| Campylobacter is the important bacterial foodborne pathogen responsible for human and animal.Campylobacteriosis,is an illness characterized by human gastroenteritis and a series of complications.GBS is the most serious complication and enteritis caused by certain serotypes of Campylobacter jejuni is considered to be an important precursor.Campylobacter is widely found in the intestines of many warm-blooded animals and in nature.Livestock and poultry are important hosts for Campylobacter.Infected animals usually have asymptomatic infection obviously,but they can continuously discharge the bacteria to the outside,contaminating food and drinking water through feces or deliversy,and thus causing human and other animal infections.Therefore,controlling or reducing Campylobacter from livestock and poultry,reducing the risk of people being exposed to this pathogen is an important prerequisite for the prevention and control of Campylobacter infection.The main objectives of this study are:(1)To investigate and analyze systematically the prevalence of Campylobacter and distribution characteristics of tissues in the bodies in livestock and poultry;(2)Multi-site sequencing technology molecular subtyping(Multilocus sequence typing,MLST)is used to explore the population structure of Campylobacter isolates and reveal the transmission patterns among different hosts and in their bodies,providing data for the effective prevention and control of Campylobacterosis stand by.1 Investigation of tissue distribution characteristics of Campylobacter in livestock and poultry with diseasesFrom October 2016 to September 2017,835 cases of livestock and poultry were collected in an animal hospital in Yangzhou City.Information of these cases,such as different types of livestock and poultry,whether they were diagnosed with different diseases and whether or not they were diarrhea,etc were recorded in detail.Among them,there were 796 poultry cases with 5885 samples of cases of chickens,ducks,geese,475,78,235,respectively.A total of 275 samples were collected in 39 cases of livestock.Using the national standard method as a control,the multiplex PCR method established in this laboratory was used to isolate and identify Campylobacter jejuni and Campylobacter coli for the above samples.The results showed that the positive rates of Campylobacter varied from 48.72%to 72.00%on different kinds of poultry.The Campylobacter positive rate of chickens,ducks and geese was 72.00%,48.72%,2.34%,respectively.The results showed that the Campylobacter positive rate of chickens was significantly higher than that of ducks and geese(P<0.01),the results indicated that among diseased poultry,chickens were the most susceptible,followed by geese.18 cases were positive for Campylobacter among 39 cases of livestock with the positive rates were 46.15%,of which 17 cases were positive for Campylobacter coli among 33 cases of swine with the Campylobacter coli positive rate 51.52%.The results of different tissues in livestock and poultry showed that both Campylobacter jejuni and Campylobacter coli from hearts,livers,spleens,lungs,kidneys,cecal contents,cloacal swabs and gallbladders of chickens were found with the detection rate of Campylobacter jejuni and Campylobacter coli simultaneously 0.52%,2.32%,0.44%,0.85%,0.63%,3.17%,5.07%,and 0.30%,respectively.In the duck samples,Campylobacter jejuni from spleens,lungs,kidneys,cecal contents,cloacal swabs and gallbladders were foud,except heart and livers.And Campylobacter coli was found in the lungs,cecal contents and cloacal swabs.Moreover,Campylobacter jejuni and Campylobacter coli both detected in these three sites with the isolation rate 2.67%,4.11%,and 1.33%,respectively.In the geese samples,both Campylobacter jejuni and Campylobacter coli from hearts,livers,spleens,lungs,kidneys,cecal contents,cloacal swabs and gallbladders were also foud.Differentiation from chicken test results,only in the kidneys and cecal contents were detected Campylobacter jejuni and Campylobacter coli simultaneously with the detection rate 0.43%and 2.99%,respectively.In swine samples,the detection rate of Campylobacter coli from the hearts,livers,spleens,lungs,kidneys,caecal contents,cloacal swabs and gallbladders was 7.14%,9.09%,6.45%,6.45%,9.68%,36.67%,41.94%and 5.88%,respectively.The results showed that Campylobacter jejuni and Campylobacter coli can be detected simultaneously in different tissues of poultry,especially in chickens.Besides,the detection rate of Campylobacter from gallbladders was higher than that of ducks and geese.In addition,there was a significant difference in positive poultry detected in 1-6 sampling sites(P<0.05),and among the positive