Received: 9 September 2022 | Revised: 8 December 2022 | Accepted: 6 January 2023 DOI: 10.1111/zph.13026 S H O R T C O M M U N I C AT I O N Seroepidemiological study of Toxoplasma gondii in equids in different European countries David Cano-­Terriza1,2 | Juan J. Franco3 | Eduard Jose-­Cunilleras4 | Francesco Buono5 | Sonia Almería6 | Vincenzo Veneziano5 | Eduardo Alguacil7 | Jesús García8 | Isabelle Villena9 | Jitender P. Dubey10 | Débora Jiménez-­Martín1 | Ignacio García-­Bocanegra1,2 1 Departamento de Sanidad Animal, Grupo de Investigación GISAZ, UIC Zoonosis y Enfermedades Emergentes ENZOEM, Universidad de Córdoba, Córdoba, Spain 2 CIBERINFEC, ISCIII -­CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain 3 Inmunología y Genética Aplicada, S.A. (EUROFINS -­INGENASA), Madrid, Spain 4 Servei de Medicina Interna Equina, Departament de Medicina Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain 5 Department of Veterinary Medicine and Animal Productions, Università degli Studi di Napoli Federico II, Naples, Italy 6 Division of Virulence Assessment, Department of Health and Human Services, Food and Drug Administration, Center for Food Safety and Nutrition (CFSAN), Office of Applied Research and Safety Assessment (OARSA), Laurel, Maryland, USA 7 Uplands Way Vets, Diss, UK 8 Fethard Equine Hospital, Tipperary, Ireland 9 University of Reims Champagne-­Ardenne, EA 7510, National Reference Centre for Toxoplasmosis, Laboratory of Parasitology, Reims Hospital, Reims, France 10 Animal Parasitic Disease Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland, USA Correspondence Débora Jiménez-­Martín, Departamento de Abstract Sanidad Animal, Grupo de Investigación Toxoplasmosis, caused by the obligate intracellular protozoan Toxoplasma gondii, is a GISAZ, UIC Zoonosis y Enfermedades Emergentes ENZOEM, Universidad de worldwide parasitic zoonosis. A cross-­sectional study was carried out to determine Córdoba, Córdoba 14014, Spain. the exposure to T. gondii in equids in Europe. Serum samples from 1399 equids (1085 Email: v42jimad@uco.es horses, 238 donkeys, and 76 mules/hinnies) bred in four European countries (Italy, Funding information Spain, United Kingdom [UK], and Ireland) were collected during the period of 2013–­ CIBER -­Consorcio Centro de Investigación Biomédica en Red; 2021. The overall seroprevalence of T. gondii was 18.9% (95% Confidence Interval Instituto de Salud Carlos III; Ministerio [CI]: 16.9–­21.0) by using the modified agglutination test (MAT) at a cut-­off of 1:25. de Ciencia e Innovación; Unión Europea –­NextGenerationEU; University of Seropositivity by country was 27.1% in Italy, 16.6% in Spain, 12.0% in UK and 7.0% Cordoba; Universidad de Córdoba in Ireland. Anti-­T. gondii antibodies were detected in 12.8% of the horses, 43.7% of the donkeys, and in 28.9% of the mules/hinnies. A Generalized Estimating Equation (GEE) analysis was carried out to study the associations between seropositivity and explanatory variables related to individuals, herds, and management measures on these herds, selected based on the bivariate analysis. The risk for being seropositive for T. gondii was 5.3 and 2.7 times higher in donkeys and mules/hinnies than in horses, respectively. In addition, significantly higher seropositivity was observed in horses This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2023 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH. 276 |  wileyonlinelibrary.com/journal/zph Zoonoses Public Health. 