Detection of Echinococcus multilocularis and other foodborne parasites in fox, cat and dog faeces collected in kitchen gardens in a highly endemic area for alveolar echinococcosis.

Echinococcus multilocularis, Toxoplasma gondii and Toxocara spp. are foodborne parasites whose eggs or oocysts are spread in the environment via canid or felid faeces. They can cause infections in humans following the raw consumption of contaminated fruit or vegetables. In this study, their occurrence was investigated by quantitative polymerase chain reaction (qPCR) in 254 carnivore faeces deposited in 94 kitchen gardens of northeastern France that were sampled between two and six times from October 2011 to April 2013. Less than 25% of the sampled kitchen gardens contained more than 75% of the collected faeces. Of the 219 faeces that could be attributed to an emitter, cat accounted for 58%, fox for 32% and dog for 10%. Echinococcus multilocularis was detected in 35%, 11% and 7% of fox, dog and cat faeces, respectively, and Toxocara spp. in 33%, 12% and 5.5% of cat, fox and dog faeces, respectively. Toxoplasma gondii was detected in 2/125 cat faeces and 2/21 dog faeces. The 34 faeces that tested positive for E. multilocularis were found in only 19 out of the 94 sampled kitchen gardens, and the 40 faeces that tested positive for Toxocara spp. were found in 28 of them. Consequently, some kitchen gardens appeared particularly at risk of human exposure to foodborne parasites, including E. multilocularis responsible for alveolar echinococcosis (AE), which is a serious zoonosis. In endemic areas, kitchen garden owners should be informed about the zoonotic risk linked to carnivore faeces deposits and encouraged to set up preventive measures.

Detection of Toxoplasma gondii DNA by qPCR in the feces of a cat that recently ingested infected prey does not necessarily imply oocyst shedding.

Detection of Toxoplasma gondii DNA in cat feces is considered indicative of the presence of T. gondii oocysts. This study aims to demonstrate that the high sensitivity of qPCR can lead to T. gondii DNA detection in cat feces in the absence of oocysts. A cat immune to toxoplasmosis was fed with a mouse experimentally infected with T. gondii. Detection of DNA of this parasite was performed by qPCR on feces passed: (i) on the day the cat ingested the infected prey; (ii) during the three previous days; and (iii) during the three following days. The kinetics of qPCR results are clearly not linked to oocyst shedding and this result demonstrates that qPCR can detect T. gondii DNA related to bradyzoites from an infected prey, in the absence of oocysts. Caution is thus recommended when interpreting T. gondii qPCR results for samples of cat feces.

Spatial distribution of Toxoplasma gondii oocysts in soil in a rural area: Influence of cats and land use.

Toxoplasma gondii is the protozoan parasite responsible for toxoplasmosis, one of the most prevalent zoonoses worldwide. T. gondii infects humans through the ingestion of meat containing bradyzoites or through soil, food or water contaminated with oocysts. Soil contamination with oocysts is increasingly recognized as a major source of infection for humans, but has rarely been quantified directly. In this study, we investigated the spatial pattern of soil contamination with T. gondii over an area of 2.25 km(2) in a rural area of eastern France. The frequency and spatial distribution of T. gondii in soil was analyzed in relation with the factors that could influence the pattern of contamination: cats' frequency and spatial distribution and land use. According to a stratified random sampling Scheme 243 soil samples were collected. The detection of T. gondii oocysts was performed using a recent sensitive method based on concentration and quantitative PCR. Sensitivity was improved by analyzing four replicates at each sampling point. T. gondii was detected in 29.2% of samples. Soil contamination decreased with increasing distance from the core areas of cat home ranges (households and farms). However, it remained high at the periphery of the study site, beyond the boundaries of the largest cat home ranges, and was not related to land use. This pattern of contamination strongly supports the role of inhabited areas which concentrate cat populations as sources of risk for oocyst-induced infection for both humans and animals. Moreover, soil contamination was not restricted to areas of high cat density suggesting a large spatial scale of environmental contamination, which could result from T. gondii oocysts dissemination through rain washing or other mechanisms.

Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil.

Toxoplasma gondii oocysts spread in the environment are an important source of toxoplasmosis for humans and animal species. Although the life expectancy of oocysts has been studied through the infectivity of inoculated soil samples, the survival dynamics of oocysts in the environment are poorly documented. The aim of this study was to quantify oocyst viability in soil over time under two rain conditions. Oocysts were placed in 54 sentinel chambers containing soil and 18 sealed water tubes, all settled in two containers filled with soil. Containers were watered to simulate rain levels of arid and wet climates and kept at stable temperature for 21.5 months. At nine sampling dates during this period, we sampled six chambers and two water tubes. Three methods were used to measure oocyst viability: microscopic counting, quantitative PCR (qPCR), and mouse inoculation. In parallel, oocysts were kept refrigerated during the same period to analyze their detectability over time. Microscopic counting, qPCR, and mouse inoculation all showed decreasing values over time and highly significant differences between the decreases under dry and damp conditions. The proportion of oocysts surviving after 100 days was estimated to be 7.4% (95% confidence interval [95% CI] = 5.1, 10.8) under dry conditions and 43.7% (5% CI = 35.6, 53.5) under damp conditions. The detectability of oocysts by qPCR over time decreased by 0.5 cycle threshold per 100 days. Finally, a strong correlation between qPCR results and the dose infecting 50% of mice was found; thus, qPCR results may be used as an estimate of the infectivity of soil samples.

Development of a sensitive method for Toxoplasma gondii oocyst extraction in soil.

Toxoplasmosis is a world-wide infection caused by Toxoplasma gondii. Oocysts disseminated in the environment by infected cats provide a major source of infection for humans and intermediate hosts. The level of soil contamination and the dynamics of this contamination are mostly unknown due to the lack of sensitivity of detection method. Our aim was to improve the detection of T. gondii oocysts in soil samples by comparing three extraction protocols (A, B and C) on unsporulated and sporulated oocysts of different strains and ages, and by testing the effect of sporulation and soil characteristics on oocyst recovery using the most efficient method. The oocyst recovery obtained using protocol C, in which the flotation solution was placed under the sample solution after the dispersion step, was at least ten-fold higher than protocols A and B, in which the sample was just filtered before flotation. The efficiency of protocol C, tested on five artificial soil matrices and four natural soils inoculated with oocysts, was lowest in soils with high proportions of sand. We recommend the protocol C for field investigations, and we advise that results should be interpreted with caution, considering the effect of soil characteristics, especially sand content, on oocyst recovery.