- Email: [email protected]
Analysis of risk factors for Campylobacter species infection in broiler flocks
Analysis of risk factors for Campylobacter species infection in broiler flocks G. Näther, T. Alter, A. Martin, and L. Ellerbroek1 Federal Institute for Risk Assessment, Unit Food Hygiene and Safety Concepts, 14195 Berlin, Germany tained from questionnaires, we identified 3 risk factors for Campylobacter colonization. Campylobacter prevalence was significantly higher in flocks from free-range and organic farms, in flocks with a size up to 15,000 birds and with more than 25,000 birds, and in flocks using nipple drinkers with trays. We found no evidence of an effect of slaughter age, time interval between successive flocks, hygiene measures, number of broiler houses on a farm, partial slaughter, source of water supply, and number of farm employees on the Campylobacter infection rate.
Key words: Campylobacter species, broiler flock, risk factor, colonization 2009 Poultry Science 88:1299–1305 doi:10.3382/ps.2008-00389
INTRODUCTION Campylobacter spp. are one of the most important human bacterial pathogens causing diarrhea and other diseases like septicemia, meningitis, and as complications reactive arthritis and Guillain-Barré syndrome (Hughes and Cornblath, 2005; Leirisalo-Repo, 2005; Uzoigwe, 2005; RKI, 2006). Most human campylobacteriosis cases are foodborne. Handling or the consumption of raw or undercooked poultry meat is regarded as a risk factor for human infection (Loewenherz-Lüning et al., 1996; RKI, 2006; Adak et al., 2005). Low numbers of Campylobacter cells are sufficient to cause human infections (Robinson, 1981). Once introduced into a flock, Campylobacter spp. spread quickly, and birds carrying Campylobacter spp. are asymptomatic without any clinical symptoms (Evans and Sayers, 2000; Van Gerwe et al., 2005). Birds shed a large number of this pathogen in feces, and leaking intestinal content contaminating the slaughter carcass is regarded as the main source of cross-contamination at the abattoir. Together with the control of Campylobacter contamination at the slaughter stage, a prevention of Campylobacter colonization at flock level seems to be the best option ©2009 Poultry Science Association Inc. Received September 10, 2008. Accepted February 13, 2009. 1 Corresponding author: [email protected]
to reduce Campylobacter contamination of poultry meat. Possible risk factors for Campylobacter colonization in broiler flocks are reported in some studies. The Campylobacter status is associated with season (JacobsReitsma, 1994; Berndtson et al., 1996; Wallace et al., 1997; Wedderkopp et al., 2000; Refrégier-Petton et al., 2001; Bouwknegt et al., 2003; Barrios et al., 2006), age of birds at slaughter (Berndtson et al., 1996; Evans and Sayers, 2000; Bouwknegt et al., 2003; Barrios et al., 2006), production type (Fernández et al., 1993; Heuer et al., 2001), and on-farm biosecurity measures (Humphrey et al., 1993; Berndtson et al., 1996; van de Giessen et al., 1996; Gregory et al., 1997; Evans and Sayers, 2000; Cardinale et al., 2004).
MATERIALS AND METHODS Epidemiological Information During the period from May 2004 to April 2005, one hundred forty-six flocks of 75 broiler farms including 60 conventional farms, 5 farms with Louisiana broiler houses (which are characterized by the absence of forced-air ventilation), 7 free-range farms, and 3 organic farms were investigated. From every farm, 1 flock was investigated per summer and per winter period. The study was based on the voluntary cooperation of the production companies and the farmers and true answers to the questions. A review of information was
1299
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
ABSTRACT We have sampled 146 German broiler flocks at slaughter from May 2004 to April 2005 to determine the prevalence of Campylobacter spp. and to investigate risk factors for the presence of Campylobacter spp. at flock level. Cecal samples were tested in accordance to ISO 10727, and potential risk factors were analyzed using farm- and flock-specific information obtained from questionnaires. Of the flocks tested, 44% were Campylobacter-positive, and most were infected with Campylobacter jejuni. Higher Campylobacter prevalence was found during the months of May to October (52%). Using farm- and flock-specific information ob-
1300
Näther et al.
not always possible, especially concerning hygiene measures. To identify potential risk factors for the presence of Campylobacter spp. at flock level, each farm was analyzed using farm- and flock-specific information obtained from questionnaires. Data concerned house surroundings, house characteristics, staff, sanitary practice, control of wild birds and rodents, dead bird management, feeding and watering practice, and various herd parameters.
Campylobacter Sampling and Analysis
Outcome Variable The outcome variable was the flock. A flock was declared as Campylobacter-positive if Campylobacter spp. were isolated from the pooled sample. If no Campylobacter spp. were detected, the corresponding flock was considered as Campylobacter-negative. Due to known seasonality in Campylobacter presence, all variables were tested per half-year and year.
All statistical analyses were done using SPSS software, version 12.0 (SPSS Inc., Chicago, IL). Table 1 lists variables under study. The number of categories per variable was limited to a maximum of 3 (for variables: house number, flock size, service period, and production type). All variables were analyzed using Fisher’s exact test. This was done by comparing Campylobacterpositive flocks to Campylobacter-negative flocks. For variables associated significantly with Campylobacter colonization (Fisher’s χ2, P < 0.05), odds ratios were calculated. If a variable consisted of 3 categories, 1 category was nominated as a main category. The other categories were separately involved in calculation of odds ratios with reference to the main category. The variable age was analyzed using Mann-Whitney U-test (Table 2). Bilateral relationships between detected risk factors were checked (Fisher’s exact test).
RESULTS AND DISCUSSION Of the 146 flocks studied, 44% were tested positive for Campylobacter spp. The most prevalent species was C. jejuni (Table 3). Higher Campylobacter prevalence was found during the summer months May to October (53%). From November to April (winter months), only 34% of flocks were colonized by Campylobacter spp. (Table 3). When analyzing single seasons (summer-winter), no variable was recognized as a risk factor. This may reflect the fact that Campylobacter prevalence is generally high in the summer period. In our study, 53% of the broiler flocks were Campylobacter-positive in the summer season. That seasonal variation in the Campylobacter prevalence was reported before. In many studies, the highest proportion of Campylobacter-positive flocks was found from June to November (Jacobs-Reitsma, 1994; Berndtson et al., 1996; Wallace et al., 1997; Wedderkopp et al., 2000; Refrégier-Petton et al., 2001; Bouwknegt et al., 2003; Barrios et al., 2006). In contrast, Humphrey et al. (1993) and Evans and Sayers (2000) found no seasonality in Campylobacter prevalence. In agreement with other studies (Cardinale et al., 2004; EFSA, 2006), C. jejuni was the dominant species. In contrast to this result, C. coli was the dominant Campylobacter species isolated from poultry in Nigeria and Thailand (Aboaba and Smith, 2005; Padungtod and Kaneene, 2005).
Factors With an Influence on the Campylobacter Status Only 3 variables tested in our analysis were significantly associated with the Campylobacter status of the flock at the end of the rearing period (Table 4). The risk of flock colonization with Campylobacter spp. increased in flocks from free-range and organic farms and
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
One flock of each farm was tested twice a year (summer season: May to October, winter season: November to April). Two flocks were excluded during the summer and winter samplings. From each flock, 10 intestinal convolutes with intact ceca were collected at abattoir by the local veterinarian. This sample size ensured that Campylobacter spp. could be detected with 95% confidence of the within-flock prevalence of a population of more than 2,000 broilers. According to Luechtefeld et al. (1981), intestinal samples were stored at 4°C and transported within 3 d to the laboratory. Isolation of Campylobacter spp. was performed in general accordance with the ISO 10727 guideline (ISO, 1995). In short, content of 10 ceca was aseptically removed and pooled in 100 mL of Preston broth (Oxoid CM67, SR 232 E, SR 104, SR 48, Basingstoke, Hampshire, UK) and cultured for 24 h at 42°C. One loop of pooled sample was streaked on Karmali agar (Oxoid CM 935, SR 205 E) and incubated for 48 h at 42°C. From each positive pooled sample, one suspected Campylobacter colony was subcultured on Mueller-Hinton agar (Oxoid CM 337, 5% sheep blood) and biotyped. The biotyping consisted of gram-staining, observation of motility in phase contrast microscope, production of catalase, hippurate hydrolysis, indoxyl acetate hydrolysis, growth at 25 and 43°C, and susceptibility to nalidixic acid and cephalothin. All incubations were carried out under microaerobic conditions (approximately 5% O2, 10% CO2, 85% N2). All strains were stored at −80°C in Brucella broth (Difco 0495-17, Franklin Lakes, NJ) with 10% glycerol. Campylobacter jejuni DSMZ 4688, Campylobacter coli DSMZ 4689, and Campylobacter lari DSMZ 11375 were used as control strains.
