Multi-organ spreading of Actinobacillus pleuropneumoniae serovar 7 in weaned pigs during the first week after experimental infection.

Actinobacillus (A.) pleuropneumoniae is normally considered strictly adapted to the respiratory tract of swine. Despite this, scattered case reports of arthritis, osteomyelitis, hepatitis, meningitis or nephritis exist, in which A. pleuropneumoniae remained the only detectable pathogen. Therefore, the aim of this study was to investigate whether spreading to other organs than the lungs is incidental or may occur more frequently. For this, organ samples (blood, liver, spleen, kidney, tarsal and carpal joints, meninges, pleural and pericardial fluids) from weaners (n = 47) infected experimentally with A. pleuropneumoniae serovar 7 by aerosol infection (infection dose: 10.9 × 103 cfu/animal) were examined by culture during the first week after infection. In addition, tissue samples of eight weaners were examined by histology and immunohistochemistry (IHC). A. pleuropneumoniae was isolated in all examined sample sites (86.7% pleural fluids, 73.3% pericardial fluids, 50.0% blood, 61.7% liver, 51.1% spleen, 55.3% kidney, 14.9% tarsal joints, 12.8% carpal joints, 27.7% meninges). These results were also obtained from animals with only mild clinical symptoms. IHC detection confirmed these findings in all locations except carpal joints. Histological examination revealed purulent hepatitis (n = 2), nephritis (n = 1) and beginning meningitis (n = 2). Isolation results were significantly correlated (p < 0.001) with the degree of lung colonization and, to a lower extent, with the severity of disease. Detection of A. pleuropneumoniae in peripheral tissues was significantly correlated to spleen colonization. In conclusion, multi-organ spreading of A. pleuropneumoniae serovar 7 strain AP 76 seems to occur more frequently during acute infection following effective lung colonization than previously thought.

[TLR-4 involvement in pyroptosis of mice with pulmonary inflammation infected by Actinobacillus pleuropneumoniae].

OBJECTIVE: Pyroptosis is a caspase-1 dependent programmed cell death and involves pathogenesis of infectious diseases by releasing many pro-inflammatory cytokines to induced inflammation. TLR-4 plays an important role in mediating pathogenesis of some infectious diseases. In this study, we detected the expression of TLR-4 and some molecules (e. g caspase-1, TNF-α, IL-1ß, IL-6, IL-18 ) related with pyroptosis to determine its involvement and mechanisms of pulmonary inflammation in mice infected by A. pleuropneumoniae. METHODS: Mice were intranasally infected by A. pleuropneumoniae and killed 48 hours post infection. Pulmonary gross lesion and histological pathology by H-E were observed. Expression levels of caspase-1 , caspase-3, TNF-α, IL-1ß, IL-6, IL-18, and TLR-4 in lung of mice were detected by RT-PCR and qPCR. RESULTS: Serious pulmonary hemorrhage and inflammation in infected mice were observed. Expression levels of caspase-1, caspase-3, TNF-α, IL-1ß, IL-6, IL-18 and TLR-4 increased, and expression levels of caspase-3 were not changed in lung of infected mice. CONCLUSION: TLR-4 might be involved in pulmonary inflammation of mice infected by A. pleuropneumoniae. After induced by activated TLR-4 some cells in this lesion expressed pro-inflammatory cytokines. These cytokines would induce pulmonary inflammation. This lesion might involve pyroptosis with caspase-1 expression.

Differential gene expression profiling of Actinobacillus pleuropneumoniae during induction of primary alveolar macrophage apoptosis in piglets.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae-PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use.

Acute and subacute response of iron, zinc, copper and selenium in pigs experimentally infected with Actinobacillus pleuropneumoniae.

