Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, 4 Stigbøljen, DK-1870 Frederiksberg C, Copenhagen, Denmark1
Author for correspondence: Kamille D. Petersen. Tel: +45 70260096. Fax: +45 70260097. e-mail: madsogkamille{at}mobilixnet.dk
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ABSTRACT |
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Keywords: phylogeny, ATP synthase, clonal lines, evolution, aetiology
Abbreviations: MLEE, multilocus enzyme electrophoresis
The GenBank accession numbers for the 16S rRNA sequences of strain HIM 830-7T (NCTC 10204T) and 77179 of P. multocida subsp. gallicida and HIM 746-6T (NCTC 11619T) of P. multocida subsp. septica are AF326323, AF326324 and AF326325, respectively; those for the atpD nucleotide sequences are in Fig. 3.
a Present address: Cureon A/S, Fruebjergvej 3, DK-2100 Copenhagen, Denmark.
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INTRODUCTION |
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In 1985, the genus Pasteurella Trevisan 1887 was reclassified on the basis of DNADNA hybridization studies. Eleven species were defined and P. multocida was divided into three subspecies (subsp. multocida, subsp. septica and subsp. gallicida) (Mutters et al., 1985a , b
). DNADNA hybridization experiments showed 84100%, 89100% and 91100% DNA binding within P. multocida subsp. multocida, P. multocida subsp. septica and P. multocida subsp. gallicida, respectively, whereas DNA bindings were as low as 5582% between P. multocida subspp. multocida and septica, 67% between P. multocida subspp. septica and gallicida and 77% between P. multocida subspp. gallicida and multocida. Although the DNADNA hybridization results obtained justified separation into three species, P. multocida was kept as a single species. Phenotypically, the three subspecies could be separated by differences in fermentation of sorbitol and dulcitol. P. multocida subsp. multocida and P. multocida subsp. septica are dulcitol-negative (sorbitol-positive and sorbitol-negative, respectively) and isolated from mammals and birds, while the epithet gallicida was preserved for dulcitol-positive P. multocida, which has been described as characteristic for strains isolated from avian sources (Mutters et al., 1985a
). Strains in the above-mentioned study included a limited number of avian P. multocida subsp. multocida (4/11) and P. multocida subsp. septica (1/4), just as the source of the three P. multocida subsp. gallicida strains included was not specified. In addition, only selected DNADNA hybridizations were performed. All three subspecies of P. multocida have subsequently been isolated from outbreaks of fowl cholera (Snipes et al., 1989
; Hirsh et al., 1990
; Fegan et al., 1995
) and a considerable heterogeneity seems to exist among these isolates as determined by DNA-based typing methods (Snipes et al., 1989
; Christiansen et al., 1992
; Wilson et al., 1993
, 1995
). Different genotypic methods have been used to estimate the phylogenetic relationship between bacterial taxa, including rRNADNA hybridizations, and rRNA sequence and atpD DNA sequence comparisons (Amann et al., 1988
; Ludwig et al., 1993
; Vandamme et al., 1996
). According to De Ley et al. (1990)
, strains belonging to a well-defined genus are all on the same rRNA branch. However, the rRNA branch of the type strain of P. multocida NCTC 10322T only contained two additional strains (Pasteurella species A, strain HIM 789-5, and P. multocida, strain NCTC 3195) out of five strains investigated and accepted as belonging to the genus Pasteurella sensu stricto (Mutters et al., 1985a
, b
). P. multocida subsp. gallicida, strain SSI P426T, and Pasteurella species B, strain SSI P683, were located outside the Pasteurella branch, indicating heterogeneity of the genus Pasteurella sensu stricto as well as the species P. multocida.
16S rRNA sequence comparisons have subsequently shown that members of the genus Pasteurella sensu stricto were contained in three disconnected clusters (Dewhirst et al., 1993 ), underlining that the genus Pasteurella may represent more than one genus. Dewhirst et al. (1993)
found that Pasteurella species A, Pasteurella avium, Pasteurella volantium and Pasteurella gallinarum, all bird-associated species, were included in cluster 3A. Cluster 3B contained the type strains for P. multocida subsp. multocida, Pasteurella canis, Pasteurella stomatis, Pasteurella dagmatis and Pasteurella species B. Thus all strains included in cluster 3B are characterized by having a mammalian host, except for P. multocida, which can be isolated from both birds and mammals (Bisgaard, 1993
). The rRNA sequence of P. multocida subsp. septica and P. multocida subsp. gallicida was not included in the study by Dewhirst et al. (1993)
. Cluster 3D contained Pasteurella langaaensis and Pasteurella anatis, two species also found to be associated with birds (Bisgaard, 1993
).
