Pulmonary and Gastrointestinal Parasitic Infections in Small Ruminant Autochthonous Breeds from Centre Region of Portugal-A Cross Sectional Study.

The production of small ruminant autochthonous breeds in the Centre region of Portugal is practiced in a semi-extensive husbandry system, exposing animals to parasitic infections. The main objective of this study was to estimate the prevalence of lungworm infection and identify risk factors. Fecal samples of 203 goats and 208 sheep from 30 herds were collected per rectum and subjected to the modified Baermann test. The overall prevalence of infection was 57.7%, significantly higher in goats (95.6%) than in sheep (20.7%) (p < 0.001). According to the binary logistic regression model, sheep dewormed with albendazole, mebendazole plus closantel, or ivermectin plus clorsulon presented a risk of Protostrongylidae infection 29.702, 7.426, or 8.720 times higher, respectively, than those dewormed with eprinomectin. Additionally, the presence of gastrointestinal parasites was investigated in 307 fecal samples using Mini-FLOTAC®. The overall prevalence of infection was 86.3%, also significantly higher in goats (93.2%) than in sheep (79.9%) (p < 0.001). Strongyle-type eggs were the most frequently identified, both in sheep (69.8%) and goats (87.8%), followed by Eimeria oocysts (40.3% in sheep and 68.9% in goats). Considering the high prevalence and the burden of lungworm parasitic infection, it is urgent to determine its economic impact and the repercussions in animal health in the Centre region of Portugal to establish appropriate therapeutic guidelines.

Seroprevalence study of brucellosis in wild boar hunted for private consumption in northeast Portugal.

Brucellosis is an important infectious disease caused by bacteria of the genus Brucella. In the northeast region of Portugal, infection with Brucella melitensis is endemic in small ruminants, and there are also humans' cases. However, the epidemiological role of the wild boar in the dynamics of this disease in this region is unknown. In this study, a total of 332 blood samples were collected from wild boar hunted in thirty-six hunting areas during the 2022/2023 hunting season. All were taken by the hunters for private consumption, with no evisceration or examination in the field. Serum samples were tested by indirect ELISA (i-ELISA). It was observed that 88 wild boars were exposed to Brucella spp., pointing to a seroprevalence of 26.5% (95% CI: 21.8 - 31.3%). This high prevalence underlines the importance that wild boar may have in the dynamics of this disease in the region and its potential transmission to other animals, and to humans (for example, during the handling of carcasses). Increased awareness and knowledge of brucellosis in wild boar is essential for the implementation of effective practices and habits and, consequently, for the control and prevention of this important zoonosis.

Prickly Connections: Sociodemographic Factors Shaping Attitudes, Perception and Biological Knowledge about the European Hedgehog.

The modern lifestyle of humans is leading to a limited exposure to nature. While several wild species are adapting and thriving in anthropic environments, natural history knowledge is declining, and positive attitudes and behaviours towards nature are facing challenges. Because anticipating attitudes and engendering broad-based support for nature-related measures requires a good grasp of social contexts, we set out to evaluate the sociodemographic factors driving the perception, attitudes towards, and natural history knowledge of a keystone species-the European hedgehog. In 2022, we conducted a questionnaire answered by 324 Portuguese adults. We found generally positive feelings and attitudes towards this species. A higher degree of academic qualifications and previous personal experience with the species seem to play a role in (i) people's perception about human impacts on hedgehogs and (ii) positive attitudes, especially during encounters where the animals were in difficulty. Despite this, the extent of natural history knowledge was low overall, and the study population was self-aware of this. Our insights underline the need to tailor educational programmes if we are to encourage people to re-establish meaningful connections with nature, to foster social support for biodiversity stewardship, and to implement the One Health approach in a way that resonates with distinct social groups.

A Preliminary Investigation of Salmonella Populations in Indigenous Portuguese Layer Hen Breeds.

