Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis.

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6% concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8% (134/137) and 94.9% (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95% and 100% with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Immune response of heifers against a Staphylococcus aureus CP5 whole cell vaccine formulated with ISCOMATRIX™ adjuvant.

The shortcomings of Staphylococcus aureus vaccines to control bovine mastitis have been attributed to insufficient capacity of the vaccines to induce opsonizing antibodies and to stimulate cellular immune responses. Types of antigen, administration route and adjuvant used in a vaccine formulation have been identified as critical factors for the development of opsonic antibodies. Current commercially available vaccines for Staph. aureus bovine mastitis control are formulated with Al(OH)3 and oil-based adjuvants. The aim of this study was to evaluate the immune response of heifers immunized with a Staph. aureus CP5 whole cell vaccine formulated either with Al(OH)3 or ISCOMATRIX™. Twenty primigravid Holstein dairy heifers in the last trimester of gestation were immunized either with a vaccine formulated with ISCOMATRIX™ (n = 6), Al(OH)3 (n = 7), or saline solution (placebo) (n = 7). Immunization was carried out 38 and 10 d before calving. Heifers vaccinated with Staph. aureus adjuvanted with ISCOMATRIX™ responded with significantly higher levels of anti-bacterin and anti-CP5 IgG and IgG2 in sera than animals in the Al(OH)3 or control groups. Animals in the ISCOMATRIX™ group responded with significantly higher anti-bacterin specific IgG in whey than animals in the Al(OH)3 and control groups, detected from the first week post calving until 60 d of lactation. Sera from animals inoculated with Staph. aureus in ISCOMATRIX™, obtained 7 d post partum, significantly increased both the number of neutrophils ingesting bacteria and the number of bacteria being ingested by the neutrophils, compared with sera obtained from heifers vaccinated with Al(OH)3 or non-vaccinated controls. These features coupled to safety of the ISCOMATRIX™ formulation, warrant additional studies.

Activity-Structure Study on the Peptide Fraction of AG2: a Potent In Vitro Transfection Agent.

Gemini-based amphiphiles are candidates for biomedical applications. In fact, most of the gemini compounds described in the literature have been prepared to be used as new synthetic vectors in gene transfection. Our group carried out an activity-structure study starting from the structure of the gemini [AG2-C18/]2, which is an effective in vitro transfection reagent. We synthesized a series of novel amphiphilic amino acid derivatives of low molecular weight, named AGn-Cm (N), in which the same apolar region (m) of oleic or palmitic acid was maintained and the peptide region was modified by amino acid insertions, deletions, and substitutions. We also determined the transfection efficiency, critical micelle concentration, particle size, and ζ-potential for these derivatives. Amphiphiles AG10-C16 and AG10-C18 were more active at a lower N/P ratio than AG2-C18. These amphiphiles showed no activity when lysine was replaced by ornithine, and the activity of all derivatives increased when there were more ornithine residues and a W/O = 1 ratio in the peptide region. It can be said that for AG10-C16, these two structural requirements on the amino acid portion predominated over the type of aliphatic chain used.

Effect of two synthetic peptides mimicking conserved regions of equine infectious anemia virus proteins gp90 and gp45 upon cytokine mRNA expression.

Gp90 and gp45 synthetic peptides, which mimic conserved sequences of native viral proteins, are recognized by antibodies to equine infectious anemia virus (EIAV) in asymptomatic carrier horses and generate humoral and cellular responses in immunized mice. Cytokine mRNA levels were evaluated in equine peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with gp90 and gp45 with the aim of determining the cytokine profile associated with the proliferative response. Stimulation index (SI) values indicate that 100 and 60% of EIAV-infected horses recognized gp90 and gp45, respectively. A strong positive correlation was found between IL-12p40 and SI, IFN-gamma and SI, and IL-12p40 and IFN-gamma (p < 0.001). These results suggest the presence of specific memory cells that would contribute to maintain reinfection resistance and that conserved viral regions represented by gp90 and gp45 synthetic peptides may be good candidates for inclusion in vaccine strategies against EIAV.

Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant.

Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)3. However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine.

Genotipificación y estudio de los genes pauA y sua de aislamientos de Streptococcus uberis de mastitis bovina / Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6 % concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8 % (134/137) and 94.9 % (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95 % and 100 % with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Este estudio pretendió determinar la relación clonal entre 137 aislamientos de S. uberis obtenidos de leche de bovinos con mastitis clínica o subclínica en la Argentina, como así también la prevalencia y la conservación de los genes sua y PauA entre dichos aislamientos. Esta información es crítica para el diseño racional de una vacuna que prevenga la mastitis bovina por S. uberis. Los aislamientos se tipificaron molecularmente por amplificación al azar del ADN polimórfico (RAPD) y mediante electroforesis de campos pulsados (PFGE). Los 137 aislamientos mostraron 61 pulsotipos mediante PFGE y 25 tipos de RAPD diferentes. Los índices de Simpson calculados fueron 0,983 por PFGE y 0,941 por RAPD; esto evidencia el elevado poder discriminatorio de ambas técnicas. El análisis de la relación entre pares de aislamientos mostró un 92,6 % de concordancia entre ambas técnicas, lo que indica que cualquier par de aislamientos que fue distinguido por un método tendió a ser distinguido por el otro. La prevalencia de los genes sua y puaA fue del 97,8 % (134/137) y 94,9 % (130/137), respectivamente. Las secuencias de nucleótidos y de aminoácidos codificados por los genes sua y pauA de los 20 aislamientos de S. uberis seleccionados sobre la base de su tipo de PFGE y RAPD y origen geográfico tuvieron un porcentaje de identidad de entre 95 % y 100 % con respecto a todas las secuencias de referencia registradas en GenBank. Estos resultados demuestran que, a pesar de la diversidad clonal de S. uberis, los genes sua y pauA son prevalentes y están altamente conservados y deberían ser incluidos en futuros estudios de vacunas para prevenir mastitis bovina causada por S. uberis.

Expression of HLA-G and MICA mRNA in renal allograft.

HLA-G is a nonclassical MHC class I antigen that displays tolerogenic functions; MICA is a stress-regulated molecule recognized by NKG2D cytotoxicity-activating receptor expressed by NK and T cells subsets. We evaluated HLA-G isoforms and MICA mRNA levels in peripheral blood mononuclear cells (PBMCs) and in biopsies from kidney allograft recipients with acute rejection (AR), chronic rejection (CR), and stable graft evolution (SE). HLA-G1 was the only transcript resulted from amplification, both in PBMCs as in biopsy samples. HLA-G1 mRNA levels in PBMCs from 9/10 patients with CR, 7/9 with AR and 8/10 healthy volunteers were below the median value of SE patients. The analysis of biopsies revealed that patients with AR (n=6), who overcame rejection had a tendency towards higher HLA-G1 levels than those with nephrotoxic acute tubular necrosis (ATN) (n=3). Similar levels of MICA expression were observed in PBMCs from AR, CR, SE and C groups; MICA expression levels were similar also in biopsy specimens from AR and nephrotoxic ATN patients. No correlation was found between MICA expression and the graft state. These preliminary results suggest that HLA-G1 isoforms, but not MICA mRNA levels, may provide a marker for measuring the state of kidney allograft, and be the basis for further studies that may establish the influence of these molecules in renal allograft rejection or acceptance.