2023;70:276–283. | 18632378, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/zph.13026 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [10/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CANO-­TERRIZA et al. 277 from herds that used disinfectants less than once a week (13.9%; p = 0.038, odds ratio [OR] = 1.6; 95% CI: 1.03–­2.62) compared with those from herds that performed weekly disinfection of the facilities (9.4%). This is the first large-­scale seroepidemio- logical study on T. gondii comprising horses, donkeys, and mules/hinnies in Europe and the first report of T. gondii exposure in horses from Ireland and UK. We found a wide- spread distribution of T. gondii among equid populations in different European coun- tries. The seroprevalence found in these species, especially in donkeys and mules/ hinnies, highlights the potential risk of human infection through the consumption of their raw/undercooked milk or meat. KEYWORDS equids, food-­borne pathogens, toxoplasmosis, zoonosis 1 | I NTRO D U C TI O N Impacts Toxoplasmosis is a worldwide parasitic zoonosis caused by Toxoplasma gondii. Its sexual reproduction occurs in felid (definitive) • Toxoplasma gondii is widespread in equid populations in hosts, which excrete oocysts in faeces, and involves virtually all ho- different European countries. meotherm species as intermediate hosts (Dubey, 2022). Humans or • Donkeys and mules/hinnies are at the highest species animals typically become infected by three main modes of transmis- risk for T. gondii positivity. sion: consumption of tissue cysts from infected raw or undercooked • Raw or undercooked foods from equids could be of pub- meat, ingestion of food or water contaminated with sporulated T. lic health concern in many European regions. gondii oocysts and by transplacental or blood transmission with tachyzoites. Toxoplasma gondii infection is usually sub-­clinical in im- munocompetent individuals, however, this opportunistic pathogen can lead to abortion, stillbirth, and congenital abnormalities as well horses and other equids are among the most resistant domestic as serious disease and even death in immunocompromised people species to clinical toxoplasmosis (Dubey, 2022), the association be- (Dubey, 2022). Moreover, the relationship between T. gondii la- tween T. gondii seropositivity and clinical equine protozoal myelo- tent infections and chronic neurological and psychiatric conditions encephalitis in horses has also been suggested (James et al., 2017; (mainly schizophrenia and bipolar disorder) has been previously sug- Schale et al., 2018). Recently, fatal toxoplasmosis was diagnosed in a gested (Fabiani et al., 2015; Fuglewicz et al., 2017; Milne et al., 2020). horse in the USA (Kimble et al., 2021). Equids were domesticated 5000–­6000 years ago, and since In farm animals, including equids, serological tests are the first-­ then, they have been used by humans for different purposes in- line strategies for diagnosis of T. gondii, while PCR methods are spe- cluding transportation of people and goods, hunting, war, farming cific, sensitive, and useful for detection of parasite DNA in animal or leisure. In addition, equids have also served as a food source since tissues (Almeria & Dubey, 2021). Indirect diagnosis methods have the late Palaeolithic (50,000–­12,000 before the present [B.P.]) (Li few drawbacks because antibody detection only indicates exposure et al., 2020; Stanciu, 2015). Currently, equine meat is still consumed to the parasite and not the presence of active infection. However, in many countries, and in the European Union (EU) alone, the average serological tests are useful tools for survey studies since they allow consumption is approximately 110,000 tons per year (FAO, 2013). to identify Toxoplasma-­positive farms and animals as well as to anal- Viable T. gondii cysts has been isolated by mouse bioassay from yse the related risk factors associated with the exposure to the horse and donkey meat (Al-­Khalidi & Dubey, 1979; Dubey et al., 2020; parasite (Dubey, 2022). Several serological studies on T. gondii expo- Evers et al., 2013; Paştiu et al., 2015) and clinical toxoplasmosis has sure in equids have been carried out worldwide (reviewed in Dubey been epidemiologically related with horse meat consumption in hu- et al., 2020). However, there is no information about T. gondii sero- mans (Pomares et al., 2011). Also, donkey milk consumption is be- prevalence in equids in many European countries and updated infor- coming progressively more popular in recent years, and it has been mation in these species is