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B Amorena

Publications and source records attributed to B Amorena.

At least 19 recordsLinked to original sources

PCR detection of colostrum-associated Maedi-Visna virus (MVV) infection and relationship with ELISA-antibody status in lambs.

A recent large-scale experimental study showed that bottle-feeding ovine colostrum from seropositive ewes results in high MVV-seroconversion in lambs. In contrast, relatively few lambs that naturally suckled colostrum from seropositive dams seroconverted as a result of it. Furthermore, lambs fed uninfected bovine colostrum readily seroconverted when mixed with ovine-colostrum lambs indicating that horizontal MVV transmission between lambs was efficient. MVV-infection was further investigated in the same samples using two PCR tests targeting sequences in the long-terminal repeats (LTR) and POL MVV genes. PCR-tests confirmed previous serological findings. However, the LTR-PCR was more sensitive and allowed detecting infection earlier than the other tests, including 5-8% of new-born lambs from seropositive dams, providing more evidence that prenatal MVV-infection may be more important than considered. The degree of agreement between PCR and antibody tests in individual samples was low up to 6 months of age and moderate at 10 months-old. Nine percent of lambs were always PCR-negative but seroconverted and 19% of lambs were PCR-positive at least once and did not seroconvert. However, seroconversion was associated with increasing number of times lambs were PCR-positive and ovine colostrum-fed lambs were more frequently PCR-positive than other lambs. The significance of these findings in terms of MVV-infection, epidemiology and control is discussed.

Aging↗

Horizontal Maedi-Visna virus (MVV) infection in adult dairy-sheep raised under varying MVV-infection pressures investigated by ELISA and PCR.

A three year long experimental study was carried out to investigate horizontal MVV-infection by PCR and ELISA, in 191 one year-old latxa dairy-sheep raised in two separate groups under low and high MVV-infection pressure, respectively. Sheep originated from a previous MVV-transmission study in lambs and seroprevalence among one year-old sheep in both groups was 15% approximately. The high infection-pressure group (H-group) consisted of 147 replacement ewes that joined a milk-producing, housed dairy-flock with 42-66% MVV-seroprevalence and the low infection-pressure group (L-group) were castrated males raised in a separate shed. In contrast to results obtained when infection was investigated in lambs, the overall degree of agreement between ELISA and PCR results was very good and there was some indication that it increased further as sheep became older. MVV-prevalence did not change in the L-group and increased to 57% in three year-old sheep in the H-group (p<0.001). Random effects logistic regression confirmed seroconversion was significantly higher in the H-group compared to the L-group and was highest during the year after the sheep were introduced in the dairy flock and did not increase with age as in previous studies using less sensitive antibody assays. The evidence that horizontal transmission can be very low in spite of prolonged close contact between infected and non-infected sheep is valuable for MVV-control purposes. Furthermore it highlights the need to investigate virus excretion dynamics in infected animals and animal to animal transmission to improve our overall understanding of horizontal MVV transmission in MVV endemic populations.

Aging↗

Relative contribution of colostrum from Maedi-Visna virus (MVV) infected ewes to MVV-seroprevalence in lambs.

Maedi-visna virus (MVV) seroprevalence associated with consumption of colostrum from seropositive ewes was investigated in 276 housed lambs from birth to 300 days-old. At birth, lambs were allocated to five experimental groups according to the maternal MVV-serological status, source and mode of feeding colostrum (bovine or ovine and bottle fed or suckled from the dam) and type of horizontal MVV-exposure (raised with the dam or separately with other lambs). The risk of being seropositive at 300 days-old was associated with feeding ovine colostrum from seropositive ewes and increased with intake of bottle-fed ovine colostrum and was higher in lambs separated from their dams and raised with other experimental lambs compared to lambs raised with their dams. Approximately 75-87% of ELISA-positive results in lambs that had ovine colostrum was attributable to colostrum itself. However, approximately only 16% of naturally raised and 29-61% of bottle-fed ovine colostrum lambs were ELISA-positive as a result feeding ovine colostrum. These results confirm that ovine colostrum from seropositive ewes can be a major source of MVV but its overall contribution to seroprevalence in natural conditions is relatively low, and shows that horizontal MVV transmission can be an important source of infection in new-born lambs.

Aging↗

Visna/maedi virus Env protein expressed by a vaccinia virus recombinant induces cell-to-cell fusion in cells of different origins in the apparent absence of Env cleavage: role of glycosylation and of proteoglycans.

