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Subclinical mastitis by Staphylococcus aureus (SAU) and by non-aureus staphylococci (NAS) is a major issue in the water buffalo. To understand its impact on milk, 6 quarter samples with >3,000,000 cells/mL (3 SAU-positive and 3 NAS-positive) and 6 culture-negative quarter samples with

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of -lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

The cows receiving antibiotics for intra-mammary infection (IMI) produce milk that cannot be marketed. This is considered waste milk (WM), and a convenient option for farmers is using it as calf food. However, adding to the risk of selecting resistant bacteria, residual antibiotics might interfere with the gut microbiome development and influence gastrointestinal health. We assessed the longitudinal effect of unpasteurized WM containing residual cefalexin on calf intestinal health and fecal microbiota in an 8-week trial. After 3 days of colostrum, six calves received WM and six calves received bulk tank milk (BM) for 2 weeks. For the following 6 weeks, all 12 calves received milk substitute and starter feed. Every week for the first 2 weeks and every 2 weeks for the remaining 6 weeks, we subjected all calves to clinical examination and collected rectal swabs for investigating the fecal microbiota composition. Most WM calves had diarrhea episodes in the first 2 weeks of the trial (5/6 WM and 1/6 BM), and their body weight was significantly lower than that of BM calves. Based on 16S rRNA gene analysis, WM calves had a lower fecal microbiota alpha diversity than that in BM calves, with the lowest p-value at Wk4 (p < 0.02), 2 weeks after exposure to WM. The fecal microbiota beta diversity of the two calf groups was also significantly different at Wk4 (p < 0.05). Numerous significant differences were present in the fecal microbiota taxonomy of WM and BM calves in terms of relative normalized operational taxonomic unit (OTU) levels, affecting five phyla, seven classes, eight orders, 19 families, and 47 genera. At the end of the trial, when 6 weeks had passed since exposure to WM, the phyla Bacteroidetes, Firmicutes, and Saccharibacteria were lower, while Chlamydiae were higher in WM calves. Notably, WM calves showed a decrease in beneficial taxa such as Faecalibacterium, with a concomitant increase in potential pathogens such as Campylobacter, Pseudomonas, and Chlamydophila spp. In conclusion, feeding pre-weaned calves with unpasteurized WM containing antibiotics is related to a higher incidence of neonatal diarrhea and leads to significant changes in the fecal microbiota composition, further discouraging this practice in spite of its short-term economic advantages.

Pentraxins are a superfamily of conserved molecules with immune functions such as complement activation and opsonization. PTX3 is the prototypic long pentraxin and is produced by different cell populations after pro-inflammatory stimuli. Different studies have demonstrated the up-regulation of PTX3 during ruminant mastitis, but its role is still unknown. The aim of this study was to elucidate the role of PTX3 in the immune response to S. aureus intra-mammary infection (IMI). Given that no data are available on PTX3 expression in goat tissues, we first studied its pattern of expression in goat normal tissues. Then we investigated the role of PTX3 during mammary infection, comparing its expression in healthy and infected blood, milk and tissues. Six healthy goats were infused with PBS in the right udder and with S. aureus in the left udder. Mammary biopsies from udders were collected 30h post infection, formalin fixed and routinely processed for microscopic evaluation or immediately stored in RNAlater. Tissue samples were collected at the slaughterhouse from healthy goats and were immediately stored in RNAlater. Blood and milk were collected from healthy and infected goats; cells from blood and milk were isolated and processed for RNA extraction or for cytospins; milk fat globules were obtained through milk centrifugation and immediately processed for RNA extraction. Total RNA from different organs, blood or milk cells, milk fat globules and mammary tissues was extracted and used as template in qPCR for PTX3. PTX3 expression was investigated by immunohistochemistry on formalin fixed paraffin embedded mammary tissue samples and on cytospins of isolated goat blood and milk cells. PTX3 mRNA was expressed with very high levels in bone marrow, mammary gland, aorta, pancreas, skin and lung. Given the high expression in the mammary gland, we investigated which cell population expressed PTX3. PTX3 was mainly expressed in the apical cytoplasmic portion of mammary gland epithelial cells, and in macrophages. During S. aureus infection PTX3 was up-regulated by epithelial cells. Macrophages and mammary secretum didn’t show PTX3 modulation, but PMNs recruited during infection were variably intensely positive. PTX3 mRNA expression was low in healthy organs and tissues of goats as has been reported indeed the molecules commonly induced after pro-inflammatory stimulation. As expected, PTX3 was constitutively expressed in bone marrow, rich in PMNs and monocytes, in aorta covered by endothelium and in the skin. PTX3 was up-regulated in epithelial mammary cells and in milk cells after S. aureus infection, demonstrating that it represents a first line of immune defense in goat udder. No modulation was observed in macrophages, in the secretum and in the ductal epithelial cells. Further experiments are needed to elucidate the role of PTX3 in the pathogenesis of S. aureus infection., International Journal of Health, Animal Science and Food Safety, Vol 2, No 1s (2015): Proceeding of Veterinary and Animal Science Days

