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Campylobacter in broiler slaughter samples assessed by direct count on mCCDA and Campy-Cefex agar

ABSTRACT

Campylobacter spp. cause foodborne illnesses in humans primarily through the consumption of contaminated chicken. The aim of this study was to evaluate the United States Department of Agriculture's (USDA) recommended methodology, protocol MLG 41.02, for the isolation, identification and direct plate counting of Campylobacter jejuni and C. coli samples from the broiler slaughtering process. A plating method using both mCCDA and Campy-Cefex agars is recommended to recover Campylobacter cells. It is also possible to use this method in different matrices (cloacal swabs and water samples). Cloacal swabs, samples from pre-chiller and post-chiller carcasses and samples of pre-chiller, chiller and direct supply water were collected each week for four weeks from the same flock at a slaughterhouse located in an abattoir in southern Brazil. Samples were analyzed to directly count Campylobacter spp., and the results showed a high frequency of Campylobacter spp. on Campy-Cefex agar. For the isolated species, 72% were identified as Campylobacter jejuni and 38% as Campylobacter coli. It was possible to count Campylobacter jejuni and Campylobacter coli from different samples, including the water supply samples, using the two-agar method. These results suggest that slaughterhouses can use direct counting methods with both agars and different matrices as a monitoring tool to assess the presence of Campylobacter bacteria in their products.

Keywords:
Campylobacter; Agar plate count; mCCDA; Campy-Cefex; Broiler

Introduction

Campylobacter bacteria are a major cause of foodborne illness in humans and are the most common gastroenteritis-causing bacteria in the world. In both developed and developing countries, they cause more cases of gastroenteritis than does foodborne Salmonella. The high incidence of Campylobacter diarrhea and its duration and possible sequelae make it highly important from a socio-economic perspective. In developing countries, Campylobacter infections in children under the age of two are particularly frequent and occasionally result in death.11 WHO. World Health Organization; 2014. Available at: http://www.who.int/mediacentre/factsheets/fs255/en/ [Accessed: 24.11.2014].
http://www.who.int/mediacentre/factsheet...

In the USA, Campylobacter is the second most isolated agent of foodborne illness,22 CDC. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food - 10 States. Morbidity and Mortality Weekly Report, v. 19, n. 14, 2010, p. 418-422. Available at: http://www.cdc.gov/mmwr/pdf/wk/mm5914.pdf. [Accessed 15.07.2014].
http://www.cdc.gov/mmwr/pdf/wk/mm5914.pd...
and in the European Union (EU), Campylobacter is the main pathogen that causes human gastroenteritis,33 EFSA. European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter on broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J. 2009;8(3):1503. with approximately 198,252 cases in 2009 alone.44 EFSA. European Food Safety Authority. Reducing Campylobacter in EU chickens; 2011. Available at: http://www.foodprocessing.com.au/articles/46692-Reducing-Campylobacter-in-EU-chickens [Accessed 10.03.2014].
http://www.foodprocessing.com.au/article...

Campylobacter bacteria can be spread by contaminated food and water, and chicken was implicated as the main contamination source.55 Wagenaar JA, Mevius DJ, Havelaar AH. Campylobacter in primary animal production and control strategies to reduce the burden of human campylobacteriosis. Rev Sci Tech Off Int Epiz. 2006;25(2):581-594. During the poultry slaughtering process, carcass contamination can occur not only by high bacterial loads present in the poultry's gastrointestinal tract but also by the bacterial loads present in skin and feathers.66 Boufleur R. Campylobacter jejuni em Frangos de Corte, Carne e Vísceras de Frango no Rio Grande do Sul e Efeito do Congelamento sobre a Contaminação nos Cortes. Santa Maria, RS, Brasil; 2009, 47p. (M.Sc. Dissertation. Centro de Ciências Rurais. UFSM). In addition to initial flock contamination, hygienic and sanitary conditions during both the slaughtering process and carcass conservation can influence the presence and level of Campylobacter in the final product.77 Fonseca BB. Transmissão vertical de Campylobacter sp em um sistema de produção avícola. Uberlândia, MG, Brasil; 2006. 80p. (M.Sc. Dissertation. Universidade Federal de Uberlândia).

