THE ANATOMICAL RECORD 228:237-246 (1990)

Effects of Purified Pasfeurella rnulfocida Dermonecrotoxin on Cartilage and Bone of the Nasal Ventral Conchae of the Piglet BEATRICE MARTINEAU-DOIZk, JOSEPH C. FRANTZ AND GUY-PIERRE MARTINEAU Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte St. Hyacinthe, Quebec, Canada J2S 7C6 (B.M.-D., G.-P.M.) and Norden Laboratories Inc., Lincoln, Nebraska 68501 (J.C.F.)

ABSTRACT The effect of intramuscular injection of purified dermonecrotoxin (DNT) from Pasteurella multocida type D on the nasal ventral conchae of piglets was studied. Severe atrophy of the conchae was observed 4, 6, and 10 days after injection (p.i.d.). Lesions were observed in conchae cartilage and bone. Cartilage changes observed were the absence of chondrocyte maturation and hypertrophy, hyaline cartilage invasion by fibroblast-like and multinucleated cells, and endothelium damage with haemorrhages along the cartilage. Intramembranous bone was absent on p.i.d. 4, 6, and 10. Lamellar bone trabeculae were rarefied on p.i.d. 4 and almost absent on p.i.d. 10. Trabeculae were either normal or had the aspect of a dissolved bone matrix, leaving only irregularly oriented collagen fiber bundles. The number of osteoclasts was increased, especially the subperiosteal osteoclasts at the eccentric side of the scrolls. The osteoblasts appeared normal or their cytoplasm was dilated by vacuoles. It is concluded that the macroscopic conchae atrophy results from histological alterations and subsequent loss of both cartilage and bone. Further investigation is necessary to know whether the toxic effect of DNT on cells and matrix is direct or dependent of the vascular damage. In pigs atrophic rhinitis is a n upper respiratory disease caused by two different aetiologic agents, Bordetella bronchiseptica and Pasteurella multocida. The pathological macroscopic and microscopic changes caused by B . bronchiseptica have been well documented (Duncan et al., 1966; Shimizu et al., 1970). They include atrophy of the nasal ventral conchae bones and shortening or distortion of the snout. Ultrastructural studies (Fetter and Capen, 1971; Fetter et al., 1975; Silveira et al., 1982) and a dynamic study using fluorochrome markers (Trepanier et al., 1988a) suggest that the nasal ventral conchae osteoporosis induced by B. bronchiseptica is due to functional alterations of their osteoblasts. B . bronchiseptica induces moderate and transient atrophic rhinitis, while toxigenic P. multocida is responsible for progressive severe and irreversible lesions (Elling and Pedersen, 1985; Pedersen et al., 1988). The reason for this irreversibility of the conchae lesions is unknown. Purified P. multocida type D dermonecrotoxin (DNT) given experimentally to piglets produces osteoporosis of the nasal conchae by inhibition of osteogenesis and accelerated osteoclastic bone resorption (Dominick and Rimler, 1986, 1988).It also produces systemic effects, including damage to liver and kidney (Rutter and Mackenzie, 1984; Cheville and Rimler, 1989). Using a n immunohistochemical technique, Riischoff et al., (1987) observed the presence of toxin in liver, kidney and lymphatic organs. 0 1990 WILEY-LISS, INC.

The present article is a first step of a long-term goal which is to elucidate the mechanism by which P. multocida dermonecrotoxin provokes progressive and irreversible osteoporosis. This experiment was designed to characterize the lesions induced by DNT in the nasal ventral conchae of the piglet. It was known from a previous study (Trkpanier et al., 198813) that the nasal ventral conchae contain several types of tissues (hyaline cartilage, calcified cartilage, woven bone, and lamellar bone). Thus, in order to be able to observe all these tissues, it was decided to examine serial sections cut through the entire length of the nasal ventral conchae. MATERIALS AND METHODS Animals

Nine piglets born from a conventional sow derived from a breeding farm without any clinical signs of atrophic rhinitis were used. The sow was maintained in isolation from day 95 of gestation and was fed with a commercial gestation diet. At the day of transfer to isolation, nasal swabs were obtained from the nasal cavities in order to test for presence of Bordetella bronchiseptica and Pasteurella multocida by microbiological

Received November 28, 1989; accepted March 16, 1990. Address reprint requests to Dr. B. Martineau-Doize, GREMIP, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, St. Hyacinthe, Quebec, Canada J2S 7C6.

