ABSTRACT:

In order to detect and identify Campylobacter spp. in broiler chicken carcasses, and to compare detection methods, 43 chilled and 43 frozen carcasses were collected and analyzed. Three methodologies were evaluated: an automated Enzyme Linked Fluorescent Assay (ELFA) VIDAS^®30, Polymerase Chain Reaction (PCR) and real-time PCR. Only four chilled carcasses (4.6%) were considered positive for Campylobacter spp. by VIDAS^®30 and no sample was positive when the conventional PCR technique was used. However, real-time PCR showed a higher incidence of contamination by Campylobacter spp. in broiler carcasses, with 45 (52.3%) positive samples. C. jejuni was the species most frequently reported in the samples (88.8%). No differences in the frequencies of Campylobacter spp. were observed between the chilled and frozen broiler carcasses. In conclusion, real-time PCR was the most sensitive method for the detection of Campylobacter spp. in chilled or frozen broiler carcasses, which were mainly contaminated by C. jejuni.

Key words:
C. jejuni; C. coli; detection methods; VIDAS^®30.

RESUMO:

Com o objetivo de detectar e identificar Campylobacter spp. em carcaças de frango de corte utilizando três metodologias distintas - ensaio imunoenzimático VIDAS^®30, Reação em Cadeia da Polimerase (PCR) e PCR em tempo real - foram coletadas e analisadas 43 carcaças de frango resfriadas e 43 congeladas. Quatro carcaças refrigeradas (4,6%) foram consideradas positivas para Campylobacter spp. pelo VIDAS®30 e nenhuma amostra positiva foi identificada quando utilizada a técnica de PCR. Porém, ao analisar as carcaças pela metodologia da PCR em tempo real, foi observada uma maior incidência de Campylobacter spp., com 45 amostras (52,3%) positivas, sendo que Campylobacter jejuni foi a espécie mais frequentemente encontrada nas amostras (88,8%). Não foi observada diferença na frequência do micro-organismo entre carcaças de frangos resfriadas e congeladas. Concluiu-se que a técnica de PCR em tempo real apresentou maior sensibilidade na detecção de Campylobacter spp. em carcaças de frangos de corte e que foi encontrada elevada presença de carcaças contaminadas, especialmente por C. jejuni.

Palavras-chave:
C. jejuni; C. coli; métodos de detecção; VIDAS^®30.

INTRODUCTION:

Campylobacteriosis is a foodborne disease associated with human infection by thermophilic Campylobacter spp., mainly C. jejuni and C. coli (BUTZLER, 2004BUTZLER, J.P. Campylobacter, from obscurity to celebrity. Clinical Microbiology and Infectious, v.10, p. 868-876, 2004. Available from: http://dx.doi.org/10.1111/j.1469-0691.2004.00983.x>. Accessed:Jul. 08, 2016. doi: 10.1111/j.1469-0691.2004.00983.x.
http://dx.doi.org/10.1111/j.1469-0691.20... ). These bacteria are some of the most widespread causative agents of human gastroenteritis, with many animals, wild and domestic, serving as potential reservoirs (CDC, 2014CDC. 2014. Campylobacter. Atlanta.Available from: http://www.cdc.gov/foodsafety/diseases/campylobacter/index.html>.Accessed: Jul. 08, 2016. Epub 03-Jun-2014.
http://www.cdc.gov/foodsafety/diseases/c... ). Birds, especially broiler chickens, are the main natural reservoir of Campylobacter spp., and there by consumption and handling of poultry and poultry products have been implicated as the main source of infection to humans (HUE et al., 2010HUE, O. et al. Prevalence of and risk factors for Campylobacter spp. contamination of broiler chicken carcasses at the slaughterhouse. Food Microbiology. v.27, p.992-999, 2010. Available from: http://dx.doi.org/10.1016/j.fm.2010.06.004>. Accessed:Jul. 08, 2016. doi: 10.1016/j.fm.2010.06.004.
http://dx.doi.org/10.1016/j.fm.2010.06.0... , EFSA, 2011EFSA - European Food Safety Authority. Scientific opinion on Campylobacter in broiler meat production: control options and performance objectives and/or targets at different stages of the food chain. EFSA Journal. v.9, p. 1-141. 2011. Available from: https://www.efsa.europa.eu/en/efsajournal/pub/2105>. Accessed: May. 25, 2017. doi:10.2903/j.efsa.2011.2105.
https://www.efsa.europa.eu/en/efsajourna... , Oliveira et al., 2013OLIVEIRA, A.L., OLIVEIRA, R.B.P. Enumeration of Campylobacter spp. and presence of Campylobacter jejuni in broiler carcasses in the state of Minas Gerais, Brazil.Ciência Rural. v.43, p.480-484, 2013. Available from: http://dx.doi.org/10.1590/S0103-84782013005000007>. Accessed: Jan. 05, 2016. doi:10.1590/S0103-84782013005000007.
http://dx.doi.org/10.1590/S0103-84782013... ). The species that are pathogenic towards humans have a narrow temperature range for growth (30-46°C) and cannot survive the heating processes used for food preparation (Humphrey et al., 2007HUMPHREY, T. et al. Campylobacters as zoonotic pathogens: a food production perspective. International Journal of Food Microbiology. v.117, p.237-257, 2007. Available from: https://doi.org/10.1016/j.ijfoodmicro.2007.01.006>. Accessed:Jul. 08, 2016. doi:10.1016/j.ijfoodmicro.2007.01.006.
https://doi.org/10.1016/j.ijfoodmicro.20... ).

