**5. A sylvatic cycle for** *T. gondii***?**

Being able to infect many species, *T. gondii* is common in wildlife, both in DHs and IHs [2, 98, 46, 1]. The question of a sylvatic cycle, and of whether such a cycle would be separated from the transmission in the domestic area, has been raised. A possible interpenetration between domestic and wild cycles would have important consequences for the management of *T. gondii*, since limiting the propagation of the parasite among domestic animals is only feasible if there is no major wild source. The main tools available to investigate this question are the analysis of genotypes that are present in both areas, and the understanding of transmission pathways through epidemiological surveys.

However, the situation differs between temperate and tropical areas. Section 5.1 reviews the factors associated with *T. gondii* infection in wild animals in temperate areas, and assesses the risk of inter-transmission between domestic and wild cycles with its consequences in terms of zoonotic hazard, animal health and population dynamics of highly susceptible wild species. Section 5.2 shows that in Europe, and to a lesser extent in North America, strains found in wildlife are similar to local strains found in domestic animals and the environment. Finally, section 5.3 deals with the specific situation of tropical areas, where the separation between domestic and wild cycles is clearer than in the temperate areas.

#### **5.1. The dynamics of** *T. gondii* **in wildlife in temperate climates**

14 Toxoplasmosis – Recent Advances

studied.

the county [91].

points [96].

*gondii* to mix domestic and sylvatic cycles.

**5. A sylvatic cycle for** *T. gondii***?** 

transmission pathways through epidemiological surveys.

between domestic and wild cycles is clearer than in the temperate areas.

environmental conditions probably also act at a very local scale, for example to affect soil contamination in South-oriented versus North-oriented slopes, however this has not been

Within villages, the spatial organization of host populations leads to heterogeneities of *T. gondii* between central villages, farm areas and fields. In particular, farm buildings and their surroundings, which shelter both cats and IHs, constitute an important source of infection for the surrounding areas. The spatial distribution of cases around a pig farm [72], and a mathematical modelling approach [92] both confirmed that farms represent a source for the whole rural environment. Farms also represent a source of infection for the surrounding wild areas: in Corsica, the seroprevalence in wild boar increased with the density of farms in

Finally, the dynamics of *T. gondii* is also variable among farms. As expected, the presence of cats often determines the risk of a farm being infected [93, 94], thus cat control is a key to the control of toxoplasmosis in farms [95], as well as rodent control [96]. Other factors reported to influence *T. gondii* prevalence are related to herd management: size and isolation of herd, presence of a water point, type of feeding [97, 93]. These factors represent interesting control

Overall, the rural environment, and in particular farms and their surroundings, are a major source of infection, including for other areas. In particular, rural areas and farms are the gate for *T. gondii* to circulate between the wild and domestic environments, thus their spatial distribution, management and level of biosecurity are determinant in the possibility for *T.* 

Being able to infect many species, *T. gondii* is common in wildlife, both in DHs and IHs [2, 98, 46, 1]. The question of a sylvatic cycle, and of whether such a cycle would be separated from the transmission in the domestic area, has been raised. A possible interpenetration between domestic and wild cycles would have important consequences for the management of *T. gondii*, since limiting the propagation of the parasite among domestic animals is only feasible if there is no major wild source. The main tools available to investigate this question are the analysis of genotypes that are present in both areas, and the understanding of

However, the situation differs between temperate and tropical areas. Section 5.1 reviews the factors associated with *T. gondii* infection in wild animals in temperate areas, and assesses the risk of inter-transmission between domestic and wild cycles with its consequences in terms of zoonotic hazard, animal health and population dynamics of highly susceptible wild species. Section 5.2 shows that in Europe, and to a lesser extent in North America, strains found in wildlife are similar to local strains found in domestic animals and the environment. Finally, section 5.3 deals with the specific situation of tropical areas, where the separation *T. gondii* infection in wildlife does not occur with the same probability in any species or place. Wild-living species first have variable levels of susceptibility and exposure. Exposure is largely determined by life history traits, especially feeding behaviour. In birds, where *T. gondii* infection can be present at a high level in many wild birds without any clinical impact, exposure to *T. gondii* is highest in carnivorous species [99]. High *T. gondii* seroprevalence is also reported in large predator species as Lynx and the European wildcat [100, 101] which is of epidemiological significance because infected felids shed oocysts in the wild environment.

