Veterinary Microbiology, 26 ( 1991 ) 1 | 5 - 1 2 4 Elsevier Science Publishers B.V., A m s t e r d a m
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Reclassification of German, British and Dutch isolates of so-called Pasteurella rnultocida obtained from pneumonic calf lungs M. Bisgaard a, S.B. Houghton b, R. Mutters c and A. Stenzel ~ alnstitute of Avian Diseases, The Royal Veterinary and Agricultural University, 13 Biilowsvej, DK- 1870 Copenhagen V, Denmark bHoechst Animal Health, A Division of Hoechst UK Ltd, Walton Manor, Milton Keynes, Buckinghamshire MK7 7AJ, UK Clnstitute of Medical Microbiology, Klinikum der Philipps-Universitiit, 2 Pilgrimstein, D-3550 Marburg, Germany (Accepted 14 June 1990)
ABSTRACT Bisgaard, M., Houghton, S.B., Mutters, R. and Stenzel, A., 1991. Reclassification of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumonic calf lungs. Vet Microbiol., 26:115-124. The taxonomic relationship of 131 strains previously identified as Pasteurella multocida obtained from calf pneumonia in West Germany, United Kingdom and Netherlands was investigated by extended phenotypic and limited genotypic characterization. Twenty-four strains were classified as P. multocida ssp. multocida, 15 strains as P. avium biovar 2 and 13 strains as P. canis biovar 2. Sixtyfive and five strains were tentatively classified as ornithine negative P. multocida ssp. multocida and P. multocida ssp. septica, respectively. Genetic investigations showed that ornithine negative strains of P. multocida were related on species level. Less genomic binding was found between an ornithine negative strain ofP. multocida ssp. septica and the type strains of the three subspecies ofP. multocida. The taxonomic position of ornithine negative strains ofP. multocida is still under investigation. The taxonomic position of the remaining nine strains is uncertain underlining the need for genotypic characterization within the genus Pasteurella to aid in defining single species by phenotypic tests.
INTRODUCTION
Pneumonic "pasteurellosis" is a major cause of economic loss and mortality in cattle (King et al., 1958; Jensen et al., 1976; Martin et al., 1980, 1981 ). The disease complex seems to be caused by a combination of respiratory viruses, bacterial infections, stress and other environmental factors (Collier, 1968; Carter, 1973). Although respiratory viruses and stress may predispose the host to bacterial infection, the importance ofPasteurella spp. is well established as an etiologic 0378- l 1 3 5 / 9 1 / $ 0 3 . 5 0
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M. BISGAARD ET AL.
factor in the development of pneumonia in cattle and sheep (Carter, 1973; Gilmour, 1978; Biberstein, 1981 ). According to Collier (1968) and Frank (1980) P. haemolytica biovar A serovar 1 and, to a lesser extent, P. multocida Heddleston type 3 are the serovars most commonly isolated from pneumonic bovine lungs. It is quite conceivable, however, that other groups of P. haemolytica and P. multocida may also be involved in the disease. Significant differences in colonial morphology, biochemical activity, antigenic structure and pathogenicity exist among isolates of P. multocida, and different bio- and serotyping systems have been proposed to aid in their differentiation (Frederiksen, 1973; Carter, 1976; Heddleston, 1976; Brogden and Packer, 1979 ). During taxonomic investigations of organisms obtained from calf pneumonia in Denmark, which by simplified routine bacteriological examinations were P. multocida, two other species in addition to P. multocida sensu stricto were identified (Madsen et al., 1985 ). These species, tentatively designated P. multocida biovar 6 (orn + taxon 13 ) and V-factor independent Haernophilus avium ( o r n - taxon 13 ), were later named P. canis (Mutters et al., 1 9 8 5 b ) a n d P . avium (Mutters et al., 1985a,b), respectively. Considering the economic impact of pneumonic "pasteurellosis" in ruminants and the recent investigations of the taxonomy of apparently typical P. multocida (Madsen et al., 1985 ) and P. haemolytica (Bisgaard et al., 1986 ) it was decided to investigate in detail the taxonomy ofP. multocida obtained from pneumonia in calves in different European countries. MATERIAL AND METHODS
A total of 135 strains were investigated (Table 1 ). These included four reference strains and 130 strains, previously identified as P. multocida, obtained from bovine pneumonia; these were from West Germany (114), United Kingdom ( 1 ! ) and Netherlands (5). A single Danish isolate obtained from mastitis in a cow was also included. All strains had been kept at - 70 ° C or in a lyophilized state since the original isolation. Ampoules were opened under sterile conditions, and contents dissolved in veal infusion broth (Difco) which afterwards was inoculated on blood agar (Tryptose blood agar base (Difco) containing 5% citrated bovine blood) and incubated aerobically at 37 °C for 24 h. Cloned subcultures were phenotypically characterized by standard methods as described previously (Bisgaard, 1975, 1982). Examination for symbiotic growth was performed on blood agar cross-inoculated with a V-factor producing strain of Staphylococcus aureus. The porphyrin test was carried out as suggested by Kilian and Frederiksen ( 1981 ). Detection of o~-fucosidase, fl-galactosidase, c~-glucosidase, fl-glucosidase, fl-glucuronidase and fl-xylosidase was done according to Kilian and Billow ( 1976 ). Tetrathionate reductase, alanine aminopeptidase,
RECLASSIFICATION OF PASTEURELLA MULTOfTDA ISOLATES
117
TABLE 1 Identification of strains investigated Taxon
No. of strains examined
Strain
Origin
Pneumonia in calves ( F R G ) Pneumonia in calves ( U K ) Pneumonia in calves ( F R G )
P. multocida ssp. m ultocida P. multocida ssp. multocida
21 3
2a, 2c, 7,8,9c, 1 8 , 2 6 , 6 0 , 9 9 , 1 0 0 , 1 0 3 , 1 0 7 , 110,123,143,179,183,222,263,277,289 12/25, B 8 0 / I I , B 8 0 / 8 4
D( - ) m a n n i t o l -
3
62,129,214
3
B80/15, C136/11, R A I 2 / 2
Pneumonia in calves ( U K ) Pneumonia in calves ( F R G )
2
1,3,4,21,22,24,28,30,32,36,38,39,40,41, 42,43,44,47,48,51,52,53,54,55,56,57,58, 59,61,63,64,69,70,71,95,97,102,120,166, 168,169,171,172,185,186,187,188,189, 190,206,215,240,249,251,253,261,267, 268,270,287 B80/20 ( i n d o l e - , x y l o s e - , s u c r o s e - ) , B80/26 ( i n d o l e - , x y l o s e - , s u c r o s e - ) , C12/122, C57/ 12, C325 160,164
Orn . P.
