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Risk factors for Campylobacter infection in Norwegian cats and dogs
Preventive Veterinary Medicine 55 (2002) 241±253
Risk factors for Campylobacter infection in Norwegian cats and dogs M. Sandberga,c,*, B. Bergsjùa, M. Hofshagenb, E. Skjerveb,c, H. Kruseb a
The National Veterinary Institute, P.O. Box 8156 Dep., N-0033 Oslo, Norway The Norwegian Zoonosis Center, P.O. Box 8156 Dep., N-0033 Oslo, Norway c The Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway b
Received 15 January 2002; accepted 31 May 2002
Abstract Rectal swabs from healthy cats and dogs, and from dogs and cats with clinical diarrhoea were collected approximately every third month from May 2000 to June 2001 from six small-animal practices throughout Norway. A questionnaire was ®lled in for each animal. Of the 301 healthy cats sampled, 54 (18%) were positive for Campylobacter, compared to 5 out of 31 (16%) cats with diarrhoea. Campylobacter jejuni was isolated from 11 (3%), C. upsaliensis from 42 (13%) and C. coli from 2 (0.6%) of the cats sampled. Isolates from four cats (1%) could not be speci®ed. Of the 529 healthy dogs, 124 (23%) were positive for Campylobacter, compared to 18 of 66 (27%) dogs with diarrhoea. C. jejuni was isolated from 20 (3%) and C. upsaliensis from 117 (20%) of the dogs sampled. Isolates from ®ve dogs (0.8%) could not be speci®ed. Eighteen out of the 20 investigated C. upsaliensis samples were resistant to streptomycin. The clinically healthy animals were included in the analysis to identify factors associated with Campylobacter prevalence. The cat model had low classi®cation ability. The dog-data model indicated increased odds of infection with Campylobacter for dogs 1 year, and in dogs sampled during the spring. No difference was observed between the prevalence of Campylobacter infections in cats and dogs with diarrhoea and healthy animals. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Campylobacter upsaliensis; Cats; Dogs; Risk factors; Humans
* Corresponding author. Present address: Department of Pharmacology, Microbiology and Food Hygiene, The Norwegian College of Veterinary Medicine, P.O. Box 8146 Dep., N-0033 Oslo, Norway. Tel.: 47-22-59-70-53; fax: 47-22-96-48-50. E-mail address: [email protected] (M. Sandberg).
0167-5877/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 5 8 7 7 ( 0 2 ) 0 0 0 9 5 - 8
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1. Introduction Campylobacteriosis became the most-frequently reported gastrointestinal infectious disease in humans in Norway in 1998 when it surpassed salmonellosis for the ®rst time (Anon., 2001a). In 2000, the reported human incidence was 58 cases per 100,000, an almost 100% increase as compared to 1997. The proportion of human cases reported for which the infection was acquired in Norway has been stable at 40% (Anon., 2001b). Most of the Campylobacter infections seem to be sporadic (Anon., 2001b). Campylobacter jejuni subsp. jejuni (C. jejuni) is the most-frequently isolated species from humans with campylobacteriosis (Bourke et al., 1998). The transmission of campylobacters appears to be by a faecal±oral route through contaminated food and water or by direct contact with faecal material from infected animals and humans. C. jejuni, C. coli, C. upsaliensis and C. lari (all potentially pathogenic to man) can be isolated from various domestic and wild birds and animals (Rosef et al., 1983; Nachamkin and Blaser, 2001). Campyobacter survives in fresh water (Brennhovd et al., 1992), fresh food and (to some extent) in frozen food (Rosef et al., 1984; Kapperud et al., 1992; Anon., 1994; Nachamkin and Blaser, 2000) and in meat packed in modi®ed atmosphere (Fossum et al., 1999). The carrier state in birds and animals is relatively long and the infectious dose is low. The incidence of campylobacteriosis in humans and the prevalence of campylobacters in chicken ¯ocks in Norway usually peak in late summer (Anon., 1994, 2001b; Nachamkin and Blaser, 2000). Seasonal variation in the prevalence of campylobacters in other animals, foods, and environmental sources is not fully investigated (Jones, 2001). C. upsaliensis was reported for the ®rst time in humans by Steele et al. (1985) and occurs in dogs and cats (Bourke et al., 1998; Fox et al., 1989; Sandstedt et al., 1983; Steinhauserova et al., 2001). As is the case in children, puppies and kittens are more likely to acquire clinical campylobacteriosis than adults, and more-commonly shed the organism (Hald and Madsen, 1997). In 1983, 33 of the 147 (22%) dogs and 10 of the 85 (12%) cats from the small-animal clinic at the Norwegian School of Veterinary Science were positive for C. jejuni and C. coli (Gondrosen et al., 1985). No signi®cant differences between the prevalences in dogs and cats with diarrhoea as compared with virtually healthy animals were observed . The selective broth used was the original Skirrow's (Skirrow, 1977), which today is known to have an inhibitory effect on some Campylobacter species (Steele et al., 1985)Ðamong them, C. upsaliensis. A case±control study conducted in the USA indicated an increased risk of C. jejuni and C. coli infection in humans after direct contact with diarrhoeic animals during the week before the onset of their illnesses (Saeed et al., 1993). Different case reports also suggest a transmission of C. upsaliensis from dogs and cats to humans (Bourke et al., 1998; Nachamkin and Blaser, 2000). Results from case±control studies in humans with a con®rmed C. jejuni diagnosis conducted in Norway in 1991±1992 pointed to drinking untreated water, consumption of poultry and having contact with a dog or cat as the mostimportant risk factors (Kapperud, 1995). There are thus clear indications that dogs and cats might play an important role in the epidemiology of Campylobacter infections in humans. Our objective was to investigate the prevalence of Campylobacter in Norwegian healthy cats and dogs in relation to age, season, geographical area and other potential risk factors.
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In addition, the association between Campylobacter infection and diarrhoea was investigated and a selection of isolates was tested for antimicrobial susceptibility. 2. Materials and methods 2.1. Study population Faecal swabs from healthy dogs and cats were collected every third month from May 2000 to June 2001 to cover the four different seasons in Norway. Individual animals were sampled by veterinary surgeons in six small-animal practices chosen by convenience but situated throughout Norway. Based upon earlier studies, the sample sizes were calculated by EpiInfo (Anon., 2001c) to detect a prevalence of 10±15% in 1- and >1-year-old cats and a prevalence of 20±25% in 1- and >1-year-old dogs with a precision of 10% at an a of 0.05 (did not account for the within-cluster correlation). Based upon these conditions, about 110 samples from healthy cats and about 145 from healthy dogs were planned to be collected in each season (a total of 430 and 580, respectively). The veterinarians at the six clinics were asked to sample about 18 healthy cats and 24 healthy dogs in each of the four periods by convenience (ideally, four cats and ®ve dogs per week). The criteria of inclusion for the healthy animals were ideally: that they came in for e.g. vaccination, to have their nails cut or for being spayed. They could not have diarrhoea or have an infectious disease or be on antibiotic treatment. In addition, the veterinarians were asked to sample about 50 diarrhoeic dogs and 50 diarrhoeic cats during the study period. Three hundred and one healthy cats (70% of the planned number), 529 healthy dogs (91% of the planned number), 31 diarrhoeic cats and 66 diarrhoeic dogs were sampled. 2.2. Sampling, culturing and identification The animals were sampled with rectal swabs (Amies 114 C, with charcoal, Copan Brescia, Italy). The specimens were cooled in a refrigerator before being sent to the laboratory the same day. Samples were refrigerated and analysed within 2 days after arrival at the diagnostic laboratory at the National Veterinary Institute. The samples were plated on a Campylobacter blood-free selective agar (Oxoid CM739, Basingstoke, UK) supplemented with cephoperazone 8.0 mg/l, teicoplanin 4.0 mg/l, and amphotericin B 10.0 mg/l (Oxoid SR174) (CAT), and the plates were incubated in a microaerophilic atmosphere (Oxoid, CampyGenTM) at 37 1 8C for 72±96 h. Presumptive Campylobacter colonies were con®rmed by phase-contrast microscopy and sub-cultured on 5% bovine blood±agar plates for 48±72 h under the same conditions as described above. The different species were identi®ed by phenotypic assays including growth pattern at 42 8C, catalase production, hippurate hydrolysis (Rosco, Taastrup, Denmark), indoxyl acetate hydrolysis (Rosco), production of H2S in triple-sugar±iron agar (Difco, Detroit, USA), and susceptibility or resistance to nalidixic acid and cephalotin determined by standard diffusion techniques with 30 mg disks (E-test; AB Biodisk, Solna, Sweden) incubated under micro-aerobic conditions at 37 8C for 48±72 h. The possibility of Arcobacter was ruled out by testing for growth aerobically at 30 8C.
