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1 Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark
2 Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Philipps-Universität, Marburg, Germany
Correspondence
Henrik Christensen
hech{at}kvl.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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is transferred to the new genus as Gallibacterium anatis gen. nov., comb. nov. Genomospecies 1 of Gallibacterium is proposed to include the former biovars 5 and 8 of the avian [P. haemolytica]–‘A. salpingitidis’ complex. The type strain of Gallibacterium anatis is F 149T (=ATCC 43329T=NCTC 11413T) and the reference strain of Gallibacterium genomospecies 1 is CCM 5974.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences determined in this investigation are AF228001–AF228018, AY038519 and AY038592–AY038594, as listed in Table 1
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A DNA–DNA binding matrix for the strains included in this paper is available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org/).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Similar organisms were later classified as ‘Pasteurella salpingitidis’ by Kohlert (1968). Mráz et al. (1976) suggested that these organisms should be reclassified as ‘Actinobacillus salpingitidis’ on the basis of their phenotypic features. Several papers have subsequently been published on avian isolates identified as either [Pasteurella haemolytica] or ‘A. salpingitidis’ (Asnani & Pathak, 1975; Bisgaard, 1977; Bisgaard & Dam, 1981; Édes et al., 1994; Greenham & Hill, 1962; Hacking & Pettit, 1974; Harbourne, 1962; Harry, 1962; Hinz, 1969; Janetschke & Risk, 1970; Matthes et al., 1969; Mráz et al., 1976; Nicolet & Fey, 1965; Shaw et al., 1990). These organisms seem to constitute part of the normal upper-respiratory-tract flora of chickens (Bisgaard, 1977), but are also associated with pathological lesions, including salpingitis with or without peritonitis, septicaemia, pericarditis, hepatitis, respiratory tract lesions and enteritis. In addition to chickens, these organisms have also been isolated from turkeys, web-footed birds, pigeons, pheasants, partridges and psittacine birds (Bisgaard, 1993).
Bisgaard (1982) reported similar phenotypic characters for ‘A. salpingitidis’ and avian P. haemolytica-like strains. Strains tentatively designated taxon 1 by Bisgaard (1982) and subsequently classified as Pasteurella anatis by Mutters et al. (1985) were also reported to be phenotypically related to ‘A. salpingitidis’ and avian P. haemolytica-like organisms. In contrast to P. anatis, however, the avian [P. haemolytica]–‘A. salpingitidis’ complex was found to show major variations in many of the characters examined. DNA–DNA hybridizations performed by Piechulla et al. (1985) showed that groups of organisms named avian P. haemolytica-like and ‘A. salpingitidis’ seem to form a common DNA relatedness cluster that was linked to the genus Actinobacillus by 48 % DNA relatedness. DNA binding at the genus level was, however, not observed between taxon 1 of Bisgaard (P. anatis) and ‘A. salpingitidis’ and P. haemolytica-like organisms. Subsequent DNA–rRNA hybridizations confirmed that these organisms belong to the family Pasteurellaceae Pohl 1981, but they did not cluster with any of the seven rRNA branches outlined (De Ley et al., 1990).
According to 16S rRNA gene sequence analysis, the proposed type strain of ‘A. salpingitidis’ clustered with P. anatis. Although deeply branching, these organisms formed a monophyletic group (cluster 3D) with Bisgaard taxa 2 and 3 and Pasteurella langaaensis (Dewhirst et al., 1993) confirming previous studies, which showed that Bisgaard taxa 2 and 3 are only distantly related to the avian [P. haemolytica]–‘A. salpingitidis’ complex (Bisgaard et al., 1993). In addition, [P. haemolytica] from ruminants, pigs, rabbits and hares has recently been shown to represent a new genus, Mannheimia, containing several species (Angen et al., 1999).
