DSA - Teses de Doutoramento
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- Safebed : improving safety and sustainability of recycled manure solids as bedding material for dairy cowsPublication . Pires, Ana José de Oliveira Nunes; Oliveira, Maria Manuela Castilho Monteiro de; Bexiga, José Ricardo Dias; Fangueiro, David PauloABSTRACT - SAFEBED: IMPROVING SAFETY AND SUSTAINABILITY OF RECYCLED MANURE SOLIDS AS BEDDING MATERIAL FOR DAIRY COWS - Antimicrobial resistance (AMR) and the environmental impact of livestock production pose significant challenges to sustainable dairy farming. The use of recycled manure solids (RMS) as bedding material offers economic and environmental benefits but raises concerns regarding microbial safety, including the persistence of antimicrobial-resistant bacteria and potential pathogens. This study evaluated the effects of biochar supplementation in RMS to mitigate AMR risks, enhance microbial safety, and reduce environmental emissions. Fresh RMS samples were collected from a commercial dairy farm and divided into five treatments: non-supplemented RMS (C-), acidified RMS (C+), and RMS supplemented with 2.5%, 5%, or 10% biochar. Samples were incubated for 30 days at ambient temperature, with assessments on Days 0, 5, 15, and 30. Two assays were conducted: one in a humid period (April–May) and another in a dry period (June–July). Microbial analyses included bacterial quantification, Escherichia coli and Enterococcus spp. isolation, AMR and virulence profiling, and 16S rDNA sequencing for microbial community shifts. In parallel, the environmental study examined RMS characteristics, nutrient retention, and greenhouse gas (GHG) emissions. Results from the bacterial quantification revealed no statistically significant reduction in E. coli and Enterococcus spp. counts with biochar supplementation. However, trends suggested a potential decrease in AMR and virulence factors, with reductions observed in oxytetracycline resistance and biofilm formation. In contrast, the microbiome analysis demonstrated a significant reduction in bacterial loads, with 5% biochar leading to a 59.50% decrease under humid conditions. However, Brucella spp. and Pseudomonas spp. increased in biochar amended RMS. Environmentally, the supplementation with 10% biochar significantly reduced CO₂ (32%) and N₂O (47%) emissions, stabilized nitrogen, and minimized volatilization losses. However, methane emissions were largely unaffected. Addicionally, Biochar supplementation increased micronutrient and heavy metal concentrations (except for molybdenum), as well as potassium and magnesium levels, while decreasing sodium, phosphorus, and total organic carbon content. This study highlights biochar’s potential for improving microbial safety and environmental sustainability in dairy farming. Further research should optimize biochar concentrations, assess long-term effects, and evaluate field-scale applications to fully harness its role in sustainable manure management
- Integrated control of avian gastrointestinal parasites : optimizing their coprological diagnosis and biocontrol using predatory fungiPublication . Lozano, João; Lozano, João Miguel Pestana; Carvalho, Luís Manuel Madeira de; Oliveira, Maria Manuela Castilho Monteiro de; Paz-Silva, AdolfoIn the last 30 years, several improvements were achieved in the control of animals’ gastrointestinal (GI) parasitism, including the development of more sensitive and precise coprological diagnosis techniques, like Mini-FLOTAC (MF), as well as the use of predatory fungi as a sustainable solution for parasite control. This project aimed to optimize the MF method for the diagnosis of avian GI parasitism, and to isolate predatory fungi and assess their potentialities in avian parasite control. First, the diagnosis of GI parasitism was performed in a poultry farm and three exotic bird collections, in Portugal mainland, using the MF technique. Eimeria spp. infections were identified in laying hens and peacocks, Capillaria spp., Trichostrongylus tenuis and Strongyloides pavonis in peacocks, as well as Libyostrongylus douglassii in ostriches and emus. Seven filamentous fungi were isolated from Galliformes feces, furtherly identified as Mucor circinelloides (n=6) and M. lusitanicus (n=1). All fungi were capable of destroying Eimeria spp. oocysts in vitro, with M. circinelloides isolate FR1 achieving the highest coccidicidal efficacy, 22%, after 14 days of exposure. All fungi were checked for potential in vitro susceptibilities to seven antiparasitic drugs (albendazole, fenbendazole, levamisole, ivermectin, lasalocid, amprolium and toltrazuril), and all were not susceptible to the tested antiparasitic drugs, independently of their concentrations. The characterization of the virulence profile of M. circinelloides FR1 revealed only the presence of genes coding for iron receptors (FOB1 and FOB2), iron permease (FTR1), ADP-ribosylation factors (ARF2, ARF6) and GTPase (CDC42), having the isolate only tested positive for lecithinase production. Oral administrations of spores of this fungal isolate were performed in laying hens and peacocks, which revealed that this fungus did not affect birds’ feces appearance and consistency (70 100% of normal feces), neither the relative abundances and alfa-diversities of their native gut bacteria and fungi. Finally, it was verified that the administration of this fungus spores to peacocks reduced significantly their Eimeria spp. fecal shedding up to 92%, after two months of trial. Results from this project point out the usefulness of MF in the coprological diagnosis of GI parasitism in birds, and provide the first report regarding the isolation of predatory fungi from bird feces. Finally, the safety and efficacy of using M. circinelloides FR1 in the integrated control of domestic and exotic birds GI parasitism was demonstrated
- Antibiotic-lipid based nanosystem as a tool to specifically target Staphylococcus aureus biofilmsPublication . Ferreira, M.C. Magda Sofia Catroga Ferreira; Aguiar, Sandra Isabel Rodrigues de; Gaspar, Maria Manuela de Jesus Guilherme; Bettencourt, Ana Francisca de Campos SimãoAbstract - 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
