Research Note Polymerase chain reaction detection of naturally occurring Campylobacter in commercial broiler chicken embryos K. L. Hiett,1 N. A. Cox, and M. J. Rothrock Jr. USDA, Agricultural Research Service, Poultry Microbiological Safety Research Unit, Richard B. Russell Research Center, 950 College Station Road, Athens, GA 30605 ABSTRACT Campylobacter, a foodborne pathogen closely associated with poultry, is recognized as a leading bacterial etiologic agent of human gastroenteritis in the United States. In this investigation, 2 trials were performed where tissues from 7-, 14/15-, and 19-d-old commercial broiler chicken embryos were tested for the presence of Campylobacter using both culturing methodology and PCR. Conventional culturing methods failed to detect Campylobacter from any samples tested during this investigation. Using a set of primers specific for the Campylobacter flagellinA short variable region (flaA SVR), Campylobacter DNA was amplified in 100, 80, and 100% of gastrointestinal tracts from 7-, 15-, and 19-d-old embryos, respectively, in the first trial. Similarly, Campylobacter DNA was detected in 100, 70, and
60% of gastrointestinal tracts of 7-, 14-, and 18-d-old embryos, respectively, in the second trial. In both trials, yolk sac, albumin, and liver/gallbladder samples from 19-d-old embryos all failed to produce amplicons indicative of Campylobacter DNA. Subsequent DNA sequence analyses of the flaA SVR PCR products were consistent with the amplicon arising from Campylobacter. Although a determination of whether the Campylobacter was living or dead within the embryos could not be made, these results demonstrate that Campylobacterspecific DNA is present within the gastrointestinal tract of broiler chicken embryos; however, the means by which it is present and the relative contribution to subsequent Campylobacter contamination of poultry flocks requires further investigation.
Key words: Campylobacter, broiler, embryo, polymerase chain reaction, gastrointestinal tract 2013 Poultry Science 92:1134–1137 http://dx.doi.org/10.3382/ps.2012-02812
INTRODUCTION Campylobacter, a gram-negative, microaerophilic bacteria, is a leading bacterial etiologic agent of acute gastroenteritis in the human population; it is estimated that Campylobacter enteritis affects approximately 1 to 2% of the US population per year (Tauxe, 1992; Humphrey et al., 2007; Scallan et al., 2011). Handling and consumption of poultry or poultry-related products are considered to be a primary sources for Campylobacterinduced disease in humans (Park et al., 1981; Kinde et al., 1983; Bryan and Doyle, 1995; Batz et al., 2012). In terms of the poultry production continuum, Campylobacter has been cultured from as many as 75% of the live broiler population and from as much as 80% of processed poultry meat samples sold commercially (Zhao et al., 2001; Hiett et al., 2007; Luber and Bartelt, 2007). The high colonization incidence of poultry and the resultant clinical infections in humans have ©2013 Poultry Science Association Inc. Received September 28, 2012. Accepted December 2, 2012. 1 Corresponding author:
[email protected]
prompted several investigations focused on identifying and subsequently eliminating sources of Campylobacter contamination in chickens. However, the routes of transmission involved in Campylobacter contamination of broiler flocks continue to remain unclear. Transmission of Campylobacter from breeder flocks to broiler offspring has traditionally been dismissed as a source of contamination due to the lack of culturebased detection of Campylobacter from newly hatched chicks (Acuff et al., 1982; Doyle, 1984; Shanker et al., 1986). However, several studies have demonstrated the presence of Campylobacter in both breeder hen reproductive tracts and in breeder rooster semen (Buhr et al., 2002; Hiett et al., 2003; Cox et al., 2005), which indicates the potential for the spread of Campylobacter from parent lines to progeny. Additionally, recent investigations demonstrated the ability of Campylobacter to penetrate artificially inoculated eggshells leading to the contamination of the embryo (Fonseca et al., 2011). Lastly, the detection and recovery of Campylobacter from hatchery-associated environmental samples, including hatchery debris (fluff), eggshells, and trayliners from commercial broiler hatcheries (Hiett et al., 2002b; Byrd et al., 2007; Messelhäusser et al., 2011),
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further supports the hypothesis that the fertilized egg may contribute to subsequent broiler flock contamination. In an effort to investigate natural Campylobacter contamination of fertilized eggs, tissues from commercial broiler chicken embryos were tested for the presence of Campylobacter using both traditional culturing methodology and molecular-based PCR.
