Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms

Abstract

Background: Pasteurella multocida type A (PmA) is considered a secondary agent of pneumonia in pigs. The role of PmA as a primary pathogen was investigated by challenging pigs with eight field strains isolated from pneumonia and serositis in six Brazilian states. Eight groups of eight pigs each were intranasally inoculated with different strains of PmA (1.5 mL/nostril of 10e7 CFU/mL). The control group (n = 12) received sterile PBS. The pigs were euthanized by electrocution and necropsied by 5 dpi. Macroscopic lesions were recorded, and swabs and fragments of thoracic and abdominal organs were analyzed by bacteriological and pathological assays. The PmA strains were analyzed for four virulence genes (toxA: toxin; pfhA: adhesion; tbpA and hgbB: iron acquisition) by PCR and sequencing and submitted to multilocus sequence typing (MLST).

Results: The eight PmA strains were classified as follows: five as highly pathogenic (HP) for causing necrotic bronchopneumonia and diffuse fibrinous pleuritis and pericarditis; one as low pathogenic for causing only focal bronchopneumonia; and two as nonpathogenic because they did not cause injury to any pig. PCR for the gene pfhA was positive for all five HP isolates. Sequencing demonstrated that the pfhA region of the HP strains comprised four genes: tpsB1, pfhA1, tpsB2 and pfhA2. The low and nonpathogenic strains did not contain the genes tpsB2 and pfhA2. A deletion of four bases was observed in the pfhA gene in the low pathogenic strain, and an insertion of 37 kb of phage DNA was observed in the nonpathogenic strains. MLST clustered the HP isolates in one group and the low and nonpathogenic isolates in another. Only the nonpathogenic isolates matched sequence type 10; the other isolates did not match any type available in the MLST database.

Conclusions: The hypothesis that some PmA strains are primary pathogens and cause disease in pigs without any co-factor was confirmed. The pfhA region, comprising the genes tpsB1, tpsB2, pfhA1 and pfhA2, is related to the pathogenicity of PmA. The HP strains can cause necrotic bronchopneumonia, fibrinous pleuritis and pericarditis in pigs and can be identified by PCR amplification of the gene pfhA2.

Keywords: Bronchopneumonia; MLST; Pigs; Polyserositis; Respiratory diseases; pfhA.

Fig. 1

Lesions caused by Pasteurella multocida type A in experimentally challenged pigs. a. Lung, group 2. Focally extensive hemorrhagic pleuropneumonia in the cardiac lobe with fibrin on the pleura. b. Thoracic cavity, group 7. Diffuse fibrinous pleuritis and pericarditis (*). c. Heart, group 3. Diffuse fibrinous pericarditis. d. Abdominal cavity, group 3. Fibrinous peritonitis. e. Lung, group 2. Coagulation necrosis area in the lung parenchyma (*), surrounded by abundant inflammatory cells, mild proliferation of connective tissue and suppurative exudate in the bronchioles (thin arrow). HE. Bar, 100 μm. f. Lung, group 2. Abundant (+++) inflammatory exudate, predominantly suppurative, intra-alveolar in a coagulation necrosis area on the lung parenchyma. HE. Bar, 10 μm. g. Spleen, group 5. Multiple splenic infarcts with fibrin threads on the capsule. h. Lung, group 2. Abundant antigen labeling of P. multocida (red labeling) in a coagulation necrosis area in the lung and between degenerated inflammatory cells. Bar, 50 μm. i. Lung, group 2. Coagulation necrosis area in the lung with P. multocida antigen labeling (red spots) in the cytoplasm of phagocytic cells. Bar, 5 μm. j. Spleen, group 5. Moderate (++) antigen labeling of P. multocida (red labeling) in a necrotic area. Bar, 20 μm. Immunohistochemistry, streptavidin-biotin-peroxidase method (LSAB™) with 3-amino-9-ethylcarbazole (AEC) and counterstaining with Mayer’s hematoxylin

Fig. 2

Schematic representation of the pfhA gene region of P. multocida type A according to pathogenic classification

Fig. 3

Dendrogram representative of the 7 concatenated gene sequences used for the MLST of the 8 isolates of P. multocida type A. The MLST was generated by joint analysis of seven housekeeping genes (adk, aroA, deoD, gdhA, g6pD, mdh, and pgi) using the RIRDC MLST database