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Year: 2008
Occurrence and genotypes of Campylobacter in broiler flocks,other farm animals, and the environment during several rearing
periods on selected poultry farms.
Zweifel, C; Scheu, K D; Keel, M; Renggli, F; Stephan , R
Zweifel, C; Scheu, K D; Keel, M; Renggli, F; Stephan , R (2008). Occurrence and genotypes of Campylobacter inbroiler flocks, other farm animals, and the environment during several rearing periods on selected poultry farms.International Journal of Food Microbiology, 125(2):182-187.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:International Journal of Food Microbiology 2008, 125(2):182-187.
Zweifel, C; Scheu, K D; Keel, M; Renggli, F; Stephan , R (2008). Occurrence and genotypes of Campylobacter inbroiler flocks, other farm animals, and the environment during several rearing periods on selected poultry farms.International Journal of Food Microbiology, 125(2):182-187.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:International Journal of Food Microbiology 2008, 125(2):182-187.
Occurrence and genotypes of Campylobacter in broiler flocks,other farm animals, and the environment during several rearing
periods on selected poultry farms.
Abstract
On 15 poultry farms, broiler flocks, other farm animals, and the environment were examined forCampylobacter. Flocks were examined weekly for six or three rearing periods. Of the 5'154 collectedsamples, 311 (6%) from 14 farms were Campylobacter positive. Positive samples originated frombroiler flocks, the broiler houses, cattle, pigs, bantams, a horse, a laying hen flock, and a mouse.Amongst them, 288 tested positive for C. jejuni and 92 for C. coli. The analysis of 917 isolates byflagellin gene typing and pulsed-field gel electrophoresis, and of 15 assorted strains by amplifiedfragment length polymorphism allowed the following conclusions: (i) on eight farms (A, D, H, K, L, M,O, P) identical genotypes were isolated from broilers and other farm animals emphasizing theirimportance as reservoirs and risk factors for flock colonization and retrieving the role of personnelmoving between areas as potential vectors; (ii) on four farms (C, D, I, L), indications of persistentcontamination of the broiler house were evident and thereby the importance of efficient cleaning anddisinfection was underlined; (iii) the previously described sources for broiler flock colonization could beexcluded for certain genotypes from eight farms suggesting the existence of more potential vectors orniches; (iv) especially on farms with extensive outdoor flocks, multiple genotypes were found within arearing period; and (v) some genotypes were identical across farms. The significance of such strainsremains to be elucidated.
1
Epidemiological examinations of Campylobacter transmission routes in 1
broiler flocks on selected poultry farms in Switzerland 2
3
4
Abbreviated running title: Campylobacter sources in broiler flocks 5
6
7
Claudio Zweifel, Kathrin Daniela Scheu, Michaela Keel, Roger Stephan* 8
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10
Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, 11
Winterthurerstrasse 272, 8057 Zurich, Switzerland 12
13
14
15
16
17
18
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20
*Corresponding author. Mailing address and correspondence address: Prof. Dr. R. Stephan, 21
Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, 22
Winterthurerstrasse 272, 8057 Zurich, Switzerland. Phone: +41-44-635-8651. Fax: +41-44-23
635-8908. E-mail: [email protected] 24
25
2
Abstract 25
On 15 poultry farms, broiler flocks, other farm animals, and the environment were 26
examined for Campylobacter. Flocks were examined weekly for six or three rearing periods. 27
Of the 5’154 collected samples, 311 (6%) from 14 farms were Campylobacter positive. 28
Positive samples originated from broiler flocks, the broiler houses, cattle, pigs, bantams, a 29
horse, a laying hen flock, and a mouse. Amongst them, 288 tested positive for C. jejuni and 30
92 for C. coli. The analysis of 917 isolates by flagellin gene typing and pulsed-field gel 31
electrophoresis, and of 15 assorted strains by amplified fragment length polymorphism 32
allowed the following conclusions: (i) on eight farms (A, D, H, K, L, M, O, P) identical 33
