Journal of Applied Microbiology 2003, 95, 10701079

doi:10.1046/j.1365-2672.2003.02083.x

Detection and survival of Campylobacter in chicken eggs

O. Sahin, P. Kobalka and Q. Zhang
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, and Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, USA
2003/0120: received 12 February 2003, revised 6 July 2003 and accepted 7 July 2003

ABSTRACT
O . S A H I N , P . K O B A L K A A N D Q . Z H A N G . 2003.

Aims: Campylobacter jejuni, a food-borne human pathogen, is widespread in poultry; however, the sources of infection and modes of transmission of this organism on chicken farms are not well understood. The objective of this study was to determine if vertical transmission of C. jejuni occurs via eggs. Methods and Results: Using a temperature differential method, it was shown that Campylobacter had limited ability to penetrate the eggshell. When C. jejuni was directly inoculated into the egg yolk and the eggs were stored at 18C, the organism was able to survive for up to 14 days. However, viability of C. jejuni was dramatically shortened when injected into the albumen or the air sac. When freshly laid eggs from Campylobacter-inoculated specic pathogen-free (SPF) layers were tested, C. jejuni-contamination was detected in three of 65 pooled whole eggs (510 eggs in each pool) via culture and PCR. However, the organism was not detected from any of the 800 eggs (80 pools), collected from the same SPF ock, but kept at 18C for 7 days before testing. Likewise, Campylobacter was not recovered from any of 500 fresh eggs obtained from commercial broiler-breeder ocks that were actively shedding Campylobacter in faeces. Also, none of the 1000 eggs from broiler breeders obtained from a commercial hatchery were positive for Campylobacter. Conclusions: These results suggest that vertical transmission of C. jejuni through the egg is probably a rare event and does not play a major role in the introduction of Campylobacter to chicken ocks. Signicance and Impact of the Study: Control of Campylobacter transmission to chicken ocks should focus on sources of infection that are not related to eggs. Keywords: Campylobacter, chicken, egg, prevalence, transmission.

INTRODUCTION Campylobacter jejuni is the leading cause of bacterial foodborne illnesses in the United States and other developed countries (Friedman et al. 2000; Adak et al. 2002), and is also frequently associated with diarrhoea in patients <6 months of age in developing countries (Coker et al. 2002). An estimated 2125 million cases of human campylobacteriosis, characterized by watery and/or bloody
Present address: Orhan Sahin, Veterinary Faculty, Department of Microbiology, Mustafa Kemal University, Hatay, Turkey.
Correspondence to: Qijing Zhang, Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, 1116 Veterinary Medicine Building, Ames, IA 50011, USA (e-mail: zhang123@iastate.edu).

diarrhoea, occur annually in the United States, exceeding the cases of salmonellosis (Friedman et al. 2000). In addition, C. jejuni is recognized as the most frequent antecedent of GuillainBarre Syndrome, an acute demyelinating disorder of peripheral nerves, which may lead to respiratory muscle compromise and death (Nachamkin et al. 1998; Wassenaar and Blaser 1999). The vast majority of human campylobacteriosis cases occur sporadically, and primarily result from consumption of undercooked poultry or other foods cross-contaminated with raw poultry meat during food preparation (Friedman et al. 2000; JacobsReitsma 2000; Corry and Atabay 2001). However, other risk factors besides poultry such as contact with house pets, or consumption of raw milk, untreated water, and undercooked
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beef or pork have also been linked to human infections (Corry and Atabay 2001; Park 2002). As poultry is considered a major reservoir for human campylobacteriosis, reduction or elimination of poultry contamination with C. jejuni would greatly reduce the risk of Campylobacter for public health. To achieve this goal, it is essential to understand the ecology of Campylobacter in the poultry production systems so that effective intervention strategies can be designed and implemented at the preharvest stage. Although numerous farm-based studies have been conducted in the past decades, the sources of ock infection, modes of transmission, and the host and environmental factors affecting the spread of Campylobacter on poultry farms are still poorly understood (Sahin et al. 2002). There has been a major debate on whether vertical or horizontal transmission is responsible for the introduction of Campylobacter into chicken ocks. For years, the prevailing theory has been that horizontal transmission from the environment is the major source of C. jejuni infection for broiler ocks, and that vertical (egg-borne) transmission is unlikely. Potential sources of ock infection include used litter, untreated drinking water, other farm animals, domestic pets, wildlife species, house ies, insects, farm equipment and workers, and transport vehicles (Kazwala et al. 1990; van de Giessen et al. 1992; Kapperud et al. 1993; Berndtson et al. 1996; Jacobs-Reitsma 1997; Evans and Sayers 2000). However, none of these suspected sources has been conclusively identied as the formal source of infection for broilers farms. In many cases, it was difcult to determine which of the events (the ock infection or environmental contamination) occurred rst as no study plan was included to monitor the direction of Campylobacter transmission. Circumstantial evidence has accumulated suggesting that vertical transmission of C. jejuni from breeder ocks to the broiler farms via the egg may occur. Earlier studies showed that, even if at a low level, C. jejuni could be isolated from both outer (Doyle 1984) and inner (Shanker et al. 1986) shell surfaces of eggs laid by naturally infected commercial layers or broiler breeders. Shane et al. (1986) isolated the organism from both interior surface of egg shell and egg contents after swabbing faeces containing C. jejuni onto the egg surface. Following experimental infection of eggs with C. jejuni by either temperature differential method (Clark and Bueschkens 1985) or inoculation of egg albumen via direct injection (Shanker et al. 1986), the organism was recovered from both the contents of unhatched eggs and from the newly hatched chicks. Investigations using sensitive molecular detection methods demonstrated the presence of Campylobacter DNA in embryos and newly hatched chicks (Chuma et al. 1994, 1997), and in hatcheries (Hiett et al. 2002a). Furthermore, C. jejuni has been isolated from the reproductive tracts of healthy laying and broiler breeder hens (Jacobs-Reitsma 1997; Camarda et al. 2000; Buhr et al.

