Study On Genetic Polymorphism And Laboratory Diagnosis Of Echinococcus Granulosus Infections In Xinjiang

Echinococcosis is a serious disease caused by the larvae of Echinococcus granulosus (E.granulosus) and Echinococcus multilocularis (E.multilocularis) living autoeciously in the hosts of humans and animals. Both humans and livestock can be infected accidentally. The farming and stockbreeding areas in the northwest part of China are the major endemic areas for human echinococcosis. Cystic Echinococcosis (CE) caused by E.granulosus is especially prevalent in Xinjiang. Since echinococcosis is comparatively hard to be cured clinically if late stage or recurrence. It has become an important barrier in guaranteeing both the health of farmers and herdsmen in the northwest China and economic development of this area. In view of this, the study of immunity and prevention of CE has been a focused research item. But the problem was that, due to different geographical situations and different hosts, there were substantial gene hereditary variations in E.granulosus, so the differences of the various strains' growth (for example, the development of larvae and eggs) may affect its sensitivity to the chemotherapy, in the meantime and the antigen mutation also influences the precise inspection and immune precaution for echinococcosis, It can be reasoned that the verification of the typical Xinjiang strains of E.granulosus and the study on its genetic diversity are directly related to the epidemiological investigation and precaution of Echinococcosis.Part 1Study on genetic polymorphism and Echinococcus granulosus strains in XinjiangThis dissertation aims firstly at the study on the differences of gene sequence amongstrains of E.granulosus isolated from different hosts in different areas in Xinjiang, and then the confirmation of the genotypes and geographical distribution of the E.granulosus, and the further study on the systemic development, evolutional mechanism, genetic variation and differentiation of E.granulosus species sorted on different classification level. A series study may play an instructive role in clinical and prophylactic treatment of echinococcosis. This study adopted DNA sequencing technique to analyze the gene sequence of mtDNA CO1 and mtDNA Cyt b of E.granulosus, and detected accurately E.granulosus genotypes and genetic mutations. Based on the nucleotide sequences of E.granulosus the cladogram was constructed. The E. granulosus gene fragment size of CO1 and Cyt b has been subjected in this experiment was 789 bp and 498 bp, respectively. According to the analysis of CO1 gene sequence, there were 3 genotypes (G1, G2, G3), and 23 heliotypes of E.granulosus in Xingjiang. The G3 genotype (Buffalo strain)of E.granulosus firstly found can be which may infect to humans, and result from some genetic mutation of G3 genotype. And a G6 genotype (camel strain) was found in these samples isolated from the patients, testifying that G6 genotype of E.granulosus could cause human CE. G1 genotype (sheep strain) of E.granulosus was the most prevalent strain in Xinjiang. The separated strains collected in different areas revealed 99.2%～100% homologous sequence without obvious distinctions between the E.granulosus gene sequences in the different hosts. There were 20 different haplotypes and 25 mutant positions of nucleotide, covering 3.2% of the whole mutant places, which caused 15 amino acids substitution at protein level. The nucleotide substitutions were mainly transition rather than the transversion. The nucleotide substitutions took place first on the third position of codon, occupying 48% of all mutations; next on the first position, covering 28%; and the second position, the most conservative one, 24%. The ratio of transition and transversion happening on the first and second codon positions was 2.5(5/2) and 5(5/1) respectively, third codon positions was only transition. The analysis of the E.granulosus CO1 and Cyt b gene sequences isolated from patients and sheep, revealed that the gene sequences of separated strains from different infected parts of the same host may still be different, that is, different G1 gene sequence. In homology comparison between 23 detected Xinjiang E.granulosus haplotypes and haplotypes in Genbank, 9 Xinjiang E.granulosus G1 haplotypes showed the 100% homologous sequences in their gene sequence mutations, but others, totally 14, without counterparts in Genbank were the newly discovered E. granulosus haplotypes.Cyt b gene is suitable for differentiation and classification analysis of species and subspecies, reinforcing the study on the species development of E.granulosus. The analysis of the Cyt b gene sequence showed that the three genotypes (G1, G2, G3) of E.granulosus in Xinjiang were different in sequence. The rate of difference was 10.7%. The phylogenetic tree drawn in light of gene sequences conformed with the results of CO1 gene sequence.Part 2Study on identifying genotypes of Echinococcus granulosus by microsatellite markersThis research used Sca, Emsk and C106 microsatellite sequence of E.granulosus as typing markers and identified the genotypes