL, infected group (c): 107 CFU/mL in the seawater during
depuration with bacteriophage VPp1 treatment (MOI = 0.1) at 12 °C. Same letters in
one line indicate no significant difference (P N 0.05).
4.1. Optimize the appropriate seawater temperature and time of accumulation
An artificially infected oyster model, using different concentra-
tions of V. parahaemolyticus, was established to evaluate the effect
of the phage on oyster depuration. V. parahaemolyticus counts in oys-
ters decreased from 6 to 12 h (Fig. 1) of accumulation, which may
have been due to the self-cleaning of oysters; only a certain level of
bacteria could colonize in the body after accumulation. Therefore,
the accumulation of V. parahaemolyticus was performed for 12 h,
when accumulated bacterial counts were comparatively steady.
Moreover, higher mortality rates of the oysters were observed at
22 °C and 20 °C (Table 1). Similar results were found in previous
studies, indicating that higher temperatures increase the mortality
of oysters (Cheney et al., 2000; Soletchnik et al., 2007). Malham
et al. (2009) reported that oysters were immuno-compromised at
21 °C by reducing the numbers of hemocytes, and by decreasing the
phagocytosis activity in blood. Furthermore, oysters were kept in aerat-
ed natural seawater without continuous flow, which is not an optimal
environment for oysters. Thus, these results not only showed that ap-
propriate seawater temperature for oyster rearing should be lower
than 20 °C, but also showed that accumulation of V. parahaemolyticus
had adverse impacts on the survival of oysters (Karunasagar et al.,
2007; Martínez-Díaz and Hipólito-Morales, 2013). Our investigation of
a shellfish purification company revealed that the water temperature
applied in depuration was approximately 15–16 °C. Therefore, 16 °C
and 12 °C were selected as the depuration temperature for this study.
4.2. The influence of water temperature on the depuration results
To identify the most appropriate depuration temperature, the
depuration results at 12 °C (Fig. 5) and 16 °C (Figs. 2, 3 and 4) were
compared. When oysters were depurated at 16 °C, all treatment groups
achieved a 2–3 log CFU/g reduction of V. parahaemolyticus in oysters,
but when depurated at 12 °C, the reductions were only 1–2 log CFU/g.
These results revealed that a temperature of 12 °C failed to achieve a
better depuration effect than that at 16 °C under the same infected con-
centration of V. parahaemolyticus. Additionally, the optimum growth
temperature of the bacteriophage VPp1 is 37 °C (Yong et al., 2013).
Therefore, compared with 16 °C, the lower temperature of 12 °C was
less favorable for bacteriophage activity. Stelios et al. (2011) also
 R. Rong et al. / Aquaculture 418–419 (2014) 171–176
reported that low temperature (4 °C) resulted in reduced efficiency of
an E. coli bacteriophage mixture (its optimum temperature is also
37 °C) on hard surfaces. Moreover, phage activity and the oyster's
water-filtering activity were also affected at low temperature. At
lower temperatures, oysters could not discharge the bacteria in their
bodies effectively (Chae et al., 2009). Conversely, phages were reported
to decrease the concentration of Salmonella in some none-vitality ob-
jects, such as cheese (Bueno et al., 2012), chicken skin (Goode et al.,
2003) and melon slices (Leverentz et al., 2001), at low temperatures.
In this study, a 1.00 to 1.78 log CFU/g reduction of V. parahaemolyticus
was also achieved at 12 °C (Fig. 5). However, for the same infected
concentration of V. parahaemolyticus, the depuration process at this
temperature may need a longer time to obtain obvious reduction of
V. parahaemolyticus. Hence, the optimal depuration temperature
was determined to be 16 °C.
4.3. The influence of phage concentration on the depuration results
The phage concentration applied was another influencing factor
in depuration. The dynamics of phage-bacterium interactions differ
among various animal samples due to the complex physicochemical
environment, phage lysis activity, and host defenses. Hence, the bac-
teriophage concentration used in a particular case might show very
different effects in other cases. In this study, the relatively low dose
of bacteriophage had a significant effect on bacterial reduction.
According to Figs. 2–4, in depuration trials under a certain tempera-
ture, all treatment groups demonstrated that various MOIs (10, 1,
and 0.1) achieved a 2–3 log CFU/g reduction of V. parahaemolyticus
in oysters. Furthermore, significantly higher reduction was obtained
at the MOI of 0.1, as shown in Figs. 2 and 4.
Park and Nakai (2003) reported that treating ayu (Plecoglossus
altivelis) with bacteriophage at MOI 1 achieved good reduction of
Pseudomonas plecoglossicida. Nakai et al. (1999) e examined the pro-
tective effects of an anti-Lactococcus garvieae phage with an MOI b 1
by intraperitoneal injection or oral administration of phage against ex-
perimentally infected young yellowtails. Steven et al. (2011) found that
a high concentration (MOI N 10) of Salmonella typhimurium U288
phage cocktail could achieve successful therapy on artificially contami-
nated pig skins. Martínez-Díaz and Hipólito-Morales (2013) reported
that the phage vpms1 was effective in eliminating the adverse effects
of V. parahaemolyticus in brine shrimp, even at an MOI of 0.45. Specific
to our study, the application of bacteriophage at an MOI of 0.1 was
found to be the best choice. This application of a lower phage titer is
cost-effective.
4.4. Summary
In summary, the application of bacteriophage VPp1 (MOI 0.1)
produced the most significant reductions of V. parahaemolyticus on
infected oysters at 16 °C. Among all of the treatment groups, oysters
depurated with the bacteriophage at an MOI of 0.1 at 16 °C was the
most efficient condition. Under this condition, the bacteriophage VPp1
was capable of reducing V. parahaemolyticus by 2.35–2.76 log CFU/g
over a period of 36 h. These conditions produced better results
than when oysters were depurated in a pilot plant using ozone,
which reduced V. parahaemolyticus by b1 log MPN/g over 44 h
(Croci et al., 2002). Artificially contaminated oysters depurated
with UV-sterilized seawater could also reduce V. parahaemolyticus
counts by N3.0 log MPN/g, but over a much longer depuration pro-
cess (five days) (Phuvasate et al., 2012). Wang et al. (2010) reported
that all of the V. parahaemolyticus in oysters were disinfected within
6 h under the treatment of a chemical sanitizer, such as chlorine di-
oxide (ClO2), in the seawater. This retention of ClO2 in oyster tissues
may indicate new potential food safety hazards.
175
4.5. Outlook
Compared to physical and chemical depuration processing, bacterio-
phages have narrow host ranges, as most are active against a limited
range of strains within a bacterial species. In order to cover a sufficiently
wide range of bacterial strains, phage cocktails or phage lysin are still
needed. Currently, work is being carried out in our laboratory to achieve
these goals, such as preparation of the phage cocktails, DNA sequencing
of VPp1, and purification of the phage lysin of VPp1.
5. Conclusion
To our knowledge, this is the first report of a depuration trial using
bacteriophage in the oyster depuration process. In this study, the data
provide proof of the principle that the application of the bacteriophage
(VPp1) could reduce the population of V. parahaemolyticus in infected
oysters.
The depuration process described in the paper requires low initial
investment and running costs. Moreover, oysters could be maintained
alive during processing. Therefore, the application of bacteriophage
was effectively proven to be useful for shellfish depuration.
Acknowledgments
This work was supported by the National Natural Science Funding of
China (Grant No. 31071540) and the “National Science & Technology
Pillar Program” (2012BAD28B05).
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