Candidate genes and gene markers for the resistance to porcine pleuropneumonia.

Actinobacillus (A.) pleuropneumoniae is one of the most important respiratory pathogens in global pig production. Antimicrobial treatment and vaccination provide only limited protection, but genetic disease resistance is a very promising alternative for sustainable prophylaxis. Previous studies have discovered multiple QTL that may explain up to 30% of phenotypic variance. Based on these findings, the aim of the present study was to use genomic sequencing to identify genetic markers for resistance to pleuropneumonia in a segregating commercial German Landrace line. 163 pigs were infected with A. pleuropneumoniae Serotype 7 through a standardized aerosol infection method. Phenotypes were accurately defined on a clinical, pathological and microbiological basis. The 58 pigs with the most extreme phenotypes were genotyped by sequencing (next-generation sequencing). SNPs were used in a genome-wide association study. The study identified genome-wide associated SNPs on three chromosomes, two of which were chromosomes of QTL which had been mapped in a recent experiment. Each variant explained up to 20% of the total phenotypic variance. Combined, the three variants explained 52.8% of the variance. The SNPs are located in genes involved in the pathomechanism of pleuropneumonia. This study confirms the genetic background for the host's resistance to pleuropneumonia and indicates a potential role of three candidates on SSC2, SSC12 and SSC15. Favorable gene variants are segregating in commercial populations. Further work is needed to verify the results in a controlled study and to identify the functional QTN.

Establishment and comparison of Actinobacillus pleuropneumoniae experimental infection model in mice and piglets.

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, a disease responsible for substantial losses in the worldwide pig industry. In this study, outbred Kunming (KM) and Institute of Cancer Research (ICR) mice were evaluated as alternative mice models for APP research. After intranasal infection of serotype 5 reference strain L20, there was less lung damage and a lower clinical sign score in ICR compared to KM mice. However, ICR mice showed more obvious changes in body weight loss, the amount of immune cells (such as neutrophils and lymphocytes) and cytokines (such as IL-6, IL-1ß and TNF-α) in blood and bronchoalveolar lavage fluid (BALF). The immunological changes observed in ICR mice closely mimicked those found in piglets infected with L20. While both ICR and KM mice are susceptible to APP and induce pathological lesions, we suggest that ICR and KM mice are more suitable for immunological and pathogenesis studies, respectively. The research lays the theoretical basis for determine that mice could replace pigs as the APP infection model and it is of significance for the study of APP infection in the laboratory.

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.

Transcriptomic analysis of porcine PBMCs in response to Actinobacillus pleuropneumoniae reveals the dynamic changes of differentially expressed genes related to immuno-inflammatory responses.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, for which the mortality rate is high. Host peripheral blood is a body site for the immune clearance of pathogens mediated by release of inflammatory factors. However, "out of control" inflammatory factor release can contribute to host death. To further understand the changes in the transcription level of immune-related effectors, samples of peripheral blood mononuclear cells (PBMCs) collected from piglets at different stages of infection (0, 24 and 120 h) were sequenced on an Illumina HiSeq™ 4000 platform. We found 3818 differentially expressed genes (DEGs) in the 24 h-infection group compared to the 0 h-infection group (Pb24-Vs-Pb0). DEGs mainly involved in the Gene ontology and KEGG pathways that included nucleic acid metabolism regulation, cell growth, cell differentiation, and organ morphological maintenance were not significantly enriched (P > 0.05). However, DEGs associated with protein kinase activity, receptor activation, metabolism, local adhesion and immune inflammatory responses were significantly enriched in Pb120-Vs-Pb24 (P < 0.05), as were those related to the T cell receptor signalling pathway, with most being down-regulated compared to the preceding stage (Pb24-Vs-Pb0). In PBMCs there were some changes in glucose metabolism, local adhesion and the immune inflammatory response (Pb120-Vs-Pb0). In addition, up-regulated DEGs, such as IL8, IL1ß, and CCL2, and were significantly enriched in immune-inflammatory related pathways compared to the uninfected stage, although they began to decline after 24 h.

