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Fontes ambientais de infeção por Cryptosporidium parvum em vitelos leiteiros recém nascidos

Publication . Rodrigues, Maria Leonor Frangão Rézio Falcão; Bexiga, José Ricardo Dias; Guerreiro, Dário Alexandre Nunes de Sá (tutor)

Cryptosporidium parvum é um protozoário parasita responsável pela infeção intestinal de humanos e animais, causando alta morbilidade e morte em hospedeiros imunocomprometidos, e é considerado uma das principais causas de diarreia em vitelos. A principal via de transmissão dos oocistos é fecal-oral. Este parasita representa um problema relevante tanto para a saúde e produção animal como para a saúde pública, uma vez que alguns genótipos são zoonóticos. O presente trabalho visou avaliar possíveis fontes de contaminação por C. parvum em vitelos numa vacaria leiteira, o que pode ajudar a desenvolver estratégias mais eficazes para prevenir e controlar a disseminação do parasita, protegendo a saúde e o bem-estar tanto de animais como de humanos. Amostras fecais de vacas adultas foram recolhidas nos períodos pré-parto (n=6) e pós parto (n=7), assim como ao parto (n=7). Foram colhidas diariamente amostras de fezes dos respetivos 7 vitelos desde o nascimento até ao 21º dia de vida. As amostras ambientais incluíram luvas, cama da maternidade, placas de Petri para recolha de aerossóis da limpeza com água a alta pressão e zaragatoas de diversos materiais como botas, jaulas, baldes, tetinas, comedouros e sondas esofágicas. A metodologia baseou-se na identificação de oocistos de Cryptosporidium spp. pela coloração de Ziehl-Neelsen e imunofluorescência direta. Foi extraído DNA das amostras ambientais e das amostras fecais dos vitelos ao 10º dia e analisado por PCR 18S rRNA para confirmar a presença de Cryptosporidium spp. Algumas jaulas pré-entrada (4/6), baldes de água (2/5), tetinas (1/5), comedouros (4/5), placas de Petri (2/7), cama da maternidade (1/1), todos os bezerros (7/7), jaulas de saída (7/7), luvas (2/2) e botas (2/2) testaram positivos. A presença de Cryptosporidium parvum foi confirmada por RFLP. A análise da sequência do gene gp60 confirmou o genótipo como IIaA15G2R1. Dado que as amostras das 7 vacas revelaram-se negativas, podem ser excluídas como uma potencial fonte de contaminação para os vitelos. No dia 6, 4 de 7 vitelos estavam positivos e ao 7º dia todos testaram positivo, sugerindo que a infeção ocorre entre os dias 0 e 4, considerando um período pré-patente de 3 a 6 dias. Cryptosporidium spp. foi detetado em todos os tipos de amostras ambientais (exceto nas sondas esofágicas), suportando a hipótese de que o problema está nas estratégias de maneio e limpeza. Nestas condições, os vitelos recém-nascidos são expostos ao parasita desde o dia 0, o que pode justificar a alta prevalência da infeção e doença em vacarias leiteiras

2024-10-31Master thesis

Embargoed access

When the solution becomes the problem: a review on antimicrobial resistance in dairy cattle

Publication . Pires, Ana José; Pereira, Gonçalo; Fangueiro, David; Bexiga, Ricardo; Oliveira, Manuela

Antibiotics' action, once a ‘magic bullet’, is now hindered by widespread microbial resistance, creating a global antimicrobial resistance (AMR) crisis. A primary driver of AMR is the selective pressure from antimicrobial use. Between 2000 and 2015, antibiotic consumption increased by 65%, reaching 34.8 billion tons, 73% of which was used in animals. In the dairy cattle sector, antibiotics are crucial for treating diseases like mastitis, posing risks to humans, animals and potentially leading to environmental contamination. To address AMR, strategies like selective dry cow therapy, alternative treatments (nanoparticles, phages) and waste management innovations are emerging. However, most solutions are in development, emphasizing the urgent need for further research to tackle AMR in dairy farms.