This study was performed to characterise the response of iron (Fe), zinc (Zn), copper (Cu) and selenium (Se) in bacterial-induced porcine acute phase reaction (APR). Twenty piglets were challenged by aerosolic infection with Actinobacillus pleuropneumoniae (A.pp.) serotype 2, ten piglets serving as controls. Blood sampling was done initially and at day 4 and 21 after infection, collection of liver tissue was done at day 21 (autopsy). A.pp.-infection caused fever and respiratory symptoms. APR at day 4 after infection was marked by an increase in total white blood cells, granulocytes and monocytes in whole blood samples and an increase in globulin/albumin ratio (G/A), α2-globulins, C-reactive protein, haptoglobin, ceruloplasmin (Cp), Cu and Se in serum. Concurrently, there was a decrease in haemoglobin (Hb) and packed cell volume (PCV) in whole blood as well as a decrease in albumin, transferrin, total iron binding capacity and Fe in serum and Zn in plasma. The subacute stage at day 21 was characterised by progressively increased concentrations of G/A, ß-globulins and γ-globulins reflecting the specific immune reaction. Hb and PCV showed further decreases, all other parameters returned to the initial concentrations. Glutathione peroxidase activity in plasma and liver tissue remained unaffected by A.pp.-infection. The liver concentration (day 21) of Zn was found to be higher, that of Se was lower in the A.pp.-group, whereas hepatic concentrations of Cu and Fe were not affected by A.pp.-infection. In summary, the acute and subacute stages of A.pp.-infection were accurately characterised by the APR-related parameters. Se was only marginally affected by the A.pp.-infection. The elevated plasma Cu concentration may be a side effect of the transient hepatic induction of Cp synthesis. Zn responded, being distinctly reduced in plasma and probably having been sequestered in the liver tissue. Reduction in serum Fe can be regarded as an unspecific defence mechanism in A.pp.-infection to withdraw Fe from bacterial acquisition systems.

Periodontal pathogen Aggregatibacter actinomycetemcomitans deteriorates pressure overload-induced myocardial hypertrophy in mice.

Although a relationship between periodontitis and myocardial hypertrophy has been reported, the precise mechanism has not been clarified. The purpose of this study was to investigate the association between periodontal infection and myocardial hypertrophy. Transverse aortic constriction (TAC) was performed. Mice were injected with Aggregatibacter actinomycetemcomitans (A.a.) (0.1 mL of 10(8) CFU/mL) in the infected group and PBS in the control group. Echocardiography, histopathology, and immunohistochemistry were performed. Echocardiography indicated that left ventricular fractional shortening had decreased in the infected group compared to the control group on day 28. Heart to body weight ratio increased in the infected group compared to the control group. Histopathologically, A.a.-infected mice showed markedly enhanced cardiac hypertrophy, fibrosis and arteriosclerosis 4 weeks after TAC operation. Immunohistochemistry revealed that expression of MMP-2 in the interstitial tissue was enhanced in the infected group. These results suggested that the periodontal pathogen caused a deterioration of pressure overload-induced myocardial hypertrophy through MMP activation.

Infection of human gingival fibroblasts with Aggregatibacter actinomycetemcomitans: An in vitro study.

OBJECTIVE: Aggregatibacter actinomycetemcomitans is known to be a major cause of localized aggressive periodontitis. Previous research has suggested that A. actinomycetemcomitans can damage many types of host cells. There is evidence for the ability of this organism to invade endothelial and epithelial cells, but information pertaining to its potential for invading gingival fibroblasts is very limited. Internalization of bacteria is not only responsible for damaging host tissue but also a means to evade the host immune response. It was hypothesized that A. actinomycetemcomitans can invade and reside in human gingival fibroblasts (HGF). METHODS: Primary cultures of HGF were infected with A. actinomycetemcomitans at a ratio of 1:100. Bacterial internalization was determined by an antibiotic protection assay. Bacterial-fibroblast interaction was examined using phase-contrast, scanning and transmission electron microscopy. RESULTS: It was demonstrated that A. actinomycetemcomitans was internalized into HGF at an efficiency of 0.084%. Transmission electron microscopic study showed the presence of A. actinomycetemcomitans in the cytoplasm of HGF without the surrounding membrane. Scanning electron micrographs revealed the sloughing of HGF surfaces on which A. actinomycetemcomitans adhered. Rounded cells, attachment loss and damaged cells were also observed. CONCLUSIONS: It is concluded that the attachment and invasion of A. actinomycetemcomitans into human gingival fibroblasts play a role in periodontal tissue damage and may also be a means of immune evasion.

Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1ß and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.

Invasive infections of Aggregatibacter (Actinobacillus) actinomycetemcomitans.

BACKGROUND/PURPOSE: Aggregatibacter (Actinobacillus) actinomycetemcomitans, part of the normal flora of the mouth, is frequently found in human periodontal cultures and is an important pathogen causing various invasive infections, particularly infective endocarditis. In this study, we describe the clinical course and outcome of patients with A. actinomycetemcomitans infection. METHODS: All patients suffering invasive A. actinomycetemcomitans infections at the National Taiwan University Hospital from January 1985 to December 2004 were included in this study. Relevant data regarding clinical presentation, antimicrobial treatment and outcome of these patients were analyzed. RESULTS: During the study period, there were 11 patients with invasive A. actinomycetemcomitans infections, including eight patients with infective endocarditis, one with osteonecrosis and two with pneumonia and chest wall lesions. Among the patients with infective endocarditis, four had prosthetic valve replacement, four suffered from rheumatic heart disease and one had undergone surgical repair of ventricular septal defect. Lesions in the oral cavity were the probable portals of entry of the microorganism, and included carious teeth, periodontitis or radiotherapy of the ear-nose-throat field, and were noted in nine patients. Transthoracic echocardiography and/or transesophageal echocardiography were performed on the patients with probable infective endocarditis but growth was demonstrated in only four of these patients. Blood culture yielded A. actinomycetemcomitans after prolonged incubation. Three isolates were resistant to penicillin and two of these were also resistant to ampicillin. CONCLUSION: The diagnosis of invasive A. actinomycetemcomitans infection was delayed due to the indolent clinical course, non-specific presentation and slow growth of the organism. Antibiotic therapy using amoxicillin/clavulanic acid, ampicillin, ampicillin/sulbactam, ceftriaxone, clindamycin, cefotaxime, or levofloxacin was successful in all patients. None of the patients demonstrated recurrence of infection 2-36 months following treatment.

Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms.

BACKGROUND: In pigs little is known about the role of innate immune defence in bacterial infections of the respiratory tract, despite their major role in pig production. In the present study we characterized and compared in vitro and in vivo activation of immune markers of different pig breeds 7 days before, and 4 and 21 days after an experimental aerosol infection with Actinobacillus (A.) pleuropneumoniae. RESULTS: In vitro stimulation of bronchoalveolar lavage fluid (BALF) and blood leukocytes with A. pleuropneumoniae, Streptococcus suis, PMA and LPS led to production of different amounts of H2O2, NO and TNF-alpha, depending on the stimulus, individual, breed and time of infection. Generally, significant responses to in vitro stimulation were observed only in blood leukocytes, whereas the alveolar macrophages showed a high basal activation. In addition, the production of haptoglobin and cytokines (TNF-alpha, IFN-gamma and IL-10) in vivo was measured in plasma and BALF. Plasma haptoglobin levels mirrored the clinical manifestations at 4 days post-infection. In plasma and BALF TNF-alpha could not be detected, whereas variable levels of IFN-gamma were found at pre- and post-infection times. IL-10 was found in some plasma but in none of the BALF samples. The different expression levels in individuals within the breeds correlated for some markers with the severity of clinical manifestations, e.g. H2O2, plasma haptoglobin and BALF IFN-gamma for German Landrace pigs. CONCLUSION: Our findings revealed differences in the activation of the immune markers with respect to infection time, individuals and breeds. Moreover, results showed different correlation grades between the immune markers produced in vitro or in vivo and the clinical manifestations. Further analyses will have to show whether these markers may serve as correlates of protection against porcine respiratory infections.

