- Email: [email protected]
Prevalence, quantitative load, and antimicrobial resistance of Campylobacter spp. from broiler ceca and broiler skin samples in Thailand
Prevalence, quantitative load, and antimicrobial resistance of Campylobacter spp. from broiler ceca and broiler skin samples in Thailand C. Chokboonmongkol,* P. Patchanee,* G. Gölz,† K.-H. Zessin,‡ and T. Alter†1 *Veterinary Public Health Center for Asia Pacific, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand; †Institute of Food Hygiene, Freie Universität Berlin, 14163 Berlin, Germany; and ‡Postgraduate Studies in International Animal Health, Freie Universität Berlin, 14163 Berlin, Germany ABSTRACT This study was conducted to determine the prevalence of Campylobacter spp. in broiler flocks by testing cecal contents at slaughter and to detect and quantify Campylobacter on broiler carcass skin samples of the corresponding slaughter batches, to determine antimicrobial resistance patterns of the Campylobacter isolates, and to genotype selected Campylobacter jejuni isolates using multilocus sequence typing analysis. Ninety-eight broiler flocks were included in the study. Intact ceca were randomly taken at the time of evisceration throughout a slaughter batch to detect Campylobacter spp. at the broiler flock level and one whole carcass per slaughter batch was taken for the detection of Campylobacter spp. on broiler skin. The prevalences of Campylobacter spp. in broiler ceca and broiler skin samples were 11.2% (11/98) and 51% (50/98), respectively. Even though most Campylobacter-positive broiler skin samples were contaminated with only up to 230 most probable number per gram, a substantial share (13.3%) showed very high Campylobacter numbers on
the broiler skin samples (most probable number = ∞; lower confidence limit T0 580/g). From 32 C. jejuni and Campylobacter coli isolates tested, the highest antimicrobial resistance rates were found for ciprofloxacin (81.2%), followed by tetracycline (40.6%), ampicillin (31.2%), and erythromycin (9.4%). All tested strains were sensitive to gentamicin. By multilocus sequence typing analysis, a total of 9 different sequence types were identified among 16 C. jejuni isolates. Campylobacter jejuni isolated from cecal content and carcass skin of the same farm or slaughter batch showed corresponding allelic profiles. Our data suggest that intense cross-contamination during the slaughter process led to a strong increase of Campylobacter prevalence on broiler skin compared with the prevalence in broiler ceca. To reduce Campylobacter prevalences on broiler skin, on-farm biosecurity measures need to be accompanied by control measures at the slaughterhouse to reduce fecal contamination of broiler skin and to minimize crosscontamination.
Key words: Campylobacter, chicken, prevalence, semiquantitative load, antimicrobial resistance 2013 Poultry Science 92:462–467 http://dx.doi.org/10.3382/ps.2012-02599
INTRODUCTION
different authors (Stern et al., 2001; EFSA, 2010a,b). Previous studies in Thailand showed a Campylobacter prevalence of 52% in poultry meat at retail (Vindigni et al., 2007), whereas the prevalence of Campylobacter spp. in chickens before slaughter was 48 to 64% (Meeyam et al., 2004; Padungtod and Kaneene, 2005; Boonmar et al., 2007). The prevention of Campylobacter colonization at farm level is a crucial factor to reduce the Campylobacter prevalence and quantitative load of poultry meat (Näther et al., 2009). Risk factors for Campylobacter colonization at the farm level have been discussed intensively (Adkin et al., 2006). In the past years, commercial broiler production in Thailand has been transformed almost completely from independent farms to integrated production systems. All integrated producers had to implement strict bios-
Campylobacter is one of the most important foodborne bacteria that cause gastroenteritis in humans in developed and developing countries (Coker et al., 2002; WHO, 2011). In Thailand, Campylobacter has become a leading foodborne pathogen (Bodhidatta et al., 2002). Foods of animal origin, in particular poultry, have been identified as a significant source of Campylobacter infection (Studahl and Andersson, 2000; EFSA, 2011). High prevalences in broiler ceca and high contamination rates on broiler carcasses have been reported by ©2013 Poultry Science Association Inc. Received July 10, 2012. Accepted October 8, 2012. 1 Corresponding author: [email protected]
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ecurity measures at the farm level. On-farm measures need to be accompanied by control measures at the slaughterhouse to reduce fecal contamination of carcasses and to minimize cross-contamination (Reich et al., 2008). A relation between within-flock prevalence of Campylobacter colonization and carcass contamination as well as a correlation between the number of Campylobacter in ceca and on carcasses has been described by Allen et al. (2007) and Reich et al. (2008). Antimicrobial resistance in both human and animal Campylobacter isolated has become increasingly common in Thailand and other developing countries (Bodhidatta et al., 2002; Isenbarger et al., 2002; Padungton and Kaneene, 2003). The wide use of antibiotics in primary production may select for resistant Campylobacter spp. (Smith et al., 2002). The prevalence of quinolone-resistant Campylobacter spp. was described as a main problem among diarrheic children (Bodhidatta et al., 2002; Serichantalergs et al., 2007) and poultry in Thailand (Padungtod et al., 2006). The aims of this study were (i) to determine the prevalence of Campylobacter in broiler ceca at slaughter and on broiler skin, (ii) to semiquantify the Campylobacter load on broiler skin, (iii) to determine the antimicrobial resistance patterns of Campylobacter isolates from broiler ceca, and (iv) to genotype selected C. jejuni strains using multilocus sequence typing (MLST) analysis.
