A robust PCR for the differentiation of potential virulent strains of Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis is the etiological agent of Glässer's disease in swine. H. parasuis comprises strains with heterogeneous virulence capacity, from non-virulent to highly virulent. Determination of the pathogenic potential of the strains is important for diagnosis and disease control. The virulence-associated trimeric autotransporters (vtaA) genes have been used to predict H. parasuis virulence by PCR amplification of their translocator domains. Here, we report a new and improved PCR designed to detect a different domain of the vtaA genes, the leader sequence (LS) as a diagnostic tool to predict virulence. METHODS: A collection of 360 H. parasuis strains was tested by PCR with LS specific primers. Results of the PCR were compared with the clinical origin of the strains and, for a subset of strains, with their phagocytosis and serum resistance using a Chi-square test. RESULTS: LS-PCR was specific to H. parasuis, and allowed the differential detection of the leader sequences found in clinical and non-clinical isolates. Significant correlation was observed between the results of the LS-PCR and the clinical origin (organ of isolation) of the strains, as well as with their phagocytosis and serum susceptibility, indicating that this PCR is a good predictor of the virulence of the strains. In addition, this new PCR showed a full correlation with the previously validated PCR based on the translocator domain. LS-PCR could be performed in a wide range of annealing temperatures without losing specificity. CONCLUSION: This newly described PCR based on the leader sequence of the vtaA genes, LS-PCR, is a robust test for the prediction of the virulence potential of H. parasuis strains.

Characterization of colonization-deficient mutants of Actinobacillus suis.

Actinobacillus suis is an important opportunistic pathogen of swine that can cause disease in pigs of all ages, especially in high-health status herds. Although A. suis shares many virulence factors in common with Actinobacillus pleuropneumoniae and can cause a haemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae, A. suis most often causes septicaemia and diseases such as arthritis and meningitis that are sequelae to septicaemia. In a recent signature-tagged transposon mutagenesis study, 30 colonization-essential genes of A. suis were identified. In the current study, the attachment and invasion patterns of strains harboring Tn10 insertions in ompA, pfhaB1, lcbB, and cpxR were evaluated using porcine palatine tonsil organ cultures, the swine kidney epithelial cell line, SK6, and a porcine brain microvascular endothelial cell line, PBMEC/C1-2. All of these mutants attached in lower numbers than wild type to the tonsillar explants and to the SK6 cells. The ompA mutant attached in significantly lower numbers than wild type to the porcine tonsil cells (P=0.02) and to PBMEC (P=0.0008) at 60 min time point. As well, the ompA mutant showed significantly greater sensitivity than wild type to chemical stressors and to swine serum. Using fluorescent microscopy, a GST-OmpA fusion protein could be demonstrated to interact with the crypt epithelial cells of porcine palatine tonsil.

Field evaluation of four novel enzyme immunoassays for Chagas' disease in Venezuela blood banks: comparison of assays using fixed-epimastigotes, fixed-trypomastigotes or trypomastigote excreted-secreted antigens from two Trypanosoma cruzi strains.

Many serological tests have been developed for the diagnosis of Chagas' disease, but few have been subjected to a rigorous field evaluation. We have recently described several novel enzyme immunoassays (EIAs) based on fixed-whole organisms or trypomastigote excretory-secretory antigens (TESA) from different Trypanosoma cruzi strains (Tulahuen or Brazil). This study evaluated the most promising of these novel assays (e.g. fixed-epimastigotes, fixed-trypomastigotes, TESA Brazil and TESA Tulahuen antigens) in a field study of Venezuelan blood bank specimens. The assays were tested in an operator-blinded fashion using 2038 blood bank samples obtained from low and high T.cruzi prevalence regions of Venezuela (n= 1050 and n= 988 from Bolivar and Portuguesa states, respectively). Based on National Laboratory for Chagas Immunodiagnosis (NLCI) 'gold standard' results, all novel EIAs were superior to the commercial kit currently used in Venezuela, achieving 100% sensitivity and >99% specificity at optimal cut-off values. The novel assays identified seven false-negative samples compared with the routine screening performed by the Venezuelan blood bank although two samples were also misclassified as positive. Minor differences in the performance of the four novel assays were observed at lower arbitrary cut-off values. This study confirms the potential utility of both the fixed-organism and the TESA-based assays in the diagnosis of T.cruzi infection.

