A Guinea Pig Model of Bovine Pneumonic Pasteurellosis D.W. Morck, J.W. Costerton, D.O. Bolingbroke, H. Ceri, N.D. Boyd and M.E. Olson

ABSTRACT

Une croissance bacterienne au useful in studying pathogenetic and pathological features applicable to niveau des poumons a ete notee The induction of pneumonic pas- bovine pneumonic pasteurellosis (p < 0,05) puisque le nombre de teurellosis in guinea pigs (Cavia (shipping fever pneumonia). bacteries retrouvees dans les poumons etait plus grand que le nombre de porcellus) was examined. Specific bacteries inoculees. Les variations de pathogen free male guinea pigs were RESUME anesthetized and a tracheostomy parametres hematologiques et biochiperformed to introduce 105, 104 or 103 miques etaient representatifs d'une Cette experience visait a induire une pneumonie fibrineuse et de 61% (11/ Pasteurella haemolytica-Al into the left principal bronchus. The surgical pneumonie a l'aide d'une souche de 18) des hemocultures realisees, on site was closed with tissue adhesive Pasteurella haemolytica-Al chez des demontra la presence de P. haemolytand staples and the animals were cobayes (Cavia porcellus) males ica. En conclusion, l'etude de la monitored for signs of respiratory exempts d'agents pathogenes a l'es- pathogenie de la pasteurellose induite tract infection. Within 24 hours after pece. Sous anesthesie, linoculation de chez le cobaye pourrait s'averer un inoculation they became depressed, 105, 104 et 103 agents dans la bronche modele pour l'etude de celle retrouvee anorectic, pyretic and dyspneic. principale gauche a ete realisee par chez les bovins. Fibrinous pleuropneumonia with tracheostomie. Le site dincision a ete prominent areas of necrosis and referme a l'aide de ruban adhesif et et par la suite les animaux hemorrhage was present. Pericardial d'agrafes INTRODUCTION effusion was a frequent finding. There ont ete examines afin de detecter les signes cliniques d'infection du systeme was infiltration of the pleura and respiratoire. Pneumonic pasteurellosis has been En moins de 24 heures, examined alveoli with degenerate heterophils les sujets sont in cattle devenus depressifs, using severalexperimentally and macrophages, a hyperplastic anorexiques, febriles infection includsystems dyspneiques. ing trans-pulmonic injections mesothelium and fibrin exudation on Une pleuropneumonieetfibrineuse (1), the pleura and within alveoli. Hemor- presence de foyers necrotiquesavec et intrabronchial catheterization (2), rhage, congestion, consolidation, hemorragiques etait rapportee. Dans intratracheal injections (3), aerosols of edema and fibrin exudation were ces cas, une effusion pericardique etait Pasteurella haemolytica (4) and prominent in the hilar region of the souvent presente. Les plevres et les aerosols of bovine herpesvirus-I lungs. Bacterial colonies were evident alveoles etaient infiltres de polymor- followed by nebulized suspensions of in all airways. More bacteria were phonucleaires et de macrophages P. haemolytica (5,6). A laboratory recovered from infected lungs than degeneres. Une hyperplasie du animal system, in which albino mice were inoculated (p < 0.05) indicating mesothelium et un exsudat fibrineux (Mus musculus) were infected sequenP. haemolytica was actively multiply- etaient retrouves au niveau des plevres tially with parainfluenza-l (Sendai) ing in the lungs. Hematological and et dans les alevoles. Au niveau de la virus and Pasteurella pneumotropica, clinical chemistry data were consistent region hilaire, des hemorragies, de la has been used to examine viralwith fibrinous pneumonia, however, consolidation, de l'oedeme et un bacterial synergism (7). Albino mice blood cultures were positive for P. exsudat fibrineux etaient souvent also have been used in studies in which haemolytica in 61% (11/18) of animals presents. Des colonies bacteriennes P. haemolytica was injected intraperisampled. Examination of pneumonic etaient retrouvees dans toutes les voies toneally (8-11) with various foreign, virulence enhancing agents such as pasteurellosis in guinea pigs may be respiratoires.