scarce in others. The aims of the present suggested that human consumption of raw milk from seropositive study were to determine the prevalence of T. gondii antibodies and donkeys could be a potential source of toxoplasmosis (Mancianti to identify risk factors associated with the exposure to this zoonotic et al., 2014). In addition, although scientific evidence indicates that protozoan in equids in different European countries. | 18632378, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/zph.13026 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [10/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 278 CANO-­TERRIZA et al. 2 | M ATE R I A L S A N D M E TH O DS medical check-­ups during the study period in veterinary hospitals. Samples were obtained by puncture of a jugular vein and sera were 2.1 | Sampling obtained by centrifugation at 400g for 10 min and were kept frozen at −20°C until ready to be sent to the Animal Health Department at Blood samples from 1399 equines were collected in southern/north- the University of Cordoba (Spain) for serological analysis. eastern Spain (n = 757 from 143 municipalities), south-­central Italy Whenever possible, individual and herd epidemiological infor- (n = 442 from 36 municipalities), southeastern UK (n = 100 from mation was gathered from each animal (Table 1). three municipalities) and Ireland (n = 100 from 23 municipalities) during the period 2013–­2021. Two equid species, horses (n = 1085) and donkeys (n = 238), and 2.2 | Serological analysis two equid hybrids mules/hinnies (n = 76), were sampled (Figure 1). Mules are a cross between a female horse and a male donkey, and hin- Presence of T. gondii antibodies in the sera from equids was assayed nies are a cross between a female donkey and a male horse. Samples by the modified agglutination test (MAT) following the protocol from Spain and Italy were obtained from serum banks of horses sam- described by Dubey and Desmonts (1987). A titre of 1:25, which is pled in epidemiological surveillance programs or submitted to med- recognized as highly sensitive and specific for detection of T. gondii ical check-­ups during the study period. Donkeys and mules/hinnies antibodies, was used as a cut-­off for T. gondii seropositivity as previ- were also included from these sources when possible. More than ously considered for these species (Dubey et al., 2020). All positive 385 animals, that provides the highest sample size with a CI95% and sera were retested and titrated at dilutions: 1:25, 1:50, 1:100 and accepted error of 5% in studies based on unknown prevalence as- 1:500. Although there is no validation of MAT for the detection of suming a prevalence of 50% (Thrusfield, 2018), were sampled in each T. gondii in equids, extensive studies in chickens, pigs, and sheep at- country. In addition, a convenience sampling was also carried out in test the validity of MAT for the diagnosis of toxoplasmosis in animals Ireland and the UK from equids subject to surgical interventions or (reviewed in Dubey, 2022). F I G U R E 1 Seroprevalence of Toxoplasma gondii by equid species and country. | 18632378, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/zph.13026 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [10/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CANO-­TERRIZA et al. 279 TA B L E 1 Distribution of the seroprevalence of Toxoplasma gondii in equids and horses by categories and results of the bivariate analysis EQUIDS HORSES Variable Categories % MAT positive Positive/overalla p-­value % MAT positive Number/overalla p-­value Species Horse 12.8 139/1085 Donkey 43.7 104/238 <0.001 Mule/hinnie 28.9 22/76 Sex Male 13.8 76/552 <0.001 11.4 56/491 0.332 Female 22.7 161/710 12.5 58/463 Country Ireland 7.0 7/100 7.2 7/97 Italy 27.1 120/442 13.4 33/247 United Kingdom 12.0 12/100 <0.001 12.0 12/100 0.365 Spain 16.6 126/757 13.6 87/641 Age Foal 18.4 49/266 10.3 19/185 Adult 19.9 128/643 0.730 11.7 55/470 0.360 Geriatric 18.0 65/361 14.3 44/307 Breed Mixed-­breed 15.4 44/285 0.024 13.6 35/258 