MATERIALS AND METHODS Sampling Sample size was calculated from 130 slaughter flocks, intended for slaughter during the study period, using 50% expected prevalence with a 95% confidence level and a 5% accepted error. The calculated 98 slaughter batches (originating from commercial broiler farms aimed at domestic consumption) were collected from December 2010 through June 2011 at a slaughterhouse in Chiang Mai, Thailand. Ten intact ceca were taken randomly throughout a slaughter batch at the time of evisceration. One whole carcass per slaughter batch was taken after chilling for the detection of Campylobacter spp. on broiler skin. Ceca and skin samples were collected from the same slaughter batch. The cecal samples served to estimate the prevalence of Campylobacter spp. at the broiler flock level, and broiler neck skin samples have been investigated to determine the prevalence and quantitative load on broiler skin after slaughter.
Sample Processing Cecal samples were examined according to the procedure for detection of Campylobacter spp. as described by ISO 10272–1:2006 (qualitative method; ISO, 2006). Briefly, the contents of the 10 ceca were removed,
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pooled, transferred to Bolton broth (Oxoid, Basingstoke, UK; test portion/enrichment media ratio 1:10), and homogenized. The enrichment culture was incubated under microaerobic conditions (CampyGen, Oxoid) at 37°C for 4 h, and then at 41.5°C for 44 ± 4 h. After incubation, one loop of suspension was inoculated onto modified charcoal cefoperazone deoxycholate agar (mCCD agar; Oxoid) and incubated in a microaerobic atmosphere at 41.5°C for 44 ± 4 h. Whole carcasses were treated as described in ISO 10272–3:2010 (semiquantitative method; ISO, 2010). In brief, the neck skin was removed (if present) together with the skin from one side of the carcass (avoiding any fat) to make a 15-g test portion that was placed into a stomacher bag. One hundred twenty milliliters of Bolton broth (Oxoid) was added, and the suspension was homogenized. An amount of 90 mL of that initial suspension was transferred to a 100-mL flask. Ten milliliters of the initial suspension was further transferred to a culture tube and used to create a 10-fold dilution series in Bolton broth up to 10−4. Incubation of enrichments and subsequent transfer to selective media is described above. Semiquantitative data are expressed as the most probable number (MPN) per gram according to ISO 10272–3:2010/AC:2011 (ISO, 2011). For Campylobacter identification, 3 colonies per plate presumed to be Campylobacter (greyish, metallic sheen, flat, moist with tendency to spread) were subcultured on nonselective Columbia blood agar (Oxoid) and incubated under microaerobic conditions at 41.5°C for 24 to 48 h. Isolates were confirmed by biochemical tests (oxidase test, catalase test, hippurate hydrolysis test, and indoxyl acetate test) and gram stained. Colonies from Columbia blood agar were suspended into 2 mL of Brucella broth (Oxoid) and incubated in a microaerobic atmosphere at 42°C or 37°C (24 h) for subsequent examination of motility and morphology as well as for DNA-extraction. Isolates were frozen and stored in 50% glycerol with Brucella broth (Oxoid) at −70°C.
Genus and Species Verification, and Antimicrobial Susceptibility Test After DNA extraction (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany), a multiplex PCR was performed according to Wang et al. (2002) to verify and differentiate Campylobacter spp. The disk diffusion method was performed as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2011). For that, 32 Campylobacter jejuni and Campylobacter coli isolates were examined for resistance to 5 antimicrobial agents (erythromycin, ciprofloxacin, tetracycline, gentamicin, and ampicillin). Campylobacter suspensions were adjusted to a turbidity equivalent to a 0.5 McFarland standard, equivalent to approximately 104 cfu/mL. Sterile cotton swabs were dipped into the suspension and streaked on the entire surface of Mueller–Hinton agar (Oxoid). The inoculum
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was allowed to dry for 5 min. Antimicrobial disks (Oxoid) were distributed over the inoculated plates. After 48 h of microaerobic incubation at 42°C, the diameters of inhibition zones were measured with calipers. Disk concentrations and zone diameter breakpoints were used according to the study of Luangtongkum et al. (2007).
