INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Apr. 1990, p. 148-153 0020-7713/90/020148-06$02.00/0 Copyright 0 1990, International Union of Microbiological Societies
Vol. 40, No. 2
Actinobacillus rossii sp. nov., Actinobacillus seminis Spa nov. , noma rev., Pastewella bettii sp. nov. , Pasteurella lymphangitidis sp. nova, Pastewella mairi sp. nova, and Pasteurella tvehalosi Spa nova P. H. A. SNEATH1* AND M. STEVENS2 Department of Microbiology, Leicester University, Leicester LEI 7RH,’ and Public Health Laboratory, Leicester Royal InJirmary, Leicester LEI 5 WW,2England Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. oov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.
A recent numerical taxonomic study of the genera Actinobacillus and Pasteurella (46) identified several phena that have not yet been formally named. In this paper we describe six of these phena as new species, based on evidence that the groups form statistically distinct phenotypic clusters and also on evidence from DNA studies.
showed overlap values of less than 1% except for the following pairs: Pasteurella multocida and Pasteurella canis (2.4%); P. multocida and Pasteurella stomatis (1.3%); P. canis and P. stomatis (2.2%); Pasteurella aerogenes and Pasteurella mairi (1.1%); and P. aerogenes and Actinobacillus lignieresii (1.4%). It should be noted that these analyses indicate the adequacy for diagnosis of the set of tests used in the tables; the distinctness of the taxa based on the very large number of tests described by us previously (46) is a good deal greater. The results of DNA studies (13,28, 34,35) also support the distinctness of the taxa. There has been considerable disquiet for many years about the genera Actinobacillus and Pasteurella and their relationship to the genus Haemophilus (24). The generic arrangements within the family Pasteurellaceae are currently undergoing considerable change. Thus, Haemophilus pleuropneumoniae and pasteurellae causing porcine necrotic pleuropneumonia have been transferred to the genus Actinobacillus (36), Pasteurella ureae is more closely related to A . lignieresii than to P. multocida (13), and 16s rRNA sequences indicate that Actinobacillus actinomycetemcomitnns is more closely related to Haemophilus injuenzae than to A. lignieresii (12). In this study we assigned the new species to the genus Actinobacillus or the genus Pasteurella according to whether they seem more closely related, on the basis of presently available evidence, to the type species A . lignieresii or the type species P. multocida, although we acknowledge that the generic assignments of the new species are highly tentative and perhaps dubious. These assignments are used for want of something better to get the species on record. It is increasingly difficult to differentiate the genera Actinobacillus, Haemophilus, and Pasteurella (22, 25); if these taxa are merged, it should be noted that Pasteurella, as the oldest name and a conserved name, would take precedence according to Rule 24b of the International Code of Nomenclature of Bacteria (19,23,39). We retained P. ureae in Table 2 in its more familiar position (11) rather than transfer it to the genus Actinobacillus as recently suggested (32) because we feel that the evidence from DNA (32) and phenotypic (46) studies is not sufficiently compelling in a situation where wholesale rather than piecemeal revision is needed. There is evidence that strains named Actinobacillus seminis are heterogeneous (46,50), and it is likely that more than
MATERIALS AND METHODS
Full details of the materials and methods which we used have been published previously (46). The strains were tested in a wide range of conventional tests, and Gower similarity coefficients were calculated based on 155 unit characters. The organisms were clustered by using the unweighted pair group method with averages; details of this method are described in reference 45. Overlap statistics were calculated by using the OVERMAT program (44) with 23 phena to ensure that they were phenotypically distinct. The most diagnostic characteristics of the phena were obtained from the DIACHAR prOgram (42). The salient properties of previously validly published Actinobacillus and Pasteurella species, together with the properties of the new species, are listed in Tables 1 and 2. The quality of the tables for identification was evaluated by using the OVERMAT program and the MOSTTYP program (43). For this evaluation the symbols in Tables 1 and 2 were coded as follows: f , 95%; (+) and +L, 85%; d, +w, dw, wL, dL, and NT, 50%; (-), 15%; and -, 5%. These values are the midpoints of the ranges of the scores in the tables or were coded as 85 or 50% because of gaps or difficulties in reading weak and late reactions. RESULTS AND DISCUSSION The internal evaluation of the diagnostic tables (Tables 1 and 2) was broadly satisfactory. The MOSTTYP program showed that the most typical possible strains (hypothetical median organisms) identified correctly with their own taxa with a Willcox probability value of more than 0.99, except for Pasteurella stomatis (0.98) and Pasteurella canis (0.82). The OVERMAT program showed no overlap that reached the 8.2% level, which indicates continuous variation (i.e., that two taxa run together phenotypically). All taxon pairs
* Corresponding author. 148
VOL.40, 1990
NEW ACTINOBACILLUS AND PASTEURELLA SPECIES
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-, 510% of the strains are positive; w, weak reaction; L, late reaction; NT, not tested.
