A systematic review and meta-analysis of the validation of serological methods for detecting anti-Toxoplasma gondii antibodies in humans and animals.

Toxoplasma gondii is a paradigmatic zoonotic parasite from the One Health perspective, since it is broadly distributed and virtually infects all warm-blooded species. A wide variety of serological techniques have been developed to detect T. gondii infection in humans and animals. Our aim was to describe and compare the main characteristics of these serological tests and validation processes and to critically analyze whether these tests meet the standards required to ensure an accurate serological diagnosis. The current systematic review and meta-analysis included 134 studies that were published from 2013 to 2023. QUADAS 2 tool was used to evaluate the quality of the included studies. A total of 52 variables related to the characteristics of the techniques and analytical and diagnostic validation parameters were studied. A wider panel of tests was developed for humans, including techniques exclusively developed for humans that involve costly equipment and the measurement of different Ig isotypes that are considered biomarkers of congenital toxoplasmosis. Studies conducted in humans frequently employed commercial techniques as reference tests, measured different immunoglobulin isotypes with a predominance for IgG (>50%) and discriminated between acute and chronic infections. In animals, the most commonly used reference techniques were in-house tests, which almost exclusively detected IgG. Common limitations identified in a large number of studies were some misunderstandings of the terms "gold standard" and "reference test" and the absence of information about the negative and positive control sera used or the exact cutoff employed, which were independent of the quality of the study. There is a lack of analytical validation, with few evaluations of cross-reactivity with other pathogens. Diagnostic odds ratio values showed that indirect ELISA based on native or chimeric antigens performed better than other tests. The reproducibility of serological test results in both humans and animals is not guaranteed due to a lack of relevant information and analytical validation. Thus, several key issues should be considered in the future, including interlaboratory ring trials.

Complete mitochondrial genome of Metathelazia capsulata (Pneumospiruridae) and comparison with other Spiruromorpha species.

Metathelazia capsulata (family Pneumospiruridae) is a lungworm parasitizing the bronchi and bronchioles, described in four species of wild carnivores. Very little molecular data are available on this nematode and none on other species of the Pneumospiruridae family. In this work, we describe for the first time the complete mitogenome (mitochondrial genome) of M. capsulata, being the first described of the family Pneumospiruridae. The mitogenome of M. capsulata has 13,659 bp in length, an A + T content of 79.2%. The mitogenome included 12 protein-coding genes (PCGs) (lacking the atp8 gene), 22 tRNA genes, 2 rRNA genes (all the genes are coded by the heavy strand), and an AT-rich region. The PCGs varied in size (232 bp-1645 bp). Only the tRNA-Trp has the standard cloverleaf secondary structure, while the other 21 do not. The AT-rich region, with a 90.5% A + T content and a length of 389 bp, is located between the cox3 and tRNA-Ala genes. Comparison with the mitogenomes of 29 species of Spiruromorpha infraorder, belonging to different families, demonstrates that M. capsulata mitogenome shared the common characteristics of most of them. The phylogeny constructions yielded phylogenies that were in agreement with the obtained previously by using sequences and gene order data of mitogenomes.

Effect of high-volume insecticide spraying on sand fly vectors in household gardens in Spain.

This study investigated the efficacy of high-volume spraying with the adulticide α-cypermethrin alone and in combination with the larvicide diflubenzuron on the density of sand flies in gardens of three detached households in periurban areas in southeast Spain. Treatments were applied four times between June and August 2016, and four nearby sites, two households and two non-urbanized sites, were untreated controls. The number of sand flies collected between May and October 2016 using sticky interception and light attraction traps, was 4446 specimens. Species identified morphologically included Sergentomyia minuta (n = 2101; 48%), Phlebotomus perniciosus (n = 1922; 44%), Phlebotomus papatasi (n = 173; 4%), Phlebotomus sergenti (n = 161; 4%) and Phlebotomus ariasi (n = 36; 1%). Sand flies were detected in both treated and untreated sites. The proportion of positive sticky traps and the median (range) density of sand flies in positive traps were 61% traps and 7 (2-172) sand flies/m2 /day in untreated sites, and 43% traps and 4 (1-56) sand flies/m2 /day in treated sites (p < 0.05). Similarly, for light traps, it was 96% traps and 30 (3-168) flies/trap/day, and 83% traps and 3 (1-12) sand flies/trap/day, respectively (p < 0.05). However, sand fly density followed a comparable seasonal pattern in untreated and treated sites and did not consistently decrease following insecticide applications. These results were confirmed with mixed negative binomial modelling of sand fly density adjusted for time since application, month, environmental setting and site. The limited efficacy of the treatments, added to their cost, the impact of insecticides on non-target organisms and human health, and the risk of development of insecticide resistance, should dissuade similar outdoor applications to control sand fly vector populations in residential areas.

