Granulomatous lymphadenitis and pneumonia associated with Actinobacillus porcitonsillarum in a slaughter pig.

Multiple coalescing granulomatous foci were detected in the pulmonary hilar and mediastinal lymph nodes and lung of a slaughtered pig aged 6 months. Haemolytic, Gram-negative bacilli were isolated from the lymph nodes. The isolate (strain TO17214) strongly cross-reacted with sera against Actinobacillus pleuropneumoniae serotype 12 in slide agglutination tests. Comparative 16S rDNA gene sequencing analysis identified strain TO17214 as Actinobacillus porcitonsillarum. Histologically, extensive inflammation took the form of large granulomas consisting of epithelioid cells and multinucleated giant cells in the lymph nodes and lung, and Gram-negative bacilli were discernible in the centres of the lesions. Immunohistochemically, the organisms cross-reacted with polyclonal antibodies against A. pleuropneumoniae serotypes 12 and 2. The results indicated that A. porcitonsillarum, previously considered non-pathogenic, can induce multifocal granulomatous lymphadenitis accompanied by pneumonia in the growing-finishing pig.

Specific immune response of mares and their newborn foals to Actinobacillus spp. present in the oral cavity.

Oral swab samples, serum and colostrum was taken from 15 mares and 14 of their foals, within 24 h of birth. The presence of antibody against Actinobacillus spp. isolated from the oral cavity was investigated using agar gel immunodiffusion. Antibodies against 48 out of the 77 Actinobacillus isolates from all horses in the study were present in the respective sera of 13 mares and 9 foals. In 11 mother-foal pairs, the antibody content of the foal serum was similar to that of the mare, and in 9 cases this was reflected in the antibody content of colostrum from the mare. The results indicate that an immune response to Actinobacillus spp. colonising the oral cavity is present in many adult horses and that this immune response can be transferred from mother to foal via colostrum.

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

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

Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli.

Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice.

A gene cluster for the synthesis of serotype d-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans.

The serotype d antigen of Actinobacillus actinomycetemcomitans consists of D-glucose, D-mannose, and L-rhamnose in a molar ratio of 1:2:1. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans IDH 781 (serotype d). This cluster consisted of 12 open reading frames. Insertional inactivation of six genes in this cluster resulted in loss of ability of A. actinomycetemcomitans IDH 781 cells to produce the polysaccharide. Comparing the structure of the gene cluster with similar clusters from other serotypes of A. actinomycetemcomitans, showed that eight genes are unique to serotype d; the other four genes are involved in the biosynthesis of dTDP-L-rhamnose. These results suggest that the synthesis and structure of serotype d-specific polysaccharide of A. actinomycetemcomitans is quite different from those of other serotype strains.

Actinobacillus species and their role in animal disease.

Actinobacillus species are Gram-negative bacteria responsible for several quite distinct disease conditions of animals. The natural habitat of the organisms is primarily the upper respiratory tract and oral cavity. A. lignieresii is the cause of actinomycosis (wooden tongue) in cattle: a sporadic, insidiously-developing granulomatous infection. In sharp contrast is A. pleuropneumoniae which is responsible for a rapidly spreading often fatal pneumonia, common among intensively reared pigs. Detailed investigation of this organism has provided a much clearer picture of the bacterial factors involved in causing disease. A. equuli similarly causes a potent septicaemia in the neonatal foal; growing apparently unrestricted once infection occurs. Other members of the genus induce characteristic pathogenesis in their preferred host, with one, A. actinomycetemcomitans, being a cause of human periodontal disease. This article reviews recent understanding of the taxonomy and bacteriology of the organisms, and the aetiology, pathogenicity, diagnosis and control of animal disease caused by Actinobacillus species.

Effects of Actinobacillus equuli culture supernatants on equine neutrophil functions and survival.

After exposure of equine granulocytes from both foals and adult horses to culture supernatants from clinical isolates of Actinobacillus equuli, phagocytic capacity and respiratory burst was examined by flow-cytometry and a chemiluminescence assay, respectively. One haemolytic isolate of an equine Actinobacillus was also included in the study. An average decrease of 22% in total number of granulocytes, in the flow cytometric assay (P < 0.01), and an average decrease of 26% in light emission, in the chemiluminescence assay (P < 0.001), was seen after exposure to bacterial culture supernatants of A. equuli, indicating that the supernatants contained leukotoxic bacterial products. Supernatants from the haemolytic isolate appeared to contain a higher amount or more potent leukotoxic metabolites when haemolysis was expressed, causing a decrease in total number of granulocytes of 44% (P < 0.01) and a decrease in light emission of 52% (P < 0.01). Evaluation of the stability of the methods used revealed that within-method variation was far less than the observed results. The leukotoxic effects of A. equuli culture supernatants were mainly reflected in the decreased survival of neutrophils and not in neutrophil functions.