The sustainability of agroecological systems, biodiversity protection, animal welfare, and consumer demand for higher quality products from alternative and extensive farming methods have reinforced interest in local breeds that are well adapted to low-input environments. However, food safety needs to be safeguarded to reinforce consumer confidence. The aim of this study was to conduct a preliminary investigation on the occurrence of Salmonella spp. in eggshells, hen's cloaca, and litter materials from autochthonous Portuguese laying hens raised in a semi-extensive system for small-scale production. A total of 279 samples from 31 flocks belonging to 12 farms were obtained, with 63 samples from the "Preta Lusitânica" breed, and 72 samples each from the remaining autochthonous breeds, namely, "Branca", "Amarela", and "Pedrês Portuguesa". None (0%) of the samples analyzed were positive for Salmonella spp. To the best of our knowledge, these are the first results of Salmonella evaluation from hen's cloaca, eggshells, and litter materials in autochthonous Portuguese chickens, suggesting that a semi-extensive production system can contribute to better food security and a lower risk to public health and the environment.

Avian Malaria in wild birds from a wildlife rehabilitation center in Central Portugal.

Avian haemosporidian (Haemoproteus, Leucocytozoon, Plasmodium) are vector-transmitted protozoan parasites highly prevalent in various bird species. Still, their importance for bird health, species decline, or impact on rehabilitation success is underestimated. This study aimed to determine the occurrence and diversity of haemosporidian parasites after necropsies of seventy wild birds from thirty-four species of twelve taxonomic orders. Detection of avian haemosporidian DNA was evaluated using PCR amplification of the cytochrome b gene. 48.6% of all sampled birds were positive, with 24.3% positive for Plasmodium spp./Haemoproteus spp. and 44.3% for Leucocytozoon spp. Mixed infections corresponded to 20% of all tested birds. Sequencing of several selected samples revealed the infection of Plasmodium matutinum, Plasmodium relictum and different lineages of Leucocytozoon spp. This study provides a baseline description of haemosporidian infections in wild birds from a rehabilitation center in central Portugal. The results show the necessity to test and monitor possible infections that undermine recovery processes for different birds. Further research into the occurrence of these haemosporidian species in birds kept in conservation centers is needed to understand the impact on bird health.

High Levels of Heavy Metal(loid)s Related to Biliary Hyperplasia in Hedgehogs (Erinaceus europaeus).

Heavy metal(loid) pollution of ecosystems is a current One Health problem. The liver is one of the most affected organs in cases of acute or chronic exposure to abnormal amounts of these substances, inducing histopathologic lesions. In order to assess the influence of heavy metal(loids), forty-five European hedgehogs (Erinaceus europaeus) were submitted to necropsy, and liver samples were collected for a routine histopathology exam and metal(loid)s determination (As, Cd, Co, Cr, Cu and Pb) by ICP-MS. Age was estimated during the necropsy exam. Biliary hyperplasia was the most frequent lesion observed (16/45; 35.56%). No statistically significant associations were found between biliary hyperplasia and age or sex. Metal(loid)s' concentrations were higher in animals with biliary hyperplasia (except for As). There was a statistically significant difference for both Cd and Co. For As, Cd and Co, cubs and juveniles animals showed significantly lower concentrations than elder individuals. Only for Pb were significant differences found between females and males. As described in the literature, exposure to metal(loid)s may be a cause of biliary hyperplasia, although further research (including the use of biochemical methods) is needed to support these results. To the authors' knowledge, this is the first report of this association in hedgehogs.

First Molecular Identification of Fasciola gigantica in Slaughtered Cattle in Cape Verde: Prevalence, Gross Pathological Lesions, Genetic Identification and Coprological Analysis.

A study on fasciolosis prevalence, gross pathological lesions, fluke genetic identification and coprological analysis was carried out in slaughtered cattle from one abattoir in Cape Verde. Of the 131 cattle inspected over two months, 12 (9.0%) presented fasciolosis-compatible lesions (FCL) that resulted in liver condemnation. The genetic characterization of the flukes collected, through restriction fragment length polymorphism analysis of PCR-amplified fragments (PCR-RFLP), confirmed the presence of Fasciola gigantica; therefore, being the first identification of this species in cattle from Cape Verde. Animals that released Fasciola spp. eggs and, thus, responsible for environment contamination (positive shedders), were identified through coprological analysis (natural sedimentation technique). Of the 12 animals with FCL, samples from 11 were submitted to coprological analysis and 7 (63.6%) were found to be positive shedders. Furthermore, of the 82 animals with non-FCL, randomly selected for coprological analysis, 4 (4.9%) were also found to be positive shedders for Fasciola spp. The results of this study, regarding species identification and coprological analysis, are epidemiologically important to update the information regarding fasciolosis in Cape Verde. The new data could help implement effective strategies for disease control and mitigation, consequently reducing economic loss and the level of animal and human infection from the One Health perspective.

