- Email: [email protected]
Phenotypic characterisation of Pasteurella multocida isolates from Australian pigs
veterinary microbiology ELSEVIER
Veterinary Microbiology 57 (1997) 355-360
Phenotypic characterisation of Pasteurella multocida isolates from Australian pigs P.J. Blackall
*,
J.L. Pahoff, R. Bowles
Queensland Department of Primary Industries, Animal Research Institute, 665 Fairfield Road. Yeerongpilly QLD 4105, Australia Received 13 September 1996; accepted 17 March 1997
Abstract A phenotypic characterisation of 1.50 isolates of bacteria previously identified as PasteureMu multocidu was performed. All the isolates had been obtained from Australian pigs in the three eastern States of Queensland (I 10 isolates), New South Wales (21 isolates) and Victoria (19 isolates). Seven different biochemical biovars were recognised amongst the isolates. A total of 100 isolates (67%) were assigned to biovar 3, previously shown to be the most common biovar in isolates of P. multocidu from Australian poultry [Fegan, N., Blackall, P.J., Pahoff, J.L., 1995. Phenotypic characterisation of Pasteurella multocidu isolates from Australian poultry. Vet. Microbial., 47, 281-286.1. Six of the seven biovars, including biovar 3, were identified as P. multocidu subsp. multocida, 124 isolates in total. One other biovar, consisting of thirteen isolates, was identified as P. multocida subsp. gallicida. Within the six biovars that were identified as P. multocidu subsp. multocidu, biovars 12, 13 and 14 represented unusual biochemical variants. The nine isolates assigned to biovar 12 appeared to be lactose positive variants of P. multocida subsp. multocidu. The three isolates in biovar 13 appeared to be ornithine decarboxylase (ODC) negative variants of P. multocida subsp. multocida. The single isolate in biovar 14 appeared to be an ODC negative, lactose positive variant of P. multocidu subsp. multocidu. 0 1997 Elsevier Science B.V. Keywords: Pig-bacteria, Pasteurella multocida; Phenotype: Taxonomy
1. Introduction multocida is a major pathogen of pigs, being involved in a number of disease conditions. The main diseases associated with this organism are
Pasteurella
different
* Corresponding author. Tel.: +61-7-3362-9498:
fax: + 61-7-3362-9429;
0378-l 135/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII s0378-1135~97)00111-9
e-mail: [email protected]
356
P.J. Black&
et al. / Veterinary
Microbiology
57 (19971 355-360
pneumonic pasteurellosis, one of the most common and widespread diseases of intensively housed pigs (Pijoan, 1992) and progressive atrophic rhinitis, an important disease of pigs in North America and Europe (De Jong, 1992). Since the taxonomic reclassification proposed by Mutters et al. (1985), it is now recognised that a number of taxa within the genus Pasteurella, other than P. multocida, are indole positive, P. canis. P. dagmatis, P. stomatis and an un-named species Pasteurella species B. As well, three subspecies within P. multocida (P. multocida subsp. multocida, P. multocida subsp. septica and P. multocida subsp. gallicida) are now recognised (Mutters et al., 1985). We have recently completed a phenotypic characterisation of indole positive Pasteurella-like organisms isolated from Australian poultry (Fegan et al., 1995). In the current study, we report on the characterisation of 150 P. multocida-like isolates all obtained from Australian pigs. We have included 8 isolates that have been previously described in a case report on a outbreak of systemic pasteurellosis (Cameron et al., 1996). This study has been performed to provide the first definitive knowledge on the phenotypic properties of P. multocida-like isolates from Australian pigs in the light of current taxonomic knowledge.
2. Materials
and methods
2.1. Bacteria A total of 150 isolates of organisms previously identified as P. multocida were used in this study. The isolates were obtained from pigs in the three eastern States of Australia-Queensland (110 isolates), New South Wales (21 isolates) and Victoria (19 isolates). 2.2. Phenotypic
characterisation
All the field isolates were subjected to a full phenotypic characterisation. The presence of catalase, oxidase, P-galactosidase, omithine decarboxylase (ODC) and urease activity and the ability to produce indole were determined as described by Cowan (1974). The ability to ferment L-arabinose, dulcitol, D-glucose, D-lactose, maltose, D-IIIannitOl, D-sorbitol, D-sucrose, D-trehalose and D-xylose was tested using a miCrOplate fermentation method described by Blackall et al. (1995).
