INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1986, p. 1-7 Vol. 36, No. 1 0020-771 3 /86/0 10001 -07$02.00/0 Copyright 0 1986, International Union of Microbiological Societies

Deoxyribonucleic Acid Relationships of “Histophilus ovislHaemophilus somnus,’ ’ Haemophilus haemoglobinophilus, and “Actinobacillus seminis”

K. PIECHULLA,l R. MUTTERS,’ S. BURBACH,l R. KLUSSMEIER,’ S. POHL,2 AND W. MANNHEIM1* Zentrum fur Hygiene und Medizinische Mikrobiologie der Philipps- Universitat, Abteilung Bakteriologie, 0-3550Marburg,‘ and Hygiene-Institut der Ruprecht-Karls-Universitat,0-6900 Heidelberg,2 Federal Republic of Germany

Three Australian isolates of “Histophilus ovis,?’ ten strains of “Haemophilus somnus” from North America, Australia, and Europe, and two American strains of “Haemophilus agni” were investigated by the deoxyribonucleic acid (DNA)-DNA hybridization (renaturation) method to determine their genetic interrela- tionships and their levels of relatedness to recognized wembers of the family Pasteurellaceae Pohl 1981. Our results confirmed that “Haemophilus somnus,” “Histophilus ovis,” and one of the “Haemophilus agni” strains studied represent one genetically homogeneous species. This species exhibited up to 41 % DNA relatedness to Haemophilus haemoglobinophilus, whereas only insignificant levels of relatedness or no measurable DNA binding was observed with the type species of the genera Actinobacillus, Haemophilus, and Pasteurella and with Haemophilus aphrophilus, Haemophilus ducreyi, Haemophilus paragallinarum, Haemophilus parainfluenzae, Haemophilus segnis, Pasteurella avium, Pasteurella ureae, and “Actinobucillus seminis. ” On the other hand, one of the “Haemophilus agni” strains studied (Hoerlein strain M650-1343) was included in the species “Acfinobacillus seminis” (DNA binding value, 91%). So far, only low levels of genetic relatedness with “Actinobacillus seminis” and currently recognized members of the family Pasteurellaceae have been detected. The problems of the generic affiliation of the “Histophilus ovis” group, Haemophilus haemoglobinophilus, and “Actinobucillus seminis” and the phenotypic differentiation of these organisms are discussed.

The hitherto unrecognized taxa “Histophilus ovis” “Haemophilus somnus,” and ‘Haemophilus agni” strains Roberts 1956 (28), “Haemophilus agni” Kennedy et al. 1958 are genetically related at the species level but are not (lo), “Actinobacillus seminis” Baynes and Simmons 1960 significantly related to “Actinobacillus seminis” and a strain (2), “Haemophilus somnus” Bailie 1969 (W. E. Bailie, of Haemophilus inJIuenzae (32). Thus, “Histophilus ovis” Ph.D. thesis, Kansas State University, Manhattan, 1969), and “Actinobacillus seminis” appear to be two distinct and “Haemophilus somnifer” Miles et al. 1972 (19) contain species of the Pasteurellaceae without generic affiliation. important pathogens that have been isolated from domestic In this paper we present DNA-DNA hybridization data for ruminants in Australia, North America, Europe, and South additional strains of “Histophilus ovis,” “Haemophilus Africa. “Actinobacillus actinoides’ ’ and “Haemophilus somnus,’ ’ “Haemophilus agni,” “Actinobacillus seminis, ” citreus” are probably additional synonyms for at least one of and Haemophilus haemoglobinophilus and reference cul- these taxa (30). Details of the histories of these organisms tures representing additional taxa of the Pasteurellaceae. In have been described elsewhere (3, 9, 12, 30, 32). Although this study we also considered DNA base composition and these bacteria fit in the family Pasteurellaceae Pohl on the genome masses and the significance of some phenotypic basis of their phenotypic properties (17), they were not features for differentiation of the genetic groups established. included in the genera Actinobacillus (23, 29) and Haemo- philus (12, 29). Genetically, strains representing “Haemo- MATERIALS AND METHODS philus somnus,” “Haemophilus agni,” and “Actinobacillus Bacterial strains and growth conditions. The bacterial seminis” were placed in the family Pasteurellaceae on the strains investigated and their origins are listed in Table 1. basis of 16s ribosomal ribonucleic acid-deoxyribonucleic Starting from lyophilized