Interaction between heat-stable hemolytic substance from Haemophilus pleuropneumoniae and porcine pulmonary macrophages in vitro.

Cytocidal and anti-phagocytic effects of the heat-stable hemolytic substance produced in culture supernatant by Haemophilus pleuropneumoniae serotype 2 on porcine pulmonary macrophages (PMP) were investigated in vitro. The substance was partially purified by sucrose density gradient ultracentrifugation, and it seems to be a carbohydrate. The partially purified preparation of the heat-stable carbohydrate with hemolytic activity was used as H. pleuropneumoniae hemolysin throughout this study. Viability of the PMP cells exposed to hemolysin with a hemolytic unit of 32 decreased by about 35% during the first 30-min exposure. The response was hemolysin dose- and exposure time-dependent and was neutralized by the anti-hemolysin antiserum. The PMP cells were found to be the most sensitive to hemolysin among the pulmonary, peritoneal, and peripheral blood macrophages obtained from the same animal. Heat treatment of the hemolytic substance in the culture supernatant at 121 degrees C for 2 h did not significantly (P greater than 0.05) alter the cytocidal and hemolytic activities. Pretreatment of the PMP cells with hemolysin at a sublethal dose significantly (P less than 0.01) reduced their phagocytic abilities. Thus, the heat-stable carbohydrate H. pleuropneumoniae hemolysin appears to possess the potential for both cytocidal and antiphagocytic effects on the PMP cells. Death and impaired phagocytosis of the PMP cells by the hemolysin, if it occurs in vivo, would enhance the chances of survival for an invading H. pleuropneumoniae organism. These findings promote understanding of the pathogenesis of pleuropneumonia caused by H. pleuropneumoniae in swine.

Intranasal inoculation of chickens with encapsulated or nonencapsulated variants of Haemophilus paragallinarum: electron microscopic evaluation of the nasal mucosa.

Chickens were inoculated intranasally with encapsulated or nonencapsulated strains of Haemophilus paragallinarum. The nasal mucosa of the chickens were examined, using scanning and transmission electron microscopy. In chickens given the encapsulated variant, marked loss of cilia and microvilli, infiltration of leukocytes, and deposition of a mucopurulent substance were seen on the surface of the nasal mucosa; the number of microvillous cells were markedly increased, and infiltration of mast cells into the lamina propria of the mucous membrane was observed. Numerous encapsulated organisms were found near cilia and on microvillous cells, and the capsule surrounding the organisms appeared to mediate attachment of the organisms to the cilia. In contrast, the nasal mucosa of the chickens given the nonencapsulated variants changed little morphologically. A few nonencapsulated organisms were found near cilia. Colonization of the nasal mucosa by encapsulated organisms probably was essential to induce the morphologic changes seen in the nasal mucosa.

Lesions induced in the respiratory tract of chickens by encapsulated or nonencapsulated variants of Haemophilus paragallinarum.

Lesions induced in chickens by an encapsulated or nonencapsulated strain of Haemophilus paragallinarum were investigated. In terms of lesion severity, major differences in pathogenicity were observed between the encapsulated and nonencapsulated variants. The principal lesion manifested by the encapsulated variant was an acute catarrhal inflammation of the upper respiratory tract, mainly of the nasal cavity and paranasal sinus. Infiltration of a large number of mast cells into the lamina propria of the mucous membrane of the nasal cavity was also characteristic. Numerous organisms were found on the cilia or on the surface of the epithelial cells of the nasal mucosa. Chickens with these histologic lesions had severe clinical signs of coryza, and organisms were recovered in high numbers from the nasal cavity and paranasal sinus. In contrast, chickens that were given the nonencapsulated variant did not have clinical signs of coryza, and the organisms were recovered in low numbers from the inoculated sites; slight histopathologic lesions were observed in the nasal mucosa at postinoculation day 1. Mast cell infiltration in the chickens inoculated with the encapsulated variant indicated that mast cells may be responsible for producing clinical signs of coryza via the activation of pharmacologic mediators. Adherence to and colonization of the encapsulated variant on the nasal mucosa seems to be a first step of the infection.

Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica.

Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.

Purification of dermonecrotic toxin from a sonic extract of Pasteurella multocida SP-72 serotype D.

A procedure was developed to purify dermonecrotic toxin (DNT) from a sonic extract of a serotype D strain of Pasteurella multocida. Sonic extract containing DNT was applied to a DEAE-Sephacel column and eluted by a linear gradient of NaCl. Upon rechromatographing, fractions with dermonecrotic activity for guinea pigs were applied on a second Sephacel column, and a pooled fraction with the toxic activity was filtered through a Sephadex G-200 column. Pooled fractions with the toxic activity were subjected to polyacrylamide disc gel electrophoresis (PAGE), and the toxic substance was eluted from each sliced gel. Eluted fractions with the toxic activity were rechromatographed on a second Sephadex G-200 column, and a pooled fraction with high dermonecrotic activity was referred to as a purified DNT. The activity of purified DNT was increased by 1,000 times, and the average yield was about 1.8%. The purified DNT was homogeneous as determined by Ouchterlony double immunodiffusion, crossed immunoelectrophoresis, and thin-layer isoelectric focusing in polyacrylamide gels and gave a single band on PAGE and sodium dodecyl sulfate-PAGE. The molecular weight of the toxin was ca. 160,000 as determined by sodium dodecyl sulfate-PAGE. The isoelectric point of the toxin was ca. 4.7 to 4.8. Amino acid analysis of the purified DNT revealed that the toxin was composed of characteristically high proportions of glutamic acid, aspartic acid, glycine, proline, alanine, and leucine. The minimal necrotizing dose of the toxin was about 1 ng of protein, and the 50% lethal dose per mouse was 0.2 micrograms. The purified DNT was heat labile and sensitive to inactivation by trypsin, Formalin, and glutaraldehyde.

Characterization of dermonecrotic toxin produced by serotype D strains of Pasteurella multocida.

Dermonecrotic toxin (DNT) produced by serotype D strains of Pasteurella multocida, isolated from pigs, was characterized and compared with DNT produced by Bordetella bronchiseptica. The DNT prepared by sonication from P multocida or B bronchiseptica had dermonecrotic activity and lethal toxicity for guinea pigs and mice, and also induced marked atrophy of spleens in the mice. Toxicity of P multocida or B bronchiseptica DNT was completely inactivated by heating at 70 C for 30 minutes, and was reduced by treatment with trypsin, formalin, or glutaraldehyde, indicating that the DNT may be a protein. Although biologic and toxic properties of P multocida DNT were similar to those of B bronchiseptica DNT, cross-neutralization tests between P multocida and B bronchiseptica indicated that DNT from the 2 bacterial species were serologically distinct.