Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling.

Actinobacillus pleuropneumoniae (APP) is the causative pathogen of porcine pleuropneumonia, a highly contagious respiratory disease in the pig industry. The increasingly severe antimicrobial resistance in APP urgently requires novel antibacterial alternatives for the treatment of APP infection. In this study, we investigated the effect of tea polyphenols (TP) against APP. MIC and MBC of TP showed significant inhibitory effects on bacteria growth and caused cellular damage to APP. Furthermore, TP decreased adherent activity of APP to the newborn pig tracheal epithelial cells (NPTr) and the destruction of the tight adherence junction proteins ß-catenin and occludin. Moreover, TP improved the survival rate of APP infected mice but also attenuated the release of the inflammation-related cytokines IL-6, IL-8, and TNF-α. TP inhibited activation of the TLR/MAPK/PKC-MLCK signaling for down-regulated TLR-2, TLR4, p-JNK, p-p38, p-PKC-α, and MLCK in cells triggered by APP. Collectively, our data suggest that TP represents a promising therapeutic agent in the treatment of APP infection.

N-terminus of flagellin enhances vaccine efficacy against Actinobacillus pleuropneumoniae.

BACKGROUND: Flagellin elicits potent immune response and may serve as a vaccine adjuvant. We previously reported that the N-terminus of flagellin (residues 1-99, nFliC) is sufficient for vaccine efficacy enhancement against Pasteurella multocida challenge in chickens. In this study, we futher tested the adjuvancy of nFliC in a subunit vaccine against the pig pathogen Actinobacillus pleuropneumoniae in a mice model. For vaccine formulation, the antigen ApxIIPF (the pore-forming region of the exotoxin ApxII) was combined with nFliC, either through genetic fusion or simple admixture. RESULTS: Immune analysis showed that nFliC, introduced through genetic fusion or admixture, enhanced both humoral (antibody levels) and cellular (T cell response and cytokine production) immunity. In a challenge test, nFliC increased vaccine protective efficacy to 60-80%, vs. 20% for the antigen-only group. Further analysis showed that, even without a supplemental adjuvant such as mineral salt or oil emulsion, genetically linked nFliC still provided significant immune enhancement. CONCLUSIONS: We conclude that nFliC is a versatile and potent adjuvant for vaccine formulation.

CopA Protects Actinobacillus pleuropneumoniae against Copper Toxicity.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium causing porcine pleuropneumonia and severe economic losses in the global swine industry. The toxic trace element copper is required for many physiological and pathological processes in organisms. However, CopA, one of the most well-characterized P-type ATPases contributing to copper resistance, has not been characterized in A. pleuropneumoniae. We used quantitative PCR analysis to examine expression of the copA gene in A. pleuropneumoniae and investigated sequence conservation among serotypes and other Gram-negative bacteria. Growth characteristics were determined using growth curve analyses and spot dilution assays of the wild-type strain and a △copA mutant. We also used flame atomic absorption spectrophotometry to determine intracellular copper content and examined the virulence of the △copA mutant in a mouse model. The copA expression was induced by copper, and its nucleotide sequence was highly conserved among different serotypes of A. pleuropneumoniae. The amino acid sequence of CopA shared high identity with CopA sequences reported from several Gram-negative bacteria. Furthermore, the △copA mutant exhibited impaired growth and had higher intracellular copper content compared with the wild-type strain when supplemented with copper. The mouse model revealed that CopA had no influence on the virulence of A. pleuropneumoniae. In conclusion, these results demonstrated that CopA is required for resistance of A. pleuropneumoniae to copper and protects A. pleuropneumoniae against copper toxicity via copper efflux.

Enhancement of Apx Toxin Production in Actinobacillus pleuropneumoniae Serotypes 1, 2, and 5 by Optimizing Culture Condition.

Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl2) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 µg/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl2 yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins.

The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

Galactose-1-phosphate uridyltransferase (GalT), an in vivo-induced antigen of Actinobacillus pleuropneumoniae serovar 5b strain L20, provided immunoprotection against serovar 1 strain MS71.

