Elías F. Rodríguez-Ferri

The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae: evidence from mutational analysis

The involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogen Actinobacillus pleuropneumoniae in virulence was investigated using haemolysin‐deficient mutants constructed by a mini‐Tn10 mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini‐Tn10 were cloned and sequenced. In the non‐haemolytic mutant, the transposon had inserted in apxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In the apxI operon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved in A. pleuropneumoniae pathogenicity. The non‐haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.

The insertion element IS6110 is a useful tool for DNA fingerprinting of Mycobacterium bovis isolates from cattle and goats in Spain

A total of 129 Mycobacterium bovis strains from 5 different Spanish locations were fingerprinted using the IS6110 repetitive element. We demonstrated the presence of multiple copies (from 2 to 13) of IS6110 in a large proportion (47.4%) of the M.bovis strains isolated from cattle and we showed that these strains can be successfully differentiated by means of the RFLP with IS6110. All of the M. bovis strains isolated from goats had multiple copies of IS6110 and 4 bands of 2, 1.7, 1.4 and 1.3 kb were common in all the caprine RFLP patterns. The caprine strains formed a clearly separate cluster from the bovine strains.

Kinetics of Infection and Effects on the Placenta of Clamydophila abortus in Experimentally Infected Pregnant Ewes

A Chlamydophila abortus-induced abortion model was carried out on the basis of the experimental infection of ewes at day 75 of gestation. The infection induced abortions and the birth of weak lambs during the last 3 weeks of pregnancy. To study the kinetics of the infection in the placenta and in other organs, infected ewes were killed at 105, 120, and 130 days of gestation and also several days after abortion or parturition. Infected ewes developed a systemic infection that caused a mild and transient pneumonia and focal hepatitis. Pathologic changes were observed in placentas at 120 day of gestation, although the lesions varied between animals and even between placentomes of the same placenta. The first placental area infected was the maternal stroma and epithelium next to the intercaruncular areas, where neutrophilic response seemed to control the infection. A substantial degree of multiplication of C. abortus was then observed in the trophoblast cells of the placentome, periplacentomal choriallantoic membranes, and hilius, with an inflammatory exudate composed mainly of neutrophils, some macrophages, and very scarce lymphocytes. After abortion, the lesions affected the intercotyledonary areas of the aborted placentas, whereas in the uterus significant lymphocyte infiltration was observed, together with a rapid decrease of the C. abortus antigen in the degenerated caruncular tissues.

Development and Characterization of Protective Haemophilus parasuis Subunit Vaccines Based on Native Proteins with Affinity to Porcine Transferrin and Comparison with Other Subunit and Commercial Vaccines

ABSTRACT Haemophilus parasuis is the agent responsible for causing Glässers disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was potentiated with a bacterial neuraminidase from Clostridium perfringens. The potential protective effect conferred by these two vaccines was compared to that afforded by two other vaccines, consisting of recombinant transferrin-binding protein (rTbp) A or B fragments from H. parasuis, Nagasaki strain, and by a commercially available inactivated vaccine. Five groups of colostrum-deprived piglets immunized with the vaccines described above, one group per each vaccine, and a group of nonvaccinated control animals were challenged intratracheally with a lethal dose (3 × 108 CFU) of H. parasuis, Nagasaki strain. The two vaccines containing rTbps yielded similar results with minimal protection against death, clinical signs, gross and microscopic lesions, and H. parasuis invasion. In contrast, the two vaccines composed of NPAPT antigen and commercial bacterin resulted in a strong protection against challenge (without deaths and clinical signs), mild histopathological changes, and no recovery of H. parasuis, thus suggesting their effectiveness in preventing Glässers disease outbreaks caused by serovar 5.

Haemophilus parasuis serovar 5 Nagasaki strain adheres and invades PK-15 cells.

Haemophilus parasuis is the agent responsible for causing Glässers disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. The purpose of this study was to investigate the in vitro ability of two H. parasuis serovars of different virulence (serovar 5, Nagasaki strain, highly virulent, belonging to serovar 5, and SW114 strain, nonvirulent, belonging to serovar 3) to adhere to and invade porcine kidney epithelial cells (PK-15 line). Nagasaki strain was able to attach at high levels from 60 to 180 min of incubation irrespective of the concentrations compared (10(7)-10(10)CFU), and a substantial increase of surface projections could be seen in PK-15 cells by scanning electron microscopy. This virulent strain was also able to invade effectively these epithelial cells, and the highest invasion capacity was reached at 180 min of infection. On the contrary, nonvirulent SW114 strain hardly adhered to PK-15 cells, and it did not invade these cells, thus suggesting that adherence and invasion of porcine kidney epithelial cells could be a virulence mechanism involved in the lesions caused by H. parasuis Nagasaki strain in this organ.

