Aerosol vaccination of calves with pasteurella haemolytica against experimental respiratory disease.

Three experiments were conducted on calves in which the efficacy of vaccination with live Pasteurella haemolytica in aerosol was tested by challenge with sequential aerosol exposure to bovine herpesvirus 1 and P. haemolytica. Neither single nor multiple aerosol vaccinations protected against the experimental disease. Macroscopically recognizable rhinitis, tonsillitis, tracheitis and pneumonia occurred in both controls and vaccinates. In one experiment as many as three aerosol vaccinations with live P. haemolytica for up to 20 minutes failed to elicit clinical signs in exposed calves. Pasteurella haemolytica was isolated less frequently from tissues of vaccinated calves than from those of nonvaccinated calves. Pasteurella haemolytica was isolated from deep nasal swabs of 4/14 vaccinated calves five and six days after viral exposure. It was concluded that although bovine herpesvirus 1 vaccination has been shown previously to prevent the experimental disease produced by bovine herpesvirus 1-P. haemolytica, live P. haemolytica vaccination by aerosol will not provide the same protection.     

A caprine pneumonia outbreak associated with caprine herpesvirus and Pasteurella haemolytica respiratory infections

In a field disease outbreak, 60 female goats died over a 2-3 week period. Necropsies of seven of these does revealed that six had an acute exudative necrotising broncho-pneumonia, and moderate to high numbers of Pasteurella haemolytica were isolated from their lungs. Caprine herpesvirus, identified as Bovid herpesvirus type 6, was isolated from the lungs of two of these does, including one with pneumonia, and from nasal swabs of in-contact goats. Clinical disease was only observed in does, and deaths began 3 weeks after the introduction of a mob of goat hoggets from another farm.     

The effect of Pasteurella haemolytica A2 infection on phagocytosis efficiency of caprine broncho-alveolar macrophages

An experiment was designed to study the in vivo effect of Pasteurella haemolytica A2 infection on the phagocytosis activity of caprine broncho-alveolar macrophages and the extent of pneumonic lesions. Twelve healthy local Kacang goats, about 7 months of age, were divided into two groups of six. Goats in group 1 were inoculated intratracheally with 4 ml inoculum containing 2.8 x 10(9) colony-forming units (CFU)/ml of Staphylococcus aureus. Goats in group 2 were inoculated intratracheally with 4 ml of inoculum containing 9.5 x 10(8) CFU/ml of Pasteurella haemolytica A2 isolated earlier from pneumonic lungs of goat. At intervals of 3 and 7 days post-challenge five goats from each group were killed and the lungs were washed with sterile phosphate-buffered saline. Smears were prepared from the lung washing fluid and the number of macrophages with phagocytic activity was determined. At day 3 post-infection, goats of both groups showed a similar pattern of pneumonic lesion. The lung washing fluid of goats in group 2 was found to contain numerous neutrophils and macrophages. Goats in group 2 showed significantly (P < 0.05) higher extent of lung lesions than group 1. Similarly, the average extent of lung lesions was significantly (P < 0.05) more severe in group 2 at day 7 post-infection. The lung washing fluid contained mostly macrophages. The phagocytic activity following S. aureus infection was more efficient and significantly (P < 0.01) higher compared with infection by P. haemolytica A2. There were weak correlations between the extent of pneumonic lesion and the phagocytic activity. Thus, goats with poor phagocytic activity were likely to develop more extensive lung lesions.     

Association and General Notes

In a field disease outbreak, 60 female goats died over a 2–3 week period. Necropsies of seven of these does revealed that six had an acute exudative necrotising broncho-pneumonia, and moderate to high numbers of Pasteurella haemolytica were isolated from their lungs. Caprine herpesvirus, identified as Bovid herpesvirus type 6, was isolated from the lungs of two of these does, including one with pneumonia, and from nasal swabs of in-contact goats. Clinical disease was only observed in does, and deaths began 3 weeks after the introduction of a mob of goat hoggets from another farm.     

Induction of immunity against pneumonic pasteurellosis following experimental infection in calves.

Immunity against pneumonic pasteurellosis was studied in calves after recovery from experimental respiratory disease with Pasteurella haemolytica. Nine calves were exposed to aerosols of parainfluenza-3 virus and Pasteurella haemolytica A1 six days apart to produce respiratory disease. After recovery from the disease, these nine principal and four control calves were challenged with aerosols of bovine herpesvirus 1 and P. haemolytica A1 four days apart. With this viral-bacterial challenge, the nine principal animals failed to develop clinical responses to this bacterial challenge and their lungs did not show the growth of P. haemolytica on cultures, whereas two of four control calves had elevated temperatures and developed necropurulent pneumonia with the isolation of P. haemolytica from the lungs. The principal calves had developed high levels of cytotoxin neutralizing antibodies in their sera following parainfluenza-3 virus-P. haemolytica infection. This demonstrated that immunity against pneumonic pasteurellosis can be achieved, with a suggestion that further search for an effective vaccine for P. haemolytica is warranted.     

