Chronic inﬂammatory lung lesions were studied in 58 purpose bred, clinically healthy rabbits that were found negative for Bordetella branchiseptica, Pasleurel/a multocida, Toxoplasma gondii, Encephalitozoon cuniculi, and lung worms. The changes found consisted of focal chronic interstitial pneumonia and chronic bronchitis and bronchiolitis, which were frequently associated with perivascular infiltrations of mononuclear leukocytes and vasculitis involving small lung vessels. The etiology of these alterations remains unknown

Feinstein, Ricardo E.

Rehbinder, Claes

Uzal, Francisco A.

English

Journals from University of Tartu

Scand J. Lab. Anim. Sci. No. 2. 1989 . Vol. 16Chronic inflammatory lung lesions in rabbits free ofknown respiratory pathogensby Ricardo E. Feinstein”, Francisco A. UzaF, and Claes Rehbinder‘.The National Veterinary Institute, S-750 07 Uppsala, Sweden‘. The SIDA International Postgraduate Course inVeterinary Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden, and The NationalInstitute of Agricultural Technology (INTA), Bariloche, Argentina? *: Correspondence author.INTRODUCTIONChronic inflammatory lung lesions were fre—quently observed during a health monitoringstudy of purpose bred laboratory rabbits (Fein-stein & Rehbina’er, 1988). In that investigationmany of the rabbits were infected with B.bron-chiseptica (Uzal et a1, 1989), but pulmonarychanges were also noticed in numerous rabbitsthat were free of known respiratory pathogens.The aim of the present study was to provide adescription of lung lesions found in 58 rabbitsthat were free of agents commonly associatedto respiratory disease, such as Berdetella bron-chiseptica, Pasteurella spp., Klebsiella spp,Toxoplasma gondii, and lung worms. The etio-logy of the lung lesions here described remainsunknown.MA TERIAL AND METHODSAnimals:The material examined was obtained in a pre—vious investigation (Feinstein & Rehbinder,1988). Lung tissues were collected from 58 pur-pose bred, laboratory rabbits, of both sexes, ofvarying age (young and adult), and with bodyweights ranging from 0.7 till 3.0 kg. Most ofthe rabbits were of the New Zealand whitebreed, born and maintained in 8 colonies undervarious conventional conditions, but 9 of therabbits were of the NZW/Swiss Hare breedand originated from a colony provided withstrict barriers. Information concerning stablesand sanitary conditions at the rabbitries is pre-sented elsewhere (Feinstein & Rehbinder,1988). The rabbits were clinically healthy andthey were shown to be free from Bordetellabronchiseptica, Pasteurella spp., Klebsiellaspp., and from other bacterial pathogens. Therabbits were also found negative for Toxoplas-ma gondii, Encephalitozoon cuniculi and lung74worms. The bacteriological, parasitological,and virological procedures were conducted f01-lowing the STANDARD OPERATIONALPROCEDURES of The National VeterinaryInstitute, Uppsala, where diseases of laborato-ry animals are regularly diagnosed as a part ofhealth monitoring and diagnostic activities.Procedures:The rabbits were sacrificed with 0.5 ml/kgFluanison — Fentanil (Hypnorm, Jansen Phar—maceutica), i.m., followed by 60 mg/kg pento-barbitone (Mebumal, ACO Lakemedel), intra-cardially. A complete necropsy was immediate—ly performed and samples were obtained for di-verse studies, such as bacteriology,parasitology, virology and histology, as descri-bed elsewhere (Feinstein & Rehbinder, 1988).In order to minimize the occurrence of autoly—tic changes the samples for histology were col-lected and fixed as rapidly as possible. Frag—ments of lung tissues, measuring approximate—ly 1 x 1,5 X 0,5 cm, were randomly obtained, asmacroscopieal changes were not noticed, andfixed by immersion in 10% buffered neutralformalin for 24 hs. Following