Epidemiology of Streptococcus suis serotype 5 infection in a pig herd with and without clinical disease.

The aim of this study was to describe the transmission and the kinetics of the infection caused by Streptococcus suis serotype 5 in a multisite farrow-to-finish pig herd. Most sows carried S. suis serotype 5 in their vaginal tract, but not in their nasal cavities, as demonstrated by immunomagnetic separation (IMS) technique. Their offspring became infected during farrowing, confirming vertical transmission. During the first 4 weeks of life, a low number of piglets were carriers of S. suis serotype 5 in their nasal cavities. However, when clinical signs appeared, the carrier rate significantly increased, suggesting that isolation from nasal cavities is a better indication of active transmission than of a carrier state. Clinical cases were present in animals between 4 and 8 weeks of age, when maternal antibodies were at their lowest level. Up to six different genotypes of the same serotype could be identified by random amplified polymorphic DNA; however, a single clone was responsible for all clinical cases studied. This clone could only be isolated from a single sow, indicating that its prevalence in breeding animals was low. Interestingly, 1 year later, clinical disease associated with S. suis serotype 5 spontaneously disappeared. At that time, the genotype responsible for the clinical signs was not detected in the herd and the levels of antibodies in sows and maternal antibodies in piglets were not higher than those of the previous year.

Antibody response to an autogenous vaccine and serologic profile for Streptococcus suis capsular type 1/2.

An autogenous vaccine was developed, using sonicated bacteria, with a strain of Streptococcus suis capsular type 1/2. The objectives of this study were to evaluate the antibody response following vaccination and to assess the changes in antibody levels in pigs from a herd showing clinical signs of S. suis capsular type 1/2 infection in 6- to 8-week-old pigs. An enzyme-linked immunosorbent assay using the vaccine antigen was standardized. Results from a preliminary study involving 2 control and 4 vaccinated 4-week-old pigs indicated that all vaccinated pigs produced antibodies against 2 proteins of 34 and 43 kDa, respectively, and, in 3 out of 4 vaccinated pigs, against the 117-kDa muramidase-released protein. For the serologic profile, groups of 30 pigs from the infected herd were blood sampled at 2, 4, 6, 8, and 10 weeks of age. The lowest antibody level was observed between weeks 6 and 8, presumably corresponding to a decrease in maternal immunity. A marked increase was seen at 10 weeks of age, shortly after the onset of clinical signs in the herd. For the vaccination field trial, newly weaned, one-week-old piglets were divided into 2 groups of 200 piglets each (control and vaccinated); blood samples were collected from 36 piglets in each group at 2-week intervals for 12 weeks. A significant increase (P < 0.05) in antibody response was observed 4 weeks following vaccination and the level of antibodies stayed high until the end of the experiment. In the control group, the increase was only observed at 13 weeks of age, probably in response to a natural infection. The response to the vaccine varied considerably among pigs and was attributed, in part, to the levels of maternal antibodies at the time of vaccination. No outbreak of S. suis was observed in the control or vaccinated groups, so the protection conferred by the vaccine could not be evaluated.

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.

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.

Streptococcus suis serotype 2 interactions with human brain microvascular endothelial cells.

Streptococcus suis serotype 2 is a worldwide causative agent of many forms of swine infection and is also recognized as a zoonotic agent causing human disease, including meningitis. The pathogenesis of S. suis infections is poorly understood. Bacteria circulate in the bloodstream in the nonimmune host until they come in contact with brain microvascular endothelial cells (BMEC) forming the blood-brain barrier. The bacterial polysaccharide capsule confers antiphagocytic properties. It is known that group B streptococci (GBS) invade and damage BMEC, which may be a primary step in the pathogenesis of neonatal meningitis. Interactions between S. suis and human endothelial cells were studied to determine if they differ from those between GBS and endothelial cells. Invasion assays performed with BMEC and human umbilical vein endothelial cells demonstrated that unlike GBS, S. suis serotype 2 could not invade either type of cell. Adherence assays showed that S. suis adhered only to BMEC, whereas GBS adhered to both types of cell. These interactions were not affected by the presence of a capsule, since acapsular mutants from both bacterial species adhered similarly compared to the wild-type strains. Lactate dehydrogenase release measurements indicated that some S. suis strains were highly cytotoxic for BMEC, even more than GBS, whereas others were not toxic at all. Cell damage was related to suilysin (S. suis hemolysin) production, since only suilysin-producing strains were cytotoxic and cytotoxicity could be inhibited by cholesterol and antisuilysin antibodies. It is possible that hemolysin-positive S. suis strains use adherence and suilysin-induced BMEC injury, as opposed to direct cellular invasion, to proceed from the circulation to the central nervous system.

