Epidemiology Investigation And Molecular Biology Of Cyclospora Sp. From Human And Cattle

Cyclospora cayetanensis is a kind of new pathogen of intestinal parasite which mainlyinfected the tourists, HIV/AIDS patients and residents in endemic area. The parasitesparasitize in the human intestinal epithelial cells, and it can cause human gastroenteritissuch as diarrhea, intestinal cramps, loss of appetite. Before1995, there were only sporadicreports about Cyclospora infection. During1996to1997, the United States and Canadahad a large Cyclospora outbreaks, a total of2944Cyclospora cayetanensis infection caseswere reported. Since2000, the United States, Canada, Britain, Turkey, Colombia, SouthAfrica and some other regions had Cyclospora outbreaks, especially since July2013in theUnited States more than25states including631cases of Cyclospora infection cases hadconfirmed, and at least49cases hospitalized due to severe diarrhea. The first Cyclosporainfection reported was1995in China, since then Yunnan, Anhui, Zhejiang have hadCyclospora infection cases reported. Since Cyclosporasis cause serious economic loss,even endanger the life and health, there are more and more scholars get the attention of theCyclospora, both in developing countries and the developed country.In order to realize the infection status of intestinal parasites of the non-human primateanimal, and the iodine staining and saturated sucrose floating method were used,3234feces samples were detected under the optical microscopy. The results shows that therewere1748samples were intestinal parasites positive, the overall infection rate was54.05%, including11intestinal parasites. The highest infection was Amoeba, followed bywhipworm and round worms. Only one species parasite infection rates as highest as40.41%, the two species parasites mixed infection rate was11.66%,3or above mixedinfection rate was1.98%. The highest mixed infected of intestinal parasites species wereAmoebas and whipworm. The samples were divided into the wild, the captive and the zootype according to the different methods of breeding, the infection rates were70.47%,55.99%and42.76%, respectively. There has statistically difference in different types ofbreeding between non-human primates infection situation of intestinal parasites (p <0.01),the highest infection rate was the wild type.To develop a detection method of Cyclospora cayetanensis by flow cytometry, theconcentration of the Cyclospora oocysts were set as different groups and negative control,as well as blank control, respectively. A total of20000events were analyzed each time, and a cluster representing Cyclospora oocysts could be clearly observed according to theirsize, assemble (R1), and autofluorescence (R2) under488nm. There were no differencebetween test group and negative control when the concentration below1048.6/mL,therefore, the detection limit of the Cyclospora cayetanensis oocysts were between34and90.8by flow cytometry analysis. That is, when detection of the Cyclospora oocysts in fecalspecimens from clinical patients by flow cytometry, and the20000events were analyzedin the set parameters. Samples having34to90events in analysis gate were consideredequivocal in terms of presence or absence of Cyclospora infection, while those with greaterthan91events were diagnosed as Cyclosporasis.To identify and compare the phylogenetic relationship between Cyclospora isolatesdetected by microscope in Henan province during the period of2007-2013withCyclospora isolates in different parts in the world submit by GenBank based on the PCRresults of18S ribosomal RNA gene and ITS-1gene sequencing, the biology sofeware suchas Clustal X and MEGA were used. The phylogenetic relationship shows that they were inthe same branch with human Cyclospora, what’s more, there was only one base differencewith Cyclospora cayetanensis (AF111183) based on the18S ribosomal RNA gene. Thephylogenetic relationship shows they were some differences with the Cyclospora fromGuatemala, Nepal, Peru and Haiti submitted in different areas based on the ITS-1gene.Also, it was shows it’s variability between different isolates of ITS-1gene.To investigate the prevalence and identify the species of Cyclospora in dairy cattle, atotal of1229fecal samples from11different cattle farms in Henan province werecollected and examined by using Sheater’s sugar flotation technique. The DNA of all thesamples was extracted and amplified the18S rRNA gene partial sequence. A newrestriction fragment length polymorphism (RFLP) enzyme BSpE I was used to differentiatethe Cyclospora species from cattle-associated Eimeria spp., the positive samples wereconfirmed by sequence. The results demonstrate that there were44positive samples werefound, that is the overall prevalence was3.58%. The highest infection ratio in the agegroup is8week, the ratio is9.6%. The phylogenetic analysis based on the18S rRNA genesequences revealed that the Cyclospora isolates from Henan dairy cattle were posited intothe Cyclospora sp. Guangzhou1isolated in Guangzhou and Cyclospora sp. Oita isolatedin Japan. The report about the epidemiological and biology of the Cyclospora infection indairy cattle, which would not only provide useful information for further understanding ofthe molecular characterization of Cyclospora, but also for the control of Cyclosporasis indairy cattle.This study investigate the prevalence of the non-human primate animal intestinal parasites infection status, established a detection method of Cyclospora cayetanensis byflow cytometry, and the species of huamn Cyclospora and diary cattle Cyclospora weredetected, as well as the molecular identification and sequence compared, Phylogeneticrelationship analysis.