routine proces-sing and embedding in paraffin wax, 4 mmthick sections were cut and stained with hema-toxylin & eosin. Selected sections were alsostained with Giemsa, Gram, PAS and van Gie—son.RESULTSThe fixation procedures used did not lead to ar—tifacts, such as lung collapse. Each and everyone of the rabbits examined exhibited chronicinflammatory lung changes. Fiftyseven rabbits(98.2%) displayed lesions diagnosed as focalchronic interstitial pneumonia (FCIP). Usual-ly, FCIP appeared as a variable number of dis— Fig. 1: Focal chronic interstitial pneumonia. A zone(P) displaying mixed leukocytic infiltration with pre-dominance of lymphoid cells. Giemsa, X40.  'u T.‘ ‘7,v :2 — .. .Fig. 2: Vascular changes. Marked perivascular infil—tration of, mainly, lymphoid cells (thick arrows).Notice thickened vascular wall due to vasculitis inone of the vessels (curved arrow). A hollow arrowpoints at a normal vessel. Giemsa, x 100.crete, irregularly shaped areas that varied mar—kedly in size, reaching a maximal diameter ofapproximately 500 um (Fig 1). FCIP exhibiteda patchy distribution, but in some few rabbitsthe lesions were so extensive that most of thelung tissue examined was involved. The chan—ges ranged from thickened alveolar septae toconsolidation, which was frequently associatedwith perivascular infiltrations and also withvasculitis (Fig 2).Occasionally, perivascular infiltration was soScand. J, Lab. Anim. Sci. No. 2. I989 . Vol. l6   ~ i 2 I ,1'}; iA . it * asFig. 3: Chronic bronchiolitis. A zone exhibiting mar-ked leukocytic infiltration at the level of a bronchio-loalveolar junction (curved dark arrow). Notice seve—re perivascular cuffing (curved hollow arrow) andconsolidation (short arrow). Giemsa, X 40. , u ’1‘“ Jw"? ‘ us “3“f - . ‘ '0' en Q.“ 2"w twwm ms:' F. “§§- ;fl 3 Mg»? .                  :95;‘.a-‘ ‘ dug fix, «.wfi‘ ‘31:“ \V‘' - ‘ 4:5 '3 .. .‘- e: “99322:?22“ “W: «a}, c .“w oa '7 a g .,.. sw" ‘ q“ a g ‘3 Ifi" a is .w ,. .2.» . a.. 2 3.. .f‘ ta; ti 5 t a .2222': ‘ \ “'9‘3 3- ;,~ :3“:;\lye‘a'h' a “ A f -"‘ t». 9‘- 9‘11; ‘ 1.ml! , Pr, ‘o tax! M3,: 5“.. W ' v» isXF“ :1, “WB‘K‘ '.- 4 :' 43A2 3“ “K a‘3 $‘f ’° RA?”’ l ‘T .I a'.‘ 3. IiHg. 4: Focal chronic interstitial pneumonia dis-playing a thickened alveolar septum due to, mainly,infiltration of lymphoid cells (straight arrow), alveolifilled with large macrophages (a), and perivascularcuffing arround an arteriole (curved arrow). Giemsa,X 100.severe that adjacent tissues were markedlycompressed. Perivascular infiltrates consistedmainly of lymphocytes and a low number ofplasma cells (Fig 2). In pneumonic areas thecomposition of the exudate varied between dif-ferent zones of one and the same section. Cer-tain areas displayed a mixed leukocytic infiltra-tion, with macrophages, monocytes, and mo-nonuclear cells (Fig 4), whereas other zones ex-hibited a more homogeneous infiltrate in whicheither macrophages and monocytes, or mono-75nuclears, were the dominant feature (Fig 1).The number of granulocytes was low. Alveolarmacrophages were conspicuous and not un-commonly they fused and formed large syncy-tial multinucleated cells. However, foreign bo-dies, such as plant tissues, or features of an or-ganized granuloma were not observed. Nume—rous rabbits exhibited hyperplasia of cellsresembling type II pneumocytes (epithelializa—tion). Severe epithelialization produced occlus-sion of alveoli. In the lesions mitotic figuresconstituted a usual finding. Alveolar septaefrequently displayed a moderate proliferationof fibrous tissues and deposition of a fibrilarsubstance that was stained metachromaticallywith Giemsa. Numerous rabbits also exhibiteda mild chronic inflammation of the airways,most commonly confined at the level of thebronchiolo-alveolar junction (Fig 3). Thesechanges were characterized by infiltration ofmacrophages and mononuclears, which werelocated both around and in the lumen of theairways. Sections stained with Giemsa, Gramand PAS did not reveal micro-organisms.DISCUSSIONPulmonary lesions constitue a most frequentproblem in rabbits (Feinstein & Rehbinder,1988). We have observed chronic inflammato-ry lung changes in clinically healthy rabbitsthat were free of known respiratory pathogens.Strawbridge (1960), in a study of spontaneouspulmonary emphysema of rabbits observed su-bacute or chronic interstitial pneumonia in 84per cent of cases. In the study of Strawbridge(1960) Gram or Giemsa stained sections did notreveal microorganisms and bacteriological stu—dies were not carried out. In rabbits, pulmona-ry disease is usually associated with Pasteurellamultocida, Bordetella bronchiseptica, and lessfrequently with other agents, such as Klebsiel—lae, Staphylococci, E. coli, Salmonella, and Li—steria (Oldenburg et al, 1972, Flatt, 1974, Be—mis, 1986, Lebas et a1, 1986, Uzal et a1, 1989).None of these organisms was isolated from thelungs included in the present study. Moreover,the lungs were negative for other bacteria, aswell as for mycoplasma spp. Nevertheless, theScand. J. Lab. Anim‘ Scii No. 2 1 1989 ‘ Vol. [6possibility remains that if bacterial organismswere present, albeit in exeedingly low numbers,they could have escaped detection by the cultu-ral procedures used. In addition, if bacteriawere irregularly distributed in the lung tissues,it is also conceivable that some of the culturalsamples obtained derived from areas that didnot contain microorganisms. In order to exa-mine for this possibility, further studies are inprogress. The present rabbits were tested andfound negative also for antibodies against theprotozoa Toxoplasma gondii and Encephalito-zoon cuniculi, and lesions attributable to theseparasites were not noticed. Finally, parasitolo-gical and histopathological examinations didnot reveal lung worms. Ostler (1961), conside-red that in enzootic pneumonia of rabbits bac-terial isolations could represent a secondary in-fection following a primary Virus or a proto-zoan disease, and that in certain adult rabbits,the lesions suggested a >>proliferative virus-typepneumonia<<. Viruses are frequently associatedwith respiratory disease. However, presentlyonly one virus has been linked to the genesis ofnaturally occurring pneumonia in rabbits. Re-cently, a picornavirus—associated hemorragicpneumonia has been described (Liu et a1, 1984)but the clinical signs and the pathological chan-ges characteristic of that disease are quite diffe-rent from our findings. In other species, reovi-ruses induce a variety of lesions in several or-gans, including lungs (Scott et a1, 1972, Tyler &Fields, 1986). Reovirus antibodies have beenfound in rabbits (Rosen, 1962) but we are notaware of any report on changes attributable toreoviruses in this species. In young dogsThompson et al (1970) with reovirus producedalterations such as the presence of intra-alveo-lar syncytial giant cells and macrophages withvacuolated cytoplasm, and perivascular infil-trations of lymphoid cells, which resembled so-me of our observations in rabbits. Rabbits ex—perimentally inoculated with reoviruses, howe-ver, remained clinically healthy and did notdisplay lung changes (Lou et al, 1963). In otherspecies, adenoviruses are frequently associatedwith respiratory disturbances, but inclusionbodies, a distinctive feature of adenovirus—in-77duced lesions (Kunstyr et a1, 1984, Dungworth,1985), were not noticed in the present study. Apersistent infection of rabbits with a humanadenovirus type 5 was produced experimental—ly (Reddick & Lefkowitz, 1969, Maré, 1974),but changes attributable to this