| Cystic echinococcosis (CE) is a chronic parasitic zoonosis caused by infection with the larval stage of the cestode Echinococcus granulosus (E. granulosus), resulting in the development of cysts in human and domestic animals. In domestic animals, especially in sheep, which appear to be highly susceptible to infection, CE causes considerable health problems and economical disadvantages because of production loss, as sheep infected with this disease often suffer from reductions in live weight gain, in milk yielding, in the fertility rates, in the value of wool or other products. In Xinjiang, there are more than60million sheep, with the high prevalence of CE reaching50%, which causes considerable health problems in sheep and thereby significantly influences the income of herdsman.Previuosly, we carried a lot of studies to investigate the relationship between MHC-DRB1gene polymorphism and genetic resistance or susceptibility to CE in Chinese Merino, Duolang and Kazakh sheep. Genetic markers, MHC MvaⅠbc-SacⅡab-HinlIab haplotype, on CE resistance in Kazakh sheep have been divergently selected by us. The rates of Echinococcus granulosus (E. granulosus) infection in the internal organs of Kazakh sheep with this haplotype, is confirmed to be significantly lower than sheep without this haplotype, when exposure to the same level of parasites. CE resistant sheep therefore have an increased genetic capability to respond to and subsequently reject parasites when challenged. Carrying out a deep study in these sheep was, therefore, worth doing, since it could produce some interesting clues on the mechanism of resistance to E. granulosus infection both in immunology and genetics.According to the previous methods stablished by us, we selected the genetic markers in250Kazakh sheep, again. Seventeen healthy two-year-old Kazakh sheep with different haplotype were selected and maintained during the course of the experiments. They were negative for antibodies to hydatid cyst fluid (HCF) antigen, assayed by a commercial ovine hydatidosis ELISA kit, and no hydatid cysts presented in internal organs detected by ultrasonography, prior to the experiment. The seven sheep with the MHC MvaⅠbc-SacⅡab-HinlⅠab haplotype (CE resistant haplotype) referred to group A, while the other seven sheep without this haplotype constitute group B. Each sheep in the two groups was infected perorally via a medical syringe with5000E. granulosus eggs suspended in1000ul physiological saline. Another three sheep choosed as healthy controls were named as group C. Then, the following studies were carried out:1. The first experiment described here was designed to analyze the system immune response in the very early stage of E. granulosus infection in sheep with CE resistant haplotype. In this experiment, after experimentally infected with E. granulosus, blood samples from these sheep were collected on day0(prior to infection), hours2,3,4, and9post-infection as well as days1,2,3, and7 post-infection, respectively. ELISA assay was used to measure serum levels of antibodies (IgE and IgM), cytokines (Thl:TNF-α and IFN-γ; Th2:IL-4and IL-10) and chemokines (Thl:CXCL-9; Th2:CCL17) at different time points of E. granulosus infection in the two groups. Results showed that, in the early stage of E. granulosus infection, antibodies like IgM and IgE, Thl cytokines such as IFN-γ and TNF-a, as well as Thl chemokines CXCL-9were predominant in group A, especially for IgE and Thl cytokines, which were significantly higher, most were at or began from4h post-infection, as compared with group B. Our findings revealed that the influence of the host’s genetic background on the immunopathology of E. granulosus infection in the early stage could be partially mediated by Thl-type cytokines and IgE.2. The second experiment was designed to detect differential gene expression in intestine of Kazakh sheep with different MHC haplotype after perorally infected with E. granulosus eggs. In this experiment, three sheep of each group (groups A and groups B) were sacrificed on hours2,3and4post-infection, respectively. Besides, another3healthy Kzakh sheep, referred to group C, were choosed as healthy controls. The intestine tissues of the three groups were collected, and gene expression profiles were assessed using sheep DNA microarray analysis. Gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,4712differentially expressed genes were identified between group A and C, with1959up-regulated and2753down-regulated. On the other hand,4944differentially expressed genes, with2076up-regulated and2868down-regulated, were also identified between group B and C. Then, we chose141significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including32up-regulated genes and109down-regulated genes. Among32up-regulated,6genes were significantly higher in group A than in group B, while26genes were significantly lower in group A than that in group B. Additionally, among109down-regulated genes,35genes were significantly higher in group A when compared with group B, while74genes were significantly lower in group A when compared with group B.3. The third experiment was designed to detect differential gene expression in the liver of Kazakh sheep with different MHC haplotype after perorally infected with E. granulosus eggs, at different time points. In this experiment, one sheep of each group (groups A and groups B) were sacrificed at8and24weeks post-infection, respectively. Besides, another3healthy Kzakh sheep, referred to group C, were choosed as healthy controls. The liver tissues of the three groups were collected, and gene expression profiles were assessed using sheep DNA microarray analysis.1) At8weeks post-infection, gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,3087differentially expressed genes were identified between group A and C, with1768up-regulated and1319down-regulated. On the other hand,4839differentially expressed genes, with 1888up-regulated and2951down-regulated, were also identified between group B and C. Then, we chose153significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including87up-regulated genes and66down-regulated genes. Among87up-regulated,13genes were significantly higher in group A than in group B, while74genes were significantly lower in group A than that in group b. Additionally, among66down-regulated genes,54genes were significantly higher in group A when compared with group B, while12genes were significantly lower in group A when compared with group B.2) At24weeks post-infection, gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,3092differentially expressed genes were identified between group A and C, with1374up-regulated and1718down-regulated. On the other hand,11717differentially expressed genes, with5381up-regulated and6336down-regulated, were also identified between group B and C. Then, we chose115significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including40up-regulated genes and75down-regulated genes. Among40up-regulated,3genes were significantly higher in group A than in group B, while37genes were significantly lower in group A than that in group b. Additionally, among75down-regulated genes,74genes were significantly higher in group A when compared with group B, while1genes were significantly lower in group A when compared with group B.3) The differentially expressed genes were classified with gene ontology (GO) analysis according to their functions. Results showed that most of them were related to the metabolism amd cellular processes. KEGG biological pathway analysis showed that most genes involved in the complement lectin pathway, especially in the intestine tissue and in the liver at24weeks post-infection, which indicated that different MHC genotype resistance to CE might be associated with complement levels.4) Finally, the following genes:the killer cell immunoglobulin-like receptor (KIR2DS1), lectin complement C8, serine protease inhibitors, growth inhibition of DNA damage gene (GADD45B), PLA2G2A were choosed as candidate genes asscociated with CE resistance in sheep.In conclusion, our studies revealed that the influence of the host’s genetic background on the immunopathology of E. granulosus infection in the early stage could be partially mediated by Thl-type cytokines and IgE. On the other hand, genes involved in the complement lectin pathway might participate in the resistance of CE, after E. granulosus infection in the intestine tissue (the very early stage of infection) or in the liver tissue (the late stage of infection). In addition to that, microarray analysis showed that, a large number of genes involved in CE resistance in sheep, varying from energy metabolism, immunity, to signal transduction, indicating that CE resistance is a complex physiological process. Compared with healthy controls, sheep with MHC CE resistance haplotype (group A) displayed an inactivate gene expression trend at the three time points detected, showing that most of the genes upward or downward were lower than that in sheep without this haplotype (group B), but interestingly, some genes related to CE resistance in group A showed a high expression level than that in group B. It is, therefore, a further deep study on the relationship between these genes and CE resistance should be carried out, in future. |
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