Detection and Quantification of Total and Pathogenic Vibrio parahaemolyticus in Anadara subcrenata in the Zhoushan Archipelago

This study aimed to investigate the prevalence of total and pathogenic Vibrio parahaemolyticus in Anadara subcrenata sampled from aquafarms and retail markets in the Zhoushan islands during June 2013 to March 2015, using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 265 (83.86%) samples with the density 0.3 to 2400 MPN/g. In total, 30.70% and 17.41% of the samples exceeded 100 MPN/g and 1,000 MPN/g, respectively. Both highest positive rate (98.99%) and highest prevalence (median = 210.0 MPN/g) were recorded in summer. Samples from aquafarms had a higher positive rate and median than those from retail markets. Pathogenic V. parahaemolyticus was detected both in aquafarms and retail markets in all seasons but not in winter. Among the 265 tlh-positive samples, 20 (7.55%) of the samples harbored tdh, and 5 (1.89%) of the samples harbored both tdh and trh. These results indicate that the Zhoushan archipelago is severely contaminated with V. parahaemolyticus in Anadara subcrenata; these results are applicable in risk assessment and to control the risk of food-borne disease caused by V. parahaemolyticus.


Introduction
Vibrio parahaemolyticus is a marine microorganism, recognized as a primary pathogen of seafood-borne illnesses in North America, Europe, and Asia [1][2][3][4]. In recent years, V. parahaemolyticus has also become the primary pathogen causing food-borne gastroenteritis in coastal cities of China [4]. e Zhoushan archipelago, East China Sea, is famous for its abundant fishery resources and sceneries for tourists. Seafood, especially blood clam (Anadara subcrenata), is the principal food among local residents. However, V. parahaemolyticus has been reported to be commonly present in the culture environment used to grow shellfish, including blood clam [2]. Individuals are faced with a high risk of bacterial infection via consumption of raw or uncooked shellfish, especially blood clam, which is considered the primary cause of V. parahaemolyticus infection [1].
In the United States, consumption of raw or undercooked shellfish results in approximately 35,000 domestically acquired food-borne V. parahaemolyticus infections annually [5]. Many infections are epidemiologically associated with the consumption of shellfish contaminated with pathogenic strains [6].
erefore, controlling V. parahaemolyticus contamination in seafood can effectively prevent food-borne disease, and it is important to determine the levels of contamination of V. parahaemolyticus both in aquafarms and in retail markets [7]. e pathogenesis of V. parahaemolyticus infections has been studied extensively, and the thermostable direct hemolysin (TDH) encoded by tdh and TDH-related hemolysin (TRH) encoded by trh are considered pathogenic genes which are present mostly in clinical strains [8]. Recognized as primary virulent factors of V. parahaemolyticus, either or both toxins encoded by trh and tdh can cause illness [9]. Several methods have been described to determine levels of contamination of V. parahaemolyticus; however, the results have been inconsistent in accordance with the analytical method.
us, this study aimed to detect and quantify the total and pathogenic V. parahaemolyticus in blood clam from aquafarms and retail markets in the Zhoushan islands, using the most probable number-polymerase chain reaction (MPN-PCR) method [10], analyzed the contamination levels of V. parahaemolyticus, and provide evidence for risk control of food-borne disease.

Samples.
From June 2013 to March 2015, 316 Anadara subcrenata samples were collected between 10th to 19th every month from aquafarms and retail markets in Zhoushan. Each month, 5-10 samples were purchased from the primary 2 retail markets (Donghe markets in the Putuo district and Qiandao market in the Dinghai district) and 3 seafood restaurants (Xiangnan Hotel and Jiaotong Hotel in the Dinghai district and Yihao Yugang in the Putuo district), and 5-10 samples were collected from two aquafarms (Dongshao aqua farm and Daidong aqua farm, 30.28N, 122.22E) in the Daishan island (Table 1). Each sample consisted of 10-12 individual clams, but dual to the size difference, sample weight ranges from 100 g to 300 g. All samples were placed in sterile, labeled, sealed plastic bags on ice and were immediately analyzed on the day of sampling.

