| [Objective] Molluscan shellfish which are delicious and nutritious are a valuable commodity both as a food source and as the sustaining product of an important coastal industry. However, with the rapid development of industry and agriculture in coastal cities of China, the waste emissions into the sea arises people's attention. Shellfish generally cultivated at coastal beach are filter feeders, and as a result, they may incidentally bioaccumulate pathogenic microbes from wastes to concentrations greater than present in the water column. Consequently, contamination of shellfish growing waters can create both public health and economic concerns. Thus, the practice of consuming raw or partially cooked shellfish increases the risk of contracting shellfish-related illnesses which can easily cause the symptoms of abdominal pain, diarrhea, vomiting and even threaten lives. Therefore, establishing an aquaculture environment monitoring and management system is imperative. This work is conducted to establish a host-orign fecal E. coli DNA fingerprint library by rep-PCR method from livestock and poultry farms in the shellfish culture area of East China Sea. Moreover, the efficacy of microbial source tracking based on rep-PCR for differentiating host sources of E. coli from shellfish and their growing waters is also evaluated. To the best of our knowledge, this is the first attempt to present the major sources of fecal pollution from agricultural wastes in the shellfish culture environment of East China Sea. Our findings will be useful for the water pollution control authorities in development of appropriate management plan(s) to prevent further fecal contamination by agricultural wastes in shellfish growing waters, and quality control measures of shellfish.[Methods] Fecal samples of poultry and livestock were obtained as cloacal swabs from different farm sites in Xiangshan Bay. The Xiangshan Bay includes Xieqian Harbor, Xihu Harbor and Da Mutu Harbor. Fecal samples were obtained as swabs and streaked within 12 h of collection onto mFC agar (Difco) by cultivation at 44.5℃overnight. The presumptive E. coli colonies were picked and streaked onto mFC agar plates (Difco) by cultivation at 35℃,2 h and subsequent 44.5℃, overnight. Blue colonies on all the above plates were streaked onto the surfaces of MacConkey agar (Difco) plates. Pink colonies from the MacConkey plates were restreaked onto the Eosin Methylene Blue agar (Difco) to obtain the purple black and shiny colonies. Typical E. coli-like colonies were confirmed by IMViC test. E. coli isolates were stored in 40% glycerol solution at-80℃prior to rep-PCR fingerprinting. The genomic DNA of E. coli isolates was extracted by bacterial genomic DNA purification kit in accordance with the manufacturer's instructions. Four kinds of different rep-PCR methods were performed to construct the E. coli DNA fingerprint library. Quantity One 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 shellfish growing waters were predicted using MST method by multivariate analysis of variance (MDS).[Results] Total number of 100 fecal samples,15 shellfish samples and 3 water samples were collected, and 60,68,74 and 70 suspected E. coli strains obtained from feces of swine, ducks, geese and chickens, respectively, were isolated. After biochemical identification, the numbers of E. coli isolates were 50,54,58 and 54 from swine, ducks, geese and chickens, respectively. The proporation of isolation were 83.3%,79.4%, 78.4% and 77.1% for swine, ducks, geese and chickens, respectively. The optimal condition of gel electrophoresis was confirmed as:80V,1.5% (w/v) agarose gel,4℃and 4 hours. At this condition, the lowest similarity coefficient of fingerprint was 0.98. The reproducibility and stability affected by experiment conditions of fingerprints was also evaluated by showing the lowest similarity coefficient of 0.95. The evaluation of spatial and temporal stability of strains was performed by selecting 10 of E. coli subcultured individuals and assessing the similarity coefficient. The result showed that the lowest similarity coefficient of fingerprint was 0.96. Cluster analysis of REP-PCR, BOX-PCR, ERIC-PCR and (GTG)5-PCR profiles revealed 31,42 33 and 38 clusters, respectively. The REP-PCR DNA fingerprints analyzed by jack-knife algorithm were revealed RCCs with 90.7%,66%,77.8% and 55.2% of chickens, swine, ducks and geese E. coli isolates classified into the correct host source, respectively; BOX-PCR showing RCCs with 74.1%, 80%,92.6% and 94.8% of chickens, swine, ducks and geese, respectively; ERIC-PCR showing 64.8%,80%,81.5% and 75.9% of chickens, swine, ducks and geese, respectively; and (GTG)5-PCR showing 77.8%,90%,85.2% and 84.5% of chickens, swine, ducks and geese, respectively. The average rate of correct classification (ARCC) of REP-, BOX-, ERIC-and (GTG)5-PCR patterns was 72.4%,85.4%,75.6% and 84.4%, respectively. Based on the above host-origin library by discriminant analysis,12 unknown source strains from shellfish and growing waters were successfully discriminanted.[Conclusion] 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. The stability both of rep-PCR methods and finerprints generated from E. coli isolates can ensure the stability of the fingerprint database. Our results showed that the rep-PCR techniques with cluster and MDS analysis could successfully classify E. coli isolates into the correct source groups. This work suggests that rep-PCR fingerprinting can be a promising genotypic tool applied in the shellfish and growing water management on East China Sea for source identification of fecal pollution.
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