- Email: [email protected]
Genotype dynamics of Campylobacter jejuni in a broiler flock
Veterinary Microbiology 106 (2005) 109–117 www.elsevier.com/locate/vetmic
Genotype dynamics of Campylobacter jejuni in a broiler flock Helena Ho¨o¨ka,*, Mohammed Abdel Fattahb, Henrik Ericssona, Ivar Va˚gsholmc, Marie-Louise Danielsson-Thamd a
Division of Food Hygiene and Bacteriology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, Box 7009, S-750 07 Uppsala, Sweden b National Food Administration, Box 622, S-751 26 Uppsala, Sweden c Swedish Zoonosis Center, National Veterinary Institute, S-751 89 Uppsala, Sweden d Section of Food Associated Pathogens, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, Box 7009, S-750 07 Uppsala, Sweden Received 9 July 2004; received in revised form 14 December 2004; accepted 17 December 2004
Abstract We investigated the genotype diversity and dynamics of Campylobacter in a commercial broiler flock during rearing and slaughter. In total, 220 Campylobacter jejuni isolates collected on four sampling occasions during rearing and from routine sampling during slaughter were subtyped by SmaI macrorestriction and pulsed-field gel electrophoresis, PFGE. Eight different SmaI types were found. During rearing, a subsequent addition of genotypes occurred, with two SmaI types found at 2 weeks of age and six types on the day before slaughter. All types that were detected in more than one isolate were also found on all succeeding sampling occasions, including the slaughter sampling. Two new types were found in the slaughter samples. In twothirds of the individual birds sampled the day before slaughter, more than one SmaI type were found, although there was a clear tendency for dominance of one type in individual birds. Our results show that multiple genotypes of C. jejuni may be present in a commercial broiler flock during rearing and even in gastrointestinal tracts of individual birds. Both recurring environmental exposure and genetic changes within the population may explain the genotype diversity. Although the distribution of genotypes varied between different sampling occasions, we found no indication that any subtype excluded another during the rearing of the broiler flock. # 2004 Elsevier B.V. All rights reserved. Keywords: Campylobacter jejuni; Chicken; Pulsed-field gel electrophoresis
1. Introduction * Corresponding author. Tel.: +46 18 67 23 92; fax: +46 18 67 33 34. E-mail address: [email protected] (H. Ho¨o¨k).
Broiler chickens are regarded as the most important source of human campylobacteriosis (Newell and Wagenaar, 2000). The prevalence of Campylobacter
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.12.017
110
H. Ho¨o¨k et al. / Veterinary Microbiology 106 (2005) 109–117
spp. in commercial broiler flocks is high in most industrialised countries (Newell and Wagenaar, 2000). In Sweden, the prevalence is followed by a national surveillance program since 1991. In 2002, 20% of the tested broiler flocks were Campylobacter positive at slaughter (Swedish Zoonosis Center, 2003). Horizontal transmission is the most important transmission route for campylobacters in broiler chickens (Jacobs-Reitsma et al., 1995). Most studies suggest that vertical transmission does not occur (Jacobs-Reitsma, 1995; Chuma et al., 1997; Petersen et al., 2001), although there is contradicting evidence (Pearson et al., 1996; Cox et al., 2002). The protective effect of hygiene barriers (Berndtson et al., 1996; Gibbens et al., 2001) suggests that introduction into the flock occurs when campylobacters from the outside of the house are transported by the staff, on shoes and clothes. Probable introduction with the water system in the chicken house is described (Pearson et al., 1993). However, water and feed are unlikely sources in Sweden, according to earlier studies (Lindblom et al., 1986; Berndtson et al., 1996). Both for tracing the sources of Campylobacter for broiler chickens and for understanding of transmission of the organism to humans, subtyping is essential. Campylobacters from broilers have been subtyped by many methods, both phenotyping methods such as Penner serotyping (Berndtson et al., 1996; PerkoMa¨ kela¨ et al., 2002), and genotyping such as PCRrestriction fragment length polymorphism (PCRRFLP) analysis of the flagellin locus, fla typing (Chuma et al., 1997), ribotyping (Manfreda et al., 2003) and pulsed-field gel electrophoresis, PFGE (Dickins et al., 2002; Perko-Ma¨ kela¨ et al., 2002). Investigation of the subtype composition of the Campylobacter population in broiler flocks, at a specific moment in time or over time, may give further insight in Campylobacter epidemiology: For example, is the bacterium introduced in the flock on one or several occasions during rearing? Are one or more Campylobacter subtypes introduced? Does colonisation with one Campylobacter strain prevent colonisation with others? In contrast to serotyping, genetic subtyping methods based on enzyme restriction patterns provide the possibility to link similar yet distinguishable subtypes to each other (Tenover et al., 1995). Genomic plasticity is a characteristic of campylobacters, and
genetic recombinations may become visible in a changed restriction pattern (Wassenaar et al., 2000; de Boer et al., 2002). PFGE provides the possibility to detect such changes and is one of the most discriminatory subtyping methods (Nielsen et al., 2000; Sails et al., 2003). In the present study, the genotype diversity and dynamics of Campylobacter in a commercial broiler flock were investigated by PFGE subtyping of Campylobacter isolates collected on several occasions during rearing and slaughter.
2. Materials and methods 2.1. The broiler farm The selected farm had a history of campylobacterpositive broilers. Eight separate houses were located at the same yard. One house had three units/rooms, two houses had two units and five houses had one unit. New chickens were placed in all units in a house at the same time, and the houses were populated according to a rolling schedule. The staff changed shoes but not other clothes before entering the chicken units. The chickens were raised on the floor with shavings as litter and free access to feed and water. The chicken houses were supplied by water from a deep-drilled well. The feed was delivered from Svenska Foder at intervals of 1–5 days, and was supplemented with the coccidiostatic narasin (70 ppm) until 5 days before slaughter. After depopulation, the litter was removed and the house cleaned and disinfected. Until arrival of new chickens, the houses were empty for 10 days. The all-in, all-out management system with houses left empty between flocks is normally used in Swedish broiler production. 2.2. The broiler flock One house with one unit was selected for the study. A field with wild birds and the abattoir’s waste management building were located in the vicinity of the house. A flock of 27,900 day-old chickens was placed in the unit on 28 September 2001 (day 1). At the time of population, three other houses at the farm had flocks of 18, 24 and 35 days of age. The studied flock was reared until day 39, when the whole unit was
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
emptied at the same time and all chickens were slaughtered. The death rate during rearing was 1224 chickens (4.5%), which is in the normal range for Swedish broiler flocks. 2.3. The abattoir and slaughter The abattoir was located 200–300 m from the broiler houses. Early in the morning on the slaughter day, all birds in the chicken house were placed in transport crates by temporary staff and transported to the abattoir. The time interval from placing of chickens in crates to hanging of the first chickens on the slaughter line was three to four hours. Apart from the chickens in the study flock, about 13,000 chickens from another house were slaughtered later the same day. 2.4. Samples The flock was sampled on days 7, 14, 21, 28 and 38 during rearing. On each sampling day, 30 birds were collected from different parts of the house and killed. Caeca were removed aseptically and placed in transport tubes, the caecum from each bird being placed in a separate tube, containing 10 ml CaryBlair medium with decreased agar (National Veterinary Institute) (Luechtefeld et al., 1981) and sent to the laboratory at the Department of Food Hygiene. The same person took the samples on all occasions. The samples were received and culturing started the day after sampling. Culturing of the day 38 samples, collected in the evening before slaughter, started 2 days after sampling. The flock was also sampled at slaughter according to the Swedish Campylobacter surveillance program, in which a total of 40 cloacal samples (a cotton swab inserted into the cloaca of each bird) were taken from the flock. The cotton swabs were pooled 10 and 10 together in Cary-Blair transport tubes. The cloacal sampling was done before stunning of the birds, with five to ten birds between each cloacal swab in each pooled sample, and the four pooled samples more scattered in time. After water chilling, 10 samples of neck skins were taken from the flock and pooled together in a Cary-Blair transport tube. The slaughter samples, i.e., four pooled cloacal samples
111
and one pooled neck skin sample, were sent to the laboratory at National Veterinary Institute on the sampling day. The five pooled samples were managed according to the usual routines in the laboratory at the National Veterinary Institute (Hansson et al., 2004). Thereafter, the Cary-Blair tubes were transported to the laboratory at the Department of Food Hygiene, which is located in the same building, for culturing as below. The samples were received and culturing started the day after slaughter. 2.5. Isolation and identification The isolation protocol included both direct and enrichment culturing. For direct culturing, samples were streaked onto Preston Campylobacter Selective Agar plates (Campylobacter agar base Oxoid CM 689, 5% saponin-lysed horse blood, 5000 IU/l polymyxin B, 10 mg/l trimethoprim, 10 mg/l rifampicin and in some plates 100 mg/l cycloheximide) and incubated for 48 h at 42 8C under microaerobic conditions. In the enrichment procedure the samples were cultured in Preston Campylobacter Selective Enrichment Broth (Nutrient Broth No. 2 Oxoid CM 67, 5% saponin-lysed horse blood, 5000 IU/l polymyxin B, 10 mg/l trimethoprim, 10 mg/l rifampicin, 100 mg/l cycloheximide) for 24 h at 42 8C under microaerobic conditions before streaked onto Preston Agar plates and incubated as the direct cultures. Colonies of typical appearance for Campylobacter were subcultured and tested for characteristic morphology and typical corkscrew motility in phase contrast microscopy, and for production of oxidase and catalase. From each positive sample during rearing, three to five colonies were picked and isolated from the direct-plated Preston agar plates. In addition, from each of the Preston agar plates that were plated after enrichment from days 21 and 38, three colonies were picked and isolated. From each pooled slaughter sample, 10 colonies were picked and isolated from direct-plated Preston agar plates, and five colonies from Preston agar plates that were plated after enrichment. All isolates were frozen in Brain Heart Infusion Broth (Oxoid CM 225 or Merck 1.10493) with 17% glycerol and stored at 75 8C until further analysis.
