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Campylobacter subtypes detected in broiler ceca and livers collected at slaughter1
Campylobacter subtypes detected in broiler ceca and livers collected at slaughter1 M. E. Berrang,2 R. J. Meinersmann, and N. A. Cox USDA-Agricultural Research Service, U.S. National Poultry Research Center, Athens, GA 30605
Key words: broiler, Campylobacter, ceca, liver 2019 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pez340
INTRODUCTION
(Barot et al., 1983), or searing of the surface with hot metal (Berrang et al. 2018). There have been multiple outbreaks of campylobacteriosis traced to undercooked or mishandled chicken livers both within the United States and internationally (O’Leary et al., 2009; Tompkins et al., 2013; Moffatt et al., 2016; Glashower et al., 2017). A review of US outbreaks is available by Lanier et al. (2018). Some outbreaks have been traced to whole livers, many to chicken liver pate (Lanier et al., 2018). Overwhelmingly, the issue that led to foodborne illness was inadequate cooking of the liver dish (Lanier et al., 2018). Thorough cooking of livers is required to fully eliminate Campylobacter (Whyte et al., 2006). It has been shown with Salmonella that an oral inoculation can result in cecal colonization and translocation to internal organs including the liver (Gast et al., 2013). Oral inoculation with Campylobacter has also been shown to result in liver contamination, albeit in low percent of experimental 14-day-old broilers (Knudsen et al., 2006). Some Campylobacter, notably C. hepaticus, can cause liver disease in chickens (Crawshaw et al., 2015; Van et al., 2017a). This species of Campylobacter has been shown to be transferred to chickens by the fecal oral route and to colonize the gut as well as infect livers (Van et al., 2017b).
Campylobacter is a bacterial human pathogen associated with poultry and poultry products. It can be found on feathers and skin and in the gut contents of broilers presenting for slaughter (Berrang et al., 2000). Campylobacter has been detected on chicken livers as early as 1983 (Barot et al., 1983). Campylobacter has also been reported from chicken liver at retail, with prevalence ranging from 33 to 100% of livers tested (Barot et al., 1983; Whyte et al., 2006; Noormohamed and Fakhr, 2012; Strachan et al., 2012; Nicorici and Ghoddusi, 2017; Berrang et al., 2018). Efforts have been made to sample internal liver tissue separate from the surface. In several studies, Campylobacter was detected from the inside of livers which had been sanitized by either boiling water (Whyte et al., 2006), ethanol soak Published by Oxford University Press on behalf of Poultry Science Association 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US. Received April 3, 2019. Accepted July 1, 2019. 1 Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 2 Corresponding author: [email protected]
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face samples, and 19 liver internal tissue samples were positive. For 11 of 18 carcasses from which all samples were positive, the predominant colony types were indistinguishable. However, some carcasses did have multiple subtypes of Campylobacter. Of carcasses with Campylobacter on the surface of the liver and within the ceca, it was more likely that the subtypes be the same than different (P < 0.01). However, Campylobacter subtypes detected in internal liver tissue were not more likely to be the same as those detected in ceca (P > 0.05). We detected different subtypes of Campylobacter from internal liver tissue and liver surface of seven broiler carcasses/flocks. Livers from a large percentage of broiler carcasses/flocks can have one or more subtypes of Campylobacter.
ABSTRACT Foodborne campylobacteriosis has been linked to undercooked chicken liver. We have detected Campylobacter in chicken livers available at retail. The objective of the current project was to determine the prevalence and subtype of Campylobacter associated with livers and ceca of the same broiler carcasses at commercial slaughter. Within 2 min of commercial evisceration, we collected liver and ceca of one broiler carcass from each of 70 discreet flocks over a 12-mo period. Liver surface, liver internal tissue, and cecal contents were cultured for Campylobacter using standard methods. One example of the predominant colony type was selected from each positive sample for whole genome sequencing and multilocus sequence typing. We detected Campylobacter in at least one sample from 58 of 70 (83%) carcasses/flocks; 41 ceca, 57 liver sur-
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MATERIALS AND METHODS Experimental Overview This study was designed to sample many flocks by sampling broiler carcasses for presence of Campylobacter in the ceca and associated with the liver. To accomplish this, one carcass per sample day was selected such that many flocks were examined as opposed to many broilers from one flock. On 70 replicate sample days, representing 70 unique broiler flocks, 1 broiler carcass was selected post evisceration in a commercial slaughter plant. From each selected carcass, ceca and liver were collected and cultured for Campylobacter as described below.
then used to inoculate a Campy Cefex agar plate (CCA) (Stern et al., 1992) and a 9 mL tube of CEB. Following swabbing, an approximately 12 cm2 portion of the surface of each liver was sterilized by searing with a heated metal spatula. A sterile, CEB pre-moistened cotton-tipped applicator was used to press through the seared flesh into the internal tissue of the liver taking care to not completely penetrate out of the other side of the organ. The applicator was pressed into the internal tissue in 2 to 3 spots within the seared area. Each applicator was then used to inoculate a CCA plate and then placed into a 9 mL tube of CEB as described above.
Campylobacter Culture All CCA plates and CEB tubes were incubated at 42◦ C in re-sealable bags flushed with microaerobic gas (5% O2 , 10% CO2 , and 85% N2; AirgasUSA LLC, Radnor, PA). After 24 h, all samples in CEB were used to inoculate CCA plates. One tenth mL from each tube was streaked for isolation. All CCA plates were incubated as described above for 48 h. Colonies characteristics of Campylobacter were confirmed as such by observation of cellular morphology and motility under phase-contrast microscopy and positive reaction on a latex agglutination test (Microgen Bioproducts Ltd. Camberly, UK).
