Veterinary Microbiology 146 (2010) 356–360

Short communication

Prevalence of Brachyspira pilosicoli and ‘‘Brachyspira canis’’ in dogs andtheir association with diarrhoea

Alvaro Hidalgo *, Pedro Rubio, Jesus Osorio, Ana Carvajal

Department of Animal Health, Infectious Diseases and Epidemiology, Faculty of Veterinary Science, University of Leon, Leon, Spain

A R T I C L E I N F O

Article history:

Received 5 February 2010

Received in revised form 30 April 2010

Accepted 5 May 2010

Keywords:

Brachyspira pilosicoli

‘‘Brachyspira canis’’

Prevalence

Intestinal spirochaetosis

Dog

Diarrhoea

A B S T R A C T

The aims of this study were to investigate the prevalence of colonization with intestinal

spirochaetes in dogs, and to assess their association with diarrhoea. To achieve this, faecal

samples from 311 dogs were obtained between November 2008 and April 2009 and

cultured for Brachyspira species. A total of 41 Brachyspira spp. isolates were recovered, and

these were classified into species according to their biochemical properties, and results of

a B. pilosicoli species-specific PCR, and partial amplification of the nox gene with

sequencing of the product. An overall Brachyspira spp. prevalence of 13.2% (41/311) was

obtained. The prevalence of Brachyspira pilosicoli faecal shedding was 4.8% (15/311) while

‘‘Brachyspira canis’’ was identified in 8.0% (25/311) of the sampled dogs. One dog shed an

isolate tentatively identified as B. intermedia. A statistically significant association

between the shedding of B. pilosicoli and the presence of diarrhoea in dogs was

demonstrated (P< 0.001). Risk factors for shedding of Brachyspira spp. were investigated.

Using the odds ratio, the risk of B. pilosicoli shedding was five times higher among dogs up

to 1 year of age as compared with adult dogs (older than 1 year). These findings may have

practical implications in the public and animal health fields.

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1. Introduction

The genus Brachyspira is comprised of oxygen tolerantanaerobic spirochaetes that colonize the large intestine ofanimals and humans (Hampson et al., 1997). Two differentBrachyspira spp. have been commonly isolated from dogs:‘‘B. canis’’, considered to be non-pathogenic, and B. pilosicoli,which has been proposed as a possible cause of diarrhoea indogs (Duhamel et al., 1998; Oxberry and Hampson, 2003;Johansson et al., 2004). Interestingly, B. pilosicoli is the onlyBrachypira species that has been isolated from a wide rangeof species, including humans, non-human primates, pigs,chickens, other birds, horses, rheas and dogs—and has beenassociated with disease (‘‘intestinal spirochaetosis’’) inseveral of these hosts. A number of investigations havesuggested the possible transmission of B. pilosicoli between

* Corresponding author at: Facultad de Veterinaria (Enfermedades

Infecciosas), Campus de Vegazana, 24071 Leon, Spain.

Tel.: +34 987 291306; fax: +34 987 291304.

E-mail address: alvaro.hidalgo@unileon.es (A. Hidalgo).

0378-1135/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2010.05.016

animals and human beings (Koopman et al., 1993; Trottet al., 1997, 1998; Hampson et al., 2006).

As dogs live in close contact with human beings, theyare a potential source of B. pilosicoli infection and this mayrepresent a public health risk. However, little is knownabout the prevalence of B. pilosicoli in dogs, or even aboutits association with diarrhoea. Similarly information aboutthe prevalence of ‘‘B. canis’’, the other major Brachyspira

species isolated from dogs, is sparse. Accordingly, thisstudy was undertaken to clarify the prevalence of canineintestinal spirochaetal infection, to assess risk factors forBrachyspira spp. shedding in dogs, and to investigate theassociation between the detection of Brachyspira spp. andthe occurrence of diarrhoea.

2. Materials and methods

2.1. Sampling and epidemiological survey

The number of dogs to be sampled was calculated withthe WIN EPISCOPE 2.0 computer package. Sample size was

A. Hidalgo et al. / Veterinary Microbiology 146 (2010) 356–360 357

estimated to be enough to predict the prevalence ofintestinal spirochaete shedding in dogs with an absoluteerror of �5% and a 95% confidence level. For an expectedBrachypira spp. prevalence of 18.7% (Lee and Hampson,1996) a total of 234 dogs should be included.