genotypes were isolated from broilers and other farm animals emphasizing their importance 34
as reservoirs and risk factors for flock colonization and retrieving the role of personnel 35
moving between areas as potential vectors; (ii) on four farms (C, D, I, L), indications of 36
persistent contamination of the broiler house were evident and thereby the importance of 37
efficient cleaning and disinfection was underlined; (iii) the previously described sources for 38
broiler flock colonization could be excluded for certain genotypes from eight farms 39
suggesting the existence of more potential vectors or niches; (iv) especially on farms with 40
extensive outdoor flocks, multiple genotypes were found within a rearing period; and (v) 41
some genotypes were identical across farms. The significance of such strains remains to be 42
elucidated. 43
44
Keywords: Campylobacter, broiler, environment, transmission, genotyping 45
46
3
1. Introduction 46
Campylobacter, mainly C. jejuni and C. coli, are recognized worldwide as major cause of 47
acute bacterial food-borne gastroenteritis (World Health Organization: 48
http://www.who.int/mediacentre/factsheets). In the European Union (EU), a total of 195'419 49
confirmed human cases have been reported in the year 2005 (EFSA, 2006). The incidence of 50
human campylobacteriosis in the EU (51.6/100’000 inhabitants) has increased over the past 51
years and recently exceeded that of Salmonella in many countries. Although 52
campylobacteriosis is usually a self-limiting diarrheal disease, severe complications such as 53
septicemia, reactive arthritis, and Guillain-Barré syndrome sometimes occur (Humphrey, 54
O’Brien & Madsen, 2007). 55
Campylobacter colonize the intestinal tracts of a large number of mammals and birds. 56
Broiler chickens are often carriers of C. jejuni. Chicken guts, particularly ceca, can be 57
colonized at very high levels and usually the entire flock is colonized once an infection 58
becomes established (Newell & Fearnley, 2003). In the year 2005, 0.2% to 86.3% of the 59
poultry flocks in the EU got colonized, and 23.0% of the Swiss poultry flocks tested positive 60
(EFSA, 2006). This may lead to contamination of carcasses during the slaughter process (Ono 61
& Yamato, 1999; Jørgensen et al., 2002; Stern & Robach, 2003; Allen et al., 2007). 62
Consumption and handling of poultry products has been identified as important risk factor for 63
human disease (Friedman et al., 2004; Siemer, Nielsen & On, 2005; Humphrey et al., 2007). 64
Major efforts are therefore attempted to reduce the number of colonized flocks being 65
delivered for slaughter. 66
The epidemiology of Campylobacter in broiler production is still incompletely 67
understood. There is a degree of dispute over which are the most important sources for flock 68
colonization (Humphrey et al., 2007). Vertical transmission, carryover from previous flocks, 69
and horizontal transmission via contaminated water, domestic and wild animals, personnel 70
4
working in the broiler house, and the external environment have been implicated. The 71
importance of vertical transmission from parent flocks to their offspring remains unclear 72
(Petersen, Nielson & On, 2001; Cox, Stern, Hiett, Berrang, 2002; Callicott et al., 2006). 73
Horizontal transmission is generally believed to be the common way for flock colonization 74
(Sahin, Morishita & Zhang, 2002; Newell & Fearnley, 2003; Bull et al., 2006). To reveal 75
transmission routes to the broilers, more knowledge on the diversity and stability of 76
Campylobacter in the environment and the distribution of different clones is necessary 77
(Johnsen, Kruse & Hofshagen, 2006). 78
In a previous study, the dynamics of Campylobacter spread within broiler flocks were 79
investigated (Ring, Zychowska & Stephan, 2005). The aim of the present study was to 80
investigate the prevalence and genetic diversity of Campylobacter in broilers and the 81
environment during several rearing periods. To establish genetic relationships and to reveal 82
potential transmission routes, strains were characterized by flaA restriction fragment length 83
polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and amplified fragment 84
length polymorphism (AFLP) analysis. 85
