SHORT COMMUNICATIONS Effects of Purified Pasteurella multocida Dermonecrotoxin on the Nasal Ventral Turbinates of Fattening Pigs: Histological Observations B. Martineau-Doize, J. Menard, C. Girard, J.C. Frantz and G.-P. Martineau
ABSTRACT Fattening specific pathogen-free derived pigs were injected intramuscularly with dermonecrotoxin of Pasteurella multocida, capsular ype D. Ten days later, the nasal ventral turbinates and liver were examined histologically. A moderate turbinate atrophy was observed due to an increased number of osteoclasts and the absence of intramembranous bone apposition. Liver lesions were limited to some hepatocyte necrosis, sinusoid neutrophil infiltration and Kupffer cell hypertrophy. This study demonstrated that adult pigs are sensitive to P. multocida dermonecrotoxin.
RESUME Des porcs a l'engraissement de descendance d'aminaux exempts d'agents pathogenes specifiques (SPF) ont ete injectes par voie intramusculaire avec de la dermon&crotoxine de Pasteurella multocida, type capsulaire D. Les cornets nasaux ventraux et le foie de ces animaux ont ete examines histologiquement dix jours plus tard. Une atrophie moderee des comets a ete observee. Celle-ci etait due A une augmentation du nombre d'osteoclastes et A la disparition de l'apposition osseuse intramembranaire. Des
lesions hepatiques moderees caracterisees par quelques foyers de necrose hepatocytaire, une infiltration de neutrophiles dans les sinusoides et une hypertrophie des cellules de Kupffer ont egalement ete observees. Cette etude a demontrd que les porcs adultes sont sensibles a la dermon6crotoxine de P. multocida.
Although atrophic rhinitis is a multifactorial disease associated with intense pig production, Bordetella bronchiseptica and Pasteurella multocida are the major etiological agents of the disease (1). Bordetella bronchiseptica causes moderate, nonprogressive turbinate atrophy (2,3). Infection of piglets older than four to six weeks does not induce significant turbinate atrophy (3-5). In contrast, toxigenic type D P. multocida provokes severe turbinate atrophy (6-1 1). Nielsen et al (12) showed that severe turbinate atrophy was induced in three month old pigs by mixing with pigs with endemic atrophic rhinitis. The other studies concerning lesions induced by P. multocida have been performed in piglets during their first weeks of life, but it is generally believed that P. multocida is able to induce atrophic rhinitis in older pigs. Elling and Pedersen (13) have shown that piglets inoculated intranasally with mild acetic
acid and P. multocida developed turbinate atrophy which persisted until slaughter at 90 kg. The aim of the present study was to demonstrate the ability of P. multocida type D dermonecrotoxin to induce atrophic rhinitis in fattening pigs. Pasteurella multocida dermonecrotoxin (DNT) was purified from a type D strain isolated from a pig with severe atrophic rhinitis (10). The median lethal dose for mice of the DNT was 20 ng. The study involved five (three experimentals and two controls) five month old, 100 kg, specific pathogenfree derived pigs. The study followed the guidelines of the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The experimental pigs were injected intramuscularly with 100 jtg (n = 1) or 50 Ag (n = 2) of the purified DNT. They were killed ten days later. Toxin dose and duration of the experiment were selected according to a previous study using seven day-old piglets (10). Liver samples and the noses were fixed in 10% formalin. The noses were decalcified in formic acidsodium citrate and the tissues were processed by standard histological techniques. Tissue sections, 7 itm thick, were cut from paraffin blocks and stained with hematoxilin-phloxinsafron. Sections were cut transversally through the noses at the rostral (space
Groupe de recherche sur les maladies infectieuses du porc (Martineau-Doiz6, Martineau), Departement de pathologie et microbiologie (Girard), Facult6 de M6decine veterinaire, Universite de Montreal, C.P. 5000, Saint-Hyacinthe, Quebec J2S 7C6, F. M6nard Inc., Ange-Gardien, Quebec JOE lEO (Menard) and SmithKline Beecham Animal Health, Veterinary Pharmaceuticals and Biologicals, 601 West Cornhusker Highway, P.O. Box 80809, Lincoln, Nebraska 68501-0809 (Frantz). Supported by the Natural Sciences and Engineering Research Council of Canada and the Fonds pour la Formation de Chercheurs et Aide a la Recherche. Submitted February 11, 1991.