ONPG reaction, Hydrolysis of ortho-nitrophenyl-P-galactopyranoside. Positive according to Piechulla et al. (34) and negative according to Sneath and Stevens (46). Different formulations of this medium may explain discrepancies in the reported results; the results for A . pleurupneurnoniae are for medium supplemented with NAD. f In Hugh-Leifson medium (17) containing glucose, with incubation for 48 h at 37°C. g Positive according to Sneath and Stevens (46) and negative according to Kilian and Frederiksen (21) and Phillips (33).
one species is implicated in epididymitis of sheep or that strains have been misidentified because of the unusually wide range of guanine-plus-cytosine (G+C) ratios, 37.8 to 48.8 mol% (16). A possible relationship to “Haemophilus agni” (34) is indicated by a high DNA-DNA pairing value to one strain (strain M650-1343) of this species, but interpretation is uncertain due to doubt over the status of “H. agni,” whose name at present has not been validly published, and strain M650-1343 may perhaps have been misidentified. The revival ofA. seminis fixes the species on one of the strains of Baynes and Simmons (l),who originally named it. Pastewella bettii seems to be a distinctive organism, similar to members of the P. ureae complex but less metabolically active. We have been unable to find valid publication of this name before 1980; therefore, it cannot be revived, so we proposed P. bettii below as a new species. Nor have we been able to trace the origin of the specific
epithet, but we retain it for reasons of stability. Without a valid name, this organism may not be recognized if it occurs infrequently. Similarly, Pasteurella Zyrnphangitidis may be not uncommon if it is looked for. Differences between biovars A and T of Pasteurella haemolytica have been noticed for a long time (2,11,40,41), and these organisms are distinct on the basis of phenotypic and DNA data (26, 35, 38, 46). It seems likely that Pasteurella trehalosi comprises biogroups 2 and 4 of Bisgaard and Mutters (7) and Mutters et al. (26), and P. haernolytica sensu strict0 comprises biogroups 1, 5, 6, 7, and 9, but this needs further study. A number of other groups have recently been recognized in the genus Pasteurella (4, 7, 8, 22). We have not been able to identify the new species proposed here with any of these groups. It is likely that many new species await description. However, one group, the SP group, appears to be taxon 22 of
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42.32 pm long. Gram reaction is negative. Endospores are not formed. Growth is aerobic and facultatively anaerobic; mesophilic. Surface colonies grown aerobically on sheep blood agar are round, greyish, semitransparent, and about 2 mm in diameter after 48 h at 37”C, sometimes showing weak hemolysis without marked greening of the erythrocytes. Notable for producing acid from m-inositol and sorbitol, but not from sucrose, and for reducing Janus green (46). Other properties are shown in Table 1. Isolated from the vaginas of postparturient sows and aborted piglets (37, 46). Serological cross-reactions have been reported with Actinobacillus suis, A . lignieresii, Actinobacillus equuli, and A . seminis, and P . haemolytica (37). DNA-DNA pairing values of about 50% or less to A . lignieresii, A . suis, A . equuli, P . haemolytica sensu stricto, and P . ureae have been reported (13, 35). The G+C content of the DNA of the type strain is 41.9 mol%, as determined by the thermal denaturation (T,) method (25, 34). The type strain is strain ATCC 27072 (= NCTC 10801 = Ross 192 = P624 = A55 and A68) (46). In our hands this strain was positive for the tests marked d in Table 1, except for gas production from glucose and acid production from lactose. Actinobacillus seminis sp. nov., nom. rev. (ex Baynes and Simmons 1960). Actinobacillus seminis (sem’in.is. M.L. gen. n. seminis, of semen). Phenon 14 of Sneath and Stevens (46); “Actinobacillus seminis” of Kilian and Frederiksen (21) and Phillips (33). Cells are small nonmotile bacilli and coccobacilli that are often >2 k m long. Gram reaction is negative. Endospores are not formed. Growth is aerobic and facultatively anaerobic, mesophilic, often requiring 5% CO, on first isolation. Surface colonies grown aerobically on sheep blood agar are round, greyish, semitransparent, about 1 mm in diameter after 48 h at 37”C, and without hemolysis but sometimes with greening of the erythrocytes. Notable for slow and weak fermentation of carbohydrates, for producing no acid from maltose or mannose, and for giving a negative phosphatase reaction (46). Other properties are shown in Table 1. Cause of epididymitis of sheep (1) and isolated from semen; may be involved in polyarthritis of sheep (33). Phenotypically similar to P . ureae (46), but DNA-DNA pairing values are less than 25% to P . ureae, P. multocida,
NEW ACTINOBACILLUS AND PASTEURELLA SPECIES
151
Pasteurella pneumotropica, A . lignieresii, A . equuli, A . suis, Haemophilus injluenzae, and Haemophilus aphrophilus, (13, 34). The G+C content of the DNA of the type strain is 43.7 mol% (T,n method) (25). The type strain is strain NCTC 10851 (= ATCC 15768 = Simmons K3844-C = A51 and A65 = 5 ) (34, 46). In our hands this strain was negative for the tests marked d in Table 1, except for late reactions in the tests for ornithine decarboxylase and acid production form m-inositol and maltose. Pasteurella bettii sp. nov. Pasteurella bettii (betti’i. M.L. gen. n. bettii, of Bett, named for a gentleman named Bett [Catalog of Strains, 8th ed., American Type Culture Collection, Rockville, Md., 19681, whose identity has not been traced). Phenon 13 of Sneath and Stevens (46); “Pasteurella sp. bettii” of Kilian et al. (22), Cells are small nonmotile bacilli and coccobacilli that are often
Vol. 40, No. 2
Actinobacillus rossii sp. nov., Actinobacillus seminis Spa nov. , noma rev., Pastewella bettii sp. nov. , Pasteurella lymphangitidis sp. nova, Pastewella mairi sp. nova, and Pasteurella tvehalosi Spa nova P. H. A. SNEATH1* AND M. STEVENS2 Department of Microbiology, Leicester University, Leicester LEI 7RH,’ and Public Health Laboratory, Leicester Royal InJirmary, Leicester LEI 5 WW,2England Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. oov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.
A recent numerical taxonomic study of the genera Actinobacillus and Pasteurella (46) identified several phena that have not yet been formally named. In this paper we describe six of these phena as new species, based on evidence that the groups form statistically distinct phenotypic clusters and also on evidence from DNA studies.
showed overlap values of less than 1% except for the following pairs: Pasteurella multocida and Pasteurella canis (2.4%); P. multocida and Pasteurella stomatis (1.3%); P. canis and P. stomatis (2.2%); Pasteurella aerogenes and Pasteurella mairi (1.1%); and P. aerogenes and Actinobacillus lignieresii (1.4%). It should be noted that these analyses indicate the adequacy for diagnosis of the set of tests used in the tables; the distinctness of the taxa based on the very large number of tests described by us previously (46) is a good deal greater. The results of DNA studies (13,28, 34,35) also support the distinctness of the taxa. There has been considerable disquiet for many years about the genera Actinobacillus and Pasteurella and their relationship to the genus Haemophilus (24). The generic arrangements within the family Pasteurellaceae are currently undergoing considerable change. Thus, Haemophilus pleuropneumoniae and pasteurellae causing porcine necrotic pleuropneumonia have been transferred to the genus Actinobacillus (36), Pasteurella ureae is more closely related to A . lignieresii than to P. multocida (13), and 16s rRNA sequences indicate that Actinobacillus actinomycetemcomitnns is more closely