GALT is an important antigen of Actinobacillus pleuropneumoniae (APP), which was shown to provide partial protection against APP infection in a previous study in our lab. The main purpose of the present study is to investigate GALT induced cross-protection between different APP serotypes and elucidate key mechanisms of the immune response to GALT antigenic stimulation. Bioinformatic analysis demonstrated that galT is a highly conserved gene in APP, widely distributed across multiple pathogenic strains. Homologies between any two strains ranges from 78.9% to 100% regarding the galT locus. Indirect enzyme-linked immunosorbent assay (ELISA) confirmed that GALT specific antibodies could not be induced by inactivated APP L20 or MS71 whole cell bacterin preparations. A recombinant fusion GALT protein derived from APP L20, however has proven to be an effective cross-protective antigen against APP sevorar 1 MS71 (50%, 4/8) and APP sevorar 5b L20 (75%, 6/8). Histopathological examinations have confirmed that recombinant GALT vaccinated animals showed less severe pathological signs in lung tissues than negative controls after APP challenge. Immunohistochemical (IHC) analysis indicated that the infiltration of neutrophils in the negative group is significantly increased compared with that in the normal control (P<0.001) and that in surviving animals is decreased compared to the negative group. Anti-GALT antibodies were shown to mediate phagocytosis of neutrophils. After interaction with anti-GALT antibodies, survival rate of APP challenged vaccinated animals was significantly reduced (P<0.001). This study demonstrated that GALT is an effective cross-protective antigen, which could be used as a potential vaccine candidate against multiple APP serotypes.

Impact of growth matrix on pharmacodynamics of antimicrobial drugs for pig pneumonia pathogens.

BACKGROUND: The most widely used measure of potency of antimicrobial drugs is Minimum Inhibitory Concentration (MIC). MIC is usually determined under standardised conditions in broths formulated to optimise bacterial growth on a species-by-species basis. This ensures comparability of data between laboratories. However, differences in values of MIC may arise between broths of differing chemical composition and for some drug classes major differences occur between broths and biological fluids such as serum and inflammatory exudate. Such differences must be taken into account, when breakpoint PK/PD indices are derived and used to predict dosages for clinical use. There is therefore interest in comparing MIC values in several broths and, in particular, in comparing broth values with those generated in serum. For the pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, MICs were determined for three drugs, florfenicol, oxytetracycline and marbofloxacin, in five broths [Mueller Hinton Broth (MHB), cation-adjusted Mueller Hinton Broth (CAMHB), Columbia Broth supplemented with NAD (CB), Brain Heart Infusion Broth (BHI) and Tryptic Soy Broth (TSB)] and in pig serum. RESULTS: For each drug, similar MIC values were obtained in all broths, with one exception, marbofloxacin having similar MICs for three broths and 4-5-fold higher MICs for two broths. In contrast, for both organisms, quantitative differences between broth and pig serum MICs were obtained after correction of MICs for drug binding to serum protein (fu serum MIC). Potency was greater (fu serum MIC lower) in serum than in broths for marbofloxacin and florfenicol for both organisms. For oxytetracycline fu serum:broth MIC ratios were 6.30:1 (P. multocida) and 0.35:1 (A. pleuropneumoniae), so that potency of this drug was reduced for the former species and increased for the latter species. The chemical composition of pig serum and broths was compared; major matrix differences in 14 constituents did not account for MIC differences. Bacterial growth rates were compared in broths and pig serum in the absence of drugs; it was concluded that broth/serum MIC differences might be due to differing growth rates in some but not all instances. CONCLUSIONS: For all organisms and all drugs investigated in this study, it is suggested that broth MICs should be adjusted by an appropriate scaling factor when used to determine pharmacokinetic/pharmacodynamic breakpoints for dosage prediction.

Pharmacokinetic and Pharmacodynamic Evaluation of Marbofloxacin in Pig against Korean Local Isolates of Actinobacillus pleuropneumoniae.