As part of a diagnostic exercise, clinicians from 10veterinary clinics located in the town of Leon and itssuburbs randomly sampled dogs attending for anyconsultation during the period November 2008–April2009. Faecal samples were collected directly from therectum of dogs using sterile alginate bacteriology swabs.Each faecal sample was labelled and accompanied by aquestionnaire filled in by the practitioner, including date ofbirth, gender and breed of the dog. Dogs presented to theclinics with any recent history of diarrhoea (multiple loosebowel movements per day) were confirmed through thedetection of loose stool at the sampling time. In addition, toevaluate potential risk factors for the shedding ofBrachyspira spp., clinicians also scored the origin of thedog (breeder/pet shop/private owner/animal shelter), thehousing (indoor/outdoor), the task or work (company/guard/hunting/miscellaneous), contact with other dogs(low/medium/high) and current drug treatments, if any.

2.2. Culture, biochemical characterization and diagnostic PCR

Faecal specimens were streaked on selective agar(Jenkinson and Wingar, 1981), and incubated in ananaerobic atmosphere at 39 8C for 10 days. Plates showinghaemolysis were checked for spirochaetal presence bymicroscopy and subsequently propagated until pure.Biochemical characterization was performed as previouslydescribed by Fellstrom and Gunnarsson (1995). Species-specific PCR (Rasback et al., 2006) was used to identify B.

pilosicoli isolates. B. hyodysenteriae B78T (ATCC 27164T),and B. pilosicoli P43/6/78T (ATCC 51139) were used ascontrols.

2.3. PCR amplification and sequencing of the nox gene

DNA samples obtained by the boiling method wereused to amplify Brachyspira spp. specific 939 bp DNAfragments of the nox gene as previously described (Rohdeet al., 2002). Amplicons were subsequently purified andsequenced. PHYLIP v3.6 was used to construct a dendro-gram, including nox sequences of Brachyspira spp. refer-

Table 1

Biochemical reactions and PCR identification using a species-specific PCR for the d

intestinal spirochaetes isolated from dogs. Biochemical groups were defined acco

reactions are indicated in parentheses.

Group No. of isolates Indole Hippurate

II n = 1 + �

IIIa n = 22 � �n = 3 � �

IV n = 6 � +

n = 3 � (+)

n = 3 + (+)

n = 1 � +

n = 1 + +

n = 1 + (+)

ence and type strains retrieved from GenBank: B. murdochii

56-150T (AF060813), B. innocens B256T (AF060804), B.

pilosicoli P43T (AF060807), B. alvinipulli C1T (AF060814), ‘‘B.

suanatina’’ (DQ487119), B. hyodysenteriae B204R (U19610),B. hyodysenteriae B78T (AF060800), B. intermedia PWS/AT

(AF060811), ‘‘B. canis’’ Dog A2R (EU819071) and B. alborgii

513AT (AF060816).

2.4. Statistical analysis

A univariate analysis using Yates’ Chi-square test(a = 0.05) was used to investigate the association betweenthe presence of B. pilosicoli or ‘‘B. canis’’ and the occurrenceof diarrhoea in dogs. Risk factors for Brachyspira spp., B.

pilosicoli and ‘‘B. canis’’ shedding were also assessed by theYates’ Chi-square test (a = 0.05). Fisher’s exact test waschosen when any expected value was lower than 5.Additionally, to study age as a possible confounding factorfor the shedding of B. pilosicoli or ‘‘B. canis’’ and thepresence of diarrhoea, a stratified analysis was performed.The variable ‘‘age’’ was taken as a categorical variable withtwo levels: dogs older than 1 year/dogs 1 year or younger.The programme Epi Info, version 3.5.1 (CDC, USA) was usedfor the calculations.

3. Results

3.1. Culture, biochemical characterization and diagnostic PCR

A total of 311 faecal samples were collected through thestudy. Of these, 41 (13.2%) were confirmed to containspirochaetes on primary cultures and were subcultured topurity. All the spirochaetes recovered showed weak beta-haemolysis. Subsequent determination of their biochemi-cal properties (Table 1) classified 15 out of 41 (36.6%)isolates as group IV (B. pilosicoli), 25 (61.0%) as group IIIa(‘‘B. canis’’) and one (2.4%) as group II (B. intermedia). Thespecies-specific PCR amplified a 16S rDNA fragmentspecific for B. pilosicoli in 15 DNA samples from 41 ofthe isolates (36.6%).

3.2. Phylogenetic analysis

In an evolutionary tree based on partial sequences ofthe nox, 15 canine isolates grouped together with the B.

pilosicoli type strain P43T, 25 isolates with the reference

etection of B. pilosicoli (Rasback et al., 2006) of 41 weakly beta-haemolytic

rding to Fellstrom et al. (2008) and Johansson et al. (2004). Weak positive

a-Galatosidase b-Glucosidase PCR identification

� + None

� + None

� (+) None

+ � B. pilosicoli

+ � B. pilosicoli

� � B. pilosicoli

� � B. pilosicoli

+ � B. pilosicoli

(+) � B. pilosicoli

Table 2

Faecal shedding of B. pilosicoli and ‘‘B. canis’’ among 311 dogs according to the presence of diarrhoea at the time of the sampling, age, origin, housing, task and

degree of contact with other dogs.

