Pneumonia in Calves Produced with Aerosols ofPasteurella multocida Alone and in

Combination with Bovine Herpesvirus 1

K.W.F. Jericho and G.R. Carter*

ABSTRACT

Pathological changes in respiratorytracts were studied in 30 calves follow-ing exposure to aerosols ofPasteurellamultocida or to bovine herpesvirus 1and P. multocida. Two groups of fivecalves were exposed to aerosols of oneof two types of P. multocida only,which produced lobar pneumonia inone calf of each group. Another fivegroups of four calves were exposed toaerosols of bovine herpesvirus 1 andfour to seven days later to one of thetwo types or one sub-type of P. mul-tocida. Extensive necropurulentlesions were produced throughout therespiratory tract with each type of P.multocida in all four calves in threegroups but none in the remaining twogroups. The pathological changes dif-fered from those produced followingsimilar exposures to bovine herpesvi-rus 1 and P. haemolytica, in that theexudate in air passages and alveoli wasmore purulent and streaming (oat)cells and large mononuclear eosino-philic granulocytes were absent. Thisis the first report of experimental res-piratory disease in cattle as a result ofaerosol exposure to P. multocidaalone or in combination with bovineherpesvirus 1.

Key words: Calves, experimentalpneumonia, aerosols, Pasteurella mul-tocida, bovine herpesvirus 1.

RESUME

Cette experience consistait a etudierles lesions des voies respiratoires, chez30 veaux prealablement soumis a desaerosols de Pasteurella multocida,seule ou melangee a l'herpesvirus

bovin #1. Deux groupes de veaux quien comptaient chacun cinq, subirentdes aerosols de seulement un des deuxtypes de P. multocida; il s'ensuivit unepneumonie lobaire, chez un veau dechacun des deux groupes. Cinq autresgroupes de quatre veaux chacun su-birent des aerosols de l'herpesvirusbovin #1 et, quatre a sept jours apres,de l'un des deux types ou d'un sous-type de P. multocida. Chacun des deuxtypes precites produisit des lesionsnecrotiques et purulentes extensives detout le systeme respiratoire, chez tousles veaux de trois des groupes, maischez aucun de ceux des deux autres.Les lesions differaient de celles dejaprovoquees par des aerosols del'herpesvirus bovin #1 et de P. haemo-lytica, en ce que l'exsudat des conduitsaeriens et des alveoles etait plus puru-lent, mais depourvu de macrophagesen forme de grains d'avoine et de grosgranulocytes eosinophiliques mono-nucleaires. Le present article corres-pond au premier rapport d'une mala-die respiratoire bovine experimentaleconsecutive 'a des aerosols de P. mul-tocida, seule ou melangee 'a l'herpesvi-rus bovin #1.

Mots cles: veaux, pneumonie experi-mentale, aerosols, Pasteurella multoci-da, herpesvirus bovin # 1.

INTRODUCTION

Pasteurella multocida is common incattle (1-5). In one survey P. multocidawas isolated from 61% of deep nasalswabs from 790 calves in 12 herds (4).The prevalence of Pasteurella spp. inindividuals and herds is variable andeither P. multocida or P. haemolytica(5) or both are detected (6). A long-

term study in a group of six calvesrevealed constant nasal colonizationby P. multocida (4). Mixing and ship-ment of feeder cattle produced a signif-icant increase in the number of cattlewith serum complement-fixing (CF)antibody titers to P. multocida (3).

Isolation of P. multocida frompneumonic tissue has been reported incalves (3,7,8), however the pathogenic-ity of P. multocida for the respiratorytract of calves has not been demon-strated experimentally. Pneumoniahas been recorded in a few calveswhich had been cold stressed, natu-rally infected with mucosal disease,and experimentally infected with aero-sols of myxovirus SF-4, and P. mul-tocida (7). Clinical signs of respiratorydisease only have been reported incalves experimentally exposed toparainfluenza-3 (PI-3) virus in aerosoland inoculated intratracheally with P.multocida (9,10). It was thereforethought worthwhile to expose calvesto aerosols of P. multocida or to aero-sols of bovine herpesvirus 1 (BHVI)and P. multocida to determine thepathogenicity of P. multocida in therespiratory tract of calves. Extensivepathological changes produced byaerosols of this virus and P. haemolyt-ica have been reported previously (I 1).