The detection of Campylobacter spp. in food samples is generally performed using traditional culture-based methods, immunoenzymatic assays, or Polymerase Chain Reaction (PCR)-based methods. The traditional culture-based methods for the isolation, identification, and differentiation of Campylobacter species are time-consuming and challenging because of the fastidious nature of the microorganisms, which have complex nutritional and environmental requirements (BARROS-VELAZQUEZ et al., 1999BARROS-VELAZQUEZ, J. et al. Isolation and typing methods for the epidemiologic investigation of thermotolerant campylobacters. International Microbiology, v.2, p.217-226, 1999. Available from: http://www.im.microbios.org/08december99/04%20Barros.pdf>. Accessed: Jul. 25, 2016.
http://www.im.microbios.org/08december99... ; FITZGERALD et al., 2008FITZGERALD, C. et al. Diagnosis and antimicrobial susceptibility of Campylobacter species. In: Nachamkin, I.; Szymanski, C.M. Campylobacter. Washington, DC.: ASM Press, 2008. p.227-243.Available from: http://www.asmscience.org/content/book/10.1128/9781555815554.ch12>. Accessed: Jul. 08, 2016.doi: 10.1128/9781555815554.
http://www.asmscience.org/content/book/1... ). The sandwich Enzyme-Linked Immunosorbent Assay (ELISA), which is based on the detection of an antigenic reaction against the bacteria after their cultivation on selective media, is often used for the analysis of Campylobacter spp. in food.This assay is faster, more sensitive, and more specific than the traditional culture-based methods. The Enzyme Linked Fluorescent Assay (ELFA) employs a similar principle featuring a final detection step by fluorescence and is used in the VIDAS^® automated immunoassay system (bioMérieux, Marcy-l’Étoile, France) (AOAC, 2015AOAC Performance Tested, Certificate No. 051201, 2015. Vidas®Campylobacter (CAM). Acessado em 15 feb. 2016. Available from: http://www.aoac.org/imis15_prod/AOAC_Docs/RI/16PTM/16C_051201_BMC[gwutxn].pdf.>. Accessed: Jul. 25, 2016.
http://www.aoac.org/imis15_prod/AOAC_Doc... ). Methods based on PCR and real-time PCR have also been used for the detection of Campylobacter spp. in food samples due to their high specificity and sensitivity. In addition to their great accuracy, PCR-based methods are also faster and more easily performed than the other techniques for detecting Campylobacter spp. (SCHNIDER et al., 2010SCHNIDER, A. et al. Comparison of real-time PCR assays for detection, quantification, and differentiation of Campylobacter jejuni and Campylo bactercoli in broiler neck skin samples. Journal of Food Protection. v.73, p.1057-1063, 2010. Available from: http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-73.6.1057?code=fopr-site>. Accessed:Fev. 22,2016.
http://jfoodprotection.org/doi/pdf/10.43... ).

