| Hydatidosis (Echinococcosis) is an amphixenosis which is found all over the world. After animal infection hydatidosis, it shows anorexia and extenuation, that impacts the production trait seriously. In our country, this disease had been recognized as one of the parasitic disease which needs to be prevented and cure carefully. In recent years, it was donated considerablely to prevent and cure the hydatidosis of sheep. Disease incidence was degraded, but it wasn't controlled well. Many animal breeding studies have focused on Major histocompatibility complex (MHC) genes which were taken as candidate genes for disease resistance and susceptibility.In the present study, the polymorphism of the class II MHC-DRB1 exon 2 was detected by PCR-RFLP analysis in Chinese Merino (Sinkiang Junken type) and Kazakh sheep. We characterized the relationship between the MHC-DRB1 exon 2 polymorphism and hydatidosis resistance. Choicing out the genotypes associated with hydatidosis resistance and susceptibility in these two sheep breeds. The haplotype Mvaâ… bc-Sacâ…¡ab-Hin1â… ab of Kazakh sheep which was associated with hydatidosis resistance was screened out and verified. Results of the present study may play an important role in developing new sheep breed which is resistant to hydatidosis. The specific experiment content and results as following:1. Chinese Merino (XinJiang Junken type) sheep was detected by sheep hydatidosis ELISA kit.425 hydatidosis sheep and 604 health controls were chosen. MHC-DRB1 exon 2 polymorphism was determined by PCR-RFLP. Using five restriction enzymes, i.e., Mval, Haeâ…¢, Sad, Sacâ…¡and Hin1â… , yielded 14 alleles,31 genotypes. Comparing the frequency of genotypes in hydatidosis sheep with the control group, genotypes of Haeâ…¢ab, Haeâ…¢ce, Haeâ…¢de and Haeâ…¢ee (P<0.01) were associated with hydatidosis resistance, while genotypes of Sacâ…¡aa(P<0.05), Haeâ…¢bd, Hin1â… bb, Haeâ…¢cf and Haeâ…¢ef (P<0.01) were associated with hydatidosis susceptibility.2. Kazakh sheep was detected by sheep hydatidosis ELISA kit.302 hydatidosis sheep and 400 health controls were chosen. MHC-DRB1 exon second polymorphism was determined by PCR-RFLP. Using five restriction enzymes, i.e., Mvaâ… , Haeâ…¢, Sacâ… , Sacâ…¡and Hin1â… , yielded 14 alleles,28 genotypes. Comparing the frequency of genotypes in hydatidosis sheep with the control group, genotypes of Mvaâ… bc,Hin1â… ab, Sacâ…¡ab, Haeâ…¢de, Haeâ…¢df and Haeâ…¢dd (P<0.01) were associated with hydatidosis resistance. Genotypes of Mvaâ… bb, Sacâ…¡aa, Hin1â… bb,Haeâ…¢ef(P<0.01) and Haeâ…¢ab (P<0.05) were associated with hydatidosis susceptibility.Comparing the resitant and susceptive genotypes between Chinese Merino (XinJiang JunKen type) and Kazakh sheep, it was found that genotypes of Sacâ…¡aa,Hin1â… bb and Haeâ…¢ef showed hydatidosis susceptibility in these two sheep breeds(P<0.01), and genotype of Haeâ…¢de showed hydatidosis resistance (P<0.01). Genotype of Mvaâ… bc is significant difference between these two sheep breeds(P<0.05). After analyzing haplotype with these resistant genotypes of two sheep breeds, the hydatidosis resistant haplotype Mvaâ… bc-Sacâ…¡ab-Hin1â… ab of Kazakh sheep was screened out. The result of blast the genic sequence of this haplotype was meaningful mutation in each enzyme site.3. Sixteen 2-year-old Kazakh sheep which were healthy and detected by hydatidosis ELISA kit were chosen. Among them, eight with the haplotype of Mvaâ… bc-SacIIab-Hin1â… ab were taken as the treatment group, and the other eight with the haplotypes of Sacâ…¡ab-Hin1â… ab or Mvaâ… bc-Hin1â… ab or Mvaâ… bc-Sacâ…¡ab, which were not associated with hydatidosis resistance or susceptibility, were taken as the control. All of these sheep was fed hydatidosis by mouth. After infection hydatidosis, the blood was collected in 0d,7d,14d,30d,45d,60d,75d,90d, 105d,120d, to analyse the blood index of test and control group. The result of infection hydatidosis showed that there were only 2 sheep infected by hydatidosis in the treatment group, whereas 7 sheep were infected in the control group, furthermore the infection rate in the treatment group was significantly lower than that in the control group (P<0.05). It suggested that the genic haplotype Mvaâ… bc-Sacâ…¡ab-Hin1â… ab had much more resistance to hydatidosis in Kazakh sheep.In addition, the blood of Kazakh sheep was collected after infection hydatidosis in ten different times, and the total white cells, granulocytes, lymphocyte number and lymphocyte ratio in the blood was tested by automation cytoanalyze. The result showed that the total white cell and lymphocyte in test group was significant higher than in control (P<0.05). It indicated that this disease was resisted in test group, and degraded the opportunity of forming into cyst in the early stage of infection hydatidosis.4. After infection hydatidosis, the blood and serum was collected in ten different times. The level of cytokines IL-2, IFN-y, TNF-a, IL-4 and IL-10 were evaluated by ELISA and fluorescence real time quantitation PCR (QRT-PCR). The result showed that IL-2 and TNF-a in test group were higher than in control, and serum level of IL-2 in test group significantly higher than in control (P<0.05) 45d p. i., while mRNA level of IL-2 was not significant difference between the two groups. Serum level of TNF-a in test group was significantly higher than in control on 90d p. i. and 105d p. i.. IL-10 and IL-4 mRNA in control was significantly higher than in test group on 120d p. i.. The results showed that the test group which was predominant by Thl could induce the protective immunity, and the control group which was predominant by Th2 could reinforce the susceptibility of hydatidosis.5. The serum was collected after infection hydatidosis in ten different times. The antibody of IgE, IgG, IgM and the content of glutamic pyruvic transaminase (GPT) and total protein was determined. The result showed that the antibody of IgE, IgG and IgM in test group was higher than in control, but there was no significant. It indicated that humoral immunity in this research was not the key factor to resist hydatidosis after infection hydatid. The content of GPT in control group was significantly higher than in test on 30d p. i.. That meaned the test group could inhibit the impairment of liver. The total serum protein in test group was higher than in control. Perhaps this was beneficial to recovery the liver impaired, and reflected much more resistance.All of the results indicated that the polymorphism of the class II MHC-DRB1 exon 2 in Chinese Merino (Sinkiang Junken type) and Kazakh sheep was related with hydatidosis resistance and susceptibility. After infection hydatidosis with Kazakh sheep which had the genic haplotype MvaIbc-SacIIab-Hin1â… ab, it showed that the group which was dominated by Th1 cell could induce protective immunity. At the same time, it suggested that hapjotype Mvalbc-Sacâ…¡ab-Hin1â… ab of Kazakh sheep could be used as a tagged molecule of hydatidosis resistance, and contributed to selection of a new breed which could resist hydatidosis.
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