Identification of Actinobacillus pleuropneumoniae strains of serotypes 7 and 4 using monoclonal antibodies: demonstration of common LPS O-chain epitopes with Actinobacillus lignieresii.

Monoclonal antibodies (Mabs) against Actinobacillus pleuropneumoniae serotype 7 were produced and characterized. Three Mabs directed against surface polysaccharides were selected. One of the Mabs was directed against a capsular polysaccharide epitope (CPS) of A. pleuropneumoniae serotype 7 whereas two other Mabs reacted with different epitopes of the LPS O-chain. One of the latter reacted with the reference strain of serotype 7 and the other one with serotypes 7 and 4. These three Mabs were used to test, by Dot-ELISA, 508 field strains of A. pleuropneumoniae. None of the strains belonging to other serotypes different from serotypes 4 and 7 were positive with the Mabs. Used in combination, the CPS and one of the LPS O-chain directed Mabs were shown to be suitable for serotyping since they detected 100% of serotype 7 strains. In this study, we confirm for the first time that A. pleuropneumoniae serotype 4 is present in North America. Finally, both O-chain specific Mabs also reacted with the O-chain of Actinobacillus lignieresii. The cross-reactivity between the two species was confirmed using sera from pigs experimentally infected with A. pleuropneumoniae serotype 7 and A. lignieresii, using immunoblotting and ELISA. This is the first report of a specific cross-reactivity between the LPS of these bacterial species.

Enhancement of host resistance to bacterial infections in normal and immunosuppressed mice with Actinobacillus suis.

A single intraperitoneal (ip) inoculation of heat-killed Actinobacillus suis ATCC 15,557 (AS 15,557) into normal and immunosuppressed (dexamethasone-treated) mice led to remarkable nonspecific resistance to ip challenge with lethal doses of opportunistic pathogens such as Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Candida albicans. The duration of this enhanced protective action and the minimal effective dose, in normal mice, induced by AS 15,557 were superior to those induced by other bacterial immunostimulants such as heat-killed Lavtobacillus casei YIT 9018 (LC 9018) and penicillin-treated Streptococcus pyogenes, Su (OK-432). In immunosuppressed mice; the reduced in vivo killing activity of peritoneal exudate cells (PECs) against P. aeruginosa infection was markedly augmented by ip injection of AS 15,557. The degree of PEC augmentation induced by AS 15,557 was higher than that induced by LC 9018 or by OK-432. The toxicity and histopathological changes associated with AS 15557 were very low, as compared with those by produced by LC 9018 and OK-432. The results suggest that AS 15,557, which showed a strong resistance-enhancing capacity against opportunistic bacterial infections, may be a useful bacterial immunostimulant.

Actinobacillus suis septicaemia in two foals.

A 24-hour-old Hackney ony filly developed signs of weakness, depression and a poor suck reflex, with harsh lung sounds over both fields, and a 48-hour-old Arabian colt from a normal birth which had sucked vigorously developed loose stools and became depressed, weak and anorectic. Both foals had serum IgG concentrations greater than 800 mg/dl, but each had a severe neutropenia with a left shift, and blood cultures from both of them yielded Actinobacillus suis. The A suis isolates had different antimicrobial susceptibility patterns and, in the case of the Arabian, the isolate was resistant to commonly used broad spectrum antimicrobial agents.

Nuclear-magnetic-resonance analysis of the capsular antigen of Actinobacillus pleuropneumoniae serotype 9. Its identity with the capsular antigen of Escherichia coli K62 (K2ab), Neisseria meningitidis serogroup H and Pasteurella haemolytica serotype T15.

The specific capsular antigen of Actinobacillus pleuropneumoniae serotype 9 was characterized by one-dimensional and two-dimensional high-field nuclear-magnetic-resonance methods, and by chemical analyses, as a teichoic-acid-type polymer of a repeating unit having the structure [formula: see text] The basic polymer structure is identical to capsular antigens of Neisseria meningitidis group H, Escherichia coli K62 (K2ab) and Pasteurella haemolytica serotype T15.

