http://hdl.handle.net/10174/1027 2026-05-16T06:52:10Z http://hdl.handle.net/10174/42017 Title: Species diversity, benzimidazole resistance, and phytochemical synergy in gastrointestinal nematode nemabiomes from sheep farms in Portugal Authors: Campbell, Natália Faria; Waap, Helga; Cortes, Helder Abstract: Anthelmintic resistance in gastrointestinal nematodes (GINs) threatens small ruminant production. This study assessed ovicidal activity of eight natural aromatic phytochemicals and interactions with synthetic benzimid- azoles (BZs: albendazole [ABZ], thiabendazole [TBZ]) against nemabiome eggs of 14 BZ-resistant sheep farms across Tra ́s-os-Montes, Beira Interior, Estremadura, and Alentejo regions of Portugal. Trans-cinnamaldehyde (TCL), octyl gallate (OGA), carvacrol (CRV), thymol (THY), and salicylaldehyde (SAL) achieved >90 % egg hatch inhibition (EHI) at 250 μg/mL in egg hatch tests (EHTs), unlike trans-cinnamic acid, gentisic acid, and curcumin (<90 % EHI). Effective concentrations (EC10–EC90) were determined for each compound against farm nem- abiomes. TCL (EC90 ≈ 27.0 μg/mL) and OGA (≈54.1 μg/mL) showed highest ovicidal efficacy, followed by CRV (≈120.3 μg/mL), THY (≈171.3 μg/mL), and SAL (≈155.2 μg/mL), requiring 25–220-fold higher concentrations than BZs. EC10–EC75 concentrations in checkerboard assays determined fractional inhibitory concentration indices (FICIs) to assess interactions. Compound interactions (FICI: 0.20–0.49; all ≤0.5) reduced ABZ mean EC90 by 0.73 μg/mL (89.6 %) and TBZ by 0.72 μg/mL (94.7 %), with TCL and OGA showing strongest effects (FICI ≤0.25). ITS2 metabarcoding identified six GIN species, with Haemonchus contortus, Teladorsagia circumcincta, and Trichostrongylus colubriformis most prevalent. Nemabiome diversity (H’: 0.09–1.17) and species prevalence showed no correlation with EHI EC90 values (|r| < 0.418, p > 0.05), suggesting ovicidal efficacy or BZ- phytochemical interactions are not species-driven. Synergistic mechanisms involving aromatic phytochemical- induced eggshell damage, complementing BZ β-tubulin disruption, are discussed. BZ-phytochemical combina- tions offer a promising strategy for sustainable GIN control, warranting in vivo validation. 2025-11-05T00:00:00Z http://hdl.handle.net/10174/42016 Title: In Silico–Based Investigation of the Immunogenicity and Biochemical Attributes of Toxoplasma gondii Apical Membrane Antigen 1 (TgAMA1) Authors: Foroutan, Masoud; Ghaffari, Ali Dalir; Ghaffarifar, Fatemeh; Karimipour-Saryazdi, Amir; Birgani, Arezo Arzani; Majidiani, Hamidreza; Elsheikha, Hany M.; Cortes, Helder Editors: Choudhury, Satabdi Datta Abstract: Background: Apical membrane antigen 1 (AMA1) is a highly conserved microneme protein in apicomplexan parasites. In this study, immunoinformatics tools and in silico protein structure prediction were used to characterize the structure, physicochemical properties, posttranslational modification sites, immunogenic epitopes, allergenicity, and immune simulation of the Toxoplasma gondii AMA1 (TgAMA1) protein. Methods: A comprehensive analysis was performed using multiple bioinformatics web servers to analyze the antigenicity, physicochemical features, secondary and tertiary structures, B and T cell epitopes, and in silico immune simulation of TgAMA1. Results: The analysis revealed that the AMA1 protein consists of 569 amino acid residues and has a molecular weight of approximately 63 kDa. The grand average of hydropathicity (GRAVY) was -0.531 and the aliphatic index was calculated as 64.62. Based on the GOR IV server, TgAMA1 contained 20.21% alpha helices, 58.52% random coils, and 21.27% extended strands. The Ramachandran plot of the refined model revealed that over 97% of the residues were located in the favored region. The AMA1 protein was highly immunogenic and nonallergenic in nature. In silico immune simulation using the C-ImmSim server suggested that three doses of TgAMA1 would elicit potent humoral and cell-mediated immune responses. Conclusion: These findings provide valuable insights for further in vitro and in vivo investigations of TgAMA1’s potential as a vaccine candidate against toxoplasmosis. 