Occurrence and limited zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Balantioides coli infections in free-ranging and farmed wild ungulates in Spain.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1-3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2-6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3-1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulate-adapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate species would not presumably play a significant role as source of human infections by these pathogens. Wild ruminants do not seem to be susceptible hosts for B. coli.

Top-predator carrion is scary: Fight-and-flight responses of wild boars to wolf carcasses.

Predation risk largely constrains prey behavior. However, whether predators may be scary also after death remains unexplored. Here, we describe the "fight-and-flight" responses of a prey, the wild boar (Sus scrofa), to carcasses of (a) its main predator, the gray wolf (Canis lupus) and (b) a carnivore that very rarely kills wild boars, the red fox (Vulpes vulpes), in the western Alps (Italy). We recorded the behavior of wild boars at 10 wolf and 9 fox carcass sites. We found eight "fight-and-flight" responses toward wolf carcasses, and none toward fox carcasses. Our results suggest that carnivore carcasses may indeed be scary; fear responses toward them are dependent on the species to which the carcass belongs; and animals approaching the carcasses are feared mainly when the latter are relatively fresh. This emphasizes the multiple and complex roles that carrion plays in the landscape of fear and opens exciting ecological, epidemiological, and evolutionary research avenues.

What is the sensitivity and exactness of post-mortem diagnostic method for cardiopulmonary nematodes in wild carnivores? towards the gold standard.

Cardiopulmonary nematodes cause health and fitness disorders in wild and domestic carnivores. The red fox (Vulpes vulpes) participates in the spread of these shared parasites at the domestic-wildlife interface. This study aimed to evaluate the sensitivity of post-mortem diagnostic method for detecting lungworms in carnivores, and its exactness to estimate the parasite intensity of each nematode species. Cardiorespiratory system of fifty-one foxes were examined through three consecutively methodological steps: first, the tracheobronchial tree, pulmonary arteries and their branches were opened (OT); next, lung parenchyma was immersed in water and squeezed (WS); finally, the parenchyma was artificially digested in a pepsin and chlorhydric acid solution (AD). Eucoleus aerophilus, Angiostrongylus vasorum, Crenosoma vulpis and Metathelazia capsulata were identified. The number of recovered nematodes in each step were 454 (OT), 285 (WS) and 141 (AD). The use of OT and WS helped to improve parasite intensity results and decreased false negative cases. Accordingly, when OT and WS were used together, the sensitivity in the detection of parasitized foxes was 96.1%, while the exactness of parasite intensity was 84%. When AD was performed, although sensitivity does not rise, results were more exact, increasing the total number of detected parasites by 16%. Moreover, AD improved the sensitivity in the detection of A. vasorum and M. capsulata, as well as quantifying more exactly the parasite intensity (92.5% and 92.3% of exactness without AD, respectively). Our study provides valuable information that should be taken into account when planning epidemiological studies based on cardiopulmonary nematode detection in carnivores.

Wolf Is Back: A Novel Sensitive Sentinel Rejoins the Trichinella Cycle in the Western Alps.

