The purpose of this study was to evaluate the detection limit of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct identification, without previous microbiological culture, of bovine mastitis-causing bacteria from milk samples. Milk samples (n = 15) were experimentally contaminated with Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli to have bacterial counts ranging from 10 to 10 cfu/mL. These contaminated milk samples were subjected to a preparation protocol for bacterial ribosomal protein extraction using the MALDI Sepsityper kit (Bruker Daltonik, Bremen, Germany), which allowed MALDI-TOF MS coupled with Biotyper software (Bruker Daltonik) to identify bacterial fingerprints based on intact ribosomal proteins. The ability of MALDI-TOF MS to correctly identify bacterial strains from experimentally contaminated milk (without previous microbiological culture) depended on the bacterial count of the samples and on the species of the bacteria evaluated. Adequate identification at the bacterial species level (score ≥2.0) directly from milk samples required bacterial counts in the following ranges: ≥10 cfu/mL of Staph. aureus, ≥10 cfu/mL of E. coli, and ≥10 cfu/mL of Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis. We concluded that direct identification of mastitis-causing pathogens is possible for Staph. aureus, E. coli, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis, but correct identification depended on the bacterial count in the milk samples.

Staphylococcus aureus (S. aureus) is the most prevalent infectious microorganism affecting dairy cattle worldwide, and its pathogenic characteristics facilitate its spread in dairy herds. S. aureus intramammary infections (IMI) are mainly subclinical, and associated losses can exceed average herd losses where the pathogen is not isolated. However, the extent it affects milk composition at udder and quarter levels is still unknown, and cow composite milk losses may be underestimated due to the dilution effect. The aim of this study was to investigate the effects of S. aureus subclinical mastitis on mammary quarter milk yield and composition. In order to determine the effects of the pathogen on milk yield and composition at quarter level, a pairwise comparison of infected and non-infected mammary quarters (n = 28) from two dairy herds was carried out. Quarters were individually milked, and milk production and composition were assessed. S. aureus has increased somatic cell counts at quarter level; however, no effect of S. aureus IMI on milk lactose, fat, and protein contents was observed. Fat yield from infected quarters decreased, but losses due to the infection caused by S. aureus were not associated with quarter positioning in cows.

ABSTRACT. The aim of this study was to evaluate the repeatability and performance of 4 methods of extracting DNA from Staphylococcus aureus (SAU) and the gene encoding bovine mitochondrial cytochrome B (BMCB) in milk samples from cows with subclinical mastitis for use in amplification by real-time polymerase chain reaction. Two milk samples were obtained from cows naturally infected with S. aureus and subjected to the following extraction methods: Qiagen DNA extraction kit; Axyprep DNA extraction kit; in silica column boil and in silica column method. After extraction in duplicate, eluates were subjected to purification and precipitation to determine purity (A 260 /A 280 ratio) and concentration (μg/ μL) by spectrophotometry and amplification by real-time polymerase chain reaction of target genes (SAU and BMCB). There was no effect of the DNA extraction method on DNA concentration and threshold cycle for BMCB and SAU. The purity ratio (A 260 /A 280 ) was higher when using Qiagen DNA extraction (1.76 ± 0.136) compared to the other methods tested. Our results indicate that the DNA extraction kit from Qiagen produces samples of the highest purity ratio compared to other methods.

The objective of the present study was to evaluate the qPCR for detection and enumeration of Staphylococcus aureus and Streptococcus agalactiae using different milk samplings in comparison to the conventional microbiology. Four dairy herds with a history of subclinical mastitis caused by S. aureus and S. agalactiae were selected. Sampling approach included milk samples from bulk tank (BT), cow level (composite samples, CO), and mammary quarter level (MQ) from 785 lactating cows. Three consecutive monthly milk samplings were carried out, totaling 3347 MQ milk samples, 912 CO, and 12 from BT. All collected milk samples were subjected to conventional microbiology and qPCR for detection and enumeration of S. aureus and S. agalactiae. The qPCR showed 71.5% of diagnostic sensitivity for S. aureus isolated from MQ milk samples, 71.8% for CO, and 50% for BT milk samples compared with conventional microbiology methodology. Taken together, the diagnostic sensitivity for S. agalactiae isolated from MQ milk samples was 90.2, 87.7 for CO, and 90.9% for BT milk samples. In general, the qPCR methodology enabled the detection of S. aureus and S. agalactiae, regardless of the type of milk sampling. The direct use of milk samples to estimate the counting of S. aureus by qPCR demonstrated lower sensitivity than the counting of S. agalactiae, which can be explained by the pathogen infection dynamics and differences in milk sample type.

