The genus Staphylococcus contains at least 47 species and 23 subspecies. Bacteria in this genus are ubiquitous; many are commensals on human and animal skin and can be opportunistic pathogens. In dairy cattle, staphylococci are the leading cause of intramammary infections (IMI) and mastitis. Mastitis is the inflammation of the mammary gland, and is one of the leading infectious diseases causing production losses in the dairy industry. Based on the ability to clot blood plasma in vitro, members of the genus can be divided into two groups: coagulase positive staphylococci (CPS) and coagulase negative staphylococci (CNS). In the dairy industry, Staphylococcus aureus is the most common CPS causing mastitis and is considered a major mastitis pathogen compared to the CNS, which as a group have been described as minor mastitis pathogens. The CNS species are increasingly recognized as an important cause of bovine mastitis, although the relative role of some species is still uncertain. Our understanding of the local and global epidemiology of CNS mastitis is improving with application of more accurate DNA sequence-based species identification methods and techniques to discriminate between strains within species. These factors have led to a shift in perspective, with the CNS being recognized as a heterogeneous group where some species are more important than others in bovine mastitis. The major goals of this thesis were to describe Staphylococcus mastitis epidemiology, and to identify phenotypes that may contribute to persistence in various niches on selected dairy farms in Vermont. We conducted 2 field studies on 2 groups of farms in Vermont. In the first study, we collected S. aureus isolates from bulk tank milk of 44 certified organic dairy farms. In the second field study, we completed quarter milk, cow skin, and environmental sampling of 5 herds that make farmstead cheeses. In both studies, we used non-selective and selective agar medium to isolate staphylococci from the farm sources. From these studies, we collected 1,853 Staphylococcus spp. isolates. We used PCR-amplicon sequence-based species identification to describe Staphylococcus species diversity on these selected Vermont dairy farms. S. aureus isolates were strain-typed using an established Multilocus Sequence Typing (MLST) scheme. A novel MLST scheme was developed to investigate the molecular epidemiology of S. chromogenes, one of the leading CNS species causing bovine mastitis in this and other studies. We also evaluated antibiotic resistance and biofilm formation phenotypes and genotypes of staphylococci to test the hypothesis that these phenotypes may be associated with strain types. In the study of organic dairy farms, 20 S. aureus strain types (STs) were identified, including ten novel STs. The majority of STs belonged to lineages or clonal complexes (CCs) previously identified as cattle adapted (e.g. CC97 and CC151). Associations between ST and carriage of beta-lactam resistance and biofilm forming capacity were identified among the S. aureus isolates from these farms. In the 5-herd study, a total of 27 different staphylococci species were identified from various niches including humans, but only five species; S. chromogenes, S. aureus, S. haemolyticus, S. simulans, and S. xylosus were commonly identified to cause IMI. S. aureus and S. chromogenes strain types were niche specific.

Magíster en Gestión y Dirección de Empresas / Este informe técnico tiene por objetivo presentar los lineamientos para realizar un plan de negocios con el fin de introducir al mercado un producto farmacéutico, como una nueva alternativa terapéutica para la mastitis clínica, que permita reducir o mejorar el régimen terapéutico de los antibióticos actualmente. Los pomos intramamarios que se desarrollaron para la mastitis son de dos tipos, unos contienen ceftiofur y los otros cloxacilina a bajas concentraciones, ambos asociados con los componentes inhibitorios extraídos desde las hojas de Aloe vera, comprobándose a través de metodologías experimentales, que cuando son utilizados en el tratamiento de las mastitis clínicas, tienen ventajas o primacías, en relación a los actuales pomos intramamarios que solo contienen antibiótico. El planteamiento de este informe se enfoca en reducir la incertidumbre y el riesgo que cualquier actividad no planificada tiene, muestra los objetivos que se pretenden alcanzar, las estrategias que se pretenden adoptar, los procesos operacionales, la estructura organizacional y permite conocer el rendimiento económico y financiero que se espera del negocio, va desde las estrategias generales llegando hasta las actividades cotidianas. La metodología propuesta cuenta con ocho etapas, las cuales se enfocan en conocer la situación actual del mercado de los medicamentos veterinarios, conocer cuál es la mejor estrategia para el negocio, conocer la oferta, la demanda, segmento objetivo, diseñar e implementar un plan de marketing para la comercialización del producto, Definir sus procesos de operaciones, los miembros del equipo claves, definir sus costos, precio de venta del producto y sus riesgos financiero, todo esto para asegurar el éxito en la entrada de este producto al mercado. El plan de implementación contempla definir cuáles son las actividades que se desean realizar, el costo de implementarlas y en qué tiempo se llevarán a cabo. Por lo tanto, basándonos en la oportunidad de negocio, atractivo de la industria en crecimiento, mercado objetivo relevante, necesidades no cubierta, resultados positivos obtenido en las proyecciones financieras de Estado de Resultado a seis años, VAN positivo de $ 228.551.460,85, TIR de 14,64%, Valor Económico positivo del proyecto, plazo de recuperación de la inversión menor de 5 años y medidas de rentabilidad positivas, es que podemos concluir que el plan de negocio desarrollado creará valor para el inversionista, su empresa y sus grupos de interés. Por lo tanto, quien invierta en el proyecto podrá rentabilizar su inversión en el mediano y largo plazo.

