Pneumonia in Calves Produced with Aerosols ofBovine Herpesvirus 1 and Pasteurella haemolytica

K. W. F. Jericho and E. V. Langford*

ABSTRACT

In each of 11 experiments, four calves wereexposed first to an aerosol of bovine herpes-virus 1 (BHV1, virus of infectious bovinerhinotracheitis) and second to an aerosol ofPasteurella haemolytica. The interval betweenaerosols was three to five days. In two otherexperiments, calves were exposed only to abacterial aerosol. Climate was controlled forall experiments from the day of viral exposureand for eight of the experiments it was alsocontrolled for four to six days before the firstaerosol. The concentration of infectious dosesof virus in the aerosols and the number ofbacteria in the aerosols of each calf were de-termined. Macroscopically recognizable rhinitis,tonsillitis, laryngitis, tracheitis and pneumoniaof lobar distribution in 42 lobes from 11 calveswere seen in five experiments in which bac-terial aerosol followed the viral aerosol byat least four days. One calf died with markedrespiratory disease in each of four experimentswithin four days of exposure to the bacterialaerosol. Production of pneumonia was depend-ent on an interval between aerosols of at leastfour days but not on the condition of con-trolled climate on the environmental chambereither before or after the viral aerosol nor onthe period of habituation allowed calves ofsome experiments.

ReSUMA

Cette etude comportait 11 experiences aucours de chacune desquelles quatre veauxsubirent d'abord une vaporisation avec unesuspension du virus de la rhino-tracheite infec-tieuse bovine, ainsi qu'une vaporisation ulte-rieure avec une suspension de Pasteurellahaemolytica. On separa ces vaporisations par

*Animal Pathology Division, Health of Animals Branch.Agriculture Canada, Animal Diseases Research Insti-tute (Western), P. 0. Box 640, Lethbridge, Alberta,Canada T1J 3Z4.

Submitted July 11, 1977.

un intervalle de trois a cinq jours. Au coursde deux autres experiences, des veaux ne su-birent qu'une vaporisation bacterienne. Danstoutes ces experiences, on controla les condi-tions climatiques des chambres experimen-tales, a partir du moment ou on vaporisait lasuspension virale; dans huit de ces expe-riences, on commenga a controler les condi-tions climatiques des chambres experimen-tales, environ quatre a six jours avant lapremiere vaporisation. On determina, pourchacun des veaux, la teneur des aerosols endoses virales infectieuses et en bacteries.Ces experiences provoquerent le developpe-ment de lesions macroscopiques de rhinite,d'amygdalite, de laryngite et de tracheite;elles entrainerent aussi de la pneumonie lo-baire dans 42 des lobes pulmonaires des 11veaux des cinq expe'riences oiu on laissa ecou-ler au moins quatre jours entre la vaporisa-tion de la suspension virale et celle de la sus-pension bacterienne. Au cours de quatre expe-riences, un veau de chacun des quatre groupescorrespondants mourut de troubles respira-toires marques, dans les quatre jours qui sui-virent la vaporisation bacterienne. La produc-tion de pneumonie dependait d'un intervalled'au moins quatre jours entre la vaporisationde la suspension virale et celle de la suspen-sion bacterienne; elle ne semblait cependantpas reliee au controle des conditions climati-ques des chambres experimentales, avant ouapres la vaporisation de la suspension virale,pas plus qu'a la periode d'acclimatation accor-dee aux veaux de certaines des experiences.

INTRODUCTION

The experimental production of lobarpneumonia in calves has been reported pre-viously. Hamdy et al (8) reported the ir-regular production of macroscopically re-cognizable pneumonia using myxovirusparainfluenza 3 (PI-3), PasteureUa multo-

Volume 42 - July, 1978 269

cida and P. haemolytica, pleuropneumonia-like organisms (PPLO) and climatic stress,in various combinations. All calves wereinoculated intratracheally and by aerosol.Similarly, Baldwin et al (1) reported pneu-monic lesions in some calves exposed to P.haemoolytica intratracheally and/or to PI-3aerosol. Macroscopically recognizable pneu-monia was also produced in colostrum-deprived calves given intranasal and intra-tracheal inoculations of the virus of PI-3(5).The distribution and extent of respira-

