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Campylobacter in Finnish Organic Laying Hens in Autumn 2003 and Spring 2004

J. Sulonen^*, R. Kärenlampi^*, U. Holma and M.-L. Hänninen^*,1

^* Department of Food and Environmental Hygiene, PO Box 66, FIN-00014 University of Helsinki, Finland; and University of Helsinki, Ruralia Institute, Mikkeli Unit, Lönnrotinkatu 3-5, 50100 Mikkeli, Finland

¹ Corresponding author: marja-liisa.hanninen{at}helsinki.fi

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A total of 642 fecal samples and 360 table eggs from Finnish organic laying hens were collected in autumn 2003 (19 farms) and spring 2004 (17 farms) and studied for the presence of Campylobacter. In autumn, 84% of the farms were positive for Campylobacter and in spring, 76%. The percentage of positive samples within a flock varied between 5 and 100%. In addition, Campylobacter was isolated in a single eggshell sample. Campylobacter jejuni was the species isolated most often, although Campylobacter coli was detected on 3 farms in autumn and on 4 farms in spring. KpnI pulsed-field gel electrophoresis genotyping revealed a high level of diversity among the isolates; 47 different patterns were detected among a total of 162 isolates studied. On most of the farms, the genotypes identified in autumn and spring were different, also indicating temporal diversity among colonizing isolates. However, some predominant persistent genotypes were also detected among the isolates. These results suggest that the pool of colonizing isolates may include both variants with capability for persistent intestinal colonization in hens as well as variants with short-term colonization characteristics. In antimicrobial susceptibility testing, the majority of isolates were susceptible to ciprofloxacin, tetracycline, ampicillin, and nalidixic acid. On 2 farms, isolates resistant to nalidixic acid and to ciprofloxacin were detected. In conclusion, Finnish organic laying hens are often colonized by a diversity of Campylobacter pulsed-field gel electrophoresis genotypes.

Key Words: campylobacter • organic • hen

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During the 1990s, organic table egg production increased in European Union countries, including Finland. In 12 mo, from August 2002 to August 2003, the sales of organic eggs increased by 30.5%. In 2003, 1.9% of the commercial table eggs produced in Finland were organic (http://www.finfood.fi/finfood/luomu.nsf,Luomubarometri 8/2003). European Union legislation regulating organic animal production (EEC 2092/91, European Commission, 1991) became effective in 2000, and Finland has adopted these regulations and applied them to local farming conditions.

Organic laying hens are housed in barns where they have free access on the ground and litter as well as free access to outdoor areas under favorable weather conditions (European Commission, 1991). The aim of free access to the outdoors is to increase the welfare of the hens, but it also augments the risk of contact with various vectors of enteric, zoonotic pathogens such Campylobacter and hence may pose a public health risk (Berg, 2001). To our knowledge, there are no previous reports on the prevalence of Campylobacter spp. in organic laying hens.

The resistance of Campylobacter to antimicrobial agents, particularly to fluoroquinolones, is an emerging problem worldwide. The increase in resistance to quinolones in human infections is associated with the use of these compounds in veterinary medicine (Engberg et al., 2001). Antimicrobials and other chemotherapeutics used as prophylaxis to prevent outbreaks of infectious diseases in animals are prohibited in organic farming, but antimicrobials are still acceptable for treatment purposes in such flocks (European Commission, 1991).

The aim of the present study was to determine the occurrence of Campylobacter on Finnish organic laying hen farms by collecting fecal samples in autumn 2003 and spring 2004, and to assess the potential for contamination of eggs by these enteric pathogens. In addition, the diversity of Campylobacter jejuni subtypes was assessed using pulsed-field gel electrophoresis (PFGE) genotyping, and the antimicrobial susceptibilities to 5 antimicrobials used in veterinary medicine were analyzed.

	MATERIALS AND METHODS

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Sample Collection
In autumn 2003 (from late August to early October) and spring 2004 (in March and April), samples of fresh fecal droppings were collected from 19 and 17 Finnish organic egg farms, respectively. Each farm was visited once in autumn and once in spring. Cotton swabs were rolled in fresh stool droppings or, alternatively, the samples were collected directly from the cloaca. The samples were sent refrigerated in transport medium (Probact transport swab, Technical Service Consultants Ltd., Lancashire, UK). The number of stool samples was 327 and 315 in autumn 2003 and spring 2004, respectively. The sizes of the hen farms and the number of fecal samples studied are presented in Table 1. In addition, 10 fresh eggs were collected from each farm and sent to the laboratory.

