A total of 53 farms were selected in Southern dairy region of Uruguay, in order to estimate the prevalence of subclinical mastitis, the incidence of clinical mastitis, distribution and antimicrobial susceptibility of mastitis pathogens. The prevalence of sub-clinical mastitis was determined in base on somatic cell counts in quarter’s milk samples and a threshold value of > 300,000 cells/ mL.; 9,016 milk quarters samples were collected from 2,254 dairy cows and the mean prevalence at herd level was 54.2% of affected cows. The main pathogen isolated from positive samples was Staphylococcus aureus (21.4%). During one with a mean incidence rate of clinical mastitis of 11.8 cases per 100 cow/year at risk. Milk samples were obtained from 341 clinical cases for bacteriological isolation, the most prevalent pathogen isolated was Staphylococcus aureus (27.8%). A total of 864 strains belonging to staphylococci, streptococci and enterococci isolated from clinical and subclinical mastitis cases were analyzed for their susceptibility to antimicrobial agents. The susceptibility patterns were studied by Agar disk diffusion method. The 39.1% of Staphylococcus aureus strains from subclinical cases and 36% from clinical cases were resistant to penicillin, and 29.4% and 33.3% of coagulase negative staphylococci strains, respectively. All the streptococcal isolates were susceptible to penicillin, while 12.5% of enterococci strains were resistant. In conclusion, Uruguay maintain a high level of sub clinical infection in the dairy herds, and Staphylococcus aureus is confirmed as the principal pathogen with a high percentage of penicillin resistance.

In this study we report the isolation and characterization of a heat shock protein 70 ( hsp70 ) gene, the hsp83 gene and two genes that encode small Hsps ( Lchsp23 and Lchsp24 ) from the Australian sheep blowfly, Lucilia cuprina , a major agricultural pest. Phylogenetic analyses indicate that the LcHsp23 protein is the orthologue of Drosophila melanogaster Hsp23 and LcHsp24 is the orthologue of Sarcophaga crassipalpis Hsp23. Quantitative reverse‐transcriptase PCR analysis showed that the basal level of Lchsp83 RNA is relatively high at all developmental stages and only moderately induced by heat shock. In contrast, Lchsp70 transcripts are present at low levels and strongly induced by heat shock at all stages. The basal levels of expression and degrees of heat induction of the Lchsp23 and Lchsp24 transcripts were more variable across the different developmental stages. Putative heat shock factor binding sites were identified in the Lchsp24 , Lchsp70 and Lchsp83 gene promoters. The isolation of these hsp gene promoters will facilitate constitutive or conditional expression of a gene of interest in transgenic Lucilia .

d-Lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of d-lactic acid and measured intracellular pH using 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that d-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100nM platelet-activating factor (PAF). d-Lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease l-selectin expression, and induces late apoptosis. We concluded that d-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10mM d-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis.

The All-Distances SVM is a single-objective light extension of the binary μ-SVM for multi-category classification that is competitive against multi-objective SVMs, such as One-against-the-Rest SVMs and One-against-One SVMs. Although the model takes into account considerably less constraints than previous formulations, it lacks of an efficient training algorithm, making its use with medium and large problems impracticable. In this paper, a Sequential Minimal Optimization-like algorithm is proposed to train the All-Distances SVM, making large problems abordable. Experimental results with public benchmark data are presented to show the performance of the AD-SVM trained with this algorithm against other single-objective multi-category SVMs.

The margin maximization principle implemented by binary Support Vector Machines (SVMs) has been shown to be equivalent to find the hyperplane equidistant to the closest points belonging to the convex hulls that enclose each class of examples. In this paper, we propose an extension of SVMs for multicategory classification which generalizes this geometric formulation. The obtained method preserves the form and complexity of the binary case, optimizing a single convex quadratic program where each new class introduces just one additional constraint. Reduced convex hulls and non-linear kernels, used in the binary case to deal with the non-linearly separable case, can be also implemented by our algorithm to obtain additional flexibility. Experimental results in well known datasets are presented, comparing our method with two widely used multicategory SVMs extensions.

In this paper, we study a single objective extension of support vector machines for multicategory classification. Extending the dual formulation of binary SVMs, the algorithm looks for minimizing the sum of all the pairwise distances among a set of prototypes, each one constrained to one of the convex-hulls enclosing a class of examples. The final discriminant system is built looking for an appropriate reference point in the feature space. The obtained method preserves the form and complexity of the binary case, optimizing just one convex objective function with m variables and 2m+K constraints, where m is the number of examples and K the number of classes. Non-linear extension are straightforward using kernels while soft margin versions can be obtained by using reduced convex hulls. Experimental results in well-known UCI benchmarks are presented, comparing the accuracy and efficiency of the proposed approach with other state-of-the-art methods.