No description available.

Streptococcus uberis, an environmental organism also associated with dairy animals, is a common and persistent cause of bovine mastitis. New approaches to control these infections need to be identified. One such strategy may be the application of bacteriocins; proteinaceous antimicrobials elaborated by bacteria that typically inhibit the growth of strains closely related to the producer organism. The well-characterized lactococcal bacteriocin nisin is the active ingredient in two commercial products currently in use for the prevention of mastitis. However, reports of resistance development have prompted the investigation of alternative bacteriocins to be used in conjunction with nisin in 'bacteriocin cocktails' designed to have more comprehensive inhibitory activity against mastitis pathogens. The bacteriocins of gram-positive bacteria have been divided into four distinct classes: (I) lantibiotics, (II) non-lantibiotic peptides, (III) large proteins, and (IV) circular peptides. Although it has been known for more than twenty years that S. uberis commonly produce bacteriocin-like inhibitory substances (BLIS), none had been characterised prior to the present study. The first step in the current investigation was a survey of the BLIS activities of a set of fifteen S. uberis and S. bovis strains against a set of standard indicators as well as common gram-positive mastitis pathogens. Additional tests using a deferred antagonism agar plate-based assay showed that some of the BLIS activities were heat-sensitive and their production was influenced by the presence of either blood or a fermentable carbohydrate source in the test medium. On the basis of the results obtained from these tests it became apparent that S. uberis and S. bovis may commonly produce more than a single inhibitory agent. S. uberis 42 became the focus of this study because (a) it had broad inhibitory activity against mastitis-associated bacteria, (b) it did not display cross-resistance to nisin, and (c) from the preliminary screening results it appeared to produce both heat-stable and heat-labile inhibitory agents. Acid extracts of S. uberis 42 cells yielded inhibitory activity that, when fractionated by reversed-phase HPLC, yielded a peptide of 3029 Da. Although this peptide was blocked to Edman degradation at position 2, following propanethiol-modification a 20-amino acid sequence was obtained. Degenerate primers to lantibiotic biosynthesis gene homologs were used to initiate inverse PCR and primer walking, ultimately yielding a 15-kb contiguous sequence encompassing 11 genes typical of those involved in lantibiotic synthesis, regulation and immunity. Due to the close similarities to nisin of the S. uberis 42 lantibiotic precursor (78%), and the organisation and composition of the locus, this inhibitor was named nisin U. Nucleotide sequences homologous to insertion sequences were detected in the vicinity of the nisin U locus, and indicate a possible mechanism of acquisition of this locus by S. uberis. The locus was detected in ten other S. uberis, and also in two S. agalactiae and two S. thoraltensis strains, and in one S. porcinus and one S. pluranimalium strain. The amino acid sequences of some of these differed in one or two amino acids, and these variants were named nisin U2 and nisin U3 accordingly. Nisin U, the two nisin U variants, and nisin A exhibited cross-immunity (i.e. all of the producer strains were insensitive to each form of nisin) and cross-inducibility (i.e. all of the producer strains displayed enhanced production when exposed to each form of nisin). Nisin U did not contribute to the entire spectrum of inhibitory activity of S. uberis 42. Freeze thaw extracts of S. uberis 42 agar cultures yielded heat-labile inhibitory activity that was inhibitory to L. lactis A5, a producer of nisin Z. Subsequent purification by cation-exchange chromatography, gel filtration, and reversed-phase HPLC yielded a peptide of mass 7048 Da, which was resistant to Edman degradation. Digestion with chymotrypsin released an 819 Da peptide fragment of sequence NH₂-KAQAVIW-COOH. Tn916 mutagenesis of S. uberis 42 enabled the identification of the genetic locus of the inhibitor, comprising six genes potentially involved in its biosynthesis and immunity. The detection of a pair of flanking 159-bp direct repeats indicates possible acquisition of the locus by 'long target duplication'. The inhibitor was inferred to be a circular peptide, on the basis of its behaviour to Edman degradation, and by comparison of its locus with that of other circular bacteriocins. On the basis that the purified peptide appears to induce lysis in sensitive bacteria, although by an as-yet unidentified mechanism, the inhibitor was named uberolysin. The uberolysin structural gene was detected in eight other strains of S. uberis, however not all of these appeared to be producing active inhibitor. No bacteriocins closely resembling the two reported in this thesis have been demonstrated previously to be produced by members of the genus Streptococcus. The remarkable diversity in the structures, activity spectra and basic modes of action of these two bacteriocins produced by a single strain of S. uberis, combined with the observation of apparent greater heterogeneity in properties of a preliminary sampling of BLIS-producing strains, indicates that these bacteria may be an important source of novel antimicrobials of potential value for the treatment of mixed bacterial infections and for minimising potential resistance development.

