屠宰前鸡、猪源大肠杆菌耐药性调查

本试验旨在了解屠宰前鸡、猪源食品动物体内大肠杆菌耐药情况,分析潜在的食品安全问题。从广州市畜禽交易市场随机采集待屠宰鸡和猪的粪便样品,分离鉴定大肠杆菌,并采用琼脂稀释法检测大肠杆菌对15种抗菌药物的敏感性。结果显示,从658份猪源样品和133份鸡源样品中共分离鉴定出731株大肠杆菌,其中猪源606株,鸡源125株。药敏试验结果显示,731株大肠杆菌均表现出不同程度的耐药,耐药谱广且多重耐药现象严重。对复方新诺明和四环素的耐药率为90.0%以上,仅对头孢西丁、黏菌素和阿米卡星较敏感(耐药率均低于3%)。鸡源大肠杆菌对头孢噻肟、头孢曲松、新霉素、阿米卡星、萘啶酸和环丙沙星的耐药率显著高于猪源大肠杆菌(P＜0.05)。鸡源大肠杆菌中3耐及3耐以上的菌株占97.60%,猪源大肠杆菌占94.72%。结果表明,屠宰前畜禽体内大肠杆菌对临床常用抗菌药物的耐药性非常严重,以多重耐药为主,且耐药谱丰富多样。提示屠宰前畜禽携带的耐药菌对食品安全和人类健康存在较大的安全隐患。     

Molecular diversity of Campylobacter coli and C. jejuni isolated from pigs at slaughter by flaA-RFLP analysis and ribotyping

A population of porcine isolates of Camplobacter jejuni (n = 11) and C. coli (n = 17) were examined for genotypic relatedness employing ribotyping, as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin (fla)A gene locus. PCR was employed to amplify a 533 bp fragment from the flaA gene, including the previously described short variable region (SVR), employing the novel primers, A2 and Al and successfully generated this amplicon for all wild-type strains examined (n = 28) of both C. jejuni and C. coli, as well as with both type strains, i.e. C. jejuni NCTC 11351 and C. coli NCTC 11366. Individual genotypes were assigned to each isolate typed employing the four typing methods (flaA-RFLP(Hae) III, flaA-RFLP(Pst) I ribotyping(Hae) III and ribotyping(Pst) I) and were assigned an arbitrary genotype code in ascending alphabetical order in comparison with a database of established genotypes for each of the methods employed. This study showed that several flaA-RFLP and ribopatterns existed within C. jejuni and C. coli, and demonstrated a heterogeneous diversity of strains occurring in the pigs examined. Ribotyping of strains with 16S and 23S rRNA with Pst I and Hae III digested chromosomal DNA allowed subdivision of strains into nine and eight groups, respectively. RFLP analyses with Pst I and Hae III digests probed with the flaA gene probe allowed subdivision of strains into eight and eleven subtypes, respectively. Employment of RFLP with the flaA nucleic acid probe and Hae III digests produced the greatest amount of variation of any genotyping scheme employed. Although there was a high degree of variability demonstrated by both typing methods, most isolates ( > 60%) clustered into four main genotypes, i.e. genotypes A-D. FlaA-PCR-RFLP typing demonstrated that the majority of isolates, 67.9 and 60.7%, were included in these four main genotypes for Pst I and Hae III restriction digests, respectively, although there was a high prevalence (7/11; 63.6%) of fla(Hae) III genotype A occurring within the C. jejuni isolates. Likewise, ribotyping studies demonstrated that most isolates were clustered into these four main genotypes, accounting for 81.5 and 60.7% of isolates for Pst I and Hae III restriction digests, respectively. This may indicate that the clonal population of campylobacters within this pig population is largely composed of persistent and dominant types, with a smaller number of hypervariable subtypes. Such data may useful in determining epidemiological routes of transmission of campylobacters from animal to animal, as well as helping to identify virulence determinants in persistent subtype populations.     

