JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1994, p. 2398-2403 Vol. 32, No. 100095-1137/94/$04.00+ 0Copyright C) 1994, American Society for Microbiology

Flavobacterium meningosepticum, a Pathogen in BirdsM. VANCANNEYT,l* P. SEGERS,' L. HAUBEN,' J. HOMMEZ,2 L. A. DEVRIESE,3

B. HOSTE,' P. VANDAMME,' AND K. KERSTERS'Laboratorium voor Microbiologie, Faculteit Wetenschappen,' and Laboratorium voor Bacteriologie,

Faculteit Diergeneeskunde,3 University of Ghent, Ghent, and Provinciaal Verbondvoor Dierenziektenbestnijding, Torhout, 2Belgium

Received 19 April 1994/Accepted 11 July 1994

Five bacterial isolates were recovered from various diseased birds (chickens, a pigeon, and a zebra finch) andwere identified as Flavobacterium meningosepticum. Four of them were isolated in pure or nearly pure cultureof samples from internal organs, and one strain was isolated in mixed culture of a tarsal joint fluid sample.Except for the last case, there was no evidence of other disease agents. By using phenotypic, chemotaxonomic,and genomic methods, the strains were taxonomically characterized and could not be differentiated from thehuman clinical reference strains of the species. Two avian strains were different in their phenotypic behaviorsand constituted another genotypic subgroup. In general, all F. meningosepticum strains constituted a single specieswhich was easily differentiated from biochemically similar species and phylogenetically closely related taxa.

Flavobacterium meningosepticum is a human pathogen and issometimes associated with a fatal meningitis in infants (1, 5).The organism has been isolated from their throats, spinal fluid,and blood as well as from the throats from adults (8). Neonatalmeningitis caused by F. meningosepticum was first described in1944 (15). Since then, about 120 cases have been described(20). Six serological groups (groups A to F) are recognized (9,11), of which serovar C was involved in several epidemics. Thusfar, most F. meningosepticum strains have been isolated fromhumans. Sporadic strains have been isolated from soil, water,drugs, and dog blood (16). One of us (J.H.) isolated five strainsfrom various diseased birds and tentatively identified them asF. meningosepticum using the API 20NE microtest system.These strains were compared with human clinical isolates of F.meningosepticum and with the type strains of Flavobacterium

balustinum, Flavobacterium gleum, Flavobacterium indologenes,Flavobacterium indoltheticum, Riemerella anatipestifer, andWeeksella zoohelcum. Members of the last six taxa were shownto be closely related to F. meningosepticum (14). All strainswere phenotypically characterized and were compared byone-dimensional whole-cell protein electrophoresis and cellu-lar fatty acid analysis. Genomic data were obtained fromDNA-DNA and DNA-rRNA hybridization studies and deter-mination of the DNA base compositions.

MATERUILS AND METHODS

Bacterial strains and growth conditions. Data on the originsof the strains are listed in Table 1. Strains LMG 13004 throughLMG 13008 were isolated from samples from chickens and

TABLE 1. Flavobactenium and allied strains studied and source of isolation

Organism name LMG strain no.ab Other strain designationa Source; place of isolation

F. meningosepticum 12279T CCUG 214T Cerebrospinal fluid, premature infant; MassachusettsF. meningosepticum 12280 CCUG 4310 Human blood; FloridaF. meningosepticum 12873 CCUG 4321 Cerebrospinal fluid, premature infant; FloridaF. meningosepticum 12874 CCUG 14224 Human throat; Goteborg, SwedenF. meningosepticum 12883 CCUG 26117 Human blood; Goteborg, SwedenF. meningosepticum 12967 CCUG 4218 Cerebrospinal fluid, infant; New YorkF. meningosepticum 13004 Hommez 21 Joint, chicken; Torhout, BelgiumF. meningosepticum 13005 Hommez 22 Pericardium, chicken; Torhout, BelgiumF. meningosepticum 13006 Hommez 40 Pericardium, broiler; Torhout, BelgiumF. meningosepticum 13007 Hommez 41 Liver, zebra finch; Torhout, BelgiumF. meningosepticum 13008 Hommez 42 Eye socket, pigeon; Torhout, BelgiumF. balustinum 8329T CCUG 13228T Fish blood; FranceF. gleum 8334T CCUG 14555T Vaginal swab; United KingdomF. indologenes 8337T CCUG 14556T Trachea; United StatesF. indoltheticum 4025T ATCC 27950T Marine mudR. anatipestifer 11054T CCUG 14215T Blood, duck; United StatesW. zoohelcum 8351T CCUG 12568T Sputum; United States

a Type strains are indicated by a superscript T.b ATCC, American Type Culture Collection, Rockville, Md.; CCUG, Culture Collection of the University of Goteborg, Department of Clinical Bacteriology,