Interleukin-10 inhibits gram-negative-microbe-specific human receptor activator of NF-kappaB ligand-positive CD4+-Th1-cell-associated alveolar bone loss in vivo.

To study anti-inflammatory cytokine effects on RANKL+-T-cell-mediated osteoclastogenesis in vivo, we injected human interleukin-10 (hIL-10) into pathogen-infected HuPBL-NOD/SCID mice. The results show significantly decreased RANKL+ Th1-associated alveolar bone loss and coexpression of human gamma interferon (hIFN-gamma) and human macrophage colony-stimulating factor, but not hIL-4, in RANKL+ Th cells compatible with those from successfully treated aggressive periodontitis subjects. Thus, there are critical cytokine interactions linking hIFN-gamma+ Th1 cells to RANKL-RANK/OPG signaling for periodontal osteoclastogenesis in vivo.

The concentration of apolipoprotein A-I decreases during experimentally induced acute-phase processes in pigs.

In this work, apolipoprotein A-I (ApoA-I) was purified from pig sera. The responses of this protein after sterile inflammation and in animals infected with Actinobacillus pleuropneumoniae or Streptococcus suis were investigated. Decreases in the concentrations of ApoA-I, two to five times lower than the initial values, were observed at 2 to 4 days. It is concluded that ApoA-I is a negative acute-phase protein in pigs.

Actinobacillus actinomycetemcomitans-induced periodontal disease in mice: patterns of cytokine, chemokine, and chemokine receptor expression and leukocyte migration.

Although the pathogenesis of periodontal disease (PD) is not well known, cytokines, chemotactic factors and inflammatory cells are certainly involved in the disease outcome. Here, we characterized the evolution of the PD induced by Actinobacillus actinomycetemcomitans in mice, showing that oral inoculation of these bacteria leads to the migration of leukocytes to periodontal tissues and marked alveolar bone resorption. We found the expression of pro-inflammatory and Th1-type cytokines including TNF-alpha, IFN-gamma and IL-12 in periodontal tissues after infection with A. actinomycetemcomitans, from the early stages after infection and throughout the course of the disease. Similar kinetics of expression were found for the chemokines CCL5, CCL4, CCL3 and CXCL10 and for the receptors CCR5 and CXCR3, all of them linked to the Th1-type pattern. The expression of the Th2-type mediators IL-10, CCL1 and their receptors CCR4 and CCR8 was detected only after 30 days of infection, determining a time-dependent mixed pattern of polarized immune response. The chemokine expression was correlated with the presence of polymorphonuclear leukocytes, macrophages, CD4 and CD8 lymphocytes, and B cells in the inflammatory infiltrate. Interestingly, during the predominance of the Th1-type response, a sharp increase in the number of inflammatory cells and intense bone loss was seen. By contrast, after the increased expression of Th2-type mediators, the number of inflammatory cells remained constant. Our data demonstrate that mice subjected to oral inoculation of A. actinomycetemcomitans represent a useful model for the study of PD. In addition, our results suggest that expression of cytokines and chemokines can drive the selective recruitment of leukocyte subsets to periodontal tissues, which could determine the stable or progressive nature of the lesion.

Effects of oral administration of tilmicosin on pulmonary inflammation in piglets experimentally infected with Actinobacillus pleuropneumoniae.

