Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The effect of the bacteriocin-like peptide AS-48, produced by Enterococcus faecalis FAIRE 92, was tested against a mastitis isolate of Staphylococcus aureus in an in vivo and in vitro study. During initial tests peptide AS-48 showed no significant activity towards S. aureus, even with a ten-fold concentrated cell-free supernatant. Activity was obtained only after purification with Triton X-114 phase partitioning, followed by cation exchange chromatography. Titers for the purified peptide varied between 3200 and 12800 AU/ml. The purified peptide also exhibited activity towards Streptococcus agalactiae and Streptococcus dysgalactiae, but not against Escherichia coli. The size of peptide AS-48 was determined at 7150 Da, based on electronspray mass spectrometry and SDS-PAGE. Complete inhibition of cell growth was obtained by adding 1ml of the purified peptide (3200 AU/ml) to 100 ml of cells of S. aureus in the lag growth phase. When the same concentration of peptide AS-48 was added to a culture of S. aureus in mid-exponential growth, a slight decrease in viable cell numbers was recorded, which lasted for only 30 min. Cell growth commenced thereafter. In situ experiments in cows were done with purified peptide AS-48, encapsulated in liposomes. These in vivo studies were conducted by administering peptide AS-48 (6400 AU/ml) to different udder quarters. In a prevention trial, i.e. where quarters were pretreated with peptide AS-48, a reduction close to 90% in the viable cell numbers of S. aureus was recorded relative to the control quarters, which were not treated with the peptide. A 50% reduction in somatic cell count (SCC) was recorded. In the treatment trial, i.e. infected quarters treated with peptide AS-48, a reduction of up to 94% in viable cell numbers of S. aureus was recorded. In the same quarters, a reduction in SCC amounted to almost 80%. A recombinant strain was constructed by conjugating plasmid 92 (p92), encoding peptide AS-48, from Enterococcus faecalis FAIRE 92 to E. faecalis FA2/Ent, which produces enterocins 1071A and 1071B. Southern blot hybridization experiments revealed thepresence of plasmid p92 in the recipient strain without the loss of plasmid pEF1071, which encodes enterocins 1071A and 1071B. All three antimicrobial peptides, i.e. enterocin 1071A, enterocin 1071B and peptide AS-48, were produced in transconjugant FA2/Ent/AS-48. The spectrum of antimicrobial activity of the transconjugant was greater than that recorded for strains FA2/Ent and FAIRE 92, respectively and included E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus and S. aureus. These organisms are not inhibited by strain FA2/Ent. However, low levels of peptide AS-48 was produced by strain FA2/Ent/AS-48. Further research in fermentation and gene expression will be needed before the transconjugant E. faecalis FA2/Ent/AS-48 may be used in the treatment of mastitis. / AFRIKAANSE OPSOMMING: Die effek van die bakteriosien-agtige, peptied AS-48, geproduseer deur Enterococcus faecalis FAIRE 92, is gedurende ‘n in vivo en in vitro studie teen ‘n mastitiese Staphylococcus aureus-isolaat getoets. Aanvanklike toetse met peptied AS-48, selfs tienvoudig gekonsentreerde selvrye supernatant, het geen beduidende aktiwiteit teen S. aureus getoon nie. Aktiwiteit is eers verkry na suiwering met Triton X-114 fase-skeiding gevolg deur katioon uitruilingschromatografie. Titers vir die gesuiwerde peptied het tussen 3200 en 12800 AE/ml gewissel. Die gesuiwerde peptied het ook aktiwiteit teen Streptococcus agalactiae en Streptococcus dysgalctiae getoon, maar nie teen Escherichia coli nie. Peptied AS-48 het ‘n molekulêre massa van 7150 Da, soos bepaal met elektronsproeimassa spektrometrie en SDS-PAGE. Totale inhibisie van selgroei is verkry deur 1 ml gesuiwerde peptied AS-48 (3200 AE/ml) by ‘n 100 ml kultuur van S. aureus in die sloerfase te voeg. Dieselfe konsentrasie peptied AS-48, toegevoeg tydens die mideksponensiële groeifase, het egter slegs ‘n klein vermindering in die aantal lewende selle teweeg gebring en het ook vir slegs ‘n 30 min geduur. Selgroei het hierna weer normaal voort gegaan. In situ eksperimente op koeie is uitgevoer met gesuiwerde peptied AS-48, geenkapsuleerd in liposome. Hierdie In vivo studies is onderneem deur peptied AS-48 (6400 AE/ml) in verskillende kwarte van die uier, kunsmatig of reeds geïnfekteerd met S. aureus, toe te dien. In ‘n voorkomings-eksperiment waar kwarte vooraf met peptied AS- 48 behandel is, is ‘n verlaging van byna 90% in die lewende seltelling van S. aureus relatief tot die kontrole kwarte, sonder behandeling met peptied AS-48, verkry. ‘n 50% verlaging in die somatiese seltelling (SST) is verkry. In die behandelings-eksperiment, waar geïnfekteerde kwarte met peptied AS-48 behandel is, is ‘n verlaging van byna 90% in lewende S. aureus selle gevind. In dieselfde kwarte is ‘n verlaging van byna 80% in die SST genoteer.