The present uncertain taxonomic situation of the avian P. haemolytica–‘A. salpingitidis’-like organisms is undesirable, as it makes interpretation of studies on these organisms difficult and it may hinder progress in studies on epidemiology and pathogenesis. In the present investigation, selected strains were initially characterized by PFGE, AFLP, internal transcribed spacer (ITS) and plasmid profiling to obtain more information about their genotypic relationships. Subsequently, strains representing existing biovars of the avian [P. haemolytica]–‘A. salpingitidis’ complex and selected strains of P. anatis were characterized by sequencing of 16S rRNA genes and by DNA–DNA hybridization. Based on the results obtained, a proposal for a revised taxonomy is presented.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Piechulla et al., 1985). Based upon differences in acid production from (+)L-arabinose, (+)D-xylose, m-inositol, (-)D-sorbitol, maltose, trehalose and dextrin, a total of 24 different biovars has been recorded. However, only strains representing 15 biovars have been kept or survived and were consequently available for the present investigation. In addition to strains of avian origin, strains of bovine origin tentatively classified as P. anatis were included (Tables 1 and 2).
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with digestion of DNA of 19 strains by SalI or XbaI (Table 1).
Plasmid analysis.
Analysis of presence and size-distribution of plasmids was performed with 25 strains according to Kado & Liu (1981) and Sorensen et al. (1991) (Table 1).
AFLP.
With a few modifications, DNA extraction and AFLP analysis were performed as described recently (Kokotovic et al., 2000). Briefly, DNA was extracted from cell pellets of 19 strains (Table 1) grown in heart infusion broth (Difco). DNA solutions were digested simultaneously with BglII and BspDI and ligation to adaptors was performed before amplification of the modified restriction fragments. The non-selective BglII primer was labelled at the 5'-end with 6-carboxyfluorescein and the non-selective BspDI primer was unlabelled. Amplification products were detected on an ABI 377 automated sequencer (PE Biosystems). Fragment size determination and pattern analysis were done by using GeneScan 3.1 fragment analysis software (Applied Biosystems). Only AFLP profiles comprising fragments in the size range 50–500 bp were considered for numerical analysis. Numerical analysis was performed on data files comprising both bacterial AFLP profiles and internal size standards by the use of the program Bionumerics 2.0 (Applied Maths). Normalized AFLP fingerprints were compared using the Dice similarity coefficient and clustering analysis was performed by UPGMA. All isolates were analysed by carrying out the entire procedure at least twice.
Sequencing of 16S rRNA genes.
Bacteria were cultured overnight in brain/heart infusion broth (Difco). DNA was extracted as described previously (Leisner et al., 1999). PCR amplification was performed as described by Vogel et al. (1997). Oligonucleotides for both PCR amplification and sequencing were synthesized according to sequences and 16S rRNA positions given by Dewhirst et al. (1989) and Paster & Dewhirst (1988). In addition, the primer 26r (5'-CAGGGCATCCACCGT) located on the 23S rRNA gene was used for PCR amplification just as the primers 37f (5'-GGCTCAGRWYGAACGC) and 1540r (5'-GGAGGTGWTCCARCCGC), both complementary to the 16S rRNA gene sequence, were used for sequencing. PCR-amplified fragments were purified on Microspin columns (Amersham Pharmacia Biotech) and cycle-sequenced using the Thermo Sequenase fluorescent-labelled primer cycle-sequencing kit (Amersham Pharmacia Biotech) on an ALF Sequencer (Pharmacia Biotech) using fluorescein-labelled primers.
Analysis of sequence data.
Searches for 16S rRNA sequences in public databases were performed by FASTA and BLAST (Wisconsin sequence analysis package, Genetics Computer Group). Sequences were aligned manually to the Escherichia coli rrnB DNA sequence and to the consensus sequence (Lane, 1991). Maximum-likelihood analysis including bootstrap analysis was performed by fast DNAml (Felsenstein, 1981, 1995; Olsen et al., 1994). Parsimony and neighbour-joining phylograms were computed by PHYLIP (Felsenstein, 1995). The phylogenetic analysis was performed with 16S rRNA sequences of the avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex, Bisgaard taxa 2 and 3 and Pasteurella gallinarum with 1457 nt, of which 1338 could be analysed after removal of ambiguous positions.
ITS analysis.