MATERIALS AND METHODS Embryo Sample Collection In 2 individual trials, fertile broiler chicken eggs (7, 14/15, and 19 d of incubation) were obtained from a commercial broiler hatchery. Ten fertile eggs were obtained for each sample age in each trial. The shell, over the air cell, of each egg was aseptically removed; membranes were detached and the embryo was removed into sterile Petri dish. Embryos were immediately killed by sharp cervical dislocation. Seven- and 14/15-d-old embryos were dissected such that sampled tissues included the entire gastrointestinal (GI) tract; the yolk sac was not included. Nineteen-day-old embryos were similarly dissected. However, in addition to the GI tract, the liver (including gallbladder) and the yolk sac were separately dissected and analyzed.
Culturing Methodology Each sampled component was placed into a sterile plastic bag, diluted 1:3 (wt/vol) in 1× PBS, and stomached for 1 min. One hundred microliters of homogenate was removed, direct-plated onto Campy-Cefex agar, and incubated at 42°C for 48 h in a microaerobic atmosphere (5% O2, 10% CO2, 85% N2) according to previous protocols developed within our laboratory (Stern et al., 1992). Additionally, one hundred microliters of homogenate was enriched in Bolton’s enrichment broth at 37°C for 4 h followed by incubation at 42°C for 48 h in a microaerobic atmosphere (Musgrove et al., 2001). One hundred microliters of enrichment broth was plated onto Campy-Cefex agar. Plates were incubated as previously described.
Template Preparation and flaA SVR Gene Amplification by PCR Total cellular DNA was isolated from each GI and liver/gallbladder sample using the Isoquick DNA Isolation Kit (Orca Research Inc., Bothell, WA) according to the manufacturer’s specifications. Yolk sac samples were diluted 1:3 in sterile 1× PBS and thoroughly mixed. Yolk sac suspensions were centrifuged at 2,000 × g for 25 min at 20°C, and the pellets discarded. Globulins were precipitated from the supernatants using 40% (vol/vol) saturated ammonium sulfate (SAS). Following thorough mixing, the SAS-treated supernatants were allowed to stand at 4°C for 60 min. The treated samples were then centrifuged at 2,000 × g for
30 min at 20°C, and the pellets were discarded. Albumen samples were diluted 1:3 (vol/vol) in 1× PBS and thoroughly mixed. The samples were then clarified by centrifugation at 2,000 × g for 25 min at 20°C, and the pellets were discarded. Five hundred microliters of each suspension (homogenized GI or liver/gallbladder tissue, treated yolk or albumin material, or enrichment broth) was removed from each sample, placed into a sterile microcentrifuge tube, and placed at 100°C for 10 min. Boiled homogenates and total cellular DNA extracts were used as template for the flagellinA short variable region (flaA SVR) PCR as previously described (Meinersmann et al., 1997). The limit of detection for this primer set was found to be ~102 cfu/mL (Ridley et al., 2008).
flaA SVR DNA Sequence Analyses Sequence data were generated using either the FLA242FU primer or the FLA625RU primer with the Big-Dye Dye-Terminator Cycle Sequencing Kit (Meinersmann et al., 1997; ABI-PE, Foster City, CA). Data were assembled with Sequencher 4.8 (GeneCodes Corp., Ann Arbor, MI) and aligned using ClustalX (Thompson et al., 1994). Aligned sequences were compared and dendrograms generated using the NeighborJoining algorithm with HKY85 distance measurements in PAUP*4.0 (Phylogenetic Analysis Using Parsimony) (Swofford, 1998).
