Atrophic Rhinitis:Appraisal of Infection Pressure on GnotobioticPiglets Infected with Bordetella bronchiseptica

G.P. Martineau, A. Broes and B. Martineau-Doize*

ABSTRACT

With the use of 16 gnotobioticpiglets inoculated at day 4,5 and6 of life with defference concen-trations (3.104 to 3.109 colonyforming units of Bordetellabronchiseptica per mL), it waspossible to establish a minimalinfective dose (3.105 CFU/mL).With a lower dose, it was not pos-sible to induce any of the typicalgross lesions of atrophic rhinitis.The authors discuss some factorswhich can modify the infectionpressure in the field.

R:SUMnCette Etude portait sur 16 por-

celets gnotox(niques que lesauteurs inoculArent dis l'&ge dequatre, cinq et six jours, avec desdoses de Bordetella bronchisep-tica qui variaient de 3,104 A3,109bact6ries/mL. Ils d6termin6rentainsi la dose minimale efficacede cette souche, laquelle sesituait A 3,105 bact6ries/mL; endeck de cette concentration, ils'av6ra impossible de provoquerles lesions macroscopiques ca-ract6ristiques de la rhinite atro-phique. Les auteurs commen-tent aussi certains facteurs sus-ceptibles de modifier la pressiond'infection, dans les conditionsde la pratique.

INTRODUCTION

Atrophic rhinitis can be consi-dered a major economic swine dis-ease (1, 2). Its etiology has still notbeen fully elucidated and the dis-ease should be regarded as aninfectious disease (3), with Borde-tella bronchiseptica and Pasteu-rella multocida as the major causa-tive agents.Bordetella bronchiseptica is con-

sidered to be the principal etiologi-cal agent. Experimental repro-duction is as easily accomplishedin gnotobiotic, specific-pathogen-free pigs as it is in conventionalpigs (4), provided intranasal inocu-lation is performed in animals lessthan three to four weeks old.As a result of studies by Dirks,

Schoss, De Jong and ourselves (5-9), P. multocida may be considereda possible etiological agent.Lactating sows and especially

six to ten week old piglets aremainly responsible for the trans-mission of the disease (10). Theeradication can only be achievedby stamping-out and restockingwith specific-pathogen-free ani-mals (10), or with pigs withoutsuspicion of atrophic rhinitis (11).The control of atrophic rhinitisrequires various measures, mainlyhigh quality management andhousing conditions (12), but alsochemotherapy (13-17) and/or vac-cination (18-22) can lead to clinicalimprovement or even to cure. Themain purpose for all these mea-

sures is to decrease the infectionpressure on the piglets.The objective of the present

work is to demonstrate the notionof infection pressure with B. bron-chiseptica on gnotobiotic piglets.

MATERIALSAND METHODSANIMALS

Nineteen germ-free piglets wereobtained by closed hysterotomyand reared in plastic isolators (23).The control methods of the germ-free status and ventilation insidethe isolators was previously de-scribed (23, 24).INOCULUM

Our strain 73B2 is the B. bron-chiseptica 6060 of the Czechos-lovak Collection of Microorga-nisms (25), already used in otherstudies on atrophic rhinitis (17,26). The lyophilized culture wasthawed and incubated for 48 hoursat 37°C in broth brain heart infu-sion (BHI) broth, Oxoid CM 225;afterwards, colonies grown wereisolated on BHI agar (Oxoid CM375). A typical S phase colony (27)was transferred into BHI brothand incubated for 48 hours at 37°Cand then onto a Roux flask withBHI agar incubated likewise for48 hours at 37°C. Bacteria werecollected under laminar flow in 10mL of sterile saline. The suspen-sion of bacteria was filtered after-wards to avoid the presence of pos-

*Laboratory of Pathology, Faculty of Veterinary Medicine, Rue des Vdterinaires, 45, 1070 Brussels, Belgium. Present address of seniorauthor: Department of Medicine, Faculty of Veterinary Medicine, C.P. 5000, Saint-Hyacinthe, Qu6bec, Canada J2S 7C6.This study was supported by a grant from the "Institut pour l'Encouragement de la Recherche dans l'Industrie et l'AgricultureI.R.S.I.A.", section for "Recherches sur l'Alimentation et les Maladies du Porc, C.S.V.V.Z.", Brussels, Belgium.Submitted January 12, 1982.

