| Background:Brucellosis caused by Brucella infection,also called Malta fever,is a zoonotic disease.In developing countries Brucellosis is one of the most distributed,dangerous and common worldwide diseases,such as in Mediterranean Region,the south and the center of America,Africa,Asia,Latin America and the Middle East.Human brucellosis is mainly due to the direct or indirect contact with infected annimals,especially the sheep,cattle,etc.,as well as consumption of disease animal products(such as lamb,goat’s milk,etc.)without sufficient disinfection.The cases of human brucellosis has increased to more than 500000 a year in the world,and the trend has increased faster.According to statistical datas from the national health and family planning commission bureau of disease prevention and control after 1995,it indicated that the amount of the lowest morbidity of human brucellosis began to increase rapidly,and there was more than 30000 new cases every year from 2008.In China,there was 43486 cases in 2013 and more than 57%increase compared with the number of 2008.According to the newest data,this number has increased to 56989 in 2015,which means the incidence rate raised to 4.1828/100000.It proves that the situation of brucellosis is increasingly serious in China,and it need to take more positive and effective measures to prevent and control.Brucella can invade human by people’s digestive tract,respiratory tract or injured skin and some other way,like slaughtering infected cow and sheep,to help animals childbirth,consuming unsterilized dairy product,so the occupations(veterinarians,herdsmen,butchers for example),are more likely to be infected.Most of the Brucella invaded in host would be killed by the immune system,the surviving part of the bacterium could parasite in host cells through five step:adhesion,biochemical,invasion,survival and reproduction.Brucella could get into the lymph node and be swallowed by macrophage.Because brucella could reside in Brucella-containing vacuole and resistant to intracellular acid condition and lysosome,the bacterium can persist and reproduct in cells.Once macrophage cells infected by Brucella died and many bacteria were discharged in periodic blood and lymph,then new infection will come up in the liver,spleen and some other organs,as as well complications.Because the clinical symptoms of Brucellosis are variety and complexity,it is easy to be confused with influenza,rheumatoid arthitis and some other diseases.According to the ministry of health’s diagnosis and treatment guidelines,the diagnosis of brucellosis should consider epidemiology,clinical feature,and laboratorial tests’results.The bacterium blood culture is the golden standard of brucellosis diagnosis,but for considering biosafety and test condition,most of hospitals can not do blood culture for principled time(10~15 days).So,serological detection became a widely prescribed detection methodssuch as SAT,RBPT and ELISA,because serological detection can’t distinguish vaccination and infection.Moreover,it was still risk to misdiagnose of Brucellosis,especially in northern China.ELISA sensitive is much higher than SAT(10~100 times)to detect antibodies of Brucella which is andit is easy to operate towidely be used in brucellosis screeming and diagnose.But the standard is only established in B.abortus.However,most of serologic methods target to detect specific antibodies directly,so it’s hard to find positive results in early infection,espectioally within 12~16 days.On the other hand,many general patients,besides herdsmen,veterinarians etc.who are routine and regular access with sheep and cattles,have no clear epidemic contact history,so that it maybe delay or clinical misdiagnosis,especially in some non-epidemic areas.Other tests like PCR had not generalized yet,although it has higher specificity,sensitivity,rapid and simple procedure.Brucellosis is a severe public health problem since the foundation of China,and it’s no time to delay to build a validity method for brucellosis detection.Objective:Aim to establish the brucella detection method by the monoclonal antibodys to outer membrane proteins of Brucella melitensis and assess its feasibility in clinical brucellosis diagnosi.Method:1.Meterials:Monoclonal antibodies to outer membrane proteins of Brucella melitensis,and recombinant proteins(OMP25,BP26,OMP31)expressed by lentivirus were all prepared and produced in our labs.Blood samples collected from 154 brucellosis patientsin General Hospital of Heilongjiang agricultural reclamation bureau,Harbin,China.Bacterial smears with Brucella were donated by Guangzhou CDC.2.Method:(1)Immunoenzymic straining test with brucella:F irst get the Brucella smear from Guangzhou Centers for Disease Control and Prevention and use the antibodies made by our laboratory to do immunoenzyme strain to test whether these antibodies can react to the native Brucella melitensis.