cultures, cloned working cultures acid (DNA) hybridization data (M. Van Houcke, Ph.D. on enriched chocolate agar (see below) were used to inoculate thesis, University of Gent, Gent, Belgium, 1983). However, mass cultures and identification test media. “Haemophilus this study did not discriminate among generic groups of the somnus,” “Haemophilus agni,” and “Histophilus ovis” Pasteurellaceae De Ley, personal communication). (J. strains were grown in tryptic soy broth (Difco Laboratories, Attempts to classify the organisms in question at the Detroit, Mich.) supplemented with 0.4% (wt/vol) yeast species level have been more successful. Workers have extract (Oxoid Ltd., London, United Kingdom), 2.0% presented biochemical, serological, and cytochemical evi- (vol/vol) horse serum (Oxoid), 2 mg of thiamine monophos- dence that bacterial strains labeled “Histophilus ovis,” phate (TMP) per liter or 2 mg of pyrophosphate (Sigma “Haemophilus agni,” and “Haemophilus somnus” are Chemical Co., Munich, Federal Republic of Germany) per highly interrelated and may represent a single taxon which is liter (1, 18), and 0.5 g of cysteine hydrochloride (Roth, antigenically related to “Actinobacillus seminis” and Hue- Karlsruhe, Federal Republic of Germany) per liter (30). All mophilus haemoglobinophilus to some extent, but is not supplements were filter sterilized before use. Precultures related to the type species of the genera Haemophilus and were incubated statically in 1-liter batches at 37°C for 8 to 16 Actinobacillus (30). Recent DNA-DNA hybridization stud- h and then adjusted with filter-sterilized 1 M NaHC03 to pH ies have shown that a collection of “Histophilus ovis,” 7.0 and transferred in 500-ml volumes into 2-liter Fernbach flasks. The cultures were shaken moderately until the early * Corresponding author. stationary phase of growth and harvested as described

1 2 PIECHULLA ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Bacterial strains used in this study and their DNA base compositions

~~ Serial G+C content Taxon (as received) Collection no.“ Other designation(s) and origin no. (mol%)h 1 Actinobacillus actinomyceterncomitans NCTC 9710T‘ P. Holm 277l49-50; human 47.1 2 Actinobacillus equuli NCTC 8529T W. C. Miller PM30753; foal 42.9 3 Actinobacillus lignieresii NCTC 4189T L. Thompson 1; cattle, glands, United States 43.4 4 Actinobacillus lignieresii NCTC 4976 P. H. Martin CM2; human 43.4 5 “Actinobacillus seminis’’ ATCC 15768 G. C. Simmons K3844-C; ovine, epididymitis, Australia 43.7 (2)“ 6 Actinobacillus sp. ATCC 27072T R. F. Ross 192; sow, vaginal discharge, United States 41.9 7 ActinobacilluslPasteurella-like HIM 744-4 M. Bisgaard F283 (taxon 2); White Pekin duck, 39.4 peritonitis, Denmark (24) 8 ActinobacilluslPasteurella-like HIM 797-5 M. Bisgaard 3.3 (taxon 5); guinea pig, throat, Denmark 42.1 (5) 9 HIM 794-718 M. Bisgaard 10.4 (taxon 7); guinea pig, throat, Denmark 42.1 (5) 10 ActinobacilluslPasteurella-like NCTC 11412 M. Bisgaard F151 (taxon 3); White Pekin duck, 39.3 salpingitis, Denmark (24) 11 Haemophilus aegyp t ius ATCC 11116T M. Pittman 180-a; human, conjunctiva, United States 38.9 12 Haernophilus aphrophilus NCTC 5886 0. Khairat PM1; human, endocarditis, United Kingdom 43.5 13 Haernophilus aphrophilus NCTC 5906T 0. Khairat 320; human, endocarditis, United Kingdom 43.6 14 Huemophilus ducreyi CIP 54.2T P. Kirsche X2; human, Viet Nam 38.0 15 Haemophilus ducreyi ATCC 27722 C. Zierdt 1X: human, United States 37.6 16 Haernophilus haemoglobinophilus NCTC 1659T P. Fieldes XIII; dog. prepuce, Denmark 37.9 17 Haemophilus haemoglobinophilus NCTC 8540 K. Zinnemann; greyhound, United Kingdom 37.5 18 Haemophilus influenzae NCTC 4560 H. J. Benstedt RAMC 18; human, United Kingdom 38.8 19 Haemophilus injluenzae NCTC 8143T A. B. Rosher 680; human, sputum, United Kingdom 39.0 20 Haemophilus paraphrophilus-like ATCC 7901 M. Pittman 429; human, United States 42.8 21 Haernophilus paragallinarum ATCC 29545’‘ K.