Bovine mastitis is one of the most costly dairy-based diseases worldwide. Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. New strategies need to be developed to control this pathogen. However, a fundamental understanding of the complex relationships that exist between the cow, the pathogen and the environment are required in order to advance the development of prevention strategies. Microarray technology was used to evaluate the complex transcriptional changes which occur in the bovine mammary gland following the onset of clinical S. uberis mastitis. A 22,000 bovine cDNA microarray indicated that S. uberis mastitis led to the up-regulation of 1,283 genes and the down-regulation of 1,237 genes by greater than 1.5 fold. Gene ontology analysis demonstrated that S. uberis mastitis was typically associated with the up-regulation of genes that are involved in the immune response and homeostasis and a down-regulation of genes involved in lipid metabolism. Quantitative real-time analyses for a selection of genes associated with the immune response validated the microarray data. Mammary epithelial cell cultures did not show an increase in the expression of any of these immune factors in response to the same S. uberis strain used to induce clinical mastitis. This indicates that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not S. uberis. The application of bacteriocins, proteinaceous antimicrobials produced by bacteria which typically inhibit the same or closely-related species to that of the producer organism, has been suggested as one possible approach in the control of mastitis. S. uberis have been previously found to commonly produce bacteriocin-like inhibitory substances (BLIS). The BLIS activities of a set of fifteen S. uberis and S. bovis strains were assessed. The results confirmed the prolific and varied nature of BLIS production by S. uberis and S. bovis and also indicated that these strains may commonly produce more than one inhibitory agent. This survey of BLIS production led to the detection and characterisation of a novel circular bacteriocin, uberolysin, produced by S. uberis strains 233 and 42. The structural gene of uberolysin was subsequently identified in nine (64%) of the fifteen test strains. Multiplex PCR analysis showed that 93% of 158 New Zealand S. uberis isolates contained the structural genes of at least one of the four known S. uberis bacteriocins (uberolysin, nisin U, ubericin A and ubericin 63). However, no apparent direct association was identified between any one of these bacteriocin-related loci and apparent ability to cause mastitis on New Zealand dairy farms. The uberolysin structural gene was detected in 91% of the isolates and this widespread distribution prompted the advancement and evaluation of a potential role for uberolysin in immunomodulation within the bovine mammary gland. Two different preparations of uberolysin were found to have different stimulatory effects on monocytes, neutrophils and epithelial cells. The less highly purified preparation appeared to diminish the production of TNF-α by monocytes in the presence of a bacterial stimulus and to decrease neutrophil phagocytosis. By contrast, the relatively more highly purified preparation of uberolysin itself induced a significant immune response by monocytes. Consistent with this, the purer preparation of uberolysin induced an increase in C3, IL-1β, IL-6, IL-8, the β-defensin LAP, the acute-phase protein MSAA, the calcium-binding protein S100A12 and TLR2 by quantitative real-time analysis. Although currently only two S. uberis bacteriocins (uberolysin and nisin U) have been fully characterised, the present study has shown that this species may be an important source of novel antimicrobials. Furthermore, bacteriocin production by S. uberis may have an immunomodulation role within the mammary gland. A better understanding of the complex immune response initiated at the onset of clinical S. uberis mastitis and of the role that bacteriocins have in S. uberis pathogenesis may lead to development of improved strategies to combat this disease.