Sensitivity of Direct Culture,Enrichment and PCR for Detection of Campylobacter jejuni and C. coli in Broiler Flocks at Slaughter

Broiler chicken flocks are a significant source of Campylobacter jejuni and Campylobacter coli that result in the major public health problem of campylobacteriosis. Accurate estimates of the prevalence of both C. coli and C. jejuni in flocks would enhance epidemiological understanding, risk assessment and control options. This study combined results from a panel of 10 detection tests (direct culture, enrichment and PCR) on caecal samples from flocks at slaughter. A parallel interpretation approach was used to determine the presence of Campylobacter spp. and for C. jejuni and C. coli individually. The sample was considered positive if at least one method detected the target and this interpretation was taken to represent a ‘proxy gold standard’ for detection in the absence of a gold standard reference test. The sensitivity of each individual method to detect Campylobacter spp., C. jejuni and C. coli was then estimated relative to the proxy gold standard. Enrichment in adapted Exeter broth (deficient in polymyxin B) with a resuscitation step was 100% sensitive, whilst direct culture on modified charcoal cefoperazone deoxycholate agar (mCCDA) was highly sensitive (97.9%). Enrichment methods using Preston broth and Bolton broth were significantly less sensitive. Enrichment in Exeter broth promoted the recovery of C. jejuni, whilst enrichment in Bolton broth favoured C. coli. A RT‐PCR detection test could identify 80% of flocks that were co‐colonised with both species. This study found that 76.3% (n = 127) of flocks were colonised with Campylobacter spp. The majority (95.9%) of Campylobacter‐positive flocks were colonised with C. jejuni; however, approximately one‐third of positive flocks were simultaneously colonised with both C. jejuni and C. coli. The findings highlight the impact of different detection methodologies on the accuracy of the estimated incidence of both C. jejuni and C. coli entering the abattoir within broiler flocks and the associated public health risks.     

Serotypes of Campylobacter jejuni and C coli isolated from dogs

The serotypes of Campylobacter jejuni and C coli isolated from 56 dogs were established by the Penner serotyping scheme. A total of 37 C jejuni and 19 C coli were typed. Only two of the C coli strains were typable by the Penner method compared to 29 of 37 C jejuni strains. Pen 2 and 4 were the most predominant serotypes, constituting 41-4 per cent of the typable C jejuni strains. All but one of the C jejuni strains belonging to serotypes pen 1 and 2 were isolated from dogs with diarrhoea.     

Heterogeneity of Campylobacter jejuni and Campylobacter coli strains from healthy sheep

The objectives of this study were to identify, at species level, thermophilic campylobacters isolated from clinically healthy sheep by a multiplex polymerase chain reaction (mPCR). The heterogeneity among Campylobacter jejuni and C. coli isolates was also investigated using a restriction fragment length polymorphism (RFLP) analysis of the flagellin (flaA) gene. Samples of intestinal contents, gall bladders and faeces were collected from 610 healthy sheep. While gall bladder samples were plated directly onto Preston agar, an enrichment stage was applied for intestinal and faecal samples. Of the 610 samples, 302 (49.5%) were positive for Campylobacter spp. Using a mPCR assay for species identification, 103 (34.1%) were positive with C. jejuni-specific primers, while 100 (33.1%) were positive with C. coli-specific primers. Additionally, 16 (11.9%) of the intestinal content samples were positive for both species by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of 203 isolates tested, 48 different flaA types were found. Twenty-six flaA types were identified among C. jejuni isolates and the remaining 22 from C. coli isolates.     

鸡源空肠弯曲菌和结肠弯曲菌的临床分离及多重PCR鉴定

弯曲菌(Campylobacter)尤其是空肠弯曲菌(C.jejuni)是重要的人畜共患病病原菌,在人可引起急性胃肠炎,感染严重者伴有心内膜炎、肺炎、败血症、血栓静脉炎、脑膜炎、多发性关节炎以及格林-巴利综合征等疾病[1-3].家禽、家畜、宠物等动物是弯曲菌的常见宿主,因此研究动物源弯曲菌具有重要的公共卫生意义.     

PFGE and PCR/RFLP typing of Campylobacter jejuni strains from poultry

1. Pulsed-field gel electrophoresis (PFGE) and PCR-restriction fragment length polymorphisms of the flagellin gene (fla-RFLP) were used to analyse 92 poultry and 110 human strains of Campylobacter jejuni. 2. Among poultry strains, 11 fla-RFLP and 11 PFGE subtypes were found, while human strains could be divided into 23 fla-RFLP and 32 PFGE subtypes. Altogether, 31 fla-RFLP and 32 PFGE subtypes were found. 3. The results show that individual flocks in farms are mostly infected with a single C. jejuni clone, while during subsequent colonisation their genotypes altered. fla-RFLP and PFGE profiles in poultry and humans were identical in less than 6% of cases. The results found so far confirm previous findings that chicken meat does not represent as important a source of campylobacteriosis as was previously believed. 4. The typing of Campylobacter sp. forms the basis for an evaluation of the current state and risk assessment of various Campylobacter sp. sources in relation to humans. Examination of samples with only one method is insufficient for epidemiology studies, because apparently different clones identified with one method could originate from a single clone, which could be proved with the other method.     