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2.3. Antimicrobial susceptibility A random sample of 22 C. jejuni (9 from cats and 13 from dogs) and 20 C. upsaliensis (5 from cats and 15 from dogs) isolates was selected with the random selection procedure in Excel1 (Anon., 2001d). The minimum inhibitory concentration (MIC-values) for C. jejuni and C. upsaliensis isolates to ampicillin, cipro¯oxacin, chloramphenicol, erythromycin, gentamicin, nalidixic acid, streptomycin, and tetracycline were investigated using the E-test with breakpoints of S 16 and R 32 for nalidixic acid and S 4, R 8 for streptomycin (Ane Nùdtvedt, personal communication, The Norwegian Zoonosis Centre) also used in the Danish integrated antimicrobial resistance monitoring and research programme (Danmap) (Anon., 2001e). The breakpoints for the other antimicrobials are shown in NORMVET 2000 (Anon., 2001f). 2.4. Epidemiological information The pet owners and the veterinary surgeons ®lled in a simple questionnaire with information about the name and address of the owner, why they came to see the veterinary surgeon and age, sex, breed and name of the animal. The healthy cat questionnaire had an additional six main questions: (1) are there other animals in the household? If yes, what kind of animals? (2) Is the cat kept only indoors? If yes, does it have contact with other cats through events such as shows or similar activities? (3) Is the cat allowed to be outdoors and if yes, in what kind of environment? (4) Has the cat stayed in a cattery during the last month? (5) Has the cat been abroad in past 3 months? (6) What kind of feed is the cat fed? The healthy dog questionnaire had an additional seven main questions: (1) are there other animals in the household? (2) In what kind of environment do you walk your dog? (3) Do you keep the dog in a dog-yard? (4) Does your dog have contact with other dogs through different events? (5) Has your dog been abroad during the past 3 months? (6) Has the dog stayed in a kennel during the last month? (7) What kind of feed is the dog fed? The questionnaires for the cats and dogs with diarrhoea also included questions about the signs. All the questions had speci®ed choices, and all variables except age had dichotomous outcomes. Most of the variables are shown in Tables 1 and 2 (exceptions are those that did not vary or those that had too many missing values). No small pilot project to evaluate the questionnaires was conducted, but the questionnaires were based on a questionnaire used in a similar project conducted in Sweden and was also discussed and evaluated by the project group and sent to the veterinarians at the six clinics for evaluation before the project was started. Only the healthy animals were included in the risk factor analyses. 2.5. Statistical analyses The dataset was established in Excel1. The descriptive and analytic analyses of the data was conducted in EpiCalc1 (Anon., 2001g) and SAS-PC system1, version 6.12 for Windows (Anon., 2001h). Throughout the analyses, detection of any Campylobacter in faeces was the case de®nition. Preliminary descriptive and univariable analyses of the
Table 1 Campylobacter in healthy cats in Norway 2000/2001 Factors
Outcomes
N
Positive
C. jejuni
C. upsaliensis
C. coli
C. spp.
No.
%
No.
%
No.
%
No.
%
No.
%
± ±
301 168 123
54 36 17
18 21 14
11 6 5
4 4 4
38 28 9
13 17 7
2 1 1
0.7 0.6 0.8
3 1 2
1 0.6 2
Season
May±June July±September October±November December±March
± ± ± ±
60 91 76 74
15 12 16 11
25 13 21 15
1 3 6 1
2 3 8 1
13 6 9 10
22 7 12 14
± 2 ± ±
± 2 ± ±
1 1 1 ±
2 1 1 ±
Clinic
1 2 3 4 5 6
± ± ± ± ± ±
69 49 66 23 41 53
5 9 14 2 12 12
7 18 21 9 29 23
1 2 2 2 1 3
1 4 3 9 2 6
4 5 12 ± 11 6
6 10 18 ± 27 11
± 1 ± ± ± 1
± 2
2
± 1 ± ± ± 2
± 2 ± ± ± 4
Norwegian mix Pure-bred
Yes No
188 82
40 8
21 10
8 1
4 1
27 7
9 9
2 ±
1 ±
3 ±
2 ±
Several animalsa
Yes No
151 146
25 29
17 20
6 5
4 3
16 22
11 15
1 1
0.7 0.7
2 1
1 0.7
Outdoorb
Yes No Yes No Yes No Yes No Yes No
217 82 34 197 149 78 55 171 7 220
41 13 3 41 29 15 14 30 ± 44
19 16 9 21 20 19 26 18 ± 20
9 2 ± 10 6 4 4 6 ± 10
4 2 ± 5 4 5 7 4 ± 5
28 10 2 27 20 9 9 20 ± 29
13 12 6 14 13 12 16 12 ± 13
2 ± 1 1 1 1 1 1 ± 2
0.9 ± 3 0.5 0.7 1.3 1.8 0.6 ± 0.9
2 1 ± 3 2 1 ± 3 ± 3
0.9 1 ± 1.5 1.3 1.3 ± 2 ± 1
Breed
City Suburb Countryside Other
245
1 year >1 year
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Total no. of cats Age
Category
246
Factors
Category
Outcomes
N
Positive
C. jejuni
C. upsaliensis
C. coli
C. spp.
No.
%
No.
%
No.
%
No.
%
No.
%
Indoorc
Yes No
13 69
2 12
15 17
± 2
± 3
± 10
± 15
± ±
± ±
2 ±
15 ±
Abroad
Yes No
5 292
1 52
20 18
± 11
± 4
1 36
20 12
± 2
± 0.7
± 3
± 1
Yes No Yes No Yes No Yes No
279 20 66 233 57 242 173 126
50 4 10 44 3 51 32 22
18 20 15 19 5 21 19 18
10 1 1 10 1 10 6 5
4 5 2 4 2 4 4 4
35 3 8 30 2 36 23 15
13 15 12 13 4 15 13 12
2 ± ± 2 ± 2 2 ±
0.7 ± ± 0.9 ± 0.8 1 ±
3 ± 1 2 ± 3 1 2
1 ± 2 0.9 ± 1 0.6 2
Feed
Pellets Leftovers Mixed Canned
a
Several animals in the household (specified: dogs, cats, reptiles, birds, dairy cows, cattle, pigs, sheep). Kept outdoor and indoor. c Kept only indoor. b
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
Table 1 (Continued )
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
247
Table 2 Campylobacter in cats and dogs with diarrhoea in Norway 2000/2001 Factors
Total no. cats Age Total no. of dogs Age
Category
N
Positive
C. jejuni
C. upsaliensis
C. spp.
No.