OBJECTIVES: To determine the effects of oral administration of tilmicosin in piglets experimentally infected with Actinobacillus pleuropneumoniae. ANIMALS: Forty 3-week-old specific-pathogen free piglets. PROCEDURES: Piglets were assigned to 1 of 4 groups as follows: 1) uninfected sham-treated control piglets; 2) infected untreated piglets that were intratracheally inoculated with 10(7) CFUs of A pleuropneumoniae; 3) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received tilmicosin in feed (400 ppm [microg/g]) for 7 days prior to inoculation; or 4) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received chlortetracycline (CTC) in feed (1100 ppm [microg/gl) for 7 days prior to inoculation. Bronchoalveolar lavage (BAL) fluid and lung tissue specimens of piglets for each group were evaluated at 3 or 24 hours after inoculation. For each time point, 4 to 6 piglets/group were studied. RESULTS: Feeding of CTC and tilmicosin decreased bacterial load in lungs of infected piglets. Tilmicosin delivered in feed, but not CTC, enhanced apoptosis in porcine BAL fluid leukocytes. This was associated with a decrease in LTB4 concentrations in BAL fluid of tilmicosin-treated piglets, compared with untreated and CTC-treated piglets, and also with a significant decrease in the number of pulmonary lesions. Tilmicosin inhibited infection-induced increases in rectal temperatures, as measured in untreated and CTC-treated piglets. Pulmonary neutrophil infiltration and prostaglandin E2 concentrations in the BAL fluid were not significantly different among groups at any time. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of tilmicosin to infected piglets induces apoptosis in BAL fluid leukocytes and decreases BAL fluid LTB4 concentrations and inflammatory lung lesions.

Detection of nuclear factor-kappaB and inducible nitric oxide synthase in the lungs of pigs naturally infected with Actinobacillus pleuropneumoniae.

Activated nuclear factor-kappaB (NF-kappaB) and inducible nitric oxide synthase (iNOS) were detected immunohistochemically in pleuropneumonic lungs from 20 pigs naturally infected with Actinobacillus pleuropneumoniae. NF-kappaB was detected mainly in nuclei of inflammatory cells, confirming its activation. Intense immunolabelling for NF-kappaB 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-kappaB and iNOS. This suggests that NF-kappaB plays a key role in triggering the activation of iNOS in porcine pleuropneumonia.

Actinobacillus actinomycetemcomitans endocarditis.

Among the bacteria of the HACEK group, Actinobacillus actinomycetemcomitans is the organism involved most commonly in infective endocarditis. However, the epidemiological and clinical features specifically associated with this species have not been evaluated adequately. Three patients with infective endocarditis caused by A. actinomycetemcomitans seen at the Hospital La Timone (Marseille, France) between 1994 and 2001 are reported. Of 99 cases in the literature, 75% of patients had previous heart disease before infective endocarditis, the portal of entry of which was usually the oral cavity. Among the total of 102 cases, 27 had prosthetic valves. Intermittent fever was observed in all cases, and weight loss and peripheral signs of endocarditis were noteworthy in this study. Anaemia and microscopic haematuria were frequently noted. The disease is insidious, with a mean duration of symptoms of 13 weeks before diagnosis, as confirmed by blood cultures incubated for > 5 days. The aortic valve is most commonly involved, and echocardiographic findings were non-specific. Complications occurred in 63% of patients, with emboli being the most common. The surgery rate was 23.5%. The overall mortality rate was 18%. Of the cases, 76.5% were cured with antibiotics alone, including a simple third-generation cephalosporin or a combination of ampicillin and an aminoglycoside. An antibiotic therapy duration of at least 4 weeks is recommended. Surgical therapy is usually required for haemodynamic reasons. Prophylaxis of A. actinomycetemcomitans endocarditis relies on antibiotic therapy for all cardiac patients at risk before dental procedures. Among 17 patients undergoing dental manipulations, only eight received amoxycillin before the procedure, demonstrating that prophylaxis is far from being systematically prescribed. In conclusion, A. actinomycetemcomitans endocarditis should be highly suspected in patients with previous cardiac disease and for whom symptoms have evolved over a number of weeks or even months.

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.

Sequence diversity in the major fimbrial subunit gene (flp-1) of Actinobacillus actinomycetemcomitans.