Cluster Analysis of Campylobacter jejuni Genotypes Isolated from Small and Medium‐Sized Mammalian Wildlife and Bovine Livestock from Ontario Farms

Using data collected from a cross‐sectional study of 25 farms (eight beef, eight swine and nine dairy) in 2010, we assessed clustering of molecular subtypes of C. jejuni based on a Campylobacter‐specific 40 gene comparative genomic fingerprinting assay (CGF40) subtypes, using unweighted pair‐group method with arithmetic mean (UPGMA) analysis, and multiple correspondence analysis. Exact logistic regression was used to determine which genes differentiate wildlife and livestock subtypes in our study population. A total of 33 bovine livestock (17 beef and 16 dairy), 26 wildlife (20 raccoon (Procyon lotor), five skunk (Mephitis mephitis) and one mouse (Peromyscus spp.) C. jejuni isolates were subtyped using CGF40. Dendrogram analysis, based on UPGMA, showed distinct branches separating bovine livestock and mammalian wildlife isolates. Furthermore, two‐dimensional multiple correspondence analysis was highly concordant with dendrogram analysis showing clear differentiation between livestock and wildlife CGF40 subtypes. Based on multilevel logistic regression models with a random intercept for farm of origin, we found that isolates in general, and raccoons more specifically, were significantly more likely to be part of the wildlife branch. Exact logistic regression conducted gene by gene revealed 15 genes that were predictive of whether an isolate was of wildlife or bovine livestock isolate origin. Both multiple correspondence analysis and exact logistic regression revealed that in most cases, the presence of a particular gene (13 of 15) was associated with an isolate being of livestock rather than wildlife origin. In conclusion, the evidence gained from dendrogram analysis, multiple correspondence analysis and exact logistic regression indicates that mammalian wildlife carry CGF40 subtypes of C. jejuni distinct from those carried by bovine livestock. Future studies focused on source attribution of C. jejuni in human infections will help determine whether wildlife transmit Campylobacter jejuni directly to humans.     

不同生长期猪源大肠埃希菌年度耐药趋势分析

采用微量肉汤稀释法对新疆等规模化猪场2013年分离的不同生长期猪源大肠埃希菌进行药敏试验,对2010年和2013年新疆某养殖场不同生长期猪源大肠埃希菌的耐药情况结果应用SPSS软件进行统计学分析。结果显示,2013年不同生长期猪源大肠埃希菌存在不同程度耐药;与2010年相比,2013年该养殖场总体耐药呈上升趋势,不同生长期分离菌对环丙沙星、恩诺沙星、诺氟沙星和氨苄西林抗菌药物耐药率明显升高;哺乳仔猪源菌和妊娠猪源菌对阿莫西林/克拉维酸的耐药率分别高出13.0%和65.4%;保育猪源菌对恩诺沙星的耐药率高出44.0%。多药耐药结果显示,多药耐药率按年份呈快速增长趋势,妊娠猪源菌多药耐药上升明显,由0耐上升为6耐;哺乳猪源菌由5耐上升为7耐,保育猪源菌由5耐上升为6耐。与2010年耐药结果相比,2013年不同生长期猪源大肠埃希菌耐药性除安普霉素外耐药率均呈现上升趋势,提示细菌耐药形势严峻,临床应联合兼或交替使用敏感的抗菌药物。     

Subspecies Characterization of Porcine Campylobacter coli and Campylobacter jejuni by Multilocus Enzyme Electrophoresis typing

Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated foodstuffs. C. jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and L-phenylalanyl-L-leucine peptidase giving 5, 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C. coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C. jejuni isolates and 19 unique ETs were detected for the 23 C. coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C. coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C. jejuni and C. coli, respectively. Alleles were shared by C. jejuni and C. coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C. coli in porcine livers. In certain cases, up to four phenotypically different strains of C. coli were isolated from one liver, indicating multiple infections.     