5
14,16,239,255,256
mu[tocida ssp. septica P. canis biovar 2
Pneumonia in calves ( F R G )
9
6, 25, 66, 217,248,264, 283, 286, A554/86
P. canis biovar 2
1
70II
P. canis biovar 2
3
35269V, 35445111, 35445III2
P. avium biovar 2
10
P. avium biovar 2
3
P. avium biovar 2
2
5, 65, 192, 193, 194, 195, 196, 198,242, 288 (indole + and sucrose- ) A283/86, A284/86 (D( - )mannitol ~+~, A285/86 31866/83, 35269V2
Not classified
1
152
Pneumonia in calves ( F R G ) Mastitis, cow (DK) Cattle, pneumonia (Netherlands) Pneumonia in calves ( F R G ) Pneumonia in calves ( F R G ) Cattle, pneumonia (Netherlands) Pneumonia, calf (FRG)
1
NCTC 10322v= Carter W9217
1
CIP A125 v
1
C C U G 16498 = K 2 6 7
1
C C U G 16497=K117
P. multocida ssp. multocida
D ( - ) mannitol P. multocida ssp. multocida
O r n - . P.
60
multocida ssp. multocida
Orn . P.
5
multocida ssp. multocida
D( - )mannitoland O r n - . P. multocida ssp.
Pneumonia in calves ( U K ) Pneumonia in calves ( F R G )
multocida
Reference strains P. multocida ssp. multocida P. multocida ssp. septica P. canis biovar 2 P. avium biovar 2
Total
Human, cat bite (France) Calf, pneumonia (DK) Ibid
135
,Abbreviations: CCUG, Culture Collection University of G6teborg, Sweden; CIP, Collection de l'Institut Pasteur, Paris, France; NCTC, National Collection of Type Cultures, London, UK; T, type strain.
1 18
M. BISGAARD ET AL.
o~-galactosidase and c~-mannosidase were demonstrated as indicated by the manufacturer (Rosco, D e n m a r k ) . When variants were found the relevant tests were repeated in test media originating from the same batch as well as in media prepared from a new batch. Eleven English isolates were characterized serologically. Capsular types were determined by the indirect hemagglutination test as originally described by Carter ( 1955, 1972 ). Gel diffusion precipitin test was conducted as described by Heddleston et al. ( 1972 ). To investigate the taxonomic significance of ornithine decarboxylation within the subspecies ofP. multocida, two strains representing ornithine negative P. multocida ssp. multocida (Strain 56) and P. multocida ssp. septica (Strain 14), respectively, were selected for genotypical investigations. Determinations of D N A base composition and genetic relatedness were performed as described previously (Mutters et al., 1985b). RESULTS
Twenty-four strains were classified with P. multocida ssp. multocida. Phenotypical characters of some of these organisms, however, differed in oxidase, glycerol, D ( - )arabinose, D ( + )xylose, L ( - )fucose, lactose, ONPG, trehalose, raffinose, and/or/~-glucuronidase when compared with those obtained with P. multocida ssp. multocida NCTC 10322 v. Six and sixty-five strains, respectively, were tentatively diagnosed as D ( - )mannitol negative P. multocida ssp. multocida and ornithine negative P. multocida ssp. multocida. Two strains differed from P. multocida ssp. multocida in both ornithine and mannitol. In excess of differences in ornithine and mannitol some of these isolates also differed from the type strain in some of the characters previously mentioned for strains classified with P. rnultocida ssp. rnultocida. Two isolates classified as ornithine negative P. multocida ssp. multocida did not produce indole. Nor did one of these strains produce acid from sucrose. Five strains were classified as ornithine negative P. multocida ssp. septica. Some of these strains had phenotypical characters which deviated from those obtained with P. rnultocida ssp. septica CIP A125 v in oxidase, glycerol, D( - )arabinose, L ( - )fucose, lactose and ONPG. All five strains were indole negative and D ( + )xylose and raffinose positive in contrast to the type strain ofP. multocida ssp. septica. Thirteen and fifteen strains, respectively, were identified as biovar 2 of P. canis and P. aviurn. Some of these isolates also had phenotypical characters which differed from those obtained with the respective reference strains. These characters included oxidase, indole, glycerol, D( + )xylose, lactose, ONPG, trehalose and raffinose. An indole positive and sucrose negative strain of P. avium biovar 2 in addition to a late D ( - )mannitol positive strain of
5
80 + -t- / ( + )
-]- / ( + )
-
13
92 83
--
69
-
+
15
7 + 73
-
~
~, ~ ~ ~
~
~ 73
72 80
,~
~
-
biovar 2
P. avium
~m
2
(+ ) + -
--
)
P. canis biovar 2
P. multocida ssp. septica (Orn)
~
65
+ 26 + 123
+ /(+ ) + 6
-}-
--
+ _
(Orn , Mannitol
P. multocida ssp. multocida
+ . all strains positive within 1-2 days; ( + ), all strains late positive ( > 3 days); + / ( + ), all strains positive, s o m e strains positive w i t h i n 1-2 days,
24
Total no. of strains
97 95
(Orn-)
(Mannitol)
+ + -
P. multocida ssp. multocida
P. multocida ssp. multocida
s o m e strains late positive: - , all strains negative within 14 days. ~Pcr cent strains positive. 2An indole + and s u c r o s e - strain has been observed. 3kate positive: weak positive reactions are seen.
+ + 54 ~ + + 21 25 83
O r n i t h i n e decarb. Indole D ( + )xylose D ( - )mannitol D ( - )sorbitol O N PG Trehalose Raffinosc
P. multocida ssp. multocida
S o m e characters s e p a r a t i n g the strains investigated
TABLE 2
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M. BISGAARDET AL.