Sample Collection and Preparation Carcasses were selected that included complete viscera packs with an intact liver and intestinal tract. From each selected carcass, the liver and intestines were removed immediately after inspection. Timing depended on operational parameters and line speed on any given sample day; generally, samples were collected within 120s of automated evisceration. Entire viscera packs, just as presented on carcasses, were placed into clean new re-sealable plastic bags, covered with ice, and transported to the lab. Upon arrival, within 20 min, the liver and the ceca were separated from the viscera. Ceca were prepared for Campylobacter culture as previously described (Berrang et al., 2016). Briefly, using a clean nitrile glove, one cecum was transferred to a sample bag (Nasco, Fort Atkinson, WI), weighed, smashed to release contents, and diluted with 3 times volume to weight sterile phosphate buffered saline. Each cecum was subjected to blending in a paddle blender (Stomacher 80, Seward, Port St Lucie, FL) for 30 s before an aliquot was removed for dilution and direct plating as described below. The surface and inner tissue of livers was sampled as previously described (Berrang et al., 2018). Briefly, each liver was placed onto a sterile absorbent paper napkin. The outer surface was sampled by use of a sterile cotton-tipped applicator previously moistened in Campylobacter enrichment broth (CEB Accumedia Neogen Corp., Lansing, MI). Each applicator was rubbed over the entire ventral surface of a liver and
Campylobacter Subtyping Well-isolated representatives of the most common Campylobacter colony type found on each positive sample were subcultured and used for DNA sequencebased subtyping as previously described (Berrang et al., 2016). Each isolate was re-streaked for a lawn onto CCA and grown for 24 h at 42◦ C in a microaerobic atmosphere as described above. Bacterial growth was removed from plates with a sterile cotton tipped applicator and added to 1 mL of sterile freezing medium (blood and additive free CEB with 15% glycerol) in a freezing vial (Cryovial, Simport, Beloeil, QC, Canada). Cultures were frozen and held at –80◦ C until all isolates were collected. Isolates were revived from frozen storage by streaking for isolation onto the surface of tryptic soy agar with 5% sheep’s blood (Remel, Lenexa KS), and incubated under microaerobic conditions at 42◦ C for 48 h. One isolated colony was selected and streaked for a lawn on the surface of Brucella agar (Accumedia, Neogen Corp.), and incubated at 42◦ C for 24 h under microaerobic conditions. All growth was removed from Brucella agar, and DNA was extracted using a kit and manufacR turer’s instructions (UltraClean microbial DNA isolation Kit, Mo Bio Laboratories Inc., Carlsbad, CA). Libraries were prepared using the Nextera XT sample preparation kit (Illumina, San Diego, CA). Genomic DNA of each isolate was sequenced using the Illumina MiSeq platform with a 2 × 250 paired end run according to the manufacturer’s instructions (Illumina).
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The prevalence of Campylobacter in the livers of healthy broilers presenting for slaughter is not known. Nor is it known if the same subtypes of Campylobacter are present in the gut and liver of broiler chickens. The objective of this study was to compare frequency and subtype of Campylobacter detected from liver and ceca of broilers presented for commercial slaughter. To achieve this, we sampled both liver and ceca of one broiler from each of 70 flocks over the course of a 12-month period.
CECA AND LIVER CAMPYLOBACTER
Statistical Analysis Prevalence of Campylobacter from liver surface, inner tissue, and cecal contents were compared by chi square test for independence. Significance was assigned at P ≤ 0.05.
RESULTS AND DISCUSSION The number of broiler carcasses with different possible cultural outcomes from examination of ceca and liver is presented in Table 1. For 18 of 70 carcasses/flocks (25.7%), Campylobacter was detected in all samples (liver surface, internal liver tissue, and ceca). Campylobacter was significantly (P < 0.01) more likely to be detected inside the liver when the liver surface and/or ceca were also positive. This is in line with the finding that bacteria (Salmonella and Campylobacter) can invade a chicken’s liver following oral inoculation and intestinal colonization. (Knudsen et al., 2006; Gast et al., 2013). Campylobacter was detected in the ceca significantly (P < 0.01) more often than inside the liver. Campylobacter was detected the most often (P < 0.01) on liver surface (57/70, 81%) and was more likely to be detected on the liver surface when the ceca was also positive. In fact, every time the ceca was positive (41/70), the liver surface from the same carcass was also positive. It is possible that Campylobacter could have been on the liver surface in vivo as Campylobacter is related to liver disease in the chicken (Shane, 1991; Crawshaw et al., Table 1. Number of broiler carcasses1 with each possible Campylobacter culture outcome from cecal contents, liver surface, and liver inner tissue (n = 70). Campylobacter culture outcome by sample type Ceca + + + + – – – – 1
Outer liver
Inner liver
Number of samples
+ – + – + – + –
+ – – + + + – –
18 (25.7%) 0 23 (32.8%) 0 0 1 (1.4%) 16 (22.8%) 12 (17.1%)
Each carcass represents a unique broiler flock.
2015; Van et al., 2017a,b). Knudsen et al. (2006) detected orally inoculated Campylobacter associated with the liver of some broilers within 8 D post inoculation. However, it seems likely that liver surface Campylobacter could also be due to leakage of gut contents or other cross contamination during evisceration, presentation on the carcass, or transport to the lab. Campylobacter leaks from the alimentary tract during broiler processing (Berrang et al., 2001) and evisceration is linked to bacterial contamination (Byrd et al., 2002). From 16/70 (23%) of carcasses, liver surface was the only sample in which Campylobacter was detected. This suggests that the outer surface of some livers may be contaminated by a source other than cecal contents of the same carcass. In the current study, depending on daily plant line speed, broiler carcasses remained on the shackle line for 60 to 120 s between evisceration and liver collection. During that time, liver surface may have been exposed to Campylobacter-contaminated surfaces. In 4 instances, flocks sampled on consecutive days (April 10 and 11; August 28 and 29; October 23 and 24; December 4 and 5) had the same MLST subtype of Campylobacter (Table 2) which may suggest lingering machine surface contamination in the plant. It is also possible that liver surface contamination could be due to air sac contamination. Air sacs, which are unavoidably torn during evisceration, can be contaminated with Campylobacter (Berrang et al., 2003). Even when aseptically hand eviscerated, a broiler carcass cavity can have detectable numbers of Campylobacter due to air sac contamination (Berrang et al., 2002). Regardless of the source of the organism, current data show that the surface or internal tissue of chicken liver collected shortly after evisceration can be Campylobacter positive. Species and MLST subtype of Campylobacter detected from each sample from all 70 broiler carcasses/flocks are presented in Table 2. It is important to remember that the methods applied in the current study were not exhaustive. We selected just one example of the most prevalent colony type from each positive sample for whole genome sequencing and MLST subtyping; more subtypes may have been present in each sample. Most Campylobacter that we isolated were either C. jejuni or C. coli. From one broiler carcass, we detected a strain of C. upsaliensis from ceca and liver surface that were indistinguishable by genomic sequence. Overall, 58 carcasses (83%) yielded Campylobacter from at least 1 of 3 samples. For 11 of the 18 carcasses in which Campylobacter was detected in all 3 samples, the predominant colony types were indistinguishable suggesting a systemic colonization of the live broiler. From 6 broiler carcasses, a different subtype was detected inside the liver than in the ceca suggesting carcasses can be colonized with more than one subtype of Campylobacter. From 7 broiler carcasses, a different subtype was detected inside the liver than on the surface, showing that there can be multiple subtypes of
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Gene sequences were analyzed by aligning multilocus sequence typing (MLST) results for loci described by Dingle et al. (2001). Raw sequence read files for each isolate were mapped to reference sequences of each locus using Geneious Mapper (Biomatters Ltd, Aukland, NZ). Strict consensus for each mapping was trimmed to match the reference and then submitted to the website pubmlst.org/campylobacter/ (Jolley and Maiden, 2010) to obtain allelic identifiers and the clonal complex and sequence type of the isolate.
