1 Department of Veterinary Pathobiology, the Royal Danish Veterinary and Agricultural University, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark
2 Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen V, Denmark
Correspondence
Henrik Christensen
hech{at}kvl.dk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank accession numbers for the 16S rRNA gene sequences of the strains reported in this paper are AY316314, AY316315, AY316316, AY316317 and AY507110.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Significant variations in the phenotypic properties of P. multocida have been reported (Heddleston, 1976), leading to confusion in the definition and identification of this organism. Based upon DNA hybridization studies, the genus Pasteurella was reclassified by Mutters et al. (1985b)
. DNA-binding data for P. multocida identified three clusters, showing 84100 %, 91100 % and 89100 % DNA binding between strains subsequently described as P. multocida subsp. multocida, subsp. gallicida and subsp. septica, respectively. However, DNA binding as low as 55 % between subspp. multocida and septica, 67 % between subspp. septica and gallicida and 77 % between subspp. gallicida and multocida was observed (Mutters et al., 1985b
). The low levels of DNA binding observed between the three groups would have allowed separation at the species level; however, for clinical and epidemiological purposes, the species status of P. multocida was maintained (Mutters et al., 1985b
). Multilocus enzyme electrophoresis and ribotyping subsequently showed a close relation between the type strains of P. multocida subspp. multocida and gallicida, whereas subsp. septica was distantly related to these taxa (Blackall et al., 1998
). These results were confirmed by 16S rRNA sequence comparison (Boerlin et al., 2000
; Petersen et al., 2001
).
P. multocida has been separated from other members of the genus Pasteurella mainly by positive reactions for ornithine decarboxylase, indole and D-mannitol, and negative reactions for maltose and dextrin, while the subspecies of P. multocida can be separated by differences in acid production from D-sorbitol and dulcitol (Table 1). Variations in phenotypes within the subspecies of P. multocida were subsequently reported by Fegan et al. (1995)
, who assigned five biotypes to P. multocida subsp. multocida, two to P. multocida subsp. septica, and one to P. multocida subsp. gallicida, but two other biotypes could not be allocated to any of the recognized subspecies of P. multocida. The study of Blackall et al. (1998)
showed by genotyping that these biovars were indeed related to P. multocida, although they could not be related to any recognized subspecies by phenotypic criteria. The study of Petersen et al. (2001)
showed a great diversity of ribotypes among strains classified as P. multocida subsp. multocida, subsp. gallicida and subsp. septica. However, 16S rRNA and atpD gene (encoding the
subunit of ATP synthase) sequence comparisons confirmed the homogeneity of P. multocida. The study of Kuhnert et al. (2000)
also showed that variant phenotypes of P. multocida shared at least 98·5 % 16S rRNA sequence similarity with the recognized subspecies of this species.
|
Since, as stated above, routine characterization and identification of the redefined taxa of Pasteurella presently rest on a single or a few phenotypic characters, and strains aberrant for these characters have been reported (Bisgaard et al., 1991a, b
), specific genetic tools or improved phenotypic tests are urgently needed to improve diagnostic methods within this area of bacteriology, which is of importance for both the veterinary and medical professions.
PCR tests specific for the detection of P. multocida have been reviewed (Christensen et al., 2003a). P. multocida can be detected by a PCR test targeting the 23S rRNA gene (Miflin & Blackall, 2001
) or an unknown gene (Townsend et al., 1998
). Specific detection is also possible based on the psl gene combined with hybridization (Kasten et al., 1997
). Problems with the non-specificity of PCR and in situ hybridization tests for P. multocida have recently been reported for biovar 2 of P. avium and biovar 2 of P. canis (Mbuthia et al., 2001
; Miflin & Blackall, 2001
; Townsend et al., 2001
).
