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Identification of members of the Pasteurellaceae isolated from birds and characterization of two new taxa isolated from psittacine birds M. Bisgaard , K.-H. Hinz , K.D. Petersen & J.P. Christensen Published online: 17 Jun 2010.
To cite this article: M. Bisgaard , K.-H. Hinz , K.D. Petersen & J.P. Christensen (1999) Identification of members of the Pasteurellaceae isolated from birds and characterization of two new taxa isolated from psittacine birds, Avian Pathology, 28:4, 369-377, DOI: 10.1080/03079459994632 To link to this article: http://dx.doi.org/10.1080/03079459994632
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Avian Pathology (1999) 28, 369±377
Identi® cation of members of the Pasteurellaceae isolated from birds and characterization of two new taxa isolated from psittacine birds M. Bisgaard 1* K.-H. Hinz2, K.D. Petersen1 & J.P. Christensen 1 1
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Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Copenhagen, Denmark, and 2Klinik fuÈr Ge¯ uÈgel, TieraÈrztliche Hochschule Hannover, 30559 Hannover, Germany
A collection of 43 unclassi® ed members of the Pasteurellaceae, most of which were obtained from lesions, were investigated using an extensive battery of phenotypical tests as well as by ribotyping. The isolates had been made from Anser anser forma (f) domestica (d), Agapornis ® scher, Amazona spp., Ara macao, Columba livia f.d., Melanopsittacus undulatus, and Psittacus erithacus. Comparison of results with those obtained from reference strains allowed classi® cation of 25 strains. Three strains were identi® ed as Pasteurella dagmatis, P. sp. A, and [P.] aerogenes, respectively. Twenty strains were classi® ed as taxon 3 and two as taxon 14. Eighteen strains, all originating from psittacine species, belonged to two new taxa, tentatively named Bisgaard taxon 33 and taxon 37. Characters obtained with taxon 33 allowed classi® cation within the family Pasteurellaceae, while the ® nal classi® cation of taxon 37 remains to be investigated. The present investigation underlines the problems confronting diagnostic laboratories attempting to identify members of the family Pasteurellaceae isolated from birds.
Introduction The genomic relationships of some recently described avian Pasteurella/Actinobacillus -like organisms were investigated by Piechulla et al. (1985a) to evaluate the taxonomic resolution of diagnostically useful phenotypic criteria. Careful collection and characterization of unclassi® ed strains, tentatively classi® ed as members of the Pasteurellaceae, have subsequently resulted in many new species or taxa, the prevalence and pathogenic potential of which are only partially known (Bisgaard, 1995). Members of the genus Pasteurella have been associated with disease in Phasianiformes, Anatiformes, Psittaciformes, Columbiformes and Passeriformes, with Pasteurella multocida and Pasteurella gallinarum being the most commonly isolated species (Gerlach, 1994; Reavill, 1996; Dorrestein, 1997). Members of the Pasteurellaceae were, however, not obtained from the cloacae of clinically healthy psittacine birds by Flammer & Drewes (1988), and Morishita et al. (1996) were unable to isolate P. multocida * To whom correspondenc e should be addressed. Tel: 1 Received 30 July 1998. Accepted 12 February 1999.
from the pharynx, choanae, or cloacae. However, P. multocida was obtained from ® ve dead psittacines with septicemia or cutaneous lesions. Reports of isolation of Pasteurella sp. from psittacine species with or without lesions have also been reported by Rolf (1977), Baker (1980), Dorrestein et al. (1985), and Drew et al. (1993). Although disease in pigeons caused by P. multocida seems to be rare, P. multocida was isolated from three out of 15 wild pigeons sampled by MacDonald et al. (1981). Recently, Dorrestein (1997) reported isolation of [P.] pneumotropicalike organisms from collared turtledoves. Taxa classi® ed within the genus Actinobacillus have been reported from members of the Psittaciformes, Columbiformes, Anatiformes and Fringillidae. However, knowledge of the biology and pathogenicity of members of this genus is limited (Gerlach, 1994). Actinobacillus spp. have been isolated from more than 1% of normal psittacine corneas and ocular conjunctivas (Zenoble et al., 1983). Chronic sinus infections associ-
45 35282760 . Fax: 1 45 35282757 . E-mail: [email protected] or [email protected]
ISSN 0307-945 7 (print)/ISSN 1465-3338 (online)/99/040369-0 9
Ó
1999 Houghton Trust Ltd
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ated with Actinobacillus spp. have been reported in parrots (Avery, 1982). True species of the genus Actinobacillus have not yet been reported from birds, according to Christensen & Bisgaard (1997). Three new avian Haemophilus-like taxa were reported by Piechulla et al. (1985b). None of these, however, belonged to recognized genera. Haemophilus-like organisms have subsequently been reported from psittacines (Devriese et al., 1988). Members of a new group of organisms, tentatively designated taxon 14, were isolated from different lesions in ducks, turkeys and a pigeon by Bisgaard & Mutters (1986a). These organisms were subsequently shown to belong to the family Pasteurellaceae by De Ley et al. (1990) and Dewhirst et al. (1993). Supplying the market with pet birds depends increasingly on the success of avicultural breeding programmes, due to restrictions and legislation placed on importation of wild caught birds into the European Union. Most members of the family Pasteurellaceae appear to be commensals of freeliving hosts. However, high density and con® ned rearing facilities developed for the captive breeding of these species might change the parasite±host balance, which seems to exist on mucous membranes under normal conditions, and result in disease. The ecology and signi® cance of members of the Pasteurellaceae were reviewed by Bisgaard (1993), who stressed that a more accurate basis for diagnosis is a prerequisite for improving our knowledge of the complexity of factors that govern the ecological preferences and virulence of these organisms. Consequently, the aim of the present investigation was to characterize a collection of organisms provisionally classi® ed within the Pasteurellaceae and isolated from pigeons, a goose, and psittacine birds to extend our knowledge of the signi® cance of these organisms, and to establish criteria for their identi® cation. Material and Methods Source of strains investigated Fifteen strains from pigeons were investigated, ® ve of which were obtained from birds with septicaemia, while 10 strains were isolated from respiratory tract lesions (Table 1). A single isolate originated from a goose, while the remaining 27 isolates originated from different species of psittacine birds. Four strains were associated with septicaemic conditions, 14 with respiratory tract lesions, while four strains were associated with other lesions. Six isolates were not associated with macroscopi c lesions (Table 1). Bacteriological investigations A total of 43 strains were investigated (Table 1). All strains had been kept as unclassi® ed member s of the Pasteurellaceae at ±80°C or as freeze-dried cultures since their original isolation. These strains can be obtained from the corresponding author. Before phenotypi c characterization, all strains were plated on blood agar (Tryptose blood agar base (Difco, Detroit, USA) containing 5% citrated bovine blood), and
incubated aerobically at 37 °C for 24 h. Pure cultures were subsequently characterized phenotypicall y by standard methods as described previousl y (Bisgaard et al., 1991) and compared with those from type or reference strains of Pasteurellaceae (Mutters et al., 1989).
Ribotyping and band pattern analysis Nineteen strains representing two new taxa, as well as different biovars of taxon 3 from different species of birds, were selected for ribotyping (Table 1). Isolation of DNA, digestion of DNA by restriction endonucleases , separation of DNA fragments in agarose gels, DNA blotting and hybridization at 56 °C with a digoxigenin-labele d probe (Boehringer-Mannheim , Mannheim, Germany) complementar y to 16S and 23S rRNA of Escherichi a coli (Sigma, St. Louis, MO, USA) were carried out as reported previously by Christensen et al. (1993). HpaII was used to digest DNA as it provided an appropriate number of distinct bands (from 7 to 14) for all strains investigated on ribotyping. Digoxigenin-labelle d phage lambda-DNA (Boehringer Mannheim) cut with HindIII was used as a molecular size marker. Ribotyping based upon new DNA preparations was repeated at least twice for each strain. Band patterns obtained with the strains investigated were analyzed using GelCompar version 4.0 (Applied Maths BVBA, Kortrijk, Belgium). Band positions were analyzed using ® ne optimization and a position tolerance of 1%. The similarity between the band pattern of individual strains was estimated using the Dice coef® cient, and dendrograms were drawn using the similarity matrix obtained (unweighted pair group method with averages) .