TABLE 3
Serotyping results of English isolates of so-called P. multocida Strain P. multocidassp, multocida 12/25 B80/I1 B80/84
Capsular typing (Carter)
Gel diffusion precipitin test (Heddleston)
D A A
3,4 3,4,7,12 3,4,7,12
~ multocidassp, m u l t o c i d a ( D ( - )mannitol ) B80/15 A C136/11 A R A I 2 / 2 (sucr.-) A
3,4,7, 12 3,4,7, 12 3, 4, 7, 12
P. multocida ssp. multocida ( o r n . ) B80/20 (indole-, xyl. , sucr.-) B80/26 (indole , xyl.-, sucr.- ) C12/122 C57/12 C325
3, 3, 3, 3 3,
A A A A A
4, 7, 12 4, 7, 12 4, 7 4, 9
TABLE 4 G + C content (tool %) of ornithine negative strains of P. multocida ssp. multocida (Strain 56) and P. multocida ssp. septica (Strain 14)
Strain 56 Strain 14
G + C content (tool %)
Standard deviation
40.0 40.4
_+0.3 _+0.4
P. avium biovar 2 was also discriminated. A single strain was found to belong to Pasteurellaceae, but could not be allocated to known species or taxa. Some characters separating the strains investigated are given in Table 2. Serotyping results of the English isolates are shown in Table 3. With the exception of a single D type all strains belonged to Carters capsule type A. Gel diffusion precipitin tests showed that all eleven strains investigated possessed Heddleston 3 antigen, ten strains Heddleston 4, eight strains Heddleston 7, seven strains Heddleston 12 and a single strain Heddleston 9, irrespective of their phenotype. The guanine plus cytosine contents (% G + C) of DNA of ornithine negative strains of P. multocida ssp. multocida and P. multocida ssp. septica are given in Table 4. Deoxyribonucleic acid relatedness of an ornithine negative strain ofP. multocida ssp. multocida and P. multocida ssp. septica, respectively, and the type strains of the three subspecies of P. multocida is given in Table 5. It appears
RECLASSIFICATIONOF PASTEURELLA MULTOCIDA ISOLATES
121
TABLE 5 DNA relatedness ofornithine negative strains ofP. multocida ssp. multocida (Strain 56 ) and P. multocida ssp. septica (Strain 14) and the type strains of the three subspecies ofP. multocida
Strain 56 Degree of binding (%) P. multocida ssp. multocida NCTC 10322 T P. multocida ssp. multocida Gerlach 1449/8 | P. multocida ssp. septica CIP A125 ~r P. multocida ssp. gallicida NCTC 10204 x P. multocida ssp. multocida ( O r n - ) Strain 56 P. muhocida ssp. septica ( O r n - ) Slrain 14
Strain 14 Standard deviation (%)
Degree of binding (%)
81
2.8
74
75
4.0
ND
Standard deviation (%) 2.3
ND
44
7.1
ND
70
1.4
100
90
5.5
90
5.5
100
ND, not done.
from this Table that Strain 14, representing the ornithine negative strains classified with P. multoeida ssp. septica showed 90% DNA homology with ornithine negative strains of P. multocida ssp. rnultocida represented by Strain 56. Eighty-one per cent DNA binding was found between the last group and the type strain ofP. rnultocida ssp. multocida. Seventy-four, seventy and fortyfour per cent D N A homology was demonstrated between ornithine negative P. rnultocida ssp. septica and the type strains ofP. multocida ssp. multocida, ssp. gallicida and ssp. septica, respectively. DISCUSSION
The principal Pasteurella spp. involved in pneumonia in ruminants are normally considered to be P. haernolytica and P. multocida (Carter, 1973; Corstvet et al., 1973; Gilmour, 1978; Biberstein, 1981 ). When 50 selected strains considered as P. multocida were reinvestigated only seven strains were P. multocida ssp. multocida. Thirty-one and 10 strains, respectively, were classified with P. canis biovar 2 and P. avium biovar 2. Two strains remained unclassified (Madsen et al., 1985; Mutters et al., 1985b). All strains originated from cases of pneumonia in Danish calves. The present investigation showed that P. canis biovar 2 and P. avium biovar 2 also occur in association with calf pneumonia in other countries. The reservoir of these organisms seems to be limited to calves unlike biov-
122
M. BISGAARD ETAL.
ars 1 of P. canis and P. avium which are associated with dogs and birds, respectively (Mutters et al., 1985b ). Decarboxylation of ornithine seems to be a key-character in distinguishing species within the genus Pasteurella (Mutters et al., 1985b). Phenotypic separation between P. canis and P. avium and also between P. canis and P. stomatis is based exclusively upon differences in ornithine decarboxylation. During the present investigation sixty-five (50%) and five strains (4%) out of 131 strains investigated were tentatively classified as ornithine negative P. multocida spp. rnultocida and P. multocida ssp. septica, respectively. D N A : D N A hybridizations showed that ornithine negative strains were related on species level. Less, but still high genomic binding, was found between an ornithine negative strain (14) of P. multocida ssp. septica and the type strains ofP. multocida ssp. multocida and P. multocida ssp. gallicida. Surprisingly, only 44% DNA-binding was observed between the ornithine positive and negative strains of P. multocida ssp. septica. Genetic investigations of additional strains are necessary to understand the taxonomic position of ornithine negative strains ofP. multocida. Production of acid from D ( - )mannitol is also of taxonomic importance within the genus Pasteurella. Eight D ( - ) mannitol negative strains were recognized among 131 strains investigated. Two of these strains were also ornithine negative. The taxonomic position of these organisms remains to be investigated. The same is true of two ornithine and sucrose negative strains. Phenotypic separation between P. stomatis and P. avium biovar 2 depends on indole formation (Mutters et al., 1985b). Indole production also separates P. gallinarum from taxon 16 (Bisgaard and Mutters, 1986). An indole positive, but sucrose negative strain was provisionally allocated P. avium biovar 2 during the present investigation. The taxonomic position of this strain, however, remains uncertain as does a late D ( - ) m a n n i t o l positive strain of P. avium biovar 2. Extended DNA:DNA hybridizations including many more aberrant strains are needed so that phenotypic tests can be defined that will delineate the species found by genomic characterization. Serologic characterization and pathogenicity testing in gnotobiotic calves should be performed with P. canis biovar 2, P. avium biovar 2 and ornithine negative strains ofP. multocida ssp. multocida and P. multocida ssp. septica. Such studies should lead to greater accuracy in the diagnosis of "pasteurellosis" and enable clarification of the role of the related taxa within this group in the pathogenesis of disease. ACKNOWLEDGMENTS