- Development of novel antibody-drug conjugated molecules for treatment of B-cell maliganciesPublication . André, Ana Filipa Santos; Silva, Frederico Nuno Castanheira Aires da; Tavares, Luís Manuel Morgado; Gonçalves, João Manuel BrazCancer is one of the leading causes of death worldwide, and is expected to continue increasing, with a projected 28.4 million cases by 2040. Dogs are also significantly affected by cancer, including NHL, which is one of the most common hematological malignancies in both species. Dogs are considered excellent models to accelerate the translation of treatments for human patients due to their similarities with humans. While immunotherapies, particularly monoclonal antibodies (mAbs), have brought specificity to cancer therapies, they are still mostly used in combination with conventional chemotherapy, which remains the standard of care for both species. However, conventional treatments still do not fully cure and have numerous adverse effects, highlighting the need for further improvement. To address this need, using the promising advantages of rabbit-derived sdAbs, we aimed to develop a platform for a novel sdAb drug delivery system for NHL treatment using the dog as an animal model. For this purpose, we explored three different drug delivery methods: ADCs, immunotoxins and immunoliposomes. For the development of ADC, we explored the potential of rabbit derived single-domain antibodies (sdAb) to selectively conjugate a payload towards cysteine at position 80. First, a rabbit-derived sdAb library against canine B-cell lymphoma receptors was subjected to in vitro and in vivo phage display. Then, VL sdAb that specifically targeted canine lymphoma cells in vitro and presented a good tumor uptake was selected for SN-38 site selective payload conjugation via its Cys80 and generated a stable and homogeneous C5- DAB-SN-38. This study validated a platform to develop novel ADCs that combine rabbit sdAbs benefits with the advantages of canine lymphoma model. Furthermore, this previously characterized and validated VL sdAb was also used to develop a new immunotoxin for the treatment of canine B-cell lymphoma. For that purpose, VL sdAb was conjugated with the PE38 toxin truncated form and tested in vitro in a canine B-cell lymphoma cells and in in vivo in a xenograft mouse model of canine lymphoma. This study validated immunotoxins as a potential treatment for canine lymphoma. Lastly, to validate a new liposome for canine lymphoma, we aimed to develop a liposome-based nanocarrier for panobinostat, using folate-targeted and non-targeted formulations. Both formulations were evaluated in canine lymphoma cells, validating liposomes as an effective treatment for canine lymphoma. In the future, our goal is to conjugate our VL sdAb to the liposome to obtain an immunoliposome for canine B-cell lymphoma. In conclusion, all the work developed contributed to the understanding of the importance of using the dog as a model and how these animals can contribute for clinical translation in the immune-oncology field
- Evaluation of morbillivirus and herpesvirus infection in cetaceans stranded along the portuguese coastlinePublication . Bento, Maria Carolina Rocha de Medeiros; Duarte, Ana Isabel Simões Pereira; Eira, Catarina Isabel Costa SimõesABSTRACT - Cetacean strandings are frequent along the Portuguese coastline and stranded specimens provide a unique opportunity to study disease and infectious disease agents in cetacean populations. Morbillivirus is particularly important in cetaceans for its ability to cause disease and for its high mortality rates. Herpesviruses are also particularly interesting since they are known to have close co-evolutionary paths with their hosts and for establishing latent infections that can resurge with any stressors of the immune system. This work aimed at investigating the epidemiology and pathophysiology of cetacean morbillivirus and herpesvirus. Furthermore, we aimed at establishing a profile of cytokine genes expression in healthy common dolphins and evaluate the possibility of using that tool to assess immunological shifts in this species. A molecular survey for dolphin morbillivirus revealed striped dolphins stranded in the Portuguese and Galician coasts have a prevalence of approximately 20% of positive individuals, higher than prevalence found for common dolphins (1%). A phylogeographic pattern was detected in the phylogenetic analysis with viral sequences from the Canary Islands more similar to those from the Mediterranean than samples from Portugal and Galicia. Herpesvirus infection was also detected in cetaceans, and both alpha and gammaherpesvirus were detected. Our work reported infection by herpesvirus in common dolphins for the first time. Co-infection with different strains of herpesvirus was detected, as well as co-infection with morbillivirus and Toxoplasma gondii. Relative expression of cytokine genes (IL1β, IL6, TNFα, IL12, IL4,IL10, and IFN-γ) was also determined for a group of healthy common dolphins. Relative expression was also determined in two sick common dolphins. Our results showed that IL1β, TNFα and IFN-γ were the cytokines with higher relative expression in healthy individuals and that Il-10 was the most expressed gene in the two sick animals, with a downregulation of all other cytokines. This work clarified the epidemiology of morbillivirus in cetacean species stranded along the Portuguse coastline, contributed to the variability of herpesvirus sequences in the gamma and alphaherpesvirus subfamilies and supports the evidence that profiling cytokine markers may serve as a tool to assess immunological shifts in stranded cetaceans
- Impact of leporid viral diseases in Iberian ecosystems : emergence, pathophysiology, prophylaxis and diagnosisPublication . Santos, Fábio Alexandre Abade dos; Duarte, Margarida Dias; Peleteiro, Maria da Conceição Cunha e Vasconcelos; Parra Fernández, FranciscoABSTRACT - 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. ...