‘n Rekombinante ras is gekonstrueer deur plasmied 92 (p92), wat kodeer vir peptied AS- 48, vanaf Enterococcus faecalis FAIRE 92 na E. faecalis FA2/Ent, wat enterosien 1071A en 1071B produseer, te konjugeer. Southern-klad hibridisasie het die teenwoordigheid van plasmied p92 in die ontvanger ras, sonder die verlies van plasmied pEF1071 wat enterosien 1071A en 1071B kodeer, getoon. Al drie antimikrobiese peptiede, nl. enterosien 1071A, enterosien 1071B en peptied AS-48, is deur die transkonjugant FA2/Ent/AS-48 geproduseer. Die spektum van antimikrobiese aktiwiteit van die transkonjugant vand die transkonjugant is breër as dié van rasse FA2/Ent en FAIRE 92, onderskeidelik en het ook E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus en S. aureus ingesluit. Hierdie organismes word nie deur ras FA2/Ent geïnhibeer nie. Lae vlakke van peptied AS-48 is egter deur ras FA2/Ent/AS-48 geproduseer. Verdere navorsing in fermentasie en geenuitdrukking is nodig voordat E. faecalis FA2/Ent/AS-48 in die behandeling van mastitis gebruik kan word.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Mastitis is the most prevalent and costly production disease of dairy cattle; hence mastitis incidence is a distinctly important trait in dairy cattle. The primary objective of the study was to determine the economic value, and develop a model for genetic prediction of clinical mastitis in South African Holstein cattle. These procedures are a prerequisite to including this trait in the breeding objective. The cost of clinical mastitis per incident was calculated as the sum of revenue loss due to discarded milk during the infection period and the associated treatment costs. Economic value (ZAR/incident) was calculated as the change in profit (increase in costs) resulting from a simulated marginal increase in mastitis incidence in an average herd. Average economic losses due to clinical mastitis were estimated at ZAR919.96/cow/year and the average incidence was 0.9cases/cow/year. The economic value of clinical mastitis was ‐ZAR1079.51/incident. A model for predicting estimated breeding values (EBVs) for clinical mastitis using somatic cell score (SCS), fore teat length (FTL), udder depth (UD) and rear udder height (RUH) was developed, using genetic (co)variances among these traits. Since EBVs for SCS, FTL, UD and RUH are routinely estimated under the national genetic evaluation programme, EBVs for clinical mastitis can be predicted from the model developed in the current study. Thus, the results of the study provide the basis for including clinical mastitis in the breeding objective for South African Holstein cattle. / AFRIKAANSE OPSOMMING: Mastitis is die mees algemeenste en duursteproduksie siekte wat voorkom by melkbeeste, daarom is die voorkoms van mastitis 'n belangrike eienskap in melkbeeste. Die primêre doel van die studie was om die ekonomiese waarde te bepaal, asook die ontwikkeling van 'n model vir genetiese voorspelling van kliniese mastitis in Suid‐Afrikaanse Holstein beeste. Hierdie prosedures is 'n voorvereiste vir insluiting van hierdie eienskap as ‘n teeldoelwit in seleksie programme. Die koste van kliniese mastitis per voorval is bereken as die som van die inkomste verlies weens melk weggegooi tydens die infeksie periode en die gepaardgaande koste vir die behandeling. Ekonomiese waarde (ZAR / voorval) is bereken as die verandering in wins (toename in koste) wat voortspruit uit 'n gesimuleerde marginale toename in mastitis voorkoms in 'n gemiddelde kudde. Gemiddelde ekonomiese verliese as gevolg van kliniese mastitis was beraam op ZAR919.96/koei/jaar en die gemiddelde voorkoms was 0.9gevalle/koei/jaar. Die ekonomiese waarde van kliniese mastitis was ‐ ZAR1079.51/geval. 'n Model vir die voorspelling van beraamde teelwaardes (EBV’s) vir kliniese mastitis is ontwikkel deur gebruik te maak van die ko‐variansies tussen die onderskeie eienskappe: somatiese sel telling (SST), voorspeen lengte (VSL), uier diepte (UD) en agter uier hoogte (AUH). Aangesien teelwaardes vir SST, VSL, UD en AUH gereeld beraam word onder die Nasionale genetiese evaluasie program, kan teelwaardes vir kliniese mastitis voorspel word vanuit die model wat ontwikkel is in die huidige studie. Dus verskaf die resultate van hierdie studie ‘n basis vir die insluiting van kliniese mastitis as ‘n teeldoelwit in seleksie programme van die Suid‐Afrikaanse Holstein beeste.