Broiler carcasses contaminated with Campylobacter have been detected in many countries. A study conducted in Brazil88 Kuana SL, Santos LR, Rodrigues LB, et al. Occurrence and characterization of Campylobacter in the Brazilian production and processing of broilers. Avian Diseases. 2008;52(4):680-684. showed that 95 of the 96 broiler carcasses examined tested positive for Campylobacter at the end of the slaughter line. According to the World Health Organization,99 FAO/WHO. Technical meeting on Salmonella and Campylobacter in chicken meat; 2009. URL: http://www.who.int/foodsafety/publications/micro/MRA19.pdf.
http://www.who.int/foodsafety/publicatio...
reducing the prevalence or concentration at a specified point in the production chain has the potential to reduce the risk of human incidences if intervention is taken.

Quantitative microbial risk assessment is a well-recognized component of modern risk analysis and is used to estimate the impact of a particular hazard/product combination and/or changes in processing on public health. In this regard, two methods for Campylobacter quantification were published by leading authorities, one by the International Standard Organization, ISO-TS 10272-2,1010 ISO 10272-1:2006. Microbiology of food and animal feeding stuffs - Horizontal method for detection and enumeration of Campylobacter spp. and another by the United States Department of Agriculture, MLG 41.02.1111 USDA. MLG 41.02. 2013. In: isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples; 2014. Available at: http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES. [Accessed 14.07.2014].
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The latter describes a method for direct plating and qualitative and quantitative evaluations using Campy-Cefex agar for the isolation, identification and counting of Campylobacter spp. present in rinsed poultry carcasses, sponges and raw product samples.

The present study aimed to test the MLG 41.021111 USDA. MLG 41.02. 2013. In: isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples; 2014. Available at: http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES. [Accessed 14.07.2014].
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methodology on carcass samples and in alternative matrices, such as cloacal swabs and water samples, from the broiler slaughtering process to compare the number of recovered Campylobacter cells on Campy-Cefex and mCCDA agar plates.

Materials and methods

Sampling

Samples were taken once a week from a slaughterhouse located in southern Brazil for four weeks in April 2013. Each time, three cloacal swabs and three samples from pre-chiller carcasses, post-chiller carcasses, pre chiller water, chiller water and direct supply water were collected, with 12 samples of each type and 72 in total. The samples were placed immediately into thermic boxes with ice and sent to the laboratory for microbiological analysis.

Quantitative and qualitative analysis

Controls

Campylobacter jejuni ATCC 33291 and Campylobacter coli ATCC 43578 were used as positive controls for un-inoculated agar plates and broth sample sets. One colony from each positive control sample was confirmed.

Carcass rinse procedure

Carcass samples were collected prior to placement in the pre-chiller tank on the evisceration line (pre-chiller samples) and immediately after the post-chiller (post-chiller samples).

To perform quantitative analysis on the carcass rinse samples, carcasses were put into sterile plastic bags with 400 mL of 1% BPW (1% Buffered Peptone Water, Oxoid®, Basingstoke, Hampshire, UK) and mixed thoroughly by gently shaking for 3 min. From the rinse solution, 250 µL was streaked onto four Campy-Cefex agar plates with antimicrobial selective supplement (SR0155, Oxoid®, Basingstoke, Hampshire, UK) and 5% sterile laked equine blood (Ebefarma®, Cachoeira de Macacu, RJ, Brazil) using sterile glass Drigalski loops. Another 100 µL from the rinse solution was streaked onto two Campy-Cefex agar plates. The procedure was performed in duplicate with streaking onto mCCDA agar plates containing an antimicrobial selective supplement (SR0155 Oxoid®, Basingstoke, Hampshire, UK). The agar plates were incubated at 42 ± 1.0 °C for 48 ± 2 h under microaerobic conditions (Microerobac, Probac®, São Paulo, SP, Brazil).