Results Bacteriological investigations All strains investigated had identical reactions in the following tests: porphyrin test, alanine aminopeptidase, Hugh and Leifson (fermentative), and production of acid from D(±)-fructose, D( 1 )glucose, and sucrose (all positive), Gram staining, motility (37 and 22° C), symbiotic growth, b haemolysis, growth on Simmons citrate, formation of base from malonate, H2S formation, KCN growth, Voges±Proskauer test at 37°C, production of gas from nitrate, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, formation of indole, gelatinase, hydrolysis of Tween 20 and 80, formation of pigment, production of acid from meso-erythritol, adonitol, xylitol, L(±)-xylose, D( 1 )-fucose, L ( 1 )-rhamnose, L (±)-sorbose, cellobiose, D( 1 )melezitose, D-glycogen, inulin, aesculin, amygdalin, arbutin, gentiobiose, salicin, D( 1 )-turanose, and b -N-CH3-glucosamide, a -fucosidase, b -glucuronidase, and a -mannosidase (all negative). Characters separating the strains investigated are shown in Table 2. One strain from a scarlet macaw was classi® ed as P. dagmatis (Bisgaard & Mutters, 1986b), while another isolate from a budgerigar was classi® ed as P. sp. A (Mutters et al., 1985). The goose isolate was classi® ed as [P.] aerogenes biovar 8 (Bisgaard, 1993). Two strains from pigeons were identi® ed as taxon 14 biovar 1 (Bisgaard &
Table 1. Information on the 43 strains investigated during this study
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Characterization of avian Pasteurellaceae 371
b
: selected for ribotyping : same ¯ ock c : forma domestica
a
Table 1. Information on the 43 strains investigated during this studyÐ (continued)
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1
1
1
1
) )
1
)
)
1
1
1
d d d ± ± /( 1 d ± /( 1 1 1 ± /( 1 ± /( 1 ± d d ± ± ± d ± ± ± d
1
± d ± ± d /( 1 /( 1 ± 1 1 1 d /( 1 ± 1 ± d d ± /( 1 d 1 ± 1 d d
1
1
1
1
)
)
)
)
±
d 1 /( 1 ) d ± (1 ) 1 d ± 1 1 ± 1 /( 1 ) 1 ± 1 ± 1 /( 1 ) 1 ± ± ± d ± ± ± d
1
1
1
d d
1
d d
1
±
1
1
d d ±
Taxon 3 biovar 4
Taxon 3 biovar 2
1
(1
1
1
1
1
1
1
(1
1
1
1
1
1
± ± ± ± ± ± ± d
±
±
± d
±
± ± d
d
± ±
)
)
Taxon 3 biovar 5
a
1 , all strains positive within 1 to 2 days; ( 1 ), all strains late positive ( $ within 14 days; d, different; w, weak positive.
Catalase Oxidase, TMPD Mucate, acid MR, 37°C Nitrate, red. Urease Phosphatase McConkey,growth Glycerol D( 1 )-Arabitol L( 1 )-Arabinose D(-)-Arabinose D(-)-Ribose D( 1 )-Xylose Dulcitol Meso-inositol D (-)-Mannitol D(-)-Sorbitol L(-)-Fucose D( 1 )-Galactose D( 1 )-Glucose, gas D( 1 )-Mannose b -Glucosidase Lactose ONPG Maltose D( 1 )-Melibiose Trehalose Raf® nose Dextrin a -Galactosidase a -Glucosidase b -Xylosidase
Taxon 3 biovar 1
±
d w
3 days); 1
d 1 /( 1 ) ± ± (1 ) 1 1 ± 1 1 1 1 /( 1 ) 1 ± 1 ± (1 ) 1 ± ± ± ± ± ± ± ±
1
1
1
1
Taxon 3 biovar 6
/( 1
±
±
± ± ± ±
±
± ± ±
±
±
± ±
)
)
1
1
1
1
1
± ± ± ± ± ± ± ± ± ±
± ±
± ± ± ± ± ± d ± ± ± ± ±
±
± ± ±
Taxon 14 biovar 1
± ± ±
± d ±
)
)
d ± ± ± ± ± 1 /( 1 ) ± 1 ± ± ± ± ± ± (1 ) ± ± ± ±
(1
(1
1
1
d d ±
Taxon 33
1
1
± ± d ± ± ± ± ±
d ±
)
± ± ± ± ± ± ± ± /( 1 ) ± ± ± /( 1 ) ± d d ± ± 1 ±
(1
1
1
Taxon 37
(1
1
1
1
1
(1
(1
(1
1
1
1
1
1
1
± ± ± ±
± ±
±
±
± ± ± ± ± ±
± ± ±
±
±
)
)
)
)
P. dagmatis
±
±
± ±
(1 ) ± (1 ) ± (1 ) ± ± ± ± ± ± (1 ) ± 1 ± 1 1 1 ± 1 (1 ) 1 ± ± ±
1
1
1
1
P. sp. A
1
(1
1
1
(1
1
1
(1
1
1
1
1
1
1
1
1
1
1
1
± ± ±
± ±
±
± ±
±
± ±
± w ±
)
)
)
P. aerogenes biovar 8
), all strains positive, some strains within 1 to 2 days, others late positive; ±, all strains negative
1
1
1
1
(1
1
1
1
1
1
1
1
1
1
(1
1
1
1
1
Taxon 2/3 biovar 5
Table 2. Phenotypic characters separating the taxa investigateda
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Characterization of avian Pasteurellaceae 373
374
M. Bisgaard et al.
Table 3. Results obtained with 43 strains tentatively classi® ed with the family Pasteurellaceae Pohl 1981 Taxon
Goose
P. dagmatis P. sp. A P. aerogenes biovar 8 Taxon 3 biovar 1 Taxon 3 biovar 2 Taxon 3 biovar 4 Taxon 3 biovar 5 Taxon 3 biovar 6 Taxon 2/3 biovar 5 Taxon 14 biovar 1 Taxon 33 Taxon 37a
1 1 ± 4 2 ± ± ± 1 ± 12 6
± ± ± 1 1 5 3 3 ± 2 ± ±
± ± 1 ± ± ± ± ± ± ± ± ±
Total
27
15
1
a
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Psittacine birds Pigeons
Does not belong to the family Pasteurellaceae sensu stricto.
Mutters, 1986a). Twenty strains from pigeons and psittacine species were found to belong to the taxon 3 complex (Piechulla et al., 1985a), which can be divided into several biovars based upon differences in production of indole, urease and acid from L( 1 )-arabinose, dulcitol, D(±)-sorbitol and D( 1 )-melibiose (Bisgaard, 1993). Twelve strains of psittacine origin formed a group tentatively designated Bisgaard taxon 33. On bovine blood agar, surface cultures of strains belonging to taxon 33 were circular, slightly raised and regular with an entire margin. The surface of the colonies was smooth, shiny and opaque with a grayish tinge. A colonial diameter of 1.0 to 1.5 mm was observed after aerobic incubation at 37° C for 24 h. After further incubation for 24 h, the diameter of the colonies reached 2.0 mm. The colonies had a buttery consistency and did not adhere to the agar surface. Growth on bovine blood agar was not accompanied by haemolysis or a speci® c odor. A greenish colour was sometimes associated with heavy inoculation of the blood agar. Phenotypic characters separating taxon 33 from the other species/taxa investigated are given in Table 2. Six strains from budgerigars and parrots formed another group, designated Bisgaard taxon 37. Surface cultures of this taxon on bovine blood agar were generally smaller (0.5 to 1.0 mm). A greenish colour was also sometimes associated with heavy growth of taxon 37 on blood agar. Differences in nitrate reduction, production of acid from glycerol, D( 1 )-galactose and maltose, and a -glucosidase separate taxon 33 and taxon 37. Both taxa are phenotypically related to organisms identi® ed as P. dagmatis, P.sp. A. and taxon 14. Characters separating these species/taxa are given in Table 2. A summary of the species/taxa identi® ed and their origin is given in Table 3.
Ribotyping Ribotyping and cluster analysis of the results obtained resulted in four major clusters designated I to IV (Figure 1). Cluster I included all strains classi® ed as taxon 33. Two strains (95 and 97) isolated from Amazona aestiva, and originating from the same ¯ ock, had identical ribotypes.Cluster II contained ® ve pigeon isolates classi® ed as biovars 1, 2, 4, 5 and 6 of taxon 3. Two budgerigar isolates, one of which was classi® ed as taxon 3 biovar 1, formed cluster III, underlining the heterogeneity of taxon 3. Three budgerigar isolates classi® ed as taxon 37 ® nally made up cluster IV. Less than 30% similarity was observed between cluster IV and clusters I to III.