The 16S–23S rRNA ITS sequence was amplified by three nested primer sets with four strains. The primer for reverse amplification was located on the rrl gene with similarity to the E. coli rrnB positions 115–130 (23S–130, 5'-GGGTTTCCCCATTCGG) (Christensen et al., 2000b). The primers for forward amplification of the ITS region were located on the rrs gene at E. coli rrnB positions 1525–1541 (16S–1525, 5'-GGTTGGATCACCTCCTT) (Christensen et al., 2000b), 1485–1504 (16S1, 5'-TGGGGTGAAGTCGTAACAAG; Privitera et al., 1998) and 1337–1353 (1337f, 5'-GGAATCGCTAGTAATCG; Fussing et al., 1998). Fragments generated with the three primer sets are 149, 189, 337 bp longer than the 16S–23S ITS. PCR amplification and denaturing PAGE were performed as described in Christensen et al. (1999). Overhangs with the 16S and 23S rRNA genes were subtracted from fragment lengths and peak intensities were represented as relative values. Mean lengths of ITS fragments were calculated for each strain.
DNA–DNA hybridization.
DNA–DNA hybridizations were performed by the spectrophotometric and micro-well methods according to Mutters et al. (1985) and Christensen et al. (2000a), respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) representing 15 biovars of the avian [P. haemolytica]–‘A. salpingitidis’ complex and P. anatis were distantly related, since no common fragments for all strains were present after digestion with either SalI or XbaI (data not shown). Plasmids were not found in nine strains, while 16 strains contained plasmids of different sizes varying between 1·9 and 14·5 kb (Table 3). A total of 21 different plasmid sizes was demonstrated. Single isolates contained from one to four different plasmids (Table 3). The diversity in plasmid profiles confirmed that the strains were genotypically different.
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). All strains were considered genotypically different, as less than 88 % similarity was observed (Fig. 1), confirming results obtained by PFGE and plasmid profiling. AFLP confirmed the results obtained by 16S rRNA phylogenetic analysis and DNA–DNA hybridization (see below), with a deep cluster of four strains including the former proposed type strain of ‘A. salpingitidis’ (72 % similarity) and a main cluster of 15 strains including the type strain of P. anatis (54 % similarity) (Fig. 1).
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The database search showed only three published sequences for which the similarity to sequences of the avian [P. haemolytica]–‘A. salpingitidis’ complex was higher than 95 %. These included P. anatis ATCC 43329T (accession no. M75054), ‘A. salpingitidis’ CCUG 23139 (L06077) and the 16S rRNA sequence of strain CCUG 28018 (=F 114) (AF224310), classified as ‘A. cf. salpingitidis’, which showed 99·9 % similarity to strain 10672/9Salp. Differences were due to inter-operon polymorphisms or sequencing errors, since both strains represent the same isolate, 10672/9Salp. The greatest similarities to representatives outside the group were 94·6, 94·5 and 93·9 %, between strain IPDH 697/78 and the 16S rRNA sequences of Bisgaard taxon 3 strain CCUG 15563 (L06079), Pasteurella avium NCTC 11297T (M75058) and Bisgaard taxon 2 strain CCUG 15564 (L06078). The sequence of the previously suggested type strain of ‘A. salpingitidis’, CCM 5974, diverged by five positions from the published sequence for this strain (L06077). For P. anatis, the published sequence of the type strain ATCC 43329T (M75054) diverged by three bases. These differences were assumed to be related to differences between rrs operons and sequencing errors, and only sequences of the present study were included for phylogenetic analysis. The similarity between the type strain and strain F 279 of P. anatis was 99·7 %. The 16S rRNA sequences of the bovine strains Hom.3, B 96/20 and B 96/24, classified with P. anatis, showed high similarity to the type strain of P. anatis (98·1–99·7 %), underlining that these organisms might exist in bovine as well as avian hosts.
The phylogenetic analysis based on the sequences of the avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex and published sequences for ‘A. salpingitidis’, P. anatis and Bisgaard taxa 2 and 3 is shown on Fig. 2, with P. gallinarum as an outgroup. P. anatis and the avian [P. haemolytica]–‘A. salpingitidis’ complex formed a monophyletic unit related to Bisgaard taxa 2 and 3. The avian [P. haemolytica]–‘A. salpingitidis’ complex, however, formed two monophyletic groups. Group 1 included the previously suggested type strain of ‘A. salpingitidis’ and four additional strains (Fig. 2). The monophyly of this group was supported strongly by a high bootstrap value of 100 % of the maximum-likelihood analysis, as well as by parsimony and neighbour-joining analysis. The sequences of these strains showed 98·4–99·9 % similarity. The remaining strains of the avian [P. haemolytica]–‘A. salpingitidis’ complex and the strains of P. anatis included formed another monophyletic group (group 2) supported by a bootstrap value of 71 % and neighbour-joining and parsimony analysis. Within this group, a large monophyletic group included the type strain of P. anatis and nine more strains (Fig. 2). The 16S rRNA similarities of group 2 ranged from 98·1 %, between Hom.3 and B 96/20, to 100 % between the following pairs, CCM 5995 and 12158/5Salp., 12158/5Salp. and F 149T, BJ 3453.2 and F 149T and BJ 3453.2 and 12158/5Salp., with a mean similarity of 99·2 % of all strains in group 2. The similarity between groups 1 and 2 (‘A. salpingitidis’-like and P. anatis-like) was 95·7–97·1 %.
