HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Johansson, K.-E. Articles by Fellström, C. Search for Related Content PubMed PubMed Citation Articles by Johansson, K.-E. Articles by Fellström, C. Agricola Articles by Johansson, K.-E. Articles by Fellström, C.

Identification of three clusters of canine intestinal spirochaetes by biochemical and 16S rDNA sequence analysis

¹National Veterinary Institute, Department of Bacteriology, SE-75189 and Department of Veterinary Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden ²Department of Veterinary and Biochemical Sciences, University of Nebraska-Lincoln, USA ³Department of Bacteriology, National Veterinary Institute, Oslo, Norway ⁴Department of Disease Control and Biosecurity, National Veterinary Institute, Uppsala, Sweden ⁵Department of Biochemistry and Biotechnology, The Royal Institute of Technology, Stockholm, Sweden ⁶Department of Large Animal Clinical Sciences, Swedish University of Agricultural Sciences, Box 7018, SE-75007, Uppsala, Sweden

Correspondence Claes Fellström claes.fellstrom{at}kirmed.slu.se

Received September 29, 2003
Accepted November 25, 2003

It has been suggested that canine intestinal spirochaetes consist of Brachyspira pilosicoli and a group of strains that has been provisionally designated ‘Brachyspira canis'. The purpose of the present study was to compare 22 spirochaete isolates that were obtained from intestinal specimens of dogs in Sweden (n = 12), Norway (n = 4), the United States (n = 3), Australia (n = 2) and Germany (n = 1) with type and reference strains, as well as field isolates, of Brachyspira species by five biochemical tests and determination of almost-complete 16S rDNA sequences. In an evolutionary tree derived from 16S rDNA sequences, the canine isolates grouped into three clusters. One cluster included the type strain of porcine B. pilosicoli, whereas a second larger cluster, which was monophyletic, contained a canine strain that was identified previously as ‘B. canis'. The third cluster consisted of three canine isolates of Scandinavian origin, which grouped together with the type strain of the species Brachyspira alvinipulli (pathogenic to chicken). These three genotypes, which were identified on the basis of 16S rDNA sequences, corresponded to four phenotypic groups based on biochemical testing. Two biochemical tests, hippurate hydrolysis and -galactosidase production, were sufficient for rapid identification of each canine cluster.

The GenBank/EMBL/DDBJ accession numbers for the new 16S rDNA sequences determined in this study are AY349934–AY349949.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION Conclusions ACKNOWLEDGEMENTS REFERENCES

 
The genus Brachyspira (formerly Serpulina) consists of spirochaete bacteria that are found in the hindgut of humans and animals. The complement of Brachyspira spp. present in the hindgut of any host is best characterized for the pig. Pathogenic Brachyspira spp. of pigs consist of Brachyspira hyodysenteriae (Ochiai et al., 1997), the cause of swine dysentery (Harris & Glock, 1981), and Brachyspira pilosicoli (Trott et al., 1996a), the cause of porcine colonic or intestinal spirochaetosis (Duhamel, 1997, 2001). Additionally, there are currently three, presumably non-pathogenic, Brachyspira species that are present in pigs, namely Brachyspira intermedia (Stanton et al., 1997), Brachyspira innocens (Kinyon & Harris, 1979) and Brachyspira murdochii (Stanton et al., 1997). These and other species of the genus Brachyspira have also been found in hosts other than pigs, including dogs, humans, non-human primates and birds (Duhamel, 1997, 2001).

It has been suggested that the complement of canine intestinal spirochaetes consists of B. pilosicoli and a novel species that has been provisionally designated ‘Brachyspira canis’ (Duhamel et al., 1998; Oxberry & Hampson, 2003). As intestinal spirochaetes are found in both healthy and diarrhoeic dogs (Pindak et al., 1965; Turek & Meyer, 1978; Meier et al., 1982; Weber & Schramm, 1989; Lee & Hampson, 1994; Duhamel et al., 1995, 1996, 1998; Lee & Hampson, 1996; Fellström et al., 2001; Oxberry & Hampson, 2003), their aetiological role in disease is still uncertain. However, correlations between clinical presentation and presence of each Brachyspira species suggest that B. pilosicoli is pathogenic, whereas ‘B. canis’ may be non-pathogenic (Oxberry & Hampson, 2003). Diarrhoeal disease with B. pilosicoli appears as either a severe infection in puppies or when intestinal function is compromised by a concurrent disease problem. As human infection with B. pilosicoli is similar to the disease in animals, canine colonic spirochaetosis may be of public health significance (Lee & Hampson, 1994; Trott et al., 1996b).

