Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs.

In order to demonstrate the possible role of aerosol in the transmission of Actinobacillus pleuropneumoniae, an experiment including 18 specific pathogen-free (SPF), 10-week-old piglets, randomly distributed into 2 adjacent units, was carried out. In these facilities, air was forced through absolute filters to prevent any contact with infectious agents. During the first 6 d post inoculation, the 2 units were connected by a rectangular opening and the air circulation was forced by the ventilation system from unit A (inoculated pigs) to unit B (non-inoculated pigs). The A. pleuropneumoniae strain (biovar 1 serovar 9) was isolated in France from an outbreak of porcine pleuropneumonia. Two different infecting doses, 10(7) cfu/animal and 10(8) cfu/animal, were inoculated by intranasal route in 6 pigs of unit A. The infection spread quickly from the inoculated pigs to the non-inoculated pigs. Clinical signs were acute during the 4 d post inoculation: hyperthermia, respiratory distress and, sometimes, death (6 pigs of the unit A and 2 pigs of the unit B). All pigs seroconverted against A. pleuropneumoniae serovar 9 within 2 weeks. Lung lesions were severe: fibrinous pleurisy and lung hemorrhages in the acute stage, pleural adherences and focal pulmonary necrosis in the chronic stage. Actinobacillus pleuropneumoniae was isolated from the tonsils and/or lungs in 16 animals. It could be also isolated from the air of the experimental unit. This study showed that A. pleuropneumoniae was readily transmitted through aerosol over a distance of at least 2.5 m.     

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.     

Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species?

Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. 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.     

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).     

Pathogenesis of porcine Actinobacillus pleuropneumonia, part II: roles of proinflammatory cytokines.

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.     

Experimental reproduction of acute lesions of porcine pleuropneumonia with a haemolysin-deficient mutant of Actinobacillus pleuropneumoniae.

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.     

Differential proteomic analysis reveals increased cathelicidin expression in porcine bronchoalveolar lavage fluid after an Actinobacillus pleuropneumoniae infection

Accurate definition of respiratory health in pigs is an important problem for swine producers and veterinarians. In an approach to identify potential biomarkers, two-dimensional gel electrophoresis and mass spectrometry on bronchoalveolar lavage fluid (BALF)-derived proteins from pigs experimentally infected with Actinobacillus pleuropneumoniae were performed at different time points post infection. Mock-infected pigs were used as a control. It was shown that the antimicrobial peptides, prophenin-2 and PR-39, and the calcium-binding protein calgranulin C were reproducibly upregulated in BALF of pigs chronically infected with A. pleuropneumoniae. Concentrations of PR-39 were significantly (p<0.05) increased in BALF (median of 4.8 nM) but not in serum (median of 2.5 nM) on day 21 after infection. A Receiver Operating Characteristics (ROC) plot showed that PR-39 in BALF is an accurate and easily accessible marker to detect clinically healthy pigs convalescent from an experimental A. pleuropneumoniae infection. These results imply that PR-39 might have a potential as a general biomarker to determine porcine respiratory health.     

猪胸膜肺炎调查及地方适用型灭活疫苗的研制

对广东珠江三角洲地区31个猪场450份血清用IHA方法进行猪胸膜肺炎放线杆菌(APP)抗体检查,阳性猪场和阳性血清分别为96.8%和58.4%;分离鉴定了7株APP,血清分型为1、7、3和4型,且以1、7型毒力较强;用平板凝集法对263份APP抗体阳性血清进行抗体反向分型试验,结果1型、7型共占76.4%。确定了APP最佳液体培养条件,培养菌液CFU为2.4×109/mL;研制的APP 1型、7型二价灭活疫苗安全、稳定。兔和仔猪免疫抗体曲线显示,油乳剂疫苗明显优于铝胶疫苗,二次免疫优于一次免疫;APP双价油乳剂灭活疫苗一次免疫仔猪,能100%抵抗APP同源1、7型强毒菌株的攻击,保护期达110 d;田间试验明显提高猪群的生产成绩。     

An immunohistochemical study of the tonsils in pigs with acute African swine fever virus infection

An immunohistochemical study of the tonsils was carried out to gain further insight in the pathogenesis of acute African swine fever (ASF). Twenty-one pigs were inoculated by intramuscular route with a highly virulent isolate of ASF virus and painlessly killed at 1-7dpi. Viral antigen was highly distributed in the tonsil from 3 to 4dpi and an increase in the number of monocyte-macrophages was very evident at the same days post inoculation. This phenomenon was observed together with an increase of the expression of proinflammatory cytokines (Tumour necrosis factor alpha and Interleukin-1 alpha) and the apoptosis of lymphocytes studied by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) technique and haemorrhages. With these results, we can conclude that the tonsil is suffering similar lesions than those observed in other lymphoid organs in acute African swine fever, even when the route of inoculation is the intramuscular and not oral-nasal.     

