Classification of taxa of Bisgaard
(For references please address international peer-reviewed papers)
Taxon 1
All 11 isolates of this taxon originally reported by Bisgaard (20)* were demonstrated in mixed flora from the intestinal tract of apparently normal ducks. These isolates were later classified with Pasteurella sensu stricto based upon DNA:DNA hybridizations (27) and named Pasteurella anatis by Mutters et al. (Int. J. Syst. Bact. 1985, 35, 309-322). P. anatis has subsequently also been obtained from ducks suffering from respiratory disease (50).
Crossed immunoelectrophoresis applied to representative strains from 11 different Pasteurella spp. showed that P. anatis formed a separate cluster branching deeply with P. langaa showing less than 70% similarity with the core group of genus Pasteurella (Schmid et al. Zbl. Bakt. 1991, 275, 16-27). A new genus, Gallibacterium gen. nov., suggested to include the avian [P.] haemolytica – [A.] salpingitidis – [P.] anatis complex was reported by Christensen et al. (119), since these taxa formed a monophyletic unit with 16S rRNA similarities above 95%. Strains originally reported as [P.]anatis are now classified as G. anatis biovar anatis. Korczak et al. (Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399) demonstrated that rpoB gene sequence analysis was useful for separating Gallibacterium spp. from other taxa of Pasteurellaceae
Taxon 2 and 3 complex
Organisms associated with salpingitis and peritonitis in ducks were first reported as atypical
Actinobacillus lignieresii by Bisgaard (4). Comparative investigations of avian Pasteurellaceae including additional isolates of atypical Actinobacillus lignieresii obtained from ducks, geese and pigeons subsequently resulted in classification of these organisms as taxa 2 and 3 based upon differences in production of acid from (+)-L-arabinose and dulcitol (20).
A total of 35 strains isolated from the respiratory tract, liver, heart and spleen of pigeons and different species of Psittaciformes were reported by Beichel (1986). Nineteen of these were reinvestigated by Bisgaard et al. (90) and biotyped as previously described (50). The host spectrum was extended to include galliform birds like partridges and pheasants (50). The importance of taxon 2 and taxon 3 in salpingitis in web-footed birds was subsequently confirmed by Bisgaard (61).
On the basis of DNA-DNA hybridizations these taxa were shown to form a large distinct group which seems to represent a new genus with several species within the family Pasteurellaceae Pohl 1981 (27). However, full matrix DNA-DNA hybridizations were not carried out.
De Ley et al. (36) performed hybridizations between labelled rRNA from seven representative members of the family Pasteurellaceae to 53 strains of Pasteurellaceae and showed that strain HIM 730-3 (F420) of taxon 2/3 (according to Bisgaard et al. (51) and not taxon 2 as listed by De Ley
et al., (36) and strain NCTC 11412 of taxon 3 biovar 2 clustered with different nodes of the rRNA branches outlined, indicating major genetic diversity within taxon 2 and 3.
Different polyamine patterns were obtained with a duck isolate of taxon 2 biovar 1 (F150T) and a parakeet isolate of taxon 3 biovar 1 (F450T) according to Busse et al. (Int. J. Syst. Bact. 1997, 47, 698-708). Both isolates had profiles different from the newly established genera Gallibacterium (119) and
Avibacterium (138) of avian origin. Comparison of phena defined by protein profiling with species/groups previously established by DNA-DNA hybridization, carbohydrate profiling and biovar typing showed that the best correlation existed between DNA-DNA hybridization and biovar typing. A correlation between results obtained from DNA-DNA hybridizations and protein profiling was not observed. Protein profiling, however, indicated a connection between protein profiles and hosts of isolation (51). Host related bacterial lineages of the taxon 2 and 3 complex have subsequently been demonstrated by amplified fragment length polymorphism (AFLP) typing (158).
Phylogenetic analysis by 16S rRNA gene sequence comparison has shown that taxon 2 biovar 1 (NCTC 11414) and taxon 3 biovar 2 (CCUG 15565T, wrongly referred to as CCUG 15563 in the article) cluster together forming subcluster 3D of Dewhirst together with strains subsequently classified as Gallibacterium and [Pasteurella] langaaensis (Dewhirst et al., Zbl. Bakt. 1993, 279, 35-44). In a subsequent investigation including avian taxa, Bisgaard taxon 2 and taxon 3 formed a separate entity within the avian cluster (127). A single isolate from septicaemia in a budgerigar classified as trehalose positive taxon 3 biovar 1 only showed 94.5% 16S rRNA gene sequence similarity to biovar 2 of taxon 3 underlining the heterogeneity of this complex group.
The accumulated evidence outlined above underlines the uncertain taxonomic position of the taxon 2 and taxon 3 complex of avian origin and the lack of unambiguous diagnostic possibilities associated with these organisms which makes interpretation of studies on these organisms difficult and prevents progress in understanding of their epidemiology and pathogenesis.