-H. Hinz IPDH 2403; chicken, sinus, Federal 42.2 Republic of Germany 22 Haernophilus parahaemolyticus NCTC 8479T M. Pittman 536; human, pneumonia, United States 41.4 23 Huemophilus parainguenzae biotype I1 HIM 170-1 G. Leidy Bossy no. 7; human, United States 41.5 24 Haernophilus parainfluenzae biotype 1 HIM 793-8 M. Kilian HK128; human, sputum, Demark 41.7 25 Haemophilus parainfluenzae biotype I1 HIM 572-1 W. Mannheim (Marburg) 20677-74; humam, sputum, 41.9 Federal Republic of Germany 26 Haernophilus parasuis NCTC 4557T R. E. Shope 1374; swine, United Kingdom 40.4 27 Haernophilus segnis NCTC 10977T M. Kilian HK316; human, dental plaque, Denmark 45.3 28 “Haemophilus agni” HIM 911-2 E. L. Biberstein 902; ovine, septicemia, United States 37.7 (32) 29 “Haemophilus agni” HIM 494-5/6 A. D. Hoerlein M650-1343; ovine, septicemia, United 41.3 States 30 “Haernophilus somnus” HIM 734-5 L. Corboz 719; bull, semen, Switzerland (6) 37.5 31 “Haernophilus somnus’ ’ HIM 734-4 L. Corboz 606; calf, lung, Switzerland (6) 38.1 32 “Haernophilus somnus” HIM 734-6 L. Corboz 43; bovine, uterus, Switzerland (6) 37.0 33 “Haernophilus somnus” HIM 734-7 L. Corboz 679; calf, joint, Switzerland (6) 38.7 34 “Haemophilus somnus” HIM 734-8 L. Corboz 8025; bovine, brain, Switzerland (6) 37.5 35 “Huernophilus somnus” HIM 738-5 R. F. Ross IVDL 1080; bovine, trachea, United States 38.0 36 “Haemophilus somnus” HIM 673-516 R. F. Ross IVDL 1229; bovine, brain, United States 38.8 37 “Haernophilus somnus” HIM 849-1 L. R. Stephens 43826; bovine, brain, Canada (30) 38.6 38 “Haemophilus somnus” HIM 911-3 E. L. Biberstein 805; bovine, lung, United States (34) 37.1 39 “Haemophilus somnus”-like HIM 849-5 APCC UQV 179; bovine, lung. Australia (30)’ 38.4 40 “Histophilus ovis” HIM 874-5 L. R. Stephens 43803, ovine, brain, Canada (30) 38.1 41 “Histophilus ovis” HIM 896-7 R. S. Rahaley H989; ovine, kidney, Australia (26, 27, 38.0 30)” 42 “Histophilus ovis ” HIM 896-6 R. Higgins 9L; ovine, vagina, Canada (30, 32)” 38.1 43 Haemophilus-like NCTC 11877 K.-H. Hinz IPDH 312; pigeon, repiratory tract, Federal 37.0 Republic of Germany (Piechulla et al., in press) 44 Haemophilus-like NCTC 11876 K.-H. Hinz IPDH 195; budgerigar, trachea, Federal 38.1 Republic of Germany (Piechulla et al., in press) 45 Haemophilus-like NCTC 11878 K.-H. Hinz IPDH 2176; kestrel, lung, Federal Republic 45.9 of Germany (Piechulla et al., in press) 46 Pasteurella avium ATCC 29546T K.-H. Hinz IPDH 2654; chicken, infraorbital sinus, 44.7 Federal Republic of Germany (21) 47 ‘ ‘Pasteurella bettii” NCTC 10535 D. G. Hollis CDC 41-5568; human, United States 38.6 48 Pusteurelfa canis NCTC 11621T J. E. Smith 182; dog, United Kingdom (20) 37.8 49 Pasteurella dagrnatis NCTC 11617T S. D. Henriksen 953/60; human, Norway (20) 40.0 50 Pasteurella haemolytica NCTC 10610 B. J. Shreeve S5R; sheep, United Kingdom 42.3 51 Pasteurella multocida NCTC 10322’ G. R. Carter W-9217; porcine, Canada 41.6 52 Pasteurella multocida NCTC 3195 H. Schutze HS; bovine, United Kingdom 40.8 53 “Pasteurella multocida” SSI P614 W. Frederiksen P614, Namioka, TS8; chicken, Viet 40.9 Nam (20) Continued on .following page VOL.36, 1986 DNA RELATIONSHIPS 3

TABLE 1-Continued Serial G+C content Taxon (as received) Collection no.” Other designationfs) and origin no. (mol%Ih 54 Pasteurella pneumotropica NCTC 8284 M. T. Parker 54818; mouse, United Kingdom 42.8 55 Pastewella stomatis NCTC 11624 W. Mannheim PE4; cat, Federal Republic of Germany 40.4 (20) 56 Pasteurella ureae NCTC 10219T S. D. Henriksen 3529/59; human, ozeana, Norway 41.2 57 Pasteurella volantium NCTC 343gT R. Love11 6; fowl, United Kingdom (21) 43.8 58 Pasteurella volantium NCTC 4101 A. Fleming 1; human, United Kingdom (21) 44.6 59 Pasteurella species A HIM 789-5 K.-H. Hinz IPDH 280; chicken, infraorbital sinus, 45.9 Federal Republic of Germany (21) 60 “Pasteurella’’ sp. HIM 821-6 W. Frederiksen P434; mouse, Denmark 47.9 APCC, Animal Pathogen Culture Collection, University of Queensland, Australia; ATCC, American Type Culture Collection. Rockville. Md.; CIP, Collection de l’lnstitut Pasteur. Paris, France; HIM, Zentrum fur Hygiene und Medizinische Mikrobiologie, Marburg. Federal Republic of Germany; IPDH, Institute for Poultry Disease, Hannover. Federal Republic of Germariy; NCTC. National Colleciton of Type Cultures. Central Public Health Laboratory, London, England; SSI, Statens Seruminstitut, Copenhagen, Denmark. Averages of nine assays. ‘ T = type strain. * The numbers in parentheses are reference numbers. ‘ Strain obtained from L. R. Stephens.