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
112
Table 1 Distribution of types among subtyped isolates Specimen
Day for sample
Number of subtyped isolates
Rearing Caecum Caecum Caecum Caecum
14 21 28 38
30a 30a 30a 90b
Slaughter Cloacal swab Neck skin
39 39
Total a b c
Number of isolates with each SmaI type 1
2a
2b
3a
3b
3c
28 25 15 48
2 5 12 10
3 6
24
1
1
32c 8c
5
3 1
1
14 7
220
121
33
10
45
1
1
3d
4
1
8
1
8
One isolate from each individual bird. Three isolates from each of 30 individual birds. Eight isolates from each pooled sample, pooled from 10 birds.
2.6. Subtyping by pulsed-field gel electrophoresis One isolate from each positive caecum sample and eight isolates from each pooled sample were subtyped by pulsed-field gel electrophoresis after cleavage with SmaI (Table 1). From the day 38 samples, three isolates from each individual bird were subtyped. All subtyped isolates originated from direct isolation. Freeze-stored isolates were streaked onto blood agar plates (blood agar base Oxoid CM 55, 5% defibrinated bovine blood) and cultured for at least one passage (1–3 days, 40–42 8C) before being subcultured on blood agar plates and incubated at 42 8C in microaerobic environment for 18–20 h. Cells were harvested from the plate and resuspended in Pett IV buffer (10 mM Tris–HCl, 1 M NaCl), diluted to an optical density of 1.2 at 405 nm and mixed with an equal amount of 1.5% low-melting temperature agarose (InCert1 Agarose, BioWhittaker Molecular Applications, Maine, USA). Gel plugs were incubated in ESP (0.5 M EDTA, 1% N-Lauroyl sarcosine, 0.2% Pronase E (Sobral and Atherly, 1989)) at 50 8C for 24 h, with refreshing of the solution after 1.5 h, and washed in TE buffer (10 mM Tris, 1 mM EDTA) at least four times. Plugs were stored in either ESP or TE at 4 8C until restriction digestion and electrophoresis. Plug slices (1–2 mm) were digested in SmaI (Roche) enzyme solution, prepared according to the manufacturer, for 4–5 h at 28 8C. For each slice, 33 units of SmaI were used.
The restriction fragments were separated on a Pharmacia GeneNavigator system (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) in a 0.9% agarose gel (SeaKem1 Gold agarose, BioWhittaker Molecular Applications, Maine, USA) in 0.5X TBE buffer (44.5 mM Tris, 44.5 mM Boric acid, 1 mM EDTA) at 200 V with a constant pulse of 25 s for 19 h at 11 8C. A molecular size marker, Lambda Ladder PFG Marker N0340S (New England BioLabs Inc., Massachusetts, USA), was cut into slices and placed in four of the 23 gel wells, evenly distributed throughout the well row. The gels were stained with ethidium bromide, visualised with GelDoc (Bio-Rad Laboratories AB, Sundbyberg, Sweden) and saved in TIFF format. 2.7. Gel analysis The SmaI profiles were assessed and assigned to different types by visual inspection. To compare band positions between different types, profiles were run in wells close to each other on the same gel. Suspicions of double bands were investigated by checking of plotted densitometric curves of the profiles and summing of fragment sizes. A clearly more intense band (about twice the amplitude of neighbouring bands in the densitometric curve) was interpreted as consisting of two fragments if the genome size, calculated from the sum of computed fragment sizes, was between about 1.6 and 1.8 Mbp. When uncer-
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
113
tainty whether a band consisted of one or two fragments remained, additional PFGE runs with adjusted electrophoretic running conditions were performed. Software used for image analysis was Scion Image (Beta 4.0.2; Scion corporation, http:// www.scioncorp.com). 2.8. Species identification Three, or all when there were fewer than three, isolates of each found PFGE pattern on each sampling occasion were identified to species. Hippurate hydrolysis was examined according to Hwang and Ederer (1975) to differentiate between Campylobacter jejuni (hippurase positive) and other thermophilic campylobacters, presumably Campylobacter coli. 2.9. Statistical analysis Time trends in type distribution were evaluated by the x2-test for trend and the x2-test for departure. Statistical significance of changes in distribution from one sampling to another was tested by Pearson’s x2test. Possible clustering of PFGE types in individual birds was tested by Fisher’s Exact Test. Software used for statistical analysis was Stata (version 7, Stata Corporation). The ptrend command (P Royston, http:// econpapers.hhs.se/software/bocbocode/s426101.htm) for Stata was used for the trend analysis.
3. Results
Fig. 1. Representative PFGE patterns, from two different gels, of the eight SmaI types found in the broiler flock. M indicates the molecular size marker, run on the same gel as the three and four lanes next to the left, respectively. The second largest band in pattern 2a and the largest bands in patterns 3a, 3b and 3c were interpreted as double bands, each consisting of two fragments, according to the criteria in 2.7. Gel analysis. Alternative runs revealed that the largest band in pattern 2b represents a fragment of about 600 kbp, while the largest band in pattern 4 represents a fragment between about 700 and 800 kbp.