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culture techniques (Leblanc et al., 1986). Sulfamethoxazol(800 mgiday) and trimethoprim (160 mgiday) were added to the sow diet. Toxin Purification

Pasteurella multocida serotype D strain NOR-1, isolated from a pig with severe atrophic rhinitis, was cultured in a semisynthetic medium for 6 h at 37°C (Herriot e t al., 1970). Cells were harvested by centrifugation at 30,OOOg for 60 min and suspended in distilled water to 10% of the original volume. Cells were disrupted by sonication and a sterile lysate was obtained following centrifugation (30,OOOg, 60 min at 4"C), ultracentrifugation, (150,OOOg, 120 min at 4"C), and filtration through a sterile 0.22 nm membrane filter. Ethylenediaminetetraacetic acid (EDTA) was added to the sterile lysate for a final concentration of 5 nM. Proteins in the sterile lysate were isolated and resolved by size exclusion and ion-exchange HPLC. Size exclusion HPLC was performed using a Spherogel TSK 3000SW (21.5 mm x 60 cm) preparative column (Beckman Instruments, Palo Alto, CA) equilibrated with size exclusion buffer (SEB) comprising 50 mM Tris-acetic acid, 100 mM NaC1, and 5 mM EDTA, pH 7.5 a t ambient temperature. Samples of sterile lysate (3 ml) were injected automatically permitting sequential unattended runs. Mobile phase flow rate for size exclusion was 6 ml/min. Pooled fractions (100-200 ml) from size exclusion HPLC were applied to a Spherogel TSK-G IEX DEAE-5PW (21.5 mm x 15 cm) preparative column (Beckman Instruments, Palo Alto, CA). Nonadherent proteins were washed through the ionexchange column with SEB at a flow rate of 5 ml/min. Proteins were eluted by a linear sodium chloride p a dient (from SEB 100 mM NaCl to SEB 750 mM NaC1) which changed a t a rate of 0.83% per min. Elution of proteins from both columns was monitored with a spectrophotometer at 280 nm. Fractions containing DNT were collected from both columns using a "Foxy" fraction collector (ISCO, Lincoln, NE). Toxin-containing peaks were pooled and characterized for purity by SDS polyacrylamide gel electrophoresis (SDS-PAGE) in which proteins were visualized by silver stain (Wray et al., 1981). Protein was estimated by BAC Protein As-

TABLE 1. Experimental design adopted for the iniection and sacrifice of the Diglets Group 1 2

3

Piglet Control Experimental Experimental Control Experimental Experimental Control Experimental ExDerimental

Dermonecrotic toxin' (pg)

Postinjection day (p.i.d.)

-

4

3 6

4

-

3 6 -

3 6

4 6 6 6 10 10 10

'Injected intramuscularly a t day 7 of age.

say Reagent (Pierce Chemical Company, Rockford, IL) using crystallized bovine serum albumin (BSA) as standard. Toxicity

Toxicity of DNT-containing preparations was determined by mouse lethality and dermonecrotic reactions in guinea pigs. For assay in mice, 0.5 ml samples diluted in physiologic saline were injected into 14-16 g female CF-1 mice by the intraperitoneal route. Mice were observed for 7 days following injection and a LD,, was determined by the method of Reed and Muench (1938). Dermonecrotic activity in guinea pigs was determined by injecting 0.1 ml samples diluted in physiologic saline into depilated sites on guinea pigs by the intradermal route. Inoculation sites were observed for 48-72 h for signs of necrosis. A reaction site was scored positive when a zone of necrosis exceeded 5 mm diameter. Experimental Design

At day 7 of age, piglets were injected intramuscularly with 3 or 6 pg of DNT suspended in isotonic saline (20 pg of toxin in 1 ml of saline). Control piglets were injected with the same amount of isotonic saline. They were killed on postinjection day (p.i.d.1 4, 6, and 10 (Table 1).Prior to sacrifice, nasal secretions of the piglets were collected for microbiological culture.

