**4. Heterogeneity in the rural environment**

Rural areas, and in particular agricultural landscapes, are suitable for *T. gondii* transmission, due to the high densities of both DHs and IHs [72], and to the high level of predation. However, this does not mean that rural areas are evenly infected. Spatial and temporal variations have been detected at several scales. We first present the temporal dynamics to identify mechanisms of heterogeneity that may also explain spatial variations.

#### **4.1. Temporal dynamics**

10 Toxoplasmosis – Recent Advances

**3. Urban toxoplasmosis** 

Urban landscapes are characterized by highly fragmented natural or semi-natural habitats resulting in a mosaic of patches varying in size and quality. Dispersal abilities of animal species between patches are generally affected by roads or by distance to the nearest patches [53, 54]. Many animal species are thus restricted to parks, artificial forest fragments or recreation areas [55]. This results in local extinctions, increased local population density, or social disturbance. Natural populations of domestic cats are present in urban areas in various sites including hospital gardens [56], parks [57, 58], cemeteries or squares [59], taking advantage of the abundance of shelters, food wastes linked to human activity or food provided by cat lovers [59, 17, 54]. Densities regularly exceed 250 cats per km² [16], and can

In urban areas, rodent densities are heterogeneous and generally strongly related to vegetation cover, predation pressure [29], and/or on how the presence of rodents is controlled by trapping or poisoning. For example, the density of field mouse *Apodemus sp*. can range from no individual in areas occupied by dense populations of predators, to 20,000 individuals per km² in isolated patches [28, 29]. Communities of small mammals can persist at high density in small habitat patches sparsely settled by predators [28]. It is therefore unlikely that cat and IHs populations coexist at high densities in the same habitat patches. In addition, urban cats are attracted by food provided by humans, easily accessible all over the year, and that requires no effort of predation. The presence of such a resource can reduce the motivation to hunt in cats [16, 59]. Observations made on urban cats in hunting activity are thus rare in such areas [41]. The altered predator-prey dynamics limits IH-HD *T. gondii* transmission, however toxoplasmosis does occur in urban hosts. Most surveys conducted in stray cats show low prevalences ranging from 5 to 20% [60, 61, 62, 41]. However, high values have occasionally been found: 35.4% in Sao Paolo [62], 51.9 Barcelona [63], 70.2 in Ghent, Belgium [64]. These cases may correspond to areas where cats have access to predation.

The high local densities of cats also entail a high local level of environmental contamination by *T. gondii* oocysts. Beside density, in such areas, cats often use the same place to defecate where they burry or expose their faeces as scent marks [16, 65], and a single location may be used by several cats when cat density is high [66]. Moreover, this behaviour is expected to favour the direct contamination of cats by oocysts while defecating, since oocyst load in defecating areas is extremely high, and cats are exposed through scratching the soil, before cleaning their paws and fur. These defecation sites spread over cat territory cumulate a high concentration of oocysts in areas closed to humans. A study of a cat population living in the Croix-Rousse hospital (Lyon, France) showed that defecation sites were the areas most often found to be positive for *T. gondii* DNA, and may be viewed as hot spots of environmental risk to humans [50]. Similarly, in Poland and in China, contaminated soil samples have been found in public parks and sand pits [67, 68]. Contact with soil, and particularly gardening and consumption of raw vegetables have been demonstrated to be significant risk factors for toxoplasmosis in humans [69, 7, 70]. Contact with defecation sites is thus expected to result in a high risk of infection, but, because contaminated sites represent a low proportion of the

reach up to 2000 cats per km² like in urban parks in Italy [58].

A temporal variability in the dynamics of *T. gondii* life cycle has been detected, both at the year-to-year level and between seasons. It is first important to notice that temporal variability is uneasy to study using serological data, because of the lifelong persistence of antibodies. In long-lived species, temporal variations in the rate of appearance of new cases (incidence) may be masked by the persistence of antibodies. The easiest ways to study temporal dynamics of *T. gondii* should be to consider short-lived species, species where antibody response does not persist lifelong, individual serological follow-up, or to consider indicators of acute infection, *i.e*., type M immunoglobulins or oocyst excretion in cats.

Due to the difficulty to organize long-term surveys, year-to-year variations have been found in a few populations only: in roe deer *Capreolus capreolus* in Spain [73] and in Sweden [74], in red deer *Cervus elaphus* in Scotland [75], as well as in Canadian seals [76]. Tizard *et al.* [77] performed the largest survey to our knowledge, with nearly 12,000 persons studied over 14 years. This survey revealed inter-annual 6-year cycles and showed that year-to-year variations follow rainfall levels with a correlation coefficient as strong as 0.71. Accordingly, a longitudinal survey of rural populations of domestic cats in France showed important interannual variations in incidence among years, related to variations in the level of rainfall [32]. In an urban site, seroprevalence in cats was highest during years with a hot and moist weather or with a moderate and less moist weather [41]. The same trend was observed during a long-term follow-up of two populations of roe deer, with maximal seroprevalence under cold/dry, or cool/moist years [78].

