Evaluation Of An ELISA For Detection Of Trichinellosis And Research On Biological Characteristics Of WN10

Trichinellosis is a food-borne disease caused by eating raw or undercooked pork or wild game contaminated with Trichinella spp. It is considered as a large threat to public health and animal husbandry. According to the harmful effects on human health and economic losses, WHO/FAO declare that trichinellosis was the most severe food-borne parasitic desease in2012.Life cycle of Trichinella spp. contain three developmental stages:muscle larvae, adult worm, new-born larvae. Especially, muscle larvae which invaded intestine epithelium before being adult worm are also known as’intestinial infective larvae’. Due to different levels of different antigen expression at different developmental stage of Trichinella spiralis, it is difficult to find a single antigenic protein for diagnosis of trichinellosis. Excretory/secretory (ES) products of muscle larvae were considered as a good candidate, but there is a fatal weakness, termed’diagnosis window’, that trichinellosis cann’t be detected until3-4weeks post infection. Because of this, although immunological diagnosis is fast, specific and sensitive, it is not authorized to using in the animal husbandry. In construct, the traditional, manual diagnosis method has to continue until now, which is the reason of high costs putting into the slaughter of livestock.In this study, we chose four antigens from each developmental stage of Trichinella spiralis, including intestinial muscle larvae. They are WN10from intestinal larvae, Ts-Serpin from muscle larvae, Ts-Adsp from adult worm and Ts-NBLsp from newborn larvae. WN10、Ts-Serpin and Ts-Adsp were cloned in pET-28a prokaryotic expression system. After expression and purification, these recombinant proteins were collected. Two epitopes were selected from Ts-NBLsp using overlapping. An indirect ELISA method was established when the protocol and reaction conditions had been optimized. Moreover, we found that WN10contained cystatin domain. As we known, cystatins of parasitic worms can not only inhibit the activity of the cysteine protease, but also play an important role in evasion of host immune response. WN10had no classical ’Q×V×G’ sequence motif in the cystaitin domain. Our study demonstrated that WN10had no activity of cystatin, but we obversed that WN10can lead to NO, TNF-a and IL-10increase in marcophage which may create proper microenvironment for parasitism.