3. Results We have already reported that, in our hands, seven reference strains of Pasteurella gave the reactions recorded by Mutters et al. (1985) with one (Fegan et al., 1995). The reference strains were as follows-NCTC 10204 (P. subsp. gallicida), NCTC 10322 (P. multocida subsp. multocida), CIP
the genus exception multocida Al25 (P.
357
P.J. Blackall et al. / Veterinay Microbiology 57 (1997) 355-360
multocidu subsp. septica), NCTC 11621 (P. canis), NCTC 11617 (P. dugmatis), NCTC 11623 (P. stomutis) and SSI P683 (Pasteurella species B) (all obtained from Dr. R. Mutters (Institut fur Medizinische Mikrobiologie, Klinikum der Philipps-Universitt, Marburg, Germany). The exception was that, despite extensive retesting, the reference strain for P. multocidu subsp. gullicidu did not produce acid from dulcitol or xylose. Full details of the results of the reference strains are provided in our previous publication (Fegan et al., 1995). All 150 field isolates were Gram negative rods that produced indole, were oxidase and catalase positive and P-galactosidase and urease negative and fermented glucose. mannitol, sorbitol and sucrose but not maltose. The field isolates differed in their ability to ferment arabinose, dulcitol, lactose, trehalose and xylose and the presence of ODC activity. These differences allowed the recognition of seven distinct biochemical types. Four of these biochemical types matched biovars 1, 2, 3 and 8 as defined in our study of avian P. multocidu (Fegan et al., 1995). Three new biovars, termed biovars 12, 13 and 14, were recognised in this study. Full details of the properties of the biovars detected in the field isolates are given in Table 1. As we have reported previously (Fegan et al., 19951, biovars 1 to 3 match the described properties of P. multocidu subsp. multocidu while biovar 8 matches the property of P. multocidu subsp. gullicidu as reported by Mutters et al. (1985). All isolates of biovar 8 have been re-checked by Dr. Magne Bisgaard (Royal Veterinary and Agricultural University, Copenhagen) and confirmed as P. multocidu subsp. gullicidu (Bisgaard, pers. comm.). Biovar 12, which has identical properties to biovar 3 except for the fermentation of lactose, appears to represent lactose positive variants of P. multocidu subsp. multocidu. Biovar 13, which has identical properties to biovar 3 except for
Table I Differential
properties
Property
ODC’ Acid from Arabinose Dulcitol Lactose Sorbitol Trehalose Xylose Identification (subsp. of P. multocida)
and identity of 150 Australian
isolates of Pasteurella”
Biovarb
1 (5)
2 (19)
3 (loo)
8 (13)
12 (9)
13 (3)
14(l)
+
+
+
+
+
_
_
_ _
_ _ -
_ _
+ + _
_ _
_ _ _
_ _
+ + + + + + + + _ _ _ _ + + + + + + + multocida multocida multocida gallicida Lactose ODC Lactose positive; positive negative ODC negative multocida multocida multocida + _ _
“All the field isolates were Gram negative rods that were oxidase and catalase positive, lacked urease and haemolytic activity and all failed to grow on MacConkey agar. All the isolates also fermented glucose. mannitol and sucrose but failed to ferment maltose. ‘Number in parenthesis is the number of isolates in the biovar. ‘ODC = Ornithine decarboxylase activity.
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P.J. Blackall et al. / Veterinary Microbiology 57 (1997) 355-360
ODC activity, multocida.
appears
ODC negative variants of P. multocida subsp. 14, which differs from biovar 3 in being lactose appears to represent a lactose positive, ODC negative
to represent
The single isolate in biovar
positive and ODC negative, variant of P. multocida subsp. multocida.
