Experimental model of swine pneumonic pasteurellosis using crude Actinobacillus pleuropneumoniae cytotoxin and Pasteurella multocida given endobronchially.

This study was designed to develop and characterize a swine pneumonic pasteurellosis model by concurrent introduction of Pasteurella multocida type A and Actinobacillus pleuropneumoniae crude cytotoxin. After a series of preliminary experiments, a combination of 4 x 10(9) P. multocida and 4,000 toxic units of A. pleuropneumoniae crude cytotoxin was determined to produce optimal results. A total of 48 pigs were divided into four groups of 12 pigs each. The control group received buffered saline only. Four pigs from each group were randomly selected for necropsy 3, 7 and 14 days postinoculation (PI). Inoculation of pigs with P. multocida and A. pleuropneumoniae cytotoxin (group 1) resulted in moderate to severe pneumonia. Pasteurella multocida was isolated from pneumonic lesions, grossly normal lung, and bronchial lymph nodes of all group 1 pigs throughout the 14 day experimental period. Pathological changes typical of field cases of swine pneumonic pasteurellosis were produced. Pigs inoculated with P. multocida alone (group 2) had pneumonic lesions and P. multocida was reisolated from lungs at three days PI. Pasteurella multocida was not isolated from these pigs at 7 and 14 days PI, except for one pig in which an abscess developed in the thorax. Pulmonary lesions induced by A. pleuropneumoniae crude cytotoxin alone (group 3) were transient and resolved by seven days PI. Group 1 pigs had significantly greater lung lesion volumes than group 2 and 3 pigs at 3, 7 and 14 days PI. Statistical analysis indicated a significant interactive effect of P. multocida and A. pleuropneumoniae cytotoxin on the development of lung lesion volumes at 7 and 14 days PI (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay for the quantitation of Actinobacillus pleuropneumoniae cytotoxin.

Using swine neutrophils as target cells, two MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay systems, one with and one without phorbol 12-myristate 13-acetate (PMA) stimulation were established for the quantitation of Actinobacillus pleuropneumoniae cytotoxin. The MTT assays were optimized for the number of neutrophils, incubation time, and PMA concentration by a series of experiments. The optimal conditions were 25 x 10(4) cells/well incubated for four hours for the assay system without PMA stimulation, and 12.5 x 10(4) cells/well incubated for two hours for the assay system with PMA stimulation. One culture supernatant of a toxigenic Pasteurella multocida strain and five A. pleuropneumoniae cytotoxin preparations produced from three A. pleuropneumoniae strains were used to test assay reproducibility. Results showed both assays were reproducible with a coefficient of variation ranging from 7.8 to 18% for the assay system without PMA stimulation and from 10.7 to 18.2% for the assay system with PMA stimulation. The PMA-stimulated assay had 40 to 60-fold higher sensitivity than the nonstimulated MTT assay. The MTT assay also was applied to the measurement of neutralizing antibody titers against A. pleuropneumoniae cytotoxin.

A comparison of different challenge methods for induction of atrophic rhinitis in pigs.

Transmission and development of atrophic rhinitis (AR) was studied in 5- to 15-week-old pigs (Groups 2-7) originating from a herd free of AR, and compared to unexposed healthy pigs (Group 1), and pigs from a herd with endemic AR (Group 8). At the start of the trial, pigs in Groups 2-5 were challenged intranasally twice a week for 3 weeks with pure cultures of bacteria originating from the endemic AR herd: Nontoxigenic Pasteurella multocida type A (PmA) plus Bordetella bronchiseptica phase I (Bb) (Group 2); PmA + toxigenic Pm type D (PmD) (Group 3); PmD only (Group 4); and PmD + Bb (Group 5). Group 6 pigs were challenged with nasal wash of pigs from the endemic AR herd, and Group 7 pigs were challenged by being housed together in the same pen with Group 8 pigs throughout the study. Nasal swabs of all pigs were cultured 5 times during the study. Serum was collected at 6 weeks post challenge. Average daily gain (ADG) and turbinate lesions (turbinate gross lesions by visual scoring and by Turbinate Perimeter Ratio, TPR, scoring, and histopathological lesions) were measured at the time of slaughter at 15 weeks of age. Mean TPR value for the Group 1 pigs was 1.64, which was significantly (P less than 0.05) different from the mean TPR value of 0.58 for the pigs from the endemic AR herd (Group 8), the 0.79 value for Group 6 pigs, and 1.03 value for Group 7 pigs. Of pigs challenged with pure bacterial cultures, only Group 5 (PmD + Bb) developed significant AR (mean TPR = 1.24). Only one pig in each of Groups 2 and 3, and two pigs in Group 4 showed TPR values indicative of AR (TPR less than 1.30). However, histopathological examination showed that those pigs were recovering from the infection 7 weeks post challenge. Constant exposure to certain bacteria or other factors in nasal washings, stress of crowding or poor environmental conditions might be required to experimentally produce AR in 5-week and older pigs similar to that in naturally infected pigs. There was no relationship between turbinate lesions and the isolation frequency or quantity of PmA, PmD, or Bb. Antibody levels against PmA or PmD had moderate to high correlation with TPR values (r = -0.694 and -0.503 respectively). ELISA values also corresponded well with the type of bacteria inoculated in each group of pigs and appeared to be a sensitive test for PmA, PmD, and Bb infections in pigs.(ABSTRACT TRUNCATED AT 400 WORDS)

