Epidemiological Investigation And Molecular Epidemiological Characteristics Of Campylobacter From Genital Tract Of Different Hosts

Campylobacter is one of the most important zoonotic pathogens worldwide.The Campylobacter infection of human causes mild acute gastroenteritis,which can lead to various complications in severe cases.In ruminants,Campylobacter fetus subspecies fetus(C.ff)and Campylobacter jejuni(C.j)cause sporadic abortion and local epidemics in sheep.Campylobacter fetus subsp.venerealis(C.fv),as the causative agent of bovine genital Campylobacter disease,causes infertility,early embryonic death and abortion,which causing serious economic losses to the livestock industry.Previous studies showed that C.fv can colonize in genital tract of cattle,cause Campylobacter infection through artificial insemination or animal mating.However,there are few reports about whether other Campylobacter species and subspecies exist in the reproductive tract of cattle or other hosts.Therefore,it is of great significance for the prevention and control of Campylobacter disease in human and animals to clarify the prevalence and transmission of Campylobacter in the reproductive tract.In this study,the PCR detection method of Campylobacter fetus was first established,and Campylocter strains were isolated and identified from the genital tract samples of human,monkey,cattle and sheep in combination with the existing method for the isolation and identification of Campylobacter.The epidemic of Campylobacter in primates and ruminants in China was analyzed preliminarily.The biological characteristics of Campylobacter isolates from the reproductive tract were analysed,and the differences between Campylobacter isolates from genital tract and Campylobacter isolates from intestinal tract were studied on the gene level.It provide data support for the prevention and control of Campylobacter in human and animal genital tract.1 Development and application of PCR method for the detection of Campylobacter fetusIn according to the documents,the Campylobacter fetus surface protein gene sapB2 was selected as a target gene,and a pair of specific primers were designed by application software.Through optimization of reaction conditions,primer sensitivity,specificity and sensitivity tests and detection of simulated contaminated samples,a rapid PCR detection method for Campylobacter fetus was established and applied to clinical samples.The results showed that the sapB2(789 bp)gene fragment,a species-specific product could be detected after amplification of the DNA template of Campylobacter fetus,while other 17 reference strains could not be detected.The detection limit of the PCR assay was 0.23 pg/μL DNA template of Campylobacter fetus pure cell culture.And it could detect as few as 0.9 CFU/mL and 20 CFU/g of Campylobacter fetus in artificial contaminated sewage and cow feces respectively.400 of fresh cow anal swab samples and 125 of vaginal swab samples of parturient women were detected for Campylobacter fetus by the PCR,and the result was consistent with that of the national standard method,and both were Campylobacter fetus negative.To the control samples of these two types of samples,the recovery rate of Campylobacter fetus was 100%by PCR method,the recovery rate of Campylobacter fetus by the national standard method was 80%in cow anal swab samples,and the recovery rate by the national standard method was 100%in vaginal swab samples of parturient women.These results suggested that the PCR assay can be used to rapidly detect Campylobacter fetus and show high specificity,sensitivity and being easy to handle.It provides technical support for the rapid detection of Campylobacter fetus,and it is of great significance to the diagnosis and control of Campylobacter fetus diseases.2 Epidemiological Survey of Campylobacter in Reproductive Tract of Different HostsFrom March 2017 to June 2018,genital tract swabs and anal swabs of 1128 pregnant women were collected from a hospital in Yangzhou,including 269 abortion cases and 269 abortion placentas.Genital tract swabs and anal swabs of 101 female monkeys were collected from Suzhou and Shanghai.Genital tract swabs and anal swabs of 455 healthy cows and 17 abortion ows wcere collected from a dairy farm in Yangzliou,and the heart,liver,spleen,lung,kidney and stomach contents from 17 stillbirths and placentas from 17 aborted