0.204 Purebred 21.0 174/830 11.3 63/559 Activity Sport 12.3 40/325 12.4 40/322 Milk production 44.7 80/179 —­ —­ Work 18.8 9/48 <0.001 9.1 2/22 0.972 Breeder 22.5 50/222 12.3 18/146 Family leisure 13.2 37/281 11.9 30/252 Presence of cats No 25.4 106/417 0.030 16.0 46/288 0.121 Yes 19.9 93/467 12.3 38/309 Census of equids >60 21.9 57/260 9.8 16/164 14–­6 0 24.9 67/269 0.136 10.8 17/158 0.084 <14 18.0 51/284 16.5 42/255 Shelter (spring–­ Indoor/Outdoor 14.2 16/113 9.9 9/91 summer) Outdoor 19.8 113/570 0.107 12.5 52/417 0.610 Indoor 23.4 69/295 14.0 29/207 Shelter (autumn-­ Indoor/Outdoor 17.4 16/92 14.1 11/78 winter) Outdoor 21.3 80/375 0.703 11.4 27/236 0.778 Indoor 20.7 106/511 13.0 49/377 Cleaning of Occasionally 25.6 126/492 <0.001 9.5 23/242 0.045 facilities Weekly 15.6 77/493 14.3 65/456 Disinfection of Occasionally 22.9 173/756 0.001 13.9 70/505 0.069 facilities Weekly 13.2 30/228 9.4 18/192 Rodent control No 22.6 145/642 0.016 11.9 49/411 0.354 Yes 16.6 56/337 13.2 37/281 Well water to No 19.9 185/931 0.068 12.6 86/682 0.418 drink Yes 15.4 37/240 11.7 24/205 Water from No 18.3 199/1088 0.028 12.1 103/848 0.199 ponds and Yes 27.7 23/83 17.9 7/39 streams to drink Tap water to No 15.1 97/644 <0.001 12.6 73/578 0.434 drink Yes 23.7 125/527 12.0 37/309 Reproductive No 22.1 145/656 0.521 11.7 51/435 0.586 disorders in Yes 14.3 1/7 14.3 1/7 the last year a Missing values omitted. | 18632378, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/zph.13026 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [10/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 280 CANO-­TERRIZA et al. 2.3 | Statistical analysis 43.7%, and 28.9% of the 1085 horses, 238 donkeys and 76 mules/ hinnies analysed, respectively (Figure 1, Table 1). Toxoplasma gondii The overall prevalence of antibodies was estimated from the ratio titres observed were 1:25 in 50.9%, 1:50 in 24.2%, 1:100 in 23.8% of positives to the total number of analysed samples, with a 95% and ≥1:500 in 1.1% of the tested samples. At least one seropositive confidence interval (CI). The continuous variables were categorized animal was detected in 80 of 205 (39.0%) municipalities sampled. considering percentiles 33 and 66 as cut-­off points. Associations Table 1 shows the distribution of the seroprevalence of T. gondii between explanatory variables and seropositivity for T. gondii were in equids and horses by the explanatory variables obtained from the tested in three steps. First, a bivariate Pearson's chi-­square test or epidemiological questionnaire and results of the bivariate analysis. Fisher's exact test, as appropriate, was performed to obtain the rel- A total of 8 out of 17 explanatory variables associated (p < 0.10) evance of the explanatory variables in the risk of an animal being with T. gondii seropositivity in equids were selected after data ex- T. gondii seropositive. Variables that showed biological plausibility ploration and bivariate analyses and included in the multivariable and with p ≤ 0.10 in the bivariate analysis were selected as potential analysis. The final GEE multivariable analysis showed the species risk factors. Secondly, collinearity between pairs of variables was as a risk factor potentially associated with the exposure to T. gondii tested by Cramer's V coefficient. When collinearity between vari- (Table 2). Significantly higher seropositivity was found in donkeys ables was detected (p < 0.05), only the variable more clearly linked to (43.7%; p < 0.001, OR = 5.3; 95% CI: 3.65–­7.57) and mule/hinnies T. gondii from an epidemiological point of view was retained. Finally, (28.9%; p < 0.001, OR = 2.7; 95% CI: 1.79–­4.15) compared with a multivariable Generalized Estimating Equations (GEE) model was horses (12.8%). carried out to study the effect of potential explanatory variables To avoid potential bias associated to co-­linearity, horses, the previously selected in the bivariate analysis (Hanley et al., 2003). The species with the most homogeneous sampling among countries, number of seropositive animals was