Can. J. comp. Med. 46: 376-381 (October 1982)376

sible medium debris. The opacityof the suspension was first roughlyadjusted to the opacity of Brown'stube No. 3, and then more accu-rately measured with a photome-ter (Lambda = 540). The dilutionwas adjusted at a 53% transmit-tance, corresponding to 3.109 Col-ony Forming Units of bacteria permL (CFU/mL). After a tenfolddilution, the suspensions weresealed in 5 mL vials containing3.109, 3.108, 3.107, 3.106, 3.105 and3.104 CFU/mL respectively. Con-trol vials were kept in the isolatoruntil the third day of inoculation,and then tested for viability andconcentration.

INOCULATION

Sixteen germ-free piglets (TableI) were infected with 0.5 mL of thebacterial suspension per nostril atthe fourth, fifth and sixth days oflife. Piglets Al and Bi receivedonly one instillation because of theonset of respiratory distressaccompanied by hyperthermiaand prostration after 24 hours. Weused only one piglet in group A.However, in another report on theexperimental reproduction ofatrophic rhinitis on gnotobioticpiglets (26), 17 piglets were inocu-lated in the same conditions, withthe same strain and at a dose cor-responding to our highest concen-tration. A 5 mm long plastic tubemounted on a syringe was used forinstillation. The suspension wasgently injected into the nostrils ofthe unanesthetized piglets.NECROPSY

The animals were necropsiedbetween 27 to 37 days after inocu-lation, as indicated in Table I. Theywere tranquilized with azaperonel(2 mg/kg IM), removed from theisolator and anesthetized withmethomidate2 (4 mg/kg, IV). Acatheter was placed into thecarotid artery and the piglets werebled aseptically. A blood samplewas taken for subsequent serologi-cal examination. A cross section ofthe snout was carried out in frontof the nasal angle of the eyes, and it

TABLE I. Experimental Protocol for Gnotobiotic Pigs Inoculated Intranasally withBordetella bronchiseptica

NecropsyInoculum Days Post-

Group Isolator Piglets (CFU/mLr inoculation AgeA 1 Al 3.109 31 35B 2 B1/B2 3.108 32/31 36/35C 3 Cl/C2/C3 3.107 27/30/31 31/34/35D 4 Dl/D2 3.106 29/29 33/33E 5 El/E2 3.105 27/27 31/31F 6 Fl/F2/F3 3.104 29/29/35 33/33/39

7 F4/F5/F6 35/36/36 39/40/40G 8 G1/G2/G3 saline 30/31/33 34/35/37aColony forming unit of Bordetella bronchiseptica per mL

was then decalcified according to amethod already described (28).EXAMINATIONS

A clinical examination was per-formed every day and with partic-ular attention to the onset, dura-tion and intensity of the clinicalsigns, especially those of acute rhi-nitis (sneezing, sniffling, etc.).Before making the cross section ofthe snout, the distance between theupper and lower central incisorswas measured according to themethod described by Done (29),and Bercovich and De Jong (30).The extent of brachygnathia wasexpressed as being negative (-) ifthe lower central incisor was cau-dal to the upper one, and positive(+) and quantified (in mm) in thissituation. Turbinate atrophy wasdetermined by gross examinationof a cross section of the decalcifiedsnout at the level of the first premo-lar tooth, and the lesion grade wasevaluated according to the classifi-cation of the "Institut Techniquedu Porc" (Paris, France) (31). Theventral turbinates were separatedfrom the nasal bones after fixation,and were thereafter embedded inparaffin. Three 6 Aum sections werecut at the level of the first premolartooth, and stained with hematox-ylin and eosin. Sterile swabs forbacteriological examination wereintroduced into the nasal cavitiesjust before bleeding, and the eth-moid turbinates were swabbedafter the snout was cross sectioned.Other bacteriological sampleswere taken from the high and low

trachea. Each swab was firststreaked onto a modified McCon-key agar (32), and then transferredto BHI broth and incubated for 48hours at 37°C. If the first isolationwas negative, a reisolation wasperformed from this BHI broth onMcConkey agar. The colonies wereidentified as previously reported(32). The sera were stored at -20°Cuntil examination. Seroagglutina-tion was performed following themodified Kang method (33).Agglutination titers were ex-pressed as the highest dilution ofserum which provoked a totalagglutination after 48 hours at370C.