TABLE 2. Summary of the observed histological alterations of the nasal ventral conchae of the experimental piglets

Matrix

Cells Invading cells Vascular alterations Mesenchyme tissue

Endochondral Cartilage bone formation Progressive Progressive disappearance disappearance of hyaline and hypertrophic cartilage Disappearance of hyaline and hypertrophic chondrocytes Multinucleated and Increased number of fibroblast-like cells chondroclasts Haemorrhages, red blood Red blood cells surrounding cells along cartilage chondroclasts Pyknotic cells, leukocytes

Intramembranous woven bone Rapid disappearance

Lamellar bone Progressive disappearance

N'

Osteoblasts with vacuolated cytoplasm

N

Increased number of osteoclasts Red blood cells surrounding osteoclasts Pyknotic cells, leukocytes

N N

'N, not observed since intramembranous woven bone was already almost absent on p.d.i. 4.

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Histology

Piglets were anesthetized with azaperone (Stresnil, Pitman-Moore, Mississauga, Ontario, Canada) and metomidate hydrochloride (Hypnodil, Janssen Pharmaceutica, Montreal, Quebec, Canada). Lactated Ringer’s solution was perfused through the two common carotids for approximately 45 sec. The two superficial jugular veins were opened to allow elimination of blood and perfusion solution. This primary washing was followed immediately by perfusion with 2.5%glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.05% CaCl, (pH 7.3) for 10 min. Immediately after perfusion, the noses were taken and immersed for 4 additional h in the same fixative at 4°C. Finally, the noses were decalcified in 10% disodium EDTA, pH 7.3 for 4 weeks at 4°C. Transverse sections were made, dividing the noses in approximately 10 mm thick blocks. They were washed for 24 h in the same buffer and processed by routine paraffin embedding. Serial 7 pm thick tranverse sections were cut and 3 successive sections were collected every 50 sections. Each section was stained respectively with either Masson’s trichrome (MT), hematoxylin phloxin safron (HPS), or Mallory-Heidenhain and examined microscopically. The nasal ventral conchae were divided, as previously described (Martineau-Doize and Martineau, 1986), into dorsal and ventral scrolls, attached a t the central zone to the transverse lamina, itself joined to the wall of the nasal cavity by the articular lamina. RESULTS Toxin Purity and Activity

M.W. Marker

200K

-b

92.5K

*-p

46K

_$

30K

,-b

21.5K

.-b

Crude

Pure

0

Analysis of toxin purity by SDS-PAGE is shown in Figure 1. A single band visualized by silver stain is interpreted as evidence for a very high level of purity. The LD,, of purified DNT for mice was 36 ng and the minimal amount of toxin required for dermonecrotic activity in guinea pigs was 7 ng.

Fig. 1. SDS polyacrylamide gel of crude (approximately 50 pg) and purified dermonecrotic toxin (approximately 2 pg). Following resolution in a linear 10% cross-linked gel, proteins were visualized by silver stain as described in Materials and Methods.

Histology

lamellar bone, trabeculae of woven bone, and trabeculae of lamellar bone.

The rostral extremity of the nasal ventral conchae of the control piglets contained hyaline cartilage. The most rostral sign of bone formation appeared at the level of the third incisor tooth in the ventral scroll where two ossification mechanisms took place simultaneously a t different sites of the cartilage: endochondral ossification at its concentric side and intramembranous woven bone apposition a t its eccentric side (Fig. 2). The same processes occurred at the level of the canine tooth in the connecting zone. In the rest of the ventral scroll and in the complete dorsal scroll, the process of endochondral ossification was incomplete, since vascular invasion did not occur. The cartilage was almost completely resorbed from its concentric side by chondroclasts, while intramembranous bone apposition at its eccentric side resulted in centrifugally oriented trabeculae of woven bone (Fig. 3). Lamellar bone was observed in the caudal extremity of the conchae, from the level of the first premolar tooth. Thus the nasal ventral conchae of the control piglets contained calcified cartilage, and four different types of trabeculae: trabeculae of both calcified cartilage and woven bone, trabeculae of both calcified cartilage and