86
2. Materials and methods 87
2.1. Farms 88
Between March and December 2006, chicken flocks and the environment of 15 poultry 89
farms (Table 1) were examined for Campylobacter. All farms were part of an integrated 90
system (parent animal farms, hatcheries, broiler units, defined feedstuff, slaughterhouse). As 91
usual in Switzerland, each farm maintained other branches of animal farming and only 92
contained one broiler house with one flock at a time. A hygiene barrier was located in the 93
anteroom and required change of clothes and boots, putting on a headdress, and washing and 94
sanitizing hands. On the farms A to I, M, and N, each broiler stable had a winter garden built 95
5
adjacent to it. This is a paved, fenced, roofed, and sealed up outdoor area. The broiler houses 96
of the farms K, L, and P were connected to an additional outdoor enclosure measuring 1 m2 97
per chick (extensive outdoor flocks). At the age of 21 days, chickens were allowed to use the 98
outdoor areas during daytime. All houses were equipped with automatic feeding and drinking 99
systems. As litter, wood shavings, straw chaff, and straw pellets were used. According to the 100
broiler houses and rearing types, flock sizes varied between 3’000 and 14’800 birds. Rearing 101
periods lasted between 35 and 58 days. 102
103
2.2. Sample collection 104
Sampling comprised three to six rearing periods (Table 1) and each flock was sampled 105
weekly from the third or fifth week of age (extensive outdoor flocks) until slaughter. In the 106
broiler stable, each sampling included three fresh fecal samples (cecal droppings), three dust 107
samples, and water from the nipples. In the anteroom, the floor on both sides of the hygiene 108
barrier, the boots used for entering the flock, dust, drinking water, and feed were sampled. 109
From other farm animals, fecal samples were collected regularly from cattle (farms A to O), 110
pigs (farms A, C, D, I, P), sheep (farms B, C, G), horses (farms B, K, P), rabbits (farm B), 111
laying hens (farm L), bantams (farm G), and sporadically from dogs (farms B, C, G, I, O). 112
Moreover, mice (feces), earthworms, arthropods, and the supply air to the broiler houses were 113
examined. In the idle time, litter and surfaces in the stable were sampled. Feces, dust, and 114
surface samples were collected with swabs (Transwab, MW 170, Medical Wire & Equipment, 115
Wiltshire, UK). Flies were caught by sticky strips placed in the anteroom, winter garden, 116
broiler house, or cattle stable. Other arthropods and earthworms were examined if found in 117
the outdoor areas. Air samples were obtained by the air sampler MAS 100 (MBV AG, 118
Microbiology & Bioanalytic, Stäfa, CH). 119
120
6
2.3. Isolation of Campylobacter 121
Swabs were put in 10 ml Campylobacter Selective Broth (CSB, Difco 0495-17-3, Becton 122
Dickinson, Sparks, USA) with Campylobacter Enrichment Supplement (Oxoid SR84, Oxoid 123
Ltd., Hampshire, UK) and Skirrow Campylobacter Selective Supplement (Oxoid SR69, 124
Oxoid Ltd.). The 40 ml of water samples were mixed with 40 ml CSB with supplements. 125
Feed, litter, earthworms, and arthropods were mixed up in a weight ratio 1:10 with CSB with 126
supplements and 10 ml of this suspension were processed. All enrichments were incubated at 127
42°C during 48 h under microaerophilic conditions (CampyPack Plus, Becton Dickinson). 128
Subsequently, samples were plated on Campylobacter Selective Agar (CSA, Difco 0964-17-5, 129
Becton Dickinson) with lyzated horse blood (Oxoid SR48, Oxoid Ltd.) and Butzler 130
Campylobacter Selective Supplement (Oxoid SR85, Oxoid Ltd.) and incubated as described. 131
In the air sampler, CSA with supplements and sheep blood agar (Difco Laboratories, Becton 132
Dickinson; 5% sheep blood, Oxoid Ltd.) were used. The later were sampled with moistened 133
cotton swabs, which were enriched as described. From every sample with presumptive 134
colonies, three colonies were subcultured on CSA with supplements. These isolates were 135
verified by PCR as C. jejuni (hipO) or C. coli (gene encoding aspartokinase) (On & Jordan, 136
2003). 137
138
2.4. Genotyping 139
Fla-RFLP was performed using primers A1 and A2 (Nachamkin, Ung & Patton, 1996). 140