The in vivo productive infection by the ovine Visna/maedi lentivirus (VISNA) is restricted to cells of the monocyte/macrophage lineage. The basis for this restriction is not understood. Although the VISNA envelope (Env) glycoprotein is the main target for virus neutralization, studies on the role of this protein in virus infection are limited. A vaccinia virus recombinant (VV- env-MV) containing the entire VISNA env sequence was generated and shown to produce in infected cells a protein of about 165 kDa (referred to as gp150). During VV- env-MV infection, expression of env caused extensive cell-to-cell fusion in cell lines of different origins. Pulse-chase and Western blot analyses revealed that gp150 is not cleaved in VV- env-MV infected cells. The glycoprotein gp150 formed oligomers held by disulfide bonding. Cell-to-cell fusion was prevented in the presence of the inhibitor of glycosilation, tunicamycin, but it was markedly enhanced by an inhibitor of proteoglycan synthesis, beta-D-xyloside. These findings showed that the receptor for VISNA Env is widely distributed within cells, that fusion-from-within of cells can occur in the apparent absence of proteolytic cleavage of gp150, and that fusion require a glycosylated Env but not the addition of proteoglycan chains at the cell surface. This recombinant virus could have utility as a potential vaccine against VISNA.

3T3 Cells↗

Immunoprecipitation and isoelectric focusing of sheep MHC class I antigens reveal higher complexity than serology.

Biochemical and serological methods were used to characterize sheep MHC class I polymorphism at the product level. The cells of 65 selected animals were subjected to immunoprecipitation and one-dimensional isoelectric focusing (IEF) with two cross-reacting monoclonal antibodies, B1.1G6 and HC-10. We were able to define up to 18 distinct haplotypes in the Latxa breed sample. Most of the locally defined serological specificities were confirmed or subdivided by biochemical typing. As IEF detected antigens not yet defined by serology, it provided us with a guide to producing more specific antisera by appropriate immunizations. Lastly, evidence of expression of two class I loci products was found.

Animals↗

Effect of slime on adherence of Staphylococcus aureus isolated from bovine and ovine mastitis.

The interactions between slime, Staphylococcus aureus and ovine mammary gland epithelial cells (MGEC) were studied in vitro. Suspensions of radiolabelled bacteria incubated with slime significantly increased the ability of S. aureus strains to adhere to a filter. When suspensions of radiolabelled bacteria were incubated with MGEC treated with trypsin, the ability of slime to improve S. aureus adherence was also shown, indicating that it was not dependent on cell membrane proteins. The interaction of radiolabelled bacteria with slime prior to the adherence test with MGEC demonstrated that the adherence process requires the interaction between slime and bacteria. This interaction is inhibited by anti-slime antibodies. This study provides evidence that a specific interaction between bacteria coated with slime and MGEC could be a critical part of mammary gland infection.

Animals↗

A simple infection model using pre-colonized implants to reproduce rat chronic Staphylococcus aureus osteomyelitis and study antibiotic treatment.

Staphylococcus aureus biofilms formed on medical implants represent a serious problem, being difficult to eradicate with antibiotic therapy and leading to chronic infections. Simplified in vivo and in vitro antibiotic susceptibility assays using biofilm bacteria are needed. In this work, a novel chronic osteomyelitis infection model was developed in rats in the absence of bacterial suspension, requiring the use of only 10(6) bacteria in biofilms at the site of surgery, with a full success in reproducing infection. Stainless-steel implants pre-colonized for 12 h with a highly adherent S. aureaus isolate were introduced into the rat tibiae. In animals not submitted to antibiotic treatment, infection was found in the implants and spread to bone in all cases, indicating the high efficacy of the model to reproduce osteomyelitis. The effect of a 21-day treatment with cefuroxime, vancomycin, tobramycin or ciprofloxacin on infection was studied in this model 42 days after surgery. Bone colonization was inhibited by vancomycin and cefuroxime. Cefuroxime (the most efficient antibiotic, able to sterilize 1 out of 8 implants) reduced the number of bacteria in biofilms adhered to implants at a higher extent than vancomycin, trobramycin and ciprofloxacin. Analogous observations were made in this work in vivo and in vitro on the relative antibiotic efficacy against S. aureus biofilm bacteria. suggesting the usefulness of both tests as a potential tool to study antibiotic suceptibility, and the need for new antimicrobials against these bacteria.

Animals↗

Synergy of different antibiotic combinations in biofilms of Staphylococcus epidermidis.