3.1. Isolation No campylobacters were isolated from the samples collected in week 1. From week 2 and subsequently, i.e., from samples collected on days 14, 21, 28, 38 and at slaughter on day 39, all samples were positive for Campylobacter spp. Both direct plating and plating after enrichment gave positive results in all cases. 3.2. Pulsed-field gel electrophoresis PFGE typing of 220 isolates (Table 1) resulted in eight SmaI types, one type defined as one specific pattern with perfect congruence in number and positions of all detected bands (Fig. 1). Each type
was named with a number, followed by a small letter when two or more types did not differ by more than four bands. Types 2a and 2b differed by four bands, and each of the types 3b, 3c and 3d differed by two bands from type 3a. 3.3. Distribution of types Table 1 shows the distribution of types. Type 1 and type 2a were found on all sampling occasions when Campylobacter was isolated, including slaughter. On day 28, type 2b was found for the first time, and the type was found in small numbers also on day 38 and in slaughter samples. On day 38 (the day before
114
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
slaughter), type 3a, not found earlier, constituted 27% of the typed isolates. The same type constituted more than 50% of the typed isolates from slaughter samples. One isolate of each was found of types 3b, 3c and 3d. The proportions of isolates with the initially dominating type 1 decreased during the whole study (p < 0.0001). Type 2a was found in increasing proportions of isolates from weeks 2 to 4 and in decreasing proportions from week 4 to slaughter (p values for trends <0.01). On the two occasions when new types were detected during rearing, on day 28 (type 2b) and day 38 (types 3a, 3b, 3c), a significant change in the distribution of earlier found types occurred (p = 0.022 for day 28 and p = 0.017 for day 38). Eight isolates of type 4, which did not share any bands with any other type in the study, were found in one of the four pooled cloacal samples collected at slaughter. Type 4 was not detected in any other samples. Seven additional isolates (two from direct isolation and five from isolation after enrichment, not included in Table 1) from this pooled sample were typed, and all were of type 4. 3.4. Types from individual birds In the samples from day 38, where three isolates from each individual bird were subtyped, more than one type were detected in 20 of the 30 birds (Table 2). Three different types (1, 3a and 3c) were found in one individual. Given the total distribution of types in the day 38 samples, the observed frequency of individuals with only one type was about two times higher than expected, whereas the frequency of individuals with three types was lower than expected (Table 2). Hence, a clustering tendency towards clean culture in individual samples was found.
3.5. Species identification Totally 52 isolates, three isolates of each found type on each sample occasion, were tested for hippurate hydrolysis. All isolates gave a positive reaction, and consequently all types were identified as C. jejuni.
4. Discussion In the present study, the dynamics of Campylobacter colonisation of a broiler flock were followed by PFGE typing. During rearing, a subsequent addition of genotypes occurred, with two different SmaI types found at 2 weeks of age and six types on the day before slaughter. All types that were detected in more than one isolate were also found on all succeeding sampling occasions, including the slaughter sampling. Some studies show that the broiler flock at the time of slaughter most often is colonised by one or a few subtypes (Berndtson et al., 1996; Chuma et al., 1997). Other authors found several subtypes in most broiler flocks (Pokamunski et al., 1986; Jacobs-Reitsma et al., 1995; Hiett et al., 2002). Two hypotheses may explain the subsequent addition of genotypes: either subsequent introductions of Campylobacter or frequent mutations of the dominant clones. Although no firm conclusion can be drawn from our study, we would suggest that both processes took place in this flock. For example, the SmaI pattern of type 3a, which first appeared on day 38, did not share any bands with patterns of types found earlier. This indicates that type 3a entered the flock at a later point than the first Campylobacter introduction, and established itself in competition with the preexisting subtypes. Three isolates had types (3b, 3c, 3d) that differed from type 3a by two bands,
Table 2 Expected and observed frequency of distribution of types in samples from individual birds (day 38) Number of types in each individual bird
Expected frequencya
Expected numberb
Observed numberb
Quota observed frequency/ expected frequency
One type Two types Three types
0.172 0.597 0.230
5 18 7
10 19 1
1.93 1.06 0.14
a
Expected frequency calculated (by combinatorics) from the observed total distribution of types in the 90 subtyped isolates from day 38. p-value = 0.038 according to Fisher’s Exact Test, calculated from comparison of observed numbers with expected numbers, rounded to integers. b
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
suggesting a close genetic relationship. The appearance of type 2b on day 28 may be due to a new introduction or a genetic recombination of the preexisting type 2a. Genetic changes may have occurred in the bacterial population before colonisation of the flock or in the intestine of an individual bird, subsequently spreading to other birds in the flock. Alternatively, they may have occurred in vitro, during isolation. Changes in restriction patterns have been found experimentally in some Campylobacter strains during both intestinal colonisation of chickens (Korolik et al., 1998; Ha¨ nninen et al., 1999; de Boer et al., 2002) and in vitro growth (On, 1998; Dickins et al., 2002). However, changes in vitro were detected after extensive subculturing, which was not performed in our study. We found no very similar, possibly genetically changed, variants of type 1, despite this type being isolated throughout the study and in the greatest numbers of all types. Type 1 obviously coexisted with other Campylobacter subtypes in the intestines of individual birds, which may have offered the opportunity to exchange genetic material. Although genetic instability occurs in campylobacters, some strains may remain genetically stable over long periods in both natural and laboratory environments (Manning et al., 2001). In our study, the distribution of types varied between different sampling occasions. However, the interpretation of fluctuations is precarious as new types occurred on some occasions. Type 2a was found in increasing proportions from day 14 to day 28, but in a smaller proportion, totally and compared with type 1, on day 38. One reason for this could be that the newly introduced type 3a competitively restricted the occurrence of some types but not others. However, as slaughter terminated the course we cannot reach any conclusion on this. Type 3a first appeared the day before slaughter, and thus the proportion of this type cannot be compared with other sampling occasions during rearing. The apparent change in proportions of types from day 38 to slaughter may be due to the different sampling methods between rearing and slaughter. In conclusion, we found no evidence for competitive exclusion between different genotypes in our study, although type 3a possibly could have this property. Also Petersen et al. (2001) and Thomas et al. (1997) found that different Campylobacter genotypes
115
in poultry flocks coexisted rather than excluded each other. In contrast, other authors showed experimentally that some C. jejuni strains were able to exclude or prevent colonisation by other strains in chickens (Korolik et al., 1998; Barrow and Page, 2000). Our results show that multiple C. jejuni genotypes may be present in the gastrointestinal tracts of individual birds. Despite that, a clear tendency for dominance of one type in individual birds was seen. Thomas et al. (1997) found five fla genotypes among 300 isolates from a commercial broiler flock, and more than one genotype in 37% of the individual faecal samples. However, the authors could not exclude cross-contamination because of sample collecting from the floor of the poultry shed. Also several different serotypes have been found in the same individual (Pokamunski et al., 1986). In contrast, in a study where different Campylobacter strains were given to chickens that then were mixed to allow crossinfection, co-colonisation with different strains of C. jejuni in individual birds was a rare finding (Korolik et al., 1998). In the present study, four rearing types were found in the slaughter samples, along with two types not found before: type 3d, found in one isolate, and type 4, found in all isolates from one of the pooled cloacal samples. This should be viewed with caution, as the pooling of slaughter samples may have resulted in domination of certain types in the transport and culture media. Moreover, Campylobacter types shed in faeces may not be representative of the types present in caeca. However, contamination during transport or slaughter is a possible explanation. Contamination of previously campylobacter-free broiler flocks with campylobacters during transport (Newell et al., 2001; Slader et al., 2002) and slaughter (Rivoal et al., 1999; Miwa et al., 2003) has been shown to occur in several studies. In conclusion, longitudinal PFGE typing may generate hypotheses about Campylobacter colonisation in poultry flocks. Our results show that multiple genotypes of C. jejuni may be present in a commercial broiler flock during rearing and even in gastrointestinal tracts of individual birds. New types appeared subsequently during rearing, and indications of both recurring environmental exposure and genetic changes within the population were found. The distribution of genotypes varied between different sampling occasions, but no type found in substantial numbers was
116
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
reduced to a level near extinction. Studies of Campylobacter epidemiology in poultry, such as investigations of possible infection sources for chickens, should take Campylobacter population dynamics into account. Likewise, studies investigating risk factors for campylobacteriosis in humans should consider the diversity of campylobacters in broilers, often assumed the main infection source for sporadic campylobacteriosis.
Acknowledgements This study was financially supported by the Swedish Farmers’ Foundation for Agricultural Research. We thank Erik Helmersson for excellent technical assistance with laboratory work. We also thank the farmer for amiable cooperation, and Ingrid Hansson at National Veterinary Institute for delivering the slaughter samples.
