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- The impact of genetic groups (Alentejano and F1 Landrace x Large White pigs) and body weight (90, 120 and 160kg) on blood metabolitesPublication . Amaral, Andreia; Cordeiro, Daniela; Bettencourt, Carlos; Moreira, Olga; Bressan, Maria Cristina; Belo, Ana Teresa; Boari, Cleube Andrade; van Harten, Sofia; Gama, Luis; Almeida, Joao MarquesThis research work was carried out with the goal of studying the impact of genetic groups-GG (Alentejano-AL, n = 30, and F1 Landrace*Large White-F1 pigs, n = 30) and body weight-BW (90, 120 and 160 kg) on plasma metabolites. Blood parameters were correlated with animal production traits, carcass measurements and meat quality. Individual records for feed conversion index (CI) and daily feed intake were recorded on a weekly basis, for a period of 15 weeks. Compared to the F1, AL pigs displayed (P <0.05) higher average levels of glucose, total cholesterol-TC, triglycerides-TG, HDL-cholesterol (HDL), LDL-cholesterol (LDL) and total protein (TP), by about 14.0, 21.0, 42.2, 18.2, 21.2 and 5.0%, respectively. AL pigs (120–160 kg) showed higher TG levels, when compared to the values at 90 kg (2.6 and 1.6 times higher). High TG levels occurred when animals exhibited high daily feed intake (0.450 and 1.810 kg, for AL and F1 pigs). In the AL high TG levels were correlated with high fat deposition, at 120 kg (r = 0.51). At 90 kg, however, high fat deposition was related to HDL (r = 0.59), a lipoprotein associated to cholesterol transport. A progressive increase in ALB was found in the F1, as expected, but AL pigs showed higher and similar ALB means at 90, 120 and 160 kg. As for meat color, AL pigs with high cholesterol were negatively associated to L*, while high TG levels were associated to low b*. Animals with high ALB produced more tender meats (low shear force). Pigs with higher levels of lipid metabolism showed Longissimus thoracis muscles with decreased luminosity and yellowness (meats of a less attractive appearance). However, these meats were tenderer.
- Clonal relatedness of Proteus mirabilis strains causing urinary tract infections in companion animals and humansPublication . Marques, Cátia Filipa Saraiva; Belas, Adriana; Aboim, Catarina; Trigueiro, Graça; Cavaco-Silva, Patrícia; Gama, Luis; Pomba, C.ABSTRACT - Proteus mirabilis is a major cause of urinary tract infection (UTI) in humans and companion animals. This study aimed to evaluate the antimicrobial resistance, virulence and clonal relatedness of P. mirabilis isolated from dogs, cats and humans with UTI. P. mirabilis isolated from companion animals (N = 107) and humans (N = 76) with UTI were compared by PFGE analysis after overnight Nod macro-restriction using Dice/UPGMA with a 1.5% tolerance. Strains were characterized for antimicrobial resistance by disk diffusion. Twenty-four resistance genes and four virulence genes were screened by PCR. Thirty-nine clusters (similarity > 80%) and 73 single pulse-types were detected. Nine clusters included P. mirabilis isolated from community and hospital patients, including strains with 100% similarity. A high number of clusters (43.6%, n = 17/39) included strains from companion animals and humans. Similarity between some companion animal and human strains varied between 80-100%. One strain from a dog was 100% similar to one human community-acquired P. mirabilis. One P. mirabilis from a cat was found to be 94.7% and 92.4% similar to community and hospital patient strains, respectively. P. mirabilis CMY-2-producers did not cluster all together. Nevertheless, cluster C36 included five P. mirabilis from companion animals (similarity 85.8%-95.7%), of which, four (80%) were multidrug-resistant CMY-2-producers. This study shows that companion animals and humans become infected with closely related P. mirabilis strains. The high number of clusters containing companion animals and human strains points to the zoonotic nature of P. mirabilis. These results underline the potential role of companion animals as reservoirs and in the dissemination of uropathogenic P. mirabilis to humans and vice versa.