The amplicon, a 1.7 kb flaA fragment, was then digested with DdeI. Macrorestriction 141
profiling using SmaI and subsequent PFGE were performed as outlined by Ribot, Fitzgerald, 142
Kubota, Swaminathan and Barrett (2001). Fifteen assorted isolates were further analyzed by 143
AFLP (Duim et al., 2000). HindI and HhaI were applied for DNA digestion and staining was 144
performed using a DNA Silver Staining Kit (Amersham Biosciences, Freiburg, D). Visually 145
7
identical patterns on the same gel were grouped. One isolate of each group was used to 146
compare with groups from other gels. Thereby, isolates were divided into final groups. 147
Moreover, patterns analyzed by BioNumerics 3.0 (Applied Maths, Kortrijk, B) and the 148
unweighted paired group method (arithmetic means) was used for dendrogram construction. 149
150
3. Results 151
3.1. Prevalence and distribution of Campylobacter 152
Of the 5’154 samples, 311 originating from 14 farms were Campylobacter positive. The 153
proportion of positive samples ranged from 0.0% to 23.4% (Table 1). Positive broiler flocks 154
were detected on 11 farms, including all farms with extensive outdoor flocks (Figure 1). 155
Beside the broiler flock, Campylobacter were detected on nine farms in the broiler house 156
(dust from the flocks, water from the nipples, clean floor and boots in the anteroom). In the 157
environment, positive samples were detected in fecal samples from bantams, cattle, laying 158
hens, pigs, a horse, and a mouse. Amongst the positive samples, 228 tested positive for C. 159
jejuni and 92 for C. coli. Highest proportions of C. jejuni or C. coli positive samples were 160
seen in those compartments in which fecal samples had been collected (Figure 2). 161
162
3.2. Fla-RFLP and PFGE 163
Of the 311 positive samples, 917 isolates were genotyped. Where the same genotype was 164
observed among the subcultures, only one isolate was used for cluster-analysis. Fla-RFLP 165
was successfully performed on 804 isolates, generated 46 different profiles, and 18, 17, and 166
11 profiles belonged to C. jejuni, C. coli, and both species, respectively. PFGE was 167
successfully performed on 807 isolates, generated 47 different profiles, and 25, 14, and 8 168
profiles belonged to C. jejuni, C. coli, and both species, respectively. 169
3.2.1. Matching genotypes from broilers and other farm animals 170
8
Fla-RFLP and PFGE profiles initially present in the cattle (C. jejuni) appeared in the 171
broilers and on the boots on farm H (Table 2), in the broilers, dust from the flock, and water 172
from the nipples on farm K, and in the broilers on farm M. Similarly, bovine PFGE profiles 173
(C. jejuni) from farm L were detected in the broilers during two rearing periods. Furthermore 174
profiles initially present in the pigs (C. coli) appeared in the broilers on farm D (Table 3), and 175
in the broilers and on the boots on farm P. Another fla profile (C. coli) from farm P was 176
detected in pigs during the first and on the boots during the second rearing period. Finally, 177
certain profiles persisting in the laying hens (farm L) were consecutively found in the broilers 178
and the anteroom. Interestingly, the genotype from a horse (farm P) was simultaneously 179
present in the broilers and the anteroom. 180
On the other hand, fla-RFLP and PFGE profiles (C. jejuni) from broilers and boots on 181
farms A and D, and broilers, water and the anteroom on farm O were subsequently detected in 182
the cattle. Similarly, a certain fla-RFLP profile (C. jejuni) from the broilers and the anteroom 183
on farm L was also detected in the laying hens. 184
3.2.2. Matching genotypes from consecutive broiler flocks 185
Identical genotypes were observed in the broilers (i) on farm D (Table 3), (ii) between the 186
second and third rearing period on farms C (fla-RFLP; C. coli) and L (fla-RFLP; C. jejuni), 187
and (iii) between the third and fourth rearing period on farm I (PFGE; C. jejuni). 188
3.2.3. Genotypes from broilers and the broiler house 189
On farms C, D, H, I, L, N, O, and P, broiler flocks harbored certain isolates with 190
genotypes not present in samples from the environment. Even though genotypes from the 191
broiler house were generally also found in the flocks, additional profiles were obtained during 192