References Barrow, P.A., Page, K., 2000. Inhibition of colonisation of the alimentary tract in young chickens with Campylobacter jejuni by pre-colonisation with strains of C. jejuni. FEMS Microbiol. Lett. 182, 87–91. Berndtson, E., Emanuelson, U., Engvall, A., Danielsson-Tham, M.L., 1996. A 1-year epidemiological study of campylobacters in 18 Swedish chicken farms. Prev. Vet. Med. 26, 167–185. Chuma, T., Makino, K., Okamoto, K., Yugi, H., 1997. Analysis of distribution of Campylobacter jejuni and Campylobacter coli in broilers by using restriction fragment length polymorphism of flagellin gene. J. Vet. Med. Sci. 59, 1011–1015. Cox, N.A., Stern, N.J., Hiett, K.L., Berrang, M.E., 2002. Identification of a new source of Campylobacter contamination in poultry: transmission from breeder hens to broiler chickens. Avian Dis. 46, 535–541. de Boer, P., Wagenaar, J.A., Achterberg, R.P., van Putten, J.P.M., Schouls, L.M., Duim, B., 2002. Generation of Campylobacter jejuni genetic diversity in vivo. Mol. Microbiol. 44, 351– 359. Dickins, M.A., Franklin, S., Stefanova, R., Schutze, G.E., Eisenach, K.D., Wesley, I., Cave, M.D., 2002. Diversity of Campylobacter isolates from retail poultry carcasses and from humans as demonstrated by pulsed-field gel electrophoresis. J. Food Prot. 65, 957–962. Gibbens, J.C., Pascoe, S.J., Evans, S.J., Davies, R.H., Sayers, A.R., 2001. A trial of biosecurity as a means to control Campylobacter infection of broiler chickens. Prev. Vet. Med. 48, 85–99.
Ha¨ nninen, M.-L., Hakkinen, M., Rautelin, H., 1999. Stability of related human and chicken Campylobacter jejuni genotypes after passage through chick intestine studied by pulsed-field gel electrophoresis. Appl. Environ. Microbiol. 65, 2272–2275. Hansson, I., Engvall, E.O., Lindblad, J., Gunnarsson, A., Va˚ gsholm, I., 2004. Surveillance programme for Campylobacter species in Swedish broilers July 2001 to June. Vet. Rec. 155, 193–196. Hiett, K.L., Stern, N.J., Fedorka-Cray, P., Cox, N.A., Musgrove, M.T., Ladely, S., 2002. Molecular subtype analyses of Campylobacter spp. from Arkansas and California poultry operations. Appl. Environ. Microbiol. 68, 6220–6236. Hwang, M.N., Ederer, G.M., 1975. Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol. 1, 114–115. Jacobs-Reitsma, W.F., 1995. Campylobacter bacteria in breeder flocks. Avian Dis. 39, 355–359. Jacobs-Reitsma, W.F., van de Giessen, A.W., Bolder, N.M., Mulder, R.W., 1995. Epidemiology of Campylobacter spp. at two Dutch broiler farms. Epidemiol. Infect. 114, 413–421. Korolik, V., Alderton, M.R., Smith, S.C., Chang, J., Coloe, P.J., 1998. Isolation and molecular analysis of colonising and noncolonising strains of Campylobacter jejuni and Campylobacter coli following experimental infection of young chickens. Vet. Microbiol. 60, 239–249. Lindblom, G.B., Sjo¨ gren, E., Kaijser, B., 1986. Natural campylobacter colonization in chickens raised under different environmental conditions. J. Hyg. (Lond.) 96, 385–391. Luechtefeld, N.W., Wang, W.L., Blaser, M.J., Reller, L.B., 1981. Evaluation of transport and storage techniques for isolation of Campylobacter fetus subsp. jejuni from turkey cecal specimens. J. Clin. Microbiol. 13, 438–443. Manfreda, G., De Cesare, A., Bondioli, V., Franchini, A., 2003. Ribotyping characterisation of Campylobacter isolates randomly collected from different sources in Italy. Diagn. Microbiol. Infect. Dis. 47, 385–392. Manning, G., Duim, B., Wassenaar, T., Wagenaar, J.A., Ridley, A., Newell, D.G., 2001. Evidence for a genetically stable strain of Campylobacter jejuni. Appl. Environ. Microbiol. 67, 1185– 1189. Miwa, N., Takegahara, Y., Terai, K., Kato, H., Takeuchi, T., 2003. Campylobacter jejuni contamination on broiler carcasses of C. jejuni-negative flocks during processing in a Japanese slaughterhouse. Int. J. Food Microbiol. 84, 105–109. Newell, D.G., Wagenaar, J.A., 2000. Poultry infections and their control at the farm level. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter. ASM Press, Washington, DC, USA, pp. 497–509. Newell, D.G., Shreeve, J.E., Toszeghy, M., Domingue, G., Bull, S., Humphrey, T., Mead, G., 2001. Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs. Appl. Environ. Microbiol. 67, 2636–2640. Nielsen, E.M., Engberg, J., Fussing, V., Petersen, L., Brogren, C.-H., On, S.L.W., 2000. Evaluation of phenotypic and genotypic methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle. J. Clin. Microbiol. 38, 3800–3810. On, S.L.W., 1998. In vitro genotypic variation of Campylobacter coli documented by pulsed-field gel electrophoretic DNA
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117 profiling: implications for epidemiological studies. FEMS Microbiol. Lett. 165, 341–346. Pearson, A.D., Greenwood, M., Healing, T.D., Rollins, D., Shahamat, M., Donaldson, J., Colwell, R.R., 1993. Colonization of broiler chickens by waterborne Campylobacter jejuni. Appl. Environ. Microbiol. 59, 987–996. Pearson, A.D., Greenwood, M.H., Feltham, R.K., Healing, T.D., Donaldson, J., Jones, D.M., Colwell, R.R., 1996. Microbial ecology of Campylobacter jejuni in a United Kingdom chicken supply chain: intermittent common source, vertical transmission, and amplification by flock propagation. Appl. Environ. Microbiol. 62, 4614–4620. Perko-Ma¨ kela¨ , P., Hakkinen, M., Honkanen-Buzalski, T., Ha¨ nninen, M.-L., 2002. Prevalence of campylobacters in chicken flocks during the summer of 1999 in Finland. Epidemiol. Infect. 129, 187–192. Petersen, L., Nielsen, E.M., On, S.L.W., 2001. Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks. Vet. Microbiol. 82, 141– 154. Pokamunski, S., Kass, N., Borochovich, E., Marantz, B., Rogol, M., 1986. Incidence of Campylobacter spp. in broiler flocks monitored from hatching to slaughter. Avian Pathol. 15, 83–92. Rivoal, K., Denis, M., Salvat, G., Colin, P., Ermel, G., 1999. Molecular characterization of the diversity of Campylobacter spp. isolates collected from a poultry slaughterhouse: analysis of cross-contamination. Lett. Appl. Microbiol. 29, 370–374.
117
Sails, A.D., Swaminathan, B., Fields, P.I., 2003. Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing correlate with strain associations identified by multilocus enzyme electrophoresis. J. Clin. Microbiol. 41, 4058–4067. Slader, J., Domingue, G., Jørgensen, F., McAlpine, K., Owen, R.J., Bolton, F.J., Humphrey, T.J., 2002. Impact of transport crate reuse and of catching and processing on Campylobacter and Salmonella contamination of broiler chickens. Appl. Environ. Microbiol. 68, 713–719. Sobral, B.W., Atherly, A.G., 1989. A rapid and cost-effective method for preparing genomic DNA from gram-negative bacteria in agarose plugs for pulsed-field gel electrophoresis. Biotechniques 7, 938. Swedish Zoonosis Center, 2003. Zoonoses in Sweden 2002. National Veterinary Institute, Uppsala. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gelelectrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239. Thomas, L.M., Long, K.A., Good, R.T., Panaccio, M., Widders, P.R., 1997. Genotypic diversity among Campylobacter jejuni isolates in a commercial broiler flock. Appl. Environ. Microbiol. 63, 1874–1877. Wassenaar, T.M., On, S.L.W., Meinersman, R.J., 2000. Genotyping and the consequences of genetic instability. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter. ASM Press, Washington, DC, USA, pp. 369–380.
