Actinobacillus Pleuropneumoniae Infection Research Articles

Prevalence of Actinobacillus pleuropneumoniae Infection in Hunted Wild Boars (Sus scrofa) in Germany

Actinobacillus pleuropneumoniae is an important pathogen of the porcine respiratory tract. Subclinically and chronically infected pigs may enter a permanent carrier state, harboring A. pleuropneumoniae in lungs or tonsils. Despite the enormous role of A. pleuropneumoniae in domestic swine production, no published data about prevalence and distribution of A. pleuropneumoniae infections in wild boars (Sus scrofa) are available. Antibodies against A. pleuropneumoniae have been detected in serum of wild boars from Slovenia. The aim of the present study was to evaluate A. pleuropneumoniae infection status in wild boars in Germany. Tissue samples from lungs and tonsils of 531 wild boars from 52 hunts during the hunting seasons 2004-05 to 2006-07 were examined for A. pleuropneumoniae by polymerase chain reaction (PCR). Although gross pathologic lesions typical for pleuropneumonia were not detected, 35.8% of the wild boars were PCR-positive. Tonsils were involved in 94.3% and lungs in 15.5%. Actinobacillus pleuropneumoniae infection prevalence increased with age and body weight. Further research is needed to understand the involvement of different A. pleuropneumoniae serotypes and to understand the interrelationship of A. pleuropneumoniae infection between wild boars and domestic pigs.

Risk factors associated with pleuritis and cranio-ventral pulmonary consolidation in slaughter-aged pigs

Examination of lung lesions at the slaughterhouse is a useful tool to estimate the importance of respiratory disease at farm, regional or national level. The objective of the present work was to describe the prevalence of gross lung lesions at slaughter, with a special focus on pleuritis and cranio-ventral pulmonary consolidation, and to identify major risk factors for these lesions. Data from 107 farms involving approximately 11,000 pigs enabled gross lung lesions to be correlated with serology to different swine respiratory pathogens as well as with production system characteristics and vaccination schedules. Pleuritis and cranio-ventral pulmonary consolidation lesions were recorded in 26.8% and 55.7% of slaughter-aged pigs, respectively. Among lungs with pleuritis, 50.1% had lesions compatible with Actinobacillus pleuropneumoniae (App) infection. Antibodies to porcine reproductive and respiratory syndrome (PRRSV), three subtypes (H1N1, H1N2 and H3N2) of swine influenza virus (SIV), App and Mycoplasma hyopneumoniae (Mhyo) were highly prevalent (>82%) in most of the farms. In a multivariable analysis, it was estimated (R(2)=0.40) that the percentage of animals with pleuritis compatible with App infection depended on the existence of an all in-all out by room management system and App and PRRSV herd seroprevalence. Moreover, it was possible to foresee (R(2)=0.59) that cranio-ventral pulmonary consolidation lesions (EP-like lesions) were affected by the type of farm ventilation, the presence of respiratory symptoms during the fattening period and Mhyo and SIV H1N2 herd seroprevalence.

Porcine Lung Surfactant Protein B Gene (SFTPB): cDNA Sequencing, Chromosomal Location, and Expression Studies

The porcine surfactant protein B (SFTPB) is a single copy gene on chromosome 3. Three different cDNAs for the SFTPB have been isolated and sequenced. Nucleotide sequence comparison revealed six nonsynonymous single nucleotide polymorphisms (SNPs), four synonymous SNPs and an in-frame deletion of 69 bp in the region coding for the active protein. Northern analysis showed lung-specific expression of three different isoforms of the SFTPB transcript. The expression level for the SFTPB gene is low in 50 days-old fetus and it increases during lung development. Quantitative real-time polymerase chain reaction (qPCR) showed significant down regulation of the SFTPB in pigs with Actinobacillus pleuropneumoniae lung infection.