To perform qualitative analysis, 30 mL of the rinsing solution was added to 30 mL of double strength blood-free Bolton enrichment broth (2X BF-BEB, Oxoid®, Basingstoke, Hampshire, UK) with selective supplement (SRE183, Oxoid® Basingstoke, Hampshire, UK) and homogenized as described above. After incubation, 10 µL from the BF-BEB was streaked onto Campy-Cefex and mCCDA agar plates and incubated under microaerobic conditions at 42 ± 1.0 °C for 48 ± 2 h.

Cloacal swabs

Swabs were collected during broiler hanging, one swab per bird, and stored in a 10 mL flask with 1% BPW (Oxoid® Basingstoke, Hampshire, UK) at 4 °C. The tubes were homogenized, and the quantitative analysis followed the same methodology as described above for the carcasses. To perform the qualitative analysis, 3 mL from the BPW homogenized solution was added to 30 mL of double strength blood-free Bolton enrichment broth (2X BF-BEB, Oxoid®, Basingstoke, Hampshire, UK) supplemented with a selective supplement (SRE183, Oxoid®, Basingstoke, Hampshire, UK) and analyzed as described above for the carcasses.

Water samples

Water samples from the pre chiller, chiller and direct water supply were collected in 50 mL sterile flasks and stored at 4 °C in the laboratory. The water samples were homogenized and quantitative and qualitative analysis was performed as described above for the carcasses.

Plate analysis and results

After incubation, all typical Campylobacter colonies were counted if the total number of cells fell within a range of 15-300 colonies. The interpretation of typical colonies followed the instructions in the MLG 41.02 protocol.1111 USDA. MLG 41.02. 2013. In: isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples; 2014. Available at: http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES. [Accessed 14.07.2014].
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Briefly, if the four plates with 250 µL were countable, the sum of the counts from the four plates was determined. If the only countable plates were the two plates with 100 µL of sample, the total number of cells on both plates was averaged and multiplied by ten. If both dilutions were within the countable interval, the final count was determined by averaging the results calculated above. All cell count results are represented as CFU/mL. If the final count was >300 CFU in each of the six plates, “TNTC” (to numerous to count) was recorded, or an estimated counting of >2100 CFU/mL was used.1111 USDA. MLG 41.02. 2013. In: isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples; 2014. Available at: http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES. [Accessed 14.07.2014].
http://www.fsis.usda.gov/wps/wcm/connect...

Examination and confirmation of colonies

A typical colony from each sample was picked to confirm and test motility, morphology, oxidase and catalase activity (Probac®, São Paulo, SP, Brazil) and latex agglutination (Dryspot, DR0150, Oxoid®, Basingstoke, Hampshire, UK), as described in MLG 41.02.1111 USDA. MLG 41.02. 2013. In: isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples; 2014. Available at: http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES. [Accessed 14.07.2014].
http://www.fsis.usda.gov/wps/wcm/connect...
These colonies were also used for the polymerase chain reaction (PCR) technique (see below).