No. of dogs (%) No. of positive dogs (%) P-Value OR (95% CI)

B. pilosicoli

Diarrhoea

Presence 44 (14.1) 8 (18.2) <0.001 8.25 (2.53–27.25)

Absence 267 (85.9) 7 (2.6)

Age

�1 year 71 (22.8) 9 (12.7) 0.002 5.66 (1.76–18.73)

>1 year 240 (77.2) 6 (2.5)

Origin

Breeder 30 (9.6) 3 (10) 0.047

Pet shop 13 (4.2) 0 (0)

Private owner 258 (83) 10 (3.9)

Animal shelter 10 (3.2) 2 (20)

Housing

Indoor 170 (54.7) 5 (2.9) 0.151 0.4 (0.11–1.30)

Outdoor 141 (45.3) 10 (7.1)

Task/work

Company 287 (92.3) 12 (4.2) <0.001

Guard 7 (2.3) 0 (0)

Hunting 11 (3.5) 0 (0)

Miscellaneous 6 (1.9) 3 (50)

Degree of contact with other dogs

Low 58 (18.6) 2 (3.4) 0.738

Medium 173 (55.7) 8 (4.6)

High 80 (25.7) 5 (6.3)

‘‘B. canis’’

Diarrhoea

Presence 44 (14.1) 4 (9.1) 0.765 1.17 (0.32–3.87)

Absence 267 (85.9) 21 (7.9)

Age

�1 year 71 (22.8) 8 (11.3) 0.373 1.67 (0.63–4.33)

>1 year 240 (77.2) 17 (7.1)

Origin

Breeder 30 (9.6) 5 (16.7) 0.167

Pet shop 13 (4.2) 0 (0)

Private owner 258 (83) 20 (7.8)

Animal shelter 10 (3.2) 0 (0)

Housing

Indoor 170 (54.7) 11 (6.5) 0.364 0.63 (0.26–1.53)

Outdoor 141 (45.3) 14 (9.9)

Task/work

Company 287 (92.3) 21 (7.3) 0.086

Guard 7 (2.3) 1 (14.3)

Hunting 11 (3.5) 3 (27.3)

Miscellaneous 6 (1.9) 0 (0)

Degree of contact with other dogs

Low 58 (18.6) 5 (8.6) 0.792

Medium 173 (55.7) 15 (8.7)

High 80 (25.7) 5 (6.3)

A. Hidalgo et al. / Veterinary Microbiology 146 (2010) 356–360358

strain of ‘‘B. canis’’, Dog A2R, and one isolate with the typestrain of B. intermedia, PWS/AT.

3.3. Prevalence and risk factors for faecal shedding of

intestinal spirochaetes

A prevalence of 13.2% (41 out of 311) was obtained forfaecal shedding of Brachyspira spp. The prevalence of B.

pilosicoli shedding dogs was 4.8% (15 out of 311), while ‘‘B.

canis’’ was detected in faecal samples from 25 dogs (8.0%).A single B. intermedia isolate was recovered (0.3%).

Data and statistical analysis regarding shedding andrisk factors for B. pilosicoli and ‘‘B. canis’’ are presented inTable 2. Isolation of B. pilosicoli was more frequent in dogs 1year or younger (12.7%) than in older dogs (2.5%). Inaddition, the prevalence of B. pilosicoli shedding wassignificantly higher among animals with diarrhoea at thetime of the sampling (P< 0.001). The stratified analysis

A. Hidalgo et al. / Veterinary Microbiology 146 (2010) 356–360 359

using Chi-square for homogeneity of the odds ratios bystratum showed that the results were similar between thetwo categories defined by the age (x2 = 0.032, P = 0.858).Significant associations were identified between theshedding of B. pilosicoli and the origin of the dog(P = 0.047) and the task or work it performed (P< 0.001),but no statistical association was detected between any ofthe studied variables and ‘‘B. canis’’ colonization.

4. Discussion

Routine identification of canine Brachyspira spp.isolates has been mainly based on their phenotypicproperties and species-specific PCR results, when avail-able (Fellstrom et al., 2001; Oxberry and Hampson, 2003;Johansson et al., 2004). In the present work, thebiochemical pattern for Spanish ‘‘B. canis’’ isolates wasstable, although the B. pilosicoli isolates presented moreheterogeneous results. Similar atypical patterns for B.

pilosicoli recovered from dogs have been reportedpreviously (Fellstrom et al., 2001; Johansson et al.,2004). However, the shortened diagnostic scheme pro-posed by Johansson et al. (2004) for rapid identification ofcanine Brachyspira spp. strains using two biochemicaltests was found to be inadequate for accurately classifyingour isolates, and this required a complete description oftheir phenotypic properties.