DEVELOPMENT OF PROTEIN CHIP BASED ON SURFACE PLASMON RESONANCE FOR THE DETECTION OF ACTINOBACILLUS PLEUROPNEUMONIAE ANTIBODY

ABSTRACT A protein chip based on surface plasmon resonance (SPR) was developed for measuring the Actinobacillus pleuropneumoniae (APP) antibody (Ab) titers using recombinant transferrin‐binding protein B (Tbp B) as an antigen. The diagnostic potential of this SPR assay for detecting the Ab titers to APP Tbp B was compared with that of enzyme‐linked immunosorbent assay (ELISA) using 70 serum samples obtained from six pig farms. The SPR assay was found to be highly specific and sensitive. Moreover, there was a strong positive correlation between the SPR and ELISA titers (n = 70, r = 0.858, P < 0.01). In conclusion, the SPR, which is a label‐free method, is expected to be a valuable and reproducible tool in the serodiagnosis of an APP infection.

Influence of rearing conditions and respiratory disease on haptoglobin levels in the pig at slaughter

Associations between serum concentrations of haptoglobin, pathological lung lesions indicative of Mycoplasma hyopneumoniae (EP) or Actinobacillus pleuropneumoniae (PL) infection at slaughter and previous rearing environment were investigated in 510 pigs (90–100 kg live weight) from 17 farms in England. Haptoglobin concentrations were significantly higher in pigs showing pathological signs of EP infection compared to those without signs of this disease (EP positive median 0.43 mg ml −1 vs. EP negative median 0.26 mg ml −1, p < 0.01). However, there were no significant associations between serum haptoglobin concentrations and pathological signs of PL. The presence of solid partitions compared with barred or similar open partitions was associated with a decrease of 0.44 mg ml −1 farm mean haptoglobin concentration, whilst an increase in pen size of 10 m 2 was associated with a decrease of 0.08 mg ml −1 farm mean haptoglobin concentration. The findings indicate that pathological signs of EP were associated with increased serum haptoglobin at slaughter, which in turn was influenced by components of the farm environment.

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.

Experimental Actinobacillus pleuropneumoniae infection in piglets with different types and levels of specific protection: Immunophenotypic analysis of lymphocyte subsets in the circulation and respiratory mucosal lymphoid tissue

Actinobacillus pleuropneumoniae (APP) infection in piglets results in severe and fatal fibrinous hemorrhagic necrotizing pneumoniae. The aim of our study was to analyze changes in lymphocyte subset distribution in peripheral blood, bronchoalveolar lavage fluid (BALF) and tracheobronchal lymph nodes (TLN) in non-immune piglets upon a challenge with a high dose of APP and to compare the quality of such changes in unprotected piglets with counterparts exhibiting specific immunity mediated by high titers of colostrum-derived APP-specific antibodies and/or a low dose APP infection in the early postnatal period. Challenge with APP resulted in a massive increase in CD8-negative γδ T-cells in parallel with a reduction in numbers of CD3 −CD8 low cells in BALF independent of the type and level of immunity and this seems to be a general phenomenon associated with experimental infection. An increase in B-lymphocyte numbers in TLN was another characteristic feature accompanying APP infection in all experimental groups. In piglets with colostrum-derived APP-specific antibodies, this was associated with higher relative numbers of IgM +CD2 + lymphocytes in TLN, while B-cells with the CD2 − surface phenotype apparently expanded in the absence of passive humoral immunity.

Significance of different types and levels of antigen-specific immunity to Actinobacillus pleuropneumoniaeinfection in piglets

The aim of the study was to verify under experimental conditions the hypothesis that a reliable protection of piglets against Actinobacillus pleuropneumoniae (App) infection can be obtained by colostrum-derived antibodies and/or a low-dose infection from sows naturally infected with App. Twenty-eight piglets were allocated into four groups. Piglets of groups 1A and 1B were the progeny of App-seronegative sows, piglets of groups 2A and 2B originated from App-seropositive sows. At the age of 4 weeks, piglets in groups 1A and 2A were exposed to a low infection dose with the pathogenic strain of App serotype 9 (4 &amp;times; 10&lt;sup&gt;4&lt;/sup&gt; CFU/ml). At the age of 8 weeks, all four groups of piglets were exposed to the App serotype 9 challenge (2.5 &amp;times; 10&lt;sup&gt;7&lt;/sup&gt; CFU/ml). We compared the differences in clinical, radiological, pathological changes and cytological findings in blood and bronchoalveolar lavage samples between groups with different levels of antigen-specific immunity after challenge. Piglets that obtained colostrum-derived antibodies and experienced a low-dose App infection were best protected against App challenge. More apparent clinical and pathological changes were observed in groups protected with either only colostrum-derived antibodies or active immunity induced by a preceding low-dose infection. However, none of the types of protection prevented developing of the disease and characteristic changes in the lungs. Cell counts changes in blood and lavage gave evidence of only bacterial infection in progress; however, between-group differences were not marked. Antibodies (IgG, IgA) present in the respiratory tract at the time of challenge played a major role in protection against App infection.