PCR methodology

Multiplex PCR was performed as described by Perdoncini et al.1212 Perdoncini G, Tejkowski T, Sierra-Arguello Y, et al. Identificação de Campylobacter jejuni e Campylobacter coli de origem avícola através do ensaio multiplex PCR. In: III Congresso Sul Brasileiro de Avicultura, Suinocultura e Laticínios. Bento Gonçalves, RS Anais, Bento Gonçalves: AVISULAT; 2012. to identify C. jejuni and C. coli. Briefly, 30 µL reactions containing 10× buffer, 1.5 mM MgCl2, 5 mM dNTPs, two units of Taq polymerase, thermo DNA extract, 4 pmol/µL of each 16S rRNA primer, 2 pmol/µL specific primers and ultra-pure water up to 30 µL were made. The specific primers used amplify the following genes: mapA (F a-CTATTTTATTTTTGAGTGCTTGTG, R b-GCTTTATTTGCCATTTGTTTTATTA with 589 pb, 50 N) (Invitrogen®, São Paulo, SP, Brazil) and ceuE (F a-AATTGAAAATTGCTCCAACTATG, R b-TGATTTTATTATTTGTAGCAGCG with 462 pb and a common region between species (16S rRNA), 50 N (Invitrogen®, São Paulo, SP, Brazil) and F a-ATCTAATGGCTTAACCATTAAAC, R b-GGACGGTAACTAGTTTAGTATT with 857 pb, 50 N (Invitrogen®, São Paulo, SP, Brazil)). Amplification reactions were carried out in a thermal cycler (Swift MaxPro®, Esco, Hatboro, PA, USA) under the following conditions: denaturation for 10 min at 95 °C, 35 cycles at 95 °C for 30 s, annealing at 59 °C for 1 min and 30 s and a final extension at 72 °C for 10 min. Arcobacter spp. were used as negative controls and C. jejuni ATCC 33291 and C. coli ATCC 43578 as positive controls. Ten microliter aliquots of the reaction mixtures were electrophoresed through 1.5% agarose gels (with the addition of 20% ethidium bromide) with a 100-bp DNA ladder (Invitrogen®, São Paulo, SP, Brazil) to determine the molecular weight. Fragments were transilluminated with UV light.

Data statistical analysis

Data were submitted for ANOVA analysis using BioStat Version 2009 (Analyst Soft. Inc., Alexandria, VA, USA).

Results

The Campylobacter direct plate count frequency was higher on the Campy-Cefex agar than on the mCCDA agar for different samples from broiler slaughtering process as described in Table 1.

Table 1
Frequency of Campylobacter spp. colonies directly isolated from mCCDA and Campy-Cefex agar plates with or without broth enrichment in different samples from the broiler slaughtering process.

Table 2 presents the results of quantitative analysis of Campylobacter spp. directly counted on Campy-Cefex and mCCDA agar plates.

Table 2
Direct count averages of Campylobacter spp. from Campy-Cefex and mCCDA agar plates plated with different samples from the broiler slaughtering process.

There was a high frequency of C. jejuni in all PCR-analyzed samples (Table 3). In addition, 18% contained both C. jejuni and C. coli in the same sample.

Table 3
Percentage of isolated Campylobacter jejuni and Campylobacter coli on Campy-Cefex and mCCDA agar plates as identified by PCR analysis.

With respect to all PCR samples, 2% contained none of the specific genes used to identify C. jejuni or C. coli, although they were identified as a Campylobacter species.

Discussion

Campylobacter spp. are recognized as a main cause of human enteritis outbreaks in both developed and developing countries. Although Brazil is the world's largest poultry meat exporter, data regarding this pathogen are limited, and at present, there is no legislation pertaining to Campylobacter risk analyses or control methods.

The Campylobacter isolation methodologies are laborious, and there are many broths and agars available. Some studies have evaluated the effectiveness of different broths and agar plates for their ability to isolate Campylobacter from several matrices to develop more efficient and lower cost methods.1313 Borsoi A, Nascimento VP. Campylobacter em produtos avícolas esua importância na saúde pública; 2011. Available at: http://pt.engormix.com/MA-avicultura/54administracao/artigos/campylobacter-produtos-avicolas-sua-t777/124-p0.htm. [Accessed 03.08.2014].
http://pt.engormix.com/MA-avicultura/54a...
Oyarzabal et al.1414 Oyarzabal AO, Macklin KS, Barbaree JM, Miller RS. Evaluation of agar plates for direct enumeration of Campylobacter spp. from poultry carcass rinses. Appl Environ Microbiol. 2005;71:3351-3354. evaluated 240 samples of broiler carcass rinse samples by recovering Campylobacter on Campy-Cefex, mCCDA and CLA (Campy-Line agar) agar plates. The authors concluded that with regards to time, preparation, performance and cost, Campy-Cefex and mCCDA agar obtained better Campylobacter counting results from carcass rinse samples. In another poultry study1515 Potturi-Venkata LP, Backert S, Lastovica AJ, et al. Evaluation of different plate media for direct cultivation of Campylobacter species from live broilers. Poultry Sci. 2007;86:1304-1311. was compared five agar plates that were used to isolate Campylobacter as of cecal and fecal samples obtained from 60 broiler chicken. The mCCA agar was more efficient at isolating the bacteria than the mCCDA, CLA, CAP (Campylobacter agar plates) and Campylobacter agars.