Genotype dynamics of Campylobacter jejuni in a broiler flock Helena Ho¨o¨ka,*, Mohammed Abdel Fattahb, Henrik Ericssona, Ivar Va˚gsholmc, Marie-Louise Danielsson-Thamd a
Division of Food Hygiene and Bacteriology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, Box 7009, S-750 07 Uppsala, Sweden b National Food Administration, Box 622, S-751 26 Uppsala, Sweden c Swedish Zoonosis Center, National Veterinary Institute, S-751 89 Uppsala, Sweden d Section of Food Associated Pathogens, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, Box 7009, S-750 07 Uppsala, Sweden Received 9 July 2004; received in revised form 14 December 2004; accepted 17 December 2004
Abstract We investigated the genotype diversity and dynamics of Campylobacter in a commercial broiler flock during rearing and slaughter. In total, 220 Campylobacter jejuni isolates collected on four sampling occasions during rearing and from routine sampling during slaughter were subtyped by SmaI macrorestriction and pulsed-field gel electrophoresis, PFGE. Eight different SmaI types were found. During rearing, a subsequent addition of genotypes occurred, with two SmaI types found at 2 weeks of age and six types on the day before slaughter. All types that were detected in more than one isolate were also found on all succeeding sampling occasions, including the slaughter sampling. Two new types were found in the slaughter samples. In twothirds of the individual birds sampled the day before slaughter, more than one SmaI type were found, although there was a clear tendency for dominance of one type in individual birds. Our results show that multiple genotypes of C. jejuni may be present in a commercial broiler flock during rearing and even in gastrointestinal tracts of individual birds. Both recurring environmental exposure and genetic changes within the population may explain the genotype diversity. Although the distribution of genotypes varied between different sampling occasions, we found no indication that any subtype excluded another during the rearing of the broiler flock. # 2004 Elsevier B.V. All rights reserved. Keywords: Campylobacter jejuni; Chicken; Pulsed-field gel electrophoresis
1. Introduction * Corresponding author. Tel.: +46 18 67 23 92; fax: +46 18 67 33 34. E-mail address: [email protected] (H. Ho¨o¨k).
Broiler chickens are regarded as the most important source of human campylobacteriosis (Newell and Wagenaar, 2000). The prevalence of Campylobacter
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.12.017
110
H. Ho¨o¨k et al. / Veterinary Microbiology 106 (2005) 109–117
spp. in commercial broiler flocks is high in most industrialised countries (Newell and Wagenaar, 2000). In Sweden, the prevalence is followed by a national surveillance program since 1991. In 2002, 20% of the tested broiler flocks were Campylobacter positive at slaughter (Swedish Zoonosis Center, 2003). Horizontal transmission is the most important transmission route for campylobacters in broiler chickens (Jacobs-Reitsma et al., 1995). Most studies suggest that vertical transmission does not occur (Jacobs-Reitsma, 1995; Chuma et al., 1997; Petersen et al., 2001), although there is contradicting evidence (Pearson et al., 1996; Cox et al., 2002). The protective effect of hygiene barriers (Berndtson et al., 1996; Gibbens et al., 2001) suggests that introduction into the flock occurs when campylobacters from the outside of the house are transported by the staff, on shoes and clothes. Probable introduction with the water system in the chicken house is described (Pearson et al., 1993). However, water and feed are unlikely sources in Sweden, according to earlier studies (Lindblom et al., 1986; Berndtson et al., 1996). Both for tracing the sources of Campylobacter for broiler chickens and for understanding of transmission of the organism to humans, subtyping is essential. Campylobacters from broilers have been subtyped by many methods, both phenotyping methods such as Penner serotyping (Berndtson et al., 1996; PerkoMa¨ kela¨ et al., 2002), and genotyping such as PCRrestriction fragment length polymorphism (PCRRFLP) analysis of the flagellin locus, fla typing (Chuma et al., 1997), ribotyping (Manfreda et al., 2003) and pulsed-field gel electrophoresis, PFGE (Dickins et al., 2002; Perko-Ma¨ kela¨ et al., 2002). Investigation of the subtype composition of the Campylobacter population in broiler flocks, at a specific moment in time or over time, may give further insight in Campylobacter epidemiology: For example, is the bacterium introduced in the flock on one or several occasions during rearing? Are one or more Campylobacter subtypes introduced? Does colonisation with one Campylobacter strain prevent colonisation with others? In contrast to serotyping, genetic subtyping methods based on enzyme restriction patterns provide the possibility to link similar yet distinguishable subtypes to each other (Tenover et al., 1995). Genomic plasticity is a characteristic of campylobacters, and
genetic recombinations may become visible in a changed restriction pattern (Wassenaar et al., 2000; de Boer et al., 2002). PFGE provides the possibility to detect such changes and is one of the most discriminatory subtyping methods (Nielsen et al., 2000; Sails et al., 2003). In the present study, the genotype diversity and dynamics of Campylobacter in a commercial broiler flock were investigated by PFGE subtyping of Campylobacter isolates collected on several occasions during rearing and slaughter.
2. Materials and methods 2.1. The broiler farm The selected farm had a history of campylobacterpositive broilers. Eight separate houses were located at the same yard. One house had three units/rooms, two houses had two units and five houses had one unit. New chickens were placed in all units in a house at the same time, and the houses were populated according to a rolling schedule. The staff changed shoes but not other clothes before entering the chicken units. The chickens were raised on the floor with shavings as litter and free access to feed and water. The chicken houses were supplied by water from a deep-drilled well. The feed was delivered from Svenska Foder at intervals of 1–5 days, and was supplemented with the coccidiostatic narasin (70 ppm) until 5 days before slaughter. After depopulation, the litter was removed and the house cleaned and disinfected. Until arrival of new chickens, the houses were empty for 10 days. The all-in, all-out management system with houses left empty between flocks is normally used in Swedish broiler production. 2.2. The broiler flock One house with one unit was selected for the study. A field with wild birds and the abattoir’s waste management building were located in the vicinity of the house. A flock of 27,900 day-old chickens was placed in the unit on 28 September 2001 (day 1). At the time of population, three other houses at the farm had flocks of 18, 24 and 35 days of age. The studied flock was reared until day 39, when the whole unit was
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
emptied at the same time and all chickens were slaughtered. The death rate during rearing was 1224 chickens (4.5%), which is in the normal range for Swedish broiler flocks. 2.3. The abattoir and slaughter The abattoir was located 200–300 m from the broiler houses. Early in the morning on the slaughter day, all birds in the chicken house were placed in transport crates by temporary staff and transported to the abattoir. The time interval from placing of chickens in crates to hanging of the first chickens on the slaughter line was three to four hours. Apart from the chickens in the study flock, about 13,000 chickens from another house were slaughtered later the same day. 2.4. Samples The flock was sampled on days 7, 14, 21, 28 and 38 during rearing. On each sampling day, 30 birds were collected from different parts of the house and killed. Caeca were removed aseptically and placed in transport tubes, the caecum from each bird being placed in a separate tube, containing 10 ml CaryBlair medium with decreased agar (National Veterinary Institute) (Luechtefeld et al., 1981) and sent to the laboratory at the Department of Food Hygiene. The same person took the samples on all occasions. The samples were received and culturing started the day after sampling. Culturing of the day 38 samples, collected in the evening before slaughter, started 2 days after sampling. The flock was also sampled at slaughter according to the Swedish Campylobacter surveillance program, in which a total of 40 cloacal samples (a cotton swab inserted into the cloaca of each bird) were taken from the flock. The cotton swabs were pooled 10 and 10 together in Cary-Blair transport tubes. The cloacal sampling was done before stunning of the birds, with five to ten birds between each cloacal swab in each pooled sample, and the four pooled samples more scattered in time. After water chilling, 10 samples of neck skins were taken from the flock and pooled together in a Cary-Blair transport tube. The slaughter samples, i.e., four pooled cloacal samples
111
and one pooled neck skin sample, were sent to the laboratory at National Veterinary Institute on the sampling day. The five pooled samples were managed according to the usual routines in the laboratory at the National Veterinary Institute (Hansson et al., 2004). Thereafter, the Cary-Blair tubes were transported to the laboratory at the Department of Food Hygiene, which is located in the same building, for culturing as below. The samples were received and culturing started the day after slaughter. 2.5. Isolation and identification The isolation protocol included both direct and enrichment culturing. For direct culturing, samples were streaked onto Preston Campylobacter Selective Agar plates (Campylobacter agar base Oxoid CM 689, 5% saponin-lysed horse blood, 5000 IU/l polymyxin B, 10 mg/l trimethoprim, 10 mg/l rifampicin and in some plates 100 mg/l cycloheximide) and incubated for 48 h at 42 8C under microaerobic conditions. In the enrichment procedure the samples were cultured in Preston Campylobacter Selective Enrichment Broth (Nutrient Broth No. 2 Oxoid CM 67, 5% saponin-lysed horse blood, 5000 IU/l polymyxin B, 10 mg/l trimethoprim, 10 mg/l rifampicin, 100 mg/l cycloheximide) for 24 h at 42 8C under microaerobic conditions before streaked onto Preston Agar plates and incubated as the direct cultures. Colonies of typical appearance for Campylobacter were subcultured and tested for characteristic morphology and typical corkscrew motility in phase contrast microscopy, and for production of oxidase and catalase. From each positive sample during rearing, three to five colonies were picked and isolated from the direct-plated Preston agar plates. In addition, from each of the Preston agar plates that were plated after enrichment from days 21 and 38, three colonies were picked and isolated. From each pooled slaughter sample, 10 colonies were picked and isolated from direct-plated Preston agar plates, and five colonies from Preston agar plates that were plated after enrichment. All isolates were frozen in Brain Heart Infusion Broth (Oxoid CM 225 or Merck 1.10493) with 17% glycerol and stored at 75 8C until further analysis.