MLST Sixteen C. jejuni strains were genotyped by MLST analysis according to Dingle et al. (2001). Genomic DNA was amplified with primers for 7 conserved housekeeping genes: aspA (aspartase A), glnA (glutamine synthetase), gltA (citrate syntase), glyA (serine hydroxymethyltransferase), pgm (phosphoglucomutase), tkt (transketolase), and uncA (ATP synthase α subunit). The PCR products were purified and sequenced in both directions (Tech Dragon, Hong Kong). The MLST data were collected in Bionumerics v6.1 (Applied Maths, Sint Martens-Latem, Belgium). Sequences were compared with existing alleles in the MLST C. jejuni database (http://pubmlst.org) to assign allele numbers and sequence types (ST). A dendrogram was created based on concatenated sequences using pairwise alignment and UPGMA for cluster analysis (Bionumerics).
RESULTS AND DISCUSSION Prevalence of Campylobacter The prevalence of Campylobacter spp. in broiler ceca was 11.2%, whereas on broiler skin the prevalence was 51% (Table 1). Compared with a study by Padungtod and Kaneene (2005), who detected a prevalence of 64% in fecal samples, that broiler ceca prevalence is very low. The reduction might be explained by the application of antimicrobials (enrofloxacin was applied to less than 3-d-old chicks) and by the strict biosecurity measures implemented in the broiler farms included in this study within the last years as a response to the avian influenza outbreak in 2004. The prevalence of Campylobacter on broiler skin (51.0%) is comparable with the report of Padungtod and Kaneene (2005), where 38% of the broiler skin samples were contaminated with Campylobacter. The low prevalence of Campylobacter on broiler ceca compared with the higher prevalence on broiler skin indicates that intense cross-contamination during the slaughtering process has occurred. A similar phenomenon (high-
er prevalence on broiler skin than in ceca) has already been described by Hue et al. (2010). These authors explain the increased prevalence by cross-contamination at different stages of the slaughter line. Such slaughterhouse effects are related to technological and hygienic slaughter practices that influence cecal and fecal contamination of carcasses (Corry and Atabay, 2001; Alter et al., 2005).
Semiquantitative Load In almost 50% of the broiler skin samples, a quantitative Campylobacter load was detectable. Even though most Campylobacter-positive skin samples were contaminated with only up to 230 MPN/g, a substantial share (13.3%) showed very high Campylobacter numbers on the skin (MPN = ∞; lower confidence limit T0 580/g; Figure 1). Only limited data on the quantitative Campylobacter load on broiler skin from Thailand are available for comparison. When comparing our data with US data from Stern and Pretanik (2006) and with a large data set generated by the European Union (EU) baseline survey in 2008 (EFSA, 2010a), a similar distribution of the Campylobacter counts can be observed. Stern and Pretanik (2006) showed that among 4,200 broiler carcass rinses tested, 74% yielded no countable Campylobacter and 3.6% of the samples contained more than 5 log cfu/g. In the EU, almost one-half of the broiler skin samples contained less than 10 Campylobacter/g. When summarized, over 47% contained 1 to 4 log cfu/g. A small portion of 5.8% of the broiler skin samples showed very high counts of over 4 log cfu/g (EFSA, 2010a). Comparable data from Thailand are only available for carcass rinse samples. Osiriphun et al. (2011) detected a Campylobacter concentration of 0.85 ± 0.95 log cfu in carcass rinse samples after chilling. The heavily contaminated skin samples originated mostly from Campylobacter-positive flocks. Such heavily contaminated flocks might act as a source of crosscontamination during the slaughter process. Based on
Table 1. Prevalence of Campylobacter in broiler ceca and broiler skin Item Broiler ceca Broiler skin samples
Prevalence (%)
95% CI
11.2 (11/98) 51 (50/98)
4.97 to 17.47 41.12 to 60.92
Figure 1. Level of Campylobacter concentrations on broiler skin samples. MPN = most probable number.
CAMPYLOBACTER IN BROILER CECA AND SKIN SAMPLES IN THAILAND
Figure 2. Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli.
the data from the EU Campylobacter baseline survey, it was concluded that a Campylobacter-colonized broiler batch was about 30 times more likely to have the sampled carcass contaminated with Campylobacter compared with a noncolonized batch, and a higher Campylobacter count on carcasses was strongly associated with Campylobacter colonization of the batch (EFSA, 2010b). Risk assessments have shown that the risk of human campylobacteriosis related to consumption of broiler meat is largely determined by tails of the concentration distribution (Nauta et al., 2009; Habib et al., 2012). Other studies showed that the risk of human illnesses correlates with carcasses from slaughter lots containing higher Campylobacter concentrations (Callicott et al., 2008). When comparing different risk assessments, Nauta et al. (2009) concluded that the most effective intervention measures aim at reducing the Campylobacter concentration, rather than reducing the prevalence.