Trichinella is a foodborne parasite whose wildlife reservoirs are represented by carnivores and omnivores with predatory and scavenger behavior. The aim of the present study was to investigate the occurrence of Trichinella infection in grey wolves (Canis lupus) that recolonized the Western Alps from the end of the past century, and discuss the epidemiological role played by this apex predator in the early phases of its return. During the period 2017-2022, diaphragm samples were obtained from 130 individuals collected in the frame of a wolf mortality survey. Trichinella larvae were found in 15 wolves (11.53%) with a parasite intensity of 11.74 larvae per gram. Trichinella britovi was the only species identified. This is the first prevalence survey of Trichinella in wolves recolonizing the Alps. Results suggest that, in this particular biotope, the wolf has rejoined the Trichinella cycle and has the potential to play an increasingly important role as maintenance host. Arguments in favor and against this perspective are discussed and knowledge gaps highlighted. The calculated Trichinella larval biomass in the estimated wolf population roaming in Northwest Italy will serve as baseline value to explore possible shifts in the relative importance of wolves as Trichinella reservoir within the regional carnivore community. Finally, wolves re-colonizing the Alps already appear as sensitive sentinels to monitor the risk of Trichinella zoonotic transmission by infected wild boar meat.

Prevalence of Oestrus ovis in small ruminants from the eastern Iberian Peninsula. A long-term study.

Oestrus ovis is an obligate parasite that causes myiasis in domestic ruminants, being commonly found in the Mediterranean area. From 2009 to 2019 a total of 3476 heads of culling sheep and goats from the Mediterranean coast of Spain were examined for the presence of O. ovis. The total prevalence was 56.3%, significantly higher in sheep than in goats (61.2% and 43%, respectively). Differences were found in the mean annual prevalence, with the highest value being registered in 2018 (61.7%) and the lowest in 2012 (50.3%). Autumn, for sheep, and winter, for goats, were the seasons with the highest number of infested specimens. Temperature, but not rainfall, was found to be associated with prevalence (p < 0.05). Most L1 were found in the anatomic region I (septum, meatus, and ventral conchae), while L2 and L3 were mainly located in regions II (nasopharynx, ethmoid labyrinth, and dorsal conchae), and III (sinuses). The overall intensity was 12.8 larvae per head, significantly higher in sheep (13.3) than in goats (3.5). Our results confirm the high prevalence of O. ovis in sheep and goats in this geographic area over the last decade, with the trend increasing in recent years in association with higher mean temperatures.

A systematic review and meta-analysis of the serological diagnosis of Toxoplasma gondii infection highlight the lack of a One Health integrative research.

Toxoplasma gondii is a globally distributed food-borne zoonotic parasite with numerous infection sources. The control of this zoonosis requires a One Health response that partially depends on serological monitoring in humans and animals. Herein, a systematic review and a meta-analysis were performed to analyse and compare the transdisciplinary and integrative research under the One Health approach. We searched for articles published between January 1st 2014 and September 5th 2022, focused on the development and evaluation of serological techniques for the diagnosis of T. gondii infection in humans and animals. After an exhaustive search on three scientific databases, a quality assessment was performed on 291 articles by QUADAS-2 tool, and 113 articles were finally selected. A total of 18 variables were extracted and analysed, including bibliometric characteristics, study aims and methodology. Remarkably, none of the studies included in the meta-analysis explicitly quoted the words "One Health", and only 23.9% of them alluded to the principles underlying the One Health approach; in particular, none were conducted by physician-only teams, with the majority of these studies involving interdisciplinary research teams, followed by veterinarians and by non-physician or non-veterinarian researchers. The One Health approach followed in the serodiagnosis of T. gondii still needs further integration among scientific disciplines, which is essential to design effective control strategies.

Monitoring for Anguillicoloides crassus, Anguillid herpesvirus 1, aquabirnavirus EVE and rhabdovirus EVEX in the European eel population of southern Spain.