(2)Construction of lentivirus and immunofluorescence of the intracellular protein:Then construct the lentivirus that can express recombinant outer membrane proteins of Brucella melitensis and infect 293FT cell line,to make the proteins express intracellular.And then consider all the results of WB and ELISA,choose 4 monoclonal antibodies:anti BP26 2A4,5A5 and anti OMP31 2C1,5H3,anti HCV NS3 2E12 as negative control.(3)The detection of brucellosis patients’ PBMC by immunofluorescence:Separate peripheral blood mononuclear cells from the brucellosis patients’blood,and use immunofluorescence technology to test these cells with the antibodies.We also use ELISA Kit,NEST-PCR technology to detect serum and peripheral blood mononuclear cells as well.And compared all the results to evaluate whether these antibodies could be used in clinical condition or not.Results:1.The result of immunoenzymic straining test:Use five anti-OMP25 and twenty two anti-OMP31 to do the immunoenzyme strain test.Three fifthes anti OMP25 antibodies’ result are positive,4A12,6C12,8F3;two are negative,2B10,4F10.Eighteen twenty-second anti OMP31 antibody are positive,containing six are strong positive reaction,2D2,5B1,2C1,4E9,5B3,5H3;six are commonly positive reaction,2G9,7A3,8F11,2A8,2B6,5F11;six are weakly positive reaction,2E7,6D8,4H6,5B11,6F9,11C4;the other four are negative reaction,112,2H5,4H10,6E10.The positive rate of antibodies of OMP31 are 81.8%,higher than antibodies of OMP25,is only 60%.And the strength of OMP31’s antibodies is higher than OMP25’s.2.The result of construction of lentivirus and immunofluorescence of the intracellular protein:Use the lentivirus system to impress the proteins(OMP25,BP26 and OMP31)in the 293FT cell,and screeming the monoclonal antibodies which can react to these proteins.All the five antibodies of OMP25 can react to the reconstructed protein OMP25,and the 6C12 is highly reactive.Two third antibodies of BP26 are positive,2A4 and 5A5;only 3H5 is negative.Among the twenty two monoclonal antibodies of OMP31,there are seventeen positive result:2C1,2E7,4E9,4H10,8F11,6E10,6F9,11C4 are higher reactive;1H2,2D2,2G9,7A3,5B1,2A8,6D8,5B3,5H3 are commonly reactive,and the other five are negative.3.The result of immunofluorescence:all positive result of patients is 106,as 68.83%of all.There are 65(42.21%)positive in 2C1 of OMP31,89(57.79%)positive in 5H3 of OMP31,95(61.89%)positive result in 2A4 of BP26,88(57.14%)positive result in 5A5 of BP26.4.Result of PCR:Forty seven samples are positive in one hundred fifty four serum DNA samples.The percentage of positive is 30.52%.Fifty one samples are positive in one hundred fifty four cells’ DNA samples.The percentage is 33.12%.5.Result of the ELISA Kit:only four samples’ results are lower than 0.9,which means negative.Three samples’ results are weakly positive.And one hundred forty seven samples’ results are high than 1.1,it is positive.Conclusion:This study uses immunofluorescence to test the existence of Brucella in the patients’ monoclonal phagocyte.The percentage of positive in all patients is 68.4%.It is much higher than the result of bacterium culture(it is only 2.60%).And the procedure of immunofluorescence is more secure,and reduce the risk of detection personnel during the test procedure.And the antibodies can be enlarge cultivated,produced and purified,which would use lower cost during testing large sample size.In a conclusion,this research established the immunofluorescence method can detect the bacteria in patients’blood mononuclear phagocytes,makes it a beneficial supplement of bacterial culture in clinical diagnose. |
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