Rafael Calero-Bernal

Toxoplasma gondii is a major cause of reproductive failure and severe gestational complications in both humans and small ruminants and early diagnosis is essential to prevent adverse outcomes. In sheep, congenital toxoplasmosis can trigger early fetal death (EFD) during the acute phase of the infection (days 7-14 post-infection) when conventional diagnosis methods are ineffective. Therefore, this study aimed to identify early infection biomarkers using a machine learning approach to support EFD diagnosis and providing a translational foundation for early diagnostic strategies in human toxoplasmosis. For that, 27 serum biomarkers (cytokines, acute-phase proteins, oxidative stress and hemostatic parameters) were evaluated during the early stage of infection in 22 pregnant sheep experimentally challenged with T. gondii and 6 uninfected controls. To assess diagnostic specificity, models were tested using sera from animals experimentally infected with other abortifacient agents ( Neospora caninum and Chlamydia abortus ) and validated with samples from 19 natural abortion outbreaks. A four-biomarkers panel, encompassing CXCL9, CXCL10, IFN-γ, and serum amyloid A, achieved over 74% accuracy in the specific detection of early T. gondii infection and 100% of sensitivity when studying natural infection cases. This approach might support the development of rapid diagnostic strategies, improving early intervention and disease management in livestock production systems. Its translation to human medicine could also aid in addressing current diagnostic gaps and facilitate timely interventions to prevent congenital toxoplasmosis and pregnancy loss in women. Importance: This study addresses a critical veterinary and public health challenge: the diagnostic gap in early infection during congenital toxoplasmosis. In sheep, early fetal death (EFD) is a clinical outcome than remain underdiagnosed under field conditions due to the absence of maternal IgG antibodies and placental or fetal parasite DNA at the time of abortion. By pioneering the integration of host-response biomarkers with machine learning, this work successfully predicted acute infection with 100% diagnostic sensitivity and 85% accuracy under real-world field conditions (natural infections). Furthermore, this study provides clues for unraveling the pathogenic mechanism involved by demonstrating that EFD is driven mainly by oxidative processes rather than by the initially proposed increase in pro-inflammatory cytokines. In addition, the proposed approach may have a translational potential, both in terms of the selected biomarkers and the machine learning-based methodology, with the purpose of enabling early therapeutic interventions in human congenital toxoplasmosis.

Many uncertainties exist on the epidemiology of the Sarcocystis species infecting wild ruminants. In this study, tongue and/or diaphragm tissues of free-ranging wild Caprinae species (54 Pyrenean chamois, 52 mouflons, 146 Iberian ibexes, and 19 Balearean wild goats) residing in the mountain areas of Spain were selected for Sarcocystis spp. detection and species identification. By light microscopy, Sarcocystis sarcocyst frequency rates were considerably high (85.7% in chamois, 82.7% in mouflon, 52.7% in Iberian ibexes), except for the Balearean wild goat (5.3%), which was investigated for the very first time. One excised sarcocyst of Iberian ibex was subjected to transmission electron microscopy, revealing a sibling S. capracanis ultrastructure. Genomic DNA isolated from 57 sarcocysts excised from the hosts’ tissues was investigated through PCR amplicon sequencing of the 18S rRNA and cox1 genes. As a result, sequences of S. capracanis, S. cornagliai, S. rossii and S. tenella were detected, sharing high identity with conspecific sequences. Phylogenetic analyses confirmed a remarkable clustering of all Sarcocystis species in clades encompassing species transmitted via canids, except S. cornagliai, which is allocated in a clade with species using corvid birds as their definitive hosts. Therefore, a significant degree of interconnection between sylvatic and domestic Sarcocystis epidemiological cycles can be drawn in the study area.

Sarcocystis spp. (Apicomplexa: Sarcocystidae) are globally distributed protozoan parasites with an obligatory two-host prey–predator life cycle involving intermediate (IHs) and definitive hosts (DHs). Canids, including the red fox (Vulpes vulpes), often serve as DHs for species infecting various ungulates. This study identified Sarcocystis species in red foxes from Croatia, Lithuania, and Portugal. Between 2021 and 2024, 164 faecal samples (80 from Croatia, 50 from Portugal, and 34 from Lithuania) were analysed using nested PCR targeting the cox1 gene. Twelve Sarcocystis species were detected: S. arieticanis, S. capracanis, S. capreolicanis, S. cruzi, S. gracilis, S. hjorti, S. iberica, S. linearis, S. miescheriana, S. morae, S. rossii, and S. tenella. The overall prevalence was highest in Croatia (78%) and Lithuania (62%) and lowest in Portugal (30%). Phylogenetic and haplotype analyses revealed high homogeneity and absence of geographic structuring. These results confirm the red fox as a key DH for multiple Sarcocystis species infecting European ungulates and underscore its epidemiological importance in parasite transmission across diverse ecosystems.