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Table 2. Species, MLST sequence type, and clonal complex of Campylobacter recovered from liver surface, internal liver tissue, or ceca from one commercially eviscerated broiler carcass from each of 70 unique flocks Flock (collection date)
Liver internal
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C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
– – – coli, 9448 (1150)2 coli, 1119 (828) jejuni, 48 (48) coli, 829 (828) jejuni, 353 (353) jejuni, 353 (353) jejuni, 939 (353) jejuni, 2517 (353) jejuni, 48 (48) jejuni, 353 (353) jejuni, 939 (353) coli, 9449 (1150) jejuni, 353 (353) – jejuni, 353 (353) jejuni, 467 (49) jejuni, 6647 (49) jejuni, 467 (49) coli, 3262 (828) coli, 3753 (828) – coli, 3262 (828) jejuni, 353 (353) coli, 9450 (1150) jejuni, 353 (353) coli, 1063 (828) jejuni, 467 (49) jejuni, 353 (353) jejuni, 457 (353) coli, 3262 (828) jejuni, 353 (353) – coli, 3262 (828) coli, 3262 (828) coli, 3262 (828) upsaliensis3 coli, 9448 (1150) jejuni, 9451 (n/a)4 jejuni, 353 (353) – – jejuni, 467 (49) jejuni, 6175 (21) jejuni, 6647 (49) jejuni, 6647 (49) jejuni, 6175 (21) jejuni, 2132 (353) – coli, 9448 (1150) – jejuni, 457 (353) jejuni, 50 (21) jejuni, 50 (21) Nonviable5 jejuni, 50 (21) jejuni, 467 (49) Nonviable5 coli, 1063 (828) coli, 41 (41) coli, 9448 (1150) jejuni, 353 (353) jejuni, 467 (49) coli, 1119 (828) – – jejuni, 939 (353)
C. C.
C. C.
C.
C. C. C. C. C. C.
C.
C.
C.
C.
C. C. C.
C.
– – – – coli, 9448 (1150) coli, 1119 (828) – – – – – jejuni, 12 (353) jejuni, 48 (48) – – – – – jejuni, 6475 (21) – – – – coli, 3753 (828) – – jejuni, 353 (353) – jejuni, 353 (353) coli, 1063 (828) – jejuni, 353 (353) – jejuni, 457 (353) – – – – – – – jejuni, 50 (21) – – – jejuni, 50 (21) – – – – – jejuni, 353 (353) – – – – jejuni, 50 (21) – – – – – coli, 9448 (1150) coli, 9448 (1150) – jejuni, 467 (49) – – – jejuni, 467 (49)
A—represents a sample in which no Campylobacter was detected. Campylobacter species, sequence type (clonal complex). The 2 C. upsaliensis isolates detected from flock 40 were indistinguishable by genomic sequence typing. 4 These isolates did not belong to any clonal complex at the time of publication. 5 These samples were positive but the isolates were not subtyped because they were nonviable from frozen storage. 1 2 3
Ceca
C. C. C. C. C. C. C. C. C. C. C.
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
– – – – coli, 9448 (1150) coli, 1119 (828) jejuni, 48 (48) – – jejuni, 353 (353) jejuni, 939 (353) jejuni, 2517 (353) jejuni, 48 (48) – – coli, 9449 (1150) jejuni, 353 (353) – jejuni, 353 (353) jejuni, 467 (49) – – – coli, 3753 (828) – – jejuni, 353 (353) – jejuni, 353 (353) coli, 1063 (828) jejuni, 467 (49) jejuni, 457 (353) – coli, 1063 (828) – – jejuni, 939 (353) – coli, 3262 (828) upsaliensis3 coli, 9448 (1150) jejuni, 939 (353) jejuni, 353 (353) – – jejuni, 467 (49) jejuni, 6175 (21) jejuni, 6647 (49) jejuni, 6647 (49) coli, 9452 (n/a)4 jejuni, 2132 (353) – coli, 9448 (1150) – jejuni, 457 (353) jejuni, 457 (353) jejuni, 50 (21) – jejuni, 50 (21) – – coli, 9453 (n/a)4 coli, 9448 (1150) coli, 9448 (1150) – jejuni, 467 (49) coli, 1119 (828) – – jejuni, 939 (353)
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1 (3/6/2017) 2 (3/7) 3 (3/13) 4 (3/14) 5 (3/20) 6 (3/27) 7 (4/3) 8 (4/4) 9 (4/10) 10 (4/11) 11 (4/17) 12 (4/18) 13 (4/24) 14 (4/25) 15 (5/1) 16 (5/2) 17 (5/8) 18 (5/9) 19 (5/15) 20 (6/5) 21 (6/12) 22 (6/13) 23 (6/19) 24 (6/20) 25 (6/21) 26 (7/17) 27 (7/18) 28 (7/24) 29 (7/25) 30 (7/31) 31 (8/1) 32 (8/7) 33 (8/8) 34 (8/14) 35 (8/15) 36 (8/22) 37 (8/28) 38 (8/29) 39 (9/5) 40 (9/18) 41 (9/19) 42 (9/25) 43 (9/26) 44 (10/2) 45 (10/3) 46 (10/16) 47 (10/17) 48 (10/23) 49 (10/24) 50 (10/30) 51 (10/31) 52 (11/13) 53 (11/14) 54 (11/27) 55 (11/28) 56 (12/4) 57 (12/5) 58 (12/11) 59 (12/18) 60 (1/2/18) 61 (1/16) 62 (1/23) 63 (1/29) 64 (1/30) 65 (2/5) 66 (2/12) 67 (2/20) 68 (2/26) 69 (2/27) 70 (3/12)
Liver surface
CECA AND LIVER CAMPYLOBACTER
ACKNOWLEDGMENTS The authors gratefully acknowledge expert technical assistance by Steven W. Knapp and Linda L. Genzlinger both of U.S. National Poultry Research Center, Athens, GA.
