| Molluscan shellfish have always been regarded as top grade foods because of their rich nutrition and delicacy, particularly for their low-fat, high protein, and abundant in polyunsaturated fatty acids and zinc and other trace elements, known as "treasures of sea". However, with the rapid development both in economy of coastal cities and density of population in China, more and more agricultural wastes are directly discharged to the sea, which produced great threat to shellfish cultural industry along the coastal beaches of ports, because shellfish are filter feeders, and able to enrich various contaminants, including pathogenic micro-organisms and other toxic inorganic or organic compounds in cultural environment. Once their growing environment was polluted, consumer groups as the top of the food chain will directly face the threat of disease, particularly those people who like eating raw shellfish in the coastal cities. Thus, the practice of consuming raw or partially cooked shellfish increases the risk of contracting shellfish-related illnesses which can easily lead to many symptoms, such as abdominal pain, bloody diarrhea, vomiting and sometimes serious or even life-threatening. Therefore, we believe that there is need for establishing a systematic, effective and scientific shellfish aquaculture regulatory system to ensure that the production of high-value aquaculture products, and then to ensure consumers’safety and reliability when consuming. This paper intend to establish a host-origin fecal E. coli DNA fingerprint patterns library by PFGE method from livestock and poultry farms in the shellfish cultural area of East China Sea for exploring the feasibility of this efficient MST method applied to safe traceability field of shellfish food and surrounding shellfish cultural environment. Moreover, theoretical support will be provided for establishing system of aquaculture products and their related growing environment monitoring, thereby ensuring the safety of shellfish aquaculture production and consumption.Xianshan Bay was selected as experimental area. Fecal samples of poultry and livestock were obtained as clonal swabs from different farm sites in Xiangshan Bay. The Xiangshan Bay includes Xieqian Harbor, Xihu Harbor and Damutu Harbor. The poultry and livestock farms near these three harbors were selected as sampling sites, while shellfish and water samples were picked near (upstream, midstream and downstream) the fecal sampling sites. Subsequently, isolates from all kinds of samples were obtained by selective culture mediums (MAC, LTT and EMB mediums). Consequently, E. coli isolates were confirmed by IMViC test. E. coli isolates. The genomic DNA of E. coli isolates were extracted by CHEF Genomic DNA Plug Kit in accordance with the manufacturer’s instruction. PFGE was performed after the genomic DNA of E. coli isolates digestion with Xba I. Quantity One, InfoQuest?, and SPSS softwares were used to analyze the similarities, rate of correct classification (RCC) and the stability of library. Finally, host sources of E. coli obtained from shellfish and growing waters were predicted using MST method combined with Multidimensional Scaling (ALSCAL) analysis.Of total number of160fecal samples, duck-, goose-, chicken-and swine-sourced were collected to be each40. And suspected E. coli strains were112,77,86and98respectively after isolated. After biochemical identification, the numbers of E. coli strains were91,60,70and83obtained from ducks, geese, chickens and swine, respectively. The percentage of isolation was81.25%,84.42%,91.86%and84.69%for ducks, geese, chickens and swine, respectively. In addition, shellfish samples were collected in various ports of20or40, a total of100shellfish samples, and each sampling point of water samples according to the upper, middle and down streams were collected to be60altogether. The optimization of experimental methods drawn test: The optimal condition of experimental methods was confirmed:DNA entrapped volume of2.3～5.04*108/ml plugs, which OD6oo of between1.0and1.4. And proteinase K digestion time was selected lOhrs. PFGE parameters were confirmed:voltage gradient is6V/cm, pulse angle is120°, the IST (Initial Switch Time) is0.1s and FST (Final Switch Time)39s, a value of0(linear correlation), buffer temperature keep in14℃, and running time is around21.3hrs. The evaluation of spatial and temporal stability of strains were performed. The results showed that either CICC21524or XQ201006SWINE01, after15generations, their PFGE fingerprints were in the lowest similarity coefficient of about0.90. The reproducibility and stability may affect by Xba I enzyme digestion and PFGE test of fingerprints were also evaluated, and results showed good to us. The construction of fingerprint patterns showed that after cluster analysis, the PFGE profiles reached69clusters in all, including8wrong clusters. The PFGE DNA fingerprints analyzed by jack-knife algorithm were revealed RCC with91.21%,92.31%,88.61%and92.77%of ducks, geese, chickens and swine-sourced E. coli isolates classified into the correct host source, respectively, and then ARCC was got subsequently. Based on the above host-origin library by discriminant analysis,26and13unknown-sourced strains respectively isolated from shellfish samples and related water samples were successfully traced, and the percentage of correct tracing reached29.89%(26/87) and17.57%(13/74), respectively.Some conclusions are obtained:1. The methods of E. coli isolation and biochemical identification used in this study were reliable and effective, and can provide a large number of strains for establishing the fingerprint library.2. The stability PFGE methods and fingerprint patterns generated from E. coli isolates can ensure the stability of the fingerprints library, at least within20generations.3. The series of optimized PFGE operational methods are good in reproducibility and operability.4. Our results showed that the PFGE techniques with Cluster and ALSCAL analysis could successfully classify E. coli isolates into the correct source groups. This paper suggests that PFGE fingerprinting can be a promising genotypic method applied in shellfish and growing water management on East China Sea for source tracking of fecal pollution. |
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