MATERIALS AND METHODS

ANIMALS

Thirty, three to eight month old beefcalves were used in seven groups offour or five calves each (Table I).Calves ofgroups A and B were derivedfrom one calf-crop and calves ofgroups F and G from another calf-crop of a semiclosed herd raised ongrassland as described previously (12).Calves in groups C,D and E of another

Can J Comp Med 1985; 49: 138-144.

*Agriculture Canada, Animal Diseases Research Institute, P.O. Box 640, Lethbridge, Alberta, Canada TIJ 3Z4 (Jericho) and Division of Pathobiologyand Public Practice, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia, U.S.A. 24061 (Carter).Submitted August 30, 1984.

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TABLE I. Experimental Design, Calves and Viral and Bacterial Suspensions Aerosolized

Cultures aerosolized

Calves Dayaof P. multocida

Age Bacterial BHV1Group Husbandry No. (months) Weaning Exposure Slaughter TCID50/ mL Cells/ mL TypeA Grassland 4 3½Y2 0 5 9 2 x 108 7 x 1010 A:3,4B Grassland 4 3½Y2 0 7 11 2 x108 9 x 109 A:3,4C Intensive 4 5 -9 4 8 l07 3 x 1010 A:3D Intensive 4 6 -30 5 8 108.5 7 x 108 A:12E Intensive 4 8 -51 4 7 1o9.6 3 x 108 A:3,4F Grassland 5 7 -8 0 4 - 6 x 107 A:3G Grassland 5 7 -8 0 4 - 3.8 x 108 A:12

aDay 0 = Day of exposure to BHVI in groups A-E and to P. multocida in groups F and G

calf-crop were raised intensively inoutdoor pens (39 x 42 m). During theexperiments groups A and B were keptin an indoor pen 4 x 2.75 x 2.5 m,while the other groups were kept inoutdoor pens, 80 M2. All pens hadconcrete floors. Calves were fed alfalfahay and given water ad lib.

EXPERIMENTAL DESIGN

Calves of groups A-E (n = 20) wereexposed to an aerosol of BHVI (iso-late 108) (13) on day 0 and on days 4, 5or 7 to an aerosol of P. multocida.Groups F and G calves (n = 10) werenot exposed to BHV I but only, on day0, to one of two types of P. multocidaaerosol. The experimental designdetails are given in Table I. Rectaltemperatures were taken daily.The experimental conditions used

have previously produced disease incalves inoculated in a similar mannerwith BHV1 and P. haemolytica (bio-type A, serotype 1) (11,14). Conse-quently, the following variablesamong groups were accepted: inten-sive versus grassland management; agerange; interval from weaning to day 0;indoor versus outdoor environment;four to seven days between viral andbacterial exposure and concentrationof viral and bacterial cultures used foraerosolization.

No-treatment control calves andcontrol calves exposed to BHV 1 only,were not included in these studies forthe following reasons: 1) neither clini-cal manifestations nor pathologicalchanges of natural respiratory diseasehave been observed in about 1000calves from this herd over a 12-yearperiod. 2) The changes produced fol-lowing aerosol exposure to BHV 1 onlyin the same type of calf have been des-cribed (13). The clinically normal sta-

tus of the calves on day 0, the natureand age of lesions produced and themicrobiological findings facilitatedthe definition of the pathologicalresponse produced by the aerosols.