Activation of Porcine Alveolar Macrophages by Actinobacillus pleuropneumoniae Lipopolysaccharide via the Toll-Like Receptor 4/NF-κB-Mediated Pathway.

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia. Overproduction of proinflammatory cytokines, like interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and resistin, in the lung is an important feature of A. pleuropneumoniae infection. These proinflammatory cytokines enhance inflammatory and immunological responses. However, the mechanism that leads to cytokine production remains unclear. As a major virulence factor of A. pleuropneumoniae, lipopolysaccharide (LPS) may act as a potent stimulator of Toll-like receptor 4 (TLR4), triggering a number of intracellular signaling pathways that lead to the synthesis of proinflammatory cytokines. Porcine alveolar macrophages (PAMs) are the first line of defense against pathogenic microbes during pathogen invasion. The results of the present study demonstrate that A. pleuropneumoniae LPS induces PAMs to produce inflammatory cytokines in time- and dose-dependent manners. Moreover, PAMs were activated by A. pleuropneumoniae LPS, resulting in upregulation of signaling molecules, including TLR4, MyD88, TRIF-related adaptor molecule, and NF-κB. In contrast, the activation effects of A. pleuropneumoniae LPS on PAMs could be suppressed by specific inhibitors, like small interfering RNA and Bay11-7082. Taken together, our data indicate that A. pleuropneumoniae LPS can induce PAMs to produce proinflammatory cytokines via the TLR4/NF-κB-mediated pathway. These findings partially reveal the mechanism of the overproduction of proinflammatory cytokines in the lungs of swine with A. pleuropneumoniae infection and may provide targets for the prevention of A. pleuropneumoniae-induced pneumonia. All the data could be used as a reference for the pathogenesis of respiratory infection.

Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia and causes significant losses in the pig industry worldwide. Early host immune response is crucial for further progression of the disease. A. pleuropneumoniae is either rapidly eliminated by the immune system or switches to a long-term persistent form. To gain insight into the host-pathogen interaction during the early stages of infection, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 and humanely euthanized eight hours after infection. Gene expression studies of inflammatory cytokines and the acute phase proteins haptoglobin, serum amyloid A and C-reactive protein were carried out by RT-qPCR from the lung, liver, tonsils and salivary gland. In addition, the concentration of cytokines and acute phase proteins were measured by quantitative immunoassays in bronchoalveolar lavage fluid, serum and saliva. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. RESULTS: Significant cytokine and acute phase protein gene expression was detected in the lung and the salivary gland however this was not observed in the tonsils. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter investigations, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. The bacteria isolated from the upper and lower respiratory tract showed distinct IR spectral patterns reflecting the organ-specific acute phase response of the host. CONCLUSIONS: In summary, this study implies a metabolic adaptation of A. pleuropneumoniae to the porcine upper respiratory tract already during early infection, which might indicate a first step towards the persistence of A. pleuropneumoniae. Not only in lung, but also in the salivary gland an increased inflammatory gene expression was detectable during the acute stage of infection.

Frequency of Th17 cells correlates with the presence of lung lesions in pigs chronically infected with Actinobacillus pleuropneumoniae.

Porcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) remains one of the major causes of poor growth performance and respiratory disease in pig herds. While the role of antibodies against APP has been intensely studied, the porcine T cell response remains poorly characterized. To address this, pigs were intranasally infected with APP serotype 2 and euthanized during the acute phase [6-10 days post-infection (dpi)] or the chronic phase of APP infection (27-31 dpi). Lymphocytes isolated from blood, tonsils, lung tissue and tracheobronchial lymph nodes were analyzed by intracellular cytokine staining (ICS) for IL-17A, IL-10 and TNF-α production after in vitro stimulation with crude capsular extract (CCE) of the APP inoculation strain. This was combined with cell surface staining for the expression of CD4, CD8α and TCR-γδ. Clinical records, microbiological investigations and pathological findings confirmed the induction of a subclinical APP infection. ICS-assays revealed the presence of APP-CCE specific CD4+CD8αdim IL-17A-producing T cells in blood and lung tissue in most infected animals during the acute and chronic phase of infection and a minor fraction of these cells co-produced TNF-α. APP-CCE specific IL-17A-producing γδ T cells could not be found and APP-CCE specific IL-10-producing CD4+ T cells were present in various organs but only in a few infected animals. The frequency of identified putative Th17 cells (CD4+CD8αdimIL-17A+) in lung and blood correlated positively with lung lesion scores and APP-specific antibody titers during the chronic phase. These results suggest a potential role of Th17 cells in the immune pathogenesis of APP infection.

