Comparative, Collaborative, and On-Site Validation of a TaqMan PCR Method as a Tool for Certified Production of Fresh, Campylobacter-Free Chickens

Certified Campylobacter-free poultry products have been produced in Denmark since 2002, the first example of fresh (unprocessed and nonfrozen) chickens labeled “Campylobacter free.” This success occurred partly through use of a 4-hour gel-based PCR testing scheme on fecal swabs. In this study, a faster, real-time PCR approach was validated in comparative and collaborative trials, based on recommendations from the Nordic system for validation of alternative microbiological methods (NordVal). The comparative real-time PCR trial was performed in comparison to two reference culture protocols on naturally contaminated samples (99 shoe covers, 101 cloacal swabs, 102 neck skins from abattoirs, and 100 retail neck skins). Culturing included enrichment in both Bolton and Preston broths followed by isolation on Preston agar and mCCDA. In one or both culture protocols, 169 samples were identified as positive. The comparative trial resulted in relative accuracy, sensitivity, and specificity of 98%, 95%, and 97%, respectively. The collaborative trial included nine laboratories testing neck skin, cloacal swab, and shoe cover samples, spiked with low, medium, and high concentrations of Campylobacter jejuni. Valid results were obtained from six of the participating laboratories. Accuracy for high levels was 100% for neck skin and cloacal swab samples. For low levels, accuracy was 100% and 92% for neck skin and cloacal swab samples, respectively; however, detection in shoe cover samples failed. A second collaborative trial, with an optimized DNA extraction procedure, gave 100% accuracy results for all three spiking levels. Finally, on-site validation at the abattoir on a flock basis was performed on 400 samples. Real-time PCR correctly identified 10 of 20 flocks as positive; thus, the method fulfilled the NordVal validation criteria and has since been implemented at a major abattoir.     

Evaluation of PCR for detection of Campylobacter in a national broiler surveillance programme in Denmark

AIMS: To develop and evaluate a rapid and sensitive PCR method for detection of Campylobacter spp. directly from chicken faeces. METHODS AND RESULTS: DNA was isolated from faecal swabs using magnetic beads followed by PCR using a prealiquoted PCR mixture, which had been stored in the freezer. The result could be obtained in <6 h. The method was evaluated on 1282 samples from the Danish surveillance programme for Campylobacter in broilers by comparing with conventional culture. The diagnostic specificity was calculated to be 0.99. The detection limits of the PCR method and of the conventional culture were compared using spiked control material. For both methods the detection limit was 36 CFU ml-1. CONCLUSIONS: It was concluded that the PCR proved useful for detection of Campylobacter in pooled cloacal swabs from broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: By taking cloacal samples in the broiler flocks the technique can be used as an important tool for planning and directing the broiler slaughtering process. This will be a great help in minimizing the risk of contaminating Campylobacter-free flocks at the abattoir.     

Validation of a same-day real-time PCR method for screening of meat and carcass swabs for Salmonella

Background  

One of the major sources of human Salmonella infections is meat. Therefore, efficient and rapid monitoring of Salmonella in the meat production chain is necessary. Validation of alternative methods is needed to prove that the performance is equal to established methods. Very few of the published PCR methods for Salmonella have been validated in collaborative studies. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, non-commercial real-time PCR method for detection of Salmonella in meat and carcass swabs.     

Effect of preslaughter events on prevalence of Campylobacter jejuni and Campylobacter coli in market-weight turkeys

The effects of events which occur prior to slaughter, such as loading, transport, and holding at an abattoir, on the prevalence of Campylobacter species, including Campylobacter jejuni and Campylobacter coli, were examined. Cloacal swabs from market-weight turkeys in each of five flocks were obtained on a farm prior to loading (time 1; 120 swabs per flock) and after transport and holding at the abattoir (time 2; 120 swabs per flock). A statistically significant increase in the overall prevalence of Campylobacter spp. was observed for cloacal swabs obtained from farm 3 following transport (P < 0.01). At time 2, an increase in the prevalence of C. coli was also noted for cloacal swabs from farms 3, 4, and 5 (P < 0.01). Neither the minimum time off of feed nor the distance transported from the farm to the abattoir was correlated with the increase in C. coli prevalence. Similarly, responses to an on-farm management questionnaire failed to detect any factors contributing to the observed changes in Campylobacter sp. prevalence. A SmaI macrorestriction analysis of Campylobacter sp. isolates recovered from flock 5 indicated that C. coli was more diverse than C. jejuni at both time 1 and time 2 (P < 0.01), based on a comparison of the Shannon indices of diversity and evenness.     