For the same reason a subset of 23 strains representing existing biovars characterized by AFLP (158) were investigated by phylogenetic analysis of the 16S rRNA, rpoB, infB and recN gene sequences. Moreover, the recN gene sequences were used for estimation of whole genome similarity to allow comparison with previously published DNA-DNA hybridizations. Selected strains were additionally characterized by polyamine profiling. Clusters outlined by
Bojesen et al. (158) and shown not to belong to Gallibacterium sensu stricto were excluded from the investigation and these taxa will be published separately. These studies allowed the identification of five groups with unique properties allowing the suggestion of five new species of Gallibacterium: G. melopsittaci, G. trehalae, G. columbinum, G. salpingitigis and a not yet named species (181). Isolates of taxon 2 and 3 sharing the properties of Volucribacter are under investigation.
Taxon 4
Isolates classified as taxon 4 were originally obtained in mixed culture from the respiratory tract of apparently healthy chickens (20). These organismes shared the cultural and biochemical characters of bacteria previously reported as Pasteurella sp. by Clark & Godfrey (Av. Dis. 1960, 4, 280-290). These organisms were later classified with Pasteurella sensu stricto based upon DNA:DNA hybridizations (27) and named Pasteurella langaa by Mutters et al. (Int. J. Syst. Bact. 1985, 35, 309-322). Crossed immunoelectrophoresis applied to representative strains from 11 different Pasteurella spp. showed that [P.] langaa formed a separate cluster branching deeply with [P.] anatis showing less than 70% similarity with the core group of genus Pasteurella (Schmid et al. Zbl. Bakt. 1991, 275, 16-27). Based upon 16S rRNA similarities Dewhirst et al. (Zbl. Bakt. 1993, 279, 35-44) showed that the type strain of P. langaa branched deeply with [P.] anatis and the taxon 2 and 3 complex Phylogenetic relationships between taxa of Pasteurellaceae based upon maximum likelihood analysis of 16S rRNA gene sequences demonstrated the highest relationship between [P.] langaa and [P.] caballi (127) while Korczak et al. (Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399) showed that [P.] langaa was phylogenetically related with [A.] rossii. However, the phylogenetic tree based upon partial rpoB sequences showed that [P.] langaa formed a separate group together with [P.] caballi, [A.] rossii and [A.] porcinus. The final taxonomic position of [P.] langaa remains to be determined.
Taxon 5, 6 7 and 8
Organisms tentatively classified as taxon 5, 6, 7 and 8 were obtained from the posterior pharynx of healthy guinea pigs originating from two different colonies of well-managed and conventionally reared guinea pigs (22). The G + C content (mol %) of DNA from these taxa were 40.0-42.1%, 51.2%, 44.1% and 39.6%, respectively. DNA:DNA hybridizations did not allow classification with known taxa. rRNA cistron similarities of taxa belonging to Pasteurellaceae showed that taxon 7 remained ungrouped on the common root of [A.] actinomycetemcomitans, [H.] aphrophilus, H. influenzae, A. lignieresii and P. multocida (36). Comparison of 16S rRNA gene sequences showed that taxon 5 and 7 were unique while taxon 6 and 8 were related with taxon 10 and Actinobacillus, respectively (129). Similar results have been obtained for rpoB (Korczak et al. Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399). Additional isolates of taxon 6 and 8 from guinea pigs misclassified as Actinobacillus sp. have been reported by Bisgaard (20) and Boot & Bisgaard (58). These taxa remains to be classified and named.
Taxon 9
Trehalose negative isolates of Actinobacillus equuli were established as a separate group tentatively named taxon 9 in 1993 (50). These organisms had been obtained from the mucous membrane of oropharynx of apparently normal horses in addition to arthritis. Additional investigations have also associated these bacteria with cases of septicaemia in a horse and foal (70, 85). Subsequent investigations of 18 isolates showed that taxon 9 represents two novel species (115), A. arthritidis and Actinobacillus genomospecies 2, both of which have been obtained from as well diseased as normal horses. The closest 16S rRNA sequence based relationships for these species were A. ureae and A. hominis, respectively. According to the phylogenetic tree of the family Pasteurellaceae based on partial rpoB sequences A. lignieresii, A. pleuropneumoniae, A. arthritidis and A. genomospecies 1 and 2 form a separate cluster with a bootstrap value of 100 (Korczak et al. Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399).
Taxon 10
Organisms tentatively classified with taxon 10 remains to be properly published. However, strains classified with this group have previously been included in other studies for comparison (127, 129, 133, 136). Taxon 10 produces gas from glucose, and has so far only been obtained from horses and horse bites in humans (Bisgaard, unpublished data). 16S rRNA gene sequence phylogeny demonstrated a close relationship with taxon 6 and [P.] aerogenes biovars 17 and 18 (136).
These organisms remain to be classified and named to improve proper identification and general understanding on the importance of these bacteria.