2002; Hiett et al. 2002b) and from semen of commercial broiler breeder roosters (Cox et al. 2002a). The same genotypes of C. jejuni have been identied from breeder ocks and their progeny ocks (Pearson et al. 1996; Cox et al. 2002b), suggesting that breeder hens might be the source of Campylobacter infection for progeny ocks. Despite these observations, vertical transmission of C. jejuni is still questionable because live Campylobacter have not been detected in the content of commercial breeder eggs, young hatchlings or hatcheries under natural conditions (Doyle 1984; Shane et al. 1986; Shanker et al. 1986; Baker et al. 1987; Kazwala et al. 1990; Chuma et al. 1994; Hiett et al. 2002a). Therefore, the exact role of vertical transmission in introducing Campylobacter to broiler ocks remains unclear. One key question that needs to be addressed is if eggs can carry Campylobacter from breeders to their progenies. The purpose of this study was to evaluate the importance of vertical transmission via eggs as a source of ock infection by C. jejuni on broiler farms. To this end, we determined the ability of C. jejuni to penetrate eggshells and to survive in different compartments of the egg. More importantly, prevalence of the organism in eggs obtained from multiple broiler ocks, a hatchery and laboratoryinfected hens was determined using both culture and PCR methods.

MATERIAL AND METHODS Methods for detection of Campylobacter in eggs To develop a sensitive detection method that would recover low numbers of Campylobacter from eggs, specic pathogenfree (SPF) eggs free of Campylobacter were articially infected with relatively low doses of C. jejuni and various detection techniques were tested to assess the sensitivity of each method. Eggs were obtained from Campylobacternegative SPF White Leghorn layers maintained at our departments poultry facilities. Outer surface of the egg shell was disinfected with 70% ethanol and air-dried. Contents and shell membranes of 10 eggs were separated under aseptic conditions and pooled separately. Approximately 10 ml of pooled egg content or 5 g of pooled shell membranes were then mixed with 90 or 45 ml, respectively, of MuellerHinton (MH) broth containing Preston Campylobacter Selective Supplement (containing polymixin B, rifampicin, trimethoprim and cycloheximide; Oxoid) and Campylobacter Growth Supplement (containing sodium pyruvate, sodium metabisulphite and ferrous sulphate; Oxoid) in sterile bottles. Each mixture was spiked with varying CFU numbers (Table 1) of a stationary phase culture of C. jejuni strain S3B (a chicken isolate), and subjected to an enrichment step for 48 h at 42C under microaerophilic conditions produced by CampyPack Plus

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Spiking dose (CFU)* 56 5 6 0 5

Egg contents Culture + + + Culture + Percoll + + + PCR + + +

Eggshell membranes Culture + + ) Culture + Percoll + + + PCR + + +

Table 1 Detection of Campylobacter jejuni in articially inoculated eggs using culture and PCR

*The numbers represent the estimated CFUs that were spiked into 10 ml of egg content or 5 g eggshell membranes resuspended in 90 ml (for egg content) or 45 ml (for shell membranes) enrichment broth, respectively.