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Crawshaw, T. R., J. I. Chanter, S. C. L. Young, S. Cawthraw, A. M. Whatmore, M. S. Koylass, A. B. Vidal, F. J. Salguero, and R. M. Irvine. 2015. Isolation of a novel thermophilic Campylobacter from cases of spotty liver disease in laying hens and experimental reproduction of infection and microscopic pathology. Vet. Microbiol. 179:315–321. Dingle, K. E., F. M. Colles, D. R. Wareing, R. Ure, A. J. Fox, F. E. Bolton, H. J. Bootsma, R. J. Willems, R. Urwin, and M. C. Maiden. 2001. Multilocus sequence typing system for Campylobacter jejuni. J. Clin. Microbiol. 39:14–23. Gast, R. K., R. Guraya, D. R. Jones, and K. E. Anderson. 2013. Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages. Poult. Sci. 92:468–473. Glashower, D., J. Snyder, D. Welch, and S. McCarthy. 2017. Outbreak of Campylobacter jejuni associated with consuming undercooked chicken liver mousse – Clark County, Washington, 2016. MMWR. Morb. Mortal. Wkly. Rep. 66:1027. Gunther, N. W., A. Abdul-Wakeel, O. J. Scullen, and C. Sommers. 2019. The evaluation of gamma irradiation and cold storage for the reduction of Campylobacter jejuni in chicken livers. Food Microbiol. 82:249–253. Jolley, K., and M. Maiden. 2010. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics. 11:595. Knudsen, K. N., D. D. Bang, L. O. Andersen, and M. Madsen. 2006. Campylobacter jejuni strains of human and chicken origin are invasive in chickens after oral challenge. Avian Dis. 50:10–14. Lanier, W. A., K. R. Hale, A. L. Geissler, and D. Dewey-Mattia. 2018. Chicken liver-associated outbreaks of campylobacteriosis and salmonellosis, United States, 2000–2016: identifying opportunities for prevention. Foodborne Pathog. Dis. 15:726–733. Moffatt, C. R. M., A. Greig, M. Valcanis, W. Gao, T. Seeman, B. P. Howden, and M. D. Kirk. 2016. A large outbreak of Campylobacter jejuni infection in a university college caused by chicken liver pate, Australia, 2013. Epidemiol. Infect. 144:2971–2978. Nicorici, G., and H. B. Ghoddusi. 2017. Prevalence of Campylobacter contamination in raw chicken and chicken liver at retail. Food Safety Mag. 23:44–49. Noormohamed, A., and M. K. Fakhr. 2012. Incidence and antimicrobial resistance profiling of Campylobacter in retail chicken livers and gizzards. Foodborne Pathog. Dis. 9:617–624. O’Leary, M. C., O. Harding, L. Fisher, and J. Cowden. 2009. A continuous common-source outbreak of campylobacteriosis associated with changes to the preparation of chicken liver pate. Epidemiol. Infect. 137:383–388. Shane, S. 1991. Campylobacteriosis. Pages 236–246 in Diseases of Poultry. B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder, eds. Jr Iowa State Press, Ames, IA. Stern, N. J., B. Wojton, and K. Kwiatek. 1992. A Differentialselective medium and dry ice-generated atmosphere for recovery of Campylobacter jejuni. J. Food Prot. 55:514–517. Strachan, N. J. C., M. MacRae, A. Thomson, O. Rotariu, I. D. Ogden, and K. J. Forbes. 2012. Source attribution, prevalence and enumeration of Campylobacter spp. from retail liver. Int. J. Food Microbiol. 153:234–236. Tompkins, B. J., E. Wirsing, V. Devlin, L. Kamhi, B. Temple, K. Weening, S. Cavallo, L. Allen, P. Brinig, B. Goode, C. Fitzgerald, K. Heiman, S. Stroika, and B. Mahon. 2013. Multistate outbreak of Campylobacter jejuni infections associated with undercooked chicken livers – Northeaster United States, 2012. Morbid. Mortal. Weekly Rep. 62:874–876. Van, T. T. H., E. Elshagmani, M-C. Gor, A. Anwar, P. C. Scott, and R. J. Moore. 2017. Induction of spotty liver disease in layer hens by infection with Campylobacter hepaticus. Vet. Microbiol. 199:85–90. Van, T. T. H., M-C. Gor, A. Anwar, P. C. Scott, and R. J. Moore. 2017. Campylobacter hepaticus, the cause of spotty liver disease in chickens, is present throughout the small intestine and caeca of infected birds. Vet. Microbiol. 207:226–230. Whyte, R., J. A. Hudson, and C. Graham. 2006. Campylobacter in chicken livers and their destruction by pan frying. Lett. Appl. Microbiol. 43:591–595.
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Campylobacter associated with one liver shortly after evisceration. Of 41 carcasses in which Campylobacter was detected on both the liver surface and in the ceca, it was significantly more likely (P < 0.01) for both samples to have the same subtype than not. This relationship was not evident with internal liver tissue as it was not significantly more likely to yield the same colony type as ceca (P > 0.05). Interestingly, when both external and internal liver samples had detectable Campylobacter, it was just as likely that they be different subtypes as the same (P > 0.05). Multiple subtypes of Campylobacter have been previously reported on chicken livers (Berrang et al., 2018); however, that work was conducted at retail and may have reflected some liver-to-liver cross contamination in the package. The current data show a large percentage (83%) of carcasses/flocks can have Campylobacter in or on the liver at slaughter. It may be wise to apply antimicrobial treatments to liver at the slaughter plant prior to packing for retail sale. A recent report suggests that irradiation is effective to control Campylobacter numbers in chicken livers (Gunther et al., 2019). More research is needed to test practical and immediately applicable methods of chicken liver decontamination.
