On the Eastern Tibetan Plateau region (Sichuan province, China) dogs are regarded as important definitive hosts of Echinococcus multilocularis. We studied dog spatial behaviour in 4 Tibetan villages in order to determine the role of dogs in environmental contamination and their potential interactions with small mammal intermediate hosts. We identified definitive host species and Echinococcus spp. infection status of feces collected in the field by PCR methods and analysed the spatial distribution of canid feces. Nocturnal space utilization of GPS collared dogs in and around villages was also undertaken. E. multilocularis DNA was amplified in 23% of dog feces (n=142) and in 15% of fox feces (n=13) but this difference was not significant. However, dog feces were more frequently observed (78% of collected feces) than fox feces and are therefore assumed to largely contribute to human environment contamination. Feces were mainly distributed around houses of dog owners (0-200 m) where collared dogs spent the majority of their time. Inside villages, the contamination was aggregated in some micro-foci where groups of dogs defecated preferentially. Finally, small mammal densities increased from the dog core areas to grasslands at the periphery of villages occasionally used by dogs; male dogs moving significantly farther than females. This study constitutes a first attempt to quantify in a spatially explicit way the role of dogs in E. multilocularis peri-domestic cycles and to identify behavioural parameters required to model E. multilocularis transmission in this region.

Deforestation is a major environmental issue driving the loss of animal and plant species. Afforestation has recently been promoted to conserve and restore Chinese forest ecosystems. We investigated the distribution of small-mammal assemblages in an area where forest and associated deforestation habitats dominate and in an agricultural area where afforestation is ongoing in the Loess Plateau of southern Ningxia Autonomous Region, P.R. China. Multiple trapping was used. Assemblages were defined based on the multinomial probability distribution and information theory. Species turnover between assemblages of deforested and afforested habitats was high, although no clear effect on species richness was observed. The two assemblages described along the deforestation gradient displayed higher diversity, whereas diversity was lower in assemblages identified in afforested habitats where Cricetulus longicaudatus, known agricultural pest in various areas of China, clearly dominated. The threatened Sorex cylindricauda and Eozapus setchuanus were recorded along the deforestation gradient but not in plantations. Therefore, habitats present along a deforestation succession in this part of Ningxia sustain a high diversity of small mammals and include species of conservation concern. At the present stage of its process (maximum 15 years), afforestation in southern Ningxia favours the dominance of an agricultural pest.

A hybridization probe-based real-time multiplex-nested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 μl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70-100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871-1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.