gas-generating envelopes (BBL Microbiology System, Cockeysville, MD, USA). One hundred microlitres of the enrichment culture was then spread onto MH agar plates containing the same Preston Campylobacter Selective Supplement and Campylobacter Growth Supplement as described above (Oxoid), and incubated microaerobically for 48 h at 42C to isolate Campylobacter. Besides direct plating of enrichment cultures onto MH agar, the enrichment cultures were also subjected to a density gradient centrifugation step to separate egg materials from bacteria using gradient medium Percoll (Pharmacia Biotech, Stockholm, Sweden) as described elsewhere (Uyttendaele et al. 1999; Wang et al. 1999). This method is based on the fact that bacterial cells have higher buoyant densities than most food homogenates, and thus can be separated from foods in a gradient medium. Briey, a standard isotonic medium (SIM) was made by adding 100 mg peptone and 850 mg NaCl to 100 ml of Percoll. Bacterial cells in the enriched egg samples were separated from the enrichment mixtures by carefully layering 09 ml of sample over the top of 06 ml of 40% SIM in peptone water (85 g NaCl and 10 g peptone in 1 l Millipore water; Millipore, Billerica, MA, USA) and centrifuging at 16 000 g for 1 min. The supernatant was gently removed down to a volume of 01 ml. The remaining 01 ml dense gradient medium in the bottom of the tube was mixed gently with 1 ml saline solution. The tube was centrifuged again at 9500 g for 5 min, after which the supernatant was removed down to a nal volume of ca 100 ll. This volume was then spread onto MH agar plates for isolation of the bacterium. For PCR detection of Campylobacter from eggs, enrichment cultures were processed by a density gradient centrifugation using Percoll as described above to remove egg materials and PCR inhibitors. The nal volume of ca 100 ll of suspension obtained after the second centrifugation was used to isolate bacterial DNA using Dneasy Tissue Kit (Qiagen, Valencia, CA, USA). A PCR assay targeted to amplify a region of 16S rRNA gene specic for C. jejuni and C. coli was employed as described previously (Linton et al. 1997). The forward primer (CCCJ609F; 5-AAT CTA ATG GCT TAA CCA TTA-3) and the reverse primer (CCCJ1442R; 5-GTA ACT AGT TTA GTA TTC CGG-

3) were designed from the published sequences (Linton et al. 1997). Amplication was performed in a 100-ll reaction volume containing 10 ll of sample DNA or 100 ng positive control DNA, 05 lmol l)1 of each primer, 15 mmol l)1 MgCl2, 200 lmol l)1 of each oligonucleotide, and 25 U of Taq DNA polymerase (Progmega, Madison, WI, USA). The PCR amplication was performed with a PC-200 thermocycler (GeneAmp PCR System 2700; Applied Biosystems, Foster City, CA, USA). The reactions were subjected to an initial denaturation for 2 min at 95C, followed by 30 amplication cycles, each consisting of 94C for 1 min, 58C for 1 min and 72C for 1 min. A nal primer extension at 72C for 10 min was included for each reaction. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Experimental egg penetration studies The ability of C. jejuni to penetrate the eggshell and also outer and inner eggshell membranes was investigated by using a temperature-differential process as described previously with minor modications (Clark and Bueschkens 1985). This method works based on the assumption that a warm egg contracts while cooling, and the resultant negative pressure can draw bacteria into/through the eggshell. Briey, a total of 50 fertile broiler eggs were obtained from a local commercial hatchery, and equilibrated at 42C in an incubator for 3 h. A stationary phase culture of C. jejuni strain S3B was added to freshly collected faecal suspension (in MH broth) obtained from Campylobacter-free SPF hens. The nal amount of the organism in this suspension was 2 105 CFU ml)1 as determined by viable CFU counting. The eggs that were held at 42C for 3 h were placed in the faecal suspension inoculated with C. jejuni, and kept there for 30 min at 22C. The eggs were then withdrawn, rinsed three times by immersing in sterile water, and allowed to air dry for 1 h at room temperature before being placed in an incubator for hatching. The isolation of C. jejuni from eggs was carried out on days 2, 6, 10 and 15 of incubation. At each time of examination, ve eggs were removed, and each whole egg (shell and contents) was mixed thoroughly using a blender. Twenty millilitres of blended egg was then added

