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1.
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.  相似文献   

2.
Comparative virulence of porcine Haemophilus bacteria.   总被引：9，自引：2，他引：7       下载免费PDF全文
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)  相似文献   

3.
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)  相似文献   

4.
Hemolysin, cell-free autolysate, and lipopolysaccharide (LPS) prepared from Escherichia coli (O141) were parenterally administered to 113 weaned pigs. Both the hemolysin and the cell-free autolysate were crude preparations which probably contained several biologically active substances. Pigs in all groups which die less than 72 hours after injection had similar gross and microscopic lesions. The pigs which survived (chronically affected pigs) were killed 3 to 12 days after injection. Of the pigs that lived more than 72 hours after injection, those given hemolysin and autolysate had generalized vascular myolysis and fibrinoid necrosis, whereas those given LPS had morphologically normal blood vessels. The vascular changes produced by hemolysin and autolysates of E coli (O141) were the same as the histologic angiopathy of naturally occurring edema disease of pigs. The LPS produced acute lesions of endotoxin shock in the pigs, but did not produce the angiopathy characteristic of edema disease. Typical clinical signs of naturally occurring edema disease were not a consistent observation in any of the treatment groups.  相似文献   

5.
To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions.  相似文献   

6.
Toxicity of Haemophilus pleuropneumoniae to porcine lung macrophages   总被引：10，自引：0，他引：10  
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.  相似文献   

7.
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.  相似文献   

8.
Bacterial counts (per lung) were made from Swiss White and/or C3H mice, inoculated intranasally with either Haemophilus pleuropneumoniae or Serratia marcescens and killed at specific times. The percentages of bacterial survival and clearance at each time were determined. Serratia marcescens was cleared progressively, but not completely, from the lungs of C3H and Swiss White mice. The overall pulmonary clearance of S marcescens by Swiss White mice was significantly greater than that of C3H mice (P less than 0.01). The pulmonary clearance pattern of H pleuropneumoniae in C3H mice differed according to the dose inoculated. With a larger H pleuropneumoniae median lethal dose (0.1 LD50), the organism multiplied consistently up to 25 times by 12 hours after the inoculations were done, when clinical signs and lesions appeared in a few of the mice. The clearance rates at 24 and 48 hours after inoculations were 60.65% and 10.3%, respectively. Mice given 0.04 LD50 H pleuropneumoniae showed a 10-fold increase of H pleuropneumoniae in the first 4 hours, with clearances reaching 33%, 66%, and 99% at 8, 12, and 24 hours, respectively.  相似文献   

9.
The in vitro production of proinflammatory cytokines after stimulation with Actinobacillus pleuropneumoniae and the relation of these cytokines in vivo with the disease caused by A. pleuropneumoniae were investigated. Within 24 h, in vitro stimulation by A. pleuropneumoniae (serotype 1) preparations, including killed bacteria, bacterial culture supernatant, lipopolysaccharide, and bacterial extracts, porcine pulmonary alveolar macrophages (PAM) produced significant (P < 0.05) amounts of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) as measured by bioassays. The supernatants containing interleukin-8 from PAM after stimulation by bacterial preparations showed significant neutrophil chemotaxis, while bacterial preparations alone did not. After in vivo infection with A. pleuropneumoniae, the mean levels of TNF-alpha and IL-1 in serum, as measured by bioassays, were elevated 37- to 27836-fold for TNF-alpha and 11- to 5941-fold higher for IL-1 within 4 d post-infection, depending on the treatments, and remained elevated up to Day 7. Both cytokines were also detected in porcine lungs by bioassays and immunocytochemistry. The results indicated that both secreted and surface components of A. pleuropneumoniae can stimulate PAM to produce proinflammatory mediators. Neutrophil chemoattractants rather than bacterial components are the major factor causing acute lung inflammation. The elevation of TNF-alpha and IL-1 in pigs occurred coincident with the onset of acute clinical disease.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
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.  相似文献   

13.
The role of the heat-labile haemolysin of Actinobacillus pleuropneumoniae in acute porcine pleuropneumonia was examined. A virulent strain was compared with an isogenic haemolysin-deficient mutant in experimental infections. The pigs which received the virulent strain showed clinical signs of acute respiratory disease whereas the animals infected with the mutant strain appeared to be less severely affected. At post mortem examination, both groups showed similar acute pulmonary lesions and pleurisy typical of A pleuropneumoniae infection. The bacterial antigen representing the haemolysin was detected in lung lesions infected with the parent strain but not in those infected with the mutant. These results demonstrate that the haemolysin of serotype 2 A pleuropneumoniae is not an essential factor for the production of the lesions of pleuropneumonia in pigs.  相似文献   

14.
Recently weaned pigs were infected aerogenically with Haemophilus (Actinobacillus) pleuropneumoniae, serotype 5. At three, six, 12, and 18 hours and one, two, four and seven days after exposure to haemophili a pair of animals were killed and necropsied. Pulmonary oedema with multifocal petechial haemorrhages and a diffuse neutrophilic bronchiolitis and alveolitis were observed at three and six hours after infection. Focal areas of coagulative necrosis developed in areas of intense suppuration at 12 and 18 hours after infection. At one and two days after infection, necrotic areas were surrounded by dense bands of degenerating leucocytes and contained unidentifiable round and elongated cells characteristic of this disease. In subacute lesions a thick layer of granulation tissue formed around the outer margin of developing abscesses. Most of the round and elongated cells in alveolar exudates could not be identified by enzyme histochemistry or electron microscopic examination. Neutrophils in various stages of degeneration and deterioration provided strong evidence that some of the cells were of this type. These findings suggest that neutrophils may play an early and significant role in development of lesions.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

18.
Haemophilus pleuropneumoniae serotypes--cross protection experiments   总被引：19，自引：0，他引：19  
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.  相似文献   

19.
Three experiments were done to evaluate some antibiotic therapies that are used commonly to treat pigs infected with Haemophilus pleuropneumoniae. Haemophilus-free piglets, 12 weeks of age, were challenged in a chamber with an aerosol of H. pleuropneumoniae serotype 1 and were medicated with antibiotics at various times before or after challenge. Antibiotic formulations which are commonly used to treat pneumonia in swine were used. They were chloramphenicol, penicillin, and a long-acting formulation of oxytetracycline given intramuscularly; and oxytetracycline, chloramphenicol and spiromycin (investigated as a potentially useful antibiotic) given in solution as the sole source of drinking water. Infection, disease (death, fever, gross lung lesions) and growth rate were measured in pigs following experimental challenge.  相似文献   

20.
One group of six pigs and another group of three pigs were separately exposed in a polyethylene enclosed chamber for ten minutes, respectively, to Haemophilus pleuropneumoniae serotype 1 and Bacillus subtilis aerosols generated by an ultrasonic nebulizer. Haemophilus pleuropneumoniae and B. subtilis were deposited throughout the lungs immediately following aerosol exposure. The number of H. pleuropneumoniae and B. subtilis deposited varied within and between lungs in each group. The mean numbers of both organisms deposited in the posterior (caudal and accessory) lobes were significantly greater than those in the anterior (cranial and middle) lobes (P less than 0.001). The four principals that received H. pleuropneumoniae aerosols and the two contact controls developed fatal fibrinous pneumonia which simulated that seen in natural infections. Since this exposure system consistently resulted in clinical disease it has good potential as a model for the study of pathogenesis of the disease and more specifically for the evaluation of vaccines.  相似文献   

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