University of Goteborg, Goteborg, Sweden; Hommez, J. Hommez, Provinciaal Verbond voor Dierziektenbestrijding, Torhout, Belgium; LMG, Culture CollectionLaboratorium voor Microbiologie, University of Ghent, Ghent, Belgium.

* Corresponding author. Mailing address: Laboratorium voor Mi-crobiologie, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Phone:32 9 264 5115. Fax: 32 9 264 5346.

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F. MENINGOSEPTICUM FROM BIRDS 2399

other birds autopsied in a regional veterinary diagnostic labo-ratory on Columbia blood agar base (Oxoid CM 331) supple-mented with 5% sheep blood. The strains were incubated at 30,37, and 42°C in air and in air enriched with 5 to 10% CO2. Allcultures were maintained aerobically on nutrient agar (OxoidCM3) slants at 28°C.

Phenotypic characterization. The strains studied were bio-chemically characterized by using the API 20NE gallery. Cellswere cultivated on nutrient agar (Oxoid CM3) slants for 24 hat 28°C. The inoculation procedure, incubation, and reading ofthe API 20NE system were performed according to theinstructions of the manufacturer (bioMerieux, Brussels, Bel-gium).PAGE of whole-cell proteins. All strains were grown on

Trypticase soy agar (TSA) plates containing 30 g (wt/vol) ofTrypticase soy broth (BBL) supplemented with 15 g (wt/vol) ofBacto Agar (Difco) per liter of distilled water and were

incubated at 28°C for 24 h. Protein extracts were prepared, andsodium dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis (PAGE) was performed as described by Pot et al.(13). Similarities between protein patterns were interpreted as

described by Vauterin and Vauterin (19).Fatty acid analysis. The strains were streaked onto TSA

plates, and the plates were incubated at 28°C for 24 h. Wefollowed the procedure of the Microbial Identification System(Microbial ID Inc., Newark, Del.) to harvest the cells, saponifythe lipids, methylate the fatty acids, and purify and separatethe fatty acid methyl esters (FAMEs) by gas chromatography.FAME fingerprints were identified and quantified by using theMicrobial Identification System software package (MIS ver-

sion 3.7) and a calibration mix of known standards (Hewlett-Packard Co., Avondale, Pa.).

Determination of DNA base compositions. The guanine-plus-cytosine (G+C) contents were determined by thermaldenaturation and were calculated by using the equation ofMarmur and Doty (10), as modified by De Ley (6).DNA-DNA hybridization experiments. The degree of DNA-

DNA binding, expressed as a percentage, was determinedspectrophotometrically by using the initial renaturation meth-od of De Ley et al. (7).DNA-rRNA hybridization experiments. 3H-labeled rRNA

from the type strain of F. meningosepticum was isolated andpurified as reported earlier (17). Radioactively labeled rRNAsfrom F. indologenes, R. anatipestifer, and W. zoohelcum were

prepared in previous studies (14). Hybridization between therRNA probe and the single-stranded filter-fixed DNA was

carried out as described previously by Van Landschoot and DeLey (18). Each DNA-rRNA hybrid was characterized by thetemperature at which half of the DNA-rRNA hybrid is ther-mally denatured [Tm(e)]. A homologous duplex was formedbetween the DNA and rRNA of the same strain; a heterolo-gous hybrid was formed between the DNAs and rRNAs ofdifferent strains. The Tm(e) values from the reciprocal hybrid-ization experiments obtained by using all strains of each rRNAbranch were used to calculate the average linkage level be-tween each pair of rRNA branches.

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Pathological and bacteriological findings in avian cases ofinfection. Strains LMG 13006 and LMG 13005 were isolated inpure culture of samples from chickens with pericarditis. The

first case was in a 2-week-old chicken with severe lesions whichyielded profuse growth. The second case was in an adultchicken with more discrete lesions that yielded less abundantbacteria. No evidence of other disease agents was found. A

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J. CLIN. MICROBIOL.2400 VANCANNEYT ET AL.

