Studies On The Identification Method Of Anisakid Nematodes In Sea Fish And Investigates On The Infection Of Fujian Coast

Anisakis are the main parasitic nematodes in major marine fish in the world, and anisakiasis is a natural marine disease. Human beings catch anisakiasis was mainly because of ingesting some species of anisakis of larva 3. In 1993, anisakiasis was included in the"prohibited the entry of animal infectious diseases and parasitic diseases list of The People's Republic of China". Because of being a big fish consumption country, accurate identification of the types of Anisakis are not only the premise and foundation in the study of anisakis,but also has the great significance in the prevention and treatment of human and animal anisakiasis.In this study, anisakis infection in farmed fish on the coast of Fujian were investigated, Pagrosomus,Bass,Hairtail and Sciaenops ocellatus have the highest infection rates, the time mainly concentrated in July to September, and the infection rates of sample can be reached 100.00%. Among them, Pagrosomus have the highest intensity of infection, in early August, infection intensity achieved 43.00 per tail. The positive detection rate of Pacific saury and marine mollusks such as squid, cuttlefish is zero, but they couldn't rule out the possibility of infection probably anisakis. For the import of sea fish of Fuzhou port,hairtail and eels had the highest infection rate, even up to 100.00%, and the infection intensity of eels could up to 59.43 per tail. And the infection rate of the squid, cuttlefish and other marine mollusks was zero, which was consistent with the survey result of the Fujian coast. The vitro survival test showed that:under the different temperatures, anisakis had the longest survival time under 6℃, that was similar to fish survive temperatures, under this temperature, anisakis could survive up to a month's time, that reflected persistent infection of anisakis in fish body. At optimum survival temperature in different spices of trials, anisakis had the shortest survive time in 56°white wine, followed by garlic juice. Unexpectedly,in other spices, anisakis can survive long time, which proved that is impossible to kill anisakis larvae after being pickled in short-time.Traditionally, the classification of anisakis ways mainly morphological identification. Based on previous work experience, Chapter II of this study introduced how to use morphological method to make a preliminary identification to anisakis. Collected Anisakis larvae had been identified to genus (individual types can be identified to species). The results of morphological identification showed that in the body of Pagrus major, Yellowtail fish and other breeding fish on the coast of Fujian, there were Anisakis simplex, and the anisakis could be divided into type-Ⅰand type-Ⅱ. The anisakis collected from the hairtail of Malaysia,the bigeye snapper of Indonesia and the Chicken grunt, American red guppy of Fujian coast are almost all Hysterothylacium Larvae.Moreover, anisakis collected from the hairtail of Malaysia, the bigeye snapper of Indonesia and the American red guppy are almost all Contracaecum murenesoxi. In this study, Molecular Identification and morphological classification method had been combined to get the better identification result, and set up a rapid, accurate specific PCR detection methods. First of all, used the universal primers to amplify the spacer fragments, and send to sequence. Then comparised the acquired sequence with the corresponding sequence in Genbank in NCBI net, most of the anisakis could be indetified to species. Through sequencing, in this study, samples of anisakis could be identified as:A. pegreffii, C. murenesoxi, A. typica, A. physeteris and H. aduncum. There were suspected about some samples, then used molecular methods to identify. The samples such as:FJ-1, FJ-2, FJ-3, SD, SH and HY-1 couldn't determined to species only identified by sequence, in this study, using PCR-RFLP and mismatch PCR method to identify separately To:A. pegreffii, A. pegreffii, A. pegreffii, A. pegreffii, A. pegreffii and Hybrid genotype.Secondly, it was the design of specific primer, then set up specific PCR detection methods of anisakis. In this study, designed the specific primers of Family, Genus and Species, among them, primer-K1 is merger primer. Experiments had proven that all the primers had good specificity, other comparison DNA samples of worm and the host were non-amplified. In this study, the sensitivity results of specific PCR showed that, the sensitivity range can up to 0.00689ng - 1.03906 ng, mainly with the design of the primers, the sensitivity at a high level.In this study, anisakis infection in farmed fish on the coast of Fujian were investigated, the results showed that different species fish infected different species anisakis and had different intensity also, there was a obvious tendency of the season. Those results could play a guiding role to the disease prevention and control in marine fish in the actual production. At the establishment of specific PCR method, this study made specific PCR identifications to Genus and Family, this is novel and characteristics places in this study. The method established in this study could be applied in inspection and quarantine department to identify anisakis quickly and accurately. The results of this study laid the foundation for further study of anisakis.