%
No.
%
No.
%
No.
%
1 year >1 year
31 15 13
5 2 1
16 13 8
± ± ±
± ± ±
4 2 1
13 13 8
1 ± ±
3 ± ±
1 year >1 year
66 26 39
18 11 7
27 42 18
2 2 ±
3 8 ±
15 8 7
23 31 18
1 1 ±
2 4 ±
results from both groups (healthy and diarrhoeic) animals were done using the chi-square and Fisher-exact tests for categorical factors such as season (1±4) and geographical area (clinic 1±6). The continuous variable age was analysed by the Kruskal±Wallis test. For the modelling, age was dichotomised in the analysis (1 and >1 year) partly because there was no effect gained by dividing the data into four groups and partly because of the low number of animals in some of the four groups. The period from May to June was de®ned as spring, July to September as summer, October to November as autumn and December to the mid of March as winter. Most of the samples were collected in the ®rst 2 months of the each of above-described periods. The multivariable logistic model was built in a backward-elimination procedure as described by Hosmer and Lemenshow (2000). The outcome variable was Campylobacterpositive animals. The risk factors tested were age, season and clinic. These and other factors with a P 0:20 from univariable analyses were candidates for the multivariable logistic model. Only factors present in 5 individuals were included in the multivariable model. The ®tting of the models was basically conducted by observing the change in the deviance. As we wanted to generalize, clinic was to be taken into the model as a random effect by using the GLIMMIX procedure in SAS. The ®t of the models was assessed as described by Hosmer and Lemenshow (2000) using the summary measure of ®t; the Hosmer and Lemenshow test. Logistic regression diagnostics were conducted to identify possible outliers by investigating the hat matrix (leverage-values) and change in deviance (DD) and b (Db). The healthy and diarrhoeic dogs were matched on age and tested for association in regard to Campylobacter by a chi-square test. Due to the small number of samples from cats with diarrhoea, the potentially confounding effect of age on the Campylobacter carriage could not be investigated. Possible differences between the diarrhoeic animals and the healthy animals regarding season and clinic also were not possible to investigate because of the low number of samples collected from animals with diarrhoea. 3. Results Of the 301 healthy cats sampled, 18% were positive for Campylobacter (Table 1), compared to 5 out of 31 (16%) cats with diarrhoea (Table 2). Of the 529 healthy dogs
248
Table 3 Campylobacter in healthy dogs in Norway 2000/2001
Total no. of dogs Age
Category
Outcomes
N
Positive
C. jejuni
C. upsaliensis
C. coli
%
No.
%
No.
%
No.
%
102 63 38
19 24 15
± ± ±
± ± ±
4 ± 3
0.8 ± 2
No.
%
No.
C. spp
1 year >1 year
± ±
529 262 258
124 80 42
23 31 16
18 13 4
3 5 2
Season
May±June July±September October±November December±March
± ± ± ±
129 155 128 117
40 42 23 19
31 27 18 16
3 10 1 4
2 7 0.8 3
34 31 22 15
26 20 17 13
± ± ± ±
± ± ± ±
1 ± ± ±
0.6 ± ± ±
Clinic
1 2 3 4 5 6
± ± ± ± ± ±
93 88 98 76 81 92
11 20 18 22 29 24
12 23 18 29 36 26
3 2 1 2 3 7
3 2 1 3 4 8
8 18 17 20 22 17
9 21 17 26 27 19
± ± ± ± ± ±
± ± ± ± ± ±
± ± ± 4 ± 3
± ± ± 5 ± 1
Yes No
277 252
71 53
26 21
12 6
4 2
56 46
20 18
± ±
± ±
1 2
0.4 3
City
Yes No
77 452
21 103
27 23
2 16
3 4
17 85
22 19
± ±
± ±
2 2
0.4 0.7
Suburb
Yes No
282 247
61 63
22 26
8 10
3 4
51 51
18 21
± ±
± ±
2 ±
0.8 ±
Countryside
Yes No
275 254
63 61
23 24
9 9
3 4
54 48
20 19
± ±
± ±
± 3
± 1
Other
Yes No
37 492
8 116
22 24
2 16
5 3
6 96
16 20
± ±
± ±
± 4
± 0.8
Several animalsa Outdoor
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
Factors
Yes No
129 398
35 88
27 22
4 14
3 4
31 70
24 18
± ±
± ±
± 4
± 1
Contact other dogs
Yes No
321 208
77 47
24 23
7 ±
2 ±
66 36
21 17
± ±
± ±
4 ±
1 ±
Abroad
Yes No
24 501
3 121
13 24
11 18
5 4
3 99
13 20
± ±
± ±
± 4
± 0.8
Yes No Yes No Yes No Yes No
502 27 167 362 34 495 77 452
119 5 30 94 7 117 14 110
24 19 18 26 21 24 18 24
15 3 1 17 ± 18 3 15
3 11 0.6 5 ± 4 4 3
100 2 29 73 7 95 11 91
20 7 17 20 21 19 14 20
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ±
4 ± ± 4 ± 4 ± 4
0.8 ± ± 1 ± 0.8 ± 0.9
Feed
Pellets Leftovers Etrails Other
a
Several animals in the household (specified: dogs, cats, reptiles, birds, dairy cows, cattle, pigs, sheep).
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
Outdoor yard
249
250
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
sampled, 124 (23%) were positive for Campylobacter (Table 3), compared to 18 out of 66 (27%) dogs with diarrhoea (Table 2). 3.1. Risk factors for Campylobacter infection in healthy cats We offered age, season, clinic, breed and being outdoors in the countryside, city or other place. The ®nal model included mixed Norwegian breed (as opposed to a pure-bred breed) (Table 4) and clinic (the random effect). The model was poor at cross-classifying the same dataset, but no outliers were identi®ed. 3.2. Risk factors for Campylobacter infection in healthy dogs Offered to the multivariable model were age, season and clinic and ``not being fed leftovers''. The ®nal model included age, season and clinic (Table 4). (The latter was the random effect.) The model was poor at cross-classifying the same dataset, but no outliers were identi®ed. 3.3. Possible association between Campylobacter and diarrhoea in cats and dogs No difference between cats with diarrhoea and healthy cats in regard to Campylobacter infection was detected. Neither was there a positive association between diarrhoea and Campylobacter infection in dogs detected when matching the diarrhoeic and healthy dogs on age (data not shown). 3.4. Antimicrobial susceptibility One of the 22 C. jejuni and 18 of the 20 C. upsaliensis were categorized as resistant to streptomycin. One of C. upsaliensis was categorized as resistant to nalidixic acid. For the other antimicrobials, all isolates were susceptible. Table 4 Results from the final multivariable analysis of the Campylobacter data from healthy cats and dogs in Norway 2000/2001 with clinic as a random effect Factors Cats Intercept Mixed Norwegian breed Dogs Intercept Age Season
Category
May±June July±September October±November December±March