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shows a neighbour-joining dendrogram of 17 strains hybridized in a full matrix, including results published by Piechulla et al. (1985). A full DNA–DNA binding matrix is available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org/). The three strains CCM 5974, Gerl. 2740/89 and CCM 5975 were linked to each other at 78–100 % DNA binding. The highest binding of strain CCM 5976 was to CCM 5975 and Gerl. 2740/89, with 75 %. This group corresponds to 16S rRNA group 1, while the remaining strains were included in another group corresponding to 16S rRNA group 2 (see Fig. 2).
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). The P. anatis-like cluster included 29 strains, 13 of which were hybridized in a complete matrix. All of these strains were linked to each other at 79–100 % DNA binding; however, single DNA binding values as low as 31 % were observed. Three strains, referred to below as Gallibacterium genomospecies 1, were linked at 87 %; however, within this group, binding as low as 48 % was observed. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Bisgaard, 1993). The application of molecular biological methods, especially DNA–DNA hybridization and 16S rRNA sequence analysis, has contributed to an improved classification (Mutters et al., 1989; Dewhirst et al., 1993). The three original genera, Pasteurella, Actinobacillus and Haemophilus, have been found to be polyphyletic and, within the past decade, three new genera Lonepinella, Mannheimia and Phocoenobacter have been proposed (Osawa et al., 1995; Angen et al., 1999; Foster et al., 2000). Although outlining of ‘true’ monophyletic groups of Pasteurella, Actinobacillus and Haemophilus is in progress, many taxa still need reclassification, as demonstrated in the present study, where the new genus Gallibacterium is proposed to include taxa previously included within both Actinobacillus and Pasteurella.
This study confirms previous findings by phylogenetic analysis of 16S rRNA sequences and rRNA–DNA hybridizations (De Ley et al., 1990; Dewhirst et al., 1993) that bacteria of the avian [P. haemolytica]–‘A. salpingitidis’ complex represent true members of the Pasteurellaceae. Additional 16S rRNA sequence comparison and DNA–DNA hybridizations showed that the avian [P. haemolytica]–‘A. salpingitidis’ complex should be classified as a separate genus within the Pasteurellaceae Pohl 1981. The new genus is proposed to be named Gallibacterium gen. nov. The present investigations also showed that P. anatis is unrelated to other species of Pasteurella, including Mannheimia haemolytica (formerly Pasteurella haemolytica; Angen et al., 1999), just as ‘A. salpingitidis’ is unrelated to other species of Actinobacillus, including Actinobacillus sensu stricto (Christensen et al., 2002).
The avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex formed two monophyletic groups by 16S rRNA sequence comparison and the same groups could be identified by AFLP analysis and DNA hybridizations. Strains were related either to the previously suggested type strain of ‘A. salpingitidis’ or to P. anatis. Strains classified in biovars 5, 8 and 9 were related to ‘A. salpingitidis’, while the remaining biovars were related to P. anatis. The similarity between the two groups was 96–97 % by 16S rRNA sequence comparison. In addition, a group of bovine strains tentatively classified with P. anatis was related to the P. anatis group at 98–100 % 16S rRNA similarity.
The 16S rRNA phylogenetic tree was based on maximum-likelihood analysis, which was found to be most accurate for phylogenetic inference (Yang et al., 1994); however, comparison was made to phylograms generated by neighbour-joining and parsimony methods, since these tests behave better than the maximum-likelihood method on some occasions (Hillis et al., 1994). Nodes supporting the genus-like group of the avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex were supported by all three methods, suggesting a very high likelihood for the grouping. A unique 258 bp ITS fragment, not found in other members of the Pasteurellaceae investigated (Fussing et al., 1998; Gu et al., 1998; Leys et al., 1994; Privitera et al., 1998), was present in four strains representing the avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex, supporting the unique genotypic nature of the group.