tory disease in calves exposed to aerosolsof bovine herpesvirus 1 (BHV1) and P.haemolytica have not been described. Onestudy described the clinical disease only(3) and another study (2) included macro-scopically recognizable changes in calvesexposed to intratracheal injections of 2 mlof tissue culture of BHV1 and to aerosol ofP. haemolytica. There is one report of dualinfection of calves by PI-3 and/or BHV1and P. haemolytica when exposure was byaerosols (18). These calves were subjectedto severe climatic stress during ten days ofexperimentation. Without specifying whichanimals were given only BHV1, it was re-ported that 16% of the calves died ofstress alone and 50% died of respiratorydisease (18).The association of stress and respiratory

disease in cattle is generally recognized.However, the definition and measurementof stressors which may contribute to di-sease production are poorly documented.The purpose of our experiments was tostudy the influence of climatic conditionson the effects of BHV1 and P. haemolyticaon respiratory tracts of calves. The reportdescribes both failure and success in theproduction of respiratory disease in groupsof calves raised under similar conditionsof management, maintained under con-trolled climatic conditions during experi-mentation and exposed to aerosols of thevirus and the bacterium at different in-tervals.

previously described (15). Calves were notrestrained in the chamber and were givenalfalfa hay and water ad lib. The fresh airinput was constant at one changeover everyten minutes. For experiments A, B, C, Dand K, temperature and humidity were con-stant in the chamber. The controlled condi-tions were different between the hours of9:00 a.m. to 4:00 p.m. and 4:00 p.m. to9:00 a.m. (Table II) for the other experi-ments.

ANIMALS

All calves were derived from the sameherd and all except those of experiment Hwere raised by the same methods as calvesdescribed by Jericho and Magwood (15).Calves of experiment H were from thesame herd as the others but were raisedon an irrigated and fertilized pasture in-stead of natural prairie grassland. Therange of body weights of calves was 86 to158 kg. Thirty-three males and 15 females,66-158 days of age, were selected for theexperiments. Evaluation of the final datashowed no relationship as to the sex of theanimals, thus, the gender is ignored in thisreport. All but one of the 12 experimentswere done in either one of two consecutivesummer seasons. The mean ambient tem-peratures for July, August and Septemberwere 190, 180 and 13°C, respectively. Ex-periment E was started in January at am-bient temperatures of -25°C.

EXPERIMENTAL DESIGN

Some experiments began with a periodof habituation of the calves to the cham-ber while it was not in operation (TableI). The time of exposure of the animals toBHV1 and to P. haemolytica is also givenin Table I.

AEROSOL EXPOSURE

MATERIALS AND METHODS

ENVIRONMENTAL CHAMBER

The operation of the chamber was as

A modified Henderson apparatus wasused for all of the viral exposures and forbacterial exposures of experiments A andB as previously described (13). The tem-perature and humidity of the lumen of thebrass tube (100 cm x 10 cm) into whichall viral aerosols were expelled were re-corded. The bacterial aerosols of experi-

Can. J. comp. Med.270

TABLE I. Chronological Events and Loss of Weight in kg for all Experimente

Day of Day of Day of Day of Loss InControl Viral Bacterial Killing Weight

Experiment of Climate Aerosol Aerosol of Calves kgABCDEFGHIJKL

555434000303

66545000303

889878435747

131414131314888

11811

552490

39488955164120

TABLE II. Temperature and Relative Humidity During Periods of Habituation and ClimaticControl of Experiments

HabituationControlled

Temperature 'C Humidity %Temperature Humidity

Experiment Max. Min. Mean Max. Min. Mean °C %A 28 22 24.8 69 59 65.4 2 90B 28 20 24.0 72 60 64 2 92C 24 18 22 70 60 66 29 92D 22 11 16 80 60 68 2 72E 13 8 10 78 72 74 28-2' 48-90F 29 24 26 81 75 77 30-2 40-90G . .no habituation .. 30-2 90-70H . _ no habituation .. 30-2 40-90I __._____. no habituation . . 30-2 40-90J 28 14 21 88 68 74 30-2 90-60K . .- no habituation . .29 90L 24 18 21 78 70 72 30-2 90-65

"Day conditions first night conditions last

ments A and B were also passed throughthis brass tube. The relative humidity was65% for experiments B and C and 90%for experiments D and E. The temperaturefor experiment C was 230C and for experi-ments D and E it was 190C. During pas-sage of the bacterial aerosol of experimentB the temperature of the interior of thetube was 19°C. For experiments F to Lrange of temperature of the air in the ex-posure room was 20-25°C and the relativehumidity was 65-70%. This narrow rangeensured a similar rate of respiration ofall calves for the time of aerosol exposure.For experiments C to L the bacterial aero-sols were blown directly into the facemask. Its total volume was 30 liters in-cluding the attached rubberized envelopewhich encircles the neck of the calf. Theaerosol was expelled from the face maskinto the chamber air via an opening (6 cmin diameter) at the side of the mask. Thisopening served as a port for the intake of

air and discharge of exhaled air. All ex-posures to aerosols were of five minutesduration.