RESULTS

CLINICAL EXAMINATION

The results are to be found inTable II. Piglets Al and Bi, respec-tively inoculated with 3.109 and3.108 CFU/mL, showed dyspnea,prostration, and fever 24 hourspostinoculation. These signs per-sisted for four to five days. Theanimals ofgroupA and B, infectedwith the larger doses of bacteria(3.109 and 3.108 CFU/mL) weresneezing and repeatedly coughingfor one week postinfection. At theend of the experiment, these ani-mals no longer exhibited any clini-cal signs, except for a diminishedgrowth rate, especially piglet Al.No clinical signs appeared in theanimals of group E and F infectedwith the two lower concentrations

'Stresnil®, Janssen Pharmaceutica, Belgium.2Hypnodil@, Janssen Pharmaceutica, Belgium.

377

3.v 1 0O 3.10 3.1O.;

Fig. 1. Cross sections of the snout at the level of the first premolar teeth of thegnotobiotic piglets inoculated with Bordetella bronchiseptica at different concen-trations (T6moin = Control).

(3.104 and 3.105 CFU/mL). In theother piglets (group C and D),sneezing appeared about one weekafter inoculation and disappearedcompletely by the 25th day. Thecontrol animals (group G) did notshow any clinical signs.Two piglets only (Al and C1) had

a slight superior brachygnathia(Table II).

PATHOLOGY

There was a distinct atrophy of

the turbinates in the animals ofgroups A, B, C, and D. The inten-sity of the atrophy is reported inTable II and illustrated in Fig. 1.Piglets ofgroup E, inoculated with3.105 CFU/mL exhibited a slightmodification of the outline of thedorsal scroll of the ventral turbi-nate. In the six piglets of group F,inoculated with the lower concen-tration, no macroscopic differenceof the turbinate as compared to thecontrol animals (group G) wasnoted.

TABLE II. Results of Clinical Examination, Measurements of Superior Brachygna-thia, Gross Lesions, Bacteriological Findings and Serological Examination of Gnoto-biotic Pigs Inoculated Intranasally with Bordetella bronchiseptica

BacteriologicalBrachy- Gross Findingsc Serological

Group Piglets Symptoms8 gnathia Lesionsb NC ETH TRA TitersA Al D.C.S. +1 mm +++ + + + 1/640B Bi D.C.S. - ++ + + + 1/1,280

B2 C.S. - ++ + + + 1/640C Cl S +2 mm ++ + + + NDd

C2 S - + + + + 1/640C3 - - + + + + 1/1,280

D Dl S - + + + + 1/320D2 - - + + + + 1/320El - - ? + + + 1/160

E E2 - - ? + + + 1/320F Fl toF6 - - - - - - -G Gl toG3 - - - - - - -'= Dyspnea, C = Coughing, S = SneezingbClassification of the Institut Technique du Porc, Paris, France (31)CNC = Nasal cavities, ETH = Ethmoid, TRA = TracheadNot done

HISTOPATHOLOGY

Table II summarizes the differ-ent lesions.

Epithelial lesions - Twelvepiglets had no epithelial lesions.Piglets Al and Dl showed ahyperplasia of the nasal epithe-lium, especially obvious in one ofthem (piglet Al). Squamous meta-plasia was present in two animals,one of which was piglet F6 inocu-lated with 3.104 CFU/mL. Thelesions were restricted to a verysmall area of the epithelium of theventral turbinate.