European eel is critically endangered in Europe. Among other stressors, pathogens are well-known to harm eels' fitness. One hundred and eighty-two eels were captured in three Eel Management Units in Andalucía (SE Spain) and analysed for Anguillicoloides crassus, Anguillid herpesvirus 1 (AngHV1), the rhabdovirus Eel Virus European X (EVEX) and the aquabirnavirus Eel Virus European (EVE). A. crassus adults and preadults were isolated and morphometrically identified, and the eel swimbladders were artificially digested to count A. crassus larvae. Also, eel tissues were examined by PCRs for the presence of viruses. EVEX and EVE were not detected in any of the eels. The estimated prevalence (95% confidence limits) was 71 (64-78)% for A. crassus and 35 (28-42)% for AngHV-1, varying these prevalences significantly between and within EMUs. Moreover, A. crassus prevalence was highest in smaller eels, in sites closest to the sea and eels sampled in the autumn. By contrast, AngHV-1 prevalence was highest in biggest eels, in sites far from the sea and sampled in the summer or winter. However, in mixed effects logistic models including site as a random variable, the risk of infection was associated with distance to the sea in both A. crassus and AngHV-1 infections and also to winter sampling in the case of AngHV-1 and not to other variables. These results are evidence that both pathogens are highly endemic in eels from Andalusian habitats. Further studies are needed to better understand the risk factors associated with these pathogens on eel populations.

Predictive factors for flea occurrence in red foxes (Vulpes vulpes) from semi-arid Mediterranean environments.

The role of red fox as host for a wide range of parasites, particularly fleas and other arthropods causing vector-borne diseases, in combination with its capability to adapt to anthropized environments, makes this wild canid an epidemiologically remarkable species at the wildlife-domestic-human interface, especially in the present time of rise of emerging and re-emerging diseases. This study evaluated the prevalence and parasite intensity of fleas in 88 foxes from Murcia Region (Southeastern Spain) and determined the geographic distribution of areas with the highest potential risk of flea presence. Pulex irritans, Ctenocephalides felis, Spilopsyllus cuniculi and Nosopsyllus fasciatus were identified. The overall prevalence was 76.13%. This is the first time that N. fasciatus has been reported in foxes from Murcia Region. The predictive model established a certain pattern to determine the areas with the highest risk of acquiring fleas. Positive correlation of daily potential evapotranspiration (ET0 ) in winter and the opposite effect occurring for ET0 in summer were obtained, as well as positive correlations for mean daily temperature (Tmean ) in summer and mean precipitation (Pmean ) in winter and summer. The model was also found positively correlated in the forest habitat ecotone areas and the anthropized areas.

Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

The fungal-related intracellular parasite Enterocytozoon bieneusi is a worldwide public health and veterinary problem. Here we demonstrated that it was present in wild boar, and wild and farmed red deer in Spain, with genotypes potentially capable of infecting humans, posing a public health risk.

Gastrointestinal parasites in red-legged partridges (Alectoris rufa) hunted in Spain: a warning to game managers.

Red-legged partridge (Alectoris rufa) populations are currently declining in the Iberian Peninsula, mainly due to habitat degradation and hunting pressure. In addition, the release of farm-reared partridges may introduce pathogens, including parasites, to wild populations. The presence of digestive parasites in red-legged partridges hunted in fifteen Spanish provinces was studied. Fecal samples and gastrointestinal tracts were collected, analyzed, and the morphometric identification of parasites was carried out. Eimeria spp. oocysts, nematode, cestode and trematode eggs were observed in fecal samples. Adult nematodes (Ascaridia galli, Ascaridia compar, Heterakis gallinarum, Heterakis tenuicauda, Trichostrongylus tenuis, Subulura spp., Cyrnea spp. and Aonchotheca caudinflata), tapeworms (Raillietina tetragona, R. echinobothrida, R. micracantha, Rhabdometra nigropunctata, and Choanotaenia infundibulum), and trematodes (Brachylaima spp., Brachylecithum spp., Dicrocoelium spp.) were identified in the gastrointestinal tracts. Significant statistical differences were found among climatic regions in the prevalence and intensity of Eimeria spp. infection, median intensity and the prevalence of indirect life cycle helminths, with Southern areas always showing higher infection values. The study provides information of the health status of red-legged partridges in Spain, highlighting the risk associated with the release of farm-reared partridges for restocking purposes. This should be taken into account to improve management strategies for the long-term conservation of the species.