桑天牛幼虫感染白僵菌后的组织病理学研究

桑天牛(Apriona germari)幼虫被球孢白僵菌(Beauveria bassiana)Bb00菌株感染后,虫体颜色及外部形态发生相应的变化。组织切片观察表明:白僵菌Bb00菌株处理后24 h,分生孢子即可以附着于桑天牛幼虫体表,48 h即可以萌发并侵入幼虫体表,使内表皮分解;菌丝进入血腔后开始增殖并向侵入点附近的脂肪、肌肉、气管等组织入侵。随着菌丝在幼虫体内的增殖,各组织器官遭到不同程度的破坏,4 d后感染幼虫死亡,5 d后菌丝突破体表在虫体外形成菌丝层并有分生孢子产生。     

The comparative pathogenicity of four serovars of Actinobacillus (Haemophilus) pleuropneumoniae

The pathogenicity of 2 isolates of each of serovars 7, 3, 1 and 2 of Actinobacillus pleuropneumoniae was tested by intranasal inoculation into 60, 6-week-old large white pigs. Four dose rates varying from 0.27 to 560 x 10(6) organisms per pig with 10-fold serial dilutions were used. Surviving pigs were necropsied 7 days after inoculation. The proportion of pigs dying and developing gross lesions following infection was significantly greater for pigs given serotype 1 than for each of the other 3 serotypes, which did not differ significantly from each other. Twelve of 16 pigs given either of the 2 isolates of serovar 1 died after acute illness and 1 of 44 pigs given either of the 2 isolates each of serovars 7, 3 and 2 died. Pigs given serovar 1 showed high temperatures, severe respiratory distress, frothy haemorrhagic nasal discharge and weight loss. Lung lesions were produced in all 16 pigs given serovar 1, in 7 of 14 pigs given serovar 7, 7 of 14 pigs receiving serovar 3 and in 5 of 16 pigs given serovar 2. The lethal infections were characterised by a severe acute fibrinohaemorrhagic necrotising pleuropneumonia, whereas non-lethal cases had lung lesions ranging from necrotising purulent pleuropneumonia to abscessation. Significant differences between isolates in proportions of tissues culture positive for A. pleuropneumoniae for serovars 7 and 2, but not for serovars 3 and 1 suggested that isolates may vary in virulence within serovars, but more detailed studies are needed to clarify this point.     

Detection of Actinobacillus pleuropneumoniae in the porcine upper respiratory tract as a complement to serological tests.

Attempts were made to isolate Actinobacillus pleuropneumoniae from the nasal cavities and tonsils of 442 healthy pigs from 15 herds. Samples were streaked onto different media formulations. Serum samples were assayed for antibodies to A. pleuropneumoniae by enzyme-linked immunosorbent assay and complement fixation test. Actinobacillus pleuropneumoniae was isolated from the nasal cavities only in 24 pigs, from tonsils only in 90 pigs, and from both the nasal cavities and the tonsils in 11 pigs. A PPLO medium supplemented with lincomycin, bacitracin and crystal violet allowed recovery of A. pleuropneumoniae from more animals than a tryptic soy agar medium from both sites. Incubation of plates in an enriched CO2 atmosphere did not affect the recovery rate. Actinobacillus pleuropneumoniae belonging to serotypes 1, 2, 3, 5a, 5b, 7, 8, 10 and 12 were isolated, and, in several herds, more than one serotype were recovered. Serotypes of A. pleuropneumoniae were isolated from nine herds which were found seronegative to these. The isolation of A. pleuropneumoniae from the upper respiratory tract can be useful for detection of carrier pigs and complements serological screening.     

Comparative virulence of NAD-dependent and NAD-independent Actinobacillus pleuropneumoniae strains.

The virulence of a NAD-independent Actinobacillus pleuropneumoniae serotype 2 strain and NAD-dependent serotype 2, 3 and 9 strains was compared under experimental conditions. Hysterectomy-derived piglets were inoculated endobronchially with 50-500 cfu of these strains. All 23 piglets inoculated with the NAD-dependent strains developed acute disease within 12 hours post inoculation. Twenty-two of these piglets died within 24 hours after the first clinical signs. Three of nine piglets inoculated with the NAD-independent strain did not develop clinical disease. In the other six piglets, disease signs were similar as in the piglets inoculated with the NAD-dependent strains. No differences in clinical disease were observed between colostrum deprived piglets and piglets that obtained colostrum from a SPF sow.     