Statistical Analysis
1301
RISK FACTORS FOR CAMPYLOBACTER INFECTION Table 1. Explanatory variables included in the analysis of Campylobacter colonization Number of flocks Summer Variable
Negative
Positive
Negative
Positive
23 12
31 (57%) 7 (37%)
33 15
22 (38%) 5 (25%)
7 22 6
13 (65%) 16 (42%) 9 (69%)
9 33 6
12 (57%) 5 (13%) 8 (57%)
13 8 9 4
13 14 7 5
(50%) (64%) (44%) (56%)
18 19 8 3
24 11
17 (42%) 21 (66%)
27 21
14 (34%) 11 (34%)
3 32
2 (40%) 36 (53%)
3 45
2 (40%) 23 (34%)
7 28
12 (63%) 26 (48%)
12 36
7 (37%) 18 (33%)
6 29
8 (57%) 30 (51%)
11 37
3 (21%) 22 (37%)
21 14
19 (48%) 19 (58%)
27 21
14 (34%) 11 (34%)
29 6
28 (49%) 10 (63%)
39 9
18 (32%) 7 (44%)
2 33
3 (60%) 35 (52%)
2 46
3 (60%) 22 (32%)
1 34
1 (50%) 37 (52%)
1 47
1 (50%) 24 (34%)
2 33
3 (60%) 35 (52%)
3 45
2 (40%) 23 (34%)
4 31
2 (33%) 36 (54%)
6 42
0 (0%) 25 (37%)
1 34
5 (83%) 33 (49%)
3 45
3 (50%) 22 (33%)
18 16
24 (57%) 15 (48%)
26 22
17 (40%) 8 (27%)
14 20
15 (52%) 24 (55%)
21 27
8 (28%) 17 (39%)
1 33
2 (67%) 37 (53%)
2 46
1 (33%) 24 (34%)
10 25
9 (47%) 29 (54%)
13 35
6 (32%) 19 (35%)
7 21 7
14 (67%) 19 (48%) 5 (42%)
12 28 8
8 (40%) 13 (32%) 4 (33%)
15 20
14 (48%) 24 (55%)
18 30
11 (38%) 14 (32%)
0 35
1 (100%) 37 (51%)
1 47
0 (0%) 25 (35%)
1
0 (0%)
1
0 (0%)
8 8 2 7
(31%) (30%) (20%) (70%)
Continued
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
Number of broiler houses <8 ≥8 Flock size ≤15,000 15,001 to 25,000 >25,000 Slaughter age 30 to 33 d 34 to 37 d 38 to 41 d >42 d Animals on nearby farms Within 1 km <1 km Separation of operational areas No Yes Black and white areas1 No Yes Cemented ways on farm No Yes Cemented and clean access around the house No Yes Other animals on the farm No Yes Intact floor No Yes Intact ceiling No Yes Intact walls No Yes Clean anteroom No Yes Boot dip at the entrance to the changing room No Yes Cleaning and disinfection of hands No Yes Flock-specific clothes used No Yes Flock-specific shoes used No Yes Flock-specific tools used No Yes Number of employees on the farm 1 person/1 house 1 person/houses >1 persons Presence of rodents, insects No Yes Control of wildlife No Yes Cleaning Cold water
Winter
1302
Näther et al. Table 1 (Continued). Explanatory variables included in the analysis of Campylobacter colonization Number of flocks Summer Variable
Winter
Positive
Negative
Positive
10 24
15 (60%) 23 (49%)
14 33
12 (46%) 13 (28%)
25 10
25 (50%) 13 (57%)
31 17
19 (38%) 6 (26%)
31 4
33 (52%) 5 (56%)
45 3
18 (29%) 7 (70%)
22 13
20 (48%) 18 (58%)
30 18
11 (27%) 14 (44%)
34 1
34 (50%) 4 (80%)
46 2
21 (31%) 4 (67%)
16 19
19 (54%) 19 (50%)
23 25
13 (36%) 12 (32%)
18 17
21 (54%) 17 (50%)
27 21
12 (31%) 13 (38%)
23 11
30 (57%) 5 (31%)
31 14
22 (42%) 2 (13%)
19 3 13
25 (57%) 1 (25%) 12 (48%)
27 4 17
17 (39%) 0 (0%) 8 (32%)
27 7
33 (55%) 6 (46%)
41 7
19 (32%) 6 (46%)
15 19
22 (60%) 17 (47%)
25 23
12 (32%) 13 (36%)
33 2
36 (52%) 2 (50%)
45 3
24 (35%) 1 (25%)
18 17
20 (53%) 18 (51%)
23 15
15 (40%) 10 (29%)
Cold water (high pressure) Hot water (high pressure) Service period ≤10 d >10 d Production system Conventional and Louisiana Free range and organic Partial slaughter No Yes Ventilation system Mechanical Natural Additional food No Yes Type of drinking water Public Private Type of nipple drinkers With trays Without trays Litter Straw Shavings Straw and shavings Litter storage Closed Open Dead bird storage Not frozen Frozen Dung storage Outside the farm On the farm Dung disposal Sale Own farmland 1
Black and white areas correspond to hygienic areas where chicken are kept (white) and the surrounding area (black).
in flocks with a size up to 15,000 and more than 25,000 birds. The use of nipple drinkers with trays also increased the risk of contamination. In contrast, no other factor under study showed a significant influence on Campylobacter infection. The production type had a significant influence on Campylobacter status of broiler flocks. However, the basis for this result is a difference in the number of houses (63 conventional, 10 free range), which may have influ-
enced the outcome. Free-range and organic broiler flocks showed a significantly higher Campylobacter prevalence in contrast to conventional broiler flocks and flocks with Louisiana houses. Fernández et al. (1993) and Heuer et al. (2001) also reported a significant difference in Campylobacter prevalence depending on production systems, with higher prevalence in flocks from freerange farming compared with conventional farming. In contrast, Wittwer et al. (2005) could not identify a spe-
Table 2. Influence of the slaughter age on the Campylobacter status at slaughter (Mann-Whitney U-test) Summer
Winter
Campylobacter status
n
Middle rank
P-value1
n
Middle rank
P-value1
Negative Positive
34 39
36.41 37.51
0.824
48 25
35.49 39.90
0.396
1
Fisher’s exact test.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
Negative
1303
RISK FACTORS FOR CAMPYLOBACTER INFECTION Table 3. Percentage of flocks colonized with Campylobacter spp. at the end of the rearing period Number of flocks Campylobacter status and species
Summer
Positive Campylobacter jejuni Campylobacter coli Campylobacter lari C. jejuni and C. coli Negative
39 25 12 1 1 34
25 17 7 0 1 48
(34%) (23%) (10%) (0%) (1%) (66%)
Year 64 42 19 1 2 82
(44%) (29%) (13%) (1%) (1%) (56%)
tion of Campylobacter spp. load but could not eliminate Campylobacter spp. entirely within broiler flocks. The flock size was also significantly associated with Campylobacter status. Flocks with a size up to 15,000 birds and more than 25,000 birds showed a higher Campylobacter prevalence. Berndtson et al. (1996) and Barrios et al. (2006) reported of a higher Campylobacter prevalence in larger flocks. Other studies found no link between flock size and Campylobacter status (Humphrey et al., 1993; Evans and Sayers, 2000; Cardinale et al., 2004). Larger flocks might give Campylobacter spp. more chances of entry because of larger volumes of water, food, litter, and air as well as of increased personnel movements. The effect of small flock size on the increasing Campylobacter status could be due to specific production systems and farm managements. Flocks of free and organic farms were of small size and the production systems might explain the increasing risk. Single farms with small flock sizes kept other animals on the farm. Animals such as pigs, cattle, and sheep are carriers of Campylobacter spp. and might serve as a reservoir (Gregory et al., 1997; EFSA, 2006). The presence of other animals on the farm has been previously reported as a risk factor (van de Giessen et al., 1996; Bouwknegt et al., 2003; Cardinale et al., 2004). Handling of Campylobacter-positive birds (e.g., during thinning) increases the risk of flock colonization. Campylobacter prevalence increased significantly when nipple drinkers with trays were used. Water in the trays is an excellent habitat for Campylobacter spp. Amoebae and algae in this water support survival of
Table 4. Risk factors for Campylobacter infection of broiler flocks Summer Variable Production system Conventional and Louisiana3 Free range and organic Flock size ≤15,000 15,001 to 25,000 >25,000 Type of nipple drinkers With tray Without tray 1
P-value1
OR2
1.000
1 1.17
0.247
2.55 1 2.06
0.093
Fisher’s exact test. OR = odds ratio. 3 Characterized by absence of forced-air ventilation. 2
2.87 1
Winter 95%
(0.29; 4.78) (0.831; 7.81)
P-value1
OR
95%
<0.05
1 5.83
(1.36; 25.09)
0.000
(0.786; 9.616) (0.87; 9.43)
<0.05
8.80 1 8.80 4.98 1
(2.45; 31.25) (2.136; 36.26) (1.02; 24.39)
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
cific production type as a risk factor for Campylobacter colonization. Broilers from free-range and organic farms have an increased contact with the environment, representing a large number of possible sources of infection for the broiler flocks. Wild animals such as deer, foxes, hares, badgers, ducks, gulls, pigeons, and falcons are known as carriers of Campylobacter spp. (Glünder et al., 1991; Oyarzabal et al., 1995; Newell and Fearnley, 2003; Lillehaug et al., 2005). Even domestic animals and pets can serve as a reservoir for Campylobacter spp. (Gregory et al., 1997; Bender et al., 2005; Wieland et al., 2005; EFSA, 2006). Rivoal et al. (2005) identified soil in the area around the poultry houses as a potential source of Campylobacter contamination, possibly infected by previous flocks. They also demonstrated the existence of multiple sources of contamination at the same time, by isolating different Campylobacter strains in 1 flock during 1 rearing period. Vaccination strategies for the prevention of Campylobacter colonization in poultry, co-colonization with nonpathogen Campylobacter strains or vaccination with nonvirulent Salmonella strains carrying Campylobacter antigens, showed no or only a limited protection against Campylobacter colonization (Weber, 2000; Chen and Stern, 2001; Wyszyńska et al., 2004; Sizemore et al., 2005). Feeding acidified feed to prevent Campylobacter spp. infection was also unsuccessful (Heres et al., 2004). The use of bacteriophages (Connerton et al., 2004; Atterbury et al., 2005; Wagenaar et al., 2005), bacteriocins (Stern et al., 2005; Svetoch et al., 2005), prebiotics, and probiotics (Rastall, 2004; Ding et al., 2005) resulted in a reduc-
(53%) (34%) (17%) (1%) (1%) (47%)
Winter
1304
Näther et al.