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Key words: broiler, Campylobacter, ceca, liver 2019 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pez340
INTRODUCTION
(Barot et al., 1983), or searing of the surface with hot metal (Berrang et al. 2018). There have been multiple outbreaks of campylobacteriosis traced to undercooked or mishandled chicken livers both within the United States and internationally (O’Leary et al., 2009; Tompkins et al., 2013; Moffatt et al., 2016; Glashower et al., 2017). A review of US outbreaks is available by Lanier et al. (2018). Some outbreaks have been traced to whole livers, many to chicken liver pate (Lanier et al., 2018). Overwhelmingly, the issue that led to foodborne illness was inadequate cooking of the liver dish (Lanier et al., 2018). Thorough cooking of livers is required to fully eliminate Campylobacter (Whyte et al., 2006). It has been shown with Salmonella that an oral inoculation can result in cecal colonization and translocation to internal organs including the liver (Gast et al., 2013). Oral inoculation with Campylobacter has also been shown to result in liver contamination, albeit in low percent of experimental 14-day-old broilers (Knudsen et al., 2006). Some Campylobacter, notably C. hepaticus, can cause liver disease in chickens (Crawshaw et al., 2015; Van et al., 2017a). This species of Campylobacter has been shown to be transferred to chickens by the fecal oral route and to colonize the gut as well as infect livers (Van et al., 2017b).
Campylobacter is a bacterial human pathogen associated with poultry and poultry products. It can be found on feathers and skin and in the gut contents of broilers presenting for slaughter (Berrang et al., 2000). Campylobacter has been detected on chicken livers as early as 1983 (Barot et al., 1983). Campylobacter has also been reported from chicken liver at retail, with prevalence ranging from 33 to 100% of livers tested (Barot et al., 1983; Whyte et al., 2006; Noormohamed and Fakhr, 2012; Strachan et al., 2012; Nicorici and Ghoddusi, 2017; Berrang et al., 2018). Efforts have been made to sample internal liver tissue separate from the surface. In several studies, Campylobacter was detected from the inside of livers which had been sanitized by either boiling water (Whyte et al., 2006), ethanol soak Published by Oxford University Press on behalf of Poultry Science Association 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US. Received April 3, 2019. Accepted July 1, 2019. 1 Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 2 Corresponding author: [email protected]
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face samples, and 19 liver internal tissue samples were positive. For 11 of 18 carcasses from which all samples were positive, the predominant colony types were indistinguishable. However, some carcasses did have multiple subtypes of Campylobacter. Of carcasses with Campylobacter on the surface of the liver and within the ceca, it was more likely that the subtypes be the same than different (P < 0.01). However, Campylobacter subtypes detected in internal liver tissue were not more likely to be the same as those detected in ceca (P > 0.05). We detected different subtypes of Campylobacter from internal liver tissue and liver surface of seven broiler carcasses/flocks. Livers from a large percentage of broiler carcasses/flocks can have one or more subtypes of Campylobacter.
ABSTRACT Foodborne campylobacteriosis has been linked to undercooked chicken liver. We have detected Campylobacter in chicken livers available at retail. The objective of the current project was to determine the prevalence and subtype of Campylobacter associated with livers and ceca of the same broiler carcasses at commercial slaughter. Within 2 min of commercial evisceration, we collected liver and ceca of one broiler carcass from each of 70 discreet flocks over a 12-mo period. Liver surface, liver internal tissue, and cecal contents were cultured for Campylobacter using standard methods. One example of the predominant colony type was selected from each positive sample for whole genome sequencing and multilocus sequence typing. We detected Campylobacter in at least one sample from 58 of 70 (83%) carcasses/flocks; 41 ceca, 57 liver sur-
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MATERIALS AND METHODS Experimental Overview This study was designed to sample many flocks by sampling broiler carcasses for presence of Campylobacter in the ceca and associated with the liver. To accomplish this, one carcass per sample day was selected such that many flocks were examined as opposed to many broilers from one flock. On 70 replicate sample days, representing 70 unique broiler flocks, 1 broiler carcass was selected post evisceration in a commercial slaughter plant. From each selected carcass, ceca and liver were collected and cultured for Campylobacter as described below.
then used to inoculate a Campy Cefex agar plate (CCA) (Stern et al., 1992) and a 9 mL tube of CEB. Following swabbing, an approximately 12 cm2 portion of the surface of each liver was sterilized by searing with a heated metal spatula. A sterile, CEB pre-moistened cotton-tipped applicator was used to press through the seared flesh into the internal tissue of the liver taking care to not completely penetrate out of the other side of the organ. The applicator was pressed into the internal tissue in 2 to 3 spots within the seared area. Each applicator was then used to inoculate a CCA plate and then placed into a 9 mL tube of CEB as described above.
Campylobacter Culture All CCA plates and CEB tubes were incubated at 42◦ C in re-sealable bags flushed with microaerobic gas (5% O2 , 10% CO2 , and 85% N2; AirgasUSA LLC, Radnor, PA). After 24 h, all samples in CEB were used to inoculate CCA plates. One tenth mL from each tube was streaked for isolation. All CCA plates were incubated as described above for 48 h. Colonies characteristics of Campylobacter were confirmed as such by observation of cellular morphology and motility under phase-contrast microscopy and positive reaction on a latex agglutination test (Microgen Bioproducts Ltd. Camberly, UK).