infection werenot reported. Caliciviruses have been involvedin respiratory disease (Dungworz‘h, 1985), butto our knowledge Caliciviruses have not beenisolated from rabbits. Sendai, a parainfluenza1 virus which is widespread among colonies ofmice, rats, hamsters and guinea pigs has notbeen recognized in rabbits.The complex lesions that we noticed exhibitedcertain similarities with changes observed inrabbits after experimental infection by a chla-mydia organism (Flatt & Dungworth, 1971),but such agents were not noticed in our mate-rial.Pulmonary lesions could be produced also byinhalation of airborne contaminants such asfungal spores, particles from bedding or fod-der, dust mites and their metabolites, and no—xious gases like ammonia (Clarke, 1987).Generally, deposition of particles in lungs givesrise to granulomas and severe proliferation offibrous tissue, which was not the case in thepresently investigated rabbits. In some of thecolonies, long term exposure to ammoniacould have played a certain role, but the bestrabbitries were characterized by high hygienicstandards and good ventilation of the stables,which excluded ammonia. Farmer’s lung andchronic interstitial pneumonia in cattle is origi—nated by exposure to spores of thermophilic ac-tinomycetes (Micropolyspora faerzi and Ther—moactinomyces vulgaris) and moulds (Asperi—gillus 51217.), which elicits an allergic alveolitisand bronchiolitis. Briefly, in cattle the lesionsconsist of bronchial and bronchiolar infiltra—tions of lymphocytes and plasma cells, thicke-ned alveolar septae due to inflammatory infil-tration, presence of granulomas composed ofepithelioid and giant cells, and severe intersti-tial fibrosis (Hogg, 1982, Dungworth, 1985).Although we noticed some of these changes inour material, bronchial lesions and interstitialfibrosis were mild and organized granulomasScand. J, Lab‘ Anim Sci, N0. 2. 1989.Vol.16did not occurr. It cannot be excluded, howevver, that, if present, the histological features ofan allergic alveolitis in rabbits may differ fromother species.Chronic inflammatory lung lesions do consti-tute a very important problem of laboratoryrabbits, and diverse pathogens are frequentlyfound in association with these changes. Ho-wever, we have now described pulmonary alte-rations in rabbits free of known respiratory pa—thogens. The changes mentioned do not consti-tute a normal feature of rabbit lungs. It ap—pears unlikely that they were a consequence ofthe continuous stimulation from ubiquitousagents. Rabbits maintained under conventio-nal conditions that were submitted at The Na—tional Veterinary Institute for health monito-ring and diagnosis, at other occasions, did notexhibit lung changes. The etiology of the le-sions found remains unknown. The environ-mental and hygienic conditions at the rabbi-tries were very heterogeneous, ranging fromvery good to bad (Feinstein & Rehbinder,1988), but the lung changes observed in rabbitsfrom the different groups were of a similar na—ture. This could indicate that the same factorproducing the lesions was present at the va-rious colonies. It is also possible that in rabbitsdiverse injuries could elicit a similar morpholo-gical lung response. Further studies are requi-red in order to characterize these changes andto understand their pathogenesis.AknowledgementsWe thank Prof. H. J. Hansen, SIDA-course directorfor his encouragement and support, to the NationalInstitute of Agricultural Technology (INTA), Ar-gentina, for economical support of one of us, and toMrs. S. Westerberg, for secretarial work.SummaryChronic inflammatory lung lesions were studied in58 purpose bred, clinically healthy rabbits that werefound negative for Bordetella branchiseptica, Pa~sleurel/a multocida, Toxoplasma gondii, Encephali-tozoon