Atrophic Rhinitis Caused by Pasteurella multocida Type D: Morphometric Analysis B. Martineau-Doize, G. Dumas, R. Larochelle, J.C. Frantz and G.-P. Martineau
ABSTRACT In order to study the distribution and the extent of atrophy caused by Pasteurella multocida in the nasal conchae, experimental piglets were injected intramuscularly at seven days of age with either two or four 50% mouse lethal doses per kg body weight of P. multocida type D dermonecrotoxin. Experimental and control piglets were killed four, six and ten days postinjection. Serial transverse paraffin embedded sections of the noses were cut throughout the entire length of the nasal conchae. The area of the nasal ventral conchae was measured and the morphometric index of the nasal cavity was calculated. It was observed that P. multocida type D dermonecrotoxin induced severe atrophy of the nasal ventral conchae. This atrophy was present along the entire conchae. However, it was most severe at the level of the first and second premolar teeth.
RESUME Dans le but d'etudier la distribution et l'importance de l'atrophie des cornets nasaux causee par Pasteurella multocida, des porcelets ages de sept jours ont ete injectes par voie intramusculaire avec de la dermonecrotoxine de P. multocida tpe D Ala dose d-e deux ou quatre doses lethales 50 N (souris) par kg de poids vif. Les porcelets experimentaux ainsi que les temoins ont e't sacrifies quatre, six et dix jours apres l'injection. Des coupes seriees transversales ont ete6 realisees a travers toute la longueur des nez enrobes a la paraffine. La surface des cornets nasaux ventraux a ete mesurie et
1'index morphometrique des cavites nasales a ete calculi. 11 a ete observe que la dermonecrotoxine de P. multocida, type D induit une atrophie severe des comets nasaux ventraux. Cette atrophie etait prisente dans tout le cornet. Cependant, elle etait plus sevire au niveau de la premiire et deuxieme
Atrophic rhinitis is an infectious disease characterized by chronic rhinitis resulting in snout deformity and in severe cases, in reduced growth rates. The major pathological change is atrophy of the nasal conchae. Two infectious agents are associated with atrophic rhinitis: Bordetella bronchiseptica and Pasteurella multocida. Although both agents induce conchae osteoporosis, their mechanism of action is different. Bordetella bronchiseptica seems to act by modifying the synthesis and secretion of prebone components by the osteoblasts (1-3), while P. multocida induces a rapid increase in the number of chondroclasts and osteoclasts (4). Lesions in the liver and urinary tract have been induced experimentally with toxigenic P. multocida (5,6). Conventionally, the clinical diagnosis of atrophic rhinitis is confirmed by postmortem examination of a transverse section of the nasal cavity at the level of the first or second premolar tooth (7-11). However, it is known that the nasal ventral conchae (NVC) contain different mineralized tissues, which have a specific arrangement throughout its length (12). Thus, the observation of a single section of the nose at that site does not allow the
examination of all the conchal mineralized tissues, nor the rostral cartilage. The aim of the present study was to observe the distribution and the extent of atrophy throughout the entire length of the NVC of piglets injected with P. multocida type D dermonecrotoxin, in order to establish standardized transverse sections for the diagnosis of P. multocida atrophic rhinitis. MATERIALS AND METHODS ANIMALS
Nine piglets from a conventional sow derived from a minimal disease herd were used. The herd as well as the sow were without any clinical signs of atrophic rhinitis. The sow was maintained in isolation from day 95 of gestation and was fed with a commercial gestation diet. On the day of transfer to isolation, nasal cavities were swabbed and cultured to verify the absence of B. bronchiseptica and P. multocida (13). From day 95 to farrowing, sulfamethoxazol (800 mg/day) and trimethoprim (160 mg/day) were added to the sow diet. Piglets were given an iron-dextran injection at the
third day of life. TOXIN
Dermonecrotic toxin (DNT) was
purified from lysates of a toxigenic strain of type D P. multocida as previously described (4). Toxicity of the DNT-containing preparation was determined by mouse lethality and dermonecrotic reactions in guinea pigs. For assay in mice, 0.5 mL samples diluted in physiological saline were
Groupe de Recherche sur les Maladies Infectieuses de Porc (GREMIP), Faculte de Medecine veterinaire, Universite de Montreal, 3200 Sicotte, Saint-Hyacinthe, Quebec J2S 7C6 (Martineau-Doize, Dumas, Larochelle, Martineau) and SmithKline Beecham Animal Health, 601 West Cornhusker Highway, P.O. Box 80809, Lincoln, Nebraska 68501-0809 (Frantz). Supported by FCAR (Fonds pour la Formation de Chercheurs et l'Aide a la Recherche) Grants 89-EQ-3725 and 90-NC-002, Cafir (Universite de Montreal) Grant 82-81 and the Ministere des Etudes Superieures de la Science et de la Technologie du Quebec, Programme des Actions Structurantes. Submitted July 16, 1990.
