Jianhua Qin

Induced Susceptibility of Host Is Associated with an Impaired Antioxidant System Following Infection with Cryptosporidium parvum in Se-Deficient Mice

Background Susceptibility or resistance to infection with Cryptosporidium parvum (C.parvum) correlates with Selenium (Se) deficiency in response to infection. Both adult Se-adequate and Se-deficient mouse models of cryptosporidiosis were used to study the cell-mediated immune response during the course of C. parvum infection. Methodology/Principal Findings Blood samples from mouse models were used for Se status. The concentration of MDA, SOD, GPx and CAT in blood has revealed that lower Se level exist in Se-deficient mice. Mesenteric lymph node (MLN) lymphocytes from both mouse models were proliferated after ex vivo re-stimulation with C. parvum sporozoite antigen. The study of the cytokine profiles from the supernatant of proliferated MLN cells revealed that Se-adequate mice produced higher levels of Th1 (IFN-γ and IL-2) and moderate amounts of Th2 (IL-4) cytokines throughout the course of infection. Whereas, MLN cells from Se-deficient mice produced lower levels of IFN-γ, IL-2 and IL-4 cytokines. The counts of total white cell and CD3, CD4, CD8 cell in Se-adequate were higher than that in Se-deficient mice. Significance These results suggest that Cell immunity is affected by Se status after infection with C.parvum from kinetic changes of different white cells and cytokine. In conclusion, induced susceptibility of host is associated with an impaired antioxidant system following infection with C.parvum in C57BL/6 Selenium deficient mice.

PARASITE SPECIES ASSOCIATED WITH WILD PLATEAU PIKA (OCHOTONA CURZONIAE) IN SOUTHEASTERN QINGHAI PROVINCE, CHINA

A survey was conducted to determine the prevalence and seasonal abundance of egg, larval, and adult stages of helminths; oocyts of protozoans; and ectoparasites of plateau pikas (Ochotona curzoniae) in seven areas of southeastern Qinghai Province, China, during August 2006 and May 2007. Fecal samples collected from 430 plateau pikas were examined by the modified McMaster technique, which revealed that 83% of the samples contained eggs from two or more helminth species. Mean fecal egg counts were generally moderate and showed the same trend irrespective of the age or sex of the pikas. The prevalence and counts of cestode eggs showed strong seasonal relationships that corresponded with the rainfall pattern in the study area during the study period. Of the 430 plateau pika examined at necropsy, 89% contained adult nematode or cestode species, but none of these contained adult trematode species or protozoans. Overall, six genera of adult nematodes including Oesophagostomum sp., Cephaluris coloradensis, Eugenuris schumakowiescsi, Haemonchus sp., Trichuris sp., and Chbertiinae sp.; three genera of adult cestodes including Schizorchis sp., Ochotonae sp., and Hymenolepis nana; three ectoparasite species including Hypoderma curzonial, Pulex sp., and Ixodes ovatus; and one proscolex stage of a cestode, Echinococcus multilocularis or Echinococcus shiquicus, were encountered during the study. Other genera examined occurred in low numbers, which did not allow any meaningful comparisons. Overall, results suggest that four parasite species, Hypoderma curzonial, Pulex sp., Ixodes ovatus Neumann, and Cephaluris coloradensis, may be regulating factors in controlling future numbers of plateau pika in this study area. These data provide evidence of a natural biologic control mechanism of plateau pika on grassland habitats, and may be of use for identifying the mechanism of transmission of parasites between plateau pika, livestock, and humans.

Biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken.

To evaluate the biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken, two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E at dose of 50 μg/chicken. At the 15 days post-injection, the tissue samples were collected, the total DNA was extracted, and the 3-1E gene was amplified by PCR. Genomic DNA was first purified away from free plasmid using gel electrophoresis, and then assayed for integrated plasmid using PCR amplification of the 3-1E gene. Simultaneously, the environmental dejection samples were collected, the total bacterial DNA was extracted and then transfer of the pcDNA3-1E gene was detected by PCR amplification of the 3-1E gene. Two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E with three dosage groups of 100 μg, 500 μg and 2500 μg/chicken for 14 days respectively, and with physiological saline at dose of 2500 μL/chicken as control group for acute toxicity test. A target band of 583 bp was obtained by PCR with chicken genomic DNA as template. If the chicken genomic DNA was purified, no target band could be obtained. It showed that the recombinant plasmid pcDNA3-1E existed in tissues, and no genomic integration of DNA plasmid was detected in the immunized chickens. No target band was found by PCR with environmental dejection bacteria genomic DNA as template. It showed that integration and transfer phenomenon did not exist in environment. The acute toxicity results showed the typical clinical symptoms did not occur in the inoculated chickens, the blood biochemical indices and viscera configuration were not affected significantly in the inoculated group and control group (P>0.05). The results showed that the plasmid pcDNA3-1E was safe and suitable for chicken clinical trials.

Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite.

A single-chain antibody library against Eimeria acervulina merozoites was constructed by phage display approach. Antibody-displaying phage was selected in four panning rounds against cryopreserved E. acervulina merozoites. Five clones were randomly selected from the fourth panning round, and their nucleotide sequences were aligned and compared to mouse germ-line sequences. Soluble antibody was produced in a non-suppressor Escherichia coli strain, purified by protein A affinity chromatography, and characterized by Western-blotting. Immunofluorescence assay showed localization of the produced recombinant antibody fragment on the surface E. acervulina merozoites. These resultant antibody fragments showed high specificity and binding capacity for soluble antigens and intact fixed merozoites which seems promising as diagnostic, therapeutic and/or vaccine tools against coccidiosis.