In the present study, genotypic methods were used to evaluate key characters employed for phenotypic characterization and identification of P. multocida. The study has focused on phenotypically variant strains of P. multocida and related taxa, mainly isolated from cases of bovine pneumonia. The study has aimed at a more strict definition of P. multocida, leading to improved identification and consequently better understanding of epidemiology and clinical implications.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The 16S23S rRNA internal transcribed spacer (ITS).
ITS was amplified for 58 strains, including reference strains, by three nested primer-sets, as recently described (Christensen et al., 2003b). Fragments generated with the three primer-sets were respectively 149, 189 and 337 bp longer than the 16S23S ITS. PCR amplification and denaturing PAGE were performed as described in Christensen et al. (1999)
. Mean lengths of ITS fragments were calculated for each strain.
PCR.
The procedure of Miflin & Blackall (2001) was followed. Briefly, a loopful of an overnight culture was taken from the surface of a blood agar plate and suspended in 200 µl sterile water. The suspension was boiled for 10 min, the cells were spun down at 13 000 g for 2 min and the supernatant was used as template for PCR. PCR was performed in a total volume of 50 µl, containing 10 mM Tris/HCl (pH 8·3), 3·0 mM MgCl2, 50 mM KCl, 200 µM of each dNTP, 25 pmol of each of the primers PM23F1 (5'-ggc tgg gaa gcc aaa tca aag-3') and PM23R2 (5'-cga ggg act aca att act gta-3'), 1·25 U Taq polymerase (Perkin-Elmer) and 1 µl lysate. DNA was amplified for 30 cycles, using the following settings: denaturation at 94 °C for 1 min, annealing at 61 °C for 1 min and extension at 72 °C for 1 min, followed by 10 °C. After electrophoresis and ethidium bromide staining, an amplicon of about 1400 bp could be visualized under UV light in positive strains.
Capsular type was determined by the PCR method of Townsend et al. (2001).
Sequencing of 16S rRNA genes.
The 16S rRNA gene sequence was determined for 11 strains: 160, 214, RA12/2, 5, A284/86, A285/86, 25, X225, K323, W208 and 16 (Table 2). Bacteria were cultured overnight in brain heart infusion broth (Difco) at 37 °C. PCR-amplified fragments were purified and cycle sequenced, as recently reported (Christensen et al., 2002
). Searches for 16S rRNA sequences were performed by FastA and BLAST in GenBank by the Wisconsin Sequence Analysis Package (GCG). Pairwise similarities were calculated by Bestfit (GCG).
DNADNA hybridization.
DNA binding was investigated by the micro-well method (Christensen et al., 2000) for 11 strains, including type strains, to represent pheno- and genotypic diversity. DNA preparations were only used if they were of high concentration (>150 µg ml1). To limit the amount of capsular material formed, cells were cultured in brain heart infusion broth with 1 mM EDDA (ethylenediamine-N,N'-diacetic acid; Sigma-Aldrich) (Ogunnariwo & Schryvers, 1990
) and 1 mg hyaluronidase ml1 (from bovine testes; Sigma-Aldrich), since the capsular material of type A is susceptible to hyaluronidase treatment (Rimler, 1994
).
GenBank accession numbers.
The 16S rRNA gene sequences of strains 5, 214, RA12/2, W208 and X225 were deposited with GenBank under the accession numbers AY316314, AY316315, AY316316, AY316317 and AY507110, respectively. The sequence of strain 5 was identical to that of strains 25, A285/86 and K323, the sequence of strain W208 was identical to that of strains 16 and A284/86 and the sequence of strain 160 was identical to that of strain RA12/2.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ribotyping
Between 6 and 11 bands were registered at 20 positions in 13 ribotypes (Fig. 1). The most conserved ribotype (RT5) was shared between 20 strains of P. avium biovar 2 and P. canis biovar 2 from Germany and Belgium. All 11 Danish isolates of P. avium biovar 2 and P. canis biovar 2 investigated also belonged to the same ribotype (RT2). Ornithine decarboxylase- and indole-negative strains of P. multocida subsp. septica belonged to RT1 and RT4. These two clusters also included strains of P. avium biovar 2.
|
Overall, the genotypic relationship demonstrated by ribotyping between atypical bovine strains previously reported as atypical P. multocida or as biovar 2 variants of P. canis and P. avium was high, compared to the reference strains included.