Expression of secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6) and membrane-bound mucin (MUC4) in the lungs of pigs experimentally infected with Actinobacillus pleuropneumoniae

The expression patterns of different secreted (MUC2, MUC5AC, MUC5B, and MUC6) and membrane-bound (MUC4) mucins were determined immunohistochemically in the lungs of pigs experimentally infected with Actinobacillus pleuropneumoniae. Forty-seven-week-old colostrum-deprived pigs were randomly allocated to infected (n=20) or control groups (n=20). Five infected and uninfected pigs were euthanized at 0, 6, 12, and 48 h post-inoculation (hpi). In the infected pigs, the expression of both types of mucins, which were invariably observed, was associated with bronchiolar and respiratory bronchiolar lesions. Strong positive mucin signals were seen on the surface of bronchiolar and respiratory bronchiolar epithelium with neutrophil infiltration. The mean mucin-positive area peaked at 6 hpi and decreased significantly to control levels by 48 hpi on the surface of the bronchiolar and respiratory bronchiolar epithelium. Further studies are needed to establish the functional relationship between mucin expression and the host defense mechanism against A. pleuropneumoniae in the lungs of infected pigs.     

Animal studies of potential chronic lung disease of workers in swine confinement buildings

The pathologic changes in laboratory animals housed in a swine confinement building were studied to predict potential chronic health effects on persons working in these buildings. Rabbits and guinea pigs were maintained for 12 months either in a confined nursery-grower unit (test animals) or in conventional laboratory animal housing (controls). Necropsies and histopathologic examinations were performed on animals that died prematurely or were killed at termination of the study. Blood serum from the animals was examined for antibodies to extracts from swine confinement house dust. The confinement atmosphere was monitored for environmental pollutants. Concentrations of gases and dust in the air of the confinement building were comparable to those found in a typical swine confinement building. Pulmonic lesions seen in the test rabbits and guinea pigs included diffuse interstitial histiocytic pneumonia. Tracheal and nasal turbinate lesions included epithelial hyperplasia and metaplasia, with submucosal infiltration of plasma cells and heterophils. Blood from the test animals contained serum precipitins to dust extract from confinement houses. The presence of precipitins combined with the microscopic appearance of the lungs indicated that an immunologic process, such as hypersensitivity pneumonitis, may be used to explain the basis of the observed lung lesions. The tracheal and turbinate lesions are best explained as a reaction to a chronic low-grade irritation.     

Early in vivo interactions of Actinobacillus pleuropneumoniae with tonsils of pigs.

Twenty gnotobiotic piglets were inoculated with 5 x 10(8) colony forming units of an Actinobacillus pleuropneumoniae biotype 1-serotype 9 strain onto their tonsils. Five other piglets (controls) were inoculated with phosphate-buffered saline solution. Pigs were euthanized at 30 min, 90 min, 180 min, 6 h, 9 h, 12 h or 24 h after inoculation. At necropsy, samples were taken from the tonsils for bacteriological, histological, immuno-histochemical and electron microscopical examination. A. pleuropneumoniae was isolated from tonsils of all the infected pigs, but not from tonsils of the control pigs. Early after inoculation bacteria were mainly associated with the stratified squamous epithelium and detached epithelial cells. Vacuolization and desquamation of the epithelium was observed and many transmigrating neutrophils were present. At later times after inoculation, bacteria were found closely associated with the crypt-walls and with detached cells present in the crypts. A strong neutrophil migration was observed mainly in the deeper parts of the crypts. It is concluded that attachment of A. pleuropneumoniae to tonsillar epithelial cells probably constitutes a first step in establishing bacteria at this body site.     

Effects of subminimal inhibitory concentrations of amoxicillin against Actinobacillus pleuropneumoniae

The bactericidal effects of amoxicillin at below minimum inhibitory concentration (MIC) against Actinobacillus pleuropneumoniae NB001 were studied in vitro and in vivo. In vivo, the efficacy of amoxicillin on experimentally induced A. pleuropneumoniae infection in disease-free pigs was evaluated. Nine pigs were divided into three groups and all three groups were housed in the same room. Group I pigs were given long-acting amoxicillin injection 22 h prior to A. pleuropneumoniae challenge. Group II pigs were also A. pleuropneumoniae challenged but not given long-acting amoxicillin. Group III pigs were not treated. In vitro, A. pleuropneumoniae growth was suppressed in porcine blood with amoxicillin at below MIC. In vivo, clinical signs of disease were absent or mild in group I during 50 h post-challenge, and serum amoxicillin concentration was already less than MIC from 15 h post-challenge. Infected group II controls were severely affected by the infection, and mortality reached 100% within 50 h post-challenge. All non-treated pigs in group III became infected with NB001 from infected control pigs, and they displayed severe clinical signs of disease within 24 h post-challenge of groups I and II, and died within 50 h post-challenge of groups I and II.     

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.