Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle from farms in China

Fecal samples of 2,056 dairy cattle from 14 farms were collected in three geographical regions of China and stained using a modified acid-fast staining technique to identify Cryptosporidium oocysts. A total of 387 (18.82%) positive samples were identified and further analyzed by polymerase chain reaction (PCR) using primers designed to amplify DNA fragments from the small subunit ribosomal RNA. The PCR products were sequenced and the sequences were deposited in the GenBank database under accession numbers EU369377-84 and GU070730-33. Phylogenetic analysis was performed and a distances matrix generated from these sequences confirmed the existence of Cryptosporidium (C.) parvum 'mouse' genotype, C. bovis, C. andersoni, C. hominis, and C. serpentis in cattle. These results represent the first report on the prevalence and genetic identification of Cryptosporidium species, and may contribute to a better understanding of the epidemiology of Cryptosporidium in cattle in China.

Recombinant goat pox virus expressing PPRV H protein / 生物工程学报

The purpose of the study is to construct recombinant goat pox virus (GPV) expressing Peste des petits ruminants virus (PPRV) H protein, and to evaluate the immunization effect. Recombinant GPV containing PPRV H gene (rGPV-PPRV-H) was selected and purified by gpt and eGFP utilizing plaque purification, and the final selected recombinant GPV was proved to be purified by PCR. Immunofluorescence and Western blotting showed that the recombinant virus could express H protein of PPRV while infecting lamb testis cells. Six goats were immunized with 2 x 10(6) PFU rGPV-PPRV-H through intradermal injection, and were immunized for the second time at 28 days with the same dose recombinant virus after first immunization. Serum was collected after immunization, and was analyzed for the neutralization antibodies. 21 days after first immunization, the neutralization antibodies of GPV were 40, 80, > or = 80, > or = 80, 40, > or = 80 in turn, and neutralization antibodies of PPRV were 80, 80, 80, 80, 40, 40, 10 in turn; 14 days after second immunization, the neutralization antibodies of GPV were all > or = 80, and the neutralization antibodies of PPRV were > 80, 80, > 80, 80, 80 and 40 in turn. This study established a foundation for the industrialization of the PPRV recombinant GPV vaccine.

EFFECTS OF SELENIUM-ENRICHED PROBIOTICS ON IMMUNITY AND ANTIOXIDATION FUNCTION IN MICE / 营养学报

Objective: To investigate the effects of selenium-enriched probiotics on immunity and antioxidation function in mice. Method: One hundred healthy KM mice, female and male in half, were randomly divided into control, sodium selenite, selenium-enriched yeast (Se yeast), selenium-enriched probiotics (Se probiotics) and probiotics groups. One milliliter of water, sodium selenite (2 ?g Se/ml), Se yeast (2 ?g Se/ml), Se probiotics (2 ?g Se/ml) or probiotics were respectively supplemented to five groups in oral (ig) every day. Whole experiment lasted for 28 d. During the experiment, immunity and antioxidation functions were measured respectively. Results: The activation of peritoneal macrophage and thymus index in Se probiotics group were significantly higher than those of other four groups. The spleen index of Se probiotics group was higher than that of control, sodium selenite and probiotics group. The spleen lymphocyte transformation rate in Se probiotics group was significantly or very significantly higher than that in control or probiotics group. In addition, blood GSH-Px and plasma SOD activity in Se probiotics group were significantly higher than those in control and sodium selenite group. Plasma MDA concentration in Se probiotics group was very significantly lower than that in control and sodium selenite group. Conclusion: Selenium-enriched probiotics supplementation could significantly enhance immunity and antioxidation function in mice.

Effects of hydrogen peroxide on infections of cryptosporidium parvum in vitro / 中国病理生理杂志

Effects of hydrogen peroxide (H2O2)on Cryptosporidium parvum infection in vitro were studied in this paper, using an oocyst excystation assay, a cell culture model, and a free radical inhibition technique. H2O2 treatment at 500 and 1 000 μmol/L significantly inhibited excystation of bleach-treated oocysts (P＜0.01). Concentrations of H2O2 at 500 and 750 μmol/L resulted in a significant decrease in C. Parvum infection at 35.77% and 58.16% respectively, when compared with the untreated control at 48 hours postinoculation. Surprisingly, C. parvum infection were significantly increased by 22.21% to 39.33% following treatment with 50 (P＜0.01), 100 (P＜0.01) or 200 (P＜0.05) μmol/L H2O2, respectively. Stimulatory effect with treatment of 100 μmol/L H2O2 was most obvious, compared with the untreated control at 48 hours postinoculation. Effects of H2O2 on C. parvum at 24, 48 and 96 hours postinoculation were similar, the highest infection being infection at 48 hours postinoculation, with the maximum inhibitory effect being seen at 96 hours postinoculation. The stimulatory and inhibitory effects of H2O2 treatment on C. parvum infection were, to a certain extent, abolished in the presence of free radical scavengers, reduced glutathione or mannitol. These observations indicate that reactive oxygen species (ROS), such as H2O2, may play a role in the course of C. parvum infection. This is the first time to demonstrate a significant action of ROS in C. parvum infection in vitro.