previously (24). For these preparations chocolate agar ranges; the mean guanine-plus-cytosine (G +C) content was (tryptic soy agar containing 5% [vol/vol] sheep blood heated 38 5 0.6 mol%, and the mean genome mass was 1.5 x lo9. for 15 min to 80°C before pouring) was enriched with TMP and A somewhat lower G+C content was reported by Bailie cysteine as described above. The other organisms investi- (Ph.D. thesis). gated were grown on chocolate agar without TMP or cysteine Our genomic data confirm the statement of Walker et al. or in Difco Proteose Peptone medium (33) supplemented with (32) that “Haemophilus somnus” and “Histophilus ovis” 0.5% (wthol) yeast extract, P-nicotinamide adenine dinucle- have to be regarded as one well-defined species. UnfQrtu- otide, and hemin, when necessary (17). nately, none of the original “Histophilus” strains of Roberts Phenotypic characterization. Most of the media and re- (28) seems to have survived in culture collections. However, agents used for testing biochemical properties were those the isolates described by Rahaley and White (27), e.g., strain described previously by Mannheim et al. (17). Exceptions H989 (26), may be used to represent this taxon. were the catalase reaction, which was examined by the Only two strains labeled “Haemophilus agni” were inves- method of Kilian (ll), and the ortho-nitrophenyl-p- tigated, and these strains were found to be genetically galactopyranoside test, which was performed with ortho- heterogeneous. One strain (strain Biberstein 902) had a G+C nitrophenyl-P-galactopyranoside disks (Institut Pasteur, content of 37.7 mol% and was placed in the “Histophilus Paris, France). Other hydrolase reactions were tested by ovis” group, in hgreement with Walker et al. (32) (Table 3), using a commercial set of substrates (API ZYM; APT whereas the other strain (Hoerlein strain M650-1343) had a System SA, La Balme les Grottes, Montalieu-Vercieu, G+C content of 41.3 mol% and no measurable level of DNA France) as suggested by the manufacturers and late expo- relatedness to this group. This strain also differed phenotyp- nential cells from chocolate agar cultures suspended in saline ically from the “Histophilus ovis” group strains in some to a concentration of about 0.5 mg of bacterial dry weight per respects (see below). However, Hoerlein strain M650-1343 ml . was placed with the other species incertae sedis, Molecular methods. DNA was prepared and characterized “Actinobacillus seminis,” on the basis of its high level of and DNA-DNA hybridization by the renaturation method DNA relatedness to the reference strain of that taxon (Table was carried out as described previously (20, 21, 24, 31; K. 3). Since Hoerlein strain M650-1343 is one of the earliest Piechulla, K.-H. Hinz, and W. Mannheim, Avian Dis., in reference strains of “Haemophilus agni” available (7), we press). The pooled standard deviation of the DNA binding concluded that this taxon consists of organisms previously data (see Tables 2 through 4) was 5.5%. classified as “Histophilus ovis” or, later, as “Actinobacillus semin is.’ ’ RESULTS AND DISCUSSION Generic affiliation of the “Histophilus ovis” group and of Classification to the species level. The cultures of “Haemo- “Actinobacillus seminis.” Although both the “Histophilus philus somnus” and “Histophilus ovis” which we investi- ovis” group and “Actinobacillus seminis” fit well in the gated formed a homogeneous genetic cluster with a level of family Pasteurellaceae on the basis of genome DNA base DNA relatedness of more than 91% (DNA bindifig value) composition and genome mass, our attempts to place these (Table 2). These findings agree with the results of Walker taxa by using DNA-DNA hybridization into one of the and co-workers (32), who used the S1 nuclease-trichloroac- presently described genera or genus-like groups of the family etic acid precipitation method for DNA hybridization. Ex- failed. cept for strains Biberstein 805, Biberstein 902, and Higgins In particular, low levels of DNA relationship or no mea- 9L, the collection of strains used by Walker et al. was surable DNA relationship was detected between different from that used in this study. “Histophilus ovis” and the type strains of Haemophilus The DNA base compositions (Table 1) and the genome injuenz a e , Haem op h il us uph rop h il us, Haem op hi1 us masses (data not shown) of the DNAs of the members of the paragallirtarum, and Haemophilus segnis, as well as be- “Histophilus ovis” group studied varied within narrow tween “Histophflus ovis” and members of the genera Pas- 4 PIECHULLA ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Levels of DNA homology for “Huemophilus somnus” TABLE 3. Levels of DNA relatedness for strain Corboz 719 “Actinobucillus seminis” ATCC 1576gT