Nasal immunization with M cell-targeting ligand-conjugated ApxIIA toxin fragment induces protective immunity against Actinobacillus pleuropneumoniae infection in a murine model.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and severe economic loss in the swine industry has been caused by the infection. Therefore, the development of an effective vaccine against the bacteria is necessary. ApxII toxin, among several virulence factors expressed by the bacteria, is considered to be a promising vaccine candidate because ApxII toxin not only accompanies cytotoxic and hemolytic activities, but is also expressed in all 15 serotypes of bacteria except serotypes 10 and 14. In this study, we identified the peptide ligand capable of targeting the ligand-conjugated ApxIIA #5 fragment antigen to nasopharynx-associated lymphoid tissue. It was found that nasal immunization with ligand-conjugated ApxIIA #5 induced efficient mucosal and systemic immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. More importantly, the nasal immunization induced protective immunity against nasal challenge infection of the bacteria, which was confirmed by histopathological studies and bacterial clearance after challenge infection. Collectively, we confirmed that the ligand capable of targeting the ligand-conjugated antigen to nasopharynx-associated lymphoid tissue can be used as an effective nasal vaccine adjuvant to induce protective immunity against A. pleuropneumoniae infection.

Infection dynamics and acute phase response of an Actinobacillus pleuropneumoniae field isolate of moderate virulence in pigs.

Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia (PCP), causes significant economic losses associated mainly with growth stunting of animals. Although serotypes can be distinguished according to their virulence, most of the studies are focused in A. pleuropneumoniae infections with virulent serotypes. There is little information regarding the role of acute phase proteins (APPs) and proinflammatory cytokines in infections with isolates of mild or moderate virulence. Thus, the present study aims to evaluate the kinetics of infection with an A. pleuropneumoniae serotype 6 (Ap6) field isolate of moderate virulence and the changes in the serum concentration of specific antibodies and different APPs and proinflammatory cytokines. Control animals showed no clinical signs or lesions throughout the study. Infected animals showed increased rectal temperature, respiratory distress and depression from 24hpi, and typical gross and microscopic lesions of PCP from 6hpi onwards. Ap6 was isolated from nasal swabs of four out of five inoculated animals at 24hpi, and from nasal swabs, tonsil and lung samples from all inoculated animals at 72hpi. Specific antibodies against Ap6 or changes in the serum concentration of IL-1ß, IL-10 and TNF-α were not detected throughout the study. The serum concentration of IL-6 increased from 6hpi as well as serum A amyloid, C-reactive protein and haptoglobin from 24hpi onwards. Our results highlight the onset of the acute phase response after the infection with a field isolate of A. pleuropneumoniae of moderate virulence from 24hpi onwards which may be of interest in the study of the pathogenesis of this disease.

Antibiotic treatment following a dog bite in an immunocompromized patient in order to prevent Capnocytophaga canimorsus infection: a case report.

