Cross-protection between Actinobacillus pleuropneumoniae biotypes-serotypes in pigs

Four groups of hysterectomy-derived and colostrum-deprived pigs were intranasally inoculated with an Actinobacillus pleuropneumoniae biotype 1-serotype 2 strain (producing RTX toxins ApxII and ApxIII. 6 pigs), an A. pleuropneumoniae biotype 1-serotype 10 strain (producing ApxI. 5 pigs), an A. pleuropneumoniae biotype 2-serotype 2 strain (producing ApxII, 5 pigs) or saline (controls, 7 pigs). All pigs were exposed to A. pleuropneumoniae biotype 1-serotype 2 endobronchial challenge. After challenge, severe clinical signs were observed in all control pigs, one pig immunized with the A. pleuropneumoniae biotype 1-serotype 10 strain and two pigs immunized with the A. pleuropneumoniae biotype 2-serotype 2 strain. These pigs died within 36 h after challenge and 20 to 50% of the lungs were macroscopically affected. In the other pigs, clinical signs were mild or absent and no or only small, focal lung lesions were observed when euthanized at 48 h after challenge. At the time challenge neutralizing antibodies against ApxI only. ApxII only and both ApxII and III were present in sera of pigs immunized with the A. pleuropneumoniae biotype 1-serotype 10 strain, the A. pleuropneumoniae biotype 2-serotype 2 strain and the A. pleuropneumoniae biotype 1-serotype 2 strain, respectively. These results indicate that immune mechanisms other than Apx neutralizing antibodies were involved in partial cross-protection of pigs immunized against A. pleuropneumoniae biotype 1-serotype 10 and challenged with the A. pleuropneumoniae biotype 1-serotype 2.     

Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease

The urease gene cluster from the virulent Actinobacillus pleuropneumoniae serotype 1 strain CM5 was cloned and sequenced. The urease activity was associated with a 6.3-kbp region which contains eight long open reading frames (ORFs). The structural genes, ureABC, are separated from the accessory genes, ureEFGD, by a 615-bp ORF of unknown function, ureX. Homologies were found with the structural and accessory urease gene products of Haemophilus influenzae and, to a lesser extent, with those of other organisms. The urease enzyme subunits had predicted molecular masses of 61.0, 11.3, and 11.0 kDa, and the size of the holoenzyme was estimated to be 337 +/- 13 kDa by gel filtration chromatography. Urease activity was maximal but unstable at 65 degrees C. In cell lysates, the A. pleuropneumoniae urease was stable over a broad pH range (5.0 to 10.6) and the optimal pH for activity was 7.7. The Km was 1.5 +/- 0.1 mM urea when it was assayed at pH 7.7. The low Km suggests that this enzyme would be active in the respiratory tract environment, where urea levels should be similar to those normally found in pig serum (2 to 7 mM).     

Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

Restriction endonuclease analysis and plasmid profiling of Actinobacillus pleuropneumoniae serotype 7 strains

The use of adriamycin, an antitumour agent, is restricted by its cardiotoxicity. The objective of this study was to investigate the role of mitochondrial Ca2+ in adriamycin-induced cardiotoxicity and the effect of either cyclosporin A (CsA) or tacrolimus (FK506) on that cardiotoxicity. A single dose of adriamycin (10 mg/kg body weight) caused myocardial damage that was manifested by elevation of serum enzymes, glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), lactate dehydrogenase isoenzyme (LDH-iso) and creatine phosphokinase isoenzyme (CPK2-MB). The permeability of heart inner mitochondrial membrane of adriamycin-treated rats was examined. Tetraphenyl phosphonium ion (TPP+) uptake, estimated with a TPP+-sensitive electrode was used to monitor changes in heart inner mitochondrial membrane potential. Ca2+ efflux was measured spectrophotometrically with the Ca2+ indicator arsenazo III. The ability of heart mitochondria isolated from adriamycin treated rats to retain accumulated Ca2+ or TPP+ was sharply reduced. The increase of diagnostic serum enzymes and isoenzymes and the reduced ability to retain Ca2+ or TPP+ by heart mitochondria were restored to almost the normal levels when (500 microg/kg body weight) of CsA or FK506 were injected with adriamycin. The data suggested that adriamycin cardiotoxicity might be due to the increase of inner membrane permeability in heart mitochondria as a result of increasing the sensitivity of a Ca2+ dependent-pore of the inner mitochondrial membrane to calcium, leading to dissipation of membrane potential and release of pre-accumulated Ca2+. Suitable antagonists of Ca2+-dependent pore formation such as CsA or FK506 may improve heart tolerance to adriamycin.     

Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo

The aim of this study was to evaluate the quality of pain management in prehospital emergency care and to get more information about the administration of analgesics in prehospital patients. METHODS: Patients with painful diseases or injuries who had been brought to Munich hospital's were included in the study. Immediately after having reached the hospitals' emergency department, they were evaluated using a 101-point visual analogue scale for the severity of pain at four predefined periods. Information about the patient, the diagnosis, and the analgesic treatment used by the emergency teams were drawn from the patient's chart. RESULTS: A total of 462 patients were included in the study. The mean pain score on arrival of the emergency team was 64 points; 36.5% of the patients were treated with analgesics. In 28.1% the emergency team tried to reduce pain through external measures (i.e., setting of fractures). In 35.3% there was no therapeutic intervention. In cases in which analgesic therapy was initiated, a definite reduction in pain was achieved during emergency care. Visual analogue scores decreased from 70 points at the beginning to 29 points at arrival to the hospital's emergency department. Analgesics were most frequently used for patients with cardiopulmonary diseases (47.2%), followed by patients with traumatic accidents (35.5%) and patients with acute abdominal pain (25.2%). Of the analgesics, opioids were given most frequently (87.0%). Nonopioid analgesic agents were used in 32.1%. The results of our investigation demonstrate that in many cases the administration of analgesics is not individualized to the patients needs. CONCLUSION: During the prehospital period of emergency care many patients suffer from severe pain. The development of patient-oriented concepts concerning pain management could contribute to improvement of pain therapy in prehospital emergency medicine.     

Development and evaluation of a selective and indicative medium for isolation of Actinobacillus pleuropneumoniae from tonsils

In order to isolate Actinobacillus pleuropneumoniae from mixed bacterial flora a selective and indicative medium was developed. The optimal concentrations of antibiotics were determined for selective chocolate agar (S-TSA) and selective blood agar (S-MBA) using a set of 25 strains of A. pleuropneumoniae. Following optimization of the media 96 isolates of A. pleuropneumoniae were tested on S-TSA and S-MBA. For isolation of A. pleuropneumoniae from 101 pig tonsils collected at slaughter the S-MBA proved significantly better than both S-TSA and the two non-selective agars tested. Furthermore, contaminating to S-TSA and the two non-selective media.     

Serological characterisation and antimicrobial susceptibility of Actinobacillus pleuropneumoniae strains isolated from pigs in Spain

Seventy-one isolates of Actinobacillus pleuropneumoniae isolated from the lungs of pigs in outbreaks of pleuropneumonia in Spain were serotyped by indirect haemagglutination. Serotype 4 (42.2 per cent), serotype 7 (22.5 per cent) and serotype 2 (12.8 per cent) were predominant, whereas serotypes 1, 3, 6, 8, 9, 12 and untypable isolates were present only in small numbers. Serotypes 1, 2, 4 and 7 originated mainly from cases of acute pleuropneumonia, whereas serotypes 3, 6, 8, 9 and 12 were associated with chronically infected herds. The susceptibility of the isolates to 20 antimicrobial agents was determined by agar disc diffusion. Most were susceptible to cefuroxime, cefaclor, cefazolin, kanamycin, tobramycin, gentamicin, oxolinic acid, ciprofloxacin, enoxacin, thiamphenicol, colistin and trimethoprim/sulphamethoxazole. Marked resistance was found with amoxicillin, ticarcillin, oxytetracycline, doxycycline and metronidazole. Rifampicin, fosfomycin and tiamulin were the agents most effective against the isolates tested.     