related to Haemophilus injuenzae than to A. lignieresii (12). In this study we assigned the new species to the genus Actinobacillus or the genus Pasteurella according to whether they seem more closely related, on the basis of presently available evidence, to the type species A . lignieresii or the type species P. multocida, although we acknowledge that the generic assignments of the new species are highly tentative and perhaps dubious. These assignments are used for want of something better to get the species on record. It is increasingly difficult to differentiate the genera Actinobacillus, Haemophilus, and Pasteurella (22, 25); if these taxa are merged, it should be noted that Pasteurella, as the oldest name and a conserved name, would take precedence according to Rule 24b of the International Code of Nomenclature of Bacteria (19,23,39). We retained P. ureae in Table 2 in its more familiar position (11) rather than transfer it to the genus Actinobacillus as recently suggested (32) because we feel that the evidence from DNA (32) and phenotypic (46) studies is not sufficiently compelling in a situation where wholesale rather than piecemeal revision is needed. There is evidence that strains named Actinobacillus seminis are heterogeneous (46,50), and it is likely that more than
MATERIALS AND METHODS
Full details of the materials and methods which we used have been published previously (46). The strains were tested in a wide range of conventional tests, and Gower similarity coefficients were calculated based on 155 unit characters. The organisms were clustered by using the unweighted pair group method with averages; details of this method are described in reference 45. Overlap statistics were calculated by using the OVERMAT program (44) with 23 phena to ensure that they were phenotypically distinct. The most diagnostic characteristics of the phena were obtained from the DIACHAR prOgram (42). The salient properties of previously validly published Actinobacillus and Pasteurella species, together with the properties of the new species, are listed in Tables 1 and 2. The quality of the tables for identification was evaluated by using the OVERMAT program and the MOSTTYP program (43). For this evaluation the symbols in Tables 1 and 2 were coded as follows: f , 95%; (+) and +L, 85%; d, +w, dw, wL, dL, and NT, 50%; (-), 15%; and -, 5%. These values are the midpoints of the ranges of the scores in the tables or were coded as 85 or 50% because of gaps or difficulties in reading weak and late reactions. RESULTS AND DISCUSSION The internal evaluation of the diagnostic tables (Tables 1 and 2) was broadly satisfactory. The MOSTTYP program showed that the most typical possible strains (hypothetical median organisms) identified correctly with their own taxa with a Willcox probability value of more than 0.99, except for Pasteurella stomatis (0.98) and Pasteurella canis (0.82). The OVERMAT program showed no overlap that reached the 8.2% level, which indicates continuous variation (i.e., that two taxa run together phenotypically). All taxon pairs
* Corresponding author. 148
VOL.40, 1990
NEW ACTINOBACILLUS AND PASTEURELLA SPECIES
149
TABLE 1. Differential characteristics of the species belonging to the genus Actinobacillus” 0,
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-, 510% of the strains are positive; w, weak reaction; L, late reaction; NT, not tested.
ONPG reaction, Hydrolysis of ortho-nitrophenyl-P-galactopyranoside. Positive according to Piechulla et al. (34) and negative according to Sneath and Stevens (46). Different formulations of this medium may explain discrepancies in the reported results; the results for A . pleurupneurnoniae are for medium supplemented with NAD. f In Hugh-Leifson medium (17) containing glucose, with incubation for 48 h at 37°C. g Positive according to Sneath and Stevens (46) and negative according to Kilian and Frederiksen (21) and Phillips (33).