In the present study, the quantitative methods from the MLG 41.021111 USDA. MLG 41.02. 2013. In: isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples; 2014. Available at: http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES. [Accessed 14.07.2014].
http://www.fsis.usda.gov/wps/wcm/connect...
protocol showed reduced levels of Campylobacter in samples collected along the slaughter line, from cloacal swabs from live birds to post chiller carcasses, suggesting that the process of slaughtering can have a beneficial effect on the microbiological status of carcasses at the end of the slaughtering line. Accordingly, Berrang et al.,1616 Berrang ME, Bailey J, Altekruse S, et al. Prevalence and numbers of Campylobacter on broiler carcasses collected at rehang and postchill in 20 U.S. processing plants. J Food Protect. 2007;70(7):1556-1560. reported that improved hygiene in the slaughtering process and constant evaluations of such hygiene measures allowed for a reduction in Campylobacter spp. numbers in carcasses prior to shipping to markets. Thus, the number of bacteria from infected flocks can be reduced during processing in the slaughterhouse.

The presence of Campylobacter in the water supply samples (Table 1) was not expected because chemical treatment of the water should eliminate bacteria; further, if bacteria were present, a low level should not have been detected by a direct count method. It is known that Campylobacter is able to compose biofilms and that their formation has been proposed as a survival mechanism outside the host for protection against chemical products, physical cleaning processes and environmental stress, among others.1717 Jarquin R, Slavik M. Campylobacter jejuni as a secondary colonizer of poultry biofilms. J Appl Microbiol. 2008;105:1199-1208. Thus, the presence of biofilms and the contamination of the water sources are possible explanations for these data.

A high (100%) prevalence of Campylobacter in cloacal swabs was also found in this study (Table 1) by direct counting on both types of agar. These data are in agreement with another study,1818 Chaves SOC. Pesquisa de Campylobacter spp. em granjas e abatedouro avícolas na mesorregião metropolitana de Belém - PA. (M.Sc. Dissertation. Universidade Federal do Pará); 2003. who found that 96.6% from 30 samples of cloacal swabs contained Campylobacter. Additionally, Evans and Sayers1919 Evans SJ, Sayers AR. A longitudinal study of Campylobacter infection of broiler flocks in Great Britain. Preventive Veterinary Med. 2000;46:209-223. identified these bacteria in 91% of chicken cloacal swabs (20 total samples) in Great Britain, and Franchin et al.2020 Franchin PR, Aidoo KE, Batista CRV. Sources of poultry meat contamination with thermophilic Campylobacter before slaughter. Brazilian J Microbiol. 2005;36:157-162. reported that 75% of the swabs from broiler flocks in southern Brazil were positive for these bacteria.

Regarding carcass contamination, we found Campylobacter contamination in 83% of post-chiller carcasses, and the isolation frequency by direct plate counting on Campy-Cefex agar was high (Table 1). After enrichment in Bolton broth to boost low cell numbers in some samples, all pre- and post-chiller carcass samples tested positive. However, these data were higher than those reported by the Europeans, who had an average poultry carcass contamination level of 75.8%,2121 EFSA, European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter on broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2009. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J. 2010;8(3):1503. and by Kuana et al.,88 Kuana SL, Santos LR, Rodrigues LB, et al. Occurrence and characterization of Campylobacter in the Brazilian production and processing of broilers. Avian Diseases. 2008;52(4):680-684. who reported that 98.3% of 60 broiler carcasses were contaminated after chiller processing. In the present work, a significant difference was found between the Campy-Cefex and mCCDA plates used for cell recovery in the analysis of pre-chiller carcass samples, where Campy-Cefex had higher Campylobacter cell numbers (Table 2). Furthermore, it is important to note that despite the high isolation percentage from the Campy-Cefex agar, no C. coli was recovered in pre- and post-chiller carcass samples, a fact that is not consistent with other matrices (Table 3).