Among the control measures that have been recommended to prevent the growth of Campylobacter spp. in broiler carcasses, cold treatment is one of the most important. Several studies have reported a significant reduction in the percentage of carcasses contaminated by C. jejuni after freezing (EL-SHIBINY et al., 2009EL-SHIBINY, A. et al. Survival at refrigeration and freezing temperatures of Campylobacter coli and Campylobacter jejuni on chicken skin applied as axenic and mixed inoculums. The International Journal of Food Microbiology, v.131, p.197-202, 2009. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2009.02.024>. Accessed:Jul. 08, 2016. doi: 10.1016/j.ijfoodmicro.2009.02.024.
http://dx.doi.org/10.1016/j.ijfoodmicro.... ; SAMPERS et al., 2010SAMPERS, I. et al. Survival of Campylobacter spp. in poultry meat preparations subjected to freezing, refrigeration, minor salt concentration, and heat treatment. International Journal of Food Microbiology. v.137, p.147-153, 2010. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2009.11.013>. Accessed:Fev. 05, 2015. doi: 10.1016/j.ijfoodmicro.2009.11.013.
http://dx.doi.org/10.1016/j.ijfoodmicro.... ). However, the effects of cooling and freezing on the survival of Campylobacter spp. in broiler carcasses have not been completely elucidated.

Therefore, the aims of the present study were to detect and identify Campylobacter spp. in chilled and frozen broiler carcasses, and to compare the performance of three detection methods, namely the VIDAS^®30 immunoassay method, PCR, and real-time PCR.

MATERIAL AND METHODS:

Samples and experimental conditions

Eighty-six broiler carcasses from 43 different production batches were collected in a slaughterhouse industry under the Federal Inspection Service located in Minas Gerais State, Brazil. From each of the 43 batches, one frozen (n = 43) and one chilled (n = 43) broiler carcass were collected and analyzed on the second day after processing. Hence, each carcass sample represented one batch or one repetition.

Microbiological analyses

VIDAS^®30 immunoenzymatic assay

The detection of Campylobacter spp. in the broiler carcasses was performed using three distinct methodologies, namely an automated ELFA (VIDAS^®30) (AOAC, 2015), PCR (HARMON et al., 1997HARMON, K.M. et al. Differentiation of Campylobacter jejuni and Campylobacter coli by polymerase chain reaction. Molecular and Cellular Probes, v.11, p.195-200, 1997. Available from: http://dx.doi.org/10.1006/mcpr.1997.0104>. Accessed:Jul. 05, 2016. doi: 10.1006/mcpr.1997.0104
http://dx.doi.org/10.1006/mcpr.1997.0104... ; VILARDO et al., 2006VILARDO, M.D.C.B. et al. Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates. Memórias do Instituto Oswaldo Cruz. v.101, p.499-501, 2006. Available from: http://dx.doi.org/10.1590/S0074-02762006000500003>. Accessed:Fev. 10, 2015. doi: 10.1590/S0074-02762006000500003.
http://dx.doi.org/10.1590/S0074-02762006... ), and real-time PCR (SCHNIDER et al., 2010SCHNIDER, A. et al. Comparison of real-time PCR assays for detection, quantification, and differentiation of Campylobacter jejuni and Campylo bactercoli in broiler neck skin samples. Journal of Food Protection. v.73, p.1057-1063, 2010. Available from: http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-73.6.1057?code=fopr-site>. Accessed:Fev. 22,2016.
http://jfoodprotection.org/doi/pdf/10.43... ).

Each carcass was aseptically placed in a sterile bag containing 100mL of Bolton broth with a selective supplement (SR183E, Oxoid, Basingstoke, UK) and was subjected to a washing process by rinse friction for approximately 1min (bioMérieux, 2015). After rinsing, the solution was incubated at 37 ± 0.5°C for 4h and then at 42 ± 0.5°C for 24h under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂) [Combibag with microaerophilia generator GENbox (bioMérieux, Hazelwood, USA)]. After incubation, 2mL aliquots of the Bolton broth were transferred to sterile tubes to perform the immunoassay by VIDAS^®30 (bioMérieux) and 1.5mL aliquots were transferred to centrifuge microtubes for PCR assays.