ANIPHI: An innovative pedagogical platform based on the Delphi method to support animal welfare teaching.

As a teaching subject, animal welfare is challenging for educators and learners, as was recently shown in a recent survey on the evolution of animal welfare teaching in Europe. Among several suggestions to overcome the current resistance to implementing animal welfare education, we highlight two. The first is that animal welfare education should be based on learner-centred approaches; the second is that it should encompass both animal welfare science and ethics and law. To the best of our knowledge, there are no learner-centred pedagogical approaches that can simultaneously explore scientific and ethical concepts. Furthermore, when exploring ethical concepts within the educational context, there is the additional challenge of being able to depart from discussion and debate to a systematic organization of knowledge. Our work simultaneously addresses these two challenges, presenting the design and implementation of a novel web-based learner-centred pedagogical platform for farm animal welfare teaching. The platform, named ANIPHI, uses the Delphi method's iterative nature as a learning process to generate both reflection and (online) debate among learners. ANIPHI can be used by educators in an online environment, in a classroom environment, or in a combination of the two environments. ANIPHI was developed within the ERASMUS+ ANICARE project and is an open web-based platform for all educators interested in teaching farm animal welfare. Given ANIPHI's flexible and user-friendly nature, the platform simultaneously exposes learners to ethical and scientific concepts in different educational realities, according to the educator's objectives. Furthermore, videos depicting different husbandry practices across different types of animal production and countries are embedded in the platform. These videos are commented on by the farmer himself and by animal scientists, which enriches the learner's experience. Educators across the ANICARE consortium have already successfully tested the ANIPHI platform for different farm animal welfare topics. We conclude this article by presenting one example of using ANIPHI in a real-life educational context, where we discuss some aspects of the design and use of our pedagogical platform.

Seroprevalence of SARS-CoV-2 in Client-Owned Cats from Portugal.

The close contact between humans and domestic cats raises concerns about the potential risks of SARS-CoV-2 transmission. Thus, this study aims to investigate anti-SARS-CoV-2 seroprevalence in client-owned cats from Portugal and evaluate the infection risk of cats that maintain contact with human COVID-19 cases. A total of 176 cats, belonging to 94 households, were sampled. Cat owners answered an online questionnaire, and cats were screened for antibodies against SARS-CoV-2 using a commercial ELISA. Twenty (21.3%) households reported at least one confirmed human COVID-19 case. Forty cats (22.7%) belonged to a COVID-19-positive and 136 (77.3%) to a COVID-19-negative household. The seroprevalences of cats from COVID-19-positive and -negative households were 5.0% (2/40) and 0.7% (1/136). The two SARS-CoV-2-seropositive cats from COVID-19-positive households had an indoor lifestyle, and their owners stated that they maintained a close and frequent contact with them, even after being diagnosed with COVID-19, pointing towards human-to-cat transmission. The SARS-CoV-2-seropositive cat from the COVID-19-negative household had a mixed indoor/outdoor lifestyle and chronic diseases. Owners of the three SARS-CoV-2-seropositive cats did not notice clinical signs or behavior changes. This study highlights the low risk of SARS-CoV-2 transmission from COVID-19-positive human household members to domestic cats, even in a context of close and frequent human-animal contact.

First Report of Echinococcus ortleppi in Free-Living Wild Boar (Sus scrofa) from Portugal.

Cystic echinococcosis (CE) is a zoonosis that is prevalent worldwide. It is considered endemic in Portugal but few studies have been performed on Echinococcus granulosus sensu lato and their hosts. In this study, CE cysts are reported for the first time in a free-living wild boar (Sus scrofa) in Portugal. The presence of the metacestodes in the liver of the wild boar was identified by morphological features, microscopic examination and molecular analysis. The sequencing of part of the DNA nuclear ribosomal internal transcribed spacer-1 (ITS-1) region revealed a G5 genotype that presently corresponds to Echinococcus ortleppi. This is the first report of E. ortleppi in Portugal and to the best of the authors' knowledge, in Europe. These results suggest that wild boar may be a host of CE, namely, crossing the livestock-wildlife interface, which has important public health implications. Wildlife reservoirs must be taken into account as CE hosts and surveillance of game as well as health education for hunters should be implemented using a One Health approach, with implementation of feasible and tailor-made control strategies, namely, proper elimination of byproducts in the field.