Discussion Within the past few years, new insights have emerged into the taxonomy of the Pasteurellaceae family and hence into the classi® cation of isolates. Careful collection and phenotypic characterization of unclassi® ed and well-classi® ed isolates have led to the recognition of new species and even larger taxonomic groups, which appear to belong to, or have been de® nitely incorporated into, the family (Mutters et al., 1989; Bisgaard, 1995). Identi® cation of species/taxa belonging to this family, however, still presents considerable problems for the clinical bacteriologist. Thirty strains from rodents were recently reinvestigated and reclassi® ed after comparison with reference strains (Boot & Bisgaard, 1995). Strains of P. gallinarum isolated from guinea pigs were reclassi® ed as the SP-group and taxon 25, con® rming the statement of Bisgaard & Mutters (1986b) that publications on isolation of P. gallinarum from species other than poultry should be questioned. Re-examination of an Australian collection of strains previously identi® ed as A. equuli also highlighted the problems confronting diagnostic laboratories attempting to identify members of the family Pasteurellaceae (Blackall et al., 1997). The present investigation showed that 25 strains could be classi® ed into previously reported species/taxa, while 12 and six strains, respectively, were shown to represent new taxa, tentatively named Bisgaard taxon 33 and taxon 37. P. dagmatis has so far mainly been associated with the oral and nasal mucosa of dogs, cats and rodents (Bisgaard, 1993). Isolation of this species from trachea and lung of an Ara macao suffering from aspergillosis represents the ® rst isolate from birds. Isolation of NAD-independent strains of P. sp. A was ® rst reported by Bisgaard (1993) and subsequently con® rmed by Bragg et al. (1997). Isolation of NAD-independent strains from the heart, liver and spleen of a budgerigar with septicaemia con® rms that this species seems to exist in
Figure 1. Hpa II ribotype clusters obtained with nine strains classi® ed as taxon 33, seven strains belonging to the taxon 3 complex of Bisgaard, and three strains classi® ed as taxon 37. Molecular weight markers were HindIII digested phage l (l -HindIII).
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Characterization of avian Pasteurellaceae 375
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several species of birds. However, none of the NAD-independent isolates have yet been con® rmed genetically to represent P. sp. A. The ecology and signi® cance of the [P.] aerogenes-complex is only incompletely known (Bisgaard, 1993). Biovar 8 has been isolated from pigs (Bisgaard, unpublished results). Isolation of biovar 8 from a goose suffering from pneumonia and airsacculitis represents the ® rst isolate from birds. Organisms belonging to the taxa 2 and 3 complex have been obtained from ducks, geese, pigeons, partridges, pheasants and psittacine birds (Bisgaard, 1993). These organisms seem to represent a new genus with several species within the family Pasteurellaceae (Piechulla et al., 1985a; Dewhirst et al.,1993). Seven of the present strains classi® ed with the taxon 3 complex originated from psittacine species and 13 were isolated from pigeons. All of these isolates were associated with lesions. Ribotyping of selected strains showed that isolates from pigeons and budgerigars belonged to different clusters. With a few exceptions, a relationship has previously been demonstrated between protein pro® les of organisms classi® ed within this complex and the hosts from which the strains were isolated (Bisgaard et al., 1993). Strains classi® ed as taxon 33 shared the common phenotypic characteristics of the new avian taxa of Pasteurellaceae reported by Piechulla et al. (1985a). Eight strains with similar characters originating from budgerigars, parrots and a chicken from Belgium, Denmark and Germany exist in the culture collection of one of the authors. Five of these strains were associated with lesions, while information on association with disease is lacking for three strains. Ribotyping of selected strains showed that these strains formed a separate cluster. The ® nal classi® cation of these organisms will depend on subsequent 16S rRNA sequencing and DNA:DNA hybridization. Although strains classi® ed as taxon 37 shared many of the common characteristics of the family Pasteurellaceae (Piechulla et al., 1985a), these organisms do not reduce nitrate and thus should not be classi® ed as members of Pasteurellaceae. Ribotyping showed that strains belonging to this taxon formed a separate cluster, branching deeply with taxon 3 and taxon 33. Subsequent sequencing of 16S rRNA might enable the ® nal classi® cation of these organisms. Only two of six isolates classi® ed as taxon 37 were associated with lesions. Two additional strains exist in one of the author’ s strain collection, one isolated from a parakeet, and the other from a parrot. It is not known whether these strains were associated with lesions. The present ® ndings underline the importance of extended characterization of isolates and comparison with reference strains to avoid misclassi® cation within the family Pasteurellaceae Pohl 1981. In addition, atypical isolates should always be kept and sent to reference laboratories to
support further taxonomic progress. To improve our understanding of the epidemiology and virulence of these organisms, co-ordinated efforts in developing reliable, commercially available kits for phenotypic identi® cation, and speci® c, sensitive and reproductive DNA-based tests should be given higher priority.
References Avery, P. (1982) . Preliminary bacteriology in psittaciforme birds with some procedures in the identi® cation of the bacteria present. Proceedings of the Association of Avian Veterinarians, pp. 58±90. Baker, J.R. (1980) . A survey of causes of mortality in budgerigar s (Melanopsittus undulatus).Veterinary Record, 106, 10±12. Bisgaard, M. (1993). Ecology and signi® cance of Pasteurellaceae in animals. Zentralblatt fuÈr Bakteriologie, 279, 7±26. Bisgaard, M. (1995). Taxonomy of the family Pasteurellaceae Pohl 1981. In W. Donachie, F.A. Lainson & J.C. Hodgson (Eds.), Haemophilus , Actinobacillus, and Pasteurella (pp. 1±7). New York: Plenum Press. Bisgaard, M. & Mutters, R. (1986a). A new facultatively anaerobic Gram-negative fermentative rod obtained from different pathological lesions in poultry and tentatively designated taxon 14. Avian Pathology, 15, 117±127. Bisgaard, M. & Mutters, R. (1986b) . Characterization of some previously unclassi ® ed ª Pasteurellaº spp.obtained from the oral cavity of dogs and cats and description of a new species tentatively classi® ed with the family Pasteurellaceae Pohl 1981 and provisionally called taxon 16. Acta Pathologica Microbiologica Immunologica Scandinavica Section B, 94, 177±184. Bisgaard, M., Houghton , S.B., Mutters, R. & Stenzel, A. (1991). Reclassi® cation of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumoni c calf lungs. Veterinary Microbiology, 26, 115±124. Bisgaard, M., Brown, D.J., Costas, M. & Ganner , M. (1993) . Whole cell protein pro® ling of Actinobacillus-like strains classi® ed as taxon 2 and taxon 3 according to Bisgaard. Zentralblatt fuÈr Bakteriologie, 279, 92±103. Blackall, P.J., Bisgaard, M. & Mckenzie, R.A. (1997) . Characterization of Australian isolates of Actinobacillus capsulatus, Actinobacillus equuli, Pasteurella caballi and Bisgaard taxa 9 and 11. Australian Veterinary Journal, 75, 52±55. Boot, R. & Bisgaard, M. (1995). Reclassi® cation of 30 Pasteurellaceae strains isolated from rodents. Laboratory Animals, 29, 314± 319. Bragg, R.R., Greyling, J.M. & Verschoor, J.A. (1997) . Isolation and identi® cation of NAD-independen t bacteria from chickens with symptoms of infectious coryza. Avian Pathology, 26, 595±606. Christensen, J.P. & Bisgaard, M. (1997) . Avian pasteurellosis: taxonomy of the organisms involved and aspect s of pathogenesis . Avian Pathology, 26, 461±483. Christensen, J.P., Olsen, J.E. & Bisgaard, M. (1993). Ribotypes of Salmonella enterica serovar Gallinarum biovars gallinarum and pullorum. Avian Pathology, 22, 725±738. De Ley, J., Mannheim, W., Mutters, R., Piechulla, K., Tytgat, R., Segers, P., Bisgaard, M., Frederiksen, W., Hinz, K.-H., & Vanhoucke, M. (1990). Inter- and intrafamilial similarities of rRNA cistrons of the Pasteurellaceae. Internationa l Journal of Systematic Bacteriology, 40, 126±137. Devriese, L.A., Viaene, N., Uyttebroek, E., Froyman, R. & Hommez, J. (1988) . Three cases of infection by Haemophilus-like bacteria in psittacines. Avian Pathology, 17, 741±744. Dewhirst, F.E., Paster, B.J., Olsen, I. & Fraser, G.J. (1993) . Phylogeny of the Pasteurellaceae as determined by comparison of 16S ribosomal ribonucleic acid sequences. Zentralblatt fuÈr Bakteriologie, 279, 35±44. Dorrestein, G.M. (1997). Bacteriology. In R.B. Altman, S.L. Clubb, G.M. Dorrestein & K. Quesenberr y (Eds.), Avian Medicine and Surgery (pp. 255±280). Phildelphia: W.B.Saunders Company.