AEROSOL EXPOSURE

Each aerosol exposure lasted 5 min.Each calf of groups A-E was exposedto 850 L of viral aerosol (1 1) using amodified Henderson apparatus (15).The P. multocida exposures were doneby letting the Collison nebulizer of theabove apparatus discharge directlyinto the face mask (I 1). The nebulizerdelivered 1.6 ± 0.3 mL in 5 min, thevariation being dependent on theambient temperature. The concentra-tions of the cultures aerosolized aregiven in Table I. The aerosols withinthe face mask were sampled by all-glass impingers (Ace Glass, Vineland,New Jersey) in 0.1% gelatine saline tomeasure the concentration of P. mul-tocidal L of aerosol presented to eachcalf of groups A and B.The three bacterial types used were

isolates from separate field outbreaksof respiratory disease in cattle. TypeA:3 is isolate 1062 (Carter), type A:3,4was isolated by the Regional Veteri-nary Laboratory, Lethbridge, Albertaand type A: 12 was isolated at thisInstitute.

VIROLOGICAL STUDIES

Viral cultures were prepared andstored as described previously (13).Aliquots of one culture were used ingroups A and B and of another culturein groups C-E. Bovine herpesvirus 1isolation was attempted on trachealand lung tissues of calves in groupsC-G and nasal swabs in groups F andG (13). Sera were collected from calvesin all groups on day 0 and at slaughter

and tested for serum neutralizing (SN)antibodies to BHV1 (13). In groups Fand G sera of day 0 were also tested forantibodies to bovine virus diarrhea[BVD, Standard Departmental Pro-tocol (Animal Diseases ResearchInstitute, Nepean, Ontario)], PI-3virus (16) and bovine respiratory syn-cytial virus (RSV) (17).

BACTERIOLOGICAL STUDIES

The preparation of cultures and theisolation procedures for Pasteurellaspp. and Mycoplasma spp. (excludingMycoplasma dispar) from tissue havebeen described previously (11,12).Aliquots of one culture were used ingroups A and B and of different cul-tures in each of the other groups.In groups F and G the method forisolating mycoplasma was modified byreplacing inactivated horse serum withfresh horse serum and by using puri-fied agar. Mycoplasma studies werepart of a routine mycoplasma surveyof cattle at this laboratory. Pasteurellaand mycoplasma isolations wereattempted on deep nasal swabs of day0 from groups A, B, of swabs takenbefore bacterial exposure in groups Fand G as well as deep nasal swabs ofthe day of slaughter, and tracheal andlung tissue of all calves. Pasteurellaisolation was also attempted fromswabs of pharyngeal tonsil taken atslaughter in groups F and G. Sera werecollected from calves in all groups onday 0 and at slaughter and tested forCF antibodies to P. haemolytica andP. multocida. The capsular typing ofP. multocida isolates was done byindirect hemagglutination (18) andsomatic typing by the gel diffusionprecipitin test (19).

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PATHOLOGICAL STUDIES

The postmortem procedures, exam-ination of the respiratory tract (12)and the estimation of percent ofpneumonic tissue in each lung weredone as described previously (20).Lung tissues for histological examina-tion were immersed in 10% bufferedformal saline, trimmed and embeddedin paraffin and sections (5 .tm) cut andstained with hematoxylin-oesin(H&E).

RESULTS

TEMPERATURE RESPONSES

The ranges of the daily means ofminimum-maximum ambient temper-atures for groups C, D and E were11-15, 5-15 and 9-13°C respectively.Daily rectal temperature readingsrevealed that all calves of groups A-Ehad elevated rectal temperature indic-ative of BHVI infection (13). In thesegroups the temperature range was 39.5to 41.7° C on the day of bacterial expo-sure, except in group B in which thebacterial exposure was carried out onday 7 by which time rectal tempera-tures had returned to normal. Twocalves of each of groups F and G withP. multocida exposure only, hadabove normal temperatures (39.5-40.20C) on day 3 after the bacterialaerosol. Three of these had macro-scopic lesions of pneumonia.

