Investigation Of Salmonella Infection In Several Chicken Farms In Sichuan Province And Antimicrobial Susceptibility Test Of The Screened Strains

Avian Salmonella infection may result in acute or chronic poultry diseases.Avian Salmonellosis is the common disease in the poultry industry in China.Prevalent serotypes of Salmonella in different areas vary greatly,and their pathogenicity is obviously different in different regions.Avian salmonella can be spread horizontally through the digestive tract,mating,artificial insemination,and also through vertical transmission.Once poultry was infected with Salmonella,growth restriction,laying rate reduction,fertility rate decrease,hatchability decline,chicken death,diarrhoea,tissue degeneration and necrosis,andmortality rise may happen.It may bring great loss to poultry industry.Due to misuse of antibiotics,Salmonellosis could still not be controlled effectually and drug-resistant Salmonella may spread out of control.In order to investigate salmonella infection inchicken farms in Sichuan Province,915 samples were collected(anal swab,liver,yolk sac,etc.)in 11 regions from December 2014 to October 2015.96 strains of salmonella,belonging to 4serumgroups and 8 serotypes,were identified by conventional isolation and purification of bacteria,TSI biochemical test,PCR and serotype identification.Antimicrobialdisk susceptibility test for 21 kinds of common clinical drug on the 96 isolated salmonella was conducted.Ciprofloxacin,florfenicol and doxycycline were screened out with high sensitivity,which are convenient for clinical application.At the same time,drugresistant salmonella and multiple drug resistant salmonella were identified.The research and achievements are as follows:1.Isolation,purification and serological identification of Salmonella from 11 regions of Sichuan ProvinceIsolation and identification of salmonella from the samples by conventional methodsThe strains that cultured in advance were subcultured to SS,HE culture medium,and then the suspected bacteria colonies were purified according to the morphological characteristics.TSI test to isolated strainsThe isolated and purified strains were detected by TSI test.The strains which wereconformed as Salmonella by TSI test were used in the further tests.Detecting of salmonella invasion genes(inv A)by PCRAccording to sequences of salmonella inv A gene in Gene Bank database,PCR was carried out to 915 samples that cultured by SC medium.722 bp nucleotide fragment were amplified using the specific primers designed by Lina Zhuwith the PCR reaction system under the optimized PCR amplification condition established by Lina Zhu.103 positive strains were detected and the detection rate was 11.26%(103/915).Salmonella serotype identificationThe strains identified as positive strain by PCR were detected by agglutination test with multivalence A-F O serum,and 96 strains were identified.In order to investigate the serotype of Salmonella from chicken in Sichuan Province in recent years,serological identification test were implemented to 96 strains of Salmonella which were isolated from 12 cities of Sichuan province from 2014 to 2015.The results showed that the isolated bacteria belonged to B,C,D and E 4 serumgroups and 8 serumtypes.The S.sendai(46/96)was the main serotype,followed by S.braenderup(9/96),S.abortusequi(8/96),S.bardo(6/96),S.choleraesuis(5/96),S.newport(3/96),S.gatuni(3/96)and S.senftenberg(2/96).There were14 strainsof Salmonella which could not be identified according to serotype.Serotype distribution was regional obviously.9 strains of S.newport were only isolated in Xinjin,and other 9 strains of S.braenderup were isolated from Pengzhou only.It has public health significance that the pathogenicity of S.newport and S.choleraesuis to multiple hosts.Salmonella infectious status of the sample collection siteFrom December 2014 to October 2015,915 samples(cloacae,liver,yolk sac,etc.)were