- Myeloperoxidase inhibition decreases the expression of collagen and metallopeptidase in mare endometria under in vitro conditionsPublication . Amaral, Ana; Fernandes, Carina; Rebordão, Maria Rosa; Szostek-Mioduchowska, Anna; Lukasik, Karolina; Pinto-Bravo, Pedro; Gama, Luis; Skarzynski, Dariusz Jan; Ferreira-Dias, GraçaABSTRACT - Neutrophils can originate neutrophil extracellular traps (NETs). Myeloperoxidase (MPO) is a peroxidase found in NETs associated to equine endometrosis and can be inhibited by 4-aminobenzoic acid hydrazide (ABAH). Metallopeptidases (MMPs) participate in extracellular matrix stability and fibrosis development. The objectives of this in vitro work were to investigate, in explants of mare’s endometrium, (i) the ABAH capacity to inhibit MPO-induced collagen type I (COL1) expression; and (ii) the action of MPO and ABAH on the expression and gelatinolytic activity of MMP-2/-9. Explants retrieved from the endometrium of mares in follicular or mid-luteal phases were treated with MPO, ABAH, or their combination, for 24 or 48 h. The qPCR analysis measured the transcription of COL1A2, MMP2, and MMP9. Western blot and zymography were performed to evaluate COL1 protein relative abundance and gelatinolytic activity of MMP-2/-9, respectively. Myeloperoxidase elevated COL1 relative protein abundance at both treatment times in follicular phase (p < 0.05). The capacity of ABAH to inhibit MPO-induced COL1 was detected in follicular phase at 48 h (p < 0.05). The gelatinolytic activity of activated MMP-2 augmented in mid-luteal phase at 24 h after MPO treatment, but it was reduced with MPO+ABAH treatment. The activity of MMP-9 active form augmented in MPO-treated explants. However, this effect was inhibited by ABAH in the follicular phase at 48 h (p < 0.05). By inhibiting the pro-fibrotic effects of MPO, it might be possible to reduce the development of endometrosis. Metallopeptidase-2 might be involved in an acute response to MPO in the mid-luteal phase, while MMP-9 might be implicated in a prolonged exposition to MPO in the follicular phase.
- Human and companion animal proteus mirabilis sharingPublication . Marques, Cátia Filipa Saraiva; Belas, Adriana; Menezes, Juliana; da Silva, Joana Moreira; Cavaco-Silva, Patrícia; Trigueiro, Graça; Gama, Luis; Pomba, C.Proteus mirabilis is an important pathogen that is associated with urinary tract infections. This study aims to determine the colonization and sharing of P. mirabilis between healthy companion animals and humans that are living together and to evaluate the clonal relatedness of the fecal and clinical stains. Eighteen households (24 humans, 18 dogs, 8 cats) with at least one human–animal pair were studied. Fecal samples were plated onto MacConkey and Hektoen agar and P. mirabilis PFGE analysis (NotI; Dice/UPGMA; 1.5% tolerance) was conducted for the households with multiple positive participants. Antimicrobial-resistance was tested according to CLSI. The fecal P. mirabilis pulse-types were compared with uropathogenic clinical strains (n = 183). Forty-nine P. mirabilis were isolated from eight households. The percentage of colonization in the dogs (44.4%, n = 8/18) was significantly higher (p = 0.0329) than in the humans (12.5%, n = 3/24). Three households had multiple colonized participants. One human–dog pair shared related P. mirabilis strains, which clustered with a clinical strain of animal origin (82.5%). One fecal P. mirabilis strain, from a dog, clustered with two human community-acquired clinical strains (80.9%, 88.9%). To our knowledge, this is the first report of dogs and humans living in close contact and sharing related P. mirabilis strains. The high frequency of colonization in the dogs underlines their possible role as P. mirabilis reservoirs for humans and other dogs.