Evidence that blood-borne infection is involved in the pathogenesis of bovine pneumonic pasteurellosis

Five calves were inoculated intravenously with 10(8) colony forming units (cfu) of Pasteurella haemolytica A1; the mean score for pneumonic consolidation 3 days post-inoculation was 28%, and the mean clinical score was 7.8. Five calves inoculated intratracheally with 10(9) cfu of the same strain of P. haemolytica had comparable scores (34% and 8.8). Histological lesions of fibrinous pneumonia were similar in all calves. P. haemolytica was recovered from all but one of the affected lungs. From one calf killed in extremis 3 hours after intravenous inoculation, numbers of bacteria recovered from lung were 1,000-fold greater than from liver and spleen. A similar difference in bacterial numbers was also obtained from a gnotobiotic calf killed in extremis, 12 hours after intravenous inoculation of 10(8) cfu P. haemolytica. Evidence from these experiments supports the hypothesis that the blood-borne route is important in the pathogenesis of bovine pneumonic pasteurellosis.     

Experimental infections of goats with Mycoplasma mycoides subspecies mycoides, LC type

In five experiments 29 goats were infected experimentally by five different routes with a strain of Mycoplasma mycoides subspecies mycoides, LC type, isolated from a contagious caprine pleuropneumonia-like outbreak on a farm in northern Sweden. All the goats were colonised except those inoculated subcutaneously with small doses. In its pattern of pathogenicity this strain was similar to other experimentally tested strains except that peroral infection in kids produced no clinical signs. A 'contact' goat was also colonised but the clinical signs seen in it were probably due to a concomitant infection with Pasteurella haemolytica.     

The effect of parainfluenza virus type 3 and Pasteurella haemolytica on oxygen-dependent and oxygen-independent bactericidal mechanisms of ovine pulmonary phagocytic cells

Groups of caesarean-derived, colostrum-deprived lambs were inoculated by the intratracheal route with Pasteurella haemolytica, either alone or 4 or 6 days after the inoculation of parainfluenza virus type 3 (PI3). Other groups were inoculated with PI3 followed by veal infusion broth, or with uninfected cell culture fluid followed by veal infusion broth (controls). All lambs were killed 24 h after the second inoculation. Pulmonary phagocytic cells were recovered by lavage and separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation. Bacterial proliferation was detected in the lungs of all five lambs inoculated with P. haemolytica 6 days after PI3 but in only one of five inoculated with P. haemolytica 4 days after PI3 and one of five inoculated with P. haemolytica alone. The number of phagocytic cells recovered from the lungs was highest in animals inoculated with P. haemolytica 6 days after PI3 and, overall, a greater number of both AM and neutrophils was recovered from the lungs of animals where bacterial proliferation occurred (greater than 10(5.0) P. haemolytica 100 g-1 lung) than from those that controlled the bacterial infection. Oxygen-dependent bactericidal activity of AM and neutrophils was measured by chemiluminescence. Infection with PI3 and P. haemolytica increased the chemiluminescence responses. The highest responses were recorded from lambs inoculated with P. haemolytica 6 days after PI3, the group where pulmonary clearance was poorest. Overall, responses were higher in lambs in which bacterial proliferation occurred than in those that controlled the infection. On the other hand, oxygen-independent bactericidal activity, measured by the direct effects of neutrophil lysates on Escherichia coli, was lowest in lambs inoculated with P. haemolytica 6 days after PI3 and was lower in lambs where bacterial proliferation occurred.     

Properties of a respiratory syncytial virus isolated from a sheep with rhinitis

A virus isolated from a yearling cross-bred ewe was identified as respiratory syncytial virus (RSV) by indirect immunofluorescence and by virus neutralization with bovine RSV antisera. The virus caused a mild conjunctivitis in 3-month-old lambs when inoculated alone. Although clinical signs of pneumonia were not observed, there was gross and microscopic evidence of pulmonary inflammation in the lungs of lambs inoculated with either the sheep RSV isolate alone or in conjunction with Pasteurella haemolytica. Lung lesions in the dual infection were more severe, with approximately 10% of the total lung mass affected. Lavage fluids from lambs inoculated with virus and bacteria contained approximately 3 times more inflammatory cells than from control lambs or lambs inoculated with virus only. The sheep RSV isolate was classified as a mild respiratory pathogen in lambs of this age. Speculations on the potential importance of this virus in interspecies transmission to cattle and goats were discussed.     