and heterozygosity of E.granulosus isolated from CE patients in different areas of Xinjiang. Analysis of many microsatellite markers may enhance identical ability of genotyping. PCR products of homozygote were composed of DNA fragments of same nucleotide sequence and otherwise PCR products of heterozygote enclosed two kinds of DNA fragments with different lengths, which resulted from different tandem repeat number in core DNA sequence. Therefore, microsatellite genotyping can be confirmed by refined measuring of length of PCR products.Two different fluorescent dyes of FAM (carboxy fluorescein) and HEX (hexacho rofluorescein) can be used markers for three pairs of primers of microsatellite sequences. After amplification of satellites by PCR, amplified products were based on capillary electrophoresis by using 310 auto DNA analysis machine ,and then basic number of amplified products were calculated by Genescan 2.1 software. PCR products with different microsatellite markers could be added in same sample well and electrophoresis. Genotypes of 3 genetic locus could be finely tested at same time because different size and different color peaks represented different genetic locus of PCR products, such as single peak represent homozygote, otherwise double peak represent heterozygote. Our result showed that 66 isolated strains from 44 CE patients were homozygote of E. granulosus, in which 65 isolated strains from 43 CE patients were identified as G1 genotype by PCR and microsatellite markers ,and 1 isolated strains from 1 CE patient were identified as G6 genotype by PCR and microsatellite markers. The results of genotypes identified by microsatellite markers was consensus with result of DNA sequence analysis.In our prevpoius study, mixed infection and heterozygote of G1 (sheep strain) and G6 (camel strain) genotypes were found in a dog intestine in Xinjiang, but in this study nor E.granulosus metacestode heterozygote was found. It may indicate that E.granulosus was autofertilization, and therefore, few chance of heterozygote take place in the patient. Microsatellite DNA markers can be used to finely and quickly identify the genotypes and heterozygosity of E.granulosus in genetic level ,and possessed the important value in study of genetic polymophosim, epidemiology and pathogenicity of E.granulosus.Part 3 Genetic identification of Echinococcus granulosus by using PCR-RFLP methodWith the purpose of verifying the typical strains of E. granulosus and clarifying the geographical distribution of its genotypes, this study adopted the PCR-RFLP method to make the PCR amplification of the separated strains samples from CE patients in different areas in Xinjiang. With mtDNA rrnl as the special attractant, a trail to digest the mtDNA rrnl by restrictive enzymes SspI and BglII were carried out after PCR amplification of this gene, and the digenstion results were analyzed by gelose gel electrophoresis. As the result, the PCR amplificons of CE patients' separated strains could not be cut by the restrictive enzyme SspI. The PCR amplificons of the separated strain samples of 43 CE patients could not be cut by the restrictive enzyme BglII, thus verified as G1 genotype. From one patient PCR amplification product was cut into two DNA fragments at 158 bp and 403 bp ,respectively, thus confirmed as G6 genotype. This further proved that G6 genotype could also infect humans and cause human echinococcosis. It was testified by the results of the experiment that the gene verification of Echinococcus granulosus by PCR-RFLP method confirmed totally with the verification by DNA sequence analysis, therefore, it can be considered as a simple, timesaving and effective method for investigate the Echinococcus granulosus genotypes.Part 4Serological and Clinical Analysis forthe patients with Cystic Echinococcosis in XinjiangDiagnosis of human echinococcosis is commonly based on the image examination. However, the serological tests are also very important for early diagnosis and epidemiological survey of the echinococcosis in the remote areas. In this study, 31 serum samples were obtained and analyzed from surgically confirmed patients with cystic echinococcosis (CE) by serology using E.granulosus recombinant antigen B (rAgB). The serodiagnostic value of rAgB was discussed by using rAgB-ELISA and rAgB-Immunoblotts combining with clinical data. Our data demonstrated that the both of rAgB-ELISA and rAgB Immunoblotts showed 90.3% (28/31) positives with these CE sera. The clinical data revealed that the remaining 3 sero-negative samples were from CE patients with single cyst classified as type CE. A comparative analysis indicated that the antibody titers against rAgB in these sera appeared to be increase through increasing the number of cysts found from the corresponding patients, however we could not found apparent statistical relationship (t-test P>0.05) between this kind of trend. This may be due to the solitary liver cyst which could evoke lover antibody titers or due to the insufficient numbers of CE cases analyzed in our study. This should be further analyzed using sufficient serum samples from post surgical CE cases.