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kok, poultry products come from commercial farms, whereas in rural areas people obtain poultry meat from their own farms where few or no antimicrobials are used. Antimicrobial resistance to erythromycin ranged from 0 to 20%. In our study, most of the isolates were resistant to a single antimicrobial substance (42.9%), followed by resistance to 2 antimicrobial substances (32.1%), 3 antimicrobial substances (21.4%), and 4 antimicrobial substances (3.6%). The most common combination of multidrug resistance was to ampicillin, tetracycline, and ciprofloxacin (17.9%). During the rearing period, chicken were exposed routinely to antimicrobials such as enrofloxacin. That might explain the high resistance to ciprofloxacin.
MLST A total of 9 different ST were identified from 16 C. jejuni isolates that were included in the MLST analysis (Figure 3). Of that, 3 sequence types were reported for the first time in Thailand (ST 305, ST 1075, and ST 5213). Despite the limited number of isolates included in the MLST analysis, C. jejuni isolated from cecal content and broiler skin of the same farm or slaughter batch showed corresponding allelic profiles (e.g., carcass skin isolates from flock 2 and 3 of farm V; isolates from
Antimicrobial Resistance Highest resistance rates were found for ciprofloxacin (81.2%), followed by tetracycline (40.6%), ampicillin (31.2%), and erythromycin (9.4%), respectively. All tested strains were sensitive to gentamicin (Figure 2). The high resistance rates to selected antimicrobials correspond to earlier data from Padungtod et al. (2006) showing resistance rates of 54.2 to 90.6% to ciprofloxacin and 37.5 to 81.3% to tetracycline in chicken isolates from Thailand. In their study, a low erythromycin resistance (1.9 to 5.8%) was detected and all strains were sensitive to gentamicin. This corresponds well with our data. Comparable data were generated by Serichantalergs et al. (2007), who tested Campylobacter isolates from diarrheic children in Thailand. Antimicrobial resistance rates for ciprofloxacin varied from 47.4% (rural areas) to 100% (Bangkok area) in the year 2000. These discrepancies in the antimicrobial resistance rate between rural and urban areas are explained by these authors by the origin of poultry meat at retail. In Bang-
Figure 3. Dendrogram of selected Campylobacter jejuni isolates based on multilocus sequence typing analysis. Sample identification (ID): first letter—farm ID; number—slaughter batch ID of corresponding farm; s = carcass skin; c = cecal content. ST = sequence type.
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carcass skin and cecal content of farm T or P, respectively). Further MLST studies need to include more C. jejuni strains to verify that observation. ST2274 is the predominant sequence type identified in our study. That rather uncommon ST (currently accounting for 0.2% of the isolates deposited in the pubMLST database) is dominated by human and chicken isolates originating from the United Kingdom, Thailand, and China (Zhang et al., 2010).
Conclusions Our data suggest that the implementation of strict biosecurity measures at the farm level in an integrated production system is able to reduce the Campylobacter prevalence in broiler flocks. Nonetheless, intense crosscontamination at the slaughterhouse might have led to higher Campylobacter prevalences on broiler skin. The results highlight the need to accompany on-farm biosecurity measures with improvements in slaughter hygiene. Further investigations on genotype distribution along the slaughter line are needed to identify points of cross-contamination and to weigh the influence of different cross-contamination points. Based on such data, targeted hygienic and decontamination measures during slaughter and processing can be implemented efficiently (Rosenquist et al., 2009).
ACKNOWLEDGMENTS The investigators acknowledge Khun Adirek Sripratak and Narin Romlumduan, CPF Co. Ltd., Bangkok, Thailand, for their funding support in this project. The authors thank Tongkorn Meeyam and Duangporn Pichpol, Chiang Mai University, Faculty of Veterinary Medicine, Chiang Mai, Thailand, for their excellent assistance. We also acknowledge the slaughterhouse staff, animal husbandry, and farm-consulting veterinarians for sampling and data collection.
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CAMPYLOBACTER IN BROILER CECA AND SKIN SAMPLES IN THAILAND Nauta, M., A. Hill, H. Rosenquist, S. Brynestad, A. Fetsch, P. van der Logt, A. Fazil, B. Christensen, E. Katsma, B. Borck, and A. Havelaar. 2009. A comparison of risk assessments on Campylobacter in broiler meat. Int. J. Food Microbiol. 129:107–123. Osiriphun, S., P. Iamtaweejaloen, P. Kooprasertying, W. Koetsinchai, K. Tuitemwong, L. E. Erickson, and P. Tuitemwong. 2011. Exposure assessment and process sensitivity analysis of the contamination of Campylobacter in poultry products. Poult. Sci. 90:1562–1573. Padungtod, P., and J. B. Kaneene. 2005. Campylobacter in food animals and humans in northern Thailand. J. Food Prot. 68:2519– 2526. Padungtod, P., J. B. Kaneene, R. Hanson, Y. Morita, and S. Boonmar. 2006. Antimicrobial resistance in Campylobacter isolated from food animals and humans in northern Thailand. FEMS Immunol. Med. Microbiol. 47:217–225. Padungton, P., and J. B. Kaneene. 2003. Campylobacter spp. in human, chickens, pigs and their antimicrobial resistance. J. Vet. Med. Sci. 65:161–170. Reich, F., V. Atanassova, E. Haunhorst, and G. Klein. 2008. The effects of Campylobacter numbers in caeca on the contamination of broiler carcasses with Campylobacter. Int. J. Food Microbiol. 127:116–120. Rosenquist, H., L. Boysen, C. Galliano, S. Nordentoft, S. Ethelberg, and B. Borck. 2009. Danish strategies to control Campylobacter in broilers and broiler meat: Facts and effects. Epidemiol. Infect. 137:1742–1750. Serichantalergs, O., A. Dalsgaard, L. Bodhidatta, S. Krasaesub, C. Pitarangsi, A. Srijan, and C. Mason. 2007. Emerging fluoroquinolone and macrolide resistance of Campylobacter jejuni and Campylobacter coli isolates and their serotypes in Thai children from 1991 to 2000. Epidemiol. Infect. 135:1299–1306.