Campylobacter spp. (Axelsson-Olsson et al., 2005; Snelling et al., 2005). In addition, cleaning and disinfection of trays after each rotation must be carried out profoundly to prevent colonization of the successive flock.
care of the broiler houses. Our study could not prove that assumption.
Factors Without an Influence on the Campylobacter Status
Aboaba, O. O., and S. I. Smith. 2005. Occurrence of Campylobacter species in poultry forms in Lagos area of Nigeria. J. Environ. Biol. 26:403–408. Adak, G. K., S. M. Meakins, H. Yip, B. A. Lopman, and S. J. O’Brian. 2005. Disease risks from foods, England and Wales, 1996–2000. Emerg. Infect. Dis. 11:365–372. Atterbury, R. J., E. Dillon, C. Swift, P. L. Connerton, J. A. Frost, C. E. R. Dodd, C. E. D. Rees, and I. F. Connerton. 2005. Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca. Appl. Environ. Microbiol. 71:4885–4887. Axelsson-Olsson, D., J. Waldenstrom, T. Broman, B. Olsen, and M. Holmberg. 2005. Protozoan Acanthamoeba polyphaga as a potential reservoir for Campylobacter jejuni. Appl. Environ. Microbiol. 71:987–992. Barrios, P. R., J. Reiersen, R. Lowman, J. R. Bisaillon, P. Michel, V. Fridriksdóttir, E. Gunnarsson, N. Stern, O. Berke, S. McEwen, and W. Martin. 2006. Risk factors for Campylobacter spp. colonization in broiler flocks in Iceland. Prev. Vet. Med. 74:264–278. Bender, J. B., S. A. Shulman, G. A. Averbeck, G. C. Pantlin, and B. E. Stromberg. 2005. Epidemiologic features of Campylobacter infection among cats in the upper midwestern United States. J. Am. Vet. Med. Assoc. 226:544–547. Berndtson, E., U. Emanuelson, A. Engvall, and M. L. DanielssonTham. 1996. A 1-year epidemiological study of campylobacters in 18 Swedish chicken farms. Prev. Vet. Med. 26:167–185. Bouwknegt, M., A. W. van de Giessen, W. D. C. Dam-Deisz, A. H. Havelaar, N. J. D. Nagelkerke, and A. M. Henken. 2003. Risk factors for the presence of Campylobacter spp. in Dutch broiler flocks. Prev. Vet. Med. 62:35–49. Cardinale, E., F. Tall, E. F. Guèye, M. Cisse, and G. Salvat. 2004. Risk factors for Campylobacter spp. infection in Senegalese broiler-chicken flocks. Prev. Vet. Med. 64:15–25. Chen, H. C., and N. J. Stern. 2001. Competitive exclusion of heterologous Campylobacter spp. in chicks. Appl. Environ. Microbiol. 67:848–851. Connerton, P. L., C. M. Loc Carrillo, C. Swift, E. Dillon, A. Scott, C. E. D. Rees, C. E. R. Dodd, J. Frost, and I. F. Connerton. 2004. Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens. Appl. Environ. Microbiol. 70:3877–3883. Ding, W., H. Wang, and M. W. Griffiths. 2005. Probiotics downregulate flaA sigma28 promoter in Campylobacter jejuni. J. Food Prot. 68:2295–2300. EFSA. 2006. The community summary report on trends and source of zoonoses, zoonotic agents and antimicrobial resistance in the European Union in 2004. European Food Safety Authority, Parma, Italy. Evans, S. J., and A. R. Sayers. 2000. A longitudinal study of Campylobacter infection of broiler flocks in Great Britain. Prev. Vet. Med. 46:209–223. Fernández, H., R. Salazar, and E. Landskron. 1993. Occurrence of thermotolerant species of Campylobacter in three groups of hens maintained under different environmental conditions. Rev. Microbiol. 24:265–268. Glünder, G., U. Neumann, S. Braune, J. Prüter, S. Petersen, and G. Vauk. 1991. Zum Vorkommen von Campylobacter spp. und Salmonella spp. bei Möwen in Norddeutschland. Dtsch. Tierärztl. Wochenschr. 98:152–155. Gregory, E., H. Barnhart, D. W. Dreesen, N. J. Stern, and J. L. Corn. 1997. Epidemiological study of Campylobacter spp. in broilers: Source, time of colonization, and prevalence. Avian Dis. 41:890–898. Hansson, I., M. Ederoth, L. Andersson, I. Vågsholm, and E. Olsson Engvall. 2005. Transmission of Campylobacter spp. to chickens during transport to slaughter. Appl. Environ. Microbiol. 99:1149–1157.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
In our study, hygienic measures, such as the use of separate clothes in every broiler house, disinfectant baths for shoes, and cleaning and disinfection of hands before entering the broiler houses, did not have a significant influence on the Campylobacter status (Table 1). This is in agreement with prior studies (Humphrey et al., 1993; Refrégier-Petton et al., 2001; Bouwknegt et al., 2003). Nonetheless, some studies demonstrated an influence of hygienic measures on the Campylobacter prevalence in broiler flocks (Humphrey et al., 1993; Berndtson et al., 1996; van de Giessen et al., 1996; Gregory et al., 1997; Evans and Sayers, 2000; Cardinale et al., 2004). The different results obtained by different studies might be caused by noncomparable levels of hygienic measures or inconsistencies in keeping to the hygienic practice throughout the whole rearing period. Keeping strict hygienic practices might be a good measure to reduce Campylobacter colonization in broiler flocks, but a complete elimination of Campylobacter spp. cannot be expected (van de Giessen et al., 1998). In contrast to some studies that have demonstrated a relationship between slaughter age and Campylobacter prevalence as stated by Berndtson et al. (1996), Evans and Sayers (2000), Bouwknegt et al. (2003), and Barrios et al. (2006), our study showed no association between the Campylobacter status and the slaughter age (Table 2). That is in agreement to reports by Jacobs-Reitsma et al. (1994) and Wedderkopp et al. (2000) Some authors identified partial slaughter as an infection risk. Both catchers and inadequately cleaned and disinfected transport crates are regarded as possible vectors (Berndtson et al., 1996; Newell and Fearnley, 2003; Ramabu et al., 2004; Hansson et al., 2005). In agreement to our results, Russa et al. (2005) and Barrios et al. (2006) did not demonstrate a significant correlation between partial slaughter and an increased risk of Campylobacter colonization. Refrégier-Petton et al. (2001) and Bouwknegt et al. (2003) reported an increasing risk of Campylobacter colonization when more than 3 broiler houses were on the farm. We could not detect any association between the number of broiler houses and the Campylobacter prevalence. The kind of water supply (public or private) had no influence on the Campylobacter colonization. This is in agreement to other studies (Berndtson et al., 1996; Humphrey et al., 1993; Cardinale et al., 2004). Refrégier-Petton et al. (2001) determined an increasing Campylobacter prevalence if 2 or more people took
REFERENCES
RISK FACTORS FOR CAMPYLOBACTER INFECTION