Mastitis is one of the most costly diseases to dairy producers around the world with milk yield loss being the biggest contributor to economic losses. The objective of first study of this thesis was to determine the impacts of high somatic cell counts on milk yield loss. To accomplish this, over one million cow data records were collected from Southeastern US dairy herds. The objective of the second study was to determine optimum treatment cost of clinical mastitis by combining two economic modeling approaches used in animal health economics. The last objective of this thesis was to determine how much Southeastern US dairy producers are spending to control milk quality on farm and determine if they understand how milk quality affects them economically. This was accomplished through a collaborative project within the Southeast Quality Milk Initiative.

Memoria para optar al Título Profesional de Médico Veterinario / Las mastitis, en todas sus formas de presentación, es una enfermedad que generar numerosas pérdidas en la industria lechera. Estas pueden deberse a la menor cantidad de leche producida por parte de la vaca, la alteración en la calidad de ésta y los costos de tratamientos, eliminación y reemplazo de animales. La fertilidad en lecherías de alta producción es un aspecto muy importante de los manejos que se deben realizar en los predios. Esto se debe principalmente a que luego de paridas, es importante preñar a las vacas lo antes posible (siempre considerando un tiempo de espera voluntario) para que de esta manera se aproveche de manera más eficiente la curva de la lactancia de las vacas. Dicho de otra manera, es importante tener preñadas a las vacas para que su lactancia no se alargue demasiado y de esta manera no tener animales con bajas producciones producto de lactancias muy largas. Algunos autores han propuesto que la mastitis podría tener un efecto en la fertilidad de las vacas lecheras de alta producción. Esto sería válido para el cuadro clínico y subclínico de la enfermedad. Por lo tanto el presente estudio tuvo como propósito determinar el efecto de alteraciones en la sanidad mamaria (mastitis clínica o subclínica) en la fertilidad a la inseminación de vacas lecheras de alta producción. Para este estudio se utilizaron los registros de más de 5000 inseminaciones provenientes de 2 predios lecheros ubicados en la región de Valparaíso. Para el caso de las mastitis clínicas se consideraron inseminaciones que estuvieran asociadas a un caso de mastitis entre 10 días previos a ésta y entre la IA y el diagnóstico de gestación. Se dividieron los datos de acuerdo al periodo en que se presentó el caso en relación a la IA en antes de la IA, durante la IA y después de la IA. También se analizó el efecto de la severidad del cuadro sobre la fertilidad a la IA. Para analizar el efecto de la mastitis subclínica sobre la fertilidad se utilizó el puntaje lineal de células somáticas como indicador de mastitis subclínica (≥ 4,5) y se registró este puntaje en el control más cercano a la IA considerando que para ambos predios se realizaban muestreos periódicamente (cada 30 días aproximadamente). Para analizar los datos se compararon las tasas de concepción de animales que presentaron la enfermedad (el cuadro clínico y subclínico por separado) con animales sanos mediante el método de chi cuadrado. Además se realizaron análisis de regresión logística para determinar si existía un efecto de la enfermedad en la probabilidad de preñez comparado con animales sanos. Para el caso de la mastitis clínica los resultados mostraron que cuando se consideraba a las inseminaciones asociadas a mastitis en un período 10 días antes de la IA hasta el diagnóstico de gestación no se presentaron diferencias significativas en la tasa de concepción (TC) de animales sanos comparado con animales enfermos (TC= 35,4% vs 32,8% respectivamente; p=0,13). Para determinar de mejor manera el efecto que podría tener la mastitis se decidió restringir el periodo en que se consideraban inseminaciones asociadas a mastitis entre los 5 días previos a la IA y los 20 días posteriores a ésta. Esta vez los resultados mostraron que existía un efecto marcado de la enfermedad en la tasa de concepción (35,6% en animales sanos vs 29,7% para vacas enfermas; p=0,002) y en la probabilidad de preñez (OR: 0,76; IC 95%= 0,64 - 0,91; p=0,002). Otros parámetros estudiados, una vez restringido los periodos seleccionables para considerar una inseminación asociada a mastitis clínica, fueron el efecto del momento de presentación del cuadro y la severidad de éste. Para el efecto del momento de presentación de la enfermedad sobre la fertilidad se pudo apreciar que animales enfermos tenían menores tasas de concepción cuando la enfermedad se presentaba durante la IA y después de ésta (Durante: TC=25,7%, p= 0,006; Después: TC=30,7%, p= 0,05 comparado con TC=35,6% de animales sanos). Para la severidad de la mastitis se vio que las mastitis grado 2 y 3 presentaban menores