VIROLOGICAL FINDINGS

All calves were without SN antibo-dies to BHV1 on day 0. At slaughterseven calves (three in group A and fourin group B) had BHV I SN titers whichranged from 1:4 to 1:32. In groups Fand G five calves had BVD serumantibody (1:27-1:729), but none hadantibody to PI-3 virus or RSV. Ingroups C, D and E, BHV I was isolatedfrom 10/ 12 tracheas and 1I / 12 lungs,but no isolates were obtained fromtissues of calves in groups F and G.

BACTERIOLOGICAL FINDINGS

Nasal swabs taken before bacterialexposure were negative for Pasteurellaspp. The average number of P. multo-cida/ L of aerosol in the face maskpresented to calves of groups A and Bwas 6 x 104. In groups exposed toBHV1 (A-E) P. multocida was isolated

on the day of slaughter from 19/20nasal swabs, 17/20 tracheas and 13/20lungs (seven negative in groups A andB). In groups F and G P. multocidawas isolated from all nasal swabstaken on the day of slaughter, all pha-ryngeal tonsils, 5/ 10 tracheas and 5/10lungs. Pasteurella haemolytica wasnot isolated from calves in any exper-imental group. Serum CF antibodiesto P. multocida were detected only in3/10 calves of groups F and G on theday of slaughter (titer of 1:8).Complement-fixing antibodies to P.haemolytica were detected only ingroups F and G in which 8/10 calveshad antibodies before bacterial expo-sure (titer of 1:8-1:16).Mycoplasma were not isolated from

animals in groups A and B. In theremaining calves M. bovirhinis wasisolated on the day of slaughter, from9/22 nasal swabs, 15/22 tracheas(three in group F and two in group G)and 10/22 lungs (one calf in each ofgroups F and G). Ureaplasma was iso-lated from two calves in group C, threecalves in group E, two calves in groupF and one calf in group G.

MACROSCOPIC OBSERVATIONS

Results are summarized in Table II.Changes in the upper respiratory tractwere confined to groups with BHVIexposure. Four palatine sinuses ingroups D and F were filled with muco-purulent exudate. Similar exudatecovered the turbinates of six calves ingroups A and E. Ten calves hadnecrotic-mucopurulent pharyngealtonsillitis and seven tracheas had sim-ilar changes in groups C-E (Fig. 1). Ingroups C and E in particular the majorair passages of the upper respiratorytract were lined by pus. All calves ingroups C, D and E with viral and bac-terial aerosol exposures had purulentlobar pneumonia (Fig. 2). However

calves in groups A and B with expo-sures similar to group E were withoutpneumonia. Bacterial aerosol expo-sure only in groups F and G producedpurulent lobar pneumonia in one calfof each group and four lobular lesionsin a lung of a third calf. Pneumonic

Fig. 1. Extensive necrotic-mucopurulent trachei-tis in group C.

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TABLE II. Macroscopically Recognizable Changes in Tissues of Calves

Number of calves with % of lungpneumonic

Palatine Pharyngeal (No. lobesGroup sinusitis Rhinitis tonsillitis Tracheitis Pneumonia affected)A 0 2 0 0 0/4 0BCDEFG

0 0 0 0 0/4 00 0 4 2 4/4 4(3)40(8)1 0 2 1 4/4 25(8)-50(8)3 4 4 4 4/4 15(8)-55(8)0 0 0 0 2/5 < 1-20(4)0 0 0 0 1/5 20(4)

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tissue ofgroup F had septal edema andpus in bronchi and bronchioli. Twolungs in group D and one each ingroups E, F and G had pleural fibri-nous adhesions. The pleural cavity ofone calf in each group C and E hadexcessive amber-colored fluid.

MICROSCOPIC OBSERVATIONS

The pneumonic lesions in calves ofgroups F and G, with P. multocidaexposure only, were histologically sim-ilar. Lymphoid tissue aggregates inperibronchiolar and peribronchialposition had proliferated. Theremainder of the tissue reaction wasessentially an acute, severe exudativealveolitis. There was focal hemorrhagein alveoli, hyperaemia of alveolarcapillaries, edema of alveoli, lobularseptae and pleura and distention oflymphatics by lymph, polymorphonu-clear (PMNL) and mononuclear leuk-ocytes and alveolar macrophages.These same cell types were also verynumerous in alveoli and, by extension,filled many bronchioli. The PMNL'swere the most numerous cell type.There was fibrin in exudate of alveoli,septae and pleura. Necrosis of cellswas not seen at this stage of thereaction.