Among non-carnivorous species, the risk of infection is related to the risk of encountering oocysts, thus to the level of contact with potentially contaminated soil. In rodents and lagomorphs, home-range size, energy requirements and life expectancy are all expected to be related to the probability to encounter *T. gondii* oocysts. As these traits are correlated to body size [102, 103, 104, 46], large rodents species are more often found positive than small ones [46]. Body size is thus a relevant indicator of prevalence in a given species, and also an indicator of the risk for predators to get infected by preying on that species. Finally, omnivores such as wild boar can acquire toxoplasmosis by incidentally ingesting infected rodents and mainly by rooting and feeding from soil contaminated with oocysts excreted by cats, as shown for other species with similar behaviour, *e.g*., poultry [105]. On the same way, nutria *Myocastor coypus* is a terrestrial herbivorous but can also eat small insects that can disseminate oocysts and mussels, which can accumulate oocysts [106, 107, 108]. Nutria and wild boar are thus particularly exposed to infection by *T. gondii* [109, 110]. Besides being potential source of *T. gondii* for scavengers, they constitute relevant species to monitor the burden of oocysts in the wild environment and to study factors associated with the dynamic of *T. gondii* infection.

The relationship between feeding behaviour and *T. gondii* infection may also act within species: in a predator species for example, differences of feeding behaviour between genders can lead to a *T. gondii* infection higher in one group compared to others. In an insular population, male cats were more often infected than females, which may be related to the fact that males are heavier and may feed on lagomorphs more often than females, which prey mainly on small mammals [46].

Beside species feeding ecology, wild-living populations also have a spatially and temporally structured risk. Like for rural populations (see 4), climatic and meteorological conditions are significant factors explaining the spatio-temporal variations of *T. gondii* in wild populations [111, 91, 90, 78, 112]. Another determinant factor is the proximity of agricultural activity: in Corsican wild boar, seroprevalence was highest in counties with high farm densities [91]. The presence of domestic cats, including farm cats or feral cats essentially living on predation, and wildcats and hybrid that may live close to rural areas [113], is probably an important factor explaining the connection between the wild and domestic life-cycles of *T. gondii*. Cats roaming into forest or rural landscape searching for preys may shed oocysts and contaminate the soil grazed by herbivorous or omnivorous IHs. The domestic-wild interconnection is thus expected to increase with the proportion of predatory cats in populations and their densities. This connection is also expected to increase with landscape fragmentation, which increases the surfaces where wild animals may come in contact with cats habitat. Generally, the level of anthropization is a relevant proxy for the presence of domestic cats and risk of toxoplasmosis in wild-living populations: in Sweden and Finland, the north-South gradient found in ungulates and hares, respectively, has been interpreted as the result of a declining presence of human settlements and cats in the North [74, 114, 115]. In Chile, the prevalence in American minks *Neovison vison* was highest at proximity of human settlements [116]. Finally, when wild-living cats are present, they constitute a strong determinant of *T. gondii* in wildlife: at the national level, *T. gondii* prevalence in French wild boar is high in the area of presence of the European wildcat *Felis silvestris* [90]; in Alaska, the prevalence of infection in herbivorous species reflects the distribution of lynx *Felis canadensis* in the area [117]. Here it is important to underline that, in order to find relevant explanatory spatial factors, these have to be measured at the appropriate scale. For example, farm density may not be an appropriate estimator of the domestic cat population when considered at the country level [90]. Similarly, as stated earlier (4.2.), oocyst survival depends on the conditions experienced by oocysts in their microenvironment, whose range can be lower than the home range of the studied host population, and also lower than the scale at which meteorological data are usually obtained [90]. The difficulty to obtain data at the right spatial scale may explain that some spatial patterns were not elucidated [118, 119].

The Life Cycle of *Toxoplasma gondii* in the Natural Environment 17

unapparent, provoking only mild symptoms. However, a limited number of highly susceptible species have been discovered, in which *T gondii* infection leads to frequent clinical disease and mortality. Marsupials and New World monkeys, which have evolved largely separately from cats, are among the most vulnerable species [2, 125]. Fatal toxoplasmosis is also well-documented in hares (*Lepus sp.*), in northern Europe [114] and Japan [126]. Hares that die of toxoplasmosis are in general in a normal nutritional state and the disease is acute. The explanations for the failure to achieve equilibrium between the host and parasite mainly focus on the host characteristics: a possible lack of cellular immune response [127, 128], the negative impact of stress (food and diet disturbances, exposure to cold, concurrent infections) on the immune response of this species, or even the cumulative effect of immunosuppression induced by toxoplasmosis and stress [129] have been proposed. A clinical expression of toxoplasmosis is also observed in a felid, the Pallas' cats *Otocolobus manul* when raised in captivity [130, 131]. In fact, wild Pallas' cats have minimal opportunity for exposure to *T. gondii* in their isolated natural habitat in Central Asia and, typically, do not become infected with this parasite until being brought into captivity. This could explain their extreme susceptibility to toxoplasmosis [132], which could threaten conservation programs devoted to this species [133]. Although no specific case has been documented in the wild, *T. gondii* may threaten local wild-living populations, for example when new human settlements come in contact with isolated endangered

Despite the presence of a sexual cycle, *T.gondii* maintains a highly clonal population structure. The majority of isolates found belong to one of the three clonal lineages referred to as type I, II and III [134]. Recently, a fourth clonal lineage, called haplogroup 12, has been

In Europe, the majority of isolates from wildlife contain type II strains, with a few type III strains. From 26 *T. gondii* positive extracts from red fox *Vulpes vulpes* from Belgium submitted to a genotyping analysis with 15 microsatellite markers [136], 25 were type II and only one type III [137]. Similarly, using six loci microsatellite analysis, only type II strains were observed in 46 French isolates including 21 from wild boar [138], 12 from roe deer, 9 from foxes, one from mouflon *Ovis aries*, red deer and mallard *Anas platyrhynchos* [139] and one from tawny owl *Strix aluco* [140]. Using the same molecular technique, Jokelainen *et al*. [141] also identified the clonal type II in 15 DNA extracts from hare (*Lepus sp.*) in Finland. In a recent study in Central and in Eastern Germany, Hermann *et al*. [87] determined the complete genotype has been determined for twelve samples tissues from red foxes, using nine PCR-RFLP markers. In addition to *T. gondii* clonal type II apico II and apico I, type III and *T. gondii* showing non-canonial allele pattern were observed. Interestingly, this study showed evidence of a mixed infection, as well as infection with a *T. gondii* genotype that

populations.

**5.2.** *T. gondii* **strains in wildlife at temperate latitudes** 

may represent a recombination of *T. gondii* types II and III.

identified based on isolates from wildlife in the United States [135].

On the other hand, the presence of cats or anthropized area is not the single way remote areas may be contaminated. Within the natural environment, long-distance dispersal of *T. gondii* is possible either as oocysts or cysts within IHs. An example of the first process is the contamination of marine mammals along the Northern Pacific American coast. Genetic as well as epidemiological studies suggest that southern sea otters *Enhydra lutris nereis* may be contaminated following fecal contamination of soil by domestic and wild felids flowing from land to sea through surface runoff, followed by the accumulation of oocysts in filterfeeding marine invertebrates [120]. The dispersal of oocysts within the marine environment is poorly known, but Massie and al. [121] recently proposed that migratory filter-feeding fish, like northern anchovies *Engraulis mordax* and Pacific sardines *Sardinops sagax*, may spread *T. gondii* throughout the ocean. On the other hand, the long-distance dispersal of *T. gondii* within IHs may be illustrated in the case of the isolated archipelago of Svalbard, where cats are absent. In this area, arctic foxes were found to carry *T. gondii*, whereas 751 grazing herbivores tested were all seronegative, indicating that contamination by oocysts is uncommon in the area. Prestrud *et al*. [122, 123] proposed that *T. gondii* may have been transported to arctic area by migratory birds.

All these processes act to spread *T. gondii* from domestic to wild, and within the wild environment. A possible consequence of this large transmission is the threat on conservation efforts of highly susceptible species [124]. In most species, *T. gondii* infection is generally unapparent, provoking only mild symptoms. However, a limited number of highly susceptible species have been discovered, in which *T gondii* infection leads to frequent clinical disease and mortality. Marsupials and New World monkeys, which have evolved largely separately from cats, are among the most vulnerable species [2, 125]. Fatal toxoplasmosis is also well-documented in hares (*Lepus sp.*), in northern Europe [114] and Japan [126]. Hares that die of toxoplasmosis are in general in a normal nutritional state and the disease is acute. The explanations for the failure to achieve equilibrium between the host and parasite mainly focus on the host characteristics: a possible lack of cellular immune response [127, 128], the negative impact of stress (food and diet disturbances, exposure to cold, concurrent infections) on the immune response of this species, or even the cumulative effect of immunosuppression induced by toxoplasmosis and stress [129] have been proposed. A clinical expression of toxoplasmosis is also observed in a felid, the Pallas' cats *Otocolobus manul* when raised in captivity [130, 131]. In fact, wild Pallas' cats have minimal opportunity for exposure to *T. gondii* in their isolated natural habitat in Central Asia and, typically, do not become infected with this parasite until being brought into captivity. This could explain their extreme susceptibility to toxoplasmosis [132], which could threaten conservation programs devoted to this species [133]. Although no specific case has been documented in the wild, *T. gondii* may threaten local wild-living populations, for example when new human settlements come in contact with isolated endangered populations.