tasas de concepción comparado con la ausencia de mastitis (Grado 2: TC=29%, p=0,006; Grado 3: TC=29,7%, p=0,05). Los resultados para el análisis de la mastitis subclínica no arrojaron diferencias significativas en la tasa de concepción vacas con mastitis subclínica comparado con animales sanos (TC= 35,8 vs. 35,4 respectivamente; p=0,61). El análisis de regresión logística tampoco mostró diferencias significativas en la probabilidad de preñez de animales sanos comparado con animales enfermos (OR: 0,94; IC 95% = 0,76 - 1,18; p=0,6). Existiría un efecto negativo de la mastitis clínica en la tasa de concepción de vacas lecheras, en un periodo cercano a la IA. También se aprecia un efecto negativo dependiendo del momento de presentación de la enfermedad en la tasa de concepción. La severidad del cuadro también afectó negativamente la fertilidad a la inseminación. Para el caso de la mastitis subclínica no existiría un efecto negativo de esta en la tasa de concepción de vacas lecheras

The goal of my thesis was to evaluate origin of mastisis diseases and specify the best way to cure this disease with antibiotics. Secondary goal was to examin origin of things that cause these diseases, efficiency of the cure and how it is influenced by the stage of the illness. This research was made in cowshed, fron april to december 2018 with 136 specimen. During this period I took samples of milk, that had more than 800 000 somatic cells in 1ml of milk. Based of possitve NK test, samples of mammary gland were taken and cultivated on one used only Petri dish. Depending of the origin of the infection, the proper atibitics were chosen for the cure. After 7 to 14 days after antibiotics treatment, milk sample was taken from the cured part od mammary gland and It was tested to see, if the treatment was successful. 77% of the specimen were sick because of microorganisms and most of the specimen were afflicted the most during 1st lactation (36.8% to 47.1%). Following microorganisms caused mastisis - Staphylococcus spp. (KNS), Streptococcus uberis, Klebsiella oxytoca, Escherichia coli, Candida rugosa a Enterococcus faecalis. 36% of mastisis was caused by Staphylococcus spp. (KNS), followed by coliform bacteria (22%). 17% of mastisis was caused by Streptococcus uberis. 10.5% of specimen was infected by Enterococcus faecalis. Candida rugosa was found in 7.4% of the samples and in 9.5% of samples the diseasehad no specific microorganism responsible. From 105 infected cows, 68% of them fully recovered. At the end, based on the stage of the mastisis, efectivness of the treatmend was statistically evaluated. The evaluation showed, that stage of infection massively influence the result of the treatment.

Orientador: Márcio Garcia Ribeiro / Resumo: Escherichia coli é o principal agente de mastite clínica bovina de origem ambiental, caracterizado pela complexidade de fatores de virulência (FV). O patógeno causa sinais clínicos que variam desde alterações exclusivamente no leite (grau 1 ou leve), no quarto afetado (grau 2 ou moderado), até manifestações sistêmicas (grau 3 ou grave). No entanto, até o momento, não está estabelecido o perfil de genes deste patógeno relacionados à virulência em infecções mamárias em vacas, tampouco com a gravidade clínica dos casos. Neste cenário, o presente estudo investigou 18 genes associados com E. coli extraentérica (ExPEC), o perfil “in vitro” de motilidade swimming e swarming, e a sensibilidade/resistência aos antimicrobianos em 114 isolados de E. coli obtidos de vacas com mastite clínica com escores de gravidade 1 (45/114=39,5%), 2 (62/114=54,4%) e 3 (7/114=6,1%). Os principais genes codificadores de FV detectados foram de adesinas (fimH, 114/114=100%; ecpA, 73/114=64,0%; fimA, 36/114=31,6%), resistência ao soro (traT, 93/114=81,6%; ompT, 40/114=35,1%), sideróforos (irp2, 11/114=9,6%) e hemolisina (hlyA, 8/114=7%). Os isolados apresentaram 99,1% (113/114) de resistência in vitro a bacitracina e cloxacilina, 98,2% (112/114) a lincosamina e 54,4% (62/114) a eritromicina. Do total de isolados, 98,2% (n=112/114) foram multirresistentes pelo cálculo do índice de resistência múltipla aos antimicrobianos (IRMA). Não houve diferença estatística significante entre as medianas para motilidade ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Escherichia coli is the major pathogen involved in the etiology of bovine mastitis from the environment origin. This pathogen is characterized by a complexity of virulence factors (VF). Mammary infections by E. coli has shown a wide range of clinical signs causing changes in milk (score 1 or mild), quarters (score 2 or moderate), and systemic signs (score 3 or severe). Nevertheless, to date, the profile of the genes related to the virulence of this pathogen in mammary infections and the severity scores of the cases are not fully understood. In this scenario, a panel of 18 genes associated with extra-intestinal E. coli (ExPEC) were investigated, in addition to in vitro swimming