Species

F. meningosepticum

F. indoltheticumF. gleumF. indologenesR. anatipestiferF. balustinumW. zoohelcum

LMG no.

13004

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FIG. 1. Normalized photograph of SDS-protein electrophoretograms of the strains studied and the corresponding dendrogram obtained on thebasis of average-linkage cluster analysis of correlation coefficients (r values). The positions of the following molecular weight markers (track labeledLWM) are from left to right: lysozyme, 14,500; trypsin inhibitor, 20,100; carbonic anhydrase, 29,000; glyceraldehyde-3-phosphate dehydrogenase,36,000; egg albumin, 45,000; bovine albumin, 66,000; 3-galactosidase, 116,000.

third strain, LMG 13004, was isolated in mixed culture withEscherichia coli from the tarsal joint fluid of a 4-week-oldchicken. LMG 13008 originated from an inflamed hemorrhagictraumatic lesion in the eye socket of a young homing pigeon,and LMG 13007 originated from the liver of a 2-week-oldzebra finch (Taeniopygia guttata). Both strains were isolated innearly pure profuse culture.

Phenotypic characterization. All except two F. meningosep-ticum strains showed good growth on Columbia blood agarwhen incubated aerobically at 30 and 37°C; strains LMG13004 and LMG 13005 grew weakly at 37°C. The latter twostrains were also differentiated by a strong beta-hemolyticactivity and by their inability to grow in a 5 to 10% C02-enriched atmosphere. None of the strains grew at 42°C. Thebiochemical characteristics of all strains were determined byusing the API 20NE gallery and are presented in Table 2. AllF. meningosepticum strains produced indole, exhibited oxidaseand ,-galactosidase activities, hydrolyzed gelatin and esculin,and grew on glucose, mannose, mannitol, N-acetylglucos-amine, and maltose. None of the strains reduced nitrate,produced acid from glucose, showed arginine dihydrolaseactivity, or assimilated gluconate, caprate, adipate, malate, or

phenylacetate. Three reactions were strain dependent: urease

activity and assimilation of L-arabinose and citrate. F. menin-gosepticum could be discriminated from F. balustinum, F.gleum, F. indologenes, F. indoltheticum, R. anatipestifer, and W.zoohelcum by its assimilation of mannitol and N-acetylglu-cosamine.

Comparative protein gel electrophoresis. The protein pat-terns of the F. meningosepticum strains were determined alongwith the profiles of type strains of closely related flavobacteria

and allied taxa. Figure 1 shows the protein profiles and theresulting dendrogram obtained after average-linkage clusteranalysis of the strains analyzed. All F. meningosepticum strainsgrouped in a separate electrophoretic cluster (r 0.88). Allother reference strains occupied separate positions in thedendrogram.FAME analysis. The fatty acid profiles are compiled in

Table 3. All F. meningosepticum strains were characterized bythe major fatty acids 15:0 iso (33.8 to 42.8%), iso 17:1 w9c (5.3to 9.1%), 17:0 iso 30H (14.4 to 16.6%), and summed feature4 (18.1 to 23.9%; see Table 3 for the interpretation of"summed feature"). Both qualitative and quantitative differ-ences in FAME fingerprints were observed for strains of otherFlavobacterium species and related taxa.DNA base compositions. The DNA base ratios which were

determined are given in Table 4. All strains studied had G+Ccontents of between 35 and 38 mol%.DNA-DNA hybridization experiments. DNA-DNA hybrid-

ization data are presented in Table 5. One representative ofthe two major hybridization groups of Ursing and Bruun (16)and two field bird isolates were included. Three strains,including the type strain, showed a high degree of homology(80 to 87%) and grouped at a level of about 50% with strainLMG 13005.DNA-rRNA hybridization experiments. The results of the

DNA-rRNA hybridization experiments are included in Table 4and are presented as a dendrogram in Fig. 2. These data showthat the field isolates and the F. meningosepticum referencestrains form a distinct and narrow cluster at the top of the F.meningosepticum rRNA branch.

