Establishment Of Immunological Diagnostic Method For Anisakis And Construction Of Phase Ⅲ Larval CDNA Library

Anisakiasis is a foodborne parasitic disease caused by anisakid nematodes, when infective stage larvae (III stage larvae, L3) are ingested from sea fish that humans eat raw or undercooked. The main pathogenic species are Anisakis simplex and Anisakis pegreffii. The both of parasites can cause gastric, intestinal and non-gastrointestinal anisakiasis. The disease is distributed all over the world, and the cases were reported in Asia, especially in Japan, but not reported in China so far. However, the anisakid parasite can be found in seafish from yellow sea、bohai sea、 north sea and the coastal cities, in China. Since eating raw or undercooked sea fish is becoming more common, the number of cases with anisakiasis will be more and more. Up to now, the methods for diagnosing anisakiasis is still based on fiber endoscopy to find anisakis larvae. However, the anisakiasis diagnosis for atypical clinical amanifestation of vomiting, nausea, abdominal pain and other clinical symptoms could be still difficult. Although immunology, molecular biology technology have been applied in anisakiasis auxiliary diagnosis and identification, there is still lack of effective diagnostic reagents and rapid diagnostic methods.In this thesis, with the aim of obtaining the valuable diagnostic antigens and efficient and rapid immune diagnosis method, we complete the following researches:To establish ELISA method and Western Blot with third stage larvae of anisakis, to produce colloidal gold immunochromatographic strips for detecting anisakiasis, and to construct cDNA library of Anisakis L3larvae for screening effective diagnostic antigens in the future.In the present research, the hairtail from the East China Sea were manually dissected to collect the third stage larvae(L3) of Anisakis spp. in cavity、mesenterium and liver, at the same time the morphological observation and molecular identification of the larvae were carried out Next, the soluble antigens of third-stage larvae of anisakis (SAA) were extracted from the third-stage larvae of anisakis by repeated freezing and thawing method. Experiments with indirect ELISA method and Western Blot were done to detect the antibodies against anisakis in the experimental animals infected with anisakis. Meanwhile, colloidal gold immunochromatographic strips for detecting anisakiasis was established and tested it. Finally the cDNA library of third stage larvae of anisakis was constructed and evaluated.The results showed that, a total of132of the hairtail were dissected and1695larvae of anisakis were obtained. On average each hairtail has more than10third-stage larvae of anisakis, which infection rate was100%. Through morphology observation, we can find that there was a characteristic of the mastoid and drilling teeth in the head, and a small spine at the tail. The worm body squirmed like waves. The gene fragment of the identified sample obtained showed99%homology with Anisakis pegreffiii accession number JQ900763.1)in BLAST search. After the larvae were collected for morphological observation and molecular identification, it could be provided reliable experimental materials for the immunology and molecular biology identification.The sensitivity and the specificity were100%and100%with infected rat serum, respectively, and the cross reaction rate was10%with the sera of patients with clonorchiasis and cysticercosis, respectively by ELISA. The SAA of Anisakis was separated into12distinct bands of Mr. from10kDa to170kDa in SDS-PAGE gel, including2main bands (Mr.38、55kDa). Western blot showed the14distinct reaction bands with the serum of the rat infected with anisakis, and the bands of170kDa、130kDa、73kDa.38kDa、22kDa and15kDa were stronger. Furthermore, it was at about Mr.70kDa, Mr.93kDa where found slightly cross-reactions with paragonimiasis and trichuriasis, but was no cross-reactions with others.The SAA antigen was diluted10times for making colloidal gold immunochromatographic strips. The strips were used to test serum samples of the three rabbits immunized with SAA, the ten rats infected with anisakis larvae, the161patients with other parasitosis and twenty normal people. The results showed that all of the immunized rabbit sera and the infected rat sera were positive, and the sera of the normal people were negtive. Cross-reaction indicated that with the exception of one serum from patients with clonorchiasis, all sera from patients suffering from other parasitic diseases, such as schisotosomiasis, paragonimiasis, cysticercosis, trichinosis, ascariasis, hookworm diasease, toxoplasmosis, trichuriasis, pentastomiasis were negative. So both of the sensitivity and specificity of the strip were100%, and cross-reactivity with clonorchiasis was5.6%(1/18). It proved that colloidal gold immunochromatographic strips could be used for the rapid detection of aniskiasis.It was first successfully constructed cDNA library of the third stage larvae of anisakis with SMART technique. The SMARTTM cDNA library control kit provides a method for producing high-quality, full-length cDNA libraries from nanograms of total or poly A+RNA, and preserves the complete5’ends. Clontech’s λ TriplEx2Vector provides all of the following advantages for cloning in a phagemid vector:high titer libraries, blue/white screening for recombinants, regulated expression of cloned inserts. The Plaque number was292. The titer of the plaque library was2.92X106pfu/ml. Through the blue and white plaque screening, the recombination rate was95.7%. The29plaque randomly selected showed that the insert rate was97.9%. Among of them, the biggest fragment length was2000bp, minimum fragment length was400bp, and the average fragment length was850bp.In summary, the third stage larvae of anisakis in this research was Anisakis pegreffii, which was Anisakis simplex sibling species. Established method of indirect ELISA and Western Blot with the SAA from third stage larvae of Anisakis had good immunodiagnosis effects. The colloidal gold immunochromatographic strip is also successfully established, and has high sensitivity, specificity, and low cross-reactivity. The experiment indicates the strip could be used for rapid detectioft of anisakiasis. Finally, The cDNA library was successfully constructed by SMART technology. It laid a solid foundation for researching gene functions and screening antigens of immunodiagnosis.