Center for Interdisciplinary Research in Animal Health

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Modulation of ruminal biohydrogenation in sheep through dietary tannins or energy sources

Publication . Costa, Mónica Mendes da; Bessa, Rui José Branquinho de

In the present thesis, four experiments were conducted to study how ruminal biohydrogenation pathways can be modulated through dietary inclusion of tannin sources and to acquire a better comprehension about the occurence of t10-shifted biohydrogenation pathways. In the first experiment, in vitro batch incubations with 100 g/kg dry matter (DM) of extracts of chestnut tannins (mostly hydrolysable tannins) and quebracho, grape seed or rockrose (Cistus ladanifer) condensed tannins, as well as a control treatment were incubated for 6 h with ruminal fluid from fistulated sheep and a dehydrated lucerne-based substrate with 60 g/kg DM of sunflower oil. Grape seed and, to a lesser extent, C. ladanifer led to a higher disappearance of 18:2n-6 with a consequent higher production of c9,t11-18:2 and t11-18:1 than chestnut, quebracho and control. There was no clear innibition of 18:0 production with any of the extracts comparing with control. In the second experiment, rumen fistulated sheep were fed tannin extracts from mimosa condensed tannins, chestnut hydrolysable tannins or their mixture (100 g/kg DM) in a complete diet with sunflower and linseed oils (40 g/kg DM), following a change-over design (3 treatments, 4 sheep and 4 periods). There was a variable inhibition of ruminal biohydrogenation and a lower “trans-/cis-18:1” ratio in bacterial fractions with mimosa than with chestnut. Mimosa led to a lower fermentative activity, as well as a lower abundance of Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio proteoclasticus and higher abundance of Selenomonas ruminantium with a lower bacterial biomass estimate of dimethylacetals than chestnut. In the third experiment, two rumen fistulated rams were housed in metabolic cages and adapted to a wheat-based diet with 41 g/kg DM of sunflower oil. During the first two weeks of trial, the t10-shift occurred temporarily in both animals but in different moments. These results were probably due to individual variability of rumen microbiota, since, for a selected period of the trial, a lower bacterial diversity was found for ram 1 compared to ram 2. Moreover, the t10-shift was associated with an increase of total trans-18:1 and a decrease of 18:0. There was no clear association of t10-shift with rumen pH or its expression in blood plasma. In the fourth experiment, 40 lambs were fed, for 6 weeks, with complete diets containing barley or barley completely replaced for dehydrated citrus pulp, dehydrated beet pulp or soybean hulls. All diets were supplemented with an oil blend (soybean:fish oils, 59:10 g/kg DM). Overall, the t10-/t11-18:1 ratio was above 3 in meat and subcutaneous fat, although soybean hulls increased t11-18:1 and c9,t11-18:2 comparing with the other treatments. Citrus pulp led to the lowest gene expression of fatty acid synthase, while that of stearoyl-CoA desaturase was inferior for soybean hulls and beet pulp.