Actinobacillus pleuropneumoniae infections in pigs: the role of virulence factors in pathogenesis and protection

This paper discusses the possible role of virulence factors of Actinobacillus pleuropneumoniae in pathogenesis and protection. Special attention is paid to the Apx-exotoxins and to adhesins.     

Cross-protection experiments in pigs vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B

Cross-protection experiments were conducted to determine whether antigenic differences located within the lipopolysaccharides (LPS) of Actinobacillus pleuropneumoniae subtypes 1A and 1B were important with respect to the efficacy of whole cell, formalin-inactivated bacterins. Based on clinical signs, lung lesions scores and mortality rates, pigs immunized with A. pleuropneumoniae subtype 1A were partially protected against severe challenge with both subtypes 1A and 1B. In contrast, 1B vaccinated pigs were not protected against severe challenge with subtype 1A but were partially protected against 1B challenge. Cross-reactive serum antibody levels were measured with an ELISA using outer membranes of subtype 1A or 1B as the coating antigen. Serum antibodies were detected against both subtypes within 2 weeks after the first immunization. Antibody levels increased with time and were generally higher against the homologous subtype coating antigen. We conclude that antigenic variation within a capsular serotype, due to antigenic variation within LPS, can result in the failure of whole cell bacterins to provide protection against challenge with the same capsular serotype. This lack of cross-protection within a capsular serotype provides partial explanation for vaccination failures observed under field conditions.     

Proteins found within porcine respiratory tract secretions bind lipopolysaccharides of Actinobacillus pleuropneumoniae

Affinity for porcine respiratory tract secretions was found in some isolates of Actinobacillus pleuropneumoniae and involved lipopolysaccharides (LPS) (M. Bélanger, S. Rioux, B. Foiry, and M. Jacques, FEMS Microbiol. Lett. 97:119-126, 1992). In the present study, the affinity for a crude preparation of porcine respiratory tract mucus of isolates of the Pasteurellaceae family, i.e., Actinobacillus, Haemophilus, and Pasteurella spp., and of some unrelated gram-negative bacteria was examined. Affinity for crude porcine respiratory tract mucus was not a property shared by all Pasteurellaceae isolates tested. Furthermore, affinity for the porcine crude mucus preparation was not unique to the Pasteurellaceae group and did not seem to be restricted to bacteria originating from pigs. Different surface properties of A. pleuropneumoniae isolates in relation to their adherence to crude mucus were examined. The capsular layer seemed to mask the adhesin and interfered with adherence to crude mucus. Two poorly capsulated isolates, which had a more hydrophobic surface and bound Congo red, were also heavily labeled by gold particles coated with polymyxin, which is known to interact with the lipid A-core region of LPS, and adhered strongly to respiratory tract secretions. Tetramethylurea, charged polymers, divalent cations, chelators, monosaccharides and amino sugars, or lectins were unable to inhibit adherence of A. pleuropneumoniae to the crude mucus preparation. To identify the receptor(s) recognized by the lipopolysaccharidic adhesin of A. pleuropneumoniae, affinity chromatography was used. Two bands, which were proteinaceous in nature, of 10 and 11 kDa were recovered. Our results suggest that two low-molecular-mass proteins present in porcine respiratory tract secretions bind A. pleuropneumoniae LPS.     