AIR SAMPLING

All-glass impingers placed 15 cm fromthe nostrils of each calf were used forsampling all aerosols for their duration.A total of 30 liters of air were thus sam-pled for each calf (16). The virus titrationwas as described by Jericho and Darcel(16). The bacterial aerosols were collectedin similar impingers containing 20 ml of0.1% gelatin saline at room temperature.For experiments A, B, C, D and E theseimpingers were placed in iced water im-mediately after the completion of samp-ling. Impingers for the other experimentswere left at room temperature until bac-teriological analysis was initiated, usuallywithin two hours of sampling.

Volume 42 - July, 1978 271

OTHER TECHNICAL PROCEDURES

These were essentially the same as re-ported previously (15). Calves were re-strained only during aerosol exposure andfeed and water were provided in utensilscommon to all calves for each experiment.Two types of nasal swabs (15) were takenfrom left nostrils, one for the isolation ofBHV1 and the other for the isolation ofP. haemolytica. The swabs were taken onfour separate days before and on threeseparate days after bacterial aerosol ex-posure in experiments A, B, C, D and E.Swabs for the remainder of the experi-ments were taken on two separate daysprior to bacterial aerosol exposure and onthe last day of the experiments. However,in experiment F additional swabs werecollected one day after exposure to the bac-terial aerosol. Sera for the determinationof the neutralizing titres to BHV1 werecollected from all calves at the same timeas the swabs.

VIROLOGICAL STUDIES

Virus suspensions used to produce aero-sols of experiments B, C, D, E, F and Hwere from the same stock as that describedby Jericho and Darcel (16). For the re-mainder of the experiments the virus andthe preparation of its suspension were thesame but the virus had been passaged twomore times. The procedures used to isolateBHV1 from nasal swabs and from suspen-sions of tracheal tissue and lung tissue inEagle's medium were as described previous-ly (16). Viral isolation and titration andserological studies of all experiments weredone in three batches. A different tissueculture cell line was used for each batch.Experiments B, C and D were batch 1, expe-riment E batch 2 and the remainder of theexperiments were batch 3. The neutralizingtitre of sera for BHV1 were also determinedby the method described (16). The concen-tration of the virus per ml of suspensionfor each experiment was determined afterthe last of the four aerosols had been pro-duced. These concentrations ranged from alow of 106 (experiments B, C, D) to a highof 1010 Tissue Culture Infective Dose50(TCID5o)/ml (experiment G).

The suspensions used for the productionof the bacterial aerosols of experiments A,B, C and D were four aliquots of the 40 pre-pared from the suspension described by Je-richo et al (14). Five percent foetal calfserum (FCS) was added to the aliquot foreach of experiments C and D only. A cultureof P. haemolytica for use in the remainderof the experiments was produced by addingone of the original aliquots to brain heartinfusion broth with 5% FCS added andincubation at 37°C for 18 hours. The num-ber of crganisms per ml of suspension ofeach experiment was determined after thelast of four aerosols was produced. The con-centrations ranged from 5.5 x 105 per ml(experiment B) to 1.8 x 1010 per ml (experi-ment L). Deep nasal swabs, tracheal swabsand lung tissue were examined for the pre-sence of P. haemolytica by the direct in-oculation of plates containing trypticase soyagar with 5% bovine blood (BAP). Aftertwo days of incubation, colonies resemblingthose of P. haemolytica were transferredonto new BAP and three days later theseplates were examined for the presence ofcolonies producing hemolysis indicative ofP. haemolytica. Representative colonies wereidentified by biochemical methods. Themethod of titration of each bacterial sus-pension was done as previously described(14). The retropharyngeal lymph nodes ofcalves of experiment L were also examinedfor the presence of P. haemolytica.

