Toxoplasma gondii, the etiologic agent of toxoplasmosis, is an apicomplexan obligate intracellular protist of major medical and veterinary relevance. The complex life cycle of T. gondii is defined as facultative heteroxenous, with virtually all warm-blooded animals as intermediate hosts (including humans, domestic and wild mammals and birds), and members of the Felidae family acting as definitive hosts (Dubey, 2021a) (Figure 1). Toxoplasmosis is a zoonosis of global distribution (Robert-Gangneux and Dardé, 2012; Dubey, 2021a) and represents an excellent example of the One Health concept, since T. gondii is present and circulates through all compartments defined in this paradigm (Aguirre et al., 2019; Djurković-Djaković et al., 2019). Due to its wide host range the parasite is of importance not only in public health, but also in livestock industry and wildlife management programs. A FAO/WHO report considered T. gondii as the fourth most important foodborne parasite in the world (FAO and WHO, 2014). In addition, globalization and trade could contribute to the inter-regional and intercontinental spread of new parasite strains (Bertranpetit et al., 2017; Galal et al., 2019).

In humans, this parasite infects up to a third of the total global population (Bigna et al., 2020; Rostami et al., 2020). The infection is usually asymptomatic and results in chronicity; however, a primary infection in pregnant women could cause congenital transmission and consequent serious damage to the fetus (Jones et al., 2001). In immunocompromised individuals, severe neurologic and pulmonary clinical signs are frequently observed consequences of a re-activated or new infection (Wang et al., 2017; Robert-Gangneux et al., 2018). Finally, ocular toxoplasmosis is an increasingly recognized clinical issue in some parts of the world, also in immunocompetent patients (Shobab et al., 2013; Maenz et al., 2014).

In livestock, T. gondii infection is associated with significant economic losses linked to reproductive failure in several domestic species such as sheep and goats (Stelzer et al., 2019; Dubey et al., 2020a; Dubey et al., 2020b). Infection by T. gondii in livestock is also a risk to public health when animals destined for human consumption are involved (Opsteegh et al., 2016). Moreover, the parasite is a cause of concern in wildlife and zoo animals since T. gondii may cause lethal infection in particular species (Dubey, 2021a).

Although important oocyst-associated human toxoplasmosis outbreaks have been documented in the past few years (Pinto-Ferreira et al., 2019; Dardé et al., 2020; Dubey, 2021b), the relevance of the environmental route remains poorly investigated. Toxoplasma gondii oocysts have been detected in a wide spectrum of matrices worldwide, including fresh produce, water, soil or even bivalves (e.g., mussels and oysters), which can accumulate T. gondii oocysts by water filtration (Shapiro et al., 2019; Marquis et al., 2019; Almeria and Dubey, 2021).

Strategies to reduce the disease burden of toxoplasmosis should be based on close collaboration between both medical practitioners and veterinarians under the One Health umbrella. The relative contributions of the different transmissible stages, sources, and transmission pathways (Figure 1) remain partly unknown. This lack of information on the attribution to specific infection sources has hampered the development of effective intervention strategies. That fact could be partly due to the absence of a systematic surveillance system for this zoonotic foodborne pathogen (van der Giessen et al., 2021). In addition, there are major geographical differences in the epidemiology of the infection as well as in food consumption habits around the world, which affect the importance of different transmission routes and specific food products for the occurrence of the infection (Galal et al., 2019).

In Europe, T. gondii is considered an important foodborne parasite that ranked high according to the multiple-criteria decision analyses (MCDA) (Bouwknegt et al., 2018) and disease-burden estimations for toxoplasmosis (Havelaar et al., 2015). Congenital toxoplasmosis is notifiable in 29 of 35 European countries surveyed, with routine testing of pregnant women in some countries such as Austria, Belgium, and France; nevertheless, underreporting is a major problem in most countries. ln animals, risk-based surveillance system of EU livestock needs to be improved to reduce human meat-borne infections; there is a lack of standardization and validation of diagnostic techniques as well as significant limitation in the number of animals tested and the information associated with them (e.g., age and breeding system) (van der Giessen et al., 2021; EFSA and ECDPC, 2021).