b
S.E. (b) 2.24 0.87
0.41 0.41
1.19 0.79 0.72 0.49 0.03 ±
0.30 0.22 0.32 0.32 0.35 ±
Deviance 240.26
530.56
P
ORlogistic 95% CI
0.003 0.035
± 2.39
± 1.06±5.38
0.011 0.0003 0.03 0.13 0.9 ±
± 2.20 2.05 1.63 1.03 1.0
± 1.43±3.39 1.09±3.89 0.87±3.08 0.51±2.04 ±
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4. Discussion The indicated higher risk for healthy dogs being Campylobacter-positive when 1-yearold corresponds well to the results from the studies by Burnens et al. (1992) and by Hald and Madsen (1997), which revealed an increased risk of shedding Campylobacter for young dogs. The animals investigated in their studies were somewhat younger (<1 year or 6 months) than in our study. The indications of a higher risk for cats of a mixed Norwegian breed was weak but could be explained by the fact that most of these cats probably are allowed to be outdoors as opposed to the cats of a pure-bred breed. A higher risk of being infected from other animals and the environment should therefore be expected for these cats. The ``seasonal'' variation of Campylobacter shedding among cats and dogs was investigated for the ®rst time in this study as far as we can ascertain, but no distinct differences was revealed for cats. More positive dogs were found in May±June and in July± September. The estimates were, however, not very robust (Table 4). The more-frequent isolation of Campylobacter from cats and dogs from the clinic situated in the capital of Norway (Oslo clinic 5), could be explained by the higher density of animals and people in this area. It also simply could be explained by better survival of Campylobacter in the sample (and therefore fewer false negatives) because of the short transport time from the Oslo clinic to the laboratory. As a curiosity, we mention that only three of the 31 dogs and one out of the ®ve cats that had been abroad (mainly Sweden and for few animals, Spain) the last 3 months before the sampling were shedding Campylobacter. 4.1. Campylobacter as a risk factor for diarrhoea in cats and dogs We found no association between Campylobacter infection and diarrhoea and this was in accord with the results from the Norwegian study by Gondrosen et al. (1985). Several other studies have revealed higher proportions of campylobacters in animals with diarrhoea (Burnens et al., 1992; Ferreira et al., 1979; Fleming, 1983; Nair et al., 1985) while others reveal similar proportions in cats and dogs without diarrhoea (Baker et al., 1999; Fox et al., 1989; McOrist and Browning, 1982; Olson and Sandstedt, 1987). Higher risk of infection in diarrhoeal versus healthy juvenile dogs has been reported (Burnens et al., 1992; Fleming, 1983). 4.2. Antimicrobial resistance That neither of the isolates tested was resistant to cipro¯oxacin and only one of the C. upsaliensis was resistant to nalidixic acid probably re¯ects that there is a restriction on use of quinolones in cats and dogs in Norway. The Norwegian monitoring program for resistance in microbes (NORM) reports an increase in the resistance of quinolones only in Campylobacter isolated from humans in Norway (Melby, 2001). The Danish integrated antimicrobial resistance monitoring and research programme (Danmap) reports streptomycin resistance in C. jejuni isolated from Danish broilers (12%), cattle (2%) and humans (two isolates). From broilers and pigs in Denmark, 25 and 48% of
252
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the C. coli isolated in 2000 were resistant to streptomycin (Anon., 2001e). Only one of the investigated C. jejuni from the present study was resistant to streptomycin as opposed to the high number of C. upsaliensis which suggests that C. upsaliensis is intrinsically resistant to the antimicrobial. 5. Conclusion The risk for Campylobacter infection was increased for dogs1-year-old. There were also differences across months and among clinics. No difference was observed between the prevalence of Campylobacter in cats and dogs with diarrhoea and healthy animals. Acknowledgements We gratefully acknowledge the following small-animal practises for providing samples: Bergen SmaÊdyrklinikk, Bodù Dyreklinikk, ByaÊsen Dyreklinikk, Gjùvik Dyreklinikk, Poliklinikken at The Norwegian School of Veterinary Science, and Sandnes Dyreklinikk. We also acknowledge the Section of Epidemiology at the National Veterinary Institute for help in the initial phase of the study, and with the GLIMMIX procedures in SAS, and Andreas Hole for the data entry, and help with the quality assurance of the data. References Anon., 1994. Smittestoffer i nñringsmidler. SNT rapport 5, Statens nñringsmiddeltilsyn, Oslo, pp. 29±30. http:// www.snt.no/dokumentasjon/index.html. Anon., 2001a. WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe. http://www.who.it/docs/fdsaf/fs_survprog.htm. Anon., 2001b. Surveillance of Communicable Diseases in Norway 1999, National Institute of Public Health, Oslo, Norway. http://www.folkehelsa.no/Ler/pdf/smittevern4.pdf. Anon., 2001c. EpiInfo. http://www.cdc.gov/. Anon., 2001d. Microsoft Office, Excel. http://www.microsoft.com/norge/products/office/productinfo/products/ excel/. Anon., 2001e. The Danish integrated antimicrobial resistance monitoring and research programme (Danmap). http://www.svs.dk/uk/Organization/Frm_org.htm. Anon., 2001f. NORM/NORMVET 2000. http://www.vetinst.no/Zoonosesenteret/NORM-NORMVET_2000.pdf. Anon., 2001g. EpiCalc. http://www.myatt.demon.co.uk/epicalc.htm. Anon., 2001h. SAS Institute, Cary, NC, USA, 1996. http://www.sas.com/rnd/app/paper/nlmixedsugi.pdf176.0KB-GLIMMIX:1. Baker, J., Barton, M.D., Lanser, J., 1999. Campylobacter species in cats and dogs in South Australia. Aust. Vet. J. 77, 662±666. Bourke, B., Chan, V.L., Sherman, P., 1998. Campylobacter upsaliensis: waiting in the wings. Clin. Microbiol. Rev. 11, 440±449. Brennhovd, O., Kapperud, G., Langeland, G., 1992. Survey of thermotolerant Campylobacter spp. and Yersinia spp. in three surface water sources in Norway. Int. J. Food Microbiol. 15, 327±338. Burnens, A.P., Angeloz-Wick, B., Nicolet, J., 1992. Comparison of Campylobacter carriage rates in diarrheic and healthy pet animals. Zentralbl. Veterinarmed. [B] 39, 175±180.