For enrichment, 1 to 3 fecal swabs were pooled from 1 farm, suspended in 5 mL of Bolton enrichment broth (CM 983, Oxoid Ltd.), with Bolton selective supplement (Oxoid Ltd., SR183E) containing cefoperazone (20 mg/ L), trimethoprim (20 mg/L), vancomycin (10 mg/L), cycloheximide (25 mg/L), and 50 mL/L of horse blood, and incubated at 37°C under a microaerobic atmosphere for 48 h. A 10-µL loopful of the enrichment culture was streaked on the mCCDA and incubated as described above.

For analysis of Campylobacter in eggshells, 5 eggs were broken aseptically and the eggshells were enriched in 250 mL of Bolton broth. In addition, 5 eggs from each farm were cleaned with 70% ethanol to prevent contamination of the contents from the shells. The whites were aseptically separated from the yolks and the yolks were enriched in 250 mL of Bolton broth and cultivated further, as described above.

Bacteria showing typical growth on mCCDA plates (spiral-shaped morphology in gram stain and positive catalase test) were regarded as Campylobacter spp. and stored at –70°C in skim milk with 15% glycerol. From each Campylobacter-positive farm, 1 to 12 isolates were selected and tested for hippurate hydrolysis. Hippurate-positive isolates were regarded as C. jejuni. Certain hippurate-negative isolates were further tested for indoxyl acetate hydrolysis and H₂S production in triple-sugar iron medium.

Pulsed-Field Gel Electrophoresis
One to 13 C. jejuni isolates were chosen from each positive farm for pulsed-field gel electrophoresis (PFGE) analysis. A total of 61 isolates from autumn 2003 and 101 from spring 2004 were analyzed by PFGE. The DNA plugs for PFGE were prepared as described previously (Maslow et al., 1993; Hänninen et al., 2000) and digested with KpnI (New England Biolabs Inc., Beverly, MA; 20 U per sample). The restriction fragments were separated in a 1% SeaKem Gold (Cambrex Life Sciences, East Rutherford, NJ) agarose gel in 0.5x TBE (45 mmol Tris, 45 mmol boric acid, 1 mmol EDTA) at 200 V with Gene Navigator (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) with an increased pulse of 1 to 25 s for 19 h. The gels were stained with ethidium bromide, visualized in UV light, photographed, and saved as TIFF images (AlphaImager 2000, 3.3i, Alpha Innotech Corp., San Leandro, CA). The PFGE patterns were analyzed using BioNumerics version 4.01 software (Applied Maths NV, Sint-Martens-Latem Belgium). Lambda Ladder PFG markers (New England Biolabs Inc.) were used to normalize the TIFF images of the PFGE gels. Similarity calculations were performed using the Dice similarity coefficient with 0.5% optimization and 1% tolerance. Clustering was performed using the unweighted pair group method using arithmetic averages dendrogram type.

Patterns with 4 or more band differences were defined as distinct genotypes and assigned capital letters (A, B, C, . . .). The isolates were considered a subtype of a genotype if they differed by 1 to 3 fragments (Tenover et al., 1995). The subtypes of a genotype were assigned by capital letter, with a number designating the subtype (A₁, A₂, A₃, . . .).

Antimicrobial Susceptibility Testing
The first phase of antimicrobial susceptibility testing was performed using the disk diffusion method (15) for 1 to 3 C. jejuni isolates from each positive farm. The following antimicrobials were used: ampicillin (25 µg), erythromycin (ERY; 15 µg), nalidixic acid (NAL, 30 µg), ciprofloxacin (CIP, 5 µg), and tetracycline (10 µg; all from Oxoid Ltd.). The isolates were cultured on Brucella blood agar plates for 24 to 48 h, transferred to Mueller-Hinton (MH) broth (Oxoid Ltd.) and incubated for 48 h at 37°C. Bacterial suspensions diluted to MacFarland 0.5 in MH broth were evenly swabbed on plates containing MH agar supplemented with 5% horse blood. The plates were incubated at 37°C in a microaerobic atmosphere and the growth inhibition zone diameter was measured after 24 and 48 h of incubation. The results were interpreted according to the National Committee for Clinical Microbiology Laboratory Standards 2000 (NCCLS, 2000) general break points. The total number of isolates tested with the disk diffusion method was 27 in autumn 2003 and 25 in spring 2004.