Department of Biological Sciences (Morck, Costerton, Ceri, Olson), Department of Pathology (Boyd), The University of Calgary, Calgary, Alberta T2N IN4 and Alberta Agriculture, Regional Veterinary Laboratory, Airdrie, Alberta (Bolingbroke). Reprint requests to Dr. M.E. Olson. This investigation was supported in part by Alberta Agriculture Farming for the Future project #88-0279 and the Natural Sciences and Engineering Research Council. Submitted March 10, 1989.

Can J Vet Res 1990; 54: 139-145

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porcine gastric mucin, microbial polysaccharides, dextrans or hemoglobin. In addition to mice, Syrian hamsters (10,11) and guinea pigs (11) have been inoculated intraperitoneally with P. haemolytica, but the results were not highly promising. Intrabronchially induced respiratory tract infection of a laboratory animal which models pneumonic pasteurellosis of calves has never been described. This investigation was undertaken to determine if intrabronchial infection of the guinea pig with P. haemolytica-A 1 could serve as a useful representation of bovine pneumonic pasteurellosis.

MATERIALS AND METHODS EXPERIMENTAL ANIMALS

Fifty-nine outbred strain Crl: (HA)Br male guinea pigs (Cavia porcellus) (Charles River Canada Inc., St-Constant, Quebec) were used in this investigation. The guinea pigs weighed 300-350 g and were acclimatized for ten days prior to infection. They were housed in suspended stainless steel cages (51 x 56 x 23 cm), fed alfalfa pellets (Unifeed, Calgary, Alberta) ad libitum and received vitamin C (Fisher Scientific, Fair Lawn, New Jersey) daily in the drinking water (400 mg/L). Environmental temperature was 20+/-1°C, relative humidity was a constant 40% and a photoperiod of 12 h was maintained. Animal care and manipulations were conducted according to the Canadian Council on Animal Care Guidelines for the Care and Use of Experimental Animals.

TABLE I. Experimental groups

Group 2 3 4

Number (CFU) of P. haemolytica inoculated 5.0 x 105 5.0 x 104 5.0 x 103 Nil

INOCULATION PROCEDURE

No. of animals 13 13 25 8

suspension was aseptically injected into the peritoneal cavity of a guinea pig. After 48 h the guinea pig was stunned and then euthanized by cervical dislocation and the peritoneal exudate was cultured aerobically for bacteria. The isolate was identified as P. haemolytica and stored in ovine blood (sodium citrate anticoagulant) at -70°C. The peritoneally passaged Pasteurella isolate was grown on chocolate agar, inoculated into BHI broth, grown as above, and an aliquot of this broth culture was inoculated into a second BHI broth. This culture was incubated at 37° C without agitation to late-logarithmic phase. The bacteria were harvested as above, washed in sterile PBS and resuspended at concentrations of 107, 106 and 105 CFU/mL. These inocula concentrations were given to experimental groups 1, 2 and 3, respectively, as illustrated in Table I. A sterile PBS solution served as a control inoculum and guinea pigs receiving this were designated group 4.

A procedure previously described (12) was used to infect the guinea pigs. Briefly, diethyl ether was used in a closed chamber to induce a surgical plane of anesthesia. The anesthetized animal was placed in a "V-trough" and anesthesia maintained using a gauze mask soaked in ether. The ventral cervical area was disinfected with 70% ethanol, a skin incision (1.5-2.0 cm) made and the trachea isolated by blunt dissection. The trachea was entered by making a small incision (1-3 mm) in the soft tissue between two cartilaginous rings and a specially fabricated, curved, blunt needle (Fig. 1) was used to inoculate 0.05 mL of suspension into the left principal bronchus. The tracheostomy site, subcutaneous tissue and epidermis were closed with tissue adhesive (Animal Care Products/3M, St. Paul, Minnesota) and the integument was apposed with stainless steel 9 mm wound clips (Becton Dickinson and Co., Parsippany, New Jersey). CLINICAL ASSESSMENT

The animals were observed hourly after experimental inoculation for clinical signs of respiratory tract infection. Rectal temperatures were taken twice daily throughout the experiment. Immediately before euthanasia, blood samples were taken aseptically from selected animals for

INOCULA PREPARATION AND EXPERIMENTAL GROUPS

Pasteurella haemolytica-Al (strain B122) isolated from a calf which died from pneumonic pasteurellosis was grown for 24 h on chocolate agar at 37°C in 5% CO2. A single colony was inoculated into brain heart infusion (BHI) broth and grown without agitation to early-logarithmic phase. Bacteria were harvested by centrifugation (4080 x g), washed in sterile phosphate buffered saline (PBS), pH 7.2, and suspended in PBS at a concentration of 106 colony forming Fig. 1. Microsyringe and specially fabricated, curved, units (CFU)/ mL. A 1.0 mL aliquot of inoculations. Scale = cm. 140

blunt needle used for the challenge

blood culture, complete blood counts (CBC) and serum biochemistry profiles (SBP), which included lactate dehydrogenase, gamma-glutamyl transferase, creatinine, sodium, potassium, chloride and carbon dioxide.