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
112
Table 1 Distribution of types among subtyped isolates Specimen
Day for sample
Number of subtyped isolates
Rearing Caecum Caecum Caecum Caecum
14 21 28 38
30a 30a 30a 90b
Slaughter Cloacal swab Neck skin
39 39
Total a b c
Number of isolates with each SmaI type 1
2a
2b
3a
3b
3c
28 25 15 48
2 5 12 10
3 6
24
1
1
32c 8c
5
3 1
1
14 7
220
121
33
10
45
1
1
3d
4
1
8
1
8
One isolate from each individual bird. Three isolates from each of 30 individual birds. Eight isolates from each pooled sample, pooled from 10 birds.
2.6. Subtyping by pulsed-field gel electrophoresis One isolate from each positive caecum sample and eight isolates from each pooled sample were subtyped by pulsed-field gel electrophoresis after cleavage with SmaI (Table 1). From the day 38 samples, three isolates from each individual bird were subtyped. All subtyped isolates originated from direct isolation. Freeze-stored isolates were streaked onto blood agar plates (blood agar base Oxoid CM 55, 5% defibrinated bovine blood) and cultured for at least one passage (1–3 days, 40–42 8C) before being subcultured on blood agar plates and incubated at 42 8C in microaerobic environment for 18–20 h. Cells were harvested from the plate and resuspended in Pett IV buffer (10 mM Tris–HCl, 1 M NaCl), diluted to an optical density of 1.2 at 405 nm and mixed with an equal amount of 1.5% low-melting temperature agarose (InCert1 Agarose, BioWhittaker Molecular Applications, Maine, USA). Gel plugs were incubated in ESP (0.5 M EDTA, 1% N-Lauroyl sarcosine, 0.2% Pronase E (Sobral and Atherly, 1989)) at 50 8C for 24 h, with refreshing of the solution after 1.5 h, and washed in TE buffer (10 mM Tris, 1 mM EDTA) at least four times. Plugs were stored in either ESP or TE at 4 8C until restriction digestion and electrophoresis. Plug slices (1–2 mm) were digested in SmaI (Roche) enzyme solution, prepared according to the manufacturer, for 4–5 h at 28 8C. For each slice, 33 units of SmaI were used.
The restriction fragments were separated on a Pharmacia GeneNavigator system (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) in a 0.9% agarose gel (SeaKem1 Gold agarose, BioWhittaker Molecular Applications, Maine, USA) in 0.5X TBE buffer (44.5 mM Tris, 44.5 mM Boric acid, 1 mM EDTA) at 200 V with a constant pulse of 25 s for 19 h at 11 8C. A molecular size marker, Lambda Ladder PFG Marker N0340S (New England BioLabs Inc., Massachusetts, USA), was cut into slices and placed in four of the 23 gel wells, evenly distributed throughout the well row. The gels were stained with ethidium bromide, visualised with GelDoc (Bio-Rad Laboratories AB, Sundbyberg, Sweden) and saved in TIFF format. 2.7. Gel analysis The SmaI profiles were assessed and assigned to different types by visual inspection. To compare band positions between different types, profiles were run in wells close to each other on the same gel. Suspicions of double bands were investigated by checking of plotted densitometric curves of the profiles and summing of fragment sizes. A clearly more intense band (about twice the amplitude of neighbouring bands in the densitometric curve) was interpreted as consisting of two fragments if the genome size, calculated from the sum of computed fragment sizes, was between about 1.6 and 1.8 Mbp. When uncer-
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
113
tainty whether a band consisted of one or two fragments remained, additional PFGE runs with adjusted electrophoretic running conditions were performed. Software used for image analysis was Scion Image (Beta 4.0.2; Scion corporation, http:// www.scioncorp.com). 2.8. Species identification Three, or all when there were fewer than three, isolates of each found PFGE pattern on each sampling occasion were identified to species. Hippurate hydrolysis was examined according to Hwang and Ederer (1975) to differentiate between Campylobacter jejuni (hippurase positive) and other thermophilic campylobacters, presumably Campylobacter coli. 2.9. Statistical analysis Time trends in type distribution were evaluated by the x2-test for trend and the x2-test for departure. Statistical significance of changes in distribution from one sampling to another was tested by Pearson’s x2test. Possible clustering of PFGE types in individual birds was tested by Fisher’s Exact Test. Software used for statistical analysis was Stata (version 7, Stata Corporation). The ptrend command (P Royston, http:// econpapers.hhs.se/software/bocbocode/s426101.htm) for Stata was used for the trend analysis.
3. Results
Fig. 1. Representative PFGE patterns, from two different gels, of the eight SmaI types found in the broiler flock. M indicates the molecular size marker, run on the same gel as the three and four lanes next to the left, respectively. The second largest band in pattern 2a and the largest bands in patterns 3a, 3b and 3c were interpreted as double bands, each consisting of two fragments, according to the criteria in 2.7. Gel analysis. Alternative runs revealed that the largest band in pattern 2b represents a fragment of about 600 kbp, while the largest band in pattern 4 represents a fragment between about 700 and 800 kbp.