Concerning the genetic diversity of T. gondii circulating in Europe, type II strains and, to a lesser extent, type III strains, are the dominating populations, both in domestic and wild environments (Khan et al., 2007; Lorenzi et al., 2016). However, the current globalization of trade seems to be causing risk situations that pose new research and public health challenges (Galal et al., 2019). For instance, cases of severe human toxoplasmosis have been reported in France due to the consumption of imported South and North American horsemeat contaminated with non-archetypal strains of the parasite (Elbez-Rubinstein et al., 2009; Pomares et al., 2011).

Because of the importance of a genetic characterization of T. gondii strains for epidemiological and clinical studies, this work is aimed to summarize present knowledge on the genetic population structure of T. gondii in Europe and the distribution of genotypes within the different compartments comprised in the One Health concept (i.e., human, domestic and wild animals, and environment).

Available genotyping methodologies, PCR-RFLP, PCR-sequencing, MS-typing among others, have been irregularly applied in different areas, over different matrices and in a different manner by distinct research groups. The present section aims to examine the use of common methodologies within the European context. PubMed database was searched combining the terms “Toxoplasma gondii”, “genotyping”, “typing”, “type” and each different possible host designations or categories (e.g., human, goat, fox, marine mammals, etc.) or environmental matrices (e.g., water, soil, fresh produce, etc.) considered. Both T. gondii strain genotyping studies involving isolated viable parasites or DNA positive specimens/clinical samples from Europe were included. Nevertheless, data from overseas territories in other continents and zoo-kept animals were not covered, in order to better limit the origin of infections to continental Europe. Finally, 101 and 43 studies including PCR-RFLP/PCR-Sequencing or MS typing, respectively, were selected (see Supplementary Tables S1, S2). Despite the large number of studies aiming at a genetic characterization of European T. gondii strains, the data are limited due to several factors. After analysis of the extracted data, it seems to be apparent that there is a notable variance in the identity and number of markers used among the studies (Figures 2A–C). The selected studies comprised the use of up to 15 different PCR-RFLP (Figure 2A), PCR-Seq or MS (Figure 2B) markers. The use of an insufficient number of molecular markers may represent a problem because a large part of diversity might be missed or genotypically different parasites not efficiently distinguished. This is especially worrying in the case of PCR-RFLP and PCR-sequencing, since an important proportion (40%, 40/101) of these studies implemented a single-locus typing method, therefore involving major limitations for reliable strain classification (Figure 2C). The most frequently used marker was SAG2 (5’ and 3’ ends of the gene) probably because it was among the first PCR-RFLP markers described, setting a milestone on T. gondii genetic studies (Figure 2A) (Sibley and Boothroyd, 1992; Howe and Sibley, 1995). On the other hand, comparison between studies is hardly possible if assays are based on infrequently used genes, such as ROP1 (Haque et al., 1999; Turčeková et al., 2013), or on markers, like the B1 gene, mostly applied in a certain type of environmental specimens (i.e., water, soil, air, vegetables, or fruit) (Figure 2A) (Burg et al., 1989; Sroka et al., 2008; Sroka et al., 2009; Sroka et al., 2010). Regarding MS typing procedures, the number of markers has not been observed as a problematic issue since the use of five “genotyping” markers or the complete panel of eight “genotyping” plus seven “fingerprinting” MS markers is quite widely used (Figure 2B).

Furthermore, it is observed that regardless of the typing technique used, the collected information (from 21 different countries) is highly unbalanced between countries, and there is a lack of data for large areas of the European continent (Figure 2D). There are cases in which the same samples have been analyzed by different techniques (PCR-RFLP, MS and/or PCR-sequencing), with matching results although with of course different resolution power (Prestrud et al., 2008; Stajner et al., 2013; Verma et al., 2015).