Characterization of Streptococcus agalactiae isolates of bovine and human origin by randomly amplified polymorphic DNA analysis.

Streptococcus agalactiae is considered one of the major causes of bovine intramammary infections. It is also found in the vaginas of women without any apparent clinical symptoms, but reports of neonatal infections, causing significant morbidity, are relatively frequent. The aim of this study was to evaluate the genetic diversity of S. agalactiae strains isolated from bovine milk and from asymptomatic women in Québec, Canada, by randomly amplified polymorphic DNA (RAPD) analysis. A total of 185 bovine isolates and 38 human isolates were first serotyped for capsular polysaccharide by double diffusion in agarose gel (bovine isolates) and coagglutination (human isolates). Strains were then studied by RAPD using 3 primers, designated OPS11, OPB17, and OPB18, which were selected from 12 primers. Thirty-eight percent of bovine isolates and 82% of human isolates could be serotyped. Prevalent serotypes were type III (28%) for bovine isolates and types V (26%) and III (24%) for human isolates. RAPD results showed that, taken together, all isolates (of bovine and human origin) shared 58% similarity. Ninety-four percent of these isolates were clustered in four groups (I, II, III, and IV) with 70% similarity among them. Three clusters, A (48 isolates), B (14 isolates), and C (32 isolates), with 79 to 80% similarity were identified within group IV, whereas the three other groups did not present any clusters. Despite some clustering of human isolates, relatively high diversity was seen among them. Relatively high heterogeneity was observed with the RAPD profiles, not only for field strains belonging to different serotypes but also for those within a given serotype.

Immunomagnetic isolation of Streptococcus suis serotypes 2 and 1/2 from swine tonsils.

Isolation of specific serotypes of Streptococcus suis from the tonsils, nasal cavities, and genital tract is difficult, since low-pathogenic serotypes and untypeable strains also inhabit these sites. An immunomagnetic separation (IMS) technique for the selective isolation of S. suis serotypes 2 and 1/2 was standardized. Superparamagnetic polystyrene beads (immunomagnetic beads or IMB) were coated with either a purified monoclonal antibody (MAb) directed to a capsular sialic acid-containing epitope or purified rabbit immunoglobulin G (polyclonal antibody [PAb]), both specific for S. suis serotypes 2 and 1/2. The amount of antibodies required for optimum coating of the beads, the number of IMB required for optimum bacterial recovery, and the nonspecific carryover were considerably higher with the MAb-IMS technique than with the PAb-IMS technique. The sensitivity of the IMS technique was 10(1) CFU/0.1 g of tonsil. The presence of serotype 1/2 bacteria did not considerably affect the recovery rate of a serotype 2 strain and vice versa. To validate the technique, PAb-coated beads were used to study 192 tonsils from animals from S. suis serotype 2- or 1/2-infected herds. Results showed that significantly more positive tonsils were detected by the IMS technique than by the standard procedure. This method represents an innovative and highly sensitive approach for the isolation of S. suis serotypes 2 and 1/2 from carrier animals.

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.

Development of an immunomagnetic method for selective isolation of Actinobacillus pleuropneumoniae serotype 1 from tonsils.