Pasteurella multocida and Bordetella bronchiseptica in atrophic rhinitis and pneumonia in swine.

A total of 163 pigs from nine farrow-to-finish herds representing various levels of atrophic rhinitis (AR) were selected for postslaughter examination of AR and pneumonia. Nasal swabs and lungs were cultured for detection of Bordetella bronchiseptica and Pasteurella multocida. Seventy-three pigs were examined at eight weeks of age and 90 contemporaries at six months of age. Mean AR scores were 1.21 and 1.11 for the eight week and six month old pigs, respectively (0 = normal, 3 = severe). In individual pigs increasing AR score was related to increasing pneumonia score in eight week old pigs but not in six month old hogs. In eight week old pigs, B. bronchiseptica and P. multocida were isolated more frequently from pigs with higher AR scores. From nasal swabs of six month old hogs, Bordetella was almost never recovered while Pasteurella was frequently isolated score. Toxigenic type DP. multocida was isolated from nasal cultures of only seven (4%) pigs and from lung cultures of only one pig. Pasteurella was never isolated from lungs of the eight week old pigs and Bordetella never from the six month old hogs. The isolation rate of P. multocida, predominantly type A, from lungs of six month old pigs increased from 11% in grossly normal lungs to 86% in lungs with severe pneumonia. Pigs from one herd free from lesions of AR and pneumonia were also examined; type AP. multocida was isolated from nasal cultures of one of six eight week old pigs. Somatic antigens of P. multocida were determined for 94 nasal and 20 lung isolates. Somatic serovar 3 was found in 93% of the nasal isolates and in all lung isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bordetella bronchiseptica and toxigenic type D Pasteurella multocida as agents of severe atrophic rhinitis of swine

Bordetella bronchiseptica and toxigenic type-D Pasteurella multocida were cultured from pigs in each of five herds diagnosed as having severe atrophic rhinitis (AR). B. bronchiseptica alone, P. multocida alone, or both organisms isolated from four herds were inoculated intranasally into 1-week-old gnotobiotic pigs which were necropsied 4 weeks post-inoculation (PI). Nasal turbinate atrophy in B. bronchiseptica-inoculated pigs was moderate to severe, while P. multocida-inoculated pigs had slight to severe atrophy. Pigs inoculated with both organisms had moderate to complete turbinate atrophy. P. multocida was reisolated at necropsy from all pigs receiving the organism except those having no turbinate damage. B. bronchiseptica and P. multocida from a fifth herd were simultaneously inoculated into six naturally farrowed 6-day-old SPF pigs. Necropsy performed 4 weeks PI revealed severe to complete turbinate atrophy. Nasal turbinates were normal for control pigs in both experiments.     

The effect of parainfluenza virus type 3 on the phagocytic cell response of the ovine lung to Pasteurella haemolytica

Groups of Caesarean-derived, colostrum-deprived lambs were inoculated by the intratracheal route with Pasteurella haemolytica 4 to 6 days after the inoculation of parainfluenza virus type 3 (PI3). Some were killed immediately (0 h) and others 24 h later. Control groups were inoculated with PI3 alone, P. haemolytica alone or media alone. Pulmonary phagocytic cells, P. haemolytica and PI3 were recovered by pulmonary lavage. The phagocytes were separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation and examined biochemically and microbiologically. Twenty-four hours after the inoculation of P. haemolytica bacterial proliferation to greater than 0 h levels had occurred in four of six animals inoculated with P. haemolytica alone, two of eight inoculated with P. haemolytica 4 days after PI3 and all of eight inoculated with P. haemolytica 6 days after PI3. Mean bacterial numbers in animals inoculated with P. haemolytica 6 days after PI3 and killed at 24 h (10(9.1 +/- 1.9)) were significantly higher than they were in the other two groups killed at this time (PI3 4 days, P. haemolytica 24 h, mean = 10(5.3 +/- 1.7); P. haemolytica alone 24 h, mean = 10(4.5 +/- 2.9)). Pneumonic lesions were also more severe in the first group. This defect in pulmonary clearance and increase in the severity of pneumonia in animals inoculated with P. haemolytica 6 days after PI3 coincided with a 1000-fold decrease in virus titres in the lung between Day 6 and Day 7 after virus inoculation and the first detectable evidence of the host's immune response. The virus infection resulted in a significant increase in the number of AM that could be recovered from the lung and an increase in the number of AM with cytoplasmic vacuolation. However, there was no difference in the total number of AM or the number of vacuolated AM between animals that controlled the P. haemolytica infection and those in which proliferation of P. haemolytica occurred. The inoculation of P. haemolytica resulted in a 100-fold increase in the number of neutrophils in the lavage fluid, but there were no differences between virus-infected and uninfected animals, nor was there a difference between animals that controlled the P. haemolytica infection and those that did not.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pneumonia in calves produced with aerosols of Pasteurella multocida alone and in combination with bovine herpesvirus 1.

