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- 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
- Parasitas gastrointestinais em produção de frango ao ar livrePublication . Lozano, João; Gomes, Lídia; Madeira de Carvalho, LuísA investigação em sanidade animal realizada a nível nacional em sistemas extensivos de produção de frango é essencial, atendendo à frequente longa exposição dos animais ao ambiente exterior e aos parasitas que o contaminam. Estudos parasitológicos são extremamente relevantes de forma a conhecer quais as espécies parasitárias mais prevalentes e ajudar os produtores a definirem melhores medidas profiláticas. Esta pesquisa procurou identificar os parasitas gastrointestinais associados a este tipo de produção aviária e ocorreu numa exploração agropecuária localizada na extremidade noroeste do distrito de Lisboa, onde foram avaliados Frangos do Campo através de amostragem fecal aos 75 e 85 dias de idade. O parasitismo gastrointestinal foi analisado através de métodos coprológicos qualitativos, quantitativos e coproculturas para obtenção de oocistos esporulados e larvas L3 de nematodes estrongilídeos. Na 1ª amostragem, a prevalência de Eimeria spp. totalizou 40,0%, sendo Eimeria mitis (13,3%) e E. maxima (11,7%) as mais prevalentes, tendo-se identificado também ovos de Capillaria sp., (1,7 % de prevalência). Nas coproculturas de oocistos, esporularam as seguintes espécies: E. necatrix, E. mitis, E. praecox, E. maxima, E. acervulina e E. tenella. Na 2ª amostragem, as coccídeas continuaram a ser o grupo mais prevalente, 90,0% de amostras positivas com oito espécies identificadas, sendo de novo E. mitis a espécie mais frequente (41,7%), seguida por E. acervulina (26,7%) e E. tenella (25%). Foram também observados ovos de Heterakis sp. (3,33%) e novamente de Capillaria sp. (10,0%) A carga parasitária fecal de oocistos na 1ª e 2ª amostragens foi de 370,8 ± 2055,3 OoPG e 599,2 ± 1289,2 OoPG, respetivamente, verificando-se um aumento superior a 60% do valor médio deste parâmetro parasitológico da 1ª para a 2ª colheita. O crescimento da erva, a idade e densidade animal, bem como o tempo de exposição a parasitas com períodos pré-patentes curtos, constituíram fatores chave para o aumento da prevalência de oocistos de Eimeria spp. e ovos de helmintes entre as duas amostragens, em particular de espécies com reconhecida patogenicidade. Isto permite-nos concluir que no final da fase de engorda, as cargas parasitárias são as mais elevadas detetadas neste sistema de produção de Frango do Campo, podendo desencadear patologia associada e interferir com a sua performance produtiva.
- Long-term survey of Eimeria spp. prevalence and faecal shedding in a traditional Portuguese free-range broiler farmPublication . Lozano, João; Palomero Salinero, Antonio; Anaya, Adriana; Lux Hoppe, Estevam G.; Gomes, Lídia; Oliveira, Manuela; Paz-Silva, Adolfo; Rebelo, Maria Teresa; Madeira de Carvalho, LuísIn free-range broiler production, animals are highly exposed to gastrointestinal parasitism, namely by coccidia, due to their long contact with the outdoor environment. Also, the prevalence of gastrointestinal parasites in extensive poultry production, is frequently influenced by biotic and abiotic factors. The current national pioneer long-term survey aimed to assess the prevalence and faecal shedding of Eimeria spp. oocysts in a traditional Portuguese free-range broiler farm, on different production conditions. Between July 2018 and March 2019, a total of 350 faecal samples were collected from different groups of free-range broilers at the fattening phase, in a poultry farm located in North-western Lisbon district, Portugal. Quantitative and qualitative coprological techniques were performed, aiming to quantify the Eimeria spp. prevalence and faecal shedding level. Coccidia prevalence was higher in Summer and Autumn groups (72% and 80%, respectively), although faecal shedding levels were higher in the Autumn and Winter groups (1191 and 1562 Oocysts Per Gram, OPG). Animals showed clinical signs of coccidiosis during the survey, especially in Autumn’s 1st sampling, in which 42% of the faecal samples had blood. The lack of sanitary depopulations, length of the fattening phase (1 month), short pre-patent period of coccidia, exposure to different environmental conditions and irregular drug treatments with amprolium, were key factors responsible for the different parasitic scenarios observed during this survey.
- Biocontrol of Avian Gastrointestinal Parasites Using Predatory Fungi: Current Status, Challenges, and OpportunitiesPublication . Lozano, João; Almeida, Cristina; Oliveira, Manuela; Paz-Silva, Adolfo; Madeira de Carvalho, LuísThis review describes the current research status regarding the implementation of predatory fungi in the biological control approach of bird gastrointestinal (GI) parasitosis. The main GI parasites of Galliformes (e.g., broilers, layers, peacocks, pheasants) and Ratites (e.g., ostriches, emus, rheas) are addressed, as well as their impact on farms, zoos, and private collections. The main characteristics regarding biocontrol with predatory fungi are briefly described, such as their mode of action and efficacy against GI parasites of different animal hosts. The state of the art regarding the use of predatory fungi in birds is reviewed here by describing all associated articles already published in the main databases, techniques, and their main findings. Ovicidal fungi such as Pochonia chlamydosporia, Metarhizium spp. and Acremonium spp., and larvicidal fungi, namely Duddingtonia flagrans, Arthrobotrys spp. and Monacrosporium thaumasium, have shown promising predacious activity against ascarid eggs and nematode larvae from chickens and ostriches, both in vitro and in vivo, also revealing tolerance to the GI passage in chickens and maintenance of predacious capacity. Further studies are needed to understand the fungi–parasite–host gut microbiota interactions and target other avian GI parasitic species, such as nematodes, coccidia, cestodes, and trematodes.
