Simultaneous Presence of Multiple Campylobacter Species in Dogs

The prevalence of coinfection of Campylobacter species in dogswas determined using four isolation methods. In 26% of the positive-testingstools, multiple Campylobacter species were identified. Theuse of multiple isolation methods as well as the time lapsebetween sampling and processing are important for detectionof coinfection.

INTRODUCTION

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Introduction

In humans, Campylobacter is considered the most frequent bacterialcause of enteritis. The species Campylobacter jejuni is theprime etiologic agent, with minor contributions of Campylobactercoli and Campylobacter lari (1, 19). The role of Campylobacteras an enteric pathogen in dogs is much less evident. It is frequentlyisolated both from animals with symptoms of enteritis and fromhealthy animals (3, 9, 12, 16). The poor identification of Campylobacterspecies as animal pathogens may result from the simultaneouspresence of multiple strains or species with various pathogenicitycharacteristics. In routine diagnostic laboratories, typingof Campylobacter is usually restricted to one colony per stoolsample. In humans, simultaneous infection with more than oneCampylobacter strain is found to be rare and is not consideredto impair epidemiological analyses (15). With respect to dogs,however, no studies have been described that investigated thesimultaneous presence of multiple species or strains. As thepossibility to discriminate by colony morphology between Campylobacterspecies (13) or even between Campylobacter and Helicobacterspecies (6, 17) is limited, the prevalence of coinfection incompanion animals could well be underestimated. Because thesimultaneous presence of multiple strains or species is crucialto establish the role of Campylobacter in clinical disease aswell as for epidemiological studies, we examined multiple coloniesfrom a total of 30 fecal samples from diarrheic and nonsymptomaticdogs. We used a variety of culture media, as antimicrobialspresent in selective medium may selectively inhibit distinctCampylobacter species or strains. As Campylobacter is sensitiveto oxygen and dryness, we also assessed the effect of the timeinterval between sampling and processing (transport time) onthe isolation of Campylobacter.

Samples were obtained from September 2000 until February 2001from household dogs of different ages (see Table 2) and weretransported to the laboratory without cooling. The average samplesize was about 25 g (minimum, 5 g). The isolation methods employedincluded a filtration method and three different selective media.Before processing the samples were homogenized and directlyplated to the selective medium. In the filtration method, 10to 12 drops of a fecal suspension in brain heart infusion wereplaced on a 0.65-µm-pore-size cellulose acetate filter(Sartorius, Goettingen, Germany) on blood agar base no. 2 (Oxoid)supplemented with 5% sheep blood. After 1 h of incubation at37°C under aerobic conditions, the filter was removed andthe plates were incubated microaerobically. We used as selectiveagar plates the following media: (i) modified-charcoal cefoperazonedeoxycholate agar (mCCDA), a blood-free selective agar basesupplemented with 32 µg of cefoperazone/ml and 10 µgof amphotericin/ml (Oxoid CM 739 with Oxoid supplement SR 155)which is widely used in medical, veterinary, and food microbiologylaboratories for the isolation of Campylobacter; (ii) cefoperazoneamphotericin teicoplanin selective medium (CAT), a blood-freecharcoal based agar containing 8 µg of cefoperazone/ml,4 µg of teicoplanin/ml, and 10 µg of amphotericin/ml(Oxoid supplement SR 174); and (iii) Karmali, a blood-free charcoal-basedagar containing 32 µg of cefazolin/ml, 20 µg ofvancomycin/ml, and 100 µg of cycloheximide/ml (Oxoid supplementSR 167). The latter two types of plates were used because theyare recommended for the detection of Campylobacter upsaliensisand Campylobacter helveticus (2, 10). All plates were incubatedat 37°C under microaerobic conditions in jars (Anoxomat,Mart, Lichtenvoorde, The Netherlands) (5% O₂, 10% CO₂, 85% H₂)and examined daily for growth for 4 to 6 days. From plates withgrowth of Campylobacter suspected (on the basis of colony morphology,catalase, oxidase, and Gram staining results), multiple subculturesof separate colonies (an average of 12 per sample) were grownon blood agar base no. 2 (Oxoid) supplemented with 5% sheepblood. The cultures were identified by PCR-restriction fragmentlength polymorphism with a method that distinguishes Campylobacter,Arcobacter, and Helicobacter by analysis of the 16S rRNA gene(11). Campylobacter species were identified by PCR-restrictionfragment length polymorphism analysis of a highly polymorphicpart of the 23S rRNA gene (5) and of the 16S rRNA gene (11).The patterns were compared with those of reference strains obtainedfrom the CCUG/LMG (Culture Collection Universiteit of Ghent,Ghent, Belgium).