assumed to follow a binomial were statistically assessed separately. In this species, three variables distribution and ‘municipalities’ was defined as the subject variable. (‘census of equids’, ‘cleaning of facilities’ and ‘disinfection of facili- A Poisson error distribution and a logarithmic link function were ties’) were selected from the bivariate analyses (p ≤ 0.10) (Table 1), considered. Biologically plausible confounding factors were tested and the final GEE model identified the ‘occasionally disinfection’ of using Mantel–­Haenszel analysis and confounding was considered to facilities (less than once a week) as the only risk factor associated be potentially significant if odd ratios shifted appreciably. Changes in with T. gondii exposure in horses (Table 2). Significantly higher sero- the OR greater than 30% were considered indicative of confounding. positivity was observed in horses from herds that used disinfectants Potential two-­way interactions between the variables were tested <1 week (13.9%; p = 0.038, OR = 1.6; 95% CI: 1.03–­2.62) compared for significance in the model. The multivariable model was re-­run with those from herds that performed weekly disinfection of the fa- until all remaining variables presented statistically significant values cilities (9.4%). (p < 0.05), and potential causal relationship with the response vari- able existed. The choice of the best model was based on the Quasi-­ likelihood under independence model criterion (QIC). Statistical 4 | DISCUSSION analyses were performed using SPSS 25.0 software (IBM Corp.). The present study is the first large-­scale seroepidemiological study on T. gondii comprising horses, donkeys, and mules/hinnies in 3 | R E S U LT S Europe, and to the best of the authors' knowledge, this is also the first report of T. gondii exposure in equids from the UK and Ireland. Anti-­T. gondii antibodies were detected in 265 of 1399 equids The results indicate a wide exposure of T. gondii in equid populations (18.9%; 95% CI: 16.9–­21.0). Seropositivity was found in 12.8%, in the European countries analysed. TA B L E 2 Results of the generalized estimating equations model of potential risk factors associated with T. gondii seropositivity in equines and horses Variable Categories β p-­value OR 95% CI EQUIDS a a a a Species Horse Donkey 1.66 <0.001 5.26 3.65–­7.57 Mule/hinnie 1.00 <0.001 2.72 1.79–­4.15 HORSES a a a a Disinfection of facilities Weekly Occasionally 0.49 0.038 1.64 1.03–­2.62 a Reference category. | 18632378, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/zph.13026 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [10/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CANO-­TERRIZA et al. 281 Wide fluctuations in T. gondii seropositivity have been reported Due to the high seroprevalence detected in donkeys in the pres- among equids from different countries and continents (Dubey ent study, the risk associated with donkey raw milk consumption et al., 2020). The overall seroprevalence observed in horses in the should also be considered, especially taking into account that in the present study (12.8% of 1085) is within the range previously re- last decade, due to its properties, donkey milk has become very pop- ported in Europe (Dubey et al., 2020). Similar antibody prevalences ular in industrialized countries as a food source (Dubey et al., 2014; against T. gondii were found in this species in Spain (10.8% of 454) Machacova et al., 2014; Mancianti et al., 2014; Martini et al., 2014). (García-­Bocanegra et al., 2012) and Portugal (13.3% of 173) (Lopes When only horses were assessed in the statistical analyses, et al., 2013). Lower T. gondii seroprevalence values were detected the occasional disinfection of facilities (less than once a week) was in Greece (1.7% of 753) (Kouam et al., 2010), Italy (3.0% of 643) shown to be a risk factor associated with T. gondii seropositivity. (Bártová et al., 2015) and Ukraine (7.9% of 76) (Rissanen et al., 2019). Accordingly, it has been widely suggested that a good hygienic