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Smith, D. L., A. D. Harris, J. A. Johnson, E. K. Silbergeld, and J. G. Morris Jr. 2002. Animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria. Proc. Natl. Acad. Sci. USA 99:6434–6439. Stern, N. J., P. Fedorka-Cray, J. S. Bailey, N. A. Cox, S. E. Craven, K. L. Hiett, M. T. Musgrove, S. Ladely, D. Cosby, and G. C. Mead. 2001. Distribution of Campylobacter spp. in selected U.S. poultry production and processing operations. J. Food Prot. 64:1705–1710. Stern, N. J., and S. Pretanik. 2006. Counts of Campylobacter spp. on U.S. broiler carcasses. J. Food Prot. 69:1034–1039. Studahl, A., and Y. Andersson. 2000. Risk factors for indigenous Campylobacter infection: A Swedish case-control study. Epidemiol. Infect. 125:269–275. Vindigni, S. M., A. Srijan, B. Wongstitwilairoong, R. Marcus, J. Meek, P. L. Riley, and C. Mason. 2007. Prevalence of foodborne microorganisms in retail foods in Thailand. Foodborne Pathog. Dis. 4:208–215. Wang, G., C. G. Clark, T. M. Taylor, C. Pucknell, C. Barton, L. Price, D. L. Woodward, and F. G. Rodgers. 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus ssp. fetus. J. Clin. Microbiol. 40:4744–4747. WHO. 2011. Fact sheet No. 255 Campylobacter. World Health Organization, Geneva, Switzerland. Zhang, M., Y. Gu, L. He, L. Ran, S. Xia, X. Han, H. Li, H. Zhou, Z. Cui, and J. Zhang. 2010. Molecular typing and antimicrobial susceptibility profiles of Campylobacter jejuni isolates from north China. J. Med. Microbiol. 59:1171–1177.
the broiler skin samples (most probable number = ∞; lower confidence limit T0 580/g). From 32 C. jejuni and Campylobacter coli isolates tested, the highest antimicrobial resistance rates were found for ciprofloxacin (81.2%), followed by tetracycline (40.6%), ampicillin (31.2%), and erythromycin (9.4%). All tested strains were sensitive to gentamicin. By multilocus sequence typing analysis, a total of 9 different sequence types were identified among 16 C. jejuni isolates. Campylobacter jejuni isolated from cecal content and carcass skin of the same farm or slaughter batch showed corresponding allelic profiles. Our data suggest that intense cross-contamination during the slaughter process led to a strong increase of Campylobacter prevalence on broiler skin compared with the prevalence in broiler ceca. To reduce Campylobacter prevalences on broiler skin, on-farm biosecurity measures need to be accompanied by control measures at the slaughterhouse to reduce fecal contamination of broiler skin and to minimize crosscontamination.