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F. MENINGOSEPTICUM FROM BIRDS 2401

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labeled rRNA of F. meningosepticum LMG 12279T

Organism used for LMG strain G+C content Tm(e)DNA isolation no. (mol%) (OC)

F. meningosepticum 12279T 37 75.4F. meningosepticum 12873 36 76.1F. meningosepticum 12967 37 77.3F. meningosepticum 13005 36 75.3F. meningosepticum 13007 37 NDaF. gleum 8334T 37 73.3F. indologenes 8337T 38 70.6R anatipestifer 11054T 35 72.6W. zoohelcum 8351T 37 71.8

a ND, not determined.

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DISCUSSION

Five strains isolated from diseased birds were tentativelyidentified as F. meningosepticum by using the API 20NEmicrotest system. Considering this unusual source, we decidedto compare these fowl isolates with "normal" human clinicalisolates in order to determine if both groups of strains repre-

sent separate taxonomic units. In addition, all strains were

compared with reference strains of closely related taxa (i.e.,F. balustinum, F. gleum, F. indologenes, F. indoltheticum, R.anatipestifer, and W zoohelcum).The commercial API 20NE database comprises F. meningo-

septicum and F. indologenes but not F. balustinum, F. gleum, F.indoitheticum, and R. anatipestifer. In the present study we alsoincluded reference strains of the latter four taxa because theyshare a considerable number of phenotypic characteristics withF. meningosepticum (8). F. meningosepticum strains could bedifferentiated from these taxa by their ability to assimilatemannitol and N-acetylglucosamine. The other species were

differentiated by means of various tests such as nitrate reduc-tion, urease activity, and hydrolysis of gelatin. Bruun andUrsing (4) already discussed the phenotypic characteristics of52 different F. meningosepticum strains and concluded that allstrains were phenotypically homogeneous. Furthermore, theystated that this species may be separated from the biochemi-cally similar Flavobacterium group lIb species (i.e., F. gleumand F. indologenes) by its ability to produce P-galactosidaseafter 2 h (o-nitrophenyl-3-D-galactopyranoside test) and by theabsence of pigmented colonies (Flavobacterium group IIbstrains produce a bright yellow pigment). When comparingdata from Bruun and Ursing (4) with our phenotypic data(Table 2), similar results were obtained for most of theoverlapping tests. Indeed, all F. meningosepticum strains stud-ied exhibited oxidase, catalase, gelatinase, and 1-galactosidaseactivities and produced indole. However, in contrast to thedata of Bruun and Ursing (4), in our study, none of the strainsproduced acid from glucose, whereas two bird isolates, LMG13004 and LMG 13005, showed urease activity (Table 2). Both

TABLE 5. DNA relatedness of F. meningosepticum strains

% DNA relatednessStrain

LMG 12279T LMG 13007 LMG 12873 LMG 13005

LMG 12279T 100LMG 13007 86 100LMG 12873 80 87 100LMG 13005 51 49 56 100

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2402 VANCANNEYT ET AL.

Flavobacterium meningosepticum

,r-j Flavobacterium indologenes'Flavobacterium gleumFlavobacterium indoitheticum'Flavobacterium balustinum

Weeksella zoohelcum

Riemerella anatipestifer

65 70 75 80 Tm(e) (OC)

FIG. 2. Position of F. meningosepticum in a partial dendrogram ofrRNA superfamily V based on the Tm(e) values of the DNA-rRNAhybrids. Data are from the present study and from the study of Segerset al. (14). The solid bar indicates the Tm(e) range of the strains withinthe F. meningosepticum branch.

strains also differed from all other F. meningosepticum strainsby their growth characteristics on Columbia blood agar: onlyweak growth was obtained aerobically at 37°C, no significantgrowth occurred in a CO2-enriched atmosphere, and a strongbeta-hemolytic activity was demonstrated. Furthermore, thelatter strains and strain LMG 12280 differed from other F.meningosepticum strains by their ability to assimilate citrate.Only one strain, LMG 12967, assimilated L-arabinose.