Can J Vet Res 1991; 55: 377-379
377
Sl
j~~~~~
Fig. 1 Transverse histological section of the nasal ventral turbinate of a control (A) and an experimental (B) pig. In the experimental pig, the number of bone trabeculne is diminished and the free nasal space is increased. Arrows indicate the bone thickness; S: nasal free space.
between the second incisor tooth and the canina) and mid-caudal regions (rostral extremity of the second premolar tooth). When examined macroscopically, the nasal ventral turbinates did not show a severe atrophy. However, the thickness of the turbinates in the experimental pigs was affected. This resulted in a larger free space between the scrolls and the nasal wall as well as the space within the curved parts of the scrolls. This observation was obvious when the experimental animals were compared to the controls. Light microscope examination of the nose sections demonstrated that the macroscopically reduced thickness of the scrolls was due to a reduction of their bone thickness. The number of bone trabeculae was diminished and 378
P. multocida dermonecrotoxin at seven days of age (10). However, although the experimental conditions (dose and time-interval between injection and euthanasia) were the same in both experiments, turbinate atrophy was less severe in the fattening pigs. Thus, fattening pigs seem to be less susceptible to P. multocida dermonecrotoxin. This is probably due to the fact that in fattening pigs turbinate growth is reduced and that their turbinates contain essentially lamellar bone. In the present study, pigs were infected at the age of five months and the delay between infection and euthanasia was short (ten days). In contrast, in other published studies, infection was performed in young animals (up to a maximum of three months) and the postinfection time was longer (up to several months) (11-13). Liver lesions were present in the animal injected with 100 Ag DNT. They consisted of a limited amount of hepatocyte necrosis, especially of hepatocytes at the periphery of the lobules. Adjacent sinusoids contained neutrophil infiltrations and some Kupffer cells were hypertrophied. Although the amount of injected DNT was high and thus did not reflect field conditions, this study demonstrated that adult pig nasal turbinates are sensitive to P. multocida dermonecrotoxin.
ACKNOWLEDGMENTS was limited to one single thin trabecule in the region of the scrolls parallel to The authors are grateful to Isabelle the nasal septum (Fig. 1). This resulted Caya for her assistance. in fragile turbinates which had a tendency to fold during the histological REFERENCES manipulations. A closer examination demonstrated two modifications. Firstly, the intramembranous bone 1. RUTTER JM. Atrophic rhinitis in swine. Vet Sci Comp Med 1985; 29: 239-279. apposition present along the excentric 2. Adv DUNCAN JR, ROSS RF, SWITZER WP, side of the control turbinates was RAMSEY FK. Pathology of experimental absent in the experimentals (Fig. 2). Bordetella bronchiseptica infection in swine: atrophic rhinitis. Am J Vet Res 1966; 27: Secondly, the number and location of 457-466. the osteoclasts was modified: 1) their 3. SHIMIZU T, NAKAGAWA M, SHIBATA number was increased along the conS, SUZUKI K. Atrophic rhinitis produced centric scroll side, 2) many osteoclasts by intranasal inoculation of Bordetella bronwere also present along the excentric chiseptica in hysterectomy produced colostrum-deprived pigs. Cornell Vet 1971; scroll side, where in the control pig 61: 696-705. osteoclasts were rarely observed 4. BRASSINE M, DEWAELE A, GOUFFAUX (Fig. 2). These lesions were similar M. Intranasal infection with Bordetelia bronto those observed in the turbinates chiseptica in gnotobiotic piglets. Res Vet Sci 1976; 20: 162-166. of piglets similarly infected with