Sample Collection and Preparation Carcasses were selected that included complete viscera packs with an intact liver and intestinal tract. From each selected carcass, the liver and intestines were removed immediately after inspection. Timing depended on operational parameters and line speed on any given sample day; generally, samples were collected within 120s of automated evisceration. Entire viscera packs, just as presented on carcasses, were placed into clean new re-sealable plastic bags, covered with ice, and transported to the lab. Upon arrival, within 20 min, the liver and the ceca were separated from the viscera. Ceca were prepared for Campylobacter culture as previously described (Berrang et al., 2016). Briefly, using a clean nitrile glove, one cecum was transferred to a sample bag (Nasco, Fort Atkinson, WI), weighed, smashed to release contents, and diluted with 3 times volume to weight sterile phosphate buffered saline. Each cecum was subjected to blending in a paddle blender (Stomacher 80, Seward, Port St Lucie, FL) for 30 s before an aliquot was removed for dilution and direct plating as described below. The surface and inner tissue of livers was sampled as previously described (Berrang et al., 2018). Briefly, each liver was placed onto a sterile absorbent paper napkin. The outer surface was sampled by use of a sterile cotton-tipped applicator previously moistened in Campylobacter enrichment broth (CEB Accumedia Neogen Corp., Lansing, MI). Each applicator was rubbed over the entire ventral surface of a liver and
Campylobacter Subtyping Well-isolated representatives of the most common Campylobacter colony type found on each positive sample were subcultured and used for DNA sequencebased subtyping as previously described (Berrang et al., 2016). Each isolate was re-streaked for a lawn onto CCA and grown for 24 h at 42◦ C in a microaerobic atmosphere as described above. Bacterial growth was removed from plates with a sterile cotton tipped applicator and added to 1 mL of sterile freezing medium (blood and additive free CEB with 15% glycerol) in a freezing vial (Cryovial, Simport, Beloeil, QC, Canada). Cultures were frozen and held at –80◦ C until all isolates were collected. Isolates were revived from frozen storage by streaking for isolation onto the surface of tryptic soy agar with 5% sheep’s blood (Remel, Lenexa KS), and incubated under microaerobic conditions at 42◦ C for 48 h. One isolated colony was selected and streaked for a lawn on the surface of Brucella agar (Accumedia, Neogen Corp.), and incubated at 42◦ C for 24 h under microaerobic conditions. All growth was removed from Brucella agar, and DNA was extracted using a kit and manufacR turer’s instructions (UltraClean microbial DNA isolation Kit, Mo Bio Laboratories Inc., Carlsbad, CA). Libraries were prepared using the Nextera XT sample preparation kit (Illumina, San Diego, CA). Genomic DNA of each isolate was sequenced using the Illumina MiSeq platform with a 2 × 250 paired end run according to the manufacturer’s instructions (Illumina).
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The prevalence of Campylobacter in the livers of healthy broilers presenting for slaughter is not known. Nor is it known if the same subtypes of Campylobacter are present in the gut and liver of broiler chickens. The objective of this study was to compare frequency and subtype of Campylobacter detected from liver and ceca of broilers presented for commercial slaughter. To achieve this, we sampled both liver and ceca of one broiler from each of 70 flocks over the course of a 12-month period.
CECA AND LIVER CAMPYLOBACTER
Statistical Analysis Prevalence of Campylobacter from liver surface, inner tissue, and cecal contents were compared by chi square test for independence. Significance was assigned at P ≤ 0.05.
RESULTS AND DISCUSSION The number of broiler carcasses with different possible cultural outcomes from examination of ceca and liver is presented in Table 1. For 18 of 70 carcasses/flocks (25.7%), Campylobacter was detected in all samples (liver surface, internal liver tissue, and ceca). Campylobacter was significantly (P < 0.01) more likely to be detected inside the liver when the liver surface and/or ceca were also positive. This is in line with the finding that bacteria (Salmonella and Campylobacter) can invade a chicken’s liver following oral inoculation and intestinal colonization. (Knudsen et al., 2006; Gast et al., 2013). Campylobacter was detected in the ceca significantly (P < 0.01) more often than inside the liver. Campylobacter was detected the most often (P < 0.01) on liver surface (57/70, 81%) and was more likely to be detected on the liver surface when the ceca was also positive. In fact, every time the ceca was positive (41/70), the liver surface from the same carcass was also positive. It is possible that Campylobacter could have been on the liver surface in vivo as Campylobacter is related to liver disease in the chicken (Shane, 1991; Crawshaw et al., Table 1. Number of broiler carcasses1 with each possible Campylobacter culture outcome from cecal contents, liver surface, and liver inner tissue (n = 70). Campylobacter culture outcome by sample type Ceca + + + + – – – – 1
Outer liver
Inner liver
Number of samples
+ – + – + – + –
+ – – + + + – –
18 (25.7%) 0 23 (32.8%) 0 0 1 (1.4%) 16 (22.8%) 12 (17.1%)
Each carcass represents a unique broiler flock.
2015; Van et al., 2017a,b). Knudsen et al. (2006) detected orally inoculated Campylobacter associated with the liver of some broilers within 8 D post inoculation. However, it seems likely that liver surface Campylobacter could also be due to leakage of gut contents or other cross contamination during evisceration, presentation on the carcass, or transport to the lab. Campylobacter leaks from the alimentary tract during broiler processing (Berrang et al., 2001) and evisceration is linked to bacterial contamination (Byrd et al., 2002). From 16/70 (23%) of carcasses, liver surface was the only sample in which Campylobacter was detected. This suggests that the outer surface of some livers may be contaminated by a source other than cecal contents of the same carcass. In the current study, depending on daily plant line speed, broiler carcasses remained on the shackle line for 60 to 120 s between evisceration and liver collection. During that time, liver surface may have been exposed to Campylobacter-contaminated surfaces. In 4 instances, flocks sampled on consecutive days (April 10 and 11; August 28 and 29; October 23 and 24; December 4 and 5) had the same MLST subtype of Campylobacter (Table 2) which may suggest lingering machine surface contamination in the plant. It is also possible that liver surface contamination could be due to air sac contamination. Air sacs, which are unavoidably torn during evisceration, can be contaminated with Campylobacter (Berrang et al., 2003). Even when aseptically hand eviscerated, a broiler carcass cavity can have detectable numbers of Campylobacter due to air sac contamination (Berrang et al., 2002). Regardless of the source of the organism, current data show that the surface or internal tissue of chicken liver collected shortly after evisceration can be Campylobacter positive. Species and MLST subtype of Campylobacter detected from each sample from all 70 broiler carcasses/flocks are presented in Table 2. It is important to remember that the methods applied in the current study were not exhaustive. We selected just one example of the most prevalent colony type from each positive sample for whole genome sequencing and MLST subtyping; more subtypes may have been present in each sample. Most Campylobacter that we isolated were either C. jejuni or C. coli. From one broiler carcass, we detected a strain of C. upsaliensis from ceca and liver surface that were indistinguishable by genomic sequence. Overall, 58 carcasses (83%) yielded Campylobacter from at least 1 of 3 samples. For 11 of the 18 carcasses in which Campylobacter was detected in all 3 samples, the predominant colony types were indistinguishable suggesting a systemic colonization of the live broiler. From 6 broiler carcasses, a different subtype was detected inside the liver than in the ceca suggesting carcasses can be colonized with more than one subtype of Campylobacter. From 7 broiler carcasses, a different subtype was detected inside the liver than on the surface, showing that there can be multiple subtypes of
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Gene sequences were analyzed by aligning multilocus sequence typing (MLST) results for loci described by Dingle et al. (2001). Raw sequence read files for each isolate were mapped to reference sequences of each locus using Geneious Mapper (Biomatters Ltd, Aukland, NZ). Strict consensus for each mapping was trimmed to match the reference and then submitted to the website pubmlst.org/campylobacter/ (Jolley and Maiden, 2010) to obtain allelic identifiers and the clonal complex and sequence type of the isolate.
