| Campylobacter is an important zoonotic pathogen,which can cause acute gastroenteritis and various complications after the body is infected.In ruminants such as cattle and sheep,Campylobacter jejuni and Campylobacter coli often cause intestinal diseases such as diarrhea,while Campylobacter fetus is related to infertility and abortion of cattle,causing serious economic losses to the livestock industry.It has been reported that Campylobacter from bovine origin is an important factor in human infection of Campylobacter.Campylobacter can cause a variety of human diseases,such as bacteremia,sepsis,and acute purulent encephalitis(meningoencephalitis,brain Myelitis),acute septic arthritis,can also cause miscarriage and infertility.Elderly people with immune insufficiency,weakened immunity,or patients with serious diseases are prone to cause Campylobacter disease after infection.There are related pathological reports around the world,such as Russia,the United States,Canada,the United Kingdom,etc.,all have different degrees of this disease Sporadic cases have also been reported in some areas of my country.In recent years,in order to ascertain the potential risks of Campylobacter to the cattle breeding industry and humans,epidemiological studies have been carried out in various aspects of breeding,slaughter,production and sales abroad,while domestic research on Campylobacter has started relatively late,and mainly focused on the breeding stage,the prevalence of Campylobacter in the production chain from breeding to slaughter is still unclear,and further research is needed.The main purpose of this study is to establish a multiple PCR method for Campylobacter and apply this method to investigate the prevalence of Campylobacter in the cattle breeding and slaughter production chain,and to analyze the characteristics of Campylobacter isolates in the cattle breeding and slaughter production chain from the perspective of genomics and drug resistance,analyzed the prevalence and spread of Campylobacter in the cattle breeding production chain from the perspective of biological characteristics.1 Establishment of multiplex PCR detection method for Campylobacter in cattleUsed 16SrRNA gene,ask gene,mapA gene and cstA gene as target genes,primer 5.0 was used to design primers,and a multiple PCR method was established for simultaneous detection of C.jejuni,C.coli and C.fetus.Cjejuni,C.coli and C.fetus could amplify their own specific bands,but other reference strains did not amplify bands,which showed that this method has strong specificity.The sensitivity of multiplex PCR detection reached 19.23 pg/?L of Campylobacter jejuni,16.93 pg/?L of Campylobacter coli and 14.37 pg/?L of Campylobacter fetus at the DNA level The verification results of the three repetitive tests were consistent,which showed that the method has high SensitivityThe multiple PCR detection method established in this study was used to identify samples from cattle breeding slaughterhouses.At the same time,the traditional plate separation method and other PCR methods currently reported were used for comparison and verification.Among 1375 samples,the multiple PCR detection method of this study detected 75 strains of Campylobacter were positive,with an average positive rate of 5.45%,which was higher than 4.87%(67/1375)of the traditional plate separation method and 5.31%(73/1375)of other PCR methods,the detection rates of the three methods are not statistically significant(p>0.05).The minimum detection limits of this method and the existing PCR methods reported at the DNA level are:Campylobacter jejuni 19.23 pg/?L,0.93 pg/?L,Campylobacter coli 16.93 pg/?L,1.05 pg/?L and Campylobacter fetus 14.37 pg/?L,0.23 pg/?L.Compared with the traditional plate separation method,the method established in this study takes less time to detect and compares other Compared with the PCR method,the method established in this study can simultaneously detect the three common species of Campylobacter from cattle in one system,and the cost of consumables is lower,so it is more suitable for the needs of large-scale detection of Campylobacter from cattle.2 Epidemiological study of Campylobacter in the production chain of cattle breeding and slaughterFrom June 2019 to October 2020,this study collected bovine anal wipes,environmental samples,and slaughter production samples from 5 dairy cattle farms and 1 beef cattle slaughterhouse in Jiangsu and 1 dairy cattle farm in Tieling,Liaoning.A total of 6681 samples were collected,including 4125 samples of cattle anal swabs and 1500 samples of environmental samples during breeding link;20 samples of pre-slaughter cattle anal swabs,150 samples of dirty meat and 410 environmental samples during slaughter link;100 samples of breast wipes and 376 samples of environmental samples during production link.The results showed that a total of 366 positive samples of Campylobacter were detected in the process of breeding,slaughter and production,with a positive rate of 5.48%.A total of 338 strains of Campylobacter jejuni and 28 strains of Campylobacter coli were isolated.Campylobacter fetus was not isolated.In each link,C.jejuni was dominant species,accounting for 91.41%(298/326),100%(16/16)and 100%(24/24)respectively.During the breeding process,in small-scale farms(9.47%),the positive rate of Campylobacter in cattle was