Russa, A. D., A. Bouma, J. C. M. Vernooij, W. Jacobs-Reitsma, and J. A. Stegeman. 2005. No association between partial depopulation and Campylobacter spp. colonization of Dutch broiler flocks. Appl. Environ. Microbiol. 41:280–285. Sizemore, D. R., B. Warner, J. Lawrence, A. Jones, and K. P. Killeen. 2005. Live, attenuated Salmonella Typhimurium vectoring Campylobacter antigens. Vaccine 24:3793–3803. Snelling, W. J., J. P. McKenna, D. M. Lecky, and J. S. G. Dooley. 2005. Survival of Campylobacter jejuni in waterborne protozoa. Appl. Environ. Microbiol. 71:5560–5571. Stern, N. J., E. A. Svetoch, B. V. Eruslanov, Y. N. Kovalev, L. I. Volodina, V. V. Perelygin, E. V. Mitsevich, I. P. Mitsevich, and V. P. Levchuk. 2005. Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens. J. Food Prot. 68:1450–1453. Svetoch, E. A., N. J. Stern, B. V. Eruslanov, Y. N. Kovalev, L. I. Volodina, V. V. Perelygin, E. V. Mitsevich, I. P. Mitsevich, V. D. Pokhilenko, V. N. Borzenkov, V. P. Levchuk, O. E. Svetoch, and T. Y. Kudriavtseva. 2005. Isolation of Bacillus circulans and Paenibacillus polymyxa strains inhibitory to Campylobacter jejuni and characterization of associated bacteriocins. J. Food Prot. 68:11–17. Uzoigwe, C. 2005. Campylobacter infections of the pericardium and myocardium. Clin. Microbiol. Infect. 11:253–255. van de Giessen, A. W., B. P. M. Bloemberg, W. S. Ritmeester, and J. J. H. C. Tilburg. 1996. Epidemiological study on risk factors and risk reducing measures for Campylobacter infections in Dutch broiler flocks. Epidemiol. Infect. 117:245–250. van de Giessen, A. W., J. J. H. C. Tilburg, W. S. Ritmeester, and J. van der Plas. 1998. Reduction of Campylobacter infections in broiler flocks by application of hygiene measures. Epidemiol. Infect. 121:57–66. Van Gerwe, T. J. W. M., A. Bouma, W. F. Jacobs-Reitsma, J. van den Broek, D. Klinkenberg, J. A. Stegeman, and J. A. P. Heesterbeek. 2005. Quantifying transmission of Campylobacter spp. among broilers. Appl. Environ. Microbiol. 71:5765–5770. Wagenaar, J. A., M. A. P. Van Bergen, M. A. Mueller, T. M. Wassenaar, and R. M. Carlton. 2005. Phage therapy reduces Campylobacter jejuni colonization in broilers. Vet. Microbiol. 109:275– 283. Wallace, J. S., K. N. Stanley, J. E. Currie, P. J. Diggle, and K. Jones. 1997. Seasonality of thermophilic Campylobacter populations in chickens. J. Appl. Microbiol. 82:219–224. Weber, R. 2000. Prüfung wechselseitiger Hemmeffekte verschiedener Campylobacter jejuni-Stämme bei der Kolonisation des Huehnerdarmes. PhD Diss. Tieraerztliche Hochschule Hannover, Germany. Wedderkopp, A., E. Rattenborg, and M. Madsen. 2000. National surveillance of Campylobacter in broilers at slaughter in Denmark in 1998. Avian Dis. 44:993–999. Wieland, B., G. Regula, J. Danuser, M. Wittwer, A. P. Burnens, T. M. Wassenaar, and K. D. Stärk. 2005. Campylobacter spp. in dogs and cats in Switzerland: Risk factor analysis and molecular characterization with AFLP. J. Vet. Med. B Infect. Dis. Vet. Public Health 52:183–189. Wittwer, M., J. Keller, T. M. Wassenaar, R. Stephan, D. Howald, G. Regula, and B. Bissig-Choisat. 2005. Genetic diversity and antibiotic resistance patterns in a Campylobacter population isolated from poultry farms in Switzerland. Appl. Environ. Microbiol. 71:2840–2847. Wyszyńska, A., A. Raczko, M. Lis, and E. K. Jagusztyn-Krynicka. 2004. Oral immunization of chickens with avirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild-type Campylobacter. Vaccine 22:1379– 1389.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
Heres, L., B. Engel, H. A. P. Urlings, J. A. Wagenaar, and F. van Knapen. 2004. Effect of acidified feed on susceptibility of broiler chickens to intestinal infection by Campylobacter and Salmonella. Vet. Microbiol. 99:259–267. Heuer, O. E., K. Pedersen, J. S. Andersen, and M. Madsen. 2001. Prevalence and antimicrobial susceptibility of thermophilic Campylobacter in organic and conventional broiler flocks. Lett. Appl. Microbiol. 33:269–274. Hughes, R. A. C., and D. R. Cornblath. 2005. Guillain-Barré syndrome. Lancet 366:1653–1666. Humphrey, T. J., A. Henley, and D. G. Lanning. 1993. The colonization of broiler chickens with Campylobacter jejuni: Some epidemiological investigations. Epidemiol. Infect. 110:601–607. ISO. 1995. Microbiology of food and animal feeding stuffs–Horizontal method for detection and enumeration of Campylobacter spp. Part 1: Detection method. ISO 10272 1995/cor.1:1996(E). International Organization for Standardization, Geneva, Switzerland. Jacobs-Reitsma, W. F. 1994. Campylobacter and Salmonella in breeder flocks. Pages 25–32 in Epidemiology of Campylobacter in poultry. W. F. Jacobs-Reitsma, ed. ID-DLO, Beekbergen, the Netherlands. Jacobs-Reitsma, W. F., N. M. Bolder, and R. W. A. W. Mulder. 1994. Cecal carriage of Campylobacter and Salmonella in Dutch broiler flocks at slaughter: A one-year study. Poult. Sci. 73:1260–1266. Leirisalo-Repo, M. 2005. Early arthritis and infection. Curr. Opin. Rheumatol. 17:433–439. Lillehaug, A., C. Monceyron Jonassen, B. Bergsjo, M. Hofshagen, J. Tharaldsen, L. L. Nesse, and K. Handeland. 2005. Screening of feral pigeon (Columba livia), mallard (Anas platyrhynchos) and graylag goose (Anser anser) populations for Campylobacter spp., Salmonella spp., avian influenza virus and avian paramyxovirus. Acta Vet. Scand. 46:193–202. Loewenherz-Lüning, K., M. Heitmann, and G. Hildebrandt. 1996. Untersuchungen zum Vorkommen von Campylobacter jejuni in verschiedenen Lebensmitteln tierischen Ursprungs. Fleischwirtschaft 76:958–961. Luechtefeld, N. W., W. L. Wang, M. J. Blaser, and L. B. Reller. 1981. Evaluation of transport and storage techniques for isolation of Campylobacter fetus subsp. jejuni from turkey cecal specimens. J. Clin. Microbiol. 13:438–443. Newell, D. G., and C. Fearnley. 2003. Sources of Campylobacter colonization in broiler chickens. Appl. Environ. Microbiol. 69:4343– 4351. Oyarzabal, O. A., D. E. Conner, and F. J. Hoerr. 1995. Incidence of campylobacters in the intestine of avian species in Alabama. Avian Dis. 39:147–151. Padungtod, P., and J. B. Kaneene. 2005. Campylobacter in food animals and humans in northern Thailand. J. Food Prot. 68:2519– 2526. Ramabu, S. S., N. S. Boxall, P. Madie, and S. G. Fenwick. 2004. Some potential sources for transmission of Campylobacter jejuni to broiler chickens. Lett. Appl. Microbiol. 39:252–256. Rastall, R. A. 2004. Bacteria in the gut: Friends and foes and how to alter the balance. J. Nutr. 134:2022S–2026S. Refrégier-Petton, J., N. Rose, M. Denis, and G. Salvat. 2001. Risk factors for Campylobacter spp. contamination in French broilerchicken flocks at the end of the rearing period. Prev. Vet. Med. 50:89–100. Rivoal, K., C. Ragimbeau, G. Salvat, P. Colin, and G. Ermel. 2005. Genomic diversity of Campylobacter coli and Campylobacter jejuni isolates recovered from free-range broiler farms and comparison with isolates of various origins. Appl. Environ. Microbiol. 71:6216–6227. RKI. 2006. Aktuelle Statistik meldepflichtiger Infektionskrankheiten. Epidemiol. Bull. 3:26–27. Robinson, D. A. 1981. Infective dose of Campylobacter jejuni in milk. Br. Med. J. (Clin. Res. Ed.) 282:1584.