BACKGROUND: Capnocytophaga canimorsus is a commensal bacterium found in the saliva of dogs and cats. Clinically significant infections in humans after a bite are often associated with the presence of immune deficiency. Early recognition and appropriate treatment are crucial for patient survival. In addition, patients with immune deficiency are susceptible to serious life-threatening nosocomial infections, which may also influence the prognosis of patients with Capnocytophaga canimorsus infection. CASE PRESENTATION: A 62-year-old Caucasian female was admitted with septic shock, acute respiratory distress syndrome, acute renal failure, metabolic acidosis and disseminated intravascular coagulation after suffering two small bites from her dog. She had received a splenectomy during childhood. The patient survived after early empiric treatment with antibiotics and intensive supportive care, including ventilation support, a high dose of noradrenalin, and continuous venovenous hemodialysis applied prior to the definitive diagnosis of Capnocytophaga canimorsus sepsis. She improved within 2 weeks but, despite all efforts to prevent nosocomial infection, her hospital course was complicated by Enterococcus species and Candida albicans pleuropneumonia that prolonged her stay in the intensive care unit, and necessitated ventilation support for 2 months. CONCLUSION: Severe Capnocytophaga canimorsus sepsis may be complicated by life-threatening nosocomial infection in immunocompromized patients. The prophylactic application of antibiotics after a dog bite should be considered in high-risk individuals with immune deficiency in order to prevent both Capnocytophyga canimorsus sepsis and serious nosocomial complications.

Adhesion protein ApfA of Actinobacillus pleuropneumoniae is required for pathogenesis and is a potential target for vaccine development.

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia, which causes serious economic losses in the pig farming industry worldwide. Due to a lack of knowledge of its virulence factors and a lack of effective vaccines able to confer cross-serotype protection, it is difficult to place this disease under control. By analyzing its genome sequences, we found that type IV fimbrial subunit protein ApfA is highly conserved among different serotypes of A. pleuropneumoniae. Our study shows that ApfA is an adhesin since its expression was greatly upregulated (135-fold) upon contact with host cells, while its deletion mutant attenuated its capability of adhesion. The inactivation of apfA dramatically reduced the ability of A. pleuropneumoniae to colonize mouse lung, suggesting that apfA is a virulence factor. Purified recombinant ApfA elicited an elevated humoral immune response and conferred robust protection against challenges with A. pleuropneumoniae serovar 1 strain 4074 and serovar 7 strain WF83 in mice. Importantly, the anti-ApfA serum conferred significant protection against both serovar 1 and serovar 7 in mice. These studies indicate that ApfA promotes virulence through attachment to host cells, and its immunogenicity renders it a promising novel subunit vaccine candidate against infection with A. pleuropneumoniae.

Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia.

DNA vaccine encoding type IV pilin of Actinobacillus pleuropneumoniae induces strong immune response but confers limited protective efficacy against serotype 2 challenge.

Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes swine pleuropneumonia, a highly contagious and often fatal disease that occurs worldwide. Our previous study showed that DNA vaccines encoding Apx exotoxin structural proteins ApxIA and/or ApxIIA, are a promising novel approach for immunization against the lethal challenge of A. pleuropneumoniae serotype 1. Vaccination against A. pleuropneumoniae is impeded by the lack of vaccines inducing reliable cross-serotype protection. Type IV fimbrial protein ApfA has been shown to be present and highly conserved in various serotypes of A. pleuropneumoniae. A novel DNA vaccine encoding ApfA (pcDNA-apfA) was constructed to evaluate the protective efficacy against infection with A. pleuropneumoniae serotype 2. A significant antibody response against pilin was generated following pcDNA-apfA immunization, suggesting that it was expressed in vivo. The IgG subclass (IgG1 and IgG2a) analysis indicates that the pcDNA-apfA vaccine induces both Th1 and Th2 immune responses. The IgA analysis shows that mucosal immunity could be enhanced by this DNA vaccine. Nevertheless, the strong antibody response induced by pcDNA-apfA vaccine only provided limited 30% protective efficacy against the serotype 2 challenge. These results in this study do not coincide with that the utility of type IV pilin is a good vaccine candidate against other infectious pathogens. It indicates that pilin should play a limited role in the development of a vaccine against A. pleuropneumoniae infection.

Identification of QTL for dorso-caudal chronic pleuritis in 12 crossbred porcine families.