3.1. Isolation No campylobacters were isolated from the samples collected in week 1. From week 2 and subsequently, i.e., from samples collected on days 14, 21, 28, 38 and at slaughter on day 39, all samples were positive for Campylobacter spp. Both direct plating and plating after enrichment gave positive results in all cases. 3.2. Pulsed-field gel electrophoresis PFGE typing of 220 isolates (Table 1) resulted in eight SmaI types, one type defined as one specific pattern with perfect congruence in number and positions of all detected bands (Fig. 1). Each type
was named with a number, followed by a small letter when two or more types did not differ by more than four bands. Types 2a and 2b differed by four bands, and each of the types 3b, 3c and 3d differed by two bands from type 3a. 3.3. Distribution of types Table 1 shows the distribution of types. Type 1 and type 2a were found on all sampling occasions when Campylobacter was isolated, including slaughter. On day 28, type 2b was found for the first time, and the type was found in small numbers also on day 38 and in slaughter samples. On day 38 (the day before
114
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
slaughter), type 3a, not found earlier, constituted 27% of the typed isolates. The same type constituted more than 50% of the typed isolates from slaughter samples. One isolate of each was found of types 3b, 3c and 3d. The proportions of isolates with the initially dominating type 1 decreased during the whole study (p < 0.0001). Type 2a was found in increasing proportions of isolates from weeks 2 to 4 and in decreasing proportions from week 4 to slaughter (p values for trends <0.01). On the two occasions when new types were detected during rearing, on day 28 (type 2b) and day 38 (types 3a, 3b, 3c), a significant change in the distribution of earlier found types occurred (p = 0.022 for day 28 and p = 0.017 for day 38). Eight isolates of type 4, which did not share any bands with any other type in the study, were found in one of the four pooled cloacal samples collected at slaughter. Type 4 was not detected in any other samples. Seven additional isolates (two from direct isolation and five from isolation after enrichment, not included in Table 1) from this pooled sample were typed, and all were of type 4. 3.4. Types from individual birds In the samples from day 38, where three isolates from each individual bird were subtyped, more than one type were detected in 20 of the 30 birds (Table 2). Three different types (1, 3a and 3c) were found in one individual. Given the total distribution of types in the day 38 samples, the observed frequency of individuals with only one type was about two times higher than expected, whereas the frequency of individuals with three types was lower than expected (Table 2). Hence, a clustering tendency towards clean culture in individual samples was found.
3.5. Species identification Totally 52 isolates, three isolates of each found type on each sample occasion, were tested for hippurate hydrolysis. All isolates gave a positive reaction, and consequently all types were identified as C. jejuni.
4. Discussion In the present study, the dynamics of Campylobacter colonisation of a broiler flock were followed by PFGE typing. During rearing, a subsequent addition of genotypes occurred, with two different SmaI types found at 2 weeks of age and six types on the day before slaughter. All types that were detected in more than one isolate were also found on all succeeding sampling occasions, including the slaughter sampling. Some studies show that the broiler flock at the time of slaughter most often is colonised by one or a few subtypes (Berndtson et al., 1996; Chuma et al., 1997). Other authors found several subtypes in most broiler flocks (Pokamunski et al., 1986; Jacobs-Reitsma et al., 1995; Hiett et al., 2002). Two hypotheses may explain the subsequent addition of genotypes: either subsequent introductions of Campylobacter or frequent mutations of the dominant clones. Although no firm conclusion can be drawn from our study, we would suggest that both processes took place in this flock. For example, the SmaI pattern of type 3a, which first appeared on day 38, did not share any bands with patterns of types found earlier. This indicates that type 3a entered the flock at a later point than the first Campylobacter introduction, and established itself in competition with the preexisting subtypes. Three isolates had types (3b, 3c, 3d) that differed from type 3a by two bands,
Table 2 Expected and observed frequency of distribution of types in samples from individual birds (day 38) Number of types in each individual bird
Expected frequencya
Expected numberb
Observed numberb
Quota observed frequency/ expected frequency
One type Two types Three types
0.172 0.597 0.230
5 18 7
10 19 1
1.93 1.06 0.14
a
Expected frequency calculated (by combinatorics) from the observed total distribution of types in the 90 subtyped isolates from day 38. p-value = 0.038 according to Fisher’s Exact Test, calculated from comparison of observed numbers with expected numbers, rounded to integers. b
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
suggesting a close genetic relationship. The appearance of type 2b on day 28 may be due to a new introduction or a genetic recombination of the preexisting type 2a. Genetic changes may have occurred in the bacterial population before colonisation of the flock or in the intestine of an individual bird, subsequently spreading to other birds in the flock. Alternatively, they may have occurred in vitro, during isolation. Changes in restriction patterns have been found experimentally in some Campylobacter strains during both intestinal colonisation of chickens (Korolik et al., 1998; Ha¨ nninen et al., 1999; de Boer et al., 2002) and in vitro growth (On, 1998; Dickins et al., 2002). However, changes in vitro were detected after extensive subculturing, which was not performed in our study. We found no very similar, possibly genetically changed, variants of type 1, despite this type being isolated throughout the study and in the greatest numbers of all types. Type 1 obviously coexisted with other Campylobacter subtypes in the intestines of individual birds, which may have offered the opportunity to exchange genetic material. Although genetic instability occurs in campylobacters, some strains may remain genetically stable over long periods in both natural and laboratory environments (Manning et al., 2001). In our study, the distribution of types varied between different sampling occasions. However, the interpretation of fluctuations is precarious as new types occurred on some occasions. Type 2a was found in increasing proportions from day 14 to day 28, but in a smaller proportion, totally and compared with type 1, on day 38. One reason for this could be that the newly introduced type 3a competitively restricted the occurrence of some types but not others. However, as slaughter terminated the course we cannot reach any conclusion on this. Type 3a first appeared the day before slaughter, and thus the proportion of this type cannot be compared with other sampling occasions during rearing. The apparent change in proportions of types from day 38 to slaughter may be due to the different sampling methods between rearing and slaughter. In conclusion, we found no evidence for competitive exclusion between different genotypes in our study, although type 3a possibly could have this property. Also Petersen et al. (2001) and Thomas et al. (1997) found that different Campylobacter genotypes
115
in poultry flocks coexisted rather than excluded each other. In contrast, other authors showed experimentally that some C. jejuni strains were able to exclude or prevent colonisation by other strains in chickens (Korolik et al., 1998; Barrow and Page, 2000). Our results show that multiple C. jejuni genotypes may be present in the gastrointestinal tracts of individual birds. Despite that, a clear tendency for dominance of one type in individual birds was seen. Thomas et al. (1997) found five fla genotypes among 300 isolates from a commercial broiler flock, and more than one genotype in 37% of the individual faecal samples. However, the authors could not exclude cross-contamination because of sample collecting from the floor of the poultry shed. Also several different serotypes have been found in the same individual (Pokamunski et al., 1986). In contrast, in a study where different Campylobacter strains were given to chickens that then were mixed to allow crossinfection, co-colonisation with different strains of C. jejuni in individual birds was a rare finding (Korolik et al., 1998). In the present study, four rearing types were found in the slaughter samples, along with two types not found before: type 3d, found in one isolate, and type 4, found in all isolates from one of the pooled cloacal samples. This should be viewed with caution, as the pooling of slaughter samples may have resulted in domination of certain types in the transport and culture media. Moreover, Campylobacter types shed in faeces may not be representative of the types present in caeca. However, contamination during transport or slaughter is a possible explanation. Contamination of previously campylobacter-free broiler flocks with campylobacters during transport (Newell et al., 2001; Slader et al., 2002) and slaughter (Rivoal et al., 1999; Miwa et al., 2003) has been shown to occur in several studies. In conclusion, longitudinal PFGE typing may generate hypotheses about Campylobacter colonisation in poultry flocks. Our results show that multiple genotypes of C. jejuni may be present in a commercial broiler flock during rearing and even in gastrointestinal tracts of individual birds. New types appeared subsequently during rearing, and indications of both recurring environmental exposure and genetic changes within the population were found. The distribution of genotypes varied between different sampling occasions, but no type found in substantial numbers was
116
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117
reduced to a level near extinction. Studies of Campylobacter epidemiology in poultry, such as investigations of possible infection sources for chickens, should take Campylobacter population dynamics into account. Likewise, studies investigating risk factors for campylobacteriosis in humans should consider the diversity of campylobacters in broilers, often assumed the main infection source for sporadic campylobacteriosis.
Acknowledgements This study was financially supported by the Swedish Farmers’ Foundation for Agricultural Research. We thank Erik Helmersson for excellent technical assistance with laboratory work. We also thank the farmer for amiable cooperation, and Ingrid Hansson at National Veterinary Institute for delivering the slaughter samples.
