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Objective: To investigate the levels of cytokines in the sera of mice chronically infected with the larvae of Echinococcus granulosus , and explore the mechanisms of immune regulation against parasite infection., Methods: The protoscoleces were isolated from the livers and lungs of sheep infected with E. granulosus , and then inoculated intraperitoneally to BALB/c mice (2 000 for each mouse), to establish the mouse model of E. granulosus infection. The mice in the control group were injected intraperitoneally with an equal volume of PBS. The sera of both control and infected mice were collected to test the levels of multiple cytokines by using the Cytometric Bead Array (CBA) 5 months post-infection., Results: In contrast to the control group, the multiple cysts were found in the abdominal cavity, livers and lungs of the infected mice. Moreover, the levels of inflammatory cytokines such as IL-17A, IL-6, IFN-γ, MCP-1, IL-12P70 and TNF-α in the sera of the infected mice were significantly higher than those in the control group ( t = 2.713-9.255, all P < 0.05) while the levels of anti-inflammatory cytokine IL-10 were elevated post-infection ( t = 3.936, P < 0.001)., Conclusions: Higher inflammatory cytokines in the mice chronically infected with the larvae of E. granulosus , may benefit for the limitation of parasite growth.

Objective: To evaluate the diagnostic potential of 5 native antigens obtained from Echinococcus granulosus., Methods: The cyst fluid and protoscoleces were collected from infected livers of slaughtered sheep near Urumqi. The protoscolex somatic soluble antigen (EgPS), crude hydatid cyst fluid antigen (EgHF), partially purified hydatid fluid antigen (Burstein's antigen, EgBu) and the native antigen B (EgAgB) were prepared. Echinococcus granulosus adult worms were collected from experimentally infected dogs, and then used to prepare adult somatic soluble antigen (EgAS). Serum samples from patients with cystic echinococcosis (369 cases), alveolar echinococcosis (14 cases), cysticercosis (20 cases), schistosomiasis (50 cases), paragonimiasis westermani (10 cases), visceral leishmaniasis (5 cases), and healthy individuals (366 cases) were examined for the antibodies against 5 native antigens with indirect ELISA. A ROC analysis was performed to determine a cut-off value for each native antigen., Results: EgPS presented the highest AUC(BOC) (0.958 +/- 0.067), sensitivity (88.7%), positive predictive value (0.96), positive likelihood ratio (16.61), diagnostic efficiency (91.7%), and odds ratio (138.475). The specificity of EgBu (96.1%) was the highest, followed by EgAgB (95.7%), EgPS (94.7%), EgAS (89.0%), and EgHF (80.4%). The Youden index was ranked in order of EgPS>EgBu>EgHF>EgAS>EgAgB. The antibody reactive intensity of EgBu-ELISA was highest (4.055 +/- 1.365) and the EgAS-ELISA was lowest (2.947 +/- 1.276), and there was no significant difference between them (P > 0.05). The five native antigens presented high cross-reactivity with sera from alveolar echinococcosis (9/14-12/14) and from cysticercosis (8/20-16/20)., Conclusion: There are significant differences among the five native antigen-ELISA. The EgPS antigen has the best diagnostic performance , which is followed by EgBu and EgAgB.

Objective: To compare the effects of two ELISA kits on IgG antibody detection of human Echinococcus granulosus., Methods: A Total of 134 sera of patients with echinococcosis, paragonimiasis westermani, clonorchiasis sinensis, schistosomiasis japonica, and cysticercosis cellulosae, and normal persons were detected by two IgG ELISA kits produced by different companies. Furthermore, the specificity, sensitivity and cross reactivity were counted and analyzed statistically., Results: The sensitivity and specificity were extremely high of the two kits as 100.00%. The cross-reactivity rates were 25.00% (paragonimiasis westermani), 26.09% (clonorchiasis sinensis), 10.00% (schistosomiasis japonica), and 87.5% (cysticercosis), respectively, by using the kit produced by the Combined Company in Shenzhen; the cross-reactivity rates were 5.00% (paragonimiasis westermani), 13.04% (clonorchiasis sinensis), 20.00% (schistosomiasis japonica), and 93.75% (cysticercosis) respectively, by using the kit produced by Haitai Company in Zhuhai. In addition, there was a significant difference of Paragonimus westermani detection (P < 0.05), but the rests had no statistically significant differences (all P > 0.05) between the two kits., Conclusion: Both ELISA kits on IgG antibody detection of human Echinococcus granulosus have the advantages of a high sensitivity, specificity, convenience and high-speed. However, it is also in urgent need to further solve the cross-reactivity of Echinococcus granulosus with other parasites, in order to improve the accuracy of early diagnosis.