- Effect of high hydrostatic pressure challenge on biogenic amines, microbiota, and sensory profile in traditional poultry- and pork-based semidried fermented sausagePublication . Borges, Ana F.; Cozar, Almudena; Patarata, Luis; Gama, Luis; Alfaia, Cristina; Fernandes, Maria J.; Fernandes, Maria H.; Perez Vergara, Herminia; Fraqueza, M. J.The processing of traditional poultry- and pork-based semidried fermented smoked sausages needs to bemodernized to improve product quality and further extend its shelf life. The aim of the present study was to applydifferent combinations of high pressure (300 to 600 MPa) and time (154 to 1,800 s) on the sausages using an experimentaldesign based on response surface methodology. The chemical, microbial, and sensory characteristics of sausages treatedwith high-pressure processing (HPP) were investigated. HPP application to semidried fermented sausages resulted incolor changes, which could be dependent on the ingredients, formulation, and smoking conditions used. Nevertheless,none of the HPP treatments applied resulted in detectable changes in sensory properties, as tested in a triangle test andconfirmed by the analysis of focus groups assessment. Significant differences were detected for lactic acid bacteria (LAB)counts from 344 MPa and 1,530 s onward, with a marked decrease for the combination of 600 MPa and 960 s (P<0.05).Coagulase-negativestaphylococcishowed higher tolerance to the increase in pressure than LAB. HPP induced a microbialreduction onEnterobacteriaceae, molds, and yeasts, minimizing the production of the main biogenic amines. However,the polyamines (spermine and spermidine) increased since their metabolic use by microorganisms did not occur. Giventhe reduction of the main spoilage microbial indicators with no detectable sensory changes observed with the binomialcondition of 600 MPa and 960 s, this was chosen as the optimal combination to be further applied.
- Genomic tools for the conservation and genetic improvement of a highly fragmented breed : the ramo grande cattle from the AzoresPublication . Amaral, A J; Pavao, Ana L.; Gama, LuisRamo Grande is a local cattle breed raised in the archipelago of Azores, with a small and dispersed census, where inbreeding control is of utmost importance. A single nucleotide polymorphism (SNP) Beadchip array was used to assess inbreeding, by analysis of genomic regions harboring contiguous homozygous genotypes named runs of homozygosity (ROH), and to estimate past e ective population size by analysis of linkage disequilibrium (LD). Genetic markers associated with production traits were also investigated, exploiting the unique genetic and adaptation features of this breed. A total of 639 ROH with length >4 Mb were identified, with mean length of 14.96 Mb. The mean genomic inbreeding was 0.09, and long segments of ROH were common, indicating recent inbred matings. The LD pattern indicates a large e ective population size, suggesting the inflow of exotic germplasm in the past. The genome-wide association study identified novel markers significantly a ecting longevity, age at first calving and direct genetic e ects on calf weight. These results provide the first evidence of the association of longevity with genes related with DNA recognition and repair, and the association of age at first calving with aquaporin proteins, which are known to have a crucial role in reproduction.
- The inhibition of Cathepsin G on endometrial explants with endometrosis in the marePublication . Amaral, Ana; Fernandes, Carina; Faes Morazzo, Sofia; Rebordão, Maria Rosa; Szóstek-Mioduchowska, Anna; Lukasik, Karolina; Gawronska-Kozak, Barbara; Gama, Luis; Jan Skarzynski, Dariusz; Ferreira-Dias, GraçaAlthough proteases found in neutrophil extracellular traps (NETs) have antimicrobial properties, they also stimulate collagen type 1 (COL1) production by the mare endometrium, contributing for the development of endometrosis. Cathepsin G (CAT), a protease present in NETs, is inhibited by specific inhibitors, such as cathepsin G inhibitor I (INH; beta-keto-phosphonic acid). Matrix metallopeptidases (MMPs) are proteases involved in the equilibrium of the extracellular matrix. The objective of this study was to investigate the effect of CAT and INH (a selective CAT inhibitor) on the expression of MMP-2 and MMP-9 and on gelatinolytic activity. In addition, the putative inhibitory effect of INH on CAT-induced COL1 production in mare endometrium was assessed. Endometrial explants retrieved from mares in follicular phase or midluteal phase were treated for 24 or 48 h with CAT, inhibitor alone, or both treatments. In explants, transcripts (quantitative polymerase chain reaction) of COL1A2, MMP2, and MMP9, as well as the relative abundance of COL1 protein (Western blot), and activity of MMP-2 and MMP-9 (zymography) were evaluated. The protease CAT induced COL1 expression in explants, at both estrous cycle phases and treatment times. The inhibitory effect of INH was observed on COL1A2 transcripts in follicular phase at 24-h treatment, and in midluteal phase at 48 h (P < 0.05), and on the relative abundance of COL protein in follicular phase and midluteal phase explants, at 48 h (P < 0.001). Our study suggests that MMP-2 might also be involved in an earlier response to CAT, and MMP-9 in a later response, mainly in the follicular phase. While the use of INH reduced CAT-induced COL1 endometrial expression, MMPs might be involved in the fibrogenic response to CAT. Therefore, in mare endometrium, the use of INH may be a future potential therapeutic means to reduce CAT-induced COL1 formation and to hamper endometrosis establishment.
- The In vitro inhibitory effect of sivelestat on elastase induced collagen and metallopeptidase expression in equine endometriumPublication . Amaral, Ana; Fernandes, Carina; Rebordão, Maria Rosa; Szóstek-Mioduchowska, Anna; Lukasik, Karolina; Gawronska-Kozak, Barbara; Gama, Luis; Skarzynski, Dariusz J.; Ferreira-Dias, GraçaAbstract: Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the e ects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory e ect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory e ect was detected in MMP9 transcripts in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development.
- Discrimination of meat produced by Bos taurus and Bos indicus finished under an intensive or extensive systemPublication . Bressan, Maria C.; Rodrigues, Erika C.; Rossato, Lizandra V.; Neto-Fonseca, M. Isabel; Alves, Susana P.; Bessa, R.J.B.; Gama, LuisMeat obtained under commercial conditions shows considerable variability, mostly due to genetic background and production system. In this study, meat physicochemical properties and fatty acid profiles were analysed to investigate the feasibility of using them as tools to discriminate between meats produced by different genetic groups and finishing systems. Samples of the Longissimus thoracis were collected from 160 commercial bulls of the B. taurus (n = 75) and B. indicus (n = 85) groups, finished either on pasture (n = 46) or with grain supplementation (n = 114) and analysed by standard procedures. Data were analysed by discriminant analysis using a stepwise procedure, to select the meat characteristics that better contribute to discriminate the various groups. Our results indicate that fatty acid profiles of meat had better discriminating ability than physicochemical properties, especially to identify meat from animals finished on grain or pasture. The overall discrimination of meat from different genetic groups was achieved with a slightly lower reliability. Nonetheless, our results show that reliability of allocation to genetic group can be improved if prior information on finishing system is considered. These results are of high importance because they can be incorporated as tools to assess the authenticity of beef, particularly in meat certification programs.
- Evidence of sharing of Klebsiella pneumoniae strains between healthy companion animals and cohabiting humansPublication . Marques, Cátia Filipa Saraiva; Belas, Adriana; Aboim, Catarina; Cavaco-Silva, Patrícia; Trigueiro, Graça; Gama, Luis; Pomba, C.This study aimed to characterize the fecal colonization and sharing of Klebsiella pneumoniae strains between companion animals and humans living in close contact. Fecal samples were collected from 50 healthy participants (24 humans, 18 dogs, and 8 cats) belonging to 18 households. Samples were plated onto MacConkey agar (MCK) plates with and without cefotaxime or meropenem supplementation. Up to five K. pneumoniae colonies per participant were compared by pulsed-field gel electrophoresis (PFGE) after XbaI restriction. K. pneumoniae strains with unique pulse types from each participant were characterized for antimicrobial susceptibility, virulence genes, and multilocus sequence type (MLST). Fecal K. pneumoniae pulse types were compared to those of clinical K. pneumoniae strains from animal and human patients with urinary tract infections (n = 104). K. pneumoniae colonization was detected in nonsupplemented MCK in around 38% of dogs (n = 7) and humans (n = 9). K. pneumoniae strains isolated from dogs belonged to sequence type 17 (ST17), ST188, ST252, ST281, ST423, ST1093, ST1241, ST3398, and ST3399. None of the K. pneumoniae strains were multidrug resistant or hypervirulent. Two households included multiple colonized participants. Notably, two colonized dogs within household 15 (H15) shared a strain each (ST252 and ST1241) with one coliving human. One dog from H16 shared one PFGE-undistinguishable K. pneumoniae ST17 strain with two humans from different households; however, the antimicrobial susceptibility phenotypes of these three strains differed. Two main virulence genotypes were detected, namely fimH-1 mrkD ycfM entB kfu and fimH-1 mrkD ycfM entB kpn. These results highlight the potential role of dogs as a reservoir of K. pneumoniae to humans and vice versa. Furthermore, to our best knowledge, this is the first report of healthy humans and dogs sharing K. pneumoniae strains that were undistinguishable by PFGE/MLST.