40.~~~~~~~~~~4
~~~~~~
~~~~~~~~~~~~ C~~~~~~~~~~~~~~~~~~~~~'
J~~~~~~£o
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Fig 2 Drsal scoll ofthe nasl vental turbnate o a contol (A)and an xpenmetal (B)pig Inthe conrol scoll, oberve te intene intr the membranous bone apposition (straight arrows) along the excentnc scroll side and the presence of some osteoclasts (cnrved arrows) atconcentric~~~~~~~~~~6
sid. n heexermetalscol, on apoitin s bsnt wil oseolatsar pesnt log ot srol ide. C:cocetrc crol id; X:exen tric scroll side.~~~~~
5. MARTINEAU GP, BROES A, MARTINEAU-DOIZE B. Appraisal of infection pressure on gnotobiotic piglets infected with Bordetella bronchiseptica. Can J Comp Med 1982; 46: 376-381. 6. DOMINICK MA, RIMLER RB. Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida. Am J Vet Res 1986; 47: 1532-1536. 7. DOMINICK MA, RIMLER RB. Turbinate osteoporosis in pigs following intranasal inoculation of purified Pasteurella toxin: histomorphometric and ultrastructural studies. Vet Pathol 1988; 25: 17-27. 8. KAMP EM, KIMMAN TG. Induction of turbinate atrophy in germ-free pigs, using
Pasteurella multocida as well as bacteriumfree crude and purified dermonecrotic toxin of P. multocida. Am J Vet Res 1988; 49: 1844-1849. 9. DE JONG MF, DE WACHTER JC, VAN DE MAREL GM. Investigation into the pathogenesis of atrophic rhinitis. II. AR induction and protection after intramuscular injections of cell-free filtrates and emulsions containing AR toxin of Pasteurella multocida. Vet Q 1986; 8: 215-224. 10. MARTINEAU-DOIZE B, FRANTZ JC, MARTINEAU GP. Effect of purified Pasteurella multocida dermonecrotoxin on cartilage and bone of the nasal ventral conchae of the piglet. Anat Rec 1990; 228: 237-246.
11. CHUNG WB, BACSTROM LR, CONRAD T, COLLINS MT. A comparison of different challenge methods for induction of atrophic rhinitis in pigs. APMIS 1990; 98: 442-542. 12. NIELSEN NC, RIISING HJ, BILLE N. Experimental reproduction of atrophic rhinitis in pigs reared to slaughter weight. Proc 4th Int Pig Vet Soc Congr 1976: P1. 13. ELLING F, PEDERSEN KB. The pathogenesis of persistent turbinate atrophy induced by toxigenic Pasteurella multocida in pigs. Vet Pathol 1985; 22: 469-474.
379
ABSTRACT Fattening specific pathogen-free derived pigs were injected intramuscularly with dermonecrotoxin of Pasteurella multocida, capsular ype D. Ten days later, the nasal ventral turbinates and liver were examined histologically. A moderate turbinate atrophy was observed due to an increased number of osteoclasts and the absence of intramembranous bone apposition. Liver lesions were limited to some hepatocyte necrosis, sinusoid neutrophil infiltration and Kupffer cell hypertrophy. This study demonstrated that adult pigs are sensitive to P. multocida dermonecrotoxin.
RESUME Des porcs a l'engraissement de descendance d'aminaux exempts d'agents pathogenes specifiques (SPF) ont ete injectes par voie intramusculaire avec de la dermon&crotoxine de Pasteurella multocida, type capsulaire D. Les cornets nasaux ventraux et le foie de ces animaux ont ete examines histologiquement dix jours plus tard. Une atrophie moderee des comets a ete observee. Celle-ci etait due A une augmentation du nombre d'osteoclastes et A la disparition de l'apposition osseuse intramembranaire. Des
lesions hepatiques moderees caracterisees par quelques foyers de necrose hepatocytaire, une infiltration de neutrophiles dans les sinusoides et une hypertrophie des cellules de Kupffer ont egalement ete observees. Cette etude a demontrd que les porcs adultes sont sensibles a la dermon6crotoxine de P. multocida.