DEDICATÓRIAAos meus pais, Roseli e Osmin, pelo amor, ensinamentos, educação, respeito, companheirismo e preocupação recebidas em todos os momentos dessa longa jornada.A minha irmã, Caroline, pela amizade, risadas, intrigas e conhecimentos compartilhados durante todos os momentos.As minhas tias, Rosangela e Edileni, e as minhas avós, Antônia e Wilma, pelo amor, amizade, companheirismo, ajuda e ensinamentos recebidos, compartilhados e retribuídos durante todos esses anos.O meu Muito Obrigada!!!!! AGRADECIMENTOSAgradeço primeiramente a Deus pela minha família, pelas oportunidades que me foi cedida, pelos ensinamentos sem respostas e por colocar pessoas incríveis no meu caminho.Agradeço aos meus pais, Osmin Cesar Jacon Dibbern e Roseli Jeronymo Gerato Dibbern, a minha irmã, Caroline Gerato Dibbern, as minhas tias, Edileni Jeronymo Gerato e Rosangela Jeronymo Gerato e as minhas avós, Wilma Jeronymo Gerato e Antônia Maria Jacon Dibbern pelo amor, ensinamentos e princípios de vida construídos. Ao Prof. Marcos Veiga dos Santos, meu orientador, pela oportunidade de elaborar mais um trabalho juntos, pela confiança, ensinamentos, conselhos e amizade.Ao CNPq pela concessão da bolsa de mestrado durante todo esse período.À amiga Juliana Regina Barreiro Raspantini (Ju) que esteve sempre presente em todos os momentos do meu mestrado com ensinamentos, palavras amigas, conselhos e risadas.Certamente sua presença foi fundamental para execução e elaboração deste experimento.Muito obrigada por ter estado ao meu lado e espero ter ajudado a expandir seus conhecimentos e contribuído de alguma forma com seu crescimento pessoal e profissional.Ao amigo Bruno Garcia Botaro, que mesmo longe sempre esteve ao meu lado compartilhando seus conhecimentos, sua amizade e seu respeito.À amiga e filha Mariana Pallú Viziak pela amizade, conselhos, risadas e por permitir que eu pudesse compartilhar meus ensinamentos com você. Espero ter contribuído com seu crescimento pessoal e profissional durante meu mestrado e sua iniciação científica. CAPÍTULO -INTRODUÇÃOA mastite é uma das principais doenças de rebanhos leiteiros, pois é apresenta alta prevalência e causa grandes prejuízos aos produtores, em razão da redução de produção, alterações da composição do leite, penalizações devido à contagem de células somáticas (CCS) elevada e dos gastos com tratamento (CUNHA et al., 2008 Os métodos convencionais de identificação bacteriana para diagnóstico da mastite baseiam-se, essencialmente, na cultura microbiológica do microrganismo, nas características fenotípicas, bioquímicas e perfil enzimático Em relação à composição do leite, em virtude do aumento de permeabilidade vascular secundário ao processo inflamatório ocorre redução do teor de proteínas sintetizadas na glândula mamária (α e β caseína, α-lactoalbumina e β-lactoglobulina) e aumento das proteínas de origem sanguínea (albumina sérica e imunoglobulinas) . O teor de proteína total do leite tem pouca variação, mas a concentração de cada tipo de proteína varia acentuadamente .Normalmente, existe uma rel...