Classification of taxa of Bisgaard
(For references please address international peer-reviewed papers)
Taxon 11
Evidence obtained to indicate that equine isolates of organisms previously reported as A. suis or haemolytic variants of A. equuli might constitute a separate taxon provisionally named taxon 11 was first published by Bisgaard et al. (25). Four biovars were initially reported, and selected DNA:DNA hybridizations did not support classification with A. suis. Additional biovars have been published, and several reports have associated these organisms with pathological lesions. However, other papers seem to point to a true predilection of taxon 11 for the respiratory tract of horses (50). Subsequent studies demonstrated that A. equuli and taxon 11 represent two different genotypes which differ with respect to disease pattern and epidemiology. For the same reasons two subspecies of A. equuli have been proposed: A. equuli subsp. equuli (former A. equuli) and A. equuli subsp. haemolyticus (former taxon 11) (111).
RTX toxin activity (eqx) has been demonstrated in isolates of A. equuli subsp. haemolytica (Berthoud et al. Vet. Microbiol. 2002, 87, 159-174), and a stronger cytotoxicity towards horse-compared to pig lymphocytes was reported by Kuhnert et al. (Vet. Microbiol. 2003, 92, 161-167). So far, eqx has not been demonstrated in A. equuli subsp. equuli associated with sleepy foal disease and septicaemia in piglets.
Taxon 12
Several unclassified isolates of Pasteurellaceae were obtained during the investigation of the aerobic pharyngeal flora of healthy white laboratory mice originating from four different colonies. Nineteen isolates formed a group tentatively designated taxon 12. The G + C content of DNA and genome size of the reference strain were 46.9 mol % and 1.4 x 109 Dalton, respectively.
Taxon 12 was linked to Actinobacillus via the avian haemolytic Actinobacillus-like complex (taxon 26) by DNA:DNA hybridizations (27) and named A. muris (29). Subsequent studies excluded this taxon from Actinobacillus sensu stricto (133). Recent taxonomic investigations classified [A.] muris with the rodent cluster of Olsen et al. (Bergeys Manual of Systematic Bacteriology 2005 vol 2B p. 855). Phylogeny based on rpoB sequences, however, failed to group this taxon with other groups (Korczak et al. Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399). Further investigations are needed to clarify the taxonomy and significance of this taxon.
Taxon 13
Routine phenotypic characterization of P. multocida-like organisms obtained from pneumonic calf lungs often result in a fermentation pattern different from what is generally accepted for P.multocida sensu stricto. A group of indole-, mannitol- and sorbitol-negative isolates was tentatively designated taxon 13 (26). Selected DNA:DNA hybridizations classified ornithine positive taxon 13 isolates with P. multocida biovar 6 (P. canis) while ornithine negative isolates were classified as V-factor independent isolates of [H.] avium. Additional DNA:DNA hybridizations demonstrated 80-92% DNA binding with P. canis for ornithine positive isolates of taxon 13 while ornithine negative isolates demonstrated 88% DNA binding with V-factor requiering isolates of [H.] avium biovar 1 classified as [P.] avium (now Avibacterium avium). Ornithine negative isolates of taxon 13 also demonstrated 69-81% DNA binding with P. stomatis (Mutters et al. Int. J. Syst. Bact. 1985, 35, 309-322). Additional information of the distribution and importance of these organisms have been published (39, 41,50).
Since routine identification of taxa belonging to Pasteurella rest on a single or a few phenotypic characters, and isolates aberrant for these characters have been reported more focus has been made on improvement of specific diagnostic genetic tools of importance for both the veterinary and medical professions. New diagnostic tools developed, however, questioned the correct classification of taxon 13 as discussed by Christensen et al. (131) who finally demonstrated that taxon 13 represents P. multocida sensu stricto, explaining positive reactions with probes (107) and in PCR- methods developed for detection of P. multocida (see 131 for references). Based upon MLST these organisms form a separate group (Unpublished data).
Taxon 14
A phenotypically homogeneous group of avian Pasteurella-like organisms including two biovars were reported by Bisgaard & Mutters (28). DNA base composition of biovars 1 and 2 varied between 53.1 (biovar 1) and 52.0 mol % (biovar 2) while both biovars demonstrated a genome mass of 1.9 x 109 Dalton. Ninety-seven % DNA similarity was observed between biovars 1 and 2. DNA:DNA hybridizations, however, failed to classify these organisms, tentatively named taxon 14. DNA:rRNA hybridizations, classified taxon 14 at the common root of the seven rRNA cistrons outlined for Pasteurellaceae (36). These organisms also demonstrated a separate polyamine pattern (Busse et al. Int. J. Syst. Bact. 1997, 47, 698-708). 16S rRNA gene sequence phylogeny classified taxon 14 with taxon 32 and 40, and [P.] testudinis (127, 129, 133). Similar observations have been reported for rpoB (Korczak et al. Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399).
Taxon 14 has been obtained from upper respiratory tract lesions in ducks, turkeys, pigeons, a goose and a peafowl (28, 50) and blepharoconjunctivitis in turkeys (Bisgaard et al. WPSA, 4th Int. Symp. on Turkey Prod. 2007 pp. 59-50). These organisms still remain to be classified and named, to improve proper identification and general understanding on the epidemiology and importance of these bacteria.
Taxon 15
Reports on [P.] haemolytica (M. haemolytica) in hosts other than ruminants were questioned by Bisgaard (24). Isolates from pigs were sufficiently different from bovine isolates to constitute a new taxon, tentatively designated taxon 15. Additional investigations including mol % G + C in DNA, genome mass, and DNA:DNA hybridizations confirmed that taxon 15 constitutes a separate group within the family Pasteurellaceae (33). Based upon quantitative evaluation of phenotypic data Angen et al. (74) outlined two biovars of taxon 15 and demonstrated that taxon 15 biovar 1 and biogroup 6 of [P.] haemolytica from ruminants could not be separated by phenotypic tests. The same was valid for taxon 15 biovar 2 and biogroup 7 of [P.] haemolytica from ruminants. Subsequent genotypic studies including ribotyping and multilocus enzyme electrophoresis (MLEE) showed that [P.] haemolytica biogroup 7 did not cluster with taxon 15, and with a few exceptions, porcine isolates formed a separate cluster by ribotyping while MLEE results did not allow a clear separation between bovine and porcine isolates (78). Based upon maximum-likelihood analysis of 16S rRNA gene sequences taxon 15 and [P.] haemolytica biogroup 6 formed a separate cluster branching into a porcine and bovine line. Strains representing these lines, however, demonstrated a DNA:DNA relationship on species level. For the same reason these organisms were classified with the same species, Mannheimia varigena (87). Taxon 36 demonstrated 88 % DNA binding with M. varigena confirming the high genetic affiliation between these groups demonstrated by ribotyping (78). rpoB based phylogeny also clustered porcine and bovine isolates of M. varigena together (Korczak et al. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399).
So far M. varigena has been isolated from pneumonia, mastitis and septicaemia as well as the oral cavity, rumen and intestines of cattle. Isolates of porcine origin have been associated with septicaemia, enteritis or pneumonia, but isolates from the upper respiratory tract have also been reported (50, 87).
Taxon 16
During characterization of Pasteurella-like organisms obtained from the oral cavity of dogs and cats Bisgaard & Mutters (31) identified a homogeneous group of bacteria which might represent urease negative P. dagmatis or indole positive [P.] gallinarum (Av. gallinarum). Mol % G + C in DNA varied between 43.5 and 44.3 % while the genome mass varied between 1.6-1.8 x 109 Dalton. However, DNA:DNA hybridizations did not allow classification with known species of Pasteurellaceae. Although strains investigated included both Danish and a UK isolate the authors desisted from naming the new taxon tentatively called taxon 16 (31). A total of 30 isolates were reported by Muhairwa et al. (99). Two major clusters showing approximately 35 % similarity were observed by ribotyping, the closest neighbours being P. dagmatis and P. canis. rRNA:DNA hybridizations left this taxon ungrouped at the common root of the rRNA branches of P. multocida, A. lignieresii, H. Influenzae, [H.] aphrophilus and [A.] actinomycetemcomitans (36). The 16S rRNA relationship of taxon 16 has remained ambiguous (127, 129, 133). These organisms have also been obtained from bite wounds in humans inflicted by dogs (Frederiksen, personal communication). These organisms remain to be classified and named.
Taxon 17
Organisms received as A. equuli or unclassified Pasteurellaceae have been provisionally named taxon 17 (50). This group, however, differs from A. equuli in D(+) xylose, D(-) mannitol, β-glucuronidase and ONPX. A mouse isolate (P434) received from W. Frederiksen deviating from taxon 17 in indole, trehalose and a-fucosidase demonstrated 44 % DNA binding wit P. dagmatis NCTC 11617 (Ryll: Untersuchungen zur genetischen Klassification und praktischen Identifizierung der Pasteurellaceae der Nagetiere. Thesis, Justus-Liebing-Universität, Giessen, 1989). Phylogenetic investigations as determined by comparison of 16S rRNA gene sequences showed that [P.] pneumotropica NCTC 8141T (Jawetz type) made up a separate cluster together with [A.] muris, [H.] influenza-murium, taxon 17 and taxon 22 (127). rpoB based phylogeny confirmed the close relationship between taxon 17 and P. pneumotropica type Jawetz (Korczak et al. Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399). This group is further characterized by a deletion of five bases through E. coli rrnB pos. 204-208 compared with other members of Pasteurellaceae (Unpublished data). The significance of taxon 17 associated with mice and rats remains to be investigated.