RESULTS AND DISCUSSION All samples, in both trials, were negative for Campylobacter using culturing methodology (direct plating and selective enrichment). Polymerase chain reaction, using a set of primers specific for the Campylobacter flagellinA short variable region (flaA SVR), was performed directly on all sampled material homogenates, as well as on the extracted total cellular DNA from GI and liver/gallbladder samples. In the first trial, positive amplicons were detected in 40, 60, and 20% of the direct homogenate of GI tracts from the 7-, 15-, and 19-d-old embryos, respectively (Table 1). Comparatively, when using the total cellular DNA extracted from the GI tracts, flaA SVR amplicons were detected in 100, 80, and 100%, of the 7-, 15-, and 19-d-old embryos, respectively (Table 1). These results show a 33 to 400% increase in detection when using total cellular DNA GI extracts compared with the direct GI homogenate analyses. In the second trial, flaA SVR amplicons were detected in all but one sample, and were detected as frequently, or more frequently (>100% increase), when using total cellular DNA extracts as the PCR template (100, 70, and 60%) compared with the direct GI homogenates (50, 70, and 0%) from the 7-, 14-, and 19-d-old embryos, respectively (Table 1). These results indicate that the ability to detect Campylobacter DNA from embryonic GI samples is reduced when using a homogenate as the PCR template, mostly likely due
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Table 1. flaA
SVR1
PCR amplification data Gastrointestinal
Item Trial 1 7 d 15 d 19 d Trial 2 7 d 14 d 19 d
Liver/gallbladder
Direct
DNA
4/10 (40%) 6/10 (60%) 2/10 (20%) 5/10 (50%) 7/10 (70%) 0/10 (0%)
10/10 (100%) 8/10 (80%) 10/10 (100%) 10/10 (100%) 7/10 (70%) 6/10 (60%)
Direct nd* nd* 0 nd* nd* 0
DNA nd* nd* 0 nd* nd* 0
Yolk sac
Albumin
Direct
Direct
nd* nd* 0 nd* nd* 0
nd* nd* 0 nd* nd* 0
1SVR
= short variable region. * = not done.
to the 3-fold dilution of the sample with prepping the sample for stomaching; therefore, total DNA extraction is preferred for subsequent Campylobacter flaA SVR detection from embryonic tissue samples. A strong correlation between culturing and PCR-based detection methods within eggs has been previously shown (Sahin et al., 2003), but Campylobacter detected from those eggs were from naturally contaminated and artificially inoculated whole egg samples (not limited to the GI tract), and culturing methods were based on selective enrichments, not direct plating. The enhanced detection of Campylobacter within embryonic chicks using PCR-based methods is in agreement with what has been more recently described in artificially inoculated vitellus samples of SPF and heavy breeder eggs (Fonseca et al., 2011). The liver/gallbladder, yolk sac, and albumin samples from the 19-d-old embryos (both trials) were all PCR negative (Table 1). These results suggest that liver/ gallbladder, yolk sac, and albumen samples were not contaminated by Campylobacter, which was not entirely unexpected. Whereas Campylobacter has been detected culturally, molecularly, or both in the contents of unfertilized table eggs (Adesiyun et al., 2005; Messelhäusser et al., 2011), an extensive survey of thousands of fertilized eggs from 5 commercial broiler breeder flocks and a commercial hatchery was unable to detect Campylobacter within the egg contents (including the yolk and albumin) by culturing or PCR-based methods, even when the broiler breeder flocks themselves demonstrated >50% Campylobacter prevalence (Sahin et al., 2003). Alternatively, the extraction methods used for these samples in this investigation may have been inadequate. Further investigations are necessary to optimize the sample extraction/purification techniques for use in subsequent flaA SVR PCR assays. For both trials, sequences from all amplicons were approximately 400 base pairs in length, consistent with the amplicon size expected for the flaA SVR genetic region of Campylobacter. In trial 1, all GI tract flaA SVR sequences (regardless of age of embryonic development) were identical. Amplicons sequenced in Trial 2 revealed the presence of 2 closely related (based on DNA sequence similarity) Campylobacter clones from the in-
testinal samples. Comparison of flaA SVR sequences between the 2 trials revealed that different Campylobacter clones were amplified during each trial (data not shown). The fact that unique flaA SVR clones were found between the 2 trials and within the second trial was unsurprising given the wealth of evidence describing the diversity of Campylobacter flaA SVR clones found throughout the poultry production continuum (Hiett et al., 2002a,c, 2003; O’Mahony et al., 2011). The findings in this study demonstrate that Campylobacter DNA is indeed present within the GI tract of embryonic chickens; however, a determination of whether the organism is living, dead, or in a nonculturable state could not be made. Additional studies are needed to further elucidate the mechanisms by which Campylobacter colonization occurs within fertile chicken embryos. Information such as that presented in this report is necessary to provide a basis for refining or adjusting intervention strategies to produce safer poultry food products, thereby reducing the risk of human exposure.