Key words: Campylobacter, chicken, prevalence, semiquantitative load, antimicrobial resistance 2013 Poultry Science 92:462–467 http://dx.doi.org/10.3382/ps.2012-02599
INTRODUCTION
different authors (Stern et al., 2001; EFSA, 2010a,b). Previous studies in Thailand showed a Campylobacter prevalence of 52% in poultry meat at retail (Vindigni et al., 2007), whereas the prevalence of Campylobacter spp. in chickens before slaughter was 48 to 64% (Meeyam et al., 2004; Padungtod and Kaneene, 2005; Boonmar et al., 2007). The prevention of Campylobacter colonization at farm level is a crucial factor to reduce the Campylobacter prevalence and quantitative load of poultry meat (Näther et al., 2009). Risk factors for Campylobacter colonization at the farm level have been discussed intensively (Adkin et al., 2006). In the past years, commercial broiler production in Thailand has been transformed almost completely from independent farms to integrated production systems. All integrated producers had to implement strict bios-
Campylobacter is one of the most important foodborne bacteria that cause gastroenteritis in humans in developed and developing countries (Coker et al., 2002; WHO, 2011). In Thailand, Campylobacter has become a leading foodborne pathogen (Bodhidatta et al., 2002). Foods of animal origin, in particular poultry, have been identified as a significant source of Campylobacter infection (Studahl and Andersson, 2000; EFSA, 2011). High prevalences in broiler ceca and high contamination rates on broiler carcasses have been reported by ©2013 Poultry Science Association Inc. Received July 10, 2012. Accepted October 8, 2012. 1 Corresponding author: [email protected]
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ecurity measures at the farm level. On-farm measures need to be accompanied by control measures at the slaughterhouse to reduce fecal contamination of carcasses and to minimize cross-contamination (Reich et al., 2008). A relation between within-flock prevalence of Campylobacter colonization and carcass contamination as well as a correlation between the number of Campylobacter in ceca and on carcasses has been described by Allen et al. (2007) and Reich et al. (2008). Antimicrobial resistance in both human and animal Campylobacter isolated has become increasingly common in Thailand and other developing countries (Bodhidatta et al., 2002; Isenbarger et al., 2002; Padungton and Kaneene, 2003). The wide use of antibiotics in primary production may select for resistant Campylobacter spp. (Smith et al., 2002). The prevalence of quinolone-resistant Campylobacter spp. was described as a main problem among diarrheic children (Bodhidatta et al., 2002; Serichantalergs et al., 2007) and poultry in Thailand (Padungtod et al., 2006). The aims of this study were (i) to determine the prevalence of Campylobacter in broiler ceca at slaughter and on broiler skin, (ii) to semiquantify the Campylobacter load on broiler skin, (iii) to determine the antimicrobial resistance patterns of Campylobacter isolates from broiler ceca, and (iv) to genotype selected C. jejuni strains using multilocus sequence typing (MLST) analysis.
MATERIALS AND METHODS Sampling Sample size was calculated from 130 slaughter flocks, intended for slaughter during the study period, using 50% expected prevalence with a 95% confidence level and a 5% accepted error. The calculated 98 slaughter batches (originating from commercial broiler farms aimed at domestic consumption) were collected from December 2010 through June 2011 at a slaughterhouse in Chiang Mai, Thailand. Ten intact ceca were taken randomly throughout a slaughter batch at the time of evisceration. One whole carcass per slaughter batch was taken after chilling for the detection of Campylobacter spp. on broiler skin. Ceca and skin samples were collected from the same slaughter batch. The cecal samples served to estimate the prevalence of Campylobacter spp. at the broiler flock level, and broiler neck skin samples have been investigated to determine the prevalence and quantitative load on broiler skin after slaughter.
Sample Processing Cecal samples were examined according to the procedure for detection of Campylobacter spp. as described by ISO 10272–1:2006 (qualitative method; ISO, 2006). Briefly, the contents of the 10 ceca were removed,
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pooled, transferred to Bolton broth (Oxoid, Basingstoke, UK; test portion/enrichment media ratio 1:10), and homogenized. The enrichment culture was incubated under microaerobic conditions (CampyGen, Oxoid) at 37°C for 4 h, and then at 41.5°C for 44 ± 4 h. After incubation, one loop of suspension was inoculated onto modified charcoal cefoperazone deoxycholate agar (mCCD agar; Oxoid) and incubated in a microaerobic atmosphere at 41.5°C for 44 ± 4 h. Whole carcasses were treated as described in ISO 10272–3:2010 (semiquantitative method; ISO, 2010). In brief, the neck skin was removed (if present) together with the skin from one side of the carcass (avoiding any fat) to make a 15-g test portion that was placed into a stomacher bag. One hundred twenty milliliters of Bolton broth (Oxoid) was added, and the suspension was homogenized. An amount of 90 mL of that initial suspension was transferred to a 100-mL flask. Ten milliliters of the initial suspension was further transferred to a culture tube and used to create a 10-fold dilution series in Bolton broth up to 10−4. Incubation of enrichments and subsequent transfer to selective media is described above. Semiquantitative data are expressed as the most probable number (MPN) per gram according to ISO 10272–3:2010/AC:2011 (ISO, 2011). For Campylobacter identification, 3 colonies per plate presumed to be Campylobacter (greyish, metallic sheen, flat, moist with tendency to spread) were subcultured on nonselective Columbia blood agar (Oxoid) and incubated under microaerobic conditions at 41.5°C for 24 to 48 h. Isolates were confirmed by biochemical tests (oxidase test, catalase test, hippurate hydrolysis test, and indoxyl acetate test) and gram stained. Colonies from Columbia blood agar were suspended into 2 mL of Brucella broth (Oxoid) and incubated in a microaerobic atmosphere at 42°C or 37°C (24 h) for subsequent examination of motility and morphology as well as for DNA-extraction. Isolates were frozen and stored in 50% glycerol with Brucella broth (Oxoid) at −70°C.