Submucosal lesions - They werepresent in only three piglets. Inpiglet B2, a characteristic acuteinflammation was present. PigletAl had a hyperplasia of the vascu-lar walls, while piglet Bi showed aheavy infiltration of round nucle-ated inflammatory cells.

Bone lesions - Characteristiclesions were observed. The threepiglets infected with the highestconcentrations demonstrated asubtotal cartilaginous metaplasiaof the turbinate bones. In piglet C3,two foci of metaplasia were foundin the turbinates. In the areas ofintense metaplasia, there was alack of osteoblasts. Piglets ofgroups C, D and E exhibited rela-tively fewer calcified areas in theventral turbinate bones as com-pared to the periostic tissue. Theseosteoporosis lesions are all themore important as the infectionpressure increases. The relativedecrease of the calcified areas inthe ventral turbinate bones of thepiglets ofgroups D, E, F and G waspreviously estimated by planime-try (34). In the piglets of group F,the surface of the calcified areaswas significantly less than in thecontrol piglets. No inflammatorycells were identified in the bonelesions in any of the piglets.Bacteriological samples col-

lected before inoculation were allnegative. The bacteriologicalresults are summarized in TableII. The animals of groups A, B, C,D, and E had B. bronchiseptica inthe nasal cavities, ethmoid and

trachea. In the piglets of group F,B. bronchiseptica was not reco-vered from the same site. In oneisolator where the piglets had beeninoculated with 3.104 CFU/mL, B.bronchiseptica was found in themanure. Bacteriological examina-tion of the supernumerary controlvials in the isolators comfirmed theviability and the concentration ofthe inoculum.

SEROLOGY

Sera of animals belonging togroups other than F and G, whichwere inoculated with high ormedium concentrations of B. bron-chiseptica, showed a seroconver-sion against it. Agglunitationtiters vary from 1/1,280 to 1/160(Table II). On the other hand, theagglutinin titer remained nega-tive, at the threshold of 1/10, in thesera of the piglets from group Fwhich received the lower concen-tration of B. bronchiseptica (3.104CFU/mL), as well as in the sera ofthe control piglets.

DISCUSSION

Despite the obvious lesions of thenasal turbinates, the clinical signswere only minimal in comparison

with those observed in piglets withnatural atrophic rhinitis. This canbe attributed to our experimentalconditions (gnotobiotic colostrum-deprived animals, optimal biocli-matic environment). Indeed, someauthors have emphasized theimportance of the secondary bac-teria (35) and housing (12) in theseverity of the disease. With thetwo lowest concentrations, no clin-ical signs were noted, whereaswith the highest concentrationsthe intensity of the clinical signswere paralleled by the concentra-tion of the inoculum . There were infact more obvious clinical signs inthe piglets of groups A and Bwhich were inoculated with thetwo highest concentrations of B.bronchiseptica. Like Brassine andhis coworkers (26), we were able todemonstrate that, in spite of severeatrophy of the nasal turbinates, nofacial distorsion of the piglets wasobserved, although they wereinfected with the highest concen-tration of B. bronchiseptica.Measurement of the cranio-

caudal distance between the lowerand the upper central incisor teethdoes not appear to be in relationwith the turbinate atrophy. This isin agreement with the opinion ofBercovich and De Jong (30) who

noted that superior brachygnathiawas only related to the atrophy ofthe nasal turbinates after eightweeks of age.

In the piglets of groups A and B,there was a very important andpredominant atrophy of the ven-tral nasal turbinate, particularlyevident 30 days postinoculation. In1976, Brassine and his coworkers(26) noticed that such atrophy wasrarely observed in the naturallyinfected piglets of the same age.These results are consistent withthe gnotobiotic status of our ani-mals and may be related to the lackof any specific or nonspecific colo-stral passive immunity. Our gno-tobiotic animals were inoculatedwith massive doses directly intothe nasal cavities during theperiod of maximal receptivity. Inthe field, conventional piglets aresubmitted to a continuous expo-sure to aerosol infection duringseveral weeks.The gradation of the lesions is

narrowly linked to the concentra-tion of the inoculum. In our exper-imental conditions, the minimuminfective dose was 3.105 CFU/mL.The gradation of the histological

lesions of the osseous tissue whichare characteristic of the disease,was parallel to the concentration of

