Morphological Observation And Genetic Variability Study Of Thelazia Callipaeda In Zunyi And Surrounding Regions

Objective: Thelazia callipaeda(T.cp) specimens were collected from human infection cases in Zunyi and surrounding regions. By analyzing epidemiological data, observing T.cps’ morphology and studing of their genetic variability, we obtained the preliminary epidemiological characteristics, the within-population and interspecific variations of T.cp in Zunyi and surrounding regions. The results could have implications for studying the epidemiology and ecology, and of provides an informative foundation to the basis for the prevention of Zunyi and surrounding regions T.cp which enriches the understanding of thelaziasis genomic sequence.Methods: 1. Department of Ophthalmology in Zunyi Medical College Hospital and Zunyi Aerospace Hospital collected the T.cp clinical data of infected adult specimes. 2. Scanning the morphology of female and male T.cp under Optical microscope, and their ultrastructure under scanning electron microscopy. 3. The transverse and longitudinal of both T.cp female and male organization structural features were determined by HE stained. 4. Total genomic DNA was isolated from individual worms using DNA extraction kit to gathering DNA of different pathogenic parasite, two gene: 18 S r RNA and cox1 were amplified and products were determined by agarose gel electrophoresis. 5. Sequenced PCR products were aligned used software Clustal X 1.83, and the evolutional relationship were analyzed by the software MEGA 7.01 based optimal maximum likelihood(ML) methods.Results: 1. The cases analysis showed an increasing and sporadic trend in case load of human thelaziasis in Zunyi and surrounding regions recent years. Cases of the period concentrated in August to November every year. High-risk groups were concentrated at age from 35 to 60 years old, most of which were female. Majority of patients lived in rural region, especially in Zheng’an County in Zunyi. Moreover, about one-third of the infected patients raise pets at home, for instance, dog or cat; 2. Morphology of collected T.cps in Zunyi and surrounding regions were basically the same as that of collected parasites from other region according to the results of ultra structure by the light microscoy, while under the scanning electron, it can be found that some differences existing between the T.cps. The main difference is two plica semilunaris fauciumtas at the bottom of buccal capsule, adult ventral tail end of the tail-to-tail on the mastoid process was not observed to have a crescent-shaped openings. 3. HE staining method was employed to dected T.cp, there is serrated surface structure folds on the surface, body wall by the angle of the cortex, the subcutaneous layer and longitudinal muscle, the body has a digestive system, reproductive system. 4.Sequences of 18 S r RNA from different areas in Zunyi and surrounding regions, for instance, Zheng’an County, Suiyang County, Tongzi County, Xishui County show similarity among 99% to 100%. Compared with other regions in China, sequence orgined from Oita Japan(AB538282.1) and other places T.cp 18 S r RNA genes were showed homology of100%, in a branch. 5. Sequences of cox1 from different areas of Zunyi and surrounding regions showed homology of 98% to 100%. Moreover, the samples sequence origined from Romanian(KP087796.1), Oita Japan(AB538283.1) and other regions showed a homology of 95% to 98%. And Zheng’an 1, Oita Japan(AB538283.1) were clustered in a same branch of Phylogenetic tree. Most of sequences from samples in Xishui showed close relative to sequences of Zunyi 2 and Zheng’an 2, 3. In addition, samples sequences of Zunyi 1 and Romania(KP087796.1) and Serbia(KJ433983.1) were clustered in the same branch.Conclusion: 1. Recent years an significant increasing in case load of human thelaziasis was sporadic in Zunyi and surrounding regions. 2. Morphological characteristics of T.cps in Zunyi and surrounding regions were consistent from other areas, but there are some differences of ultrastructure under scanning electron microscope. 3. Contrast to the high homology of 18 S r RNA, can be useful to effectively detect gene; cox1 gene showed more species-specific and region-specific,can be useful to identify different species,epidemiological analysis and genetic diversity.