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1 Food Safety and Quality National Program, Agriculture and Agri-Food Canada Research Centre, 5403 1st Avenue S, Lethbridge, AB, T1J 4B1, Canada
2 Department of Biological Sciences, Faculty of Science, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada
3 Faculty of Veterinary Medicine, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada
4 Calgary Zoo Animal Health Centre, 1625 Centre Avenue E, Calgary, AB, T2E 8K2, Canada
Correspondence
G. Douglas Inglis
inglisd{at}agr.gc.ca
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ABSTRACT |
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92 % sequence similarity to recognized Campylobacter species) and genomic DNA (no detectable relatedness) analyses were consistent with novel species status. Cells of the Campylobacter from whooping cranes were uniflagellar and typically sigmoid to allantoid in shape (0.48 µm wide and 2.61 µm long), but also spheroid to coccoid (0.59 µm wide and 0.73 µm long). The bacterium was oxidase-positive, able to reduce nitrite, able to grow at 3 ° and 42 °C, and grew anaerobically, as well as in an atmosphere devoid of H2, and on MacConkey agar. It was not 
-haemolytic and was negative for hippurate and indoxyl acetate hydrolysis and alkaline phosphatase. It also was susceptible to cephalotin and was unable to grow on nutrient agar, on a medium containing 3.5 % NaCl or in ambient O2. The bacterium was unable to grow at 25 °C and growth was negative or very restricted at 30 °C. Fluorescent amplified fragment length polymorphism analysis indicated that nine of the recovered isolates were genetically distinct. A species-specific primer set targeting the cpn60 gene was developed. The name Campylobacter canadensis sp. nov. is proposed for the novel species, with the type strain L266T (=CCUG 54429T =LMG 24001T).
The GenBank/EMBL/DDBJ accession numbers for the 23S rRNA, 16S rRNA, rpoB and cpn60 gene sequences of the novel isolates determined in this study are EF621904 and EF621905 (23S rRNA), EF621894–EF621903 (16S rRNA), EF621885–EF621893 (rpoB) and EF621906–EF621915 (cpn60), as detailed in Figs 2–5; the accession numbers for strain L266T are respectively EF621904, EF621894, EF621885 and EF621906.
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Three unidentified isolates of a Campylobacter-like bacterium were recovered from cloacae of three whooping cranes in 2004 during a study to characterize the normal enteric bacterial flora of captive whooping and sandhill cranes at the Calgary Zoo (Hoar et al., 2007
). Seven additional isolates were recovered from whooping cranes in 2006. Isolations were made on a Campylobacter-selective Preston medium containing selective supplement SR117 (Oxoid), Karmali agar containing selective supplement SR167 (Oxoid) or Campylobacter charcoal-deoxycholate medium (CCDA) containing selective supplement SR155 (Oxoid) incubated anaerobically (10 % CO2, 10 % H2 and 80 % N2) or microaerobically (5 % O2, 10 % CO2, 3 % H2 and 83 % N2) at 37 or 42 °C. Cultures were examined after 2, 3 and 7 days incubation. The physiological characters of all ten isolates along with selected reference strains were determined using standard methods (e.g. On et al., 1996 and references therein); all tests were conducted at least two times. Morphological characteristics of the bacterium were determined using light and transmission electron microscopy (TEM). For TEM, cells (48 h old) were fixed in 2 % glutaraldehyde, stained with uranyl acetate and examined with a Hitachi H-7100 microscope operated at 30 kV.
