| Brucellosis,also called Mediterranean remittent fever,heat waves caused by an allergic people and animals suffering from infectious diseases.It's first found in 1887 by David Bruce,separation and identification in the spleen.Our country is reported this epidemic disease for the first time in 1905.At present,except a few developed countries,brucellosis occurred in more than 160 countries around the world.Brucellosis is one of important zoonosis,its infected animals such as cattle,sheep,pigs and dogs,of which the most serious infection situation of cattle and sheep.Brucella can spread through the digestive tract,skin and mucous membrane and the vertical infection caused by a variety of ways,such as animal,one of the most dangerous sources of infection for pregnancy sick animals,brucella abortion or childbirth can be associated with fetal,afterbirth and amniotic fluid,breast milk is also one of the important sources of infection.The most significant clinical symptoms of reproductive barriers and cause pregnant female abortion,common in mid and late pregnancy,and infertility for a long time;for sire,cause epididymitis and orchids,serious impact on reproductive performance.Brucella is often associated with brucellosis,arthritis and other clinical symptoms occur.In addition,parasitic brucella as cell bacteria,once infected individuals through antibiotic treatment,even animals self-healing,still is in danger of continuous discharge bacterium,so the immune and quarantine is the basic way of control and prevention of brucella.Brucella is currently the law of the People's Republic of China on the prevention and control of infectious diseases of 35 kinds of infectious diseases in the B class infectious diseases,and is listed as the second class infectious disease at the same time.Nowadays,for brucellosis detected methods are mostly based on humoral immunity,the traditional detection methods have rose bengal plate agglutination test(RBPT)and standard tube agglutination test(SAT),complement fixation test(CFT)and so on,but the traditional method takes longer time,method cumbersome and judgment is difficult,the result judgement is very dependent on the experience of the relevant personnel.In the 1970 s,with the development of related technologies,enzyme-linked immunosorbent assay(ELISA)and fluorescence polarization experiments(FPA)is also used for the detection of brucella.Due to technical limitations,however,the use of animals in the ELISA test kit has been monopolized by foreign manufacturers,has the high testing cost;Moreover,our country existing animal brucella vaccine prevention,like brucella S2,A19 and M5 vaccine strains are smooth type strains,after the immune antibodies and natural infection between interference,makes the specific status of the existing method cannot distinguish between animals.Some foreign countries(such as the United States,Canada,etc.)by using the rough strain vaccine to solve the problem,but because our country has not effectively use the rough strain vaccine,make our country of brucella effective quarantine for animals and eliminated in time more difficult.In order to solve these problems,this study established a competitive ELISA for animal brucellosis antibody detection method and bovine brucellosis indirect ELISA antibody detection method,and set up for cattle brucella subtype IgM antibody detection method,and defined the different stages of antibodies.Under different infection/immune state and build the animal model,and draw lessons from the previous studies,identified the different situations of cattle the humoral immune condition,based on different cattle brucella antibody subtypes in the differential diagnosis methods.At the same time,this study aimed at the existing testing methods is limited to test the limits of the humoral immune status,through the selection of stimuli,and means of mycobacterium tuberculosis disease,established for cattle brucella IFN-gamma release test method.This study by monoclonal antibody technology to screen resistance to against the brucella specific monoclone antibody cell lines(4c3 strain),and antigen coating concentration was determined by the board the crossover,substrate concentration,sample diluent,sample dilution degree,set up the animal brucella antibody competitive ELISA detection method.Under the condition of the sensitivity of this method was 97.9%,specificity of 96.3%.This study also in purification of brucella lipopolysaccharide(LPS)antigen,according to the above methods were established for brucella specific subtype IgG and IgM antibody detection method,the sensitivity of 97.7% and 97.7% respectively,specificity of 95.5% and 92% respectively.Moreover,the indirect ELISA method based on similar as brucella antigen of Yersinia enterocolitica O:9 and E.coli O:157 antiserum had no cross reactivity,proved good specificity of the method in this study.In this study,respectively for brucella subtype IgG and IgM antibody detection method,on the basis of(cows)model by building a susceptible animals,to gain the vaccine is commonly used in China(brucella suis S2 vaccine strain and brucella abortus A19 strain)as well as standard virulent strain(brucella abortus 2308 strains)antibodies.By testing found that S2 after oral vaccine during the testing period(0 ~ 150 days)levels are lower than the critical value,the IgG and IgM has reached the highest in immune after 15 days after the decline gradually,and lower than the critical value in immune after 60 days;The subcutaneous injection A19 immune group had its IgG and IgM in 15 days after the peak,after all fell rapidly,Ig G 90 days after infection,IgM is lower than the critical value after 60 days;Unlike S2 and A19 group,tapping poison control in 21 ~ 30 days after its IgG has always remained stable level,while subtype IgM antibody in time than the immune group delay,rapidly declining after the peak.This shows that between the vaccine and its subtype antibody is an obvious difference between wild strains,through these differences,can detect antibody subtypes methods to different states of the cattle were identified.In order to verify the feasibility of this method,this research clearly 50 samples for testing to the background,through the establishment of the differential diagnosis of the above method,to determine its true state,in addition to the individual by being cross range,other samples(46/50)differential diagnosis can be carried out,the accuracy is 92%.In our study,we found that at the same time,in the early stage of the infection of infected individuals through antibody detection method cannot achieve timely diagnosis,compared with humoral immunity,cellular immunity effect time is shorter,stronger reactivity.This study by drawing from bovine tuberculosis IFN-? related detection method,determines the 1.0 ×107 cfu/ml of inactivated brucella abortus 2308 antigen as the best antigen for bovine brucellosis IFN-? release test and to test the infected cattle,the results found that IFN-? release test can detect time point to infection after 5 days in advance,compared to traditional antibody detection method,the 2 ~ 3 weeks in advance.Show that the method can increase to the detection effect of brucellosis is of great significance for early detection and treatment positive individuals.Finally,this study set up animal brucella competitive ELISA detection method,bovine brucella indirect ELISA detection method can effectively detect the brucellosis infection,compared with the traditional detection method is more convenient and rapid,and coincidence rate;and the indirect ELISA detection method for the IgM subtype of the bovine disease is important for the accurate analysis of infection.And the differential diagnosis method based on antibody subtype solves the problem that the vaccine immune and natural infection cannot be identified;The IFN-? release is important for early diagnosis of the disease. |
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