Forty Wistar rats were randomly divided into 4 groups with 10 in each group. Group A served as normal control. The other 3 groups were injected intraperitoneally with Echinococcus granulosus protoscoleces 5 x 10(4) each. Six months later, group D was injected intraperitoneally with antibody against IL-10 2.0 microg/time, twice a day for 2 d. Two days later, rats in groups C and D were injected intraperitoneally with cyst fluid 5 ml each to induce allergic reaction. 30 min later, all the rats were sacrificed to observe the infection status and obtain peripheral blood. The level of IL-10, IgE and histamine in the sera was detected by ELISA. The results showed that 5, 6 and 5 rats in groups B, C and D were infected successfully, respectively, with 2 deaths in group A. The rats in groups C and D appeared nasal itching, sneezing and declined activity. 30 min later, the symptoms in group C got improved, but not for group D. The levels of IL-10 and IgE in groups C and D increased significantly compared to group B (P < 0.05). The levels of IL-10 and IgE in group D were (142.61 +/- 43.58) pg/ml and (20.67 +/- 1.58) microg/ml, respectively, lower than those in group C (P < 0.05). The level of histamine was (17.69 +/- 3.90) ng/ml, higher than that in groups B and C (P < 0.05). There was no significant difference in histamine level between B and C (P > 0.05).

Objective: To understand the prevalence of echinococcosis in some areas of Jiangsu Province, so as to provide the evidence for making practical control measures., Methods: The cases were determined by a retrospective survey and case-clues survey. All canine feces were tested by immunological fecal antigen in the villages of the local infection suspected cases. The antigen-positive feces were detected for E. chinococcus eggs by Kato-Katz technique. A serological investigation was administrated to search infected persons by double methods of enzyme-labeled immune assay and point immuno-gold filtration assay in the focus groups around the local infection suspected cases, and some students aged from seven to twelve years in their townships and in five different-orientation townships sampled randomly in their counties. The antibody-positive people were confirmed further by using imaging detection., Results: There were 10 imported cases and 16 local infection suspected cases. Twenty canine feces were positive among 1 938 samples, and the positive rate was 1.03%. However, no pathogen was found. Of 12 473 serum samples, 72 were positive in sera immunological antibody test (0.58%), but no cystic nodules were found by imaging detection., Conclusion: There is no direct evidence for confirming the presence of infectious source and foci of echinococcosis in Jiangsu Province. However, the surveillance of echinococcosis is still needed.

Objective: To express the recombinant antigen B (rAgB) of Echinococcus granulosus (Eg) and investigate its immunoreactivity., Methods: The rAgB gene fragments were inserted into pET41a (+) prokaryotic vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by isopropyl beta-D-1-thiogalactopyranoside (IPTG). The protein was purified with sepharose 4B by affinity chromatography, and tested by SDS-PAGE electrophoresis. Its immunoreactivity was examined by Western blotting, and a rapid diagnosis kit for human echinococcosis was used as control., Results: The constructed recombinant plasmid pET41a-rAgB was identified by PCR, digestion with restriction enzyme and sequencing. The recombinant rAgB-GST was about Mr 40 800 with a purity of 78.4%. Western blotting showed that the positive rate of rAgB-GST reacting with sera of cystic echinococcosis (CE), alveolar echinococcosis (AE), paragonimiasis westermani and clonorchiasis sinensis patients, and healthy persons is 79.2%(95/120), 51.1% (23/45), 0 (0/32), 0 (0/20), and 0 (0/24), respectively. Its overall sensitivity and specificity were 79.2% (95/120) and 81.0% (98/121), respectively, slightly higher than the sensitivity (72.8%, 75/103) and specificity (76.9%, 30/39) of the rapid diagnosis kit for human echinococcosis., Conclusion: The rAgB-GST recombinant protein is recognized by the sera of CE and AE patients, showing a proper immunoreactivity.