PATHOLOGICAL STUDIES

The postmortem procedures and examin-ation of the entire respiratory tracts wereconducted as previously described (15). Theventral aspects of all lungs and other siteswith pathological changes were photo-graphed. Any macroscopic pathologicalchanges in nose, larynx, trachea and lungswere recorded.

RESULTS

CLINICAL OBSERVATIONS

Similar clinical observations to experi-

Can. J. comp. Med.

BACTERIOLOGICAL STUDIES

272

ments with climatic stress (15) and to ex-periments with climatic stress and virusexposure (16) were observed in these ex-periments. There were, however, some note-worthy differences. Calves of experimentE were moved from outside temperaturesof -25°C to 12°C within the nonoperationalchamber. Four hours later one calf had arespiratory rate of 130 per minute whereasthe others had a rate of 68 per minute. Twodays later all respiratory rates were atexpected levels (15). Increases in rectaltemperatures were similar to experimentswith calves infected with the virus only(16). However, regression of rectal tem-perature to normal by day 6 postinfectionhad not occurred in two calves in each ofexperiments G, I, J, K and L. In thesecalves, temperatures of up to 41.6°C wererecorded. In some calves of all experimentsexcept A and B, mucoid and muco-purulentnasal discharge was evident two days afterexposure to the bacterial aerosol. This dis-charge was usually from the left nostril,the one which was routinely used in thenasal swabbing technique. Two calvesraised on an irrigated-fertilized pasture inexperiment H developed very severe diar-rhea and one of these calves died. Oneanimal died in each of experiments G, I, Kand L. All these animals, with one excep-tion, died four days after exposure to thebacterial aerosol. One calf from experimentL died two days after exposure to the bac-terial aerosol.

VIROLOGICAL STUDIES

The ranges of TCID50 for BHV1 re-covered from 30-liter samples of the fouraerosols per experiment were as follows:within experiments B, C and D (batch 1)4.1 x 102 to 4.0 x 103, the range within ex-periment E (batch 2) was 4.0 x 106 to 4.0 x107, the maximum range within any of theother experiments (batch 3) was 5.12 x104 to 1.02 x 105 (experiment I). Amongexperiments of batch 3 the maximum rangewas 1.28 x 104 (experiments I and G) to3.28 x 106 (experiment J). The means ofTCID50 of the virus recovered from 30-litersamples of the four aerosols per experimentare given in Table III.

Bovine herpesvirus 1 was isolated fromonly one nasal swab (F10) taken beforethe exposure of the calves to the virus.Nasal swabs were negative one day after

exposure to the virus in experiments B,C and D but the virus was isolated withincreasing frequency from later swabs. Allnasal swabs were positive on the last dayof all experiments. All nasal swabs fromthe remainder of the experiments weretaken at least three days after virus ex-posure and all of these yielded BHV1. Thevirus was isolated from the tracheas ofall animals except those in experiments C,E and F. The virus was also isolated fromone lung in experiment C, three lungs inexperiments B, F and A and from all otherlungs. The virus was not isolated fromany lungs in experiment E. The sera of 11animals had neutralizing titres for BHV1,nine (all of experiment F, two of each ex-periments J and I and one of experimentL) were positive only on the final day.The sera of three calves, one from experi-ment K and two from experiment E, werepositive throughout. The highest titre was1/12.

BACTERIOLOGICAL STUDIES

The maximum range of numbers of or-ganisms of P. haemolytica in 30-litersamples of four aerosols for all experi-ments was 4.0 x 105 to 1.0 x 106 in experi-ment E. The mean bacterial counts in 30-liter samples of four aerosols per experi-ment are given in Table III. All nasalswabs taken before aerosol exposure werenegative for P. haemolytica. Of the 20nasal swabs (experiments A, B, C, D, E)taken immediately after aerosol exposure,all but two (experiment B) were positive.Of 16 nasal swabs taken three or five daysafter the bacterial aerosol in experimentsA and B only one swab was positive for P.haemotytica. The remainder of the nasalswabs from all experiments were positivefor this organism. P. haemolytica was isol-ated with the following frequency from tra-cheas: none from experiment A, one fromeach of experiments B and D, two fromexperiments C and F, three from experi-ments E and I and from all tracheas of ex-periments G, H, J, K and L. All lungs inexperiments A, B, C and D were negativefor P. haemolytica, one was positive ineach of experiments E, F, H, J and L. Alllungs yielded P. haemolytica in experimentsG, I and K. One retropharyngeal lymphnode from a calf in experiment J waspositive.