The first explanation that has been proposed for the correlation between meteorological conditions and *T. gondii* dynamics involves the survival of oocysts. The free stage of *T. gondii* is subject to hard environmental conditions: in the terrestrial environment, its survival in soil depends on temperature and moisture. Oocyst survival is maximal (> 200 days) for

temperatures comprised between -6°C and +20 °C [79]. Above +20°C, dessication of oocysts may occur [41, 80, 81], but moisture should prevent it [82, 83]. Under-6°C, the survival of oocysts is reduced and their capacity to sporulate is lost [79], although one may hypothesize that snow cover may protect them from cold. Meteorological variations are thus expected to determine the survival of oocysts. Oocyst survival has also been demonstrated as one of the parameters that most influence predictions given by a mathematical model [10]. However, other factors may also vary with meteorological conditions and influence *T. gondii* life cycle. In particular, the population dynamics of rodents is affected by climate-driven vegetation growth [84]. Specifically, when winter is mild, survival is high and rodent populations comprise many adult or old individuals, which are the age groups most often infected. Thus the risk of encountering an infected prey is expected to increase after mild winters [32]. This mechanism would contribute to the high transmission of *T. gondii* after mild winters, in combination with high oocyst survival.

The Life Cycle of *Toxoplasma gondii* in the Natural Environment 13

**Figure 4.** Possible seasonal pattern of the transmission of *T. gondii*.

**4.2. Spatial heterogeneity** 

surrounding areas.

scenario, however, more detailed data and/or a theoretical approach are needed to fully confirm the proposed pattern. Should this pattern be confirmed, the variability of risk with time should be taken into account for management and prevention recommendations.

Spatial heterogeneity of the infection in the rural environment has been demonstrated both between regions and between areas within and around villages. Farm animals, being restrained to agricultural areas, are particularly relevant to analyse spatial heterogeneities of the circulation of *T. gondii*. However, studies concerning other species or humans may provide useful information when the studied processes concern farms as well as

At the between-region scale, heterogeneity has essentially been shown to correlate with climatic variations. The hypothesis of relationships between humidity, temperature and *T. gondii* prevalence has been suggested in cattle in Serbia [88] and in sheep in Spain [89]: regions with high humidity and moderate temperatures are considered as most favourable for the sporulation and survival of oocysts. However, few data allowed authors to formally analyse these relationships in farm animals. When comparing the incidence of toxoplasmosis in rural cats living in three villages from distinct regions in France, Afonso *et al.* found that the difference between the villages was explained by their level of rainfall [32]. Other surveys, considering wild-living species and humans, are in accordance with the hypothesis of a climate-driven dynamics. Regional seroprevalences in woman in France vary with temperature: they increase when mean temperature increases, but decrease when the mean number of days below -5°C increases in the region [13]. In a national survey on the wild boar *Sus scrofa*, the number of 10-day periods below -6°C was also found as a determinant of *T. gondii* seroprevalence [90]. Still in the wild boar, in Corsica, prevalence was highest at high altitude, where rainfalls are abundant and temperatures are low [91]. Overall, these and previous results (4.1) underline the importance of climate and meteorological conditions in driving the temporal and spatial dynamics of *T. gondii*. These

Meteorological conditions are also expected to act at the seasonal level. Oocyst survival should be lowest during dry, hot summer periods, and during very cold winters. Moreover, the population dynamics of hosts follows seasonal cycles: most births of rodents and cats occur in spring and summer. However, since many kittens carry maternal derived antibodies [41], the susceptible populations may increase in summer for rodents and in fall for cats. We thus propose the following pattern (Figure 4): in summer, the low survival of oocysts would lead to a low level of environmental contamination. However, the renewal of the pool of susceptible rodents at the same period may boost *T. gondii* transmission. The proportion of infected rodents would increase during summer and fall, thus increasing the risk for cats to get infected. During fall and winter, kittens would have a maximal risk to get infected and excrete oocysts. Finally, in spring, most cats born during the previous year and highly exposed through hunting would have terminated their oocyst excretion thus the rate of soil contamination would decrease. However, due to the survival of oocysts, the prevalence in rodents would continue to rise, and would reach its maximal value at the beginning of spring when reproduction starts again, giving birth to naïve rodents. Following this scenario, the infection of domestic herbivores would increase at the end of fall and in winter, when cats excrete oocysts, specifically within farm buildings [85], but could continue up to the following spring, due to the high survival of oocysts. The risk for infection of people would thus be maximal in winter when oocyst contamination and herbivore infections are frequent, and may persist up to the following spring.

Because of the methodological difficulties presented above, many studies do not find any seasonal pattern [47], and few data come in support of this hypothesis. Tizard *et al.*, considering only high titres, found a clear decrease of human infections in fall [77]. This decrease was interpreted as a consequence of the dry summer period, corresponding to low oocyst survival. In Serbia, new infections occurred more often between October and April than the rest of the year [86]. A seasonal pattern was also found in the proportion of cat faeces presenting oocysts in Germany. Faeces collected between January and June (0.09%) were significantly less often infected than those collected during the second part of the year, between July and December (0.31%) [87]. These observations are concordant with the above

**Figure 4.** Possible seasonal pattern of the transmission of *T. gondii*.

scenario, however, more detailed data and/or a theoretical approach are needed to fully confirm the proposed pattern. Should this pattern be confirmed, the variability of risk with time should be taken into account for management and prevention recommendations.