- Unraveling the replication process of Toxoplasma gondii through the MOB1 proteinPublication . L. S. Delgado, Inês; Narciso, Sofia Bizarro Nolasco da Silva; Leitão, José Alexandre da Costa Perdigão e CameiraABSTRACT - 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.
- Mesophilic Aeromonas in threatened Iberian leuciscids : conservation and public health implicationsPublication . Grilo, Miguel; Oliveira, Maria Manuela Castilho Monteiro de; Robalo, Joana Isabel Espírito SantoDespite the high risk of extinction of the Iberian leuciscids, the impact of bacterial diseases on their conservation is unknown. A comprehensive knowledge of the interaction between these species and pathogens, such as mesophilic Aeromonas, as well as the development of biosafety measures for ex situ programs, are considered essential for the success of their conservation. The main objectives of this thesis are the evaluation of the epidemiology of mesophilic Aeromonas in Iberochondrostoma lusitanicum, as well as the characterization of their antimicrobial resistance and virulence profiles, the detection of differences in the diversity of Aeromonas spp. of I. lusitanicum and Squalius pyrenaicus, the evaluation of the potential of biosecurity measures in the control of Aeromonas spp. in ex situ programs and testing the effect of climate change on Aeromonas spp. The results showed that I. lusitanicum presented lower general health status in the dry season, varying with location. Aeromonas communities varied between seasons and locations, while their pathogenic potential increased in the dry season. Relevant resistance phenotypes (carbapenems and fluoroquinolones) were detected. Furthermore, I. lusitanicum and S. pyrenaicus showed different levels of skin lesions, with S. pyrenaicus being the most affected species. Aeromonas communities differed between the two fish species. Additionally, the use of biosecurity measures suggests a decrease in the prevalence of Aeromonas spp. in ex situ programs, as well as its pathogenicity to fish, while an increase in antimicrobial resistance was observed throughout the program. Finally, climate change predicted by the International Panel on Climate Change for water temperature and pH influenced the growth, biofilm production and antimicrobial resistance profiles of Aeromonas spp. The current results clarify the epidemiology of mesophilic Aeromonas in threatened Iberian leuciscids and contribute to the establishment of adequate conservation measures.