Genomic DNA was extracted using the Qiagen DNeasy tissue kit. Extracted genomic DNA was subjected to genus Campylobacter- and Campylobacter species-specific PCR (Inglis & Kalischuk, 2003; Lawson et al., 2001). The 23S rRNA genes of two isolates, L266T and L268, were amplified using the basic primers and PCR conditions published by Sallen et al. (1996) with slight modifications to the primer sequences (Inglis et al., 2006). The 16S rRNA gene was amplified using the eubacterial primers UNI27F and UNI1492R (Lane, 1991), and sequences were obtained using the primers UNI27F, UNI338F, UNI1100R and UNI1492R. The rpoB and cpn60 genes (synonyms groEL and hsp60) were sequenced as described by Korczak et al. (2006) and Inglis et al. (2006), respectively. All sequences were compared directly with the NCBI non-redundant nucleotide database using BLASTN. Subsequently, the nucleotide sequences for the unidentified isolates were aligned along with data retrieved from NCBI using the multialignment program CLUSTAL W, and the alignments were refined visually using GeneDoc (Nicholas & Nicholas, 1997). Sequence data were analysed using programs contained within the phylogenetic software PHYLIP (Felsenstein, 2005). Phylogenetic estimates were obtained based on the neighbour-joining distance method. Divergence (or distance) of each pair of sequences was calculated by DNADIST using Kimura's two-parameter model. The NEIGHBOR program was used to carry out the neighbour-joining method for estimating phylogenies from the distance matrices. Support for internal branches within the resulting trees was obtained by bootstrap analysis (1000 replicates) generated by SEQBOOT, and a majority-rule consensus tree was constructed using the CONSENSE program. TreeView (Page, 2001) was used to generate rooted phylogenetic trees. Species-specific primers targeting the cpn60 gene were developed using the COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) program (http://bioinformatics.weizmann.ac.il/blocks/codehop.html). The primers developed were CraneF (5'-GAAAAAGTAGGCAAAGATGGTGTTA-3') and CraneR (5'-CAATTATTAAAAGCGGTC-3'). The predicted annealing temperature was 46.5 °C, no hairpin loops or primer dimers were predicted and the projected amplicon was 253 bases long. Each primer was subjected to BLAST analysis using the NCBI website (short but nearly exact matches). The forward primer produced identical alignments to the cpn60 genes of Campylobacter jejuni, Campylobacter coli, Helicobacter pullorum, Helicobacter pametensis and Wolinella succinogenes. In contrast, the reverse primer was predicted to be specific for the crane isolates. The PCR mixture (20 µl) consisted of 2 µl 10x buffer, 0.4 µl 10 mM dNTPs, 0.4 µl 25 mM MgCl2, 1.0 µl each of 10 µM solutions of CraneF and CraneR, 2.0 µl BSA, 0.1 µl Qiagen HotStar Taq polymerase (5 U µl–1), 11.1 µl Optima water and 2 µl DNA (20–100 ng). PCR conditions applied were 15 min at 95 °C followed by 35 cycles of 30 s at 94 °C, 60 s at 47 °C and 60 s at 72 °C, followed by 10 min at 72 °C. For DNA–DNA hybridization, the method of Mehlen et al. (2004) was employed with DNA from L266T and L284 used as the digoxigenin-labelled probes. The G+C content of reference strains was determined from Vandamme et al. (1991) and Logan et al. (2000). The clonality of the isolates was investigated by fluorescent amplified fragment length polymorphism (AFLP) analysis using the basic procedure described by Kokotovic & On (1999). Amplified DNA products were separated using POP7 polymer with an ABI 3130 Automated Genetic Analyzer (Applied Biosystems). Only AFLP profiles comprising fragments detected in the size range 80 to 500 bp were considered for numerical analysis. GENESCAN-processed data files comprising both bacterial AFLP profiles and the internal molecular mass standard (GENESCAN-500 LIZ) were imported into the program BioNumerics 4.01 (Applied Maths). After registration of strain details, profiles were normalized within and between runs by linking profiles for analysis to their respective molecular mass standard, as described in the software manufacturer's instructions. Normalized AFLP profiles were compared using the Dice coefficient and clustered by the unweighted pair group with mathematical averages (UPGMA) method.