TABLE III. Gross and Microscopic Lesions of Gnotobiotic Pigs Inoculated Intranasally with Bordetella bronchi8eptica

Gross HistopathologyGroup Piglets Lesionsa Epithelial Lesions Scorec Submucosal Lesions Score Osseous Lesions Score

A Al +++ Hyperplasia +++ Hyperplasia of +++ Cartilaginous +++Vascular Walls Metaplasia

B Bi ++ Nb N Cartilaginous +++B2 +++ Squamous ++ Micro-abcess + Metaplasia +++

MetaplasiaC Cl ++ N N Osteoporosis +++

C2 + N Infiltration of Osteoporosis +++Round Nucleated H+Inflammatory Cells

C3 + N N Idem and ++Cartilaginous +Metaplasia

D Dl + Hyperplasia N Osteoporosis +D2 + N N Osteoporosis +

E El/E2 ? N N Osteoporosis +F Flto F5 N N Osteoporosis +

F6 - SquamousMetaplasia N Osteoporosis +

G Gl to G3 - N N N

aClassification of the Institut Technique du Porc, Paris, France (31)bN = NormalcScore +++ = modifications which can be observed in all the turbinate

++ = modifications which can be observed in more than half the turbinate+ = modifications which can be observed in any focus of the turbinate

379

the inoculum. In piglets Al, Biand B2, we observed a subtotalcartilaginous metaplasia of theventral turbinate bones. In thepiglets of group F, the surface ofthe calcified areas was smallerthan in the control piglets. Thus,lower concentrations ofB. bronchi-septica have been able to producemicroscopic osteoporosis of theventral nasal turbinates. However,it was not possible to detect thebacteria 29 to 36 days after inocu-lation in the six piglets of group F.A possible explanation is the exist-ence of a natural clearing mecha-nism of B. bronchiseptica from thenasal cavities before the rhinopa-thogenic effect of bacteria can bemacroscopically induced. How-ever, in conventional piglets inocu-lated under similar conditions atthe same age, it is possible to dem-onstrate the presence of this bacte-ria in the nasal cavities, ethmoidand trachea (unpublished data).This difference can be request inthe environmental conditionsand/or in the gnotobiotic status.Although the agglutination

titers were not proportional to theinfective dose, it was possible todistinguish three groups of ani-mals. The piglets of the first grouphad titers varying from 1/640 to1/1,280, corresponding to thepiglets infected with the threehighest concentrations (3.109,3.108, and 3.107 CFU/mL). Thepiglets inoculated with 3.106 and3.105 CFU/mL represent thesecond group, in which titers varyfrom 1/160 to 1/320. In the lastgroup, including piglets inocu-lated with the lower concentration,it was not possible to demonstratethe presence of anti-B. bronchisep-tica agglutinin.Our results confirm those of

other workers on the well-definedrole of B. bronchiseptica as an etio-logical factor of atrophic rhinitisusing gnotobiotic piglets.Our model is a theorical ap-

proach designed to quantify theinfection pressure. However, in thefield, it is very difficult to quantifyit. Indeed, some factors arise withthe infection pressure. They aresummarized in Table IV.Nevertheless, these results

380

TABLE IV. Factors Influencing theMinimal Infectious Dose of Bordetellabronchiseptica in Conventional PigletsBefore Three Weeks of Age

Factors1 - Related to the animals:

Natural resistance (heredity).Acquired resistance (passive bycolostrum, and active byvaccination).

2 - Related to the environment andmanagement:Temperature, moisture, venti-lation,Number and density of pigs,All-in all-out system,Antibacterial treatment,Air dust

3 - Related to the etiological agent:Rhinopathogenicity,Synergy between microorganisms,

quantify the notion of infectionpressure and demonstrated theexistence of a minimal infectivedose which was, in our experimen-tal model with gnotobiotic piglets,3.105CFU/mL ofB. bronchisepticaper day on the fourth, fifth andsixth days of life.Therefore, all factors likely to

decrease the infection pressure canlead to clinical improvement of thedisease.