Nonbinding Site-Directed Mutants of Transferrin Binding Protein B Exhibit Enhanced Immunogenicity and Protective Capabilities

ABSTRACT Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

Distribution of Tetracycline Resistance Genes in Actinobacillus pleuropneumoniae Isolates from Spain

ABSTRACT Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia. Tetracycline is used for therapy of this disease, and A. pleuropneumoniae carrying the tet(B) gene, coding for an efflux protein that reduces the intercellular tetracycline level, has been described previously. Of the 46 tetracycline-resistant (Tcr) Spanish A. pleuropneumoniae isolates used in this study, 32 (70%) carried the tet(B) gene, and 30 of these genes were associated with plasmids. Eight (17%) isolates carried the tet(O) gene, two (4%) isolates carried either the tet(H) or the tet(L) gene, and all these genes were associated with plasmids. This is the first description of these tet genes in A. pleuropneumoniae. The last two Tcr isolates carried none of the tet genes examined. Except for tet(O)-containing plasmids, the other 34 Tcr plasmids were transformable into an Escherichia coli recipient. Two plasmids were completely sequenced. Plasmid p11745, carrying the tet(B) gene, was 5,486 bp and included a rep gene, encoding a replication-related protein, and two open reading frames (ORFs) with homology to mobilization genes of Neisseria gonorrhoeae plasmid pSJ7.4. Plasmid p9555, carrying the tet(L) gene, was 5,672 bp and, based on its G+C content, consisted of two regions, one of putative gram-positive origin containing the tet(L) gene and the other comprising four ORFs organized in an operon-like structure with homology to mobilization genes in other plasmids of gram-negative bacteria.

Characterization of a recombinant transferrin-binding protein A (TbpA) fragment from Haemophilus parasuis serovar 5

Haemophilus parasuis, the etiological agent of Glässers disease in pigs, possesses iron acquisition pathways mediated by a surface receptor that specifically bind porcine transferrin. This receptor is composed of transferrin-binding protein A (TbpA) and TbpB. As it has been reported for other gram-negative organisms, H. parasuis TbpA could be useful as a candidate target for H. parasuis vaccination. In this study, a 600-bp tbpA fragment of the gene encoding TbpA from H. parasuis serovar 5, the Nagasaki strain, was amplified by PCR and cloned into a pBAD/Thio-TOPO expression vector, generating the pBAD-Thio-TbpA-V5-His (TbpA-His) construction. Escherichia coli LMG194-competent cells were transformed with this construction, followed by the induction of protein expression with arabinose. A band (38.5 kDa) corresponding to a 200-amino acid recombinant TbpA (rTbpA) fragment was seen on the sodium dodecyl sulfate polyacrylamide gel electrophoresis and confirmed by immunoblotting. Polyclonal antibodies raised against this fragment were specific for H. parasuis and Actinobacillus pleuropneumoniae, reacted at the cell surface with H. parasuis, and a significant bactericidal activity was also detected. Therefore, this rTbpA fragment induces an immunological response and might be useful as an antigen for vaccination against Glässers disease.

Epidemic infection caused by Citrobacter rodentium in a gerbil colony

Non-motile, Gram-negative rods, isolated from the intestinal tract and kidney of several dead animals in a gerbil colony, were identified as Citrobacterrodentium (formerly included in C freundii species) on the basis of 31 biochemical tests. The isolates were tested against 40 antimicrobial agents and were all susceptible to ticarcillin plus clavulanate, ceftazidime and most of the quinolones studied, but were all resistant to most of the penicillins and aminoglycosides tested, and to fosfomycin, metronidazole and tiamulin. This bacterial species has been primarily associated with transmissible murine colonic hyperplasia, and this appears to be the first report of an epidemic infection in a gerbil colony with a fatal outcome in most of the animals affected.

Evaluation of efficacy of several disinfectants against Campylobacter jejuni strains by a suspension test

The objective of this study was to determine the efficacy of 16 active compounds and 11 commercial disinfectants against Campylobacter jejuni. Two reference strains (one of avian origin and the other isolated from bovine) and two avian field strains were tested in suspension test in the presence and absence of serum. Chloramine-T, povidone-iodine (1% available iodine), cetylpiridinium chloride, ethanol, isopropanol, chlorhexidine digluconate, formaldehyde, phenol, and 10 of the 11 commercial formulations (eight of them based on quaternary ammonium compounds) showed an excellent disinfectant capability, resulting in the highest level of reduction (>6-log(10)) in colony-forming units of the four C. jejuni strains compared regardless of the presence or absence of organic material. These compounds might be helpful in the adoption of environmental control measures against C. jejuni.