Patrones electroforéticos y antigénicos de los serotipos 1, 2, 5 y 7 de Actinobacillus pleuropneumoniae / Elecrtophoretic and antigenic patterns of Actinobaccillus pleuropneumoniae serotypes 1, 2, 5 and 7

Se estudiaron extractos bacterianos solubilizados con Triton X-100 de los serotipos 1,2, 5 y 7 de Actinobacillus pleuropneumoniae. Los extractos se analizaron mediante electroforesis en geles de sodio-duodecil-sulfato (SDS) poliacrilamida e inmunotransferencia con sueros hiperinmunes de conejo y 9 sueros de cerdo A. pleuropneumoniae positivos. Los patrones electroforéticos mostraron las siguientes proteínas comunes a todos los serotipos: 26-29, 30-33, 43, 50, 66 y 81-83 Kd. En cuanto a los patrones antigénicos con sueros hiperinmunes de conejo, se observó un antígeno de 30-31 Kd de peso molecular reconocido por todos los antisueros con todos los serotipos (con excepción del serotipo 7, cuando reaccionó con el antisuero contra el serotipo 5).En relación con los antígenos reconocidos por sueros de cerdo, se onservó un grupo de antígenos cuyo peso molecular osciló entre 29 y 33 Kd, reconocido por todo los sueros con todos los serotipos, salvo la reacción de 3 sueros con el serotipo 2.

The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.

Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS.

6-Deoxy-D-talan and 6-deoxy-L-talan. Novel serotype-specific polysaccharide antigens from Actinobacillus actinomycetemcomitans.

Serotype-specific polysaccharide antigens from Actinobacillus actinomycetemcomitans ATCC 29523 (serotype a) and NCTC 9710 (serotype c) were extracted from whole cells by autoclaving and purified by ion-exchange chromatography and gel filtration. Analysis of component sugars by gas-liquid chromatography-mass spectrometry, high performance liquid chromatography, and NMR together with optical rotation data showed that the serotype a antigen was composed solely of 6-deoxy-D-talose, whereas the serotype c antigen consisted of 6-deoxy-L-talose. Structural analysis indicated that both of these antigens were composed of closely related repeating units, -3)-6-deoxy-alpha-D-Talp-(1-2)-6-deoxy-alpha-D-Talp-(1-(sero type a) and -3)-6-deoxy-alpha-L-Talp-(1-2)-6-deoxy-alpha-L-Talp-(1-(sero type c). 1H and 13C NMR analysis showed that both of these serotype antigens contained one acetyl group/2 sugar residues. These acetyl groups localized at the O-2 position of 3-linked 6-deoxy-D-talose (serotype a) or O-4 position of 3-linked 6-deoxy-L-talose residues (serotype c), respectively. These results coupled with our previous findings on the serotype b antigen (Amano, K., Nishihara, T., Shibuya, N., Noguchi, T., and Koga, T. (1989) Infect. Immun. 57, 2942-2946) showed that the serotype antigens from A. actinomycetemcomitans are a group of novel polysaccharides with structural features closely related biosynthetically.

Actinobacillus pleuropneumoniae-induced thymic lesions in mice and pigs.

Actinobacillus pleuropneumoniae produces several hemolysins/cytotoxins that may be important in the pathogenesis of acute lesions. Little is known, however, about the role of these virulence factors in chronic disease or the carrier state. We investigated the effects of live bacterial infection and transthoracic injection of a sterile culture supernatant on primary lymphoid organs and lymphocyte populations. Transthoracic inoculation of mice or intranasal inoculation of pigs with virulent A. pleuropneumoniae serotypes 1 and 7 induced thymic cortical lymphoid necrosis. These lesions were reproduced in mice by transthoracic injection of a concentrated sterile culture supernatant. The cytotoxic effect of this culture supernatant was also demonstrated in vitro by using a tetrazolium dye reduction assay. Both porcine and murine thymic lymphocytes as well as splenic T lymphocytes were susceptible to the toxin. Porcine convalescent serum, but not preimmune serum, prevented thymic lesions and neutralized the in vitro cytotoxic effect of the culture supernatant on murine thymic lymphocytes. Thymic lesions also were reproduced in mice by using purified lipopolysaccharide (LPS) from Escherichia coli O111:B4; however, LPS had no in vitro cytotoxic effect on either porcine or murine thymic lymphocytes. These results suggest that secreted A. pleuropneumoniae toxin(s) is capable of affecting host T-lymphocyte populations and may affect host immune function.

In vitro killing of Actinobacillus actinomycetemcomitans and Capnocytophaga spp. by human neutrophil cathepsin G and elastase.