and swarming motility profile, and antimicrobial susceptibility/resistance pattern among 114 E. coli strains isolated from cows with clinical mastitis showing severity scores 1 (45/114=39.5%), 2 (62/114=54.4%) and 3 (n=7/114=6.1%). The main genes related to VF harbored by isolates were adhesins (fimH, 114/114=100%; ecpA, 73/114=64.0%; fimA, 36/114=31.6%), serum resistance (traT, 93/114=81.6%; ompT, 40/114=35.1%), siderophores (irp2, 11/114=9.6%) and hemolysin (hlyA, 8/114=7%). Among studied isolates, 99.1% (113/114) showed in vitro resistance to bacitracin and cloxacillin, 98.2% (112/114) to lincosamin, and 54.4% (62/114) to erytromycin. Out of the total isolates, 98.2% (112/114) were considered multidrug resistant based on multiple antimicrobial resistance (MAR) index. No statistical difference was obs... (Complete abstract click electronic access below) / Doutor

O objetivo deste estudo foi avaliar a técnica de espectrometria de massas por ionização/dessorção a laser assistida por matriz tempo-de-vôo (MALDI-TOF) para a identificação direta em amostras de leite (sem cultivo microbiológico) de bactérias causadoras de mastite. Para tanto foram realizados dois experimentos (1 e 2). No experimento 1, o objetivo foi determinar a sensibilidade diagnóstica da técnica MALDI-TOF MS para a identificação direta em amostras de leite de Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae e Escherichia coli. Foram realizadas contaminações experimentais de S. aureus, S. uberis, S. agalactiae, S. dysgalactiae e E. coli em amostras de leite, para a obtenção de contagens de 103 a 109 ufc/mL. As amostras de leite contaminadas foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Espectros de massas foram coletados na faixa de massas de 2.000-20.000 m/z e foram analisados pelo programa MALDI Biotyper 3.0 (Bruker Daltonics) com as configurações padrão para obtenção da identificação bacteriana. A identificação direta de patógenos causadores de mastite a partir de amostras de leite foi possível em contagem ≥106 ufc/mL para S. aureus, ≥107 ufc/mL para E. coli, e ≥108 ufc/mL para S. agalactiae, S. dysgalactiae e S. uberis. No experimento 2, o objetivo foi avaliar o efeito da pré-incubação de amostras de leite de quartos mamários com mastite subclínica sobre a eficácia da identificação sem cultivo de patógenos causadores de mastite por MALDI-TOF MS. Foram selecionados 2 rebanhos leiteiros para coletas de leite de quartos mamários de todas as vacas em lactação. As amostras de leite foram submetidas às análises de: a) cultura microbiológica; b) pré-incubação seguida de identificação por espectrometria de massas diretamente do leite; c) contagem bacteriana total (CBT). Para a realização da CBT, as amostras de leite foram submetidas à citometria de fluxo; e para a identificação direta de patógenos causadores de mastite a partir do leite, as amostras foram submetidas ao desnate por centrifugação (10.000 Xg por 10 minutos), e pré-incubação a 37ºC por 12 horas. Posteriormente, as amostras foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Do total de 810 amostras de leite analisadas por cultura microbiológica (método referência), 347 apresentaram crescimento bacteriano, sendo 305 identificadas como agentes de interesse na identificação direta pelo método MALDI-TOF MS: Staphylococcus coagulase negativa (n=191), S. aureus (n=31), S. agalactiae (n=42), S. uberis (n=37), S. dysgalactiae (n= 4). Sendo assim, 305 amostras foram analisadas pelo método de idenfificação direta MALDI-TOF MS, a qual apresentou baixa sensibilidade quando comparado com a cultura microbiológica (método referência): Staphylococcus coagulase negativa (14,08%), S. agalactiae (15,25%), S. uberis (1,69%) S. aureus (6,12%) e S. dysgalactiae (0%). A pré-incubação de amostras de leite não aumentou a sensibilidade de identificação direta de microrganismos causadores de mastite pelo método MALDI-TOF MS. / The purpose of the present study was to evaluate the technique of mass spectrometry by desorption / ionization assisted laser array time-of-flight (MALDI-TOF MS) for the direct identification in milk samples (no microbiological culture) of mastitis causing bacteria. Therefore, we carried out two experiments (1 and 2). In experiment 1, we determined the diagnostic sensitivity of MALDI-TOF MS technique for the direct identification in milk samples of Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli. Experimental contamination of S. aureus, S. uberis, S. agalactiae, S. dysgalactiae and E. coli in samples of milk to a concentration of 103-109 cfu/mL were performed. The contaminated milk samples were processed using kit Maldi Sepsityper® (Bruker Daltonics) and subjected to bacterial lysis protocol for analysis by MALDI-TOF MS. Mass spectra were collected in the mass range of 2000-20000 m/z and were analyzed by MALDI Biotyper 3.0 software (Bruker Daltonics) with default settings to obtain bacterial identification. Direct identification of mastitis causing