Research of the Isolation of E. coli in Broilers and Drug Susceptibility Testing.doc

1、1Research of the Isolation of E. coli in Broilers and Drug Susceptibility TestingAbstract. E. coli disease in broilers epidemiological survey conducted this test for higher mortality chicken clinical examination， inspection and disease pathogens necropsy to determine the chickens infected with E. co

2、li. The results of susceptibility testing， said Zhuang chloramphenicol， neomycin， and the ofloxacin is highly sensitive drugs. Keywords： Broiler Susceptibility testing Isolation and identification of pathogenic E. coli 1. Introduction It is understood that the chicken E. coli disease is a general te

3、rm caused by a variety of pathogenic E. coli illness. Broiler E. coli disease has become one of the major infectious diseases harm aquaculture. Broiler easy season in large temperature has difference suffering from the disease. In the winter， the usual lack of good breeding management is more likely

4、 to cause disease， and for this reason is often overlooked， thus causing unnecessary economic losses. 2E. coli is a Gram-negative bacillus spore-free， the size of 0.4-0.7m 2-3m， Rounded at both ends， scattered or in pairs 2， flagellated， and some strains can form capsular easy proliferation in norma

5、l medium， strong adaptability. The bacteria sensitive to general disinfectant， but their mucus and feces will reduce the effect of these disinfectants. The bacteria to antibiotics and other drugs can easily develop resistance. In the animal building， E. coli in water， faeces and dust can survive for

6、 several weeks or months. 2. The related materials 2.1 Diseased Material from the onset of the disease in chickens has obvious symptoms of clinical diagnosis， initial diagnosis of pathological autopsy Colibacillosis chickens. （Dead time less than 24 hours in winter， summer is not over 20 hours） or d

7、ying chicken heart， liver， spleen， lymph， fallopian tubes and other organizations. 2.2 Medium Medium containing glucose， maltose， urea， hydrogen sulfide， peptone， lactose and other biochemical fermentation medium MacConkey agar and nutrient agar medium （peptone 17g， Xiu peptone 3g， porcine bile 5g，

8、NaCl 5g， 17g agar， 3distilled water 1000mL， lactose 10g， 0.01% crystal violet solution 10mL， 0.5% neutral red solution 5mL， ph7.3） and making plates according to the requirements. 2.3 Test equipment Experimental equipment has balance， pressure steam sterilizer， sterile laminar flow， the number of ba

9、cteria biochemical reactor and test centrifuges and other commonly used equipment. 3. The separation of E. coli in broilers 3.1 Isolated and cultured We hooking the dead chickens liver， lungs， and pericardial fluid in a sterile environment were inoculated on a MacConkey agar plate and ordinary nutri

10、ent agar plates. The inoculated agar plates were placed in a constant temperature incubator at 37 and 24 hours on agar plates to pick from a single colony， for pure culture. The colonies were then placed in the incubator spare at 4 . 3.2 Biochemical tests Triple sugar iron agar test： Take inoculated

11、 bacteria in pure culture triple sugar iron agar slant culture tubes， 37 cultured 24h， observe the results. Carbohydrate fermentation tests： 4Pure culture of bacteria inoculated taken sugars （glucose， lactose， maltose， sucrose） fermentation micro tube， 37 cultured 24h， observe the results. Indole te

12、st： Take pure culture of bacteria inoculated into peptone water broth， 37 culture 3d. Before adding a small amount of ether， shake the tube to extract and concentrate indole， let it float on the surface of the culture medium， and then along the wall slowly try Jia Ruou - Wave IIs reagent few drops，

13、observe the results. Methyl red （MR） test： Take pure culture of bacteria inoculated in glucose peptone water broth， 37 culture 3d， add two drops of methyl red reagent， and immediately observe the results. VP test： Pure cultured bacteria were inoculated in taking glucose peptone water broth， 37 cultu

14、re 3d， an equal amount of VP reagent （ added first a solution VP， VP solution B added） ， after mixing， observe the results. Citrate utilization test： Pure culture of bacteria taken citrate seeded on a slant medium， 37 cultured 24h， observe the results. Ureolytic test： 5Take pure culture of bacteria

15、inoculated into urea medium， 37 cultured 24h， observe the results. H2S production test： Take a pure culture of bacteria inoculated in H2S puncture culture tubes， 37 cultured 24h， observe the results. The isolation rate of E. coli test broilers is 85.00%. 3.3 Serum test Agglutination antigen is first

16、 prepared： Identification of the isolated pure culture of E. coli strains were inoculated in nutrient broth at 37 for 24h， in a sterile environment with cotton and autoclaved forceps tube dipped bacteria was coated on a prepared agar after three 37 for 24h， under sterile conditions with a pipette pi