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Ferreira, M.C., Ribeiro, V.L., Ricciardi, I.D., 1979. Campylobacter, dogs and human enteritis. Vet. Rec. 105, 451. Fleming, M.P., 1983. Association of Campylobacter jejuni with enteritis in dogs and cats. Vet. Rec. 113, 372± 374. Fossum, H., Kapperud, G., Underdal, B., Nordal, J., 1999. Overlevelse av Campylobacter pakket i normal og modifisert atmosfñre. SNT-Draft, Statens Nñringsmiddeltilsyn, 20 pp. http://www.snt.no/dokumentasjon/ index.html. Fox, J.G., Maxwell, K.O., Taylor, N.S., Runsick, C.D., Edmonds, P., Brenner, D.J., 1989. ``Campylobacter upsaliensis'' isolated from cats as identified by DNA relatedness and biochemical features. J. Clin. Microbiol. 27, 2376±2378. Gondrosen, B., Knaevelsrud, T., Dommarsnes, K., 1985. Isolation of thermophilic campylobacters from Norwegian dogs and cats. Acta Vet. Scand. 26, 81±90. Hald, B., Madsen, M., 1997. Healthy puppies and kittens as carriers of Campylobacter spp., with special reference to Campylobacter upsaliensis. J. Clin. Microbiol. 35, 3351±3352. Hosmer, D.W., Lemenshow, S., 2000. Applied Logistic Regression, 2nd ed. Wiley, New York, 375 pp. Jones, K., 2001. Campylobacters in water, sewage and the environment. Symp. Ser. Soc. Appl. Microbiol. 30, 68±79. Kapperud, G., 1995. Risikofaktorer for campylobacterinfeksjon i Norge. Norsk Epidemiologi. 5, 44±49. Kapperud, G., Skjerve, E., Bean, N.H., Ostroff, S.M., Lassen, J., 1992. Risk factors for sporadic Campylobacter infections: results of a case±control study in southeastern Norway. J. Clin. Microbiol. 30, 3117±3121. McOrist, S., Browning, J.W., 1982. Carriage of Campylobacter jejuni in healthy and diarrhoeic dogs and cats. Aust.Vet. J. 58, 33±34. Melby, K., 2001. UllevaÊl hospital, Oslo. Campylobacter og antibiotikaresistens, norske forhold. In: Proceedings of the Campylobacter hva vet vi hva gjùr vi. The Norwegian Zoonosis center. http://www.vetinst.no/ zoonose.htm. Nachamkin, I., Blaser, J., 2000. Campylobacter, 2nd ed. ASM Press, Washington, DC, 545 pp. Nair, G.B., Sarkar, R.K., Chowdhury, S., Pal, S.C., 1985. Campylobacter infection in domestic dogs. Vet. Rec. 116, 237±238. Olson, P., Sandstedt, K., 1987. Campylobacter in the dog: a clinical and experimental study. Vet. Rec. 121, 99± 101. Rosef, O., Gondrosen, B., Kapperud, G., Underdal, B., 1983. Isolation and characterization of Campylobacter jejuni and Campylobacter coli from domestic and wild mammals in Norway. Appl. Environ. Microbiol. 46, 855±859. Rosef, O., Gondrosen, B., Kapperud, G., 1984. Campylobacter jejuni and Campylobacter coli as surface contaminants of fresh and frozen poultry carcasses. Int. J. Food Microbiol. 1, 205±215. Saeed, A.M., Harris, N.V., DiGiacomo, R.F., 1993. The role of exposure to animals in the etiology of Campylobacter jejuni/coli enteritis. Am. J. Epidemiol. 137, 108±114. Sandstedt, K., Ursing, J., Walder, M., 1983. Thermotolerant Campylobacter with no or weak catalase activity isolated from dogs. Curr. Microbiol. 4, 209±213. Skirrow, M.B., 1977. Campylobacter enteritis: a ``new'' disease. Br. Med. J. 2, 9±11. Steele, T.W., Sangster, N., Lanser, J.A., 1985. DNA relatedness and biochemical features of Campylobacter spp. isolated in central and South Australia. J. Clin. Microbiol. 22, 71±74. Steinhauserova, I., Fojtikova, K., Klimes, J., 2001. Identification of thermophilic Campylobacter spp. by phenotypic and molecular methods. J. Appl. Microbiol. 90, 470±475.
Risk factors for Campylobacter infection in Norwegian cats and dogs M. Sandberga,c,*, B. Bergsjùa, M. Hofshagenb, E. Skjerveb,c, H. Kruseb a
The National Veterinary Institute, P.O. Box 8156 Dep., N-0033 Oslo, Norway The Norwegian Zoonosis Center, P.O. Box 8156 Dep., N-0033 Oslo, Norway c The Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway b
Received 15 January 2002; accepted 31 May 2002
Abstract Rectal swabs from healthy cats and dogs, and from dogs and cats with clinical diarrhoea were collected approximately every third month from May 2000 to June 2001 from six small-animal practices throughout Norway. A questionnaire was ®lled in for each animal. Of the 301 healthy cats sampled, 54 (18%) were positive for Campylobacter, compared to 5 out of 31 (16%) cats with diarrhoea. Campylobacter jejuni was isolated from 11 (3%), C. upsaliensis from 42 (13%) and C. coli from 2 (0.6%) of the cats sampled. Isolates from four cats (1%) could not be speci®ed. Of the 529 healthy dogs, 124 (23%) were positive for Campylobacter, compared to 18 of 66 (27%) dogs with diarrhoea. C. jejuni was isolated from 20 (3%) and C. upsaliensis from 117 (20%) of the dogs sampled. Isolates from ®ve dogs (0.8%) could not be speci®ed. Eighteen out of the 20 investigated C. upsaliensis samples were resistant to streptomycin. The clinically healthy animals were included in the analysis to identify factors associated with Campylobacter prevalence. The cat model had low classi®cation ability. The dog-data model indicated increased odds of infection with Campylobacter for dogs 1 year, and in dogs sampled during the spring. No difference was observed between the prevalence of Campylobacter infections in cats and dogs with diarrhoea and healthy animals. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Campylobacter upsaliensis; Cats; Dogs; Risk factors; Humans
* Corresponding author. Present address: Department of Pharmacology, Microbiology and Food Hygiene, The Norwegian College of Veterinary Medicine, P.O. Box 8146 Dep., N-0033 Oslo, Norway. Tel.: 47-22-59-70-53; fax: 47-22-96-48-50. E-mail address: [email protected] (M. Sandberg).
0167-5877/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 5 8 7 7 ( 0 2 ) 0 0 0 9 5 - 8
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1. Introduction Campylobacteriosis became the most-frequently reported gastrointestinal infectious disease in humans in Norway in 1998 when it surpassed salmonellosis for the ®rst time (Anon., 2001a). In 2000, the reported human incidence was 58 cases per 100,000, an almost 100% increase as compared to 1997. The proportion of human cases reported for which the infection was acquired in Norway has been stable at 40% (Anon., 2001b). Most of the Campylobacter infections seem to be sporadic (Anon., 2001b). Campylobacter jejuni subsp. jejuni (C. jejuni) is the most-frequently isolated species from humans with campylobacteriosis (Bourke et al., 1998). The transmission of campylobacters appears to be by a faecal±oral route through contaminated food and water or by direct contact with faecal material from infected animals and humans. C. jejuni, C. coli, C. upsaliensis and C. lari (all potentially pathogenic to man) can be isolated from various domestic and wild birds and animals (Rosef et al., 1983; Nachamkin and Blaser, 2001). Campyobacter survives in fresh water (Brennhovd et al., 1992), fresh food and (to some extent) in frozen food (Rosef et al., 1984; Kapperud et al., 1992; Anon., 1994; Nachamkin and Blaser, 2000) and in meat packed in modi®ed atmosphere (Fossum et al., 1999). The carrier state in birds and animals is relatively long and the infectious dose is low. The incidence of campylobacteriosis in humans and the prevalence of campylobacters in chicken ¯ocks in Norway usually peak in late summer (Anon., 1994, 2001b; Nachamkin and Blaser, 2000). Seasonal variation in the prevalence of campylobacters in other animals, foods, and environmental sources is not fully investigated (Jones, 2001). C. upsaliensis was reported for the ®rst time in humans by Steele et al. (1985) and occurs in dogs and cats (Bourke et al., 1998; Fox et al., 1989; Sandstedt et al., 1983; Steinhauserova et al., 2001). As is the case in children, puppies and kittens are more likely to acquire clinical campylobacteriosis than adults, and more-commonly shed the organism (Hald and Madsen, 1997). In 1983, 33 of the 147 (22%) dogs and 10 of the 85 (12%) cats from the small-animal clinic at the Norwegian School of Veterinary Science were positive for C. jejuni and C. coli (Gondrosen et al., 1985). No signi®cant differences between the prevalences in dogs and cats with diarrhoea as compared with virtually healthy animals were observed . The selective broth used was the original Skirrow's (Skirrow, 1977), which today is known to have an inhibitory effect on some Campylobacter species (Steele et al., 1985)Ðamong them, C. upsaliensis. A case±control study conducted in the USA indicated an increased risk of C. jejuni and C. coli infection in humans after direct contact with diarrhoeic animals during the week before the onset of their illnesses (Saeed et al., 1993). Different case reports also suggest a transmission of C. upsaliensis from dogs and cats to humans (Bourke et al., 1998; Nachamkin and Blaser, 2000). Results from case±control studies in humans with a con®rmed C. jejuni diagnosis conducted in Norway in 1991±1992 pointed to drinking untreated water, consumption of poultry and having contact with a dog or cat as the mostimportant risk factors (Kapperud, 1995). There are thus clear indications that dogs and cats might play an important role in the epidemiology of Campylobacter infections in humans. Our objective was to investigate the prevalence of Campylobacter in Norwegian healthy cats and dogs in relation to age, season, geographical area and other potential risk factors.