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to 200 ml of MH broth containing Campylobacter growth supplements (Oxoid). Following the enrichment step for 2 days under microaerophilic atmosphere at 42C, isolation of Campylobacter colonies was attempted using the culture methods as described above. Also, the 17 hatched chicks were placed on a wire-oored cage in a steam-cleaned and formaldehyde-fumigated isolation room, and provided with Campylobacter-free water and feed ad libitum. Cloacal swabs were taken right after placement of the birds, and then once per week for the next 6 weeks to isolate Campylobacter as described previously. To enumerate Campylobacter cells in faeces, cloacal swabs were suspended and serially diluted in MH broth. One hundred microlitres of each faecal suspension was directly plated onto MH plates containing the aforementioned Campylobacter selective and growth supplements (Oxoid). The plates were incubated for 23 days under microaerobic atmosphere at 42C. Survival of Campylobacter in experimentally infected eggs The viability of C. jejuni inoculated into the egg yolk, albumen and air sac was examined as follows. For the egg yolk inoculation, a total of 50 eggs, 25 from a commercial broiler hatchery and 25 from Campylobacter-free SPF hens, were surface disinfected with 70% ethanol and air-dried. Using a sterile 19G needle, 01 ml aliquots (ca 45 102 CFU for each egg) of a stationary phase culture of C. jejuni strain S3B were injected into the yolk. The injection hole was sealed with tape, and the eggs were kept at 18C. At different time points, i.e. 2, 5, 8, 11 and 14 days after inoculation, ve eggs from each group were sampled and the egg contents were collected under aseptic conditions for detecting Campylobacter using both direct plating or the enrichment culture as described above. For inoculation of Campylobacter into the egg albumen and the air sac, eggs obtained from Campylobacter-free SPF hens were used (25 eggs for each group). The procedure of egg inoculation, storage conditions and isolation methods were similar to that of the egg yolk injection, except that the inoculation was into the albumen or air sac. The inoculum dose was ca 7 102 CFU for each egg. When testing for Campylobacter in the air sac following storage at 18C, a whole egg (instead of just the contents) was rst mixed using a blender and then cultured for isolation of Campylobacter via the enrichment method as described above. Detection of C. jejuni in eggs laid by experimentally infected layers To determine the extent of contamination by Campylobacter, eggs laid by C. jejuni-infected hens were surveyed. An SPF layer ock free of Campylobacter was maintained in the laboratory animal facilities of our department. The absence

of colonization of these birds (15 pullets and four cockerels) with Campylobacter was conrmed by culturing of cloacal swabs as described previously. After they started laying eggs, hens were orally inoculated with C. jejuni strain S3B (5 107 CFU for each bird) at the age of 22 weeks. Following establishment of Campylobacter-colonization (as determined by cloacal swabbing) in the hens, eggs were collected daily over a period of a year. The birds were re-inoculated several times during the egg collection period with the same strain of C. jejuni via drinking water to ensure that they would constantly shed Campylobacter in their faeces. Fresh faecal droppings were cultured weekly to isolate Campylobacter. Of the eggs collected throughout the study period, ca 500 eggs were examined on the day of collection, while about 800 were kept at 18C for 7 days before bacteriological examination. In both cases, eggs were not washed or disinfected before examination. To isolate Campylobacter, a pool of ve to 10 whole eggs was mixed using a blender. Twenty millilitres of the blended eggs was then added to 200 ml of MH broth containing the previously mentioned Campylobacter selective and growth supplements (Oxoid). Following an enrichment step, detection of Campylobacter was attempted using both the culture and the PCR methods. In order to determine whether the source of C. jejuni isolated from eggs was the Campylobacterinfected laying hens, isolates recovered from the eggs and those from the faeces of layers were compared by sequencing of the cmp gene (encoding a major outer membrane protein), which is divergent among different Campylobacter strains as described previously (Zhang et al. 2000). Besides testing of eggs laid by Campylobacter-infected layers, we also investigated the presence of C. jejuni in young chicks. In two instances, eggs (70 eggs each time) were placed in an incubator for hatching when the shedding of organism in faeces of hens was at high levels (ca log10 65 CFU g)1 of faeces). The newly hatched chicks (a total of 95) were tested via cloacal swabbing on day 2 of age to determine whether they had Campylobacter as described earlier. Survey of commercial broiler breeders and eggs A local broiler breeder operation consisting of multiple ocks was chosen to survey the level of Campylobacter contamination in eggs laid by naturally infected hens. Five commercial breeder ocks at the age of 2550 weeks were investigated during the fall of 2002. Each ock was located at different premises, with a minimum distance of 20 miles between them. Fifty fresh faecal droppings were collected from each of the ock, and cultured within few hours via direct plating as described in the previous section. Colonies with Campylobacter-like morphology were further identied using a PCR specic for C. jejuni and C. coli (Linton et al.