80

60

POSTMORTEM EXAMINATION

Animals which became moribund, and all surviving guinea pigs at the end of the experiment were stunned and exsanguinated, and the hearts and respiratory tracts immediately removed. Sterile PBS was used to rinse the surface of these tissues prior to examination for grossly evident pathological changes and bacteriological sampling. Tissue specimens from three standard areas of the lung (left cranial, middle and caudal) were aseptically removed and each divided for histological examination and electron microscopy. The specimen for histological processing was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) using standard methodology. These specimens were used to subjectively quantify the degree of hemorrhage, consolidation and inflammation as follows: for the presence of lesions (0 = not affected, 1 = one lobe affected, 2 = two lobes affected, 3 = three lobes affected) and the severity of lesions (0= absent, 1 = mild, 2 = moderate, 3 severe). These scores were totalled to arrive at an individual animal score (I.A.S.). Several specimens were also fixed in Carnoy's fixative (13) and embedded in methacrylate (JB-4 or ImmunoBed, Polysciences, Inc., Warrington, Pennsylvania). Sections (1.5 ,um) were cut on an LKB Rotary-One microtome and stained with Lee's methylene blue-basic fuchsin (14) to show bacterial colonies more clearly. Additionally, sections from selected animals were stained with immunoperoxidase to confirm the presence of P. haemolytica. Polyclonal antiserum raised in New Zealand white rabbits intravenously inoculated with P. haemolytica (B 122), nonimmune serum and peroxidase labelled, affinity purified goat antibody against rabbit IgG (heavy and light chains) (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Maryland) were

4)

-0

-03-

40 -U-0-

Group 1 Group 2 Group 3 Group 4

20

0 0

20

40

60

80

1 00

Time (hours) Fig. 2. Percent dead (euthanized) vs time after experimental inoculation (Group 1 n = 13, Group 2 n = 13, Group 3 n = 25, Group 4 n = 8). The 12.5% mortality in group 4 represents the single animal that died of pneumonia due to a Streptococcus sp. Note the comparatively high mortality in group 1 within the first 24 hours of the experiment.

used. The samples for electron dehyde in 0.1 M cacodylate buffer microscopy were fixed in 5% glutaral- containing 0.15% ruthenium red and

Fig. 3. A photograph of severely affected lungs from a group 2 guinea pig. Hemorrhage, consolidation and atelectasis are present and thick sheets of fibrinous exudate are evident on the pulmonary pleura (arrows). This exudate has been pulled away from the pulmonary tissue by hemostats (upper left).

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estimate the number of viable bacteria present. Major isolates were identified by standard techniques which included Gram stain and cellular morphology, growth on blood agar and MacConkey agar, glucose fermentation, indole production and motility as well as urease, catalase and oxidase activities. STATISTICAL ANALYSES

A two-tailed Student's t-test and one-way analysis of variance were used to compare means of two groups (p < 0.05). When comparing three or more means, one-way analysis of variance followed by StudentNewman-Keuls test for multiple comparisons were used to determine statistical significance (p < 0.05). Fig. 4. A photomicrograph of a formalin fixed, paraffin embedded, H&E stained section of pulmonary tissue from a group 2 guinea pig. Note the extreme inflammation and consolidation of pulmonary tissue as well as fibrin exudation (F) on the pleura. Bar equals 100 ,um. Inset -Formalin fixed, paraffin embedded, H&E stained section of pulmonary tissue from a group 4 guinea pig. The mesothelium is slightly thickened but all other histological features appear normal. Bar equals 500 ,um.