The purpose of this study was to compare the killing of Actinobacillus actinomycetemcomitans with that of Capnocytophaga spp. by purified cathepsin G and elastase in vitro. Both were sensitive to killing by purified cathepsin G, but only the Capnocytophaga spp. were killed by elastase. Killing by cathepsin G exhibited logarithmic kinetics, was enhanced slightly by alkaline pH, and was enhanced greatly under hypotonic conditions. Treatment of cathepsin G with diisopropyl fluorophosphate significantly reduced its bactericidal activity against Capnocytophaga spp. but not against Escherichia coli or A. actinomycetemcomitans. The bactericidal effects of cathepsin G against Capnocytophaga sputigena and A. actinomycetemcomitans were inhibited by alpha-1-antichymotrypsin, alpha-1-antitrypsin, and alpha-2-macroglobulin but not by bovine serum albumin. We conclude that (i) cathepsin G kills Capnocytophaga spp. and A. actinomycetemcomitans, (ii) elastase kills Capnocytophaga spp., (iii) the bactericidal activity of cathepsin G is enzyme dependent against Capnocytophaga spp. and enzyme independent against A. actinomycetemcomitans, and (iv) natural plasma antiproteases may control both enzyme-dependent and enzyme-independent bactericidal activities of cathepsin G.

Lethal effects of Actinobacillus actinomycetemcomitans leukotoxin on human T lymphocytes.

The majority of strains of Actinobacillus actinomycetemcomitans isolated from patients with periodontal diseases secrete a leukotoxin that destroys human myeloid cells within minutes but has no effect on viability of peripheral blood lymphocytes in culture for 1.5 h. However, since this organism persists in the gingival crevice and thus may continuously release toxin over extended periods of time, we assessed the viability of T cells cultured with leukotoxin (0 to 250 ng/ml) for up to 2 days. Although the total numbers of cells recovered from cultures with or without leukotoxin were equivalent, leukotoxin killed up to 70% of the T cells in a time- and concentration-dependent manner. Cell death was associated with uptake of propidium iodide, release of 51Cr from the cytoplasm, and morphological evidence of damage to the plasma membrane and apoptosis. Leukotoxin also induced increased cleavage of chromosomal DNA into nucleosome-sized fragments, suggesting activation of an endogenous nuclease in the T cells. These data suggest that leukotoxin kills T cells by pathways resembling necrosis and programmed cell death. Leukotoxin-induced lymphotoxicity may represent a critical mechanism by which A. actinomycetemcomitans suppresses the host local immune response and contributes to the pathogenesis of diseases involving this microorganisms.

Cross-reactivity and antigenic heterogeneity among Actinobacillus pleuropneumoniae strains of serotypes 4 and 7.

Actinobacillus pleuropneumoniae strains of serotypes 4 and 7 were studied for their antigenic properties by means of agglutination, coagglutination, indirect hemagglutination, immunodiffusion, and counterimmunoelectrophoresis tests. Strains of serotype 4 showed cross-reactivity with those of serotype 7 in various serological tests. Serotype 7 strains were antigenically heterogeneous and shared common antigens with several other serotypes. By using boiled whole-cell saline extract as the antigen in the immunodiffusion test, serotype 7 strains could be divided into four subgroups. Subgroup I strains did not have antigens in common with other serotypes, whereas subgroup II strains had antigens in common with serotype 4; subgroup III strains had antigens in common with serotype 10, and subgroup IV had antigens in common with serotypes 1, 9, and 11. The indirect hemagglutination test using unheated whole-cell saline extract as the antigen detected serotype-specific activity. Quantification of serotype-specific and group-specific antigens by coagglutination and immunodiffusion tests was found useful for identifying strains that belonged to serotype 4 or 7.

Immunological properties of Actinobacillus pleuropneumoniae hemolysin I.

The 105 kDa hemolysin I protein from Actinobacillus pleuropneumoniae serotype I type strain 4074 (HlyI) was shown by immunoblot analysis to be the predominant immunogenic protein if convalescent field sera or sera from pigs experimentally infected with A. pleuropneumoniae serotype 1 were used. SDS gel- and immunoblot-analysis using total culture, washed cells or culture supernatant showed that HlyI is essentially secreted and is not found attached to the bacteria. Proteins in the 105 kDa range that react strongly with anti-HlyI antibody, are produced by all serotypes and are presumed to be their hemolysins. Sera from pigs experimentally infected with each of the 12 serotypes strongly reacted with HlyI. In addition, some sera from pigs that were confirmed to be negative for A. pleuropneumoniae, also reacted with HlyI as well as with related proteins from Actinobacillus rossii and Actinobacillus suis. These two species produce proteins in the 105 kDa range which cross-react strongly with HlyI. They could be the source of the immunological reactions of the A. pleuropneumoniae-negative sera with HlyI. However, no cross-reactions could be found between HlyI and the Pasteurella haemolytica leukotoxin, the Escherichia coli alpha-hemolysin or related proteins from various hemolytic E. coli strains isolated from pigs. The immunological cross-reactions of HlyI with related proteins from A. rossii, A. suis and possibly from other bacterial species may create uncertainty in interpretation if HlyI is used as the antigen in serodiagnosis of A. pleuropneumoniae.