1305
Key words: Campylobacter species, broiler flock, risk factor, colonization 2009 Poultry Science 88:1299–1305 doi:10.3382/ps.2008-00389
INTRODUCTION Campylobacter spp. are one of the most important human bacterial pathogens causing diarrhea and other diseases like septicemia, meningitis, and as complications reactive arthritis and Guillain-Barré syndrome (Hughes and Cornblath, 2005; Leirisalo-Repo, 2005; Uzoigwe, 2005; RKI, 2006). Most human campylobacteriosis cases are foodborne. Handling or the consumption of raw or undercooked poultry meat is regarded as a risk factor for human infection (Loewenherz-Lüning et al., 1996; RKI, 2006; Adak et al., 2005). Low numbers of Campylobacter cells are sufficient to cause human infections (Robinson, 1981). Once introduced into a flock, Campylobacter spp. spread quickly, and birds carrying Campylobacter spp. are asymptomatic without any clinical symptoms (Evans and Sayers, 2000; Van Gerwe et al., 2005). Birds shed a large number of this pathogen in feces, and leaking intestinal content contaminating the slaughter carcass is regarded as the main source of cross-contamination at the abattoir. Together with the control of Campylobacter contamination at the slaughter stage, a prevention of Campylobacter colonization at flock level seems to be the best option ©2009 Poultry Science Association Inc. Received September 10, 2008. Accepted February 13, 2009. 1 Corresponding author: [email protected]
to reduce Campylobacter contamination of poultry meat. Possible risk factors for Campylobacter colonization in broiler flocks are reported in some studies. The Campylobacter status is associated with season (JacobsReitsma, 1994; Berndtson et al., 1996; Wallace et al., 1997; Wedderkopp et al., 2000; Refrégier-Petton et al., 2001; Bouwknegt et al., 2003; Barrios et al., 2006), age of birds at slaughter (Berndtson et al., 1996; Evans and Sayers, 2000; Bouwknegt et al., 2003; Barrios et al., 2006), production type (Fernández et al., 1993; Heuer et al., 2001), and on-farm biosecurity measures (Humphrey et al., 1993; Berndtson et al., 1996; van de Giessen et al., 1996; Gregory et al., 1997; Evans and Sayers, 2000; Cardinale et al., 2004).
MATERIALS AND METHODS Epidemiological Information During the period from May 2004 to April 2005, one hundred forty-six flocks of 75 broiler farms including 60 conventional farms, 5 farms with Louisiana broiler houses (which are characterized by the absence of forced-air ventilation), 7 free-range farms, and 3 organic farms were investigated. From every farm, 1 flock was investigated per summer and per winter period. The study was based on the voluntary cooperation of the production companies and the farmers and true answers to the questions. A review of information was
1299
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
ABSTRACT We have sampled 146 German broiler flocks at slaughter from May 2004 to April 2005 to determine the prevalence of Campylobacter spp. and to investigate risk factors for the presence of Campylobacter spp. at flock level. Cecal samples were tested in accordance to ISO 10727, and potential risk factors were analyzed using farm- and flock-specific information obtained from questionnaires. Of the flocks tested, 44% were Campylobacter-positive, and most were infected with Campylobacter jejuni. Higher Campylobacter prevalence was found during the months of May to October (52%). Using farm- and flock-specific information ob-
1300
Näther et al.
not always possible, especially concerning hygiene measures. To identify potential risk factors for the presence of Campylobacter spp. at flock level, each farm was analyzed using farm- and flock-specific information obtained from questionnaires. Data concerned house surroundings, house characteristics, staff, sanitary practice, control of wild birds and rodents, dead bird management, feeding and watering practice, and various herd parameters.
Campylobacter Sampling and Analysis
Outcome Variable The outcome variable was the flock. A flock was declared as Campylobacter-positive if Campylobacter spp. were isolated from the pooled sample. If no Campylobacter spp. were detected, the corresponding flock was considered as Campylobacter-negative. Due to known seasonality in Campylobacter presence, all variables were tested per half-year and year.
All statistical analyses were done using SPSS software, version 12.0 (SPSS Inc., Chicago, IL). Table 1 lists variables under study. The number of categories per variable was limited to a maximum of 3 (for variables: house number, flock size, service period, and production type). All variables were analyzed using Fisher’s exact test. This was done by comparing Campylobacterpositive flocks to Campylobacter-negative flocks. For variables associated significantly with Campylobacter colonization (Fisher’s χ2, P < 0.05), odds ratios were calculated. If a variable consisted of 3 categories, 1 category was nominated as a main category. The other categories were separately involved in calculation of odds ratios with reference to the main category. The variable age was analyzed using Mann-Whitney U-test (Table 2). Bilateral relationships between detected risk factors were checked (Fisher’s exact test).
RESULTS AND DISCUSSION Of the 146 flocks studied, 44% were tested positive for Campylobacter spp. The most prevalent species was C. jejuni (Table 3). Higher Campylobacter prevalence was found during the summer months May to October (53%). From November to April (winter months), only 34% of flocks were colonized by Campylobacter spp. (Table 3). When analyzing single seasons (summer-winter), no variable was recognized as a risk factor. This may reflect the fact that Campylobacter prevalence is generally high in the summer period. In our study, 53% of the broiler flocks were Campylobacter-positive in the summer season. That seasonal variation in the Campylobacter prevalence was reported before. In many studies, the highest proportion of Campylobacter-positive flocks was found from June to November (Jacobs-Reitsma, 1994; Berndtson et al., 1996; Wallace et al., 1997; Wedderkopp et al., 2000; Refrégier-Petton et al., 2001; Bouwknegt et al., 2003; Barrios et al., 2006). In contrast, Humphrey et al. (1993) and Evans and Sayers (2000) found no seasonality in Campylobacter prevalence. In agreement with other studies (Cardinale et al., 2004; EFSA, 2006), C. jejuni was the dominant species. In contrast to this result, C. coli was the dominant Campylobacter species isolated from poultry in Nigeria and Thailand (Aboaba and Smith, 2005; Padungtod and Kaneene, 2005).
Factors With an Influence on the Campylobacter Status Only 3 variables tested in our analysis were significantly associated with the Campylobacter status of the flock at the end of the rearing period (Table 4). The risk of flock colonization with Campylobacter spp. increased in flocks from free-range and organic farms and
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
One flock of each farm was tested twice a year (summer season: May to October, winter season: November to April). Two flocks were excluded during the summer and winter samplings. From each flock, 10 intestinal convolutes with intact ceca were collected at abattoir by the local veterinarian. This sample size ensured that Campylobacter spp. could be detected with 95% confidence of the within-flock prevalence of a population of more than 2,000 broilers. According to Luechtefeld et al. (1981), intestinal samples were stored at 4°C and transported within 3 d to the laboratory. Isolation of Campylobacter spp. was performed in general accordance with the ISO 10727 guideline (ISO, 1995). In short, content of 10 ceca was aseptically removed and pooled in 100 mL of Preston broth (Oxoid CM67, SR 232 E, SR 104, SR 48, Basingstoke, Hampshire, UK) and cultured for 24 h at 42°C. One loop of pooled sample was streaked on Karmali agar (Oxoid CM 935, SR 205 E) and incubated for 48 h at 42°C. From each positive pooled sample, one suspected Campylobacter colony was subcultured on Mueller-Hinton agar (Oxoid CM 337, 5% sheep blood) and biotyped. The biotyping consisted of gram-staining, observation of motility in phase contrast microscope, production of catalase, hippurate hydrolysis, indoxyl acetate hydrolysis, growth at 25 and 43°C, and susceptibility to nalidixic acid and cephalothin. All incubations were carried out under microaerobic conditions (approximately 5% O2, 10% CO2, 85% N2). All strains were stored at −80°C in Brucella broth (Difco 0495-17, Franklin Lakes, NJ) with 10% glycerol. Campylobacter jejuni DSMZ 4688, Campylobacter coli DSMZ 4689, and Campylobacter lari DSMZ 11375 were used as control strains.
