Identification Of Four Geographical Strains Of Trichinella Spiralis By COI Gene And Analysis Of Immune Components

Trichinellosis is an important zoonotic parasitic disease caused by eating raw or undercooked meat which infected with Trichinella spiralis. Trichinella spiralis and Trichinella native are the only two pathogens presented in our country, while Trichinella spiralis is the major one causing human trichinellosis. Different symptoms, such as diarrhea, nausea, fever, fatigue; periorbital edema, myalgia, diffuse paralysis symptoms, even encephalitis and myocarditis--the main cause of death, are showed in Trichinellosis, due to the different infected species, different degree of infection or immune state of different individuals.Because of nonspecific clinical symptoms for the diagnosis of trichinosis, the definite diagnosis of it need support from laboratory examination results. Biopsy is recommended as the gold diagnostic criteria of Trichinellosis by the International Commission on trichinellosis, but the patients’compliance is low because it is invasive examination. Biopsy is widely used only in the animal husbandry and the slaughtering industry, though it may result in missed diagnosis for insensitive in early stage of infection, useless sampling site. The sensitivity of ELISA is highest among current serological method for the detection of Trichinella antibody, with advantages such as economical, standardized, and stable and reliable of sensitivity and specificity, which has been listed as a recommended method of serological method for the detection of Trichinellosis by International Commission on trichinellosis. Because study of the effective antigen is the basis of the research on immunological diagnosis of Trichinellosis, in this study, the crude muscle larvae antigens (CLA) and the excretory-secretory antigens (ESA) of Trichinella were analyzed by enzyme linked immunosorbent assay (ELISA) and immunoblot analysis (Western blot, WB), in order to understand the difference among different geographical strains of antigens and their immune effect.In this study, Kunming mice were infected with 4 Trichinella isolates (about 500 larvae each) to establish the animal model, and the newborn muscle larvae were collected at 45 days after infection. We observed the morphology of different muscle larvae and identified the species of the 4 isolates with mitochondrial cytochrome oxidase I (COI). The crude muscle larvae antigens(CLA) were prepared by artificial digestion method and excretory-secretory antigens (ESA) of muscle larvae were prepared with culture. Through ELISA test, the sensitivity and specificity of different antigens were analyzed, and their cross reactivity with other parasites was also detected. Useful components and immune bands of CLA and ESA of four Trichinella isolates were identified by polyacrylamide gel electrophoresis and Western blotting.The results, up to 40000 larvae were collected each isolates, the cysts of them were fusiform shape under microscope, body curled, after washing the body was "C" shape or curvature, rounded at both ends, with no projections and appendages, the stichosome accounted for about 2/3 of total length. The COI gene amplification products of four trichinella isolates appeared obvious belt at about 400bp, and the sequencing results showed that product of Yunnan isolate was 380bp length、 Hubei isolate 380 bp、Henan isolate 380 bp、Dongbei isolate 380 bp. By blast with Trichinella spiralis ISS413 strains AF 129486.1 gene alignment, homology was 99%; the similarity of the sequence of four isolates was 100%, and this meant they may belong to Trichinella spiralis.The results of ELISA test showed that the sensitivity and specificity of different antigens to detect trichinellosis were both 100%. There were cross reactions between different antigens and sera of patient with schitosomiasis, echinococcosis, cysticercoids, and no cross reactions with sera of patient with clonorchiasis, paragonimiasis, and hookworm. The positive reaction rate between sera of schistosomiasis patients and CLA of Hubei isolate was 2.85%, Dongbei isolatell.4%; the rate between sera of echinococcosis patients and CLA of Yunnan isolate was 11.7%, Henan isolate5.8%; the rate between sera of cysticercosis patients and CLA of Yunnan isolate was 12%, Hubei isolate 16%, Henan isolate32%, Dongbei isolatel2%. The positive reaction rate between sera of schistosomiasis patients and ESA of Yunnan isolate was2.85%, Dongbei isolate 11%; the rate between sera of cysticercosis patients and ESA of Yunnan isolate was 12%, Hubei isolate 8%, Henan isolate24%, Dongbei isolate 12%.Analysis of different antigens with SDS-PAGE showed that the antigens of CLA were distributed among molecular weight of 100 000-10 000, while contains 8 main bands, Mr was 100 000,66 000,49 000,45 000,43 000,30000,18 000,12 000,respectively; and 3 minor bands, Mr82 000,80 000,53 000, and the excretory secretory antigens (ESA) were distributed in the range of Mr 70 000-10 000, also with 8 main bands, Mr 66000,53 000,49000,43000,40000,34000,30000, 16000.Western blot analysis showed that the CLA had reactions with sera of patients with trichinellosis and sera of infected mice, bands with Mr 95000,72000,53000,49000,43000; and bands of ESA with Mr53000,49000,43000,40000. The CLA of Yunnan isolate had a positive band in Mr 95 000 with sera of patients with schistosomiasis, sparganosis, trichuriasis, cysticercosis, hookworm dieases. The ESA of Dongbei isolate had a positive band in Mr 45 000 with sera of patients with cysticercosis, paragonimiasis.To sum up, the four Trichinella isolates were identified as the same species of trichinella spilaris by COI gene. The CLA and ESA of each isolate was testified to has a good effect on the diagnosis of trichinellosis; in addition, SDS-PAGE and Western blot analysis also found that Mr 40000 components of ESA and Mr 95 000,72 000 components of CLA should be the focus of further research.