The immunoassay was performed with the VIDAS^®30 system, using the Campylobacter (CAM) kit (bioMérieux), according to the manufacturer’s instructions. From the aliquot after incubation in Bolton broth, 0.5mL was heated at 100°C for 15min and then transferred to a test well of a reagent strip and inserted into the VIDAS^®30 system. For the confirmation of positive results, a loop from the remaining Bolton broth, that was not heated, was plated on modified charcoal cefoperazone deoxycholate agar (mCCDA) (Oxoid) and incubated at 42 ± 0.5°C for 48h under microaerophilic conditions (bioMérieux, 2015). Subsequently, colonies with growth typical of Campylobacter spp. were selected and plated on blood agar and incubated at 42 ± 0.5°C for 24h. Then, they were transferred to a Neisseria-Haemophilus (NH) card for biochemical tests using the VITEK 2^® system (bioMérieux) (VALENZA et al., 2007VALENZA, G. et al. Microbiological Evaluation of the New VITEK 2Neisseria-Haemophilus Identification Card. Journal of Clinical Microbiology. v.45, p.3493-3497,2007. Available from: https://doi.org/10.1128/JCM.00953-07>. Accessed: Fev. 05. 2015. doi: 10.1128/JCM.00953-07.
https://doi.org/10.1128/JCM.00953-07... ).

PCR analyses

For Campylobacter spp. determination by PCR, the microtubes containing 1.5mL of inoculated Bolton broth were centrifuged at 20,000 ×g at room temperature and submitted to DNA extraction (PITCHER et al., 1989PITCHER, D.G. et al. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in Applied Microbiology. v.8. p.151-156, 1989. Available from: http://dx.doi.org/10.1111/j.1472-765X.1989.tb00262.x>. Accessed:Fev. 05, 2015. doi: 10.1111/j.1472-765X.1989.tb00262.x.
http://dx.doi.org/10.1111/j.1472-765X.19... ). DNA purity and concentration were determined by spectrophotometry (GeneQuant™ 100 Classic, GE Healthcare Life Sciences, Little Chalfont, UK)(SAMBROOK & RUSSELL, 2001SAMBROOK, J., RUSSELL. D.W. Molecular Cloning: A Laboratory Manual. New York : Cold Spring Harbor Laboratory Press, 2001. ).

In the multiplex PCR assays, the primers pg3 (5′- GAACTTGAACCGATTTG-3′) and pg50 (5′-ATGGGATTTCGTATTAAC-3′) were used for the amplification of the flaA gene (460 bp), which is present in Campylobacter species, especially C. jejuni and C. coli. Additionally, the primers C1 (5′-CAAATAAAGTTAGAGGTAGAATGT-3′) and C4 (5′-GGATAAGCACTAGCTAGCTGAT-3′) were used for the amplification of a non-determined gene of C. jejuni (160 bp), according to HARMON et al. (1997HARMON, K.M. et al. Differentiation of Campylobacter jejuni and Campylobacter coli by polymerase chain reaction. Molecular and Cellular Probes, v.11, p.195-200, 1997. Available from: http://dx.doi.org/10.1006/mcpr.1997.0104>. Accessed:Jul. 05, 2016. doi: 10.1006/mcpr.1997.0104
http://dx.doi.org/10.1006/mcpr.1997.0104... ) and VILARDO et al. (2006VILARDO, M.D.C.B. et al. Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates. Memórias do Instituto Oswaldo Cruz. v.101, p.499-501, 2006. Available from: http://dx.doi.org/10.1590/S0074-02762006000500003>. Accessed:Fev. 10, 2015. doi: 10.1590/S0074-02762006000500003.
http://dx.doi.org/10.1590/S0074-02762006... ) (Table 1). The reactions were performed in a total volume of 25µL containing 20ng of DNA, 1× buffer (10mM Tris-HCl, 50mM KCl) (Phoneutria, Belo Horizonte, MG, Brazil), 5.5mM of MgCl₂ (Phoneutria), 200µM of each deoxyribonucleotide (dATP, dCTP, dGTP, and dTTP), 1.0µM of each primer (Eurofins Scientific, Luxembourg), and 1.25 units of Taq DNA polymerase (Phoneutria). The thermocycling conditions (Veriti^® Thermal Cycler, Thermo Fisher Scientific, Waltham, MA, USA) were as follows: initial denaturation step at 94°C for 4min followed by 25 cycles of denaturation at 94°C for 1min, primer annealing at 45°C for 1min, and elongation at 72°C for 1min. The final extension step was performed at 72°C for 7min. The amplified products were submitted to electrophoresis in a 1.5% agarose gel in Tris-borate-EDTA (TBE) buffer (1.0M Tris, 0.01M boric acid, 0.01M EDTA, pH 8.2) and stained with 0.5mg mL^-1 ethidium bromide. Products were visualized under ultraviolet light and photographed (L-Pix EX, Loccus Biotecnologia, Cotia, SP, Brazil). A 100-bp DNA size marker (Life Technologies, Carlsbad, CA, USA) was used to estimate amplicons length.