significantly higher than that of medium-scale(4.93%)and large-scale(4.42%)farms.Among the different breeding modes,the positive rate of the mixed breeding mode of adult cattle and calves(9.47%)was significantly higher than that of the adult cattle and calves divided into stalls(4.93%).Among the farms with different cleaning methods,the positive rate of Campylobacter(0.88%,0.20%)in farms that use mechanized cleaning(manure removal trucks,scrapers)was significantly lower than that of farms that were manually cleaned(6.20%).Among different ages,the positive rate of Campylobacter in calf samples(10.14%)was significantly higher than that in adult cattle samples(4.07%).The positive rate of beef cattle isolates(8.49%)was significantly higher than that of dairy cattle(5.31%).During the slaughter process,16 positive samples of Campylobacter were isolated,with a positive rate of 2.76%(16/580).A total of 16 strains of Campylobacter jejuni were isolated.No positive Campylobacter coli and Campylobacter fetus were detected.Three strains of Campylobacter jejuni were isolated from the meat samples with a positive rate of 0.52%;7 strains of Campylobacter jejuni were isolated from organs such as the large intestine and hairy belly with a positive rate of 1.21%;One strain of Campylobacter jejuni was isolated from both hands after the slaughter,with a positive rate of 0.17%,and one strain of Campylobacter jejuni was isolated with a positive rate of 0.17%after the knife used for slaughter.During the milking process,24 positive samples of Campylobacter were isolated,with a positive rate of 4.17%(24/576).A total of 24 strains of Campylobacter jejuni were isolated.No positive for Campylobacter coli and Campylobacter fetus.10 strains of Campylobacter jejuni were isolated from anal swabs of milking cows,with a positive rate of 1.74%;5 strains of Campylobacter jejuni were isolated from swabbing samples of dairy cows,with a positive rate of 0.87%;5 strains of Campylobacter jejuni were isolated from the milking machine in the milking workshop,with a positive rate of 0.87%;4 strains of Campylobacter jejuni were isolated from the work floor,with a positive rate of 0.69%.In order to evaluate the impact of antibiotics on the resistance of Campylobacter in cattle farms,248 C.jejuni isolates from 6 cattle farms were selected for drug susceptibility tests against 12 antibiotics.The results showed that 89.92%(223/248)of Campylobacter isolates showed different degrees of drug resistance,and 78.63%(195/248)of isolates showed multi drug resistance;the isolates were resistant to 6 categories and 12 species Highly sensitive antibiotics are:Amoxicillin(AMC)100%,Gentamicin(CN)98.39%,Erythromycin(E)96.77%,Kanamycin(K)93.55%,Azithromycin(AZM)92.74%,Ceftriaxone(CRO)89.11%,Cefotaxime(CTX)84.27%and Streptomycin(S)81.45%,highly resistant are:Ciprofloxacin(CIP)72.18%,Tetracycline(TET)44.76%,Levofloxacin(LEV)44.35%,Penicillin(P)36.69%.Among different scale farms,the resistance rate of small farms to high antibiotic resistance was significantly higher than that of large and medium-sized farms(p<0.01);among different breeds of cattle,the resistance rate of beef cattle to high antibiotic resistance was significantly higher than that of dairy cows(p<0.01),the resistance rate of some highly sensitive antibiotics was significantly lower than that of dairy cows(p<0.01).3 Traceability analysis of isolates based on whole-genome sequencing in cattleThrough whole-genome sequencing methods,molecular traceability analysis of isolates from the same batch of beef cattle breeding and slaughter stages to be slaughtered,combined with the results of epidemiological field research,we will further explore the spreading law of Campylobacter in the cattle breeding and slaughter production chain from the molecular and genetic level.The genetic evolution relationship shows that the same batch of individual Campylobacter has a certain genetic relationship.The isolates from calves and adult cattle are widely distributed and have low homology,indicating that Campylobacter has higher genetic diversity in cattle,and adult cattle are higher than calves,and isolates from the same evolutionary branch(YZU2602/YUZ2610,YZU2607/YZU2600)were separated from the breeding and slaughter stages,indicating that the isolates from the breeding and slaughter stages were cross-distributed.MLST results showed that Campylobacter isolates contained 11 kinds of homologous complexes,and Campylobacter isolates with homologous complexes accounted for 93.48%(43/46)of the total.The 37 strains of Campylobacter jejuni were divided into 17 ST types,belonging to 10 homologous complexes.The sequence type ST 4526 is the dominant ST type of Campylobacter jejuni,accounting for 21.62%;9 strains of Campylobacter coli were divided into 4 ST types,belonging to the same homologous complex ST-828 CC,sequence type ST 827 is the dominant ST type of Campylobacter coli,accounting for 44.44%.The detection of isolates from human-derived cloning complexes ST-21 CC and ST-61 CC indicates that Campylobacter can be transmitted from cattle to humans.The isolates of the sequence types ST 61 and ST 4526 can be isolated in two batches of samples from the same farm,indicating that Campylobacter can exist in the farm for a long time and spread through contact between cattle. |
PDF Full Text Request