Statistical Analysis
1301
RISK FACTORS FOR CAMPYLOBACTER INFECTION Table 1. Explanatory variables included in the analysis of Campylobacter colonization Number of flocks Summer Variable
Negative
Positive
Negative
Positive
23 12
31 (57%) 7 (37%)
33 15
22 (38%) 5 (25%)
7 22 6
13 (65%) 16 (42%) 9 (69%)
9 33 6
12 (57%) 5 (13%) 8 (57%)
13 8 9 4
13 14 7 5
(50%) (64%) (44%) (56%)
18 19 8 3
24 11
17 (42%) 21 (66%)
27 21
14 (34%) 11 (34%)
3 32
2 (40%) 36 (53%)
3 45
2 (40%) 23 (34%)
7 28
12 (63%) 26 (48%)
12 36
7 (37%) 18 (33%)
6 29
8 (57%) 30 (51%)
11 37
3 (21%) 22 (37%)
21 14
19 (48%) 19 (58%)
27 21
14 (34%) 11 (34%)
29 6
28 (49%) 10 (63%)
39 9
18 (32%) 7 (44%)
2 33
3 (60%) 35 (52%)
2 46
3 (60%) 22 (32%)
1 34
1 (50%) 37 (52%)
1 47
1 (50%) 24 (34%)
2 33
3 (60%) 35 (52%)
3 45
2 (40%) 23 (34%)
4 31
2 (33%) 36 (54%)
6 42
0 (0%) 25 (37%)
1 34
5 (83%) 33 (49%)
3 45
3 (50%) 22 (33%)
18 16
24 (57%) 15 (48%)
26 22
17 (40%) 8 (27%)
14 20
15 (52%) 24 (55%)
21 27
8 (28%) 17 (39%)
1 33
2 (67%) 37 (53%)
2 46
1 (33%) 24 (34%)
10 25
9 (47%) 29 (54%)
13 35
6 (32%) 19 (35%)
7 21 7
14 (67%) 19 (48%) 5 (42%)
12 28 8
8 (40%) 13 (32%) 4 (33%)
15 20
14 (48%) 24 (55%)
18 30
11 (38%) 14 (32%)
0 35
1 (100%) 37 (51%)
1 47
0 (0%) 25 (35%)
1
0 (0%)
1
0 (0%)
8 8 2 7
(31%) (30%) (20%) (70%)
Continued
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
Number of broiler houses <8 ≥8 Flock size ≤15,000 15,001 to 25,000 >25,000 Slaughter age 30 to 33 d 34 to 37 d 38 to 41 d >42 d Animals on nearby farms Within 1 km <1 km Separation of operational areas No Yes Black and white areas1 No Yes Cemented ways on farm No Yes Cemented and clean access around the house No Yes Other animals on the farm No Yes Intact floor No Yes Intact ceiling No Yes Intact walls No Yes Clean anteroom No Yes Boot dip at the entrance to the changing room No Yes Cleaning and disinfection of hands No Yes Flock-specific clothes used No Yes Flock-specific shoes used No Yes Flock-specific tools used No Yes Number of employees on the farm 1 person/1 house 1 person/houses >1 persons Presence of rodents, insects No Yes Control of wildlife No Yes Cleaning Cold water
Winter
1302
Näther et al. Table 1 (Continued). Explanatory variables included in the analysis of Campylobacter colonization Number of flocks Summer Variable
Winter
Positive
Negative
Positive
10 24
15 (60%) 23 (49%)
14 33
12 (46%) 13 (28%)
25 10
25 (50%) 13 (57%)
31 17
19 (38%) 6 (26%)
31 4
33 (52%) 5 (56%)
45 3
18 (29%) 7 (70%)
22 13
20 (48%) 18 (58%)
30 18
11 (27%) 14 (44%)
34 1
34 (50%) 4 (80%)
46 2
21 (31%) 4 (67%)
16 19
19 (54%) 19 (50%)
23 25
13 (36%) 12 (32%)
18 17
21 (54%) 17 (50%)
27 21
12 (31%) 13 (38%)
23 11
30 (57%) 5 (31%)
31 14
22 (42%) 2 (13%)
19 3 13
25 (57%) 1 (25%) 12 (48%)
27 4 17
17 (39%) 0 (0%) 8 (32%)
27 7
33 (55%) 6 (46%)
41 7
19 (32%) 6 (46%)
15 19
22 (60%) 17 (47%)
25 23
12 (32%) 13 (36%)
33 2
36 (52%) 2 (50%)
45 3
24 (35%) 1 (25%)
18 17
20 (53%) 18 (51%)
23 15
15 (40%) 10 (29%)
Cold water (high pressure) Hot water (high pressure) Service period ≤10 d >10 d Production system Conventional and Louisiana Free range and organic Partial slaughter No Yes Ventilation system Mechanical Natural Additional food No Yes Type of drinking water Public Private Type of nipple drinkers With trays Without trays Litter Straw Shavings Straw and shavings Litter storage Closed Open Dead bird storage Not frozen Frozen Dung storage Outside the farm On the farm Dung disposal Sale Own farmland 1
Black and white areas correspond to hygienic areas where chicken are kept (white) and the surrounding area (black).
in flocks with a size up to 15,000 and more than 25,000 birds. The use of nipple drinkers with trays also increased the risk of contamination. In contrast, no other factor under study showed a significant influence on Campylobacter infection. The production type had a significant influence on Campylobacter status of broiler flocks. However, the basis for this result is a difference in the number of houses (63 conventional, 10 free range), which may have influ-
enced the outcome. Free-range and organic broiler flocks showed a significantly higher Campylobacter prevalence in contrast to conventional broiler flocks and flocks with Louisiana houses. Fernández et al. (1993) and Heuer et al. (2001) also reported a significant difference in Campylobacter prevalence depending on production systems, with higher prevalence in flocks from freerange farming compared with conventional farming. In contrast, Wittwer et al. (2005) could not identify a spe-
Table 2. Influence of the slaughter age on the Campylobacter status at slaughter (Mann-Whitney U-test) Summer
Winter
Campylobacter status
n
Middle rank
P-value1
n
Middle rank
P-value1
Negative Positive
34 39
36.41 37.51
0.824
48 25
35.49 39.90
0.396
1
Fisher’s exact test.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
Negative
1303
RISK FACTORS FOR CAMPYLOBACTER INFECTION Table 3. Percentage of flocks colonized with Campylobacter spp. at the end of the rearing period Number of flocks Campylobacter status and species
Summer
Positive Campylobacter jejuni Campylobacter coli Campylobacter lari C. jejuni and C. coli Negative
39 25 12 1 1 34
25 17 7 0 1 48
(34%) (23%) (10%) (0%) (1%) (66%)
Year 64 42 19 1 2 82
(44%) (29%) (13%) (1%) (1%) (56%)
tion of Campylobacter spp. load but could not eliminate Campylobacter spp. entirely within broiler flocks. The flock size was also significantly associated with Campylobacter status. Flocks with a size up to 15,000 birds and more than 25,000 birds showed a higher Campylobacter prevalence. Berndtson et al. (1996) and Barrios et al. (2006) reported of a higher Campylobacter prevalence in larger flocks. Other studies found no link between flock size and Campylobacter status (Humphrey et al., 1993; Evans and Sayers, 2000; Cardinale et al., 2004). Larger flocks might give Campylobacter spp. more chances of entry because of larger volumes of water, food, litter, and air as well as of increased personnel movements. The effect of small flock size on the increasing Campylobacter status could be due to specific production systems and farm managements. Flocks of free and organic farms were of small size and the production systems might explain the increasing risk. Single farms with small flock sizes kept other animals on the farm. Animals such as pigs, cattle, and sheep are carriers of Campylobacter spp. and might serve as a reservoir (Gregory et al., 1997; EFSA, 2006). The presence of other animals on the farm has been previously reported as a risk factor (van de Giessen et al., 1996; Bouwknegt et al., 2003; Cardinale et al., 2004). Handling of Campylobacter-positive birds (e.g., during thinning) increases the risk of flock colonization. Campylobacter prevalence increased significantly when nipple drinkers with trays were used. Water in the trays is an excellent habitat for Campylobacter spp. Amoebae and algae in this water support survival of
Table 4. Risk factors for Campylobacter infection of broiler flocks Summer Variable Production system Conventional and Louisiana3 Free range and organic Flock size ≤15,000 15,001 to 25,000 >25,000 Type of nipple drinkers With tray Without tray 1
P-value1
OR2
1.000
1 1.17
0.247
2.55 1 2.06
0.093
Fisher’s exact test. OR = odds ratio. 3 Characterized by absence of forced-air ventilation. 2
2.87 1
Winter 95%
(0.29; 4.78) (0.831; 7.81)
P-value1
OR
95%
<0.05
1 5.83
(1.36; 25.09)
0.000
(0.786; 9.616) (0.87; 9.43)
<0.05
8.80 1 8.80 4.98 1
(2.45; 31.25) (2.136; 36.26) (1.02; 24.39)
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
cific production type as a risk factor for Campylobacter colonization. Broilers from free-range and organic farms have an increased contact with the environment, representing a large number of possible sources of infection for the broiler flocks. Wild animals such as deer, foxes, hares, badgers, ducks, gulls, pigeons, and falcons are known as carriers of Campylobacter spp. (Glünder et al., 1991; Oyarzabal et al., 1995; Newell and Fearnley, 2003; Lillehaug et al., 2005). Even domestic animals and pets can serve as a reservoir for Campylobacter spp. (Gregory et al., 1997; Bender et al., 2005; Wieland et al., 2005; EFSA, 2006). Rivoal et al. (2005) identified soil in the area around the poultry houses as a potential source of Campylobacter contamination, possibly infected by previous flocks. They also demonstrated the existence of multiple sources of contamination at the same time, by isolating different Campylobacter strains in 1 flock during 1 rearing period. Vaccination strategies for the prevention of Campylobacter colonization in poultry, co-colonization with nonpathogen Campylobacter strains or vaccination with nonvirulent Salmonella strains carrying Campylobacter antigens, showed no or only a limited protection against Campylobacter colonization (Weber, 2000; Chen and Stern, 2001; Wyszyńska et al., 2004; Sizemore et al., 2005). Feeding acidified feed to prevent Campylobacter spp. infection was also unsuccessful (Heres et al., 2004). The use of bacteriophages (Connerton et al., 2004; Atterbury et al., 2005; Wagenaar et al., 2005), bacteriocins (Stern et al., 2005; Svetoch et al., 2005), prebiotics, and probiotics (Rastall, 2004; Ding et al., 2005) resulted in a reduc-
(53%) (34%) (17%) (1%) (1%) (47%)
Winter
1304
Näther et al.