Volume 42 - July, 1978 273

PATHOLOGICAL STUDIES

It is learned from the data in Tables Iand III that the weight loss of the calvesin each experiment was not related to theduration of the experimental periods, or tothe type of climatic conditions or to thenumber of lobes with pneumonia.The number of calves with macroscopi-

cally recognizable purulent rhinitis (Fig.1) of at least one nasal cavity, with puru-lent tonsillitis, inflammation of the middletrachea, number of lobes with pneumoniaof at least one-third of the tissue (hence-forth referred to as lobar pneumonia)(Figs. 2 and 3) and the number of calveswith lobar pneumonia are given in TableIII. Frequently inflammation of the tonsilsextended to involvement of the larynx. Noneof these macroscopically recognizablechanges were evident in the animals of thefirst six experiments. In four experiments(G, J, K and L) all the above changes wereseen but not in all calves. In experiment Lall sites except the middle trachea wereaffected. In experiment H, one calf hadpurulent rhinitis and middle tracheitis.Macroscopically recognizable inflammationof the middle trachea was characterized byeither whitish foci of necrosis or by apurulent exudate on the epithelium. Atsites of the middle or lower trachea in ex-periment H, changes occurred independent-ly of tonsillitis, laryngitis or lobar pneu-monia. Tonsillitis, laryngitis and lobarpneumonia were present in animals of ex-periments I, J and K, although tracheitiswas absent. However, in experiments G and

L, macroscopically recognizable inflamma-tion of the middle trachea was associatedwith lobar pneumonia, tonsillitis and laryn-

Fig. 1. Purulent rhinitis (arrow, right) with necrosisof epithelium (arrow, left) in calf of experiment K.

Fig. 2. Pneumonia of cardiac lobe in experiment K.This tissue was severely congested and consolidated andcovered by fibrinous adhesions. Lung tissue on the rightwas affected only at its distal point (arrow).

TABLE III. Mean TCID50 of Virus in 30 Liter Samples of Four Aerosols per Experiment, MeanBacterial Count in 30 Liter Samples of Four Aerosols per Experiment, Number of Calves withRhinitis, Tonsillitis and Middle Tracheitis and Number of Lobes with Pneumonia in Each Ex-periment (Number of Calves with Lobar Pneumonia Per Experiment)

Average Calves Calves Calves LobesAverage Bacterial with with with with

Experiment TCID,0 Count Rhinitis Tonsillitis Tracheitis Pneumonia1.4 X 103

1.3 X 103 Nonerecovered

2.2 X 103 9.0 X 1044.0 X 102 9.6 X 1042.2 X 107 3.0 X 1052.3 X 105 5.2 X 1061.9 X 104 5.0 X 1044.1 X 105 5.6 X 1054.8 X 104 1.1 X 1061.84 x 106 4.0 X 1051.0 x 106 9.26 X 105

4.26 X 105Notatermined

0

0

0

0

0

0

32244

4

0

0

0

0

0

0

30

244

4

0

0

0

0

0

0

2

1

0

0

0

1

0

0

0

0

0

0

11(3)0

6(2)3(1)

14(3)

8(2)

Can. J. comp. Med.

AB

CDEFGHIJKL

de

274

versely, in experiments where lobar pneu-monia occurred, some lungs without lobarpneumonia had numerous BHVY-like lobu-lar lesions.One calf died in each of experiments G,

H, I, K and L. All these calves had lobarpneumonia with the exception of the calffrom experiment H which died with numer-ous lobular lesions in its lungs and severehemorrhagic diarrhea. The extent of pneu-monia in the other four calves which diedwas 25 to 80% of the lung tissue, whereasfor calves which did not die 5 to 40% ofthe lung tissue was pneumonic.