Cells of the periodontal pathogen Actinobacillus actinomycetemcomitans exhibit tight adherence to surfaces such as glass, plastic and hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize teeth and other surfaces. Tight adherence is mediated by long fibrils of bundled pili (fimbriae) that form on the surface of the cell. The flp-1 gene encodes the major pilin protein component of A. actinomycetemcomitans fimbriae. In this study we compared flp-1 DNA sequences from 43 strains of A. actinomycetemcomitans isolated in Europe, Japan and the United States and identified seven distinct flp-1 allelic classes. DNA and predicted protein sequences were almost completely conserved within each flp-1 class but were highly divergent between classes. Most amino acid substitutions occurred in the C-terminus of the pilin protein, a region that has been shown to be important for the bundling and adhesive properties of the pili. flp-1 classes correlated with serotypes and 16S rRNA genotypes in most strains. At least five strains showed evidence of horizontal transfer of flp-1 between strains of different serotypes and 16S rRNA genotypes. Four of the seven flp-1 classes were present in geographically diverse isolates. Strains representing all seven flp-1 classes, but not a strain carrying a transposon insertion in flp-1, bound avidly to polystyrene in an in vitro adherence assay. Strains representing six of the seven flp-1 classes were isolated from localized juvenile periodontitis patients, suggesting that phylogenetically diverse strains carry pathogenic potential. Our findings provide a framework for future biochemical, immunological and genetic studies of A. actinomycetemcomitans fimbriae.

Expression of nitric oxide synthase 2 and tumor necrosis factor alpha in swine naturally infected with Actinobacillus pleuropneumoniae.

Nitric oxide synthase 2 (NOS2) and tumor necrosis factor alpha (TNF-alpha) were detected and localized in 15 pigs with naturally occurring pleuropneumonia by use of in situ hybridization with a nonradioactive digoxigenin-labeled cDNA probe. Two cDNA probes 491 and 219 base pairs for NOS2 and TNF-alpha, respectively, were generated by reverse transcription polymerase chain reaction. All 15 pigs infected with Actinobacillus pleuropneumoniae had distinct positive hybridization signals for NOS2 and TNF-alpha. Strong hybridization signals for both NOS2 and TNF-alpha were evident in degenerate alveolar leukocytes bordering zones of coagulative necrosis and in alveolar spaces. NOS2 nucleic acids were detected in neutrophils and macrophages. In situ hybridization of serial sections of lung tissue revealed numerous cells positive for NOS2 and TNF-alpha, suggesting that NOS2 and TNF-alpha expression may play a role in the pathophysiology of pleuropneumonia.

Comparison of serologic testing and slaughter evaluation for assessing the effects of subclinical infection on growth in pigs.

OBJECTIVE: To compare serologic testing with slaughter evaluation in assessing effects of subclinical infection on average daily weight gain (ADG) in pigs. DESIGN: Cohort study. ANIMALS: 18 cohorts (30 to 35 pigs/cohort) of pigs on/farms. PROCEDURE: Blood samples were collected, and pigs were weighed at 8, 16, and 24 weeks of age. Sera were tested for antibodies to porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), transmissible gastroenteritis virus (TGEV), pseudorabies virus, Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae. At slaughter, skin, nasal turbinates, lungs, and liver were examined. Associations between ADG and results of serologic testing and slaughter evaluation were examined by use of multiple linear regression. RESULTS: Pathogens that had a significant effect on any given farm during any given year and the magnitude of that effect varied. However, at 16 and 24 weeks of age, a higher antibody titer was consistently associated with a lower ADG. Mean differences in ADG between seropositive and seronegative pigs were 18 g/d (0.04 lb/d) for SIV, 40 g/d (0.09 lb/d) for PRRSV, 38 g/d (0.08 lb/d) for M hyopneumoniae, and 116 g/d (0.26 lb/d) for TGEV. Of the evaluations performed at slaughter, only detection of lung lesions was consistently associated with a decrease in ADG. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that subclinical infection with any of a variety of pathogens commonly found in swine herds was associated with a decrease in ADG. Serologic testing was more effective than slaughter evaluation in assessing the impact of subclinical infection on ADG in these pigs.