Campylobacter spp. (Axelsson-Olsson et al., 2005; Snelling et al., 2005). In addition, cleaning and disinfection of trays after each rotation must be carried out profoundly to prevent colonization of the successive flock.
care of the broiler houses. Our study could not prove that assumption.
Factors Without an Influence on the Campylobacter Status
Aboaba, O. O., and S. I. Smith. 2005. Occurrence of Campylobacter species in poultry forms in Lagos area of Nigeria. J. Environ. Biol. 26:403–408. Adak, G. K., S. M. Meakins, H. Yip, B. A. Lopman, and S. J. O’Brian. 2005. Disease risks from foods, England and Wales, 1996–2000. Emerg. Infect. Dis. 11:365–372. Atterbury, R. J., E. Dillon, C. Swift, P. L. Connerton, J. A. Frost, C. E. R. Dodd, C. E. D. Rees, and I. F. Connerton. 2005. Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca. Appl. Environ. Microbiol. 71:4885–4887. Axelsson-Olsson, D., J. Waldenstrom, T. Broman, B. Olsen, and M. Holmberg. 2005. Protozoan Acanthamoeba polyphaga as a potential reservoir for Campylobacter jejuni. Appl. Environ. Microbiol. 71:987–992. Barrios, P. R., J. Reiersen, R. Lowman, J. R. Bisaillon, P. Michel, V. Fridriksdóttir, E. Gunnarsson, N. Stern, O. Berke, S. McEwen, and W. Martin. 2006. Risk factors for Campylobacter spp. colonization in broiler flocks in Iceland. Prev. Vet. Med. 74:264–278. Bender, J. B., S. A. Shulman, G. A. Averbeck, G. C. Pantlin, and B. E. Stromberg. 2005. Epidemiologic features of Campylobacter infection among cats in the upper midwestern United States. J. Am. Vet. Med. Assoc. 226:544–547. Berndtson, E., U. Emanuelson, A. Engvall, and M. L. DanielssonTham. 1996. A 1-year epidemiological study of campylobacters in 18 Swedish chicken farms. Prev. Vet. Med. 26:167–185. Bouwknegt, M., A. W. van de Giessen, W. D. C. Dam-Deisz, A. H. Havelaar, N. J. D. Nagelkerke, and A. M. Henken. 2003. Risk factors for the presence of Campylobacter spp. in Dutch broiler flocks. Prev. Vet. Med. 62:35–49. Cardinale, E., F. Tall, E. F. Guèye, M. Cisse, and G. Salvat. 2004. Risk factors for Campylobacter spp. infection in Senegalese broiler-chicken flocks. Prev. Vet. Med. 64:15–25. Chen, H. C., and N. J. Stern. 2001. Competitive exclusion of heterologous Campylobacter spp. in chicks. Appl. Environ. Microbiol. 67:848–851. Connerton, P. L., C. M. Loc Carrillo, C. Swift, E. Dillon, A. Scott, C. E. D. Rees, C. E. R. Dodd, J. Frost, and I. F. Connerton. 2004. Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens. Appl. Environ. Microbiol. 70:3877–3883. Ding, W., H. Wang, and M. W. Griffiths. 2005. Probiotics downregulate flaA sigma28 promoter in Campylobacter jejuni. J. Food Prot. 68:2295–2300. EFSA. 2006. The community summary report on trends and source of zoonoses, zoonotic agents and antimicrobial resistance in the European Union in 2004. European Food Safety Authority, Parma, Italy. Evans, S. J., and A. R. Sayers. 2000. A longitudinal study of Campylobacter infection of broiler flocks in Great Britain. Prev. Vet. Med. 46:209–223. Fernández, H., R. Salazar, and E. Landskron. 1993. Occurrence of thermotolerant species of Campylobacter in three groups of hens maintained under different environmental conditions. Rev. Microbiol. 24:265–268. Glünder, G., U. Neumann, S. Braune, J. Prüter, S. Petersen, and G. Vauk. 1991. Zum Vorkommen von Campylobacter spp. und Salmonella spp. bei Möwen in Norddeutschland. Dtsch. Tierärztl. Wochenschr. 98:152–155. Gregory, E., H. Barnhart, D. W. Dreesen, N. J. Stern, and J. L. Corn. 1997. Epidemiological study of Campylobacter spp. in broilers: Source, time of colonization, and prevalence. Avian Dis. 41:890–898. Hansson, I., M. Ederoth, L. Andersson, I. Vågsholm, and E. Olsson Engvall. 2005. Transmission of Campylobacter spp. to chickens during transport to slaughter. Appl. Environ. Microbiol. 99:1149–1157.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
In our study, hygienic measures, such as the use of separate clothes in every broiler house, disinfectant baths for shoes, and cleaning and disinfection of hands before entering the broiler houses, did not have a significant influence on the Campylobacter status (Table 1). This is in agreement with prior studies (Humphrey et al., 1993; Refrégier-Petton et al., 2001; Bouwknegt et al., 2003). Nonetheless, some studies demonstrated an influence of hygienic measures on the Campylobacter prevalence in broiler flocks (Humphrey et al., 1993; Berndtson et al., 1996; van de Giessen et al., 1996; Gregory et al., 1997; Evans and Sayers, 2000; Cardinale et al., 2004). The different results obtained by different studies might be caused by noncomparable levels of hygienic measures or inconsistencies in keeping to the hygienic practice throughout the whole rearing period. Keeping strict hygienic practices might be a good measure to reduce Campylobacter colonization in broiler flocks, but a complete elimination of Campylobacter spp. cannot be expected (van de Giessen et al., 1998). In contrast to some studies that have demonstrated a relationship between slaughter age and Campylobacter prevalence as stated by Berndtson et al. (1996), Evans and Sayers (2000), Bouwknegt et al. (2003), and Barrios et al. (2006), our study showed no association between the Campylobacter status and the slaughter age (Table 2). That is in agreement to reports by Jacobs-Reitsma et al. (1994) and Wedderkopp et al. (2000) Some authors identified partial slaughter as an infection risk. Both catchers and inadequately cleaned and disinfected transport crates are regarded as possible vectors (Berndtson et al., 1996; Newell and Fearnley, 2003; Ramabu et al., 2004; Hansson et al., 2005). In agreement to our results, Russa et al. (2005) and Barrios et al. (2006) did not demonstrate a significant correlation between partial slaughter and an increased risk of Campylobacter colonization. Refrégier-Petton et al. (2001) and Bouwknegt et al. (2003) reported an increasing risk of Campylobacter colonization when more than 3 broiler houses were on the farm. We could not detect any association between the number of broiler houses and the Campylobacter prevalence. The kind of water supply (public or private) had no influence on the Campylobacter colonization. This is in agreement to other studies (Berndtson et al., 1996; Humphrey et al., 1993; Cardinale et al., 2004). Refrégier-Petton et al. (2001) determined an increasing Campylobacter prevalence if 2 or more people took
REFERENCES
RISK FACTORS FOR CAMPYLOBACTER INFECTION