Efficacy of vaccines against bacterial diseases in swine: what can we expect?

This paper discusses what can be expected with regard to efficacy of antibacterial vaccines used in swine, based on the present knowledge of pathogen–host interactions. First, vaccination against bacteria that mainly cause disease by production of exotoxins is considered. Vaccines containing the inactivated toxin or a non-toxic but antigenic recombinant protein derived from the exotoxin can be expected to provide protection against disease. The degree of protection induced by such vaccines varies, however, depending amongst other things on the pathogenesis of the disease. Vaccination against clostridial infections, Actinobacillus pleuropneumoniae infections, progressive atrophic rhinitis and enterotoxigenic Escherichia coli, is considered. The second part of the article deals with vaccination against extracellular bacteria. Protection against these bacteria is generally mediated by antibodies against their surface antigens and certain secreted antigens, but cellular immunity may also play a role. Efficacy of vaccines against swine erysipelas, Streptococcus suis infections, Mycoplasma hyopneumoniae infections and swine dysentery is discussed. Finally, vaccination against facultatively intracellular bacteria is considered. For protection against these bacteria cell-mediated immunity plays an important role, but antibodies may also be involved. It is generally accepted that live-attenuated vaccines are more suitable for induction of cell-mediated immunity than inactivated vaccines, although this also depends on the adjuvant used in the vaccine. As an example, vaccination against Salmonella enterica serotype Typhimurium is discussed.

IP004 Presence of two antibiotic peptides in bronchoalveolar lavage fluid of convalescent pigs after an Actinobacillus pleuropneumoniae infection

IP004 Presence of two antibiotic peptides in bronchoalveolar lavage fluid of convalescent pigs after an Actinobacillus pleuropneumoniae infection

Interference of outer membrane protein PalA with protective immunity against Actinobacillus pleuropneumoniae infections in vaccinated pigs

The role of antibodies to the outer membrane protein PalA of Actinobacillus pleuropneumoniae in protective immunity was studied in pigs vaccinated with purified PalA alone and PalA in combination with toxoids of the RTX toxins ApxI and ApxII using an established challenge model with the virulent serotype 1 of A. pleuropneumoniae. Pigs that developed antibody titers against PalA after immunization were more significantly affected by challenge with A. pleuropneumoniae serotype 1. Following challenge, pigs that were immunized with PalA showed more severe respiratory symptoms, had a higher mortality rate and died faster. They also displayed much more severe lung lesions after necropsy than animals not immunized with PalA. Pigs that were immunized with toxoids of the two cytotoxins ApxI and ApxII were protected against challenge with A. pleuropneumoniae. In contrast, the protective efficacy of the ApxI and ApxII vaccine was completely lost when it was supplemented with PalA. Hence, antibodies induced against the outer membrane protein PalA of A. pleuropneumoniae aggravated the consequences of infection and counteracted the protective effect of anti-ApxI and anti-ApxII antibodies. Due to the high similarity between protein analogues of PalA from various bacteria of the Pasteurellaceae family such as P6 of Haemophilus influenzae or 16kDa Omp of Pasteurella multocida, this deleterious effect of PalA in vaccination should be taken into consideration in the development of vaccines against infections with other Pasteurellaceae.