We wish to thank the following colleagues for collecting and forwarding strains: Prof. Dr. G. Amtsberg, Institut ffir Mikrobiologie und Tierseuchen der Tier~irztlichen Hochschule Hannover, 15 Bischofsholder Damm, D-3000
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Hannover 1, Bundesrepublik Deutschland. Prof. Dr. H. Blobel, Institut ffir Bakteriologie und Immunologie Fachbereich Veterin~irmedizin der JustusLiebig Universit~it Giessen, 107 Frankfurter Strasse, D-6300 Giessen, Bundesrepublik Deutschland. Dr. Ed ter Laak, Central Veterinary Institute, 15 Edelhertweg, NL-8219 PH Lelystad, Netherlands. REFERENCES Biberstein, E.L., 1981. Haernophilus - Pasteurella - Actinobacillus: their significance in veterinary medicine. In: M. Kilian, W. Frederiksen and E.L. Biberstein (Editors), Haemophilus, Pasteurella and Actinobacillus. Academic Press, London, pp. 61-73. Bisgaard, M., 1975. Characterization of atypical Actinobacillus lignieresii isolated from ducks with salpingitis and peritonitis. Nord. Vet. Med., 27: 378-383. Bisgaard, M., 1982. Isolation and characterization of some previously unreported taxa from poultry with phenotypical characters related to Actinobacillus and Pasteurella species. Acta Pathol. Microbiol. Immunol. Scand. Sect. B, 90: 59-67. Bisgaard, M. and Mutters, R., 1986. Characterization of some previously unclassified "'Pasteurella" spp. obtained form the oral cavity of dogs and cats and description of a new species tentatively classified with the family Pasteurellaceae Pohl 1981 and provisionally called taxon 16. Acta. Pathol. Microbiol. Immunol. Scand. Sect. B, 94:177-184. Bisgaard, M., Mutters, R. and Frederiksen, W., 1986. Reinvestigations of so-called Pasteurella haemolytica obtained from ruminants. Taxonomic and diagnostic consequences, lVth International Symposium of Veterinary Laboratory Diagnosticians, 2-6 June, Amsterdam, Netherlands. Abstracts, pp. 568-571. Brogden, K.A. and Packer, R.A., 1979. Comparison of Pasteurella multocida serotyping systems. Am. J. Vet. Res., 40: 1332-1335. Carter, G.R., 1955. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types. Am. J. Vet. Res., 16: 481-484. Carter, G.R., 1972. Improved hemagglutination test for identifying type A strains of Pasteurella multocida. J. Appl. Microbiol., 24: 162-163. Carter, G.R., 1973. Pasteurella infections as sequelae to respiratory viral infections. J. Am. Vet. Med. Assoc., 163: 863-864. Carter, G.R., 1976. A proposal for five biotypes of Pasteurella multocida. 19th Annu. Proc. American Association of Veterinary Laboratory Diagnosticians, pp, 189-196. Collier, J.R., 1968. Pasteurellae in bovine respiratory disease. J. Am. Vet. Med. Assoc., 152: 824-828. Corstvet, R.E., Panciera, R.J., Rinker, H.B., Starks, B.E. and Howard, C., 1973. Survey of tracheas of feedlot cattle for Haemophilus somnus and other selected bacteria. J. Am. Vet. Med. Assoc., 163: 870-873. Frank, G.H., 1980. Pasteurella haemolytica and respiratory disease in cattle, Proc. US Anim. Health Assoc., 83: 153-160. Frederiksen, W., 1973. Pasteurella taxonomy and nomenclature. Contrib. Microbiol. Immunol., 2: 170-176. Gilmour, N.J.I_., 1978. The role of Pasteurellae in the respiratory diseases of cattle. In: W.B. Martin (Editor), Respiratory Diseases of Cattle. A seminar in the EEC programme of coordination of research on beef production held at Edinburgh 8-10 Nov. 1977. M. N ijhoff, Den Haag, pp. 356-361. Heddleston, K.L., 1976. Physiologic characteristics of 1,268 cultures ofPasteurella multocida. Am. J. Vet. Res., 37: 745-747.
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Heddleston, K.L., Gallagher, J.E. and Rebers, P.A., 1972. Fowl cholera: Gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis., 16: 925-936. Jensen, R., Pierson, R.E., Braddy, P.M., Sarri, D.A., Lavermann, L.H., England, J.J., Key-Vanfar, H., Collier, J.R., Horton, D.P., McChesney, A.E., Benitz, A. and Christie, R.M., 1976. Shipping fever pneumonia in yearling feedlot cattle. J. Am. Vet. Med. Assoc., 169: 500-506. Kilian, M. and Btilow, P., 1976. Rapid diagnosis of Enterobacteriaceae. 1. Detection of bacterial glycosidases. Acta Pathol. Microbiol. Scand. Sect. B, 84:245-251. Kilian, M. and Frederiksen, W., 1981. Identification tables for the Haemophilus-l'asteurellaActinobacillus group. In: M. Kilian, W. Frederiksen and E.L. Biberstein (Editors), ttaernophilus, Pasteurella and Actinobacillus. Academic Press, London, pp. 281-290. King, N.B., Gale, C., Smith, H.R., Hamdy, A.H., Sanger, V.L., Pounden, W.D. and Klosterman. E.W., 1958. Stress factors in shipping fever. Vet. Med., 53: 67-72. Madsen, E.B., Bisgaard, M., Mutters, R. and Pedersen, K.B., 1985. Characterization of Pasteurella species isolated from lungs of calves with pneumonia. Can. J. Comp. Med., 49: 63-67. Martin, S.W., Meek, A.H., Davis, D.G., Thompson, R.G., Johnson, J.A., Lopez, A., Stephens, L., Curtis, R.A., Prescott, J.F., Rosendal, S., Saran, M., Zubaidy, A.J. and Bolton, M.R., 1980. Factors associated with mortality in feedlot cattle: The Bruce county beef cattle project. Can. J. Comp. Med., 44: 1-10. Martin, S.W., Meek, A.H., Davis, D.G., Johnson, J.A. and Curtis, R.A., 1981. Factors associated with morbidity and mortality in feedlot calves: The Bruce county beef project, year two. Can. J. Comp. Med., 45:103-112. Mutters, R., Piechulla, K., Hinz, K.-H. and Mannheim, W., 1985a. Pasteurella avium (Hinz and Kunjara, 1977) comb. nov. and Pasteurella volantium sp. nov. Int. J. Syst. Bact., 35: 59. Mutters, R., Ibm, P., Pohl, S., Frederiksen, W. and Mannheim, W., 1985b. Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis and Pasteurella langaa. Int. J. Syst. Bact., 35: 309-322.