Detection and genotypic differentiation of Campylobacter jejuni and Campylobacter coli strains from laying hens by multiplex PCR and fla-typing

In total, 26 Campylobacter (C.) strains, isolated from liver, spleen, caecal or jejunal content of laying hens from different flocks were examined. In these flocks a drop in egg production, an increasing mortality and livers with whitish-grey lesions as post-mortem finding were observed. Suspected Campylobacter colonies were differentiated using a modified m-PCR in 13 Campylobacter jejuni and 13 Campylobacter coli strains. All isolates were characterised by typing of the flaA and flaB gene each with two restriction enzymes. To compare the four different profiles for all strains an artificial “fla-type” was generated. Different and identical fla-types of C. jejuni and C. coli were recovered from both intestinal and extra-intestinal organs of the laying hens and even from individual birds. One significant observation is that some fla-types of C. jejuni or C. coli were detected in intestinal and systemic sites but not all fla-types of both species appeared to be equally able to invade internal organs.     

Detection and genotypic differentiation of Campylobacter jejuni and Campylobacter coli strains from laying hens by multiplex PCR and fla-typing

In total, 26 Campylobacter (C.) strains, isolated from liver, spleen, caecal or jejunal content of laying hens from different flocks were examined. In these flocks a drop in egg production, an increasing mortality and livers with whitish-grey lesions as post-mortem finding were observed. Suspected Campylobacter colonies were differentiated using a modified m-PCR in 13 Campylobacter jejuni and 13 Campylobacter coli strains. All isolates were characterised by typing of the flaA and flaB gene each with two restriction enzymes. To compare the four different profiles for all strains an artificial “fla-type” was generated. Different and identical fla-types of C. jejuni and C. coli were recovered from both intestinal and extra-intestinal organs of the laying hens and even from individual birds. One significant observation is that some fla-types of C. jejuni or C. coli were detected in intestinal and systemic sites but not all fla-types of both species appeared to be equally able to invade internal organs.     

Occurrence of virulence genes among Campylobacter jejuni and Campylobacter coli isolates from domestic animals and children

The presence of the flaA, cadF, cdtB and iam genes of Campylobacter spp. was determined with the PCR method. The materials to investigate were 56 C. jejuni and 23 C. coli strains isolated from clinical samples (children and domestic animals). It was found that all of the Campylobacter spp. isolates from children with diarrhoea and domestic animals had cadF gene, responsible for adherence. The flaA gene was present in all Campylobacter spp. isolates derived from children and cats. Occurrence of flaA gene was confirmed in 100% of C. jejuni strains obtained from dogs. The high prevalence of the cdtB gene associated with toxin production was observed in this study (100%-Campylobacter spp. isolates obtained from dogs and cats, 97.9%-Campylobacter spp. isolates from children). The isolates showed a wide variation for the presence of iam gene. The lowest prevalence (23.5%) was detected in Campylobacter spp. obtained from dogs. The highest rates of iam detection (91.6%) were revealed in C. coli isolates from children.     

Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction

In this study, 100 gall bladder samples of sheep slaughtered at an abattoir in Elazi? province were examined for Campylobacter jejuni and Campylobacter coli by culture and polymerase chain reaction (PCR). Preston Campylobacter Agar supplemented with 7% horse blood and Preston Selective Supplement (Oxoid, Hampshire, UK) were used for isolation of the agents. Campylobacter spp. were isolated in 66 samples, and they were identified as 34% C. jejuni and 32% C. coli. A multiplex PCR based upon the use of ceuE gene-specific primers was applied on DNA samples extracted from C. jejuni and C. coli isolates. All C. jejuni and C. coli strains that were positive by culture were also detected to be positive by PCR. This study shows that PCR can be used an alternative, rapid and sensitive test for the identification of C. jejuni and C. coli which threaten human and animal health.     

Intestinal carriage of Campylobacter jejuni and Salmonella by chicken flocks at slaughter.

Campylobacter jejuni were isolated in large numbers from the majority of birds sampled in colonic swabs from 28 of 60 flocks at slaughter. By contrast only small numbers of birds from 11 of the same 60 flocks yielded Salmonella enteritidis serotypes. Three C. jejuni isolates from each flock were serotyped on the basis of their heat-stable antigens, using antisera prepared against 16 serotypes common in Campylobacter diarrhea in man. The majority (72 of 83) of the chicken isolates could be serotyped.     