ITS fragment-length typing
All strains, except five (66, 248, RA12/2, 21474/76, X225), shared an ITS profile with fragments of 388 and 612 bp (Fig. 2). Strain 248 of P. canis biovar 2 possessed four fragment lengths, two of which were also shared with the majority of strains, while the other two were also found in strain 66 of P. canis biovar 2. None of these strains had a deviating phenotype. A unique fragment profile was found in strain 21474/76 of P. avium biovar 2, which deviated in indole and D-xylose. Finally, a D-mannitol-negative strain of P. multocida subsp. multocida (RA12/2) contained an extra fragment not found in the other strains. The type strain of P. canis had ITS fragments different from the other strains investigated; however, strain X225 of P. canis biovar 1 (bovine isolate) shared a profile with the majority of strains, as did the type strain of P. gallinarum. ITS profile comparison does not therefore seem to represent a reliable criterion to separate Pasteurella species.
|
Of the strains tested, 52 of 53, including the type strains for the subspecies of P. multocida (Table 2), were found to be capsular type A. Strain 77179, isolated from a chicken and representing P. multocida subsp. gallicida, belonged to capsular type F.
16S rRNA sequence comparison
Eleven strains were selected for sequencing to represent isolates identified as P. multocida subsp. multocida and P. avium biovar 2 (three strains each), P. canis biovar 2 and P. multocida subsp. septica (two strains each), and strain X225, identified as P. canis biovar 1, including phenotypically aberrant strains. This selection covered 10 out of the 13 ribotypes determined for the strains characterized. For strains 5, 16, 25, 160, 214, K323, RA12/2, A284/86, A285/86 and W208, the 16S rRNA gene sequence determined covered the positions 281491 (Escherichia coli rrnB), resulting in 1464 bp. In strain X225, the 16S rRNA gene sequence covered the positions 281459 (E. coli rrnB), resulting in 1432 bp. The sequences for strains 5, 25, K323 and A285/86 were identical to the published sequence of taxon 13 of Bisgaard (CCUG 16497, accession no. L06085). In addition, the 16S rRNA gene sequences of strains W208, 16 and A284/86 were identical, just as strains RA12/2 and 160 were identical. All strains showed 99·9 % similarity to the type strain of P. multocida subsp. multocida and 98·6 % similarity to the type strain of P. multocida subsp. septica. Surprisingly, strains 16 and W208, phenotypically identified as P. multocida subsp. septica, showed the highest similarity to P. multocida subspp. multocida and gallicida and only a more distant relationship to P. multocida subsp. septica. The highest 16S rRNA gene sequence similarity to the strains of P. multocida, biovar 2 of P. canis and biovar 2 of P. avium was found for the type strain ATCC 43327T of P. stomatis (accession no. M75050) with 97·8 % similarity to strain RA12/2 and a slightly lower similarity of 97·7 % to strains 5, 214 and W208. The highest similarity to the type strain of P. canis (biovar 1) was 97·8 % for strain RA12/2, and the highest similarity to the type strain of P. avium (biovar 1) was 93·7 % for strains 214, W208 and RA12/2.
DNADNA hybridization
Six strains were selected for DNADNA hybridization from the strains analysed by 16S rRNA sequencing to represent the taxa identified as P. avium biovar 2, P. canis biovar 2 and P. multocida subsp. septica. All investigated strains of P. multocida, biovar 2 of P. canis and biovar 2 of P. avium and mannitol-negative variants of P. multocida subsp. multocida were closely related, at 79 % DNA binding, to the type strain of P. multocida. DNA binding between the strains of biovar 2 of P. canis and biovar 2 of P. avium ranged from 81 to 92 % (Table 3, Fig. 3
). The DNA binding between the type strains of P. multocida and P. canis was only 25 %.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The strains investigated in the present study were mostly isolated from cases of bovine pneumonia, and included strains that diverged from P. multocida in key phenotypic properties. 16S rRNA sequence-based comparisons, in addition to DNADNA hybridizations, were able to confirm the genotypic relationships of the strains.