pathogens from milk samples was possible at ≥106 cfu/mL for S. aureus, ≥107 cfu/mL for E. coli and ≥108 cfu/mL for S. agalactiae, S. dysgalactiae and S. uberis. In experiment 2, the objective was to evaluate the effect of pre-incubation of milk samples from mammary quarters with subclinical mastitis on the effectiveness of identification without cultivation, of mastitis-causing pathogens by MALDI-TOF MS. We selected mammary quarter milk samples from all lactating cows on two dairy herds. The milk samples were subjected to analyzes of: a) microbiological culture; b) pre-incubation followed by identification by mass spectrometry directly from milk; c) total bacterial count (TBC). Flow cytometry was used to determine TBC and; to directly identify the mastitis-causing pathogens from milk, fat was separated by centrifugation (10,000 Xg for 10 minutes) and; samples were pre-incubated at 37°C for 12 hours. Subsequently, the skim milk samples were submitted to kit Maldi Sepsityper® (Bruker Daltonics) and to the bacterial lysis protocol for analysis by MALDI-TOF MS. A total of 810 milk samples were analyzed by microbiological culture (reference method), of which 347 showed bacterial growth. Considering all culture positive samples 305 were identified as agents of interest in the direct identification by MALDI-TOF MS method: coagulase negative staphylococci (n = 191), S. aureus (n = 31), S. agalactiae (n = 42), S. uberis (n = 37) and S. dysgalactiae (n = 4). Therefore, 305 samples were directly identified by MALDI-TOF MS, which presented low sensitivity when compared to microbiological culture (Reference method): coagulase-negative staphylococci (14.08%), S. agalactiae (15.25 %), S. uberis (1.69%), S. aureus (6.12%) and S. dysgalactiae (0%). Pre-incubation of milk samples did not increase the sensitivity of the MALDI-TOF MS method directly identify mastitis-causing microorganisms.

O controle de mastite consiste em um conjunto de medidas de manejo e prevenção da doença em um rebanho, pois estudos realizados em todo estado de São Paulo estimaram um prejuízo de 17% da produção por propriedade devido à mastite considerando perdas como os gastos com tratamentos; descarte de leite; queda na produção; além de perdas por resíduos de antibióticos. O trabalho mostra que a população microbiana da mucosa dos tetos não varia conforme a sua posição no úbere do animal e a identificação dos microorganismos predominantes foram como sendo de Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae e Acinetobacter junii. O objetivo deste trabalho foi determinar a eficácia do muco de escargot nos tetos de vacas leiteiras utilizando-o como \"pré e pós dipping\" e o comparado com a aplicação de solução de iodo, visando à prevenção ou a redução da incidência de infecções intramamárias. O muco de escargot possui uma propriedade antibacteriana bastante efetiva, conhecida como Achacin. Sua ação bactericida ocorre principalmente na fase de crescimento da bactéria agindo como agente quimioterápico. O muco de escargot mostrou ser igualmente eficiente no controle da população de microorganismo presente na superfície dos tetos, quando comparados ao uso da solução de iodo, além de que o seu efeito sobre a pele e mucosa dos tetos foi de deixá-los mais hidratados e com melhor elasticidade evitando rachaduras e focos de infecção, quando comparado ao agente mineral, podendo ser até mesmo um produto alternativo no controle e prevenção de mastite em vacas leiteiras. / The control of mastitis is a set of measures for management and prevention of disease in a herd, because studies from every state of Sao Paulo have estimated a loss of 17% in a property considering losses due to mastitis as spending on treatments; discarded milk, drop in production, and losses due to antibiotic residues. The study shows that the microbial population of the mucosa of the teats doesnt vary according to their position in the animal\'s udder and identification of microorganisms were predominant as Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae and Acinetobacter junii. The objective of this study was to determine the effectiveness of snail mucus teats of dairy cows using it as a \"pre and post dipping\" and compared with the application of iodine solution, aiming at preventing or reducing the incidence of mammary infections. The mucus of snail has a very effective antibacterial property, known as Achacin. Its bactericidal action occurs mainly in the growth of bacteria by acting as a chemotherapeutic agent. The mucus of snail proved to be equally effective in controlling the population of microorganisms present on the surface of the teats, compared to the use of iodine solution, and that its effect on the skin and mucous membrane of the teats was leaving them more hydrated and with improved elasticity avoiding cracks and foci of infection, when compared to mineral agent, which may even be an alternative product in the control and prevention of mastitis in dairy cows.