Can J Vet Res 1991; 55: 224-228
Fig. 1. Transverse sections through the nose of a control piglet at the level of the rostral (A) and caudal (B) extremities of the first premolar tooth, to show the structures from which measurements were taken and the boundary (straight and curved lines) drawn between the dorsal concha (ndc) and the nasal wall. S: free surface; nvc: nasal ventral concha; si: sinus of the dorsal concha.
injected intraperitoneally into 14-16 g female mice. The median lethal dose (LD50) was determined by the method of Reed and Muench (14). Dermonecrotic activity was assayed by intradermal injection of 0.1 mL of twofold serial dilutions in physiological saline into depilated sites of guinea pigs. Inoculation sites were observed for 48 to 72 h for signs of necrosis. A reaction site was scored positive when a zone of necrosis exceeded 5 mm in diameter. EXPERIMENTAL DESIGN
At seven days of age, piglets were injected intramuscularly with two or four mouse LD50/kg body weight DNT suspended in isotonic saline. They were killed on postinjection day (p.i.d.) 4, 6 or 10. Control piglets were
injected with the same amount of isotonic saline. Prior to euthanasia, nasal secretions were collected for microbiological culture. The procedures followed the guidelines of the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. HISTOLOGY
Piglets were anesthetized and perfused with lactated Ringer's solution through the two common carotids for 45 s. The superficial veins were opened to allow elimination of blood and perfusion solution. This primary washing was followed by perfusion with 2.507o glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.05% CaC12 (pH 7.3) for 10 min. The noses were immersed for four additional
hours in the same fixative at 4°C. Finally, the noses were decalcified in 1001o disodium ethylenediaminetetraacetate (EDTA), pH 7.3 for three weeks at 4°C. The noses were cut transversely in 10 mm long blocks, and embedded in paraffin. Serial transverse sections, 7 Am thick, were cut and three successive sections were collected every 350 Am. Each section was stained respectively with either Masson's trichrome, hematoxylin phloxin safron or MalloryHeidenhain. For morphometric analysis, all the collected sections per animal were photographed (Aristophot, Leitz, Canada) at a low magnification to obtain a complete picture of all the transverse sections of the nasal conchae. Sections located at four levels selected at topographic reference levels were used. These levels were the first incisor tooth, the canine tooth, the first and the second premolar teeth. All the sections cut per level were used for analysis. The photographs were placed on a digitizing tablet linked to a microprocessor (Bioquant System IV, R & M Biometrics, Inc., Canada) and the outlines of the nasal structures were traced by a pointer. The computer was programmed to calculate the areas of the structures as well as the morphometric index (MI) according to Done et al (9). The MI being a ratio of free space to total cross-sectional area of the nasal cavity, the parameters measured were the transverse area of the nasal cavity (NS) and the NVC. The area of free space (S) was obtained by subtraction (S = NS-NVC). These measurements were repeated but with the exclusion of the dorsal concha (Fig. IA, B). In this way, two morphometric indexes were calculated, one which included the dorsal concha and one without the dorsal concha. Actual measurements were limited to the right nasal cavity. STATISTICAL ANALYSIS
The mean of the NVC area and the MI were established to calculate their mean per level (mean of 10 to 20 transverse sections). The experimental unit was the nose of the piglet. The effects of treatment and level were analyzed according to a completely random design. Statistical analysis was per225
nounced at the level of the first and second premolar teeth than at the level of the canine and first incisor teeth. However, this difference was not significant (p > 0.05). MORPHOMETRIC INDEXES
In order to measure the transverse area of the nasal cavity without taking the dorsal concha into account, it was necessary to make an arbitrary boundary between the dorsal concha and the nasal wall. At the rostral levels, this boundary was made by drawing a straight line from the angle formed by the dorsal extremity of the nasal septum and the dorsal concha to the angle between the dorsal concha and the lateral wall (Fig. IA). At the more caudal regions, where the dorsal concha includes a large sinus, the line ran from the same angles, but surrounded the sinus (Fig. IB). This division was Fig. 2. Area of the nasal ventral concha according to the level of transverse section, the dose of not accurate for the caudal extremity. injected toxin and the postinoculation time. I1: level of first incisor tooth; C: level of canine tooth; PM1: level of first premolar tooth; PM2: level In the control animals, the MI, calculated for the dorsal concha included, of second premolar tooth. Vertical axis: area of the NVC in mm2. did not exceed 72% (varying from control piglets El 5307o to 720/o). However, the MI was not uniform, since it changed from piglets injected with 2 LD50 animal to animal and also from one transverse level to another (Fig. 3). piglets injected with 4 LDs5 a In the experimental animals, the MI exceeded 75% (varying from 75% to formed using the GLM (General piglets, the area (surface) of the NVC MI of the experimental 93%). The Linear Model) procedure of the SAS varied according to the level of transpiglets was always significantly higher rostral its at Small verse section. system (Statistical Analysis System Institute Inc., Cary, North Caroline). extremity, its area increased by three statistically than the control MI times between the first incisor tooth (p 0.05). more extensive in pigs injected with BACTERIOLOGY DISCUSSION 4 LD50 than in the animals injected When the sow arrived in the isola- with 2 of The severity atrophy LD50. tion room, nasal swabs were negative increased also with the time between present results show that for B. bronchiseptica