The in vitro effect of nine antibiotic combinations was investigated in Staphylococcus epidermidis biofilms using ATP-bioluminescence for viable bacterial cell quantification. Four slime-producing (SP) strains were used to form biofilms 6, 24 and 48 h old. These biofilms were exposed for 24 h to antibiotics at 4 x, 2 x, 1 x and 0.5 x MIC. Combinations involving tetracycline together with another antibiotic were the most efficient at the biofilm age and concentration range under study. The combination vancomycin-rifampicin produced the highest bactericidal effect on 6 h biofilms at 4 x MIC, but this effect decreased dramatically in older biofilms. To detect possible antibiotic synergy in combinations that had a significant killing effect, antibiotics were studied not only in combination but also individually. Synergic effects were observed in all the combinations tested. Differences between the effect in combination and the sum of individual antibiotic effects (degree of synergy) were significant (mostly P< 0.001) and exceeded 1 log10 cfu/mL in the majority of cases. In 48 h biofilms, antibiotics caused a significant bactericidal effect when applied in combination, but never when used individually. These results indicate that the biofilm test applied allows the detection of synergy between antibiotics and suggests that this assay could be useful in clinical and extensive synergy studies on S. epidermidis biofilms.

Anti-Bacterial Agents↗

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Bacterial Adhesion↗

Bap, a Staphylococcus aureus surface protein involved in biofilm formation.

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.

Adhesins, Bacterial↗

Early detection of maedi-visna (ovine progressive pneumonia) virus seroconversion in field sheep samples.

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples (n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (K value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animal's life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.

Animals↗

Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro.

Four slime-producing isolates of Staphylococcus aureus were used in an antibiotic susceptibility assay for biofilms developed on 96-well polystyrene tissue culture plates. The study involved 11 antibiotics, two biofilm ages (6 and 48 h), two biofilm growth media (tryptone soy broth (TSB) and delipidated milk) and three antibiotic concentrations (4 x MBC, 100 mg/L and 500 mg/L). ATP-bioluminescence was used for automated bacterial viability determination after a 24 h exposure to antibiotics, to avoid biofilm handling. Under the conditions applied, viability in untreated biofilms (controls) was lower when biofilm growth was attempted in milk rather than in TSB. Various antibiotics had a greater effect on viability when used at higher (> or =100 mg/L) antibiotic concentrations and on younger (6 h) biofilms. Increased antibiotic effect was observed in milk-grown rather than TSB-grown biofilms. Phosphomycin and cefuroxime, followed by rifampicin, cefazolin, novobiocin, vancomycin, penicillin, ciprofloxacin and tobramycin significantly affected biofilm cell viability at least under some of the conditions tested. Gentamicin and erythromycin had a non-significant effect on cell viability. Transmission electron microscopy revealed that cells at the inner biofilm layers tend to remain intact after antibiotic treatment and that TSB-grown biofilms favoured a uniformity of cell distribution and increased cell density in comparison with milk-grown biofilms. A reduced matrix distribution and enhanced cell density were observed as the biofilm aged. The S. aureus biofilm test discriminated antibiotics requiring shorter (3 h or 6 h) from those requiring longer (24 h) exposure and yielded results which may be complementary to those obtained by conventional tests.

Adenosine Triphosphate↗

A new sensitive serological assay for detection of lentivirus infections in small ruminants.

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99. 8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.

Amino Acid Sequence↗

Application of a rat osteomyelitis model to compare in vivo and in vitro the antibiotic efficacy against bacteria with high capacity to form biofilms.

A rat experimental osteomyelitis model was used to study the efficiency of antibiotics on biofilm bacteria adhered to implants in relation to the efficiency obtained in vitro. In the osteomyelitis model, 10(4) bacteria of the strain variant used for the in vitro studies (a slime-producing variant of Staphylococcus aureus) were inoculated into the rat tibia at surgery, after implanting a stainless steel canula precolonized for 12 h with this strain. After 5 weeks, a 21-day antibiotic treatment was applied (using cefuroxime, vancomycin, or tobramycin). Subsequently, implant and tibia were studied for presence of bacteria. In this osteomyelitis model, cefuroxime inhibited bone colonization and reduced the number of bacteria in metal and bone at a higher degree (P < 0.05) than vancomycin and trobramycin (the latter antibiotic did not have this reduction effect). The in vitro assay was applied using three concentrations of each antibiotic (8, 100, and 500 microg/ml) and 6-, 24-, and 48-h biofilms. Bacterial viability was evaluated by ATP-bioluminescence after 24 h of antibiotic treatment. In this in vitro assay, cefuroxime significantly (P < 0.05) reduced in all cases the number of viable bacteria in biofilms, tobramycin did not affect viability, and vancomycin affected viability except at the lowest concentration used (8 microg/ml, i.e., 8x the minimal bactericidal concentration of this antibiotic) when facing the oldest (48 h) biofilm. These results demonstrate the usefulness of the osteomyelitis model applied in providing evidence for a close correlation between the in vitro and in vivo findings on the effect of three antibiotics under study.

Animals↗

Effect of in vitro maedi-visna virus infection on adherence and phagocytosis of staphylococci by ovine cells.