Toward an international standard for PCR-based detection of Escherichia coli O157. Part 1. Assay development and multi-center validation

As part of a major European research project, a diagnostic PCR assay, including an internal amplification control, was developed and validated in a collaborative trial for the detection of Escherichia coli O157. The assay is based on amplification of sequences of the rfbE O157 gene. The collaborative trial, including 12 international laboratories, was carried out in two phases: phase (a) was performed with identical PCR reagents, including the internal control, provided by the sending laboratory; phase (b) was performed on the same samples and internal control but using in-house PCR reagents of own choice. Phase (a) showed an inclusivity (detection of target strains) of 96.8% and the exclusivity (negative response from nontarget strains) was 100%. The overall performance resulted of phase (a) in an accordance of 98.8, concordance of 98.6, and a concordance odds ratio of 1.11. Phase (b) results showed an accuracy of 100% with all partners and by using different polymerase types and thermocycler models. This indicates that the assay, under consideration as an international standard, was just as reproducible between laboratories, as repeatable within a laboratory. The assay is taken further for validation on carcass-rinse samples.     

Prevalence of thermotolerant Campylobacter in partridges (Perdix perdix)

Aim:  To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges ( Perdix perdix ) in southern Italy.
Methods and Results:  Cloacal swabs of partridges ( n  =   240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49·2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12·7%) out of the 118 positive samples were also positive for Campylobacter jejuni . In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence ( P  <   0·05) than younger ones.
Conclusion:  These results reinforce the assumption that game birds may be considered as potential carriers of Campylobacter spp. for human being and other animal species.
Significance and Impact of the Study:  Although an earlier 1986 publication described the prevalence of Campylobacter coli in commercially reared partridges, this is the first report to confirm the species of Campylobacter using a PCR test.     

Optimising recovery of Campylobacter spp. from the lower porcine gastrointestinal tract

To determine the incidence of campylobacters in Northern Ireland pigs, ileal contents and anal swabs were taken shortly after death. Direct streaking onto Preston agar, and modified charcoal cefoperazone desoxycholate agar (mCCDA), were compared, as was enrichment in selective broths prior to streaking onto the corresponding solid medium. For anal swabs direct plating on mCCDA was most efficient, with 100% of samples positive, whilst for ileal contents enrichment in mCCD broth was best with 86% of samples positive. Although only 34% of ileal samples enriched in Preston broths were positive they yielded three species not isolated from mCCD broth, and hence indicated that some pigs were infected by at least two species of Campylobacter. Overall, the number of samples found to contain campylobacters, and the range of species isolated, was seen to be markedly affected by both the choice of selective medium and the isolation procedures.     

Development of a m-PCR assay for simultaneous identification of Campylobacter jejuni and C. coli

Multiplex PCR assay (m-PCR) with three sets of primers was developed for simultaneous identification of Campylobacter jejuni and C. coli. Poultry faecal samples were enriched in Preston broth for 24 h and streaking on selective media was performed before and after enrichment. m-PCR was applied on bacterial cultures harvested from media plates. The data showed a selective effect of Preston broth which favoured the growth of C. coli. Identification of the species by the hippurate hydrolysis test and by the m-PCR was performed on 294 isolates of Campylobacter. The efficiency of the identification by the biochemical test is only 34% in comparison to 100% efficiency with the PCR. The use of our m-PCR in combination with the culture method allowed reliable detection and identification of C. jejuni and C. coli within 3-4 d.     

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: validation in a multicenter collaborative trial

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (Campylobacter jejuni, C. coli, and C. lari) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.     