Adherence of Bordetella bronchiseptica and Pasteurella multocida to swine nasal ciliated epithelial cells in vitro.

A bacterial adherence assay using swine nasal turbinate fragments was established. Turbinate fragments were incubated with Bordetella bronchiseptica or Pasteurella multocida type D at different concentrations or for different incubation times at 37 degrees C on a shaker at 120 rev/min. B. bronchiseptica phase I strains exhibited strong adherence to swine nasal ciliated epithelial cells. The number of adherent bacteria per cell increased when the bacterial concentration or incubation time increased (0, 15, 30, and 60 min); however, the number of adherent bacteria decreased after 3 or 6 hours' incubation due to the loss of cilia from cells. The optimal bacterial concentration and incubation time were 1 x 10(9) organisms/ml and one hour respectively, which resulted in 7.48 +/- 0.66 (Mean +/- SEM; B. bronchiseptica strain 03) and 9.31 +/- 0.54 (B. bronchiseptica strain 013) adherent bacteria per cell. In contrast to B. bronchiseptica phase I strains, rough phase strains of B. bronchiseptica and all P. multocida strains tested showed no adherence to swine nasal ciliated epithelial cells. All B. bronchiseptica phase I strains could agglutinate calf RBC but rough phase strains could not. Furthermore, pretreatment of B. bronchiseptica phase I organisms with 1 mg/ml or 2 mg/ml of trypsin significantly inhibited the adherence of B. bronchiseptica to ciliated epithelial cells; however, trypsin (2 mg/ml) treatment of bacteria did not decrease their ability to agglutinate calf RBC. From these results we conclude that, in addition to hemagglutinin, other proteinaceous components exist on the surface of virulent B. bronchiseptica that are sensitive to 2 mg/ml trypsin; these are suggested to be the adhesins for the adherence of B. bronchiseptica to swine nasal ciliated epithelial cells.

Blood gas and hematological changes in experimental peracute porcine pleuropneumonia.

The effect of experimental, peracute, porcine pleuropneumonia on arterial blood gases, acid base status, the leukogram, and gross and microscopic lung structure was studied in nine growing pigs (mean weight +/- SD 10.6 +/- 2.0 kg). Pigs were inoculated intranasally with a virulent serotype 5 isolate of Actinobacillus pleuropneumoniae, and all showed signs typical of the disease within four hours. Death occurred in all pigs from 4.5 to 32 hours postinoculation (mean 14 hours). Gross and microscopic changes were typical of porcine pleuropneumonia in all pigs. Changes in the leukogram included a rapid decline in total white cells, segmented neutrophils, lymphocytes, monocytes, and eosinophils. Pigs maintained alveolar ventilation throughout the study as arterial CO2 tension was unchanged; however, arterial O2 tension and pH decreased from (mean +/- SD) 95.2 +/- 5.7 torr and 7.463 +/- 0.018 at baseline to 62.1 +/- 12.3 torr and 7.388 +/- 0.045, respectively, within 90 minutes prior to death. The data showed that in this model of peracute porcine pleuropneumonia, progressive ventilatory failure was not a feature of the disease, and the blood gas values and acid base status were maintained within physiological ranges. The histopathological hematological and physiological findings were consistent with the hypothesis that peracute porcine pleuropneumonia resembles septic shock.

Comparison of conventional and long-acting oxytetracyclines in prevention of induced Actinobacillus (Haemophilus) pleuropneumoniae infection of growing swine.

These experiments tested the hypothesis that long-acting oxytetracycline (oxytetracycline-LA) was more effective than regular oxytetracycline in preventing porcine pleuropneumonia when administered either 24 or 48 h prior to experimental challenge with virulent strains of Actinobacillus pleuropneumoniae. Two experiments (1 and 2) were conducted using growing pigs (average weight 12-15 kg). Antibiotic treatments were administered once intramuscularly at 20 mg/kg body weight; controls received an equivalent volume of saline. Clinical signs were recorded over seven days, and mortality rates and pathological lesions were analyzed using analysis of variance. Serum oxytetracycline levels were compared 48 and 72 h postinjection. All pigs developed clinical disease following experimental infection. Actinobacillus pleuropneumoniae was recovered from 42% of experiment 1 pigs and all of experiment 2 pigs. The data showed that both oxytetracycline and oxytetracycline-LA given at the same dose protected pigs against experimental infection when given 24 h prior to challenge, and there was no difference between the efficacy of the two drugs in this experiment. When administered 48 h prior to challenge, only oxytetracycline-LA reduced the clinical signs and pathological changes following A. pleuropneumoniae challenge. Between 48 and 72 h postinjection, oxytetracycline-LA blood levels were significantly greater compared to oxytetracycline-treated pigs.

