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Associate Laboratory for Animal and Veterinary Sciences
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Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocysts
Publication . Lozano, João; Louro, Mariana; Almeida, Cristina; Victório, Ana Cláudia; Melo, Pedro; Rodrigues, João Paulo; Oliveira, Manuela; Paz-Silva, Adolfo; Madeira de Carvalho, Luís
Fungal 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
Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial community
Publication . Lozano, João; Cunha, Eva; Almeida, Cristina; Nunes, Mónica; Dias, Ricardo; Vicente, Eduardo; Sebastião, Daniela; Henriques, Sérgio; Madeira de Carvalho, Luís; Paz-Silva, Adolfo; Oliveira, Manuela
Parasiticide 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.
Hygiene and biosecurity conditions of initial examination on-spot in Portugal: one step toward game meat safety
Publication . Abrantes, Ana Carolina; Ferreira, Maria Pureza; Ruano, Zita; Vinhas, Bruno; Vaz, Yolanda; Vieira-Pinto, Madalena
Background 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.
Characterization of insect bite hypersensitivity in a population of Lusitano horses : contribution for future implementation of skin prick tests (SPT) in IBH diagnosis
Publication . Pessoa, Vera Purificação Carvalho; Tilley, Paula Alexandra Botelho Garcia de Andrade Pimenta; Ferreira, Manuel Branco
Insect Bite Hypersensitivity (IBH) is a recurrent seasonal pruritic dermatitis affecting many horses worldwide. It is mainly a type I hypersensitivity, but type IV may also occur, to allergens present in the saliva of Culicoides spp. It is a multifactorial disease affecting different breeds, with a variable prevalence. Treatment options are very limited, but being an allergic disease IgE-mediated, there is the possibility of developing specific immunotherapy. This study aimed to characterize IBH in Lusitano horses living in Portugal mainland, through a case control study involving 30 IBH-affected animals and 30 healthy controls. Questionnaires were performed for all the horses involved in the study in order to characterize the studied population. In vivo allergen testing was evaluated by performing skin tests, both skin prick tests (SPT) and intradermal tests (IDT), using 14 specific allergens, including 13 different recombinant (r-) proteins from Culicoides nubeculosus (Cul n) or Culicoides obsoletus (Cul o) salivary glands (termed Cul n 1 to Cul n 11, Cul o 1P and Cul o 2P) as well as Cul n whole body extract (WBE). Moreover, a cluster of 12 of these horses, 6 IBH-affected and 6 from the control group, were also tested with allergens produced in insect cells (Cul n 3Bac, Cul n 4Bac) and barley (Cul n 3 Bar, Cul n 4Bar) as well as Cul o 3 and Cul o whole body extract (WBE). Furthermore, in vitro diagnostic tests have been performed, namely, serum IgEs that were measured by ELISA for the allergens Cul n 3, 4, 9, 10 and Cul o 2, 3, as well as Cul o1P and Cul o2P and in vitro sulfidoleukotriene (sLT) release assay which were carried out with Cul n WBE and Cul o WBE. In our study SPTs presented a higher discriminatory diagnostic potential than IDTs, and should be preferred, being a potential breakthrough in the establishment of more suitable allergen avoidance measures in IBH diagnosis. Regarding the in vitro tests, even though serology measurement of specific IgEs showed that IBH-affected horses had higher serum IgE levels for the significant allergens, sLT release assay performed best. It was also possible to study and identify Culicoides spp found in the stud farms, which may be related to IBH in the studied horses. The most frequently found were Obsoletus group species, C. nubeculosus and C. imicola. This study contributed to improve IBH diagnosis, representing a step forward for the future implementation of locally relevant diagnostic allergen panels and eventually for the establishment of patient tailored, component resolved specific immunotherapy
Impact of high Spirulina diet, extruded or supplemented with enzymes, on blood cells, systemic metabolites, and hepatic lipid and mineral profiles of broiler chickens
Publication . Spínola, Maria P.; Alfaia, Cristina M.; Costa, Mónica M.; Pinto, Rui M. A.; Lopes, Paula A.; Pestana, José M.; Tavares, João C.; Mendes, Ana R.; Mourato, Miguel; Tavares, Beatriz; Carvalho, Daniela F. P.; Martins, Cátia F.; Ferreira, Joana I.; Lordelo, Madalena; Prates, José A. M.
The impact of 15% dietary inclusion of Spirulina (Arthrospira platensis) in broiler
chickens was explored, focusing on blood cellular components, systemic
metabolites and hepatic lipid and mineral composition. From days 14 to
35 of age, 120 broiler chickens were divided and allocated into four dietary
treatments: a standard corn and soybean meal-based diet (control), a 15%
Spirulina diet, a 15% extruded Spirulina diet, and a 15% Spirulina diet super-dosed
with an enzyme blend (0.20% porcine pancreatin plus 0.01% lysozyme). The
haematological analysis revealed no significant deviations (p > 0.05) in blood
cell counts across treatments, suggesting that high Spirulina inclusion maintains
haematological balance. The systemic metabolic assessment indicated an
enhanced antioxidant capacity in birds on Spirulina diets (p < 0.001), pointing
toward a potential reduction in oxidative stress. However, the study noted a
detrimental impact on growth performance metrics, such as final body weight
and feed conversion ratio (both p < 0.001), in the Spirulina-fed treatments, with
the super-dosed enzyme blend supplementation failing to alleviate these effects
but with extrusion mitigating them. Regarding hepatic composition, birds on
extruded Spirulina and enzyme-supplemented diets showed a notable increase
in n-3 fatty acids (EPA, DPA, DHA) (p < 0.001), leading to an improved n-6/n-3
PUFA ratio (p < 0.001). Despite this positive shift, a reduction in total hepatic lipids
(p = 0.003) was observed without a significant change in cholesterol levels. Our
findings underscore the need for further exploration into the optimal inclusion
levels, processing methods and potential enzymatic enhancements of Spirulina
in broiler diets. Ultimately, this research aims to strike a balance between
promoting health benefits and maintaining optimal growth performance in
poultry nutrition.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
LA/P/0059/2020