Modifications in the cartilage of the experimental nasal ventral conchae were of four categories. They consisted of (1)disappearance of the rostral cartilage, (2) modification in the cartilage composition, (3) presence of cartilage invading cells, and (4)vascular alterations. Disappearance of the rostral cartilage was progressive. On p.i.d. 4, cartilage was absent in the dorsal and ventral scrolls at the level of the third incisor tooth, while on p.i.d. 10 i t was absent up to the level of the canine tooth (Fig. 4A,B). In the control conchae, regressive changes of cartilage hypertrophy occurred in the rostral cartilage at the sites of endochondral ossification. These changes were absent in the experimental conchae on p.i.d. 10. In the latter, cartilage seemed to remain “immature” a s chondrocytes did not hypertrophy, but were small. Their pericellular matrix did not increase and was not metachromatic (Fig. 5). On p.i.d. 4 and 6, cartilage hypertrophy was present, but limited to a small area in the ventral scroll.

Cartilage

Fig. 2. Light micrograph of the ventral scroll of the nasal ventral concha of a control piglet at the level of the canine tooth (MalloryHeidenhain staining method). x 105. The hypertrophied cartilage (hc) is invaded by blood vessels and by chondroclasts (arrows) a t its concentric side, while its eccentric side is the site of intramembranous woven bone apposition (wb).

Fig. 3. Light micrograph of the dorsal scroll of the nasal ventral concha of a control piglet a t the level of the canine tooth (MalloryHeidenhain staining method). X 57. The hypertrophied cartilage (hc) is almost completely resorbed, while intramembranous bone apposition results in centrifugally oriented trabeculae of woven bone (wb).

Fig. 4. Light micrograph of the nasal ventral concha a t the level of the third incisor tooth of a control (A, x 32) and an experimental piglet (B, x 41)(HPS stained). Atrophy of the experimental concha is due to the disappearance of the cartilage in the dorsal scroll (straight arrows) and the reduction of the cartilage size in the rest of the concha (curved arrows).

Fig. 5. Central zone of the nasal ventral concha a t the level of the third incisor tooth from a n experimental piglet (A, x 408), (HPS stained). Cartilage (c) is not hypertrophied, but many rnultinucleated cells (arrows) are located between the perichondrium and the carti-

lage and seem to invade the latter. (B) Enlarged view to show multinucleated cells along and within the cartilage. p, perichondrium; v, vessel; c, cartilage. x 1220.

The third modification concerned the cartilage invading cells. In the control conchae, vascular and chondroclastic invasion occurred only in “mature” cartilage, after hypertrophy. However, the experimental cartilage was invaded by multinucleated cells, although it remained “immature” (Fig. 5A,B). Moreover, a second type of cell seemed to penetrate into this cartilage, fibroblast-like cells arising from the perichondrium (Fig. 6). Finally, vascular alterations were observed in venules from the submucosa surrounding the cartilage. Several haemorrhages were observed, where blood cells and fibrin deposited along the surface of the cartilage (Fig. 7). These vessels contained multinucleated cells which apparently were cartilage invading multinucleated cells. Large eosinophilic formations which contained pyknotic material were also found within these interrupted vessels (Fig. 8). Whether these formations were macrophages that had phagocytized red blood cells could not be determined. Endochondral Ossification

Endochondral ossification was observed in the experimental nasal ventral conchae on p.i.d. 4 and p.i.d. 6, Fig. 6. Light micrograph of the nasal ventral concha cartilage at the level of the third incisor tooth of a n experimental piglet (HPS stained). x 1000. Although not hypertrophied, cartilage (c) is invaded by fibroblast-like cells (arrows) that seem to come from the perichondrium (p).

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Figs. 7 and 8. A portion of a blood vessel adjacent to cartilage (c) of a n experimental piglet (HPS stained). In Fig. 7 ( x 554), the endothelial cell layer is interrupted between the two straight arrows allowing red blood cell to accumulate within the perichondrium. Multinucleated cells are observed within the blood vessel (curved closed arrows). The typical appearance of the perichondrium is modified (*) and

multinucleated cells invade the cartilage (triangle). In Fig. 8 ( X 674), cartilage is in direct contact with red blood cells (bl). Large eosinophilic formations containing pyknotic material are present within the interrupted vessel (open arrow). Triangle: multinucleated cell in contact with cartilage.