Real-time PCR analysis

Real-time PCR assays were performed using the primers Ccj FusA-L1 (5′-GCCTTGAAGAGATTAAAACAGGGATT-3′) and Ccj FusA-R1 (5′-TTTAAACGCTGTACCGCAAAGCA-3′) to amplify the fusA gene of C. jejuni (83 bp). In addition, the primers Ccc FusA-L2 (5′-GCCTTGAGGAAATTAAAACTGGTATT-3′) and Ccc FusA-L2 (5′-TTTAAATGCAGTTCCACAAAGCA-3′) were used to amplify the fusA gene of C. coli(83 bp).The probes used were Cj_fusA-probea (FAM- AAGTCTTTCTATCGTTCC -MGBNFQ) for C. jejuni and Cc_fusA-probea (NED- AAGTCTTTCTATTGTTCC -MGBNFQ) for C. coli (SCHNIDER et al., 2010SCHNIDER, A. et al. Comparison of real-time PCR assays for detection, quantification, and differentiation of Campylobacter jejuni and Campylo bactercoli in broiler neck skin samples. Journal of Food Protection. v.73, p.1057-1063, 2010. Available from: http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-73.6.1057?code=fopr-site>. Accessed:Fev. 22,2016.
http://jfoodprotection.org/doi/pdf/10.43... ) (Table 1). Real-time PCR assays were performed with the two primer pairs and probes simultaneously, in triplicate. Reactions were performed in a total volume of 25µL containing 1× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 0.3µM of each primer, 0.2µM of each probe, and 2µL of genomic DNA. Amplification was carried out at 50°C for 2min and 95°C for 10min, followed by 40 cycles of 95°C for 15s and 60°C for 1min using anABI 7500 Thermal Cycler Real-Time PCR System (Applied Biosystems). The result was considered positive when at least two of the triplicate samples were defined as positive, with a threshold cycle (Ct) value less than 40.

Limits of detection of the PCR and real-time PCR assays

To assess the limits of detection of the PCR and real-time PCR assays, two standard curves were generated. One standard curve was performed with serial dilutions of DNA from C. jejuni and C. coli in ultrapure water. To obtain the other standard curve, DNA from C. jejuni and C. coli were diluted in Bolton broth that had been used to rinse a broiler carcass. The DNA of C. jejuni and C. coli were subjected to serial tenfold dilutions from 20ng to 2fg, corresponding to 10⁷ to 10⁰ genome copies of Campylobacter spp., respectively (GenBank accession numbers: C. jejuni, CP000814 and C. coli, CP006702) (DOLEZEL et al., 2003DOLEZEL J. et al. Nuclear DNA content and genome size of trout and human. Cytometry A, v. 51, p. 127-128, 2003. Available from: http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.10013/epdf>. Accessed:Jul. 10, 2016. doi: 10.1002/cyto.a.10013.
http://onlinelibrary.wiley.com/doi/10.10... ). Reference strains, which were used as controls in the PCR and real-time PCR assays, were C. jejuni (NCTC 11351) and C. coli (NCTC 11366). These reference strains were cultivated in blood agar at 42 ± 0.5°C for 48h under microaerophilic conditions.

Statistical analysis

The experiments were conducted using a completely randomized 2 × 3 factorial design, including two types of broiler carcasses (chilled and frozen) and three analytical methodologies (immunoenzymatic VIDAS^®30, PCR, and real-time PCR) with 43 repetitions each. Comparisons among the analytical methodologies were performed by the McNemar test and the analyses of chilled and frozen samples were performed by the Fisher test at the 5% significance level. Statistical analysis was performed using R Statistical Software version 3.1.2 (R Foundation for Statistical Computing, Vienna, Austria).