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243
In addition, the association between Campylobacter infection and diarrhoea was investigated and a selection of isolates was tested for antimicrobial susceptibility. 2. Materials and methods 2.1. Study population Faecal swabs from healthy dogs and cats were collected every third month from May 2000 to June 2001 to cover the four different seasons in Norway. Individual animals were sampled by veterinary surgeons in six small-animal practices chosen by convenience but situated throughout Norway. Based upon earlier studies, the sample sizes were calculated by EpiInfo (Anon., 2001c) to detect a prevalence of 10±15% in 1- and >1-year-old cats and a prevalence of 20±25% in 1- and >1-year-old dogs with a precision of 10% at an a of 0.05 (did not account for the within-cluster correlation). Based upon these conditions, about 110 samples from healthy cats and about 145 from healthy dogs were planned to be collected in each season (a total of 430 and 580, respectively). The veterinarians at the six clinics were asked to sample about 18 healthy cats and 24 healthy dogs in each of the four periods by convenience (ideally, four cats and ®ve dogs per week). The criteria of inclusion for the healthy animals were ideally: that they came in for e.g. vaccination, to have their nails cut or for being spayed. They could not have diarrhoea or have an infectious disease or be on antibiotic treatment. In addition, the veterinarians were asked to sample about 50 diarrhoeic dogs and 50 diarrhoeic cats during the study period. Three hundred and one healthy cats (70% of the planned number), 529 healthy dogs (91% of the planned number), 31 diarrhoeic cats and 66 diarrhoeic dogs were sampled. 2.2. Sampling, culturing and identification The animals were sampled with rectal swabs (Amies 114 C, with charcoal, Copan Brescia, Italy). The specimens were cooled in a refrigerator before being sent to the laboratory the same day. Samples were refrigerated and analysed within 2 days after arrival at the diagnostic laboratory at the National Veterinary Institute. The samples were plated on a Campylobacter blood-free selective agar (Oxoid CM739, Basingstoke, UK) supplemented with cephoperazone 8.0 mg/l, teicoplanin 4.0 mg/l, and amphotericin B 10.0 mg/l (Oxoid SR174) (CAT), and the plates were incubated in a microaerophilic atmosphere (Oxoid, CampyGenTM) at 37 1 8C for 72±96 h. Presumptive Campylobacter colonies were con®rmed by phase-contrast microscopy and sub-cultured on 5% bovine blood±agar plates for 48±72 h under the same conditions as described above. The different species were identi®ed by phenotypic assays including growth pattern at 42 8C, catalase production, hippurate hydrolysis (Rosco, Taastrup, Denmark), indoxyl acetate hydrolysis (Rosco), production of H2S in triple-sugar±iron agar (Difco, Detroit, USA), and susceptibility or resistance to nalidixic acid and cephalotin determined by standard diffusion techniques with 30 mg disks (E-test; AB Biodisk, Solna, Sweden) incubated under micro-aerobic conditions at 37 8C for 48±72 h. The possibility of Arcobacter was ruled out by testing for growth aerobically at 30 8C.
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2.3. Antimicrobial susceptibility A random sample of 22 C. jejuni (9 from cats and 13 from dogs) and 20 C. upsaliensis (5 from cats and 15 from dogs) isolates was selected with the random selection procedure in Excel1 (Anon., 2001d). The minimum inhibitory concentration (MIC-values) for C. jejuni and C. upsaliensis isolates to ampicillin, cipro¯oxacin, chloramphenicol, erythromycin, gentamicin, nalidixic acid, streptomycin, and tetracycline were investigated using the E-test with breakpoints of S 16 and R 32 for nalidixic acid and S 4, R 8 for streptomycin (Ane Nùdtvedt, personal communication, The Norwegian Zoonosis Centre) also used in the Danish integrated antimicrobial resistance monitoring and research programme (Danmap) (Anon., 2001e). The breakpoints for the other antimicrobials are shown in NORMVET 2000 (Anon., 2001f). 2.4. Epidemiological information The pet owners and the veterinary surgeons ®lled in a simple questionnaire with information about the name and address of the owner, why they came to see the veterinary surgeon and age, sex, breed and name of the animal. The healthy cat questionnaire had an additional six main questions: (1) are there other animals in the household? If yes, what kind of animals? (2) Is the cat kept only indoors? If yes, does it have contact with other cats through events such as shows or similar activities? (3) Is the cat allowed to be outdoors and if yes, in what kind of environment? (4) Has the cat stayed in a cattery during the last month? (5) Has the cat been abroad in past 3 months? (6) What kind of feed is the cat fed? The healthy dog questionnaire had an additional seven main questions: (1) are there other animals in the household? (2) In what kind of environment do you walk your dog? (3) Do you keep the dog in a dog-yard? (4) Does your dog have contact with other dogs through different events? (5) Has your dog been abroad during the past 3 months? (6) Has the dog stayed in a kennel during the last month? (7) What kind of feed is the dog fed? The questionnaires for the cats and dogs with diarrhoea also included questions about the signs. All the questions had speci®ed choices, and all variables except age had dichotomous outcomes. Most of the variables are shown in Tables 1 and 2 (exceptions are those that did not vary or those that had too many missing values). No small pilot project to evaluate the questionnaires was conducted, but the questionnaires were based on a questionnaire used in a similar project conducted in Sweden and was also discussed and evaluated by the project group and sent to the veterinarians at the six clinics for evaluation before the project was started. Only the healthy animals were included in the risk factor analyses. 2.5. Statistical analyses The dataset was established in Excel1. The descriptive and analytic analyses of the data was conducted in EpiCalc1 (Anon., 2001g) and SAS-PC system1, version 6.12 for Windows (Anon., 2001h). Throughout the analyses, detection of any Campylobacter in faeces was the case de®nition. Preliminary descriptive and univariable analyses of the
Table 1 Campylobacter in healthy cats in Norway 2000/2001 Factors
Outcomes
N
Positive
C. jejuni
C. upsaliensis
C. coli
C. spp.
No.
%
No.
%
No.
%
No.
%
No.
%
± ±
301 168 123
54 36 17
18 21 14
11 6 5
4 4 4
38 28 9
13 17 7
2 1 1
0.7 0.6 0.8
3 1 2
1 0.6 2
Season
May±June July±September October±November December±March
± ± ± ±
60 91 76 74
15 12 16 11
25 13 21 15
1 3 6 1
2 3 8 1
13 6 9 10
22 7 12 14
± 2 ± ±
± 2 ± ±
1 1 1 ±
2 1 1 ±
Clinic
1 2 3 4 5 6
± ± ± ± ± ±
69 49 66 23 41 53
5 9 14 2 12 12
7 18 21 9 29 23
1 2 2 2 1 3
1 4 3 9 2 6
4 5 12 ± 11 6
6 10 18 ± 27 11
± 1 ± ± ± 1
± 2
2
± 1 ± ± ± 2
± 2 ± ± ± 4
Norwegian mix Pure-bred
Yes No
188 82
40 8
21 10
8 1
4 1
27 7
9 9
2 ±
1 ±
3 ±
2 ±
Several animalsa
Yes No
151 146
25 29
17 20
6 5
4 3
16 22
11 15
1 1
0.7 0.7
2 1
1 0.7
Outdoorb
Yes No Yes No Yes No Yes No Yes No
217 82 34 197 149 78 55 171 7 220
41 13 3 41 29 15 14 30 ± 44
19 16 9 21 20 19 26 18 ± 20
9 2 ± 10 6 4 4 6 ± 10
4 2 ± 5 4 5 7 4 ± 5
28 10 2 27 20 9 9 20 ± 29
13 12 6 14 13 12 16 12 ± 13
2 ± 1 1 1 1 1 1 ± 2
0.9 ± 3 0.5 0.7 1.3 1.8 0.6 ± 0.9
2 1 ± 3 2 1 ± 3 ± 3
0.9 1 ± 1.5 1.3 1.3 ± 2 ± 1
Breed
City Suburb Countryside Other
245
1 year >1 year
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
Total no. of cats Age
Category
246
Factors
Category
Outcomes
N
Positive
C. jejuni
C. upsaliensis
C. coli
C. spp.
No.