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Table 2. Species, MLST sequence type, and clonal complex of Campylobacter recovered from liver surface, internal liver tissue, or ceca from one commercially eviscerated broiler carcass from each of 70 unique flocks Flock (collection date)
Liver internal
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C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
– – – coli, 9448 (1150)2 coli, 1119 (828) jejuni, 48 (48) coli, 829 (828) jejuni, 353 (353) jejuni, 353 (353) jejuni, 939 (353) jejuni, 2517 (353) jejuni, 48 (48) jejuni, 353 (353) jejuni, 939 (353) coli, 9449 (1150) jejuni, 353 (353) – jejuni, 353 (353) jejuni, 467 (49) jejuni, 6647 (49) jejuni, 467 (49) coli, 3262 (828) coli, 3753 (828) – coli, 3262 (828) jejuni, 353 (353) coli, 9450 (1150) jejuni, 353 (353) coli, 1063 (828) jejuni, 467 (49) jejuni, 353 (353) jejuni, 457 (353) coli, 3262 (828) jejuni, 353 (353) – coli, 3262 (828) coli, 3262 (828) coli, 3262 (828) upsaliensis3 coli, 9448 (1150) jejuni, 9451 (n/a)4 jejuni, 353 (353) – – jejuni, 467 (49) jejuni, 6175 (21) jejuni, 6647 (49) jejuni, 6647 (49) jejuni, 6175 (21) jejuni, 2132 (353) – coli, 9448 (1150) – jejuni, 457 (353) jejuni, 50 (21) jejuni, 50 (21) Nonviable5 jejuni, 50 (21) jejuni, 467 (49) Nonviable5 coli, 1063 (828) coli, 41 (41) coli, 9448 (1150) jejuni, 353 (353) jejuni, 467 (49) coli, 1119 (828) – – jejuni, 939 (353)
C. C.
C. C.
C.
C. C. C. C. C. C.
C.
C.
C.
C.
C. C. C.
C.
– – – – coli, 9448 (1150) coli, 1119 (828) – – – – – jejuni, 12 (353) jejuni, 48 (48) – – – – – jejuni, 6475 (21) – – – – coli, 3753 (828) – – jejuni, 353 (353) – jejuni, 353 (353) coli, 1063 (828) – jejuni, 353 (353) – jejuni, 457 (353) – – – – – – – jejuni, 50 (21) – – – jejuni, 50 (21) – – – – – jejuni, 353 (353) – – – – jejuni, 50 (21) – – – – – coli, 9448 (1150) coli, 9448 (1150) – jejuni, 467 (49) – – – jejuni, 467 (49)
A—represents a sample in which no Campylobacter was detected. Campylobacter species, sequence type (clonal complex). The 2 C. upsaliensis isolates detected from flock 40 were indistinguishable by genomic sequence typing. 4 These isolates did not belong to any clonal complex at the time of publication. 5 These samples were positive but the isolates were not subtyped because they were nonviable from frozen storage. 1 2 3
Ceca
C. C. C. C. C. C. C. C. C. C. C.
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
– – – – coli, 9448 (1150) coli, 1119 (828) jejuni, 48 (48) – – jejuni, 353 (353) jejuni, 939 (353) jejuni, 2517 (353) jejuni, 48 (48) – – coli, 9449 (1150) jejuni, 353 (353) – jejuni, 353 (353) jejuni, 467 (49) – – – coli, 3753 (828) – – jejuni, 353 (353) – jejuni, 353 (353) coli, 1063 (828) jejuni, 467 (49) jejuni, 457 (353) – coli, 1063 (828) – – jejuni, 939 (353) – coli, 3262 (828) upsaliensis3 coli, 9448 (1150) jejuni, 939 (353) jejuni, 353 (353) – – jejuni, 467 (49) jejuni, 6175 (21) jejuni, 6647 (49) jejuni, 6647 (49) coli, 9452 (n/a)4 jejuni, 2132 (353) – coli, 9448 (1150) – jejuni, 457 (353) jejuni, 457 (353) jejuni, 50 (21) – jejuni, 50 (21) – – coli, 9453 (n/a)4 coli, 9448 (1150) coli, 9448 (1150) – jejuni, 467 (49) coli, 1119 (828) – – jejuni, 939 (353)
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1 (3/6/2017) 2 (3/7) 3 (3/13) 4 (3/14) 5 (3/20) 6 (3/27) 7 (4/3) 8 (4/4) 9 (4/10) 10 (4/11) 11 (4/17) 12 (4/18) 13 (4/24) 14 (4/25) 15 (5/1) 16 (5/2) 17 (5/8) 18 (5/9) 19 (5/15) 20 (6/5) 21 (6/12) 22 (6/13) 23 (6/19) 24 (6/20) 25 (6/21) 26 (7/17) 27 (7/18) 28 (7/24) 29 (7/25) 30 (7/31) 31 (8/1) 32 (8/7) 33 (8/8) 34 (8/14) 35 (8/15) 36 (8/22) 37 (8/28) 38 (8/29) 39 (9/5) 40 (9/18) 41 (9/19) 42 (9/25) 43 (9/26) 44 (10/2) 45 (10/3) 46 (10/16) 47 (10/17) 48 (10/23) 49 (10/24) 50 (10/30) 51 (10/31) 52 (11/13) 53 (11/14) 54 (11/27) 55 (11/28) 56 (12/4) 57 (12/5) 58 (12/11) 59 (12/18) 60 (1/2/18) 61 (1/16) 62 (1/23) 63 (1/29) 64 (1/30) 65 (2/5) 66 (2/12) 67 (2/20) 68 (2/26) 69 (2/27) 70 (3/12)
Liver surface
CECA AND LIVER CAMPYLOBACTER
ACKNOWLEDGMENTS The authors gratefully acknowledge expert technical assistance by Steven W. Knapp and Linda L. Genzlinger both of U.S. National Poultry Research Center, Athens, GA.