Since indications have been published that P. multocida might represent different species and most species of the Pasteurellaceae seem to have a restricted host reservoir, the aim of the present study was to investigate the genetic diversity of avian strains of all three subspecies of P. multocida, one of which, the dulcitol-positive P. multocida subsp. gallicida, seems mainly associated with avian hosts. The hypothesis was that P. multocida subsp. gallicida and fowl-cholera-associated P. multocida subspp. multocida and septica might represent different phylogenetic lines, some of which might show a relationship with the bird-associated rRNA sequence clusters 3A and 3D published by Dewhirst et al. (1993) . Initially the correlation between phenotypic classification of strains into subspecies and genotype clusters obtained by ribotyping was investigated. Based upon ribotyping results, strains were selected for 16S rRNA and atpD sequence comparison.
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METHODS |
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Ribotyping.
Ribotyping was carried out as previously described (Christensen et al., 1993 ) with the following modifications. Instead of 1·5 ml overnight culture, 6 ml was centrifuged and the pellet was frozen before being resuspended in TE buffer (50 mM Tris, 50 mM EDTA). HpaII (Boehringer Mannheim Biochemicals) was used to digest DNA, and before digestion each sample was adjusted to contain 17 µg DNA by spectrophotometry (Gene Quant; Pharmacia Biotech). The probe was derived from 16S and 23S Escherichia coli rRNA (Sigma) and labelled with digoxigenin. A digoxigenin-labelled lambda phage digested with HindIII and EcoRI (Boehringer Mannheim Biochemicals) served as molecular size marker. Strain 4147/S7/88 was included as a reference on every gel. Band patterns were analysed using GelCompar version 4.0 (Applied Maths, Kortrijk, Belgium). Only bands with a molecular mass of 0·8 kb or higher were included and analysed using a fine optimization and a band position tolerance of 1%. The similarity between band patterns of individual strains was estimated using the Dice coefficient, and dendrograms were derived from the similarity matrix by UPGMA.
Determination of partial 16S rRNA and atpD gene sequences.
To investigate the phylogenetic relationship between the clusters obtained by ribotyping, three strains were selected for 16S rRNA sequencing. These strains included the type strain of P. multocida subsp. septica, NCTC 11619T, and P. multocida subsp. gallicida strains NCTC 10204T and 77179 (representing ribotype cluster VI). Together with the 16S rRNA sequence of P. multocida NCTC 10322T obtained from GenBank (accession no. M35018), these four strains represented three of the six obtained ribotype clusters. atpD DNA sequence determination included the type strains of P. multocida, P. multocida subsp. gallicida strain 77179, P. canis strain NCTC 11621T and P. gallinarum strain ATCC 13361T, the last two representing 16S rRNA clusters 3B (mammalian) and 3A (avian) of Dewhirst et al. (1993) . PCR amplification was performed as described by Vogel et al. (1997)
. Oligonucleotides for both PCR amplification and rrs DNA sequence determination were synthesized according to Dewhirst et al. (1988)
and Paster & Dewhirst (1988)
. PCR-amplified fragments were purified on Microspin columns (Pharmacia Biotech) and cycle sequenced (Thermo Sequenase fluorescent labelled primer cycle sequencing kit; Amersham) on an ALF sequencer (Pharmacia Biotech) by use of fluorescein-labelled primers. Primers for atpD gene DNA sequence determination were chosen after comparison of the published genome sequence of Haemophilus influenzae strain KW20 with the unpublished sequence of P. multocida strain Pm 70 determined at the University of Minnesota and available from the TIGR database (http://www.tigr.org). Gene positions are listed according to the putative atpD sequence of H. influenzae. For PCR amplification, the primers pmkatpf3w (20 nt upstream atpD, 5'-GCT CGT CAA GCA AGT ATY AC-3') and pmkatpr3w (1356 nt of atpD, 5'-CTA AYA CTT CRT CGA TKG WAC-3') were used. For DNA sequence determination, primers pmkatpf3w, pmkatpr3w, pmkatp4wf (158 nt of atpD, 5'-CGC AAT GGG ATC WTC WG-3'), pmkatpf5 (338 nt of atpD, 5'-RAG YTA TGA AGA ACA AKC-3'), pmkatpf7 (1057 nt of atpD, 5'-TAY RAY GTT GCH CGT GGY G-3'), pmkatp4wr (1135 nt of atpD, 5'-GWA CCR TTA AAT ACT TCM GC-3') and pmkatpr5 (1054 nt of atpD, 5'-GTT CTT SAC CMA CRA C-3') were used with a fluorescein label at the 5' end.
