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. 2002 Feb;70(2):481-90.
doi: 10.1128/iai.70.2.481-490.2002.

Role of the Dermonecrotic Toxin of Bordetella Bronchiseptica in the Pathogenesis of Respiratory Disease in Swine

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Free PMC article

Role of the Dermonecrotic Toxin of Bordetella Bronchiseptica in the Pathogenesis of Respiratory Disease in Swine

Susan L Brockmeier et al. Infect Immun. .
Free PMC article

Abstract

Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT(+) parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.

Figures

FIG. 1.
FIG. 1.
Construction of dnt mutant B58GP. (A) Map of the 5-kb insert of pBH1 containing the dnt gene. (B) Map showing the insert after replacement of the internal NsiI fragment (1.8 kb) with the kanamycin resistance cassette (1.2 kb).
FIG. 2.
FIG. 2.
PCR products separated by agarose gel electrophoresis. Lanes: 1, pBHI control; 2, B58GP genomic DNA. The molecular size of the markers is indicated in base pairs on the left.
FIG. 3.
FIG. 3.
Expression of DNT, FHA, adenylate cyclase toxin, and pertactin. Proteins contained in bacterial cell extracts prepared from strain B58 (lanes 1, 3, 5, and 7) or B58GP (lanes 2, 4, 6, and 8) (A) or KM22 (lanes 1, 3, 5, and 7), or KB24 (lanes 2, 4, 6, and 8) (B) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. DNT, FHA, adenylate cyclase toxin, and pertactin were detected by incubation with monoclonal antibodies AE9 (lanes 1 and 2), X3C (lanes 3 and 4), 9D4 (lanes 5 and 6), and BPE3 (lanes 7 and 8), respectively.
FIG. 4.
FIG. 4.
Colonization of the nasal cavity of pigs inoculated with B58 (□), B58GP (▪), KM22 (○), or KB24 (•), as determined by nasal washes performed 7, 14, 21 (and 28 in the first experiment) days postinfection. The difference in the level of colonization between the parent and isogenic mutant strains was statistically significant for B58-B58GP on days 14 (P = 0.046), 21 (P = 0.016), and 28 (P = 0.003) and for KM22-KB24 on days 7 (P = 0.001), 14 (P = 0.001), and 21 (P = 0.01).
FIG. 5.
FIG. 5.
Colonization of the turbinate, trachea, and lungs of pigs inoculated with B58(formula image), B58GP (▤), KM22 (formula image), or KB24 (▥) at 28 days postinfection. The difference in the level of colonization between B58 and B58GP was statistically significant for the turbinate (P = 0.013).
FIG. 6.
FIG. 6.
Cross sections of snouts showing the degree of turbinate atrophy at 28 days postinfection in pigs inoculated with KM22 (A) or KB24 (B).
FIG. 7.
FIG. 7.
Pathological examination of the lungs of pigs 28 days after infection with KM22. (A) Gross photograph showing an area of tan, fibrous consolidation of the right cranial, middle, and part of the caudal lung lobes. (B) Photomicrograph showing moderate to marked thickening of the alveolar septa with fibrillar material, fibroblasts, and macrophages; marked type II pneumocyte hyperplasia and pneumocytes which contain abundant vacuolated cytoplasm; and alveolar lumina which contain variable quantities of fibrin, sloughed epithelium, and macrophages. Hematoxylin and eosin stain; 1 cm = 55 μm. (C) Photomicrograph showing abscessed lung lobule. Note the core of necrosis surrounded by a thick dense band of collagenous connective tissue. Hematoxylin and eosin stain. 1 cm = 350 μm. (D) Photomicrograph showing extensively focal obliteration of the alveolar lumina with infiltrates of large numbers of neutrophils and neutrophilic bronchiolitis. Note the infolding of the bronchiole mucosa and epithelial hyperplasia. Hematoxylin and eosin stain. 1 cm = 110 μm.
FIG. 8.
FIG. 8.
B. bronchiseptica immunoreactivity in the cilia of epithelial cells of a bronchus in a pig infected with KB24 (A) and in the cilia of epithelial cells of a bronchus (arrow) (B) as well as an area of abscessation of the lung (C) in a pig infected with KM22.

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