To the authors’ knowledge, there are no availablespecies-specific PCR assays for identification of ‘‘B. canis’’.Therefore, to confirm suspicious isolates, we sequencedthe nox gene, which is suitable for discriminating betweenBrachyspira species (Rohde et al., 2002; Rasback et al.,2007; Fellstrom et al., 2008). Nox sequences of the canineBrachyspira spp. isolates were in agreement with previousclassifications based on phenotypes and species-specificPCR.

The prevalence of Brachyspira spp. faecal shedding(13.2%) reported here among Spanish dogs is slightlylower than the 18.7% of positive animals previouslyreported in dogs from Australia (Lee and Hampson,1996). In Sweden, 21 out of 32 dogs (65.6%) in a beaglecolony and 3 out of 17 pet dogs (17.6%) were colonizedby Brachyspira spp., but in both cases, animals weresuffering acute or chronic diarrhoea problems (Fellstromet al., 2001).

B. pilosicoli was recovered from 4.8% of the sampleddogs. This prevalence is similar to that reported previouslyin dogs from Papua New Guinea (5.3%) (Trott et al., 1997).Colonization with B. pilosicoli was more frequent amongpups or young dogs (1 year or less) than in older dogs.Moreover, the prevalence detected in our study amongdogs 1 year or younger (12.7%) was very similar to thatreported among pet shop puppies in Australia (14.2%)(Oxberry and Hampson, 2003). On the other hand, ‘‘B.

canis’’ was identified in 8.0% of the Spanish dogs.Interestingly, no dogs were found harbouring more thanone Brachyspira species.

To the best of our knowledge, the present study is thefirst confirmation of a statistically significant associationbetween the shedding of B. pilosicoli and the presence ofdiarrhoea in dogs. In recent reports, Fellstrom et al. (2001)

were not able to confirm a causal relationship betweendiarrhoea and isolation of spirochaetes from dogs, whilstOxberry and Hampson (2003) failed to statisticallydemonstrate this relationship due to a small sample size.No association between the shedding of ‘‘B. canis’’ and thepresence of diarrhoea was identified in our study,supporting the likelihood that this species is a commensal.Moreover, this fact further supports the idea of anassociation between B. pilosicoli colonization and diarrhoeasince it excludes the possibility of a passive shedding ofspirochaetes in dogs suffering from diarrhoea caused byother aetiologies, as previously proposed (Leach et al.,1973). However, the confirmation of the role of B. pilosicoli

as a primary or concurrent aetiological agent in dogsrequires further studies, including examining biopsies orundertaking necropsies of naturally or experimentallyinfected dogs. Although in the present study it was notpossible to obtain any colorectal biopsies to studypathological changes, B. pilosicoli attachment to themucosa of dogs, consistent with intestinal spirochaetosis,has been previously reported (Duhamel et al., 1996), andmacro- and micro-scopic changes have been observed indogs that were likely to be associated with B. pilosicoli

infection (Fellstrom et al., 2001).Two significant risk factors were found in the univariate

analysis for the shedding of B. pilosicoli. The first was theorigin, with dogs from animal shelters having a higher risk,following by those that came from breeders. A high densityof dogs, together with a lack of knowledge about control ofB. pilosicoli compared to other pathogens, could favour itsspread in these animals. The second risk factor was thetask/work of the dog, with a higher prevalence of B.

pilosicoli shedding among dogs classified as miscellaneous.However, no differences were found among company,guard and hunting dogs.

In summary, shedding of Brachyspira spp. in faeces iscommon among Spanish dogs. Although ‘‘B. canis’’ ismore prevalent, B. pilosicoli was detected in 4.8% of dogsof all ages, being associated with diarrhoea. In addition,the prevalence of B. pilosicoli shedding among dogs 1year or younger was 12.7%. Hence, dogs should beconsidered as a likely reservoir of B. pilosicoli, which mayhave practical implications in the public and animalhealth fields.

Acknowledgements

The authors express their thanks to Gloria FernandezBayon and Idoia Portillo Arias for excellent technicalassistance as well as to all the clinicians who contributedduring the sampling. Alvaro Hidalgo is supported by agrant from Consejerıa de Educacion of the Junta deCastilla y Leon and the European Social Fund. This workwas funded by the Ministerio de Educacion y Ciencia(Spanish Ministry of Education and Science) and co-financed by the European Regional Development Funds(ERDF) as Project AGL2005-01976/GAN (January 2006).We acknowledge Professor David Hampson of MurdochUniversity for assistance with English grammar andorthography.

A. Hidalgo et al. / Veterinary Microbiology 146 (2010) 356–360360

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