Strains selected for 16S rRNA sequencing represented isolates identified as P. multocida subsp. multocida, P. avium biovar 2, P. canis biovar 1, P. canis biovar 2 and P. multocida subsp. septica (aberrant strains included), and allowed for geographic diversity as well as representing the major ribotypes. All strains showed at least 99·9 % similarity to the type strain of P. multocida subsp. multocida, but only 98·6 % similarity to the type strain of P. multocida subsp. septica. Even the two strains phenotypically identified as P. multocida subsp. septica showed the highest similarity to P. multocida subsp. multocida. At least 98·5 % similarity has been found between strains of P. multocida subsp. multocida and P. multocida subsp. septica (Kuhnert et al., 2000; Petersen et al., 2001
). This indicates that the five strains phenotypically identified as P. multocida subsp. septica actually represented variant strains of P. multocida subsp. multocida, not only in indole and ornithine decarboxylase, used for separating species, but also in sorbitol, used for separating the two subspecies P. multocida subsp. multocida and P. multocida subsp. septica. For the eleven strains sequenced, a maximum of 97·8 % similarity was found to related species of Pasteurella such as P. stomatis and P. canis, and only 93·7 % to P. avium.
The DNADNA hybridizations documented the close genotypic relationship between the strains of P. multocida and biovar 2 variants of P. canis and P. avium. A value of 79 % DNA binding between the strains investigated is only slightly lower than the 85 % species limit outlined for Pasteurella (Mutters et al., 1989). Binding as low as 49 % was also observed. Low levels of binding between certain strains of P. multocida have previously been reported (Mutters et al., 1985b
). Strain 77149 of P. multocida subsp. gallicida (isolated from chicken) bound at 85 % to the type strain of P. multocida subsp. multocida.
According to the present genotypic characterization, the strains identified as biovar 2 variants of P. canis and P. avium all belong to P. multocida. Their classification with taxa other than P. multocida was based on a few DNADNA hybridizations (Mutters et al., 1985a, b
). A single strain of P. avium biovar 2 (K117) was found to bind at 88 % to the type strain of the species (Mutters et al., 1985a
), while the only strain of biovar 2 of P. canis investigated (K267) bound at 80 % to the type strain of the species (Mutters et al., 1985b
).
The high genotypic diversity among isolates of P. multocida investigated by ribotyping (see Fig. 1) is in accordance with previous investigations. Fussing et al. (1999)
, using HindIII for digestion of DNA, found high genotypic diversity among Danish porcine strains of P. multocida subsp. multocida. P. multocida subsp. multocida isolated from fowl, cats and dogs were separated into at least four ribotype clusters by Muhairwa et al. (2001)
, and Petersen et al. (2001)
found at least six ribotype clusters among avian isolates of P. multocida. Petersen et al. (2001)
also showed that P. multocida subsp. multocida and subsp. septica might share ribotypes. Previous investigations by Petersen et al. (1998)
showed by ribotyping that maltose-positive strains of P. multocida also exist. Unpublished data from the present investigation also showed that ribotyping represents a useful tool to separate atypical P. multocida and non-haemolytic bovine isolates of Gallibacterium anatis.
ITS fragment-length profiling showed that 90 % of strains shared a profile with the type strain of P. multocida. The presence of an identical profile for the type strains of P. multocida and P. gallinarum, however, shows that ITS profiling cannot be used on its own for species separation within Pasteurella as presently defined.