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Crawshaw, T. R., J. I. Chanter, S. C. L. Young, S. Cawthraw, A. M. Whatmore, M. S. Koylass, A. B. Vidal, F. J. Salguero, and R. M. Irvine. 2015. Isolation of a novel thermophilic Campylobacter from cases of spotty liver disease in laying hens and experimental reproduction of infection and microscopic pathology. Vet. Microbiol. 179:315–321. Dingle, K. E., F. M. Colles, D. R. Wareing, R. Ure, A. J. Fox, F. E. Bolton, H. J. Bootsma, R. J. Willems, R. Urwin, and M. C. Maiden. 2001. Multilocus sequence typing system for Campylobacter jejuni. J. Clin. Microbiol. 39:14–23. Gast, R. K., R. Guraya, D. R. Jones, and K. E. Anderson. 2013. Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages. Poult. Sci. 92:468–473. Glashower, D., J. Snyder, D. Welch, and S. McCarthy. 2017. Outbreak of Campylobacter jejuni associated with consuming undercooked chicken liver mousse – Clark County, Washington, 2016. MMWR. Morb. Mortal. Wkly. Rep. 66:1027. Gunther, N. W., A. Abdul-Wakeel, O. J. Scullen, and C. Sommers. 2019. The evaluation of gamma irradiation and cold storage for the reduction of Campylobacter jejuni in chicken livers. Food Microbiol. 82:249–253. Jolley, K., and M. Maiden. 2010. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics. 11:595. Knudsen, K. N., D. D. Bang, L. O. Andersen, and M. Madsen. 2006. Campylobacter jejuni strains of human and chicken origin are invasive in chickens after oral challenge. Avian Dis. 50:10–14. Lanier, W. A., K. R. Hale, A. L. Geissler, and D. Dewey-Mattia. 2018. Chicken liver-associated outbreaks of campylobacteriosis and salmonellosis, United States, 2000–2016: identifying opportunities for prevention. Foodborne Pathog. Dis. 15:726–733. Moffatt, C. R. M., A. Greig, M. Valcanis, W. Gao, T. Seeman, B. P. Howden, and M. D. Kirk. 2016. A large outbreak of Campylobacter jejuni infection in a university college caused by chicken liver pate, Australia, 2013. Epidemiol. Infect. 144:2971–2978. Nicorici, G., and H. B. Ghoddusi. 2017. Prevalence of Campylobacter contamination in raw chicken and chicken liver at retail. Food Safety Mag. 23:44–49. Noormohamed, A., and M. K. Fakhr. 2012. Incidence and antimicrobial resistance profiling of Campylobacter in retail chicken livers and gizzards. Foodborne Pathog. Dis. 9:617–624. O’Leary, M. C., O. Harding, L. Fisher, and J. Cowden. 2009. A continuous common-source outbreak of campylobacteriosis associated with changes to the preparation of chicken liver pate. Epidemiol. Infect. 137:383–388. Shane, S. 1991. Campylobacteriosis. Pages 236–246 in Diseases of Poultry. B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder, eds. Jr Iowa State Press, Ames, IA. Stern, N. J., B. Wojton, and K. Kwiatek. 1992. A Differentialselective medium and dry ice-generated atmosphere for recovery of Campylobacter jejuni. J. Food Prot. 55:514–517. Strachan, N. J. C., M. MacRae, A. Thomson, O. Rotariu, I. D. Ogden, and K. J. Forbes. 2012. Source attribution, prevalence and enumeration of Campylobacter spp. from retail liver. Int. J. Food Microbiol. 153:234–236. Tompkins, B. J., E. Wirsing, V. Devlin, L. Kamhi, B. Temple, K. Weening, S. Cavallo, L. Allen, P. Brinig, B. Goode, C. Fitzgerald, K. Heiman, S. Stroika, and B. Mahon. 2013. Multistate outbreak of Campylobacter jejuni infections associated with undercooked chicken livers – Northeaster United States, 2012. Morbid. Mortal. Weekly Rep. 62:874–876. Van, T. T. H., E. Elshagmani, M-C. Gor, A. Anwar, P. C. Scott, and R. J. Moore. 2017. Induction of spotty liver disease in layer hens by infection with Campylobacter hepaticus. Vet. Microbiol. 199:85–90. Van, T. T. H., M-C. Gor, A. Anwar, P. C. Scott, and R. J. Moore. 2017. Campylobacter hepaticus, the cause of spotty liver disease in chickens, is present throughout the small intestine and caeca of infected birds. Vet. Microbiol. 207:226–230. Whyte, R., J. A. Hudson, and C. Graham. 2006. Campylobacter in chicken livers and their destruction by pan frying. Lett. Appl. Microbiol. 43:591–595.
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Campylobacter associated with one liver shortly after evisceration. Of 41 carcasses in which Campylobacter was detected on both the liver surface and in the ceca, it was significantly more likely (P < 0.01) for both samples to have the same subtype than not. This relationship was not evident with internal liver tissue as it was not significantly more likely to yield the same colony type as ceca (P > 0.05). Interestingly, when both external and internal liver samples had detectable Campylobacter, it was just as likely that they be different subtypes as the same (P > 0.05). Multiple subtypes of Campylobacter have been previously reported on chicken livers (Berrang et al., 2018); however, that work was conducted at retail and may have reflected some liver-to-liver cross contamination in the package. The current data show a large percentage (83%) of carcasses/flocks can have Campylobacter in or on the liver at slaughter. It may be wise to apply antimicrobial treatments to liver at the slaughter plant prior to packing for retail sale. A recent report suggests that irradiation is effective to control Campylobacter numbers in chicken livers (Gunther et al., 2019). More research is needed to test practical and immediately applicable methods of chicken liver decontamination.
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