Pathophysiologic correlates of acute porcine pleuropneumonia.

OBJECTIVE: To develop and evaluate an in vivo model to study early events in the pathogenesis of acute porcine pleuropneumonia. ANIMALS: Thirty-six 6- to 8-week-old pigs. PROCEDURE: Pigs were inoculated intranasally or endotracheally with Actinobacillus pleuropneumoniae; inoculation routes were compared by evaluation of clinical signs, gross and microscopic lung lesions, hematologic changes, serum zinc, iron, and haptoglobin concentrations, and inflammatory cytokines. RESULTS: The 2 inoculation routes resulted in similar findings, although intranasal inoculation caused unilateral gross lung lesions, whereas endotracheal inoculation caused bilateral gross lesions. Clinical signs of disease were observed < 2 hours after endotracheal inoculation and 6 to 8 hours after intranasal inoculation. Total WBC counts did not differ significantly after inoculation by either inoculation route, although band neutrophils increased significantly. The earliest findings associated with A pleuropneumoniae inoculation, irrespective of route, were decreased serum zinc and iron concentrations. Serum haptoglobin concentrations were significantly increased after inoculation. Inoculation induced rapid influx of macrophages into the lung and local induction of proinflammatory cytokines. Northern blot analysis of total RNA from lung tissue indicated that inoculated pigs had increased concentrations of interleukin (IL)-1beta, IL-1alpha, and IL-8; tumor necrosis factor messenger RNA concentration was not increased. CONCLUSIONS: Endotracheal inoculation with A pleuropneumoniae rapidly and consistently induced diffuse bilateral pneumonia; thus, this method may be useful for the study of acute pathophysiologic changes associated with bacterial pneumonia and may provide an experimental model for testing modalities for prevention and treatment of this and other respiratory tract diseases of pigs.

Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues.

Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.

Association of tumour necrosis factor and acute phase reactant changes with post arrival disease in swine.

Severe weight loss in the absence of respiratory, enteric or systemic clinical disease or gross pathologic lesions is often observed when immunologically naive boars are placed in conventional health swine facilities. Affected animals develop this weight loss in spite of receiving pre-entry vaccinations against common swine pathogens, such as Haemophilus parasuis or Mycoplasma hyopneumoniae. In many cases, the weight loss is non-responsive to long term antibiotic therapy. In order to determine the relationships between the severity of post arrival weight loss and disease and its potential immunological or physiological indicators, tumour necrosis factor (TNF) and acute phase reactant levels were correlated with the clinical status in immunologically naive boars following their transfer to a conventional facility. Boars had higher TNF (P < 0.0001) and plasma protein (P = 0.0054) levels and decreased zinc (P = 0.0004) levels during periods of clinical sickness. Likewise, peak and average plasma TNF, serum haptoglobin, and serum zinc were correlated indicating a prolonged stress or pathogenic insult (r = 0.89, P < 0.0001 for TNF; r = 0.67, P = 0.01 for haptoglobin; r = 0.73, P = 0.005 for zinc). An acute phase response, a systemic TNF increase and the development of a lymphopenia were observed in post arrival disease in swine. This is the first time cytokines and acute phase reactants have been investigated in a field study involving immunologically naive or high health swine.

Inflammatory cytokines in animal health and disease.

Inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and IL-8, are rapidly induced early in a disease or injury process. They mediate and modulate myriad healing processes but, if overexpressed, may exacerbate the severity of a disease condition. In order to test this concept and to establish a foundation for the role of inflammatory cytokines in the pathogenesis of gram-negative bacterial infections in the respiratory tract of animals, the patterns of inflammatory cytokine expression were determined in experimental porcine pleuropneumonia. We observed that IL-1 and IL-6, but not TNF, were rapidly and dramatically elevated in the lavage fluid of the lung within 24 h of infection. The increased levels of IL-1 might contribute to increased severity of disease, but elevated IL-6 levels were consistent with a protective acute phase response. Additional studies were performed to examine the hypothesis that IL-4 expression later in infection might be involved in turning off the inflammatory response and promoting an antigen-specific humoral immune response. Interleukin-4 efficiently suppressed inflammatory cytokine production in alveolar macrophages. Its expression was induced in peripheral blood mononuclear cells by TNF, IL-4, and by reexposure to a specific antigen. To obtain the maximum amount of information on the role of inflammatory cytokines in animals of veterinary significance it will be useful to perform studies in species such that evolutionary relatedness will allow widespread application of the findings. Furthermore, the variety of molecules involved in inflammatory cytokine regulation will require much more extensive investigations of the relevant enzymes, inhibitors and receptors in veterinary species. Finally, the complexity and redundancy of immune defenses in animals mean that attempts to modulate health status through manipulation of inflammatory cytokines must be performed with caution and that a multiplicity of processes will be affected.