115
Reclassification of German, British and Dutch isolates of so-called Pasteurella rnultocida obtained from pneumonic calf lungs M. Bisgaard a, S.B. Houghton b, R. Mutters c and A. Stenzel ~ alnstitute of Avian Diseases, The Royal Veterinary and Agricultural University, 13 Biilowsvej, DK- 1870 Copenhagen V, Denmark bHoechst Animal Health, A Division of Hoechst UK Ltd, Walton Manor, Milton Keynes, Buckinghamshire MK7 7AJ, UK Clnstitute of Medical Microbiology, Klinikum der Philipps-Universitiit, 2 Pilgrimstein, D-3550 Marburg, Germany (Accepted 14 June 1990)
ABSTRACT Bisgaard, M., Houghton, S.B., Mutters, R. and Stenzel, A., 1991. Reclassification of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumonic calf lungs. Vet Microbiol., 26:115-124. The taxonomic relationship of 131 strains previously identified as Pasteurella multocida obtained from calf pneumonia in West Germany, United Kingdom and Netherlands was investigated by extended phenotypic and limited genotypic characterization. Twenty-four strains were classified as P. multocida ssp. multocida, 15 strains as P. avium biovar 2 and 13 strains as P. canis biovar 2. Sixtyfive and five strains were tentatively classified as ornithine negative P. multocida ssp. multocida and P. multocida ssp. septica, respectively. Genetic investigations showed that ornithine negative strains of P. multocida were related on species level. Less genomic binding was found between an ornithine negative strain ofP. multocida ssp. septica and the type strains of the three subspecies ofP. multocida. The taxonomic position of ornithine negative strains ofP. multocida is still under investigation. The taxonomic position of the remaining nine strains is uncertain underlining the need for genotypic characterization within the genus Pasteurella to aid in defining single species by phenotypic tests.
INTRODUCTION
Pneumonic "pasteurellosis" is a major cause of economic loss and mortality in cattle (King et al., 1958; Jensen et al., 1976; Martin et al., 1980, 1981 ). The disease complex seems to be caused by a combination of respiratory viruses, bacterial infections, stress and other environmental factors (Collier, 1968; Carter, 1973). Although respiratory viruses and stress may predispose the host to bacterial infection, the importance ofPasteurella spp. is well established as an etiologic 0378- l 1 3 5 / 9 1 / $ 0 3 . 5 0
© | 991 - - Elsevier Science Publishers B.V.
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M. BISGAARD ET AL.
factor in the development of pneumonia in cattle and sheep (Carter, 1973; Gilmour, 1978; Biberstein, 1981 ). According to Collier (1968) and Frank (1980) P. haemolytica biovar A serovar 1 and, to a lesser extent, P. multocida Heddleston type 3 are the serovars most commonly isolated from pneumonic bovine lungs. It is quite conceivable, however, that other groups of P. haemolytica and P. multocida may also be involved in the disease. Significant differences in colonial morphology, biochemical activity, antigenic structure and pathogenicity exist among isolates of P. multocida, and different bio- and serotyping systems have been proposed to aid in their differentiation (Frederiksen, 1973; Carter, 1976; Heddleston, 1976; Brogden and Packer, 1979 ). During taxonomic investigations of organisms obtained from calf pneumonia in Denmark, which by simplified routine bacteriological examinations were P. multocida, two other species in addition to P. multocida sensu stricto were identified (Madsen et al., 1985 ). These species, tentatively designated P. multocida biovar 6 (orn + taxon 13 ) and V-factor independent Haernophilus avium ( o r n - taxon 13 ), were later named P. canis (Mutters et al., 1 9 8 5 b ) a n d P . avium (Mutters et al., 1985a,b), respectively. Considering the economic impact of pneumonic "pasteurellosis" in ruminants and the recent investigations of the taxonomy of apparently typical P. multocida (Madsen et al., 1985 ) and P. haemolytica (Bisgaard et al., 1986 ) it was decided to investigate in detail the taxonomy ofP. multocida obtained from pneumonia in calves in different European countries. MATERIAL AND METHODS
A total of 135 strains were investigated (Table 1 ). These included four reference strains and 130 strains, previously identified as P. multocida, obtained from bovine pneumonia; these were from West Germany (114), United Kingdom ( 1 ! ) and Netherlands (5). A single Danish isolate obtained from mastitis in a cow was also included. All strains had been kept at - 70 ° C or in a lyophilized state since the original isolation. Ampoules were opened under sterile conditions, and contents dissolved in veal infusion broth (Difco) which afterwards was inoculated on blood agar (Tryptose blood agar base (Difco) containing 5% citrated bovine blood) and incubated aerobically at 37 °C for 24 h. Cloned subcultures were phenotypically characterized by standard methods as described previously (Bisgaard, 1975, 1982). Examination for symbiotic growth was performed on blood agar cross-inoculated with a V-factor producing strain of Staphylococcus aureus. The porphyrin test was carried out as suggested by Kilian and Frederiksen ( 1981 ). Detection of o~-fucosidase, fl-galactosidase, c~-glucosidase, fl-glucosidase, fl-glucuronidase and fl-xylosidase was done according to Kilian and Billow ( 1976 ). Tetrathionate reductase, alanine aminopeptidase,
RECLASSIFICATION OF PASTEURELLA MULTOfTDA ISOLATES
117
TABLE 1 Identification of strains investigated Taxon
No. of strains examined
Strain
Origin
Pneumonia in calves ( F R G ) Pneumonia in calves ( U K ) Pneumonia in calves ( F R G )
P. multocida ssp. m ultocida P. multocida ssp. multocida
21 3
2a, 2c, 7,8,9c, 1 8 , 2 6 , 6 0 , 9 9 , 1 0 0 , 1 0 3 , 1 0 7 , 110,123,143,179,183,222,263,277,289 12/25, B 8 0 / I I , B 8 0 / 8 4
D( - ) m a n n i t o l -
3
62,129,214
3
B80/15, C136/11, R A I 2 / 2
Pneumonia in calves ( U K ) Pneumonia in calves ( F R G )
2
1,3,4,21,22,24,28,30,32,36,38,39,40,41, 42,43,44,47,48,51,52,53,54,55,56,57,58, 59,61,63,64,69,70,71,95,97,102,120,166, 168,169,171,172,185,186,187,188,189, 190,206,215,240,249,251,253,261,267, 268,270,287 B80/20 ( i n d o l e - , x y l o s e - , s u c r o s e - ) , B80/26 ( i n d o l e - , x y l o s e - , s u c r o s e - ) , C12/122, C57/ 12, C325 160,164
Orn . P.
