Porcine Pleuropneumonia Research Articles

Actinobacillus pleuropneumoniae metalloprotease: cloning and in vivo expression

The complete amino acid and nucleotide sequence of a secreted metalloprotease produced by Actinobacillus pleuropneumoniae serotype 1 is reported. A clone showing proteolytic activity in cell-free culture media was selected from a genomic library of A. pleuropneumoniae serotype 1 in pUC 19. The sequence obtained contained an open reading frame encoding a protein with 869 amino acids. This protein was identified as a zinc neutral-metalloprotease belonging to the aminopeptidase family, with a predicted molecular weight of approximately 101 kDa. This sequence showed high homology with other predicted or sequenced aminopeptidases reported for different Gram-negative bacteria. Expression of the protease was observed in lung tissue from pigs that died of porcine pleuropneumonia suggesting a role in pathogenesis.

Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N′-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N′-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App™ were serologically negative in the ApxIV-ELISA.

Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation—ProteinChip™ (SELDI) technology

Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia. An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3–20 kDa. However, conventional techniques to study bacterial protein expression, such as SDS–PAGE and 2-dimensional electrophoresis, typically focus on the 15–200 kDa range. In this study we have evaluated Surface Enhanced Laser Desorption Ionisation—ProteinChip™ (SELDI) technology for the analysis of protein expression, in particular those of <20 kDa, of APP grown under different environmental conditions. Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium. Optimum conditions for SELDI profiles included a sample size of 1 μg and the use of sinapinic acid as the energy absorbing matrix. In the <20 kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33–66% pig serum were most similar to those grown in defined medium. The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences. The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20 kDa mass range.

Growth phase mediated regulation of the Actinobacillus pleuropneumoniae ApxI and ApxII toxins

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal respiratory tract disease of pigs. The Apx toxins are primary virulence factors of this pathogen, with ApxI and ApxII being produced by all highly virulent strains in North America. Further characterization of these hemolytic toxins is needed to fully understand their role in disease and elucidate the environmental signals and genes that affect their production during infection. Many bacteria regulate genes in response to growth phase, and in this report we examined the effect of growth phase on ApxI and ApxII gene expression. Batch cultures of ApxI- and ApxII-producing strains were grown in heart infusion broth supplemented with β-NAD, and samples were prepared throughout the growth curve. Maximal gene expression occurred in late exponential or early stationary phase, as indicated by a peak in apx mRNA concentration in Northern blot analysis. The amount of accumulated Apx protein and Apx hemolytic activity confirmed this increase in gene expression. These findings suggest a novel transcriptional regulatory mechanism that enhances Apx gene expression under in vitro conditions of high cell density and/or slow growth rate.

Detection of Nuclear Factor-κB and Inducible Nitric Oxide Synthase in the Lungs of Pigs Naturally Infected with Actinobacillus pleuropneumoniae

Activated nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) were detected immunohistochemically in pleuropneumonic lungs from 20 pigs naturally infected with Actinobacillus pleuropneumoniae. NF-κB was detected mainly in nuclei of inflammatory cells, confirming its activation. Intense immunolabelling for NF-κB and iNOS was seen within the lung lesions, but labelling was minimal in unaffected portions of the lung of infected pigs and in normal lung from uninfected (control) pigs. Examination of serial sections from the 20 infected lung samples demonstrated a close association between NF-κB and iNOS. This suggests that NF-κB plays a key role in triggering the activation of iNOS in porcine pleuropneumonia.

Identification of dimethyl sulfoxide reductase in Actinobacillus pleuropneumoniae and its role in infection.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in an A. pleuropneumoniae serotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein of Haemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinished A. pleuropneumoniae genome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of the dmsA ORF. In order to investigate the role of the A. pleuropneumoniae DmsA protein in virulence, an isogenic deletion mutant, A. pleuropneumoniae DeltadmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniae DeltadmsA was attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniae virulence.

Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host.

Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is the causative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis (STM) to identify genes required for survival of the organism within the pig. A total of 2,064 signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used to inoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated in vivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type, whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced from competitive index experiments. Inverse PCR was used to generate DNA sequence of the chromosomal domains flanking each transposon insertion. Only one sibling pair of mutants was identified, but three apparent transposon insertion hot spots were found--an anticipated consequence of the use of a Tn10-based system. Transposon insertions were found within 55 different loci, and similarity (BLAST) searching identified possible analogues or homologues for all but four of these. Matches included proteins putatively involved in metabolism and transport of various nutrients or unknown substances, in stress responses, in gene regulation, and in the production of cell surface components. Ten of the sequences have homology with genes involved in lipopolysaccharide and capsule production. The results highlight the importance of genes involved in energy metabolism, nutrient uptake and stress responses for the survival of A. pleuropneumoniae in its natural host: the pig.