#### **4.2. Spatial heterogeneity**

12 Toxoplasmosis – Recent Advances

combination with high oocyst survival.

temperatures comprised between -6°C and +20 °C [79]. Above +20°C, dessication of oocysts may occur [41, 80, 81], but moisture should prevent it [82, 83]. Under-6°C, the survival of oocysts is reduced and their capacity to sporulate is lost [79], although one may hypothesize that snow cover may protect them from cold. Meteorological variations are thus expected to determine the survival of oocysts. Oocyst survival has also been demonstrated as one of the parameters that most influence predictions given by a mathematical model [10]. However, other factors may also vary with meteorological conditions and influence *T. gondii* life cycle. In particular, the population dynamics of rodents is affected by climate-driven vegetation growth [84]. Specifically, when winter is mild, survival is high and rodent populations comprise many adult or old individuals, which are the age groups most often infected. Thus the risk of encountering an infected prey is expected to increase after mild winters [32]. This mechanism would contribute to the high transmission of *T. gondii* after mild winters, in

Meteorological conditions are also expected to act at the seasonal level. Oocyst survival should be lowest during dry, hot summer periods, and during very cold winters. Moreover, the population dynamics of hosts follows seasonal cycles: most births of rodents and cats occur in spring and summer. However, since many kittens carry maternal derived antibodies [41], the susceptible populations may increase in summer for rodents and in fall for cats. We thus propose the following pattern (Figure 4): in summer, the low survival of oocysts would lead to a low level of environmental contamination. However, the renewal of the pool of susceptible rodents at the same period may boost *T. gondii* transmission. The proportion of infected rodents would increase during summer and fall, thus increasing the risk for cats to get infected. During fall and winter, kittens would have a maximal risk to get infected and excrete oocysts. Finally, in spring, most cats born during the previous year and highly exposed through hunting would have terminated their oocyst excretion thus the rate of soil contamination would decrease. However, due to the survival of oocysts, the prevalence in rodents would continue to rise, and would reach its maximal value at the beginning of spring when reproduction starts again, giving birth to naïve rodents. Following this scenario, the infection of domestic herbivores would increase at the end of fall and in winter, when cats excrete oocysts, specifically within farm buildings [85], but could continue up to the following spring, due to the high survival of oocysts. The risk for infection of people would thus be maximal in winter when oocyst contamination and

herbivore infections are frequent, and may persist up to the following spring.

Because of the methodological difficulties presented above, many studies do not find any seasonal pattern [47], and few data come in support of this hypothesis. Tizard *et al.*, considering only high titres, found a clear decrease of human infections in fall [77]. This decrease was interpreted as a consequence of the dry summer period, corresponding to low oocyst survival. In Serbia, new infections occurred more often between October and April than the rest of the year [86]. A seasonal pattern was also found in the proportion of cat faeces presenting oocysts in Germany. Faeces collected between January and June (0.09%) were significantly less often infected than those collected during the second part of the year, between July and December (0.31%) [87]. These observations are concordant with the above Spatial heterogeneity of the infection in the rural environment has been demonstrated both between regions and between areas within and around villages. Farm animals, being restrained to agricultural areas, are particularly relevant to analyse spatial heterogeneities of the circulation of *T. gondii*. However, studies concerning other species or humans may provide useful information when the studied processes concern farms as well as surrounding areas.

At the between-region scale, heterogeneity has essentially been shown to correlate with climatic variations. The hypothesis of relationships between humidity, temperature and *T. gondii* prevalence has been suggested in cattle in Serbia [88] and in sheep in Spain [89]: regions with high humidity and moderate temperatures are considered as most favourable for the sporulation and survival of oocysts. However, few data allowed authors to formally analyse these relationships in farm animals. When comparing the incidence of toxoplasmosis in rural cats living in three villages from distinct regions in France, Afonso *et al.* found that the difference between the villages was explained by their level of rainfall [32]. Other surveys, considering wild-living species and humans, are in accordance with the hypothesis of a climate-driven dynamics. Regional seroprevalences in woman in France vary with temperature: they increase when mean temperature increases, but decrease when the mean number of days below -5°C increases in the region [13]. In a national survey on the wild boar *Sus scrofa*, the number of 10-day periods below -6°C was also found as a determinant of *T. gondii* seroprevalence [90]. Still in the wild boar, in Corsica, prevalence was highest at high altitude, where rainfalls are abundant and temperatures are low [91]. Overall, these and previous results (4.1) underline the importance of climate and meteorological conditions in driving the temporal and spatial dynamics of *T. gondii*. These

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 studied.

The Life Cycle of *Toxoplasma gondii* in the Natural Environment 15

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

wild environment.

of *T. gondii* infection.

prey mainly on small mammals [46].

*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

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

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

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

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 the county [91].

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 points [96].

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. gondii* to mix domestic and sylvatic cycles.