In the second phase, the minimum inhibitory concentration (MIC) of CIP was studied in 17 and 14 isolates from farms 14 and 49, respectively. The plates were incubated microaerobically at 37°C and MIC values were assessed after 24 and 48 h. The break points applied were adapted from the National Committee for Clinical Microbiology Laboratory Standards 2000 (NCCLS, 2000), and isolates with MIC of 1 mg/L were regarded as susceptible, between >1 mg/L and <4 mg/L were regarded as intermediate, and 4 mg/L were regarded as resistant. The reference strain used was local control strain C. jejuni 143483 (Hakanen et al., 2002).

	RESULTS

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Sample Collection
Of the 19 farms studied in autumn and 17 in spring, 3 (farms 10, 13, and 20) participated in the project only in autumn 2003 and 1 (farm 22) participated only in spring 2004 (Table 1). The number of birds on the farms varied from 92 to 4,000, and the number of samples obtained from each farm varied between 5 and 50, depending on the size of the farm. The farms included in this study represented approximately 80% of Finnish farms producing commercial organic table eggs.

Cultivation of Campylobacter
In autumn 2003 84% (16/19) and in spring 2004 76% (13/17) of the farms were positive for Campylobacter (Figure 1). The percentage of positive samples within a flock varied between 5 and 100% in autumn 2003 and between 31 and 98% in spring 2004. Farms 18 and 25 were Campylobacter-negative in both autumn 2003 and spring 2004 in both direct culture and enrichment. On farms 7 and 28, Campylobacter was detected only in autumn 2003. Only 1 eggshell sample was found to be positive for C. jejuni in spring 2004 (on farm 14). All other eggshell samples and all yolk samples examined were negative.

View larger version (23K): [in this window] [in a new window]   Figure 1. Campylobacter-positive farms in autumn 2003 and spring 2004 and percentage of positive samples on the farms.

Old laying hens were replaced by new birds between the 2 samplings on farms 1, 7, 24, and 34. Three of these farms (farms 1, 24, and 34) were Campylobacter-positive in both spring and autumn, whereas on farm 7, Campylobacter was detected only in autumn.

PFGE Analysis
The KpnI PFGE genotypes detected on different farms are presented in Table 1. In autumn 2003, 14 different genotypes, including a total of 25 different subtypes of C. jejuni, were detected among 61 isolates. Among the samples collected during spring 2004, 13 different PFGE genotypes and a total of 31 different subtypes among 101 isolates were identified. Four of the C. jejuni isolates were not digested with KpnI. The total number of different PFGE genotypes and subtypes was 47. The most common genotype detected was A₁, which was found on farms 1, 31, and 49 in autumn 2003 and on farms 1 and 24 in spring 2004 (Table 1). In addition, KpnI PFGE genotypes related to A₁ were isolated in autumn 2003 and spring 2004 on 5 farms. Other PFGE genotypes with several subtypes differing by only a few fragments were C₁ to C₈ and B₁ to B₄. Seven of the genotypes were unique.

A high diversity among genotypes was detected on some of the farms; for example, 12 C. jejuni isolates from farm 14 represented 9 different KpnI PFGE subtypes. The positive eggshell sample on this farm had a unique PFGE genotype. The genotypes detected in spring 2004 on farms 24 and 34, which had acquired new birds, were not similar to those identified in autumn 2003. However, on farm 1 genotype A₁ persisted from the previous to the following flock. The new flock on farm 7 was negative in spring 2004 (Table 1).