certain rearing periods (i) from the boots on farms H and P, (ii) from the clean floor in the 193
anteroom on farms I and P, and (iii) from the water on farm I. Interestingly, the mentioned 194
boot profiles from farms H and P were also detected in cattle and pigs, respectively. 195
9
3.2.4. Different genotypes within one positive sample 196
Twenty-three (7.4%) of the Campylobacter positive samples showed multiple profiles 197
during a certain rearing period and 13 of them originated from farms P and L. Multiple 198
genotypes were found in broilers from farms D, L, N, and P, in pigs from farms A and D, in 199
cattle from farm M, in laying hens from farm L, in a mouse from farm A, in the water from 200
farm I, and on the clean floor and the boots from farm P. 201
3.2.5. Matching genotypes across farms 202
Fla-typing revealed eight profiles and PFGE nine profiles that proofed to be identical and 203
widely distributed across different farms and compartments. 204
205
3.3. AFLP 206
AFLP analysis of 15 isolates generated patterns from 23 to 25 bands and divided C. jejuni 207
and C. coli into two clusters with an average similarity of 27% (Figure 3). Groups of isolates 208
sharing more than 90% similarity were considered as genetically related and grouped to form 209
a phenon (Olive & Bean, 1999). Among C. jejuni, four phena were identified and similarity 210
was between 95.2% and 99.2% within these phena. 211
212
4. Discussion 213
During the investigated rearing periods, Campylobacter were detected in 6% of the 214
samples, especially in fecal samples. The majority of isolates were identified as C. jejuni. 215
Following fecal shedding, Campylobacter could be found in the dust from the flocks and the 216
water from the nipples became contaminated. The role of water and the water system as 217
source of chicken colonization remains controversial (Pearson et al., 1993; Herman, 218
Heyndrickx, Grijspeerd, Vandekerchove, Rollier & De Zutter, 2003; Zimmer, Barnhart, Idris 219
& Lee, 2003). Boots became contaminated the same way and led to the contamination on the 220
10
clean site of the hygiene barrier. Comparable to other studies, the organisms were not detected 221
in feed or in fresh litter (Jacobs-Reitsma, van de Giessen, Bolder & Mulder, 1995; Ring et al., 222
2005; Bull et al., 2006). Although the role of airborne transmission in the spread of 223
Campylobacter was recently raised (Bull et al., 2006), all samples from the supply air to the 224
broiler house tested negative. As indicated by several studies, insects and other wild animals 225
might play an important role as vectors for (Jacobs-Reitsma et al., 1995; Berndtson, 226
Emanuelson, Engvall & Danielsson-Tham, 1996; Newell & Fearnley, 2003; Hald et al., 2004; 227
Waldenström, On, Ottvall, Hasselquist & Olsen, 2007). In the present study, Campylobacter 228
were not detected in any of the examined arthropods, especially flies. Low detection rates in 229
flies were also reported in two recent studies (Johnsen et al., 2006; Hansson, Vågsholm, 230
Svensson & Olsson Engvall, 2007). However, due to the desiccation sensitivity of 231
Campylobacter, collecting of flies by sticky strips must be reconsidered. 232
Several methods have been compared for Campylobacter subtyping but the genetic 233
instability makes it difficult to establish universally applicable methods. Applying fla-RFLP 234
and PFGE, which both were successfully used for studies in the broiler production (Ring et 235
al., 2005; Bull et al., 2006; Hansson et al., 2007), resulted in a comparable discriminatory 236
power but the distribution of the RFLP and PFGE profiles was slightly different. The 237
untypability of about 12% of the isolates might result in PFGE from the production of DNAse 238
and in RFLP from a fla type that could not be recognized by the primers used (Thomas, Long, 239
Good, Panaccio & Widders, 1997). AFLP on the other hand is almost insensitive to genome 240
rearrangements and ensures reliable results over a long time. Consequently, subtyping 241
methods should be selected according to the epidemiological problem addressed. 242
Identification of the sources of flock colonization would enable measures to be targeted 243