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TABLE 2. Campylobacter species detected in fecal samples from dogs by filter, mCCDA, CAT, and Karmali

Efficiency of isolation.

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Efficiency of isolation.

The use of different kinds of growth media increased the sensitivityof Campylobacter isolation (Table 1), as described before (4,14). Overall, 23 (77%) of the samples were found positive forCampylobacter by one or more of the methods. Of the 23 positive-testingdog samples, 16 (70%) harbored C. upsaliensis, 12 (52%) harboredC. jejuni, and 3 (13%) harbored C. lari. This distribution isconsistent with other reports on dogs (3, 9, 12, 16).

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TABLE 1. Incidence of Campylobacter species grown from dog feces as determined by four isolation protocols

Coinfection.

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Coinfection.

By analysis of an average of 12 single colonies from each positive-testingsample, multiple Campylobacter species were observed in 6 (26%)of the 23 positive-testing stool samples (Table 2). In two samples,as many as three species could be isolated. No systematic studyon coinfection with Campylobacter species in dogs has been reportedbefore to our knowledge, although the presence of two Campylobacterspecies (7, 20) or serotypes (7) in a single sample has beenmentioned. However, our results are comparable with those foundin investigations of cats, for which it has been demonstratedthat 34% of the Campylobacter-positive samples from healthyanimals contained more than one Campylobacter species (17).

Healthy versus diarrheic animals.

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Healthy versus diarrheic...

We examined fecal samples from diarrheic animals (n = 8) aswell as from healthy animals (n = 22). Multiple Campylobacterspecies were detected only in samples from healthy animals (Table2). No conclusions can be drawn, however, because of the limitednumber of samples and since most samples of diarrheic animalshad a sampling-to-processing time that exceeded 4 h.

Media.

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Media.

The selective media showed comparable isolation rates, whereasthe filtration method was less sensitive (Table 1), possiblybecause samples with low numbers of bacteria have been shownto give negative results (8, 18). The distribution of speciesisolated with the various methods is shown in Table 2. Our resultsdo not confirm that CAT and Karmali media are better suitedfor detection of C. upsaliensis, notably, as we found a highernumber of mCCDA plates positive for C. upsaliensis than resultedusing the Karmali method, CAT method, and filtration method,which were specifically introduced for the detection of thisCampylobacter species (2, 10). Although the filtration methodis recommended for the detection of Campylobacter strains thatmight be inhibited by antibiotics present in selective media,we did not detect additional Campylobacter species using thismethod.

Karmali medium performed best in the detection of multiple speciesfrom single samples, as two species were recovered from 5 (28%)of the 18 positive-testing samples. The filter method failedto detect any coinfection. As expected, the chance of detectingcoinfection was increased by the use of multiple isolation methods.

Transport time.

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Transport time.

An influence of the time interval between sampling and processingof the sample in the laboratory was observed, as coinfectionwas detected only in samples processed within 4 h after collection(Table 2). Some samples yielded a single species even after3 days, on the other hand, indicating a considerable variationin the survival times of campylobacters between samples and/ordifferences in the viability of Campylobacter species.

In conclusion, despite a relatively low number of samples ourdata clearly show that coinfections of different Campylobacterspecies are quite common in dogs. The possibility cannot beexcluded that the diversity might be even more extensive, asdifferent strains might exist within a single species. Thisnotion is important for epidemiological studies, e.g., the tracingof sources of human infections and studies on the pathogenicityof Campylobacter in dogs.

For studying Campylobacter infections in dogs and cats multiplecolonies should be examined, preferably from a combination ofmedia and from specimens that are processed within 4 h aftersampling.

ACKNOWLEDGMENTS

The help of the technicians from the Veterinary MicrobiologicalDiagnostic Center of the Veterinary Faculty is gratefully acknowledged.We also thank Alan Rigter for the helpful assistance in thespeciation of the strains and Jos van Putten for his criticalreading of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Animal Health Service, P.O. Box 9, 7400 AA Deventer, The Netherlands. Phone: 31 570 660177. Fax: 31 570 660176. E-mail: m.koene{at}gdvdieren.nl.

REFERENCES

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