Identification, cloning and characterization of rfaE of Actinobacillus pleuropneumoniae serotype 1, a gene involved in lipopolysaccharide inner-core biosynthesis

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and its lipopolysaccharides (LPS) have been identified as important adhesins involved in adherence to host cells. To better understand the role of LPS core in the virulence of this organism, the aim of the present study was to identify and clone genes involved in LPS core biosynthesis by complementation with Salmonella enterica serovar Typhimurium mutants ( rfaC, rfaD, rfaE and rfaF). Complementation with an A. pleuropneumoniae 4074 genomic library was successful with Salmonella mutant SL1102. This Salmonella deep-rough LPS mutant is defective for the rfaE gene, which is an ADP–heptose synthase. Novobiocin was used to select transformants that had the smooth-LPS type, since Salmonella strains with wild-type smooth-LPS are less permeable, therefore more resistant to hydrophobic antibiotics like novobiocin. We obtained a clone that was able to restore the wild-type smooth-LPS Salmonella phenotype after complementation. The wild-type phenotype was confirmed using phage (Felix-O, P22c.2 and Ffm) susceptibility and SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). One of the open reading frames contained in the 3.3-kb insert in the plasmid encoded a 475-amino-acid protein with 71% identity and 85% similarity to the RfaE protein of S. enterica. We then attempted to generate an A. pleuropneumoniae rfaE mutant by gene replacement. The rfaE gene seems essential in A. pleuropneumoniae viability as we were unable to isolate a heptose-less knockout mutant.

Induction of protective immunity in pigs after immunisation with CpG oligodeoxynucleotides formulated in a lipid-based delivery system (Biphasix™)

A large number of studies demonstrated the immunostimulatory effects of CpG oligonucleotides (ODN), particularly in mice. In the present study, we evaluated the ability of lipid-based delivery systems to enhance the adjuvant effect of CpG-ODN and protect against infection in a porcine pleuropneumonia model. Increased levels of OmlA-specific antibody were detected in animals immunised with OmlA and CpG-ODN formulated in the delivery system Biphasix™–vaccine targeting adjuvant (VTA), compared to pigs immunised with VTA without CpG-ODN or CpG-ODN alone. In addition, the responses induced by VTA/CpG formulation were similar to those induced by the commercial adjuvant VSA; however, VTA formulations caused significantly less tissue damage than VSA.

Comparison of Protection Efficacy of Toxoid and Whole-Cell Vaccines Against Porcine Pleuropneumonia Caused by Endotracheal Infection with Actinobacillus pleuropneumoniae

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Detection, identification, and subtyping of Actinobacillus pleuropneumoniae.

Abstract Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes significant losses in industrialized swine production worldwide. Bacterial diagnosis of contagious porcine pleuropneumonia is generally done by bacteriological isolation and cultivation of A. pleuropneumoniae, followed by serological typing which differentiates 15 serotypes (1–3). There are significant differences in virulence among the 15 serotypes. Serotypes 1,5, and also 9 and 11 are involved in particularly severe outbreaks of disease with high mortality and severe pulmonary lesions. Serotypes 2-4, 6-8, 12, and 15 are generally less virulent, causing moderate mortality but relatively strong lung lesions. Serotype 3 seems to be mostly of low epidemiological importance, while the remaining serotypes are isolated only very rarely. The degree of virulence seems to be mainly due to the presence of one or two of the pore-forming RTX toxins ApxI, ApxII, and ApxIII, found in A. pleuropneumoniae, which characterize the different serotypes of A. pleuropneumoniae (4,5) (see Table 1). Toxin ApxI is encoded by the genes apxICABD, with gene A specifying the structural protein toxin, C coding for its activator, and B and D coding for the corresponding type I secretion pathway. ApxI is produced and secreted by the highly pathogenic serotypes 1, 5, 9, and 11, and also in serotype 10 and 14 of A. pleuropneumoniae. ApxII, encoded by apxIICA, is produced by all serotypes except 10 and 14. Table 1 Recipe for Preparation of pH 4–8 IPG IEF GelFull size table KeywordsPolymerase Chain ReactionLung TissueNest Polymerase Chain ReactionPolymerase Chain Reaction AmpliconSerological TypingThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Tissue pharmacokinetics of florfenicol in pigs experimentally infected with Actinobacillus pleuropneumoniae.