Objective: To investigate the variation of lymphocytes and cytokines in patients with cystic echinococcosis (CE)., Methods: 80 CE patients who were diagnosed for the first time (60 of Han and 20 of Uygur nationality), and 37 patients who were to accept the second surgical operation(24 Han and 13 Uygur nationality) were included in the study. The peripheral lymphocytes of patients before operation were analyzed by flow cytometry (FCM) to detect T and B lymphocytes, NK cells bearing surface markers, as well as Thl cytokine IFN-gamma and Th2 cytokine IL-4 in the cytoplasm of lymphocytes. 179 healthy persons served as control., Results: In the group of Han patients who were diagnosed for the first time, the percentage of total T cells(CD3+) was lower than the control (P<0.05), while among patients accepting the second operation, the ratio of total T cells showed no difference to the control. For the helper T cells (CD3+/CD4+), NK cells (CD3+/CD16,56+) and B cells (CD3+/CD19+), their ratio were significantly lower in both groups than the control (P<0.01), but their cytotoxic T cells (CD3+/CD8+) were higher than the control. In Uygur patients diagnosed for the first time, B cell ratio was lower than that in control (P<0.05). The NK cell level in both groups of patients was lower than control (P<0.01 and P<0.05 respectively). The level of ThO and Th1 showed no statistical difference among the three groups (P>0.05). The Th2 level was significantly higher in the first diagnosed patients than control (P<0.01), but no statistical difference between the group with the second operation and the other two groups., Conclusion: The immune status of echinococcosis patients is inhibited and the level of Th1 and Th2 shows difference in the first and second operation groups.

The survey was carried out in July, 2006 in Zhiduo County. The IHA and ELISA positive rate in human population was 4.5% (42/933) and 8.2% (76/931) respectively. Ultrasonography revealed a morbidity of 3.4% (33/979) with 3.2% Echinococcus granulosus and 0.2% of E. multilocularis respectively. Animal dissection showed an infection rate of 15.1% (14/93) in pikas with one infected by E. shiquicus proved by molecular biology. Coproantigen rate by ELISA was 62% (12/193) in dogs and 35.7% (5/14) in wolves. The results indicated that Zhiduo County is a mixed endemic area for echinococcosis.

Objective: To investigate the reduction of hydatid cyst weight and change of splenocyte lymphokines in mice immunized with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg)., Methods: BALB/C mice were subcutaneously, intranasally, orally and intramuscularly vaccinated respectively, with BCG and PBS served as controls. The mice were challenged with Eg protoscolices 8 weeks after vaccination and sacrificed in 18 weeks after infection. The weight of hydatid cyst was measured and reduction of the weight was obtained, spleens were used to separate splenocytes which were cultured under stimulation with EgAg or ConA. The supernatant was collected to measure the level of IL-2, IFN-gamma, TNF-alpha and IL-4 by ELISA., Results: The hydatid cyst weight reduced by 45.77%, 18.20%, 88.05% and 92.46% respectively in the 4 immunization groups. In comparison with PBS control, the level of IL-2, IFN-gamma and TNF-alpha was (30.0+/-0) pg/ml, (65.0+/-0) pg/ml and (425.0+/-10.7) pg/ml respectively in the intramuscular group with a significant increase, but that of IL-4 decreased, with a value of (10.0+/-0) pg/ml., Conclusion: The recombinant BCG-Eg95 vaccination induces Thl response in mice against challenge infection.

Objective: To assess the immune status and metabolic reaction of hepatic enzymes on mice with alveolar echinococcosis (AE) after treatment with matrine (Mat) and albendazole (ABZ)., Methods: Mice with AE were treated with single dose Mat, ABZ and combined Mat and ABZ respectively for 60 days. The serum level of IL-2, IL-4, IL-6 and TNF-alpha was detected before and after treatment. NO, LD content and LDH, NOS, iNOS, Na+-K+ ATPase, Ca 2+ Mg+2 ATPase activities in hepatic tissue homogenate from the mice were measured by chromatometry., Results: The level of IL-4, IL-6 and TNF-alpha decreased in the treated groups than infected control (P<0.05), and especially that of IL-4 and TNF-alpha in the drug-combination group. While the level of IL-2 increased in the combination group than the infected control (P<0.05). The content of NO and the activity of NOS and iNOS in hepatic tissue decreased after treatment than control (P<0.05). iNOS in Mat and combination groups decreased more significantly than that of ABZ group (P<0.05). The LDH activity also decreased after treatment (P<0.05), however, the decrease of LD occurred only in the combination group (P<0.05). The activity of Na+-K+ ATPase and Ca2+-Mg2+ ATPase increased in the treated groups (P<0.05) with more considerable increase of Ca2+-Mg2+ ATPase in Mat and combination groups than the ABZ group (P<0.05)., Conclusion: Matrine may strengthen the immunity of the mice and shows an inhibitory effect on the growth of Echinococcus multilocularis in vitro and in vivo, which is posssibly related to the intervention of calcium ion passages and reversion of the multi-drug resistance.