#### **5.2.** *T. gondii* **strains in wildlife at temperate latitudes**

16 Toxoplasmosis – Recent Advances

contaminate the soil grazed by herbivorous or omnivorous IHs. The domestic-wild interconnection is thus expected to increase with the proportion of predatory cats in populations and their densities. This connection is also expected to increase with landscape fragmentation, which increases the surfaces where wild animals may come in contact with cats habitat. Generally, the level of anthropization is a relevant proxy for the presence of domestic cats and risk of toxoplasmosis in wild-living populations: in Sweden and Finland, the north-South gradient found in ungulates and hares, respectively, has been interpreted as the result of a declining presence of human settlements and cats in the North [74, 114, 115]. In Chile, the prevalence in American minks *Neovison vison* was highest at proximity of human settlements [116]. Finally, when wild-living cats are present, they constitute a strong determinant of *T. gondii* in wildlife: at the national level, *T. gondii* prevalence in French wild boar is high in the area of presence of the European wildcat *Felis silvestris* [90]; in Alaska, the prevalence of infection in herbivorous species reflects the distribution of lynx *Felis canadensis* in the area [117]. Here it is important to underline that, in order to find relevant explanatory spatial factors, these have to be measured at the appropriate scale. For example, farm density may not be an appropriate estimator of the domestic cat population when considered at the country level [90]. Similarly, as stated earlier (4.2.), oocyst survival depends on the conditions experienced by oocysts in their microenvironment, whose range can be lower than the home range of the studied host population, and also lower than the scale at which meteorological data are usually obtained [90]. The difficulty to obtain data at the right spatial scale may explain that some spatial patterns were not elucidated [118, 119].

On the other hand, the presence of cats or anthropized area is not the single way remote areas may be contaminated. Within the natural environment, long-distance dispersal of *T. gondii* is possible either as oocysts or cysts within IHs. An example of the first process is the contamination of marine mammals along the Northern Pacific American coast. Genetic as well as epidemiological studies suggest that southern sea otters *Enhydra lutris nereis* may be contaminated following fecal contamination of soil by domestic and wild felids flowing from land to sea through surface runoff, followed by the accumulation of oocysts in filterfeeding marine invertebrates [120]. The dispersal of oocysts within the marine environment is poorly known, but Massie and al. [121] recently proposed that migratory filter-feeding fish, like northern anchovies *Engraulis mordax* and Pacific sardines *Sardinops sagax*, may spread *T. gondii* throughout the ocean. On the other hand, the long-distance dispersal of *T. gondii* within IHs may be illustrated in the case of the isolated archipelago of Svalbard, where cats are absent. In this area, arctic foxes were found to carry *T. gondii*, whereas 751 grazing herbivores tested were all seronegative, indicating that contamination by oocysts is uncommon in the area. Prestrud *et al*. [122, 123] proposed that *T. gondii* may have been

All these processes act to spread *T. gondii* from domestic to wild, and within the wild environment. A possible consequence of this large transmission is the threat on conservation efforts of highly susceptible species [124]. In most species, *T. gondii* infection is generally

transported to arctic area by migratory birds.

Despite the presence of a sexual cycle, *T.gondii* maintains a highly clonal population structure. The majority of isolates found belong to one of the three clonal lineages referred to as type I, II and III [134]. Recently, a fourth clonal lineage, called haplogroup 12, has been identified based on isolates from wildlife in the United States [135].

In Europe, the majority of isolates from wildlife contain type II strains, with a few type III strains. From 26 *T. gondii* positive extracts from red fox *Vulpes vulpes* from Belgium submitted to a genotyping analysis with 15 microsatellite markers [136], 25 were type II and only one type III [137]. Similarly, using six loci microsatellite analysis, only type II strains were observed in 46 French isolates including 21 from wild boar [138], 12 from roe deer, 9 from foxes, one from mouflon *Ovis aries*, red deer and mallard *Anas platyrhynchos* [139] and one from tawny owl *Strix aluco* [140]. Using the same molecular technique, Jokelainen *et al*. [141] also identified the clonal type II in 15 DNA extracts from hare (*Lepus sp.*) in Finland. In a recent study in Central and in Eastern Germany, Hermann *et al*. [87] determined the complete genotype has been determined for twelve samples tissues from red foxes, using nine PCR-RFLP markers. In addition to *T. gondii* clonal type II apico II and apico I, type III and *T. gondii* showing non-canonial allele pattern were observed. Interestingly, this study showed evidence of a mixed infection, as well as infection with a *T. gondii* genotype that may represent a recombination of *T. gondii* types II and III.

