Overall prevalence of cercariae in snails.

A total of 12103 snails examined in Central Kashmir for determining the population dynamics of cercariae revealed overall prevalence to be 4.03%. Gymnocephalus (0.13%), furcocercous (0.28%), echinostome (0.34%) and xiphido-cercaria (3.26%) were recorded. The prevalence of cercaria was recorded highest in summer (4.28%) followed by spring (4.05%) and autumn (3.32%). None of the cercaria was recorded during winter season. Morphologically identified cercariae of Veterinary importance were subjected to molecular analysis using genus (28S rDNA and ITS-2) specific primers. The isolates of gymnocephalus cercaria (FA28, FC28, FZ28, FC2) were identified as cercarial stages of Fasciola spp. The phylogenetic trees revealed that the isolates FA28 and FC28 belonged to Fasciola gigantica and FZ28 and FC2 isolate to F. hepatica. The isolate of Fasciola gigantica (FA28, FC28, FZ28) showed 2, 4 and 13 nucleotide polymorphisms. There was addition and deletion of 1 and 8 nucleotides at various positions in case of Fasciola isolates respectively. Besides this, there were nucleotide substitutions at 4 positions along with presence of nucleotide T at 475 position which confirmed it be Fasciola hepatica. The isolates of echinostome cercaria (B1, BD13 and GY) were identified as cercarial stages of Moliniella anceps, Echinoparyphium recurvatum and family Echinostomatidae respectively. The Moliniella anceps isolate showed prominent differences at 8 positions with respect to other Echinostomatidae spp. The insertion of C at position 612 confirmed it to be Moliniella anceps, while as other two isolates showed 2 nucleotide polymorphisms each after 28S gene amplification. On ITS-2 rDNA analysis, the isolate B1 showed 7 nucleotide polymorphisms and phylogenetic tree revealed that the isolate B1, also belonged to Echinoparyphium recurvatum. The study made it very clear that molecular characterization employing internal transcribed spacer (ITS-2) and 28S ribosomal DNA sequences are reliable approach for genetic differentiation of cercarial stages of trematodes. The phylogenetic taxonomy of echinostomes is still unclear and molecular diversity found in this study is perhaps the first study from India as well as in Indian subcontinent. So, focus should be made more on echinostomes for understanding their morphological, biological and molecular diversity for clarifying their taxonomic position.本研究共检测克什米尔中部地区的12103枚蜗牛，以明确尾蚴的种群动态，总体感染率达4.03%。本次检出的尾蚴类型包括裸茎尾蚴（Gymnocephalus，占比0.13%）、叉尾尾蚴（furcocercous，0.28%）、棘口类尾蚴（echinostome，0.34%）以及剑尾尾蚴（xiphido-cercaria，3.26%）。尾蚴感染率以夏季最高（4.28%），其次为春季（4.05%）与秋季（3.32%）；冬季未检出任何尾蚴。经形态学鉴定的具有兽医学价值的尾蚴样本，采用属特异性引物（28S核糖体DNA（28S rDNA）与内转录间隔区2（ITS-2））开展分子生物学分析。裸茎尾蚴分离株（FA28、FC28、FZ28、FC2）被鉴定为片形吸虫属（Fasciola spp.）尾蚴阶段。系统发育树分析结果显示，FA28与FC28分离株属于大片形吸虫（Fasciola gigantica），而FZ28与FC2分离株属于肝片形吸虫（Fasciola hepatica）。大片形吸虫分离株（FA28、FC28、FZ28）分别存在2、4及13个核苷酸多态位点；不同片形吸虫分离株分别存在1个与8个核苷酸的插入及缺失突变位点。此外，肝片形吸虫分离株存在4个核苷酸替换位点，且在475位点存在核苷酸T，以此可明确其为肝片形吸虫。棘口类尾蚴分离株B1、BD13及GY分别被鉴定为安氏莫林尼吸虫（Moliniella anceps）、卷曲棘口吸虫（Echinoparyphium recurvatum）及棘口科（Echinostomatidae）的尾蚴阶段。安氏莫林尼吸虫分离株与其他棘口科虫种相比，在8个位点存在显著差异；612位点的C碱基插入可明确其为安氏莫林尼吸虫，其余两株分离株经28S基因扩增后，各存在2个核苷酸多态位点。经内转录间隔区2（ITS-2）核糖体DNA分析，分离株B1存在7个核苷酸多态位点，系统发育树结果显示其同样属于卷曲棘口吸虫。本研究明确证实，采用内转录间隔区2（ITS-2）与28S核糖体DNA序列进行分子鉴定，是区分复殖吸虫尾蚴遗传特征的可靠方法。目前棘口类吸虫的系统发育分类学研究仍不明确，且本研究发现的分子多样性特征，可能是印度乃至整个印度次大陆地区针对此类吸虫的首项相关研究。因此，未来应重点关注棘口类吸虫，深入探究其形态学、生物学及分子多样性特征，以明确其分类学地位。