Induction of interleukin (IL)-1beta and IL-8 mRNA expression in porcine macrophages by lipopolysaccharide from Serpulina hyodysenteriae.

Lipopolysaccharide (LPS) is a classic inducer of inflammatory cytokines and is a key virulence factor for most gram-negative pathogens. The effect of phenol-water (LPS) and butanol-water (endotoxin) extracts from Serpulina hyodysenteriae on inflammatory cytokine mRNA expression from porcine alveolar macrophages was investigated. The LPS and endotoxin extracts from S. hyodysenteriae induced a dose-dependent expression of interleukin 1beta (IL-1beta) and IL-8 which was weak compared with the responses induced by Escherichia coli LPS. In addition, the spirochetal extracts induced no detectable upregulation of mRNA expression for either IL-6 or tumor necrosis factor alpha.

Regulation of interleukin-6 expression in porcine immune cells.

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates the immune response, acute-phase reaction, and hematopoiesis. As a first step in studying the actions of IL-6 in pigs, the regulation of IL-6 expression was examined in various swine cells, including a fibroblast cell line, peripheral blood mononuclear cells (PBMC), and alveolar macrophages. IL-6 expression in transformed swine testicular (TST) fibroblasts was enhanced by TNF and IL-1 beta and to a lesser extent by poly(I).(C) and LPS. IL-6 was induced in porcine PBMC by either LPS or PHA; however, the combination of LPS plus PHA resulted in maximal IL-6 expression. Furthermore, in PBMC cells separated by adherence, LPS was a more potent inducer than PHA in adherent cells, whereas PHA was more potent in nonadherent cells. Alveolar macrophages collected from different pigs could be divided into low and high responders with respect to IL-6 induction by LPS. IL-6 mRNA induction by LPS could be detected in only 6 of 20 donor animals. Other inflammatory cytokines (IL-8, IL-beta, and TNF) were readily induced by LPS in alveolar macrophages from both low and high responders. Treatment of low-responder alveolar macrophages with conditioned medium containing IFN-gamma did not significantly alter the capacity of these macrophages to synthesize IL-6 mRNA in response to LPS. Comparison of IL-6 production capacity by the cell types in this study revealed the following order: PBMC = high-responder alveolar macrophages >> TST.cells > low-responder alveolar macrophages. Thus, PBMC appear to be quantitatively the most significant source of IL-6 in swine on a per cell basis.

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.

Class specific antibody response to influenza A H1N1 infection in swine.

Early detection of swine influenza A outbreaks is essential to understand the true cause and effect relationship that exists between this disease and other serious respiratory or herd health problems. Enzyme-linked immunosorbent assays (ELISAs) for the early detection of H1N1 subtype specific serum IgM, IgG and secretory IgA were compared to direct virus detection in in embryonated eggs. Elevated levels of H1 hemagglutinin (HA) specific IgM and IgG were detected as early as 3 days post experimental infection with a field strain of swine influenza A (H1N1). Influenza specific IgA in nasal mucous samples was detected on day 4 post infection (PI). This compared favorably with egg inoculation methods which detected virus 2-4 days PI. Identification of elevated H1 HA specific IgM in test herds could signify a recent influenza outbreak. Alternatively, ELISA analysis of nasal mucous samples for H1 HA specific IgA could provide a noninvasive method of obtaining similar information on the influenza specific immune status of the herd.

Interleukin-4 suppresses inflammatory cytokine gene transcription in porcine macrophages.