MPN-PCR Method.
e MPN-PCR method was used to estimate V. parahaemolyticus in 3 density levels in the samples [11]. Twenty-five Gram samples were fully cut into pieces with aseptic scissors in homogeneous cup after separated from shells and combined with 225 mL of alkaline peptone water (APW) containing 3% NaCl (Huankai, Guangzhou, China) to generate a 1 : 10 dilution. ree serial 10-fold dilutions were applied (10 − 1 to 10 − 3 ), and all dilutions were incubated at 37°C for 16-18 h. In total, 1.5 vmL of the MPN culture was centrifuged and resuspended twice. DNA was extracted by resuspending samples in 200 μL of DNA extraction buffer (Zhuocheng, Shanghai, China) and heated in 100°C for 10 min. e suspension was centrifuged at 3,000 ×g for 1 min, and the supernatant was used as the template DNA. V. parahaemolyticus tlh, tdh, and trh were detected via Roche LightCycler ® 480II real-time PCR system. e primer pairs and probes used for detection are enlisted in Table 2, and the reactions were performed as described by Ward et al. [12].
e PCR cycling conditions were as follows: denaturationat 95°C for 120 s, followed by amplification for 45 cycles, each comprising denaturation at 95°C for 20 s, annealing at 56°C for 20 s, and extension at 72°C for 30 s. e fluorescence signal was measured and recorded at the end of the annealing step of each cycle.

Statistical Analysis.
e MPN value was normalized before analysis. e MPN value was normalized as follows: the MPN value under 0.30 MPN/g was estimated to be 0.15 MPN/g; over 110 MPN/g, 240 MPN/g; and over 1100 MPN/g, 2400 MPN/g [10]. In accordance with the month of sampling, all samples were classified into four seasons: spring (March to May), summer (June to August), autumn (September to November), and winter (December to February). Samples were also determined on the basis of two sampling resources: aquafarm and retail market. e results were analyzed using Pearson's chi-squared test for the prevalence of total and pathogenic V. parahaemolyticus. To compare differences among sampling seasons and methods, considering nonnormal distribution of the resulting data, two non-parametric tests (the Levene's robust test and Kruskal-Wallis nonparameter test) were performed for analysis. e correlation coefficient between total and pathogenic V. parahaemolyticus contamination levels was determined via Pearson's correlation (Spearman's correlation) analysis. A P value of 0.05 was considered to determine statistical significance. Statistical analysis was performed using Microsoft Excel add-in tools and the SPSS 20.0 software.