%
No.
%
No.
%
No.
%
No.
%
Indoorc
Yes No
13 69
2 12
15 17
± 2
± 3
± 10
± 15
± ±
± ±
2 ±
15 ±
Abroad
Yes No
5 292
1 52
20 18
± 11
± 4
1 36
20 12
± 2
± 0.7
± 3
± 1
Yes No Yes No Yes No Yes No
279 20 66 233 57 242 173 126
50 4 10 44 3 51 32 22
18 20 15 19 5 21 19 18
10 1 1 10 1 10 6 5
4 5 2 4 2 4 4 4
35 3 8 30 2 36 23 15
13 15 12 13 4 15 13 12
2 ± ± 2 ± 2 2 ±
0.7 ± ± 0.9 ± 0.8 1 ±
3 ± 1 2 ± 3 1 2
1 ± 2 0.9 ± 1 0.6 2
Feed
Pellets Leftovers Mixed Canned
a
Several animals in the household (specified: dogs, cats, reptiles, birds, dairy cows, cattle, pigs, sheep). Kept outdoor and indoor. c Kept only indoor. b
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
Table 1 (Continued )
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
247
Table 2 Campylobacter in cats and dogs with diarrhoea in Norway 2000/2001 Factors
Total no. cats Age Total no. of dogs Age
Category
N
Positive
C. jejuni
C. upsaliensis
C. spp.
No.
%
No.
%
No.
%
No.
%
1 year >1 year
31 15 13
5 2 1
16 13 8
± ± ±
± ± ±
4 2 1
13 13 8
1 ± ±
3 ± ±
1 year >1 year
66 26 39
18 11 7
27 42 18
2 2 ±
3 8 ±
15 8 7
23 31 18
1 1 ±
2 4 ±
results from both groups (healthy and diarrhoeic) animals were done using the chi-square and Fisher-exact tests for categorical factors such as season (1±4) and geographical area (clinic 1±6). The continuous variable age was analysed by the Kruskal±Wallis test. For the modelling, age was dichotomised in the analysis (1 and >1 year) partly because there was no effect gained by dividing the data into four groups and partly because of the low number of animals in some of the four groups. The period from May to June was de®ned as spring, July to September as summer, October to November as autumn and December to the mid of March as winter. Most of the samples were collected in the ®rst 2 months of the each of above-described periods. The multivariable logistic model was built in a backward-elimination procedure as described by Hosmer and Lemenshow (2000). The outcome variable was Campylobacterpositive animals. The risk factors tested were age, season and clinic. These and other factors with a P 0:20 from univariable analyses were candidates for the multivariable logistic model. Only factors present in 5 individuals were included in the multivariable model. The ®tting of the models was basically conducted by observing the change in the deviance. As we wanted to generalize, clinic was to be taken into the model as a random effect by using the GLIMMIX procedure in SAS. The ®t of the models was assessed as described by Hosmer and Lemenshow (2000) using the summary measure of ®t; the Hosmer and Lemenshow test. Logistic regression diagnostics were conducted to identify possible outliers by investigating the hat matrix (leverage-values) and change in deviance (DD) and b (Db). The healthy and diarrhoeic dogs were matched on age and tested for association in regard to Campylobacter by a chi-square test. Due to the small number of samples from cats with diarrhoea, the potentially confounding effect of age on the Campylobacter carriage could not be investigated. Possible differences between the diarrhoeic animals and the healthy animals regarding season and clinic also were not possible to investigate because of the low number of samples collected from animals with diarrhoea. 3. Results Of the 301 healthy cats sampled, 18% were positive for Campylobacter (Table 1), compared to 5 out of 31 (16%) cats with diarrhoea (Table 2). Of the 529 healthy dogs
248
Table 3 Campylobacter in healthy dogs in Norway 2000/2001
Total no. of dogs Age
Category
Outcomes
N
Positive
C. jejuni
C. upsaliensis
C. coli
%
No.
%
No.
%
No.
%
102 63 38
19 24 15
± ± ±
± ± ±
4 ± 3
0.8 ± 2
No.
%
No.
C. spp
1 year >1 year
± ±
529 262 258
124 80 42
23 31 16
18 13 4
3 5 2
Season
May±June July±September October±November December±March
± ± ± ±
129 155 128 117
40 42 23 19
31 27 18 16
3 10 1 4
2 7 0.8 3
34 31 22 15
26 20 17 13
± ± ± ±
± ± ± ±
1 ± ± ±
0.6 ± ± ±
Clinic
1 2 3 4 5 6
± ± ± ± ± ±
93 88 98 76 81 92
11 20 18 22 29 24
12 23 18 29 36 26
3 2 1 2 3 7
3 2 1 3 4 8
8 18 17 20 22 17
9 21 17 26 27 19
± ± ± ± ± ±
± ± ± ± ± ±
± ± ± 4 ± 3
± ± ± 5 ± 1
Yes No
277 252
71 53
26 21
12 6
4 2
56 46
20 18
± ±
± ±
1 2
0.4 3
City
Yes No
77 452
21 103
27 23
2 16
3 4
17 85
22 19
± ±
± ±
2 2
0.4 0.7
Suburb
Yes No
282 247
61 63
22 26
8 10
3 4
51 51
18 21
± ±
± ±
2 ±
0.8 ±
Countryside
Yes No
275 254
63 61
23 24
9 9
3 4
54 48
20 19
± ±
± ±
± 3
± 1
Other
Yes No
37 492
8 116
22 24
2 16
5 3
6 96
16 20
± ±
± ±
± 4
± 0.8
Several animalsa Outdoor
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
Factors
Yes No
129 398
35 88
27 22
4 14
3 4
31 70
24 18
± ±
± ±
± 4
± 1
Contact other dogs
Yes No
321 208
77 47
24 23
7 ±
2 ±
66 36
21 17
± ±
± ±
4 ±
1 ±
Abroad
Yes No
24 501
3 121
13 24
11 18
5 4
3 99
13 20
± ±
± ±
± 4
± 0.8
Yes No Yes No Yes No Yes No
502 27 167 362 34 495 77 452
119 5 30 94 7 117 14 110
24 19 18 26 21 24 18 24
15 3 1 17 ± 18 3 15
3 11 0.6 5 ± 4 4 3
100 2 29 73 7 95 11 91
20 7 17 20 21 19 14 20
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ±
4 ± ± 4 ± 4 ± 4
0.8 ± ± 1 ± 0.8 ± 0.9
Feed
Pellets Leftovers Etrails Other
a
Several animals in the household (specified: dogs, cats, reptiles, birds, dairy cows, cattle, pigs, sheep).