Analysis of sequence data.
Searches for sequences in public databases were performed by FASTA and BLAST available in the Wisconsin Sequence Analysis Package (Genetics Computer Group, GCG, Madison). Sequences were aligned with PILEUP (GCG). Maximum-likelihood analysis including bootstrap analysis was performed by fastDNAml (Felsenstein, 1981 ; Olsen et al., 1994
) run on a HP9000/890 computer (UNI-C, Lyngby, Denmark). Parsimony and neighbour-joining analysis were performed by the use of PHYLIP (Phylogeny Inference Package, version 3.57c; Joseph Felsenstein, University of Washington, 1995).
GenBank accession numbers.
The accession numbers of the 16S rRNA sequences for strain HIM 830-7T (NCTC 10204T) and 77179 of P. multocida subsp. gallicida and HIM 746-6T (NCTC 11619T) of P. multocida subsp. septica are AF326323, AF326324 and AF326325, respectively. The GenBank accession numbers for the atpD nucleotide sequences determined in this investigation are listed in Fig. 3.
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RESULTS |
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Comparison of atpD gene sequences
DNA sequences were determined for the 341370 region of the putative sequence of H. influenzae strain KW20 (accession no. U32273). All sequences were of identical length with 431 amino acids translated. DNA sequence comparison showed 0·31·3% variation between P. multocida subsp. multocida and P. multocida subsp. gallicida and 2·12·2% variation between these two subspecies and P. multocida subsp. septica. The variations between P. multocida subsp. multocida and P. canis, P. gallinarum and H. influenzae were 10·2, 14·9 and 14·3%, respectively. At the amino acid level, identical sequences were observed between all three subspecies of P. multocida. The variations between P. multocida subsp. multocida and P. canis, P. gallinarum and H. influenzae were 1·6, 6·5 and 6·7%, respectively. By phylogenetic analysis of the nucleotide sequence of atpD it was possible to separate P. multocida subsp. septica from the other two subspecies, but not P. multocida subsp. multocida from P. multocida subsp. gallicida (Fig. 3). Thus the atpD sequence analysis supported the rRNA sequence analysis in that P. multocida is monophyletic and is more closely related to other mammal-associated Pasteurella species (Dewhirst cluster 3B) represented by P. canis than to bird-associated Pasteurella species (Dewhirst cluster 3A) represented by P. gallinarum.
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DISCUSSION |
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All P. multocida subsp. gallicida strains investigated were included in cluster VI, with the exception of the type strain of P. multocida subsp. gallicida and a Danish strain (24609/1) obtained from chicken (Fig. 2). This indicated that most strains of P. multocida subsp. gallicida are genetically related and that this subspecies may be more distantly related to other P. multocida isolates, including those of avian origin. Bowles et al. (2000)
ribotyped porcine P. multocida using the restriction enzymes HpaII and HindIII. Isolates of P. multocida subsp. gallicida investigated were contained in a separate ribotype cluster, confirming the results obtained in the present study. The type strain for P. multocida subsp. gallicida, NCTC 10204T, however, does not seem to be genetically representative of this subspecies. This supports the ribotyping results of Blackall et al. (1998)
, who investigated a limited number of avian P. multocida subsp. gallicida strains, some of which (Pm 130 and Pm 82) have been included in the present study. Consequently, the possible phylogenetic relationship of P. multocida subsp. gallicida represented by the present strain collection to other bird-associated Pasteurella species contained in the 16S rRNA cluster 3A of Dewhirst et al. (1993)
was investigated. Results obtained from 16S rRNA sequence comparison showed that P. multocida subsp. gallicida strain 77179, representing ribotype cluster VI, the type strain for P. multocida subsp. gallicida, NCTC 10204T, which branched deeply with cluster II, and the type strain for P. multocida subsp. multocida and genus Pasteurella sensu stricto, NCTC 10322T (cluster II), had identical 16S rRNA sequences, while the type strain of P. multocida subsp. septica only showed 98% similarity. These findings contradict the rRNADNA hybridization results previously published by De Ley et al. (1990)
. Maximum-likelihood including bootstrap analysis and parsimony and neighbour-joining analysis of sequence data showed that P. multocida, despite the ribotyping results and the heterogeneity of the host spectrum, is monophyletic (data not shown).