Aiming to find the right balance between reliability and robustness, and taking into account the number of studies implementing a different number of PCR-RFLP/PCR-Seq or MS genotyping markers (Figure 2C), a minimum of four and five genomic regions analyzed was established as a “cut-off”, respectively. To this end, 51 (with typing results on n=804 samples) and 42 studies (n=831 samples typed) including PCR-RFLP/PCR-Seq or MS typing, respectively, were considered to represent a robust pan-European overview (Table 1 and Figure 3).

PCR-RFLP and MS typing are the most widely used methods, but except for predominant lineages and some unique strains, equivalence between assigned genotypes by each technique remains at some extent confusing; thus, remarks will be given separately. The classification of an isolate into archetypal, recombinant or atypical, or even distinguishing between a recombinant strain and a mixed infection (co-infection) is a sensitive issue. In most cases this requires the availability of viable parasites in a sample that could be separated into different co-existing clonal populations, e.g., by limiting dilution cloning (Herrmann et al., 2010). The unambiguous identification of mixed infections is difficult in only DNA positive materials and largely depends on the number and the discriminating power of markers used for genotyping. Therefore, from a critical viewpoint, mixed infections, as well as infections with recombinant (mixture of type I, II or III alleles as a consequence of recombination events) and atypical (including unique polymorphisms at any loci) strains should be treated as a whole (MRA category), differentiating them from the widely prevalent archetypal clonal strains (e.g., types I, II and III). Based on the One Health concept, we sorted genotypic information according to samples or isolates origin into four “compartments”, namely humans, domestic animals, wildlife, and environment (Table 1 and Figure 3).

The complex biology and epidemiology of T. gondii means that researchers face not only the detection of routes or sources of transmission, as in other emerging zoonotic diseases, but also the enormous variety of susceptible hosts that makes it an underestimated and silent concern, only visible in specifically vulnerable groups of populations (immunosuppressed or pregnant hosts). This review examines the distribution of various T. gondii genotypes throughout the European continent taking into account the different One Health compartments. As a whole, the predominance of clonal type II strains is evident, but exhaustive published data collection and analysis suggests the existence of an interesting proportion of divergent strains (MRA), slightly more concentrated in the wildlife compartment. Hence, the dichotomy “domestic versus wild” so manifest in the American continent is possibly present in Europe as well (Mercier et al., 2011; Jiang et al., 2018; Galal et al., 2019). Nonetheless, the potential genetic diversity of T. gondii in wildlife has been less studied than in domestic animals, with fewer samples available, with less effort/success on parasite isolation and consequently limited PCR amplification and a limited resolution of typing assays (Herrmann et al., 2012b; Verin et al., 2013; Bacci et al., 2015; Uzelac et al., 2019). If only studies in which the isolation of the parasite was achieved (mainly in mice or cell culture) and where a sufficient number of RFLP or MS genotyping markers were applied are taken into account, the genotypes described are mainly clonal type II. On the other hand, the selection of certain strains at the expense of others during isolation procedures has been demonstrated in literature (Verma et al., 2017; Fernández-Escobar et al., 2020a). Therefore, data obtained directly from clinical samples should not be ignored but need verification, and conclusions should be drawn with caution. In short, findings should be always interpreted cautiously, as well as with interest, since strains that circulate in wildlife are a source of infection for domestic animals and humans, and have been associated with greater pathogenicity at least in North and South America (Dubey et al., 2014). Virulence characterization data of European field T. gondii strains are worryingly scarce (Uzelac et al., 2020; Fernández-Escobar et al., 2021).