Five genotyping methods were used during different parts of the investigation. PFGE was used to verify that isolates used in the taxonomic analysis were genotypically diverse. This information could not be used further for classification since not even a single fragment was shared among all strains. Analysis of plasmids further confirmed that isolates were unrelated; however, this information was used for descriptive purposes only, since plasmid content has not been recommended for classification (Gürtler & Mayall, 2001; Stackebrandt et al., 2002). 16S rRNA sequence comparison was used to confirm the monophyly of the new genus proposed and to identify species-like groups. ITS typing confirmed the unique genotype of the genus and might be used for identification in future studies. AFLP and DNA–DNA hybridization were used to genotype the isolates according to the whole-genome approach (e.g. Gürtler & Mayall, 2001) and to identify groups at the level of genomospecies. Within the dendrograms constructed by the two methods, similar groups of related strains could be identified as defined by phylogenetic analysis of 16S rRNA sequences; however, the correlation between the percentage similarity determined by AFLP (Dice) and percentages of DNA binding determined by DNA–DNA hybridization was poor (data not shown). It was impossible to define a ‘cut-off’ value of species similarity based on AFLP data, as with the DNA–DNA hybridization method (see below). The present study showed that AFLP might represent an alternative to DNA–DNA hybridization for genotypic separation of species, as suggested previously by Stackebrandt et al. (2002); however, the comparison has to be performed at the level of groups defined as clusters or monophyletic units. For example, if species have been defined by DNA–DNA hybridization, AFLP can be used to classify novel isolates with the species-like groups.
Variability of the methods used for genotyping might be related to variation with different cultures of the same isolate. Such variation has never been reported for members of the Pasteurellaceae, and the high similarity obtained by AFLP with repeated analysis of the same isolates had a good or even better reproducibility compared with previous work (Tenover et al., 1994; Huys et al., 1996; Duim et al., 1999; On & Harrington, 2000), suggesting low variation between different cultures of the same isolates as well as low experimental error. For some strains, there was minor variation between the fingerprints. Various reasons have been suggested to contribute to variation. Gel-to-gel variation, faintly stained bands, doublet bands representing two or more fragments of approximately the same size and variations in peak height due to differences in PCR efficiency may all influence the final result (Tenover et al., 1995). In this study, repeated analysis of strain F 279 on different gels resulted in 93·7 % similarity between fingerprints, although both runs showed the same number of bands at nearly the same positions. The low band-position tolerance of 0·5 % used in the comparison of fragments may give similarities lower than 100 % for identical fingerprints. The comparison of restriction patterns remains, in part, a subjective process that cannot be reduced totally to rigid algorithms (Tenover et al., 1995). Despite the minor variations, the AFLP results support the phylogenetic relationships suggested with 16S rRNA and DNA–DNA hybridizations techniques.
The methodological variation in relation to DNA–DNA hybridization was discussed recently (Christensen et al., 2000a) and data in the present study represent at least repetitions where strains used for driver and tracer had been reversed.
DNA–DNA hybridization showed that the bacteria of the avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex were related at the genus level. The closest relationships of this new genus Gallibacterium were found to Pasteurella multocida, P. langaaensis, Actinobacillus sp. and Pasteurella trehalosi, with 54, 51, 48 and 45 % DNA binding (Piechulla et al., 1985). Strains previously classified as biovars 1, 3, 4, 11, 12, 15, 17–20, 22 and 24 of the avian [P. haemolytica]–‘A. salpingitidis’ complex and avian and bovine isolates of P. anatis were linked at a DNA binding level of 79 % or higher, forming a single species, for which the name Gallibacterium anatis gen. nov., sp. nov. is proposed. Biovar 9 (CCM 5976) was linked to Gallibacterium at a DNA binding level of 75 % and might represent a separate species. Three strains (CCM 5974, CCM 5975 and Gerl. 2740/89) representing biovars 5 and 8 of the former avian [P. haemolytica]–‘A. salpingitidis’ complex showed 87 % or more DNA binding. However, phenotypic separation of this group was not possible at the species level and the group is considered to represent a novel genomospecies of Gallibacterium with phenotypic similarities to those of G. anatis. A single strain, Gerl. 3191/88, representing biovar 8, however, showed only 65 % DNA binding with CCM 5974, which also belongs to biovar 8. The final classification of biovars 5, 8 and 9 remains unsolved and must await the analysis of more strains, including extended phenotypic characterization and DNA hybridizations. However, 16S rRNA and AFLP data and complete DNA matrix data from G. anatis seem to indicate the existence of only one additional genomospecies.