Evaluation of a single dose versus a divided dose regimen of danofloxacin in treatment of Actinobacillus pleuropneumoniae infection in pigs

A single versus a divided dose regimen of danofloxacin was evaluated in treatment of porcine Actinobacillus pleuropneumoniae infection using clinical observations combined with biochemical infection markers: C-reactive protein, zinc and ascorbic acid. Twenty hours after experimental infection, the 18 pigs received danofloxacin intravenously as a single dose of 2.5 mg/kg or four doses of 0.6 mg/kg administered at 24 h intervals. These dosage regimens resulted in similar AUCs of the plasma danofloxacin vs time curve. The maximum concentration was 3.5-fold higher using the single dose regimen, while the time with concentrations above the MIC was 2.5-fold longer using the fractionated regimen. Using the single dose regimen, temperature was normalised 32 h post-infection. In contrast, normalisation was delayed until 44 h post-infection using four low doses and a relapse with elevated temperatures at 52 and 68 h was observed. No other significant differences between the treatments were found, neither regarding clinical, haematological nor biochemical observations. The use of the more convenient single dose regimen was appropriate, as it was at least equivalent to the fractionated regimen.

Putative biomarkers for evaluating antibiotic treatment: an experimental model of porcine Actinobacillus pleuropneumoniae infection

Biomarkers of infection were screened for their possible role as evaluators of antibiotic treatment in an aerosol infection model of porcine pneumonia caused by Actinobacillus pleuropneumoniae (Ap). Following infection of 12 pigs, clinical signs of pneumonia developed within 20 h, whereafter the animals received a single dose of either danofloxacin (2.5 mg/kg) or tiamulin (10 mg/kg). To test the discriminative properties of the biomarkers, the dosage regimens were designed with an expected difference in therapeutic efficacy in favour of danofloxacin. Accordingly, the danofloxacin-treated pigs recovered clinically within 24 h after treatment, whereas tiamulin-treated animals remained clinically ill until the end of the study, 48 h after treatment. A similar picture was seen for the biomarkers of infection. During the infection period, plasma C-reactive protein (CRP), interleukin-6 and haptoglobin increased, whereas plasma zinc, ascorbic acid and α-tocopherol decreased. In the danofloxacin-treated animals, CRP, interleukin-6, zinc, ascorbic acid and α-tocopherol reverted significantly towards normalisation within 24 h of treatment. In contrast, signs of normalisation were absent (CRP, zinc and ascorbic acid) or less marked (interleukin-6 and α-tocopherol) in the tiamulin-treated animals. Plasma haptoglobin remained elevated throughout the study in both groups. This indicates that CRP, zinc, ascorbic acid and to a lesser extent interleukin-6 and α-tocopherol might be used to evaluate antibiotic treatment of acute Ap-infection in pigs. The present model provides a valuable tool in the evaluation of antibiotic treatments, offering the advantage of clinical and pathological examinations combined with the use of biochemical infection markers.

Influence of porcine Actinobacillus pleuropneumoniae infection and dexamethasone on the pharmacokinetic parameters of enrofloxacin.

The impact of Actinobacillus pleuropneumoniae (APP) infection in swine on the pharmacokinetic parameters of enrofloxacin were determined. Twenty-four animals were used in a 2 x 2 factorial of treatment groups (six animals per group) to determine the impact of APP-induced inflammation and the anti-inflammatory drug dexamethasone on enrofloxacin pharmacokinetic parameters. All animals received enrofloxacin as a single intravenous dose (5 mg/kg). Administration of dexamethasone was associated with an increase in clearance of enrofloxacin Clearance of enrofloxacin was not affected by APP. Volume of distribution at steady state was significantly increased in the dexamethasone-treated pigs. Volume of distribution at steady state was decreased by APP infection. Dexamethasone significantly increased the terminal elimination half-life of enrofloxacin. APP infection decreased the terminal elimination half-life of enrofloxacin in the infected pigs. Infection and dexamethasone significantly decreased the urine enrofloxacin/creatinine and ciprofloxacin/creatinine ratios. This study shows that APP infection does affect plasma pharmacokinetic parameters. Dexamethasone and APP infection may reduce renal clearance of enrofloxacin with a compensatory increase in intestinal clearance. Neither infection nor dexamethasone altered the metabolism of enrofloxacin to ciprofloxacin, the principal metabolite of enrofloxacin.

