Dose response relationship of Haemophilus pleuropneumoniae aerosols in pigs.

The virulence of Haemophilus pleuropneumoniae was quantitated for ten and 12 week old pigs following aerosol exposure. The volume and concentration of culture aerosolized, the estimated numbers of organisms inhaled by the pigs and the mortality rates at 72 hours postexposure were computed and used to calculate the LD50. There was correlation between the concentration of culture aerosolized, the amount of the estimated inhaled dose and the mortality rates. The ten week old pigs were apparently more susceptible to aerosols of H. pleuropneumoniae than the 12 week old pigs. The LD50 value or a multiple of it appears to be a reasonable basis for a standardized aerosol challenge of the immunity of pigs vaccinated with experimental or commercial H. pleuropneumoniae vaccines.     

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.     

Mortality in swine herds endemically infected with Haemophilus pleuropneumoniae: effect of immunization with cross-reacting lipopolysaccharide core antigens of Escherichia coli

The benefit of increased immunity to cross-reacting lipopolysaccharide core antigens of gram-negative bacteria induced by vaccination with an Rc mutant of Escherichia coli 0111:B4 (strain J5) was evaluated in commercial swine herds endemically infected with Haemophilus pleuropneumoniae. Weanling pigs were vaccinated IM with E coli J5 (group 1) before the expected time of H pleuropneumoniae infection. Clinical signs, antibiotic treatment frequency, mortality, growth performance (days to market weight), and serologic responses of the pigs were monitored for approximately 5 months after vaccination. The results were compared with those of pigs vaccinated IM with a commercial H pleuropneumoniae bacterin (group 2) and with those of nonvaccinated control pigs of the same age (group 3). The treatment frequency and growth performance were similar in the 3 groups. However, vaccination with E coli J5 or with the H pleuropneumoniae bacterin lowered mortality, compared with mortality in the controls. Serum titers against E coli J5 increased after vaccination with the E coli J5 bacterin, but were not increased by vaccination with the H pleuropneumoniae. In contrast, serum titer to E coli J5 increased in all treatment groups as a result of H pleuropneumoniae infection or exposure. The protection against lethal H pleuropneumoniae infections in swine that was provided by vaccination with the E coli J5 and the H pleuropneumoniae bacterin appeared to be immunologically distinct on the basis of serologic analysis, indicating the possibility of different mechanisms of protection.     

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.     

Acute inflammatory effects of intratracheally instilled Escherichia coli endotoxin and sonicated suspension of Haemophilus pleuropneumoniae in swine.

A single bolus of either Escherichia coli endotoxin, sonicated suspension of Haemophilus pleuropneumoniae, or pyrogen-free normal saline was intratracheally instilled in six week old specific-pathogen-free pigs. Pigs exposed to E. coli endotoxin developed fever, leukopenia followed by leukocytosis, and endotoxemia. Leukocytosis was the only clinical abnormality noted in pigs receiving the sonicated suspension of H. pleuropneumoniae. At one day postexposure, focal areas of atelectasis and consolidation were observed in the caudal lung lobes of animals receiving either E. coli endotoxin or the sonicated suspension of H. pleuropneumoniae. Lesions were characterized by a neutrophilic bronchitis and bronchiolitis with alveolitis in the surrounding tissue. Increased numbers of alveolar macrophages and evidence of phagocytosis were observed by light and electron microscopy. No clinical abnormalities or lesions were observed in animals receiving normal saline. Lesions typical of acute porcine Haemophilus pleuropneumonia were not produced by either E. coli endotoxin or sonicated suspension of H. pleuropneumoniae, indicating that multiple virulence factors are probably involved in lesion development.     

Evaluation of a selective medium for isolation of Haemophilus pleuropneumoniae.