4. Discussion As this study was a retrospective study based on isolates held in culture collections in a number of Australian laboratories, there are a number of inherent limitations in the study. Toxin-producing strains of P. multocida are rare in Australian pigs with an apparent herd prevalence of 2% in one study (Gardner et al., 1989). Hence, no attempt was made to determine the toxin producing ability of our isolates. The retrospective nature of the study meant that there was a lack of information on the clinical conditions associated with the isolates. We did not determine the serotypes of our isolates as our laboratory currently lacks the capacity to serotype P. multocida. Since the first isolation of the organism we now know as P. multocida in the late 1870s (Rhoades and Rimler, 1991) there have been many studies on the phenotypic properties of isolates from a range of different animal hosts. However, as most of these studies present only brief summaries or tables of results, it is impossible to retrospectively assign the isolates used in these studies to the subspecies proposed by Mutters et al. (1985). Hence, there have been only a few reports on the classification of P. multocida isolates to the three subspecies gallicida, multocida and septica. To date, the only substantial studies have been on isolates from cattle, cats and birds (both commercial poultry and wild birds) (Madsen et al., 1985; Hirsh et al., 1990; Snipes et al., 1990; Bisgaard et al., 199la,b; Korbel et al., 1992; Mohan et al., 1994; Fegan et al., 1995). The only previous studies of the subspecies of porcine P. multocida isolates have involved only small numbers of isolates, three from pigs in Zimbabwe (Mohan et al., 1994) and eight from pigs in Australia (Cameron et al., 1996). Hence, our study is the first extended phenotypic characterisation of a large collection of indole positive Pasteurella from pigs. We have shown that the subspecies multocida is the most common of the three subspecies of P. multocida present in Australian pigs, representing 137 of the 150 isolates we examined. We have noted a similar dominance of this subspecies in Australian poultry (Fegan et al., 1995). Within the subspecies multocida, we have established the presence of six biovars (I, 2, 3, 12, 13 and 14) with the overwhelming majority of isolates belonging to biovar 3 (100 isolates, 66.7% of the total number of isolates examined). Biovar 3 is also the predominant biovar of this subspecies present in Australian poultry (Fegan et al., 1995). The presence of the subspecies gallicida in Australian pigs was first reported in the case report by Cameron et al. (1996). In the current study, a further nine isolates of this subspecies were detected, resulting in a total of 13 isolates, 8.7% in total. The subspecies gallicida has previously been regarded as being a common isolate in waterfowl with 37% of 295 P. multocida isolates from waterfowl and associated birds in
P.J. Blackall et al. / Veterinay
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57 (19971 355-360
359
California being identified as this subspecies (Hirsh et al., 1990). Further studies using the subspecies definition proposed by Mutters et al. (1985) on porcine isolates of P. multocida from geographical locations outside Australia are necessary to confirm if the subspecies gallicida is widespread in pigs. On the basis of the results of this study and those of previous studies (Madsen et al., 1985; Hirsh et al., 1990; Snipes et al., 1990; Bisgaard et al., 1991a,b; Korbel et al., 1992; Mohan et al., 1994; Fegan et al., 1995), a picture of the relative abundance of the three subspecies in a range of animals is beginning to emerge. Isolates of P. multocida subsp. multocidu are the most common subspecies in cattle, pigs, poultry and cats (Snipes et al., 1990; Bisgaard et al., 1991a,b; Korbel et al., 1992; Mohan et al., 1994; Fegan et al., 1995, this study). While the subspecies gallicida has been reported in pigs (Cameron et al., 1996, this study) and in poultry (Snipes et al., 1990; Fegan et al., 1995), it is relatively more common in wild waterfowl (Hirsh et al., 1990). The subspecies septica has been reported in cattle (Bisgaard et al., 1991b) and poultry (Snipes et al., 1990; Fegan et al., 1995) but