2025-03-18T00:00:00Z http://hdl.handle.net/10174/42014 Title: Comparison of two BCG adjuvants in enhancing the immunogenicity of a DNA vaccine encoding the Toxoplasma gondii ROP2 gene in BALB/c mice Authors: Yousefi, Hamed; Ghaffarifar, Fatemeh; Foroutan, Masoud; cortes, Helder; Khosroshahi, Kamy Hosseinian Editors: Beckingham, Lachlan Abstract: The lack of a definitive treatment against toxoplasmosis and its high prevalence encouraged us to seek ways to expand DNA vaccines. The rhoptry protein 2 (ROP2) of Toxoplasma gondii is one of the most important antigens expressed in all three stages of the parasite’s life cycle. BCG is an immune stimulator and can act as an important adjuvant for protection against infectious diseases. The recombinant pcROP2 plasmid was constructed, trans- fected into CHO cells, and protein expression was confirmed by western blotting. The recombinant plasmids (100 μg/100 μL) and two types of BCG adjuvants, namely intact BCG and BCG lysate, were injected into female BALB/c mice three times. Then, immunological factors (total IgG, IgG2a, and IgG1) and two cytokines, including IFN-γ and IL-4, were measured, and the survival rate of the mice after challenge with T. gondii tachyzoites was monitored. The group receiving pcROP2 +BCG lysate produced more IgG2a and total IgG than the other groups (p ≤ 0.05). Evaluation of spleen cytokines IFN-γ and IL-4 showed that IFN-γ levels were significantly higher in mice receiving the recombinant plasmid and BCG lysate compared to the other groups. In mice receiving pcROP2 +BCG lysate, the survival rate was higher than in the other groups, suggesting that pcROP2 +BCG lysate is more effective at inducing cellular immune responses and may be suitable for increasing the lifespan of mice with acute toxoplasmosis. 2025-12-20T00:00:00Z http://hdl.handle.net/10174/42013 Title: High burden and multi-parasite profile of gastrointestinal infections in cattle from Limpopo District, Southern Mozambique: Epidemiology, risk factors, and One Health implications Authors: Manave, Edvânia Celso; Nchowela, Guido André; Miguel, Avelino Raimundo; Matuassa, Carcésia César; Muadica, Aly Salimo; Machanja, Bendito; Fernandes, Lucel; Mavilingue, Omar Manito; Simbine, Lúnice; Tsamba, Priscília; Manuel, Ilídio Filipe; Muchanga, Izaidino Jaime; Deta, Taís; Elina Manuel, Ualema; Cortes, Helder; Alfredo, Célio Editors: Sherasiya, Anjum V. Abstract: Background and Aim: Gastrointestinal (GI) parasites significantly affect cattle productivity and animal health, especially in tropical regions where environmental and management conditions favor parasite survival. In Mozambique, most previous studies have focused on goats or individual parasite species, leaving crucial gaps in understanding multi-parasite burdens in cattle. This study aimed to determine the prevalence, diversity, and risk factors associated with GI parasites in cattle from the Limpopo district of southern Mozambique, applying a One Health lens due to the zoonotic potential of some parasites that circulate in cattle. Materials and Methods: A cross-sectional study was conducted from January to May 2025 using 200 stool samples collected directly from cattle rectums. Samples were examined using Ritchie centrifugal sedimentation for helminths and protozoa and Ziehl–Neelsen staining for Cryptosporidium spp. Epidemiological data on grazing areas, deworming practices, and animal demographics were collected to identify risk and protective factors through Fisher’s test and odds ratios (OR). Results: Overall, 88.5% of cattle harbored at least one GI parasite. Eight parasite groups were detected: Eimeria spp. (49%), Strongyle-type eggs (46.5%), ciliates (29.5%), Paramphistomum spp. (18%), Fasciola spp. (11%), Cryptosporidium spp. (3.5%), Giardia spp. (2.5%), and Entamoeba spp. (1.5%). Grazing in non-flooded areas significantly reduced Fasciola spp. infection. (OR = 0.126) and Paramphistomum spp. (OR = 0.236), whereas deworming reduced Strongyle-type infections (OR = 0.366). Conversely, dewormed animals had higher odds of Eimeria spp. and ciliate infections, likely because ivermectin was ineffective against protozoa. Co-infections were common, particularly among adult animals. Conclusion: This first multi-parasite epidemiological assessment in Mozambican cattle reveals a high burden of GI parasites, influenced by grazing conditions and suboptimal deworming practices. Avoiding flooded grazing areas, adopting coccidiostats, and implementing anthelmintic rotation are crucial for effective parasite control. Given the zoonotic relevance of Cryptosporidium, Giardia, and Fasciola, molecular studies are urgently needed to characterize circulating species and clarify the role of cattle as reservoirs. These findings provide essential evidence to strengthen veterinary surveillance and inform One Health interventions in southern Mozambique. 2025-12-23T00:00:00Z