Intravascular macrophages in lungs of pigs infected with Haemophilus pleuropneumoniae

Pigs were inoculated intratracheally with a virulent or an avirulent isolate of Haemophilus pleuropneumoniae serotype 5 and sacrificed during the first 24 hours post-inoculation. Intravascular macrophages were examined by electron microscopic and morphometric techniques. Samples of lung were taken from regions with no macroscopic lesions (Zone 0), 2.5 to 3.0 cm from lesions (Zone 1), and from the immediate edge of lesions (Zone 2). Those pigs inoculated with the avirulent isolate did not develop lesions. Pigs given the virulent isolate consistently developed necrohemorrhagic lesions in the dorsolateral aspect of the caudal and middle lung lobes. Relative volumes of intravascular macrophages in Zones 1 and 2 increased with increased time post-inoculation; in pigs given the avirulent isolate, intravascular macrophage volume decreased with increased time post-inoculation. Cytoplasmic volume to nuclear volume ratios for macrophages in Zone 2 from pigs with necrohemorrhagic lesions progressively increased with increased time post-inoculation. Enlarged intravascular macrophages had large nuclei, prominent nucleoli, and abundant cytoplasm. Increased cytoplasmic volume was the result of increased numbers of lysosomes, phagosomes, rough and smooth endoplasmic reticulum, and large Golgi complexes. Pigs inoculated with the virulent bacteria had IV macrophages with large phagosomes that contained necrotic cell debris and fibrin. Macrophages with phagosomes were more frequent in the 6-, 9-, and 24-hour sample periods of pigs with lesions than in any other group. Intercellular adhesion plaques (ICAP) were present between IV macrophages and subjacent endothelial cells. ICAP's increased in length with increased time post-inoculation in Zones 1 and 2 from pigs with necrohemorrhagic lesions. In later sample periods, multiple closely associated and interlacing IV macrophages formed a discontinuous layer over endothelial cells in Zone 2 samples near necrohemorrhagic lesions. These results suggest that the intravascular macrophage population changes from immature macrophages to mature macrophages or immature epithelioid cells within 24 hours after inhalation of a virulent Haemophilus pleuropneumoniae. Furthermore, intravascular macrophages likely function to clear cellular and acellular debris from the blood in pneumonic conditions.     

Serological cross-reactivity between a porcine Actinobacillus strain and Haemophilus pleuropneumoniae.

During serological screening of a closed SPF-herd free of pleuropneumonia, more than half of the pigs were positive for complement-fixing antibodies to Haemophilus pleuropneumoniae. Actinobacillus bacteria closely related to A. suis were isolated from tonsillar tissue of 14 out of 20 slaughtered pigs submitted for pathological and bacteriological evaluation. None of the pigs had evidence of respiratory disease. Two pigs inoculated endobronchially with a selected Actinobacillus strain developed mild focal pneumonia and complement-fixing antibodies cross-reacting with H. pleuropneumoniae. Five pigs exposed and vaccinated with the Actinobacillus strain and five pigs spontaneously infected with the strain also developed complement-fixing antibodies against H. pleuropneumoniae and appeared to be less susceptible to experimental Haemophilus pleuropneumonia than pigs not exposed to the Actinobacillus infection. The agglutination test applied on serum treated with 2-mercaptoethanol detected antibodies against H. pleuropneumoniae serotype 5 but not against serotype 1 in pigs exposed to the Actinobacillus strain. Antibodies reactive with the Actinobacillus strain were also found in pigs hyperimmunized against H. pleuropneumoniae serotypes 1-5 in 2-mercaptoethanol tube agglutination test and rabbits hyperimmunized against serotypes 1,2 and 7, and strain 73567 in the immunodiffusion test. Conversely rabbits immunized against the Actinobacillus strain had antibodies against H. pleuropneumoniae serotypes 1, 3, 4, 5 and 6. It is concluded that pigs infected with Actinobacillus organisms may become false positive reactors against H. pleuropneumoniae.     