apparently is only a rare isolate in these animal species. In contrast, isolates of the subspecies septica were found at significant levels in feral birds (18%) and cats (28%) (Korbel et al., 1992). Three of the seven biovars we recognised represent unusual biochemical variations of the subspecies multocidu. Biovars 13 and 14 appear to represent, respectively, ODC negative and combined lactose positive/ODC negative variants of P. multocidu subsp. multocida. ODC negative variants of P. multocida subsp. multocida and P. multocida subsp. septica have been obtained from cattle, poultry and a dog (Bisgaard et al., 199 la,b; Mohan et al., 1994; Fegan et al., 1995) As we have noted in our previous study on avian P. multocidu (Fegan et al., 19951, any use of the ODC as a key character in distinguishing species in the genus Pasteurella, as suggested by Mutters et al. (1985). must be tempered with a recognition that ODC negative variants exist. Preliminary DNA-DNA hybridisation studies have suggested that ODC negative variants of subspecies multocidu and septica were related at the species level (Bisgaard et al., 199 1b). Clearly. further studies on ODC negative variants of P. multocidu are required. Two of the seven biovars we recognised (biovars 12 and 14) were apparently lactose positive variants of P. multocida subspecies multocida. Lactose positive variants of P. multocidu subsp. multocida have not been widely reported before. Bisgaard et al. (1991b) have noted that some isolates of the subspecies multocida and septica obtained from calf lungs were lactose positive. The nine isolates of biovar 12 (lactose positive variants of P. multocida subspecies multocida) were obtained from all three States included in this study. This indicates that such variants, while not numerically large, are widespread in the Australian pig industry. While the combination of ODC negative and lactose positive does not appear to have been previously reported in the subspecies multocidu, Bisgaard et al. (1991b) have reported this combination in isolates of the subspecies septica from calf lungs. In summary, this study has established that the majority of the 150 P. multucidcr isolates obtained from Australian pigs belong to the subspecies multocidu (137 isolates in total). The only other subspecies recognised was gallicidu (13 isolates in total). Diagnostic laboratories should be aware of the existence of lactose and ODC variants of porcine isolates of P. multocidu.
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Acknowledgements The assistance of Michelle Adamson (Victorian Department of Agriculture), Paul Duffy (Yeerongpilly Veterinary Laboratory), Carol Stephens (Toowoomba Veterinary Laboratory) and Dr. Mark White (Allied Feeds) in providing some of the isolates used in this study is gratefully acknowledged. This work was funded, in part, by the Australian Pig Research and Development Corporation.
References Bisgaard, M., Abdullahi, M.Z., Gilmour, N.J.L., 199la. Further studies on the identification of Pasteurellaceae from cattle lungs. Vet. Rec. 128, 428-429. Bisgaard, M., Houghton, S.B., Mutters, R., Stenzel, A., 1991b. Reclassification of German, British and Dutch isolates of so-called Pasteurefiu multocida obtained from pneumonic calf lungs. Vet. Microbial. 26, 115-124. Blackall, P.J., Pahoff, J.L., Marks, D., Fegan. N., Morrow. C.J., 1995. Characterisation of Pasteurella multocida isolates from fowl cholera outbreaks on seven turkey farms, Aust. Vet. J. 72, 135-138. Cameron, R.D.A.. O’Boyle, D., Frost, A.J., Fegan, N., 1996. An outbreak of haemorrhagic septicemia associated with Pasteurella mulfocida subsp. gnllicida in a large pig herd. Aust. Vet. J. 73, 27-29. Cowan. S.T., 1974. Cowan and Steel’s Manual for the Identification of Medical Bacteria. Cambridge Univ. Press, Cambridge. De Jong, M.F., 1992. (Progressive) Atrophic Rhinitis. In: Leman, A.D., Straw, B.E., Mengeling, W.L., D’Alliare, S., Taylor. D.J. (Eds.), Diseases of Swine. Iowa State University Press, Ames. pp. 414-435. Fegan, N., Blackall, P.J., Pahoff, J.L., 1995. Phenotypic characterisation of Pasteurella multocida isolates from Australian poultry. Vet. Microbial. 47. 