Effects of Quercetin on CYP450 and Cytokines in Aroclor 1254 Injured Endometrial Cells of the Pregnant Rats

Polychlorinated biphenyls (PCBs) are widespread persistent residual environmental pollutants, which affect seriously the growth and reproductive alterations in humans and animals. Aroclor 1254 is a commercial mixture of PCBs. Quercetin is a flavonoid, which acts on estrogen receptors and causes the development of estrogen-related diseases. In this paper, the primary cultured endometrial cells in the pregnant rats were isolated and Aroclor 1254 was used to induce the injured endometrial cells model. The cells were treated with gradient quercetin, the viability of the endometrial cells, the expressions of CYP450, the contents of TNF-α, IL-6, estradiol (E2), and progesterone (P4) were measured. It showed that the viability of the cultured endometrial cells, the expression of CYP1A1 and CYP2B1, and the contents of TNF-α, E2, and IL-6 in the injured endometrial cells increased with the treatment of quercetin. It shows that quercetin has protective effect on the injured endometrial cells in the pregnant rats, this provide a basis on herbal medicine protection for animal reproductive diseases caused by environmental endocrine disruptors.

Construction and immunogenicity of the recombinant Lactobacillus acidophilus pMG36e-E0-LA-5 of bovine viral diarrhea virus.

Bovine viral diarrhea/mucosal disease (BVD/MD) is an infectious disease of cattle with a worldwide distribution, creating a substantial economic impact. It is caused by bovine viral diarrhea virus (BVDV). This research was conducted to construct the recombinant Lactobacillus acidophilus (L. acidophilus) pMG36e-E0-LA-5 of BVDV E0 gene and to test its immunogenicity and protective efficacy against BVDV infection in the mice model. The BVDV E0 gene was sub-cloned into the expression vector and then transformed into the L. acidophilus LA-5 strain by electroporation. The recombinant L. acidophilus pMG36e-E0-LA-5 was confirmed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The mice were immunized orally with the recombinant L. acidophilus pMG36e-E0-LA-5. The serum IgG antibody and fecal sIgA antibody responses, expression levels of interleukin (IL)-12 (IL-12) and interferon gamma (IFN-γ) were detected respectively. On the 7th day after the last-immunization, the mice were inoculated with BVDV to evaluate the protective efficiency of the recombinant L. acidophilus pMG36e-E0-LA-5. The results showed that the expressed products protein E0 in the L. acidophilus LA-5 resulted in single band of 27kDa by SDS-PAGE and its strong reactivity with BVDV antibody was confirmed by Western blotting. The IgG and sIgA antibodies responses, IL-12 and IFN-γ expression levels in the vaccinated mice with recombinant L. acidophilus pMG36e-E0-LA-5 were significantly higher than those in the control mice. The protective rate of the vaccinated mice against BVDV increased significantly, and a 90.00% protection rate in virulent challenge was observed. These results indicated that the recombinant L. acidophilus pMG36e-E0-LA-5 strain was successfully constructed and it could effectively improve the immune response in mice and might provide protection against BVDV.

Expression of E2 gene of bovine viral diarrhea virus in Pichia pastoris: a candidate antigen for indirect Dot ELISA.

The E2 gene containing the EcoR I and Not I sites of bovine viral diarrhea virus (BVDV) was amplified from the plasmid pMD-18T-E2 of the HB-bd isolated, and inserted into Pichia pastoris (P. pastoris) expression vector pPIC9K, and transfected into Escherichia coli DH5α. The recombinant plasmid pPIC9K-E2 was digested by the SalI restriction enzyme and transformed into the P. pastoris strain GS115 by electroporation. High copy integrative transformants were obtained by G418 screening and induced for expression with methanol. The expressed products in the culture medium were identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the Western blotting and the antibody test for immunity. An indirect Dot-ELISA for the detection of antibody against BVDV was established by the recombinant E2 protein as the coating antigen. The reaction conditions of the indirect Dot-ELISA were optimized. The coating concentration of the E2 recombinant protein antigen, the dilution of serum sample, the optimal concentration of HRP labeled antibody, the optimal blocking reagent and blocking time were studied. 100 sera samples from cows in the field were tested for the antibody against BVDV by the Dot-ELISA and the IDEXX HerdChek BVDV antibody ELISA kit simultaneously to compare the specificity, sensitivity and accuracy. The results showed that the expressed products in the culture medium resulted in single band of 44kDa by SDS-PAGE and Western blotting. The results of the immunogenicity assay indicated that the protein E2 expressed in P. pastoris could induce the experimental animals of the rabbit to produce BVDV specific antibodies. The results of the indirect Dot-ELISA showed that the optimal coating concentration of the E2 recombinant protein was 2.0μg/mL, the bovine serum dilution was 1:100, the optimal concentration of HRP-labeled rabbit anti-bovine antibody IgG was 1:500, and the optimal blocking reagent was 3% glutin-TBS and blocking for 45min. The indirect Dot-ELISA showed 96.7%, 92.5% and 95% in the terms of specificity, sensitivity and accuracy compared to the IDEXX ELISA test kit. The indirect Dot-ELISA using the E2 recombinant protein can be used for the detection of antibody against the BVDV and could be considered in the surveillance programs.