Serologic profile of a cohort of pigs and antibody response to an autogenous vaccine for Actinobacillus suis

Actinobacillus suis is a commensal opportunistic pathogen in swine. However, in recent years, an increasing prevalence of clinical signs associated with A. suis has been observed in high health status herds in North America. The objectives of the study were to assess the kinetics of antibodies to A. suis in pigs from a herd showing clinical signs of A. suis infection and, to evaluate the antibody response in gilts following vaccination with an autogenous vaccine. An enzyme-linked immunosorbent assay (ELISA) using a saline extract of boiled-formalinized whole cells of a field strain as the coating antigen was standardized. This ELISA was used as a tool for monitoring, in a comparative way, the variations in A. suis antibody levels. The herd selected for the serologic profile was negative for Actinobacillus pleuropneumoniae infection and showed clinical signs of A. suis infection in 16 to 19-week-old pigs. A cohort of 20 pigs was blood sampled at 5, 8, 12, and 16 weeks of age. The lowest level of serum antibodies was observed between weeks 8 and 12, this probably corresponding to a decrease in maternal immunity. A marked increase in the antibody response was seen at 16-week of age, at the approximate time of onset of A. suis clinical signs in the herd. The evaluation of serum antibody responses to an autogenous vaccine revealed that the humoral immunity of gilts further increased following vaccination although the level of antibodies was already high prior to vaccination. The magnitude of the response to vaccination was higher when the level of antibodies was low prior to the first injection. The ELISA test seems to detect antibodies against the O-chain LPS.

Prevention of pleuropneumonia in pigs by in‐feed medication with sulphadimethoxine and sulphamethoxazole in combination with trimethoprim

Summary The prophylactic effect of in‐feed medication of conventional pigs with sulphadimethoxine (SDM), sulphamethoxazole (SMX), and trimethoprim (TMP) was tested by using an Actinobacillus pleuropneumoniae infection model. In each of five experiments, six pigs were given medicated feed twice daily and three pigs received antibiotic‐free feed and served as positive (unmedicated, infected) controls. The following drugs or drug combinations were tested (in mg per kg feed): 500 SDM + 100 TMP, 500 SMX + 100 TMP, 125 SMX + 25 TMP, 125 SMX (alone) and 25 TMP (alone). After six days of feed medication, all animals were endobronchially inoculated with A. pleuropneumoniae in a dose of 1–3.104 colony‐forming units (CFU). The response to the challenge in all control pigs was characterized by fever, lethargy, anorexia, reduced water consumption, and laboured breathing. At autopsy all controls manifested a fibrinous haemorrhagic pleuropneumonia. In‐feed medication with 500 SDM + 100 TMP, 500 SMX + 100 TMP as well as 125 SMX + 25 TMP resulted in an effective protection against the challenge in all treated animals. After consumption of feed medicated with 125 mg per kg SMX or 25 mg per kg TMP, pleuropneumonia was evident in all challenged pigs. The results of this study indicate an in vivo potentiation of SMX and TMP in pigs against this respiratory tract pathogen.

The Porcine Acute Phase Response to Infection with Actinobacillus pleuropneumoniae. Haptoglobin, C-Reactive Protein, Major Acute Phase Protein and Serum Amyloid A Protein Are Sensitive Indicators of Infection

In an experimental infection model mimicking acute Actinobacillus pleuropneumoniae (Ap) infection in swine (Sus scrofa) by aerosol inoculation, the development of a number of typical clinical signs was accompanied by a prototypic acute phase reaction encompassing fever and an acute phase protein response peaking at around 2 days after infection. Haptoglobin, C-reactive protein (CRP), and major acute phase protein (MAP) responded with large increases in serum levels, preceding the development of specific antibodies by 4–5 days. Serum amyloid A protein (SAA) was also strongly induced. The increase, kinetics of induction and normalization were different between these proteins. It is concluded that experimental Ap-infection by the aerosol route induces a typical acute phase reaction in the pig, and that pig Hp, CRP, MAP, and SAA are major acute phase reactants. These findings indicate the possibility of using one or more of these reactants for the nonspecific surveillance of pig health status.

