Production and Deformation of Clonorchis sinensis Eggs during In Vitro Maintenance

Citation: Uddin MH, Bae YM, Choi M-H, Hong S-T ( Production and Deformation of Clonorchis sinensis Eggs during In Vitro Maintenance Md. Hafiz Uddin 0 Young Mee Bae 0 Min-Ho Choi 0 Sung-Tae Hong 0 Jason Mulvenna, Queensland Institute of Medical Research, Australia 0 Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine , Seoul , Republic of Korea Clonorchis sinensis is a carcinogenic human liver fluke. The present study monitored eggs produced by long-term maintained adult worms of C. sinensis to confirm their egg productivity in vitro. The worms from infected rabbits were incubated in vitro in 16 Locke's solution and broth media (RPMI-1640, DMEM and IMDM). Numbers of expelled eggs were counted sequentially and their morphological changes were monitored by microscopy after 1, 30, 60, and 90 days of cultivation. On the 1-3 days of cultivation, the eggs counted maximum 4,7566202 eggs/worm/day in IMDM medium. The number of eggs gradually decreased less than 1,000 at 7-14 days and below 100 at 21days but continued to pass eggs after 56 days in all media. Length of the eggs were reduced about 1 mm at 30 days, and the length/width ratio was maintained around 1.8 at 30 days but decreased to 1.7 at 60 days and 1.5 at 90 days. Faust-Meleney index (FMI) decreased as the cultivation duration increased and lowest FMI (5662.96974.7) observed in IMDM media at day 90 (P = 0.001). Microscopic findings of the eggs recognized the miracidium in most of eggs at 60 days but not in those at 90 days. Instead, the eggs contained dark granules or vacuoles in the deformed shell at 90 days. Scanning electron microscopy revealed partial loss of wrinkles on the deformed egg surface and prominent abopercular knob. Eggs viability decreased as the cultivation progressed and showed significant positive correlation with FMI and length/width ratio. In conclusion, the cultivated worms pass only the eggs which are preformed in their uterus before cultivation. One gravid C. sinensis contains about 37,000 eggs in its uterus and produces about 4,000 eggs every day. The deformed eggs with FMI less than 7,000 and length/width ratio lower than 1.7 are non-viable. - Funding: The present study was supported by a research grant from Seoul National University Hospital (21-2005-0330). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Helminthes usually produce a large number of eggs to overcome the transmission barriers in nature. Production of enough number of eggs is essential for most flukes because they survive over 3 domains of hosts. The 3 host domains include the first snail intermediate host, the second intermediate host, and the definite host. Biological transition between the hosts is a physical or biological barrier for a fluke. When an adult fluke produces many eggs, only a few of the eggs may survive by successful invasion into the snail host, the first intermediate host. The infected snails shed cercariae into water after asexual reproduction cycles in their body, and the cercariae invade the second intermediate or definitive host to continue its life. Egg production is a fundamental biological function for reproduction of flukes. Clonorchis sinensis is a liver fluke of human causing clonorchiasis, which is prevalent in East Asia. Clonorchiasis is now classified as one of food-borne nelgected tropical diseases. Since cholangiocarcinoma is a serious complication, clonorchiasis is a major health concern in endemic areas [1]. As egg production is an important process for a flukes biology and detection of eggs is a base of diagnosis, several studies observed egg productivity of C. sinensis. It has been reported that C. sinensis produce about 4,000 eggs/worm/day in humans, 2,400 eggs/ worm/day in cats, and 1,600 eggs/worm/day in guinea pigs [2]. However, it is still unknown that C. sinensis may produce their eggs by in vitro cultivation. Recently adult worms of C. sinensis were in vitro maintained long in broth media [3]. During the cultivation, the worms passed many eggs. The present study counted the eggs and observed morphological changes of eggs sequentially to investigate whether the worms are able to produce new eggs in broth media and whether they are viable. Materials and Methods Ethics Statement The animal experiment was reviewed and approved by the institutional animal care and use committee of Seoul National University (2010). Collection of Adult Worms of C. sinensis Metacercariae were collected from naturally infected fish Pseudorasbora parva according to the method described by Li et al. [4]. The collected metacercariae were preserved in cold (4uC) 16 PBS with antibiotics until use. The metacercariae were introduced to male New Zealand white rabbits and adult C. sinensis worms were recovered as described in our previous stud (...truncated)