5
14,16,239,255,256
mu[tocida ssp. septica P. canis biovar 2
Pneumonia in calves ( F R G )
9
6, 25, 66, 217,248,264, 283, 286, A554/86
P. canis biovar 2
1
70II
P. canis biovar 2
3
35269V, 35445111, 35445III2
P. avium biovar 2
10
P. avium biovar 2
3
P. avium biovar 2
2
5, 65, 192, 193, 194, 195, 196, 198,242, 288 (indole + and sucrose- ) A283/86, A284/86 (D( - )mannitol ~+~, A285/86 31866/83, 35269V2
Not classified
1
152
Pneumonia in calves ( F R G ) Mastitis, cow (DK) Cattle, pneumonia (Netherlands) Pneumonia in calves ( F R G ) Pneumonia in calves ( F R G ) Cattle, pneumonia (Netherlands) Pneumonia, calf (FRG)
1
NCTC 10322v= Carter W9217
1
CIP A125 v
1
C C U G 16498 = K 2 6 7
1
C C U G 16497=K117
P. multocida ssp. multocida
D ( - ) mannitol P. multocida ssp. multocida
O r n - . P.
60
multocida ssp. multocida
Orn . P.
5
multocida ssp. multocida
D( - )mannitoland O r n - . P. multocida ssp.
Pneumonia in calves ( U K ) Pneumonia in calves ( F R G )
multocida
Reference strains P. multocida ssp. multocida P. multocida ssp. septica P. canis biovar 2 P. avium biovar 2
Total
Human, cat bite (France) Calf, pneumonia (DK) Ibid
135
,Abbreviations: CCUG, Culture Collection University of G6teborg, Sweden; CIP, Collection de l'Institut Pasteur, Paris, France; NCTC, National Collection of Type Cultures, London, UK; T, type strain.
1 18
M. BISGAARD ET AL.
o~-galactosidase and c~-mannosidase were demonstrated as indicated by the manufacturer (Rosco, D e n m a r k ) . When variants were found the relevant tests were repeated in test media originating from the same batch as well as in media prepared from a new batch. Eleven English isolates were characterized serologically. Capsular types were determined by the indirect hemagglutination test as originally described by Carter ( 1955, 1972 ). Gel diffusion precipitin test was conducted as described by Heddleston et al. ( 1972 ). To investigate the taxonomic significance of ornithine decarboxylation within the subspecies ofP. multocida, two strains representing ornithine negative P. multocida ssp. multocida (Strain 56) and P. multocida ssp. septica (Strain 14), respectively, were selected for genotypical investigations. Determinations of D N A base composition and genetic relatedness were performed as described previously (Mutters et al., 1985b). RESULTS
Twenty-four strains were classified with P. multocida ssp. multocida. Phenotypical characters of some of these organisms, however, differed in oxidase, glycerol, D ( - )arabinose, D ( + )xylose, L ( - )fucose, lactose, ONPG, trehalose, raffinose, and/or/~-glucuronidase when compared with those obtained with P. multocida ssp. multocida NCTC 10322 v. Six and sixty-five strains, respectively, were tentatively diagnosed as D ( - )mannitol negative P. multocida ssp. multocida and ornithine negative P. multocida ssp. multocida. Two strains differed from P. multocida ssp. multocida in both ornithine and mannitol. In excess of differences in ornithine and mannitol some of these isolates also differed from the type strain in some of the characters previously mentioned for strains classified with P. rnultocida ssp. rnultocida. Two isolates classified as ornithine negative P. multocida ssp. multocida did not produce indole. Nor did one of these strains produce acid from sucrose. Five strains were classified as ornithine negative P. multocida ssp. septica. Some of these strains had phenotypical characters which deviated from those obtained with P. rnultocida ssp. septica CIP A125 v in oxidase, glycerol, D( - )arabinose, L ( - )fucose, lactose and ONPG. All five strains were indole negative and D ( + )xylose and raffinose positive in contrast to the type strain ofP. multocida ssp. septica. Thirteen and fifteen strains, respectively, were identified as biovar 2 of P. canis and P. aviurn. Some of these isolates also had phenotypical characters which differed from those obtained with the respective reference strains. These characters included oxidase, indole, glycerol, D( + )xylose, lactose, ONPG, trehalose and raffinose. An indole positive and sucrose negative strain of P. avium biovar 2 in addition to a late D ( - )mannitol positive strain of
5
80 + -t- / ( + )
-]- / ( + )
-
13
92 83
--
69
-
+
15
7 + 73
-
~
~, ~ ~ ~
~
~ 73
72 80
,~
~
-
biovar 2
P. avium
~m
2
(+ ) + -
--
)
P. canis biovar 2
P. multocida ssp. septica (Orn)
~
65
+ 26 + 123
+ /(+ ) + 6
-}-
--
+ _
(Orn , Mannitol
P. multocida ssp. multocida
+ . all strains positive within 1-2 days; ( + ), all strains late positive ( > 3 days); + / ( + ), all strains positive, s o m e strains positive w i t h i n 1-2 days,
24
Total no. of strains
97 95
(Orn-)
(Mannitol)
+ + -
P. multocida ssp. multocida
P. multocida ssp. multocida
s o m e strains late positive: - , all strains negative within 14 days. ~Pcr cent strains positive. 2An indole + and s u c r o s e - strain has been observed. 3kate positive: weak positive reactions are seen.
+ + 54 ~ + + 21 25 83
O r n i t h i n e decarb. Indole D ( + )xylose D ( - )mannitol D ( - )sorbitol O N PG Trehalose Raffinosc
P. multocida ssp. multocida
S o m e characters s e p a r a t i n g the strains investigated
TABLE 2
120
M. BISGAARDET AL.
TABLE 3
Serotyping results of English isolates of so-called P. multocida Strain P. multocidassp, multocida 12/25 B80/I1 B80/84
Capsular typing (Carter)
Gel diffusion precipitin test (Heddleston)
D A A
3,4 3,4,7,12 3,4,7,12
~ multocidassp, m u l t o c i d a ( D ( - )mannitol ) B80/15 A C136/11 A R A I 2 / 2 (sucr.-) A
3,4,7, 12 3,4,7, 12 3, 4, 7, 12
P. multocida ssp. multocida ( o r n . ) B80/20 (indole-, xyl. , sucr.-) B80/26 (indole , xyl.-, sucr.- ) C12/122 C57/12 C325
3, 3, 3, 3 3,
A A A A A
4, 7, 12 4, 7, 12 4, 7 4, 9
TABLE 4 G + C content (tool %) of ornithine negative strains of P. multocida ssp. multocida (Strain 56) and P. multocida ssp. septica (Strain 14)
Strain 56 Strain 14
G + C content (tool %)
Standard deviation
40.0 40.4
_+0.3 _+0.4
P. avium biovar 2 was also discriminated. A single strain was found to belong to Pasteurellaceae, but could not be allocated to known species or taxa. Some characters separating the strains investigated are given in Table 2. Serotyping results of the English isolates are shown in Table 3. With the exception of a single D type all strains belonged to Carters capsule type A. Gel diffusion precipitin tests showed that all eleven strains investigated possessed Heddleston 3 antigen, ten strains Heddleston 4, eight strains Heddleston 7, seven strains Heddleston 12 and a single strain Heddleston 9, irrespective of their phenotype. The guanine plus cytosine contents (% G + C) of DNA of ornithine negative strains of P. multocida ssp. multocida and P. multocida ssp. septica are given in Table 4. Deoxyribonucleic acid relatedness of an ornithine negative strain ofP. multocida ssp. multocida and P. multocida ssp. septica, respectively, and the type strains of the three subspecies of P. multocida is given in Table 5. It appears
RECLASSIFICATIONOF PASTEURELLA MULTOCIDA ISOLATES
121
TABLE 5 DNA relatedness ofornithine negative strains ofP. multocida ssp. multocida (Strain 56 ) and P. multocida ssp. septica (Strain 14) and the type strains of the three subspecies ofP. multocida
Strain 56 Degree of binding (%) P. multocida ssp. multocida NCTC 10322 T P. multocida ssp. multocida Gerlach 1449/8 | P. multocida ssp. septica CIP A125 ~r P. multocida ssp. gallicida NCTC 10204 x P. multocida ssp. multocida ( O r n - ) Strain 56 P. muhocida ssp. septica ( O r n - ) Slrain 14
Strain 14 Standard deviation (%)
Degree of binding (%)
81
2.8
74
75
4.0
ND
Standard deviation (%) 2.3
ND
44
7.1
ND
70
1.4
100
90
5.5
90
5.5
100
ND, not done.
from this Table that Strain 14, representing the ornithine negative strains classified with P. multoeida ssp. septica showed 90% DNA homology with ornithine negative strains of P. multocida ssp. rnultocida represented by Strain 56. Eighty-one per cent DNA binding was found between the last group and the type strain ofP. rnultocida ssp. multocida. Seventy-four, seventy and fortyfour per cent D N A homology was demonstrated between ornithine negative P. rnultocida ssp. septica and the type strains ofP. multocida ssp. multocida, ssp. gallicida and ssp. septica, respectively. DISCUSSION
The principal Pasteurella spp. involved in pneumonia in ruminants are normally considered to be P. haernolytica and P. multocida (Carter, 1973; Corstvet et al., 1973; Gilmour, 1978; Biberstein, 1981 ). When 50 selected strains considered as P. multocida were reinvestigated only seven strains were P. multocida ssp. multocida. Thirty-one and 10 strains, respectively, were classified with P. canis biovar 2 and P. avium biovar 2. Two strains remained unclassified (Madsen et al., 1985; Mutters et al., 1985b). All strains originated from cases of pneumonia in Danish calves. The present investigation showed that P. canis biovar 2 and P. avium biovar 2 also occur in association with calf pneumonia in other countries. The reservoir of these organisms seems to be limited to calves unlike biov-
122
M. BISGAARD ETAL.
ars 1 of P. canis and P. avium which are associated with dogs and birds, respectively (Mutters et al., 1985b ). Decarboxylation of ornithine seems to be a key-character in distinguishing species within the genus Pasteurella (Mutters et al., 1985b). Phenotypic separation between P. canis and P. avium and also between P. canis and P. stomatis is based exclusively upon differences in ornithine decarboxylation. During the present investigation sixty-five (50%) and five strains (4%) out of 131 strains investigated were tentatively classified as ornithine negative P. multocida spp. rnultocida and P. multocida ssp. septica, respectively. D N A : D N A hybridizations showed that ornithine negative strains were related on species level. Less, but still high genomic binding, was found between an ornithine negative strain (14) of P. multocida ssp. septica and the type strains ofP. multocida ssp. multocida and P. multocida ssp. gallicida. Surprisingly, only 44% DNA-binding was observed between the ornithine positive and negative strains of P. multocida ssp. septica. Genetic investigations of additional strains are necessary to understand the taxonomic position of ornithine negative strains ofP. multocida. Production of acid from D ( - )mannitol is also of taxonomic importance within the genus Pasteurella. Eight D ( - ) mannitol negative strains were recognized among 131 strains investigated. Two of these strains were also ornithine negative. The taxonomic position of these organisms remains to be investigated. The same is true of two ornithine and sucrose negative strains. Phenotypic separation between P. stomatis and P. avium biovar 2 depends on indole formation (Mutters et al., 1985b). Indole production also separates P. gallinarum from taxon 16 (Bisgaard and Mutters, 1986). An indole positive, but sucrose negative strain was provisionally allocated P. avium biovar 2 during the present investigation. The taxonomic position of this strain, however, remains uncertain as does a late D ( - ) m a n n i t o l positive strain of P. avium biovar 2. Extended DNA:DNA hybridizations including many more aberrant strains are needed so that phenotypic tests can be defined that will delineate the species found by genomic characterization. Serologic characterization and pathogenicity testing in gnotobiotic calves should be performed with P. canis biovar 2, P. avium biovar 2 and ornithine negative strains ofP. multocida ssp. multocida and P. multocida ssp. septica. Such studies should lead to greater accuracy in the diagnosis of "pasteurellosis" and enable clarification of the role of the related taxa within this group in the pathogenesis of disease. ACKNOWLEDGMENTS
