| Pullorum is an acute,systemic,and contagious disease caused by Salmonella pullorum in Salmonella enterica,usually occurring in 2-3 week-old chicks,with clinical symptoms including depression,diarrhea,and feces at the excretory port Adhesions,which have a high fatality rate;Adult chickens are mostly recessively diseased after infection,but the fertilization rate,egg production rate and hatchability of chickens in the chicken farm can be significantly reduced.Salmonella pullorum can be transmitted not only horizontally through food and water sources,but also through vertical channels such as eggs(chicken embryos),which greatly increases the difficulty of controlling pullorum.Although antibacterial drugs can effectively reduce the mortality rate of chickens after infection,the problem of multi-drug resistance of bacteria caused by long-term heavy use or abuse of antibacterial drugs is becoming more and more prominent.At present,the purification of pullorum pullorum is an effective measure to control the disease.The agglutination test is widely used to detect Salmonella pullorum worldwide because of its simplicity,low cost,accuracy and reliability.However,commercial agglutination antigens are not always reliable in practice,and the detection results have a high rate of missed detection.rate and false positive rate;Studies have shown that self-made antigens show significantly improved sensitivity and consistency in serum plate agglutination tests,and positive reactions appear faster and stronger.Pullorum occurred in a certain chicken farm in YJ County.In order to effectively control the infection of Salmonella pullorum in this farm,this study used clinical samples to isolate and identify Salmonella pullorum,and studied its biological characteristics;The self-made agglutination antigen was used from clinical isolates,and its detection effect was evaluated.In order to clarify the molecular biological characteristics of Salmonella pullorum in a certain chicken farm in YJ County,this study collected 90 unhatched dead embryo samples of 18-21 days old and 100 cloacal swabs of chickens with diarrhea.Diagnostic serum and PCR methods were used to isolate and identify pathogenic bacteria.The results showed that a total of 80 strains of Salmonella pullorum(80/90,88.89%)were isolated from 90 samples of unhatched dead embryos,but no Salmonella was isolated from the cloacal cotton swab samples;Using multilocus sequence typing technology(MLST)to identify its gene sequence type,it was found that 80 strains of Salmonella pullorum were all ST92(80/80,100%);The PCR method was used to detect the carrying status of 13 common virulence genes of the isolated strains,and it was found that 80 strains of Salmonella pullorum all carried 11 virulence genes(inv A,hil A,sef A,sip A,sip C,sop B,sop E,spv C,ssa R,ssr A,stn P1),while pef A and rck virulence genes were not detected;The Kirby-Bauer method was used to carry out the drug susceptibility test of 14 kinds of antibiotics on the isolated strains.The results showed that the isolated Salmonella pullorum had the highest resistance rate to erythromycin,which was 93.75%(75/80),while ofloxacin,Gentamicin,florfenicol,polymyxin B,doxycycline,gatifloxacin,ampicillin,amoxicillin,cefoxitin,ceftazidime,streptomycin,tetracycline,and cotrimoxazole sensitive;PCR technology was further used to detect the carrying status of six types of drug-resistant genes(β-lactams,quinolones,tetracyclines,aminoglycosides,sulfonamides,and chloramphenicol)in the isolated strains,and the results showed that they were related to the drug-resistant phenotype.Consistent,other drug resistance genes were not detected.In order to prove the detection effect of self-made antigen,this study randomly selected 8 strains of clinically isolated Salmonella pullorum as candidate strains to prepare agglutination staining antigen,and passed autoagglutination test,PBS non-specific agglutination test,SPF chicken serum specific Salmonella-negative chicken serum agglutination test,finally screened 2 isolates(SPI-1 and SPI-2)for the preparation of self-made agglutination staining antigen,and compared with the detection results of Qingdao Lijian commercial antigen and ELISA antibody kit.In order to prove the detection effect of self-made antigen,this study randomly selected 8 strains of clinically isolated Salmonella pullorum as candidate strains to prepare agglutination staining antigen,through self-agglutination test,PBS nonspecific agglutination test,SPF chicken serum specific agglutination test and pullorum pullorum Salmonella-negative chicken serum agglutination test,finally screened 2isolates(SPI-1 and SPI-2)for the preparation of self-made agglutination staining antigen,and compared with the detection results of Qingdao Lijian commercial antigen and ELISA antibody kit.100 clinical serum samples were randomly selected for serum plate agglutination test.The results showed that the positive rate of ELISA antibody kit was 61%,the positive rate of self-made antigen of clinical isolates was 50%,and the positive rate of self-made antigen of standard strain CVCC1792 was 43%.The positive rate of Lijian commercialized antigen is 15%;Further analysis showed that the results of the self-made antigen Kappa consistency test were highly consistent(0.78),the selfmade antigen Kappa consistency test result of the standard strain CVCC1792 was significant(0.615),and the commercialized antigen Kappa consistency test result of Qingdao Lijian was not significant(0.203);The area under the receiver operating characteristic curve of the self-made antigen can reach 0.910,the area under the receiver operating characteristic curve of the standard strain CVCC1792 self-made antigen is0.852,and the area under the receiver operating characteristic curve of Qingdao Lijian commercial antigen is 0.623.In summary,in this study,Salmonella pullorum pullorum could be isolated from dead chicken embryos in breeder farms,and the detection rate was as high as 88.89%(80/90).The 80 strains of Salmonella pullorum all carried 11 kinds of virulence genes,suggesting strong virulence;Except for erythromycin,they are sensitive to other drugs,suggesting that the chicken farm is relatively safe to use drugs;The sensitivity and consistency of self-made antigens are significantly improved,and positive reactions appear faster and stronger.This study supplemented the epidemiological data of pullorum salmonellosis in silky chickens in YJ County,and provided effective tool support for the purification of local chicken salmonellosis. |
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