The limits of DNA binding of 79–87 % within isolates of the species-like group of Gallibacterium are within the limits of the family. A study of Actinobacillus sensu stricto showed a limit for separation of species of around 80 % (Christensen et al., 2002), which is slightly lower than for species of the genus Mannheimia and for other species of the family, for which a limit of 85 % has been proposed (Angen et al., 1999; Mutters et al., 1989).
In agreement with the genotypic divergence observed by 16S rRNA sequence comparison and DNA–DNA hybridization, a higher putrescine content was found in cells walls of biovars 1 and 4 of the avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex than for the previously suggested type strain of ‘A. salpingitidis’, representing biovar 8 (Busse et al., 1997). It was further concluded that the heterogeneity of polyamines in cell walls of 16S rRNA cluster 3D of Dewhirst et al. (1993) (‘A. salpingitidis’, P. anatis, P. langaaensis and Bisgaard taxa 2 and 3) indicated the presence of more genera. This is in agreement with the distant relationship between the avian [P. haemolytica]–‘A. salpingitidis’ complex and taxa 2 and 3 found by SDS-PAGE (Bisgaard et al., 1993) and the 16S rRNA sequence comparison of the present study. The classification of taxa 2 and 3 as well as reclassification of P. langaaensis must await further studies.
Due to lack of information on biovars in previous reports, the sources of organisms described as G. anatis and genomospecies of Gallibacterium in the present paper are based upon strains characterized in our laboratory only. G. anatis has been isolated from chickens (287 strains), ducks (24), geese (19), cattle (14), budgerigars (7), psittacine birds (5), turkeys (4), partridges (3), pigs (2) and guinea fowl (1), while four isolates were of unknown source. Strains of G. anatis have been obtained from lesions in addition to normal mucosal membranes. Gallibacterium genomospecies 1 (biovars 5 and 8) has been isolated from chickens (8 strains) and a pigeon. Biovars 5, 8 and 9 have only been obtained from lesions.
Differentiation of members of Gallibacterium gen. nov. from related taxa
Members of Gallibacterium can be separated from other genera of Pasteurellaceae by differences in catalase, symbiotic growth, haemolysis, urease, indole, acid production without gas from (+)D-xylose, (-)D-mannitol, (-)D-sorbitol, (+)D-mannose, maltose, raffinose and dextrin and o-nitrophenyl 
-D-glucopyranoside (ONPG) and p-nitrophenyl -D-glucopyranoside (PNPG) tests. Members of Gallibacterium are (+)D-mannitol-positive, while P. gallinarum strains are negative. Gallibacterium are either non-haemolytic and negative for growth on (-)D-sorbitol, maltose and dextrin or haemolytic and variable for these characters, while P. gallinarum is non-haemolytic, (-)D-sorbitol-negative and maltose- and dextrin-positive. Gallibacterium is negative for symbiotic growth and positive in raffinose fermentation, while Pasteurella volantium shows symbiotic growth and a negative reaction for raffinose fermentation. P. avium shows symbiotic growth and is negative for acid production from (-)D-mannitol, while Gallibacterium is (-)D-mannitol-positive. Gallibacterium is catalase- and (+)D-xylose-positive while P. langaaensis and Lonepinella koalarum are negative for these characters. Phocoenobacter uteri is also catalase-negative and (-)D-mannitol-negative, while Gallibacterium is (-)D-mannitol-positive. Gallibacterium is ONPG-positive and indole-negative, whereas the members P. multocida, Pasteurella canis, Pasteurella stomatis and Pasteurella dagmatis of Pasteurella 16S rRNA cluster 3B (Dewhirst et al., 1993) are indole-positive and ONPG-negative. Gallibacterium is urease-negative and PNPG-positive whereas genuine members of Actinobacillus (see Christensen et al., 2002) are urease-positive and PNPG-negative. Gallibacterium is positive for (+)D-mannose and PNPG while Mannheimia is negative for these characters (Angen et al., 1999). Gallibacterium is urease-negative and ONPG-positive while genuine members of Haemophilus (16S rRNA clusters 1B and 1C; Dewhirst et al., 1993) are urease-positive and ONGP-negative.