Degree Strain Degree of Strain af Taxon Taxon (serial no.) binding (serial no.) binding (%I (%I“ “Haemophilus agni” 29 91 “Haemophilus agni” 28 looh Pasteurella pneumotropica 54 23 “Haemophilus agni” 28 94 Pasteurella ureae 56 22 “Histophilus ovis ” 42 100’ Haemophilus aphrophilus 12 21 “Histophilus ovis” 42 92 Actinobacillus equidi 2 20 “Histophilus ovis’ ’ 41 looJ Haemophilus haemoglobinophilus 17 19 “Histophilus ovis ” 41 99‘ Haemophilus puruinjhenzae 23 17 ‘ ‘Haernophilus somnus” 34 99‘ Haemophilus haemoglobinophilus 16 16 “Haemophilus somnus 37 98 Pasteurella multocidu 52 13 “Haernophilus somnus”-like 39 97 Actinobacillus actinomycetemcomitans 1 13 “Haernophilus somnus” 33 9d “Pasteurella bettii” 47 10 “Haemophilus somnus” 31 96 “Haemophilus somnus” 30 8 ‘ ‘Haemophilus somnus” 38 95 Actinobacillus lignieresii 4 7 “Haemophilus somnus” 36 94 Haemophilus influenzae 18 6 “Haemophilus somnus” 35 9 lJ Pasteurella volantium 58 6 “Huemophilus somnus” 31 9 lJ “Pasteurella” sp. 60 4 Huemophilus haemoglobinophilus 16 41” Taxon 7 9 0 Haemophilus haemoglobinophilus 16 38 Taxon 5 8 0 Haemophilus haemoglobinophilus 16 35“ Huemophilus ducreyi 14 0 Haemophilus haemoglobinophilus 17 39 Huemophilus ducreyi 15 0 Huemophilus ducreyi 15 24 Actinobucillus sp. 6 20 Pusteurella multocida 51 20” “Pasteurella multocida” 53 18 species (Piechulla et al., in press), which all lack generic Huemophilus segnis 23 16“ affiliation so far and may represent several additional genera. Actinobucillus lignieresii 3 14K A significant level of DNA relatedness was found only “Haemophilus agni” 29 9h with “Histophilus ovis” and Haemophilus haemoglobinoph- ‘ ‘Actinobacillus seminis” 5 8 ilus (DNA binding range, 35 to 41%). This is the only link Pasteurella avium 46 3 Pusteurella ureae 56 2 between the “Histophilus ovis” group and established mem- Pasteurellu species A 59 0 bers of the Pasteurellaceae detected so far. However, DNA Huernophilus influenzae 19 0 hybridization data suggest exclusion of Haemophilus Haemophilus parainfluenzae biotype I 24 0 haemoglobinophilus from the genus Haemophilus sensu Haemophilus ducreyi 14 0 stricto rather than generic affiliation of “Histophilus” and ActinobacilluslPasteurella-like 7 0 allied strains via Haemophilus haemoglobinophilus (Table Haemophilus uphrophilus 13 0 4). Haemophilus parainfluenzae biotype I1 25 0 Similarly, the search for genetic relationships between A ctinobacilluslPasteurella-like 10 0 “Actinobacillus seminis” and members of the Pasteurel- Haemophilus paragallinarum 21 0 Haemophilus-like 43 0 Haemophilus-like 44 0 Haemophilus-like 45 0 TABLE 4. DNA relationships of Haemophilus a Unless otherwise indicated, strains were tested with “Hmmophilus haemoglobinophilus NCTC 1659T somnus” strain Corboz 719 (serial no. 30). Tested with “Haemophilus somnus” HIM 911-3 (serial no. 38). Degree of Strain ‘ Tested with “Histophilus ovis” HIM 874-5 (serial no. 40). Taxon binding Tested with “Huemophilus somnus” HIM 849-1 (serial no. 37). (serial no.) (%I Tested with “Histophilus ovis” HIM 896-7 (serial no. 41). fTested with “Haemophilus somnus” HIM 734-6 (serial no. 32). Haemophilus haemoglobinophilus 17 84 Tested with “Huemophilus somnus”-like strain HIM 849-5 (serial no. 39). Haemophilus somnus 30 41 Tested with “Haemophilus ugni” HIM 911-2 (serial no. 28). Actinobucillus lignieresii 3 36 (32)” Haemophilus inguenzue 18 29 Pusteurella rnultocidu 48 28 teurella and Actinobacillus (Table 2). As genetically defined Pasteurella pneumotropica 49 24 genera within the family Pasteurellaceae are clusters (con- Haemophilus influenzae 19 22 structed by single-linkage clustering) of species with DNA Huemophilus aphrophilus 12 21 binding levels of more than 50% (16, 20, 24), the Haemophilus parasuis 26 20 Pasteurella volantium 58 16 “Histophilus ovis’Bgroup certainly does not belong to the Haemophilus aegyptius 11 14 genus Haemophilus, to Pasteurella sensu stricto (20), to the Haernophilus parainfluenzae 23 13 Actinobacillus group, to the genus-like cluster consisting of Haemophilus purahuemolyticus 22 11 Bisgaard taxa 2 and 3 (24), or to the Haemophilus Pasteurella haemolytica 50 8 uph rop h il u s - Ha emop h il u s pa rap h r op hi1us - Hae m op h ilu s Haemophilus paraphrophilus-like 20 7 segnis group (S. Burbach, unpublished data). Furthermore, Pasteurella volantium 57 4 the “Histophilus ovis grsup exhibited no measurable DNA Pasteurellu stornatis 55 4