The occurrence and characterization of Campylobacter jejuni and C. coli in organic pigs and their outdoor environment

The occurrence and species distribution of thermophilic Campylobacter was investigated in organic outdoor pigs. An increased exposure of outdoor pigs to C. jejuni from the environment may cause a shift from a normal dominance of C. coli to more C. jejuni, which may imply a concern of reduced food safety. Bacteriological methods for determination of Campylobacter excretion level were combined with colony-blot hybridization and real-time PCR for specific detection of C. jejuni in pigs. Campylobacter was isolated from pigs (n=47), paddock environment (n=126) and wildlife (n=44), identified to species by real-time PCR and sub-typed by serotyping (Penner) and pulse-field gel electrophoresis (PFGE) genotyping. All pigs excreted Campylobacter (10(3)-10(7) CFU g(-1) faeces) from the age of 8-13-weeks old. C. jejuni was found in 29% of pigs in three consecutive trials and always in minority to C. coli (0.3-46%). C. jejuni and C. coli were isolated from 10% and 29% of the environmental samples, respectively, while crow-birds and rats harboured C. jejuni. Individual pigs hosted several strains (up to nine serotypes). The paddock environment was contaminated with C. coli serotypes similar to pig isolates, while most of the C. jejuni serotypes differed. C. jejuni isolates of different origin comprised few similar serotypes, just one identical genotype was common between pigs, environment and birds. In conclusion, the occurrence of C. jejuni varied considerably between the three groups of outdoor pigs. Furthermore, transfer of C. jejuni to the outdoor pigs from the nearby environment was not predominant according to the subtype dissimilarities of the obtained isolates.     

Molecular and Statistical Analysis of Campylobacter spp. and Antimicrobial‐Resistant Campylobacter Carriage in Wildlife and Livestock from Ontario Farms

The objectives of this study were to (i) compare the carriage of Campylobacter and antimicrobial‐resistant Campylobacter among livestock and mammalian wildlife on Ontario farms, and (ii) investigate the potential sharing of Campylobacter subtypes between livestock and wildlife. Using data collected from a cross‐sectional study of 25 farms in 2010, we assessed associations, using mixed logistic regression models, between Campylobacter and antimicrobial‐resistant Campylobacter carriage and the following explanatory variables: animal species (beef, dairy, swine, raccoon, other), farm type (swine, beef, dairy), type of sample (livestock or wildlife) and Campylobacter species (jejuni, coli, other). Models included a random effect to account for clustering by farm where samples were collected. Samples were subtyped using a Campylobacter‐specific 40 gene comparative fingerprinting assay. A total of 92 livestock and 107 wildlife faecal samples were collected, and 72% and 27% tested positive for Campylobacter, respectively. Pooled faecal samples from livestock were significantly more likely to test positive for Campylobacter than wildlife samples. Relative to dairy cattle, pig samples were at significantly increased odds of testing positive for Campylobacter. The odds of isolating Campylobacter jejuni from beef cattle samples were significantly greater compared to dairy cattle and raccoon samples. Fifty unique subtypes of Campylobacter were identified, and only one subtype was found in both wildlife and livestock samples. Livestock Campylobacter isolates were significantly more likely to exhibit antimicrobial resistance (AMR) compared to wildlife Campylobacter isolates. Campylobacter jejuni was more likely to exhibit AMR when compared to C. coli. However, C. jejuni isolates were only resistant to tetracycline, and C.  coli isolates exhibited multidrug resistance patterns. Based on differences in prevalence of Campylobacter spp. and resistant Campylobacter between livestock and wildlife samples, and the lack of similarity in molecular subtypes and AMR patterns, we concluded that the sharing of Campylobacter species between livestock and mammalian wildlife was uncommon.     

Anti‐microbial Susceptibility for east1+Escherichia coli Isolated from Diarrheic Pigs in Korea

The in vitro susceptibilities of 128 isolates of east1⁺Escherichia coli from pre‐weaned and post‐weaned pigs with diarrhoea were tested with nine commonly used anti‐microbial agents by an agar dilution minimal inhibitory concentration (MIC) procedure according to National Committee for Clinical Laboratory Standards guidelines. For the isolates from pre‐weaned and post‐weaned pigs, most of them were susceptible to low concentrations (MIC₉₀) of tetracycline (4 and 2 μg/ml), ceftiofur (2 and 2 μg/ml), and colistin (4 and 2 μg/ml). Marked resistance was found in others.     

Biotin-streptavidin enzyme-linked immunosorbent assay for the detection of antibodies to Campylobacter jejuni and C. coli in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.