Performance and applications of polyclonal antipeptide antibodies specific for the enterococcal bacteriocin enterocin P

Polyclonal antibodies with specificity for enterocin P (EntP) have been generated by immunization of rabbits with two chemically synthesized N-terminal peptides (P1 and P2) and a C-terminal peptide (P3) of this bacteriocin conjugated to the carrier protein KLH. The sensitivity and specificity of the peptide-KLH-generated antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect (CI)-ELISA. The NCI-ELISA but not the CI-ELISA was valuable for detecting the existence of EntP specific antibodies in the sera of the P2-KLH and P3-KLH immunized animals and to detect and quantify the EntP in the supernatant of producer strains. The anti-P2-KLH sera cross-reacted with the supernatant of a strain producer of sakacin A, a bacteriocin closely related to EntP. Immunoaffinity chromatography columns with anti-P2-KLH or anti-P3-KLH immunoglobulins retained the EntP from the supernatant of the producer strain. Western blotting of EntP with the anti-P2-KLH-generated antibodies suggests that purified EntP tends to the formation of aggregates with no antimicrobial activity. Monitoring the purification of EntP with antipeptide antibodies suggests that while the performance of the evaluated purification procedures would be reasonably acceptable in terms of recovery of the antimicrobial activity of the bacteriocin, their yield is far from attractive in terms of recovery of the initial concentration of enterocin P.     

Phylogeography and conservation perspectives of an endangered macaronesian endemic: Picconia azorica (Tutin) Knobl. (Oleaceae)

The Azores provide an excellent field test for research activities aimed at developing conservation strategies for endangered tree species. In this work, the urgency to promote Picconia azorica conservation programs addressed (a) insights into the biotaxonomy of the species (including an evaluation of its origin and relationships with the only congeneric species of P. azorica: P. excelsa from the Canary and Madeira islands) and (b) the evaluation of P. azorica genetic diversity. Plastid DNA sequence analysis and molecular markers (RFLP and SSR) were used for this purpose. Phylogenetic data suggest the monophyly of Picconia and support a late Miocene divergence of the two species. Three polymorphic cpSSR loci allowed the identification of five different haplotypes in P. azorica. Uniqueness and relictuality of lineages are presented and discussed. Picconia azorica intra-specific diversity patterns revealed low genetic diversity and a weak genetic structure, which could result from long-lasting ecological stability and efficient inter-island seed movement that have been severely affected in recent times. The species survival is at risk, and we suggest management practices focusing on ex situ and in situ conservation units based on eco-genetic data. Additional measures contributing to mild erosion of the genepool and to remove barriers to seed dispersal are indicated.     

Respiration Index Determination: A Comparative Study of Different Methods

Three respirometric methods were tested using 18 organic matrices. Two methods were performed in the solid state condition using static (SRI) and dynamic approaches (DRI) while the third was performed in the liquid state (SOUR). The results obtained showed that all methods were able to describe biological stability and were well correlated. SOUR gave results that differed from DRI and SRI, which, in turn, showed good agreement. SOUR showed a good correlation with dissolved organic carbon, above all with the hydrophilic fraction, indicating that water media was responsible for the high availability of soluble organic material during initial hours of the test. The use of SOUR cumulative data (OD₁₂ and OD₂₀) reduced the effect of the soluble fraction on the measurement, thereby increasing the correlation coefficient with the solid state methods.     

Tight junction proteins in the canine epidermis: A pilot study on their distribution in normal and in high IgE-producing canines

Epidermal tight junctions (TJ) have been well-described in human medicine and are involved in many skin diseases such as atopic dermatitis (AD). In dogs, there are no data regarding the implication of TJ in skin diseases including canine AD.The aim of this study was to compare the expression and the distribution of ZO-1, occludin and claudin-1 in the epidermis of healthy and atopic dogs.Skin biopsies from 6 high IgE-producing beagles sensitized to house dust mite (atopic group) were used. Skin specimens from nine healthy dogs without skin issues were sampled (healthy group).Immunoperoxydase staining was used to study the staining pattern of zonula occludens-1 (ZO-1), occludin and claudin-1 in the epidermis of healthy and atopic dogs. Positive controls were healthy human skin samples.Labeling patterns were assessed by 2 examiners blinded to the identities of the specimens. Comparisons between groups were performed using an exact Wilcoxon-Mann-Whitney test.The mean total expression score of claudin-1 was lower in atopic dogs as compared to healthy subjects. Occludin and ZO-1 expression remained unchanged within each group.These results suggest a defect in claudin-1 expression in the nonlesional epidermis of atopic dogs.     

Performance characteristics of a monoclonal antibody cocktail-based ELISA for detection of allergen-specific IgE in dogs and comparison with a high affinity IgE receptor-based ELISA

The purpose of this study was to define the operational and performance characteristics of a commercially available monoclonal antibody based (mac) ELISA for detection of allergen-specific IgE in dogs. The average intra-assay variance over 1 year was 9.7% (range 2.5–62.7%), while the interassay variance averaged 10.8% (range 8.1–13.8%). The average positive control responses observed for grass, weed, tree and mite allergens during each month remained relatively constant; the average monthly variance was 11.6% (range 8.3–19.2%) for grass pollens, 13.3% (range 9.1–20.4%) for weed pollens, 13.3% (range 9.8–18.2%) for tree pollens and 13.6% (range 8.9–18.7%) for mite allergens. The interlaboratory concordance of results for the macELISA was approximately 91%. The interlaboratory concordance of results comparing the macELISA and a high affinity IgE receptor-based ELISA was approximately 92%. The results demonstrate that the macELISA is reproducible and the results are comparable to the high affinity IgE receptor based ELISA within and between laboratories.     

Bacteriological investigation on “Mauro” sold in Catania

Seaweeds known as “Mauro” are traditionally used fresh or to prepare omelettes in Sicily (Italy). Twenty samples sold in Catania between May 2005 and September 2007 were analyzed for Escherichia coli, Total Enterobacteria, Aeromonas spp., Pseudomonas spp., Vibrio spp., and Salmonella spp. Thirty Vibrio strains were examined for the presence of the virulence genes, toxR, toxRS, tl, tdh, and trh in the genomes of the isolates. Total Enterobacteria ranged between 2.23 and 6.85 log CFU/g, and in six samples, E. coli ranged between 0.70 and 2.74 log CFU/g. Aeromonas spp. was present in samples between 1.0 and 5.90 log CFU/g, while Pseudomonas spp. ranged between 2.70 and 7.27 log CFU/g. Vibrio spp. was present in 75% of samples at values between 1.30 and 4.60 log CFU/g. The most frequently isolated species was V. alginolyticus (76.66% of isolates), of which 82.60% were positive for toxR and the remaining 17.40% of strains were positive for toxRS. V. parahaemolyticus was identified in 13.33% of strains; all were positive for toxR and, in one case, for both toxR and toxRS. V. coralliitycus was isolated in 6.66% of strains (all positive for toxR), and one was identified as V. mimicus (positive for toxRS). The results of this study suggest that there is need for stringent quality control during harvesting and distribution of Mauro.