The pharmacokinetics of marbofloxacin in pigs after intravenous (i.v.), intramuscular (i.m.), and peroral (p.o.) administration and pharmacokinetic/pharmacodynamic indices of this drug against Korean local isolates of Actinobacillus pleuropneumoniae were determined in this study. Marbofloxacin (2.50 mg/kg of body weight) was administered, and blood samples were collected with designated time intervals. Plasma-extracted marbofloxacin was injected into the LC-MS/MS system. The in vitro and ex vivo antibacterial activities of marbofloxacin were evaluated against 20 isolates of A. pleuropneumoniae. The mean peak plasma concentrations (Cmax) after i.v., i.m., and p.o administration were 2.60 ± 0.10, 2.59 ± 0.12, and 2.34 ± 0.12 µg/mL at 0.25 ± 0.00, 0.44 ± 0.10, and 1.58 ± 0.40 h, respectively. The area under the plasma concentration-time curves (AUC0-24) and elimination half-lives were 24.80 ± 0.90, 25.80 ± 1.40, and 23.40 ± 5.00 h·µg/mL and 8.60 ± 0.30, 12.80 ± 1.10, and 8.60 ± 0.00 h, for i.v., i.m., and p.o. administration, correspondingly. The AUC0-24/MICs of marbofloxacin after i.v., i.m., and p.o. administration were 253.86 ± 179.91, 264.1 ± 187.16, and 239.53 ± 169.75 h, respectively. The Cmax/MIC values were 26.58 ± 18.84, 26.48 ± 18.77, and 23.94 ± 16.97, and T>MICs were 42.80 ± 1.01, 36.40 ± 1.24, and 38.60 ± 1.18 h, after i.v., i.m., and p.o. administration, respectively. Thus, marbofloxacin dosage of 2.50 mg/kg of body weight by i.v., i.m., and p.o. administration with 24 h dosing interval will provide effective treatment for the infection of pig by A. pleuropneumonia.

What is the true in vitro potency of oxytetracycline for the pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida?

The pharmacodynamics of oxytetracycline was determined for pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Indices of potency were determined for the following: (i) two matrices, broth and pig serum; (ii) five overlapping sets of twofold dilutions; and (iii) a high strength starting culture. For A. pleuropneumoniae, minimum inhibitory concentration (MIC) was similar for the two matrices, but for P. multocida, differences were marked and significantly different. MIC and minimum bactericidal concentration (MBC) serum: broth ratios for A. pleuropneumoniae were 0.83:1 and 1.22:1, respectively, and corresponding values for P. multocida were 22.0:1 and 7.34:1. For mutant prevention concentration (MPC) serum: broth ratios were 0.79:1 (A. pleuropneumoniae) and 20.9:1 (P. multocida). These ratios were corrected for serum protein binding to yield fraction unbound (fu) serum: broth MIC ratios of 0.24:1 (A. pleuropneumoniae) and 6.30:1 (P. multocida). Corresponding fu serum: broth ratios for MPC were almost identical, 0.23:1 and 6.08:1. These corrections for protein binding did not account for potency differences between serum and broth for either species; based on fu serum MICs, potency in serum was approximately fourfold greater than predicted for A. pleuropneumoniae and sixfold smaller than predicted for P. multocida. For both broth and serum and both bacterial species, MICs were also dependent on initial inoculum strength. The killing action of oxytetracycline had the characteristics of codependency for both A. pleuropneumoniae and P. multocida in both growth media. The in vitro potency of oxytetracycline in pig serum is likely to be closer to the in vivo plasma/serum concentration required for efficacy than potency estimated in broths.

Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

BACKGROUND: Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. RESULTS: For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. CONCLUSIONS: In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.

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.

Pharmacokinetic/pharmacodynamic evaluation of marbofloxacin in the treatment of Haemophilus parasuis and Actinobacillus pleuropneumoniae infections in nursery and fattener pigs using Monte Carlo simulations.