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ciências da Educação O objectivo deste estudo foi conhecer e analisar qual a importância que as mulheres imigrantes brasileiras residentes em Portugal atribuem à educação e acerca de sua inserção no contexto educacional português. O interesse por este tema responde à nossa vivência como estudantes imigrantes, à curiosidade em desvendar o universo da imigração passando pelas aspirações e pelos valores que cada pessoa traz consigo, assim como seus pensamentos e expectativas. Desejamos que esta investigação possa contribuir para que os olhares da educação portuguesa não restrinjam-se apenas aos filhos dos imigrantes, mas que envolvam também os pais, mais especificamente as mulheres, que ambicionam compartilhar e integrar-se na cultura de outro país. A investigação foi dividida em duas partes: a fundamentação teórica e o estudo empírico. A primeira parte da investigação esteve alicerçada num referencial bibliográfico voltado para os assuntos relacionados aos temas: mulher, imigração e educação. A segunda parte teve como foco uma metodologia fundamentada na abordagem qualitativa, mediante entrevistas semi-estruturadas com doze mulheres imigrantes brasileiras. Os critérios de amostra foram: estar morando em Portugal há no mínimo quatro anos e ter idade compreendida entre 20 e 49 anos. Como resultados, identificamos que o nível de escolaridade das mulheres ainda é baixo e aquelas que têm nível superior ainda não conseguiram dar continuidade aos estudos nem inserirem-se na nova comunidade. Para tanto essas mulheres demonstraram acreditar e apostar, apesar das dificuldades serem maiores que as possibilidades, que ter estudo ainda é um indicador de evolução, de status que possibilita o ser humano a tomar decisões certas e a trilhar novos caminhos com maior consciência e responsabilidade.

INTRODUZIONE - Gli Stafilococchi sono i microrganismi che più frequentemente vengono isolati in caso di infezioni mammarie negli allevamenti caprini italiani. Tra questi Staphylococcus aureus è considerato il più pericoloso in quanto produttore di enterotossine e quindi dannoso per la salute umana. Scopo del presente lavoro è stato il monitoraggio per un periodo di cinque mesi, mediante tecniche microbiologiche e molecolari, della presenza di Staphylococcus aureus nel latte e nei formaggi prodotti in un allevamento caprino lombardo dotato di caseificio aziendale. MATERIALI E METODI - Campioni: l'indagine è stata effettuata su prelievi di latte da singola mammella (148 campioni), di latte di massa (3), di cagliata (3) e di formaggi (3) derivati dalla lavorazione effettuata nel periodo preso in esame. Analisi microbiologiche: l'esame batteriologico è stato effettuato sui campioni di latte secondo le procedure indicate dalla FIL-IDF; sui formaggi è stata eseguita la determinazione del numero di stafilococchi coagulasi positivi in Baird Parker RPF Agar secondo norma ISO 6888 1/2. Estrazione del DNA: il DNA è stato estratto direttamente dai campioni di latte seguendo i protocolli descritti in Cremonesi et al. (2006 e 2007). PCR: il DNA estratto è stato amplificato mediante una reazione di multiplex PCR per l'identificazione simultanea dei geni codificanti per la regione 23S dell'RNA ribosomiale di Staphylococcus aureus, per il gene della coagulasi (coa), per il gene della termonucleasi (nuc) e per i geni codificanti le principali enterotossine (sea, sec, sed, seg, seh, sei, sej, sel) (Cremonesi et al., 2005). La reazione di multiplex PCR è stata effettuata con il seguente ciclo: denaturazione iniziale a 94°C per 5 minuti seguita da 30 cicli di 1 minuto a 94°C, 1 minuto a 56°C e 1 minuto a 68°C e un'estensione finale a 72°C per 7 minuti. RISULTATI E CONSIDERAZIONI - Analisi microbiologiche: nell'allevamento è stata trovata una prevalenza di infezione pari al 27.7 %, con l'isolamento di Staphylococcus aureus nel 7.4% dei casi e di Staphylococcus spp. nel 20.2 % dei casi. Anche il monitoraggio del latte di massa, della cagliata e dei formaggi ha confermato la presenza di Staphylococcus aureus. Analisi molecolari: mediante queste analisi la prevalenza di infezione da Staphylococcus aureus nell'allevamento è risultata pari all'8.1 % dei casi. La presenza di Staphylococcus aureus è stata riscontrata nei campioni di latte di massa, nella cagliata e nei formaggi derivati dalla lavorazione. Tutti i ceppi di Staphylococcus aureus isolati e caratterizzati mediante multiplex PCR sono risultati positivi per i geni codificanti per la coagulasi e la termonucleasi. Nessun ceppo di Staphylococcus aureus presente nell'allevamento è risultato enterotossinogeno. Le indagini molecolari da noi effettuate sono risultate un valido strumento d'indagine da affiancare alle analisi microbiologiche; inoltre, i metodi molecolari possono essere applicati anche nelle indagini a livello di filiera, per l'individuazione delle possibili fonti di contaminazione e per monitorare le vie attraverso le quali Staphylococcus aureus si diffonde.