The existence of phenotypic variation in key characters of P. multocida, including ornithine decarboxylase, indole and mannitol, makes routine diagnostics based on phenotypic characters difficult and uncertain (Table 1). Using the source of isolation might improve correct allocation of variant strains of P. multocida. Investigations on the population structure and diversity of avian isolates of P. multocida from Australia showed that a range of P. multocida clones are associated with fowl cholera and that many of the Australian isolates are similar to non-Australian reference strains (Blackall et al., 1998
). The strains of biovar 2 of P. canis and P. avium investigated here (previous taxon 13 of Bisgaard; Madsen et al., 1985
) seem to be genetically related and are exclusively associated with pneumonia in calves, which aids in their final classification.
The present study showed that ornithine decarboxylase, indole, mannitol and sorbitol reactions might vary for P. multocida. In addition, maltose-positive variants have recently been found (Petersen et al., 1998). As a consequence, bovine strains negative for indole and mannitol are members of P. multocida and should no longer be classified as biovar 2 of P. canis. Bovine strains negative for ornithine decarboxylase, indole and mannitol, and without a requirement for V-factor, are members of P. multocida and should not be classified as biovar 2 of P. avium. All strains of biovar 2 of P. avium and P. canis investigated have been shown to belong to P. multocida. Consequently, the existence of P. avium biovar 2 and P. canis biovar 2 is highly questionable. Finally, the presence of sorbitol-negative variants might lead to misidentification of P. multocida subsp. septica when characterization is based on a single phenotypic character. The isolation of G. anatis from bovine lungs (Christensen et al., 2003b
; Ø. Angen, unpublished results) shows that P. multocida-like organisms might still be found that are genotypically unrelated to P. multocida. The frequency of isolation of P. multocida divergent in key species characteristics is probably higher from bovine lung than from other hosts, but the occurrence of such strains is low and phenotypic identification of P. multocida will in most cases be feasible.
Development of specific DNA tools for the detection and identification of P. multocida has previously been hampered by non-specific PCR and in situ detection of P. avium biovar 2 and P. canis biovar 2 (Mbuthia et al., 2001; Miflin & Blackall, 2001
; Townsend et al., 2001
). The data presented in the present paper fully document that these organisms should be reclassified with P. multocida, since genotypically they represent genuine P. multocida. The existence of sucrose-negative variant strains of P. multocida has also been reported (Bisgaard et al., 1991a
; Busse et al., 1997
; Capitini et al., 2002
). Genotypic characterization is required to document their relationship with P. multocida.
In conclusion, P. multocida is genotypically homogeneous, although phenotypically diverse lineages exist with respect to the ornithine decarboxylase, indole and mannitol reactions, at least in association with bovine pneumonia. A formal reclassification of the species is not possible, since only about 3 % of the strains characterized during the past three decades have been found to vary in these key species characteristics, and at least 10 % are needed to change the classification. This study confirmed earlier observations (Carter, 1973; Madsen et al., 1985
; Frank, 1989
) that bovine pneumonia is mainly caused by capsular type A of P. multocida. Future diagnostics should be based on extended phenotypic characterization, PCR tests and 16S rRNA sequence comparison, or equivalent methods, just as emphasis should be put on the animal species of isolation.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bisgaard, M., Houghton, S. B., Mutters, R. & Stenzel, A. (1991a). Reclassification of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumonic calf lungs. Vet Microbiol 26, 115126.[CrossRef][Medline]
Bisgaard, M., Abdullahi, M. Z. & Gilmour, N. J. (1991b). Further studies on the identification of Pasteurellaceae from cattle lungs. Vet Rec 128, 428429.[Medline]
Blackall, P. J., Fegan, N., Chew, G. T. I. & Hampson, D. J. (1998). Population structure and diversity of avian isolates of Pasteurella multocida from Australia. Microbiology 144, 279289.[Abstract]
Boerlin, P., Siegrist, H. H., Burnens, A. P., Kuhnert, P., Mendez, P., Prétat, G., Lienhard, R. & Nicolet, J. (2000). Molecular identification and epidemiological tracing of Pasteurella multocida meningitis in a baby. J Clin Microbiol 38, 12351237.