Ten isolates of small, motile, Gram-negative bacilli were isolated from cloacal swabs of captive whooping cranes. All isolates were presumptively identified as Campylobacter based on genus-specific PCR, but were not identifiable by Campylobacter species-specific PCR. TEM revealed that cells were uniflagellate and pleomorphic, typically sigmoid to allantoid in shape but also spheroid to coccoid (Fig. 1). Sigmoid cells were 2.61±1.72 µm (mean±SD) in length and 0.48±0.09 µm in width. Spheroid cells were 0.73±0.16 µm in length and 0.59±0.09 µm in width. The following percentages of positive results for physiological characters were observed (n=10): 0 % -haemolysis, 100 % oxidase, 40 % catalase, 0 % hippurate hydrolysis, 50 % urease, 50 % nitrate reduction, 0 % alkaline phosphatase, 40 % trace H2S production, 20 % acid production on triple-sugar iron (TSI) agar, 0 % indoxyl acetate hydrolysis, 0 % growth at 25 °C (microaerobic), 100 % growth at 37 °C (microaerobic), 100 % growth at 42 °C (microaerobic), 100 % anaerobic growth at 37 °C, 0 % growth in ambient O2 at 25 or 37 °C, 0 % growth on nutrient agar, 100 % growth on CCDA, 100 % growth on MacConkey agar, 30 % growth on 1 % glycine, 10 % growth on 3.5 % NaCl, 40 % resistance to nalidixic acid, 0 % resistance to cephalotin, 0 % required H2 within the microaerobic atmosphere, 80 % 
-glutamyl transpeptidase and 100 % nitrite reduction (Table 1). The bacterium exhibited no or very restricted growth at 30 °C (microaerobically). The most useful characters that distinguished the bacterium from Arcobacter species and recognized Campylobacter taxa were negative hippurate and indoxyl acetate hydrolysis, production of -glutamyl transpeptidase, positive nitrite reduction, inability to grow in ambient 02, no growth microaerobically at 25 and 30 °C, inability to grow on nutrient agar and on 3.5 % NaCl, ability to grow anaerobically and on MacConkey agar and sensitivity to cephalotin.
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). Phylogenetic relationships were examined further using the 16S rRNA gene (approx. 1400 nt). A search of the NCBI database confirmed that the unidentified isolates were phylogenetically most closely related to Campylobacter, and pairwise sequence comparisons of L266T with the type strains of most closely related species revealed similarities of 92 % with C. jejuni and C. coli and 91 % with Campylobacter lari, C. insulaenigrae, C. helveticus and C. upsalienesis. The dissimilarity in sequence with its nearest phylogenetic neighbours was substantially greater than the 3 % divergence typically used to delineate different species based on 16S rRNA gene sequence (Stackebrandt & Ludwig, 1988). The neighbour-joining dendrogram indicated that all ten isolates from whooping cranes formed a robust clade (100 % bootstrap support) which was clearly distinct from Arcobacter and Sulfurospirillum strains and grouped with Campylobacter species (Fig. 3). However, the clade was very distinct from clades containing other Campylobacter species.
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; Kärenlampi et al., 2004; Korczak et al., 2006). Searches of the NCBI database indicated that the isolates from whooping cranes were most similar to Campylobacter species. Pairwise comparisons of sequence similarity between L266T and Campylobacter species were 81 and 93 %, respectively, for the rpoB and cpn60 genes. The unidentified isolates formed robust monophyletic groupings distinct from Campylobacter and Arcobacter species for both the rpoB (Fig. 4) and cpn60 (Fig. 5) genes. Similarly to Korczak et al. (2006), we observed good congruence between rpoB and 16S rRNA gene sequence results. In contrast, analysis of the cpn60 gene indicated that the unidentified isolates were much more closely related to Arcobacter species than was indicated by the 23S rRNA, 16S rRNA or rpoB gene sequences. The results of DNA–DNA hybridization confirmed that the unidentified isolates are genetically distinct from other Campylobacter species (Table 2); a 
Tm greater than 5.0 °C indicates two different species (Mehlen et al., 2004). Species-specific primers targeting the cpn60 gene that we developed were found to be specific for the unidentified species. As predicted, an amplicon of approximately 250 nt was observed for all ten isolates recovered from cranes but not for any reference strains of Campylobacter, Arcobacter or Helicobacter.