Pathological changes in respiratory tracts were studied in 30 calves following exposure to aerosols of Pasteurella multocida or to bovine herpesvirus 1 and P. multocida. Two groups of five calves were exposed to aerosols of one of two types of P. multocida only, which produced lobar pneumonia in one calf of each group. Another five groups of four calves were exposed to aerosols of bovine herpesvirus 1 and four to seven days later to one of the two types or one sub-type of P. multocida. Extensive necropurulent lesions were produced throughout the respiratory tract with each type of P. multocida in all four calves in three groups but none in the remaining two groups. The pathological changes differed from those produced following similar exposures to bovine herpesvirus 1 and P. haemolytica, in that the exudate in air passages and alveoli was more purulent and streaming (oat) cells and large mononuclear eosinophilic granulocytes were absent. This is the first report of experimental respiratory disease in cattle as a result of aerosol exposure to P. multocida alone or in combination with bovine herpesvirus 1.     

Experimental bovine pneumonic pasteurellosis. I. Prevention of the disease.

Fifteen three- to six-month old Hereford-cross calves were divided into three groups. The first group was inoculated with bovid herpersvirus 1 (Strain 108), the second with a commercial intranasal vaccine against bovid herpesvirus 1 and the third group acted as controls. At least three weeks after vaccination, all calves were weaned, placed in an environmental chamber at 25.0 degrees C (days) and -13.3 degrees C (nights) and challenged with an aerosol of bovid herpesvirus 1 followed four days later by an aerosol of Pasteurella haemolytica. All surviving calves were sacrificed four days after the second aerosol. None of the calves inoculated with bovid herpesvirus 1 virus or the commercial vaccine developed a generalized pneumonia, although there were one or two nodules (4--8 mm diameter) in two of the calves given the commercial vaccine. Four of the five control calves had extensive lobar pneumonia at necropsy, two of the five died from the disease. Details of the clinical, pathological, bacteriological, virological and some of the serological findings are reported.     

Exacerbation of murine respiratory mycoplasmosis in gnotobiotic F344/N rats by Sendai virus infection

Six- to eight-week-old gnotobiotic F344/N rats were inoculated intranasally with 10(5.0) colony-forming units of Mycoplasma pulmonis or were sham inoculated, then one week later were given 10(0.2) 50% tissue culture infective doses of Sendai virus or sterile medium. Groups of rats were killed immediately after virus inoculation and three, five, ten, and 20 days later. Lesions in nasal passages, middle ears, larynxes, tracheas, and lungs from half of the rats in each group were subjectively scored. Organs from the other rats were quantitatively cultured for M. pulmonis and for Sendai virus. Rats given Sendai virus alone had mild, patchy, necrotizing rhinitis, laryngitis, tracheitis, and bronchitis, but not bronchiolitis or interstitial pneumonia. M. pulmonis alone induced mild lesions of murine respiratory mycoplasmosis including mild to moderate suppurative rhinitis, otitis media, laryngitis, and tracheitis with submucosal lymphoid accumulation and epithelial hyperplasia, but not lung lesions. Rats given M. pulmonis and Sendai virus had severe lesions characteristic of advanced mycoplasmal disease throughout the respiratory tract, including suppurative bronchitis with extensive lymphoid accumulations and epithelial hyperplasia; some rats also had suppurative pneumonia and bronchiectasis. Larger numbers of M. pulmonis colony-forming units were in rats given Sendai virus, but there was no statistically significant difference in Sendai virus infectious units between rats also given M. pulmonis and those given virus only.     