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
Outdoor yard
249
250
M. Sandberg et al. / Preventive Veterinary Medicine 55 (2002) 241±253
sampled, 124 (23%) were positive for Campylobacter (Table 3), compared to 18 out of 66 (27%) dogs with diarrhoea (Table 2). 3.1. Risk factors for Campylobacter infection in healthy cats We offered age, season, clinic, breed and being outdoors in the countryside, city or other place. The ®nal model included mixed Norwegian breed (as opposed to a pure-bred breed) (Table 4) and clinic (the random effect). The model was poor at cross-classifying the same dataset, but no outliers were identi®ed. 3.2. Risk factors for Campylobacter infection in healthy dogs Offered to the multivariable model were age, season and clinic and ``not being fed leftovers''. The ®nal model included age, season and clinic (Table 4). (The latter was the random effect.) The model was poor at cross-classifying the same dataset, but no outliers were identi®ed. 3.3. Possible association between Campylobacter and diarrhoea in cats and dogs No difference between cats with diarrhoea and healthy cats in regard to Campylobacter infection was detected. Neither was there a positive association between diarrhoea and Campylobacter infection in dogs detected when matching the diarrhoeic and healthy dogs on age (data not shown). 3.4. Antimicrobial susceptibility One of the 22 C. jejuni and 18 of the 20 C. upsaliensis were categorized as resistant to streptomycin. One of C. upsaliensis was categorized as resistant to nalidixic acid. For the other antimicrobials, all isolates were susceptible. Table 4 Results from the final multivariable analysis of the Campylobacter data from healthy cats and dogs in Norway 2000/2001 with clinic as a random effect Factors Cats Intercept Mixed Norwegian breed Dogs Intercept Age Season
Category
May±June July±September October±November December±March
b
S.E. (b) 2.24 0.87
0.41 0.41
1.19 0.79 0.72 0.49 0.03 ±
0.30 0.22 0.32 0.32 0.35 ±
Deviance 240.26
530.56
P
ORlogistic 95% CI
0.003 0.035
± 2.39
± 1.06±5.38
0.011 0.0003 0.03 0.13 0.9 ±
± 2.20 2.05 1.63 1.03 1.0
± 1.43±3.39 1.09±3.89 0.87±3.08 0.51±2.04 ±
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4. Discussion The indicated higher risk for healthy dogs being Campylobacter-positive when 1-yearold corresponds well to the results from the studies by Burnens et al. (1992) and by Hald and Madsen (1997), which revealed an increased risk of shedding Campylobacter for young dogs. The animals investigated in their studies were somewhat younger (<1 year or 6 months) than in our study. The indications of a higher risk for cats of a mixed Norwegian breed was weak but could be explained by the fact that most of these cats probably are allowed to be outdoors as opposed to the cats of a pure-bred breed. A higher risk of being infected from other animals and the environment should therefore be expected for these cats. The ``seasonal'' variation of Campylobacter shedding among cats and dogs was investigated for the ®rst time in this study as far as we can ascertain, but no distinct differences was revealed for cats. More positive dogs were found in May±June and in July± September. The estimates were, however, not very robust (Table 4). The more-frequent isolation of Campylobacter from cats and dogs from the clinic situated in the capital of Norway (Oslo clinic 5), could be explained by the higher density of animals and people in this area. It also simply could be explained by better survival of Campylobacter in the sample (and therefore fewer false negatives) because of the short transport time from the Oslo clinic to the laboratory. As a curiosity, we mention that only three of the 31 dogs and one out of the ®ve cats that had been abroad (mainly Sweden and for few animals, Spain) the last 3 months before the sampling were shedding Campylobacter. 4.1. Campylobacter as a risk factor for diarrhoea in cats and dogs We found no association between Campylobacter infection and diarrhoea and this was in accord with the results from the Norwegian study by Gondrosen et al. (1985). Several other studies have revealed higher proportions of campylobacters in animals with diarrhoea (Burnens et al., 1992; Ferreira et al., 1979; Fleming, 1983; Nair et al., 1985) while others reveal similar proportions in cats and dogs without diarrhoea (Baker et al., 1999; Fox et al., 1989; McOrist and Browning, 1982; Olson and Sandstedt, 1987). Higher risk of infection in diarrhoeal versus healthy juvenile dogs has been reported (Burnens et al., 1992; Fleming, 1983). 4.2. Antimicrobial resistance That neither of the isolates tested was resistant to cipro¯oxacin and only one of the C. upsaliensis was resistant to nalidixic acid probably re¯ects that there is a restriction on use of quinolones in cats and dogs in Norway. The Norwegian monitoring program for resistance in microbes (NORM) reports an increase in the resistance of quinolones only in Campylobacter isolated from humans in Norway (Melby, 2001). The Danish integrated antimicrobial resistance monitoring and research programme (Danmap) reports streptomycin resistance in C. jejuni isolated from Danish broilers (12%), cattle (2%) and humans (two isolates). From broilers and pigs in Denmark, 25 and 48% of
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the C. coli isolated in 2000 were resistant to streptomycin (Anon., 2001e). Only one of the investigated C. jejuni from the present study was resistant to streptomycin as opposed to the high number of C. upsaliensis which suggests that C. upsaliensis is intrinsically resistant to the antimicrobial. 5. Conclusion The risk for Campylobacter infection was increased for dogs1-year-old. There were also differences across months and among clinics. No difference was observed between the prevalence of Campylobacter in cats and dogs with diarrhoea and healthy animals. Acknowledgements We gratefully acknowledge the following small-animal practises for providing samples: Bergen SmaÊdyrklinikk, Bodù Dyreklinikk, ByaÊsen Dyreklinikk, Gjùvik Dyreklinikk, Poliklinikken at The Norwegian School of Veterinary Science, and Sandnes Dyreklinikk. We also acknowledge the Section of Epidemiology at the National Veterinary Institute for help in the initial phase of the study, and with the GLIMMIX procedures in SAS, and Andreas Hole for the data entry, and help with the quality assurance of the data. References Anon., 1994. Smittestoffer i nñringsmidler. SNT rapport 5, Statens nñringsmiddeltilsyn, Oslo, pp. 29±30. http:// www.snt.no/dokumentasjon/index.html. Anon., 2001a. WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe. http://www.who.it/docs/fdsaf/fs_survprog.htm. Anon., 2001b. Surveillance of Communicable Diseases in Norway 1999, National Institute of Public Health, Oslo, Norway. http://www.folkehelsa.no/Ler/pdf/smittevern4.pdf. Anon., 2001c. EpiInfo. http://www.cdc.gov/. Anon., 2001d. Microsoft Office, Excel. http://www.microsoft.com/norge/products/office/productinfo/products/ excel/. Anon., 2001e. The Danish integrated antimicrobial resistance monitoring and research programme (Danmap). http://www.svs.dk/uk/Organization/Frm_org.htm. Anon., 2001f. NORM/NORMVET 2000. http://www.vetinst.no/Zoonosesenteret/NORM-NORMVET_2000.pdf. Anon., 2001g. EpiCalc. http://www.myatt.demon.co.uk/epicalc.htm. Anon., 2001h. SAS Institute, Cary, NC, USA, 1996. http://www.sas.com/rnd/app/paper/nlmixedsugi.pdf176.0KB-GLIMMIX:1. Baker, J., Barton, M.D., Lanser, J., 1999. Campylobacter species in cats and dogs in South Australia. Aust. Vet. J. 77, 662±666. Bourke, B., Chan, V.L., Sherman, P., 1998. Campylobacter upsaliensis: waiting in the wings. Clin. Microbiol. Rev. 11, 440±449. Brennhovd, O., Kapperud, G., Langeland, G., 1992. Survey of thermotolerant Campylobacter spp. and Yersinia spp. in three surface water sources in Norway. Int. J. Food Microbiol. 15, 327±338. Burnens, A.P., Angeloz-Wick, B., Nicolet, J., 1992. Comparison of Campylobacter carriage rates in diarrheic and healthy pet animals. Zentralbl. Veterinarmed. [B] 39, 175±180.
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