REFERENCES Acuff, G. R., C. Vanderzandt, F. A. Gardner, and F. A. Golan. 1982. Examination of turkey eggs, poults, and brooder house facilities for Campylobacter jejuni. J. Food Prot. 45:1279–1281. Adesiyun, A., N. Offiah, N. Seepersadsingh, S. Rodrigo, V. Lashley, L. Musai, and K. Georges. 2005. Microbial health risk posed by table eggs in Trinidad. Epidemiol. Infect. 133:1049–1056. http://dx.doi.org/10.1017/S0950268805004565. Batz, M. B., S. Hoffmann, and J. Morris. 2012. Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation. J. Food Prot. 75:1278–1291. Bryan, F. L., and M. P. Doyle. 1995. Health risks and consequences of Salmonella and Campylobacter jejuni in raw poultry. J. Food Prot. 58:326–344. Buhr, R. J., N. A. Cox, N. J. Stern, M. T. Musgrove, J. L. Wilson, and K. L. Hiett. 2002. Recovery of Campylobacter from segments of the reproductive tract of broiler breeder hens. Avian Dis. 46:919–924. Byrd, J., II, R. H. Bailey, R. W. Wills, and D. J. Nisbet. 2007. Recovery of Campylobacter from commercial broiler hatchery trayliners. Poult. Sci. 86:26–29. Cox, N. A., J. S. Bailey, L. J. Richardson, R. J. Buhr, D. E. Cosby, J. L. Wilson, K. L. Hiett, G. R. Siragusa, and D. V. Bourassa. 2005. Presence of naturally occurring Campylobacter and Salmonella in the mature and immature ovarian follicles of late-
RESEARCH NOTE life broiler breeder hens. Avian Dis. 49:285–287. http://dx.doi. org/10.1637/7324-011005. Doyle, M. P. 1984. Association of Campylobacter jejuni with laying hens and eggs. Appl. Environ. Microbiol. 47:533–536. Fonseca, B. B., M. E. Beletti, R. T. Melo, E. P. Mendonca, C. U. Vieira, M. A. Levenhagen, and D. A. Rossi. 2011. Transfer, viability and colonisation of Campylobacter jejuni in the chicken vitellus and in embryos. Br. Poult. Sci. 52:279–286. http:// dx.doi.org/10.1080/00071668.2011.577056. Hiett, K. L., N. A. Cox, J. Buhr, and N. J. Stern. 2002a. Genotype analyses of Campylobacter isolated from distinct segments of the reproductive tracts of broiler breeder hens. Curr. Microbiol. 45:400–404. Hiett, K. L., N. A. Cox, and N. J. Stern. 2002b. Direct polymerase chain reaction detection of Campylobacter spp. in poultry hatchery samples. Avian Dis. 46:219–223. http://dx.doi. org/10.1637/0005-2086(2002)046[0219:dpcrdo]2.0.co;2. Hiett, K. L., G. R. Siragusa, N. A. Cox, R. J. Buhr, M. T. Musgrove, N. J. Stern, and J. L. Wilson. 2003. Genotype analyses of Campylobacter isolated from the gastrointestinal tracts and the reproductive tracts of broiler breeder roosters. Avian Dis. 47:406– 414. http://dx.doi.org/10.1637/0005-2086(2003)047[0406:gaocif] 2.0.co;2. Hiett, K. L., N. J. Stern, P. Fedorka-Cray, N. A. Cox, M. T. Musgrove, and S. Ladely. 2002c. Molecular subtype analyses of Campylobacter spp. from Arkansas and California poultry operations. Appl. Environ. Microbiol. 68:6220–6236. Hiett, K. L., N. J. Stern, P. Fedorka-Cray, N. A. Cox, and B. S. Seal. 2007. Molecular phylogeny of the flaA short variable region among Campylobacter jejuni isolates collected during an annual evaluation of poultry flocks in the Southeastern United States. Foodborne Pathog. Dis. 4:339–347. http://dx.doi.org/10.1089/ fpd.2007.0008. Humphrey, T., S. O’Brien, and M. Madsen. 