cows were collected.The genital tract swabs and anal swabs of 368 ewes were collected,as well as 225 environmental samples from different farms in Haian,Yancheng,Xuyi,Jiangdu and Lianshui of Jiangsu Province.A total of 2126 genital tract samples,2126 anal swabs and 225 environmental samples were collected.The results of isolation and identification of Campylobacter showed that 7 strains of Campylobacter jejuni,2 strains of Campylobacter coli,1 strain of Campylobacter sputorum and 1 strain of Campylobacter hyointestinalis were isolated from the genital tract samples of human,monkey,cow and sheep.No Campylobacter fetus was isolated from all samples.Only 1 Campylobacter strain was isolated from the genital tract samples of pregnant women in the hospital and was Campylobacter jejuni.The total positive rate of Campylobacter was 0.09%in the genital tract samples.2 strains of Campylobacter were isolated from the anal swabs.The total positive rate of Campylobacter was 0.18%in the anal swabs.There was no significant difference in the positive rate of Campylobacter(P>0.05)between genital tract samples and anal samples of 1128 pregnant women.The isolation rates of Campylobacter in the genital tract samples of healthy pregnant women and aborted individuals were 0.12%and 0 respectively,with no significant difference(P>0.05).The isolation rates of Campylobacter in the anal samples of abortion individuals and healthy individuals were 0.37%and 0.12%respectively,and there was no significant difference(P>0.05).Campylobacter was not isolated from 269 placental samples.3 Campylobacter strains from monkey genital tract swab samples and and 24 Campylobacter strains from anal swab samples were collected,and the total positive rates were 2.57%and 20.69%respectively.The positive rate of Campylobacter in genital tract swab samples was significantly lower than that in anal swab samples(P<0.05).2 strains of Campylobacter jejuni were isolated from female monkey genital tract samples,with a positive rate of 1.72%and 1 strain of Campylobacter coli,with a positive rate of 0.86%.6 strains of Campylobacter jejuni were isolated from the anal swabs,positive rate was 5.17%,14 strains of Campylobacter coli,positive rate was 12.07%,4 strains of other Campylobacter,positive rate was 3.45%.116 female monkeys came from two different breeding modes.The total positive rates of Campylobacter in reproductive tract samples of female monkeys in large cage group and single cage group were 3.16%and 0 respectively.The total positive rates of Campylobacter from anal swab samples were 24.21%and 4.76%respectively.The positive rates of Campylobacter in reproductive tract and intestinal tract of female monkeys in large cage group were significantly 1higher than those in single cage group(P<0.05).The total posiLive rates of Cmpylobacter from sheep genital tract swabs and anal swabs were 0.73%and 13.41%respectively.The positive rates of Campylobacter from genital tracl were significantly lower than those from anal swab(P<0.05).2 strains of Campylobacter jejuni and 1 strain of Canpylobacter coli were isolated from 410 ewe genital tract swab samples and anal swab samples respectively,and positive rates were 0.49%and 0.24%respectively.Four times sampling of a female flock in 6 weeks,no Campylobacter was isolated from the genital tract samples in normal feeding group and inoculated Listeria group.The total positive rate of Campylobacter in anal swabs of the normal feeding group continued to decrease from 90.00%to 37.50%.But the total positive rate of Campylobacter in anal swabs of the inoculated Listeria groupwas 100.00%,100.00%,88.89%and 88.89%respectively,and was at a higher level of Campylobacter infection for six weeks.The isolation rates of Campylobacter from reproductive tract samples in ewes group and mixed sheep group were 0 and 1.48%respectively,and that of’Campylobacter from anal swabs was 0.97%and 26.11%respectively,with a sigfihcant difference(P<0.05).The positive rates of Campylobacter from genital tract samples of normal sheeps and long-distance transported sheeps were 1.61%and 0 respectively.The positive rates of Campylobacter from anal swab samples of normal sheeps and long-distance transported sheeps were 25.81%and 95.00%respectively.The isolation rates of Campylobacter from anal swab samples in long-distance transported sheeps were significantly higher than those