Survey And Identification Of Gnathostoma In Eel And Loach From Part Of Regional Markets And Shang Hai Entry Port

Gnathostoma, a food-borne zoonotic nematode parasites, the human cases caused by which were worldwide distribution, and its hazards can’t be underestimated. But in our country, it lacks of relevant research and attention on Gnathostomiasis, and has not been linked to the disease epidemic prevention standards, which may poses a threat to human public health security. In order to provide new evidence and idea for disease prevention and further study, it is necessary to conduct a systematic survey for Gnathostoma in fresh water from some important cities of China, and find out the species of Gnathostoma exist in our country.This study investigated the domestic7provinces and11species of intermediate hosts, which included freshwater fish, eel, loach. We found the third stage larva of Gnathostoma in eels from Shanghai, Guangzhou, Nanning, in loaches from Guangzhou, Heilongjiang. The infection rate of Gnathostoma in eels from Guangzhou was14%, which was the highest of the all. Otherwise,12batch eels were detected as Gnathostoma positively within14batch eels imported from3countries. The positive eels were all from Philippines and Indonesia. Indonesia eels had Gnathostoma nematodes highest average infection rate, and most total number, but there was slightly lower mean intensity than Philippines.To identify the species of the Gnathostoma in the present study, we observed the Gnathostoma under light microscope and scanning electron microscope(SEM), then measured and analysed datas of Morphological characteristics. Preliminary determination of the Fujian wild boar isolates were G.doloresi adults, Heilongjiang and Guangzhou loach isolates were G.nipponicum early third stage larvae(EL3). Shanghai eel isolates were G.hispidum advance third stage larvae (AL3). Nanning, Guangzhou, Philippines eel isolates were G.spinigerum AL3. Indonesia eel isolates of Gnathostoma mostly were G.spinigerum AL3, but there were still1G.nipponicum AL3and2G.hispidum AL3mixed together.Next, amplified and sequenced complete rDNA-ITS2gene and partial mitochondria CO1gene of the isolates that morphology have been studied and one isolates from Yizhou which have been studied by Wu Huifang. The results showed that it matched with the expected results of morphological identification, further confirmed the accuracy of identification. Results also showed that there was more interspecific differences in Gnathostoma ITS2, which was suitable for species identification. And obviously intraspecific differences in CO1can be used for Gnathostoma intraspecific genetic variation research. G.spinigerum CO1gene showed at least4genotypes, which had no apparent relationship with geographical distribution, but there was close relationship in Indonesia, Philippines, Vietnam and China Guangdong and Guangxi isolates of G.spinigerum. ITS2and CO1genes of G.hispidum were presented2different genotypes. The variations in Gnathostoma ITS2and CO1gene sequences have no reference to morphology.Finally, We designed3pairs of specific primers based on ITS2as target gene and established multiplex PCR method for G.spinigerum、G.doloresi and G.nipponicum identification. The experiment showed that the method was simple, fast, specific and repeatable, which can be judged the results only through the position of electrophoretic band without sequencing and other time-consuming steps.