Antimicrobial Susceptibility Testing
Fifty (96%) of the isolates were susceptible to all antimicrobials tested by the disk diffusion method (ampicillin, ERY, NAL, CIP, and tetracycline). Because 1 isolate from farm 14 and 1 isolate from farm 49 were resistant to NAL and CIP, a total of 31 additional isolates from these farms were selected for MIC determination for CIP. In autumn 2003 on farm 14, 2 isolates of the 7 tested were resistant to CIP (MIC values of 8 and 16 mg/L). In spring 2004, 1 isolate of the 10 tested was resistant to CIP (MIC 16 mg/ L). Ciprofloxacin-resistant isolate 14/2R (MIC 16 mg/L) from autumn 2003 and CIP-sensitive isolate 14/3BS (MIC <0.125 mg/L) from spring 2004 showed related PFGE patterns (S₁ and S₂). The second CIP-resistant isolate from autumn 2003 showed PFGE pattern T₁, which was identical to that of a CIP-susceptible isolate. The isolates were from the same sample after direct culture (14/3BS) or enrichment (14/3BR). In autumn 2003 on farm 49, 3 of 6 isolates were resistant to CIP (MIC 8 mg/L). All 3 resistant isolates showed identical PFGE pattern J₁. In addition, genotype J₁ was detected from 1 CIP-sensitive isolate on the farm. In spring 2004, 1 of 8 isolates studied was CIP resistant (MIC 4 mg/L). The isolate showed PFGE pattern A₇, different from all other KpnI PFGE patterns detected.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To our knowledge, our study represents one of the first investigations of the prevalence of Campylobacter spp. in organic laying hens. Our study, which included 80% of Finnish commercial organic table egg production farms, indicated that the majority of the farms were contaminated by Campylobacter spp. In autumn, after the period of free access outside during the summer season, and in spring, after 6 to 7 mo of indoor rearing, 89 and 76% of the farms were Campylobacter-positive, respectively. Campylobacter jejuni was the most common species isolated. Campylobacter coli was the only Campylobacter species detected on only 1 farm, whereas on 2 farms both C. jejuni and C. coli were detected. A Danish study in 1998 compared the prevalence of Campylobacter species in organic and conventional chicken flocks, of which 100 and 36.7%, respectively, were positive (Heuer et al., 2001). El-Shibiny et al. (2005) isolated Campylobacter in 68.5% of organic chickens and in 90% of free-range chickens. In organic laying hen and chicken farming, one of the obligatory requirements is free access to open air under favorable weather conditions, if possible for as much as one-third of their lifetime. This causes a lack of biosecurity and hygienic barriers by increasing free contacts with possible sources and vehicles of Campylobacter contamination, such as wild birds, rodents, insects, and other domestic or wild animals (Berg, 2001). Previous Finnish studies have indicated that Finnish chicken production is less heavily contaminated by Campylobacter compared with that of several other industrialized countries (Perko-Mäkelä et al., 2002; Ministry of Agriculture and Forestry, 2004). In a Finnish study conducted in 1999 by PerkoMäkelä et al. (2002), only 2.9% (33/1,132) of broiler flocks studied during the seasonal period were colonized by C. jejuni or C. coli. Comparison of these results with the results of our present study of laying hens revealed the impact of biosecurity in preventing contamination. Campylobacter detected in organic laying hens indicates that reservoirs of the bacterium exist in the Finnish environment and that the animals with access to open-air conditions in summer are easily contaminated by Campylobacter.

All farms operated under an "all in-all out" system; that is, all hens were brought to the farm simultaneously and the henhouses were cleaned and disinfected in between the flocks. Farms 18 and 25 were Campylobacter-negative in both 2003 and 2004. The number of hens on these 2 farms was greater than the mean size of the farms and new hens arrived on the farms in August 2003. No distinct feature in the managing conditions of the birds could explain the nonexistence of Campylobacter on these farms, except that the hens on these farms had not had access outside. This finding emphasizes the significance of environmental contacts for acquisition of Campylobacter in the flocks.