Detection of an Actinobacillus pleuropneumoniae serotype 2 lipopolysaccharide (LPS) variant

Until now 12 serotypes of Actinobacillus pleuropneumoniae have been recognized. The specificity of the serotypes reside in the carbohydrate composition of the capsular polysaccharides and lipopolysaccharides (LPS). The LPS of A. pleuropneumoniae serotype 2 is a smooth type LPS with O-chains of linear repeating pentasaccharide units with an O-acetyl group linked to a glucose unit. A monoclonal antibody (MAb 102-G02) directed against A. pleuropneumoniae serotype 2 was characterized in enzyme linked immunosorbent assay (ELISA) and in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The MAb 102-G02 was directed against an epitope on the O-chain of the LPS and was used to define a new. LPS variant of A. pleuropneumoniae serotype 2 (referred to as A. pleuropneumoniae serotype 2X). Investigation of the reactivity of the MAb 102-G02 against an A. pleuropneumoniae serotype 2X, field isolate (9008) and the Danish App-2 strain 4226 in electron microscopy, confirmed the different binding patterns.     

DNA extraction and stability for epidemiological studies

An outbreak of food poisoning involving most autonomous Spanish communities was detected in the first half of 1994. The causative food was infant formula milk contaminated by lactose-fermenting Salmonella virchow. It was not possible to isolate the causative strain from the manufacturer's facilities. During the same period of time, there was a significant increase in lactose-non-fermenting Salmonella virchow strains compared with the same period in previous years. Simultaneously, lactose-non-fermenting strains were recovered from clinical samples from children and from some milk samples that were involved in the outbreak. Therefore, it was speculated that the outbreak might be more extensive than initially thought. The following epidemiological markers were used for typing the Salmonella virchow strains involved in the outbreak: (i) phage typing: (ii) ribotyping, using a set of 20 different endonucleases: and (iii) pulsed-field gel electrophoresis, using three different endonucleases. The most useful markers for this serotype were phage typing and pulsed-field gel electrophoresis, since ribotyping was not able to distinguish all strains tested. The results obtained revealed that the outbreak was caused by at least two strains: one presenting phage type 4-4a and pulsed-field patterns A1 or A2 and L+ or L-, and another presenting phage type 2 and pulsed-field patterns A1 or A2 and L+ or L-. The results indicate that the outbreak was more extensive than initially thought and that the Virchow serotype is very clonal in Spain.     

Protection of mice against challenge with homologous and heterologous serovars of Actinobacillus pleuropneumoniae after live vaccination

BACKGROUND: Massive neurofilament conglomeration in motor neurons has been described to occur in the early stages of both familial and sporadic amyotrophic lateral sclerosis (ALS). Previously, neurofilament conglomerates were immunolabeled for both superoxide dismutase (SOD1) and nitrotyrosine, suggesting the potential for oxidative nitration damage to neurofilament protein by peroxynitrite. Long-lived neurofilaments may also undergo modification by advanced glycation endproducts (AGEs) with concomitant generation of free radicals, including superoxide. This radical species may then react with nitric oxide to form the potent oxidant, peroxynitrite, which in turn can nitrate neurofilament protein. Such a glycated and nitrated neurofilament protein may become resistant to proteolytic systems, forming high-molecular-weight protein complexes and cytotoxic, neuronal inclusions. MATERIALS AND METHODS: Paraffin sections containing both neurofilament conglomerates and neuronal inclusions were obtained from patients with sporadic (n = 5) and familial (n = 2) ALS and were probed with specific antibodies directed against the AGEs cypentodine/piperidine-enolone, arginine-lysine imidazole, pentosidine, and pyrraline. RESULTS: Neurofilament conglomerates, but not neuronal inclusions, were intensely immunolabeled with each of the anti-AGE antibodies tested. The immunoreactivity was selective for neurofilament conglomerates and suggested that AGEs may form inter- or intramolecular cross-links in neurofilament proteins. CONCLUSIONS: These data support the hypothesis that AGE formation affects neurofilament proteins in vivo and is associated with the concomitant induction of SODI and protein nitration in neurofilament conglomerates. AGE formation in neurofilament protein may not only cause covalent cross-linking but also generate superoxide and block nitric oxide-mediated responses, thereby perpetuating neuronal toxicity in patients with ALS.     