Turbinate perimeter ratio as an indicator of conchal atrophy for diagnosis of atrophic rhinitis in pigs.

Transverse sections of snouts from 171 cross-bred (principally Yorkshire X American Landrace) pigs were evaluated for evidence of turbinate atrophy by use of conventional (atrophic rhinitis [AR] score) and morphometric methods. Of the 171 pigs, 35 were clinically normal (AR score, 0), 65 had mild AR (AR score, 1), 41 had moderate AR (AR score, 2), and 30 had severe AR (AR score, 3). Turbinate cross-sectional area (TA) and the ratio of TA to nostril cross-sectional area, called turbinate area ratio (TAR), had the lowest correlations (r = 0.24 to 0.55) with conventional AR score. Among clinically normal pigs, TA was greater in older pigs as expected, but the TAR values also were significantly (P less than 0.0001) different between 15-week-old pigs (55 kg) and 22-week-old pigs (100 kg). Turbinate perimeter and turbinate perimeter ratio (TPR) were not influenced by pig age or source. The TPR values were closely correlated with subjective visual AR scores (r = 0.73), with AR scores derived by measuring the space between the ventral portion of the scroll and the floor of the nasal cavity (r = 0.72), and the actual size of this space in millimeters (r = 0.71). Mean TPR values for pigs assigned visual AR scores of 0, 1, 2, or 3 were 1.54, 1.25, 0.97, and 0.73, respectively. The 95% confidence intervals around these mean TPR values were discreet and did not overlap. Turbinate perimeter ratio, therefore, may be a more reliable morphometric measure of atrophic rhinitis and also provides parametric data suitable for quantitative analysis.

Blood gas stability and hematological changes in experimentally-induced acute porcine pleuropneumonia.

Blood gas and hematological responses to acute, mild Actinobacillus pleuropneumoniae infection of growing pigs was studied. Six pigs (average weight 10.1 kg) were experimentally infected intranasally with A. pleuropneumoniae serotype 5. Four pigs served as controls. Rectal temperatures and arterial blood for gas analysis and hematology were taken at 0, 8, 16, 24, 48 and 72 h postinfection. All infected pigs became febrile showing clinical signs typical of mild to moderate porcine pleuropneumonia; controls remained asymptomatic. Neutrophilia with bands and lymphopenia were observed only in infected pigs. Arterial partial pressures of O2 and CO2, and pH did not change in infected pigs. All pigs were killed after 72 h, and lungs were examined and cultured. Gross and microscopic lesions consistent with porcine pleuropneumonia were seen in 3/6 and 5/6 infected lungs, respectively. Control lungs were grossly normal with no histological evidence of pleuropneumonia. We conclude that in mild, acute porcine pleuropneumonia as established experimentally, a leukogram typical of acute inflammation and stress is seen; however, hypoxemia and alveolar hypoventilation are not features of this form of the disease.

Development of turbinate lesions and nasal colonization by Bordetella bronchiseptica and Pasteurella multocida during long-term exposure of healthy pigs to pigs affected by atrophic rhinitis.

Natural transmission of atrophic rhinitis from pigs from a herd with an endemic atrophic rhinitis problem to pigs from a herd free of atrophic rhinitis was demonstrated. Six replicates each with five pigs from the endemic atrophic rhinitis herd (Group A) and five pigs from the atrophic rhinitis-free herd (Group B) were housed together from 5 wk of age, with each replicate kept in isolation rooms maintained at optimal and controlled environmental conditions. Three replicates each with six pigs/room from the atrophic rhinitis-free herd (Group C), served as nonexposed controls. Group C pigs remained healthy and had no turbinate atrophy at either 10 or 17 wk of study (atrophic rhinitis score = 0 on a 0 to 3 scale). Group A pigs had a mean atrophic rhinitis score of 1.85 +/- 0.84, and group B pigs developed atrophic rhinitis to a mean score of 1.57 +/- 0.70. The isolation rate and quantity of Pasteurella multocida found on nasal swabs was directly related to lesions while those for Bordetella bronchiseptica were inversely related to turbinate atrophy. Of the various types of P. multocida evaluated, nontoxigenic type A and toxigenic type D were both directly related to atrophic rhinitis while nontoxigenic type D strains were not. No toxigenic type A P. multocida strains were isolated.