CIISA - Artigos em revistas internacionais
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- Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugatesPublication . Andre, Ana S; Dias, Joana N. R.; Aguiar, Sandra I; Nogueira, Sara; Bule, Pedro; Carvalho, Joana Ines; António, J.P.M.; Carrapiço, Belmira; Ferreira São Braz, Berta; Solange, GilAntibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer
- The Evolution of Cystic Echinococcosis in Humans and Ruminants in Portugal-A One Health ApproachPublication . Alho, Ana Margarida; Dias, Miguel Canhão; Cardo, Miguel; Aguiar, Pedro; Madeira de Carvalho, LuísCystic echinococcosis, also known as hydatid disease, is a significant parasitic zoonosis with public health implications, albeit often neglected. In Portugal, data on this zoonosis are scarce despite being a mandatory notifiable disease in both humans and animals. To assess the impact of cystic echinococcosis on both livestock and humans, we compiled data from slaughterhouse records of ruminants, human hospitalizations, and confirmed cases of human echinococcosis. Overall, a total of 298 cases of cystic echinococcosis were identified in ruminants slaughtered from national farms for human consumption in Portugal between 2008 and 2022, comprising 192 cases in ovines, 95 in bovines, and 11 in caprines. Echinococcosis led to 582 hospitalizations in Portuguese public hospitals, with an average hospital stay of 11 days (±15.66), and resulted in 13 deaths (2.23%) from 2008 to 2018. Each infected animal was associated with a 7% increase in the incidence rate of human hospitalization (p = 0.002, IRR = 1.070, 95% CI: 1.025–1.117). Additionally, for every 100,000 person-years observed between 2008 and 2018, the total number of hospitalizations was 0.528. Residence in the Alentejo region was associated with a 5.3-fold increase in the incidence rate of human hospitalizations and an 8-fold higher risk of death from echinococcosis.
- A Preliminary Report on the Combined Effect of Intra-Articular Platelet-Rich Plasma Injections and Photobiomodulation in Canine OsteoarthritisPublication . Alves, J.C.; Santos, Ana; Carreira, L.MiguelOsteoarthritis (OA) is highly prevalent in the canine population. Due to the multiple dimensions of the disease, a multimodal approach is usually favored by clinicians. To evaluate the combined treatment with intra-articular platelet-rich plasma (PRP) and photobiomodulation in dogs with bilateral hip OA, thirty dogs were assigned to a PRP group (PRPG, n = 10), a photobiomodulation group (PBMTG, n = 10), or a combined therapies group (PRP+PBMTG, n = 10). The PRPG received two intra-articular administrations of platelet-rich plasma 14 days apart. The PBMTG received photobiomodulation with a therapeutic laser, with three sessions every other day in week one; two sessions in week two; a single session in week three; and one session/month on follow-up evaluation days. The PRP+PBMTG received the two combined therapies. The response to treatment was evaluated with weight-bearing distribution and the Canine Brief Pain Inventory, the Liverpool Osteoarthritis in Dogs, and the Canine Orthopedic Index. Evaluations were conducted before treatment and +8, +15, +30, +60, and +90 days after initial treatment. Normality was assessed with a Shapiro–Wilk test, and the groups’ results in each evaluation moment were compared using a Mann–Whitney U test. Animals of both sexes (male n = 19, female n = 11) were included in the sample, with a mean age of 7.8 ± 2.5 years and a body weight of 26.5 ± 4.7 kg. Joints were classified as mild (n = 6, three in PRPG, two in PBMTG, and one in PRP+PBMTG), moderate (n = 18, six in PRPG, five in PBMTG, and seven in PRP+PBMTG), and severe (n = 6, one in PRPG, three in PBMTG, and two in PRP+PBMTG). No differences were found between groups at the initial evaluation. All treatments produced clinically significant improvements compared to the assessment on treatment day. The combination of PRP and photobiomodulation produced greater, longer-lasting improvements. PRP and photobiomodulation can improve objective outcomes and client-reported outcome measures in dogs with OA. Their combined use leads to greater, longer-lasting, clinically significant improvements