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Characterization of avian Pasteurellaceae Dorrestein, G.M., Buitelaar, M.N., van der Hage, M.H. & Zwart, P. (1985). Evaluation of a bacteriological and mycological examination of psittacine birds. Avian Diseases, 29, 951±962. Drew, M.L., Joyner , K. & Lobingler, R. (1993). Laboratory reference intervals for a group of captive thick-billed parrots (Rhynchopsitta pachyrhyncha) . Journal of the Association of Avian Veterinarian s, 7, 35±38. Flammer, K. & Drewes, L.A. (1988). Species-related differences in the incidence of Gram-negative bacteria isolated from the cloaca of clinically normal psittacine birds. Avian Diseases, 32, 79±83. Gerlach, H. (1994). Bacteria. In B.W. Richie, G.J. Harrison & L.R. Harrison (Eds.), Avian Medicine: Principles and Application (pp. 948±983). Lake Worth: Winger Publishers. MacDonald, J.W., Owen, D., Spencer, K.G. & Curtis, P.E. (1981) . Pasteurellosis in wild birds. Veterinary Record, 109, 58. Morishita, T.Y., Lowenstine, L.J., Hirsh, D.C. & Brooks, D.L. (1996) . Pasteurella multocida in psittacines: prevalence , pathology , and characterization of isolates. Avian Diseases, 40, 900±907. Mutters, R., Piechulla, K., Hinz, K.-H. & Mannheim, W. (1985) , Pasteurella avium (Hinz and Kunjara 1977) comb. nov. and Pasteurella volantium sp. nov. International Journal of Systematic Bacteriology, 35, 5±9. Mutters, R., Mannheim, W. & Bisgaard, M. (1989). Taxonomy of the group. In C. Adlam & J.M. Rutter, (Eds.), Pasteurella and pasteurelloses (pp. 3±34). London: Academi c Press. Piechulla, K., Bisgaard, M., Gerlach, H. & Mannheim, W. (1985a) . Taxonomy of some recently described avian Pasteurella/Actinobacillus-like organisms as indicated by deoxyribonuclei c acid relatedness. Avian Pathology, 14, 281±311. Piechulla, K., Hinz, K.-H. & Mannheim, W. (1985b) . Genetic and phenotypi c comparison of three new avian Haemophilus-like taxa and Haemophilu s paragallinaru m Biberstein and White1969 with other members of the family Pasteurellaceae Pohl 1981. Avian Diseases, 29, 601±612. Reavill, D. (1996). Bacterial Diseases. In W.R. Rosskopf Jr. & R.W. Woerpel (Eds.), Diseases of Cage and Aviary Birds (pp. 596±612). Baltimore: Williams & Wilkins. Rolf, J. (1977). Statistische Auswertung der Autopsien bei Zier-, Zoo und WildvoÈgeln. Berliner und MuÈnchene r TieraÈrztliche Wochenschrift, 90, 117±119. Zenoble, R.D., Grif® th, R.W. & Clubb, S.L. (1983). Survey of bacteriologic ¯ ora of cunjunctiva and cornea in healthy psittacine birds. American Journal of Veterinary Research, 44, 1966±1967.
REÂSUME CaracteÂrisation des souches de Pasteurella isoleÂes chez l’oiseaux Quarante trois souches, non classe es, appartenan t aux Pasteurellaceae dont la plupart ont e teÂobtenues aÁ partir de le sions, ont fait l’ objet de tests d’ identi® cation relatifs au phe notypage et au ribo typage. Les isolats ont e teÂobtenus de Anser anser forma (f) domestica (d), Agaporni s ® scher, Amazona spp., Ara macao, Columba livia f.d., Melanopsittacus undulatus, and Psittacus erithacus. La comparaiso n des re sultats aÁ ceux obtenus aÁ partir des souches de re fe rence a permis la classi® cation de 25 souches. Trois souches ont e teÂidenti® e es comme P. dagmatis, P. sp. A, et P. aerogenes. Vingt souches ont e teÂclasse es comme taxon 3 et deux comme taxon 14. Dix
377
huit souches, toutes isole es de psittacide s appartiennen t aÁ deux nouveaux taxons provisoiremen t de nomme s taxon Bisgaard 33 et taxon 37. Les caracteÁres du taxon 33 ont permis de le classer parmi les Pasteurellaceae, alors que la classi® cation ® nale du taxon 37 reste aÁ e tudier. La pre sente enqueà te souligne les probleÁmes auxquels se heurtent les laboratoires de diagnostic, en essayant d’ identi® er les membres de la famille Pasteurellaceae isole s d’ oiseaux. ZUSAMMENFASSUNG Charakterisierung einer aus VoÈgeln isolierten Kollektion von Pasteurellaceae Eine Kollektion von 43 unklassi® zierten AngehoÈrigen der Pasteurellaceae, von denen die meisten aus LaÈsionen isoliert worden waren, wurde mit Hilfe einer umfassenden Reihe von PhaÈnotyptests sowie durch Ribotypisierung untersucht . Die Isolate stammten aus Anser anser forma (f) domestica (d), Agaporni s ® scheri, Amazona spp., Ara macao, Columba livia f. d., Melanopsittacus undulatus und Psittacus erithacus. Der Vergleich der Ergebnisse mit denen von Referenzst aÈmmen ermoÈglichte die Klassi® zierung von 25 StaÈmmen. Drei StaÈmme wurden als P. dagmatis, P. sp. A und P. aerogene s identi® ziert. Zwanzig StaÈmme wurden als Taxon 3 und zwei als Taxon 14 identi® ziert. Achtzehn StaÈmme, die alle aus Psittaziden stammten, gehoÈrten zu zwei neuen Taxa, die vorlaÈu® g als Bisgaard-Taxon 33 und Taxon 37 bezeichne t wurden. Die beim Taxon 33 festgestellten Merkmale erlaubten dessen Einordnung in die Familie Pasteurellaceae, wohingegen die endguÈltige Klassi® zierung des Taxon 33 noch zu erforschen bleibt. Die vorliegende Untersuchung unterstreicht die Probleme, mit denen diagnostisch e Laboratorien konfrontier t sind, wenn sie versuchen, aus VoÈgeln isolierte AngehoÈrige der Familie Pasteurellaceae zu identi® zieren. RESUMEN Caracterizacio n de un grupo de aislados de la familia pasteurellaceae obtenidos de palomos Un grupo de 43 miembros de la familia Pasteurellaceae, la mayorõ  a de los cuales se habõ  an obtenido de lesiones tisulares, se estudiaron mediante una extensa baterõ  a de tests fenotõ  picos asõ Âcomo mediante ribotipi® cacio n. Los aislados procedõ  an de las siguientes especies Anser anser forma (f) dome stica (d), Agaporni s ® scher, Amazona spp., Ara macao, Columba livia f.d., Melopsittacus undulatus y Psittacus erithacus. La comparaci o n de los resultados con los obtenidos de las cepas de referencia, permitioÂclasi® car 25 cepas. Tres cepas fueron clasi® cadas como P. dagmatis, P. sp A, y (P.) aeroÂgenes, respectivamente . Veinte cepas fueron clasi® cadas como taxon 3 y dos como taxon 14. Dieciocho cepas, todas procedente s de psita cidas, pertenecõ  an a dos nuevos taxones, inicialmente denominado s taxon Bisgard 33 y taxon 37. Las caracterõ  sticas del taxon 33 permitieron su inclusio n en la familia Pasteurellaceae mientras que la clasi® cacio n del taxon 37 permanece sin determinar . El presente trabajo subraya los problemas existentes al confrontar diagno sticos laboratoriales, intentando identi® car miembros de la familia Pasterurellaceae aislados en aves.
Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20
Identification of members of the Pasteurellaceae isolated from birds and characterization of two new taxa isolated from psittacine birds M. Bisgaard , K.-H. Hinz , K.D. Petersen & J.P. Christensen Published online: 17 Jun 2010.
To cite this article: M. Bisgaard , K.-H. Hinz , K.D. Petersen & J.P. Christensen (1999) Identification of members of the Pasteurellaceae isolated from birds and characterization of two new taxa isolated from psittacine birds, Avian Pathology, 28:4, 369-377, DOI: 10.1080/03079459994632 To link to this article: http://dx.doi.org/10.1080/03079459994632
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Avian Pathology (1999) 28, 369±377
Identi® cation of members of the Pasteurellaceae isolated from birds and characterization of two new taxa isolated from psittacine birds M. Bisgaard 1* K.-H. Hinz2, K.D. Petersen1 & J.P. Christensen 1 1
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Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Copenhagen, Denmark, and 2Klinik fuÈr Ge¯ uÈgel, TieraÈrztliche Hochschule Hannover, 30559 Hannover, Germany
A collection of 43 unclassi® ed members of the Pasteurellaceae, most of which were obtained from lesions, were investigated using an extensive battery of phenotypical tests as well as by ribotyping. The isolates had been made from Anser anser forma (f) domestica (d), Agapornis ® scher, Amazona spp., Ara macao, Columba livia f.d., Melanopsittacus undulatus, and Psittacus erithacus. Comparison of results with those obtained from reference strains allowed classi® cation of 25 strains. Three strains were identi® ed as Pasteurella dagmatis, P. sp. A, and [P.] aerogenes, respectively. Twenty strains were classi® ed as taxon 3 and two as taxon 14. Eighteen strains, all originating from psittacine species, belonged to two new taxa, tentatively named Bisgaard taxon 33 and taxon 37. Characters obtained with taxon 33 allowed classi® cation within the family Pasteurellaceae, while the ® nal classi® cation of taxon 37 remains to be investigated. The present investigation underlines the problems confronting diagnostic laboratories attempting to identify members of the family Pasteurellaceae isolated from birds.
Introduction The genomic relationships of some recently described avian Pasteurella/Actinobacillus -like organisms were investigated by Piechulla et al. (1985a) to evaluate the taxonomic resolution of diagnostically useful phenotypic criteria. Careful collection and characterization of unclassi® ed strains, tentatively classi® ed as members of the Pasteurellaceae, have subsequently resulted in many new species or taxa, the prevalence and pathogenic potential of which are only partially known (Bisgaard, 1995). Members of the genus Pasteurella have been associated with disease in Phasianiformes, Anatiformes, Psittaciformes, Columbiformes and Passeriformes, with Pasteurella multocida and Pasteurella gallinarum being the most commonly isolated species (Gerlach, 1994; Reavill, 1996; Dorrestein, 1997). Members of the Pasteurellaceae were, however, not obtained from the cloacae of clinically healthy psittacine birds by Flammer & Drewes (1988), and Morishita et al. (1996) were unable to isolate P. multocida * To whom correspondenc e should be addressed. Tel: 1 Received 30 July 1998. Accepted 12 February 1999.
from the pharynx, choanae, or cloacae. However, P. multocida was obtained from ® ve dead psittacines with septicemia or cutaneous lesions. Reports of isolation of Pasteurella sp. from psittacine species with or without lesions have also been reported by Rolf (1977), Baker (1980), Dorrestein et al. (1985), and Drew et al. (1993). Although disease in pigeons caused by P. multocida seems to be rare, P. multocida was isolated from three out of 15 wild pigeons sampled by MacDonald et al. (1981). Recently, Dorrestein (1997) reported isolation of [P.] pneumotropicalike organisms from collared turtledoves. Taxa classi® ed within the genus Actinobacillus have been reported from members of the Psittaciformes, Columbiformes, Anatiformes and Fringillidae. However, knowledge of the biology and pathogenicity of members of this genus is limited (Gerlach, 1994). Actinobacillus spp. have been isolated from more than 1% of normal psittacine corneas and ocular conjunctivas (Zenoble et al., 1983). Chronic sinus infections associ-
45 35282760 . Fax: 1 45 35282757 . E-mail: [email protected] or [email protected]
ISSN 0307-945 7 (print)/ISSN 1465-3338 (online)/99/040369-0 9
Ó
1999 Houghton Trust Ltd
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ated with Actinobacillus spp. have been reported in parrots (Avery, 1982). True species of the genus Actinobacillus have not yet been reported from birds, according to Christensen & Bisgaard (1997). Three new avian Haemophilus-like taxa were reported by Piechulla et al. (1985b). None of these, however, belonged to recognized genera. Haemophilus-like organisms have subsequently been reported from psittacines (Devriese et al., 1988). Members of a new group of organisms, tentatively designated taxon 14, were isolated from different lesions in ducks, turkeys and a pigeon by Bisgaard & Mutters (1986a). These organisms were subsequently shown to belong to the family Pasteurellaceae by De Ley et al. (1990) and Dewhirst et al. (1993). Supplying the market with pet birds depends increasingly on the success of avicultural breeding programmes, due to restrictions and legislation placed on importation of wild caught birds into the European Union. Most members of the family Pasteurellaceae appear to be commensals of freeliving hosts. However, high density and con® ned rearing facilities developed for the captive breeding of these species might change the parasite±host balance, which seems to exist on mucous membranes under normal conditions, and result in disease. The ecology and signi® cance of members of the Pasteurellaceae were reviewed by Bisgaard (1993), who stressed that a more accurate basis for diagnosis is a prerequisite for improving our knowledge of the complexity of factors that govern the ecological preferences and virulence of these organisms. Consequently, the aim of the present investigation was to characterize a collection of organisms provisionally classi® ed within the Pasteurellaceae and isolated from pigeons, a goose, and psittacine birds to extend our knowledge of the signi® cance of these organisms, and to establish criteria for their identi® cation. Material and Methods Source of strains investigated Fifteen strains from pigeons were investigated, ® ve of which were obtained from birds with septicaemia, while 10 strains were isolated from respiratory tract lesions (Table 1). A single isolate originated from a goose, while the remaining 27 isolates originated from different species of psittacine birds. Four strains were associated with septicaemic conditions, 14 with respiratory tract lesions, while four strains were associated with other lesions. Six isolates were not associated with macroscopi c lesions (Table 1). Bacteriological investigations A total of 43 strains were investigated (Table 1). All strains had been kept as unclassi® ed member s of the Pasteurellaceae at ±80°C or as freeze-dried cultures since their original isolation. These strains can be obtained from the corresponding author. Before phenotypi c characterization, all strains were plated on blood agar (Tryptose blood agar base (Difco, Detroit, USA) containing 5% citrated bovine blood), and
incubated aerobically at 37 °C for 24 h. Pure cultures were subsequently characterized phenotypicall y by standard methods as described previousl y (Bisgaard et al., 1991) and compared with those from type or reference strains of Pasteurellaceae (Mutters et al., 1989).
Ribotyping and band pattern analysis Nineteen strains representing two new taxa, as well as different biovars of taxon 3 from different species of birds, were selected for ribotyping (Table 1). Isolation of DNA, digestion of DNA by restriction endonucleases , separation of DNA fragments in agarose gels, DNA blotting and hybridization at 56 °C with a digoxigenin-labele d probe (Boehringer-Mannheim , Mannheim, Germany) complementar y to 16S and 23S rRNA of Escherichi a coli (Sigma, St. Louis, MO, USA) were carried out as reported previously by Christensen et al. (1993). HpaII was used to digest DNA as it provided an appropriate number of distinct bands (from 7 to 14) for all strains investigated on ribotyping. Digoxigenin-labelle d phage lambda-DNA (Boehringer Mannheim) cut with HindIII was used as a molecular size marker. Ribotyping based upon new DNA preparations was repeated at least twice for each strain. Band patterns obtained with the strains investigated were analyzed using GelCompar version 4.0 (Applied Maths BVBA, Kortrijk, Belgium). Band positions were analyzed using ® ne optimization and a position tolerance of 1%. The similarity between the band pattern of individual strains was estimated using the Dice coef® cient, and dendrograms were drawn using the similarity matrix obtained (unweighted pair group method with averages) .