Comparison of atpD sequences at the nucleotide and amino acid levels has previously been used for phylogenetic studies of members of the Bacteria (Ludwig et al., 1993 ) and in a taxonomic study of Salmonella in which a genotypical resolution at the level of subspecies was found (Christensen & Olsen, 1998
). In the present study, atpD sequence analysis of members of the Pasteurellaceae was introduced to investigate whether results obtained by rRNA sequence analysis could be supported. By comparing atpD sequences of the selected P. multocida strains and Pasteurella species representing rRNA clusters 3A (P. gallinarum) and 3B (P. canis) of Dewhirst et al. (1993)
, the phylogeny of the species as determined by 16S rRNA sequence comparison was overall supported. Although P. multocida subsp. multocida and P. multocida subsp. gallicida were found to have identical 16S rRNA sequences, the atpD sequences for these two subspecies diverged by 0·31·3%. This is in agreement with the Salmonella study mentioned above where atpD sequence analysis was also found to have a higher resolution compared to rRNA sequence analysis. The separation of P. multocida subsp. septica from the type strains of the other two subspecies as demonstrated in the present study by both rRNA and atpD sequence comparison supports the previous multilocus enzyme electrophoresis (MLEE) analysis by Blackall et al. (1998)
. At the amino acid level of atpD, no differences were observed between the three subspecies of P. multocida, indicating the conserved nature of this housekeeping gene within the Pasteurellaceae. The phylogenetic analysis of the partial atpD sequence also showed that P. multocida was more closely related to P. canis than P. gallinarum or H. influenzae, confirming the 16S rRNA results (Dewhirst et al., 1993
). From the present investigation it can be concluded that the genus Pasteurella sensu stricto as defined by DNADNA hybridization needs to be redefined since atpD sequence results confirmed the existence of at least two phylogenetic clusters previously demonstrated by 16S rRNA sequence comparison. Although the three subspecies of P. multocida were found to differ at the species level (Mutters et al., 1985a
), extended DNADNA hybridization is indicated because strains may exist which will connect the existing subspecies at species level and thus further support the present findings that the species P. multocida consists of a very diverse group of bacteria.
Knowledge of the population structure for a group of bacteria is of practical importance when diagnostic characteristics for epidemiological purposes have to be defined as well as for investigating the genetic traits of pathogenesis. The genus Pasteurella and its 11 species (Mutters et al., 1985a , b
) were defined without knowing the degree of recombination within the population. The species proposed might consequently impose arbitrary divisions of a continuum (Smith, 1995
). Evidence exists that P. multocida to some extent is restricted by the host; e.g. capsular type B has mainly been associated with haemorrhagic septicaemia in cattle while capsular type A is the most important cause of fowl cholera (Carter, 1984
). Clones of P. multocida have also been shown to be restricted to a particular host and not exchanged between different animal species, even though these were kept in close contact (Muhairwa et al., 2001
). By MLEE, the population structure of avian P. multocida isolated in Australia was found to be clonal and an overall correlation between MLEE types and ribotypes has been found for P. multocida (Blackall et al., 1998
). Correlation between MLEE types and ribotypes has also been found for other groups within the family Pasteurellaceae (Angen et al., 1997
). In the present study, strains with identical ribotypes might represent clonal lines. Within clusters II, IV and VI such strains were obtained from different avian hosts, indicating that virulence for a particular bird species is not restricted to a specific genotypic line of P. multocida. Only turkey isolates from Denmark and England were contained in cluster III. Whether this reflects the existence of a turkey-associated P. multocida clone, exchange of infected birds through trade or other factors is unknown.
As demonstrated in the present investigation, the genetic diversity of organisms causing fowl cholera is considerable. Previous investigations have also shown that P. multocida associated with fowl cholera represented multiple clones (Wilson et al., 1993 , 1995
; Snipes et al., 1989
; Christiansen et al., 1992
; Blackall et al., 1998
). However, clonal outbreaks of P. multocida infections have been described in wild birds (Christensen et al., 1998
), fallow deer (Aalbaek et al., 1999
) and swine (Blackall et al., 2000
). Isolates of P. multocida from healthy flocks of poultry have also been shown to be clonal (Muhairwa et al., 2000
). These observations seem to indicate that transmission within the flock follows infection, while recombination between different clones of P. multocida seems rare during outbreaks. The extent of recombination and its possible contribution to the observed genetic variability within the P. multocida complex has not been fully elucidated. The MLEE study by Blackall et al. (1998)
investigating P. multocida fowl cholera isolates showed the index of association IA for 56 electrophoretic types to be significantly different from zero, indicating that the population was not undergoing significant horizontal gene flow. However, the population structure of the P. multocida complex remains to be further investigated.
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ACKNOWLEDGEMENTS |
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Received 9 March 2001;
revised 3 May 2001;
accepted 9 May 2001.