- Osteomyelitis caused by Aspergillus terreus complex in a dog: a case reportPublication . Abreu, Raquel; Martinho, António; Noiva, Rute; Pissarra, Hugo; Cota, João; Cunha, Eva; Tavares, Luis; Oliveira, ManuelaBackground In dogs, the most frequently reported mycosis associated with Aspergillus spp. are respiratory infections. Systemic aspergillosis is uncommon, with reported cases been associated with several Aspergillus species. Aspergillus terreus species complex are ubiquitous organisms, unfrequently associated with local or systemic disease in animals and humans, and treatment of osteomyelitis caused by this species is usually unfavorable. Case presentation This case report describes the case of a 5-year-old dog, referred to the Veterinary Hospital of the Faculty of Veterinary Medicine of the University of Lisbon, Portugal, with a history of lameness of the right thoracic limb. Radiographs and CT scan revealed two diferent lesions on right humerus and radio, which were biopsied. The samples collected were submitted to cytological and histopathological evaluation and bacterial and mycological culture. Environmental samples, including of the surgery room and of the biopsy needle were also evaluated for the presence of fungi. Regarding biopsy samples, bacterial culture was negative, but mycological analysis originated a pure culture of a fungal species later identifed as Aspergillus terreus by Sanger sequencing. Results were compatible with histopathologic examination, which revealed periosteal reaction and invasion of hyphae elements. Also, mycological analysis of both environmental samples evaluated were negative. The virulence profle of the fungal isolate was phenotypically characterized using specifc media, allowing to reveal its ability to produce several enzymes involved in its pathogenicity, namely lipase, hemolysin and DNAse, corresponding to a Virulence Index (V. Index.) of 0.43. The patient was submitted to itraconazole therapy for 8 weeks. After 3 weeks, the patient showed signifcant clinical improvement, and after 6 weeks no radiographic signs were observed. Conclusions Antifungal therapy with itraconazole can contribute to the remission of canine infections promoted by Aspergillus terreus complex with a relevant V. Index
- Hygiene and biosecurity conditions of initial examination on-spot in Portugal: one step toward game meat safetyPublication . Abrantes, Ana Carolina; Ferreira, Maria Pureza; Ruano, Zita; Vinhas, Bruno; Vaz, Yolanda; Vieira-Pinto, MadalenaBackground and Aim: Due to the particularities of the first steps of the game food chain, large game species are shot, bled, and handled in collection points (spot of evisceration and initial examination in the field). These steps of the game meat chain affect the microbiological quality of this type of meat, thus posing a risk to consumers. This study aimed to characterize the collection points in terms of central hygiene and biosecurity procedures/requirements.Materials and Methods: One survey with 16 questions was applied in 95 hunting areas throughout Portugal. It was a convenience sample obtained by direct visualization on-spot procedures. Four categories were characterized in the survey: Initial examination (performance assiduity and type of operator performing it), hygiene requirements on-spot (floor, ceiling, water, and electricity), biosecurity procedures such on initial examination (use of personal protective equipment as gloves, glasses, mask, and specific clothes), and by-products disposal (destination and packaging of by-products).Results: Sixty percentage (n = 57) eviscerated the carcasses and performed the initial examination on-spot. Moreover, most of the time (n = 71), the initial examination was carried out by veterinarians. However, the category that showed the best results was those related to the biosecurity procedures on initial examination, mainly with the use of the individual protective material (e.g., regular use of disposal clothes and specific clothes). Concerning the questions about the disposal of by-products, 66 game managers say that this was done correctly (69%), being the majority destination of the inspected carcasses was the burial (64%; n = 47).Conclusion: This survey demonstrates an immediate need in all this problematic of the standardization of hygiene and biosecurity requirements of the collection points, which requires uniform application of rules. There is a lot of resistance and limitations to the inclusion of these requirements in collection points, due to lack of structural and financial conditions. However, training all those involved in the hunting area (hunters, game managers, authorities, etc.) creating rules that promote hunting food security and setting limits on the microbiological criteria of game meat are hot points to consider in the future.