towards the areas posing the greatest risk. On eight of the 15 farms, identical genotypes were 244
isolated from broiler flocks and other farm animals. Thereby, certain genotypes present in 245
11
cattle on farms H, K, L and M, in pigs on farms D and P, and in laying hens on farm L were 246
subsequently found in the broiler flocks. These results emphasized the importance of farm 247
animals as reservoirs and risk factors for broiler flock colonization. Otherwise, genotypes 248
from the flocks appeared in cattle on three farms and in the laying hens. Because the results 249
illustrate only punctual time events, the direction of spread could not be verified in either 250
case. Despite the strict hygiene prescriptions of the poultry integrations, personnel working in 251
the broiler houses represent the probable vector for Campylobacter transmission from the 252
environment to the chickens or vice versa (Gibbens, Pascoe, Evans, Davies, & Sayers, 2001; 253
Hiett, Stern, Fedorka-Cray, Cox, Musgrove & Ladely, 2002; Sahin et al., 2002; Newell & 254
Fearnley, 2003). 255
Indications of persistent contamination of the broiler houses were observed on four farms 256
(C, D, I, L) where identical genotypes were detected in consecutive broiler flocks, but not 257
concurrently in other samples. Probably due to inadequate cleaning and disinfection of the 258
stables between the flocks, Campylobacter have survived in certain niches. Persistent 259
contamination of the broiler house is described as risk factor that needs to be further 260
investigated (Petersen & Wedderkopp, 2001; Shreeve, Toszeghy, Ridley & Newell, 2002). 261
None of the previously described sources for broiler flock colonization could be 262
confirmed for certain genotypes from eight flocks. This suggests the existence of other 263
potential vectors or niches for Campylobacter such as wild birds, bugs, flies or even mice. On 264
the other hand, the restriction of positive samples to the environment outside the broiler flocks 265
on farms B, E, and G may indicate adequate hygiene prerequisites and compliance with them. 266
Especially a more efficient and strict hygiene barrier might have averted colonization of the 267
flocks. The importance of the hygiene barrier as critical factor to either prevent or delay flock 268
colonization was underlined by several studies (Van de Giessen, Tilburg, Ritmeester & van 269
der Plas, 1998; Gibbens et al., 2001; Herman et al., 2003; Hansson et al., 2007). 270
12
In flocks from stables with adjacent winter garden, merely only one genotype was 271
detected during a certain rearing period. This might be explained (i) by a single contact during 272
a limited time span and the spread of a strain over the flock, which then maintained its 273
dominating position until slaughter, or (ii) by a constant colonization pressure of a certain 274
genotype from the environment. Multiple genotypes within flocks were especially detected in 275
extensive outdoor flocks, probably due to frequent ingress from the environment. Overall, 276
inconsistent data exist on the heterogeneity of the Campylobacter within colonized broiler 277
flocks. Similar to our results from the first rearing type, some studies revealed a low amount 278
of genetic diversity (Berndtson et al., 1996; Shreeve, Toszeghy, Pattison & Newell, 2000; 279
Ring et al., 2005), whereas others detected multiple genotypes within single flocks (Petersen 280
et al., 2001; Hiett et al., 2002; Höök, Fattah, Ericsson, Vågsholm & Danielsson-Tham, 2005; 281
Wittwer et al., 2005). 282
By fla-RFLP, PFGE, and confirmed by AFLP, some Campylobacter genotypes proofed 283
to be identical across different farms. AFLP analysis showed in different studies that there 284
exist stable strains, despite the known genome instability (Wassenaar & Newell, 2000; 285
Manning, Duim, Wassenaar, Wagenaar, Ridley & Newell, 2001). In Switzerland, such strains 286
are geographically widespread and show no host specificity (Wittwer et al., 2005). Currently 287
it is not known, how these strains survive in nature or their distribution takes places. The 288
significance of such strains remains to be elucidated. 289
290
Acknowledgments 291
We thank F. Renggli for facilitating access to the poultry farms, V. Perreten from the 292