All strains investigated were further characterized by SDS-PAGE of their whole-cell proteins (Fig. 1) and gas chromato-graphic analysis of their fatty acids (Table 3). F. meningosep-ticum strains revealed highly similar protein patterns at acorrelation level of r 2 0.88 and clearly belong to a singlespecies. Three bird isolates, LMG 13006, LMG 13007, andLMG 13008, formed a separate protein electrophoretic sub-group because of a different density of bands in the molecularweight regions of approximately 25,000 and 50,000, respec-tively (Fig. 1). Fatty acid data indicated small quantitativedifferences among strains of the species. No subgroups couldbe delineated. Both methods allowed clear-cut differentiationof F. meningosepticum strains from related taxa (Fig. 1; Table3). Major fatty acids for all F. meningosepticum strains were15:0 iso, iso 17:1 w9c, 17:0 iso 30H, and summed feature 4.Profiles of the type strain of F. balustinum, F. gleum, F.indologenes, and F. indoltheticum showed significantly loweramounts of 15:0 iso and summed feature 4 and a substantiallyhigher amount of iso 17:1 w9c. The type strain of W. zoohelcumcould be differentiated from the others by quantitative differ-ences for the major fatty acids. The type strain of R. anatipes-tifer was easily differentiated from the others by the absence ofsummed feature 4 and iso 17:1 w9c and the presence ofsignificant amounts of 13:0 iso.Two reference strains representing the two genotypic sub-

groups (16) within F. meningosepticum (LMG 12279T andLMG 12873) and two field isolates (LMG 13005 and LMG13007) were selected for DNA-DNA hybridization studies.Owen and Snell (12) demonstrated that the type strain of thespecies, which represents serovar A, showed much lower levelsof relatedness to strains of other serovars. Ursing and Bruun(16) studied 52 strains of the species and confirmed that onlythree other strains showed appreciable homology to the typestrain; the remaining strains were divided into four subgroups.Differences between the two major groups were not found inantimicrobial susceptibility patterns (2), crossed immunoelec-trophoresis results (3), or phenotypic characters (4). In our

study, three strains, including the type strain, yielded relative

DNA-binding ratios of 80 to 87% and showed 49 to 56%relatedness to strain LMG 13005. Although these valuesconfirm that all strains studied belong to a single species, thesubgroupings within this species did not correspond to thosedelineated by Ursing and Bruun (16) or Owen and Snell (12).Methodological differences may account for this discrepancy.No significant differences in DNA base compositions were

observed between the Flavobacterium, Riemerella, and Week-sella species because all strains had G+C contents of between35 and 38 mol% (Table 4).

In an ongoing study, the position of F. meningosepticum in aphylogenetic tree was determined by DNA-rRNA hybridiza-tion experiments (Table 4; Fig. 2). Previous studies havealready demonstrated that F. indologenes, W. zoohelcum, andR. anatipestifer, together with F. balustinum, F. indoltheticum,F. gleum, and F. meningosepticum, belong to the same branchin rRNA superfamily V (14). Data from the present studyshowed that the new isolates from birds together with the F.meningosepticum strains of human origin form a distinct andnarrow cluster with the type strain.

In conclusion, five bacterial isolates from birds were unam-biguously identified as F. meningosepticum. Phenotypic, che-motaxonomic, and genotypic methods revealed that this spe-cies, although well defined, is rather heterogeneous. Theintraspecific variation was not correlated with the source ofisolation. The isolation of four strains in pure or virtually purecultures of samples from severe lesions in diseased birdssuggests a pathogenic role. In view of our minimal knowledgeof the natural habitat of F. meningosepticum, it should beevaluated if birds might serve as a reservoir for infections inhumans.

ACKNOWLEDGMENTSWe thank Urbain Torck and Dirk Dewettinck for performing the

protein electrophoresis.P.V. is indebted to the National Fund for Scientific Research

(Belgium) for a position as a postdoctoral research fellow. K.K. isindebted to the Fund for Medical Scientific Research, Belgium, forresearch and personnel grants. Part of the research was performed inthe framework of the CEC-contract BIOT-CT91-0294.

REFERENCES

1. Brody, J. A., H. Moore, and E. 0. King. 1958. Meningitis caused byan unclassified gram-negative bacterium in newborn infants. Am.J. Dis. Child. 96:1-5.

2. Bruun, B. 1987. Antimicrobial susceptibility of Flavobactenummeningosepticum strains identified by DNA-DNA hybridization.Acta Pathol. Microbiol. Immunol. Scand. Sect. B 95:95-101.

3. Bruun, B., and N. H0iby. 1987. Crossed immunoelectrophoreticanalysis of Flavobacterium and allied Flavobacterium taxa. ActaPathol. Microbiol. Immunol. Scand. Sect. B 95:245-252.