Su *et al*. [142] developed a standardized restriction fragment length polymorphism (RFLP) typing scheme based on nine mostly unlinked nuclear genomic loci and one apicoplast marker. These markers enable one to distinguish the archetypal from atypical types. In addition, mixed strains in samples can be easily detected by these markers. Mixed infection of *T. gondii* strains in IHs has been previously reported [134, 143]. Detection of mixed infection is of particular interest in epidemiological studies. For genetic exchange, the DH must ingest different types of parasites from their prey at nearly the same time. The frequency of mixed infections in IHs is a relevant indicator of the likelihood of the genetic exchange to occur in the field. In Svalbard, a Norvegian arctic archipelago, 55 artic foxes *Vulpes lagopus* were found infected with *T. gondii*: 27 (49.1%) harboured clonal type II (17/27 were apico I and 10/27 apico II) and four (7.3%) had clonal type III [123]. Strains from 22 foxes (40%) could not be fully genotyped, but two (3.6%) shared more than one allele at a given locus. Again, the most prevalent genotype in this study was clonal type II (with apico alleles I and II) with a few types III genotypes.

The Life Cycle of *Toxoplasma gondii* in the Natural Environment 19

countries. As in temperate climate [150], the prevalence of *T. gondii* infection was higher in carnivorous or carrion-eaters, or those that accidentally consume oocysts while foraging for food on the ground than in arboreal animals [151]. It was also remarkably high in aquatic mammals such as free-living Amazon River dolphins *Inia geoffrensis* [152]. Remote human population living in wild environment may also exhibit high seroprevalence level for *T. gondii* infection, for example 60.4% in Amerindian tribes [153] or 38.9% in Pygmies from Central Africa [154]. As domestic cats are generally absent from this environment, wild felids are the main source of water and soil contamination. Thirty-nine species of felids have been described, of which 20 live in humid tropical areas [155, 156]. The capacity of oocyst excretion has been demonstrated in captivity, and high seroprevalences were found on freeliving felids [157]. In captive Neotropical felids from Southern Brazil, wild-caught felids were three-times more likely to be infected when compared to zoo-born animals [158]. The different species of wild felids varied in home range and resource requirements, but they generally have larger hunting areas and dietary intake than domestic cats, especially the largest ones [159]. This could result in a high opportunity to ingest *T. gondii* infected preys. So, despite the fact that the ecology of *T. gondii* in the wild tropical environment has been poorly studied, the different behaviour of wild felids compared to that of domestic cats and the number of possible IHs suggest a complex ecology of this parasite in this environment

This high genetic diversity in tropical wild-life in connection with a sylvatic life cycle has been firstly evoked in French Guiana where severe cases of human toxoplasmosis were detected after eating Amazonian undercooked game or drinking untreated river water [161, 162, 163]. These cases were due to highly atypical strains, all with unique genotype, as determined by microsatellite analysis [164]. The difference between these strains acquired from the Amazonian environment and strains from the anthropized environment of French Guiana was further documented by strain sampling in animals from the different

Compared to the strains of the anthropized environment, the "wild" strains from the Amazonian rainforest in the Guianas exhibited a remarkably high genetic diversity [162, 164, 165]. Whereas the majority of strains from the adjacent anthropized environment are clustered into a few widespread lineages, the "wild" population of strains does not exhibit any clear genetic clustering/structure nor any linkage disequilibrium, supporting the hypothesis of an important circulation and mixing in this environment. This could be connected to the high level of biodiversity in Amazonian neotropical rainforest. This biodiversity concerns the different protagonists of *T. gondii* life cycle (DH, IH and environment). This part of the world may be considered as one of the most important hotspot of diversity with at least 183 mammal species, including 8 of thirty nine known wild felid species, and 718 bird species in French Guiana [165]. The corresponding high level of diversity among *T. gondii* strains may reflect the "natural" population structure of this parasite (before the time of domestication of cats and development of farming) within the

leading to a high genetic diversity [160].

compartments [165].