DISCUSSION

Fig. 3. Ventral aspect of lunIg. Pneumonia of distalpart of apical (arrow, left), cardiac (arrow, bottom),and intermediate (arrow, top) lobes. Pneumonic tissuewas congested and consolidated. Fibrinous adhesionswere absent. Note sharp line of demarcation betweenpneumonic (left) and functional tissue of intermediatelobe (arrow, top). Lung tissue on the right of photowas not affected but has mottled appearance due to un-even distribution of blood.

gitis. With the exception of two calvesfrom experiment J, all calves used in ex-periments J, K and L had petechial changesin the upper trachea which were an exten-sion of similar laryngeal changes.Lobar pneumonia was produced in five

experiments (G, I, J, K and L) in a totalof 42 lung lobes of 11 calves. This pneu-monia was usually, but not always, as-sociated with fibrinous pleural adhesionsand the presence of straw-colored thoracicfluid. The volume of this fluid increasedwith the extent of pneumonic tissue. Thesechanges were observed in only those expe-riments where bacterial exposure occurredat least four days after viral exposure(Table I). Production of any macroscopicpathological changes was not proportionalto the concentration of infectious units ofvirus or bacterial counts in the aerosols(Table III). Similarly, the occurrence oflobar pneumonia was not proportional tothe number of lesions affecting only lob-ules. These lobular lesions were indistin-guishable from those in lungs of calvesexposed to only BHV1 (16). Lobar pneu-monia occurred in lungs which had veryfew such BHV1-like lobular lesions. Con-

Throughout these experiments every ef-fort was made to use calves of similarbackground. The only exception beingcalves in experiment E which were broughtinto the chamber during severe winter con-ditions. Variations in the management ofthe calves in the chamber were as follows:(a) time of habituation to the chamber,(b) time between virus and bacterial ex-posure, (c) length of experiment and (d)the controlled climatic conditions withinthe chamber. Additional stressors on all thecalves were anxiety and fear due to wean-ing, transport to the chamber (1/3 km),introduction into the chamber, daily hand-ling, sampling procedures and aerosalexposures. However, they did have the bene-fit of feed and water ad lib and the oppor-tunity of rest between experimental proce-dures. The sale and transport of calves bythe cattle industry is also associated withmany stressors and much respiratory di-sease in calves. It is not known how thephysiological effects of the stressors byindustry compare with the experimentalstressors noted above.The effect of constant or daily fluctua-

tions of climatic conditions on the inci-dence of respiratory disease is not elucid-ated by these studies. In some experimentswith various daily climatic conditions lobarpneumonia was not produced. In a similarstudy using only virus (BHV1) aerosol,the number of macro- or microscopicallyrecognizable lesions produced in the lungs

Volume 42 - July, 1978 275

was not influenced by variations in theambient temperature or humidity (16).Similarly, the period of habituation al-lowed calves of some experiments did notinfluence the susceptibility to this experi-mental respiratory disease.The exposure of calves in experiments A

and B to only aerosols of P. haemolyticafailed to produce any respiratory disease.This confirmed the results of reportedsimilar experiments (17).When the calves were exposed to the

bacterial aerosol at least four days afterthe viral aerosol, pneumonia was producedunder the conditions of these experiments.Evidently, this period of time is requiredby the virus infection to produce some im-pairment of pulmonary bactericidal activity(7, 12) or to inhibit the phagocytic ac-tivity of alveolar macrophages (6) or toeffectively impair the clearance functionof damaged respiratory epithelium (16). Itis noteworthy that the virus used for ex-periments with macroscopic respiratory,disease had undergone two more propaga-tions than the virus of experiments B, C,D, E, F and H which were without respira-tory disease. The significance of these twopropagations to the results is not known.The importance of the interval between

infections with parainfluenza-3 and P.haemolytica to the production of respira-tory disease in cattle has been suspectedby Heddleston et al (10), Hetrick et al(11) and Baldwin et al (1). From experi-ments with intratracheal injections withBHV1 followed three days later by anaerosol of P. haemolytica, Collier et al (3)concluded that infection with BHV1 fol-lowed by P. haemolytica did not producea "profound potentiating effect" of eitherorganism. Similarly, in six experimentsreported herein, P. haemolytica failed topotentiate the effects of BHV1 if givenwithin three days of the virus. However, aconsiderable potentiation was observed infive other experiments of this study whenthe time interval between infections wasmore than three days. This observation issupported by reports of Collier (2) inwhich four calves infected intratracheallywith BHV1 and 30 days later with an aero-sol of P. haemolytica produced clinical di-sease and bronchopneumonia of a type de-scribed in this study.The production of respiratory disease in

experiments G, I, J, K and L may be re-garded as a truly mixed infection and also