RESULTS AND DISCUSSION:

Limits of detection of the PCR and real-time PCR assays

In the present study was observed that real-time PCR proved to be more sensitive than conventional PCR to detect Campylobacter spp. in broiler carcasses (Table 2). Conventional PCR results using the primers designed by HARMON et al. (1997HARMON, K.M. et al. Differentiation of Campylobacter jejuni and Campylobacter coli by polymerase chain reaction. Molecular and Cellular Probes, v.11, p.195-200, 1997. Available from: http://dx.doi.org/10.1006/mcpr.1997.0104>. Accessed:Jul. 05, 2016. doi: 10.1006/mcpr.1997.0104
http://dx.doi.org/10.1006/mcpr.1997.0104... ) and VILARDO et al. (2006VILARDO, M.D.C.B. et al. Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates. Memórias do Instituto Oswaldo Cruz. v.101, p.499-501, 2006. Available from: http://dx.doi.org/10.1590/S0074-02762006000500003>. Accessed:Fev. 10, 2015. doi: 10.1590/S0074-02762006000500003.
http://dx.doi.org/10.1590/S0074-02762006... ) showed that those primer pairs were unsuitable for use with food samples. They were designed for the detection of C. jejuni and C. coli from cultured bacteria using a large amount of microorganisms per sample (HARMON et al. 1997, VILARDO et al., 2006). Several substances present in food and clinical materials, such as broiler carcasses, can inhibit or significantly decrease the efficiency of the PCR reactions (SCHRADER et al., 2012SCHRADER, C. et al. PCR inhibitors - occurrence, properties and removal. Journal of Applied Microbiology. v.113, p.1014-1026, 2012. Available from: https://doi.org/10.1111/j.1365-2672.2012.05384.x>. Accessed: Jul. 10, 2016. doi: 10.1111/j.1365-2672.2012.05384.x.
https://doi.org/10.1111/j.1365-2672.2012... ). Moreover, it is important to consider that besides the presence of inhibitors, the amount of Campylobacter spp. usually present in contaminated food is relatively low, owing to their high sensitivity to atmospheric oxygen concentrations and the low temperatures typically used for food preservation (BARROS-VELAZQUEZ et al., 1999BARROS-VELAZQUEZ, J. et al. Isolation and typing methods for the epidemiologic investigation of thermotolerant campylobacters. International Microbiology, v.2, p.217-226, 1999. Available from: http://www.im.microbios.org/08december99/04%20Barros.pdf>. Accessed: Jul. 25, 2016.
http://www.im.microbios.org/08december99... ).

Detection of Campylobacter spp. using VIDAS^®30, PCR, and real-time PCR assays

The detection of Campylobacter spp. in chicken skin is important for assessing the incidence of contamination among the broiler carcasses that reach the market. Table 3 summarizes results of Campylobacter spp. detection in chilled and frozen broiler carcasses. Among the total chilled carcasses, only four (9.3%) samples were considered positive for Campylobacter spp. by the VIDAS^®30 assay and no sample was considered positive when the conventional PCR technique was used. However, results of the real-time PCR showed a higher incidence of Campylobacter spp. in broiler carcasses, with 45 (52.3%) samples being considered positive, being 24 (55.8%) from chilled and 21 (48.8%) from frozen carcasses. Hence, the results of the present study highlighted the potential public health risks linked to the consumption of chicken and the usefulness of the real-time PCR assay for the detection of Campylobacter spp. in broiler carcasses.