Taxon 18
A total of 26 isolates obtained from the rumen of sheep and classified as A. lignieresii were originally received from Dr. J.E.Phillips. Further investigations showed that these isolates might represent non-haemolytic and maltose and dextrine negative [P.] haemolytica and were provisionally named taxon 18 (30). Based upon differences in D (-) sorbitol and L (+) arabinose four different biovars were outlined. An additional biovar was outlined by Angen et al. (74) who also demonstrated that these organisms clustered together with the exception of biovar 5. However, biogroup 8 D of [P.] haemolytica and biovar 2 of taxon 20 also clustered with taxon 18. Although forming a separate cluster biovar 5 of taxon 18 branched deeply with biogroup 9 of [P.] haemolytica. Ribotyping confirmed the relationship between taxon 18 biovars 1, 3 and 4 and [P.] haemolytica biogroup 8 D while other isolates of taxon 18 biovars 3 clustered separately. By MLEE a single isolate of taxon 18 biovar 1 clustered deeply with selected isolates of [P.] haemolytica biogroups 1, 8 D and 10 (78). The phylogenetic relationships of the [P.] haemolytica-complex as revealed by maximum-likelihood analysis of 16S rRNA gene sequences resulted in five major groups of which two strains of taxon 18 biovars 1 and 3 (HPA 81 and 92T) formed a separate group with HPA 98 ([P.] haemolytica biogroup 8 D) and UT 26 (atypical biogroup 1 of [P.] haemolytica).
Two strains of taxon 18 biovars 1 and 3 (HPA 92T and 109) demonstrated 92 % DNA binding and clustered with HPA 98 at 88 % DNA binding. UT 26 finally showed 83 % DNA binding with HPA 98 and 84 % binding with UT 27 (biogroup 10 of [P.] haemolytica). For the same reasons these organisms were named Mannheimia ruminalis (87). Species of Mannheimia also form a separate phylogenetic group by analysis of rpoB (Korczak et al. Int. J. Syst. Evol. Micriobiol. 2004, 54, 1393-1399).
Analysis of the leukotoxin genotype by Southern blot and the corresponding β-haemolytic phenotype on sheep blood agar plates revealed that both characters were present only in strains of biogroup 1 (UT26), biogroup 190 (HPA95, HPA114, and UT27), and taxon 18 biovar 2 (HPA113, HPA90, and UT38), whereas strains of biogroup 8D and taxon 18 biovars 1, 3, and 4 have lost the leukotoxin operon.
Taxon 19
During investigations of porcine Pasteurellaceae (24) a separate group of isolates was outlined and tentatively named taxon 19. These isolates originated from septic infections in piglets and abortion in sows. However, only NCTC 10699 was included in the publication as a reference strain. Six out of 10 isolates investigated represented original isolates from Dr. N.S. Mair after whom these organisms subsequently were named (Sneath & Stevens Int. J. Syst. Bact. 1990, 40, 148-153).
rRNA:DNA hybridizations affiliated [P.] mairii at the common root of the rRNA brances of P. multocida, A. lignieresii, H. influenza and [H.] aphrophilus (36). An emended description of [P.] mairi was reported by Christensen et al. (136). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that [P.] aerogenes sensu stricto, [P.] mairii sensu stricto and [A.] seminis formed a monophyletic group representing a new genus candidate within the family Pasteurellaceae. Similar observations had previously been observed for rpoB by Korczak et al. (Int. J. Syst. Evol. Microbiol. 2004, 54, 1393-1399).
RTX toxins are predominantly observed in taxa of Pasteurellaceae and often associated with pathogenic representatives. The pax of [P.] aerogenes has been demonstrated in isolates associated with abortion in pigs, but not in those from other clinal conditions (Kuhnert et al. Infect. Immun. 2000, 68, 6-12). The pax gene has subsequently been demonstrated in all isolates of [P.] mairii investigated (155).
Taxon 20
Organisms classified with [P.] haemolytica were reported from 44 out of 81 European hares suffering from respiratory tract lesions. Similar organisms were obtained from mammary and uterine infections and from 10 out of 42 apparently asymptomatic carriers investigated (Louzis Rec. Méd. Vét. 1984, 160, 581-584). Phenotypically related organisms provisionally named taxon 20 were subsequently reported from cases of purulent bronchopneumonia and from conjunctivitis in European brown hares (40).
Two biovars of taxon 20 have been reported. Quantitative evaluation of phenotypic data clustered biovar 1 with [P.] granulomatis while biovar 2 clustered with taxon 18 (74). Ribotyping, however, clustered biovars 1 and 2 of taxon 20 together. MLEE finally classified these organisms with [P.] granulomatis (78). Taxonomic investigations of the [Pasteurella] haemolytica-complex as evaluated by 16S rRNA sequencing and DNA:DNA hybridizations classified both isolates from ruminants and leprine species with Mannheimia granulomatis nov. rev. (87). A novel lineage of M. granulomatis has subsequently been reported from lesions in roe deer (163).