- Assessing the efficacy of the ovicidal fungus Mucor circinelloides in reducing coccidia parasitism in peacocksPublication . Lozano, João; Almeida, Cristina; Vicente, Eduardo; Sebastião, Daniela; Palomero Salinero, Antonio; Cazapal Monteiro, Cristiana; Arias, María Sol; Oliveira, Manuela; Madeira de Carvalho, Luís; Paz-Silva, AdolfoThe biological control of gastrointestinal (GI) parasites using predatory fungi has been recently proposed as an accurate and sustainable approach in birds. The current study aimed to assess for the first time the efficacy of using the native ovicidal fungus Mucor circinelloides (FMV-FR1) in reducing coccidia parasitism in peacocks. For this purpose, an in vivo trial was designed in the resident peacock collection (n = 58 birds) of the São Jorge Castle, at Lisbon, Portugal. These animals presented an initial severe infection by coccidia of the genus Eimeria (20106 ± 8034 oocysts per gram of feces, OPG), and thus received commercial feed enriched with a M. circinelloides suspension (1.01 × 108 spores/kg feed), thrice-weekly. Fresh feces were collected every 15 days to calculate the coccidia shedding, using the Mini-FLOTAC technique. The same bird flock served simultaneously as control (t0 days) and test groups (t15–t90 days). The average Eimeria sp. shedding in peacocks decreased up to 92% following fungal administrations, with significant reduction efficacies of 78% (p = 0.004) and 92% (p = 0.012) after 45 and 60 days, respectively. Results from this study suggest that the administration of M. circinelloides spores to birds is an accurate solution to reduce their coccidia parasitism.
- First insights on the susceptibility of native coccidicidal fungi Mucor circinelloides and Mucor lusitanicus to different avian antiparasitic drugsPublication . Lozano, João; Cunha, Eva; Madeira de Carvalho, Luís; Paz-Silva, Adolfo; Oliveira, ManuelaBackground The combined application of predatory fungi and antiparasitic drugs is a sustainable approach for the integrated control of animal gastrointestinal (GI) parasites. However, literature addressing the possible interference of antiparasitic drugs on the performance of these fungi is still scarce. This research aimed to assess the in vitro susceptibility of six native coccidicidal fungi isolates of the species Mucor circinelloides and one Mucor lusitanicus isolate to several antiparasitic drugs commonly used to treat GI parasites’ infections in birds, namely anthelminthics such as Albendazole, Fenbendazole, Levamisole and Ivermectin, and anticoccidials such as Lasalocid, Amprolium and Toltrazuril (drug concentrations of 0.0078–4 µg/mL), using 96-well microplates filled with RPMI 1640 medium, and also on Sabouraud Agar (SA). Results This research revealed that the exposition of all Mucor isolates to the tested anthelminthic and anticoccidial drug concentrations did not inhibit their growth. Fungal growth was recorded in RPMI medium, after 48 h of drug exposure, as well as on SA medium after exposure to the maximum drug concentration. Conclusions Preliminary findings from this research suggest the potential compatibility of these Mucor isolates with antiparasitic drugs for the integrated control of avian intestinal parasites. However, further in vitro and in vivo studies are needed to confirm this hypothesis.
- Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial communityPublication . Lozano, João; Cunha, Eva; Almeida, Cristina; Nunes, Mónica; Dias, Ricardo; Vicente, Eduardo; Sebastião, Daniela; Henriques, Sérgio; Madeira de Carvalho, Luís; Paz-Silva, Adolfo; Oliveira, ManuelaParasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds’ gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.
- Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocystsPublication . Lozano, João; Louro, Mariana; Almeida, Cristina; Victório, Ana Cláudia; Melo, Pedro; Rodrigues, João Paulo; Oliveira, Manuela; Paz-Silva, Adolfo; Madeira de Carvalho, LuísFungal strains used in the biocontrol of animal gastrointestinal parasites have been mainly isolated from pasture soil, decaying organic matter, and feces from herbivores and carnivores. However, their isolation from birds and assessment of predatory activity against avian GI parasites has been scarce thus far. This research aimed to isolate flamentous fungi from avian fecal samples and evaluate their predatory activity against coccidia. A pool of 58 fecal samples from chickens, laying hens, and peacocks, previously collected between July 2020-April 2021, were used for isolation of flamentous fungi and assessment of their in vitro predatory activity against coccidian oocysts, using Water-Agar medium and coprocultures. The Willis-fotation technique was also performed to obtain concentrated suspensions of oocysts. A total of seven Mucor isolates was obtained, being the only fungal taxa identifed, and all presented lytic activity against coccidia. Isolates FR3, QP2 and SJ1 had signifcant coccidiostatic efcacies (inhibition of sporulation) higher than 70%, while isolates FR1, QP2 and QP1 had coccidicidal efcacies (destruction of the oocysts) of 22%, 14% and 8%, respectively, after 14 days of incubation, being a gradual and time-dependent process. To our knowledge, this is the frst report regarding the isolation of native predatory fungi from avian feces and demonstration of their lytic activity against coccidia
- Silages of agro-industrial by-products in lamb diets : effct on growth performance, carcass, meat quality and in vitro methane emissionsPublication . Dentinho, M. T. P.; Paulos, K.; Costa, C.; Costa, J.; Fialho, L.; Cachucho, L.; Portugal, A. P.ABSTRACT - The use of agro-industrial by-products in animal feed is an opportunity to reduce imports, food waste and promote a clean and circular economy, turning worthless raw materials into high -quality and low-cost animal feeds, which does not compete with human food. This study aims to produce silages with by-products of carrot, sweet potato, potato, and tomato pomace and integrate them in lamb diets, replacing 50% of dry matter (DM) of a concentrate-based diet. Effects on growth performance, meat quality and methane production were evaluated. Three silages were produced using 350 g/kg tomato pomace, 200 g/kg wheat bran, 150 g/kg alfalfa hay and 300 g/kg potato (Psil) or 300 g/kg sweet potato (SPsil) or 300 g/kg carrot (Csil). Thirty-two lambs were housed individually and divided into four groups (8 animals/group) on the following diets: Control -850 g/kg concentrate and 150 g/kg hay; P -500 g/kg concentrate and 500 g/kg Psil in DM; SP - 500 g/kg concentrate and 500 g/kg SPsil in DM; C - 500 g/kg concentrate and 500 g/kg Csil in DM. The trial lasted 6 weeks after 1 week of adaptation. Methane production was assessed in vitro by the Ankom system, using as inoculum rumen content samples collected individually from 6 lambs on each diet at slaughter. Diet had no effect on DM intake, average daily gain and DM conversion ratio, averaging 1055 +/- 248 g, 320 +/- 61.1 g and 3.35 +/- 0.600, respectively. Also, carcass characteristics were not affected by the diet except for the lightness of subcutaneous fat which was increased by the silage diets (L* value, P = 0.016). The meat pa-rameters, pH, intramuscular fat, shear force, cooking losses and sensory attributes, were not affected by the diet. In meat color, the parameters a* and Chroma were highest in animals fed diet C, have an intermediate value with Control diet and are lowest with diets P and SP (P = 0.027 and P = 0.012, respectively). Diets had no impact on total methane emissions. The costs of the silage diets per kg live weight gain were lower than those of Control diet (P = 0.018). In summary, by-product silage can be a good option to replace concentrated feed in lamb diets without altering the growth performance and meat quality or methane production and thus contributing to the sustainability of livestock farms and the environment.