Although atrophic rhinitis is a multifactorial disease associated with intense pig production, Bordetella bronchiseptica and Pasteurella multocida are the major etiological agents of the disease (1). Bordetella bronchiseptica causes moderate, nonprogressive turbinate atrophy (2,3). Infection of piglets older than four to six weeks does not induce significant turbinate atrophy (3-5). In contrast, toxigenic type D P. multocida provokes severe turbinate atrophy (6-1 1). Nielsen et al (12) showed that severe turbinate atrophy was induced in three month old pigs by mixing with pigs with endemic atrophic rhinitis. The other studies concerning lesions induced by P. multocida have been performed in piglets during their first weeks of life, but it is generally believed that P. multocida is able to induce atrophic rhinitis in older pigs. Elling and Pedersen (13) have shown that piglets inoculated intranasally with mild acetic
acid and P. multocida developed turbinate atrophy which persisted until slaughter at 90 kg. The aim of the present study was to demonstrate the ability of P. multocida type D dermonecrotoxin to induce atrophic rhinitis in fattening pigs. Pasteurella multocida dermonecrotoxin (DNT) was purified from a type D strain isolated from a pig with severe atrophic rhinitis (10). The median lethal dose for mice of the DNT was 20 ng. The study involved five (three experimentals and two controls) five month old, 100 kg, specific pathogenfree derived pigs. The study followed the guidelines of the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The experimental pigs were injected intramuscularly with 100 jtg (n = 1) or 50 Ag (n = 2) of the purified DNT. They were killed ten days later. Toxin dose and duration of the experiment were selected according to a previous study using seven day-old piglets (10). Liver samples and the noses were fixed in 10% formalin. The noses were decalcified in formic acidsodium citrate and the tissues were processed by standard histological techniques. Tissue sections, 7 itm thick, were cut from paraffin blocks and stained with hematoxilin-phloxinsafron. Sections were cut transversally through the noses at the rostral (space
Groupe de recherche sur les maladies infectieuses du porc (Martineau-Doiz6, Martineau), Departement de pathologie et microbiologie (Girard), Facult6 de M6decine veterinaire, Universite de Montreal, C.P. 5000, Saint-Hyacinthe, Quebec J2S 7C6, F. M6nard Inc., Ange-Gardien, Quebec JOE lEO (Menard) and SmithKline Beecham Animal Health, Veterinary Pharmaceuticals and Biologicals, 601 West Cornhusker Highway, P.O. Box 80809, Lincoln, Nebraska 68501-0809 (Frantz). Supported by the Natural Sciences and Engineering Research Council of Canada and the Fonds pour la Formation de Chercheurs et Aide a la Recherche. Submitted February 11, 1991.
Can J Vet Res 1991; 55: 377-379
377
Sl
j~~~~~
Fig. 1 Transverse histological section of the nasal ventral turbinate of a control (A) and an experimental (B) pig. In the experimental pig, the number of bone trabeculne is diminished and the free nasal space is increased. Arrows indicate the bone thickness; S: nasal free space.
between the second incisor tooth and the canina) and mid-caudal regions (rostral extremity of the second premolar tooth). When examined macroscopically, the nasal ventral turbinates did not show a severe atrophy. However, the thickness of the turbinates in the experimental pigs was affected. This resulted in a larger free space between the scrolls and the nasal wall as well as the space within the curved parts of the scrolls. This observation was obvious when the experimental animals were compared to the controls. Light microscope examination of the nose sections demonstrated that the macroscopically reduced thickness of the scrolls was due to a reduction of their bone thickness. The number of bone trabeculae was diminished and 378
P. multocida dermonecrotoxin at seven days of age (10). However, although the experimental conditions (dose and time-interval between injection and euthanasia) were the same in both experiments, turbinate atrophy was less severe in the fattening pigs. Thus, fattening pigs seem to be less susceptible to P. multocida dermonecrotoxin. This is probably due to the fact that in fattening pigs turbinate growth is reduced and that their turbinates contain essentially lamellar bone. In the present study, pigs were infected at the age of five months and the delay between infection and euthanasia was short (ten days). In contrast, in other published studies, infection was performed in young animals (up to a maximum of three months) and the postinfection time was longer (up to several months) (11-13). Liver lesions were present in the animal injected with 100 Ag DNT. They consisted of a limited amount of hepatocyte necrosis, especially of hepatocytes at the periphery of the lobules. Adjacent sinusoids contained neutrophil infiltrations and some Kupffer cells were hypertrophied. Although the amount of injected DNT was high and thus did not reflect field conditions, this study demonstrated that adult pig nasal turbinates are sensitive to P. multocida dermonecrotoxin.