cuniculi, and lung worms. The changes foundconsisted of focal chronic interstitial pneumonia andchronic bronchitis and bronchiolitis, which were fre—quently associated with perivascular infiltrations ofmononuclear leukocytes and vasculitis involvingsmall lung vessels. The etiology of these alterationsremains unknown.79SammanfatmingKroniska inflammatoriska lungforandringar har un—dersokts hos 58 destinationsuppfodda, kliniskt fri-ska kaniner, fria fran infektion med Bordetella bron-chiseptica, Pasteurella multocida, Toxoplasma gon-dii, Encephalitozoon cuninculi och lungmask. Defunna forandringarna bestod av fokal kronisk inter-stitiell pneumoni och kronisk bronkit och bronkiolit,ofta forbundna med perivaskulara infiltrat av mo-nonukleara leukocyter och vaskulit hos sma lung—karl. Etiologien till dessa forandringar har fOrblivitokand.Yhteenveto / K. PelkonenTass'a tyossa oli tutkittu 58:sta tavoitekasvatetustakliinisesti terveesta kanista kroonisia tulehdusvau-rioita keuhkoista. Yhdesséikéia'n kanissa ei ollutosoitettavissa Bordetella bronchiseptica, Pasteu-rella multocida, Toxoplasma gondii, Encephalito-zoon cuniculi tai keuhkomatoja. Havaittuja muu—toksia olivat seuraavat: pesakkeinen interstitia-alipneumonia seka krooninen bronkiitti ja bron-kioliitti, joihin usein liittyi mononukleaaristenleukosyyttien perivaskulaarinen infiltraatio ja pi-enten keuhkosuonten tulehdus. Naiden muutostenetiologia jai epéiselvaksi.REFERENCESBemis, A. D.: Bordetella. In: >>Pathogenesis of bac-terial infections in animals<<. Eds. C L Gyles andC O Thoen. First ed., Iowa State UniversityPress, Ames, 1986, pp 137-146.Clarke, A. : Air hygiene and equine respiratory disea-se. In Practice, 1987, 9, 196-204.Dungworth, D. L. : The Respiratory system. In: >>Pa-thology of domestic animals<<. Jubb, K. V. F., P.C. Kennedy, N. Palmer. Third ed., AcademicPress, Inc., Orlando, Vol 2, 1985, 413-556.Feinstein R. E. & C. Rehbinder: Health monitoringof purpose bred laboratory rabbits in Sweden:Major findings. Scand. J. Lab. Anim. Sci. 1988,15, 49-67.Flatt, R. E.. Pasteurellosis. In: >>The biology of thelaboratory rabbit<<. Weisbroth S. H., Flatt, R.E., Kraus A. L., eds. Academic Press, NewYork, 1974, 194-205.Flatt, R. E. & D. L. Dungworth.‘ Enzootic pneumo-nia in rabbits: Microbiology and comparisonwith lesions experimentally produced by Pasteu-rella multocida and a Chlamydial organism. Am.J. Vet. Res., 1971, 32, 627637.Scand. J, Lab. Anim, Sci, No. 2. I989 . Vol. 16Hogg, J. C.: The histological appearence of farmer’slung. Chest, 1982,81, 133-134.Kunstyr, I. J. Maess, S. Naumann, F. J. Kaup, V.Kraft & K. W. Knocke: Adenovirus pneumoniain guinea pigs: An experimental reproduction ofthe disease. Lab. Anim., 1984, 18, 55-60.Lebas, F. P. Coudert, R. Rouvier & H. de Rocham-beau: The rabbit. Husbandry, health and pro—duction. FAO Animal Production and HealthSeries, No 21, Rome, 1986, pp 109-148.Liu, S. J., H. P. Xue, B. Q, Pu, & N. H. Quian: Anew viral disease in rabbits. Animal Husbandryand Veterinary medicine (Xumu yu Shouyi),1984, 16, 253-255.Lou, T. Y., H. A. Wenner, & J. Mc Millen: Naturaland experimental infection of dogs with Reovi-rus, type 1: Pathogenicity of the strain for otheranimals. Am. J. Hyg. 1963, 77, 293—304.Maré, C. J.: Viral diseases. In >>The biology of thelaboratory rabbit<<. Eds. S. H. Weisbroth, R. E.Flatt, A. L. Kraus. Academic Press, Inc., NewYork, 1974, pp 237-261.Oldenburg von, J. B. Kohler, H.-W. 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Chronic inﬂammatory lung lesions in rabbits free of known respiratory pathogens

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Chronic inﬂammatory lung lesions in rabbits free of known respiratory pathogens

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