We wish to thank the following colleagues for collecting and forwarding strains: Prof. Dr. G. Amtsberg, Institut ffir Mikrobiologie und Tierseuchen der Tier~irztlichen Hochschule Hannover, 15 Bischofsholder Damm, D-3000
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123
Hannover 1, Bundesrepublik Deutschland. Prof. Dr. H. Blobel, Institut ffir Bakteriologie und Immunologie Fachbereich Veterin~irmedizin der JustusLiebig Universit~it Giessen, 107 Frankfurter Strasse, D-6300 Giessen, Bundesrepublik Deutschland. Dr. Ed ter Laak, Central Veterinary Institute, 15 Edelhertweg, NL-8219 PH Lelystad, Netherlands. REFERENCES Biberstein, E.L., 1981. Haernophilus - Pasteurella - Actinobacillus: their significance in veterinary medicine. In: M. Kilian, W. Frederiksen and E.L. Biberstein (Editors), Haemophilus, Pasteurella and Actinobacillus. Academic Press, London, pp. 61-73. Bisgaard, M., 1975. Characterization of atypical Actinobacillus lignieresii isolated from ducks with salpingitis and peritonitis. Nord. Vet. Med., 27: 378-383. Bisgaard, M., 1982. Isolation and characterization of some previously unreported taxa from poultry with phenotypical characters related to Actinobacillus and Pasteurella species. Acta Pathol. Microbiol. Immunol. Scand. Sect. B, 90: 59-67. Bisgaard, M. and Mutters, R., 1986. Characterization of some previously unclassified "'Pasteurella" spp. obtained form the oral cavity of dogs and cats and description of a new species tentatively classified with the family Pasteurellaceae Pohl 1981 and provisionally called taxon 16. Acta. Pathol. Microbiol. Immunol. Scand. Sect. B, 94:177-184. Bisgaard, M., Mutters, R. and Frederiksen, W., 1986. Reinvestigations of so-called Pasteurella haemolytica obtained from ruminants. Taxonomic and diagnostic consequences, lVth International Symposium of Veterinary Laboratory Diagnosticians, 2-6 June, Amsterdam, Netherlands. Abstracts, pp. 568-571. Brogden, K.A. and Packer, R.A., 1979. Comparison of Pasteurella multocida serotyping systems. Am. J. Vet. Res., 40: 1332-1335. Carter, G.R., 1955. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types. Am. J. Vet. Res., 16: 481-484. Carter, G.R., 1972. Improved hemagglutination test for identifying type A strains of Pasteurella multocida. J. Appl. Microbiol., 24: 162-163. Carter, G.R., 1973. Pasteurella infections as sequelae to respiratory viral infections. J. Am. Vet. Med. Assoc., 163: 863-864. Carter, G.R., 1976. A proposal for five biotypes of Pasteurella multocida. 19th Annu. Proc. American Association of Veterinary Laboratory Diagnosticians, pp, 189-196. Collier, J.R., 1968. Pasteurellae in bovine respiratory disease. J. Am. Vet. Med. Assoc., 152: 824-828. Corstvet, R.E., Panciera, R.J., Rinker, H.B., Starks, B.E. and Howard, C., 1973. Survey of tracheas of feedlot cattle for Haemophilus somnus and other selected bacteria. J. Am. Vet. Med. Assoc., 163: 870-873. Frank, G.H., 1980. Pasteurella haemolytica and respiratory disease in cattle, Proc. US Anim. Health Assoc., 83: 153-160. Frederiksen, W., 1973. Pasteurella taxonomy and nomenclature. Contrib. Microbiol. Immunol., 2: 170-176. Gilmour, N.J.I_., 1978. The role of Pasteurellae in the respiratory diseases of cattle. In: W.B. Martin (Editor), Respiratory Diseases of Cattle. A seminar in the EEC programme of coordination of research on beef production held at Edinburgh 8-10 Nov. 1977. M. N ijhoff, Den Haag, pp. 356-361. Heddleston, K.L., 1976. Physiologic characteristics of 1,268 cultures ofPasteurella multocida. Am. J. Vet. Res., 37: 745-747.
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Heddleston, K.L., Gallagher, J.E. and Rebers, P.A., 1972. Fowl cholera: Gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis., 16: 925-936. Jensen, R., Pierson, R.E., Braddy, P.M., Sarri, D.A., Lavermann, L.H., England, J.J., Key-Vanfar, H., Collier, J.R., Horton, D.P., McChesney, A.E., Benitz, A. and Christie, R.M., 1976. Shipping fever pneumonia in yearling feedlot cattle. J. Am. Vet. Med. Assoc., 169: 500-506. Kilian, M. and Btilow, P., 1976. Rapid diagnosis of Enterobacteriaceae. 1. Detection of bacterial glycosidases. Acta Pathol. Microbiol. Scand. Sect. B, 84:245-251. Kilian, M. and Frederiksen, W., 1981. Identification tables for the Haemophilus-l'asteurellaActinobacillus group. In: M. Kilian, W. Frederiksen and E.L. Biberstein (Editors), ttaernophilus, Pasteurella and Actinobacillus. Academic Press, London, pp. 281-290. King, N.B., Gale, C., Smith, H.R., Hamdy, A.H., Sanger, V.L., Pounden, W.D. and Klosterman. E.W., 1958. Stress factors in shipping fever. Vet. Med., 53: 67-72. Madsen, E.B., Bisgaard, M., Mutters, R. and Pedersen, K.B., 1985. Characterization of Pasteurella species isolated from lungs of calves with pneumonia. Can. J. Comp. Med., 49: 63-67. Martin, S.W., Meek, A.H., Davis, D.G., Thompson, R.G., Johnson, J.A., Lopez, A., Stephens, L., Curtis, R.A., Prescott, J.F., Rosendal, S., Saran, M., Zubaidy, A.J. and Bolton, M.R., 1980. Factors associated with mortality in feedlot cattle: The Bruce county beef cattle project. Can. J. Comp. Med., 44: 1-10. Martin, S.W., Meek, A.H., Davis, D.G., Johnson, J.A. and Curtis, R.A., 1981. Factors associated with morbidity and mortality in feedlot calves: The Bruce county beef project, year two. Can. J. Comp. Med., 45:103-112. Mutters, R., Piechulla, K., Hinz, K.-H. and Mannheim, W., 1985a. Pasteurella avium (Hinz and Kunjara, 1977) comb. nov. and Pasteurella volantium sp. nov. Int. J. Syst. Bact., 35: 59. Mutters, R., Ibm, P., Pohl, S., Frederiksen, W. and Mannheim, W., 1985b. Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis and Pasteurella langaa. Int. J. Syst. Bact., 35: 309-322.