The aim of this study has been to determine the tissue pharmacokinetic parameters of florfenicol in the pigs experimentally infected with Actinobacillus pleuropneumoniae. 21 crossed-bred (Duroc x Landrace x Yorkshire) local species of pigs were infected experimentally with Actinobacillus pleuropneumoniae serotype 1 and confirmed as typical sub-acute pleuropneumonia. A single dose of 20 mg/kg body weight of florfenicol, a novel animal-using antibiotic, was administrated intramuscularly in the pigs and then samples of blood, lung, trachea with bronchi, liver, kidney and muscle were taken at scheduled time points. Drug concentrations were determined by high performance liquid chromatography (HPLC) with an ultraviolet detector via extraction with ethyl acetate under nitrogen flow. The statistic moment theory (SMT) mathematic package was applied to calculate the tissue pharmacokinetic parameters of florfenicol in the infected model. AUC of lung, trachea with bronchi, liver, kidney and muscle were 121.69, 79.37, 81.05, 181.2, and 94.07 mg/l x h, respectively, MRT were from 34.66 to 90.17 h, and t1/2beta from 24.75 to 69.34 h, respectively. Florfenicol was widely distributed in these tissues and maintained the effective therapeutic concentrations especially in the respiratory tract tissues that are the target organs of Actinobacillus pneuropneumoniae. Tissue pharmacokinetic data could be evidence for regime designing of florfenicol in treatment of porcine pleuropneumonia.

Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene ( apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase ( apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16 kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5 kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms ( Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

Immunohistochemical Detection of Cyclooxygenase-2 in Lungs of Pigs Naturally Infected with Actinobacillus pleuropneumoniae

Cyclooxygenase-2 (COX-2) protein was detected immunohistochemically in formalin-fixed, paraffin wax-embedded lung tissues from 15 pigs with naturally occurring pleuropneumonia caused by Actinobacillus pleuropneumoniae. Positive cells typically exhibited a red reaction product without background staining. Alveolar macrophages, neutrophils, and bronchial and bronchiolar epithelial cells had positive immunohistochemical signals. Immunoreactivity of COX-2 protein was intense in the clustered leucocytes with streaming nuclear chromatin that are a characteristic histological feature of porcine pleuropneumonia. COX-2 protein was always associated with macrophages and neutrophils in pleuropneumonic lung lesions but was minimal in non-lesional lung of A. pleuropneumoniae -infected pigs and in normal lung from control pigs. The results suggest that COX-2 plays a role in pathophysiological processes during A. pleuropneumoniae infection.

In situ hybridization for the detection of the apxIV gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes.

The detection of the apxlV gene in lung tissues from pigs experimentally infected with the 12 major A. pleuropneumoniae serotype (1 to 12) reference strains was studied by in situ hybridization using a non-radioactive digoxigenin-labeled DNA probe. In situ hybridization produced a distinct positive signal in all pigs inoculated with the 12 A. pleuropneumoniae serotypes. Positive hybridization typically exhibited a dark-brown to black reaction product in intracellular and extracellular locations, without background staining. A strong hybridization signal was seen in degenerated alveolar leukocytes ("oat cells") adjacent to the foci of coagulative necrosis and in the alveolar spaces. The in situ hybridization methodology developed for the detection of the apxIV gene is a valuable tool for the diagnosis of porcine pleuropneumonia caused by A. pleuropneumoniae when only formalin-fixed tissues are submitted for diagnosis.

Porcine pleuropneumonia: the first evaluation of field efficacy of a subunit vaccine in Croatia

A vaccine for porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae was studied in Croatia on a farm infected by agent serotypes 2 and 9. Vaccination with a commercial subunit vaccine was initiated in the second half of 1998 due to the immense economic damage caused on the farm by this disease. All prefattening and fattening pigs kept on the farm during the first three months of 1999 were allocated in two groups: vaccinated and control. In the control and vaccinated group, 226 and 35 animals (5.78% and 0.96% of the average number of prefattening and fattening pigs in control and vaccinated group), respectively, died from pleuropneumonia. The vaccine efficacy was 83.5%. Examination of the randomly selected lungs on the slaughter line revealed significant reduction in the lesions specific for the chronic form of pleuropneumonia in the vaccinated group (vaccine efficacy 78.6%). The tested vaccine significantly decreased the death rate and pulmonary lesions due to A. pleuropneumoniae.

In vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding

The early cytokines interferon-α (IFN-α), tumour necrosis factor-α (TNF-α), interleukin-1, -6 and -8 (IL-1, -6, -8) are produced during the most early stage of an infection. The activities of these cytokines have been studied extensively in vitro and in rodents, but in vivo studies on the role of these cytokines in infectious diseases of food animals are few. This review concentrates on in vivo studies of cytokine involvement in infectious respiratory diseases of swine, with an emphasis on viral infections. First evidence for the role of early cytokines in pneumonia in swine came from experimental infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae. The role of TNF-α and IL-1 in the symptoms and pathology of porcine pleuropneumonia has recently been proven by use of an adenovirus vector expressing the anti-inflammatory IL-10. In the authors’ laboratory, studies were undertaken to investigate the relationship between viral respiratory disease and bioactive lung lavage levels of IFN-α, TNF-α, IL-1 and IL-6. Out of three respiratory viruses—porcine respiratory coronavirus (PRCV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV)—only SIV induced acute respiratory disease and severe lung damage by itself. Disease and lung pathology were tightly associated with the simultaneous production of IFN-α, TNF-α, IL-1 and IL-6. In challenge studies of SIV-vaccinated pigs, levels of IFN-α, TNF-α and IL-6, but not IL-1 were correlated with clinical and virological protection. Multifactorial respiratory disease was reproduced by combined inoculations with PRCV or PRRSV followed by LPS from Escherichia coli. In comparison with the respective single inoculations, which were subclinical, there was a true potentiation of disease and production of TNF-α, IL-1 and IL-6. TNF-α and IL-6 were best correlated with disease. In further studies, we will use more specific strategies to dissect the role of cytokines during viral infections.

