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Actinobacillus Rossii Sp INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1990, p. 148-153 Vol. 40, No. 2 0020-7713/90/020148-06$02.00/0 Copyright 0 1990, International Union of Microbiological Societies 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 Acti- showed overlap values of less than 1% except for the nobacillus and Pasteurella (46) identified several phena that following pairs: Pasteurella multocida and Pasteurella canis have not yet been formally named. In this paper we describe (2.4%); P. multocida and Pasteurella stomatis (1.3%); P. six of these phena as new species, based on evidence that the canis and P. stomatis (2.2%); Pasteurella aerogenes and groups form statistically distinct phenotypic clusters and Pasteurella mairi (1.1%); and P. aerogenes and Actinobacil- also on evidence from DNA studies. lus lignieresii (1.4%). It should be noted that these analyses indicate the adequacy for diagnosis of the set of tests used in MATERIALS AND METHODS the tables; the distinctness of the taxa based on the very Full details of the materials and methods which we used large number of tests described by us previously (46) is a have been published previously (46). The strains were tested good deal greater. The results of DNA studies (13,28, 34,35) in a wide range of conventional tests, and Gower similarity also support the distinctness of the taxa. coefficients were calculated based on 155 unit characters. There has been considerable disquiet for many years The organisms were clustered by using the unweighted pair about the genera Actinobacillus and Pasteurella and their group method with averages; details of this method are relationship to the genus Haemophilus (24). described in reference 45. Overlap statistics were calculated The generic arrangements within the family Pasteurel- by using the OVERMAT program (44) with 23 phena to laceae are currently undergoing considerable change. Thus, ensure that they were phenotypically distinct. The most Haemophilus pleuropneumoniae and pasteurellae causing diagnostic characteristics of the phena were obtained from porcine necrotic pleuropneumonia have been transferred to the DIACHAR prOgram (42). the genus Actinobacillus (36), Pasteurella ureae is more The salient properties of previously validly published closely related to A. lignieresii than to P. multocida (13), and Actinobacillus and Pasteurella species, together with the 16s rRNA sequences indicate that Actinobacillus actino- properties of the new species, are listed in Tables 1 and 2. mycetemcomitnns is more closely related to Haemophilus The quality of the tables for identification was evaluated by injuenzae than to A. lignieresii (12). In this study we using the OVERMAT program and the MOSTTYP program assigned the new species to the genus Actinobacillus or the (43). For this evaluation the symbols in Tables 1 and 2 were genus Pasteurella according to whether they seem more coded as follows: f, 95%; (+) and +L, 85%; d, +w, dw, closely related, on the basis of presently available evidence, wL, dL, and NT, 50%; (-), 15%; and -, 5%. These values to the type species A. lignieresii or the type species P. are the midpoints of the ranges of the scores in the tables or multocida, although we acknowledge that the generic assign- were coded as 85 or 50% because of gaps or difficulties in ments of the new species are highly tentative and perhaps reading weak and late reactions. dubious. These assignments are used for want of something better to get the species on record. It is increasingly difficult RESULTS AND DISCUSSION to differentiate the genera Actinobacillus, Haemophilus, and Pasteurella (22, 25); if these taxa are merged, it should be The internal evaluation of the diagnostic tables (Tables 1 noted that Pasteurella, as the oldest name and a conserved and 2) was broadly satisfactory. The MOSTTYP program name, would take precedence according to Rule 24b of the showed that the most typical possible strains (hypothetical International Code of Nomenclature of Bacteria (19,23,39). median organisms) identified correctly with their own taxa We retained P. ureae in Table 2 in its more familiar with a Willcox probability value of more than 0.99, except position (11) rather than transfer it to the genus Actinobacil- for Pasteurella stomatis (0.98) and Pasteurella canis (0.82). lus as recently suggested (32) because we feel that the The OVERMAT program showed no overlap that reached evidence from DNA (32) and phenotypic (46) studies is not the 8.2% level, which indicates continuous variation (i.e., sufficiently compelling in a situation where wholesale rather that two taxa run together phenotypically). All taxon pairs than piecemeal revision is needed. There is evidence that strains named Actinobacillus sem- * Corresponding author. inis are heterogeneous (46,50), and it is likely that more than 148 VOL. 40, 1990 NEW ACTINOBACILLUS AND PASTEURELLA SPECIES 149 TABLE 1. Differential characteristics of the species belonging to the genus Actinobacillus” 0, ._ i 8 6 @ B .U $ -*; ._ Test $u, s .9 8 .Y ;=: .s .\h .@ ::: .s = u, 2 .Y uou .2 2 :s $ % -sE & 2 4 s 2 h Tu i 9 9 * 9 9 i .;, 9 Catalase + - d + Oxidase + + (+) + Nitrate reduction + + + + ONPG reactionC + + d - Phosphatase + + + - Ornithine decarboxylase - Indole production - Urease + Esculin hydrolysis + ,L NAD requirement - Growth on MacConkey agaf + Beta-hemolysis (sheep cells) - Fermentatiod NT Gas production from glucosef - Acid production from: L- Arabinose - Arbutin NT Cellobiose + Dulcitol - Galactose + rn-Inositol - Lactose + Maltose + Mannitol + Mannose + Melibiose + Raffinose + Salicin + Sorbitol + Sucrose + Tre halose + + - + D-Xylose + + + - G+C content (mol%, range) 42.4 40.9 41.8-43.2 46.9 a Data from references 5-8, 10, 11, 15, 20, 21, 24, 27-31, 33, 34, 46, and 47. ‘ +, 290% of the strains are positive; (+), 80 to 89% of the strains are positive; d, 21 to 79% of the strains are positive; (-), 11 to 20% of the strains are positive; -, 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 epithet, but we retain it for reasons of stability. Without a strains have been misidentified because of the unusually valid name, this organism may not be recognized if it occurs wide range of guanine-plus-cytosine (G+C) ratios, 37.8 to infrequently. Similarly, Pasteurella Zyrnphangitidis may be 48.8 mol% (16). A possible relationship to “Haemophilus not uncommon if it is looked for. agni” (34) is indicated by a high DNA-DNA pairing value to Differences between biovars A and T of Pasteurella one strain (strain M650-1343) of this species, but interpreta- haemolytica have been noticed for a long time (2,11,40,41), tion is uncertain due to doubt over the status of “H. agni,” and these organisms are distinct on the basis of phenotypic whose name at present has not been validly published, and and DNA data (26, 35, 38, 46). It seems likely that Pas- strain M650-1343 may perhaps have been misidentified. The teurella trehalosi comprises biogroups 2 and 4 of Bisgaard revival ofA. seminis fixes the species on one of the strains of and Mutters (7) and Mutters et al. (26), and P. haernolytica Baynes and Simmons (l),who originally named it. sensu strict0 comprises biogroups 1, 5, 6, 7, and 9, but this Pastewella bettii seems to be a distinctive organism, needs further study. similar to members of the P. ureae complex but less meta- A number of other groups have recently been recognized bolically active. We have been unable to find valid publica- in the genus Pasteurella (4, 7, 8, 22). We have not been able tion of this name before 1980; therefore, it cannot be to identify the new species proposed here with any of these revived, so we proposed P. bettii below as a new species. groups. It is likely that many new species await description. Nor have we been able to trace the origin of the specific However, one group, the SP
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