Clonal type III-related strains were also highlighted, mainly detected in animal hosts. Some authors claimed that type III alleles are more frequently detected in southern Europe compared to other parts of the continent (Kuruca et al., 2019; Uzelac et al., 2021), but the reality is that France, Italy, and Portugal are the countries that have published the most T. gondii genotyping studies, with a lower contribution from northern countries (Figure 2D), implying large areas without information. Type I alleles are particularly underrepresented in Europe. Most articles describing type I alleles during genotyping (Turčeková et al., 2013; Papini et al., 2015; Mancianti et al., 2015; Battisti et al., 2018; Santoro et al., 2020; Sroka et al., 2020) only involved direct genotyping from tissue samples DNA, with an often lower success in the amplification of typing markers. On the other hand, two studies (Verma et al., 2015; Moskwa et al., 2017) showed a complete clonal type I profile in two isolates obtained from an aborted bovine fetus in Portugal [firstly reported by (Canada et al., 2002)] and from an aborted fetus of European bison (Bison bonasus bonasus L.) in Poland, respectively. Clonal type I isolates fully typed by 15 MS markers have been also described infecting humans (Ajzenberg et al., 2010).

Standardization of typing methods is definitively necessary for the integration of genetic data. The BRC biobank (Biological Resource Center for Toxoplasma, www.toxocrb.com) was one of the approaches that comes closest to this objective, storing around 1500 strains from different hosts (humans or animals) and from different countries around the world, all genotyped by the widely applied 15 MS markers (Ajzenberg et al., 2010; Rocaboy et al., 2020). There are important limitations of traditional methodologies used for T. gondii typing, because only quite specific and restricted sites within a large T. gondii genome are assessed. Whole-genome sequencing (WGS) data analysis has emerged as the most suitable approach for a thorough analysis of the genetic diversity in T. gondii, its evolutionary history, and population structure. Although WGS is difficult to apply as a routine technique for strain typing, the number of studies using this technology is growing rapidly, mainly due to its enormous potential and the continued costs reduction. WGS data are publicly available only for a few isolates from Europe (namely PRU, MAS, FOU, BOF, TgH26044, TgH21016, TgH20005, Cz-H3, among others) of which only two (PRU, Cz-H3) belong to the dominant clonal type II. The others, although isolated in Europe (i.e., France, Belgium) are at least partially reminiscent of strains likely originating from other continents, like FOU and BOF (Africa) or MAS (South America) (Lorenzi et al., 2016). The European Type II isolate PRU (Pruginaud) was assorted to Clade D, a clade which was established based on WGS data and comprises, in addition to other type II strains, of North American HG12 strains and some atypical North or South American strains (Lorenzi et al., 2016). The generation of WGS data on further strains including European type II strains could help to better understand the real genetic diversity within the dominant European strains, to explore the possible exchange of sequence blocks between clonal lineages in Europe and probably to link genetic differences not covered by the traditional widely used typing methods with phenotypic differences (e.g., virulence in mice) evidenced in literature between European isolates (Fernández-Escobar et al., 2020b; Fernández-Escobar et al., 2021).

Under the light of the data exposed, authors identified some key questions that should be addressed:

Despite many important efforts on T. gondii genotyping in Europe, the situation is still blurred and in need of extra and closer look. Still many questions remain unsolved and will constitute medium term challenges for researchers. Some important facts, like the lack of consensus over the methodologies and markers applied, the huge differences in samples’ quality and concentration, the sampling disparities among regions and the fact that vast areas remain unexplored, as well as the scarcity of data from human cases and environment, are the main limitations to having a complete picture. In this respect, epidemiological surveillance systems must be strengthened at many levels, in humans and in livestock industry (for example on farms, slaughterhouses, and during veterinary inspection of hunted and home slaughtered animals). Therefore, close collaboration between the medical and veterinary sectors is crucial.

There is consensus on type II T. gondii prevailing in Europe, followed by type III, but the presence of a noticeable proportion of recombinant and atypical genotypes whose phylogenetic positioning remains obscure, deserves further investigation. Standardized, high-end typing tools and integrative strategies within the One Health approach are needed to fill the existing gaps and provide a clear picture of the T. gondii population in Europe.

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

MF-E was funded by UCM-Santander/2017 pre-doctoral Grants. MF-E, GS, PM, MJ, LO-M, and RC-B are part of the TOXOSOURCES consortium supported by the funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Grant Agreement No 773830: One Health European Joint Programme.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor OD declared a past collaboration with the author GS.

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