techniques for the brucellosis
1. Overof Brucellosis (the cloth disease), Brucella is a zoonotic infectious disease caused. The main clinical characteristics of dams abortion, mastitis, infertility and various organizations (such as testosterone, joint) inflammation. After infection of human brucellosis, longer duration, recurrent, long-term unhealed. Brucellosis is almost all over the world. Areas where animals have brucellosis epidemic. According to incomplete statistics, in 160 countries and territories, there are 123 countries and regions, brucellosis occurred. Our common in Inner Mongolia, northeast and northwest pastoral areas. Agricultural area in the north also have distributed, often has to become endemic. In adverse environments, such as under the influence of antibiotics, the bacteria mutate easily, usually formed by Brucella variation in the body a long time. Strain on the thermal resistance is not strong, 63 by 7 ~ 10 min can be killed, can survive in fresh milk from 2 days to 18 months, can survive in frozen meat in the 14 to 47 days, in dry soil and the fetus in vivo survival of 37 days, respectively 6 months. Sensitive to common disinfectants, such as minutes of 0.1% mercuric chloride, 1% or 2% of the children to the Soviet Uni5 min formalin can kill it. 4 h exposure to sunlight can kill the bacteria. And a variety of animals susceptible to brucellosis. Animals, sheep, cattle, pigs, the susceptibility of the strongest. Dams than male livestock, the incidence of adult animals more than
the young animals. In the dams, the incidence of first pregnancy more dams. Carrier animals, especially sick animals, aborted fetuses, afterbirth is the main of infection. Digestive tract, respiratory tract, reproductive tract is the major route of transmission, but also through injury of the skin, mucous membranes and other infections. Chang Cheng endemic. People, mainly through the skin, mucous membranes and respiratory infections. The incidence of the disease all year round. Disease endemic area in the peak season (spring and early summer) can be spotty outbreaks. And occupational disease closely related, such as: veterinary, animal husbandry workers, slaughterhouse workers, leather workers, and other higher chance of infection. The prevention of human brucellosis, the first awareness of occupational infection, where in the animal breeding areas, slaughtering areas, livestock processing plant workers and veterinarians, laboratory workers, etc., must maintain protection system (ie, wearing protective clothing , well disinfection), especially in the newborn animal mass production season. Also pay special attention. Sick animals must be sterilized milk after eating meat. Available when necessary, vaccination, allergy tests should be performed before inoculation. Negative responders to vaccination. 2. Laboratory Monitoring Technical Overtechnical specifications according to Brucellosis Control) 2.1 Detection of serological testing or bacterial isolates. Screening test used bengal plate agglutination test (RBPT) or whole milk dairy brucellosis ring test (MRT) (see GB / T 18646). Official selection of animal brucellosis test tube agglutination test (SAT) or animal brucellosis complement fixation test (CFT) (see GB / T 18646) 2.2 Monitoring object: cattle, sheep, pigs, deer and other animals. 2.3 Monitoring Methods: Epidemiological, serological methods, combined with monitoring of bacterial isolates. 2.4 The scope of monitoring, the number of immune region: in newborn animals, animals not immunized, immune year and a half or one year after oral immunization monitoring of livestock (pigs can be carried out in six months after oral immunization.) Monitoring at least once a year, sampling 500 pastoral county (only) above, agricultural areas and semi-agricultural and semi-pastoral areas, sampling 200 (only) over sampling in remote areas inaccessible rural 9 3 50% of the village livestock serological monitoring. Non-immune areas: monitoring at least once a year. Pastoral areas of the county to control the standard sampling 1000 (only) above, agricultural areas and semi-agricultural and semi-pastoral areas, sampling 500 (only) above; achieve stability control standard sampling 500 pastoral county (only) above, agricultural areas and semi-agricultural and semi- Pasture sampling 200 (only) above. All of the cows, milk goats and breeding stock must be twice a year serological surveillance. 