Effect of Preslaughter Events on Prevalence of Campylobacter jejuni and Campylobacter coli in Market-Weight Turkeys

The effects of events which occur prior to slaughter, such as loading, transport, and holding at an abattoir, on the prevalence of Campylobacter species, including Campylobacter jejuni and Campylobacter coli, were examined. Cloacal swabs from market-weight turkeys in each of five flocks were obtained on a farm prior to loading (time 1; 120 swabs per flock) and after transport and holding at the abattoir (time 2; 120 swabs per flock). A statistically significant increase in the overall prevalence of Campylobacter spp. was observed for cloacal swabs obtained from farm 3 following transport (P < 0.01). At time 2, an increase in the prevalence of C. coli was also noted for cloacal swabs from farms 3, 4, and 5 (P < 0.01). Neither the minimum time off of feed nor the distance transported from the farm to the abattoir was correlated with the increase in C. coli prevalence. Similarly, responses to an on-farm management questionnaire failed to detect any factors contributing to the observed changes in Campylobacter sp. prevalence. A SmaI macrorestriction analysis of Campylobacter sp. isolates recovered from flock 5 indicated that C. coli was more diverse than C. jejuni at both time 1 and time 2 (P < 0.01), based on a comparison of the Shannon indices of diversity and evenness.     

Detection of low pathogenic avian influenza viruses in wild ducks from Canada: comparison of two sampling methods

Surveillance for avian influenza viruses in wild birds was initiated in Canada in 2005. In 2006, in order to maximize detection of highly pathogenic avian influenza viruses, the sampling protocol used in Canada's Inter-agency Wild Bird Influenza Survey was changed. Instead of collecting a single cloacal swab, as previously done in 2005, cloacal and oropharyngeal swabs were combined in a single vial at collection. In order to compare the two sampling methods, duplicate samples were collected from 798 wild dabbling ducks (tribe Anatini) in Canada between 24 July and 7 September 2006. Low pathogenic avian influenza viruses were detected significantly more often (P<0.0001) in combined oropharyngeal and cloacal samples (261/798, 33%) than in cloacal swabs alone (205/798, 26%). Compared to traditional single cloacal samples, combined samples improved virus detection at minimal additional cost.     

Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Validation in a Multicenter Collaborative Trial

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (Campylobacter jejuni, C. coli, and C. lari) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.     

Campylobacter contamination in French chicken production from farm to consumers. Use of a PCR assay for detection and identification of Campylobacter jejuni and Camp. coli

AIMS: Campylobacter contamination in French chicken production from the farm to the consumer was determined using a PCR assay for bacteria detection and identification. METHODS AND RESULTS: Samples were bird droppings from poultry houses, neck skins, livers, hearts, gizzards, wings, legs and escalopes from slaughterhouses and gizzards, legs, drumstick, breast and escalopes from a supermarket. Bacterial DNA extraction was performed after an enrichment step in a broth and was followed by PCR. An internal control (IC) was used for both DNA extraction and PCR. Campylobacter were detected in 79.2% of poultry houses. Of the 303 samples, 201 were Campylobacter-positive (i.e. 66.3%) including 43.2% faecal samples, 5.6% slaughterhouse samples and 17.5% supermarket samples. There was no significant difference between the molecular method and the conventional culture technique for Campylobacter detection whatever the samples. The sensitivity was 5 UFC g(-1) of samples and 1.5 x 10(3) UFC ml(-1) of enrichment broth. The use of IC revealed PCR inhibition in 13 samples and problems in the DNA extraction in five samples. CONCLUSION: Significant Campylobacter contamination affects all stages of French chicken production. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Campylobacter contamination at different levels of chicken production and the determination of the best place(s) for intervention are important for significantly decreasing Campylobacteriosis. Our technique is rapid and can be used on different chicken samples for Campylobacter detection and identification.     

实时荧光定量PCR技术在SPF鸡四种垂直传播疾病监测中的应用

目的探讨荧光定量PCR检测技术对SPF鸡四种垂直传播病毒的检测应用。方法采集60份SPF鸡及70份普通鸡群蛋清、泄殖腔试子样品,提取样品核酸,分别进行ARV、REV、CAV、ALV四种病毒实时荧光定量PCR检测,根据标准曲线及溶解曲线分析判读样品病毒拷贝数。结果 SPF鸡ALV 2份阳性,检出率3.3%,其余病毒检测均为阴性;普通鸡样品REV检测2份阳性,检出率2.9%,ALV 10份阳性,检出率14.3%。结论荧光定量PCR检测方法最低可检测到100个拷贝核酸,检测灵敏度较高,有望应用于SPF鸡临床样品的病原检测。     

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.     

Nasal,Oral and Ear Swabs for Canine Visceral Leishmaniasis Diagnosis: New Practical Approaches for Detection of Leishmania infantum DNA

Background

The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil.

Methodology/Principal Findings

Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P = 0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05).

Conclusions

Nasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.     