Crystal violet, lincomycin, spectinomycin and bacitracin were evaluated as selective agents in media for isolation of Haemophilus pleuropneumoniae. No single antimicrobial agent or combination of two or more inhibited all non-Haemophilus strains (Escherichia coli, Pasteurella haemolytica, Pasteurella multocida, Streptococcus faecalis, Streptococcus equisimilis and Staphylococcus aureus) without marked suppression of 16 H. pleuropneumoniae strains. A medium containing 1 micrograms/mL of crystal violet, 1 microgram/mL of lincomycin, 8 micrograms/mL of spectinomycin and 128 micrograms/mL of bacitracin inhibited one E. coli strain and the Gram-positive strains while H. pleuropneumoniae strains were suppressed to a minor degree only. Haemophilus pleuropneumoniae was isolated on the selective medium on three occasions from the nose or pharynx of two out of eight experimentally inoculated pigs. Haemophilus pleuropneumoniae was recovered from the nose of only two pigs at necropsy and from tonsil of one, whereas the lower airways in most pigs and the lung lesions in all pigs were positive. There was no advantage to using the selective medium for the recovery of H. pleuropneumoniae at necropsy from these eight experimentally infected pigs, probably because other bacteria were absent or present in very low numbers in the tissues with H. pleuropneumoniae. The isolation rate on selective medium was higher than the rate on non-selective medium (p less than or equal to 0.1; chi 2 test) when the airways of slaughtered pigs were cultured. This was likely due to a high degree of contamination. Dry swabs placed in tryptone yeast extract with nicotinamide-adenine-dinucleotide gave a significantly higher recovery rate than commercial Culturette swabs in modified Stuart's transport medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actinobacillus (Haemophilus) pleuropneumoniae: use of coagglutination and complement fixation to determine the relationship between presence of organism and antibody titer in slaughterhouse pigs

The conventional culture method was compared to coagglutination for detection of Actinobacillus (Haemophilus) pleuropneumoniae in 425 sets of pig lungs. Sera from the same animals were evaluated for antibodies to A. pleuropneumoniae by the complement fixation (CF) test. All samples were collected at 2 packing plants in Iowa. In 2 nonvaccinated herds with no history of respiratory disease, the difference between standard culture results and coagglutination was highly significant (P less than 0.001). None of the 57 pigs in this group were positive for A. pleuropneumoniae by conventional culture, but 7 were positive by the coagglutination test. There were 15 animals with CF titers between 1:8 and 1:32. Animals from 6 herds vaccinated for A. pleuropneumoniae and without recent respiratory problems were evaluated. One out of 118 animals tested was positive for A. pleuropneumoniae by standard culture as compared to 9 positive by coagglutination. The difference in positive results between culture and coagglutination was highly significant (P less than 0.001). Twenty-eight animals had CF titers to A. pleuropneumoniae (1:4 to greater than or equal to 1:128). Two hundred fifty lungs and sera samples were collected from 7 herds which had recently experienced varying degrees of respiratory disease. Thirty-nine lungs were positive for A. pleuropneumoniae by culture and 182 were positive by coagglutination. The number of positives detected by coagglutination was significantly different (P less than 0.001) from the number positive by culture. There were 172 animals with antibody titers ranging from suspect to greater than or equal to 1:128. There were significantly fewer positive animals detected by standard culture than with the CF test (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with a vaccine containing Apx toxins and transferrin-binding proteins

The efficacy of a subunit vaccine containing the Apx toxins of Actinobacillus pleuropneumoniae and transferrin-binding proteins was determined. Ten pigs were vaccinated twice with the vaccine. Eight control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 10(6.5) colony-forming units (CFU) of an A. pleuropneumoniae serotype 9 strain. In the vaccine group, none of the pigs died after inoculation. Only one pig of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The mean percentage of affected lung tissue was 64% in the control group and 17% in the vaccine group. Actinobacillus pleuropneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 5.0 x 10(8) CFU/g in the control group and 3.0 x 10(6) CFU/g in the vaccine group. It was concluded that the vaccine induced partial protection against severe challenge.     

Vaccination Against Pleuropneumonia of Pigs Caused by Haemophilus pleuropneumoniae

A strain of Haemophilus pleuropneumoniae was isolated from a pig with pleuropneumonia from a herd where this condition was frequent. A formalin inactivated culture of this isolate was used as antigen in two vaccine preparations: A and B. Vaccine A had peanut oil + arlacel 80 + tween 80 as adjuvant and vaccine B had aluminum hydroxide gel as adjuvant. Twenty pigs were vaccinated twice with vaccine A and 19 with vaccine B. Twenty additional pigs were not vaccinated. All pigs were transferred to the herd. Eleven pigs in the nonvaccinated group developed pneumonia and seven of these died within eight days after exposure. None of the vaccinated pigs had signs of pneumonia. It is concluded that the vaccines prevented the acute form of pleuropneumonia due to H. pleuropneumoniae.     

Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 10 of pigs vaccinated with bacterins consisting of A. pleuropneumoniae serotype 10 grown under NAD-rich and NAD-restricted conditions

The efficacy of two bacterins containing an Actinobacillus pleuropneumoniae serotype 10 strain was evaluated. The bacterial cells constituting bacterin 1 and 2 were grown under nicotinamide adenine dinucleotide (NAD)-rich (low-adherence capacity to alveolar epithelial cell cultures) and NAD-restricted (high-adherence capacity to alveolar epithelial cell cultures) conditions, respectively. Ten pigs were vaccinated twice with the bacterin 1 and nine pigs with the bacterin 2. Ten control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 106.5 colony-forming units (CFU) of an A. pleuropneumoniae serotype 10 strain. In the bacterin 1 and 2 group, three and two pigs died after inoculation, respectively. Only two pigs of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The percentage of pigs with severe lung lesions (> 10% of the lung affected) was 100% in the control group, 70% in the bacterin 1 group and 22% in the bacterin 2 group. Actinobacillus pleuropneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 7.0 x 10(6) CFU/g in the control group, 6.3 x 10(5) CFU/g in the bacterin 1 group and 1.3 x 10(6) CFU/g in the bacterin 2 group. It was concluded that both bacterins induced partial protection against severe challenge. Furthermore, there are indications that the bacterin 2, containing A. pleuropneumoniae bacteria grown under conditions resulting in high in vitro adhesin, induced better protection than the bacterin 1.     

Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae

Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.     

Detection of strains of Haemophilus pleuropneumoniae using the coagglutination test

Three tests were used for the serological identification of the strains of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) isolated from pigs and coming from 66 sites where pleuropneumonia, caused by Haemophilus, occurred in pigs. The coagglutination test was found to be the best for the identification of the causal agent; the ring precipitation test was somewhat less sensitive, and worse results were obtained when rapid slide agglutination was used. Of all the field isolates of H. pleuropneumoniae, serovar 2 occurred most frequently (56%), followed by serovar 1 (39%); one strain was identified as serovar 7. Two strains have remained unidentified. The serological identification of the strains was performed on the basis of their comparison with eight type serovars of H. pleuropneumoniae.     

Prevalence of pig herds affected by pleuropneumonia associated with Haemophilus pleuropneumoniae in eastern England

A survey for the macroscopic lesions indicative of pneumonic infection in the pig with Haemophilus pleuropneumoniae was made in an abattoir in eastern England. A total of 78 herds located in 11 counties of eastern or central England were seen between December 1982 and August 1983. Lesions were noted in the batches submitted by 44 (56 per cent) of the 78 herds. A further 16 herds (21 per cent) submitted batches containing pigs affected by pleurisy principally of the caudal lobes but without the pneumonic lesions. Lesions suggestive of enzootic pneumonia were also seen in 61 herds (78 per cent). Circumstances restricted corroborative bacteriological examinations to 53 and serological examinations to 33 herds. Strains of H pleuropneumoniae (predominantly serotype 3 but also serotype 2) were isolated from 26 herds. These comprised 22 out of 42 (51 per cent) of those where typically affected plucks, or plucks with caudal lobe pleurisy, were encountered, and four out of 11 (36 per cent) in which there was either no observable thoracic disease or enzootic pneumonia only. Complement fixing antibodies to serotype 3 or 2 antigens occurred in 26 out of 33 herds (79 per cent). These comprised 25 (83 per cent) of 30 herds with batches exhibiting either typical pulmonary lesions and, or, caudal lobe pleurisy and one of three herds without such lesions. Collectively these data indicate that herds containing pigs with pleuropneumonia are common at least in the more easterly parts of England and that H pleuropneumoniae, usually but not always associated with disease, is also widespread.     

Survey of pleuritis and pulmonary lesions in pigs at abattoir with a focus on the extent of the condition and herd risk factors

Cranioventral pulmonary consolidation (enzootic pneumonia-like lesions) and chronic pleuritis (CP) are common findings in slaughtered pigs. Pleural lesions involving dorsocaudal lobes are suggestive of pleuropneumonia due to Actinobacillus pleuropneumoniae. In this report the results of an abattoir survey of pleuritis and pulmonary lesions in pigs is presented with a focus on herd risk factors. A total of 4889 animals, ranging in age from 9 to 10 months, from 48 batches of pigs belonging to an equal number of herds, were included in the study. Bronchopneumonic lesions suggestive of enzootic pneumonia (EP-like lesions) were detected in 46.4% of the examined lungs. The EP-like lesion average value for all lungs was 1.03 (95% CI 0.98-1.08), ranging from 0.17 to 2.56 among the 48 batches; 47.5% of lungs showed chronic pleuritis. Dorsocaudal pleuritis suggestive of recovered pleuropneumonia (SPES score ≥2) was found in 25.1% of the lungs. The mean SPES (slaughterhouse pleuritis evaluation system) value of the overall 4889 lungs was 0.83 (95% CI 0.78-0.86). The mean SPES value of the batches ranged from 0.04 to 1.87. The mean Actinobacillus pleuropneumoniae index of all studied batches was 0.61 (95% CI 0.51-0.71), ranging from 0 to 1.84. Blood samples were collected from each herd to evaluate antibody titres to Mycoplasma hyopneumoniae, A. pleuropneumoniae, Aujeszky's disease virus, porcine reproductive and respiratory syndrome virus (PRRSV), and swine influenza virus. Herd characteristics were recorded using a questionnaire given to the farmers. A multivariable analysis was conducted to identify risk factors for pleuritis and EP-like lesions. High dorsocaudal pleuritis was associated with A. pleuropneumoniae seroprevalence and history of A. pleuropneumoniae isolation from pneumonic lungs of dead animals. Vaccination of weaners at 3-5 weeks of age against PRRS using a modified live vaccine was associated with a reduction in the percentage of cranioventral pulmonary consolidation (EP-like lesions).     