- Control of Strongyles in first-season grazing ewe lambs by integrating deworming and thrice-weekly administration of parasiticidal fungal sporesPublication . Voinot, Mathilde; Bonilla, Rodrigo; Sousa, Sérgio; Sanchís, Jaime; Canhão-Dias, Miguel; Delgado, José Romero; Lozano, João; Sánchez-Andrade, Rita; Arias, Maria Sol; Madeira de Carvalho, LuísParasiticidal fungi have been used in several in vivo experiments in livestock farms worldwide, constituting an effective tool for the biocontrol of gastrointestinal parasites in grazing animals. In the first year of study, two groups of eight first-season pasturing ewe lambs infected by strongyles were dewormed with albendazole, and then, the test group received an oral dose of 106 chlamydospores of Mucor circinelloides and 106 Duddingtonia flagrans individually and thrice a week from mid-September to May (FS1), while the control group remained without fungi (CT1). In the second year, two new groups of first-season grazing ewe lambs were treated with ivermectin and subjected to the same experimental design (FS2 and CT2, respectively). The anthelmintic efficacy was 96.6% (CT1), 95.6% (FS1), 96.1% (CT2), and 95.1% (FS2). The counts of strongyle egg output increased in the control groups (CT1 and CT2) throughout the study and reached numbers higher than 600 eggs per gram of feces (EPG), while in FS1 and FS2, they were <250 EPG. The values of red blood cell parameters registered for CT1 and CT2 were lower than those of the reference standards, while a significant increment was recorded in FS1 and FS2, and values within the physiological range were attained. It is concluded that integrating efficient anthelminthic deworming with rotational pasturing and the regular intake of chlamydospores of M. circinelloides and D. flagrans provides a helpful strategy for maintaining low levels of strongyle egg output in first-season grazing ewe lambs and improves their health status.
- Gastrointestinal parasites of free-range chickens : a worldwide issuePublication . Lozano, João; Anaya, Adriana; Palomero Salinero, Antonio; Lux Hoppe, Estevam Guilherme; Gomes, Lídia; Paz-Silva, Adolfo; Rebelo, Maria Teresa; Madeira de Carvalho, LuísGastrointestinal parasites with direct and/or indirect life cycles, namely Eimeria spp., Ascaridia galli, Heterakis gallinarum and Capillaria spp., are responsible for the most frequent and heavy economic losses in aviculture. The current review aimed to collect information about gastrointestinal parasites affecting poultry, as well as, research studies regarding parasitic diseases in free-range chicken production. From the references used in this review, 20% highlight research studies correlating prevalence of gastrointestinal parasites in free-range chickens and factors such as the production system, age and animal density, hygienic conditions and weather. Coccidiosis is responsible for the major economic losses in poultry farms. Diseases have different clinical signs and result in variable impacts on animals. Control programs include mainly vaccination, anti-coccidia and anthelminthic drugs, as well as, house cleaning and disinfection. This review concluded that coccidia and helminths are a global threat to free-range poultry production. More research in this area is necessary in order to understand the major factors influencing the prevalence of parasitic diseases in this type of poultry production. Also the search for new control strategies must be a priority, mainly using natural antiparasitic compounds and biological control approaches.
- Diagnosis of coccidiosis by Eimeria spp. in free-range chickens using Mini-FLOTAC and McMaster techniques - preliminary resultsPublication . Lozano, João; Anaya, Adriana; Rinaldi, Laura; Cringoli, Giuseppe; Gomes, Lídia; Oliveira, Manuela; Paz-Silva, Adolfo; Rebelo, Maria Teresa; Madeira de Carvalho, LuísMini-FLOTAC is emerging as a more sensitive and accurate tool to identify gastrointestinal parasites in faecal samples from domestic animals, in comparison with the McMaster method. However, research regarding its specific application in poultry samples, particularly from free-range chickens, is scarce. The current research aimed to test the use of Mini-FLOTAC for the identification of Eimeria spp. in free-range chickens and compare its results with McMaster. For this study, 40 faecal samples were collected from free-range chickens in a poultry farm located in North-Western Lisbon (Portugal). Each sample was processed with McMaster and Mini-FLOTAC techniques for the detection and count of coccidian Eimeria spp. oocysts. The resulting OPG (oocysts per gram of faeces) data obtained by the two techniques were compared using the Wilcoxon Test and correlated with the Spearman Test, and Mini-FLOTAC’s relative sensitivity was assessed, using a significance level of p<0.05. The average OPG was higher with Mini-FLOTAC and doubled the one obtained using the McMaster method (2669.3 OPG and 1220 OPG, respectively), although these results were not significant. Mini-FLOTAC’s relative sensitivity obtained in this study reached 86% (70.5-95.3%, 95%CI), although this result was not statistically significant. However, correlation of OPG counts between Mini-FLOTAC and McMaster, was significant. These preliminary results suggest the potential interest in the use of Mini-FLOTAC for the diagnosis of coccidiosis by Eimeria spp. in poultry, based on its assessment in a free-range poultry production system.