References Barrow, P.A., Page, K., 2000. Inhibition of colonisation of the alimentary tract in young chickens with Campylobacter jejuni by pre-colonisation with strains of C. jejuni. FEMS Microbiol. Lett. 182, 87–91. Berndtson, E., Emanuelson, U., Engvall, A., Danielsson-Tham, M.L., 1996. A 1-year epidemiological study of campylobacters in 18 Swedish chicken farms. Prev. Vet. Med. 26, 167–185. Chuma, T., Makino, K., Okamoto, K., Yugi, H., 1997. Analysis of distribution of Campylobacter jejuni and Campylobacter coli in broilers by using restriction fragment length polymorphism of flagellin gene. J. Vet. Med. Sci. 59, 1011–1015. Cox, N.A., Stern, N.J., Hiett, K.L., Berrang, M.E., 2002. Identification of a new source of Campylobacter contamination in poultry: transmission from breeder hens to broiler chickens. Avian Dis. 46, 535–541. de Boer, P., Wagenaar, J.A., Achterberg, R.P., van Putten, J.P.M., Schouls, L.M., Duim, B., 2002. Generation of Campylobacter jejuni genetic diversity in vivo. Mol. Microbiol. 44, 351– 359. Dickins, M.A., Franklin, S., Stefanova, R., Schutze, G.E., Eisenach, K.D., Wesley, I., Cave, M.D., 2002. Diversity of Campylobacter isolates from retail poultry carcasses and from humans as demonstrated by pulsed-field gel electrophoresis. J. Food Prot. 65, 957–962. Gibbens, J.C., Pascoe, S.J., Evans, S.J., Davies, R.H., Sayers, A.R., 2001. A trial of biosecurity as a means to control Campylobacter infection of broiler chickens. Prev. Vet. Med. 48, 85–99.
Ha¨ nninen, M.-L., Hakkinen, M., Rautelin, H., 1999. Stability of related human and chicken Campylobacter jejuni genotypes after passage through chick intestine studied by pulsed-field gel electrophoresis. Appl. Environ. Microbiol. 65, 2272–2275. Hansson, I., Engvall, E.O., Lindblad, J., Gunnarsson, A., Va˚ gsholm, I., 2004. Surveillance programme for Campylobacter species in Swedish broilers July 2001 to June. Vet. Rec. 155, 193–196. Hiett, K.L., Stern, N.J., Fedorka-Cray, P., Cox, N.A., Musgrove, M.T., Ladely, S., 2002. Molecular subtype analyses of Campylobacter spp. from Arkansas and California poultry operations. Appl. Environ. Microbiol. 68, 6220–6236. Hwang, M.N., Ederer, G.M., 1975. Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol. 1, 114–115. Jacobs-Reitsma, W.F., 1995. Campylobacter bacteria in breeder flocks. Avian Dis. 39, 355–359. Jacobs-Reitsma, W.F., van de Giessen, A.W., Bolder, N.M., Mulder, R.W., 1995. Epidemiology of Campylobacter spp. at two Dutch broiler farms. Epidemiol. Infect. 114, 413–421. Korolik, V., Alderton, M.R., Smith, S.C., Chang, J., Coloe, P.J., 1998. Isolation and molecular analysis of colonising and noncolonising strains of Campylobacter jejuni and Campylobacter coli following experimental infection of young chickens. Vet. Microbiol. 60, 239–249. Lindblom, G.B., Sjo¨ gren, E., Kaijser, B., 1986. Natural campylobacter colonization in chickens raised under different environmental conditions. J. Hyg. (Lond.) 96, 385–391. Luechtefeld, N.W., Wang, W.L., Blaser, M.J., Reller, L.B., 1981. Evaluation of transport and storage techniques for isolation of Campylobacter fetus subsp. jejuni from turkey cecal specimens. J. Clin. Microbiol. 13, 438–443. Manfreda, G., De Cesare, A., Bondioli, V., Franchini, A., 2003. Ribotyping characterisation of Campylobacter isolates randomly collected from different sources in Italy. Diagn. Microbiol. Infect. Dis. 47, 385–392. Manning, G., Duim, B., Wassenaar, T., Wagenaar, J.A., Ridley, A., Newell, D.G., 2001. Evidence for a genetically stable strain of Campylobacter jejuni. Appl. Environ. Microbiol. 67, 1185– 1189. Miwa, N., Takegahara, Y., Terai, K., Kato, H., Takeuchi, T., 2003. Campylobacter jejuni contamination on broiler carcasses of C. jejuni-negative flocks during processing in a Japanese slaughterhouse. Int. J. Food Microbiol. 84, 105–109. Newell, D.G., Wagenaar, J.A., 2000. Poultry infections and their control at the farm level. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter. ASM Press, Washington, DC, USA, pp. 497–509. Newell, D.G., Shreeve, J.E., Toszeghy, M., Domingue, G., Bull, S., Humphrey, T., Mead, G., 2001. Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs. Appl. Environ. Microbiol. 67, 2636–2640. Nielsen, E.M., Engberg, J., Fussing, V., Petersen, L., Brogren, C.-H., On, S.L.W., 2000. Evaluation of phenotypic and genotypic methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle. J. Clin. Microbiol. 38, 3800–3810. On, S.L.W., 1998. In vitro genotypic variation of Campylobacter coli documented by pulsed-field gel electrophoretic DNA
H. Ho¨ o¨ k et al. / Veterinary Microbiology 106 (2005) 109–117 profiling: implications for epidemiological studies. FEMS Microbiol. Lett. 165, 341–346. Pearson, A.D., Greenwood, M., Healing, T.D., Rollins, D., Shahamat, M., Donaldson, J., Colwell, R.R., 1993. Colonization of broiler chickens by waterborne Campylobacter jejuni. Appl. Environ. Microbiol. 59, 987–996. Pearson, A.D., Greenwood, M.H., Feltham, R.K., Healing, T.D., Donaldson, J., Jones, D.M., Colwell, R.R., 1996. Microbial ecology of Campylobacter jejuni in a United Kingdom chicken supply chain: intermittent common source, vertical transmission, and amplification by flock propagation. Appl. Environ. Microbiol. 62, 4614–4620. Perko-Ma¨ kela¨ , P., Hakkinen, M., Honkanen-Buzalski, T., Ha¨ nninen, M.-L., 2002. Prevalence of campylobacters in chicken flocks during the summer of 1999 in Finland. Epidemiol. Infect. 129, 187–192. Petersen, L., Nielsen, E.M., On, S.L.W., 2001. Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks. Vet. Microbiol. 82, 141– 154. Pokamunski, S., Kass, N., Borochovich, E., Marantz, B., Rogol, M., 1986. Incidence of Campylobacter spp. in broiler flocks monitored from hatching to slaughter. Avian Pathol. 15, 83–92. Rivoal, K., Denis, M., Salvat, G., Colin, P., Ermel, G., 1999. Molecular characterization of the diversity of Campylobacter spp. isolates collected from a poultry slaughterhouse: analysis of cross-contamination. Lett. Appl. Microbiol. 29, 370–374.
117
Sails, A.D., Swaminathan, B., Fields, P.I., 2003. Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing correlate with strain associations identified by multilocus enzyme electrophoresis. J. Clin. Microbiol. 41, 4058–4067. Slader, J., Domingue, G., Jørgensen, F., McAlpine, K., Owen, R.J., Bolton, F.J., Humphrey, T.J., 2002. Impact of transport crate reuse and of catching and processing on Campylobacter and Salmonella contamination of broiler chickens. Appl. Environ. Microbiol. 68, 713–719. Sobral, B.W., Atherly, A.G., 1989. A rapid and cost-effective method for preparing genomic DNA from gram-negative bacteria in agarose plugs for pulsed-field gel electrophoresis. Biotechniques 7, 938. Swedish Zoonosis Center, 2003. Zoonoses in Sweden 2002. National Veterinary Institute, Uppsala. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gelelectrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239. Thomas, L.M., Long, K.A., Good, R.T., Panaccio, M., Widders, P.R., 1997. Genotypic diversity among Campylobacter jejuni isolates in a commercial broiler flock. Appl. Environ. Microbiol. 63, 1874–1877. Wassenaar, T.M., On, S.L.W., Meinersman, R.J., 2000. Genotyping and the consequences of genetic instability. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter. ASM Press, Washington, DC, USA, pp. 369–380.