CLINICAL FINDINGS

Immediately after experimental inoculation the guinea pigs were were processed as described elsewhere hypothermic and coughing, presumably due to anesthesia and experimen(15,16). The remaining pulmonary tissue tal manipulation because they rapidly was weighed, homogenized, asepti- recovered and appeared normal 1-2 h cally diluted with PBS and 0.1 mL after being inoculated. The infected aliquots were plated on BHI agar to animals (Groups 1, 2 and 3) became

12 -

7a11

RESULTS

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10 -

8*

L

T

. 6

a 0

4. 4

T

*

a

a

IT

aca

1

U

Group I Group 2 Group 3 U Group 4 0

2-

0* 1

2

3

4

Group

Fig. 5. Mean Individual Animal Scores (I.A.S.) of the four experimental groups of guinea pigs. (Group 1 n = 10, Group 2 n = 10, Group 3 n = 22, Group 4 n = 7), (+/-SE), (*= significantly different from Groups 1, 2 and 3. p < 0.05).

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Hemorrhage

Consolidation

Inflammation

Fig. 6. Mean histological scores of the four experimental groups of guinea pigs considering the individual pathological components of the scoring system. (Group 1 n = 10, Group 2 n = 10, Group 3 n = 22, Group 4 n = 7), (+/-SE), (Consolidation *= significantly different from Groups 2 and 3. p < 0.05) (Inflammation *= significantly different from Group 3. p < 0.05).

within the first 24 h of the experiment. Figure 2 indicates the proportion of animals in each group euthanized in relation to time after inoculation. Euthanasia was required more frequently in group 1 animals within the initial 24 h after inoculation than in other groups. Wide variation was seen in all serum chemistry and hematological parameters. No definite correlations were established in the data obtained from the CBC or SBP of infected or control animals, but infected animals were often either neutrophilic or severely neutropenic. Blood cultures were positive for P. haemolytica in 61% (11/ 18) of the infected animals tested, i.e. group 1 (1/ 1), group 2 (2/4), cup 3 Fig. 7. A micrograph of a Carnoy's fixed, methacrylate embedded, methylene blue and basic fuchsin (8/13). Blood from two control stained, 1.5 zm section of pulmonary tissue from a group 2 guinea pig. Several microcolonies of animals was also cultured and was Pasteurella haemolytica (B) are evident within an alveolar space (A). A degenerating leukocyte (L) Pasteurella negative. with many adherent bacteria is also shown. An intact capillary containing erythrocytes (C) is evident. Note the consortium or microcolony of Pasteurella haemolytica which appears to be associated with the alveolar epithelial cells (large arrow). Bar equals 10 ,Am.

POSTMORTEM EXAMINATION

There was a spectrum of severity of gross changes, but there was no obvious correlation with bacterial dyspneic, inappetent, lethargic, febrile over the next 24-48 h, but 10 of 13 in concentration in the inoculum. (39.4°C-40.5°C), and in some cases group 1, 7 of 13 group 2 animals and Hemorrhage and consolidation were cyanotic and recumbent within 24- 11 of 25 in group 3 became so severely frequently present in infected animals 48 h. Moribund animals had rectal affected that euthanasia was neces- but also occasionally in controls. temperatures as low as 34.00 C. sary. The majority of control animals Severely affected guinea pigs had a Several animals did not become (Group 4) showed no signs of infec- marked fibrinous exudate on the moribund and appeared to recover tion, but a single guinea pig died pulmonary and parietal pleurae and often pericardial effusion and mild pericarditis. Often these animals had survived 48-96 h after infection and became progressively more ill. A typical respiratory tract with grossly detectable fibrinous pleuropneumonia is shown in Fig. 3. Conventional paraffin embedded H&E stained sections of pulmonary tissue showed infiltration of the pleura and alveoli with degenerate heterophils and macrophages, a hyperplastic mesothelium and fibrin exudation (Fig. 4). Elongated "streaming" macrophages (oat cells) were not evident. The hilar and central regions of the lung (not shown) were most severely affected with congestion, edema, fibrin exudation and consolidation. The inset photomicrograph of Fig. 4 shows the pleura and several alveoli of a group 4 (PBS) guinea pig. The scoring of histopathology (Fig. 5) showed no significant difference in Fig. 8. Transmission electron micrograph of a glutaraldehyde fixed, ruthenium red stained, thin I.A.S. between groups 1, 2 and 3 section of pulmonary tissue from a group 1 guinea pig. A typical gram-negative bacterium (B) is (p