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1997). Within 2 weeks of collection of faecal samples, 100 commercial eggs were obtained from each breeder ock that shed Campylobacter (a total of 500 eggs were tested). These eggs were <2 days old, and were not subjected to any washing or disinfection procedure. Faecal contamination was apparent on the surface of many eggs. To isolate Campylobacter from eggs, a pool of 10 whole eggs (shell and contents) was mixed together using a blender. Twenty millilitres of the blended eggs was then added to 200 ml of MH broth containing Campylobacter selective and growth supplements (Oxoid). Following an enrichment step rst, detection of the organism was attempted by culture and PCR methods as aforementioned. Survey of eggs from a commercial hatchery for broilers To determine the extent of Campylobacter contamination of hatchery eggs, eggs from a commercial hatchery supplying chicks to broiler grow-out farms were tested. Hatchery eggs were supplied by multiple breeder ocks with different management conditions, and whether the breeder ocks were colonized by Campylobacter at the time of egg collection was not known. A total of 1000 eggs at monthly intervals (100 eggs each time) were obtained from the hatchery and tested for Campylobacter. The eggs were <10 days old, and subjected to standard cleaning procedures (washing and disinfecting with a detergent) used routinely in commercial hatching facilities. To determine the contamination of egg with Campylobacter, egg contents and shell membranes were aseptically separated, and the contents or the membranes from 10 individual eggs were pooled together as one sample. Each sample type (egg content or shell membranes) was treated as described above to isolate Campylobacter using the optimized enrichment and PCR detection methods as described in the earlier section. Antimicrobial susceptibility testing Fluoroquinolone (FQ) MICs of Campylobacter isolates recovered from faeces of commercial broiler breeders were determined using enrooxacin E-test strips (AB Biodisk, Solna, Sweden) following the manufacturers instructions. None of the surveyed ocks had received FQ antibiotics at any time throughout their life. A total of 63 Campylobacter isolates obtained from 5 different ocks were tested for susceptibility to enrooxacin. Briey, 100 ll of each fresh Campylobacter culture in MH broth was spread onto MH agar plates. After the inoculated agar surface was completely dry, E-test strips of enrooxacin, were applied onto individual plates. Plates were cultured at 42C under microaerophilic conditions for 24 h. MICs were read at the point of intersection between the clear zone edge and the E-test strip.

Enrooxacin sensitive and resistant C. jejuni S3B isolates described in a previous study (Luo et al. 2003) were used as internal controls. Campylobacter isolates with an enrooxacin MIC of 4 lg ml)1 were regarded as resistant.

RESULTS Sensitive recovery of C. jejuni from spiked egg samples Following articial inoculation of egg contents and shell membranes, C. jejuni could be efciently recovered with the optimized methods (Table 1). As few as a single C. jejuni cell spiked into the suspensions of egg contents or shell membranes in the enrichment broth could be recovered from both sample types following the enrichment method. Assuming that each egg has ca 50 ml of contents, and 1 g of shell membranes, the detection limit of the enrichment method would theoretically reach ca 25 CFU in egg contents and 1 CFU in shell membranes of an egg. For isolation from egg membranes, density gradient centrifugation using the gradient medium Percoll of enrichment cultures increased the sensitivity of the detection method as compared with the direct plating of enrichment cultures, especially when low numbers of bacteria were used for the inoculation (Table 1). The recovered Campylobacter colonies were conrmed by PCR using a pair of species-specic primers described by Stucki et al. (1995). Also, the PCR assay targeting a region of 16S rRNA gene specic for C. jejuni and C. coli was 100% in accordance with our culture method, indicating that both methods were sensitive and specic enough to detect low numbers of Campylobacter in eggs following an enrichment step. Repeating of the experiment yielded similar results (data not shown), indicating the high reproducibility and sensitivity of the enrichment method. Egg penetration by C. jejuni A total of 50 eggs from a commercial hatchery for broilers were used to investigate the ability of C. jejuni strain S3B to invade the eggshell using a temperature differential method, which mimics the cooling of a freshly laid egg. The presence of C. jejuni in eggs placed in an incubator to hatch, and in the newly hatched chicks was monitored. Of the eggs collected on days 2, 6, 10 and 15 of incubation, none (ve eggs per day) had the organism when individual eggs were tested separately for the presence of C. jejuni by the culture method. On incubation day 21, a total of 17 chicks hatched and were housed in a disinfected room for 6 weeks. Cloacal swabs were taken on day 1 of age and weekly thereafter to isolate the organism. Campylobacter was not isolated from any of the cloacal swabs throughout the study period.

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Table 2 Survivability of Campylobacter jejuni following injection into the egg yolk, albumen and air sac of SPF eggs stored at 18C Injection site Egg yolk Albumen Air sac

Inoculum dose (CFU per egg) 45 10 70 102 70 102

2

Presence of C. jejuni after inoculation on days (positive/total eggs) 2 5/5 5/5 4/5 5 5/5 3/5 1/5 8 5/5 0/5 0/5 11 4/5 0/5 0/5 14 3/5 0/5 0/5