Results Bacteriological investigations All strains investigated had identical reactions in the following tests: porphyrin test, alanine aminopeptidase, Hugh and Leifson (fermentative), and production of acid from D(±)-fructose, D( 1 )glucose, and sucrose (all positive), Gram staining, motility (37 and 22° C), symbiotic growth, b haemolysis, growth on Simmons citrate, formation of base from malonate, H2S formation, KCN growth, Voges±Proskauer test at 37°C, production of gas from nitrate, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, formation of indole, gelatinase, hydrolysis of Tween 20 and 80, formation of pigment, production of acid from meso-erythritol, adonitol, xylitol, L(±)-xylose, D( 1 )-fucose, L ( 1 )-rhamnose, L (±)-sorbose, cellobiose, D( 1 )melezitose, D-glycogen, inulin, aesculin, amygdalin, arbutin, gentiobiose, salicin, D( 1 )-turanose, and b -N-CH3-glucosamide, a -fucosidase, b -glucuronidase, and a -mannosidase (all negative). Characters separating the strains investigated are shown in Table 2. One strain from a scarlet macaw was classi® ed as P. dagmatis (Bisgaard & Mutters, 1986b), while another isolate from a budgerigar was classi® ed as P. sp. A (Mutters et al., 1985). The goose isolate was classi® ed as [P.] aerogenes biovar 8 (Bisgaard, 1993). Two strains from pigeons were identi® ed as taxon 14 biovar 1 (Bisgaard &
Table 1. Information on the 43 strains investigated during this study
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Characterization of avian Pasteurellaceae 371
b
: selected for ribotyping : same ¯ ock c : forma domestica
a
Table 1. Information on the 43 strains investigated during this studyÐ (continued)
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1
1
1
1
) )
1
)
)
1
1
1
d d d ± ± /( 1 d ± /( 1 1 1 ± /( 1 ± /( 1 ± d d ± ± ± d ± ± ± d
1
± d ± ± d /( 1 /( 1 ± 1 1 1 d /( 1 ± 1 ± d d ± /( 1 d 1 ± 1 d d
1
1
1
1
)
)
)
)
±
d 1 /( 1 ) d ± (1 ) 1 d ± 1 1 ± 1 /( 1 ) 1 ± 1 ± 1 /( 1 ) 1 ± ± ± d ± ± ± d
1
1
1
d d
1
d d
1
±
1
1
d d ±
Taxon 3 biovar 4
Taxon 3 biovar 2
1
(1
1
1
1
1
1
1
(1
1
1
1
1
1
± ± ± ± ± ± ± d
±
±
± d
±
± ± d
d
± ±
)
)
Taxon 3 biovar 5
a
1 , all strains positive within 1 to 2 days; ( 1 ), all strains late positive ( $ within 14 days; d, different; w, weak positive.
Catalase Oxidase, TMPD Mucate, acid MR, 37°C Nitrate, red. Urease Phosphatase McConkey,growth Glycerol D( 1 )-Arabitol L( 1 )-Arabinose D(-)-Arabinose D(-)-Ribose D( 1 )-Xylose Dulcitol Meso-inositol D (-)-Mannitol D(-)-Sorbitol L(-)-Fucose D( 1 )-Galactose D( 1 )-Glucose, gas D( 1 )-Mannose b -Glucosidase Lactose ONPG Maltose D( 1 )-Melibiose Trehalose Raf® nose Dextrin a -Galactosidase a -Glucosidase b -Xylosidase
Taxon 3 biovar 1
±
d w
3 days); 1
d 1 /( 1 ) ± ± (1 ) 1 1 ± 1 1 1 1 /( 1 ) 1 ± 1 ± (1 ) 1 ± ± ± ± ± ± ± ±
1
1
1
1
Taxon 3 biovar 6
/( 1
±
±
± ± ± ±
±
± ± ±
±
±
± ±
)
)
1
1
1
1
1
± ± ± ± ± ± ± ± ± ±
± ±
± ± ± ± ± ± d ± ± ± ± ±
±
± ± ±
Taxon 14 biovar 1
± ± ±
± d ±
)
)
d ± ± ± ± ± 1 /( 1 ) ± 1 ± ± ± ± ± ± (1 ) ± ± ± ±
(1
(1
1
1
d d ±
Taxon 33
1
1
± ± d ± ± ± ± ±
d ±
)
± ± ± ± ± ± ± ± /( 1 ) ± ± ± /( 1 ) ± d d ± ± 1 ±
(1
1
1
Taxon 37
(1
1
1
1
1
(1
(1
(1
1
1
1
1
1
1
± ± ± ±
± ±
±
±
± ± ± ± ± ±
± ± ±
±
±
)
)
)
)
P. dagmatis
±
±
± ±
(1 ) ± (1 ) ± (1 ) ± ± ± ± ± ± (1 ) ± 1 ± 1 1 1 ± 1 (1 ) 1 ± ± ±
1
1
1
1
P. sp. A
1
(1
1
1
(1
1
1
(1
1
1
1
1
1
1
1
1
1
1
1
± ± ±
± ±
±
± ±
±
± ±
± w ±
)
)
)
P. aerogenes biovar 8
), all strains positive, some strains within 1 to 2 days, others late positive; ±, all strains negative
1
1
1
1
(1
1
1
1
1
1
1
1
1
1
(1
1
1
1
1
Taxon 2/3 biovar 5
Table 2. Phenotypic characters separating the taxa investigateda
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Characterization of avian Pasteurellaceae 373
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M. Bisgaard et al.
Table 3. Results obtained with 43 strains tentatively classi® ed with the family Pasteurellaceae Pohl 1981 Taxon
Goose
P. dagmatis P. sp. A P. aerogenes biovar 8 Taxon 3 biovar 1 Taxon 3 biovar 2 Taxon 3 biovar 4 Taxon 3 biovar 5 Taxon 3 biovar 6 Taxon 2/3 biovar 5 Taxon 14 biovar 1 Taxon 33 Taxon 37a
1 1 ± 4 2 ± ± ± 1 ± 12 6
± ± ± 1 1 5 3 3 ± 2 ± ±
± ± 1 ± ± ± ± ± ± ± ± ±
Total
27
15
1
a
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Psittacine birds Pigeons
Does not belong to the family Pasteurellaceae sensu stricto.
Mutters, 1986a). Twenty strains from pigeons and psittacine species were found to belong to the taxon 3 complex (Piechulla et al., 1985a), which can be divided into several biovars based upon differences in production of indole, urease and acid from L( 1 )-arabinose, dulcitol, D(±)-sorbitol and D( 1 )-melibiose (Bisgaard, 1993). Twelve strains of psittacine origin formed a group tentatively designated Bisgaard taxon 33. On bovine blood agar, surface cultures of strains belonging to taxon 33 were circular, slightly raised and regular with an entire margin. The surface of the colonies was smooth, shiny and opaque with a grayish tinge. A colonial diameter of 1.0 to 1.5 mm was observed after aerobic incubation at 37° C for 24 h. After further incubation for 24 h, the diameter of the colonies reached 2.0 mm. The colonies had a buttery consistency and did not adhere to the agar surface. Growth on bovine blood agar was not accompanied by haemolysis or a speci® c odor. A greenish colour was sometimes associated with heavy inoculation of the blood agar. Phenotypic characters separating taxon 33 from the other species/taxa investigated are given in Table 2. Six strains from budgerigars and parrots formed another group, designated Bisgaard taxon 37. Surface cultures of this taxon on bovine blood agar were generally smaller (0.5 to 1.0 mm). A greenish colour was also sometimes associated with heavy growth of taxon 37 on blood agar. Differences in nitrate reduction, production of acid from glycerol, D( 1 )-galactose and maltose, and a -glucosidase separate taxon 33 and taxon 37. Both taxa are phenotypically related to organisms identi® ed as P. dagmatis, P.sp. A. and taxon 14. Characters separating these species/taxa are given in Table 2. A summary of the species/taxa identi® ed and their origin is given in Table 3.
Ribotyping Ribotyping and cluster analysis of the results obtained resulted in four major clusters designated I to IV (Figure 1). Cluster I included all strains classi® ed as taxon 33. Two strains (95 and 97) isolated from Amazona aestiva, and originating from the same ¯ ock, had identical ribotypes.Cluster II contained ® ve pigeon isolates classi® ed as biovars 1, 2, 4, 5 and 6 of taxon 3. Two budgerigar isolates, one of which was classi® ed as taxon 3 biovar 1, formed cluster III, underlining the heterogeneity of taxon 3. Three budgerigar isolates classi® ed as taxon 37 ® nally made up cluster IV. Less than 30% similarity was observed between cluster IV and clusters I to III.