2017-09-11Doctoral thesis

Open access

Parasitas gastrointestinais em produção de frango ao ar livre

Publication . Lozano, João; Gomes, Lídia; Madeira de Carvalho, Luís

A investigação em sanidade animal realizada a nível nacional em sistemas extensivos de produção de frango é essencial, atendendo à frequente longa exposição dos animais ao ambiente exterior e aos parasitas que o contaminam. Estudos parasitológicos são extremamente relevantes de forma a conhecer quais as espécies parasitárias mais prevalentes e ajudar os produtores a definirem melhores medidas profiláticas. Esta pesquisa procurou identificar os parasitas gastrointestinais associados a este tipo de produção aviária e ocorreu numa exploração agropecuária localizada na extremidade noroeste do distrito de Lisboa, onde foram avaliados Frangos do Campo através de amostragem fecal aos 75 e 85 dias de idade. O parasitismo gastrointestinal foi analisado através de métodos coprológicos qualitativos, quantitativos e coproculturas para obtenção de oocistos esporulados e larvas L3 de nematodes estrongilídeos. Na 1ª amostragem, a prevalência de Eimeria spp. totalizou 40,0%, sendo Eimeria mitis (13,3%) e E. maxima (11,7%) as mais prevalentes, tendo-se identificado também ovos de Capillaria sp., (1,7 % de prevalência). Nas coproculturas de oocistos, esporularam as seguintes espécies: E. necatrix, E. mitis, E. praecox, E. maxima, E. acervulina e E. tenella. Na 2ª amostragem, as coccídeas continuaram a ser o grupo mais prevalente, 90,0% de amostras positivas com oito espécies identificadas, sendo de novo E. mitis a espécie mais frequente (41,7%), seguida por E. acervulina (26,7%) e E. tenella (25%). Foram também observados ovos de Heterakis sp. (3,33%) e novamente de Capillaria sp. (10,0%) A carga parasitária fecal de oocistos na 1ª e 2ª amostragens foi de 370,8 ± 2055,3 OoPG e 599,2 ± 1289,2 OoPG, respetivamente, verificando-se um aumento superior a 60% do valor médio deste parâmetro parasitológico da 1ª para a 2ª colheita. O crescimento da erva, a idade e densidade animal, bem como o tempo de exposição a parasitas com períodos pré-patentes curtos, constituíram fatores chave para o aumento da prevalência de oocistos de Eimeria spp. e ovos de helmintes entre as duas amostragens, em particular de espécies com reconhecida patogenicidade. Isto permite-nos concluir que no final da fase de engorda, as cargas parasitárias são as mais elevadas detetadas neste sistema de produção de Frango do Campo, podendo desencadear patologia associada e interferir com a sua performance produtiva.

2018Journal article

Open access

Impact of leporid viral diseases in Iberian ecosystems : emergence, pathophysiology, prophylaxis and diagnosis

Publication . Santos, Fábio Alexandre Abade dos; Duarte, Margarida Dias; Peleteiro, Maria da Conceição Cunha e Vasconcelos; Parra Fernández, Francisco

ABSTRACT - The alarming state of conservation of leporid species in the Iberian Peninsula justifies the adoption in this Doctoral Thesis of an integrative approach that includes the study of different aspects of the virus-host-environment interfaces with respect to the main viral pathogens of the wild rabbit (Oryctolagus cuniculus algirus) and the Iberian hare (Lepus granatensis) in Mediterranean ecosystems. The studies to which this thesis refers to have used and developed methodologies of molecular and cellular biology, genetics, virology, immunology and pathology, applied to the wild rabbit and the Iberian hare, establishing as objectives of this Thesis the pathophysiological research of the diseases caused by rabbit hemorrhagic disease virus (RHDV2), myxoma virus (MYXV) and leporid gammaherpesvirus 5 (LeHV-5), their impact on affected species, the design of new diagnostic methods and the search for control measures to mitigate the effects of these agents. In addition to reflecting on the correct interpretation of molecular diagnoses in the context of the virus-host relationship, as a previous step to the presentation of the experimental results, some methodologies have been developed which were necessary for their implementation, such as the extraction of blood through the external jugular vein in wild rabbits and Iberian hares, or simple procedures in order to obtain primary cultures from leporid fibroblasts. In this thesis it has been shown that the Eurasian badger (Meles meles) is susceptible to RHDV2 and that this species can act as a potential reservoir of the rabbit hemorrhagic disease virus. It was also shown that the same RHDV2 strain isolated from badgers could infect a dwarf rabbit, who had a prolonged (atypical) illness and a history of vaccination ineffectiveness with a commercial RHDV vaccine still available on the Portuguese market. With regard to myxomatosis, this thesis includes the detection and analysis of the first cases of this disease in Iberian hares, associated with a natural recombinant strain of the virus (ha-MYXV), as well as the first demonstration that the recombinant virus of hares can infect both wild and domestic rabbits. Additionally, evidence of the first cases of co-infection of the recombinant virus ha-MYXV and the classic myxoma virus MYXV is reported, both in wild rabbits and in Iberian hares. In an attempt to provide solutions for the control of myxomatosis in wild leporids, it has been shown that commercial homologous vaccines against myxomatosis are effective in wild rabbits against the new naturally recombinant strain ha-MYXV, but failed to protect the Iberian hare against myxomatosis. ...