Results
e incidence and contamination levels of total V. parahaemolyticus in Anadara subcrenata are enlisted in Tables 3  and 4. Among the 316 samples, 265 (83.86%) were tested positive for tlh, thereby being identified as V. parahaemolyticus-positive. Levels of total V. parahaemolyticus vary from <0.3 to 2400.0 MPN/g with median 42.0 MPN/g. e samples of V. parahaemolyticus at each level (<10, 10-1 × 10 2 , 1 × 10 2 -1 × 10 3 , and >1 × 10 3 MPN/g) were 71, 97, 42, and 55. In total, 30.70% and 17.41% of samples exceeded 100 MPN/g and 1,000 MPN/g, respectively. Table 3 shows the median contamination levels of V. parahaemolyticus in Anadara subcrenata in all seasons. e positive rates among the four seasons were significantly different (χ 2 � 38.371, P < 0.01). Both the highest positive rate (98.99%) and the highest contamination level (median � 210.0 MPN/g) were observed in summer, while the lowest positive rate and contamination levels were observed in winter. In accordance with nonparametric tests, contamination levels of total V. parahaemolyticus were significantly different (χ 2 � 122.797, P < 0.001) among different seasons.
As shown in Table 4, the positive rate of V. parahaemolyticus in the samples from retail markets was 73.64%, which was lower than that of aquafarms (92.85%). e MPN/g estimate of V. parahaemolyticus in the samples from retail markets with the median 24 MPN/g was lower than that in aquafarms (53 MPN/g). For samples that exceeded 100 MPN/g and 1000 MPN/g, those fromaqua farms were 17.26% and 23.21%, greater than those in retail markets, respectively. e level of contamination was significantly different between aqua farms and retail markets total V. parahaemolyticus (P < 0.01).
Pathogenic V. parahaemolyticus strains were determined on the basis of the presence of one or both of the tdh and trh genes. Among the 265 tlh-positive samples, 7.55% (20 of 265) samples harbored tdh, and 1.89% (5 of 265) samples     harbored both tdh and trh. e total positive rate of pathogenic V. parahaemolyticus was 9.43%. e contamination rate of pathogenic V. parahaemolyticus with the median 6.4 MPN/g ranged from <0.3 to 240 MPN/g. Differently from total V. parahaemolyticus density, the highest density of pathogenic V. parahaemolyticus was generally detected in samples obtained during spring ( Table 5). None of the samples obtained during winter tested positive. e positive rate of pathogenic V. parahaemolyticus differed significantly among different seasons (P < 0.05).
Apart from total V. parahaemolyticus, both positive rate and density of pathogenic V. parahaemolyticus in samples obtained from retail markets were greater than those obtained from aquafarms, being generally lesser than 10 MPN/ g for both samples, which are shown in Table 6.
As shown in Table 7, apart from winter, pathogenic V. parahaemolyticus were detected in samples obtained from both aquafarms and retail markets in all seasons. e positive rate of pathogenic V. parahaemolyticus in aquafarms was greater than that in samples obtained from retail markets in all seasons, being significantly different only during autumn (P < 0.05). e highest density of pathogenic V. parahaemolyticus (240 MPN/g) was observed in samples obtained from retail markets during spring.

