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.     

Antimicrobial resistance in fecal Escherichia coli isolated from growing chickens on commercial broiler farms

To investigate the effects of rearing practices of commercial broiler chickens on the incidence of antimicrobial resistance in commensal Escherichia coli isolates, fecal E. coli isolates obtained in 4 farms were screened for anitimicrobial resistance. Ten E. coli isolates were recovered from each of the fecal samples collected from 10 birds in the farms at the ages of 2 days, 14-17 days, and 47-50 days. In 2 out of the 4 farms, no antimicrobials were used during the rearing period. In the other two farms, following collection of the fecal samples at 14 and 15 days of age, oxytetracycline (OTC), sulfadimethoxine (SDMX), and tylosin were given to birds on one farm and SDMX was used in the other. Isolates resistant to ampicillin and OTC that were obtained from an untreated flock at different sampling times were closely related to each other by pulsed-field gel electrophoresis patterns (PFGE) of XbaI-digested chromosomal DNA. PFGE analysis together with in vitro conjugation experiments suggested that diversity of resistance phenotypes within a clone may be resulted from the acquisition and loss of R-plasmids in an untreated and a treated flock. The numbers of resistance phenotypes observed among fecal isolates increased during the growth of the chickens in all the farms. The results in the present study suggest that persistence of commensal E. coli strains resistant to antimicrobials even in the absence of antimicrobial administration. It is also hypothesized that horizontal transmission of resistance determinants resulted in the emergence of different resistance phenotypes in those farms.     

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.     

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.     

Epidemiologic features of Campylobacter jejuni isolated from poultry broiler houses and surrounding environments as determined by use of molecular strain typing

OBJECTIVE: To genetically type Campylobacter jejuni isolates from broiler houses or the external environment to identify the source of Campylobacter organisms in broiler chickens. SAMPLE POPULATION: Environmental samples associated with broiler chickens, in commercial grow-out houses. PROCEDURE: Polymerase chain reaction (PCR) was used to amplify flaB, and the amplicon was digested with Sau3A to create a restriction fragment length polymorphism assay; PCR was also used to detect a transcribed spacer region in the 23S rRNA gene. RESULTS: Isolates possessing a 23S spacer region were more prevalent outside broiler houses than inside. Houses that had previously contained chickens or lacked biosecurity procedures were more likely to contain isolates possessing the 23S spacer. One house contained only isolates possessing the spacer, whereas an adjacent house contained only isolates lacking the spacer. The flaB type detected in broiler houses was different from the type detected in the environment; however, many isolates within the broiler houses contained untypable flaB genotypes. CONCLUSIONS AND CLINICAL RELEVANCE: Most isolates from within houses were genetically distinct from isolates from outside houses that were examined by bacteriologic culture, suggesting an undetected source of C jejuni. Detection of isolates containing the 23S spacer appeared to be an indicator of environmental contamination of the houses. The observation of completely different C jejuni genetic types simultaneously within adjacent houses suggests that some types do not compete successfully during the grow-out period. In addition, the diversity of genotypes identified within broiler houses indicates the complexity of the ecologic features of C jejuni in the chicken environment.     

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.     

Effects of diet formulations containing proteins from different sources on intestinal colonization by Campylobacter jejuni in broiler chickens

The objective of this study was to compare the effects of 3 diet formulations containing different protein sources (animal, plant, and a combination of animal and plant) on the colonization of Campylobacter jejuni in the gastrointestinal tract of broiler chickens. A freshly isolated strain of C. jejuni (biotype IV, serotype HS O:21, O:29, HL untypable) from a broiler chicken was used to infect 3-day-old chicks that had been free of C. jejuni; 0.5 mL of an inoculum containing 10⁸ colony-forming units was administered orally. Shedding of the organism was studied, and C. jejuni in the ceca, jejuni, and crop were enumerated by quantitative culture. The isolates recovered from the birds during the study period of 35 d were characterized and confirmed as C. jejuni by the use of standard methods and underwent biotyping, serotyping, antimicrobial susceptibility testing by disk diffusion and the E-test, and flagellin gene typing. A cyclical pattern of shedding of C. jejuni was observed in all the birds. Colonization was highest in the ceca. The ceca of birds receiving plant-protein-based feed had significantly less colonization then the ceca of birds receiving the other types of feed, whereas the differences in colonization of the jejuni and crops were not significant. Characterization by biotyping, serotyping, and flagellin gene typing showed that 95% of the recovered isolates were identical to the strain used for infecting the chicks. However, with the Lior-HL typing scheme, 74% of the recovered isolates were HL untypable. Antimicrobial resistance testing did not reveal significant differences between the infecting strain and the recovered isolates among the different feed groups.     

Antibiotic resistance in Escherichia coli and Enterococcus spp. isolates from commercial broiler chickens receiving growth-promoting doses of bacitracin or virginiamycin

Antibacterial agents such as zinc bacitracin (ZB) and virginiamycin (VG) are used as growth promoting agents (GP) in broiler chicken production. The objective of this study was to evaluate the effect of the use of ZB and VG on the emergence of antibacterial resistance in a commercial broiler chicken farm. Three trials were conducted using 3 different diets: one without antibacterial agents, one containing VG, and one with ZB. Escherichia coli and Enterococcus spp. strains were isolated and tested for their susceptibility to various antibacterial agents. The occurrence of the resistance genes vatD, ermB, and bcrR in Enterococcus spp. isolates was determined by polymerase chain reaction (PCR). Comparative quantification of vatD and bcrR genes in total deoxyribonucleic acid (DNA) extracts from litter was done by SYBR Green Real-Time PCR (QPCR). Escherichia coli and Enterococcus spp. isolates from diet groups had different levels of resistance to various antibacterial agents over time. These GPs did not select for specific antibacterial agent resistance (AAR) in Enterococcus spp. The use of GPs seemed to lower the percentage of E. coli isolates resistant to some antibacterial agents. The presence of the bcrR gene could not explain all resistant phenotypes to ZB. Genes other than vatD and ermB might be involved in the resistance to VG in Enterococcus spp. Use of GPs was not associated with presence of the bcrR gene in DNA extracts from litter, but use of VG was associated with vatD presence.     

Antimicrobial resistances,and molecular typing of Campylobacter jejuni isolates,separated from food-producing animals and diarrhea patients in Iran

The aims of this study were to regain new epidemiology information about frequency, drug resistance rates, and typing of Campylobacter jejuni (C. jejuni) isolates, obtained from some poultry and cattle farms, slaughterhouses, and people with diarrhea. In this regard, Minimal Inhibitory Concentration (MIC) of several antibiotics and the associated antibiotic resistance genes, including tetO, tetA, cmeB, and bla_OXA-61 were evaluated. The isolates were also typed, using the Fla-RFLP method. Generally, between 233 food animal samples, 80 (34.33%) C. jejuni were isolated. Moreover, 20 out of 74 (27%) human specimens suspected to infectious diarrhea were C. jejuni positive. High frequencies of resistance to tetracycline (100%), ciprofloxacin (95%), and nalidixic acid (86%), and low frequencies of resistance to florfenicol (0%), erythromycin (5%), and gentamicin (8%) were observed. Furthermore, in the tetracycline-resistant isolates, the existences of tetO, tetA, and cmeB were 86%, 23%, and 48%, respectively. There was a significant correlation between the cluster types obtained from Fla-RFLP method and antibiotic resistance pattern. The results suggested that the genomic link between Campylobacter spp. should be always evaluated in each country to provide an insight about the Campylobacter spp., spread in the region, in order to implement the health-controlling programs efficiently.     

Prevalence of thermophilic Campylobacter species in Swedish dogs and characterization of C. jejuni isolates

Background

The aims of this study were to investigate the prevalence of Campylobacter species in Swedish dogs, to identify the species of the Campylobacter isolates and to genotype the C. jejuni isolates. Young and healthy dogs were targeted and the sampling was performed at 11 veterinary clinics throughout Sweden from October 2011 to October 2012. Faecal swab samples were collected and sent to the laboratory at the National Veterinary Institute (SVA) for isolation of Campylobacter, speciation and genotyping.

Results

Campylobacter spp. were isolated from 67 of the 180 sampled dogs which yields an overall prevalence of 37%. The most prevalent species of Campylobacter among the participating dogs was C. upsaliensis with 52 of the 67 identified isolates. A lower prevalence was observed for C. jejuni with seven identified isolates and one isolate was identified as C. helveticus. Multi-locus sequence typing (MLST) was carried out on the seven C. jejuni isolates and all sequence types that were found are also commonly found in humans. The dogs were divided into three age groups; 1) under 12 months, 2) 12 to 23 months and 3) 24 months and older. The highest prevalence was found in the two younger age groups. Dogs shedding C. jejuni were between 3–12 months of age while dogs shedding C. upsaliensis were found in all ages.

Conclusions

The present investigation finds that Campylobacter spp. known to cause campylobacteriosis in humans are present in Swedish dogs. The results suggest an age predisposition where dogs under 2 years of age are more likely to shed Campylobacter spp. than older dogs. The most commonly isolated species was C. upsaliensis followed by C. jejuni, which was only detected in dogs up to 12 months of age. All C. jejuni isolates identified in the present study were of the same MLST types that have previously been described both in humans and in animals. The awareness of the Campylobacter risk of healthy young dogs may be an important way to reduce the transmission from dogs to infants, young children and immunocompromised adults.     

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.     

Comparative studies on competitive exclusion of three isolates of Campylobacter fetus subsp. jejuni in chickens by native gut microflora

Resistance of young chicks to Campylobacter fetus subsp. jejuni was substantially increased by early exposure to native gut microflora. Protection was demonstrated against two human isolates and a chicken isolate of C. fetus subsp. jejuni. Significant protection against the chicken isolate was observed throughout a 91-day test period. Infection reached 100% (25/25) in the untreated group at 56 days of age and only 4% (1/25) in the group treated with native gut microflora. Campylobacter fetus subsp. jejuni was isolated from the ceca and less frequently from the gall bladder and liver of chicks that actively shed the bacteria. Cultures of feces from chicks reared on wood-shavings litter were often negative, suggesting that culturing litter as an indicator of infection has limited value.     

Occurrence of Campylobacter species from broiler chickens and chicken meat in Malaysia

Tropical Animal Health and Production - Campylobacter is reported as a major cause of foodborne illness worldwide. Consumption of contaminated chicken meat is considered a significant risk factor...     

Assessing cellulitis pathogenicity of Escherichia coli isolates in broiler chickens assessed by an in vivo inoculation model.

The purpose of this study was to identify Escherichia coli isolates that could be characterized as cellulitis pathogens. Twelve E. coli isolates from diagnostic cases of cellulitis or mixed infections with various serotypes were compared for ability to produce cellulitis and internal lesions indicative of systemic infection. Ranking of isolates was based on the premise that E. coli isolates that were "cellulitis-type" would cause cellulitis lesions without causing systemic infection. A quantitative scoring system was also used so both the time required for a lesion to develop and lesion severity could be evaluated as determinants of virulence. Escherichia coli isolates were inoculated by subcutaneous injection of a standardized dose in 24 broiler chickens per isolate. Necropsy was performed on four birds per group at 6, 12, 24, 36, 48, and 60 hr postinoculation (PI). Cellulitis lesions were scored on a 0 to 5 scale based on size, migration from the inoculation site, and gross characteristics. Lesions of the pericardium, liver, joint, or body cavity were evaluated. Gross lesion scores of 1 or 2 were evident by 6 hr PI with all isolates. Mortality occurred in 4 of 12 experimental groups. Internal lesions were observed in 3 to 12 birds per group. Escherichia coli was reisolated from all lesions. The four isolates with the highest lesion score and highest lesion points as determined by the quantitative scoring system did not vary. However, the rankings of two other isolates were affected. Four isolates that were below average for mean internal lesion score and above average for mean cellulitis points were characterized as cellulitis-type. Three isolates that were above average for internal lesion score and below average for mean cellulitis points were characterized as systemic-type. The E. coli serotype was not a determining factor for cellulitis-type pathogenicity. Isolates discriminated as cellulitis-type or septicemic-type E. coli in this study are being used to further investigate virulence factors involved in the pathogenesis of cellulitis in broiler chickens.     

PCR-based restriction fragment length polymorphism (RFLP) analysis of Campylobacter jejuni isolates from humans, chickens and dogs in northern Taiwan

Two hundred and twenty strains of Campylobacter jejuni (70 human, 51 canine and 99 chicken strains) were isolated from September 2003 to September 2004 in northern Taiwan. These strains were subtyped by PCR-RFLP analysis of the flagellin (FlaA) gene. On the basis of restrictive digest, six types were identified with AfaI, seven types with MboI and five types with HaeIII. With the combination of these three enzymes, 47 distinct PCR-RFLP patterns were observed-25 each from human and chicken isolates, and 9 from canine isolates. In human strains, the most frequently occurring types were Cj-28 (14.3%), Cj-17 (10%), Cj-16 (8.6%), Cj-37 (7.1%) and Cj-46 (7.1%). In canine strains, the most prevalent types were Cj-1 (33.3%), Cj-26 (19.6%), Cj-3 (15.7%), Cj-2 (9.8%) and Cj-10 (9.8%). In chicken strains, the most frequently occurring types were Cj-46 (40.4%), Cj-29 (9.1%), Cj-45 (7.1%) and Cj-41 (5.1%). The results suggest that poultry is a source, but not the sole source, of C. jejuni infection in humans. Two RFLP types, Cj-17 and Cj-37, frequently occurring in human isolates in this study have also been found to be prevalent in human isolates in Japan, China and the Czech Republic, indicating a possible international clonal spread.     

Antimicrobial susceptibility of Salmonella isolates from the broiler chicken industry in Ontario.

Antibacterial drug resistance among 219 salmonella isolates recovered during 1974 from poultry and poultry environments at the various production stages of broiler chickens in three integrated Ontario companies are recorded. All isolates were susceptible to ampicillin, trimethroprim-sulfamethoxazole complex, furazolidone, cephaloridine and amoxicillin. A relative increase in resistance to tetracycline and streptomycin with an accompanying decrease in resistance to triple sulfa compound was recorded when compared to a previous investigation of avian salmonella isolates in Ontario. The percentage and patterns of antimicrobial resistance were comparable at the various stages of production. Resistance to tetracycline and streptomycin was the most common pattern found among both Salmonella typhimurium and other serotypes. A notably high prevalence of resistance was found among Salmonella enteritidis isolates including some isolates with R factors for chloramphenicol resistance. This latter finding is of particular concern because of the high prevalence of this serotype in poultry and in human salmonellosis.