An immunomagnetic separation technique (IMS) for the selective isolation of Actinobacillus pleuropneumoniae serotype 1 was developed. Superparamagnetic polystyrene beads (immunomagnetic beads [IMBs]) were coated with purified rabbit immunoglobulin G specific for A. pleuropneumoniae serotype 1. The antibody concentration, the number of IMBs, the incubation time, and the temperature of incubation influenced the recovery of the target bacteria. The sensitivity of the IMS technique was 1,000-fold higher than that of direct culture. When tonsils from animals from infected herds were tested, significantly more positive tonsils were detected by the IMS technique (68%) than by the standard procedures (22%). The method represents an innovative and highly sensitive approach for the isolation of A. pleuropneumoniae from carrier animals.

Serodiagnosis of swine pleuropneumonia due to Actinobacillus pleuropneumoniae serotypes 7 and 4 using long-chain lipopolysaccharides.

A saline boiled extract (SBE), capsular polysaccharides (CPS) and long-chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 7 have been evaluated in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. Mean optical densities (ODs) obtained with the 3 antigens using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotypes 7 or 4 were not statistically different. The positive ELISA reaction with anti-serotype 4 sera was unexpected with the CPS, which are supposed to be serotype-specific; LPS traces present in the CPS appeared to be responsible for this reaction. In addition, sera from animals exposed to A. pleuropneumoniae serotypes 5 or 10 presented cross-reactions with the SBE and the CPS, but not with the LC-LPS. Cross-reactions were mainly due to rough LPS, as shown by immunoblotting. The LC-LPS is easily obtainable and can be used for the detection of antibodies in animals infected with A. pleuropneumoniae serotypes 7 and 4.

Production and characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 1.

Three monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 1, designated 4.2 A11 B5, 5.1 G8 F10 and 1.5 C5 F4 (IgG3, IgG2b and IgM respectively), were produced and characterized. mAbs 4.2 A11 B5 and 5.1 G8 F10 were directed against different epitopes located in the O chain of the LPS. Both clones also recognized reference strains of A. pleuropneumoniae serotypes 9 and 11. The mAb 1.5 C5 F4 reacted with the reference strain of A. pleuropneumoniae serotype 1, with the encapsulated strain 4045 (but not with its non-capsulated mutant) and with A. pleuropneumoniae serotype 1 purified capsular polysaccharides (CPS). The epitope was sensitive to periodate oxidation, heat-labile, and located in the capsular material of A. pleuropneumoniae serotype 1, as demonstrated by immunoblotting. Treatment of the CPS with 5% ammonium hydroxide eliminated the reaction, which may indicate that the epitope recognized by 1.5 C5 F4 mAb is a O-acetyl containing determinant. When different A. pleuropneumoniae field strains were tested, the percentage of strains recognized by the mAbs varied with the mAb and the test used. Cross-reactions associated with the LPS of some A. pleuropneumoniae serotype 5 field strains could be observed with the 4.2 A11 B5 mAb. Of the three mAbs characterized, 1.5 C5 F4 seemed to be the most suitable for A. pleuropneumoniae serotype 1 detection since it reacted with 99% of serotype 1 field strains and it did not recognize any of the strains belonging to other serotypes.

Characterization of Streptococcus suis capsular type 2 haemolysin.

The production of a haemolysin by Streptococcus suis capsular type 2 was investigated. Human group O erythrocytes were the most susceptible, followed by horse, sheep, cow and pig red blood cells, which exhibited similar susceptibilities; rabbit erythrocytes were the least susceptible. The haemolysin was produced at the end of the exponential growth phase. The toxin described in this paper was purified by affinity chromatography using a thiopropyl-Sepharose 6B column. It is an extracellular protein with a molecular mass of 65 kDa. The haemolysin belongs to the family of toxins known as antigenically related cholesterol-binding cytolytic toxins, since it shares common characteristics with other members of this family, such as sensitivity to oxygen and oxidizing agents, activation by reducing agents, inhibition by low concentrations of cholesterol, formation of transmembrane pores and a 'multihit' mechanism of action. In addition, anti-streptolysin antibodies inhibited the haemolytic activity caused by the S. suis haemolysin. Antibodies against the haemolysin could not be detected in pigs experimentally infected with a haemolytic positive strain of S. suis capsular type 2. To our knowledge, this is the only Lancefield group D Streptococcus producing a haemolysin with these characteristics. The role of this haemolysin in the pathogenesis of S. suis infections remains to be investigated.