Studies using direct plate counting methods have indicated that selective enrichment does not increase the recovery of Campylobacter from fecal or cecal samples or chicken carcasses.2222 Martin WT, Patton MC, Morris GK, Potter ME, Puhr ND. Selective enrichment broth medium for isolation of Campylobacter jejuni. J Clin Microbiol. 1983;17(5):853-855.,2323 Musgrove MT, Berrang ME, Byrd JA, Stern NJ, Cox NA. Detection of Campylobacter spp. in ceca and crops with and without enrichment. Poultry Sci. 2001;80:825-828. These data differ from the present study where 100% of enriched samples were positive for Campylobacter spp. Kiess et al.2424 Kiess AS, Parker HM, Mcdaniel CD. Evaluation of different selective media and culturing techniques for the quantification of Campylobacter spp. from broiler litter. Poultry Sci. 2010;89:1755-1762. demonstrated that direct plating serves an advantage in isolating Campylobacter from poultry litter samples; 37% of the samples were positive for Campylobacter as tested by direct plating and 2% were positive following enrichment. Despite our higher Campylobacter frequency in enriched post-chiller samples, the direct count method was able to recover and quantify Campylobacter in agreement with Oyarzabal et al.,1414 Oyarzabal AO, Macklin KS, Barbaree JM, Miller RS. Evaluation of agar plates for direct enumeration of Campylobacter spp. from poultry carcass rinses. Appl Environ Microbiol. 2005;71:3351-3354. who demonstrated the value of direct plating in studying Campylobacter spp. contamination of poultry carcasses.

Multiplex PCR analysis identified 72% of samples as positive for C. jejuni and 38% as positive for C. coli (Table 3). Similar results were demonstrated in the European Union, where 60.8% of cecal samples tested positive for C. jejuni and 41.5% tested positive for C. coli.2121 EFSA, European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter on broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2009. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J. 2010;8(3):1503. In southern Brazil, a study conducted by Perdoncini et al.1212 Perdoncini G, Tejkowski T, Sierra-Arguello Y, et al. Identificação de Campylobacter jejuni e Campylobacter coli de origem avícola através do ensaio multiplex PCR. In: III Congresso Sul Brasileiro de Avicultura, Suinocultura e Laticínios. Bento Gonçalves, RS Anais, Bento Gonçalves: AVISULAT; 2012. sampled eight different points from a broiler slaughterhouse line. They identified C. jejuni in 75% of the samples and C. coli in 10% of the samples. In addition, both C. jejuni and C coli were present together in 15% of all samples. In contrast, 200 samples of broiler cecal content from a southern Brazil slaughterhouse were evaluated, and it was found that 44% were positive for C. coli and 2% were positive for C. jejuni.2525 BIOTECSUR. Informe Descritivo Final. 2013. Available at: http://www.biotecsur.org/proyectosregionales/informe_final_pi_atecnica_internacional.pdf. [Accessed: 30 .03.2014].
http://www.biotecsur.org/proyectosregion...

Studies have also been performed with regards to the different Campylobacter serotypes colonizing birds. Shibiny-El et al.2626 Shibiny-El SA, Connerton PL, Connerto IF. Campylobacter succession in broiler chickens. Microbiol Vet. 2007;125:323-332. reported that it is not common to isolate more than one type or subtype of Campylobacter from the same bird. The authors suggest that C. jejuni and C. coli compete equally and showed a decline in C. jejuni and C. coli dominance in isolates from 35-day-old birds. In contrast, the present study, as well as the study by Perdoncini et al.,1212 Perdoncini G, Tejkowski T, Sierra-Arguello Y, et al. Identificação de Campylobacter jejuni e Campylobacter coli de origem avícola através do ensaio multiplex PCR. In: III Congresso Sul Brasileiro de Avicultura, Suinocultura e Laticínios. Bento Gonçalves, RS Anais, Bento Gonçalves: AVISULAT; 2012. isolated two different serotypes from the same sample; 18% of the samples contained both C. jejuni and C. coli. In fact, both serotypes appeared in the same frequency in cloacal swabs.