BACKGROUND: Toxoplasma gondii is a zoonotic protozoan capable of infecting warm-blooded animal species and humans. Although toxoplasmosis presents mostly as mild or asymptomatic infection in immunocompetent individuals, in unborn children and people with weakened immune systems, the disease can be severe with ocular, neurological or multi-systemic manifestations and even death. AIM: We aimed to collate and analyse data on T. gondii seroprevalence in humans to model and compare age-dependent prevalence in geographic regions in Europe. METHODS: A systematic review identified 1,822 scientific publications, from which seroprevalence data were extracted from 69 studies. Data were analysed using a Bayesian hierarchical model. RESULTS: The modelling of the seroprevalence indicated the highest incidence rates in eastern (50%) and western (48%) Europe, with the lowest estimates in northern Europe (18%) and the United Kingdom (UK) (18%). Eastern and western Europe were regions where T. gondii infections occurred earliest in life, with half of the population expected to be seropositive by the age of 44 and 47 years, respectively. In contrast, in northern Europe and the UK the modelled median time to infection exceeded 170 years. CONCLUSION: Results of the study provide a robust baseline for future epidemiological research on human T. gondii infections in Europe and may be useful to validate subsequent research, such as risk assessment studies.

The European brown hare (Lepus europaeus), widely distributed across Eurasia, is a key prey species for many vertebrates. Toxoplasma gondii, a globally distributed zoonotic protist, can infect all homoeothermic animals, including lagomorphs. Hares are highly susceptible, with sporadic toxoplasmosis cases reported in central and northern Europe, but its impact on southern populations remains unclear. Lethal toxoplasmosis was confirmed in two European brown hares from Catalonia, northeastern Spain (2018 and 2021). Samples were collected for histopathology and molecular analyses. Necropsy showed good body condition but revealed splenomegaly, liver necrosis, and pulmonary oedema. Microscopically, necrotizing multiorgan lesions with protozoan-like structures were observed. Toxoplasma gondii DNA was detected in spleen, with microsatellites-genotyping identifying a type II specific variant of the ToxoDB#3 genotype. In addition, 231 serum samples from passive and active surveillance (2013–2023) from Catalonia (n = 205) and other northern Spanish regions (n = 28) were tested for anti-T. gondii IgG antibodies using an indirect ELISA kit, showing a seroprevalence of 1.73% (4/231; 95% CI: 0.05–3.41). These are the first clinical toxoplasmosis cases reported in European brown hares from southern Europe, highlighting their unusual susceptibility. The low seroprevalence, consistent with Mediterranean reports, raises questions about exposure frequency. Further studies are needed to assess the impact of T. gondii on European brown hare populations.

The growing wild ungulate populations across Europe represents an increasingly important source for the spread of zoonotic pathogens. Blastocystis is a common intestinal protist observed in humans and animals worldwide. Studies on Blastocystis occurrence and subtype (ST) diversity in free-ranging wild ruminants are lacking globally, and more data are needed to understand the epidemiological scenario in wild European ruminants. We collected 833 faecal samples from free-ranging wild ungulates across Spain (n = 699) and Portugal (n = 134) between 1998 and 2021. Using conventional PCR and next-generation amplicon sequencing, Blastocystis was found in 13.8% (115/833; 95% CI: 11.5–16.3) of the wild ruminants analysed. Its occurrence was significantly higher in Portugal (38.1%, 51/134; 95% CI 29.8–46.8) than in Spain (9.2%, 64/699; 95% CI: 7.1–11.5). Fifteen Blastocystis STs, fourteen previously recognised (ST2, ST5, ST10, ST13, ST14, ST21, ST23–ST26, ST30, and ST42–ST44), and one novel (named ST49), were detected among the surveyed wild ruminant populations. Novel ST49 was described using Oxford Nanopore sequencing to produce full-length reference sequences of the small subunit ribosomal RNA gene. A greater ST diversity was observed in Spanish samples. Mixed infections were found in 58.3% (67/115) of the total Blastocystis-positive samples. Our results have enhanced the knowledge regarding Blastocystis occurrence and ST diversity and host preference present in wild ruminants from the Iberian Peninsula, which will assist in clarifying the relationships between the sylvatic and domestic cycles of this protist and may ultimately provide tools to help manage future public health epidemiological scenarios.