dependent on factors other than the twoinfectious agents. Evidently the respiratorytract was only susceptible to infection bythe bacterial aerosol when exposure occur-red at least four days after virus exposure.This suggests that for the purpose of theseexperiments the marginal quantitative ex-posure of critical sites of the respiratorytract was most appropriate. Overwhelmingexposure with bacteria may not have re-vealed differences in susceptibility of calvesbetween experiments. Dao Trong Dat andSchimmel (4) reported the uniform pro-duction of extensive pneumonia in 11 of13 calves two to ten weeks of age byinjecting the relatively large volumes of10 and 20 ml of a culture of P haemolyticainto the noses and tracheas respectively.The interval of four days between aero-

sols required for the production of expe-rimental respiratory disease in this modelis noteworthy in relation to studies on theimportance of stressors on antibody levelsin calves following active and passive im-munization (9). It was found that humoralantibody production was impeded by re-peated or lasting stressor effect (ACTHinjections) and that the immunologicalreactions of the calves to antigen injectionsimmediately after transport into the rear-ing unit were stronger than those to an-tigen application three days after transfer(9). With this observation in mind, theimmune response of calves of this modelshould be investigated to elucidate theassociation of stressors and respiratorydisease of cattle which is based on epide-miological considerations.

Based on results by Jericho et al (14) itis concluded that the viability of bacteriain aerosols for these experiments was notsignificantly influenced by the tempera-tures and humidities of the aerosols. Theseworkers also reported that the addition ofFCS was beneficial for viability of bacteria.Experiments A and B were done withoutFCS and thus yielded very few organismsin the fluids into which the aerosols weresampled.Some or all of the noses and tracheas

were colonized by P. haemolytica in allexperiments of this study, regardless ofthe concentration of bacteria in the aero-sols. The results of virological studies andthe effect of virus exposure were in agree-ment with results of similar experimentswithout bacterial aerosols (16).Death of calves was not always directly

Can. J. comp. Med.276

related to the extent of lobar pneumonia.Although all calves with extensive lesionsdied, one with 40% of its lung tissue af-fected survived and one with only 25%involvement died. In experiment L, oneanimal, which died two days after expo-sure to the bacterial aerosol, had pneu-monic lesions involving 80% of its lungtissue.

Pathological events were not uniformin affected respiratory tracts. Three outof 11 calves with lobar pneumonia hadmacroscopically recognizable inflammationin lower and upper trachea, larynx andnose. In seven calves with lobar pneumonia,macroscopically recognizable changes werenot observed in the trachea. They had,however, rhinitis and/or tonsillitis. Thesefindings suggest that the pathological se-quence of events in the upper and lowerrespiratory tracts may be independent ofeach other, but nonetheless, similar im-munopathological events may occur at therespective sites.

ACKNOWLEDGMENTS

The authors would like to thank Mr. D.Carpenter for his assistance in all tech-nical procedures in the environmentalchamber, during aerosol exposures and atnecropsy of the calves. The bacteriologicalprocedures were conducted by Mrs. B.Larson, the virological procedures by Mrs.S. Smithson and Mrs. B. Jones and themacrophotographs were taken by Dr. L.Niilo. The viral suspensions and super-vision of technical procedures of viralstudies were provided by Dr. C. le Q.Darcel. The cattle herd from which thecalves were derived was managed by Dr. W.Dorward, Mr. J. Grisack and Mr. W.Krampl. All of the above are employees ofA.D.R.I. (W). Thanks are extended toMiss D. Pickett for the typing of themanuscript.

REFERENCES

1. BALDWIN, D. E., R. G. MARSHALL and G. E.WESSMAN. Experimental infection of calves withmyxovirus parainfluenza-3 and Pasteurella haemo-lytica. Am. J. vet. Res. 28: 1773-1782. 1967.

2. COLLIER, J. R. Pasteurellae in bovine respiratorydisease. J. Am. vet. med. Ass. 152: 824-828. 1968.

3. COLLIER, J. R., T. L. CHOW, M. M. BENJAMINand A. W. DEEM. The combined effect of infec-tious bovine rhinotracheitis virus and Pasteurellahaemolytica on cattle. Am. J. vet. Res. 21: 195-198.1960.

4. DAO TRONG DAT and D. SCHIMMEL. The signif-icance of Pasteurella infections in diseases of therespiratory tract of calves. 2. Experimental Pas-teurella infections. Arch. exp. VetMed. 28: 303-315.1974.

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