In this work, the ability to isolate C. coli from different matrices by Campy-Cefex and mCCDA agar plates was variable (Table 3). According to WHO,11 WHO. World Health Organization; 2014. Available at: http://www.who.int/mediacentre/factsheets/fs255/en/ [Accessed: 24.11.2014].
http://www.who.int/mediacentre/factsheet...
many different forms of media can be used in the recovery of Campylobacter spp., and although mCCDA agar is the recommended medium, alternatives may be used. The main difference between media is the degree to which each inhibits contaminating flora, but all selective agents allow for the growth of both C. jejuni and C. coli.

Conclusions

The direct plating methods applied in this study were able to recover Campylobacter from different poultry matrices. Only from pre-chiller water did the Campy-Cefex agar direct counting method recover statistically high Campylobacter cells numbers. Based on the results of this study, it is plausible to suggest that both Campy-Cefex and mCCDA agar plates can increase the chances of recovering C. coli from swabs, carcasses and water samples. The present work also demonstrated that direct counting of Campylobacter from samples at different sites in the broiler slaughterhouse is useful for identifying contamination points and levels and is a possible tool for controlling Campylobacter contamination at Brazilian slaughterhouses.

  • Associate Editor: Elaine Cristina Pereira De Martinis

REFERENCES

  • 1
    WHO. World Health Organization; 2014. Available at: http://www.who.int/mediacentre/factsheets/fs255/en/ [Accessed: 24.11.2014].
    » http://www.who.int/mediacentre/factsheets/fs255/en/
  • 2
    CDC. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food - 10 States. Morbidity and Mortality Weekly Report, v. 19, n. 14, 2010, p. 418-422. Available at: http://www.cdc.gov/mmwr/pdf/wk/mm5914.pdf [Accessed 15.07.2014].
    » http://www.cdc.gov/mmwr/pdf/wk/mm5914.pdf
  • 3
    EFSA. European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter on broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J 2009;8(3):1503.
  • 4
    EFSA. European Food Safety Authority. Reducing Campylobacter in EU chickens; 2011. Available at: http://www.foodprocessing.com.au/articles/46692-Reducing-Campylobacter-in-EU-chickens [Accessed 10.03.2014].
    » http://www.foodprocessing.com.au/articles/46692-Reducing-Campylobacter-in-EU-chickens
  • 5
    Wagenaar JA, Mevius DJ, Havelaar AH. Campylobacter in primary animal production and control strategies to reduce the burden of human campylobacteriosis. Rev Sci Tech Off Int Epiz 2006;25(2):581-594.
  • 6
    Boufleur R. Campylobacter jejuni em Frangos de Corte, Carne e Vísceras de Frango no Rio Grande do Sul e Efeito do Congelamento sobre a Contaminação nos Cortes. Santa Maria, RS, Brasil; 2009, 47p. (M.Sc. Dissertation. Centro de Ciências Rurais. UFSM).
  • 7
    Fonseca BB. Transmissão vertical de Campylobacter sp em um sistema de produção avícola. Uberlândia, MG, Brasil; 2006. 80p. (M.Sc. Dissertation. Universidade Federal de Uberlândia).
  • 8
    Kuana SL, Santos LR, Rodrigues LB, et al. Occurrence and characterization of Campylobacter in the Brazilian production and processing of broilers. Avian Diseases 2008;52(4):680-684.
  • 9
    FAO/WHO. Technical meeting on Salmonella and Campylobacter in chicken meat; 2009. URL: http://www.who.int/foodsafety/publications/micro/MRA19.pdf
    » http://www.who.int/foodsafety/publications/micro/MRA19.pdf
  • 10
    ISO 10272-1:2006. Microbiology of food and animal feeding stuffs - Horizontal method for detection and enumeration of Campylobacter spp.