Comparative performance of five recombinant and chimeric antigens in a time-resolved fluorescence immunoassay for detection of Toxoplasma gondii infection in cats.

Felids are definitive hosts of Toxoplasma gondii, being the only hosts that can spread the infection through oocyst shedding in their feces. The elevated presence of this parasite in the domestic cat (Felis catus), and its close contact with humans, make it necessary to obtain reliable diagnostic methods to detect positive animals as a public health measure. For this reason, in this study, the diagnostic performance of five different recombinant antigen-based techniques was assessed to diagnose T. gondii infection in cat blood plasma samples. Specifically, four T. gondii recombinant antigens (GRA7, truncated GRA7, SAG2, and truncated SAG2) and a chimeric antigen (SAG1-GRA8) were used. A time-resolved fluorescence immunoassay (TRFIA) was developed for each antigen, and the results of each of these techniques were compared with those obtained by a commercial enzyme-linked immunoassay (ELISA) and a modified agglutination test (MAT) as reference techniques. The TRFIA based on SAG1-GRA8 antigen showed better discrimination between seropositive and seronegative cats (p < 0.001), as well as a better area under the curve (0.95), sensitivity (93.6%), and specificity (89.5%) values for the optimal cut-off, versus the other TRFIAs. In addition, SAG1-GRA8 TRFIA showed substantial agreement (kappa value = 0.78) and a moderate significant correlation (Spearman's correlation: r = 0.62, p < 0.001) compared with the reference techniques. On the other hand, since plasma samples were obtained from 101 cats in Bangkok city and four of them were Neospora caninum seropositive by indirect immunofluorescence assay (IFAT), this is the first time that anti-N. caninum antibodies are detected in cats in Thailand. In conclusion, our study highlights that the TRFIA with TgSAG1-GRA8 antigen is an accurate and recommended diagnostic technique for detecting anti-T. gondii antibodies in cats.

Urban landscape and infection risk in free-roaming cats.

Despite public concern on the role of free-roaming cats as reservoirs of zoonotic agents, little is known about the influence of urban and peri-urban landscapes on the exposure risk. We evaluated the seroprevalence of three zoonotic agents (Chlamydia felis, Coxiella burnetii and Toxoplasma gondii) in domestic cats (Felis catus). Two hundred and ninety-one free-roaming cats were trapped in Murcia municipality (Southeast Spain), and their sera were tested for specific antibodies against T. gondii using a modified agglutination test (MAT), and for C. felis, C. burnetii and feline immunodeficiency virus (FIV) antibodies with ELISA technique. Pathogen seroprevalence at 95% CI was calculated for each sex and age category (up to and over 12 months) and compared with a chi-squared test. The role of human population density and urban landscape characteristics on the risk of pathogen exposure in the cat population was explored using generalized linear models. Seropositivity against a single pathogen was found in 60% of the cats, while 19% was seropositive for two or three pathogens. Seroprevalence of C. felis was 8% (CI95% : 5-11), 37% (CI95% : 31-42) for C. burnetii and 42% (CI95% : 36-47) for T. gondii. In addition to these three pathogens, FIV seropositivity was low (1%, CI95% : -0.1 to 2) and adult cats were more likely to be seropositive to C. burnetii than young individuals (OR: 2.3, CI95% : 1.2-4.2). No sex or age class differences in seroprevalence were observed for the rest of the pathogens. Seropositivity was correlated with water surface areas for C. felis, and not with crop areas. Coxiella burnetii seropositivity was correlated with the percentage of urban areas (continuous with only buildings and discontinuous, that include buildings, parks, and pedestrian and urban green areas), human population size and peri-urban areas with shrubs, and not correlated with other agricultural landscapes (orchards and crop areas). However, the seroprevalence of T. gondii was only associated with agricultural landscapes such as orchards. The detection of hotspot areas of high pathogen exposure risk is the basis for municipal services to implement surveillance and risk factor control campaigns in specific-risk areas, including (a) efficient health management of urban cat colonies by geographical location, population census and health status monitoring of the components of each cat colony, (b) improvement of hygiene and sanitary conditions at the feeding points of the cat colony and (c) free-roaming cat trapping for health monitoring and, in the long term, to know the evolution of the health status of their populations.