2024-04Journal article

Restricted access

Antibiotic-lipid based nanosystem as a tool to specifically target Staphylococcus aureus biofilms

Publication . Ferreira, M.C. Magda Sofia Catroga Ferreira; Aguiar, Sandra Isabel Rodrigues de; Gaspar, Maria Manuela de Jesus Guilherme; Bettencourt, Ana Francisca de Campos Simão

Abstract - Hospital acquired infections (HAIs) is one of the leading causes of death worldwide, with Staphylococcus aureus being among the most prevalent microorganisms implicated in these infections. The ability of S. aureus to form biofilms and evade the immune system, along with the emergence of multidrug-resistant strains (MDR), exacerbates the complexity of eradicating infections. Conventional therapies characterized by prolonged antibiotic regiments have a poor rate of success, mainly due to the reduced penetration of antibiotics through the biofilms and low accumulation levels at infected sites. The ineffectiveness of current treatments has stimulated extensive research into the development of innovative therapeutic approaches. To address this need, the aim of this thesis was the development of a nanosystem by incorporating an antibiotic into liposomes, taking advantage of the unique benefits offered by these nanotechnological platforms. For this purpose, we first explored the antibacterial activity of three common antibiotics in clinical use – levofloxacin (LEV), vancomycin (VCM) and rifabutin (RFB) – against a reference strain of S. aureus (ATCC®25923™) in both planktonic and biofilm states. Subsequently, the antibiotics were incorporated into liposomes with different lipid compositions, and their incorporations parameters were assessed. Free RFB displayed the most potent antibacterial effect with MIC and MBIC50 below 0.006 µg/mL, along with the highest antibiotic loading capacity when nanoformulated, preserving its antibacterial activity. Based on these results, RFB was selected for further in vitro studies and the influence of the different lipid compositions on bacterial biofilm interactions was assessed, using a biofilm transwell model and confocal scanning laser microscopy analysis. It was observed that the positively charged RFB liposomes (LIP3) exhibited the highest interaction with biofilms. Nevertheless, RFB incorporated in negatively charged liposomes with fusogenic properties (LIP1) displayed lower MBIC50 values. Preliminary safety assessment of RFB formulations towards osteoblast and fibroblast cell lines indicated that a reduction in cell viability was only observed for the LIP3. Taking this into account, LIP1 was selected to move forward. Following these findings, the potential of free RFB was validated in a collection of S. aureus clinical isolates to provide a more accurate reflection of the challenges faced in real-world settings, in both planktonic (n=114) and biofilm (n=40) states. Additionally, the antibacterial activity of RFB incorporated in our developed liposome (LIP1) was validated against a set of clinical isolates (n=40) in both states. In conclusion, all the work developed contributed to the pursuit of effective therapeutic strategies for planktonic and biofilm-associated S. aureus infections, by exploring the potential of antibiotic repurposing and incorporating them into liposomes

2024-09-18Doctoral thesis

Open access

Impact of high Spirulina diet, extruded or supplemented with enzymes, on blood cells, systemic metabolites, and hepatic lipid and mineral profiles of broiler chickens

Publication . Spínola, Maria P.; Alfaia, Cristina M.; Costa, Mónica M.; Pinto, Rui M. A.; Lopes, Paula A.; Pestana, José M.; Tavares, João C.; Mendes, Ana R.; Mourato, Miguel; Tavares, Beatriz; Carvalho, Daniela F. P.; Martins, Cátia F.; Ferreira, Joana I.; Lordelo, Madalena; Prates, José A. M.

The impact of 15% dietary inclusion of Spirulina (Arthrospira platensis) in broiler chickens was explored, focusing on blood cellular components, systemic metabolites and hepatic lipid and mineral composition. From days 14 to 35 of age, 120 broiler chickens were divided and allocated into four dietary treatments: a standard corn and soybean meal-based diet (control), a 15% Spirulina diet, a 15% extruded Spirulina diet, and a 15% Spirulina diet super-dosed with an enzyme blend (0.20% porcine pancreatin plus 0.01% lysozyme). The haematological analysis revealed no significant deviations (p > 0.05) in blood cell counts across treatments, suggesting that high Spirulina inclusion maintains haematological balance. The systemic metabolic assessment indicated an enhanced antioxidant capacity in birds on Spirulina diets (p < 0.001), pointing toward a potential reduction in oxidative stress. However, the study noted a detrimental impact on growth performance metrics, such as final body weight and feed conversion ratio (both p < 0.001), in the Spirulina-fed treatments, with the super-dosed enzyme blend supplementation failing to alleviate these effects but with extrusion mitigating them. Regarding hepatic composition, birds on extruded Spirulina and enzyme-supplemented diets showed a notable increase in n-3 fatty acids (EPA, DPA, DHA) (p < 0.001), leading to an improved n-6/n-3 PUFA ratio (p < 0.001). Despite this positive shift, a reduction in total hepatic lipids (p = 0.003) was observed without a significant change in cholesterol levels. Our findings underscore the need for further exploration into the optimal inclusion levels, processing methods and potential enzymatic enhancements of Spirulina in broiler diets. Ultimately, this research aims to strike a balance between promoting health benefits and maintaining optimal growth performance in poultry nutrition.

2024-03Journal article

Open access