Effect of Pasteurella multocida and Haemophilus pleuropneumoniae toxins on swine alveolar macrophages

Supernatants were obtained from 18 hr. broth cultures of Pasteurella multocida strains D82 and D62 (serotype D, toxigenic), Kobe 6 (type D, non-toxigenic), A50 and X73 (type A, non-toxigenic), Haemophilus pleuropneumoniae serotypes 1, 5 and 6, Haemophilus sp. taxon "minor group" (2 strains) and an avirulent serotype 1 strain of H. pleuropneumoniae. The supernates were filtered, pH-neutralized and tested for cytotoxicity after incubation for 18 hours in the presence of swine alveolar macrophage monolayers. Supernatants from H. pleuropneumoniae serotypes 1, 5 and 6 were cytocidal.     

Adherence of Actinobacillus pleuropneumoniae to primary cultures of porcine lung epithelial cells

To study adherence of Actinobacillus pleuropneumoniae to porcine lower respiratory epithelium, a cell culture model was developed using primary cultures of porcine lung epithelial cells (LEC). Adherence assays were performed and results were compared with data obtained with swine kidney cells (SK6). A. pleuropneumoniae efficiently adhered to LEC with up to 62 bacteria per cell after 2h of incubation. Reference strain of serotype 3 (R3) adhered better to LEC than reference strains of serotypes 1 (R1), 7 (R7) and 8 (R8). Overall the adherence to LEC was more rapid and up to 30-fold more efficient than adherence to SK6 cells. In search for the mechanism involved in the adherence event, we tested the effect of LPS which has previously been demonstrated to cause adherence of the pathogen to upper respiratory epithelium. Adherence assays with LPS transposon mutants demonstrated unaltered (mutant with modification in core/lipid A moiety) or even three-fold more adherence (mutants lacking O antigen) compared to the parent micro-organisms. Purified LPS of strains R1, R3, R7 and R8 did not inhibit adherence of R8 to LEC either, suggesting that LPS and particularly the O-antigen are not essential for adherence of A. pleuropneumoniae to LEC. The efficient, LPS-independent adherence of A. pleuropneumoniae to LEC cells indicates that A. pleuropneumoniae may carry different, cell type-specific adhesins and that primary cultures of lower respiratory epithelium are valuable infection models in studying A. pleuropneumoniae pathogenesis.     

Role of haemophilus pleuropneumoniae lipopolysaccharide endotoxin in the pathogenesis of porcine Haemophilus pleuropneumonia

Intact Haemophilus pleuropneumoniae cells (strain Shope 1, serotype 1), highly purified lipopolysaccharide (LPS) obtained from this strain of H pleuropneumoniae, as well as from Escherichia coli O111:B4, filter-sterilized H pleuropneumoniae cell-free culture supernatant fluid, and heat-inactivated supernatant fluid were given intranasally to CF1 mice and intratracheally to pigs. Pulmonary lesions induced by H pleuropneumoniae in mice were similar to those induced by H pleuropneumoniae in pigs. Histologically, lungs of mice and pigs killed 1 or 2 days after inoculation with 200 micrograms of highly purified H pleuropneumoniae LPS had lesions similar to one another and were similar to those in mice and pigs given intact H pleuropneumoniae, except that little or no necrosis or hemorrhage was observed. In mice killed 1 or 2 days after inoculation of 200 micrograms of E coli O111:B4 LPS, pulmonary lesions were similar to those in mice given H pleuropneumoniae LPS. Pulmonary lesions in mice given cell-free culture supernatant fluid obtained from a midlog-phase growth culture of H pleuropneumoniae cultivated in a chemically defined medium were severe and consisted of neutrophil infiltration and extensive necrosis. In mice, the heat-inactivated supernatant fluid produced mild lesions that consisted of foci of neutrophil aggregation and no necrosis. Extensive necrosis observed in lesions caused by cell-free culture supernatant fluid could be attributed to the action of a heat-labile component, perhaps by the extracellular heat-labile hemolysin produced by H pleuropneumoniae cultivated in chemically defined medium. A LPS endotoxin and a heat-labile factor may be involved in the pulmonary lesion development in the acute phase of porcine Haemophilus pleuropneumonia.     

Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelial cells and frozen lung sections

The ability of 23 different Actinobacillus pleuropneumoniae isolates to adhere in vitro to porcine tracheal epithelial cells and to porcine frozen lung sections was examined. It was found that A. pleuropneumoniae adhered poorly to isolated tracheal epithelial cells. On the other hand, A. pleuropneumoniae adhered to frozen lung sections and marked variations were observed between and within serotypes. Adherence to lung sections did not seem related to the hemagglutinating activity of the isolate. Two noncapsulated variants adhered to lung sections in greater numbers than their capsulated parent strains. Adherence to lung sections was not inhibited by the extracellular matrix components tested namely, laminin, fibronectin, and collagen, but was inhibited by homologous serotype-specific antiserum. The data indicated that the A. pleuropneumoniae isolates tested possess the ability to adhere to porcine lung tissue, a property which did not seem to be related to the serotype and did not seem to involve the capsular material or the hemagglutinins of the isolates.     

Haemophilus pleuropneumoniae serotypes--cross protection experiments

Pigs vaccinated with a killed 6-hour culture of Haemophilus pleuropneumoniae serotype 2 with Freund's incomplete adjuvant were not protected against challenge with serotypes 1, 5, 6 or 8. Equivalent results were obtained when pigs were vaccinated with serotypes 4 or 5 and challenged with serotype 2. In earlier studies of immunity induced by intranasal immunization with live H. pleuropneumoniae organisms, it was clearly shown that intranasal inoculation with one serotype of H. pleuropneumoniae would induce a strong immunity to both homologous and heterologous serotypes (Nielsen 1979). The present study has shown that cross immunity is not obtained with parenteral immunization. The results strongly suggest that the immune response of the pig to parenteral vaccination is different from the response seen after natural infection, and indicate that an important part of the defence mechanism against H. pleuropneumoniae infection is a local immune-barrier which is effective in preventing the bacterium from penetrating the mucosa. In earlier vaccination experiments 90 per cent of vaccinates were protected against homologous challenge (Nielsen 1976). In the present work a vaccine containing serotypes 1 through 6 was fully protective against serotypes 2 and 3 and also against serotype 8, which shares antigenic determinants with serotypes 3 and 6. These results indicate that the protection obtained by parenteral immunization is serotype-specific. Vaccines must therefore contain the serotypes existing in the swine population.     

A Stable L-form of Haemophilus pleuropneumoniae

A stable L-form of Haemophilus pleuropneumoniae (Shope) was isolated from primary pig kidney cell tissue cultures which had been inoculated 28 days previously with glycine induced spheroplasts of this organism.

H. pleuropneumoniae was definitely cytopathic in primary pig kidney cell cultures, producing cell rounding, cytoplasmic vacuolation and nuclear enlargement with peripheral condensation of nuclear DNA. By contrast, the effect of spheroplasts was much less distinct, producing only loss of cytoplasmic granularity coincident with apparent loss of some cytoplasmic RNA, and slight nuclear enlargement.

Both the organism and its L-form were shown to be related by cultural methods, antibiotic sensitivity tests, immunofluorescence and immunodiffusion.

The L-form remained stable after 90 serial passages on agar and 45 in broth, each medium being capable of supporting the growth of both forms of the organism.

     

Serologic studies on porcine strains of Haemophilus parahaemolyticus (pleuropneumoniae): extraction of type-specific antigens.

By the use of phenol water extraction it was possible to obtain strictly serotype-specific antigens from mucoid cell cultures of five serotypes of Haemophilus parahaemolyticus (pleuropneumoniae). These serotype-specific antigens did not cross-react with each other in immunodiffusion tests. The type-specific precipitating phenol-water-fractions were composed of two to four antigenic components, presumably of polysaccharide or lipopolysaccharide nature.     