Immunohistopathological characterization of pig pneumonia caused by a combined Aujeszky's disease virus and Actinobacillus pleuropneumoniae infection

Nine pigs were inoculated endobronchially with Actinobacillus pleuropneumoniae serotype 1 (App-1) 6 days after infection with Aujeszky's disease virus (ADV); four died within 3 days and the remainder were killed after 1-6 days. Immunohistopathologically, there were two types of pneumonic lesion: pleuropneumonia, characterized by coagulative necrosis, oedema and fibrinous thrombosis; and necrotizing interstitial pneumonia, characterized by bronchitis, bronchiolitis and alveolitis. The former type of lesion was associated with App-1 antigen, and the latter with ADV antigen. These results indicated that a combined ADV and App-1 infection produced severe haemorrhagic pleuropneumonia; and that ADV and App-1 each produced a characteristic pneumonic lesion.

Dual infections of PRRSV/influenza or PRRSV/ Actinobacillus pleuropneumoniae in the respiratory tract

To study the effect of a previous porcine respiratory and reproductive syndrome-infection (PRRS) of the respiratory tract on influenza virus and Actinobacillus pleuropneumoniae (App) infections, 3-week-old specific-pathogen-free (spf) piglets were intranasally infected with PRRS virus. One week later, when the lung alveolar macrophages of PRRSV infected pigs were lowest in number, a second infection was applied by intranasal aerosol of influenza virus H3N2 or by endobronchial instillation of a mildly virulent App. The first experiment consisted of two groups (only influenza infection or dual PRRSV/influenza infection). A second experiment consisted of 4 groups (only influenza infection, only PRRSV infection, dual PRRSV/influenza infection and uninfected controls). At day 2, 4, 14 and 21 after influenza infection, two pigs were killed and sampled for virological and histopathological examination. Influenza H3N2 virus caused only a mild inflammation of the smaller bronchioli. Previous PRRSV infection did not influence clinical signs during influenza infection. Next, we studied in two experiments the effect of dual PRRSV/App infection during the acute stage at two days after App infection. In a third experiment, the influence of PRRSV on more chronic stages of App infection was studied at two weeks after the App infection. At the end of the experiments, the pigs were killed. Lungs were ranked according to size and kind of the lesions. Lesions were cut and measured, samples were taken for virological and histopathological examination. Statistical analysis of the ranked lung-lesions in the first experiment showed a distinct but small effect of previous PRRSV infection on the development of App-lesions. In PRRSV infected pigs, App produced a more severe disease. The second and third experiment however failed to show any influence of the previous PRRSV infection on the App infection. We conclude that previous PRRSV infection of the respiratory tract of spf pigs does not necessarily enhance the severity of secondary infections of the respiratory tract.

Inflammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae

An Actinobacillus pleuropneumoniae infection model in swine was established to study the expression of inflammatory cytokines during acute respiratory disease. Lavage fluid, lavage cells consisting primarily of alveolar macrophages, and lung tissue were analyzed for the presence of various cytokines at 2, 4, 8, and 24 h following endotracheal inoculation of A. pleuropneumoniae. Interleukin-1 beta (IL-1) and IL-8 mRNA levels were elevated within 2 h in lavage cells of animals inoculated with A. pleuropneumonia but not in cells from controls treated with saline-bovine serum albumin, based on Northern (RNA blot) analysis. Tumor necrosis factor (TNF) mRNA was present at low levels in all animals, and the level was not increased at any time point. In situ hybridization was more sensitive than Northern blotting and revealed elevations of all three cytokines in lavage cells within 2 to 4 h of A. pleuropneumoniae inoculation. IL-6 was detected in lavage cells by in situ hybridization but not by Northern blotting. In lung tissue obtained 18 to 24 h after A. pleuropneumoniae instillation, all cytokine mRNAs, including that of IL-6, were detected by Northern blot analysis. The levels of bioactive IL-1 and IL-6 in lavage fluids increased approximately 1,000-fold following A. pleuropneumoniae inoculation, but TNF bioactivity was not detected. Morphological localization of cytokine mRNAs by in situ hybridization indicated markedly increased levels of TNF, IL-1, and IL-8 mRNAs at the periphery of focal lung lesions. These findings indicate that inflammatory cytokines, particularly IL-1 and IL-8, are associated with the development of pleuropneumonia and may contribute to disease severity.