Rapid identification of canine intestinal spirochaetes has been accomplished by determination of a few biochemical properties (Fellström et al., 2001) and by PCR based on 16S rDNA sequences (Duhamel et al., 1998; Oxberry & Hampson, 2003). Additional methods that have been used for identification of intestinal spirochaetes from other hosts include phylogenetic analysis based on 16S rDNA sequences (Pettersson et al., 1996), multilocus enzyme electrophoresis (Lee et al., 1993; Duhamel et al., 1998; Oxberry & Hampson, 2003), PFGE (Fellström et al., 1999, 2001, 2003) and random amplification of polymorphic DNA (Duhamel et al., 1995; Fellström et al., 2003). The purpose of the present study was to compare spirochaetes that were isolated from intestinal specimens of dogs in Europe, North America and Australia with type and reference strains, as well as field isolates, of Brachyspira species by five biochemical tests and determination of almost-complete 16S rDNA sequences.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION Conclusions ACKNOWLEDGEMENTS REFERENCES

 
Twenty-two canine isolates of spirochaetes that originated from intestinal specimens obtained from dogs in Australia, Germany, Norway, Sweden and the United States were classified biochemically as described previously (Fellström et al., 1999). Type and reference strains of each Brachyspira species and six previously described porcine field isolates were also included in the study. Designation, biochemical group, 16S rDNA sequence accession numbers (GenBank) and origin of spirochaetes investigated in the present study are given in Tables 1 and 2.

16S rDNA sequences were determined as described previously (Pettersson et al., 1996) with some modifications. The 16S rRNA gene was first amplified with the PCR primers listed in Table 3. The sequencing system (Pettersson et al., 1996) was adapted to an ABI Prism 3100 genetic analyser (Applied Biosystems). Labelled terminators (BigDye; Applied Biosystems) were used in cycle sequencing reactions. Contigs were created by using the ContigExpress program, included in the Vector NTI Suite (InforMax). 16S rDNA sequences were aligned manually to a prealigned set of Brachyspira sequences that were retrieved from the Ribosomal Database Project (Maidak et al., 2001) by using the GDE software (Smith, 1992). A phylogenetic tree was computed by using algorithms implemented in the PHYLIP package (Felsenstein, 1993).

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION Conclusions ACKNOWLEDGEMENTS REFERENCES

 
Biochemical tests

Although the canine spirochaetes were weakly ß-haemolytic, diverse phenotypes were identified on the basis of their biochemical reaction patterns (Table 2). Overall, the spirochaetes had biochemical reaction patterns that were similar to those of group IIIa, B. murdochii, and group IV, B. pilosicoli (Duhamel et al., 1998; Fellström et al., 2001; Oxberry & Hampson, 2003). However, isolate AN 1788/01, which was obtained from a dog in Sweden, and isolate CN 2, obtained from a dog in Norway, had the biochemical characteristics of group IIIbc, which includes porcine B. innocens. Another isolate, AN 4578/01, shared the specific biochemical reaction pattern of Brachyspira alvinipulli, i.e. it was hippurate-positive, -galactosidase-negative and ß-glucosidase-positive (Stanton et al., 1998). Additionally, isolates H98-5 and A3077, obtained from dogs in the United States and Germany, respectively, had the characteristics of group IV, B. pilosicoli, but gave positive reactions for indole production, a finding that has not been reported in previous studies of porcine B. pilosicoli (Fellström et al., 1999). However, previous reports by Weber & Schramm (1989) and Murray et al. (2003) have indicated the existence of indole-positive spirochaetes among canine intestinal isolates.

Recently, Murray et al. (2003) identified diverse phenotypes by testing indole, hippurate, -glucosidase and ß-glucosidase production among 112 weakly ß-haemolytic spirochaetes that were isolated from dogs in Scotland. However, correlation of biochemical assays with 16S rDNA sequence analysis in the present study suggests that neither indole production nor the presence of - or ß-glucosidase activity is sufficiently discriminatory for typing canine intestinal spirochaetes. Interestingly, in this study, hippurate hydrolysis and -galactosidase production appeared to be sufficient for the classification of canine intestinal spirochaetes into four biochemical groups (Table 4).

Phylogenetic analysis

In an evolutionary tree derived from 16S rDNA sequence analysis, the canine spirochaetes clustered into three major groups (Fig. 1). One group, which comprised six canine isolates that were representative of Europe, North America and Australia, clustered with the type strain of B. pilosicoli, porcine strain P43/6/78^T. Thirteen of the 22 isolates examined in the present study formed a second monophyletic cluster (with sequence similarity values between 99.8 and 100 %) that grouped with isolate Dog A2, which was obtained from a dog in Australia and was identified previously as ‘B. canis’ (Duhamel et al., 1998; Fig. 1). These values were higher than the sequence similarity values of the four porcine strains included in the B. innocens–B. murdochii cluster (99.4–99.7 %). The ‘B. canis’ cluster appeared to be homogeneous, in spite of the fact that these isolates originated from dogs on three distant continents. A third group consisted of three canine isolates of Scandinavian origin and the type strain of the pathogenic species B. alvinipulli from chicken. This group consisted of two distinct sublineages, each of which comprised two strains.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Evolutionary tree based on 16S rDNA sequence analysis, showing phylogenetic relationships between spirochaetes of the genus Brachyspira. Taxa included in the tree were isolated from intestinal specimens of dogs throughout the world or represented type and reference strains, as well as field isolates, of Brachyspira species. The tree was constructed by the neighbour-joining method (Saitou & Nei, 1987) from a distance matrix that comprised 1393 nucleotide positions, which was corrected for multiple substitutions at single locations by the two-parameter method of Kimura (1980). B. aalborgi was used as the outgroup. Solid vertical bars indicate phylogenetic clades and dashed vertical bars indicate strains of canine origin. Bootstrap values for 1000 replicates are given for some stable nodes. Biochemical groups are given in bold type, if defined. Bar, distance equivalent to 1 nucleotide substitution per 100 positions.