Discussion
e Zhoushan archipelago is rich in seafood resources, including Anadara subcrenata, a type of bivalve seashell consumed raw or semi-cooked by from the residents in Zhoushan. Owing to its special physical structure and soil environment preference, Anadara subcrenata can easily enrich V. parahaemolyticus in seawater [5]. erefore, the risk of food-borne illnesses caused by V. parahaemolyticus through Anadara subcrenata is relatively high [13]. With the rapid development of the Chinese economy, the consumption of seafood has increased greatly, not only in the coastal cities of China, but also in mainland China. V. parahaemolyticus distribution in Anadara subcrenata from the eastern coastal cities of China has been reported previously [14,15]. Using information relating to V. parahaemolyticus exposure from COVISS and CDC estimates that 62% of all V. parahaemolyticus illness cases are caused by consumption of raw oysters. US-FDA-predicted mean levels of total and pathogenic V. Parahaemolyticus in raw oysters at-harvest were 5.0-2100.0 MPN/g and 1.3-15.0 MPN/g in summer, <52.0 MPN/g and <0.1 MPN/g in winter.
In this study, we analyzed 316 Anadara subcrenata samples obtained during all four seasons from both aqua farms and retail markets in the Zhoushan area. e prevalence status of total V. parahaemolyticus in Anadara subcrenata observed in this study (83.86%) was greater than those reported previously in studies of samples from mainland China. e level of contamination of the total V. parahaemolyticus ranged from 0.3 to 2400.0 MPN/g with the median 40.0 MPN/g, concurrent with those reported previously in many other coastal cities in China [6,16,17]. e present results indicate that the positive rate of total V. parahaemolyticus in all four seasons indicated a high level of contamination (83.86%), comparing the results of Han [10] and Suffredini et al. [13], thereby reflecting a relatively high level of total V. parahaemolyticus contamination of the sea area of Zhoushan. is result shows an obvious seasonal distribution similar to those reported previously [3,10,18]. e level of contamination increased from spring and peaked in summer, reduced in autumn, and was the lowest in winter.
is temporal distribution of total V. parahaemolyticus reflected the effect of water temperature [19]. Some researchers found that in winter, V. Parahaemolyticus were not detected in seawater samples, but some were detected in seawater sediments. It could be speculated that V. Parahaemolyticus' possible survival mechanism was: V. Parahaemolyticus' survives in seawater sediments in winter, proliferates after the temperature rises in summer, and then pollutes seawater and seafood.
In contrast with many studies [20,21] reporting that the transportation and storage of seashells may increase contamination by V. parahaemolyticus; the present data indicate that aquafarms have a significantly higher abundance and prevalence (P < 0.01) of V. parahaemolyticus than retail markets.
ese findings indicate that aquafarms are the primary source of V. parahaemolyticus contamination in Zhoushan, and the spread of V. parahaemolyticus was effectively controlled during transport and storage of Anadara subcrenata. e Anadara subcrenata acquired from aquafarm was transported in 4°C to the main retail markets in Zhoushan.
e authorities shall effectively monitor the pollution of seafood and its by-products, ban fishing or close farms in highly polluted sea areas, and prevent high-risk foods from entering the retail market. V. Parahaemolyticus is sensitive to low temperatures, so iced seafood is considered to have little chance of transmitting V. parahaemolyticus. Because V. Parahaemolyticus is not heat resistant, it is the most effective way to prevent V. Parahaemolyticus poisoning when food is well cooked.
In certain cases, the prevalence of pathogenic V. parahaemolyticus tended to increase in samples contaminated with relatively high levels of total V. parahaemolyticus [22]. e positive rate of pathogenic V. parahaemolyticus in this study was 9.43%, which was relatively higher than that in numerous cities in China [18]. Pathogenic V. parahaemolyticus was detected in all the seasons except in winter. However, winter may not be considered an absolutely safe season [12]. e highest levels of tdh-and trh-positive V. parahaemolyticus were observed during spring, which may be associated with the uneven contribution of sample numbers [4]. Samples collected from retail markets presented higher levels of pathogenic V. parahaemolyticus contamination.
is result may be associated with a reduction in total V. parahaemolyticus density or with differences in the survival and tolerance of virulent and nonvirulent strains during transportation and storage.
Antimicrobials are overused in the treatment of infectious diseases in the aquaculture industry, which leads to the extensive use of antimicrobials that has led to the development of antimicrobial resistance among pathogens in aquatic products and has rendered antimicrobials ineffective. V. parahaemolyticus has been reported to have resistance to ampicillin, amikacin, kanamycin, tetracycline, ceftazidime, and cefotaxime [4,20].

Conclusions
Our study's results highlight both total and pathogenic V. parahaemolyticus in two food supply chains of Zhoushan across all four seasons, suggesting the need to improve strategies to prevent the occurrence of diseases [4,23] transmitted via consumption of Anadara subcrenata contaminated with pathogenic V. parahaemolyticus. e exceeding of restrictions set by Chinese Food Safety Standards GB29221 on ready-to-eat seafood, put Chinese consumers at a high risk with V. Parahaemolyticus-related gastroenteritis. Although most environmental V. Parahaemolyticus separations are nonpathogenic, consumers should still pay attention to and ensure that oysters are properly cooked before consumption. In addition, in order to prevent cross-contamination of wet markets and supermarkets, important measures must be taken, including the good hygiene practices in the handling of goods and the cleanliness of handlers and display areas. Furthermore, the continuous monitoring of the V. Parahaemolyticus prevalence and antibiotics should be carried on which it serves to derive data for risk assessment and to control the risk of food-borne diseases caused by V. parahaemolyticus in Zhoushan.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Additional Points
(1) e MPN-PCR method was used to estimate total and pathogenic V. parahaemolyticus. (2) High positive rate (83.86%) of total VP was detected in Anadara subcrenata in

Conflicts of Interest
e authors declare that they have no conflicts of interest.