Pleuropneumonia is a major problem in pig production. At the time of slaughter, chronic pleuritis (CP) developed from pleuropneumonia is a common finding, and breeding for a reduced incidence of CP using marker-assisted selection (MAS) would be advantageous. Before applying MAS, quantitative trait loci (QTL) or markers associated with the prevalence of CP should be identified. In this study, 7470 pigs from crosses between 12 Danish Duroc boars and 604 sows (Danish Landrace × Danish Large White) were evaluated for CP located on the dorso-caudal part of the lungs. Quantitative trait loci were identified within boar families using both a Binomial logistic regression method and a chi-square test of association. Significant QTL for CP were detected on Sus scrofa chromosomes (SSC) 2, 8, 12, 13, 14 and 18 using both methods. One QTL on SSC 8 was also detected across families. For the QTL identified within families, the odds-ratio of having CP was approximately twice as high for the unfavourable allele compared to the favourable one. These QTL and closely linked markers show promise for the development of gene-specific markers associated with a reduced incidence of CP located on the dorso-caudal part of the lungs.

[The content of T-helper antiinflammatory mediators and cytokins of type 1,2 as the immunoregulatory criterion in children with purulent-destructive pneumonia, taking in account tinctorial properties of the pathogen during the hospitalization stage].

In children, suffering purulent-destructive pneumonia in the presence of gramnegative microflora, there were observed prevalence of cytokins helpers (Tx) type 1-interleukin-2 (IL-2), interferon-gamma (IF-gamma) and inhibition of activity of Tx type 2. The cellular chain of immunity advantage is associated with the most high content of anti-inflammatory mediators.

Development of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae infection.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia which leads to high economic losses in the swine industry worldwide. Vaccination against this pathogen is hampered by the occurrence of 15 serotypes, and commonly used whole cell bacterin vaccines are not sufficiently cross-serotype protective. In addition, for generating and maintaining specified pathogen-free herds it is desirable to use DIVA (differentiating infected from vaccinated animals) vaccines. Based on a detergent wash extraction of outer membrane associated proteins and secreted proteins we developed a DIVA vaccine using the immunogenic ApxII toxin which is present in 13 of the 15 A. pleuropneumoniae serotypes as the DIVA antigen. The apxIIA gene was deleted in one strain each of serotypes 1, 2, and 5 using a single-step transconjugation system, and equal parts of detergent washes from these strains served as the vaccine antigen. After intramuscular immunisation all pigs developed a strong humoral immune response to the vaccine antigen and showed no reactivity in an ApxIIA ELISA. Upon challenge all pigs were completely protected from clinical symptoms in trials with a homologous (serotype 2) as well as with a heterologous strain (serotype 9); in addition, colonisation of the challenge strain was clearly reduced but not abolished completely. As a result of the highly efficient protection, however, immunised pigs did not develop antibodies to the DIVA-antigen at levels detectable by ELISA but only by a more sensitive Western blotting approach, thereby demonstrating the challenge in developing appropriate marker vaccines for the livestock industry.

Expression of mRNA encoding interleukin (IL)-10, IL-12p35 and IL-12p40 in lungs from pigs experimentally infected with Actinobacillus pleuropneumoniae.

The expression of mRNA encoding interleukin (IL)-10, IL-12p35 and IL-12p40 was studied, by reverse transcription-polymerase chain reaction and by in situ hybridization with a non-radioactive digoxigenin-labelled cDNA probe, in formalin-fixed, paraffin-wax-embedded lung tissue from pigs experimentally infected with Actinobacillus pleuropneumoniae. Forty-eight 7-week-old colostrum-deprived pigs were randomly allocated to infected (n = 24) or control (n = 24) groups. Three pigs from each group were euthanized at 3, 6, 9, 12, 24, 36, 48 and 60 h post inoculation (hpi). IL-10 mRNA was detected in the lung at 3 hpi, numbers of cells positive for IL-10 increasing at 36 hpi. IL-12p35 mRNA was detected in the lung at 3 hpi, numbers of cells positive for IL-12p35 increasing at 36 and 48 hpi and rapidly decreasing thereafter whereas IL-12p40 mRNA was constitutively expressed at low levels during the experiment. Hybridization signals for IL-10, IL-12p35 and IL-12p40 were always associated with inflammation, in particular with macrophages and neutrophils within alveolar spaces. Expression of these cytokines was minimal in non-lesional lung of A. pleuropneumoniae-infected pigs and in normal lung from control pigs. In situ hybridization of A. pleuropneumoniae and these cytokines in serial sections of lung tissues indicated close co-localization of A. pleuropneumoniae and these cytokines in pleuropneumonia. The results suggest that the expression of IL-10 and IL-12 play a role in pathogenesis of A. pleuropneumoniae infection.

