Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant (p<0.01), and these effects were significantly (p<0.02, p<0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant (p<0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Ultrastructural pathology of nasal and tracheal mucosa of rabbits experimentally infected with Pasteurella multocida serotype D:1.

Sixteen 8- to 9-week-old Pasteurella multocida-free New Zealand White rabbits were divided into two equal groups. The first group was inoculated intranasally with P multocida serotype D:1 strain and the second group that was inoculated with phosphate-buffered saline (PBS) only was used as a control group. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits in group 1 and from tracheal swabs of seven rabbits in this group. Four rabbits in group 1 died with clinical signs of septicaemia, two rabbits had mucopurulent nasal discharge and pneumonic lesions and the other two did not show any clinical signs or gross lesions. The ultrastructural changes detected were deciliation or clumping of cilia of ciliated epithelium, cellular swelling, vacuolation and sloughing. The subepithelial capillaries showed congestion, intravascular fibrin deposition, platelets aggregation and endothelial injury. Pasteurella multocida was observed attached to the injured endothelial cells. Heterophils, mast cells, vacuolated monocytes and macrophages infiltrated the lamina propria and between the degenerated epithelial cells.

Ultrastructural observation of nasal and pulmonary intracellular Pasteurella multocida A:3 in rabbits.

Sixteen 8- to 9-week-old Pasteurella multocida-free rabbits were divided into two equal groups. Eight rabbits in one group were inoculated intranasally with P. multoida type A:3. The other eight were inoculated intranasally with phosphate-buffered saline and used as controls. Nasal swabs taken before and after inoculation were cultured for bacterial isolation. Post-mortem nasal swabs and lung samples were cultured for bacteriological isolation. Nasal mucosa and lung samples were collected and processed for transmission electron microscopy. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits and from the lungs of four infected rabbits. Degenerative ultrastructural changes in epithelial cells and endothelial cells were seen in the infected rabbits. Deciliation of the ciliated epithelium and hyperplasia of the goblet cells in the nasal mucosa were noted. Thickening of the alveolar septa due to hyperplasia of type II pneumocytes, swelling of the endothelial lining of capillaries and infiltration of inflammatory cells were also observed. Intracellular invasion of the nasal epithelial cells and of type II pneumocytes by the organism was observed. Coccobacilli were observed in membrane-bound vacuoles in the cytoplasm of these cells. The vacuoles were adjacent to the host-cell mitochondria and some of these vacuoles appeared to be fused to the mitochondrial membrane. Some type I pneumocytes with intracellular membrane-bound vacuoles containing bacterial cells showed protrusions, which appeared to detach into the alveolar lumina. These results indicated that P. multocida serotype A:3 in rabbits can invade the epithelial cell and cause structural changes in the interstitium, epithelium and endothelium. Heterophils and macrophages appear to play important roles in tissue injury.

In vitro study of Pasteurella multocida adhesion to trachea, lung and aorta of rabbits.

In vitro experiments were undertaken to study the adhesion and colonization to tracheal mucosa, lung and aorta explants from freshly killed rabbits of two different strains of Pasteurella multocida. Serotype A:3 (capsulated, fimbriae +, haemagglutination -, dermonecrotic toxin -) isolated from a rabbit with rhinitis, and serotype D:1 (non-capsulated, fimbriae +, haemagglutination +, dermonecrotic toxin +) isolated from a dead rabbit with septicaemia, were used. When the explants were observed under the scanning electron microscope, the type D strain was highly adherent to trachea and aorta explants compared to the type A strain. Adhesion to lung explants was best achieved by the type A strain after 45 min incubation, but after 2 h incubation no significant difference was observed between the strains. Our data indicate that the presence of fimbriae and the absence of capsule seem to enhance the adherence of P. multocida type D strain to tracheal tissue. The capsular material of P. multocida type A strain and the toxin of the type D strain seem to influence the adherence to lung tissue in rabbit. Adhesion of strain D to aorta may indicate the expression of receptors on the endothelium to that strain and may also explain the ability of certain strains to cause septicaemia.

Ultrastructural pathology of the upper respiratory tract of rabbits experimentally infected with Pasteurella multocida A:3.

Twenty-four 8 to 9 week-old Pasteurella multocida -free rabbits were divided into three equal groups, the first group was pretreated with hydrocortisone and inoculated intranasally with pasteurella multocida serotype A:3. The second group was inoculated intranasally with P. multocida without hydrocortisone treatment. The third group was inoculated with phosphate buffered saline only and used as a control group. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits in group 1 and 2 and from the trachea of seven rabbits in group 1 and five rabbits in group 2. This study was conducted to observe the ultrastructural changes of the upper respiratory tract of hydrocortisone treated and non-treated rabbits infected with P. multocida serotype A:3. The ultrastructural changes detected in infected rabbits were ciliary destruction and deciliation of the ciliated epithelial cells, cellular swelling, goblet cell hyperplasia and endothelial cell damage. Pasteurella multocida was observed attached to the degenerated cilia, microvilli and mucus. Pasteurella multocida infection was associated with inflammatory responses, which may have caused tissue damage. It is possible that hydrocortisone modulates the severity of infection as an immune suppressor and an inhibitor of goblet cell secretion.

Genotypic and phenotypic relationship in Burkholderia pseudomallei indicates colonization with closely related isolates.

Seven isolates of Burkholderia pseudomallei from cases of melioidosis in human (2 isolates) and animal (2 isolates), cat (one isolate) and from soil samples (2 isolates) were examined for in vitro sensitivity to 14 antimicrobial agents and for presence of plasmid DNA. Randomly amplified polymorphic DNA (RAPD) analysis was used to type the isolates, using two arbitrary primers. All isolates were sensitive to chloramphenicol, kanamycin, carbenicillin, rifampicin, enrofloxacin, tetracycline and sulfamethoxazole-trimethoprim. No plasmid was detected in all the isolates tested. RADP fingerprinting demonstrated genomic relationship between isolates, which provides an effective method to study the epidemiology of the isolates examined.