ACKNOWLEDGMENTS was limited to one single thin trabecule in the region of the scrolls parallel to The authors are grateful to Isabelle the nasal septum (Fig. 1). This resulted Caya for her assistance. in fragile turbinates which had a tendency to fold during the histological REFERENCES manipulations. A closer examination demonstrated two modifications. Firstly, the intramembranous bone 1. RUTTER JM. Atrophic rhinitis in swine. Vet Sci Comp Med 1985; 29: 239-279. apposition present along the excentric 2. Adv DUNCAN JR, ROSS RF, SWITZER WP, side of the control turbinates was RAMSEY FK. Pathology of experimental absent in the experimentals (Fig. 2). Bordetella bronchiseptica infection in swine: atrophic rhinitis. Am J Vet Res 1966; 27: Secondly, the number and location of 457-466. the osteoclasts was modified: 1) their 3. SHIMIZU T, NAKAGAWA M, SHIBATA number was increased along the conS, SUZUKI K. Atrophic rhinitis produced centric scroll side, 2) many osteoclasts by intranasal inoculation of Bordetella bronwere also present along the excentric chiseptica in hysterectomy produced colostrum-deprived pigs. Cornell Vet 1971; scroll side, where in the control pig 61: 696-705. osteoclasts were rarely observed 4. BRASSINE M, DEWAELE A, GOUFFAUX (Fig. 2). These lesions were similar M. Intranasal infection with Bordetelia bronto those observed in the turbinates chiseptica in gnotobiotic piglets. Res Vet Sci 1976; 20: 162-166. of piglets similarly infected with
40.~~~~~~~~~~4
~~~~~~
~~~~~~~~~~~~ C~~~~~~~~~~~~~~~~~~~~~'
J~~~~~~£o
IM
{
Fig 2 Drsal scoll ofthe nasl vental turbnate o a contol (A)and an xpenmetal (B)pig Inthe conrol scoll, oberve te intene intr the membranous bone apposition (straight arrows) along the excentnc scroll side and the presence of some osteoclasts (cnrved arrows) atconcentric~~~~~~~~~~6
sid. n heexermetalscol, on apoitin s bsnt wil oseolatsar pesnt log ot srol ide. C:cocetrc crol id; X:exen tric scroll side.~~~~~
5. MARTINEAU GP, BROES A, MARTINEAU-DOIZE B. Appraisal of infection pressure on gnotobiotic piglets infected with Bordetella bronchiseptica. Can J Comp Med 1982; 46: 376-381. 6. DOMINICK MA, RIMLER RB. Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida. Am J Vet Res 1986; 47: 1532-1536. 7. DOMINICK MA, RIMLER RB. Turbinate osteoporosis in pigs following intranasal inoculation of purified Pasteurella toxin: histomorphometric and ultrastructural studies. Vet Pathol 1988; 25: 17-27. 8. KAMP EM, KIMMAN TG. Induction of turbinate atrophy in germ-free pigs, using
Pasteurella multocida as well as bacteriumfree crude and purified dermonecrotic toxin of P. multocida. Am J Vet Res 1988; 49: 1844-1849. 9. DE JONG MF, DE WACHTER JC, VAN DE MAREL GM. Investigation into the pathogenesis of atrophic rhinitis. II. AR induction and protection after intramuscular injections of cell-free filtrates and emulsions containing AR toxin of Pasteurella multocida. Vet Q 1986; 8: 215-224. 10. MARTINEAU-DOIZE B, FRANTZ JC, MARTINEAU GP. Effect of purified Pasteurella multocida dermonecrotoxin on cartilage and bone of the nasal ventral conchae of the piglet. Anat Rec 1990; 228: 237-246.
11. CHUNG WB, BACSTROM LR, CONRAD T, COLLINS MT. A comparison of different challenge methods for induction of atrophic rhinitis in pigs. APMIS 1990; 98: 442-542. 12. NIELSEN NC, RIISING HJ, BILLE N. Experimental reproduction of atrophic rhinitis in pigs reared to slaughter weight. Proc 4th Int Pig Vet Soc Congr 1976: P1. 13. ELLING F, PEDERSEN KB. The pathogenesis of persistent turbinate atrophy induced by toxigenic Pasteurella multocida in pigs. Vet Pathol 1985; 22: 469-474.
379