Serological characterization of Actinobacillus pleuropneumoniae biotype 1 strains antigenically related to both serotypes 2 and 7

Free radical formation in nucleosides and nucleotides containing cytosine as base was studied after X-irradiation at 77 K of samples prepared in frozen aqueous BeF2 glasses and in frozen aqueous solutions by means of electron spin resonance (ESR) spectroscopy. By comparison with radical patterns from the cytosine base and from 1-CH3-cytosine, by using specifically base-deuterated nucleosides and by comparison between the 2'-deoxy- and the ribonucleotide it could be demonstrated that a radical at the C1'-position of the sugar was formed in nucleosides and nucleotides in both matrices. Quantitative analysis showed that in the BeF2 glass an initial population of about 10% of substrate located species due to this radical was present at 77 K and developed to about 25% after decay of the .OH (.OD) radicals at about 140 K. This was taken as proof that at least part of these radicals were formed from .OH radicals. In frozen aqueous solutions about 20% of C1' located sugar radicals were found to be present at 77 K, the population remaining roughly constant with increasing temperature to 140 K. The mechanistic findings of these unexpected results are discussed in terms of mobile .OH radicals and/or hole transfer in the glass and in the glassy regions of the frozen aqueous solutions.     

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.     

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7

OBJECTIVE: To determine the expression of endothelial nitric oxide synthase in the endometrium during the menstrual cycle in endometriosis and adenomyosis. DESIGN: Immunohistochemical identification of endothelial nitric oxide in endometrial tissues using the monoclonal antibody. SETTING: Department of obstetrics and gynecology in a university hospital. PATIENT(S): The subjects were divided into three groups: 35 patients with endometriosis, 33 patients with adenomyosis proven histologically, and 46 fertile controls. MAIN OUTCOME MEASURE(S): Semiquantitative immunostaining (evaluation nomogram) score in endometrial cells. RESULT(S): The analyses revealed phase-dependent changes in the expression of endothelial nitric oxide synthase in the surface and glandular epithelia during the menstrual cycle in the fertile controls. The expression was weakest in the early proliferative phase, gradually increased, was most marked in the midsecretory phase, and decreased thereafter. In contrast, stromal cells did not change throughout the cycle. Contrary to expectations, the expression of endothelial nitric oxide synthase in endometriosis and adenomyosis was persistently greater than the control levels throughout the menstrual cycle. CONCLUSION(S): This study has shown that endothelial nitric oxide synthase is changed in a phase-dependent manner during the menstrual cycle. The exaggerated expression of endothelial nitric oxide synthase in the endometrium throughout the cycle suggests some pathologic role in endometriosis and adenomyosis.     

Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.     

Molecular methods for diagnosis and epidemiological studies of parasitic infections

Direct microscopy is widely used for the diagnosis of parasitic infections although it often requires an experienced microscopist for accurate diagnosis, is labour intensive and not very sensitive. In order to overcome some of these shortcomings, molecular or nucleic acid-based diagnostic methods for parasitic infections have been developed over the past 12 years. The parasites which have been studied with these techniques include the human Plasmodia, Leishmania, the trypanosomes, Toxoplasma gondii, Entamoeba histolytica, Giardia, Trichomonas vaginalis, Cryptosporidium parvum, Taenia, Echinococcus, Brugia malayi, Wuchereria bancrofti, Loa loa and Onchocerca volvulus. Early methods, which involved hybridisation of specific probes (radiolabelled and non-radiolabelled) to target deoxyribonucleic acid (DNA), have been replaced by more sensitive polymerase chain reaction (PCR)-based assays. Other methods, such as PCR-hybridisation assays, PCR-restriction fragment length polymorphism (PCR-RFLP) assays and random amplified polymorphic DNA (RAPD) analysis have also proved valuable for epidemiological studies of parasites. The general principles and development of DNA-based methods for diagnosis and epidemiological studies will be described, with particular reference to malaria. These methods will probably not replace current methods for routine diagnosis of parasitic infections in developing countries where parasitic diseases are endemic, due to high costs. However, they will be extremely useful for genotyping parasite strains and vectors, and for accurate parasite detection in both humans and vectors during epidemiological studies.