one species is implicated in epididymitis of sheep or that strains have been misidentified because of the unusually wide range of guanine-plus-cytosine (G+C) ratios, 37.8 to 48.8 mol% (16). A possible relationship to “Haemophilus agni” (34) is indicated by a high DNA-DNA pairing value to one strain (strain M650-1343) of this species, but interpretation is uncertain due to doubt over the status of “H. agni,” whose name at present has not been validly published, and strain M650-1343 may perhaps have been misidentified. The revival ofA. seminis fixes the species on one of the strains of Baynes and Simmons (l),who originally named it. Pastewella bettii seems to be a distinctive organism, similar to members of the P. ureae complex but less metabolically active. We have been unable to find valid publication of this name before 1980; therefore, it cannot be revived, so we proposed P. bettii below as a new species. Nor have we been able to trace the origin of the specific
epithet, but we retain it for reasons of stability. Without a valid name, this organism may not be recognized if it occurs infrequently. Similarly, Pasteurella Zyrnphangitidis may be not uncommon if it is looked for. Differences between biovars A and T of Pasteurella haemolytica have been noticed for a long time (2,11,40,41), and these organisms are distinct on the basis of phenotypic and DNA data (26, 35, 38, 46). It seems likely that Pasteurella trehalosi comprises biogroups 2 and 4 of Bisgaard and Mutters (7) and Mutters et al. (26), and P. haernolytica sensu strict0 comprises biogroups 1, 5, 6, 7, and 9, but this needs further study. A number of other groups have recently been recognized in the genus Pasteurella (4, 7, 8, 22). We have not been able to identify the new species proposed here with any of these groups. It is likely that many new species await description. However, one group, the SP group, appears to be taxon 22 of
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42.32 pm long. Gram reaction is negative. Endospores are not formed. Growth is aerobic and facultatively anaerobic; mesophilic. Surface colonies grown aerobically on sheep blood agar are round, greyish, semitransparent, and about 2 mm in diameter after 48 h at 37”C, sometimes showing weak hemolysis without marked greening of the erythrocytes. Notable for producing acid from m-inositol and sorbitol, but not from sucrose, and for reducing Janus green (46). Other properties are shown in Table 1. Isolated from the vaginas of postparturient sows and aborted piglets (37, 46). Serological cross-reactions have been reported with Actinobacillus suis, A . lignieresii, Actinobacillus equuli, and A . seminis, and P . haemolytica (37). DNA-DNA pairing values of about 50% or less to A . lignieresii, A . suis, A . equuli, P . haemolytica sensu stricto, and P . ureae have been reported (13, 35). The G+C content of the DNA of the type strain is 41.9 mol%, as determined by the thermal denaturation (T,) method (25, 34). The type strain is strain ATCC 27072 (= NCTC 10801 = Ross 192 = P624 = A55 and A68) (46). In our hands this strain was positive for the tests marked d in Table 1, except for gas production from glucose and acid production from lactose. Actinobacillus seminis sp. nov., nom. rev. (ex Baynes and Simmons 1960). Actinobacillus seminis (sem’in.is. M.L. gen. n. seminis, of semen). Phenon 14 of Sneath and Stevens (46); “Actinobacillus seminis” of Kilian and Frederiksen (21) and Phillips (33). Cells are small nonmotile bacilli and coccobacilli that are often >2 k m long. Gram reaction is negative. Endospores are not formed. Growth is aerobic and facultatively anaerobic, mesophilic, often requiring 5% CO, on first isolation. Surface colonies grown aerobically on sheep blood agar are round, greyish, semitransparent, about 1 mm in diameter after 48 h at 37”C, and without hemolysis but sometimes with greening of the erythrocytes. Notable for slow and weak fermentation of carbohydrates, for producing no acid from maltose or mannose, and for giving a negative phosphatase reaction (46). Other properties are shown in Table 1. Cause of epididymitis of sheep (1) and isolated from semen; may be involved in polyarthritis of sheep (33). Phenotypically similar to P . ureae (46), but DNA-DNA pairing values are less than 25% to P . ureae, P. multocida,
NEW ACTINOBACILLUS AND PASTEURELLA SPECIES
151
Pasteurella pneumotropica, A . lignieresii, A . equuli, A . suis, Haemophilus injluenzae, and Haemophilus aphrophilus, (13, 34). The G+C content of the DNA of the type strain is 43.7 mol% (T,n method) (25). The type strain is strain NCTC 10851 (= ATCC 15768 = Simmons K3844-C = A51 and A65 = 5 ) (34, 46). In our hands this strain was negative for the tests marked d in Table 1, except for late reactions in the tests for ornithine decarboxylase and acid production form m-inositol and maltose. Pasteurella bettii sp. nov. Pasteurella bettii (betti’i. M.L. gen. n. bettii, of Bett, named for a gentleman named Bett [Catalog of Strains, 8th ed., American Type Culture Collection, Rockville, Md., 19681, whose identity has not been traced). Phenon 13 of Sneath and Stevens (46); “Pasteurella sp. bettii” of Kilian et al. (22), Cells are small nonmotile bacilli and coccobacilli that are often