All canine B. pilosicoli strains had the hexa(U) loop (positions 208–211 in Escherichia coli), which has been shown to be unique for B. pilosicoli (Pettersson et al., 1996). However, canine B. pilosicoli strains appeared to be more heterogeneous in their 16S rRNA gene sequences than porcine strains (C. Fellström and K.-E. Johansson, unpublished data). The three B. alvinipulli-like canine strains had a –GCGA–tetraloop in this region, which they shared with at least reference strain 56-150^T of B. murdochii, whereas B. alvinipulli strain C1^T had a –GUGA–tetraloop. All 13 ‘B. canis’ strains had a –GUAA–tetraloop in this region, which they shared with some group II and some group III brachyspiras. At positions 224 and 590, canine B. pilosicoli strains possessed a C and a U, respectively, which are characteristic for B. pilosicoli in other hosts. Other Brachyspira strains have a U and a C, respectively, at these positions, except for Brachyspira aalborgi, which has an A at position 590. Another interesting observation was of an insertion of an A, found in ‘B. canis’ strain AN 5304/01 between positions 183 and 184. Insertions or deletions are not common in the 16S rRNA gene of Brachyspira strains, except for the above hexa(U) loop.

Nine variable nucleotide positions were found among the 16S rRNA genes of the 13 ‘B. canis’ isolates, but each of these variations was caused by a mutation in individual sequences. Two positions that were unique among members of the genus Brachyspira were found in the 16S rRNA gene sequences of the ‘B. canis’ isolates. Position 193 : 4, which indicates the fourth nucleotide after the nucleotide homologous to position 193 of E. coli, had a G in ‘B. canis’ isolates, whereas all other Brachyspira strains had a U. Similarly, there was a G at position 615 in ‘B. canis', whereas all other Brachyspira strains had an A at the same position.

Sequence similarities among the four isolates that clustered with B. alvinipulli from chicken varied between 98.8 and 99.6 %, with 99.6 % sequence similarity between the two isolates that comprised the two subclusters, designated C1 and CN2 (Fig. 1). The two subclusters could be distinguished on the basis of six positions; four of these each comprised a nucleotide that appeared to be unique for either the C1 or the CN2 cluster among members of the genus Brachyspira. On the basis of relatively few sequence differences that have accounted for classification of the genus Brachyspira into different species in previous studies, cluster CN2 might represent a novel species. Topologies of phylogenetic trees with Brachyspira spp. result from relatively few nucleotide substitutions; therefore, nodes are unstable and inclusion of additional isolates may change the topology of the tree, including the relationship of subclusters such as C1 and CN2. Thus, definitive classification of CN2 as a novel species will require biochemical and phylogenetic characterization of other CN2-related strains.

	Conclusions

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION Conclusions ACKNOWLEDGEMENTS REFERENCES

 
The results of the present study confirm that B. pilosicoli and a previously proposed species, designated ‘B. canis', are common among spirochaetes that are isolated from intestinal specimens of dogs worldwide. The identification of a canine isolate of Scandinavian origin with phenotypic and genotypic characteristics that are similar to those of the type strain of B. alvinipulli, a spirochaete that is pathogenic for chickens (Stanton et al., 1998), is important. To the best of our knowledge, only two isolates of this species, designated C1 and C2, have been isolated from chicken and partially characterized (Swayne et al., 1995). Isolate AN 4578/01 was obtained from a healthy dog living in a dog yard. Although the possibility that this dog was exposed to materials that were contaminated by birds cannot be ruled out, the presence of spirochaetes that are pathogenic for birds in dogs requires further confirmation.

The combined results of -galactosidase and hippurate hydrolysis assays corresponded to three clusters and two subclusters that were identified on the basis of 16S rDNA sequence analysis. However, caution should be exercised when using the proposed biochemical scheme, as few isolates were represented in two of the four biochemical groups identified. Also, as porcine strains of B. innocens and B. murdochii have phenotypic characteristics that are similar to those of groups IIIbc and IIIa, respectively, it is recommended that the proposed diagnostic scheme should be restricted to the identification of canine Brachyspira strains.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION Conclusions ACKNOWLEDGEMENTS REFERENCES

 
The authors thank Staffan Tamm for valuable help with the Linux computer system that was used for generating the phylogenetic trees. This project was supported by the Swedish Farmers’ Foundation for Agricultural Research, Formas and the Intervet Research Foundation. This article is paper no. 14262 of the University of Nebraska Agricultural Research Division, Lincoln, NE, USA.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION Conclusions ACKNOWLEDGEMENTS REFERENCES