Detection of C. jejuni and C. coli by real-time PCR

The real-time PCR assay allowed differentiation between C. jejuni and C. coli and showed that C. jejuni was the most frequently detected Campylobacter species reported in the tested samples (Table 4). C. jejuni was detected in 40 (88.8%) of the 45 positive samples, while C. coli was found in 15 (33.3%) samples. C. jejuni and C. coli were simultaneously reported in ten (22.2%) of the 45 positive broiler carcasses. Among the chilled carcasses that were positive for Campylobacter spp., C. jejuni was detected in 20 carcasses (83.3%) and C. coli was detected in 9 carcasses (37.5%). C. jejuni and C. coli. were simultaneously detected in five chilled carcasses (20.8%). Among the frozen carcasses that were positive for Campylobacter spp., C. jejuni was detected in 20 carcasses (95.2%) and C. coli was reported in six carcasses (28.5%). Both C. jejuni and C. coli were simultaneously detected in five frozen samples (23.8%). It is noteworthy that, usually, 95% of human cases of campylobacteriosis are related to infection by C. jejuni, while C. coli accounts for 4% of cases and C. lari accounts for about 1% (NACHAMKIN et al., 2008NACHAMKIN, I. et al. Campylobacter.Washington, DC: ASM Press, 2008. 3th ed. 716p. Available from: http://www.asmscience.org/content/book/10.1128/9781555815554>. Accessed: Fev. 22,2016. doi: 10.1128/9781555815554.
http://www.asmscience.org/content/book/1... ).

Comparison among the methods used for Campylobacter spp. Detection

A comparison among the three methods used for Campylobacter spp. detection in broiler carcasses showed a significant difference (P<0.001) between real-time PCR and the other studied techniques (Table 3). The VIDAS^®30 immunoenzymatic assay did not present superior results to those of conventional PCR for the detection of Campylobacter spp. in broiler carcasses. The low bacterial counts in the carcasses could have influenced the performance of the VIDAS^®30 assay in this study, since only four chilled carcasses were found to be positive for contamination when using this technique. These results may suggest that the VIDAS^®30 immunoenzymatic assay was only able to detect contamination in carcasses with higher loads of microorganisms. Thus, comparing results ofthe VIDAS^®30 assay with those of real-time PCR, the lower incidence of Campylobacter spp. detected by the former method may suggest that the bacterial counts in the tested broiler carcasses were below the limit of detection (> 10³ CFU mL^-1) (SAHIN et al., 2008) or that viable but non culturable bacteria predominated in the samples. Low incidences of the contamination of broiler carcasses by Campylobacter spp. recorded by PCR and VIDAS^®30 immunoenzymatic assay was unexpected, since high rates of contamination has been widely demonstrated in the world and in different Brazilian states (DIAS et al., 1990DIAS, T.C. et al. Chicken carcasses as a source of Campylobacter jejuni in Belo Horizonte, Brazil. Revista do Instituto de Medicina Tropical de São Paulo, v.32, p. 414-418, 1990. Available from: http://dx.doi.org/10.1590/S0036-46651990000600005>. Accessed: Jul. 10, 2016. doi: 10.1590/S0036-46651990000600005.
http://dx.doi.org/10.1590/S0036-46651990... ; FREITAS & NORONHA, 2007FREITAS, J.A.; NORONHA, G.N. Occurence ofCampylobacterspp. in chicken meat and giblets exposed to consumption in Northern Brazil. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.59, p.813-815, 2007. Available from: dx.doi.org/10.1590/S0102-09352007000300038>. Accessed: Jun. 12, 2016. doi: 10.1590/S0102-09352007000300038.
dx.doi.org/10.1590/S0102-093520070003000... ; KAAKOUSH et al., 2015KAAKOUSH, N.O. et al. Global Epidemiology of Campylobacter Infection. Clinical Microbiology Reviews. v.28, p.687-720, 2015. Available from: http://cmr.asm.org/content/28/3/687.full>. Accessed:Jul. 08, 2016. doi: 10.1128/CMR.00006-15.
http://cmr.asm.org/content/28/3/687.full... ).