Increasing importance of anthelmintic resistance in European livestock: creation and meta-analysis of an open database.

Helminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke (Fasciola hepatica) was collated by members of the European COST Action "COMBAR" (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980-2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0-100% (BZ and ML), 0-17% (LEV) and 0-73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread.

TITLE: Importance croissante de la résistance aux anthelminthiques chez les ruminants européens : création et méta-analyse d'une base de données ouverte. ABSTRACT: Les helminthes sont omniprésents dans les systèmes de production de ruminants au pâturage et sont responsables de coûts et de pertes de production importants. La résistance aux anthelminthiques (RA) des parasites est maintenant répandue dans toute l'Europe, bien qu'il existe encore des lacunes dans nos connaissances dans certaines régions et certains pays. La RA est une menace majeure pour la pérennité de la production moderne de ruminants, en diminuant la productivité, en compromettant la santé et le bien-être des animaux, et en augmentant les émissions de gaz à effet de serre au travers d'une augmentation du parasitisme et des intrants agricoles. Une meilleure compréhension de l'étendue de la RA en Europe est nécessaire pour développer et préconiser des approches de lutte antiparasitaire plus durables. Une base de données intégrant des informations publiées et non publiées en Europe concernant la RA des nématodes gastro-intestinaux (NGI) et de la douve du foie (Fasciola hepatica) a été compilée par les membres de l'action européenne COST « COMBAR ¼ (« Combattre la résistance aux anthelminthiques chez les ruminants ¼) et combinée avec les données d'une précédente étude systématique concernant la RA des NGI. Au total, 197 publications sur la RA des NGI étaient disponibles pour analyse, représentant 535 études dans 22 pays et couvrant la période 1980­2020. Des signalements de RA étaient présents sur tout le continent européen et certains rapports indiquaient une forte prévalence nationale. Les estimations heuristiques pondérées par la taille de l'échantillon de la prévalence de la RA en Europe sur toute la période d'étude, stratifiées par classe d'anthelminthiques, variaient de 0 à 48 %. La prévalence régionale (nationale) estimée était très hétérogène, variant entre 0 % et 100 % selon le secteur de l'élevage et la classe d'anthelminthique, et augmentait généralement avec les efforts de recherche dans le pays. Dans les quelques pays disposant de données longitudinales adéquates, il y avait une tendance à l'augmentation de la RA au fil du temps pour toutes les classes d'anthelminthiques des NGI : les résultats agrégés chez les ovins et caprins depuis 2010 révèlent une prévalence moyenne de résistance aux benzimidazoles (BZ) de 86 %, aux lactones macrocycliques sauf moxidectine (ML) de 52 %, au lévamisole (LEV) de 48 % et à la moxidectine (MOX) de 21 %. Tous les genres principaux de NGI ont survécu au traitement dans diverses études. Chez les bovins, la prévalence de la RA variait selon les classes d'anthelminthiques de 0 à 100 % (BZ et ML), 0 à 17 % (LEV) et 0 à 73 % (MOX), et Cooperia et Ostertagia ont survécu aux traitements. Une RA suspectée chez F. hepatica a été signalée dans 21 études portant sur 6 pays. Pour les NGI, et encore plus pour F. hepatica, il y avait un biais d'échantillonnage en faveur des exploitations individuelles suspectées de RA, et l'effort de recherche était biaisé vers l'Europe occidentale et en particulier le Royaume-Uni. La capture continue des résultats futurs dans la base de données, en direct, les efforts pour éviter les biais dans le recrutement des exploitations, des tests plus précis pour la RA et une meilleure appréciation de l'importance de la RA parmi l'industrie agricole et les décideurs politiques, soutiendront des analyses plus sophistiquées des facteurs contribuant à la RA, et des stratégies efficaces pour ralentir sa propagation.

A Qualitative Market Analysis Applied to Mini-FLOTAC and Fill-FLOTAC for Diagnosis of Helminth Infections in Ruminants.