  • 11
    USDA. MLG 41.02. 2013. In: isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples; 2014. Available at: http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES [Accessed 14.07.2014].
    » http://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES
  • 12
    Perdoncini G, Tejkowski T, Sierra-Arguello Y, et al. Identificação de Campylobacter jejuni e Campylobacter coli de origem avícola através do ensaio multiplex PCR. In: III Congresso Sul Brasileiro de Avicultura, Suinocultura e Laticínios Bento Gonçalves, RS Anais, Bento Gonçalves: AVISULAT; 2012.
  • 13
    Borsoi A, Nascimento VP. Campylobacter em produtos avícolas esua importância na saúde pública; 2011. Available at: http://pt.engormix.com/MA-avicultura/54administracao/artigos/campylobacter-produtos-avicolas-sua-t777/124-p0.htm [Accessed 03.08.2014].
    » http://pt.engormix.com/MA-avicultura/54administracao/artigos/campylobacter-produtos-avicolas-sua-t777/124-p0.htm
  • 14
    Oyarzabal AO, Macklin KS, Barbaree JM, Miller RS. Evaluation of agar plates for direct enumeration of Campylobacter spp. from poultry carcass rinses. Appl Environ Microbiol 2005;71:3351-3354.
  • 15
    Potturi-Venkata LP, Backert S, Lastovica AJ, et al. Evaluation of different plate media for direct cultivation of Campylobacter species from live broilers. Poultry Sci 2007;86:1304-1311.
  • 16
    Berrang ME, Bailey J, Altekruse S, et al. Prevalence and numbers of Campylobacter on broiler carcasses collected at rehang and postchill in 20 U.S. processing plants. J Food Protect 2007;70(7):1556-1560.
  • 17
    Jarquin R, Slavik M. Campylobacter jejuni as a secondary colonizer of poultry biofilms. J Appl Microbiol 2008;105:1199-1208.
  • 18
    Chaves SOC. Pesquisa de Campylobacter spp. em granjas e abatedouro avícolas na mesorregião metropolitana de Belém - PA. (M.Sc. Dissertation. Universidade Federal do Pará); 2003.
  • 19
    Evans SJ, Sayers AR. A longitudinal study of Campylobacter infection of broiler flocks in Great Britain. Preventive Veterinary Med 2000;46:209-223.
  • 20
    Franchin PR, Aidoo KE, Batista CRV. Sources of poultry meat contamination with thermophilic Campylobacter before slaughter. Brazilian J Microbiol 2005;36:157-162.
  • 21
    EFSA, European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter on broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2009. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J 2010;8(3):1503.
  • 22
    Martin WT, Patton MC, Morris GK, Potter ME, Puhr ND. Selective enrichment broth medium for isolation of Campylobacter jejuni. J Clin Microbiol 1983;17(5):853-855.
  • 23
    Musgrove MT, Berrang ME, Byrd JA, Stern NJ, Cox NA. Detection of Campylobacter spp. in ceca and crops with and without enrichment. Poultry Sci. 2001;80:825-828.
  • 24
    Kiess AS, Parker HM, Mcdaniel CD. Evaluation of different selective media and culturing techniques for the quantification of Campylobacter spp. from broiler litter. Poultry Sci 2010;89:1755-1762.
  • 25
    BIOTECSUR. Informe Descritivo Final. 2013. Available at: http://www.biotecsur.org/proyectosregionales/informe_final_pi_atecnica_internacional.pdf [Accessed: 30 .03.2014].
    » http://www.biotecsur.org/proyectosregionales/informe_final_pi_atecnica_internacional.pdf
  • 26
    Shibiny-El SA, Connerton PL, Connerto IF. Campylobacter succession in broiler chickens. Microbiol Vet. 2007;125:323-332.

Publication Dates

  • Publication in this collection
    Jul-Sep 2016

History

  • Received
    25 Feb 2015
  • Accepted
    13 Jan 2016
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br