Findings from real-time PCR showed a completely different situation, i.e., that a high incidence (52.3%) of broiler carcasses were contaminated by Campylobacter spp.. These results most probably reflect the high analytical sensitivity of the method, which has a limit of detection of 10² cells mL^-1. Lower detection limit of real-time PCR as compared to those of conventional PCR (10⁴-10⁶ cells mL^-1) and the VIDAS^®30 assay (> 10³ CFU mL^-1) (SAHIN et al., 2003SAHIN, O. et al. Detection of Campylobacter . In: Torrence, M.E.; Isaacson, R.E. Microbial Food Safety in Animal Agriculture: Current Topics. New Jersey: Wiley Online Library. 2003. Cap. 20, p.183-194. Available from: http://onlinelibrary.wiley.com/book/10.1002/9780470752616>. Accessed:Jul. 08, 2016.doi: 10.1002/9780470752616.
http://onlinelibrary.wiley.com/book/10.1... ) presumably enabled the detection of Campylobacter spp. in samples in which the concentration of microorganisms was relatively low, which is the typical situation in food samples. It should be noted that amplification by real-time PCR was observed even for low concentrations of Campylobacter spp. DNA (20fg, equivalent to 10cells mL⁻¹). Moreover, the Bolton broth showed no inhibitory effects during the assessment of the detection limit of the real-time PCR assays. However, since the efficiency of the reaction with this lowest tested concentration of DNA was substantially lower than that observed for the other tested concentrations, these results were disregarded when interpreting the limit of detection. It is also important to consider that very low quantities of C. jejuni, around 8.0× 10² CFU mL^-1, are sufficient to cause infection and diarrhea in humans (BLACK et al., 1988BLACK, R.E. et al. Experimental Campylobacter jejuni infection in humans. The Journal of Infectious Diseases, v.157, p.472-479, 1988.Available from: https://academic.oup.com/jid/article-abstract/157/3/472/859012?redirectedFrom=PDF>. Accessed: Jul. 08, 2016.doi: 10.1093/infdis/157.3.472.
https://academic.oup.com/jid/article-abs... ).

Regarding the temperature treatment of broiler carcass, it is interesting to observe that despite the higher frequency of carcasses contaminated by Campylobacter spp. among chilled samples compared to frozen ones (Table 3), this difference was not significant (P>0.05). The fact that real-time PCR detects the genetic material of the microorganism, even if it is no longer viable, could be the reason for the absence of a difference between these storage conditions, since Campylobacter spp. is usually more sensitive to freezing than refrigeration (EL-SHIBINY et al., 2009EL-SHIBINY, A. et al. Survival at refrigeration and freezing temperatures of Campylobacter coli and Campylobacter jejuni on chicken skin applied as axenic and mixed inoculums. The International Journal of Food Microbiology, v.131, p.197-202, 2009. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2009.02.024>. Accessed:Jul. 08, 2016. doi: 10.1016/j.ijfoodmicro.2009.02.024.
http://dx.doi.org/10.1016/j.ijfoodmicro.... ; SAMPERS et al., 2010SAMPERS, I. et al. Survival of Campylobacter spp. in poultry meat preparations subjected to freezing, refrigeration, minor salt concentration, and heat treatment. International Journal of Food Microbiology. v.137, p.147-153, 2010. Available from: http://dx.doi.org/10.1016/j.ijfoodmicro.2009.11.013>. Accessed:Fev. 05, 2015. doi: 10.1016/j.ijfoodmicro.2009.11.013.
http://dx.doi.org/10.1016/j.ijfoodmicro.... ). It is possible to speculate that if the immunoenzymatic technique used had shown a higher analytical sensitivity, or if we had incorporated conventional methods for the isolation of Campylobacter spp., a significantly higher proportion of positive samples among chilled carcasses would have been observed. Therefore, although the real-time PCR assay detected a high level of Campylobacter spp. contamination among broiler carcasses, it is not possible to make statements about the viability of this microorganism. Thus, it is not possible to assess the real risk to public health using real-time PCR alone. Alternative methods for the detection of viable Campylobacter spp. cells have been researched. Combination of real-time PCR with propidium monoazide was tested by Josefsen et al. (2010) and provided a fast and reliable methodology for detection and quantification of viable Campylobacter bacteria in broiler carcasses.

CONCLUSION:

Real-time PCR was more sensitive than conventional PCR and the VIDAS^®30 immunoassay for the detection of Campylobacter spp. in broiler carcasses. The level of Campylobacter spp. contamination among chilled or frozen broiler carcasses was high, especially for C. jejuni.

ACKNOWLEDGMENTS

The authors acknowledge the assistance of the Colegiado de Pós-Graduação em Ciência Animal of the Escola de Veterinária at the Universidade Federal de Minas Gerais - UFMG, the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors also thank the Instituto Mineiro de Agropecuária (IMA) for assisting with the analyses.

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