This work was aimed at studying the effect of maedi-visna virus (MVV) infection in vitro on the ability of sheep cells to adhere to staphylococci (Staphylococcus aureus and Staphylococcus epidermidis), and phagocytose these bacteria. Adherence was studied in sheep choroid plexus cells (SCPC) using an ELISA test and phagocytosis was studied in pulmonary alveolar macrophages (PAM) by chemiluminescence. A 5- and 7-day of in vitro MVV infection resulted in syncytium formation and a significant increased adherence (P < 0.01) of SCPC to bacteria. SCPC endogenous fibronectin was significantly higher (P < 0.01) on days 5 and 7 than on day 0 of MVV infection. A significantly decreased phagocytosis (P < 0.05) was also observed on days 5 and 7 of MVV infection in PAM when compared to MVV-free controls. Comparatively, phagocytosis was highest for S. aureus non-slime producing strains, followed by S. epidermidis, and S. aureus slime producing strains, in that order. Finally, increased expression of both, class I and class II major histocompatibility antigens was also observed in MVV-infected PAM on days 5 and 7, whereas SCPC only demonstrated upregulation of MHC class I. These results, indicative of an alteration of some cell functions in MVV-infected cells, may help to understand interactions between MVV-infected cells and bacteria in simultaneous infections and may provide clues to the possible in vivo interactions of both pathogens.

Animals↗

Adherence of Staphylococcus aureus slime-producing strain variants to biomaterials used in orthopaedic surgery.

The adherence of Staphylococcus aureus to biomaterials used in orthopaedic surgery (polymethylmethacrylate, fresh bone, steel and titanium alloys) and to glass was studied in vitro at 1, 2, 6, 24 and 48 h of incubation. Nonslime-producing strains (72, 80 and 510) and slime-producing variants of these strains were used. An automated and fast method of ATP-bioluminiscence was applied to determine bacterial viability. The lowest adherence corresponded to polymethylmethacrylate and bone, and the highest to metals. Significant adherence was detected in all cases after 6 h and was strain dependent, being lowest for strain 72. In most cases, adherence of nonslime-producing variants was not significant compared with controls, and slime-producing were more adherent than nonslime-producing variants. These differences were maximal at 6 h or 48 h, depending on the strain and the material. The findings suggest that the appearance of slime-producing cells within a given nonslime-producing bacterial population may jeopardise postoperative immune systems and antibiotic efficacy as a consequence of biofilm formation on implants and prostheses.

Analysis of Variance↗

Use of liposome-immunopotentiated exopolysaccharide as a component of an ovine mastitis staphylococcal vaccine.

Experiments on the development of a vaccine against staphylococcal mastitis were carried out in ewes. The vaccine (Spanish patent no. 9200223) has the following components: (i) inactivated (formalinized) bacteria (Staphylococcus aureus and a coagulase-negative staphylococcal species. Staphylococcus simulans) and S. aureus toxoid in presence of an adjuvant (dextran sulfate, Mw 500,000); and (ii) S. aureus exopolysaccharide included within liposomes. High serum antibody titres were obtained against whole cells from Staphylococcus aureus, Staphylococcus simulans, Staphylococcus hyicus and Staphylococcus epidermidis strains. However, there was no response to cells from Staphylococcus warneri and Staphylococcus chromogenes strains. An immune response (serum IgG) against the inoculated exopolysaccharide was obtained when > or = 20 micrograms of exopolysaccharide were included in liposomes and when > or = 20 mg of exopolysaccharide were adjuvanted with dextran sulfate instead of liposomes. For experimental infection assays, ewes were vaccinated during pregnancy and challenged either with a low virulence S. simulans strain or with a highly virulent S. aureus strain. In these assays, the incidence of S. simulans subclinical mastitis and of S. aureus acute mastitis was significantly lower in vaccinated animals than in unvaccinated controls. Specifically, on challenge with S. simulans, two out of 14 glands became infected among the vaccinated animals and nine out of ten glands in the unvaccinated group (p < 0.001). On challenge with S. aureus, no protection was detected when component (ii) was omitted from the vaccine; nine out of ten animals developed mastitis (two mild, two moderate and five severe).(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗

Staphylococcus aureus capsule and slime as virulence factors in ruminant mastitis. A review.

Staphylococcus aureus is one of the most prevalent causes of ruminant mastitis. The interaction of this microorganism with the host is strongly dependent on its cell surface properties, specially concerning the presence of the exopolysaccharide-containing outer layers (glycocalyx), which appear to play an important role in virulence. In this article, the definition and recognition of the types of exopolysaccharide layers are described, together with their likely role in the pathogenesis of mastitis.

Animals↗