4. Bruun, B., and J. Ursing. 1987. Phenotypic characterization ofFlavobactenum meningosepticum strains identified by DNA-DNAhybridization. Acta Pathol. Microbiol. Immunol. Scand. Sect. B95:41-47.

5. Cabrera, A. H., and G. H. David. 1961. Epidemic meningitis of thenewborn caused by flavobacteria. Am. J. Dis. Child. 101:289-295.

6. De Ley, J. 1970. Reexamination of the association between meltingpoint, buoyant density, and chemical base composition of deoxyri-bonucleic acid. J. Bacteriol. 101:738-754.

7. De Ley, J., H. Cattoir, and A. Reynaerts. 1970. The quantitativemeasurement of DNA hybridization from renaturation rates. Eur.J. Biochem. 12:133-142.

8. Holmes, B. 1992. The genera Flavobacterium, Sphingobacterium,and Weeksella, p. 3620-3630. In A. Balows, H. G. Truper, M.Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes,vol. 4, 2nd ed. Springer-Verlag, Berlin.

9. King, E. 0. 1959. Studies on a group of previously unclassified

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F. MENINGOSEPTICUM FROM BIRDS 2403

bacteria associated with meningitis in infants. Am. J. Clin. Pathol.31:241-247.

10. Marmur, J., and P. Doty. 1962. Determination of the basecomposition of deoxyribonucleic acid from its thermal denatur-ation temperature. J. Mol. Biol. 5:109-118.

11. Owen, R. J., and S. P. Lapage. 1974. A comparison of strains ofKing's group lIb of Flavobacterium with Flavobactenium meningo-septicum. Antonie van Leeuwenhoek 40:255-264.

12. Owen, R. J., and J. J. S. Snell. 1976. Deoxyribonucleic acidreassociation in the classification of flavobacteria. J. Gen. Micro-biol. 93:89-102.

13. Pot, B., P. Vandamme, and K. Kersters. 1993. Analysis of electro-phoretic whole-organism protein fingerprints, p. 493-521. In M.Goodfellow and A. G. O'Donnell (ed.), Chemical methods inprokaryotic systematics. John Wiley & Sons, Chichester, UnitedKingdom.

14. Segers, P., W. Mannheim, M. Vancanneyt, K. De Brandt, K.-H.Hinz, K. Kersters, and P. Vandamme. 1993. Description ofRiemerella anatipestifer gen. nov., comb. nov., the causative agentof septicaemia anserum exsudativa and its phylogenetic affiliationwithin the Flavobacterium-Cytophaga rRNA homology group. Int.J. Syst. Bacteriol. 43:768-776.

15. Shulman, B. H., and M. S. Johnson. 1944. A case of meningitis in

a premature infant due to a proteolytic gram-negative bacillus. J.Lab. Clin. Med. 29:500-507.

16. Ursing, J., and B. Bruun. 1987. Genetic heterogeneity ofFlavobac-terium meningosepticum demonstrated by DNA-DNA hybridiza-tion. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 95:33-39.

17. Vandamme, P., P. Segers, M. Vancanneyt, K. Van Hove, R.Mutters, J. Hommez, F. Dewhirst, B. Paster, K. Kersters, E.Falsen, L. A. Devriese, M. Bisgaard, K.-H. Hinz, and W. Mann-heim. 1994. Ornithobacterium rhinotracheale gen. nov., sp. nov.,isolated from the avian respiratory tract. Int. J. Syst. Bacteriol.44:24-37.

18. Van Landschoot, A., and J. De Ley. 1983. Intra- and intergenericsimilarities of the rRNA cistrons of Alteromonas, Marinomonas(gen. nov.) and some other gram-negative bacteria. J. Gen.Microbiol. 129:3057-3074.

19. Vauterin, L., and P. Vauterin. 1992. Computer-aided objectivecomparison of electrophoresis patterns for grouping and identifi-cation of microorganisms. Eur. Microbiol. 1:37-41.

20. von Graevenitz, A. 1985. Ecology, clinical significance and antimi-crobial susceptibility of infrequently encountered glucose-nonfer-menting gram-negative rods, p. 181-232. In G. L. Gilardi (ed.),Nonfermentative gram-negative rods. Laboratory identificationand clinical aspects. Marcel Dekker, Inc., New York.

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