The objective of the first part of this study was to observe and describe the profile of test-day somatic cell counts throughout a lactation in individual cows, and to examine the correspondence between such profiles and various lactation measures of cell count presently available. The objective of the second part of the study was to obtain estimates of the genetic and phenotypic parameters of lactation measures of cell count in different lactations, possibly for use in a breeding program. / In the first part of the study, the lactation cell count profiles of eighteen Holstein heifers of the Macdonald College Herd were individually examined. Weekly cell counts were expressed as deviations from the herd test-day average and plotted against week of test. A labile cell activity was found for most of the plotted lactations. The cellular responsiveness of cows facing external challenges seemed a trait peculiar to each individual. Lactation measures of cell count were unable to give good descriptions of cell count profiles of individual cows. However, they were better expressions of the lactation cell count performance than single test-day observations. / In the second part of the study, monthly cell count observations were obtained between February, 1977, and February, 1982, for the Holstein cows in herds enrolled on the official option of the Quebec Dairy Herd Analysis Service. Maximum likelihood, I-MINQUE (iterative Minimum Norm Quadratic Unbiased Estimation), and multivariate REML (Restricted Maximum Likelihood) procedures were used for the estimation of genetic and phenotypic parameters. / Repeatabilities of log test-day cell counts and log of test-day cell counts corrected for milk yield varied between 0.36 and 0.42 in first, second, third, fourth, and fifth and later lactations. Repeatabilities of test-day cell counts (cells/ml) in the five lactation groups varied between 0.17 and 0.25. Repeatabilities of lactation expressions for cell count between lactations ranged from 0.13 to 0.44. / Heritabilities of lactation expressions of cell count were low and varied from 0.06 to 0.14 in the five lactation groups examined; however, the genetic correlations between lactations were very close to unity, 0.90 to 0.97.

A study was performed in agricultural company “Nemunas”, Prienai district, the Departament of Non-contagious Diseases of the Lithuanian veterinary Academy and the Laboratory of Animal reproduction. The master of thesis analyses reproduction of the cowherd and mastitis prblems in the farm of agricultural “Nemunas”, Prienai district. Analysis of data shows that the yield of milk is quite low (3203 kg) despite the fact that annual introduction of first-calf heifers amounts to 21,1 % of the herd. By examination of 1227 milk samples in 2003, the general count of somatics cells was 590,550 ± 51,030 cells/ml. Only 61,2 % of the delivered milk satisfied standart milk indicators (SCCL < 400,000 cells/ml). An increase in the somatic cell count is closely related with hidden mastitis. By examination of 41 milk samples (SCCL < 400,000 cells/ml), a mixed micro-flora was isolated in 78 % of the cases and pure cultures isolated in 22 % of the cases. The mixed microorganism cultures constituted of Streptococci, Stafilococci, Enterococci and yeast-like fungi. The sensitivity of the isolated organism was determined by carrying out antibioticogram testing. Used anti-mastitis preparations were found differently effective against isolated microorganisms. The preparation “Penak Entervitol” was effective agaisnt enterobacter and Candida, the preparation “Targot mastitis Suspension” was effective against Streptococci, Stafilococci and Proteus. The preparation “Streptopen” was effective against... [to full text]

Neutrophil potential phagocytic capacity (NPPC), measured on 25 AI Canadian Holstein bulls, was investigated for evidence of association with production and type traits, SCC, and survival in dairy cows. Bulls were ranked based on different degrees of NPPC (Uptakes of 0, 1, 2, and 3 or more latex beads), using the solutions coming from an animal model. A total of 42,103 first lactation records, collected from 1985 through 1993 in 2,919 Quebec dairy herds, were used to obtain EBV's for SCC and for log SCC (LogSCC) for 697 sires. Correlations between NPPC measurements and somatic cell EBV's were null. Canadian official ETA's for type traits related with mammary system had a tendency of being positively correlated with higher NPPC and negatively with Uptake-0. Canadian official ETA's for production traits were negatively correlated with higher NPPC and positively with Uptake-0. A total of 17,202 first lactation records of daughters of the 25 AI bulls were used to study the effect of NPPC and log SCC on survival in dairy cows. Survival after first lactation was more closely related to sires' NPPC-EBV's than to LogSCC-EBV.

No description available.

Diapedesis, phagocytosis and microbicidal activity are important parameters of neutrophil functionality and thus outcome of mastitis. An in vitro model of an "alveolar pavement" using the MAC-T3 bovine mammary epithelial cell line was developed to assess neutrophil diapedesis. Features of this biologically-meaningful barrier include: characteristic transepithelial resistance, tight junction complexes and polarity. Continuous transepithelial resistance measurements showed no significant changes throughout the assay period. Neither a Staphylococcus aureus challenge ($1 times10 sp7$ and $2 times10 sp9$ cfu/ml), or the presence of neutrophils, both resting and challenged had any deleterious effects on monolayer integrity over a short term (1-2 h) exposure. Neutrophils, both resting and challenged gave no indication of causing damage to the epithelium over the short term. Neutrophils isolated from proven sires and evaluated for phagocytic activity were found to differ significantly (p $<$ 0.05) in activity, rate and capacity to uptake particles. Correlations between phagocytic parameters and production traits were negative and small in magnitude. Microbicidal activity of neutrophils isolated from proven sires showed a highly significant variation between animals due to test day (p $<$ 0.001), however variation due to source of cells (i.e. animal) was not significant. in vitro analysis of diapedesis and phagocytosis is promising as a tool for the assessment of resistance or susceptibility to mastitis.