281-286. Gardner, LA., Eamens, G.J., Turner. A.J., Homitzky, C.L., 1989. Toxigenic type D Pasteurella multocida in New South Wales pig herds-prevalence and factors associated with infection. Aust. Vet. J. 66, 318-321. Hirsh, D.C., Jessup, D.A.. Snipes, K.P., Carpenter, T.E., Hird, D.W., McCapes, R.C., 1990. Characteristics of Pasteurella multocidu isolated from waterfowl and associated avian species in California. J. Wildlife Dis. 26, 204-209. Korbel. R., Gerlach, H., Bisgaard, M., Hafez, H.M., 1992. Further investigations on Pusteurellu multocidu infections in feral birds injured by cats. J. Vet. Med. B 39, 10-18. Madsen, E.B., Bisgaard, M.. Mutters, R., Pedersen, K.B., 1985. Characterization of Pusreurellu species isolated from lungs of calves with pneumonia. Can. J. Comp. Med. 49, 63-67. Mohan, K., Sadza, M., Madsen, M., Hill, F.W.G., Pawandiwa, A.. 1994. Phenotypic characterization of Zimbabwean isolates of Pasteurella multocidu. Vet. Microbial. 38. 351-357. Mutters, R., Ihm, P.. Pohl, S., Frederiksen, W., Mannheim, W., 1985. Reclassification of the genus Pusteurellu Trevisan 1887 on the basis of deoxyribonucleic acid homology. with proposals for the new species Pusteurella dagmatis, Pasteurella cunis. Pasteurella stomatis. Pasteurella anutis. and Pasteurella langaa. Int. J. Syst. Bacterial. 35. 309-322. Pijoan, C., 1992. Pneumonic pasteurellosis. In: Leman, A.D., Straw, B.E., Mengeling, W.L., D’AIliare, S., Taylor, D.J. (Eds.1, Diseases of Swine. Iowa State University Press, Ames, pp. 552-559. Rhoades, K.R., Rimler. R.B., 1991. Pasteurellosis. In: Calnek, B.W., Barnes, H.J., Beard, C.W., Reid, W.M.. Yoder, H.W.. Jr. (Eds.), Diseases of Poultry. Iowa State University Press, Ames, pp. 145-162. Snipes, K.P.. Hirsh, D.C.. Kasten, R.W., Carpenter, T.E., Hird, D.W., McCapes. R.H., 1990. Homogeneity of characteristics of Pasteurella multocida isolated from turkeys and wildlife in California. 1985-88. Avian Dis. 34. 315-320.
Veterinary Microbiology 57 (1997) 355-360
Phenotypic characterisation of Pasteurella multocida isolates from Australian pigs P.J. Blackall
*,
J.L. Pahoff, R. Bowles
Queensland Department of Primary Industries, Animal Research Institute, 665 Fairfield Road. Yeerongpilly QLD 4105, Australia Received 13 September 1996; accepted 17 March 1997
Abstract A phenotypic characterisation of 1.50 isolates of bacteria previously identified as PasteureMu multocidu was performed. All the isolates had been obtained from Australian pigs in the three eastern States of Queensland (I 10 isolates), New South Wales (21 isolates) and Victoria (19 isolates). Seven different biochemical biovars were recognised amongst the isolates. A total of 100 isolates (67%) were assigned to biovar 3, previously shown to be the most common biovar in isolates of P. multocidu from Australian poultry [Fegan, N., Blackall, P.J., Pahoff, J.L., 1995. Phenotypic characterisation of Pasteurella multocidu isolates from Australian poultry. Vet. Microbial., 47, 281-286.1. Six of the seven biovars, including biovar 3, were identified as P. multocidu subsp. multocida, 124 isolates in total. One other biovar, consisting of thirteen isolates, was identified as P. multocida subsp. gallicida. Within the six biovars that were identified as P. multocidu subsp. multocidu, biovars 12, 13 and 14 represented unusual biochemical variants. The nine isolates assigned to biovar 12 appeared to be lactose positive variants of P. multocida subsp. multocidu. The three isolates in biovar 13 appeared to be ornithine decarboxylase (ODC) negative variants of P. multocida subsp. multocida. The single isolate in biovar 14 appeared to be an ODC negative, lactose positive variant of P. multocidu subsp. multocidu. 0 1997 Elsevier Science B.V. Keywords: Pig-bacteria, Pasteurella multocida; Phenotype: Taxonomy
1. Introduction multocida is a major pathogen of pigs, being involved in a number of disease conditions. The main diseases associated with this organism are
Pasteurella
different
* Corresponding author. Tel.: +61-7-3362-9498:
fax: + 61-7-3362-9429;
0378-l 135/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII s0378-1135~97)00111-9
e-mail: [email protected]
356
P.J. Black&
et al. / Veterinary
Microbiology
57 (19971 355-360