A mastite representa um grande desafio na pecuária leiteira, visto que é uma das afecções que mais acometem o rebanho bovino ocasionando grande impacto econômico. As bactérias são importantes agentes associados à enfermidade, sendo que as mais comumente encontradas são as do gênero Staphylococcus, associadas tanto às manifestações clínicas quanto subclínicas. A terapia antimicrobiana é usualmente requerida como tratamento, auxiliando as defesas do animal para a eliminação do agente invasor, sendo assim, de suma importância monitorar a sensibilidade dos patógenos aos antimicrobianos. Visto que a resistência aos medicamentos utilizados tem se tornado frequente, há a necessidade de estudos mais abrangentes sobre o assunto. Desta forma, o presente estudo avaliou 300 isolados de Staphylococcus provenientes de amostras de leite de bovinos com mastite clínica e/ou subclínica de propriedades de exploração leiteira. A espécie mais detectada nas análises foi S. aureus, e dentre os genes que codificam para adesinas e biofilmes os mais frequentes foram (eno, fib e fnbA) e (bap, icaA, icaD). A combinação mais frequente no tocante às adesinas foi eno-fib-fnbA, e para os biofilmes foram bap e bap-icaD. Os maiores índices de resistência foram verificados para os antimicrobianos betalactâmicos (amoxicilina, ampicilina e penicilina). Identificou-se as maiores frequências de sensibilidade para cefalotina, seguida pela oxacilina e gentamicina, e não foi detectada relação de concordância da oxacilina com os betalactâmicos. Avaliou-se a concentração mínima inibitória (MIC) para ampicilina, gentamicina, oxacilina e penicilina, para todas as cepas resistentes no antibiograma. Posteriormente, investigou-se os genes responsáveis pela codificação de resistência antimicrobiana, com os genes femA e femB sendo os mais comuns, porém o gene femA não foi detectado em todas as cepas de S. aureus. Os genes mecA e bla<//i>Z foram identificados, porém em baixa frequência, e o homólogo de mecA, o mecALGA251, somente em duas cepas. As informações obtidas podem ajudar em diferentes aspectos acerca dos perfis dos microrganismos no tocante aos fatores de virulência dos mesmos, permitindo novas abordagens relativas a terapias e medidas de prevenção à mastite. / Mastitis represents a major challenge in dairy farming, since it is one of the affections that most impact the cattle herd, causing great economic distress. Bacteria are important agents associated with the disease, and the most commonly found are those of the genus Staphylococcus, associated with both clinical and subclinical manifestations. Antimicrobial therapy is usually required as a treatment, assisting the animal\'s defenses to eliminate the invading agent, and it is therefore of paramount importance to monitor the susceptibility of pathogens to antimicrobials. Since resistance to drugs commonly used has become frequent, there is a need for more comprehensive studies on the subject. Thus, the present study evaluated 300 isolates of Staphylococcus from samples of dairy cattle from dairy farms. The most prevalent species in the analyses were S. aureus, and among the genes coding for adhesins and biofilms the most frequent combinations were (eno, fib and fnbA) and (bap, icaA, icaD). The most frequent combination for adhesins was eno-fib-fnbA, and for biofilms they were bap and bap-icaD. The highest resistance indices were verified for betalactam antibiotics (amoxicillin, ampicillin and penicillin). The highest frequencies of sensitivity were identified for cephalothin, followed by oxacillin and gentamicin, and no concordance relationship was found between oxacillin and betalactam. Minimum inhibitory concentration (MIC) for ampicillin, gentamicin, oxacillin and penicillin were performed for all strains resistant to the antibiogram. Subsequently, the genes responsible for the encoding of antimicrobial resistance were investigated, with the femA and femB genes being the most common, but the femA gene was not detected in all strains of S. aureus. The mecA and blaZ genes were identified, but at low frequency, and the mecA homolog, mecALGA251, was only found in two strains. The information obtained can help in different aspects about the microorganisms\' profiles regarding their virulence factors, allowing new approaches to therapies and mastitis prevention measures.

