An Epidemiologic Study On The Infection Rates Of Echinococcus Spp. In Tibetan Fox Population

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis(Em), is a worldwide lethal parasitic zoonosis that causes serious healthy problem to both human and animals, which is significant for public health on the vast pasturing area of eastern Tibetan plateau, China. There are three Echinococcus species, E. granulosus (Eg), Em, and E. shiquicus (Es) coexist in the locality.Tibetan foxes (Vulpes ferrilata) and domestic dogs are the mainly definitive host of Em. The Tibetan fox is also proved as a wild definitive host of Em and Es. The existing epidemiologic studies mainly focusing on the AE transmission cycle involve in domestic dogs because they have a close relationship with human beings and bring the disease to human communities. However, in fact, some reports showed that the prevalence of Em in wild foxes is the highest in all known definitive hosts of Em. The prevalence is more than44%, two or three times that of domestic dogs. Thus, Only when fully understanding the transmission mechanisms of Em in wild and domestic animals, can we truly achieve effective prevention and treatment of hydatid disease of western China pastoral area.The research evaluated the fecal prevelance of Tibetan foxes with the samples from the area of eastern Tibetan plateau, China. We collected229suspect fox feces in two times at Yunbo Gou, Shiqu County, Sichuan Province. However, Tibetan foxes and red foxes live sympatrically in our study area and the feces of Tibetan foxes can't be distinguished from those of red foxes with their appearance. Therefore, we used molecular techniques to separate them in laboratory work. We established a molecular species identification method with45fecal samples collected in August2009and28Tibetan fox and two red fox tissue samples saved in laboratory.184fecal samples collected in July and August2010were used for detecting Echinococcus infection of local foxes and calculated the copro-prevalence.The major results and conclusions are as follows:1.30tissue samples from Tibetan foxes and red foxes were sequenced in a portion of the mitochondrial cytb and the maximum difference rate of the homologous sequence between the two species reached11%. We selected suitable restriction endonucleases using Primer Premier5.0to distinguish the two foxes according to eight haplotypes of cytb identified from28Tibetan fox tissue samples (GU724772-GU724779) and eight haplotypes of cytb in red fox retrieved from GenBank. BamHI-restricted digestion sites could be found at positions145and358only in the Tibetan fox sequence while Sspl-restricted digestion sites could be found at positions121and264in the target region of red fox. The restriction digestion procedure was performed with Tibetan fox tissue samples (28) and red fox tissue samples (2) as expected, indicating the restricted digestion sites in the gene fragment with a highly intraspecific conservative and interspecfic specificity.2. Copro-DNA was extracted with Phenol and chloroform extraction method, Chelex-100boiling method and commercial kit modified method respectively. The result showed that the complete genomic DNA can not be extracted from fox feces and Cytb amplicon can not be obtained with the phenol and chloroform extraction method. The Copro-DNA extracted with Chelex-100boiling method showed a serious degradation but Cytb amplicon can be obtained in the fresh sample. Commercial kits modified method is a simple, timesaving and effective technique to overcome the problem of PCR inhibitors from faecal samples, which can extract complete genomic DNA from old and fresh feces and obtain Cytb amplicon. The sensitivity analysis showed that the lowest concentration of copro-DNA template was0.1%for the PCR amplification and1%for restricted digestion detection.3. Among the45faecal samples, the copro-DNA was successfully extracted and amplified from36. On restriction enzyme analysis,29of the36positive faecal samples originated from Tibetan foxes, while seven originated from red foxes. Copro-DNA was failed to be extracted and amplified from nine faecal samples. Copro-DNA concentrations were compared between the nine positive faecal samples and the nine negative faecal samples by a F test. Detections by spectrophotometer showed a concentration of15.45±5.10ng/μL (±SD, n=9) in the positive samples and9.05±3.95ng/μL (±SD, n=9) in the negative samples. DNA concentrations in positive samples are significantly higher than those in negative samples (F= 8.848, P=0.009).4. For the nine negative samples, we also analyzed the content of PCR inhibitors. Among which, five faecal samples were found old and decomposed in field. Therefore, both the copro-DNA and PCR inhibitors may have already decomposed when collecting in field. The other four feces contained a large number of inhibitors. The result showed that PCR inhibitors in0.4μl copro-DNA extraction from fox species are enough to fail a PCR with a template DNA quantity of at least2.0ng.5. Four different floating liquid, screen filters and double-layer sterilized gauze filters were used to compare the effect of enrichment of Echinococcus eggs before the detection of Echinococcus infection in fecal samples. The result showed lead nitrate solution with the positive detection rate of100%has the best effect of floating collection in four floating liquid. However, the shortcomings were more impurities and deformative eggs caused by lead nitrate solution. The detection rate of screen filter was only40%. The detection rate of double-layer sterilized gauze filter also reached100%with no deformative eggs. the Bertin Precellys24mechanically was used to disrupt the eggs of Echinococcus spp. and release DNA with a protocol consisting of5500rpm for15s repeated twice.6. We totally collected184fecal samples, and72of them were randomly chosen to develop and test by our copro-DNA diagnosis protocol. Copro-DNA was successfully extracted from48fecal samples which were identified as originated from the Tibetan fox. The multiplex nested PCR protocol successfully identified and distinguished the Em and Es positive controls. There are significant differences between the results using the PCR-RFLP and the multiplex nested PCR methods. Using our multiplex nested PCR method,23(48%) out of the48feces samples were detected with the existence of Echinococcus. Nine (19%of48feces) samples were found with the existence of Em, while20(42%) were found of Es, among which six (12.5%) were mixed infected with both Echinococcus species. By comparison, using PCR-RFLP, three fecal samples (6%) were identified infected with Em, four (8%) were infected with Es, and no sample was found mixed infection. All the positive samples detected by PCR-RFLP were also detected by the multiplex nested PCR method. In addition, we identified six haplotypes of Es, among which three were newly discovered (Accession No. JN706738-JN706740). Meanwhile, we found five haplotypes of Es, Among which JN706741was newly discovered.7. To all the184fecal samples, we totally identified120feces originated from Tibetan foxes and6from red foxes. In all120Tibetan fox fecal samples,74(62%) were detected with the existence of Echinococcus. No feces was found with the existence of Eg. Twenty three (19%) were examined the special amplicon of Em,32(27%) were identified with special amplicon of Es, and19(16%) had mixed infections with both Em and Es. To all the Tibetan fox fecal samples,94were successfully sexed. The sex ratio was not significantly different from1:1(43♀and51♂, χ2=0.341, P=0.559), and no significant difference in the copro-prevalence of the two parasites was found between sexes. To the6red foxes original samples, two were found infected with Em, and no sample was found infected with Es or mixed infected with the both Echinococcus spp. Five were successfully sexed (4♀and1♂).