Inflammatory cytokines, including interleukin (IL)-1 alpha, IL-1 beta, IL-8, and tumor necrosis factor alpha (TNF-alpha) are produced by macrophages in response to a variety of pathogenic stimuli. We show here that the expression of inflammatory cytokines is suppressed by IL-4 at the transcriptional level. Interleukin-4, when added together with bacterial lipopolysaccharide (LPS), suppressed LPS-induced increases in mRNA levels of IL-1 alpha, IL-1 beta, IL-8, and TNF-alpha in alveolar macrophages. The level of suppression was dependent on dose and time of exposure and reached a maximum of 75-80% of uninduced values for IL-1 alpha, IL-8, and TNF. Interleukin-1 beta expression was completely inhibited by IL-4. The amount of secreted protein, as determined by TNF-alpha bioassay, was also suppressed by IL-4. Half-maximal suppression occurred at IL-4 concentrations between 0.02 and 0.1 ng/ml for all inflammatory cytokines. Nuclear run-on assays showed that IL-4 suppressed transcriptional activity of all inflammatory cytokines. Messenger RNA stability was not changed by IL-4. The data suggest that IL-4 plays an important transcriptional role in the regulation of alveolar macrophage inflammatory activities in respiratory disease and raise the possibility that IL-4 may function in vivo as a coordinator of inflammatory and immune responses.

Regulation of interleukin-8 expression in porcine alveolar macrophages by bacterial lipopolysaccharide.

Interleukin (IL)-8 is a macrophage-derived neutrophil chemotactic factor that plays an important role in the recruitment of neutrophils to inflammatory loci. Hence, expression of IL-8 by alveolar macrophages may be a significant factor in host defense in the lung and in the pathogenesis of pneumonia in swine. To initiate molecular studies of IL-8 regulation in pigs, we cloned IL-8 cDNA and examined the regulation of its mRNA in alveolar macrophages. The porcine IL-8 cDNA consists of 1491 base pairs including a coding region of 309 base pairs. The deduced amino acid sequence was 75 and 81% similar to human and rabbit IL-8, respectively. Resting macrophages contained low levels of IL-8 mRNA, which increased markedly after exposure to bacterial lipopolysaccharide (LPS). LPS induction of IL-8 was direct, not mediated through elevation of tumor necrosis factor or interleukin-1. The effect of LPS on IL-8 expression was dose dependent, and induction was observed at a concentration of 10 pg/ml. IL-8 mRNA expression was detectable within 0.5 h after stimulation with LPS, peaked at 3-6 h at about 30-fold higher levels than in resting cells, and was maintained for 24 h. Secreted IL-8, measured by neutrophil chemotaxis, was induced within 4 h by LPS, and accumulated in the media throughout the 24-h period. The mechanism of induction of IL-8 mRNA appeared to involve transcription and RNA processing. Nuclear run-on analysis showed that the IL-8 gene was actively transcribed in noninduced cells; upon stimulation with LPS, the rate of IL-8 transcription was increased about 4-fold. A single mature mRNA species was detected by primer extension analysis. The half-life of IL-8 mRNA transcripts in aveolar macrophages was approximately 2 h and did not change after LPS stimulation. The ability of LPS to induce IL-8 expression was suppressed by recombinant human IL-4 and dexamethasone in a concentration-dependent manner. These observations indicate that the expression of IL-8 is an early event in the sequelae to bacterial infection in the lung.

Subtype specific ELISA for the detection of antibodies against influenza A H1N1 and H3N2 in swine.

A subtype specific ELISA using purified hemagglutinin (HA) from influenza A H1N1 and H3N2 was developed to detect antibodies present in swine previously exposed to either H1N1 or H3N2 influenza viruses. The HA was extracted using the detergent octylglucoside followed by ion exchange chromatography. All HA preparations were free of contaminating nucleoprotein and matrix protein contamination. Monospecific swine anti-H1N1 and swine anti-H3N2 sera were used to demonstrate the subtype specificity of the assay. Monospecific rabbit anti-H1N1 or H3N2 was used to sterically block crossreacting determinants and thus enhance assay specificity. A linear relationship between single dilution point ELISA and the hemagglutination inhibition (HI) test was established. This enabled the accurate estimation of HI titer from ELISA. Further refinement of this ELISA based HI estimation system could allow it to replace the current HI procedures in instances where identification at the subtype level of specificity is acceptable. The substantial specificity requirements associated with the detection of strain specific antibody would still necessitate the use of the HI procedure.