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) and, in addition to the genotypic uniqueness we observed, the isolates from whooping cranes also differed from these three taxa physiologically. Relative to C. lari, the crane isolates were unable to grow at 30 °C, grew anaerobically and on MacConkey and nutrient agars, were sensitive to cephalotin and were able to reduce nitrite. The inability to hydrolyse hippurate and indoxyl acetate, the inability to haemolyse blood or grow at 30 °C, the ability to grow anaerobically and on MacConkey and nutrient agars and to reduce nitrite, combined with sensitivity to cephalotin and the ability of some strains to degrade urea, distinguished the whooping crane isolates from C. jejuni and C. coli. Interestingly, a number of studies have reported unidentified hippurate-negative and urease-positive or urease-negative Campylobacter species from a variety of avian sources (e.g. Kaneko et al., 1999; Moore et al., 2002; Waldenström et al., 2002). In some of these studies, it is possible that the Campylobacter isolates in question were C. lari. However, in others, it is very possible that the unidentified isolates represented the novel taxon described in this study. The primers that we have developed to target the cpn60 gene should prove useful in this regard.
The results of the polyphasic taxonomic study conducted and described herein clearly indicate that the isolates recovered from captive whooping cranes at the Calgary Zoo represent a unique species of Campylobacter, for which we propose the name Campylobacter canadensis sp. nov. The epithet was chosen because it pertains to the restricted geography of breeding and nesting of the migratory population of whooping cranes in the wild, and the country in which the birds examined in the current study live. Physiological characters that serve to differentiate C. canadensis from Arcobacter species as well as other species within the genus Campylobacter are presented in Table 1
. These characters along with species-specific primers that we developed can be used to identify C. canadensis. It is pertinent to note that the ten isolates of C. canadensis were recovered from different individuals in 2004 and 2006. AFLP genotyping indicated that considerable genetic variability existed within the strains examined (Fig. 6). Interestingly, two isolates (L267 and L281) were determined to be identical but were isolated 2 years apart and from different individuals.
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Cells are Gram-negative, motile, polymorphic in shape (sigmoid to coccoid), 0.5–0.6 µm wide and 0.7–2.6 µm long, possessing a single polar flagellum. On Karmali agar, colonies are whitish-grey in colour with a light centre, margins are entire and the colony surfaces are typically smooth and flat. Colonies are 0.3–2.0 mm in diameter at 37 and 40 °C after 48 h. Colony appearance on CCDA, Columbia agar and trypticase soy agar is similar to that on Karmali agar, but growth on these media is slightly restricted relative to Karmali agar. Growth on Mueller–Hinton agar is very restricted. The bacterium is not -haemolytic. All strains are microaerophilic and thermophilic, able to grow well at 40 and 42 °C. Under microaerobic conditions, no isolates grow at 25 °C and no or highly restricted growth occurs at 30 °C; all isolates grow at 35 and 37 °C. All isolates also grow under anaerobic conditions. No detectable growth occurs under aerobic conditions at 37 or 40 °C. None of the isolates hydrolyse indoxyl acetate or hippurate, produce alkaline phosphatase or require H2 to grow. All isolates produce oxidase, grow on MacConkey agar and reduce nitrite. No isolates grow on nutrient agar. Some isolates reduce nitrate (50 %), produce catalase (40 %), urease (50 %) and -glutamyl transpeptidase (80 %), grow on 1 % glycine (30 %) and 3.5 % NaCl (10 %), are resistant to nalidixic acid (40 %) and produce acid (20 %) or H2S (40 %) in TSI agar. All isolates are susceptible to cephalotin. No growth occurs in the presence of 2.0 or 3.5 % (w/v) NaCl at 40 °C.
The type strain is L266T (=CCUG 54429T =LMG 24001T). This strain and other known strains were isolated from the cloacae of captive whooping cranes at the Calgary Zoo in 2004 and 2006.
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ACKNOWLEDGEMENTS |
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70 %) indicate support for the internal branches within the tree obtained by bootstrap analysis (percentages of 1000 bootstraps).