pneumonic pasteurellosis, one of the most common and widespread diseases of intensively housed pigs (Pijoan, 1992) and progressive atrophic rhinitis, an important disease of pigs in North America and Europe (De Jong, 1992). Since the taxonomic reclassification proposed by Mutters et al. (1985), it is now recognised that a number of taxa within the genus Pasteurella, other than P. multocida, are indole positive, P. canis. P. dagmatis, P. stomatis and an un-named species Pasteurella species B. As well, three subspecies within P. multocida (P. multocida subsp. multocida, P. multocida subsp. septica and P. multocida subsp. gallicida) are now recognised (Mutters et al., 1985). We have recently completed a phenotypic characterisation of indole positive Pasteurella-like organisms isolated from Australian poultry (Fegan et al., 1995). In the current study, we report on the characterisation of 150 P. multocida-like isolates all obtained from Australian pigs. We have included 8 isolates that have been previously described in a case report on a outbreak of systemic pasteurellosis (Cameron et al., 1996). This study has been performed to provide the first definitive knowledge on the phenotypic properties of P. multocida-like isolates from Australian pigs in the light of current taxonomic knowledge.
2. Materials
and methods
2.1. Bacteria A total of 150 isolates of organisms previously identified as P. multocida were used in this study. The isolates were obtained from pigs in the three eastern States of Australia-Queensland (110 isolates), New South Wales (21 isolates) and Victoria (19 isolates). 2.2. Phenotypic
characterisation
All the field isolates were subjected to a full phenotypic characterisation. The presence of catalase, oxidase, P-galactosidase, omithine decarboxylase (ODC) and urease activity and the ability to produce indole were determined as described by Cowan (1974). The ability to ferment L-arabinose, dulcitol, D-glucose, D-lactose, maltose, D-IIIannitOl, D-sorbitol, D-sucrose, D-trehalose and D-xylose was tested using a miCrOplate fermentation method described by Blackall et al. (1995).
3. Results We have already reported that, in our hands, seven reference strains of Pasteurella gave the reactions recorded by Mutters et al. (1985) with one (Fegan et al., 1995). The reference strains were as follows-NCTC 10204 (P. subsp. gallicida), NCTC 10322 (P. multocida subsp. multocida), CIP
the genus exception multocida Al25 (P.
357
P.J. Blackall et al. / Veterinay Microbiology 57 (1997) 355-360
multocidu subsp. septica), NCTC 11621 (P. canis), NCTC 11617 (P. dugmatis), NCTC 11623 (P. stomutis) and SSI P683 (Pasteurella species B) (all obtained from Dr. R. Mutters (Institut fur Medizinische Mikrobiologie, Klinikum der Philipps-Universitt, Marburg, Germany). The exception was that, despite extensive retesting, the reference strain for P. multocidu subsp. gullicidu did not produce acid from dulcitol or xylose. Full details of the results of the reference strains are provided in our previous publication (Fegan et al., 1995). All 150 field isolates were Gram negative rods that produced indole, were oxidase and catalase positive and P-galactosidase and urease negative and fermented glucose. mannitol, sorbitol and sucrose but not maltose. The field isolates differed in their ability to ferment arabinose, dulcitol, lactose, trehalose and xylose and the presence of ODC activity. These differences allowed the recognition of seven distinct biochemical types. Four of these biochemical types matched biovars 1, 2, 3 and 8 as defined in our study of avian P. multocidu (Fegan et al., 1995). Three new biovars, termed biovars 12, 13 and 14, were recognised in this study. Full details of the properties of the biovars detected in the field isolates are given in Table 1. As we have reported previously (Fegan et al., 19951, biovars 1 to 3 match the described properties of P. multocidu subsp. multocidu while biovar 8 matches the property of P. multocidu subsp. gullicidu as reported by Mutters et al. (1985). All isolates of biovar 8 have been re-checked by Dr. Magne Bisgaard (Royal Veterinary and Agricultural University, Copenhagen) and confirmed as P. multocidu subsp. gullicidu (Bisgaard, pers. comm.). Biovar 12, which has identical properties to biovar 3 except for the fermentation of lactose, appears to represent lactose positive variants of P. multocidu subsp. multocidu. Biovar 13, which has identical properties to biovar 3 except for