Swab Sample Transfer for Point-Of-Care Diagnostics: Characterization of Swab Types and Manual Agitation Methods

Background

The global need for disease detection and control has increased effort to engineer point-of-care (POC) tests that are simple, robust, affordable, and non-instrumented. In many POC tests, sample collection involves swabbing the site (e.g., nose, skin), agitating the swab in a fluid to release the sample, and transferring the fluid to a device for analysis. Poor performance in sample transfer can reduce sensitivity and reproducibility.

Methods

In this study, we compared bacterial release efficiency of seven swab types using manual-agitation methods typical of POC devices. Transfer efficiency was measured using quantitative PCR (qPCR) for Staphylococcus aureus under conditions representing a range of sampling scenarios: 1) spiking low-volume samples onto the swab, 2) submerging the swab in excess-volume samples, and 3) swabbing dried sample from a surface.

Results

Excess-volume samples gave the expected recovery for most swabs (based on tip fluid capacity); a polyurethane swab showed enhanced recovery, suggesting an ability to accumulate organisms during sampling. Dry samples led to recovery of ∼20–30% for all swabs tested, suggesting that swab structure and volume is less important when organisms are applied to the outer swab surface. Low-volume samples led to the widest range of transfer efficiencies between swab types. Rayon swabs (63 µL capacity) performed well for excess-volume samples, but showed poor recovery for low-volume samples. Nylon (100 µL) and polyester swabs (27 µL) showed intermediate recovery for low-volume and excess-volume samples. Polyurethane swabs (16 µL) showed excellent recovery for all sample types. This work demonstrates that swab transfer efficiency can be affected by swab material, structure, and fluid capacity and details of the sample. Results and quantitative analysis methods from this study will assist POC assay developers in selecting appropriate swab types and transfer methods.     

Longitudinal study of the persistence of antimicrobial-resistant campylobacter strains in distinct Swine production systems on farms, at slaughter, and in the environment

The objectives of this study were to compare and characterize the prevalence of antimicrobial-resistant (AR) Campylobacter in conventional and antimicrobial-free (ABF) production systems on farms, at slaughter, and in the environment. Fecal and environmental samples were collected from ABF farms (pigs, 1,239; environment, 797) and conventional farms (pigs, 1,650; environment, 1,325). At slaughter, we collected samples from carcasses, including postevisceration swabs, postchill swabs, and mesenteric lymph nodes from ABF systems (postevisceration swabs, 182; postchill swabs, 199; mesenteric lymph nodes, 184) and conventional systems (postevisceration swabs, 272; postchill swabs, 271; mesenteric lymph nodes, 255) at separate processing facilities. We also sampled the processing plant environment, including truck and lairage floor swab samples (ABF, 115; conventional, 90). Overall, a total of 2,908 Campylobacter isolates, including Campylobacter coli (farm, 2,557, 99.8%; slaughter, 341, 98.3%) and Campylobacter jejuni (farm, 4, 0.2%; slaughter, 6, 1.7%), were isolated in the study. There was no significant difference in the prevalence of Campylobacter between ABF and conventionally raised pigs (farrowing, P = 0.20; nursery, P = 0.06; finishing, P = 0.24) and the environment (P = 0.37). At slaughter, Campylobacter was isolated from all of the stages, including postchill. The highest frequencies of resistance were exhibited against tetracycline (ABF, 48.2%; conventional, 88.3%). Ciprofloxacin-resistant C. coli isolates were observed in conventionally raised (17.1%) and ABF (1.2%) pigs (P = 0.11). Antimicrobial use data from conventional farms indicated significant associations between oxytetracycline use and tetracycline resistance in the nursery pigs (P = 0.01), between tiamulin exposure and azithromycin and erythromycin resistance in nursery (P < 0.01) and finishing (P < 0.01) pigs, and between enrofloxacin exposure and ciprofloxacin and nalidixic acid resistance in farrowing (P < 0.01) and nursery (P < 0.01) pigs. Identical antimicrobial resistance profiles were observed in the pigs and their environments on farms and at slaughter. In summary, our results highlight the persistence and dissemination of AR Campylobacter from farm to slaughter in ABF and conventionally raised pigs and their environments.     

Validation of a PCR-Based Method for Detection of Food-Borne Thermotolerant Campylobacters in a Multicenter Collaborative Trial

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.     

Validation of a PCR-based method for detection of food-borne thermotolerant campylobacters in a multicenter collaborative trial

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.