Survival of C. jejuni in eggs Eggs were injected with a relatively low dose of C. jejuni strain S3B into the different compartments, and kept for 14 days at 18C, which is the approximate temperature at which fertile eggs are stored before being set into incubators at commercial operations. When injected into the yolk (ca 45 102 CFU for each egg), C. jejuni survived for at least 14 days, although the numbers of eggs with viable Campylobacter gradually declined with increased storage time (Table 2). Campylobacter organisms were isolated only after enrichment but not from direct culture, in agreement with the inability of the organism to multiple at temperatures below 31C (Hazeleger et al. 1998). There was no difference in the survivability of the organism between egg yolks of broiler chickens (with Campylobacter-specic maternal antibody) and SPF hens (without Campylobacter-specic maternal antibody), indicating that anti-Campylobacter antibodies present at high levels in the egg yolk (Sahin et al. 2001) did not adversely affect the viability of C. jejuni. In contrast to the egg yolk, when C. jejuni was injected into the albumen (ca 7 102 CFU for each egg) of eggs laid by SPF hens that were stored at 18C, viability of the organism was <8 days (Table 2). On day 5 postinoculation, only three of ve eggs was culture positive following the enrichment. None of the cultured eggs yielded C. jejuni on the subsequent sampling days. Similarly, C. jejuni was unable to survive for a long time when inoculated into the air sac (ca 7 102 CFU for each egg) of SPF eggs that were kept at 18C. On day 5 after inoculation, only one of ve eggs was culture positive after enrichment, and no Campylobacter was isolated on the following sampling days. Campylobacter was not detected at any time via direct culture of egg samples when inoculated into the albumen or the air sac, indicating that the organism is unable to propagate inside eggs at 18C. Detection of Campylobacter in eggs from experimentally infected SPF laying hens Oral inoculation of SPF White Leghorn laying hens at week 22 of age with C. jejuni strain S3B resulted in shedding of the organism in faeces within a week as determined by culturing cloacal swabs. Eggs laid by Campylobacter-

colonized chickens were collected over a period of a year, during which the persistence of colonization was ensured by multiple inoculations of the birds via drinking water with the same C. jejuni strain. The shedding level (log10 CFU g)1 faeces) ranged from 35 and 65 units in the majority of chickens throughout the study as determined by culturing of freshly collected faecal dropping at weekly intervals. Of 500 freshly laid eggs examined on the day of lay, Campylobacter was detected in three of 65 pooled (510 eggs for each pool) whole egg samples by both the enrichment culture and the PCR method. Samples positive by culture also gave positive results with the PCR, indicating a 100% correlation between the detection techniques. Also, none of the culture-negative egg samples were positive with the PCR. A 100% sequence identity was found in cmp gene (coding for the major outer membrane protein) sequences among Campylobacter isolates recovered from faeces of the laying hens and the positive egg samples, conrming the layers as the source of Campylobacter isolated from the eggs. As we only tested the pooled whole eggs, whether Campylobacter recovered from the eggs was on the surface or inside the egg was not known. In contrast to fresh eggs, Campylobacter was not detected in any of 800 eggs (80 pools; 10 eggs for each pool) that were rst stored at 18C for 7 days before testing by enrichment culture and PCR. In two instances when the level of Campylobacter shedding was high (ca log10 65 CFU g)1 faeces), a total of 140 eggs were placed in an incubator for hatching. Campylobacter was not isolated from any of the 95 newly hatched chicks that were tested on day 2 of age via cloacal swabbing. Prevalence of Campylobacter in breeder ocks and eggs All of the ve broiler breeder ocks were tested positive for Campylobacter colonization. As shown in Table 3, prevalence of Campylobacter was generally high reaching 100% in some ocks, although the prevalence was relatively low in other ocks. In individual birds, the shedding level ranged between log10 25 and 65 CFU g)1 faeces. There was no correlation between the age of the ock and the prevalence of Campylobacter colonization. Within 2 weeks of collection of faecal droppings from the breeder ocks, a total of 500 freshly laid (<2 days) oor and nest eggs (100 eggs from

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Table 3 Prevalence of Campylobacter in faecal droppings and eggs from ve different commercial broiler breeder ocks Prevalence in (positive/total eggs) Faecal droppings 40/50 25/50 46/50 50/50 50/50 Eggs 0/100 0/100 0/100 0/100 0/100 Average shedding level 287 269 307 304 330

Flock no 1 2 3 4 5

Age (week)* 50 43 25 42 29

*Flock age at the time of collection of faecal samples. Number of Campylobacter-positive samples/total number of samples tested. Mean log10 CFU g)1 of faecal droppings of Campylobacter-colonized birds.