but was absent on p.i.d. 10. On p.i.d. 4 and p.i.d. 6, calcified cartilage was invaded by blood vessels and resorbed by chondroclasts. However, unlike the control piglets in which resorption occurred only at the concentric side of the cartilage (Fig. 9A), in the experimental piglets multinucleated cells were observed along the entire surface of the calcified cartilage, at both its concentric and eccentric sides (Fig. 9B). Many osteoclasts were surrounded by red blood cells. Pyknotic cells and leukocytes were observed within the mesenchymal tissue and in the osteoprogenic layer of the periosteum.

the entire conchae on p.i.d. 4 (3 and 6 pg) and p.i.d. 6 (3 pg). On p.i.d. 6 (6 pg) only very few trabeculae were left, while on p.i.d. 10 they were limited to the articular lamina. Besides these rarefied but normal trabeculae, there were trabeculae which contained areas that differed from normal bone tissue. They appeared clearer than normal lamellar bone and they were formed by a thin network of irregularly oriented collagen fiber bundles that continued directly into normal appearing bone tissue. They gave the visual impression of having been partly dissolved (Fig. 10). The majority of osteoblasts had numerous vacuoles in their cytoplasm (Fig. 11).No relationship could be observed between these osteoblasts and the type of trabeculae. Several pyknotic cells, leukocytes, and eosinophilic formations were present between the mesenchyma1 cells and the osteoblasts. Although not quantified, the number of osteoclasts was increased and occurred essentially a t the eccentric side of the scrolls. A constant observation was the presence of many red blood cells surrounding osteoclasts (Fig. 12).

lntramembranous Woven Bone

In the experimental piglets intramembranous woven bone trabeculae were almost absent from the nasal ventral conchae on p.i.d. 4 and 6 and they were totally absent on p.i.d. 10. Lamellar Bone

The amount of trabeculae was decreased in the experimental conchae. They were dispersed throughout

P. MULTOCIDA TOXIN IN BONE AND CARTILAGE

243

Fig. 9. Light micrograph of the nasal ventral concha central zone at the level of the first premolar tooth of a control (A, ~ 9 4 and ) an experimental piglet (B, ~ 2 6 8 ) .(A) HPS stained; (B) Masson's trichrome staining method. In the control piglet invasion of cartilage occurs at its concentric surfaces (straight arrows), while intramem-

branous bone apposition takes place at its eccentric surface (curved arrows). In the experimental piglet, invasion occurs not only at the concentric but also at the eccentric surfaces (straight arrows) and intramembranous bone formation is absent.

DISCUSSION

by endothelial elements, multinucleated cells, and tumor cells (Eisenstein et al., 1973; Kuettner et al., 1976, 1977). This invasion resistance of hyaline cartilage is due to its content in antiinvasion factors (Langer et al., 1976; Horton et al., 1977; Kuettner et al., 1977). Pasteurella DNT induces invasion of hyaline cartilage by multinucleated and fibroblast-like cells. That invasion of uncalcified cartilage by mononucleated cells is possible has been shown in avian embryonic diaphyseal cartilage (Silvestrini et al., 1979; Sorrel1 and Weiss, 1980, 1982). In the latter, cartilage resorption is mediated by two different cells, fibroblastic cells closely associated with macrophages. In the present study, fibroblastic cells do not seem to be associated with macrophages. Moreover, no association can be observed between these fibroblastic cells and the invading multinucleated cells. The presence of hyaline cartilage invading multinucleated and fibroblastic cells a s well a s the increased number of chondroclasts suggest that the disappearance of the hyaline and hypertrophic cartilage is due to their increased resorption. Bone lesions caused by pasteurella DNT in the conchae seem to be the result of a toxic effect on both bone formation and bone resorption. The absence of intramembranous woven bone might have been caused by any or a combination of the following mechanisms: inability of woven bone apposition due to a modified cartilage, absence of osteogenesis due

A study of serial transverse sections cut through the entire nasal ventral conchae demonstrated a macroscopic atrophy resulting from histological modifications and subsequent loss of both cartilage and bone. The histological lesions were similar in both cartilage and bone: alterations in cells and matrix, increased number of invading cells, and vascular injury (Table 2). Whether cell and matrix modifications are dependent of the endothelial lesions or the result of a n independent toxic effect of DNT cannot be known from the present study. A toxic effect of pasteurella DNT on blood vessels has also been observed by Cheville and Rimler (1989). They observed rat liver sinusoidal endothelium degeneration accompanied by intravascular leukocyte accumulation after DNT administration. In the present study, vascular alterations gave rise to red blood cell accumulation a t the surface of cartilage and around chondroclasts and osteoclasts. The significance of this observation is at present unknown. It has been shown that polymorphonuclear leukocytes synthesize and secrete enzymes that can diffuse into cartilage matrix, attack both proteoglycan and collagen, and thereby degrade cartilage matrix (Janoff et al., 1976; Starkey et al., 1977). Normal, healthy hyaline cartilage resists invasion