Discussion Within the past few years, new insights have emerged into the taxonomy of the Pasteurellaceae family and hence into the classi® cation of isolates. Careful collection and phenotypic characterization of unclassi® ed and well-classi® ed isolates have led to the recognition of new species and even larger taxonomic groups, which appear to belong to, or have been de® nitely incorporated into, the family (Mutters et al., 1989; Bisgaard, 1995). Identi® cation of species/taxa belonging to this family, however, still presents considerable problems for the clinical bacteriologist. Thirty strains from rodents were recently reinvestigated and reclassi® ed after comparison with reference strains (Boot & Bisgaard, 1995). Strains of P. gallinarum isolated from guinea pigs were reclassi® ed as the SP-group and taxon 25, con® rming the statement of Bisgaard & Mutters (1986b) that publications on isolation of P. gallinarum from species other than poultry should be questioned. Re-examination of an Australian collection of strains previously identi® ed as A. equuli also highlighted the problems confronting diagnostic laboratories attempting to identify members of the family Pasteurellaceae (Blackall et al., 1997). The present investigation showed that 25 strains could be classi® ed into previously reported species/taxa, while 12 and six strains, respectively, were shown to represent new taxa, tentatively named Bisgaard taxon 33 and taxon 37. P. dagmatis has so far mainly been associated with the oral and nasal mucosa of dogs, cats and rodents (Bisgaard, 1993). Isolation of this species from trachea and lung of an Ara macao suffering from aspergillosis represents the ® rst isolate from birds. Isolation of NAD-independent strains of P. sp. A was ® rst reported by Bisgaard (1993) and subsequently con® rmed by Bragg et al. (1997). Isolation of NAD-independent strains from the heart, liver and spleen of a budgerigar with septicaemia con® rms that this species seems to exist in
Figure 1. Hpa II ribotype clusters obtained with nine strains classi® ed as taxon 33, seven strains belonging to the taxon 3 complex of Bisgaard, and three strains classi® ed as taxon 37. Molecular weight markers were HindIII digested phage l (l -HindIII).
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several species of birds. However, none of the NAD-independent isolates have yet been con® rmed genetically to represent P. sp. A. The ecology and signi® cance of the [P.] aerogenes-complex is only incompletely known (Bisgaard, 1993). Biovar 8 has been isolated from pigs (Bisgaard, unpublished results). Isolation of biovar 8 from a goose suffering from pneumonia and airsacculitis represents the ® rst isolate from birds. Organisms belonging to the taxa 2 and 3 complex have been obtained from ducks, geese, pigeons, partridges, pheasants and psittacine birds (Bisgaard, 1993). These organisms seem to represent a new genus with several species within the family Pasteurellaceae (Piechulla et al., 1985a; Dewhirst et al.,1993). Seven of the present strains classi® ed with the taxon 3 complex originated from psittacine species and 13 were isolated from pigeons. All of these isolates were associated with lesions. Ribotyping of selected strains showed that isolates from pigeons and budgerigars belonged to different clusters. With a few exceptions, a relationship has previously been demonstrated between protein pro® les of organisms classi® ed within this complex and the hosts from which the strains were isolated (Bisgaard et al., 1993). Strains classi® ed as taxon 33 shared the common phenotypic characteristics of the new avian taxa of Pasteurellaceae reported by Piechulla et al. (1985a). Eight strains with similar characters originating from budgerigars, parrots and a chicken from Belgium, Denmark and Germany exist in the culture collection of one of the authors. Five of these strains were associated with lesions, while information on association with disease is lacking for three strains. Ribotyping of selected strains showed that these strains formed a separate cluster. The ® nal classi® cation of these organisms will depend on subsequent 16S rRNA sequencing and DNA:DNA hybridization. Although strains classi® ed as taxon 37 shared many of the common characteristics of the family Pasteurellaceae (Piechulla et al., 1985a), these organisms do not reduce nitrate and thus should not be classi® ed as members of Pasteurellaceae. Ribotyping showed that strains belonging to this taxon formed a separate cluster, branching deeply with taxon 3 and taxon 33. Subsequent sequencing of 16S rRNA might enable the ® nal classi® cation of these organisms. Only two of six isolates classi® ed as taxon 37 were associated with lesions. Two additional strains exist in one of the author’ s strain collection, one isolated from a parakeet, and the other from a parrot. It is not known whether these strains were associated with lesions. The present ® ndings underline the importance of extended characterization of isolates and comparison with reference strains to avoid misclassi® cation within the family Pasteurellaceae Pohl 1981. In addition, atypical isolates should always be kept and sent to reference laboratories to
support further taxonomic progress. To improve our understanding of the epidemiology and virulence of these organisms, co-ordinated efforts in developing reliable, commercially available kits for phenotypic identi® cation, and speci® c, sensitive and reproductive DNA-based tests should be given higher priority.
References Avery, P. (1982) . Preliminary bacteriology in psittaciforme birds with some procedures in the identi® cation of the bacteria present. Proceedings of the Association of Avian Veterinarians, pp. 58±90. Baker, J.R. (1980) . A survey of causes of mortality in budgerigar s (Melanopsittus undulatus).Veterinary Record, 106, 10±12. Bisgaard, M. (1993). Ecology and signi® cance of Pasteurellaceae in animals. Zentralblatt fuÈr Bakteriologie, 279, 7±26. Bisgaard, M. (1995). Taxonomy of the family Pasteurellaceae Pohl 1981. In W. Donachie, F.A. Lainson & J.C. Hodgson (Eds.), Haemophilus , Actinobacillus, and Pasteurella (pp. 1±7). New York: Plenum Press. Bisgaard, M. & Mutters, R. (1986a). A new facultatively anaerobic Gram-negative fermentative rod obtained from different pathological lesions in poultry and tentatively designated taxon 14. Avian Pathology, 15, 117±127. Bisgaard, M. & Mutters, R. (1986b) . Characterization of some previously unclassi ® ed ª Pasteurellaº spp.obtained from the oral cavity of dogs and cats and description of a new species tentatively classi® ed with the family Pasteurellaceae Pohl 1981 and provisionally called taxon 16. Acta Pathologica Microbiologica Immunologica Scandinavica Section B, 94, 177±184. Bisgaard, M., Houghton , S.B., Mutters, R. & Stenzel, A. (1991). Reclassi® cation of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumoni c calf lungs. Veterinary Microbiology, 26, 115±124. Bisgaard, M., Brown, D.J., Costas, M. & Ganner , M. (1993) . Whole cell protein pro® ling of Actinobacillus-like strains classi® ed as taxon 2 and taxon 3 according to Bisgaard. Zentralblatt fuÈr Bakteriologie, 279, 92±103. Blackall, P.J., Bisgaard, M. & Mckenzie, R.A. (1997) . Characterization of Australian isolates of Actinobacillus capsulatus, Actinobacillus equuli, Pasteurella caballi and Bisgaard taxa 9 and 11. Australian Veterinary Journal, 75, 52±55. Boot, R. & Bisgaard, M. (1995). Reclassi® cation of 30 Pasteurellaceae strains isolated from rodents. Laboratory Animals, 29, 314± 319. Bragg, R.R., Greyling, J.M. & Verschoor, J.A. (1997) . Isolation and identi® cation of NAD-independen t bacteria from chickens with symptoms of infectious coryza. Avian Pathology, 26, 595±606. Christensen, J.P. & Bisgaard, M. (1997) . Avian pasteurellosis: taxonomy of the organisms involved and aspect s of pathogenesis . Avian Pathology, 26, 461±483. Christensen, J.P., Olsen, J.E. & Bisgaard, M. (1993). Ribotypes of Salmonella enterica serovar Gallinarum biovars gallinarum and pullorum. Avian Pathology, 22, 725±738. De Ley, J., Mannheim, W., Mutters, R., Piechulla, K., Tytgat, R., Segers, P., Bisgaard, M., Frederiksen, W., Hinz, K.-H., & Vanhoucke, M. (1990). Inter- and intrafamilial similarities of rRNA cistrons of the Pasteurellaceae. Internationa l Journal of Systematic Bacteriology, 40, 126±137. Devriese, L.A., Viaene, N., Uyttebroek, E., Froyman, R. & Hommez, J. (1988) . Three cases of infection by Haemophilus-like bacteria in psittacines. Avian Pathology, 17, 741±744. Dewhirst, F.E., Paster, B.J., Olsen, I. & Fraser, G.J. (1993) . Phylogeny of the Pasteurellaceae as determined by comparison of 16S ribosomal ribonucleic acid sequences. Zentralblatt fuÈr Bakteriologie, 279, 35±44. Dorrestein, G.M. (1997). Bacteriology. In R.B. Altman, S.L. Clubb, G.M. Dorrestein & K. Quesenberr y (Eds.), Avian Medicine and Surgery (pp. 255±280). Phildelphia: W.B.Saunders Company.