It is noteworthy that type II is also the dominant type in domestic mammals in Europe. For instance, Dumètre *et al*. [144] showed by multilocus microsatellite analysis the predominance of type II in sheep, which has also been previously described in humans. In the same way, Halos *et al*. [145] analysed 433 hearts of sheep by using PCR-restriction fragment length polymorphism and microsatellite markers on parasites isolated after bioassay in mice. All 46 genotypes belonged to type II, except for one strain from the Pyrenees mountains area, which belonged to genotype III, which is the first non-type II genotype found in sheep in Europe [146], Denmark [147] and France [144]. This similarity between strains found in wildlife and domestic species in Europe suggests that no clear separation exists between the two cycles.

In North America, strains of *T. gondii* are more diverse. A recent study [148] analysed 169 *T. gondii* isolates from various wildlife species including DHs and IHs, and revealed the large dominance of a recently designated fourth clonal type, called type 12, followed by the type II and III lineages. These three major lineages accounted for 85% of strains from wildlife in North America [148]. The strains isolated from wildlife in North America are thus more diverse, but may also be more different from strains found in the domestic environment than in Europe. Although type 12 has been identified from pigs and sheep in the USA, it may be more specifically found in wildlife [135]. The relative high diversity in *T. gondii* genotypes isolated from wildlife samples compared to those from domestic animals raised the question as to whether distinct gene pools exist for domestic and sylvatic hosts [149].

#### **5.3. The wild environment in tropical areas**

The wild environment in tropical areas is still characterized by high fauna diversity, and large areas preserved from influence of humans and of domesticated animals, including cats. Studies on *T. gondii* seroprevalence conducted on tropical wild animals, mainly in South America, show the wide circulation of *T. gondii* in the wild environment of these countries. As in temperate climate [150], the prevalence of *T. gondii* infection was higher in carnivorous or carrion-eaters, or those that accidentally consume oocysts while foraging for food on the ground than in arboreal animals [151]. It was also remarkably high in aquatic mammals such as free-living Amazon River dolphins *Inia geoffrensis* [152]. Remote human population living in wild environment may also exhibit high seroprevalence level for *T. gondii* infection, for example 60.4% in Amerindian tribes [153] or 38.9% in Pygmies from Central Africa [154]. As domestic cats are generally absent from this environment, wild felids are the main source of water and soil contamination. Thirty-nine species of felids have been described, of which 20 live in humid tropical areas [155, 156]. The capacity of oocyst excretion has been demonstrated in captivity, and high seroprevalences were found on freeliving felids [157]. In captive Neotropical felids from Southern Brazil, wild-caught felids were three-times more likely to be infected when compared to zoo-born animals [158]. The different species of wild felids varied in home range and resource requirements, but they generally have larger hunting areas and dietary intake than domestic cats, especially the largest ones [159]. This could result in a high opportunity to ingest *T. gondii* infected preys. So, despite the fact that the ecology of *T. gondii* in the wild tropical environment has been poorly studied, the different behaviour of wild felids compared to that of domestic cats and the number of possible IHs suggest a complex ecology of this parasite in this environment leading to a high genetic diversity [160].

18 Toxoplasmosis – Recent Advances

alleles I and II) with a few types III genotypes.

separation exists between the two cycles.

**5.3. The wild environment in tropical areas** 

Su *et al*. [142] developed a standardized restriction fragment length polymorphism (RFLP) typing scheme based on nine mostly unlinked nuclear genomic loci and one apicoplast marker. These markers enable one to distinguish the archetypal from atypical types. In addition, mixed strains in samples can be easily detected by these markers. Mixed infection of *T. gondii* strains in IHs has been previously reported [134, 143]. Detection of mixed infection is of particular interest in epidemiological studies. For genetic exchange, the DH must ingest different types of parasites from their prey at nearly the same time. The frequency of mixed infections in IHs is a relevant indicator of the likelihood of the genetic exchange to occur in the field. In Svalbard, a Norvegian arctic archipelago, 55 artic foxes *Vulpes lagopus* were found infected with *T. gondii*: 27 (49.1%) harboured clonal type II (17/27 were apico I and 10/27 apico II) and four (7.3%) had clonal type III [123]. Strains from 22 foxes (40%) could not be fully genotyped, but two (3.6%) shared more than one allele at a given locus. Again, the most prevalent genotype in this study was clonal type II (with apico