sta- Conversely, higher seropositivity rates were detected in Italy (17.6% tus of the farm and the implementation of proper disinfection mea- of 153) (Papini et al., 2015), Czech Republic (24.0% of 552) (Bártová sures has a protective effect against T. gondii infection in livestock et al., 2010), Romania (37.8% of 82) (Paştiu et al., 2015), and France (reviewed in Stelzer et al., 2019). (58.9% of 231) (Aroussi et al., 2015). Comparing and interpreting Of note, statistically significant differences were not found in results obtained from equines using different study designs, sero- the seroprevalence of T. gondii in horses among countries (p = 0.365) logical methods, and cut-­offs to define seropositivity is challenging, (Table 1). This result could indicate that weather conditions did not and comparisons should be carefully made. In this respect, the sur- play a major role in T. gondii seropositivity in horses. In this regard, vey study carried out by García-­Bocanegra et al. (2012) in southern in Spain, where a wider sampling was carried out, statistical differ- Spain, in 2011, was performed using the same serological technique ences (p = 0.402) between the south (14.2% of 254 horses) and the and cut-­off values, and results could, therefore, be compared. The northeast (13.2% of 387 horses) of the country were also not ob- similar seropositivity observed in horses in the present study in the served despite the weather differences. In any case, further studies, same area (14.2%; 36/254) suggest an endemic circulation of T. gon- increasing the number of animals sampled, are warranted to support dii in this region during the last decade. this hypothesis. The information on T. gondii exposure in donkeys and mules/hin- This study has some limitations. First, given the limited number nies in Europe is still very limited. In the present study, the overall of samples in Ireland and the UK, extrapolating the results to the en- seroprevalence of T. gondii in donkeys (43.7% of 238) represents the tire equine population of these countries should be carefully made. highest found in this species in Europe to date. Lower seropreva- In addition, due to the lower census of donkeys and mules/hinnies, lence values were observed in two previous studies carried out in there is a great variation in the number of individuals of the studied the Iberian Peninsula: 5.9% (11/186) (Rodrigues et al., 2019) and animal species. Finally, disinfectants used on the studied herds could 25.6% (21/82) (García-­Bocanegra et al., 2012), and in Italy: 25.0% not be individually discriminated. Future studies are warranted in- (11/44) and 5.0% (12/238) (Machacova et al., 2014; Mancianti cluding a larger number of equids (especially donkeys and mules/ et al., 2014). Regarding mules/hinnies, there have been very few hinnies) to verify results of the present investigation. Moreover, serological studies of T. gondii reported worldwide to date. Lower given T. gondii oocysts are resistant to most used disinfectants seroprevalence rates to those found in the present study (28.9% of (CFSPH, 2017; Dubey, 2022), newer compounds are needed to im- 76) were reported in the two previous serosurveys performed in plement appropriate control measures. this species in Europe. Kouam et al. (2010) detected 7.6% of anti-­T. The seroprevalence values detected in the present study indi- gondii antibodies in 13 mules from Greece, while the seropositivity cate a widespread distribution of T. gondii among equid populations observed by García-­Bocanegra et al. (2012) in this species in Spain in different European countries, with donkeys and mules/hinnies was 15.0% (12/80). being the species at highest risk of T. gondii exposure. Given the high The risk of T. gondii seropositivity was 5.3 and 2.7 times higher seroprevalences found in the present study, raw or undercooked in donkeys and mules/hinnies than in horses. The higher seroposi- foods from these species could be of public health concern in many tivity detected in donkeys, followed by mules/hinnies