Russa, A. D., A. Bouma, J. C. M. Vernooij, W. Jacobs-Reitsma, and J. A. Stegeman. 2005. No association between partial depopulation and Campylobacter spp. colonization of Dutch broiler flocks. Appl. Environ. Microbiol. 41:280–285. Sizemore, D. R., B. Warner, J. Lawrence, A. Jones, and K. P. Killeen. 2005. Live, attenuated Salmonella Typhimurium vectoring Campylobacter antigens. Vaccine 24:3793–3803. Snelling, W. J., J. P. McKenna, D. M. Lecky, and J. S. G. Dooley. 2005. Survival of Campylobacter jejuni in waterborne protozoa. Appl. Environ. Microbiol. 71:5560–5571. Stern, N. J., E. A. Svetoch, B. V. Eruslanov, Y. N. Kovalev, L. I. Volodina, V. V. Perelygin, E. V. Mitsevich, I. P. Mitsevich, and V. P. Levchuk. 2005. Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens. J. Food Prot. 68:1450–1453. Svetoch, E. A., N. J. Stern, B. V. Eruslanov, Y. N. Kovalev, L. I. Volodina, V. V. Perelygin, E. V. Mitsevich, I. P. Mitsevich, V. D. Pokhilenko, V. N. Borzenkov, V. P. Levchuk, O. E. Svetoch, and T. Y. Kudriavtseva. 2005. Isolation of Bacillus circulans and Paenibacillus polymyxa strains inhibitory to Campylobacter jejuni and characterization of associated bacteriocins. J. Food Prot. 68:11–17. Uzoigwe, C. 2005. Campylobacter infections of the pericardium and myocardium. Clin. Microbiol. Infect. 11:253–255. van de Giessen, A. W., B. P. M. Bloemberg, W. S. Ritmeester, and J. J. H. C. Tilburg. 1996. Epidemiological study on risk factors and risk reducing measures for Campylobacter infections in Dutch broiler flocks. Epidemiol. Infect. 117:245–250. van de Giessen, A. W., J. J. H. C. Tilburg, W. S. Ritmeester, and J. van der Plas. 1998. Reduction of Campylobacter infections in broiler flocks by application of hygiene measures. Epidemiol. Infect. 121:57–66. Van Gerwe, T. J. W. M., A. Bouma, W. F. Jacobs-Reitsma, J. van den Broek, D. Klinkenberg, J. A. Stegeman, and J. A. P. Heesterbeek. 2005. Quantifying transmission of Campylobacter spp. among broilers. Appl. Environ. Microbiol. 71:5765–5770. Wagenaar, J. A., M. A. P. Van Bergen, M. A. Mueller, T. M. Wassenaar, and R. M. Carlton. 2005. Phage therapy reduces Campylobacter jejuni colonization in broilers. Vet. Microbiol. 109:275– 283. Wallace, J. S., K. N. Stanley, J. E. Currie, P. J. Diggle, and K. Jones. 1997. Seasonality of thermophilic Campylobacter populations in chickens. J. Appl. Microbiol. 82:219–224. Weber, R. 2000. Prüfung wechselseitiger Hemmeffekte verschiedener Campylobacter jejuni-Stämme bei der Kolonisation des Huehnerdarmes. PhD Diss. Tieraerztliche Hochschule Hannover, Germany. Wedderkopp, A., E. Rattenborg, and M. Madsen. 2000. National surveillance of Campylobacter in broilers at slaughter in Denmark in 1998. Avian Dis. 44:993–999. Wieland, B., G. Regula, J. Danuser, M. Wittwer, A. P. Burnens, T. M. Wassenaar, and K. D. Stärk. 2005. Campylobacter spp. in dogs and cats in Switzerland: Risk factor analysis and molecular characterization with AFLP. J. Vet. Med. B Infect. Dis. Vet. Public Health 52:183–189. Wittwer, M., J. Keller, T. M. Wassenaar, R. Stephan, D. Howald, G. Regula, and B. Bissig-Choisat. 2005. Genetic diversity and antibiotic resistance patterns in a Campylobacter population isolated from poultry farms in Switzerland. Appl. Environ. Microbiol. 71:2840–2847. Wyszyńska, A., A. Raczko, M. Lis, and E. K. Jagusztyn-Krynicka. 2004. Oral immunization of chickens with avirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild-type Campylobacter. Vaccine 22:1379– 1389.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
Heres, L., B. Engel, H. A. P. Urlings, J. A. Wagenaar, and F. van Knapen. 2004. Effect of acidified feed on susceptibility of broiler chickens to intestinal infection by Campylobacter and Salmonella. Vet. Microbiol. 99:259–267. Heuer, O. E., K. Pedersen, J. S. Andersen, and M. Madsen. 2001. Prevalence and antimicrobial susceptibility of thermophilic Campylobacter in organic and conventional broiler flocks. Lett. Appl. Microbiol. 33:269–274. Hughes, R. A. C., and D. R. Cornblath. 2005. Guillain-Barré syndrome. Lancet 366:1653–1666. Humphrey, T. J., A. Henley, and D. G. Lanning. 1993. The colonization of broiler chickens with Campylobacter jejuni: Some epidemiological investigations. Epidemiol. Infect. 110:601–607. ISO. 1995. Microbiology of food and animal feeding stuffs–Horizontal method for detection and enumeration of Campylobacter spp. Part 1: Detection method. ISO 10272 1995/cor.1:1996(E). International Organization for Standardization, Geneva, Switzerland. Jacobs-Reitsma, W. F. 1994. Campylobacter and Salmonella in breeder flocks. Pages 25–32 in Epidemiology of Campylobacter in poultry. W. F. Jacobs-Reitsma, ed. ID-DLO, Beekbergen, the Netherlands. Jacobs-Reitsma, W. F., N. M. Bolder, and R. W. A. W. Mulder. 1994. Cecal carriage of Campylobacter and Salmonella in Dutch broiler flocks at slaughter: A one-year study. Poult. Sci. 73:1260–1266. Leirisalo-Repo, M. 2005. Early arthritis and infection. Curr. Opin. Rheumatol. 17:433–439. Lillehaug, A., C. Monceyron Jonassen, B. Bergsjo, M. Hofshagen, J. Tharaldsen, L. L. Nesse, and K. Handeland. 2005. Screening of feral pigeon (Columba livia), mallard (Anas platyrhynchos) and graylag goose (Anser anser) populations for Campylobacter spp., Salmonella spp., avian influenza virus and avian paramyxovirus. Acta Vet. Scand. 46:193–202. Loewenherz-Lüning, K., M. Heitmann, and G. Hildebrandt. 1996. Untersuchungen zum Vorkommen von Campylobacter jejuni in verschiedenen Lebensmitteln tierischen Ursprungs. Fleischwirtschaft 76:958–961. Luechtefeld, N. W., W. L. Wang, M. J. Blaser, and L. B. Reller. 1981. Evaluation of transport and storage techniques for isolation of Campylobacter fetus subsp. jejuni from turkey cecal specimens. J. Clin. Microbiol. 13:438–443. Newell, D. G., and C. Fearnley. 2003. Sources of Campylobacter colonization in broiler chickens. Appl. Environ. Microbiol. 69:4343– 4351. Oyarzabal, O. A., D. E. Conner, and F. J. Hoerr. 1995. Incidence of campylobacters in the intestine of avian species in Alabama. Avian Dis. 39:147–151. Padungtod, P., and J. B. Kaneene. 2005. Campylobacter in food animals and humans in northern Thailand. J. Food Prot. 68:2519– 2526. Ramabu, S. S., N. S. Boxall, P. Madie, and S. G. Fenwick. 2004. Some potential sources for transmission of Campylobacter jejuni to broiler chickens. Lett. Appl. Microbiol. 39:252–256. Rastall, R. A. 2004. Bacteria in the gut: Friends and foes and how to alter the balance. J. Nutr. 134:2022S–2026S. Refrégier-Petton, J., N. Rose, M. Denis, and G. Salvat. 2001. Risk factors for Campylobacter spp. contamination in French broilerchicken flocks at the end of the rearing period. Prev. Vet. Med. 50:89–100. Rivoal, K., C. Ragimbeau, G. Salvat, P. Colin, and G. Ermel. 2005. Genomic diversity of Campylobacter coli and Campylobacter jejuni isolates recovered from free-range broiler farms and comparison with isolates of various origins. Appl. Environ. Microbiol. 71:6216–6227. RKI. 2006. Aktuelle Statistik meldepflichtiger Infektionskrankheiten. Epidemiol. Bull. 3:26–27. Robinson, D. A. 1981. Infective dose of Campylobacter jejuni in milk. Br. Med. J. (Clin. Res. Ed.) 282:1584.
1305