each ock) were tested for Campylobacter contamination. Faecal contamination was evident on the surface of most eggs, which did not have any cleaning treatment before examination. Campylobacter was not detected in any pooled whole egg samples (50 pools; 10 eggs for each pool) by either enrichment culture or PCR method. FQ MICs of Campylobacter isolates from breeder ocks A total of 63 Campylobacter isolates recovered from faecal droppings of different breeder ocks were all found susceptible to enrooxacin as determined by E-test (results not shown). The MICs in all of the isolates were <0125 lg ml)1. Laboratory studies demonstrated that use of FQ in chickens can rapidly select for FQ-resistant Campylobacter organisms (Luo et al. 2003). None of the breeder ocks surveyed in this study had used FQ antibiotics throughout their life, suggesting that the emergence of FQ resistant Campylobacter may be directly related to use of FQ antibiotics on poultry farms. Detection of Campylobacter in eggs from a commercial hatchery The presence of Campylobacter was tested separately in the shell membranes and contents of a total of 1000 eggs obtained from a commercial hatchery over a period of a year. Campylobacter was not detected from any of the pooled samples (10 eggs for each pool) by either the culture or the PCR method. DISCUSSION In general, vertical transmission of bacteria including Campylobacter may take place by primary (contamination

of the egg content in the hens reproductive tract during the egg development) or secondary (contamination of the eggshell after the lay via faecal material containing the bacteria with the subsequent penetration of the agent inside the egg) infection of the egg. If vertical transmission of Campylobacter through the secondary egg infection occurs, the organism must rst penetrate through the eggshell and then maintain its viability once inside the egg until hatching. The limited ability of C. jejuni to penetrate the eggshell under laboratory conditions has been demonstrated in a number of earlier studies (Doyle 1984; Clark and Bueschkens 1985; Neill et al. 1985; Shane et al. 1986; Allen and Grifths 2001). Most of these investigators used a temperature differential method of various forms to test the penetration of Campylobacter. Although the temperature differential method theoretically mimics the events during cooling of freshly laid eggs, the conditions employed in these studies, such as use of broth or autoclaved faeces contaminated with rather extremely high numbers of Campylobacter, during the assay may not necessarily simulate the environment of a newly laid egg. In reality, Campylobacter (and other bacteria) must compete effectively with other microora present at high numbers in faeces to penetrate through the eggshell. Therefore, we used freshly collected faecal material obtained from Campylobacter-free SPF hens without any further treatment (such as autoclaving) to closely mimic the process of natural egg contamination through the secondary egg infection. Following the temperature differential method, Campylobacter was not isolated from any of the incubating eggs, even in those cultured on day 2 after the treatment, indicating that Campylobacter was unable to penetrate the eggshell, or it did not remain viable for 48 h in incubating eggs if penetration occurred. An earlier study by Neill et al. (1985) showed that although shell penetration by C. jejuni occurred following a temperature differential method, the organism was unable to survive for more than 6 h in any part of the eggs that were incubated in a humidied and ventilated atmosphere at 37C. However, in rare cases Campylobacter managed to survive inside the eggs following a temperature differential treatment (Clark and Bueschkens 1985, 1986a) or direct injection into the albumen (Shanker et al. 1986) for long enough in incubating eggs to contaminate a low percentage of new hatchlings. Despite the observations (Clark and Bueschkens 1985, 1986a; Shanker et al. 1986), many other investigators indicated a short period of survivability (<72 h) of Campylobacter inside eggs (Doyle 1984; Neill et al. 1985; Clark and Bueschkens 1986b; Shane et al. 1986; Maruyama et al. 1995). Shane et al. (1986) showed that although contamination of commercial table eggs with faeces containing C. jejuni resulted in a low level of penetration, viability of the organism on the shell surface was retained for only 16 h and the organism was not recovered from inside

2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 10701079, doi:10.1046/j.1365-2672.2003.02083.x

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the eggs beyond 2 h when the eggs were kept at room temperature. Similarly, Doyle (1984) using a temperature differential method demonstrated that although C. jejuni could be isolated at a low level from the inner shell surface and shell membranes for up to 72 h, the organism was never recovered from the contents of eggs that were kept at 4C. The high susceptibility of Campylobacter to desiccation and atmospheric oxygen (Park 2002) are thought to be the primary reason for its low survivability on the egg surface. The absence of Campylobacter in the chicks hatched from the surface challenged eggs (via the temperature differential method) further supports the notion that Campylobacter contamination of eggs by a secondary egg infection is rare, if it occurs at all, and is unlikely to result in chicks infected with Campylobacter. In commercial settings, newly laid eggs are usually stored up to 10 days at ca 18C before set into the incubators. The question, therefore, remains as to whether Campylobacter, when present inside eggs, could stay viable during this storage period. To test this possibility, different compartments (the yolk, the albumen and the air sac) of the egg were articially inoculated with Campylobacter, and stored at 18C for 14 days. Although the organism remained viable in egg yolk for up to 14 days, the survivability of Campylobacter inside the albumen and the air sac was <8 days. Previous studies also indicated the lower survival rate of C. jejuni in the albumen as compared with the egg yolk at different temperatures (Hanninen et al. 1984; Clark and Bueschkens 1986b; Maruyama et al. 1995). It is likely that viability of Campylobacter was adversely affected by the high pH and the presence of bactericidal compounds, such as lysozyme and conalbumin, in the albumen. In general, bacterial penetration of the egg is most likely to occur via the blunt (broad) end (where the air sac is located), since more pores in larger diameter are found at this site of the eggshell, and the suction effect when the air sac is formed could ease the bacterial penetration (Mayes and Takeballi 1983). Our results obtained from inoculation of the air sac indicated that Campylobacter is unlikely to remain viable inside the egg at 18C even if it penetrated the eggshell over the air sac. The low viability in the air sac is probably because of the high sensitivity of the organism to atmospheric oxygen (Park 2002). The inability of Campylobacter to penetrate the eggshell and/or to survive inside the egg for longer than 48 h, and its low survivability inside the albumen and air sac of inoculated eggs observed in this study would further reduce the likelihood of egg-borne transmission of Campylobacter. However, the long survival rate of Campylobacter in the yolk may have ramications for vertical transmission. In general, primary contamination of the egg yolk with bacteria occurs during egg development in the ovaries. C. jejuni has been isolated from the reproductive tract of healthy laying and broiler breeder hens (Jacobs-Reitsma 1997; Camarda