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Figs. 10-12

P. MULTOCZDA TOXIN IN BONE AND CARTILAGE

245

ACKNOWLEDGMENTS to changes in the osteoblasts, increased osteoclastic resorption, and lysis of the matrix or resorption without The authors wish to thank Jocelyne Boutin for skilosteoclasts. ful technical assistance and Claudette Beaudin for typIn the control nasal ventral conchae, deposition of ing the manuscript. This work was supported by woven bone by intramembranous bone formation is NSERC (Natural Sciences and Engineering Research very rapid and results in a centrifugal growth of the Council of Canada) Grant OGPIN 001, FCAR (Fonds scrolls. However, this occurs mainly in the rostra1 ex- pour la Formation de Chercheurs et 1’Aide a la Rechertremity of the conchae, along the eccentric side of the che) Grants 89-36-3725 and 90-NC-0002, CAFIR cartilage and only after chondrocyte hypertrophy. (Comite #Attribution des Fonds Internes de RecherSince the latter disappears progressively, intramem- che, Universite de Montreal) Grant 82-81, and the Mibranous bone formation might not have been initiated. nistgre des Etudes Superieures de la Science et de la Impaired function of osteoblasts and thus absence of Technologie du Quebec, Programme des Actions Strucosteogenesis or formation of a n abnormal matrix might turantes. have contributed to the decreased amount of bone tissue. However, the presence of many morphologically LITERATURE CITED normal osteoblasts seems to indicate t h a t this is not the N.F., and R.B. Rimler 1989 A protein toxin from Pusteurellu major cause of osteoporosis. This does not agree with Cheville, multocidu type D causes acute and chronic hepatic toxicity in the observation of severe and generalized osteoblast rats. Vet. Pathol., 26:148-157. necrosis observed by Dominick and Rimler (1988)and Dominick, MA., and R.B. Rimler 1986 Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated cannot be explained. However, dose and route of adfrom type D Pusteurellu multocidu. Am. J. Vet. Res., 47t1532ministration of DNT used in the present study were 1536. different. Dominick, M.A., and R.B. Rimler 1988 Turbinate osteoporosis in pigs The increased number of osteoclasts was due mainly following intranasal inoculation of purified Pusteurellu toxin: Histomorphometric and ultrastructural studies. Vet. Pathol., 25; to the abnormal presence of subperiosteal osteoclasts at 17-27. the eccentric side of the scrolls. They had the appear- Duncan, J.R., R.F. Ross, W.P. Switzer, and F.K. Ramsey 1966 Patholance of active resorbing cells. This observation agrees ogy of experimental Bordetellu bronchisepticu infection in swine: with already described increased numbers of preosteoAtrophic rhinitis. Am. J. Vet. Res., 27t457-466. clasts and osteoclasts caused by DNT (Dominick and Eisenstein, R., N. Sorgente, L.W. Soble, A. Miller, and K.E. Kuettner 1973 The resistance of certain tissues to invasion. Am. J . Pathol., Rimler, 1986, 1988; Kimman et al., 1987; Kamp and 73:765-774. Kimman, 1988). Elling, F., and K.B. Pedersen 1985 The pathogenesis of persistent The severity of the induced lesions, namely total disturbinate atrophy induced by toxigenic Pusteurellu multocidu in pigs. Vet. Pathol., 22t469-474. appearance of cartilage and woven bone a s well as the A.W., and C.C. Capen 1971 Ultrastructural evaluation of bone disappearance of most lamellar bone within 10 days Fetter, cells in pigs with experimental turbinate osteoporosis (atrophic postinoculation may explain the irreversibility of atrorhinitis). Lab. Invest., 24:392-403. phy observed by Elling and Pedersen (1985) and Ped- Fetter, A.W., W.P. Switzer, and C.C. Capen 1975 Electron microscopic evaluation of bone cells in pigs with experimentally induced Borersen et al., (1988). The fact that these authors used P. detellu rhinitis (turbinate osteoporosis). Am. J . Vet. Res., 36t15multocida cultures, inoculated by intranasal instilla22. tion, does not prevent this explanation, since i t has Herriott, R.M., E.Y. Meyer, M. Vogt, and M. Modan 1970 Defined been shown that broth cultures, crude DNT and purimedium for growth of Huemophilus influenza. J. Bacteriol., 101: 513-516. fied DNT, instillated intranasally, induce the same J.E., F.H. Wezeman, and K.E. Kuettner 1977 Inhibition of conchae bone loss as purified DNT injected intramus- Horton, bone resorption in uitro by a cartilage derived anticollagenase cularly (Kamp and Kimman, 1988). factor. Science, 199t1342-1345. In conclusion, DNT from P. multocida, type D in- Janoff, A,, G. Feinstein, C.J. Malemud, and J.M. Elias 1976 Degradation of cartilage proteoglycan by human leukocyte granule duces severe atrophy of the nasal ventral conchae. The neutral protease. A model ofjoint injury. I. Penetration of enzyme toxin induces similar histological lesions in bone and into rabbit articular cartilage and release of 35S0,-labeled matecartilage: modification of their matrix and cells, inrial from the tissue. J . Clin. Invest., 57t615-624. creased numbers of invading (resorbing) cells, and vas- Kamp, E.M., and T.G. Kimman 1988 Induction of nasal turbinate atrophy in germ-free pigs, using Pusteurellu multocidu as well as cular injury. Further studies are necessary to know bacterium-free crude and purified dermonecrotic toxin of P. mulwhether the toxin has a direct effect on cells and matocidu. Am. J. Vet. Res., 49:1844-1849. trix or if i t is the vascular damage that accounts for the Kimmann, T.G., C.W.G.M. Lowik, L.J.A. van de Wee-Pals, C.S. Thesbone and cartilage changes. ingh, P. Defize, E.M. Kamp, and O.L.M. Bijvoet 1987 Stimulation