This study evaluated the theoretical clinical outcome of three marbofloxacin posology regimens in two groups of pigs (weaners and fatteners) for the treatment of Actinobacillus pleuropneumoniae (App) and Haemophilus parasuis (Hp) infection and the appearance of resistant bacteria due to the antibiotic treatment. The probability of target attainment (PTA) for pharmacokinetic/pharmacodynamics (PK/PD) ratios associated with clinical efficacy and with the appearance of antimicrobial resistance for fluoroquinolones at each minimum inhibitory concentration (MIC) or mutant prevention concentration (MPC) were calculated, respectively. The cumulative fraction of response (CFR) was calculated for the three posology regimens against App and they ranged from 91.12% to 96.37% in weaners and from 93% to 97.43% in fatteners, respectively. In the case of Hp, they ranged from 80.52% to 85.14% in weaners and from 82.01% to 88.49% in fatteners, respectively. Regarding the PTA of the PK/PD threshold associated with the appearance of antimicrobial resistance, results showed that marbofloxacin would prevent resistances in most of the animals up to the MPC value of 1 µg/mL.

Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.

In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemcomitans.

The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 µg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and that this property might be utilized for exploring its therapeutic potential in treatment of A. actinomycetemcomitans-associated oral and nonoral infections.

Actinobacillus pleuropneumoniae is impaired by the garlic volatile allyl methyl sulfide (AMS) in vitro and in-feed garlic alleviates pleuropneumonia in a pig model.

Decomposition products of ingested garlic are to a certain extent excreted via the lungs. If the supposed health-supporting capacities associated with garlic extend to these exhaled sulfurous compounds, they could have an effect on the course of pneumonia. In this study, the garlic-derived volatile allyl methyl sulfide (AMS) as a lead compound of volatile garlic metabolites was shown to exhibit an antibacterial effect against the pig pathogen Actinobacillus pleuropneumoniae serotype 9. AMS caused a delay in the appearance of the optical density-monitored growth of A. pleuropneumoniae in medium when compared to unaffected growth curves, yet without lowering the stationary phase yield at the concentration range tested. At 1.1mM, AMS impaired the in vitro growth rate of A. pleuropneumoniae serotype 9 by 8% compared to unimpeded growth. In an animal trial, a garlic-fed group of 15 pigs that received a diet with 5% garlic feed component and a control group of 15 pigs that received a diet without garlic were infected with A. pleuropneumoniae serotype 2 via an aerosol and subsequently followed for 4 days. At the day of the challenge, blood AMS in the garlic-fed group amounted to 0.32 ± 0.13 µM. A beneficial, alleviating effect of garlic on the course and severity of an A. pleuropneumoniae infection in pigs was indicated by the reduced occurrence of characteristic pleuropneumonia lesions (27% of the lungs affected in the garlic-fed group vs. 47% in the control group) and a near to significant (p=0.06) lower relative lung weight post mortem in the garlic-fed group.

Responses of pigs to a re-challenge with Actinobacillus pleuropneumoniae after being treated with different antimicrobials following their initial exposure.

Four groups of six specific pathogen-free (SPF) pigs were inoculated intranasally with Actinobacillus pleuropneumoniae serotype 2 and treated with either enrofloxacin, tetracycline or penicillin at the onset of clinical disease, or left untreated. A fifth group was left uninoculated. The inoculated control and the penicillin-treated groups developed severe disease, but the groups treated with enrofloxacin and tetracycline recovered rapidly. All the inoculated pigs, except those treated with enrofloxacin developed serum antibodies to A pleuropneumoniae. On day 28, all five groups were challenged with A pleuropneumoniae without any subsequent treatment. The previously uninoculated control group and the enrofloxacin-treated group developed severe disease, but the three seropositive groups remained unaffected.