relate dne s s to Haem op h ilus du crey i , ‘ Actin o b a cill us a The number in parentheses is the value obtained with Haemophilus seminis,” or several other new avian Haemophilus-like huemoglobinophilus NCTC 8540 (serial no. 17). VOL. 36, 1986 DNA RELATIONSHIPS 5

TABLE 5. Selected phenotypic characteristics of “Actinobacillus 719 exhibited catalase activity. However, even with cata- seminis, ’’ “Histophilus ovis, ” and Haemophilus lase-negative strains, mass cultures in liquid media tolerated haemoglobinophilus moderate aeration during the exponential phase of growth, “Histophilus Hnemophilus with increasing growth yields. After prolonged incubation, “Actinobacillus ovis” serial hcremoglo- hazy zones of incomplete hemolysis developed around col- Characteristic seminis” serial no. 28 and binophilus onies on Columbia agar (Oxoid) containing 5% (vol/vol) no. 5 and 29 30 through serial no. 16 sheep blood. However, one strain (strain UQV 179) showed 42 and 17 prompt hemolysis. Demethylmenaquinones have been re- Catalase + + ported to be the only respiratory quinones produced by these Yellowish pigments - + organisms (8, 17). When we examined cells harvested from Alkaline phosphatase aerated cultures in rich media that supported growth yields TMPD oxidaseb up to 0.4 mg (bacterial dry weight) per ml, all members of the D-Sucrose reaction “Histophilus ovis” group contained ubiquinones in D-Fructose reaction m-Inositol reaction micromole amounts per gram of cell protein in addition to Hemoly sis demeth ylmenaquinones. D-Galactose reaction The conventional test media used for fermentation and D-Mannose reaction other biochemical reactions that require cell proliferation Salicin reaction (13, 17) are probably not optimal for investigating organisms Esculin hydrolysis belonging to this group, even when they are supplemented Ornithine decarboxylase with thiamine phosphate. Indole reactions were weakly - L-Arabinose reaction positive or negative; H2S (SIM agar [E. Merck AG, ONPG‘ d Darmstadt, Federal Republic of Germany]) and urease were Urease Maltose reaction not formed; nitrates were reduced to nitrites in Proteose Trehalose reaction Peptone-containing medium (33); and lysine and arginine D-Mannitol reaction media were not alkalized, whereas the ornithine D-Sorbitol reaction decarboxylase reaction was usually weakly positive after Dulcitol reaction heavy inoculation. Gelatinase, alkaline phosphatase, and Indole reaction + P-galactosidase (ortho-nitrophenyl-P-galactopyranoside)re- D-Glucose reaction (gas) actions were always negative. No growth occurred in Sim- Requirement for: mons citrate medium. Slow fermentative production of acid V factor without gas from D-glucose and D-mannose was regularly X factor TMP observed; varying reactions were obtained with D-galactose, D-fructose, D-xylose, L-arabinose, and D-mannitol. Forma- a + , Positive; (+), delayed positive; -, negative for 30 days; d, positive or tion of acid from L-sorbose, L-rhamnose, starch, D-lactose, negative; (d), delayed positive or negative; (w), weak positive. Fermentation salicin, esculin, D-sorbitol, m-inositol, adonitol, and dulcitol tests for the reference strain of “Actinobacillus seminis” (strain ATCC 15768) were performed in xenocultures with a nonfermenting Acinetobncfer strain was never observed in our standard media (17). Corboz and (15). Wild (6) obtained different results for fermentation reactions TMPD, N, N, N‘, N’-Tetramethyl-p-phenylenediamine. in their test system with some substrates by using initially ONPG, ortho-Nitropheny I-p-galactopyranoside. anaerobic incubation conditions, followed by incubation in Stephens et al. (30) described an otherwise typical strain that does not require TMP (thiamine monophosphate). air for several days. On the basis of the criteria shown in Table 5, the “Histophilus ovis” group can be differentiated from “Actinobacillus seminis,” Haemophilus haemoglobin- laceae yielded only low degrees of DNA binding (at most ophilus, and other genera or genus-like clusters of the 23%) (Table 3). Pasteurellaceae (13, 22). Phenotypic behavior of the “Histophilus ovis” group. The Furthermore, all previously described members of the organisms classified as “Histophilus ovis,” “Haemophilus Pasteurellaceae except ‘‘Actinobucillus seminis” were dif- somnus,” and “Haemophilus agni” (strain Biberstein 902) ferentiated from the “Histophilus ovis” group by their exhibited very similar phenotypic traits. These taxa con- characteristic patterns of hydrolase (API ZYM) reactions. sisted of small short, gram-negative, nonmotile, nonspore- This group and “Actinobacillus seminis” were characterized forming, rod-shaped bacteria that grew in various complex by lacking, or having weak, alkaline phosphatase reactions media at 36°C in an atmosphere containing reduced oxygen and by the constant presence of a strong P-glucuronidase and increased carbon dioxide tensions or anaerobically. and a leucine aminopeptidase (Table 6). Growth in peptone-containing media was considerably en- Phenotypic features of “Actinobacillus seminis.” Hoerlein hanced by adding whole blood, fresh yeast extract, co- strain M650-1343, which was originally classified as “Hue- carboxylase, and cysteine. After 48 h of incubation, smooth mophilus agni, grew more vigorously than “Histophilus convex colonies about 1 mm or less in diameter were ovis” strains even in unsupplemented peptone-containing obtained on unsupplemented chocolate agar (tryptic soy agar media, did not produce yellow pigments, fermented D- containing 5% [vol/vol] sheep blood heated for 15 min at glucose and D-fructose (but not D-mannose) promptly (17), 8OoC),and colonies up to 2 mm in diameter were obtained on and contained a P-galactosidase specific for ortho- Columbia agar (Oxoid) containing 5% (vol/vol) sheep blood. nitrophenyl-P-galactopyranoside.Delayed production of On chocolate agar supplemented with TMP and cysteine or acid from L-arabinose, D-xylose, maltose, and D-mannitol with Vitox (Oxoid), larger colonies appeared (up to 3 to 4 was observed. Very similar colonial and fermentation pat- mm in diameter) that contained pale yellowish pigments (30). terns were observed with the reference strain of “Ac- Colonial adherence or pitting was not observed. In our tinobacillus seminis” despite the higher degree of hands, all strains examined except one showed a slow type microaerophilia of the latter (17). The phenotypic similarity of oxidase reaction (17) and a lack of catalase; strain Corboz of the two cultures is not surprising since they are genetically 6 PIECHULLA ET AL. INT. J. SYST.BACTERIOL.

TABLE 6. API-ZYM reactions of “Actinobacillus serninis, ” the “Histophilus ovis” group, and Haemophilus haemoglobinophilus Color intensities in the following API ZYM reactions:“ Strains Taxon 9 13 16 (serial no.) 2 3 4 5 6 7 8 and 11 12 and 15 through 10 14 20 “Actinobacillus seminis” 5 and 29 0-2’ 0-1 0-1 0 34 0-1 0-2 0 1-5 0-1 0 3-5 0 Huemophilus huemoglobinophilus 16 and 17 0 0 0 01-20 0 01-20-10 0 0 “Histophilus ovis” group 28 and 30 0-1 0-1 0-1 0 2-5 0-1 0-1 0 1-3 0-2 0 3-5 0 through 42 (’ The API ZYM reactions (API System SA, La Balme les Grottes, Montalieu-Vercieu, France) were numbered as follows: 2, alkaline phosphatase; 3, esterase (CJ; 4, esterase-lipase (C8);5, lipase (CI4);6, leucine aminopeptidase; 7, valine aminopeptidase; 8, cystine aminopeptidase: 9, trypsin; 10. chymotrypsin; 11, phosphatase (acid); 12, phosphoamidase; 13, a-galactosidase; 14, P-galactosidase; 15, p-glucuronidase; 16, a-glucosidase; 17, P-glucosidase; 18, p- glucosaminidase; 19, a-mannosidase; 20, a-frucosidase. Color intensities were read as suggested by the manufacturer. The results are from at least three tests per strain. members of the same species (see above). API ZYM reac- tant to anticipate taxonomic decisions like the decision tions did not discriminate between “Actinobacillus seminis” concerning delineation of the genus “Histophilus,” which, and the “Histophilus ovis” group (Table 6). Isolates resem- in our opinion, will require considerable broadening of the bling Hoerlein strain M650-1343 may have been seen by genetic data base. other authors (14, 23). Further taxonomic considerations. Considering the data ACKNOWLEDGMENTS described above, there is no doubt that “Histophilus ovis” We are most grateful to all of our colleagues who provided valuable and synonymous taxa, “Actinobacillus seminis,” and Hae- cultures. mophilus haemoglobinophilus represent three distinct spe- This investigation was supported in part by a grant from the cies of the Pusteurellaceae. The generic affiliation of these Deutsche Forschungsgemeinschaft to W.M. taxa remains problematic. Stephens and co-workers (30) suggested on the basis of conventional descriptive data, cell LITERATURE CITED envelope protein profiles, and immunodiffusion tests that the 1. Asmussen, M. D., and C. L. Baugh. 