Busse, H.-J., Bunka, S., Hensel, A. & Lubitz, W. (1997). Discrimination of members of the family Pasteurellaceae based on polyamine patterns. Int J Syst Bacteriol 47, 698708.
Capitini, C. M., Herrero, I. A., Patel, R., Ishitani, M. B. & Boyce, T. G. (2002). Wound infection with Neisseria weaveri and a novel subspecies of Pasteurella multocida in a child who sustained a tiger bite. Clin Infect Dis 34, E7476.[CrossRef][Medline]
Carter, G. R. (1973). Pasteurella infections as sequelae to respiratory viral infections. J Am Vet Med Assoc 163, 863864.
Christensen, H. & Bisgaard, M. (2003). The genus Pasteurella. In The Prokaryotes. Edited by M. Dworkin. Release 3.14. New York: Springer. http://141.150.157.117:8080/prokWIP/index.htm.
Christensen, J. P., Olsen, J. E. & Bisgaard, M. (1993). Ribotypes of Salmonella enterica serovar Gallinarum biovars gallinarum and pullorum. Avian Pathol 22, 725738.
Christensen, H., Jørgensen, K. & Olsen, J. E. (1999). Differentiation of Campylobacter coli and C. jejuni by length and DNA sequence of the 16S23S rRNA internal spacer region. Microbiology 145, 99105.[Abstract]
Christensen, H., Angen, Ø., Olsen, J. E. & Bisgaard, M. (2000). DNADNA hybridization determined in micro-wells utilizing covalent attachment of DNA. Int J Syst Evol Microbiol 50, 10951102.[Abstract]
Christensen, H., Bisgaard, M., Angen, Ø. & Olsen, J. E. (2002). Final classification of Bisgaard taxon 9 as Actinobacillus arthritidis sp. nov., and recognition of a novel genomospecies for equine strains of Actinobacillus lignieresii. Int J Syst Evol Microbiol 52, 12391246.
Christensen, H., Bisgaard, M., Larsen, J. & Olsen, J. E. (2003a). PCR-detection of Haemophilus paragallinarum, Haemophilus somnus, Mannheimia (Pasteurella) haemolytica, Mannheimia spp., Pasteurella trehalosi, and Pasteurella multocida. In Methods in Molecular Biology, vol. 216, PCR Detection of Microbial Pathogens: Methods and Protocols, pp. 257274. Edited by K. Sachse & J. Frey. Totowa: Humana Press.
Christensen, H., Bisgaard, M., Bojesen, A. M., Mutters, R. & Olsen, J. E. (2003b). Genetic relationships among strains of biovars of avian isolates classified as Pasteurella haemolytica, Actinobacillus salpingitidis or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., comb. nov. and description of additional genomospecies within Gallibacterium gen. nov. Int J Syst Evol Microbiol 53, 275287.
Fegan, N., Blackall, P. J. & Pahoff, J. L. (1995). Phenotypic characterization of Pasteurella multocida isolates from Australian poultry. Vet Microbiol 47, 281286.[CrossRef][Medline]
Frank, G. H. (1989). Pasteurellosis of cattle. In Pasteurella and Pasteurellosis, pp. 197222. Edited by C. Adlam & J. M. Rutter. London: Academic Press.
Fussing, V., Nielsen, J. P., Bisgaard, M. & Meyling, A. (1999). Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark. Vet Microbiol 65, 6174.[CrossRef][Medline]
Heddleston, B. S. (1976). Physiologic characteristics of 1,268 cultures of Pasteurella multocida. Am J Vet Res 37, 745747.[Medline]
Kasten, R. W., Carpenter, T. E., Snipes, K. P. & Hirsh, D. C. (1997). Detection of Pasteurella multocida-specific DNA in turkey flocks by use of the polymerase chain reaction. Avian Dis 41, 676682.[Medline]
Krieg, N. R. & Garrity, G. M. (2001). On using the Manual. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 1519. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springer.
