The Establishment Of The Rapid Detection Method Of Salmonella In Feed And The Drug Resistance And Pathogenicity Analysis Of Isolates

This study established the PCR and real-time fluorescence PCR rapiddetection methods of salmonella in feed, has carried on separation andidentification of salmonella in feed, carried out resistance comparison ofsalmonella isolates separated from live poultry and feed, pathogenicityanalysis of Isolates.Traditional microbial culture method has been applied for detectionSalmonella in feed, it needs about5~7days, the steps are trival, itneeds many sorts of reagent and seriously dependant on subjectivejudgment, whice is prone to leak suspected colonies.So it demands muchto inspector. This study choosed specific genes-invA gene-for thepurpose, using molecular biology methods, established the PCR andreal-time fluorescent PCR detection technology for detection salmonellain feed, shorten test time to48hours, and has good specificity, highsensitivity, no false negative, lower cost etc. Detection limit of PCR infeed is1~3cfu/25g, minimum detectable bacteria liquid concentrationis3.2~4.0x102cfu/mL. This study has passed the nationalstandardization committee review, now become the national standard, inNovember2012promulgated formally label for GB/T28642-2012. In 2008~2012, we detected1221batches of samples from Liaoningprovince for salmonella, used polymerase chain reaction (PCR) andtraditional microbiological methods, coincidence rate of two methodswas100%.14strains of salmonella were detected, the detection rate is1.15%.The salmonellas detected contains six serotype, respectively,S.dhaka’s,S.thompson,S.reagan,S.duringtheir,S.stanley and S.paratyphi.there are8strains of S dhaka’s. Virulence factor in salmonella dhaka’swere negative. This method has been used as practical application by thefeed test center of liaoning province, henan feed test center, shandongfeed test center, jilin veterinary medicine feed supervision, the ministry ofagriculture agricultural products testing center (dalian) and shenyangveterinary drug and feed testing institutions, in the three years from2009to2011, a total of5352batch samples,935batch of feed ingredients,56batches were found contamination by salmonella, detection rate was0.89%. Prove that this method is suitable for the detection of salmonellain feed.Based on ordinary PCR method, real-time fluorescent PCR wasestablished.It eliminates the gel electrophoresis process, further shortentesting time, about10times sensitivity higher than ordinary PCR.It candetect bacteria liquid containing2.2x101cfu/mL and needs45hours fortest, further improve the detection efficiency.The practice has proved that PCR and real-time fluorescence PCR method in this study solves the rapid, sensitive, specific and accuratedetection problem.This study carried out resistance measurement on the isolates ofsalmonella.8strains of salmonella in feed and3strains separated fromlive poultry in this lab,13drugs which were used clinical commonlywere selected. Salmonellas of feed source is sensitive to10kinds ofdrugs, resistant to sulfanilamide oxazole and cotrimoxazole Ming whichwere used earlier in clinic, etc. Poultry source salmonellas are sensitive tothree kinds of drugs and is100%resistant to9kinds of drug. Feed sourceof salmonella in clinic, therefore, should be not enough to causewidespread disease, that is to say,the resistance of salmonellas from livepourtry was connected directly with clinical antibiotics abuse,andnothing to do with contamination and transport of salmonella from feed.To detect pathogenicity and risk assessment.6strains of S.dhaka,1S.thompson,1S.reagan were inoculated to SPF chicks of3days old, setcontrol group at the same time. Experimental group each chicks oralliquid inoculant0.1mL, concentration of about108cfu/mL, blank groupwith sterilized saline instead. Observation6days after inoculation, therewere no death,experimental chicks only has mild clinical symptoms andautopsy lesions, and the clinical symptoms tend to be better.To test whether the virulence will be back to strong, take the purecolonies of separation of S. dhaka’s,made5McNamara, the bacterial suspension of turbidity, simulating natural infection, oral inoculation100u L to SPF chicks of3days, and successive batches for four times, eachtime the experiment period for7days, observe the chicks clinicalsymptoms, autopsy change and tissue pathological changes. The resultshows: the experimental chicken without death, but the lesion is causedpriority to the liver and duodenum, and affects chicken growth, theweight is lower than blank group about10%. With the same dose ofbacteria liquid to intraperitoneal inoculation test chicken, the clinicalsymptoms, autopsy symptoms are serious than oral inoculation testchicks. From the batches results, the virulence of salmonella from feedwere not found to return strong, no death for all the four generation, thereis no significant differences between generations about the pathologicalresults. Thus, outbreaks of pandemic possibility in poultry industry inliaoning province is very small. So,some serotypes salmonella, no stronglethal force, but the serious influence in production performance, reducethe economic benefit. So it should avoid as far as possible in productionthe feed which contaminated with salmonella will be feeding livestockand poultry.This study has five characteristics: first of all,it is the first time indomestic than the molecular biology method is applied to detect ofpathogenic microorganism in feed, and set up the rapid PCR and thereal-time fluorescent PCR detection method of salmonella in feed. Secondly, this study selected a kind of simple, rapid, accurate, highsensitivity of detection method, the feed source in the field of research onrapid detection of salmonella in the domestic leading level. The rapidPCR and the real-time fluorescent PCR detection method of salmonella,the PCR has risen to national standards, standards of GB/T28642-2012.Thirdly,the PCR and the method (GB/T13091-2002) were used at thesame time to detect1221batch of feed for salmonella,obtained14strains,a total of six serotype. Fouth, detection of drug resistance was carried out.13clinical commonly used drugs,8strains of this laboratory separationand3strains of poultry source, the results show that the feed source ofsalmonella for only3kinds of drug resistance, and poultry source ofsalmonella is sensitive to three drugs only, resistance rate of9kinds ofdrug was100%. Fifth, poison attack experiment was carried4generation.Salmonella isolates from liaoning province were used tovaccine SPF chicks of3days, there is no death in each batch test, butthere were liver and intestinal lesions, and slow growth after7day trialperiod, the weight of control group is about10%higher than that of theexperimental group.