ELISA method for detection of influenza A infection in swine.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to monitor virus shedding associated with experimental infection with a field strain of swine influenza in pigs. The assay consisted of a monoclonal anti-nucleoprotein capture antibody and a biotinylated rabbit anti-influenza A (H1N1) sandwich antibody. The antigen-capture system was capable of detecting as little as 1 ng/ml purified virus. The ELISA system surpassed egg cultivation procedures in the detection of low levels of shedding virus. Egg cultivation procedures indicated that most viral shedding had ceased by day 10 postinfection. In contrast, antigen-capture ELISA still showed an ongoing presence of viral antigen. A virus-capture ELISA, using this capture-sandwich antibody system, is equivalent in sensitivity to conventional egg inoculation procedures for the detection of the early phases of virus shedding. The automative potential of an ELISA-based system coupled with a substantially reduced assay time requirement give this virus-capture ELISA a distinct advantage over other cell culture or egg-based diagnostic techniques.

Determination of hemagglutination-inhibition titers to influenza A virus in porcine sera by use of an enzyme-linked immunosorbent assay.

An ELISA-based method to estimate hemagglutination-inhibition (HI) titer was developed. Subtype specificity was obtained by using purified H1 and H3 hemagglutinin antigens. Using the linear relation that exists between ELISA and HI methods, regression lines for H1N1- and H3N2-monospecific porcine antisera were constructed. Approximation of actual HI titer could be obtained from insertion of ELISA values into the appropriate regression line. The HI estimations were within 50% of the actual measured HI value 84% of the time. In young pigs that had suckled immune sows, use of this ELISA revealed estimated HI titer > 320 at 2 and 4 weeks of age. After a typical farm outbreak of influenza A/swine (H1N1), estimated HI titers remain high for 4 to 6 months. Sub-type-specific estimation of the distribution frequency of positive influenza A (H1N1 or H3N2) results for sera from swine in regional herds indicated that 31.3 and 7.4% of the swine tested were positive (HI > 41) for H1N1 and H3N2, respectively. From these observations, we conclude that in many circumstances, an ELISA-based HI estimation method could be used as a substitute for the HI test.

Detection of tumor necrosis factor alpha from porcine alveolar macrophages using an L929 fibroblast bioassay.

Tumor necrosis factor plays a central role in the mediation of the pathophysiological sequelae of infection and inflammation in animals and humans. The elucidation of its role in respiratory disease of swine has not been investigated, due in part to the lack of a sensitive and specific quantitative assay for its presence in tissue and fluid samples. Here we describe the detection of porcine tumor necrosis factor utilizing L929 murine fibroblast cells and characterize various parameters affecting assay sensitivity. Plating cell density and length of exposure time to test supernatants were the most critical factors. Using standard assay conditions as described here, porcine tumor necrosis factor was detected in alveolar macrophage conditioned media diluted more than 400-fold. Specificity of the assay for porcine tumor necrosis factor was shown by inhibition of cytotoxicity with neutralizing polyclonal antibodies for human recombinant tumor necrosis factor. Furthermore, comparisons of bioactivity with tumor necrosis factor mRNA levels from lipopolysaccharide-stimulated porcine alveolar macrophages indicated that the L929 bioassay was specific for porcine tumor necrosis factor.

Immunological detection of B-DNA to Z-DNA transition of polynucleotides by immobilization of the DNA conformation on a solid support.

We studied the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) in the presence of NaCl using an enzyme immunoassay. The polynucleotides were coated on microtiter plates at varying concentrations of NaCl and treated with a monoclonal anti-Z-DNA antibody, Z22. The plates were subsequently treated with alkaline phosphatase conjugated polyvalent mouse immunoglobulins and the enzyme substrate, p-nitrophenyl phosphate. The color development due to the enzyme-substrate reaction was quantitated using a microplate autoreader. Our results show that the antibody does not recognize the polynucleotides in the B-DNA conformation and binds strongly to the Z-DNA conformation. A smooth transition curve is obtained at intermediate concentrations of the counterions. From the transition curves, we determined the concentration of the counterions at the midpoint of B-DNA to Z-DNA transition. The midpoint concentrations for poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) are 2.3 and 0.74 M NaCl, respectively. Using the immunological method, we also examined the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of naturally occurring polyamines. The midpoint concentrations of the polyamines are as follows: putrescine, 2.5 mM; spermidine, 34 microM; spermine, 1.8 microM. The midpoint values determined by the enzyme immunoassay are in good agreement with those determined by circular dichroism and ultraviolet absorption spectroscopic measurements. These results demonstrate that immobilization of a preexisting conformation or a mixture of conformations of DNA on a solid support followed by a titration of the DNA conformations using a monoclonal anti-DNA antibody is an excellent method to study the conformational dynamics of DNA.