Genus and Species Verification, and Antimicrobial Susceptibility Test After DNA extraction (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany), a multiplex PCR was performed according to Wang et al. (2002) to verify and differentiate Campylobacter spp. The disk diffusion method was performed as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2011). For that, 32 Campylobacter jejuni and Campylobacter coli isolates were examined for resistance to 5 antimicrobial agents (erythromycin, ciprofloxacin, tetracycline, gentamicin, and ampicillin). Campylobacter suspensions were adjusted to a turbidity equivalent to a 0.5 McFarland standard, equivalent to approximately 104 cfu/mL. Sterile cotton swabs were dipped into the suspension and streaked on the entire surface of Mueller–Hinton agar (Oxoid). The inoculum
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was allowed to dry for 5 min. Antimicrobial disks (Oxoid) were distributed over the inoculated plates. After 48 h of microaerobic incubation at 42°C, the diameters of inhibition zones were measured with calipers. Disk concentrations and zone diameter breakpoints were used according to the study of Luangtongkum et al. (2007).
MLST Sixteen C. jejuni strains were genotyped by MLST analysis according to Dingle et al. (2001). Genomic DNA was amplified with primers for 7 conserved housekeeping genes: aspA (aspartase A), glnA (glutamine synthetase), gltA (citrate syntase), glyA (serine hydroxymethyltransferase), pgm (phosphoglucomutase), tkt (transketolase), and uncA (ATP synthase α subunit). The PCR products were purified and sequenced in both directions (Tech Dragon, Hong Kong). The MLST data were collected in Bionumerics v6.1 (Applied Maths, Sint Martens-Latem, Belgium). Sequences were compared with existing alleles in the MLST C. jejuni database (http://pubmlst.org) to assign allele numbers and sequence types (ST). A dendrogram was created based on concatenated sequences using pairwise alignment and UPGMA for cluster analysis (Bionumerics).
RESULTS AND DISCUSSION Prevalence of Campylobacter The prevalence of Campylobacter spp. in broiler ceca was 11.2%, whereas on broiler skin the prevalence was 51% (Table 1). Compared with a study by Padungtod and Kaneene (2005), who detected a prevalence of 64% in fecal samples, that broiler ceca prevalence is very low. The reduction might be explained by the application of antimicrobials (enrofloxacin was applied to less than 3-d-old chicks) and by the strict biosecurity measures implemented in the broiler farms included in this study within the last years as a response to the avian influenza outbreak in 2004. The prevalence of Campylobacter on broiler skin (51.0%) is comparable with the report of Padungtod and Kaneene (2005), where 38% of the broiler skin samples were contaminated with Campylobacter. The low prevalence of Campylobacter on broiler ceca compared with the higher prevalence on broiler skin indicates that intense cross-contamination during the slaughtering process has occurred. A similar phenomenon (high-
er prevalence on broiler skin than in ceca) has already been described by Hue et al. (2010). These authors explain the increased prevalence by cross-contamination at different stages of the slaughter line. Such slaughterhouse effects are related to technological and hygienic slaughter practices that influence cecal and fecal contamination of carcasses (Corry and Atabay, 2001; Alter et al., 2005).
Semiquantitative Load In almost 50% of the broiler skin samples, a quantitative Campylobacter load was detectable. Even though most Campylobacter-positive skin samples were contaminated with only up to 230 MPN/g, a substantial share (13.3%) showed very high Campylobacter numbers on the skin (MPN = ∞; lower confidence limit T0 580/g; Figure 1). Only limited data on the quantitative Campylobacter load on broiler skin from Thailand are available for comparison. When comparing our data with US data from Stern and Pretanik (2006) and with a large data set generated by the European Union (EU) baseline survey in 2008 (EFSA, 2010a), a similar distribution of the Campylobacter counts can be observed. Stern and Pretanik (2006) showed that among 4,200 broiler carcass rinses tested, 74% yielded no countable Campylobacter and 3.6% of the samples contained more than 5 log cfu/g. In the EU, almost one-half of the broiler skin samples contained less than 10 Campylobacter/g. When summarized, over 47% contained 1 to 4 log cfu/g. A small portion of 5.8% of the broiler skin samples showed very high counts of over 4 log cfu/g (EFSA, 2010a). Comparable data from Thailand are only available for carcass rinse samples. Osiriphun et al. (2011) detected a Campylobacter concentration of 0.85 ± 0.95 log cfu in carcass rinse samples after chilling. The heavily contaminated skin samples originated mostly from Campylobacter-positive flocks. Such heavily contaminated flocks might act as a source of crosscontamination during the slaughter process. Based on
Table 1. Prevalence of Campylobacter in broiler ceca and broiler skin Item Broiler ceca Broiler skin samples
Prevalence (%)
95% CI
11.2 (11/98) 51 (50/98)
4.97 to 17.47 41.12 to 60.92
Figure 1. Level of Campylobacter concentrations on broiler skin samples. MPN = most probable number.
