[In vitro cultivation model of Cryptosporidium parvum in MDCK cells and its development].

OBJECTIVE: To develop an in vitro culture system for Cryptosporidium parvum in Madin-Darby canine kidney (MDCK) cell and observe its life cycle (from desquamate to oocyst). METHODS: Oocysts of C. parvum were co-cultured with MDCK cells in vitro. Culture condition was optimized and the life cycle of C.parvum investigated. RESULTS: The optimal culture conditions for C. parvum in MDCK cells were 2.0 x 10(5) cells cultured for 12 h, and infected by 1.0 x 10(5) oocysts in the Dulbecco's Modified Eagle Medium with 5% FBS. Following 72 h co-culture, desquamate, sporozoites, trophozoites, meronts, microgametocytes, macrogametocytes, zygote, thin-wall oocyst, and thick-wall oocyst appeared orderly. Between the 60th and 72nd hour, many oocysts emerged. Inoculated by the C. parvum-infected cell culture supernatant at the 48th hour, the immunosuppressed mice became infected. CONCLUSION: The culture system provides a model for propagation of the parasites and demonstrates a complete in vitro life cycle of C. parvum.

[Expression of recombinant bovine beta-interferon by baculovirus system and evaluation of its bioactivity].

In this paper, a recombinant baculovirus containing the ORF of bovine interferon-beta (BoIFN-beta) gene, rBac-BoIFN-beta, was generated to express recombinant BoIFN-beta (rBoIFN-beta) in sf9 insect cells. The expression of rBoIFN-beta in rBac-BoIFN-beta infecting sf9 cells and its supernatants was confirmed by indirect immunofluorescence assay and Western blot. The antiviral activity of rBoIFN-beta in the supernatant can reach 10(6.0) AU/mL evaluated by the antiviral assay with VSV * GFP that expressed green fluorescence protein, and rBoIFN-beta could stimulate the expression of luciferase reporter gene controlled by chicken Mx promoter. All the results showed that rBac-BoIFN-beta constructed here could express high level recombinant BoIFN-beta in secreted form that had the bioactivity of natural type I IFN.

[Study on methods for isolation and purification of Cryptosporidium parvum oocysts from mouse feces].

OBJECTIVE: To explore an applicable method for isolation and purification of Cryptosporidium parvum oocysts with high purity, recovery and vigor from mouse feces. METHODS: Four techniques were used for isolating and purifying C. parvum oocysts from mouse feces: modified saturated saline flotation, percoll gradient centrifugation, CsCl gradient centrifugation and the classical discontinuous sucrose gradient centrifugation. Oocysts received from the methods were used respectively to infect in vitro bovine fallopian tube epithelial cells (BFTE) and the development of the oocysts was examined under microscope after 48 h and 72 h cultivation. RESULTS: The number of oocysts received by the classical discontinuous sucrose gradient centrifugation [(2.86 +/- 0.08) x 10(7)] was significantly higher than that of percoll gradient centrifugation [(1.52 +/- 0.08) x 10(7)] (P<0.01) and CsCl gradient centrifugation [(2.46 +/- 0.13) x 10(7)] (P<0.05), but similar to that of the modified saturated saline flotation [(2.88 +/- 0.15) x 10(7)]. No significant difference was found on the number of oocysts by BFTE cultivation at 48 and 72 hours post-inoculation(P>0.05). Oocysts received from CsCl gradient centrifugation showed higher purity than those by discontinuous sucrose gradient centrifugation. CONCLUSION: In comparison to the classical discontinuous sucrose gradient centrifugation, operation of the modified saturated saline flotation is easier and faster, and the purity of oocysts isolated by CsCl gradient centrifugation is higher.

[Construction of the eukaryotic expression vector with IL-2 gene and VP2 gene of PPV and research on immunogenicity].

To construct gene vaccine of PPV and to investigate the effects of interleukin 2 (IL-2) as an adjuvant on immune responses in mouse, the recombinant expression plasmid of pCIneo-IL2-VP2 was constructed and transfected into PK-15 cells by lipofectamine, the expressed product was detected by immunofluore assay. To study the immune effects of DNA vaccine in vitro and in vivo, mice were used as the animal model. The recombinant plasmid pCIneo-IL2-VP2, the control plasmid pCI-neo and the PPV live vaccine were immunized by intramuscular injection. Anti-PPV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method, and the specific killing activities of CTL were assayed too. The results show that the immunized mice produced PPV antibody after one week, and reached to highest after four weeks. Compared with the control group, the pCIneo-IL2-VP2 immunized group produced significant differences in the antibody titers, the lymphocyte proliferation activity and the specific killing activities of CTL. The pCIneo-IL2-VP2 induced humoral and cellular immunity responses similarly to that the live vaccine induced. These results manifested that the PPV DNA vaccine successfully induced humoral and cellular immunity response in mice with the IL-2 gene as an adjuvant.

[Quantitative detection of Vibrio parahaemolyticus by real-time TaqMan PCR].

Vibrio parahaemolyticus is one of important human food pathogens. Traditional diagnostic tests for V. parahaemolyticus are laborious and always present false negative results. Therefore, it is important to develop a nucleic acid-based test for quantitative detection of V. parahaemolyticus. A TaqMan PCR assay was presented for quantitative detection of V. parahaemolyticus in pure cultures and oysters. The primers and probe were designed according to the gyrase B gene (gyrB) sequence of V. parahaemolyticus strains. Amplification of DNAs from 12 bacterial strains comprising 9 genera showed that all of the strains of V. parahaemolyticus tested (n = 4) were positive and all other species of strains tested (n = 8) were negative. The results of the TaqMan PCR with raw oysters inoculated with V. parahaemolyticus were comparable to those of pure cultures. The sensitivity of the assay was 1 CFU PCR Mixture(-1) and 10 CFU PCR Mixture(-1) in pure culture and inoculated raw oyster, respectively. The correlation rate was 0.99 (gamma2 = 0.99). The assay could be completed within 1h. The Real-time PCR can be used as a rapid screening tool for the presence of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.