Table I Differential
properties
Property
ODC’ Acid from Arabinose Dulcitol Lactose Sorbitol Trehalose Xylose Identification (subsp. of P. multocida)
and identity of 150 Australian
isolates of Pasteurella”
Biovarb
1 (5)
2 (19)
3 (loo)
8 (13)
12 (9)
13 (3)
14(l)
+
+
+
+
+
_
_
_ _
_ _ -
_ _
+ + _
_ _
_ _ _
_ _
+ + + + + + + + _ _ _ _ + + + + + + + multocida multocida multocida gallicida Lactose ODC Lactose positive; positive negative ODC negative multocida multocida multocida + _ _
“All the field isolates were Gram negative rods that were oxidase and catalase positive, lacked urease and haemolytic activity and all failed to grow on MacConkey agar. All the isolates also fermented glucose. mannitol and sucrose but failed to ferment maltose. ‘Number in parenthesis is the number of isolates in the biovar. ‘ODC = Ornithine decarboxylase activity.
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P.J. Blackall et al. / Veterinary Microbiology 57 (1997) 355-360
ODC activity, multocida.
appears
ODC negative variants of P. multocida subsp. 14, which differs from biovar 3 in being lactose appears to represent a lactose positive, ODC negative
to represent
The single isolate in biovar
positive and ODC negative, variant of P. multocida subsp. multocida.
4. Discussion As this study was a retrospective study based on isolates held in culture collections in a number of Australian laboratories, there are a number of inherent limitations in the study. Toxin-producing strains of P. multocida are rare in Australian pigs with an apparent herd prevalence of 2% in one study (Gardner et al., 1989). Hence, no attempt was made to determine the toxin producing ability of our isolates. The retrospective nature of the study meant that there was a lack of information on the clinical conditions associated with the isolates. We did not determine the serotypes of our isolates as our laboratory currently lacks the capacity to serotype P. multocida. Since the first isolation of the organism we now know as P. multocida in the late 1870s (Rhoades and Rimler, 1991) there have been many studies on the phenotypic properties of isolates from a range of different animal hosts. However, as most of these studies present only brief summaries or tables of results, it is impossible to retrospectively assign the isolates used in these studies to the subspecies proposed by Mutters et al. (1985). Hence, there have been only a few reports on the classification of P. multocida isolates to the three subspecies gallicida, multocida and septica. To date, the only substantial studies have been on isolates from cattle, cats and birds (both commercial poultry and wild birds) (Madsen et al., 1985; Hirsh et al., 1990; Snipes et al., 1990; Bisgaard et al., 199la,b; Korbel et al., 1992; Mohan et al., 1994; Fegan et al., 1995). The only previous studies of the subspecies of porcine P. multocida isolates have involved only small numbers of isolates, three from pigs in Zimbabwe (Mohan et al., 1994) and eight from pigs in Australia (Cameron et al., 1996). Hence, our study is the first extended phenotypic characterisation of a large collection of indole positive Pasteurella from pigs. We have shown that the subspecies multocida is the most common of the three subspecies of P. multocida present in Australian pigs, representing 137 of the 150 isolates we examined. We have noted a similar dominance of this subspecies in Australian poultry (Fegan et al., 1995). Within the subspecies multocida, we have established the presence of six biovars (I, 2, 3, 12, 13 and 14) with the overwhelming majority of isolates belonging to biovar 3 (100 isolates, 66.7% of the total number of isolates examined). Biovar 3 is also the predominant biovar of this subspecies present in Australian poultry (Fegan et al., 1995). The presence of the subspecies gallicida in Australian pigs was first reported in the case report by Cameron et al. (1996). In the current study, a further nine isolates of this subspecies were detected, resulting in a total of 13 isolates, 8.7% in total. The subspecies gallicida has previously been regarded as being a common isolate in waterfowl with 37% of 295 P. multocida isolates from waterfowl and associated birds in