Risk assessment of transmission of capsule-deficient, recombinant Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiologic agent of swine pleuropneumonia. Live, non-encapsulated vaccine strains have been shown to be efficacious in preventing acute disease in pigs. Recombinant DNA technology has the advantage of generating defined mutants that are safe, but maintain critical immunoprotective components. However, some recombinant strains have the disadvantage of containing antibiotic resistance genes that could be transferred to the animal's normal bacterial flora. Using DNA allelic exchange we have constructed attenuated, capsule-deficient mutants of A. pleuropneumoniae that contain a kanamycin resistance (Kn(R)) gene within the capsule locus of the genome. Following intranasal or intratracheal challenge of pigs the encapsulated parent strains colonized the challenge pigs, and were transmitted to contact pigs. In contrast, the capsule-deficient mutants were recovered only from the challenged pigs and not from contact pigs. Each kanamycin-resistant colony type recovered from the respiratory or gastrointestinal tracts of pigs challenged with the recombinant strain was screened with a probe specific for the Kn(R) gene. All probe-positive colonies were assayed for the specific Kn(R) gene by amplification of a 0.9 kb fragment of the antibiotic resistance gene by PCR. The 0.9 kb fragment was amplified from the recombinant A. pleuropneumoniae colonies, but not from any of the heterologous bacteria, indicating there was no evidence of transmission of the Kn(R) gene to resident bacteria. Following aerosol exposure of 276 pigs with recombinant, non-encapsulated A. pleuropneumoniae the recombinant bacteria were not recovered from any nasal swabs of 75 pigs tested or environmental samples 18 h after challenge. Statistical risk analysis, based on the number of kanamycin-resistant colonies screened, indicated that undetected transmission of the Kn(R) gene could still have occurred in at most 1.36% of kanamycin-resistant bacteria in contact with recombinant A. pleuropneumoniae. However, the overall risk of transmission to any resident bacteria was far lower. Our results indicate there was little risk of transmission of capsule-deficient, recombinant A. pleuropneumoniae or its Kn(R) gene to contact pigs or to the resident microflora.

Inflammatory cytokines, pleuropneumonia infection and the effect of dexamethasone.

OBJECTIVES: Actinobacillus pleuropneumoniae causes an often fatal infection of swine due to pleuropneumonia. To determine if inflammatory cytokines are associated with A. pleuropneumoniae-induced pneumonia, infected and noninfected animals were concomitantly administered saline or dexamethasone. METHODS: Twenty-four swine were treated with saline, A. pleuropneumoniae, dexamethasone, or A. pleuropneumoniae and dexamethasone (n = 6). The plasma levels of TNF-alpha, IL-1beta, IL-6, IL-8, and IL-10 were examined through time of necropsy (72 h). Gross pathology and histopathology was performed on all animals. RESULTS: Dexamethasone had no effect on A. pleuropneumoniae-induced increases in lung/body weight ratios. Gross pathology of the infected pigs included fibrinous pleuropneumonia with necrosis and hemorrhage in a focal to a multifocal pattern. Histopathology of infected pig lungs revealed necrotizing extensive, fibrinopurulent pneumonia with edema and fibrinopurulent pleuritis. Plasma IL-6 levels were elevated in A. pleuropneumoniae-infected animals beginning 6 h after infection. Dexamethasone treatment did not alter A. pleuropneumoniae-induced plasma IL-6 levels. A. pleuropneumoniae infection did not elicit plasma levels of TNF-alpha, IL-1beta, IL-8, or IL-10. CONCLUSION: These results suggest that the pneumonia caused by A. pleuropneumoniae infection is not due to the release of systemic inflammatory cytokines.