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Author contributions: G.Á.-G., I.F. and L.M.O.-M. conceived the study and acquired funding for the study; M.F.-Á., A.J.-M. and P.H. carried out the experimental assays; M.F.-Á., P.A.L., A.H.-L. and F.H.-L. performed the bioinformatic and statistical analyses and prepared the figures and tables. M.F.-Á., G.Á.-G. and P.H. drafted the original manuscript. All authors have read and agreed to the published version of the manuscript., Spanish Ministry of Economy and Competitiveness, Community of Madrid, Complutense University of Madrid, University of Murcia through the Margarita Salas Program of Requalification of the Spanish University System (Spanish Ministry of Universities) financed by the European Union—NextGenerationEU, Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

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 h, Universidad de Murcia, European Commission, Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia, Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results., European Commission, Comunidad de Madrid (España), National Agency of Research and Innovation (Uruguay), Universidad Complutense de Madrid (España), Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub, Pagado por el autor

Coronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available.

Parasit’Xpert es un blog sobre enfermedades parasitarias de los rumiantes domésticos para el veterinario (https://parasitxpert.es/). Su objetivo es ser un punto de encuentro y referencia para los profesionales del sector y estudiantes de veterinaria que quieran conocer y profundizar en el campo de las enfermedades parasitarias. La plataforma se inició en enero de 2021 y se estructura en diversas secciones prácticas: parásito del mes, ¿sabías qué?, diagnóstico, control, zoonosis y avances. El grupo SALUVET y la empresa de transferencia del conocimiento universitario SALUVET-Innova han puesto en marcha la web Parasit’xpert junto a la empresa farmacéutica Boehringer Ingelheim Animal Health España. El objetivo general del presente proyecto de innovación docente fue desarrollar el blog Parasit’Xpert (https://parasitxpert.es/) ya en activo e implementar un plan de difusión promocional para incrementar el número de usuarios, especialmente en países hispanohablantes.

Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8–7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0–79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection., European Union’s Horizon 2020 Research, Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

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., Comunidad de Madrid, Fundación Séneca, Horizonte 2020, Universidad de Murcia, Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

2023 Acuerdos Transformativos CRUE, Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied. All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results., European Commission, Universidad Complutense de Madrid, Banco Santander, Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Fac. de Veterinaria, TRUE, pub, APC financiada por la UCM

Author Contributions: Conceptualization, R.C.-B. and L.M.O.-M.; methodology, R.C.-B. and S.C.; software, S.C.; formal analysis, R.C.-B., S.C. and A.R.; investigation, R.C.-B., S.M.G., M.Y.S.-F. and S.C.; resources, G.Á.-G. and L.M.O.-M.; data curation, R.C.-B., S.M.G. and A.R.; writing—original draft preparation, R.C.-B. and S.M.G.; writing—review and editing, R.C.-B., S.M.G., M.Y.S.-F., G.Á.-G. and L.M.O.-M.; visualization, R.C.-B. and S.C.; supervision, R.C.-B. and L.M.O.-M.; funding acquisition, G.Á.-G. and L.M.O.-M. All authors have read and agreed to the published version of the manuscript., Toxoplasmosis has a major impact on animal and public health. Information regarding the seroprevalence of human Toxoplasma gondii infections from a European perspective has not yet been compiled to date. Thus, the present review summarized available resident data from the period 2000–2020. The overall seroprevalence of anti-T. gondii IgG was 32.1%, with great variability between countries (n = 30). The subgroup analysis identified different pooled prevalence data depending on the geographic area (p < 0.0001), target population (p = 0.0147), and serological diagnosis assays used (p = 0.0059). A high heterogeneity (I2 = 100%, p < 0.001; Q = 3.5e+05, d.f. = 135, p < 0.001) and degree of publication bias (Egger’s test = 6.14, p < 0.001) were observed among the 134 studies considered. The occurrence of anti-T. gondii IgM, which was reported in 64.7% of studies, reached a pooled seroprevalence of 0.6%. In addition, among the eight main risk factors identified, “contact with soil”, “consumption of undercooked beef”, and “intake of unwashed vegetables” were the most significantly associated with infections. The fact that one-third of the European population has been exposed to T. gondii justifies extra efforts to harmonize surveillance systems and develop additional risk-factor analyses based on detailed source attribution assessment., Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

2023 Acuerdos transformativos CRUE, The intake of Toxoplasma gondii tissue cysts through raw or undercooked pork meat is one of the main infection sources for humans. Thus, surveillance is recommended to control and prevent infection in domestic pigs. However, the lack of comparative studies hampers the updating of their performance and the comparison of seroprevalence data. Therefore, the aim of this study was to develop and validate three in-house tests and accomplish a comparative analysis of the most widely used serological tests employed in pigs. A panel of sera from pigs experimentally infected with either oocysts or tissue cysts from type II and III isolates (n = 158) was used to develop and validate a tachyzoite-based Western blot assay. Then, this technique was used as a reference to develop and preliminary validate a lyophilized tachyzoite-based enzyme-linked immunosorbent assay and an immunofluorescence antibody test. Next, a comparative study of the three in-house tests and three widely used commercial ELISAs (IDScreen®, PrioCHECK™ and Pigtype®) was accomplished with the abovementioned sera together with an additional serum panel of pigs experimentally infected with oocysts from the type II isolate (n = 44) and a panel of naturally infected pigs (n = 244). The results obtained by the majority of the tests were regarded as reference, and data analyses included TG-ROC calculations and agreement tests. Finally, the kinetics of anti-T. gondii IgGs from experimentally infected pigs was analyzed. Excellent sensitivity (Se) and specificity (Sp) values (≥ 93%) and moderate to near perfect agreement (k = 0.63–0.91) were observed using sera from experimental infections without requiring further readjustment, except for PrioCHECK (100% Se, 73% Sp). However, the Se of IDScreen® (87%) and TgSALUVET WB (71%) and the Sp of PrioCHECK (72%) were slightly or notably reduced when sera from naturally infected animals were analyzed, which also influenced the kappa values (k = 0.30–0.91). Cutoff readjustments i, European Commission, Universidad Complutense de Madrid, Banco Santander, Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub, APC financiada por la UCM