P.J. Blackall et al. / Veterinay
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California being identified as this subspecies (Hirsh et al., 1990). Further studies using the subspecies definition proposed by Mutters et al. (1985) on porcine isolates of P. multocida from geographical locations outside Australia are necessary to confirm if the subspecies gallicida is widespread in pigs. On the basis of the results of this study and those of previous studies (Madsen et al., 1985; Hirsh et al., 1990; Snipes et al., 1990; Bisgaard et al., 1991a,b; Korbel et al., 1992; Mohan et al., 1994; Fegan et al., 1995), a picture of the relative abundance of the three subspecies in a range of animals is beginning to emerge. Isolates of P. multocida subsp. multocidu are the most common subspecies in cattle, pigs, poultry and cats (Snipes et al., 1990; Bisgaard et al., 1991a,b; Korbel et al., 1992; Mohan et al., 1994; Fegan et al., 1995, this study). While the subspecies gallicida has been reported in pigs (Cameron et al., 1996, this study) and in poultry (Snipes et al., 1990; Fegan et al., 1995), it is relatively more common in wild waterfowl (Hirsh et al., 1990). The subspecies septica has been reported in cattle (Bisgaard et al., 1991b) and poultry (Snipes et al., 1990; Fegan et al., 1995) but apparently is only a rare isolate in these animal species. In contrast, isolates of the subspecies septica were found at significant levels in feral birds (18%) and cats (28%) (Korbel et al., 1992). Three of the seven biovars we recognised represent unusual biochemical variations of the subspecies multocidu. Biovars 13 and 14 appear to represent, respectively, ODC negative and combined lactose positive/ODC negative variants of P. multocidu subsp. multocida. ODC negative variants of P. multocida subsp. multocida and P. multocida subsp. septica have been obtained from cattle, poultry and a dog (Bisgaard et al., 199 la,b; Mohan et al., 1994; Fegan et al., 1995) As we have noted in our previous study on avian P. multocidu (Fegan et al., 19951, any use of the ODC as a key character in distinguishing species in the genus Pasteurella, as suggested by Mutters et al. (1985). must be tempered with a recognition that ODC negative variants exist. Preliminary DNA-DNA hybridisation studies have suggested that ODC negative variants of subspecies multocidu and septica were related at the species level (Bisgaard et al., 199 1b). Clearly. further studies on ODC negative variants of P. multocidu are required. Two of the seven biovars we recognised (biovars 12 and 14) were apparently lactose positive variants of P. multocida subspecies multocida. Lactose positive variants of P. multocidu subsp. multocida have not been widely reported before. Bisgaard et al. (1991b) have noted that some isolates of the subspecies multocida and septica obtained from calf lungs were lactose positive. The nine isolates of biovar 12 (lactose positive variants of P. multocida subspecies multocida) were obtained from all three States included in this study. This indicates that such variants, while not numerically large, are widespread in the Australian pig industry. While the combination of ODC negative and lactose positive does not appear to have been previously reported in the subspecies multocidu, Bisgaard et al. (1991b) have reported this combination in isolates of the subspecies septica from calf lungs. In summary, this study has established that the majority of the 150 P. multucidcr isolates obtained from Australian pigs belong to the subspecies multocidu (137 isolates in total). The only other subspecies recognised was gallicidu (13 isolates in total). Diagnostic laboratories should be aware of the existence of lactose and ODC variants of porcine isolates of P. multocidu.
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Acknowledgements The assistance of Michelle Adamson (Victorian Department of Agriculture), Paul Duffy (Yeerongpilly Veterinary Laboratory), Carol Stephens (Toowoomba Veterinary Laboratory) and Dr. Mark White (Allied Feeds) in providing some of the isolates used in this study is gratefully acknowledged. This work was funded, in part, by the Australian Pig Research and Development Corporation.
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