ACKNOWLEDGMENTS

We are grateful to Miss LucieDewaele, Miss Michele Piersonand Mr. Michel Renard for theirskilled technical assistance and toSuzanne Tanguay for the type-scripting of the manuscript.

REFERENCES

1. MARTINEAU GP, BROES A,MARTINEAU-DOIZE B. Inter6ts etlimites de l'assainissement des elevagesdans l'economie de la production por-cine. Ann Med Vet 1982; (In press)

2. PENNY RHC. Priorities for pigresearch. Vet Rec 1976; 99: 451.

3. DONE JT. Atrophic rhinitis and bor-detellosis. Vet Rec 1981; 109: 23.

4. MARTINEAU GP, DEWAELE A,JOSSE M. Interets et limites du diag-nostic serologique de la rhinite atro-phique. Journees Rech Porcine enFrance 1979; 387-400.

5. DIRKS, C. Untersuchungen zur Atiol-ogie der rhinitis atrophicans der

Schweines. Inaugural DissertationHannover 1972; p. 93.

6. DIRKS C, SCHOSS P, SCHIM-MELPFENNIG H. Aetiology of atro-phic rhinitis of swine. Dtsch TierztlWochenschr 1973; 80:342-345,380-382.

7. SCHOSS P. Bakteriologische Unter-tersuchung von Nasentupferproben auRhinitis atrophicans er krankter undunverdachtiger Schweines. DtschTierztl Wochenschr 1971; 13: 371-374.

8. SCHOSS P, OBERWALDER V.Atrophic rhinitis, further bacteriologi-cal studies. Proc Int Pig Vet Soc Zagreb1978; M10.

9. DE JONG MF, OEI HL, TETEN-BURG GH. AR-Pathogenicity tests forPasteurella multocida isolates. Proc IntPig Vet Soc Copenhagen 1980; IX: 211.

10. SWITZER WP, FARRINGTON DO.Infectious atrophic rhinitis. In: DunneHW, Leman AD, eds. Diseases of swine.Ames, Iowa: Iowa State UniversityPress, 1975; 34: 687-711.

11. SCHOSS P, KAYSER G, JAHNKEF, FEDKE P, DAHNS M, DITT-MAR HJ. Rhinitis atrophicans derSchweine: Ergebnisse der Bestandssa-nierungen im Weser-Ems-Gebiet seit1971. Dtsch Tierztl Wochenschr 1980;87: 425-428.

12. DE JONG MF, BARTELSE A. Theinfluence of management and housingon the isolation frequency of Bordetellabronchiseptica and Pasteurella multo-cida in piglet population. Proc Int PigVet Soc Copenhagen 1980; IX:212.

13. CASTRYCK F, MARTINEAU GP,DEWAELE A. Bestrijding van Atro-fische rhinitis bij het varken door sys-tematische voorbehoedende behadel-ing. VI Diergeneeskd Tijdschr 1979; 5:373-386.

14. DEJONG MF, OOSTERWOUD RA,WEEDA JT. Het gebruik van Oxyte-tracycline HC1 neusspray bij biggenter bestrijding en preventie van atro-fische rhinitis. Tijdschr Diergeneeskd1980; 105:519-525.

15. GILES CJ, SMITH IM, BASKER-VILLE AJ, OLIPHANT J. Chemio-therapy with a sulfadiazine-trimetho-prim in experimental Bordetellabronchiseptica infection in young pigs.Proceed Int Pig Vet Soc Copenhagen1980; IX:206.

16. GILES CJ, SMITH IM, BASKER-VILLE AJ, OLIPHANT J. Treat-ment of experimental infection inyoung pig with potentiated sulphonam-ide in the drinking water. Vet Rec 1981;2:136-139.

17. MARTINEAU GP, BROES A,MARTINEAU-DOIZE B. Rhiniteatrophique a Bordetella bronchisepticachez des porcelets gnotobiotes, essai detraitement. Ann MLd Vet 1982; 126:123-131.