and horses, European regions. Boiling of milk and an adequate cooking of meat are in accordance with those previously reported (de Oliveira (considering that tissue cysts remain viable for approximately 4 min et al., 2013; García-­Bocanegra et al., 2012; Kouam et al., 2010; at 60°C) could be suitable measures to prevent zoonotic infection by Munhoz et al., 2019). Differences in farming systems among equids this parasite (CFSPH, 2017). In addition, a frequency of disinfection could explain the differences observed in this study. In this regard, of the facilities of at least once a week could reduce the risk of T. donkeys and mules are usually raised under extensive management gondii seropositivity in horse herds. conditions, increasing the risk of seropositivity to T. gondii by in- gesting oocysts contaminating the environment (García-­Bocanegra AC K N O​W L E D ​ G E ​M E N T S et al., 2012). Moreover, Munhoz et al. (2019) suggested that donkeys This research was supported by CIBER -­Consorcio Centro de and mules could maintain detectable T. gondii antibodies titers for Investigación Biomédica en Red-­ (CB), Instituto de Salud Carlos longer than horses. In any case, further studies are needed to assess III, Ministerio de Ciencia e Innovación and Unión Europea –­ differences in the susceptibility to infection among equid species. NextGenerationEU. D. Jiménez-­Martín holds a PhD. contract | 18632378, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/zph.13026 by Consorci De Serveis Universitaris De Catalunya, Wiley Online Library on [10/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 282 CANO-­TERRIZA et al. Dubey, J. P., Ness, S. L., Kwok, O. C. H., Choudhary, S., Mittel, L. D., & granted by Own Research Plan of the University of Cordoba. Funding Divers, T. J. (2014). Seropositivity of toxoplasma gondii in domes- for open access charge: Universidad de Córdoba/CBUA. tic donkeys (Equus asinus) and isolation of T. gondii from farm cats. Veterinary Parasitology, 199(1–­2), 18–­23. https://doi.org/10.1016/j. vetpar.2013.09.027 C O N FL I C T O F I N T E R E S T Evers, F., Garcia, J. L., Navarro, I. T., Zulpo, D. L., Nino, B. D. S. L., The authors have declared no conflict of interest. Ewald, M. P. D. C., Pagliari, S., Almeida, J. C., & Freire, R. L. (2013). Diagnosis and isolation of toxoplasma gondii in horses DATA AVA I L A B I L I T Y S TAT E M E N T from Brazilian slaughterhouses. Revista Brasileira de Parasitologia The data that support the findings of this study are available from Veterinária, 22, 58–­6 3. https://doi.org/10.1590/S1984​-­29612​ 01300​5 000009 the authors upon reasonable request. Fabiani, S., Pinto, B., Bonuccelli, U., & Bruschi, F. (2015). Neurobiological studies on the relationship between toxoplasmosis and neuropsy- E T H I C S S TAT E M E N T chiatric diseases. Journal of the Neurological Sciences, 351(1–­2), 3–­8. Horse samples were obtained within the official Epidemiological https://doi.org/10.1016/j.jns.2015.02.028 FAO. (2013). Food and Agriculture Organization of The United Nations. Surveillance System Programs and from specimens subjected to La Comisión Europea propone reforzar los controles sobre la medical check-­ups or surgical interventions during the study pe- carne de caballo y vacuno. http://www.fao.org/agron​otici​as/agro-­ riod. Animal handling and sampling were performed by qualified and notic​i as/detal​l e/es/?dyna_fef%5Bbac​k uri%5D=21174​& dyna_ trained veterinarians following European (Directive 86/609/CEE). fef%5Buid​%5D=170018 Fuglewicz, A. J., Piotrowski, P., & Stodolak, A. (2017). Relationship be- Therefore, no ethical approval was necessary. tween toxoplasmosis and schizophrenia: A review. Advances in Clinical and Experimental Medicine, 26(6), 1031–­1036. https://doi. ORCID org/10.17219/​acem/61435 David Cano-­Terriza https://orcid.org/0000-0001-5657-2567 García-­Bocanegra, I., Cabezón, O., Arenas-­Montes, A., Carbonero, A., Dubey, J. P., Perea, A., & Almería, S. (2012). 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