CAMPYLOBACTER IN BROILER CECA AND SKIN SAMPLES IN THAILAND
Figure 2. Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli.
the data from the EU Campylobacter baseline survey, it was concluded that a Campylobacter-colonized broiler batch was about 30 times more likely to have the sampled carcass contaminated with Campylobacter compared with a noncolonized batch, and a higher Campylobacter count on carcasses was strongly associated with Campylobacter colonization of the batch (EFSA, 2010b). Risk assessments have shown that the risk of human campylobacteriosis related to consumption of broiler meat is largely determined by tails of the concentration distribution (Nauta et al., 2009; Habib et al., 2012). Other studies showed that the risk of human illnesses correlates with carcasses from slaughter lots containing higher Campylobacter concentrations (Callicott et al., 2008). When comparing different risk assessments, Nauta et al. (2009) concluded that the most effective intervention measures aim at reducing the Campylobacter concentration, rather than reducing the prevalence.
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kok, poultry products come from commercial farms, whereas in rural areas people obtain poultry meat from their own farms where few or no antimicrobials are used. Antimicrobial resistance to erythromycin ranged from 0 to 20%. In our study, most of the isolates were resistant to a single antimicrobial substance (42.9%), followed by resistance to 2 antimicrobial substances (32.1%), 3 antimicrobial substances (21.4%), and 4 antimicrobial substances (3.6%). The most common combination of multidrug resistance was to ampicillin, tetracycline, and ciprofloxacin (17.9%). During the rearing period, chicken were exposed routinely to antimicrobials such as enrofloxacin. That might explain the high resistance to ciprofloxacin.
MLST A total of 9 different ST were identified from 16 C. jejuni isolates that were included in the MLST analysis (Figure 3). Of that, 3 sequence types were reported for the first time in Thailand (ST 305, ST 1075, and ST 5213). Despite the limited number of isolates included in the MLST analysis, C. jejuni isolated from cecal content and broiler skin of the same farm or slaughter batch showed corresponding allelic profiles (e.g., carcass skin isolates from flock 2 and 3 of farm V; isolates from
Antimicrobial Resistance Highest resistance rates were found for ciprofloxacin (81.2%), followed by tetracycline (40.6%), ampicillin (31.2%), and erythromycin (9.4%), respectively. All tested strains were sensitive to gentamicin (Figure 2). The high resistance rates to selected antimicrobials correspond to earlier data from Padungtod et al. (2006) showing resistance rates of 54.2 to 90.6% to ciprofloxacin and 37.5 to 81.3% to tetracycline in chicken isolates from Thailand. In their study, a low erythromycin resistance (1.9 to 5.8%) was detected and all strains were sensitive to gentamicin. This corresponds well with our data. Comparable data were generated by Serichantalergs et al. (2007), who tested Campylobacter isolates from diarrheic children in Thailand. Antimicrobial resistance rates for ciprofloxacin varied from 47.4% (rural areas) to 100% (Bangkok area) in the year 2000. These discrepancies in the antimicrobial resistance rate between rural and urban areas are explained by these authors by the origin of poultry meat at retail. In Bang-
Figure 3. Dendrogram of selected Campylobacter jejuni isolates based on multilocus sequence typing analysis. Sample identification (ID): first letter—farm ID; number—slaughter batch ID of corresponding farm; s = carcass skin; c = cecal content. ST = sequence type.
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carcass skin and cecal content of farm T or P, respectively). Further MLST studies need to include more C. jejuni strains to verify that observation. ST2274 is the predominant sequence type identified in our study. That rather uncommon ST (currently accounting for 0.2% of the isolates deposited in the pubMLST database) is dominated by human and chicken isolates originating from the United Kingdom, Thailand, and China (Zhang et al., 2010).
Conclusions Our data suggest that the implementation of strict biosecurity measures at the farm level in an integrated production system is able to reduce the Campylobacter prevalence in broiler flocks. Nonetheless, intense crosscontamination at the slaughterhouse might have led to higher Campylobacter prevalences on broiler skin. The results highlight the need to accompany on-farm biosecurity measures with improvements in slaughter hygiene. Further investigations on genotype distribution along the slaughter line are needed to identify points of cross-contamination and to weigh the influence of different cross-contamination points. Based on such data, targeted hygienic and decontamination measures during slaughter and processing can be implemented efficiently (Rosenquist et al., 2009).
ACKNOWLEDGMENTS The investigators acknowledge Khun Adirek Sripratak and Narin Romlumduan, CPF Co. Ltd., Bangkok, Thailand, for their funding support in this project. The authors thank Tongkorn Meeyam and Duangporn Pichpol, Chiang Mai University, Faculty of Veterinary Medicine, Chiang Mai, Thailand, for their excellent assistance. We also acknowledge the slaughterhouse staff, animal husbandry, and farm-consulting veterinarians for sampling and data collection.
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