17、pette 5mL of physiological saline on the plate， and then autoclaved for cleaning coating bar lawn until after shave all moved into a sterile flask； inactivated by high pressure 121 2h destroy bacteria K antigen； bacterial antigens after washing with saline by centrifugation and washed four times 4mL

18、 each 4000r/min centrifugal 20min supernatant； repeated pipetting adding 2mL saline mix， McFarland turbidity standard tube 10 compared to finally get concentrated broth （4 billion bacteria / mL） of 1.8mL； finally joined the proportion of 6% alkaline methylene blue staining， 4 to save backup. This an

19、tigen is prepared 6agglutination of E. coli O antigen. With 26 kinds of E. coli single factor serum， the E. coli O antigen plate agglutination test， while doing standard strain antigen positive control and a negative control. 4. Susceptibility testing 4.1 The required artifacts Incubator， alcohol la

20、mp， vaccination stick， scissors， tweezers， ordinary nutrient agar， glass plates， beakers， glass， distilled water， saline， fine needles， general balance， medicine spoon， ruler. 4.2 During the test Liver， lung and pericardial fluid and other tissues we dipped into the ring with dead chickens were inoc

21、ulated directly on the surface of the agar evenly， which is normal because of E. coli grown in normal medium， and E. coli bacterial disease dead chickens visceral tissue quantity. In the painting process， we mean the dishes can be divided into four parts， and find the corresponding points around the

22、 dish， labeled accordingly. After finding a starting point， the intermediate position inoculated into a Petri dish. Repeated several times until inoculation is complete. Note that not to damage the agar. Then， at a position corresponding to the Petri dish on the drug name， the drug in contact with t

23、he 7agar. Finally， the plates were placed in an incubator at 37 24 hours. 4.3 Note In the course of susceptibility testing， we should pay attention to the concentration， temperature， mass and pH of the medium. But also concerned about the concentration and pH value of the susceptibility piece suitab

24、ility. In recent years， cases have occurred when E. coli infection in broilers. And support families in order to control this from happening， taken into the corresponding additives in broiler feed means. But sometimes， most of the farmers in the course of the additive into the blind use of antimicro

25、bial drugs， the results of such behavior is to produce a drug-resistant strains. The reason why the environment more resistant strains of E. coli， E. coli is faster because the resistance is formed by fimbriae of Escherichia coli resistant to pass to other E. coli plasmid. Susceptibility testing res

26、ults from the analysis of E. coli have been sensitive to the continued use of the drug with a long， leading to drug resistance in varying degrees. In summary， based on the results of susceptibility testing is best to choose the amount of drugs and medicines in the treatment of our broiler 8colibacil

27、losis， a reasonable amount of medication should be conditional. 5. Summary Statistics show that E. coli is conditional pathogens. Therefore， to prevent the occurrence of E. coli disease， we must first eliminate pathogenic conditions. Thus， in the daily feeding should be used in high-quality feed， an

28、d a sufficient amount of vitamin supplements and electrolytes； strictly enforce health and epidemic prevention measures to create a favorable breeding environment； should be reasonable and appropriate to add feed additives and so on. Currently universal vaccine to treat the disease has not yet appea

29、red on the market. The reason for this phenomenon is that the advantages of the different parts of the serum. Therefore， E. coli is the basic working immunization screening area superior immunogenicity of vaccine production strain of E. coli. Proposed conditional broiler breeder farms， the best use

30、of inactivated vaccine owns reliable this effect， more suitable for serotype complex areas. Winter use of greenhouses and chickens， are often difficult to grasp the good news is balanced ventilation with heat， the emphasis is often on the insulation and cause insufficient ventilation， making the poo

31、r 9house gases increased mucosal barrier injury， such as defensive chicken body. When the chicken defense mechanisms to reduce， it is easy to cause the occurrence of E. coli disease. Stocking density is too large， such as stress factors， and the occurrence of the disease also has a close relationshi

32、p. Meanwhile， pathogenic E. coli normally present in the intestines of healthy poultry， generally less likely to occur in normal healthy chicken disease. Although vaccines are attenuated， but still can cause certain infections and immune suppression. Therefore， prevention of E. coli disease from the

33、 roots， remove all unfavorable factors chickens， good sanitation and disinfection of all aspects of the work， the use of drugs on this basis， in order to achieve better results. As Chinas policies system has been continuous improvement and development of Chinas animal medicine and has also been a co

34、nstant development， I believe in the development of our country and susceptibility testing of E. coli in broilers will become increasingly distant. References 1 Fang Guangyuan. Guang-Wei Chen. An AA broilers Isolation and identification of E. coli D. Jinling Institute 10of Technology， 2011 2 Wu Heyun. Broiler E. Lee root disease diagnosis report J. Chinese livestock and seed industry， 2013 （06） 3 Wang Jinbo. King naturally. Treatment comparison test florfenicol ester （sodium） on broiler pathogenic Escherichia coli J. Veterinary Market Guide， 2009 （01）