Foram submetidas a prova do \"California Mastitis Test\"(CMT), 468 vacas leiteiras pertencentes a seis propiedades rurais produtoras de leite tipo B, localizadas na região do Município de Barretos, SP. Das 56 (12,0 por cento ) fêmeas reagentes a esta prova, 48 (1O,3 por cento ) foram confirmadas através do isolamento e identificação do agente etiológico. A elevada concordância (85,71 por cento ) verificada entre, a prova do CMT e o exame bacteriológico, confirma a eficiência desta prova na detecção de transtôrnos da glândula mamária não perceptíveis clínicamente, uma vez que nenhuma das fêmeas analisadas apresentou sinais da enfermidade. A contagem leucocitária e a pesquisa do teor de cloretos efetuadas nas 56 fêmeas reagentes a prova do CMT, apresentaram concordâncias de 87,5 por cento e 50,0 por cento , respectivamente, em relação ao exame bacteriológico. O Staphylococcus aureus foi o agente etiológico mais frequente (52,08 por cento ) nos casos de mastite bovina. O estudo comparativo entre a contagem padrão em placas e a prova da redução do azul de metileno, efetuado nas 56 amostras reagentes a prova do CMT, revelou que 30 (53,57 por cento ) amostras apresentaram tempo de redução não condizente com o número de microrganismos determinado, isto é, tempo de redução menor que 3 l/2 horas e contagem total de microrganismos inferior a 500.000 microrg/ml. Observou-se, ainda, que destas 30 amostras, 26 (86,67 por cento ) apreesentaram contagens leucocitárias superiores a 2.500.000 leucócitos/ml., sugerindo deste modo, uma provável interferência dos leucócitos quando em número elevado, na prova da redução do azul de metileno. O estudo comparativo entre a contagem padrão em placas e a prova da redução do azul de metileno, efetuado nas 30 amostras negativas a prova do CMT, revelou que 27 (90,0 por cento ) amostras apresentaram o tempo de redução condizente com o número de microrganismos determinado, o que confirma a importância de sua utilização na rotina do Serviço de Inspeção. A provãvel interferência dos leucócitos não compromete a eficiência desta prova, pois as variações observadas ocorrem no sentido de abreviar o tempo de redução, sendo muito pequena a probabilidade de aceitação de um produto com o número de microrganismos superior ao permitido pelo Ministério da Agricultura. Dentre as provas utilizadas pelo Serviço de Inspeção na detecção da mastite bovina a nível de Estabelecimentos Industriais a prova de Whiteside foi a que apresentou maior sensibilidade e especificidade, capaz de revelar a presença de leite anormal em rebanhos cuja prevalência desta doença foi igual ou superior a 7,0 por cento das fêmeas ou 1,4 por cento dos quartos analisados. A pesquisa do teor de cloretos apresentou menor sensibilidade e especificidade, incapaz de revelar a presença de leite anormal em todos os rebanhos analisados. Entretanto, este fato não invalida a utilidade de sua aplicação, pois esta prova pode ser de fundamental importância na detecção de fraudes no leite. A prevalência da mastite bovina em até 20,0 por cento das fêmeas leiteiras ou em ate 5,0 por cento dos quartos examinados, não foi suficiente para colocar os quesitos analisados nas amostras de leite, fora dos padrões normais. Acredita-se que estes padrões devam ser objeto de estudos posteriores. / Four-hundred and sixty-eigth milk cows, belonging to six type B milk producing dayry farms, located in the region of Barretos, SP, Brazil were submitted to \"California Mastitis Test\", of the fifty-six (12,0 per cent ) cows positive to the mentioned test, forty eigth (10,31) had the diagnosis confirmed throght isolation and identification of the etiologic agent. High agreement (85,7 per cent ) was obtained between CMT and bacteriologic examination, which confirms the efficiency of CMT in the detection of diseases of the mammary glands, not perceptible by clinical examination, as none the animals examined presented signs of disease. Leucocyte count and determination of chloride level performed on the fifty-six reactors presented agreement of 87,5 per cent and 50,0 per cent , respectively, with bacteriological examination. Staphylococcus aureus was the most frequently isolated bacterium (52,08 per cent of cases). Comparison between total plate count and reductase test, performed on thirty samples negative to CMT, showed that twenty-seven samples (90,0 per cent ) presented a reduction time compatible with the number of microorganisms present, confirming the importance of reductase test regarding inspection. The theoretical interference of leucocytes was not important in practice, as variations observed were abreviation of reduction time, rendering the propability of accepting a product with a number of microorganisms over legal limits very small. Considering tests used by the Inspection Service for detection of bovine mastitis, the Whiteside test presented highest sensivity and specificity, as it was capable of reveling abnormal milk when prevalence was 7,0 per cent and over, considering animals and 1,1 per cent considering quarters. Chloride level presented lower sensitivity and specificity as it was unable to detect abnormal milk in all dairy farms. However it is very important in the detection of frauds. Prevalence of mastitis of up to 20,0 per cent of animals or of 5,0 per cent of quarters was not sufficient to bring the total milk produced below standards.