2.5 Monitoring time monitoring of adult animals, the pigs, sheep, more than 5 months of age, cows more than 8 months of age, pregnant animals, fetal birth in 1 month to 1 month between; on S2, M5, S19 strain immunization of an animal vaccine, 18 months after vaccination (pigs 6 months after inoculation) were. 2.6 determine and report the monitoring results appear positive screening test, and epidemiological history and clinical symptoms or isolated Brucella, sentenced to sick animals. Formal test of serological positive or tube agglutination test complement fixation test was positive, judged as positive animals. Determined should pay attention to the exclusion of other suspected causes of disease and vaccine vaccination serological reactions. Required monitoring results of the use and complete and timely report. Disease treatment must be strictly enforced, "Bovine tuberculosis control technical specifications" of the requirements. 3. Laboratory tests Laboratory tests include the following methods: 3.1 bacteriological testing will be cut into small samples, clamp the clamp applied directly in the liver soup agar plate, in duplicate, a 10% carbon dioxide release culture, a culture in the general environment, generally at 37 for 10 days in the Brucella agar plate colony was colorless, transparent, round, elevated, smooth, homogeneous sample of colonies, colony center sometimes very small particles, micro-colonies began to clear, then gradually becomes turbid, colony sizes. Smear microscopy, colonies were picked to find brucellosis. Aborted fetal tissue with the dams and the afterbirth smear microscopy can detect brucellosis. 3.2.1 Methods 3.2 agglutination test for brucellosis diagnosis of the current multi-use agglutination test, serum or milk in the main anti-Brucella antibodies. Monitoring methods used bengal plate agglutination test, tube agglutination test brucellosis, whole milk ring test, complement fixation test. RBT test and whole milk ring test for brucellosis in livestock and dairy farm field screening test for brucellosis in dairy cow monitoring and diagnosis of brucellosis in the screening test; tube agglutination test and complement fixation test for diagnosis of sheep species , bovine species and breeds of livestock infected with brucellosis. 3.2.2 diseased 18.104.22.168 bengal plate agglutination test, tube agglutination test and complement fixation test materials for the serum sickness, that is prepared by conventional methods separation of blood serum, serum should be fresh, no obvious protein clot, no hemolysis, No smell of corruption. 22.214.171.124 Brucellosis whole milk cow disease testing materials for the ring fresh, clean, whole milk, the sampling of the breast should be dams with warm water washed, dried, and then squeeze into a clean vessel in the emulsion. 3.2.3 steps (only introduce common RBT test and tube agglutination test) 126.96.36.199 RBT test the operation of main processes: 188.8.131.52.1 standard negative and positive serum control and test sera were drawn The negative serum 0.03ml, respectively, drops on the glass, in the positive and negative sera plus 0.03ml of the next drop of antigen, serum and antigen stir with a toothpick to make the full mix, 2min to judge when the results of a positive serum agglutination; negative No agglutination of serum, were uniformly pink. 184.108.40.206.2 Detection of the tested serum samples were drawn each 0.03ml, drops on the glass to the side in the tested serum dropwise 0.03ml of the antigen, serum and antigen stir with a toothpick to make the full mix, 2min judge the outcome when . 220.127.116.11.3 The results determine the method and basis: in the negative, positive serum control under the conditions established before determination of serum were seized; subjects appeared in the serum 4min visible agglutination were judged as positive (), no agglutination phenomenon, who was sentenced pink uniform negative (-). 18.104.22.168 The main tube agglutination test operation: 22.214.171.124.1 serum dilution subjects: In general, cattle, horses and four camels and 1:400 dilution with 1:0,1:200 degrees. Large-scale quarantine may also be that only two dilutions of cattle, horses and camels for the 1:50 and 1:100; pigs, goats, sheep and dogs for the 1:25 and 1:50. 126.96.36.199.2 serum dilution (in sheep, pigs for example) and join antigen approach: each serum with 5 small test tubes (diameter 8-10mm), the first tube add 2.3ml phenol saline, and the second tube without, third, fourth and The fifth tube add 0.5ml. Subjects with 1ml serum straw draw 0.2ml, join the first tube and mix well. Hybrid method: a mixture of the test tube inside the suction pipette, and then blown into the original test tube along the wall, so inhaled, sucked out three or four times. After mixing, the mixture of the straw were added to draw the second tube and third tubes, each tube 0.5ml. The third tube to the mixture of straw mix (method with the former) to draw 0.5ml tubes by adding the fourth mixture, and from the fourth to join the fifth tube 0.5ml suction tube, discard the completion of the fifth tube 0.5ml mixing . After such a dilution effect from the second tube 1:12.5,1: serum dilution, and 1:100, respectively. Provisions of carbolic acid in serum diluted with normal saline with 0.5%, but when testing with 0.5% goat serum 10% carbolic acid diluted