Institute of Veterinary Bacteriology University of Berne for his help with BioNumerics, and 293
L. Ellerbroek and K. Toutounian-Mashhad from the German Federal Institute for Risk 294
Assessment for their assistance with AFLP. 295
13
296
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417
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Figure Captions 417
418
Figure 1 419
Isolation of Campylobacter (positive samples dark colored, n=313) during the examined 420
rearing periods (1-6) on 15 different farms 421
422
Figure 2 423
Proportional distribution of C. jeuni (, n=228) and C. coli (, n=92) positive samples on the 424
farms 425
426
Figure 3 427
AFLP dendrogram of 15 selected Campylobacter isolates from different farms with a cut-off 428
set at 90% similarity. Isolates No. 1-8, 10-12, 14, C. jejuni; No. 9, 13, 15, C. coli. Isolates No. 429
1, 6, 10, 15 originating from cattle; No. 2, 4, 5, 8, 9, 13, 14 originating from broilers; No. 3, 430
11 originating from boots, No. 7, 12 originating from water inside the flocks 431
432
19
Table 1 432
No. (%) of Campylobacter positive samples during the sampled rearing periods on the 433
different farms (n=5’154) 434
435
No. (%) of Campylobacter positive samples during the rearing periods Farm 1st rearing 2nd rearing 3rd rearing 4th rearing 5th rearing 6th rearing
A 12 (14.1%) 2c (4.1%) 3c (3.8%) 3c (4.5%) nsb ns B 4c (6.3%) 1c (1.0%) 6c (7.4%) 1c (1.8%) 0 (0.0%) 3c (6.1%)
C 0 (0.0%) 10d (9.6%) 2d (2.2%) 4d (4.5%) 0 (0.0%) 12d (22.6%) D 4c (4.0%) 1c (1.0%) 5 (6.2%) 8 (10.7%) 13 (16.5%) 13 (15.5%)
E 2c (3.0%) 1c (1.5%) 3c (6.4%) 0 (0.0%) 1c (1.6%) 2c (4.2%) F 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
G 3c (4.5%) 0 (0.0%) 0 (0.0%) 5c (6.3%) 0 (0.0%) 1c (2.5%) H 3c (3.4%) 2c (3.1%) 4d (5.8%) 8 (12.1%) 1c (1.1%) 2c (5.6%)
I 0 (0.0%) 0 (0.0%) 6 (13.6%) 5d (7.0%) 7d (9.9%) 0 (0.0%) Ka 2c (3.4%) 9 (9.6%) 0 (0.0%) ns ns ns
La 5c (8.1%) 14 (19.4%) 10 (11.6%) 15 (23.4%) ns ns M 7c (13.7%) 3 (8.3%) 6c (11.3%) 8c (9.2%) 2c (3.7%) 1c (1.9%)
N 0 (0.0%) 3d (4.7%) 1c (1.5%) 1c (1.5%) 2c (5.6%) 0 (0.0%) O 2c (4.4%) 0 (0.0%) 6d (9.7%) 8 (15.7%) 1c (2.6%) 0 (0.0%)
Pa 10 (9.9%) 16d (20.8%) 16 (19.5%) ns ns ns 436
a extensive outdoor flocks; b ns, not sampled; c positive samples only in the environment; d 437
positive samples only in boilers and the broiler house 438
439
20
Table 2 439
Genotypes (No. of isolates) of Campylobacter positive samples from different compartments 440
on farm H 441
442
Broilers Boots Clean floor Cattle Rearing period RFLPa PFGEb RFLP PFGE RFLP PFGE RFLP PFGE
1 H-I (6), H-V (3)
H-c (3), H-e (6)
2 H-I (3), H-II (3)
H-b (3), H-c (3)
3 H-I (9) H-b (9) H-I (3) H-b (3)
4 H-IV (9) H-a (7) H-IV (3), H-V (3)
H-a (3), H-d (3)
H-IV (3) H-a (2) H-I (2)c, H-III (1)c, H-V (3)
H-b (3)c, H-c (3)
5 H-I (3) H-b (3) 6 H-b (3),
H-c (3) 443
a fla-RFLP profiles: H-I to H-V; b PFGE profiles: H-a to H-e; c C. coli; 444
445
446
21
Table 3 446
Genotypes (No. of isolates) of Campylobacter positive samples from different compartments 447
on farm D 448
449
Broilers Boots Cattle Pigs Rearing period RFLPa PFGEb RFLP PFGE RFLP PFGE RFLP PFGE
1 D-I (3)c, D-IV (6)c, D-V (3)
D-a (9)c, D-d (3)
2 D-I (3)c D-a (3)c 3 D-II (5)c,
D-III (2)c, D-IV (2)c
D-a (5)c, D-b (4)c
D-II (3)c D-b (3)c D-I (1)c, D-II (1)c, D-IV (1)c
D-a (3)c
4 D-VI (12) D-c (8) D-V (9) D-d (4) D-VII (3)c D-e (3)c
5 D-VI (9) D-c (9) D-VI (2) D-c (2) D-V (24)d D-d (24)d D-IV (3)c D-a (3)c 6 D-III (6)c D-b (6)c D-III (3)c D-b (3)c D-V (5),
D-VI (12), D-I (3)c
D-c (12), D-d (5), D-a (3)c
D-V (3), D-VI (6)
D-c (5), D-d (3)
450
a fla-RFLP profiles: D-I to D-VII; b PFGE profiles: D-a to D-e; c C. coli; d C. jejuni and C. 451
coli 452
453 454
22
Figure 1, Zweifel et al. 454
455
456
457
458
459
460
461
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
A Mouse
B
C
D
E
F
G Bantams
H
I
Kc
Lc Laying hens
M
N
O
Pc Horse
a ns, not sampled; b na, not available; c extensive outdoor flocks
na
Boots
ns
nab
na
na
na
ns
Anteroom broiler house
Dust Water Clean floor
Farm Other positive
samples
ns
na
Cattle Pigs
nsa ns nsns
ns ns
na
ns ns ns ns
na
na
na
na
ns ns ns ns
Broilers Stable broiler house
nsnsns ns
ns ns
ns ns
23
Figure 2, Zweifel et al. 461
462
463
464
465
466
467
468
24
Figure 3, Zweifel et al. 468
469
470
471
472
473
474
475
476 477