It is noteworthy that type II is also the dominant type in domestic mammals in Europe. For instance, Dumètre *et al*. [144] showed by multilocus microsatellite analysis the predominance of type II in sheep, which has also been previously described in humans. In the same way, Halos *et al*. [145] analysed 433 hearts of sheep by using PCR-restriction fragment length polymorphism and microsatellite markers on parasites isolated after bioassay in mice. All 46 genotypes belonged to type II, except for one strain from the Pyrenees mountains area, which belonged to genotype III, which is the first non-type II genotype found in sheep in Europe [146], Denmark [147] and France [144]. This similarity between strains found in wildlife and domestic species in Europe suggests that no clear

In North America, strains of *T. gondii* are more diverse. A recent study [148] analysed 169 *T. gondii* isolates from various wildlife species including DHs and IHs, and revealed the large dominance of a recently designated fourth clonal type, called type 12, followed by the type II and III lineages. These three major lineages accounted for 85% of strains from wildlife in North America [148]. The strains isolated from wildlife in North America are thus more diverse, but may also be more different from strains found in the domestic environment than in Europe. Although type 12 has been identified from pigs and sheep in the USA, it may be more specifically found in wildlife [135]. The relative high diversity in *T. gondii* genotypes isolated from wildlife samples compared to those from domestic animals raised the question as to whether distinct gene pools exist for domestic and sylvatic hosts [149].

The wild environment in tropical areas is still characterized by high fauna diversity, and large areas preserved from influence of humans and of domesticated animals, including cats. Studies on *T. gondii* seroprevalence conducted on tropical wild animals, mainly in South America, show the wide circulation of *T. gondii* in the wild environment of these This high genetic diversity in tropical wild-life in connection with a sylvatic life cycle has been firstly evoked in French Guiana where severe cases of human toxoplasmosis were detected after eating Amazonian undercooked game or drinking untreated river water [161, 162, 163]. These cases were due to highly atypical strains, all with unique genotype, as determined by microsatellite analysis [164]. The difference between these strains acquired from the Amazonian environment and strains from the anthropized environment of French Guiana was further documented by strain sampling in animals from the different compartments [165].

Compared to the strains of the anthropized environment, the "wild" strains from the Amazonian rainforest in the Guianas exhibited a remarkably high genetic diversity [162, 164, 165]. Whereas the majority of strains from the adjacent anthropized environment are clustered into a few widespread lineages, the "wild" population of strains does not exhibit any clear genetic clustering/structure nor any linkage disequilibrium, supporting the hypothesis of an important circulation and mixing in this environment. This could be connected to the high level of biodiversity in Amazonian neotropical rainforest. This biodiversity concerns the different protagonists of *T. gondii* life cycle (DH, IH and environment). This part of the world may be considered as one of the most important hotspot of diversity with at least 183 mammal species, including 8 of thirty nine known wild felid species, and 718 bird species in French Guiana [165]. The corresponding high level of diversity among *T. gondii* strains may reflect the "natural" population structure of this parasite (before the time of domestication of cats and development of farming) within the true complexity of less disturbed ecosystems. The relative richness of potential hosts that exists within the tropics may have resulted in a correspondingly more diverse range of genotypes of the parasite that can co-exist in such an environment. Under this hypothesis, *T. gondii* would have developed a plurality of alleles to increase its colonization potential [160, 162]. In addition, the larger home ranges of wild felids compared to domestic cats can also strongly influence hybridization patterns and gene flow of the parasite and thus the genetic structure of pathogen populations. The high prevalence in IHs, added to wild felid ecology (diet and home range), could suggest that DHs are more frequently infected by multiple *T. gondii* genotypes, which then cross and recombine before transmission to a new IH. The possibility of reinfection by different strains is known for humans [166]. It has never been explored for felids, but may be hypothesized as another source of increasing diversity.

The Life Cycle of *Toxoplasma gondii* in the Natural Environment 21

and III) even in wild animals. In other temperate or cold countries, such as the U.S.A. or Canada where large territories are non-anthropized, the genotypic diversity of *T. gondii* in the wild animals is present [148, 149, 172]. The diversity is maximal in tropical areas, due to high host diversity and large non-anthropized areas. Thus the risk of transmission of toxoplasmosis from wildlife has not the same consequences everywhere. In tropical areas, specific "wild" strains may be transmitted, thus the transmission risk is relatively easy to characterize through strain genotyping, while in Europe, a case of infection acquired from wildlife would pass unnoticed due to the similarity of strains. The risk of infection from wildlife may be analyzed through genotyping strains in tropical areas, but through