Description of Gallibacterium gen. nov.
Gallibacterium (Gal'li.bac.te'ri.um. L. n. gallus chicken; N.L. n. bacterium rod; N.L. n. Gallibacterium bacterium of chicken).
A member of the family Pasteurellaceae (Pohl 1979) Pohl 1981 that includes the avian [P. haemolytica]–‘A. salpingitidis’–P. anatis complex. The phenotypic description is based on Bisgaard (1982) and characterization of subsequent isolations.
Gram-negative, non-motile, rod-shaped or pleomorphic with cells occurring singly and in pairs. Colonies on bovine-blood agar are mostly strongly 
-haemolytic, greyish, non-transparent, but eventually translucent at the periphery, with a butyrous consistency, smooth and shiny, circular, raised with an entire margin and 1·0–2·0 mm in diameter after 24–48 h at 37 °C. Endospores are not formed. Growth is mesophilic and facultatively anaerobic or microaerophilic. Catalase-, oxidase- and phosphatase-positive. Nitrate is reduced. The reaction in Hugh–Leifson medium with (+)D-glucose is fermentative. Porphyrin and alanine aminopeptidase tests are positive. Acid is formed without gas from glycerol, (-)D-ribose, (+)D-xylose, (-)D-mannitol, (-)D-fructose, (+)D-galactose, (+)D-glucose, (+)D-mannose, sucrose and raffinose. ONPG and PNPG tests are positive. Negative in symbiotic growth, Simmons citrate, mucate-acid, malonate-base, H2S/tri-sugar iron (TSI), growth in the presence of KCN, Voges–Proskauer at 37 °C and urease. Negative tests are further observed with arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, indole, gelatinase and Tweens 20 and 80. Pigment is not formed. Acid is not produced from m-erythritol, adonitol, (+)D-arabitol, xylitol, (-)L-xylose, dulcitol, (+)D-fucose, (+)L-rhamnose, (-)L-sorbose, cellobiose, (+)D-melibiose, (+)D-melezitose, (+)D-glycogen, inulin, aesculin, amygdalin, arbutin, gentiobiose, salicin, (+)D-turanose or -N-CH3–glucosamid. Reactions for p-nitrophenyl -D-glucopyranoside (NPG), o-nitrophenyl -L-fucopyranoside (ONPF), -galactosidase, p-nitrophenyl -D-glucopyranosiduronic acid (PGUA), -mannosidase and o-nitrophenyl -D-xylanopyranoside (ONPX) are also negative. Variations are observed in the methyl red reaction at 37 °C, growth on MacConkey agar and acid production from (+)L-arabinose, (-)D-arabinose, m-inositol, (-)D-sorbitol, (-)L-fucose, lactose, maltose, trehalose and dextrin. The type species is Gallibacterium anatis.
Description of Gallibacterium anatis (Mutters et al. 1985) comb. nov.
Basonym: Pasteurella anatis Mutters et al. 1985.
Isolates are haemolytic, (-)D-arabinose- and (-)L-fucose-positive (biovar haemolytica, former biovars 1, 3, 4, 11, 12, 15, 17–20, 22, 24 of the avian [P. haemolytica]–‘A. salpingitidis’ complex) or non-haemolytic, trehalose-positive and (-)D-arabinose-, (-)L-fucose-, maltose- and dextrin-negative (biovar anatis, former P. anatis) (Table 5). The G+C content ranges from 39·9 to 42·6 mol% and the genome size from 1·6 to 2·1 GDa (Mutters et al., 1985; Piechulla et al., 1985). The type strain is strain F 149T (=ATCC 43329T =NCTC 11413T), isolated from the intestinal tract of a duck.
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90 % of strains positive within 1–2 days; (+),