- Periodontal disease in dogs : an experimental approach towards prevention using antimicrobial peptidesPublication . Cunha, Eva; Oliveira, Maria Manuela Castilho Monteiro de; Veiga, Ana Salomé Rocha do NascimentoABSTRACT - Periodontal disease (PD) is one of the most widespread inflammatory diseases in dogs. This disease is initiated by a polymicrobial biofilm (dental plaque) in the teeth surface, along with a subsequent local inflammatory response leading to periodontium damage and systemic consequences. Enterococci are opportunistic bacteria that may be found in the plaque biofilm. Their ability to act as reservoirs of resistant determinants and their previously linkage to PD-systemic consequences, makes enterococci an interesting bacterial model for antimicrobial studies. Several strategies can be used for PD control, being the removal and inhibition of dental plaque the keystone for PD prevention. Antimicrobial peptides (AMPs) are promising compounds for the management of bacterial infections with low rate of resistance incidence. Nisin is an AMP with antimicrobial activity against Gram-positive and some Gram-negative bacteria, including periodontal pathogens. Considering PD high prevalence, its consequences, and the urgent need to control antimicrobial resistant (AMR) strains and develop new antimicrobial strategies, the main goal of this project was to evaluate the potential of the AMP nisin A for PD control in dogs. First, the antimicrobial activity of nisin and of nisin incorporated in two delivery systems (guar gum biogel and a veterinary toothpaste) were evaluated against a previously characterized collection of canine oral enterococci (n=20). Nisin and nisin-biogel were able to inhibit and eradicate all isolates tested in both planktonic and biofilm forms. Supplemented toothpaste was able to inhibit 95% of the isolates tested. As such, nisin-biogel was selected for the following assays, being observed that it also presented inhibition and eradication abilities against an in vitro multispecies biofilm. Then, nisin-biogel antimicrobial ability was evaluated in the presence of canine saliva and over a 24-month storage, as well as its safety towards eukaryotic cells. Saliva hampered nisin-biogel activity, but it maintained its antimicrobial activity over the storage period at freezing, cooling and room temperatures. In addition, nisin-biogel showed no cytotoxicity at concentrations up to 200 μg/mL. Later, nisin-biogel influence on the canine oral microbiome was evaluated by next generation sequencing, revealing that one dental application of nisin-biogel promoted a reduction in bacterial diversity. Still, a diversity recovery was detected after three applications, along with a reduction in some PD-related bacterial species. Finally, nisin influence in AMR development was evaluated by determination of the mutant section window, mutants AMR profiles and horizontal gene transfer (HGT). It was observed that nisin selective pressure may induce changes in the bacteria’s AMR profile, but not influencing vanA HGT between enterococci and staphylococci. Together, these results show that nisin-biogel seems to be an appropriate approach for PD control in dogs, being important to test this product in an in vivo controlled clinical trial.
- Cytokine gene expression and cellular immune response in dogs with leishmaniosis before and under the two first-line treatment protocols : new insights into the animal diseasePublication . Santos, Marcos André Ferreira; Sampaio, Isabel Maria Soares Pereira da Fonseca de; Gomes, Gabriela Maria SantosCanine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic visceral disease of worldwide concern. The drugs used for its treatment improve the animal’s clinical condition, although, in most cases, the parasites are not completely destroyed. The current study aimed to evaluate the immune response of the dog with leishmaniosis before and during treatment with first-line drugs, by analyzing the profile of cytokines and subsets of CD4+ and CD8+ T-cells in peripheral blood, lymph node and bone marrow. Two groups of six dogs diagnosed with CanL were treated with either miltefosine or meglumine antimoniate in combination with allopurinol. Simultaneously, another group of ten clinically healthy dogs was used as a control group. Upon diagnosis and during the following three months of treatment, clinical signs, hematological and biochemical parameters, urinalysis results and anti-Leishmania antibody titers using IFAT were recorded. Furthermore, peripheral blood, popliteal lymph node and bone marrow mononuclear cells were collected to evaluate the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β and IFN-γ by qPCR. In parallel, these cells were also immunophenotypically analyzed be flow cytometry, using surface monoclonal antibodies anti-CD45, CD3, CD4, CD8, CD25 and intracellular monoclonal antibody anti-nuclear factor FoxP3. Both treatment protocols promoted the remission of clinical signs, normalization of hematological and biochemical parameters and urinalysis values. Sick dogs showed a generalized increase in IFN-γ gene expression and a decrease of IL-2, IL-4, and TGF-β. The expression of IL-12, TNF-α, IL-5, and IL-10 showed variations between groups of dogs and the tissue analyzed. CanL also resulted in an overall increase in the percentage of CD8+ T-cells in all tissues. In the peripheral blood there was also a decrease in CD4+ T-cells and an increase of CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ T-cells, with the latter also increasing on the bone marrow. CD4+CD25-FoxP3- T-cells showed a marked decrease in blood and bone marrow. During treatment, a trend towards normalization of cytokine gene expression and T-cell subsets was observed. However, high levels of IFN-γ gene expression were still observed in all tissues. In turn, the treatments caused an increase in the percentage of CD4+CD25+FoxP3+ and a decrease in CD8+CD25-FoxP3- T-cells, leading to normalization of CD4+ and CD8+ T cells in all tissues. Furthermore, the effect of treatment on gene expression of cytokines that were not significantly altered by infection indicates that these combined treatment protocols directly affect cytokine production. Both combined treatments are effective in remitting clinical sings and appear to influence the dog’s immune response, sustaining a pro-inflammatory immune environment while promoting the normalization of T-cell subsets. These findings indicate that L. infantum may be able to manipulate elements of the dog's immune system to avoid differentiating an efficient protective response, preventing the rapid development of severe pathology while ensuring the parasite’s survival and securing the possibility of several transmission cycles. Allied to these results, other studies carried out in collaboration with the working group on the role of neutrophils, hepatocytes and Kupffer cells in CanL, as well as the evaluation of treatment in feline leishmaniosis, have allowed to enhance the knowledge in the area of animal leishmaniosis.
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