in normal scattered sheeps(P<0.05),but the isolation rate of Campylobacter in the genital tract is not increased after long-distance transportation of the flock.The positive rates of Campylobacter in 455 healthy cows and 17 aborted cows were 0.88%and 0 respectively,both at a lower infection level.2 strains of Campylobacter jejuni,1 strain of Campylobacter sputorum and 1 strain of Campylobacter hyointestinalis were isolated from 455 healthy cow reproductive tract samples,and the separation rates were 0.44%,0.22%and 0.22%respectively.Canpylobacter was not isolated from the genital tract swabs,placenta,and stillbirth samples of 17 aborted cows.The positive rates of Campylobacter from anal swab samples of healthy cows and abortion cows were 3.74%and 5.88%respectively,with no significant difference(P>0.05).3 Biological and molecular characteristics of Campylobacter isolates from genital tractPrevious studies showed that not all Campylobacter can be isolated in the reproductive tract of the host.In order to further investigate whether there is a difference between Campylobacter strains from the reproductive tract and other niches such as the intestinal tract,we studied the biological characteristics of Campylobacter strains,such as motility and chemotaxis,and compared the genomes of Campylobacter strains from genital tract and intestine by whole genome sequencing.At the species level,the average diameters of motility of Campylobacter jejuni isolates from the genital tract swabs and anal swabs were 4.94 cm and 5.05 cm respectively,with no significant difference.The average diameters of motility of Campylobacter coli and Ca”pylobacter sputorum isolates from the genital tract swabs were larger than those of Campylobacter isolates from the anal swabs.The lactic acid tolerance of Campylobacter jejuni,Campylobacter coli and Campylobacter sputorum from the genital tract swabs were stronger than that of anal swab isolates at the species level.The average reduction of lactate tolerance of Campylobacter jejuni,Campylobacter coli and Campylobacter sputorum isolates from the genital tract swabs and anal swabs were 1.53,1.49,1.10 orders of magnitude and 1.60,1.93,2.15 orders of magnitude,respectively.The chemotaxis of Campylobacter isolates was analyzed by hard agar method.At the species level,Campylobacterjeuni.isolates from genital tract showed stronger chemotaxis in DL-malic acid and succinic acid than anal swab isolates,Campylobacter coli genitalis isolates showed stronger chemotaxis in three chemotaxis than anal swab isolates;Campylobacter sputorum genitalis isolates showed stronger chemotaxis in a-ketoglutaric acid and succinic acid.Cell adhesion and invasion from the species level,the adhesion and invasion ability of Campylobacter jejuni,Campylobacter coli and Campylobacter sputorum isolates to Caco-2 cells and CHO cells was stronger than that of anal swab isolates,and the adhesion and invasion ability to CHO cells was stronger than that of Caco-2 cells.Therefore,we speculate that these Campylobacter can survive in the reproductive tract with some biological characteristics,so that they can survive in the adverse environment with stress factors of the reproductive tract.Evolutionary relationship analysis showed that most of the genital tract isolates and anal swab isolates belonged to similar evolutionary branches.There was no significant difference in the number of CDS between Campylobacter genitalis isolates and Campylobacter anus swab isolates.The total base number and average GC(%)content of Campylobacter genitalis isolates were lower than those of anal swab isolates,which may be related to adaptive evolution.MLST analysis showed that Campylobacter genitalis isolates and anal swab isolates had three homologous complex types(ST-21,ST-22,ST-828),indicating that the homologous complex type of Campylobacter genitalis infection in the host genital tract was not specific;There were two ST-fomis(2 and 4253)in the isolates from anal swab,which indicated that the ST-forms of Canpylobacter infection in the genital tract of the host were not specific,and the same ST-forms of Campylobacter infection in intestinal tract might occur.Statistical analysis of 11 virulence genes in bacterial gnome was carried out,but no significant difference and regularity were found between Campylobacter genitalis isolates and Campylobacter anus swab isolates.