Detection of anti-Neospora caninum antibodies in sheep's full-cream milk by a time-resolved fluorescence immunoassay.

Ovine neosporosis, caused by the Apicomplexan parasite Neospora caninum, leads to reproductive failure worldwide. Nowadays, there is a trend to develop diagnostic techniques using non-invasive samples, such as milk, in order to reduce animal stress, sample collection effort, and costs. The objective of this study was to develop and validate a highly sensitive and specific serological technique, based on a time resolved-fluorescence immunoassay using a N. caninum GRA7 antigen (GRA7-TRFIA), for the detection of anti-N. caninum immunoglobulins G on sheep' full-cream milk samples. An analytical validation was performed, including intra- and inter-assay precision, analytical sensitivity and accuracy. The diagnostic performance of the assay was evaluated by studying the positive-negative discrimination by Mann Whitney U tests. In additon optimal cut-offs, diagnostic sensitivity and specificity, and areas under the curve were calculated by three Receiver Operating Curve (ROC) analyses, using GRA7-TRFIA and a N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET-ELISA) in blood sera, and the coinciding results of both techniques, as reference techniques. Moreover, Spearman's correlation of GRA7-TRFIA in milk with the techniques in sera and agreement (kappa values) were also estimated. GRA7-TRFIA for milk samples showed an adequate precision, with high analytical sensitivity and accuracy. Regarding ROC analyses, at the optimal cut-offs, the diagnostic sensitivity and specificity were more than 90 % in all cases. In addition, GRA7-TRFIA values in milk were more positively correlated to GRA7-TRFIA values in blood sera than in the case of values obtained with NcSALUVET-ELISA. GRA7-TRFIA in milk showed an almost perfect agreement with GRA7-TRFIA in blood sera (kappa = 0.98) and with the coinciding results of GRA7-TRFIA and NcSALUVET in blood sera (kappa = 1.00), while it has a substantial agreement with NcSALUVET-ELISA (kappa = 0.69). In the light of these results, GRA7-TRFIA in full-cream milk samples is a highly sensitive technique that could be used for screening anti-N. caninum antibodies in sheep flocks.

Smart carnivores think twice: Red fox delays scavenging on conspecific carcasses to reduce parasite risk.

The recent SARS-CoV-2 epidemic has highlighted the need to prevent emerging and re-emerging diseases, which means that we must approach the study of diseases from a One Health perspective. The study of pathogen transmission in wildlife is challenging, but it is unquestionably key to understand how epidemiological interactions occur at the wildlife-domestic-human interface. In this context, studying parasite avoidance behaviours may provide essential insights on parasite transmission, host-parasite coevolution, and energy flow through food-webs. However, the strategies of avoiding trophically transmitted parasites in mammalian carnivores have received little scientific attention. Here, we explore the behaviour of red foxes (Vulpes vulpes) and other mammalian carnivores at conspecific and heterospecific carnivore carcasses using videos recorded by camera traps. We aim to determine 1) the factors influencing the probability of foxes to practice cannibalism, and 2) whether the scavenging behaviour of foxes differ when facing conspecific vs. heterospecific carcasses. We found that red foxes were generally reluctant to consume mesocarnivore carrion, especially of conspecifics. When recorded, consumption by foxes was delayed several days (heterospecific carcasses) or weeks (conspecific carcasses) after carcass detection. Other mammalian scavengers showed a similar pattern. Also, meat-borne parasite transmission from wild carnivore carcasses to domestic dogs and cats was highly unlikely. Our findings challenge the widespread assumption that cannibalistic or intra-specific scavenging is a major transmission route for Trichinella spp. and other meat-borne parasites, especially for the red fox. Overall, our results suggest that the feeding decisions of scavengers are probably shaped by two main contrasting forces, namely the nutritional reward provided by carrion of phylogenetically similar species and the risk of acquiring meat-borne parasites shared with these species. This study illustrates how the detailed monitoring of carnivore behaviour is essential to assess the epidemiological role of these hosts in the maintenance and dispersion of parasites of public and animal health relevance.