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Characterization of avian Pasteurellaceae Dorrestein, G.M., Buitelaar, M.N., van der Hage, M.H. & Zwart, P. (1985). Evaluation of a bacteriological and mycological examination of psittacine birds. Avian Diseases, 29, 951±962. Drew, M.L., Joyner , K. & Lobingler, R. (1993). Laboratory reference intervals for a group of captive thick-billed parrots (Rhynchopsitta pachyrhyncha) . Journal of the Association of Avian Veterinarian s, 7, 35±38. Flammer, K. & Drewes, L.A. (1988). Species-related differences in the incidence of Gram-negative bacteria isolated from the cloaca of clinically normal psittacine birds. Avian Diseases, 32, 79±83. Gerlach, H. (1994). Bacteria. In B.W. Richie, G.J. Harrison & L.R. Harrison (Eds.), Avian Medicine: Principles and Application (pp. 948±983). Lake Worth: Winger Publishers. MacDonald, J.W., Owen, D., Spencer, K.G. & Curtis, P.E. (1981) . Pasteurellosis in wild birds. Veterinary Record, 109, 58. Morishita, T.Y., Lowenstine, L.J., Hirsh, D.C. & Brooks, D.L. (1996) . Pasteurella multocida in psittacines: prevalence , pathology , and characterization of isolates. Avian Diseases, 40, 900±907. Mutters, R., Piechulla, K., Hinz, K.-H. & Mannheim, W. (1985) , Pasteurella avium (Hinz and Kunjara 1977) comb. nov. and Pasteurella volantium sp. nov. International Journal of Systematic Bacteriology, 35, 5±9. Mutters, R., Mannheim, W. & Bisgaard, M. (1989). Taxonomy of the group. In C. Adlam & J.M. Rutter, (Eds.), Pasteurella and pasteurelloses (pp. 3±34). London: Academi c Press. Piechulla, K., Bisgaard, M., Gerlach, H. & Mannheim, W. (1985a) . Taxonomy of some recently described avian Pasteurella/Actinobacillus-like organisms as indicated by deoxyribonuclei c acid relatedness. Avian Pathology, 14, 281±311. Piechulla, K., Hinz, K.-H. & Mannheim, W. (1985b) . Genetic and phenotypi c comparison of three new avian Haemophilus-like taxa and Haemophilu s paragallinaru m Biberstein and White1969 with other members of the family Pasteurellaceae Pohl 1981. Avian Diseases, 29, 601±612. Reavill, D. (1996). Bacterial Diseases. In W.R. Rosskopf Jr. & R.W. Woerpel (Eds.), Diseases of Cage and Aviary Birds (pp. 596±612). Baltimore: Williams & Wilkins. Rolf, J. (1977). Statistische Auswertung der Autopsien bei Zier-, Zoo und WildvoÈgeln. Berliner und MuÈnchene r TieraÈrztliche Wochenschrift, 90, 117±119. Zenoble, R.D., Grif® th, R.W. & Clubb, S.L. (1983). Survey of bacteriologic ¯ ora of cunjunctiva and cornea in healthy psittacine birds. American Journal of Veterinary Research, 44, 1966±1967.
REÂSUME CaracteÂrisation des souches de Pasteurella isoleÂes chez l’oiseaux Quarante trois souches, non classe es, appartenan t aux Pasteurellaceae dont la plupart ont e teÂobtenues aÁ partir de le sions, ont fait l’ objet de tests d’ identi® cation relatifs au phe notypage et au ribo typage. Les isolats ont e teÂobtenus de Anser anser forma (f) domestica (d), Agaporni s ® scher, Amazona spp., Ara macao, Columba livia f.d., Melanopsittacus undulatus, and Psittacus erithacus. La comparaiso n des re sultats aÁ ceux obtenus aÁ partir des souches de re fe rence a permis la classi® cation de 25 souches. Trois souches ont e teÂidenti® e es comme P. dagmatis, P. sp. A, et P. aerogenes. Vingt souches ont e teÂclasse es comme taxon 3 et deux comme taxon 14. Dix
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huit souches, toutes isole es de psittacide s appartiennen t aÁ deux nouveaux taxons provisoiremen t de nomme s taxon Bisgaard 33 et taxon 37. Les caracteÁres du taxon 33 ont permis de le classer parmi les Pasteurellaceae, alors que la classi® cation ® nale du taxon 37 reste aÁ e tudier. La pre sente enqueà te souligne les probleÁmes auxquels se heurtent les laboratoires de diagnostic, en essayant d’ identi® er les membres de la famille Pasteurellaceae isole s d’ oiseaux. ZUSAMMENFASSUNG Charakterisierung einer aus VoÈgeln isolierten Kollektion von Pasteurellaceae Eine Kollektion von 43 unklassi® zierten AngehoÈrigen der Pasteurellaceae, von denen die meisten aus LaÈsionen isoliert worden waren, wurde mit Hilfe einer umfassenden Reihe von PhaÈnotyptests sowie durch Ribotypisierung untersucht . Die Isolate stammten aus Anser anser forma (f) domestica (d), Agaporni s ® scheri, Amazona spp., Ara macao, Columba livia f. d., Melanopsittacus undulatus und Psittacus erithacus. Der Vergleich der Ergebnisse mit denen von Referenzst aÈmmen ermoÈglichte die Klassi® zierung von 25 StaÈmmen. Drei StaÈmme wurden als P. dagmatis, P. sp. A und P. aerogene s identi® ziert. Zwanzig StaÈmme wurden als Taxon 3 und zwei als Taxon 14 identi® ziert. Achtzehn StaÈmme, die alle aus Psittaziden stammten, gehoÈrten zu zwei neuen Taxa, die vorlaÈu® g als Bisgaard-Taxon 33 und Taxon 37 bezeichne t wurden. Die beim Taxon 33 festgestellten Merkmale erlaubten dessen Einordnung in die Familie Pasteurellaceae, wohingegen die endguÈltige Klassi® zierung des Taxon 33 noch zu erforschen bleibt. Die vorliegende Untersuchung unterstreicht die Probleme, mit denen diagnostisch e Laboratorien konfrontier t sind, wenn sie versuchen, aus VoÈgeln isolierte AngehoÈrige der Familie Pasteurellaceae zu identi® zieren. RESUMEN Caracterizacio n de un grupo de aislados de la familia pasteurellaceae obtenidos de palomos Un grupo de 43 miembros de la familia Pasteurellaceae, la mayorõ  a de los cuales se habõ  an obtenido de lesiones tisulares, se estudiaron mediante una extensa baterõ  a de tests fenotõ  picos asõ Âcomo mediante ribotipi® cacio n. Los aislados procedõ  an de las siguientes especies Anser anser forma (f) dome stica (d), Agaporni s ® scher, Amazona spp., Ara macao, Columba livia f.d., Melopsittacus undulatus y Psittacus erithacus. La comparaci o n de los resultados con los obtenidos de las cepas de referencia, permitioÂclasi® car 25 cepas. Tres cepas fueron clasi® cadas como P. dagmatis, P. sp A, y (P.) aeroÂgenes, respectivamente . Veinte cepas fueron clasi® cadas como taxon 3 y dos como taxon 14. Dieciocho cepas, todas procedente s de psita cidas, pertenecõ  an a dos nuevos taxones, inicialmente denominado s taxon Bisgard 33 y taxon 37. Las caracterõ  sticas del taxon 33 permitieron su inclusio n en la familia Pasteurellaceae mientras que la clasi® cacio n del taxon 37 permanece sin determinar . El presente trabajo subraya los problemas existentes al confrontar diagno sticos laboratoriales, intentando identi® car miembros de la familia Pasterurellaceae aislados en aves.