2022-12-05Doctoral thesis

Open access

Unraveling the replication process of Toxoplasma gondii through the MOB1 protein

Publication . L. S. Delgado, Inês; Narciso, Sofia Bizarro Nolasco da Silva; Leitão, José Alexandre da Costa Perdigão e Cameira

ABSTRACT - MOB1 is a conserved protein that regulates cellular proliferation versus apoptosis, centrosome duplication and cellular differentiation in multicellular eukaryotes and also cytokinesis and division axis orientation in unicellular and multicellular eukaryotes. Toxoplasma gondii, an obligate intracellular parasite of veterinary and medical importance, presents one MOB1 protein. T. gondii interconverts between several cellular stages during its life cycle, namely between fast replicating tachyzoite and slow replicating bradyzoite stages during its asexual cycle, a key ability for its success as a parasite. Bradyzoites produce tissue cysts, establishing a chronic infection that enables recrudescence. Conversion is dependent on cell cycle regulation and involves cell differentiation and regulation of replication. This led us to select MOB1 as a strong candidate to be involved in the Toxoplasma replication process. We employed reverse genetics to assess the Mob1 function in T. gondii. In opposition to what was observed in other unicellular eukaryotes, as Tetrahymena and Trypanosoma, Mob1 knockout in T. gondii showed no cytokinesis impairment in its asexual cycle. Instead, we observed an increase in replication, a decrease in parasitophorous vacuole regularity and a significant loss in tachyzoite to bradyzoite conversion. Additionally, recombinant MOB1 accumulates in a midline between the daughter nuclei at the end of mitosis, suggesting MOB1 may be involved in this process. To elucidate how MOB1 acts in T. gondii, we employed a proximity biotinylation method and identified the MOB1 interactome. This analysis detected proteins related to several functional categories, indicating a multivalent role for MOB1 regulated by the ubiquitin proteasome system. We also verified that the Mob1 locus is transcribed from both genomic strands and gives rise to alternatively spliced variants. Our results indicate that MOB1 is tightly regulated along the cell cycle and along the life cycle of T. gondii, contributing to the control of replication and tachyzoite-bradyzoite differentiation.

2022-04-29Doctoral thesis

Open access

Virulence of Salmonella typhimurium 1,4,[5],12:i:- : the new emergent strain

Publication . Seixas, Rui Emanuel Antunes de; Oliveira, Maria Manuela Castilho Monteiro de; Bernardo, Fernando Manuel d’Almeida

Salmonella serovar 1,4,[5],12:i:- is presently considered one of the major serovars responsible for human salmonellosis worldwide. A multidisciplinary approach, including the fields of epidemiology, spatial statistics, clinical and applied microbiology was used to perform an extensive characterization of Salmonella 1,4,[5],12:i:- isolates obtained by the National Health Institute Dr. Ricardo Jorge, which was lacking due to the recent emergence. It was observed that cases are reported in most districts, being more frequent in the Portuguese coastland. Spatial statistical analysis showed a significant geographic clustering, pointing out for the importance of evaluating these areas to identify risk factors, in order to establish adequate prevention programs. The most relevant antimicrobial profile in this serovar is the tetra-resistance pattern (R-type ASSuT), displaying resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. A high occurrence of R-type ASSuT isolates was observed in the isolates under study, with the majority harboring the resistance genes frequently associated with the European clone, namely blaTEM, sul2, straA-straB, tetB. Additionally, resistance to quinolones and 3rd generation cephalosporin was also detected. In Portugal, the rapid spread of Salmonella 1,4,[5],12:i:- R-type ASSuT might be related with the diversity of pulsotypes and also the presence of a core of virulence factors, including biofilm production. Biofilm-forming ability varied between sample locations and collection year, and can be one of the virulence features related with the rise of this serovar. Furthermore, biofilm formation was evaluated in vitro using a simulated human intestinal environment. In such conditions was observed an impairment of biofilm production, revealing that conditions mimicking the human intestinal tract can influence the biofilm-forming ability of the isolates under study. This research highlight the critical importance of close surveillance of Salmonella 1,4,[5],12:i:- in Portugal, including R-type ASSuT isolates. Information gathered may unravel Salmonella 1,4,[5],12:i:- features, prevent the dissemination to other regions and also benefit the medical community in order to rationalize salmonellosis antimicrobial therapeutics.

2017-04-10Doctoral thesis

Open access