In experiments A-E changes typicalof BHV1 infection (13) were seen.Necrosis of alveoli and respiratoryepithelium was most prevalent andsevere in experiments D and E.Changes attributable to viral-

bacterial synergism were seen only inexperiments C, D and E. Ulceratedepithelium of pharyngeal tonsil (Fig.3), trachea and bronchi was associatedwith purulent exudate. Such changeswere rare in walls of bronchioli (Fig.4). The epithelium of bronchi andbronchioli was frequently infiltratedby PMNL's, which were also plentifulin exudate of air passages (Fig. 5).Frequently, the exudate in bronchioliwas continuous with that in alveoli(Fig. 5). Alveolitis of variable severitywas seen with septal necrosis (attribu-table to BHVI), vascular congestion,hemorrhage (particularly in threelungs of group E), edema, fibrin andinfiltration of PMNL, mononuclearcells and alveolar macrophages (Fig.6). These cell types were usually in thatorder of frequency, except in tissue ofgroup C and one lung in group E inwhich PMNL were less prominent.

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The ratio of the fluid to cellular com-ponents of the alveolar reactions wasvariable among lungs. Three lungs ofgroup C, for example, had a particu-larly cell-rich alveolar exudate with lit-tle fluid while three lungs of group Ehad much congestion, hemorrhageand edema. Alveolar exudate (Fig. 7),was inspissated, contained mononu-clear cells and had cells lining its sur-face in groups D and E and in one lung

of group C. Such organized exudatehas been described for lungs infectedwith BHV1 for eight days (13).

Lymphatics were distended by exu-date, particularly in interlobular sep-tae (Fig. 7) and pleura. However, thisdistension was not always propor-tional to the amount of fluid in alveoli,suggesting that there was either insuf-ficient time for lymphatics to becomefilled, or clearance of alveolar fluid via

141

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Fig. 6. Alveolitis in group E. Necrosis of septae of alveoli on the right, hemorrhlinfiltration of septae is attributed to BHV1. Extensive PMNL infiltration of alveoli 4probably due to P. multocida. Note absence of streaming (oat) cells. H & E. X180.

lymphatics was inefficient.Lungs with much exudate had inter-

lobular septae thickened by edema,PMNL, mononuclear cells and, occa-sionally, red blood cells. Lymph, fibrinand inflammatory cells also distendedseptal lymphatics. These changes alsooccurred in the pleura but exudationand inflammation at the pleural sur-face was rare.

DISCUSSION

The objective of this stuestablish whether P. multoor in combination with BYproduce pneumonia. The rathe experiments was based oity of calves available and thdescription of disease prcexposure of calves to BHVI

experimental viral disease is character-ized by lobular nonpurulent pneumo-nia, with necrosis of alveoli and respi-ratory epithelium.Exposure of calves in groups F and

;*!;4 j G to aerosol of P. multocida only pro-duced pneumonia in three calves. Thefactors which made these calves sus-ceptible are not known. They did notrelate to serum antibodies to BVD, norto isolations of mycoplasma or urea-plasma. Pasteurella multocida was iso-lated from nose, tonsil and lung of all

monia.Exposure of calves to viral and bac-

terial aerosol did not produce necro-purulent pneumonia in all groups.Groups A, B and E were exposed toBHV1 and P. multocida type A:3,4aerosol and yet necropurulent lesions

Thealveolar in the upper and lower respiratorynormal walls tracts were produced only in group E.