Improvement of Amplification Method for Cryptosporidium parvum Oocysts from Mice / 中国寄生虫学与寄生虫病杂志

Objective To establish a simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from infected C57BL/6N mice. Methods All mice in the experimental groups were immunosuppressed by given different concentrations of dexamethasone phosphate added in drinking water throughout the experiment. The recovery and purity of the oocysts obtained using different purification methods was compared. The infectivity of the oocysts obtained from the same origin but different animals and different purification methods in a bovine fallopian tube epithelial cell culture system was studied. Results 4.16?10 9 oocysts were obtained in 30 mice in the 3rd group with dexamethasone of 20 ?g/ml in drinking water. No significant difference in the oocyst recovery, purity and infectivity was found between methods using saturated saline floatation and sucrose density gradient centrifugation. The infectivity of the oocysts obtained from the same origin but different animals was similar. Conclusion A simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from the infected mice was established.

Study on Methods for Isolation and Purification of Cryptosporidium parvum Oocysts from Mouse Feces / 中国寄生虫学与寄生虫病杂志

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)?107] was significantly higher than that of percoll gradient centrifugation [(1.52?0.08)?107] (P0.05). 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.

In Vitro Cultivation Model of Cryptosporidium parvum in MDCK Cells and its Development / 中国寄生虫学与寄生虫病杂志

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?105 cells cultured for 12 h, and infected by 1.0?105 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 72th 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.

EFFECTS OF SELENIUM-AND ZINC-ENRICHED PROBIOTICS ON BLOOD CONTENT OF SELENIUM AND ZINC,ANTIOXIDATION FUNCTION AND INTESTINAL MICROFLORA IN CANINES / 营养学报

Objective To investigate the effects of selenium-and zinc-enriched probiotics on the content of selenium and zinc in blood,antioxidation function and intestinal microflora in canine.Method Eight 18-month native canines,female and male in half,were randomly divided into the control and treatment groups on average.The control group received basal diet,the treatment group received basal diet supplemented with 2.0g selenium-and zinc-enriched probiotics everyday.To determine the experimental indices,the samples were collected on D0,D15 and D30,respectively.Results Compared with the control group,on D15,the content of selenium and zinc in blood,blood GPX,serum SOD,T-AOC and the amount of Lactobacillus in the experimental group were significantly increased,while the amount of Escherichia coli significantly decreased,but the serum MDA and the amount of Bifidobacterium,Staphylococcus and Enterococcus had no significant change.On D30,the content of selenium in blood,serum SOD,T-AOC and the amount of Lactobacillus were very significantly increased,while the content of zinc in blood,blood GPX and the amount of Bifidobacterium significantly increased;but serum MDA and the amount of Escherichia coli,Staphylococcus and Enterococcus very significantly decreased.Conclusion Selenium-and zinc-enriched probiotics could increase content of selenium and zinc in blood,enhance antioxidation function,improve and regulate the intestinal microflora.

EFFECTS OF DIFFERENT SELENIUM SOURCES ON THE FUNCTION OF HUMORAL IMMUNITY AND ANTIOXIDANT CAPACITY OF RABBITS IN VIVO / 营养学报

Objective: To explore the effect of different selenium sources on the function of humoral immunity and antioxidant capacity of rabbits. Method: Thirty-five rabbits were randomly divided into seven groups and vaccinated with rabbit haemorrhagic disease (RHD) dead vaccine. At the same time, rabbits were injected respectively with sodium selenite (0.1 mg/kg bw and 0.3 mg/kg bw), Kappa-selenocanageenan (0.1 mg/kgbw and 0.3 mg/kg bw), DL-selenomethionine (0.1 mg/kg bw and 0.3 mg/kg bw) and physiological saline as control. Antibody against RHD, activity of GSH-Px and content of MDA in rabbit serum were detected on 0, 10, 20, 30d after inoculation. Results: Sodium selenite (0.1 mg/kg bw), Kappa- selenocanageenan (0.3 mg/kg), and DL-selenomethionine (0.3mg/kg bw) could significantly increase the level of RHD antibody. Sodium selenite (0.3 mg/kg bw) and Kappa-selenocanageenan (0.3mg/kg bw) improved the activity of GSH-Px. All selenium groups could decrease serum MDA, but Kappa-selenocanageenan (0.3 mg/kg bw) showed the best effect. Conclusion: Kappa-selenocanageenan (0.3 mg/kg bw) was better than the lower dosage and other selenium sources in the effects on the function in humoral immunity and antioxidant capacity of rabbits.

EFFECTS OF SELENIUM ENRICHED PROBIOTICS ON ANTIOXIDATIVE ACTIVITIES AND IMMUNE FUNCTIONS IN WEANLING PIGLETS / 营养学报

Objective: To investigate the effects of selenium enriched probiotics on antioxidative activities and immune functions in weanling piglets. Methods: Twenty-four healthy weanling piglets (Duroc?Landrace?Yorkshire ) were divided into 3 groups. One served as control group (C) and two were supplemented different selenium (Se) sources namely selenium enriched probiotics (T1) and Na selenite (T2) as test groups respectively . The experiment lasted for 60 days. The blood GSH-Px activity, blood Se concentration, serum superoxide dismutase (SOD) activity, serum malonaldehyde (MDA) level, serum antibody level of swine fever, and the tissue content of selenium were determined. Results: Blood GSH-Px , Se concentration and serum SOD activity of T1, T2 groups were higher than those of control group after 60 d Se supplementation, and serum MDA content was markedly lower than control group. At the same time, the serum antibody level of swine fever, the tissue content of selenium in T1, T2 group were much higher than control group, and T1 group was higher than T2 group. Conclusion: Organic Se supplementation could significantly enhance immune function and antioxidative activities of weanling piglets.