Objective: To observe the effect of pentoxifylline (PTX), albendazole (ABZ), and a combination of PTX and ABZ in mice infected with Echinococcus multilocularis (E.M)., Methods: The first part of the experiment was to observe the in vitro effect of PTX on the cultured E.M protoscolex. In the second part, mice were infected by abdominal inoculation of E.M and divided into groups given by ABZ 50 mg/kg-d, PTX 360 mg/kg-d, PTX 180mg/kgxd, and a combined regimen ABZ 50 mg/(kgxd)+PTX 180 mg/(kgxd). Another infected group and a uninfected group served as controls which received normal saline only. 100 days post-treatment, the mice were sacrificed for further observation. Indicators included wet weight of the cyst, cyst inhibition rate, level of serum cytokines TGF-beta determined by ELISA, IL-2 and IL-10 determined by radio-immunoassay (RIA)., Results: The inhibition rate on cysts of the combined ABZ and PTX was 88%, considerably higher than 58 % of the group ABZ. The serum TGF-beta and IL-10 decreased and IL-2 increased after treatment in comparison to the controls., Conclusion: The PTX and ABZ combination shows better effect on E.multilocularis infection than that of single ABZ. PTX might help increase immunity of the mice.

Objective: To prepare specific antibodies against the recombinant Eml8 antigen (ReEml8), establish a sandwich ELISA and detect the Eml8 circulating antigen in patients' sera., Methods: Rabbits and BALB/c mice were immunized with the ReEml8 antigen, which was purified by affinity method for preparation of the specific poly-clonal and monoclonal antibodies. A sandwich ELISA was established by the specific antibodies., Results: By immunizing with the ReEml8 antigen, high antibody level was reached with a serum dilution of 1: 204 800 and above in the immunized rabbits. After double selection by ELISA using the ReEml8 antigen and block ELISA using both AE-positive and negative control sera, 14 positive cell clones were obtained with an inhibition rate of more than 50%. Those mono-and poly-clonal antibodies were matched freely in sandwich ELISA tests for detecting the ReEml8 antigen. A combination of monoclonal antibody No.9 and polyclonal antibody showed the best result. The sensitivity to detect ReEml8 antigen was at 3 ng/ml. Six of 11 AE sera were positive when tested with the sandwich ELISA system., Conclusion: Highly specific polyclonal and monoclonal antibodies have been prepared, and a sensitive sandwich ELISA established. Preliminary result is suggested that a detectable level of Eml8 circulating antigen is present in AE patients' sera.

Urea-soluble antigens (Ag-u) and water-soluble antigens (Ag-w) of Cysticercus cellulosae were studied by using ELIB and ELISA, respectively. The results showed that using Ag-u and Ag-w, 9 and 14 bands of polypeptides could be recognized by sera from cysticercosis patients; 4 and 10 bands could be recognized by sera from hydatidosis patients; whereas 4 and 8 bands could be recognized by sera from clornochiasis patients. Using ELISA at serum dilutions of 1:400, the positive rate of sera against Ag-u and Ag-w was 96.6% and 86.6%, respectively in 60 cysticercosis patients; 16.6% and 46.6% respectively in 30 hydatidosis patients and negative in 24 clornorchiasis patients and 50 normal healthy persons, indicating that both Ag-u and Ag-w share common antigen components causing cross reaction with the sera from hydatidosis and clonorchiasis patients. However, such components were fewer in Ag-u than in Ag-w. In view of the fact that Ag-u is superior to Ag-w in both sensitivity and specificity, Ag-u might be better used for the diagnosis of cysticercosis.