We reported recently that the inhibition of cysteine-proteases with E-64-d disturbs DNA replication and prevents mitosis of the early sea urchin embryo. Since E-64-d is a rather general inhibitor of thiol-proteases, to specifically target the cysteine-protease previously identified in our laboratory as the enzyme involved in male chromatin remodeling after fertilization, we injected antibodies against the N-terminal sequence of this protease that were able to inhibit the activity of this enzyme in vitro. We found that injection of these antibodies disrupts the initial zygotic cell cycle. As shown in this report in injected zygotes a severe inhibition of DNA replication was observed, the mitotic spindle was not correctly bipolarized the embryonic development was aborted at the initial cleavage division. Consequently, the injection of these antibodies mimics perfectly the effects previously described for E-64-d, indicating that the effects of this inhibitor rely mainly on the inhibition of the cysteine-protease involved in male chromatin remodeling after fertilization. These results further support the crucial role of this protease in early embryonic development.

This paper describes the modelling of fuzzy rule systems using a multiresolution strategy that handles the problem of granularization of the input space by using multiresolution linguistic terms. Models of different resolutions are chained by antecedents because linguistic terms of a level j are obtained by refinements of linguistic terms of a superior level j + 1. The models can also be chained by consequents using aggregation procedures. The family of models are called Multiresolution Fuzzy Rule Systems. A metasemantics based on linguistic operators is proposed for the interpretation of the refinements as a rule specialization. Interesting models result allowing local refinement of rules that preserve the semantic interpretation.

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B-cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis-localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs.

We postulated an essential role for a cysteine-protease in sea urchins sperm histones degradation which follows fertilization. We now report the purification of this enzyme, the determination of its N-terminal amino acid sequence and the localization of the protein with antibodies generated against this amino-terminal peptide. The immunofluorescence data confirmed the presence of this enzyme in the nucleus of unfertilized eggs. After fertilization labeling is observed both in female and male pronuclei suggesting a rapid recruitment of the enzyme to the male pronuclei. Interestingly, we have found that this cysteine-protease persists in the nucleus of the zygotes during S phase of the cell cycle and co-localizes with alpha-tubulin that organizes the mitotic spindle during the initial embryonic cell division.

The dry extract prepared from the Panax ginseng C.A. Meyer-root (total ginseng (T-ginseng)) contain ginsenosides (G-des) which were shown to have adjuvant properties as demonstrated by:(a) injecting guinea pigs with a mixture of T-ginseng and inactivated porcine parvovirus (PPV) as a conventional vaccine;(b) injecting PPV-antigen and T-ginseng simultaneously but separately at different sites on the animal and(c) injecting only the T-ginseng 1 or 2 weeks prior to immunisation with the PPV-antigen.Using a haemagglutination inhibition (HI) test in the antibody titration, it was found that the mean HI-titre for the animals injected with PPV-antigen only was 320 +/- 0. By comparison, the mean titre value was 2026 +/- 1206 for the sera from the animals injected with the same vaccine but adjuvanted with 4 mg of T-ginseng, while the antibody titre induced by a vaccine containing Al(OH)(3)-gel was 2986 +/- 1596. Interestingly, the T-ginseng and Al(OH)(3) acted synergistically and further improved the antibody response to the PPV-antigen to 6826 +/- 2413, i.e. more than 20 times the HI titre of the non-adjuvanted PPV-vaccine. Immunisations using PPV-vaccines adjuvanted with single purified G-des demonstrated that the ginseng fractions Rb1 and Rg1 are potent adjuvants inducing higher or similar antibody titres than the vaccine adjuvanted with Al(OH)(3), e.g. Rb1 tested at a concentration of 830 mug per dose induced a significantly (P = 0.009) higher antibody titre than the one adjuvanted with Al(OH)(3). Nevertheless, different than the mixture Al(OH)(3)-T-ginseng; Rb1 and Rg1 act antagonistically and partially inhibit each other. The G-des adjuvanted vaccines induced significantly (P = 0.0011) higher titres of IgG2 antibodies compared with IgG1. (C) 2002 Elsevier Science Ltd. All rights reserved.

The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization.