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- Agronomic potential of Hermetia illucens frass in the cultivation of ryegrass in distinct soilsPublication . Rehan, I.; Lopes, I. G.; Murta, D.; Lidon, F.; Fareleira, P.; Esteves, C.; Moreira, O.; Menino, R.Cropping systems are strongly dependent on mineral fertilisers, which are effective in achieving high crop productivities. However, these chemical inputs end up compromising soil quality in the long-term. Frass from black soldier fly (BSF) larvae is a novel organic fertiliser that is rich in organic matter and advocated as a material that can sustain crop productivity while increasing soil quality. This study aimed at evaluating distinct fertilisation regimes in the cultivation of ryegrass (Lolium multiflorum Lam. or annual ryegrass) in soils of different types (sandy, loamy and clay) and fertility levels. In a 7-month pot experiment conducted in a glass greenhouse, plants were cultivated with exclusive mineral (MT) or organic (OT) fertilisation, in addition to combinations between both (mineral and organic, MOTs) in different proportions (25:50; 50:50 and 75:25), considering a 140 kg per hectare N demand. Crop yield was favoured by the combination of organic and mineral fertilisers in all soils, which also had its fertility increased, especially regarding organic matter build-up and nutrient accumulation. In addition, the presence of frass in the sandy soil stimulated microbial activity, which was measured by the enzyme dehydrogenase. Frass derived from BSF larvae can be considered an adequate organic fertiliser in the cultivation of ryegrass in distinct soil types, when applied in partial (25% to 75%) replacement of mineral fertilisers, enabling high crop productivity and nutritional quality of the crop, while increasing soil fertility.
- Are reusable dry electrodes an alternative to gelled electrodes for canine surface electromyography?Publication . Ribeiro, Ana M.; Brás, I.; Caldeira, L.; Peham, C.; Silva, H. Plácido da; Requicha, João F.Despite its increasing use in veterinary rehabilitation, practical constraints—such as skin preparation and single-use electrodes—limit the wider adoption of surface electromyography (sEMG). Having conventional pre-gelled Ag/AgCl electrodes as reference, we made a pioneering comparison of the performance of reusable soft polymeric dry electrodes for recording paraspinal muscle activity in dogs during treadmill walking. Twelve clinically healthy Dachshunds from both genders were evaluated under two conditions, namely: (i) dry electrodes on untrimmed hair; and (ii) pre-gelled electrodes after trichotomy. Signals were acquired from the longissimus dorsi muscle at 1 kHz, processed with standardized filtering and rectification, and analyzed in both time and frequency domains. Dry electrodes yielded higher amplitude and Root Mean Square (RMS) values, but slightly lower power spectral density metrics when compared to pre-gelled electrodes. Nevertheless, frequency-domain results were broadly comparable between configurations. Dry electrodes reduce the preparation time, avoid hair clipping, and allow reusability without major signal degradation. While pre-gelled electrodes may still offer marginally superior stability during movement, our results suggest that soft polymeric dry electrodes present a feasible, less invasive, and more sustainable alternative for canine sEMG. These findings support further validation of dry electrodes in clinical populations, particularly for neuromuscular assessment in intervertebral disk disease.
- Assessing in vivo and in vitro biofilm development by Streptococcus dysgalactiae subsp. dysgalactiae using a murine model of catheter-associated biofilm and human keratinocyte cellPublication . Alves-Barroco, Cinthia; Botelho, Ana Maria Nunes; Américo, Marco Antonio; Fracalanzza, Sérgio Eduardo Longo; Matos, António P. Alves de; Guimaraes, Márcia Aparecida; Ferreira-Carvalho, Bernadete Teixeira; Figueiredo, Agnes Marie Sá; Fernandes, Alexandra R.Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is an important agent of bovine mastitis. This infection causes an inflammatory reaction in udder tissue, being the most important disease-causing significant impact on the dairy industry. Therefore, it leads to an increase in dairy farming to meet commercial demands. As a result, there is a major impact on both the dairy industry and the environment including global warming. Recurrent mastitis is often attributed to the development of bacterial biofilms, which promote survival of sessile cells in hostile environments, and resistance to the immune system defense and antimicrobial therapy. Recently, we described the in vitro biofilm development on abiotic surfaces by bovine SDSD. In that work we integrated microbiology, imaging, and computational methods to evaluate the biofilm production capability of SDSD isolates on abiotic surfaces. Additionally, we reported that bovine SDSD can adhere and internalize human cells, including human epidermal keratinocyte (HEK) cells. We showed that the adherence and internalization rates of bovine SDSD isolates in HEK cells are higher than those of a SDSD DB49998-05 isolated from humans. In vivo, bovine SDSD can cause invasive infections leading to zebrafish morbidity and mortality. In the present work, we investigated for the first time the capability of bovine SDSD to develop biofilm in vivo using a murine animal model and ex-vivo on human HEK cells. Bovine SDSD isolates were selected based on their ability to form weak, moderate, or strong biofilms on glass surfaces. Our results showed that SDSD isolates displayed an increased ability to form biofilms on the surface of catheters implanted in mice when compared to in vitro biofilm formation on abiotic surface. A greater ability to form biofilm in vitro after animal passage was observed for the VSD45 isolate, but not for the other isolates tested. Besides that, in vitro scanning electron microscopy demonstrated that SDSD biofilm development was visible after 4 hours of SDSD adhesion to HEK cells. Cell viability tests showed an important reduction in the number of HEK cells after the formation of SDSD biofilms. In this study, the expression of genes encoding BrpA-like (biofilm regulatory protein), FbpA (fibronectin-binding protein A), HtrA (serine protease), and SagA (streptolysin S precursor) was higher for biofilm grown in vivo than in vitro, suggesting a potential role for these virulence determinants in the biofilm-development, host colonization, and SDSD infections. Taken together, these results demonstrate that SDSD can develop biofilms in vivo and on the surface of HEK cells causing important cellular damages. As SDSD infections are considered zoonotic diseases, our data contribute to a better understanding of the role of biofilm accumulation during SDSD colonization and pathogenesis not only in bovine mastitis, but they also shed some lights on the mechanisms of prosthesis-associated infection and cellulitis caused by SDSD in humans, as well.
- Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosisPublication . Zhou, Ershun; Silva, Liliana M. R.; Conejeros, Iván; Velásquez, Zahady D.; Hirz, Manuela; Gärtner, Ulrich; Jacquiet, Philippe; Taubert, Anja; Hermosilla, CarlosBesnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.
- Besnoitia besnoiti tachyzoite replication in bovine primary endothelial cells relies on host Niemann–Pick type C protein 1 for cholesterol acquisitionPublication . Larrazabal, Camilo; Hermosilla, Carlos; Taubert, Anja; Silva, Liliana M. R.Besnoitia besnoiti is a cyst-forming apicomplexan parasite and the causal agent of bovine besnoitiosis. During early phase of infection, tachyzoites replicate within host endothelial cells in a host cell cholesterol-dependent process. By applying U18666A treatments, we here evaluated the role of Niemann–Pick type C protein 1 (NPC1) in both, intracellular B. besnoiti replication and host cellular cholesterol distribution. Additionally, B. besnoiti-driven changes in NPC1 gene transcription were studied by qPCR. Overall, U18666A treatments significantly reduced B. besnoiti proliferation and induced cholesterol accumulation in host cytoplasmic dense vesicles. However, NPC1 gene transcription was not affected by B. besnoiti infection.
- Bovine ocular squamous cell carcinoma : a descriptive epidemiological survey in the Azores, PortugalPublication . Bilhastre, Beatriz; Vala, Helena; Ribeiro, Ana Clara; Faria, Sara; Oliveira, Ana; Branco, Sandra; Pinto, CarlosBovine ocular squamous cell carcinoma (BOSCC) is the most common ocular tumour in cattle, with a multifactorial aetiology involving ultraviolet (UV) radiation, genetic factors, pigmentation, and management practices. A detailed epidemiological characterisation of BOSCC in the Azores, Portugal, is provided, with particular emphasis on its spatial distribution and potential risk determinants. Data were obtained through an epidemiological questionnaire completed by field veterinarians between August 2023 and March 2025. A total of 85 BOSCC cases were recorded across 62 farms—45 on Terceira Island and 17 on São Miguel Island. All affected animals were adult Holstein Friesian dairy cows, managed under extensive pasture-based systems. The nictitating membrane was the most frequently affected structure (69.5%), and multiple lesions occurred in 20% of the cases. Farms located at 200–400 m of altitude presented the highest number of cases. Continuous exposure to UV under pasture-based management represents the main environmental risk factor. Although periocular pigmentation may provide partial protection, other environmental and genetic factors can also contribute to tumour development. Artificial insemination is considered a promising preventive tool, enabling genetic selection for protective traits such as periocular pigmentation. This research provides the first regional epidemiological characterization of BOSCC in the Azores, highlighting the interplay among environmental, genetic, and management-related factors in disease occurrence.
- Brucella ceti in common dolphins (Delphinus delphis) in Portugal : characterization of first isolatesPublication . Cavaco, Sandra; Grilo, Miguel L.; Dias, Ricardo; Nunes, Mónica; Pascoal, Pedro; Pereira, Marcelo; Fogaça, Catarina; Costa, Ana Beatriz; Pardal, Sofia; Ferreira, Ana CristinaThis study investigates Brucella ceti infection in marine mammals stranded along the Lisbon and Tagus Valley coast between 2022 and mid-2024, marking the first report of Brucella presence in Portuguese waters. Out of 59 examined marine mammals, B. ceti was isolated in three common dolphins (5.1%), a prevalence rate consistent with previous studies from other coastlines. PCR-based detection indicated a higher infection rate (23.7%), suggesting an underestimation of the prevalence of B. ceti infection in this population. Multi-locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) revealed distinct genetic profiles and close relationships to B. ceti strains from the Atlantic, supporting the hypothesis of specific host-adapted lineages in dolphins. Virulence genes, including those for host interaction (bspE, btpB) and intracellular survival (virB7, vceA), were consistent across isolates, highlighting the pathogenic potential. Additionally, antimicrobial resistance (AMR) genes, such as mprF and efflux proteins (bepC-G), were also identified. These findings underscore the need for further research and surveillance to understand B. ceti transmission, host range, and impacts on Atlantic cetaceans, as well as to develop effective diagnostic and management strategies to mitigate infection risks in marine environments.
- Building a Portuguese coalition for biodiversity genomicsPublication . Marques, João P.; Alves, Paulo C.; Amorim, Isabel R.; Lopes, Ricardo J.; Moura, Monica; Myers, Eugene; Sim-sim, Manuela; Sousa-Santos, Carla; Alves, M. Judite; Borges, Paulo A. V.; Brown, Thomas; Carneiro, Miguel; Carrapato, Carlos; Ceríaco, Luís M. P.; Ciofi, Cláudio; Silva, Luís P. da; Diedericks, Genevieve; Diroma, Maria Angela; Farelo, Liliana; Formenti, Giulio; Gil, Fátima; Grilo, Miguel; Iannucci, Alessio; Leitão, Henrique G.; Máguas, Cristina; Mc Cartney, Ann M.; Mendes, Sofia L.; Moreno, João M.; Morselli, Marco; Mouton, Alice; Natali, Chiara; Pereira, Fernando; Rego, Rúben M. C.; Resendes, Roberto; Roxo, Guilherme; Svardal, Hannes; Trindade, Helena; Vicente, Sara; Winkler, Sylke; Alvarenga, Marcela; Amaral, Andreia J.; Antunes, Agostinho; Campos, Paula F.; Canário, Adelino V. M.; Castilho, Rita; Castro, L. Filipe C.; Crottini, Angelica; Cunha, Mónica V.; Themudo, Gonçalo Espregueira; Esteves, Pedro J.; Faria, Rui; Fernandes, Carlos Rodríguez; Ledoux, Jean-Baptiste; Louro, Bruno; Magalhaes, Sara; Paulo, Octávio S.; Pearson, Gareth; Pimenta, João; Pina-Martins, Francisco; Santos, Teresa L.; Serrão, Ester; Melo-Ferreira, José; Sousa, Vítor C.The diverse physiography of the Portuguese land and marine territory, spanning from continental Europe to the Atlantic archipelagos, has made it an important repository of biodiversity throughout the Pleistocene glacial cycles, leading to a remarkable diversity of species and ecosystems. This rich biodiversity is under threat from anthropogenic drivers, such as climate change, invasive species, land use changes, overexploitation, or pathogen (re)emergence. The inventory, characterisation, and study of biodiversity at inter- and intra-specific levels using genomics is crucial to promote its preservation and recovery by informing biodiversity conservation policies, management measures, and research. The participation of researchers from Portuguese institutions in the European Reference Genome Atlas (ERGA) initiative and its pilot effort to generate reference genomes for European biodiversity has reinforced the establishment of Biogenome Portugal. This nascent institutional network will connect the national community of researchers in genomics. Here, we describe the Portuguese contribution to ERGA’s pilot effort, which will generate high-quality reference genomes of six species from Portugal that are endemic, iconic, and/or endangered and include plants, insects, and vertebrates (fish, birds, and mammals) from mainland Portugal or the Azores islands. In addition, we outline the objectives of Biogenome Portugal, which aims to (i) promote scientific collaboration, (ii) contribute to advanced training, (iii) stimulate the participation of institutions and researchers based in Portugal in international biodiversity genomics initiatives, and (iv) contribute to the transfer of knowledge to stakeholders and engaging the public to preserve biodiversity. This initiative will strengthen biodiversity genomics research in Portugal and fuel the genomic inventory of Portuguese eukaryotic species. Such efforts will be critical to the conservation of the country’s rich biodiversity and will contribute to ERGA’s goal of generating reference genomes for European species.
- Cellular immune responses of bovine polymorphonuclear neutrophils to Calicophoron daubneyiPublication . Silva, Liliana M. R.; López-Osorio, Sara; Peixoto, Raquel; Zhou, Ershun; Espinosa, Gabriel; Gärtner, Ulrich; Taubert, Anja; Conejeros, Iván; Hermosilla, CarlosCalicophoron daubneyi infections have increased in Europe, being more frequent than fasciolosis in some areas. Infection occurs once definitive hosts ingest encysted metacercariae present on vegetation. Following excystation, juvenile flukes penetrate the small intestinal mucosa and migrate into the rumen where adults mature. Throughout the somatic migration, juveniles come across different microenvironments and tissues and encounter host leukocytes. Besides phagocytosis, production of reactive oxygen species (ROS) and degranulation, polymorphonuclear neutrophils also cast neutrophil extracellular traps (NETs), which can entrap several parasite species, including the closely related liver fluke Fasciola hepatica. In this study, we analyzed whether in vitro exposure of bovine neutrophils to C. daubneyi antigen (CdAg) and eggs triggered neutrophils activation and NET formation. Results on scanning electron microscopy (SEM) and immunofluorescence analyses show weak formation of short spread NETs upon CdAg stimulation, corroborated by increased extracellular DNA measurements. Likewise, early NETosis was confirmed via nuclear area expansion assays. Bovine neutrophil stimulation with CdAg 100 µg/mL concentration led to a significant increase in oxygen consumption rates (p = 0.0152) and extracellular acidification rates (p = 0.0022), while lower concentrations of CdAg (10 µg/mL) failed to induce neutrophil activation, suggesting a dose dependent response. Both intra- and extracellular ROS production was not affected by any CdAg concentration here studied. Bovine neutrophil total adenosine triphosphate concentration significantly decreased after exposure to CdAg 100 µg/mL, in line to the observed with the positive control (phorbol myristate acetate/ionomycin). In summary, C. daubneyi activates bovine neutrophils with rather weak responses, which might suggest that the release of C. daubneyi-specific molecules (i.e. excretory-secretory antigens, proteases, or nucleases) could interfere with neutrophil-related effector mechanisms. Further ex vivo analyses will clarify if such mechanisms are also involved in pathogenesis of paramphistomosis by demonstrating neutrophil recruitment into affected intestinal mucosa.
- Combining Hermetia illucens and Tenebrio molitor meals in diets for European seabass : effects on growth, nutrient utilisation, intestinal morphology and muscle qualityPublication . Costa, Rafaela S.; Basto, Ana; Monteiro, Marta; Pinho, Bia; Sá, Tiago; Santos, Marisa V.; Murta, Daniel; Schrama, Johan W.; Valente, Luisa M.P.This study explored the potential of an insect meal (IM) mixture of black soldier fly larvae (BSFL) and yellow mealworms (YMs) to substitute 3% (diet IM3), 25% (diet IM25) and 50% (diet IM50) of the fishmeal (FM) protein in a control diet (CTRL; 15% FM) for European seabass (Dicentrarchus labrax) juveniles. The four diets were isoproteic and isolipidic and were tested in triplicate in a recirculating aquaculture system with water at 22 °C and 35 ppt salinity, under a 12 h light/12 h dark photoperiod. After a 75-day feeding period, the fish growth performance, nutrient utilisation, intestinal morphology, muscle fatty acid profile and antioxidant capacity were evaluated. The diets containing IM ensured similar growth (DGI = 2.0) and feed efficiency (FCR = 1.0–1.1) to CTRL. Moreover, they promoted comparable nitrogen and energy retention efficiencies (38–40% and 44–47%). However, fish fed with the IM diets presented 20–27% lower faecal phosphorus losses than the control group. Anterior intestine integrity was maintained in all fish, but those fed IM50 displayed longer villi than the control. The muscle fatty acid profile of IM-fed fish also resembled that of the control, with 364–405 mg EPA + DHA per 100 g of fillet. IM25 and IM50 promoted higher lauric acid deposition in the muscle, and the muscle of fish fed with IM50 presented the highest oxygen radical absorbance capacity, suggesting higher antioxidant capacity. Overall, the study shows that the IM mixture can be an adequate protein source for seabass juveniles, also offering functional benefits.