Helminth infections, mainly by gastrointestinal nematodes (GIN), are one of the main concerns for animal health, welfare and productivity in grazing ruminant livestock worldwide. The use of a sensitive, precise, accurate, low-cost, and easy-to-perform copromicroscopic technique is of pivotal importance to perform reliable fecal egg count (FEC) and fecal egg count reduction test (FECRT), in order to determine the need of anthelmintic treatment, but also anthelmintic efficacy or resistance. This approach is fundamental to a correct and efficient control of GIN. Unfortunately, in worldwide ruminant farm practice, repeated anthelmintic treatments are carried out, without prior diagnosis of infection, contributing to the spread of Anthelmintic Resistance (AR). Tackling this phenomenon, improving mainly the GIN diagnosis and AR status in farm animals, is a priority of the European COST Action "COMBAR-COMBatting Anthelmintic Resistance in Ruminants" and of the STAR-IDAZ International Research Consortium on Animal Health. One of the specific objectives of the COMBAR Working Group 1 (WG1) is to conduct an European market analysis of new diagnostics and develop a business plan for commercial test introduction, leveraging technical know-how of participants. Since the Mini-FLOTAC in combination with the Fill-FLOTAC may be considered a good candidate for a standardized FEC and FECRT in the laboratory, as well as directly in the field, the aim of this study was to conduct SWOT (Strength-Weaknesses-Opportunities-Threats) and PESTEL (Political, Economic, Social, Technological, Environmental, and Legal) analyses of these tools in 20 European countries involved in the COMBAR WG1, in order to identify the opportunities, barriers, and challenges that might affect the Mini-FLOTAC and Fill-FLOTAC commercialization in Europe.

Phylogenetic relationships among Toxocara spp. and Toxascaris sp. from different regions of the world.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

Limited Knowledge About Hydatidosis Among Farmers in Northwest Portugal: A Pressing Need for a One Health Approach.

Hydatidosis is a re-emerging disease. Farmers are a vulnerable population; however, little is known about their awareness of this disease. The purpose of this study was two-fold: (1) to assess sheep and goat farmers' awareness of, perceptions of, and attitudes towards parasitic zoonoses and hydatidosis and (2) to identify the preferred means for promotion of information about hydatidosis. A cross-sectional study was conducted. An in-person questionnaire was constructed and administered to 279 individuals. A coprological survey in shepherd dogs was performed using 88 faecal samples. SPSS version 18.0 was used for statistical analysis. Farmers reported several risk practices (69% practice home slaughtering, 46% do not deworm the dogs, 58% of these dogs have contact with other animals) and very little knowledge about hydatidosis (97% have never heard about it). Nevertheless, 75% of the farmers demonstrated interest in receiving information, mainly from a veterinarian. A wide diversity of potentially zoonotic parasites (Trichuris spp., Ancylostomatidae, Toxocara spp., Taeniidae) was found in 61% of the dogs. This survey revealed farmers' lack of knowledge in relation to hydatidosis and a high prevalence of potentially zoonotic parasites in dogs, thus pointing to the need for health education and a closer collaboration between veterinarian and public health professionals.

Multiple zoonotic parasites identified in dog feces collected in Ponte de Lima, Portugal-a potential threat to human health.

Dogs play many roles and their presence within people's houses has increased. In rural settings dog faeces are not removed from the streets, representing an environmental pollution factor. Our aim was to evaluate the occurrence of environmental contamination with zoonotic intestinal parasites of three groups of dogs in Ponte de Lima, Portugal, with a particular emphasis on Echinococcus granulosus. We collected 592 dog faecal samples from the environment, farm and hunting dogs. Qualitative flotation coprological analysis was performed and the frequency in the positive samples ranged between 57.44% and 81.19% in different groups. We isolated up to four different parasites in one sample and detected seven intestinal parasitic species, genera or families overall. Ancylostomatidae was the most prevalent parasite, followed by Trichuris spp., Toxocara spp., Isospora spp., Dipylidium caninum, Taeniidae and Toxascaris leonina. Taeniidae eggs were analyzed with the PCR technique and revealed not to be from Echinococcus. The parasite prevalence and the diversity of zoonotic parasites found were high, which calls for a greater awareness of the problem among the population, especially hunters. Promoting research at the local level is important to plan control strategies. Health education should be developed with regard to farmers and hunters, and a closer collaboration between researchers, practitioners and public health authorities is needed.