Molecular Detection Of Nematode Parasites From Wintering Hooded Crane (Grus Monacha)

Parasites are quite common in wild birds. They can result in parasitic diseases of birds, causing growth stagnation, and even deadly serious diseases, and thus become a focused issue of endangered waterbirds’ conservation. Hooded Crane (Grus monacha) is a large fragile migrating wader, the species protection situation is very austere. In this study we used feces as sources of samples to study the parasitic infection of Hooded Cranes, considering the simplest and least damage features of feces. According to previous study, wild Hooded Crane has plenty of intestinal parasites infection, include protozoan, nematode and trematode. But the research of parasite identification is not thorough for the limitations of traditional morphological identification methods. To address this problem, a simple, rapid and accurate method for parasite examine is need to be solved. In this study, we focus on nematodes infection status of wild Hooded Crane and explore the molecular detection methods.Hooded Crane faecal samples were collect from Poyang, Caizi and Shengjin Lake in the middle and lower reaches of Yangtze river during November 2013 to February 2015. The parasite eggs were detected from Hooded Crane faecal samples using saturated salt solution floatation, followed by morphological identification. Five nematode parasites were recovered in Hooded Crane faecal samples, including Capillaria spp., Strongyloides spp., Trichostrongylus spp., Ancylostomatidae spp., Ascaridia spp.. The species of nematodes infection rate is Ascaridia sp.(6.9%), Capillaria spp.(4.6%), Strongyloides spp. (4.1%).We separated the parasite eggs from faecal samples, and treated the eggs by liquid nitrogen/85℃ water bath, ultrasonication, and cracking fluid digestion, respectively, then detected the different effects using real-time fluorescence PCR. We succeeded in getting the method to detect Capillaria by the eggs from Hooded Crane faecals using real-time fluorescence PCR. The results showed cracking fluid digestion has the best effect, this method made the eggs to meet the need of molecular detection by one step pretreatment, providing stable and sensitive detection results with simple and efficient processes. It’s able to meet the need of high detection rate in trace molecular detection, providing sensitive detection results. Faecal samples can be detected only by floating and brief centrifugation, omitting of microscopic examination can reduce the workload with non-professional technicians and make it desirable to rapid detection of parasites in bulk. Positive samples can be examined accurately by one step pretreatment using the method we built with greatly detection efficiency improvement. The Capillaria ITS primers we designed has proved to be peculiarly and no cross reaction with the common parasites in cranes, with the aid of specific real-time fluorescence PCR. This method offered a reliable molecular detection for Capillaria nematodes, and also provided a convenient way for detection of the parasitic infection in endangered wild bird species.