| Objective:In this study, the Argali hybrid sheep(AHS) and Bashibai sheep(BS) were used as Mycoplasma ovipneumoniae(MO)disease model, and the AHS was susceptible to MO,while the BS was not.To analyze the immunological response of the recombinant bactericidal/permeability-increasing protein(r BPI) in AHS infected with MO, the AHS was treated by injecting r BPI,then detecting several related cytokines concentration by ELISA,relative expression level of BPI m RNA by real-time QPCR and peripheral blood physiological indexes by blood routine test.Method:(1)Artificially infecting MO in sheep: Proliferation culture of the MO strain with special medium for MO, measuring the MO concentration and diluted to the needed concentration for artificial infection. 6 BS was taken as group A, while 12 AHS were randomly divided into group B and C. All the sheep were artificially infected with MO. Clinical symptom and temperature were recorded every day. Two weeks later, two nasal secretions were randomly taken from every group and were cultured for biochemical identification.(2) Therapeutic effect of BPI on sheep with MO: From the 4th day post-infection(DPI),group C was treated with r BPI, 150mg/sheep/day, and group A and B with the same dose placebo.At the end of the experiment, all sheep were dissected to observe lung pathological changes and to conduct pathological grading.(3) Endogenous BPI level change in AHS with injecting r BPI: collecting blood and separating neutrophil before infection(0 d) and the second day(2d), fifth day(5d), first week(7d), second week(14d),third weeks(21d)after infection,respectively. BPI m RNA relative expression level was detected by real time-QPCR method.(4)Collecting blood and separating serum in 0d, 2d, 5d, 7d, 14 d and 21 d after infection,respectively. The concentrations of IL-1β,IL-4,IL-6,TNF-α, IL-8,IL-10 and IFN-γ from serum were assayed by ELISA to compare cytokine content changes in all groups.(5) Detecting some physiological blood indexes: peripheral blood was collected and conducted by routine blood test in 0d, 7d and 14 d after infection,respectively.Result:(1) Nasal secretions samples were cultured and the medium became yellow after 5 days. Glucose fermentation test,digitalis saponins experiment, methylene blue reduction test and hemoadsorption test were positive, while hydrolyzing arginine experiment was negative. And the hemolytic test showed β-hemolysis. These suggested that all sheep were successfully infected with MO contained in nasal secretions.(2)Anorexia, transiently high temperature(40-41 ℃), moist sounds, nodead case and haemorrhage in lung were observed in group A on the 2nd and third DPI. Long high temperature(40-41℃),cough,serous nasal discharge, even serious moist sounds, diarrhea,pink meat-like and liver-like changes with haemorrhages, ecchymosesin lung and two dead case were observed in group B. Slightly high temperature, anorexia, cough and haemorrhages, ecchymosesin lung were observed in group C. Histopathological scores results: Scores of the three groups were 9.4,19.9 and 12.8,respectively. The score of group B had significant difference with group A and C(P<0.05; P<0.05). Thus implying clinical symptoms and lung inflammation relieving in AHS after injecting r BPI.(3) On the 7th DPI, the expression level of BPI gene in group C was lower than group A and B(P<0.05). From the 14 th to 21 st DPI, the BPI ingroup C was significantly higher than group B(P<0.01). These data indicated that injecting r BPI had an effect on endogenous BPI level.(4) From the 7th to 21 st days post-infection(DPI), the concentration of IL-1β in group C was significantly lower than group A and B(P<0.01). From the 7th to 21 st DPI, the concentration of IL-4 in group C was significantly higher than group A and B(P<0.01),and on the 14 th to 21 st DPI, the IL-4 in group A was higher than group B(P<0.05). From the 14 th to 21 st DPI, the concentration of IL-6 in group C was significantly lower than group A and B(P<0.01). the IL-6 in group A was lower than group B(P<0.05). On the 7th DPI, the concentration of IL-8 in group C was lower than group A(P<0.05). From the 7th to 21 st DPI, IL-8 in group B was significantly higher than group A(P<0.01). On the 5th DPI, IL-10 in group C was higher than group A(P<0.05),but lower than group B(P<0.01). On the 14 th to 21 st DPI, IL-10 in group C was lower than group A(P<0.05),but higher than group B(P<0.05). On the 14 th to 21 st DPI, IFN-r in group C was higher than group A(P<0.05). On the 14 th to 21 st DPI, TNF-α in group C was higher than group A(P<0.05; P<0.01)and lower than group B(P<0.05; P<0.05).(5) WBC, LYM increased at an early stage of DPI in two breeds. MID in group C was significantly higher than group B(P<0.05) on the 5th to 7th DPI. on the 14 th to 21 st DPI, WBC and LYM in group C increased, its LYM(%), MID and MCHC was significantly higher than that in group B(P<0.05),while the GRAN(%)was opposite. It suggested that BPI had certain effect on blood physiological indexes in AHS. |
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