poultry,87.77%of the birds found Campylobacter in 1-3 sampling sites,and only a few birds detected detected positively in 4-6 sampling sites,accounting for 12.23%of the positive poultry.The study also found that in 319 cases of Campylobacter-positive poultry,only 22 cases of positive organ-existing Campylobacter were detected,and the detection rate was 6.90%.In Campylobacter-positive poultry,the birds with both the intestinal tract and visceral samples detected positive,102 cases,the detection rate was 31.97%.There were only 195 cases of positive for Campylobacter in the intestine,the detection rate was 61.13%.The results showed that the detection rate of only the poultry whose visceral samples were only positive for Campylobacter was significantly lower than that of poultry with positive for Campylobacterin the intestine(P<0.01),and the detection rate of the poultry with positive for Campylobacter both in the intestine and visceral samples was significantly lower than that in the intestine(P<0.05).Among 297 cases of positive poultry whose intestine samples were positive,all visceral tissues were found to have different degrees of Campylobacter,of which the detection rate was the highest in lungs,17.17%,followed by kidneys,spleens,hearts and livers,the detection rates were 16.16%,11.11%,9.43%and 8.75%,respectively.The detection rate in lungs was significantly higher than that of hearts and livers(P<0.01),which was significantly higher than that of spleens(P<0.05).The results indicated that Campylobacter is more easily colonized in the gut of poultry,it can also cause extraintestinal infection.And it is easier to invade poultry's lungs or kidneys,followed by the spleens or livers.The detection rate of Campylobacter in livestock and poultry with clinical manifestations of diarrhea was significantly higher than that of animals without diarrhea(P<0.05).However,there were obvious differences between different kinds of animals.The detection rate of Campylobacter in chickens with diarrhea was significantly higher than that of chickens without diarrhea(P<0.01).And the detection rate of Campylobacter in geese with diarrhea was significantly higher than that of geese without diarrhea(P<0.05),and the detection rate of Campylobacter in swine with diarrhea was significantly higher than that of pigs without diarrhea(P<0.01).The results indicated that Campylobacter may be related to diarrhea.The statistics of the detection of Campylobacter among poultry with different diseases showed that in parasite disease group and the virus disease group,the positive rate of Campylobacter was significantly higher than that in the bacterial group(P<0.05).In detail,the detection results of chickens showed that the detection rate of Campylobacter in the bacterial group was significantly lower than that in the virus disease group and the parasite group(P<0.01).There was no significant difference in any other group(P>0.05),but the detection rate of Campylobacter in the parasite group was higher than that of the virus disease group,of which was 90.11%(82/91)and 86.75%(72/83),respectively.In ducks,there was no significant difference between the detection rate of Campylobacter in the bacterial group and the virus disease group(P>0.05),but it could be found that the bacterial group was significantly lower than the parasite group(P<0.01).The detection rates of Campylobacter were high in the virus and parasite disease group,60.00%(3/5)and 88.89%(8/9),respectively,but there was no significant difference(P>0.05).In Geese,the detection rate of Campylobacter in the bacterial group was significantly lower than that in the virus disease group(P<0.05),but there was no significant difference from other groups(P>0.05).The detection rate of Campylobacter in the virus disease group was significantly higher than that in the parasite group(P<0.05).In addition,the detection results of the bacterial group,the virus disease group and parasite disease group in swine showed that there was no significant difference in the detection rate of Campylobacter among different groups(P>0.05),and the results showed that Campylobacter in the parasite disease group was higher 58.82%(10/17).The results of showed that Campylobacter had a higher detection rate in the cases of livestock and poultry with different diseases,especially with viral diseases and parasitic infections.However,the detection rate of livestock and poultry with bacterial diseases was lower and may be concerned with competitive exclusion between bacteria.2 Analysis on the epidemiological characteristics of Campylobacter isolates from different tissues by MLSTIn this study,a set of 7 housekeeping genes including