1981. Thiamine pyrophos- “Histophilus ovis” group “be accommodated in a new phate (cocarboxylase) as a growth factor for Haemophilus genus,” which, however, could not be phenotypically delin- somnus. J.Clin. Microbiol. 14:178-183. eated. 2. Baynes, 1. D., and G. C. Simmons. 1960. Ovine epididymitis After two DNA hybridization studies (32; this study) the caused by Actinohacillus seminis, n.sp. Aust. Vet. J. 36:454459. situation is not much better. We agree with E. L. Biberstein 3. Biberstein, E. L. 1981. “Haemophilus somnus” and “Huemo- (personal communication) that the genus “Histophilus” philus agni,” p. 125-132. In M. Kilian, W. Frederiksen, and could be revived. In this study, however, we were still E. L. Biberstein (ed.), Haemophilus, Pasteurella, and unable to give an unambiguous circumscription of the genus Actinobacillus. Academic Press, Inc., London. on the basis of both the phenotypic and genetic data avail- 4. Biberstein, E. L., and C. K. Francis. 1968. Nucleic acid homol- able so far. ogies between the A and T types of Pasteurellu haemolytica. J. “Histophilus ovis” and Haemophilus haemoglobinophilus Med. Microbiol. 1:lOS-108. exhibited significant levels of DNA relatedness, whereas 5. Bisgaard, M., R. Mutters, and W. Mannheim. 1983. Character- “Actinobacillus seminis” did not. These three species pos- ization of some previously unreported taxa isolated from guinea pigs (Cavia po,rcellus) and provisionally classed with the “HPA- sess common envelope proteins and common antigens (30). group.” Les Editions INSERM 114:227-244. Supposedly discriminating features like production of yel- 6. Corboz, L., and P. Wild. 1981. Epiderniologie der Haemophilus lowish pigments and catalase, requirements for TMP and sumnus-Infektion beim Rind: Vergleich von Stammen in der COl, and some catabolic reactions appear to be unreliable Polyacrylamidgel-Elektrophorese (PAGE). Schweiz. Arch. (3, 17, 30). Therefore, in spite of lacking data on DNA Tierheilkd. 123:79-88. relatedness at higher levels, we cafino: rule out the possibil- 7. Dierks, R. E., S. A. Hanna, and R. C. Dilman. 1973. ity that the three species are genetically connected by Epizootology and pathogenesis of Haemophilus somnus infec- hitherto unconsidered intermediates (e.g., so-called “fastid- tion. J. Am. Vet. Med. Assoc. 163:86&869. ious Pasteurella multocida” strains .[3, 301) and other atyp- 8. Hollander, R., A. Hess-Reihse, and W. Mannheim. 1981. Respi- ical isolates that may exist in culture collections. ratory quinones in Haemophilus, Pasteurella, and Actinobacil- lus: pattern, function and taxonomic evaluation, p. 83-93. In M. In similar situations, we have observed that phenotypi- Kilian, W. Frederiksen, and E. L. Biberstein (ed.), Haemoph- cally related taxa that lacked significant levels of mutual ilus, Pasteurella, and Actinohacillus. Academic Press, Inc., DNA binding were linked by genetic intermediates. For London. example, biovars A and T of Pasteurella haemolytica that 9. Kennedy, P. C., E. L. Biberstein, J. A. Howarth, L. M. Frazier, had been shown to be unrelated by DNA-DNA hybridization and D. L. Dungworth. 1960. Infectious meningoencephalitis in (4,25) were connected at the generic relatedness level by the cattle, caused by a Haemophilus-like organism. Am. J. Vet. type strain of the newly described species Pusteurella Res. 21:403-409. testudinis Snipes and Biberstein 1982 (20); Pasteurella canis 10. Kennedy, P. C., L. M. Frazier, G. H. Theilen, and E. L. Biberstein. 1958. A septicemic disease of lambs caused by (so-called biovar 6 or dog type strains of Pasteurella Haemophilus agni (new species). Am. J. Vet. Res. 19:645-654. rnultocida) exhibited only 20% DNA relatedness to typical 11. Kilian, M. 1976. A taxonomic study of the genus Haemuphilus, Pasteurella multocidu strains (31) but was included in the with the proposal of a new species. J. Gen. Microbiol. 93:9-62. genus Pasteurellu sensu strict0 via Pasteurella dugmatis 12. Kilian, M., and E. L. Biberstein. 1984. Genus 11. Haemophilus (Pasteurella “gas” or the Henriksen type of Pasteurella Winslow et al. 1917, p. 558-569. In N. R. Krieg, and J. G. Holt pneumotropica) (20). After these experiences we are reluc- (ed.), Bergey’s manual of systematic bacteriology, vol. 1. The VOL. 36, 1986 DNA RELATIONSHIPS 7

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