Pulsed-field gel electrophoresis genotyping revealed a high level of diversity in C. jejuni genotypes detected on all farms, because a total of 47 genotypes and subtypes were identified among 162 isolates studied in 2003 and 2004. Most other PFGE-genotyping studies performed in Finland, on strains isolated either from chickens (Hänninen et al., 2000; Perko-Mäkelä et al., 2002) or from humans (Kärenlampi et al., 2003), have also revealed high levels of diversity among C. jejuni. Within a chicken flock, however, only 1 PFGE genotype usually existed during a rearing period (Perko-Mäkelä et al., 2002). In contrast, within each laying hen flock, several genotypes coexisted in both 2003 and 2004. The diversity of subtypes detected was most probably associated with the open management system, in which the hens may have acquired Campylobacter from diverse sources. The genotype diversity among Finnish organic hens reflects and is a good indicator of the diversity of C. jejuni genotypes in the outdoor environment. In conventional chicken production systems, successful C. jejuni strains usually spread efficiently and at slaughter, a flock is colonized by 1 or a few sero-or genotypes (Petersen and Wedderkopp, 2001; Newell et al., 2003). The percentage of positive fecal samples at the Campylobacter-positive farms varied between 5 and 100%. These results revealed that, for unknown reasons, not all hens were as susceptible to colonization by Campylobacter or that the bacterium did not spread throughout the flock.

Even though a common feature among C. jejuni isolates was a high level of diversity, there were also examples of predominant and persistent genotypes occurring within a flock and in several flocks. One such genotype was PFGE type A₁, which was predominant on farms 1 and 31 in 2003 and on farm 24 in spring 2004. In addition, it occurred on farm 49, and its related genotypes A₂ to A₇ were present on farms 1 and 31 in 2004. The genome of C. jejuni is prone to genetic changes such as point mutations and recombination (Wassenaar et al., 1998), and genotype A, with variable PFGE pattern types, could be an example of an expressed genomic instability occurring within a flock. In our previous studies on C. jejuni PFGE genotypes isolated from human domestic Campylobacter infections as well as from chickens during a 3-yr period starting from 1996, we showed that some genotypes were predominant and had persisted for several years (Hänninen et al., 2000), thus supporting the present results on persistent genotypes. Genotype A₁ was also recognized as a persistent genotype in our previous studies on human isolates (Kärenlampi et al., 2003). There is evidence from other studies that the survival patterns of C. jejuni strains are not all similar and that variability exists in the persistence under various environmental conditions (Petersen and Wedderkopp, 2001).

As expected, the antimicrobial susceptibility of most isolates to selected antimicrobials was high. Tetracycline, nalidixic acid, CIP, ERY, and amoxicillin were selected because they or their representatives are used in veterinary medicine and decreased susceptibility to them has been reported in several studies among chicken samples (Heuer et al., 2001) and in human isolates (Rautelin et al., 2003). Organic farming regulations specify that use of antimicrobials in the preventive treatment of animals is prohibited (European Commission, 1991). In general, laying hens in Finland are not treated with antimicrobials, except by some coccidiostatics. In the FINRES-Vet 2002–2003 monitoring studies, resistance was rare among Campylobacter isolates (Myllyniemi et al., 2004). Ciprofloxacin-resistant isolates were detected on only 2 of the farms, and only 6% of the 83 C. jejuni isolates studied were resistant, reflecting a rather strict Finnish policy on the use of antimicrobials in veterinary medicine. Our present results were in line with the results of the Finnish monitoring study. Despite the common feature of susceptibility among C. jejuni isolates, several CIP-resistant isolates were detected on 2 farms. Interestingly, on 1 farm the genotypes of the CIP-resistant and CIP-susceptible isolates were indistinguishable, and on the other farms the genotypes were subtypes of the same type. The resistance may have been due to contamination of some of the hens from environmental sources by a mixture of CIP-susceptible and CIP-resistant variants of C. jejuni.

Most of the egg samples were negative for Campylobacter, suggesting that this bacterium is not likely to contaminate eggs. Although C. jejuni was isolated on 1 farm from an eggshell sample, it is unlikely that it would pose a major food hygienic risk, because Campylobacter species are unlikely to persist in dry environments, such as eggshells (Doyle, 1984; Sahin et al., 2003).

In conclusion, our study revealed that organic hen flocks are commonly colonized by a diverse variety of C. jejuni genotypes. In addition, the antimicrobial resistance of Campylobacter isolates was low.

	ACKNOWLEDGMENTS

 
The research was funded by the Ministry of Agriculture and Forestry from the Project "Organic Farming in Finland."

Received for publication November 8, 2006. Accepted for publication February 10, 2007.

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