Like other IHs, humans can be infected either by cysts containing bradyzoits, or by oocysts of *T. gondii*. Tissue cysts are responsible for meat-borne infection (pork, lamb, beef or poultry are possible source of contamination), while sporulated oocysts lead to infection by ingesting particles of soil (after gardening for example) or by consuming unwashed raw fruits or vegetables, or untreated water [2, 145, 173, 174, 175]. However, the crucial question of the relative part of risk related to bradyzoits versus oocysts remains open. Different approaches have been used to estimate the relative importance of sources of contamination, using risk-factor analyses or estimation of the fraction of attributable risk, either in the general population (chronic infection) or in cases of seroconversion in pregnant women. These studies clearly identified the ingestion of undercooked meat as a risk factor [7, 13, 173, 176]. However, this result is probably partly due to this risk being easier to characterize than the risk due to oocysts. Another way to get an idea of the relative part of risk related to cysts or oocysts is to undertake a quantitative assessment of the risk of toxoplasmosis [177]. Recently, in the Netherlands, Opsteegh *et al.* performed a quantitative microbial risk assessment (QMRA) for meat-borne toxoplasmosis, which predicted high numbers of infections per year. The study also demonstrated that, even with a low prevalence of infection in cattle, consumption of beef constitutes an important source of infection [178]. However, the risk assessment remains limited by the lack of detailed information on which fraction of meat is more contaminated in carcass: although seroprevalences are available for farm animals from many countries [2], the correlation between seropositivity and detection of parasites in meat is weak. In terms of veterinary medicine, there is no surveillance system for animal toxoplasmosis and only cases of abortions (due to *T. gondii* or other causes) have to be declared. The meat-borne risk analysis is also limited by the low level of information on the food cooking practices, and on the contamination of species consumed less often,

Up to now, the risk analyses essentially used information on, and produced estimates about, meat-borne toxoplasmosis. These studies permitted to identify control points for the

**6. Conclusion: consequences for the management of zoonotic** 

epidemiological surveys in Europe.

**transmission** 

such as game [90, 91, 78].

Most tropical countries are also characterized by an ongoing anthropization with development of farming and settlement in deforested areas. At the confluence between the two environments, wild animals may penetrate in anthropized areas and domestic animals come in contact with the wild through wild game, soil or running water. The increasing pressure of anthropization reduces the hunting area of wild carnivores, including felids and favours their penetration in domestic area. The predatory activity of wild felines or stray cats around these disturbed environments (consumption of chickens, dogs, cats…) would ensure gene flow between the two populations of strains. The consequences of this interpenetration in terms of *T. gondii* genotypes are diverse: (i) detection of *T. gondii* strains with "hybrid" genotypes between the "wild" population and the anthropized population reflecting genetic exchanges, (ii) strains from the wild environment found in domestic animals, such as stray dogs, or (iii), on the opposite, strains from the anthropized environment found in wild animals [165]. In parallel, the influence of human activities with urbanization, fragmentation of landscape, deforested areas, farming, domestication of cats and other animals, modifies *T. gondii* ecology reducing the number of ecological niches. This process favours an impoverishment of *T. gondii* genetic diversity with the selection of a few strains well adapted to a small number of domestic species [167, 168]. Transportation of these strains through large distances by human trade exchange and transportation of animals lead to introduction of domestic strains in the wild environment and occasionally to expansion of clonal lineages. In tropical countries, this is evidenced by the so-called *Caribbean* genotypes found in the anthropized areas of French Guiana and in several Caribbean Islands, or in Africa, where the same African lineages were found in different countries [169, 170, 171].

Finally, the dynamics of *T. gondii* in wildlife and its interaction with domestic areas show a contrasted pattern. In most European countries, due to the large anthropization, any wildliving individual lives relatively close to domestic areas. Farming and cat domestication occurred long time ago. Farms constitute the reservoir of infection, from which a few genotypes adapted to farm species irradiate in the surrounding environment [72]. This could explain the widespread occurrence of only a few well adapted clonal lineages (types II and III) even in wild animals. In other temperate or cold countries, such as the U.S.A. or Canada where large territories are non-anthropized, the genotypic diversity of *T. gondii* in the wild animals is present [148, 149, 172]. The diversity is maximal in tropical areas, due to high host diversity and large non-anthropized areas. Thus the risk of transmission of toxoplasmosis from wildlife has not the same consequences everywhere. In tropical areas, specific "wild" strains may be transmitted, thus the transmission risk is relatively easy to characterize through strain genotyping, while in Europe, a case of infection acquired from wildlife would pass unnoticed due to the similarity of strains. The risk of infection from wildlife may be analyzed through genotyping strains in tropical areas, but through epidemiological surveys in Europe.