salt water solution. Adding antigen Methods: The 0.5% phenol saline, the antigen for 20 times diluted stock solution (0.5% if the serum was carbolic acid, 10% dilution of the antigen stock solution of salt water solution with 0.5% phenol also, 10% salt solution diluted) and then adding the above-mentioned tube (first tube without, reserved for the control of serum protein aggregation), each tube 0.5ml, shake well, add antigen, the second to the fifth pipe pipe pipe the mixture solution of the product are 1ml, dilution of serum from the third tube from the turn into 1:,1:100 and 1:200. Cattle, horses and camels and processing antigen serum dilution method, the specific method is consistent with the above, the difference is, the first tube plus 2.4ml0.5% phenol saline and 0.1ml subjects serum plus antigen after the second pipe to the five serum and 1:400 diluti:0,1:200. 188.8.131.52.3 control tube production: each test subject to the three control each. Negative serum control: negative serum antigen dilution and processing methods and subjects with serum. Positive serum control: control positive serum titers to be diluted to its original, plus serum antigen the same methods and subjects. Antigen control (antigen know whether the phenomenon of self-curing): Add 1:20 dilution of antigen in the test tube, 0.5ml, plus 0.5ml0.5% phenol saline (0.5% if the serum was carbolic acid, 10% salt water solution is also diluted 0.5% phenol, 10% salt water solution). 184.108.40.206.4 turbidity tube production: preparation of each test to be brighter than the turbidity tube to determine the level (aggregation level) is based, the preparation is as follows: Take this test with the antigen dilution (ie, 20 times the liquid antigen dilution) 5-10ml of 0.5% phenol added the same amount of saline (0.5% if the serum was carbolic acid, 10% salt solution is diluted by adding 0.5% phenol, 10% salt water solution) against dilution, and then press the table prepared turbidimetric tube.
No dilution of control antigen (ml) phenol saline (ml) 0.0 1.0 1 tag clear brightness 100% 75% 3 2 0.25 0.75 0.50 0.75 0.25 0.50 50% 4 25% 5 1.0 0.0 0% -220.127.116.11.5 all tube oscillations in the full post 37 -38 incubator 22-24 hours, then check and record the results. 18.104.22.168.6 Record the results: According to the top of the tube to clear the liquid intensity of the brightness record of agglutination (agglutination titer), especially 50% of the clear brightness (that is, "" aggregation) to determine the result of a great relationship. Management controls required to determine turbidity. : Equal to the full aggregation and precipitation, the upper liquid 100% brighter (ie 100% of the cell sink.) : Equal to almost completely aggregation and precipitation, the upper liquid 75% brighter. : Equal to a significant aggregation and precipitation, liquid 50% brighter. : Equal to precipitation significantly, 25% brighter liquid. -: No precipitation is equal to, not brighter. To determine when each titer, should be two, "" above the aggregation phenomenon (that is 50% brighter) maximum serum dilution of serum agglutination titer. 22.214.171.124.7 126.96.36.199.7.1 test results to determine cattle, horses and camels at 1:100 dilution, pigs, goats, sheep and dogs appear in the 1:50 dilution of more than two plus agglutination phenomenon, test serum determined to be positive. Cattle, horses and camels at the 1:50 dilution, pigs, goats, sheep and dogs appear in the 1:25 dilution of more than two plus agglutination was judged to be suspicious of test serum response. 188.8.131.52.7.2 livestock suspected reaction, after 3-4 weeks, blood samples to be re-tested in cattle and sheep, is still doubtful if the re-examination when the animals judged to be positive. A rein pigs and horses, if the price remained suspicious agglutination reaction level and farm animals without clinical symptoms and a large number of positive responses Huanxu the items judged to be negative in the serum of the animal. 184.108.40.206.7.3 of the individual pig serum often experience non-specific agglutination, the test results to be combined with epidemiological determination. If the serum of individual subjects appear weak positive reactions (eg agglutination price 1:0), but all the pig herd had no clinical symptoms of brucellosis (abortion, arthritis, testicular Yandeng ), consider such non-specific response by the blood 3-4 weeks to re-examination. 220.127.116.11.7.4 The test results notify pet owners, they should indicate the agglutination titer. 3.3 Direct detection of disease pathogens compound application of immunoenzyme staining direct method, indirect method and the SPA assay Brucella disease compound, applied PCR detection of Brucella should be applied in practical work. Enzyme-linked immunosorbent assay, polymerase chain reaction method for the specific operation, in accordance with operating instructions.