Use of monoclonal antibodies to serotype-specific antigens of Haemophilus pleuropneumoniae serotype 2 in passive immunization

The prophylactic value of mouse monoclonal antibodies to the pig pathogen Haemophilus pleuropneumoniae was studied. Approximately 250 mg of purified mouse monoclonal antibody specific to capsular antigens of H pleuropneumoniae serotype 2 was given IV to five 9-week-old pigs. Five additional pigs from the same litter served as controls. On the following day, all pigs were given a lethal dose (5 x 10(9)) of H pleuropneumoniae serotype 2 into the trachea. Four controls and 1 pig that was given antibodies died within 24 hours. The surviving 5 pigs developed typical signs of pleuropneumonia. After 6 days, the pigs were euthanatized and their respiratory tracts were examined for pathologic changes. All 5 pigs had pathologic changes, but they were less severe in the 4 pigs that had been given antibodies, compared with those in the control pig.     

Protective efficacy of capsule extracts of Haemophilus pleuropneumoniae in pigs and mice

Capsular extracts of Haemophilus pleuropneumoniae, Serotype 1, were mixed with AL(OH)3 gel (3 parts extract + 1 part Al(OH)3) and used as vaccines in pigs and mice. Four preparations were tested in Experiment I: NaCl and Cetavlon (hexadecyltrimethyl ammonium bromide) extracts of both low in vitro passage (LP) and high in vitro passage (HP) culture, respectively. Four pigs vaccinated with the NaCl extract of the LP strain survived, whereas one of four from each of the remaining vaccine groups and five of six from the control group died. All vaccines induced complement-fixing antibodies. No apparent boosting of titres occurred as a result of challenge with live bacteria. Mice were vaccinated in Experiment II with NaCl and Cetavlon extracts of the LP strain. Both were protective, although the Cetavlon vaccine appeared more efficacious than the NaCl extract. The use of Al(OH)3 adjuvant improved the efficacy of the NaCl vaccine in mice. In Experiment III six gnotobiotic pigs were vaccinated with a combined NaCl and Cetavlon vaccine and seven animals were given placebo. In Experiment IV seven specific pathogen-free (SPF) pigs were given the combined vaccine and eight pigs received placebo treatment. Both of these experiments indicated that the extract vaccines did not completely protect but reduced the mortality in pigs challenged with homologous virulent H. pleuropneumoniae bacteria. The results indicate that capsular antigens of H. pleuropneumoniae have some protective immunogenic efficacy in pigs and mice.     

Comparative virulence of porcine Haemophilus bacteria.

The virulence of strains of Haemophilus pleuropneumoniae serotype 1, 2, 3, 7 and strains of the "minor-group" and Haemophilus parasuis were compared by inoculating specific pathogen-free pigs into the lower airways with specified doses of bacteria. Haemophilus pleuropneumoniae, strain W, serotype 1, given in 1 X 10(8) colony-forming units, produced a lethal acute pleuropneumonia in four pigs. Nonlethal localized pulmonary necrosis was induced in four groups of two pigs given 1 X 10(7), 1 X 10(6), 1 X 10(5) and 1 X 10(4) respectively of the same strain. Two groups of four pigs developed chronic lesions when inoculated with 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain Shope 4074, serotype 1 and 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain WF83, serotype 7, respectively. Of 20 pigs given 1 X 10(8) colony-forming units of strain 1536, serotype 2, two died of acute pleuropneumonia and 18 had lesions of pulmonary necrosis or abscessation and pleuritis. A dose of 4 X 10(9) colony-forming units of strain BC181, serotype 3, induced pulmonary necrosis similar to the lesions in pigs given 10(7) colony-forming units or less of strain W, serotype 1, suggesting that the serotype 3 strain is less virulent. No clinical signs, but focal areas of pulmonary fibrosis and pleural adhesions were induced in four pigs inoculated with 4 X 10(9) colony-forming units of the "minor-group" strain 7ATS. Similarly, four pigs inoculated with "minor-group" strain 33PN did not show clinical signs, but had focal necrotic and fibrotic pulmonary lesions and pleural adhesions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of serology,bacteriological isolation and polymerase chain reaction for the detection of pigs carrying Actinobacillus pleuropneumoniae in the upper respiratory tract after experimental infection

Pigs, asymptomatically infected with Actinobacillus pleuropneumoniae in their upper respiratory tract, can transmit the infection. Detection of such animals is indispensable to prevent the intake of the disease in a herd. This study was conducted to evaluate bacteriology, polymerase chain reaction (PCR) and serology for the detection of subclinically infected pigs. Pigs were inoculated onto the tonsils with an A. pleuropneumoniae serotype 9 strain (n=12, group 1) or phosphate buffered saline solution (PBSS) (n=5, group 2). To prevent infection of the lungs, pigs of group 1 were treated three times with sodium ceftiofur as an aerosol. A third group (n=5) was inoculated intranasally with the same strain. All animals were euthanized 30 days post-inoculation (dpi). In pigs of group 1, clinical signs were not observed. A small lung lesion was found in only one pig and A. pleuropneumoniae was isolated from this lesion. The bacterium was not isolated from the lungs of animals that did not develop lung lesions. A. pleuropneumoniae was demonstrated in tonsils of 9/12 animals using bacteriological isolation, whereas it was demonstrated in mixed bacterial cultures from tonsils of all 12 animals by PCR. In non-infected animals (group 2), clinical signs were not observed and A. pleuropneumoniae was not demonstrated in any sample. All intranasally infected animals (group 3) developed disease signs and lung lesions. High antibody titers against ApxI, ApxII and heat-stable antigens were detected in animals that developed lung lesions. Antibody titers against these antigens were low or absent in all other pigs. It was concluded that pigs carrying A. pleuropneumoniae in the upper respiratory tract generally do not show measurable antibodies in serum. Therefore, sensitive methods for the detection of the etiological agent such as PCR are required to identify carrier animals, while serological methods are not suitable.     

Associations between pathogens in healthy pigs and pigs with pneumonia

The aim of this study was to evaluate the associations between different pathogens in the development of pneumonia and bronchopneumonia in pigs. Samples of bronchoalveolar lavage fluid from 100 pigs showing no clinical signs and 239 pigs with clinical signs of respiratory disease were examined for Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, US-type porcine respiratory and reproductive syndrome virus (PRRSV), EU-type PRRSV, porcine circovirus type 2 (pcv-2), influenza virus type A, alpha-haemolytic Streptococcus species, beta-haemolytic Streptococcus species, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Actinobacillus pleuropneumoniae. These potential pathogens were detected more frequently in the pigs with respiratory problems than in the pigs with no clinical signs. pcv-2 and alpha-haemolytic streptococci were the pathogens most frequently detected; A pleuropneumoniae was isolated in only two cases. There were more often associations between the organisms in the pigs with clinical signs than in the healthy pigs. In particular, alpha-haemolytic streptococci and M hyopneumoniae were both associated with the presence of M hyorhinis, EU-type PRRSV, P multocida and B bronchiseptica, and alpha-haemolytic streptococci also occurred more often in pigs that were already infected with other pathogens. P multocida and B bronchiseptica were both significantly associated with M hyopneumoniae, alpha-haemolytic streptococci, EU-type PRRSV and US-type PRRSV.     

Toxicity of Haemophilus pleuropneumoniae to porcine lung macrophages

Viable Haemophilus pleuropneumoniae bacteria were toxic for porcine alveolar macrophages in vitro. This cytotoxic effect proved to be dose-related. A cell-free extract of H. pleuropneumoniae, heat-killed bacteria, and a Pasteurella multocida field strain were nontoxic. When macrophages were cultured with H. pleuropneumoniae bacteria in a ratio of 100 macrophages to six bacteria, ultrastructural signs of cellular degeneration were observed within 1 h. This degeneration was observed in macrophages with or without phagosomes containing H. pleuropneumoniae. A cytotoxic substance was filtered from a H. pleuropneumoniae culture in Eagle's minimal essential medium supplemented with Earle's salts (EMEM) and 10% foetal calf serum that was incubated for 10 h at 37 degrees C. This substance was destroyed by heating at 65 degrees C for 30 min. Macrophages were less susceptible to the toxic effect of H. pleuropneumoniae when serum of convalescent pigs was added.