and P. multo- injection and euthanasia. However, P. The multocida type D dermonecrotoxin cida. Nasal swabs from the piglets were induces and severe nasal ventral rapid were not time also negative for these bacteria at the effects of dose and > conchae atrophy. This is in agreement In significant (p 0.05). statistically necropsy. fact, with 2 LD50 and on p.i.d. 4, with the results described by Dominick NASAL VENTRAL CONCHAE atrophy was already very pronounced, and Rimler (15) and Kamp and MORPHOMETRY the area of the NVC in the experimen- Kimman (16). This atrophy occurs All the experimental piglets had tal piglets was less than half that of the along the entire length of the concha. severe macroscopic atrophy of their controls. Atrophy was present along the However, the rostral extremity (at the NVC, consisting of an extensive reduc- entire length of the NVC, but the level of the first incisor to the canine tion of the conchae and an increase in degree of atrophy was not uniform teeth) is less atrophied than the central the nasal free space. In the control throughout. It was always more pro- and caudal (at the level of the first and
second premolar teeth respectively) regions. This can be explained 1) by the different proportion of soft to hard tissues in the rostral versus the more caudal regions of the concha and 2) the severity of the lesions. Indeed, at the level of the first incisor tooth, the ratio of soft tissues to hyaline cartilage is high, due to the presence of a thick submucosa, while in the more caudal regions the submucosa is thinner (Fig. 4). Histological examination of the sections confirmed the disappearance of both the rostral hyaline cartilage and the more caudal bone (4). The MI of the control piglets was higher than that obtained by others (9,11). This is probably due to the very young age (1 to 2 weeks) of the piglets in this study. At this age, the NVC are not fully developed yet (12,17). Nevertheless, the MI was considerably higher in the injected piglets. This was particularly the case at the level of the first and second premolar teeth. A striking observation was the irregular MI in the control animals. Indeed, the morphometric indices were not only different for the three control piglets, but varied also according to the level of transverse section. This observation has to be taken in account with care, since the number of control piglets was low. However, it confirms the observations on conchae throughout the different experiments, namely that there is a relatively high variation in the size and shape of the nasal ventral conchae of normal piglets (Martineau-Doize, unpublished). This observation is of practical importance, since diagnosis of atrophic rhinitis is done usually by the observation of only one transverse section through the central region of the ventral conchae (7-11). Normal, but small sized ventral conchae may be scored as having mild atrophy. The MI is a good tool for estimating atrophic rhinitis. However, calculation of this index with the dorsal concha included overestimated the ventral conchal atrophy, since the dorsal concha was also atrophied. Moreover, free space was probably also increased, since we observed the presence of numerous osteoclasts along the bone trabeculae of the concentric side of the nasal wall. On the other hand, calculating the MI disregarding the dorsal con-
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Fig. 3. Morphometric index according to the level of transverse section, the dose of injection and the postinoculation time. I1: level of first incisor tooth; C: level of canine tooth; PM1: level of first premolar tooth; PM2: level of second premolar tooth. Vertical axis: morphometric index in N. control piglets E
LD5E piglets injected wih 4 LD5
piglets injected with 2
M M3 ES .........
Fig. 4. Transverse section through the nasal ventral concha of a control piglet at the level of the first incisor tooth (A) and the caudal extremity of the first premolar tooth (B). Rostrally the submucosa (*) is much thicker than the cartilage (c), while caudally the submucosa is thinner than the surrounded bone (b).
cha is not recommended due to the difficulty in delineating precisely the boundary between the dorsal concha
and the nasal wall. The dose of toxin and the time between injection and euthanasia had little influence on the 227
severity of atrophy, at least for the doses used in the present study. Our finding that atrophy was less severe in the rostral extremity of the NVC is different from the results of the study of Martineau et al (18), in which atrophic rhinitis lesions were more severe and more frequent in the rostral part of the ventral conchae. However their route of administration (intranasal instillation) and the inoculum (sterile cell-free filtrate of P. multocida culture) were different. De Jong et al (19) observed that the severity of atrophic rhinitis lesions was correlated with the quantity and frequency of intramuscularly injected cell-free filtrates of P. multocida. Since DNT seems to act systemically, and since we injected purified DNT, blood concentrations of DNT were probably lower in those two studies than in the present experiment. In the present study, the lack of influence of dose of DNT and time between injection and euthanasia on the severity of atrophy is not surprising in view of the histopathological examination of the conchae. Indeed, with 2 LD50 at 4 p.i.d. the nasal ventral conchae had already lost most of their osseous trabeculae as well as their rostral cartilage (4). The present study seems to confirm the validity of using one transverse section of the nose at the level of the first or second premolar tooth for diagnosis of atrophic rhinitis caused by P. multocida at slaughter. However, since our experimental conditions do not really reflect the field conditions (age of animals, dose, inoculation route), we still recommend the use of two sections, one rostral between the incisor and canine teeth and one more caudal, at the level of the first-second premolar teeth.
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