Although isolation of BHV1 was notattempted from tissue of groups A andB, BHVI infection was shown to haveproduced the increases in temperatureand viral histopathological changesusually observed following such expo-sure (13). The same methods of expo-sure to virus in groups C-E led to sim-ilar temperature and tissue responsesand isolations of BHVI from all buttwo tracheas and one lung. Failure toproduce necropurulent pneumonia ingroups A and B is not likely due to lowbacterial dose. The concentrations ofbacterial aerosol in the face mask,were similar to concentrations of P.haemolytica aerosols in groups withBHV I -P. haemolytica pneumonia (I 1)and the cultures used in groups A andB were at least as concentrated as cul-tures used in groups C-E with bacterialdisease. Similarly the one to three daylonger interval between aerosol expo-

age and cell sures in groups A and B as comparedon the left is with groups C, D and E is not likely to

have prevented extensive bacterialinfection in groups A and B (21).Alternatively, the different pathologi-cal responses among the groups maybe explained by the nature of the bac-

dy was to terial or viral cultures used or the sus-cida alone ceptibility of calves, none of which{Vl could could be defined. Possibly the use oftionale for freshly grown, log-phase bacterial cul-n the qual- tures would have produced more uni-e previous form results among the groups.)duced by Susceptibility of calves in groups C,t(13). This D and E, to P. multocida infection

142

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Fig. 7. Alveolitis in group E. Alveoli contain organized exudate attributed to viral changes andnumerous PMNL macrophages and mononuclear cells. Septal lymphatic on the left is distended bylymph and inflammatory cells. H & E. X180.

could not be related to either grasslandor intensive husbandry or mycoplas-ma/ureaplasma status (M. bovirhiniswere found in 6/ 7, M. bovoculi in 7/7and ureaplasmas in 3 / 7 calves withoutpneumonia).The macroscopic pathological

changes described herein were similarto those produced by the same virusand P. haemolytica ( 11). Althoughpneumonia was extensive in manylungs of calves in groups C, D and E,pleural fibrinous exudation andaccumulation of amber-colored fluidin the pleural cavities were less fre-quent and less extensive than in exper-iments with P. haemolytica. Pleuralfibrinous exudate was also associatedwith pneumonic tissue in calves ofgroups F and G exposed to P. multoc-ida only. Macroscopically, the majordifference between experiments withP. haemolytica (I 1) and those reportedherein was the extensive purulent exu-date in major air passages and upperrespiratory tracts of groups C and E.

Microscopically the characteristicfeature was extensive involvement ofPMNL and mononuclear leukocytes.It is not known whether the paucity offluid exudate in lungs of calves ingroup C was related to the type of P.multocida (A:3) used or due to lesssevere reaction produced by BHV1.

This study revealed an infrequentinflammation of walls of bronchioliwhich has also been observed with P.haemolytica (22), however, streaming(oat) cells and large mononucleareosinophilic granulocytes describedfor alveolitis with P. haemolytica (22)were not seen.

It has been shown that P. multocidaaerosol exposure of calves alone or incombination with BHV1 can producemacroscopic respiratory disease. Thelesions produced by the virus and bac-terium were more extensive and severethan with the bacterium alone. In bothcases the degree of reaction was prob-ably influenced by the nature of theinocula and susceptibility of the exper-imental calves.

ACKNOWLEDGMENTS

The authors acknowledge the pro-fessional assistance of Dr L.A. Babiuk(Veterinary Microbiology, WesternCollege of Veterinary Medicine, Uni-versity of Saskatchewan, Saskatoon,Canada), the technical assistance ofMs. R. Endo, Ms. B. Jones, Mrs. S.Smithson, Mr. D. Carpenter, Mr. J.Burchak and Mr. G. Tiffin (AnimalDiseases Research Institute, Leth-bridge), and the production of photo-

micrographs by Mr. J. Kolpak andMr. K. Sinclair (Agriculture CanadaResearch Station, Lethbridge). Wethank Dr. K.R. Rhoades, NationalAnimal Diseases Laboratory, Ames,Iowa, USA for typing the bacterialcultures.

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