Selection of serotype-specific vaccine candidate genes in Actinobacillus pleuropneumoniae and heterologous immunization with Propionibacterium acnes.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a highly contagious lethal causative agent of swine pleuropneumoniae. Vaccines for this disease are usually serotype specific. In order to identify immunogenic genes specific to serotypes, two differentially expressed gene cDNA libraries of A. pleuropneumoniae CCVC259 (serotype 1) and CCVC263 (serotype 5) had been constructed by using a cDNA representational difference analysis (cDNA-RDA). From the libraries, six potential vaccine candidate genes expressed only in serotype 1 and 13 genes in serotype 5 were identified by antibody screening after gene expression in vitro with a ribosome display system. Eight sequences out of these exhibited 77-100% identity to the corresponding genes in Propionibacterium acnes. The antisera raised against A. pleuropneumoniae serotypes 1 and 5 were reactive with P. acnes at a titer of 1:6400 and vice versa (ELISA titer, 1:3200). Mice immunized with P. acnes were protected against 10 x LD50 challenge with A. pleuropneumoniae serotypes 1 and 5, and the survival rates were 90% and 95%, respectively. Pigs vaccinated with the P. acnes strain could develop high level antibody cross-reacted with A. pleuropneumoniae and obtain noticeable protection from A. pleuropneumoniae infection. These data demonstrate that there were common antigens between A. pleuropneumoniae and P. acnes, and the cross protectivity highlights the possibility of using P. acnes vaccines for preventing infection by A. pleuropneumoniae.

Clinical and microbiological effects of a combined mechanic-antibiotic therapy in subjects with Actinobacillus actinomycetemcomitans-associated periodontitis.

PURPOSE: To evaluate the clinical and microbiological effects of a combined mechanic-antibiotic periodontal therapy in subjects that were tested positive for subgingival Actinobacillus actinomycetemcomitans (A.a.). METHODS: The postoperative follow-up ranged from 12-115 months (average 39.2 months). This follow-up study analyzed the data of 53 subjects (37 females) aged from 16-59 years, who underwent systemic periodontal therapy with adjunctive systemic antibiotics between 1992-2001 and had their last re-examination including microbiological analysis done in 2003. The antibiotic regime was either amoxicillin/metronidazole or ciprofloxacine/metronidazole. During this study, A.a. was detected with two gene probe tests (IAI PadoTest 4.5 and DMDx/PathoTek) and cultivation on TSBV agar plates. The clinical situation was characterized with the help of pocket probing depths and subsequent categorization into three different groups (< or = 4 mm, 5-6 mm and > or = 7 mm). RESULTS: After therapy, A.a. was detected with IAI PadoTest 4.5 in a magnitude between 3.0 x 10(3) up to 2.06 x 10(5) counts per specimen in 9 out of 53 subjects. Only two subjects tested positive for A.a. with the DMDx/PathoTek-assays and the agar cultivation. The clinical situation improved significantly in all subjects after systemic periodontal therapy. The treatment results remained stable during the course of the postoperative follow-up. Concerning the clinical data, no differences were found between the subjects that were tested positive and negative for A.a in the postoperative period.

Systemic and local antibody responses after experimental infection with Actinobacillus pleuropneumoniae in piglets with passive or active immunity.

The objectives of the present study was to describe different dynamics of humoral immune responses to experimental infection in piglets of different stages of infection and immunity. Two groups of piglets originating from non-immune (group 1) and immune (group 2) sows at the age of 3 weeks were subdivided as follows: a half of each group of piglets was exposed to a low-dose infection with Actinobacillus pleuropneumoniae (APP) strain 9. At the age of 8 weeks, all four groups of piglets were challenged with a high infection dose of APP of the same strain. Isotype characterization of the specific antibodies in sera and in bronchoalveolar lavage fluids (BALF) to a lipopolysaccharide was carried out, besides monitoring clinical signs and post-mortem examinations. A typical primary immune response was observed in specific antibody-free piglets infected with a challenge infection. Colostrum-derived immunoglobulin-G (IgG) antibodies persisted in sera and BALF of piglets up to the age of 8 weeks. However, they did not prevent induction of specific-primary antibody response, either in 8 or 4 weeks of age, when levels of specific colostrum-derived antibodies were still high. It was demonstrated by the increase of specific IgM antibodies in sera. The infection induced an increase in the levels of IgA antibodies in BALF regardless the severity of infection and presence of specific colostrum-derived antibodies. The specific antibodies of IgG isotype increased only in BALF from piglets without colostrum-derived antibodies.