18. GOIS M, SISAK F, KUKSA F. Con-trol of porcine atrophic rhinitis by vac-cination and sanitary precautions. ProcWld Vet Cong Moscow 1979; 6:42.

19. KELLER Von H, LORETZ H. Feld-versuch zur Bekampfund der Rhinitis

Atrophicans durch Vakzination mitBordetella bronchiseptica SchweizArch Tierheilkd 1980; 122: 541-552.

20. SCHOSS P. Atrophic rhinitis: fieldtest of aBordetella-Pasteurella vaccine.Proc Int Pig Vet Soc Copenhagen 1980;IX: 204.

21. SCHULLER W, TRUBRICH H,KOSZTOLICH 0, FLATSCHER J,JAHN J. Vaccination againstatrophicrhinitis in swine with a combined Bor-detella bronchiseptica, Pasteurella mul-tocida vaccine. Zentralbl Veteri-naermed (B) 1980; 27: 125-132.

22. WISECARVER JL, GOODNOWRA. Efficacy of an intranasal live Bor-detella bronchiseptica vaccine in con-trolling swine atrophic rhinitis. ProcInt Pig Vet Soc Copenhagen 1980;IX:205.

23. MARTINEAU GP, DE COSTER R,D'IETEREN G, BROES A. Produc-tion et 6levage de porcelets axeniques.Sci Tech Anim Lab 1982; (In press)

24. MARTINEAU GP, RENARD M,HAUCHART JL. Quelques aspectspratiques de la ventilation et de son con-trole dans les isolateurs en plastic sou-

ple pour animaux gnotobiotes. AnnMed Vet 1982. (In press).

25. CATALOGUE OF CULTURES. 3rded. Czechoslovak Collection of Micro-organisms. Brno Czechoslovakia 1978;18.

26. BRASSINE M, DEWAELE A,GOUFFAUX M. Intranasal infectionwith Bordetella bronchispetica in gno-tobiotic piglets. Res Vet Sci 1976;20:162-166.

27. MARTINEAU GP, DEWAELE A.Sensibilite de Bordetella bronchisepticaa differents antibiotiques et sulfam-ides. Premiere partie: les antibiotiques.Ann Med Vet 1978; 122: 607-612.

28. EVERS-BRUAUX C. Une techniquede decalcification du nez du pore: miseau point et standardisation. InstitutPaul Lambin Universite Catholique deLouvain 1980; p. 11.

29. DONE JT. Atrophic rhinitis: use ofcontrol charts for herd monitoring.Proc Int Pig Vet Soc Hannover 1972;48.

30. BERCOVICH Z, DE JONG MF.Shortening of the upper jaw (brachyg-nathia superior) as a clinical feature of

atrophic rhinitis in approximatelyeight-week-old piglets. Tijdschr Dier-geneeskd 1976; 101: 1011-1022.

31. INSTITUT TECHNIQUE DUPORC. Classement des coupes de nezde porc. ITP, Paris France 1972.

32. FARRINGTON DO, SWITZER WP.Evaluation of nasal culturing proce-dures for the control of atrophic rhinitiscaused by Bordetella bronchiseptica. JAm Vet Med Ass 1977; 170:34-36.

33. MARTINEAU GP, BROES A,DEWAELE A. Bordetellose porcine:resultats d'une enquete serologique.Ann Med V6t 1981; 125: 293-301.

34. MARTINEAU-DOIZE B, MARTI-NEAU GP, DEWAELE A. Aspectshistomorphom6triques de la rhiniteatrophique: rapport pr4liminaire. ProcEur Vet Path Soc Ghent 1980. Medelvan de Fac Diergeneesk Rijksuniv Gent1980; 3-4: 44-45.

35. MARTINEAU GP, BROES A,KAECKENBEECK A. Rble de Pas-teurella multocida dans l'etiologie de larhinite atrophique; int6rOts de ladatermination de sa structure anti-g6nique. Ann M6d Vet 1982; (In press).

381