Fig. 10. Trabeculae of an experimental piglet to demonstrate the presence of normal (LB) and abnormal appearing lamellar bone (AB), in which the collagen fibers are visible as if the bone matrix has been partly dissolved. (Masson’s trichrome staining method). x 1240.

Fig. 11. High magnification of osteoblasts from a n experimental piglet showing the presence of numerous vacuoles in their cytoplasm (HPS stained). B, bone; ob, osteoblast. x 2580. Fig. 12. Mallory-Heidenhain stained section from a n experimental piglet to demonstrate the presence of numerous red blood cells (*) around the osteoclasts (arrows). x 1050.

of bone resorption by inflamed nasal mucosa, dermonecrotic toxin-containing medium from Pusteurellu multocidu, and purified dermonecrotic toxin from P. multocidu. Infect. Immun., 55: 2 110-2 116. Kuettner, K.E., J . Hiti, R. Eisenstein, and E. Harper 1976 Collagenase inhibition by cationic proteins derived from cartilage and aorta. Biochem. Biophys. Res. Commun., 72:40-46. Kuettner, K.E., L. Soble, R.L. Croxen, and B. Marczynska 1977 Tumor cell collagenase and its inhibition by a cartilage derived protease inhibitor. Science., 196t653-654. Langer, R., H. Brem, K. Falterman, M. Klein, and J. Folkman 1976 Isolation of a cartilage factor that inhibits tumor neovascularization. Science, 193:70-72. Leblanc, L., M. Denicourt, and G.-P. Martineau 1986 Comparison of isolation methods for the recovery of Bordetellu bronchiseptrcu and Pusteurellu multocidu from the nasal cavities of piglets. Proc. Int. Pig Vet. SOC. Congr., p. 226.

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B. MARTINEAU-DOIZE ET AL.

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Effects of purified Pasteurella multocida dermonecrotoxin on cartilage and bone of the nasal ventral conchae of the piglet.

The effect of intramuscular injection of purified dermonecrotoxin (DNT) from Pasteurella multocida type D on the nasal ventral conchae of piglets was ...
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