2007. Campylobacters as zoonotic pathogens: A food production perspective. Int. J. Food Microbiol. 117:237–257. http://dx.doi.org/10.1016/j.ijfoodmicro.2007.01.006. Kinde, H., C. A. Genigeorgis, and M. Pappaioanoi. 1983. Prevalence of Campylobacter jejuni on chicken wings. Appl. Environ. Microbiol. 45:1116–1118. Luber, P., and E. Bartelt. 2007. Enumeration of Campylobacter spp. on the surface and within chicken breast filets. J. Appl. Microbiol. 102:313–318. Meinersmann, R., L. Helsel, P. Fields, and K. L. Hiett. 1997. Discrimination of Campylobacter jejuni isolates by fla gene sequencing. J. Clin. Microbiol. 33:2810–2814. Messelhäusser, U., D. Tharigen, D. Elmer-Englhard, H. Bauer, H. Schreiner, and C. Holler. 2011. Occurrence of thermotolerant
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Campylobacter spp. on eggshells: A missing link for food-borne infections? Appl. Environ. Microbiol. 77:3896–3897. Musgrove, M. T., M. E. Berrang, J. A. Byrd, N. J. Stern, and N. A. Cox. 2001. Detection of Campylobacter spp. in ceca and crops with and without enrichment. Poult. Sci. 80:825–828. O’Mahony, E., J. F. Buckley, D. Bolton, P. Whyte, and S. Fanning. 2011. Molecular epidemiology of Campylobacter isolates from poultry production units in southern Ireland. PLoS ONE 6:e28490 http://dx.doi.org/10.1371/journal.pone.0028490. Park, C. E., Z. K. Stankiewicz, J. Lovett, and J. Hunt. 1981. Incidence of Campylobacter jejuni in fresh eviscerated whole market chickens. Can. J. Microbiol. 27:841–842. Ridley, A. M., V. M. Allen, M. Sharma, J. A. Harris, and D. G. Newell. 2008. Real-time PCR approach for detection of environmental sources of Campylobacter strains colonizing broiler flocks. Appl. Environ. Microbiol. 74:2492–2504. Sahin, O., P. Kobalka, and Q. Zhang. 2003. Detection and survival of Campylobacter in chicken eggs. J. Appl. Microbiol. 95:1070– 1079. http://dx.doi.org/10.1046/j.1365-2672.2003.02083.x. Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, M.-A. Widdowson, S. L. Roy, J. L. Jones, and P. M. Griffin. 2011. Foodborne illness acquired in the United States—Major pathogens. Emerg. Infect. Dis. 17:7–15. Shanker, S., A. Lee, and T. C. Sprrell. 1986. Campylobacter jejuni in broilers: The role of vertical transmission. J. Hyg. (Lond.) 96:153–159. Stern, N. J., B. Wojton, and K. Kwiatek. 1992. A differential-selective medium and dry ice-generated atmosphere for recovery of Campylobacter jejuni. J. Food Prot. 55:514–517. Swofford, D. 1998. PAUP*: Phylogenetic Analysis Using Parsimony and other methods, version 4.0. Sinauer Associates, Sunderland, MA. Tauxe, R. V. 1992. Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. Pages 9–19 in Campylobacter jejuni: Current Status and Future Trends, 1st ed. I. Nachamkin, M. J. Blaser, and L. S. Thompkins, ed. American Society for Microbiology, Washington, DC. Thompson, J., D. Higgins, and T. Gibson. 1994. CLUSTAL X: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice. Nucleic Acids Res. 22:4673–4680. Zhao, C., B. Ge, J. De Villena, R. Sudler, E. Yeh, S. Zhao, D. G. White, D. Wagner, and J. Meng. 2001. Prevalence of Campylobacter spp., Escherichia coli, and Salmonella serovars in retail chicken, turkey, pork, and beef from the Greater Washington, D.C. area. Appl. Environ. Microbiol. 67:5431–5436.