Epidemiological approach to nematode polyparasitism occurring in a sympatric wild ruminant multi-host scenario.

The epidemiology behind multi-host/multi-parasite systems is particularly interesting to investigate for a better understanding of the complex dynamics naturally occurring in wildlife populations. We aimed to approach the naturally occurring polyparasitism of gastrointestinal nematodes in a sympatric wild ruminant scenario present in south-east Spain. To this end, the gastrointestinal tract of 252 wild ruminants of four different species (red deer, Cervus elaphus; mouflon, Ovis aries musimon; Iberian ibex, Capra pyrenaica and fallow deer, Dama dama) were studied in Cazorla, Segura y Las Villas Natural Park (Andalusia, Spain). Of the analysed animals, 81.52% were positive for parasite infection and a total of 29 nematode species were identified. Out of these, 25 species were detected in at least two host species and 11 parasitized all ruminant species surveyed. The multi-host interaction between these nematodes and the four host species is discussed under the perspective of host family-based differences.

Understanding potential implications for non-trophic parasite transmission based on vertebrate behavior at mesocarnivore carcass sites.

High infection risk is often associated with aggregations of animals around attractive resources. Here, we explore the behavior of potential hosts of non-trophically transmitted parasites at mesocarnivore carcass sites. We used videos recorded by camera traps at 56 red fox (Vulpes vulpes) carcasses and 10 carcasses of other wild carnivore species in three areas of southeastern Spain. Scavenging species, especially wild canids, mustelids and viverrids, showed more frequent rubbing behavior at carcass sites than non-scavenging and domestic species, suggesting that they could be exposed to a higher potential infection risk. The red fox was the species that most frequently contacted carcasses and marked and rubbed carcass sites. Foxes contacted heterospecific carcasses more frequently and earlier than conspecific ones and, when close contact occurred, it was more likely to be observed at heterospecific carcasses. This suggests that foxes avoid contact with the type of carcass and time period that have the greatest risk as a source of parasites. Overall, non-trophic behaviors of higher infection risk were mainly associated with visitor-carcass contact and visitor contact with feces and urine, rather than direct contact between visitors. Moreover, contact events between scavengers and carnivore carcasses were far more frequent than consumption events, which suggests that scavenger behavior is more constrained by the risk of acquiring meat-borne parasites than non-trophically transmitted parasites. This study contributes to filling key gaps in understanding the role of carrion in the landscape of disgust, which may be especially relevant in the current global context of emerging and re-emerging pathogens.

Detection of Stephanurus dentatus in wild boar urine using different parasitological techniques.

Stephanurus dentatus is a nematode that parasitizes the urinary tract of domestic and wild Suidae, especially in tropical areas. However, there is a lack of information about stephanurosis in wild boar (Sus scrofa), thus making it necessary to develop sensitive techniques with which to diagnose this pathogen in order to carry out further research. In Spain, the high prevalence of this nematode has been evidenced in Doñana National Park (DNP). The objective of the present work is twofold. The first is to compare the efficacy of three parasitological techniques to detect S. dentatus eggs in the urine of infected wild boar: (i) gravity sedimentation, (ii) sedimentation by centrifugation, and (iii) flotation techniques, while the second is to determine whether the quantification of eggs can serve as an indicative value of the host's parasite intensity. In order to accomplish these purposes, 27 wild boars from DNP were necropsied, and the urinary system of each animal was examined in order to determine parasite intensity. While all the aforementioned techniques can be used to detect eggs in urine, the most effective in terms of egg quantification are sedimentation by gravity and by centrifugation, as they allow a greater number of S. dentatus eggs to be detected. However, none of the results obtained with these techniques significantly correlated with the number of adult nematodes parasitizing the host, signifying that counts in urine can provide guidance on only the parasite intensity of wild boar.