Identification of serotypes of Haemophilus pleuropneumoniae by counterimmunoelectrophoresis

Counterimmunoelectrophoresis, direct immunofluorescence and immunodiffusion procedures were used to serotype 15 strains of Haemophilus pleuropneumoniae isolated from the respiratory tract of pigs in southern Brazil. Antigens were prepared by extracting cultures with a saline solution or by the phenol-water method. Antisera were prepared in rabbits against serotypes 1, 2, 3 and 5. Thirteen of the isolates were type 5 and two were type 3. No differences were observed between the results obtained in serotyping with counter immunoelectrophoresis and direct immunodiffusion, but both procedures were significantly better than immunodiffusion except with the saline extracted antigen. Counterimmunoelectrophoresis was quicker, more sensitive and more easily performed than the other techniques.     

Evaluation of a Killed Vaccine Against Porcine Pleuropneumonia Due to Haemophilus pleuropneumoniae

A bacterin containing serotypes 1 and 5 of Haemophilus pleuropneumoniae was developed for the prevention and the control of porcine pleuropneumonia. It was injected intramuscularly into three groups of ten piglets, the first group with one dose, the second one with two doses and the third one with three doses at two-week intervals. Another group of ten piglets did not receive the vaccine. All the piglets were then challenged by an aerosol of mixed suspensions of H. pleuropneumoniae serotypes 1 and 5. Two and three injections of vaccine completely prevented mortality, whereas half of the control piglets and of those receiving only one dose of vaccine died. All surviving piglets, both control and vaccinated, had severe signs of respiratory disease for at least 36 hours after exposure to challenge. Moreover, vaccination did not induce the production of antibodies at high titers. Local reactions were not noted after vaccination and at postmortem; ten weeks after the challenge, there were no signs of abscess formation or induration.     

Binding patterns of five monoclonal antibodies to Actinobacillus (Haemophilus) pleuropneumoniae

Five monoclonal antibodies were obtained after immunising mice with superficial antigens of three strains representing serovars 1 to 3 of Actinobacillus (Haemophilus) pleuro-pneumoniae. When tested in ELISA against the standard strains representing serovars 1 to 10, the monoclonal antibody raised against the standard strain of serovar 1 reacted only with that strain. Of the three monoclonal antibodies raised against the standard strain of serovar 3, one reacted with serovars 3 and 8 only, another with serovar 7 only and the third with the strains representing serovars 7, 9 and 10. The monoclonal antibodies produced with the serovar 2 strain also reacted with a wide spectrum of strains, representing serovars 7 to 10.     

Biochemical and serological identification of strains of Haemophilus pleuropneumoniae

Eighteen field isolates of Haemophilus pleuropneumoniae were studied biochemically and serotyped using the complement fixation test (CFT), agglutination test and the immunodiffusion test. Three biochemical tests (V-dependency, CAMP-reaction and urease activity) were found to be very useful for the biochemical characterization of the H. pleuropneumoniae. Haemolysis on blood agar plates, although present, was not sufficiently pronounced in all cases to warrant absolute dependence on this characteristic. Serological typing revealed the isolates belong to Serotypes 1 and 5. The immunodiffusion test proved to be the most serotype specific, while a marked cross-reaction was observed with the CFT.     

Effect of porcine reproductive and respiratory syndrome virus infection on the clearance of Haemophilus parasuis by porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection in young piglets is frequently associated with secondary infection due to various pathogens, especially those of the respiratory tract. One of the most important mechanisms in respiratory diseases is related to the alteration of function of porcine alveolar macrophages (PAMs). The objective of this study was to determine how PRRS virus infection affects the capabilities of PAMs in the phagocytosis and destruction of Haemophilus parasuis. Phagocytosis percentages were determined in vitro and ex vivo, after collected PAMs were directly exposed to the virus of if PAMs were collected from piglets previously infected with PRRSV. In vitro experiments demonstrated that H. parasuis uptake by PAMs is only increased in the early stages of PRRSV infection (2 h post-infection). In contrast, in the ex vivo experiments it was shown that PAMs from PRRSV-infected piglets do not seem to change in their phagocytic rate until the later stages of infection. Together with a decrease in the phagocytic rate, a marked decrease in the functional ability of PAMs to kill bacteria was observed 7 d post-infection. It is hypothesized that when animals are exposed to PRRSV, there is a marked decrease in the functional ability of PAMs to kill bacteria through the release of superoxide anion, indicating a possible negative effect of the virus, at least at the macrophage level.