Immunohistochemical Detection and Distribution of Inducible Nitric Oxide Synthase in Pigs Naturally Infected withActinobacillus pleuropneumoniae

Inducible nitric oxide synthase (iNOS) protein was detected immunohistochemically in formalin-fixed, paraffin wax-embedded lung tissues from 10 natural cases of porcine pleuropneumonia. Positive cells typically exhibited a red reaction product without background staining. Labelling of iNOS protein was intense in “oat cells”, the clustered leucocytes with streaming nuclear chromatin that are a characteristic histological feature of porcine pleuropneumonia. Macrophages and neutrophils within alveolar spaces but not within blood vessels consistently showed iNOS labelling, but such labelling was minimal in non-lesional lung of Actinobacillus pleuropneumoniae -infected pigs and in normal lung from control pigs. The results suggest that iNOS plays a role in pathophysiological processes during A. pleuropneumoniae infection.

Ohr, Encoding an organic hydroperoxide reductase, is an in vivo-induced gene in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a disease characterized by pulmonary necrosis and hemorrhage caused in part by neutrophil degranulation. In an effort to understand the pathogenesis of this disease, we have developed an in vivo expression technology (IVET) system to identify genes that are specifically up-regulated during infection. One of the genes that we have identified as being induced in vivo is ohr, encoding organic hydroperoxide reductase, an enzyme that could play a role in detoxification of organic hydroperoxides generated during infection. Among the 12 serotypes of A. pleuropneumoniae, ohr was found in only serotypes 1, 9, and 11. This distribution correlated with increased resistance to cumene hydroperoxide, an organic hydroperoxide, but not to hydrogen peroxide or to paraquat, a superoxide generator. Functional assays of Ohr activity demonstrated that A. pleuropneumoniae serotype 1 cultures, but not serotype 5 cultures, were able to degrade cumene hydroperoxide. In A. pleuropneumoniae serotype 1, expression of ohr was induced by cumene hydroperoxide, but not by either hydrogen peroxide or paraquat. In contrast, an ohr gene from serotype 1 cloned into A. pleuropneumoniae serotype 5 was not induced by cumene hydroperoxide or by other forms of oxidative stress, suggesting the presence of a serotype-specific positive regulator of ohr in A. pleuropneumoniae serotype 1.

Effects of Pentoxifylline on Inflammatory Cytokine Expression and Acute Pleuropneumonia in Swine

Pentoxifylline, a methylxanthine derivative and nonspecific type 4 phosphodiesterase inhibitor, has been used to improve survival of animals with sepsis and to attenuate lung injury in acute lung inflammation. The purpose of this study was to examine whether pentoxifylline would inhibit the expression of inflammatory cytokines, particularly tumor necrosis factor alpha (TNF), and thereby decrease the pathophysiology of acute porcine pleuropneumonia. E. coli lipopolysaccharide (LPS) and bacterial extracts of A. pleuropneumoniae--induced elevations in TNF mRNA which were fully abrogated by addition of pentoxifylline in both alveolar macrophage and neutrophil cultures. A 30% reduction in the level of LPS-induced interleukin (IL)-1beta mRNA levels also was achieved in macrophages. Pentoxifylline did not affect either IL-1alpha or IL-8 expression in vitro. Pentoxifylline therapy in vivo significantly reduced the number of band neutrophils in swine but did not reduce the pathology associated with pleuropneumonia, including changes in serum zinc, iron, or haptoglobin. Neither did it alter TNF, IL-1, IL-6, or IL-8 expression. Measurement of pentoxifylline and its metabolites in pig sera suggested that efficacious doses of pentoxifylline were probably not achieved in vivo. However, subcutaneous doses of pentoxifylline higher than 25 mg/kg produced transient diarrhea, vomiting, and tremors. These results suggest that pentoxifylline is an effective pharmacological tool for the dissection of cytokine regulation in vitro, but inhibitory concentrations may not be achievable for in vivo pharmacological use in swine.