Immunoperoxidase evaluation of pneumonic lesions induced by Pasteurella haemolytica in calves

Calves inoculated with Pasteurella haemolytica serovar 1 developed lesions of coagulation necrosis in the lungs that were sharply demarcated by leukocytes. The P haemolytica antigen was detected in the area of coagulation necrosis in histologic sections, using an immunoperoxidase technique. In the central area of the necrotic tissue, the bacterial antigen was diffusely presented in the necrotic alveolar wall, fibrin, serous exudate, and degenerated leukocyte. The bacterial antigen also was found in some groups of degenerating leukocytes around the necrotic tissue. The bacterial colonies among these leukocytes had strong specific reactions against P haemolytica. The bacterial antigen was observed in the cytoplasm of macrophages in alveoli around the necrotic lesion. These findings confirmed that coagulation necrosis is an important lesion in calves with pneumonia caused by P haemolytica.     

Isolation of Pasteurella haemolytica from the nasal cavity of goats

Twenty transport-stressed goats were divided into two groups. The first group was further stressed with steroid. Pasteurella haemolytica was found at various sites in the nasal cavity of goats in this group as early as 2 weeks post-transportation. The successful isolations continued consistently with more goats having pure growth of P. haemolytica at later stages. Mild catarrh rhinitis, loss of epithelial cilia and erosions were the main lesions observed in the nasal cavity. Goats in the second group that were not given steroid injections had inconsistent bacterial isolation and less severe pathological lesions.     

Investigations into the possible role of Mycoplasma arginini in ovine respiratory disease

The inoculation of eight five- to seven-month-old sheep by the respiratory route with a culture of Mycoplasma arginini, administered simultaneously with or two days before a culture of Pasteurella haemolytica A2, did not lead to the pulmonary establishment of either organism. Minor lung changes found at slaughter seven days later were therefore considered not to have been induced by the inocula. Two other groups of seven sheep each were initially inoculated intratracheally with an ampicillin-treated lung lesion homogenate in which only M ovipneumoniae was detectable. After seven days one group was inoculated intranasally and intratracheally with mixed cultures of M arginini and P haemolytica A2, and one with P haemolytica A2 alone. In the M arginini-treated group pyrexia peaked earlier and one animal died, but no macroscopic or microscopic differences were apparent between the two groups at necropsy 10 to 11 days later; six sheep from each group had lung lesions indistinguishable from ovine atypical pneumonia. M arginini was isolated in high titre from the respiratory tract of two animals in the M arginini-treated group, including the sole fatality. However, an adventitious parainfluenza type 3 virus infection, identified in four animals from the M arginini-treated group and one from the other, may have been responsible for the inter-group clinical differences. It was concluded that the strain of M arginini used was capable neither of predisposing the lung to secondary invasion by P haemolytica A2, nor of exacerbating the pneumonia and effects elicited with M ovipneumoniae and P haemolytica.     

Experimental infection of sheep with Mycoplasma ovipneumoniae and Pasteurella haemolytica

A group of Caesarian-derived, colostrum-deprived lambs was inoculated intranasally and intratracheally with a virulent Mycoplasma ovipneumoniae isolate selected from ovine mammary studies and propagated in an ovine mammary gland. Other groups of lambs were inoculated with M. ovipneumoniae in combination with Pasteurella haemolytica type Al or P. haemolytica alone. The M. ovipneumoniae isolate alone did not induce any specific pneumonic lesions in the lambs and when combined with P. haemolytica type Al did not increase the severity of the P. haemolytica-type lesions. Fifty percent of lambs inoculated with P. haemolytica developed a purulent and exudative bronchopneumonia with pleurisy and high titres of P. haemolytica were recovered from these lesions.     

Presence of bacterial glycocalyx and fimbriae on Pasteurella haemolytica in feedlot cattle with pneumonic pasteurellosis.

This investigation was conducted to determine if Pasteurella haemolytica within feedlot cattle affected by pneumonic pasteurellosis express fimbriae (pili) and bacterial glycocalyx. Bacteriological culture of pulmonary tissue from three calves with fibrinous pneumonia resulted in heavy growth of P. haemolytica. Transmission electron microscopy of the lungs showed numerous microcolonies of gram-negative bacteria with morphology typical of Pasteurella haemolytica. The cells within these microcolonies possessed bacterial glycocalyces which stained with ruthenium red. Glycocalyx-encased microcolonies were also present in specimens examined by scanning electron microscopy. Typical P. haemolytica cells were evident in a tracheal specimen and these bacteria had radial glycocalyces consistent with polysaccharide and proteinaceous material condensed on linear structures suggestive of fimbriae. The pathogenetic importance of the bacterial glycocalyx and fimbriae in shipping fever pneumonia has yet to be established but their presence in clinical cases of Pasteurella pneumonia in feedlot cattle further supports a possible role in the initiation and progression of this disease as well as bacterial resistance to antimicrobial agents.