et al. 2000; Buhr et al. 2002; Hiett et al. 2002b). Therefore, egg yolk contamination by C. jejuni via infection of the organs involved in egg development is a possibility. Apart from a very low level of isolation from the outer (Doyle 1984) or inner (Shanker et al. 1986) surface of freshly laid (<14 h) nonhatching quality (soiled and/or cracked) eggs, so far live Campylobacter has not been isolated from internal contents of sound (uncracked, hatching-quality) eggs laid by Campylobacter-shedding hens (Acuff et al. 1982; Doyle 1984; Shane et al. 1986; Shanker et al. 1986; JacobsReitsma 1997). In the present study, we surveyed eggs obtained from different sources to better determine the association of Campylobacter with the eggs. Results obtained from SPF laying hens inoculated with C. jejuni showed that although a low percentage (46%) of newly laid eggs could be contaminated with Campylobacter, storage of the eggs at 18C for 7 days after laying resulted in eggs without Campylobacter-contamination at any site, suggesting that the few Campylobacter-positive eggs were likely the result of egg surface contamination. It is also possible that Campylobacter, if penetration had occurred, was unable to survive inside eggs during this storage period since viability of the organism following injection into the albumen or the air sac was <8 days (Table 2). Similarly, Clark and Bueschkens (1986a) showed that storage of experimentally infected eggs at 16C for 58 days before being set into incubators led to absence of hatched chicks carrying C. jejuni, although 4% of chicks at hatching were infected when no storage was applied. In the present study we were also unable to detect Campylobacter from commercial eggs (<2 days old) obtained from multiple, Campylobacter-colonized broiler breeder ocks although faecal contamination of eggshell was apparent on most eggs, and the hens were still likely to be shedding Campylobacter at the time of egg collection. Although these eggs were not chilled, it is likely that any Campylobacter potentially present on the egg surface would not survive for a long time because of its sensitivity to desiccation and oxygen. In agreement with a previous study (Shanker et al. 1986), we did not nd Campylobacter in eggs obtained from a commercial hatchery for broilers. Routinely employed husbandry practices at hatcheries such as egg washing and/or fumigation, and storage at cool temperatures before incubation are likely to be formidable barriers for Campylobacter survival. Thus, vertical transmission of Campylobacter via eggs does not seem to play a role in introduction of Campylobacter into chicken ocks. However, it should be pointed out that although many studies including this one were unable to detect live Campylobacter in the contents of hatching quality chicken eggs, the possibility of a an extremely low level of vertical transmission cannot be totally excluded. In summary, our results indicated that egg-borne transmission of Campylobacter via secondary infection is unlikely

2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 10701079, doi:10.1046/j.1365-2672.2003.02083.x

1078 O . S A H I N ET AL.

because of its limited capability for shell invasion and poor survivability on egg surface and in air sac and albumen. Regardless of the site of contamination (on shell surface or in egg contents), the lack of detection of Campylobacter in a large number of eggs from commercial breeders that were actively shedding Campylobacter and from a commercial hatchery further disputes the role of egg-borne transmission in introducing Campylobacter into chicken ocks. Based on these ndings, we conclude that vertical transmission of C. jejuni through the egg is probably a rare event and does not play a major role in the transmission of Campylobacter on poultry farms. Therefore, control of Campylobacter should focus on sources of infection that are not related to eggs. ACKNOWLEDGEMENTS This work is supported by a USDA NRICGP competitive grant 99-35212-8517. Orhan Sahin was supported by a scholarship from the Ministry of Education of Turkey. We thank Jerrel Meitzler, Bob Dearth and Greg Myers for technical help. DNA sequences were determined at the Molecular, Cellular and Imaging Center of OARDC. REFERENCES
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