aspA,glnA,gltA,glyA,pgm,tkt,and uncA were selected and used to subtyping 232 isolates of different origins of different animal origin and tissues by MLST.The MLST classification results showed that 232 isolates were divided into 112 different sequence types,belonging to 12 kinds of colonal complexes.Of which,105 strains of C.jejuni were divided into 66 ST types,belonging to 10 kinds of colonal complexes,127 strains of C.coli,were divided into 46 types of ST,belonging to two major colonal complexes and idiotypes.There were 86 strains of bacteria belonging to 60 new sequences,accounting for 37.07%of the total strains of Campylobacter.Among them,54 strains were Campylobacter jejuni isolated from chickens,including 12 in livers,1 from spleens,7 from gallbladders,3 from cecal contents and 10 from cloacal swabs,from ducks including 1 from gallbladders,3 from caecal contents and 2 from cloacal swabs,from geese includingl from hearts,2 from livers,2 from spleens,1 from lungs,6 from cecum contents and 3 from cloacal swabs.Other 32 strains were Campylobacter coli isolated from chickens,including 1 in hearts,7 in livers,13 in caecal contents and 4 from cloacal swabs,from ducks including 2 from cecal contents,from geese including 1 isolate of each origin including livesr,kidneys gallbladders and cloacal swabs,and from swine 1 isolate of cloacal swabs.The strains of chickens,ducks,geese,pigeons and pigs belong to 9,3,7,1 and 1 colonal complexes and idiotypes,respectively.There are 7 kinds of colonal complexes of two or more kinds of origin strains,of which colonal complexes are mostly composed of two kinds of source strains,six of them contained geese-derived isolates,five of them contained chicken-derived isolates,and all three contained duck-derived isolates.In this study,all strains of Campylobacter isolates obtained 112 types of sequence types.There are 10 known sequences containing Campylobacter isolates of two or more different origins,62 isolates in total.Among them,the sequence ST-828 contains isolates from three different sources and the remaining nine sequences contain Campylobacter isolates from two different sources,and most of them from both chickens and geese.The MLST typing results of individual detected in multiple tissues from livestock and poultry showed that the strains of different tissues in the same individual have the same sequence type,such as 170-413J5,170504E2 and 170316Z2 with ST-825,ST-828,respectively.And fewer individuals contained 2-3 sequence types.The results showed that strains of different tissues had a close relationship,and the same individual can infect more than one type of Campylobacter.Based on the Campylobacter isolates of Yangzhou city and its surrounding areas and the PubMLST database,it was found that the gene population structure of some isolates and international strains from districts mentioned above was the same,and it was also found that part of the sequence types were only found in China,such as ST-6077,ST-4259,ST-4265,ST-4268,ST-4324,ST-6521,ST-6914,ST-7268,ST-7469,ST-8089,etc.The results showed that the genetic relationship between some strains in China and international strains is relatively close,besides,this results indicated that the distribution of Campylobacter also has regional characteristics.In the PubMLST database,it showsed that ST-131 has been associated with sporadic gastroenteritis,Guillain-Barre Syndrome(GBS),and Fisher syndrome,the sequence type was also found in chicken-derived isolates this study.The results of the MLST classification of the isolates of different diseases showed that the sequence patterns of these isolates presented complexity and diversity.For example,as many as 19 kinds of sequence types were obtained from the isolates of parasites.There are also some strains that have a certain type of disease in a particular case,such as ST-872,which is distributed in strains isolated from Newcastle disease and Marek's disease case.In general,there is no obvious corresponding relationship between sequence types and disease in the poultry with different diseases.The analysis of the genetic relationship between Campylobacter isolates in different animal sources and different tissues showed that the sequence patterns of different organs and intestinal tissue isolates from different hosts and in hosts were cross-distribution.It indicatied that there was a certain degree of genetic relationship between the livestock and poultry.And the genetic relationships between the intestinal tissue isolate and the organ tissue isolates were closer.The intestinal infections were a potential risk factor for organ infectior. |
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