collected from chicken farms in 12 cities of Sichuan Province,and detection rate was 10.50%(96/915).The number of salmonella detected around the city are as follows:Qionglai 10(10/88),Lezhi 3(3/19),Jintang 11(11/115),Guanghan 16(16/173),Xinjin 9(9/78),Meishan 38(38/120)and Pengzhou 9(9/19).However,no Salmonellawere detected from samples collected from Jianyang(0/190),Yaan(0/22),Shuangliu(0/85),and Longquan(0/3).Among them,the highest detection rate of 40.91%was found in samples from Pengzhou,followed by 31.67% in Meishan,and 15.79% in Lezhi.There were regional differences.Among different ages of chicken from different cities,salmonella infection rate of chicken in brood stage(0 ~ 30 days of age)was the highest,with detection rate of 28.22%(68/241).Detection rate of 31 to 60-day-old chicks and chickens older than 120 days(mainly laying hens)were4.82%(4/83)and 4.29%(19/443),respectively.Detection rateof 61-90 day-old chicks(mainly commercial chicken)was 7.35%(68/926),and no salmonella(0/80)was detected from 91-120 day-old chickens(mainly for laying hens).Detection rate of chickens in the coop was 4.76%(28/588),which was significantly lower than detection rate of the ground scattered chicken(20.20%,58/287).2.Results analysis of drug susceptibility test to isolated SalmonellaDrug susceptibility test was carried out to 96 strains of identified salmonella with 21 kinds of commonly used clinical drugs according to Kirby Bauer‘s method which recommended by WHO.The results were analyzed according to Standard of antibiotics Susceptibility Test(WS/T125-1999)drew up by Ministry of Health.The results of drug sensitivity testThe results showed that the drugs with high sensitivity were as follows: 1.?-Lactams(Cephalosporins): ceftriaxone(CRO)91.7%(88/96),cefazolin(CEZ)88.5%(85/96),cefotaxime(CLA)67.7%(65/96).2.Aminoglycoside: amikacin(AMK)99%(95/96),gentamicin(GEN)89.6%(86/96),spectinomycin(SPT)75%(72/96),neomycin(NEO)87.5%(84/96),kanamycin(KAN)83.3%(80/96).3.Quinolones: pefloxacin(PEF)97.9%(94/96),ofloxacin(OFX)91.7%(88/96),ciprofloxacin(CIP)92.7%(89/96)and norfloxacin(NOR)72.9%(70/96).4.Tetracycline doxycycline(DOX)63.5%(61/96).5.Chloramphenicols: florfenicol(FLO)82.3%(79/96).6.Polymyxin: polymyxin B(PMB)93.8%(90 /96).The results showed that the drug with low sensitivity were as follows: 1.?-Lactams(Penicillin): amoxicillin(AMO)38.5%(37/96),ampicillin(AMP)25%(24/96).2.?-Lactams(Cephalosporins): cefradine(CRD)49% 47/96,cefalexin(CFL)54.2%(52/96).3.Aminoglycoside: streptomycin(STR)13.5%(13/96).4.Sulfanilamide: selectrin(SXT)34.4%(33/96).Drug resistance analysisThe majority of isolated Salmonella showed drug resistance to the 21 clinical commonly used drugs,and drug resistance ranging from 1% to 66.7%.Drug-resistance rates of ?-lactam(Penicillin)were as follows: amoxicillin(AMO)61.5%(60/96),ampicillin(AMP)66.7%(64/96).Drug-resistance rates of ?-lactam(Cephalosporins)were as follows: cefradine(CRD)31.2%(30/96),cefotaxime(CLA 4.2%(4/96),cefazolin(CEZ)9.4%(9/96),cefalexin(CFL)28.1%(27/96),ceftriaxone(CRO)1%(1/96).Drug-resistance rates of aminoglycosides were as follows: amikacin(AMK)1%(1/96),gentamicin(GEN)10.4%(10/96),spectinomycin(SPT)11.5%(11/96),neomycin(NEO)8.3%(8/96),kanamycin(KAN)8.3%(8/96),streptomycin(STR)63.5%(61/96).Drug-resistance rates of quinolone were as follows: pefloxacin(PEF)1.1%(1/96),ofloxacin(OFX)1%(1/96),ciprofloxacin(CIP)6.2%(6/96),norfloxacin(NOR)8.3%(8/96).Drug-resistance rate of deoxytetracycline(DOX)was 33.3%(32/96).Drug-resistance rateof florfenicol(FLO)was 12.5%(12/96).Drug-resistance rate of polymyxin B(PMB)was 6.2%(6 /96).Drug-resistance rate of selectrin(SXT)was 64.6%(62/96).53 of 96 strains of isolated Salmonella showed multiple drug resistance and the rate was 55.21%(53/96).There were 23 strains had resistance to 4 drugs.There were both 11 strains had resistance to 3 and 6 antibiotics.1 strain even had resistance to 16 drugs,with strong multi-drug resistance.