Kuhnert, P., Boerlin, P., Emler, S., Krawinkler, M. & Frey, J. (2000). Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing. Int J Med Microbiol 290, 599604.[Medline]
Madsen, E. B., Bisgaard, M., Mutters, R. & Pedersen, K. B. (1985). Characterization of Pasteurella species isolated from lungs of calves with pneumonia. Can J Comp Med 49, 6367.[Medline]
Mbuthia, P. G., Christensen, H., Boye, M., Petersen, K. M. D., Bisgaard, M., Nyaga, P. N. & Olsen, J. E. (2001). Specific detection of Pasteurella multocida in chickens with fowl cholera and in pig lung tissues using fluorescent rRNA in situ hybridization. J Clin Microbiol 39, 26272633.
Miflin, J. K. & Blackall, P. J. (2001). Development of a 23S rRNA-based PCR assay for the identification of Pasteurella multocida. Lett Appl Microbiol 33, 216221.[CrossRef][Medline]
Muhairwa, A. P., Mtambo, M. M. A., Christensen, J. P. & Bisgaard, M. (2001). Occurrence of Pasteurella multocida and related species in village free ranging chickens and their animal contacts in Tanzania. Vet Microbiol 78, 139153.[CrossRef][Medline]
Mutters, R., Piechulla, K., Hinz, K.-H. & Mannheim, W. (1985a). Pasteurella avium (Hinz and Kunjara 1977) comb. nov. and Pasteurella volantium sp. nov. Int J Syst Bact 35, 59.
Mutters, R., Ihm, P., Pohl, S., Frederiksen, W. & Mannheim, W. (1985b). Reclassification of the genus Pasteurella Trevisan 1887 on the basis of DNA homology with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis, and Pasteurella langaa. Int J Syst Bacteriol 35, 309322.
Mutters, R., Mannheim, W. & Bisgaard, M. (1989). Taxonomy of the Group. In Pasteurella and Pasteurellosis, pp. 334. Edited by C. Adlam & J. M. Rutter. London: Academic Press.
Ogunnariwo, J. A. & Schryvers, A. B. (1990). Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor. Infect Immun 58, 20912097.[Medline]
Petersen, K. D., Christensen, J. P. & Bisgaard, M. (1998). Phenotypic and genotypic diversity of organisms previously classified as maltose positive Pasteurella multocida. Zentralbl Bakteriol 288, 112.[Medline]
Petersen, K. D., Christensen, H., Bisgaard, M. & Olsen, J. E. (2001). Genetic diversity of Pasteurella multocida isolated from fowl cholera as demonstrated by ribotyping, 16S rRNA and partial atpD sequence comparisons. Microbiology 147, 27392748.
Rimler, R. B. (1994). Presumptive identification of Pasteurella multocida serogroups A, D, and F by capsule depolymerisation with mucopolysaccharidases. Vet Res 134, 191192.
Schmid, H., Hartung, M. & Hellmann, E. (1991). Crossed immunoelectrophoresis applied to representative strains from 11 different Pasteurella species under taxonomic aspects. Zentralbl Bakteriol 275, 1627.[Medline]
Stackebrandt, E., Frederiksen, W., Garrity, G. M. & 10 other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 10431047.
Townsend, K. M., Frost, A. J., Lee, C. W., Papadimitriou, J. M. & Dawkins, H. J. S. (1998). Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates. J Clin Microbiol 36, 10961100.
Townsend, K. M., Boyce, J. D., Chung, J. Y., Frost, A. J. & Adler, B. (2001). Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system. J Clin Microbiol 39, 924929.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc commitee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463464.
Received 19 August 2003;
revised 9 January 2004;
accepted 9 February 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |