Real Time PCR to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis.

Bovine venereal campylobacter infection, caused by Campylobacter fetus venerealis, is of significant economic importance to the livestock industry. Unfortunately, the successful detection and discrimination of C. fetus venerealis from C. fetus fetus continue to be a limitation throughout the world. There are several publications warning of the problem with biotyping methods as well as with recent molecular based assays. In this study, assessed on 1071 isolates, we report on the successful development of two Real Time SYBR® Green PCR assays that will allow for the detection and discrimination of C. fetus fetus and C. fetus venerealis. The sensitivity reported here for the C. fetus (CampF4/R4) and the C. fetus venerealis (CampF7/R7) specific PCR assays are 100% and 98.7% respectively. The specificity for these same PCR assays are 99.6% and 99.8% respectively.

Characteristics and comparative performance of direct culture, direct PCR and enumeration methods for detection and quantification of Campylobacter spp. in broiler caeca.

Detection and enumeration of Campylobacter spp. in broiler chicken flocks are key components of research and surveillance studies aimed at reducing Campylobacter infections in people. Direct culture of caecal contents onto selective agar is the typical method used to confirm flock colonisation. Modified charcoal cefoperazone deoxycholate agar (mCCDA) is commonly used for this method, although alternative selective media have been used. Additionally, PCR methods to detect Campylobacter DNA from caecal contents may provide a rapid alternative. However comparative performance data for these methods is limited and therefore required to ensure optimal detection methods for this sample type. In this study, 306 broiler caeca were tested for Campylobacter using direct culture on mCCDA, Skirrows and Preston agars and two real-time PCR methods, one specific for mapA/ceuE regions and another for the flaA gene region. Additionally, the suitability of spread plating and spiral plating methods for enumeration of Campylobacter and the impact of sample storage were assessed. This study confirmed modified CCDA as an optimal media for detection of Campylobacter in broiler caeca. It was significantly more sensitive than Skirrows or Preston agars. This study also demonstrated that the mapA/ceuE PCR had excellent agreement with culture on mCCDA and is a genuine alternative method. Spread plating and spiral plating methods were suitable for enumeration although spiral plating appeared more sensitive for stored samples (72 h). A 1 log reduction in viable Campylobacters was observed in stored samples, therefore storage effects should be considered for quantitative studies with broiler caeca.

Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine.

AIMS: A panel of pulsed field gel electrophoresis (PFGE) type variants of Campylobacter jejuni, previously identified as of clonal origin, were investigated to determine whether genomic instability could be observed during competitive growth. METHODS AND RESULTS: Upon recovery from frozen storage, some variants had undergone alterations in PFGE profiles, but subsequent culture produced constant genotypes. Individual variants did not display differences in colonization potential when tested in orally challenged 1-day-old chickens. However, competitive colonization using mixtures of two or three PFGE types generally resulted, by 4 weeks postchallenge, in one predominant PFGE type in all birds. For some variant mixtures, a minor population of novel PFGE types was detected in individual birds. The creation of new variants appeared to be dependent on the extent of competition and of the individual host. Genomic rearrangements most likely explain this increase in genetic diversity, apparently without the involvement of natural transformation or plasmid acquisition. In vitro cultivation of mixed inoculations were again selected for particular variants; but genetic diversity was not generated, suggesting that the selection pressures in vitro differed from those active in vivo. CONCLUSION: These observations support the hypothesis that by generating genetic diversity, C. jejuni can improve its phenotypic fitness to survive and colonize subsequent hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: The consequences of such observations for the development of campylobacter control strategies for poultry may be substantial.

Intestinal carriage of verocytotoxigenic Escherichia coli O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica, in cattle, sheep and pigs at slaughter in Great Britain during 2003.

An abattoir survey was undertaken to determine the prevalence of foodborne zoonotic organisms colonizing cattle, sheep and pigs at slaughter in Great Britain. The study ran for 12 months from January 2003, involved 93 abattoirs and collected 7703 intestinal samples. The design was similar to two previous abattoir surveys undertaken in 1999-2000 allowing comparisons. Samples were examined for VTEC O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica. The prevalence of VTEC O157 faecal carriage was 4.7% in cattle, 0.7% in sheep and 0.3% in pigs. A significant decrease in sheep was detected from the previous survey (1.7%). Salmonella carriage was 1.4% in cattle, a significant increase from the previous survey of 0.2%. In sheep, faecal carriage was 1.1% a significant increase from the previous survey (0.1%). In pigs, carriage was 23.4%, consistent with the previous study. Thermophilic Campylobacter spp. were isolated from 54.6% of cattle, 43.8% of sheep and 69.3% of pigs. Y. enterocolitica was isolated from 4.5% of cattle, 8.0% of sheep and 10.2% of pigs.

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp.

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.

The carry-over of Campylobacter isolates between sequential poultry flocks.

The carry-over of Campylobacter strains from one flock to a subsequent flock in the same broiler house has been studied using molecular epidemiological techniques. In all, 524 Campylobacter strains, isolated from two sequential broiler flocks from 60 broiler houses, were typed by restriction fragment polymorphism of the polymerase chain reaction (PCR) product of the flaA and flaB genes (fla typing). Selected strains were also typed using pulsed field gel electrophoresis (PFGE). By fla typing, 15 (21%) of the 60 houses with Campylobacter-positive sequential flocks had identical genotypes. In 10 (16% overall) of these houses the strains were also identical by PFGE. The difference in PFGE patterns in the strains from the three remaining houses may be indicative of genetic instability. Overall, these results suggest that carry-over from one flock to a subsequent flock in the same house is a relatively infrequent event and, therefore, that routine broiler house cleansing and/or disinfection is largely adequate to eliminate Campylobacter contamination. An alternative explanation of the low level carry-over is a persistent source or reservoir, external to the environment of the broiler houses.

Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs.

The recent development of simple, rapid genotyping techniques for Campylobacter species has enabled investigation of the determinative epidemiology of these organisms in a variety of situations. In this study we have used the technique of fla typing (PCR-restriction fragment length polymorphism analysis of the flaA and flaB genes) to identify the sources of strains contaminating the carcasses of five campylobacter-positive and two campylobacter-negative broiler flocks during abattoir processing. The results confirmed that, in the United Kingdom, individual broiler flocks are colonized by a limited number of subtypes of Campylobacter jejuni or C. coli. In some but not all cases, the same subtypes, isolated from the ceca, contaminated the end product as observed in carcass washes. However, the culture methodology, i.e, use of direct plating or enrichment, affected this subtype distribution. Moreover, the number of isolates analyzed per sample was limited. fla typing also indicated that some campylobacter subtypes survive poultry processing better than others. The extent of resistance to the environmental stresses during processing varied between strains. The more robust subtypes appeared to contaminate the abattoir environment, surviving through carcass chilling, and even carrying over onto subsequent flocks. From these studies it is confirmed that some campylobacter-negative flocks reach the abattoir but the carcasses from such flocks are rapidly contaminated by various campylobacter subtypes during processing. However, only some of these contaminating subtypes appeared to survive processing. The sources of this contamination are not clear, but in both negative flocks, campylobacters of the same subtypes as those recovered from the carcasses were isolated from the crates used to transport the birds. In one case, this crate contamination was shown to be present before the birds were loaded.

Sequential spread of Campylobacter infection in a multipen broiler house.

Generally, colonization with Campylobacter jejuni is first detected in broilers 2-3 wk after hatching. Once introduced into a flock, this infection spreads very rapidly. The sources and routes of transmission of C. jejuni in broilers remain debatable. In this study, the spread of infection was monitored in a commercial multipen broiler house in which birds were contained in discrete groups and sampled sequentially. Colonization was monitored in two broiler flocks up to slaughter. Serotyping and fla typing methods were applied to differentiate all the C. jejuni strains isolated. In flock 1, colonization was first detected at 32 days of age in birds located at the rear of the house. By 40 days, nearly all the birds were infected with the same strain (fla type 1.9). However, at 46 days of age, a second strain (fla type 3.7) was detected in some of the birds. These birds were also located toward the rear of the house. In flock 2, infection was detected at 5 wk of age. This infection was once again first detected in birds located at the rear of the house. In this flock, only a single fla type (1.1) was isolated throughout. A survey of the broiler house relative to the location of first point of infection indicated the use of an entrance door unprotected by boot dips. However, securing this door during the second flock study did not prevent infection.