| Cyclospora is an emerging food-borne parasitic protozoa,which parasitize in intestinal epithelial cells and caused gastroenteritis to diarrhea symptoms in humans and animals.At present,it has 20 species,and C.cayetanensis is the only species that infect humans.Since1990 s,there have been numerous cyclosporiasis reports by sporadic or outbreaks in the world.It had been documented that the infections are related to travel to the endemic areas or contaminated food consumption by source trace study.The significant seasonality(Summer or wet season)were observed about the Cyclospora infection in humans.As well as,many parasitic protozoan(including Cryptosporidium spp.,Cyclospora spp.,Giardia duodenalis,Enterocytozoon spp.,and Toxoplasma gondii)related to consumption of the raw or undercooked vegetables/fruit.In order to understand the molecular epidemiology of the Cyclospora in human and assess the population genetic structure,as well as the infection of the parasitic protozoan on the fruits and vegetables conducted this present study,it is expected to provide support for the transmission mode study of the human C.cayetanensis and its prevention and control.The average size of C.cayetanensis oocysts(n=102)in China was 8.49 ± 0.12 ?m ×8.26 ± 0.12 ?m,and the oval index was 1.03.A molecular epidemiological study of the Cyclospora infection in hospital patients in Henan(Zhengzhou and Kaifeng)showed that the overall infection rate was 1.2%(76/6579),and all the age groups were infected(P >0.05).A declining trend were showed about the C.cayetanensis infection during the 2011 to2015.The present study was similar to that in Honduras hospital patients(1.3%),higher than that in Mexico pediatric hospital(0.67%),and lower than that in Nepal school children and and the Italy hospital patients.Molecular phylogenetic analysis showed that the obtained C.cayetanensis isolates in present study were clustered in the same clade with that isolates in humans from Mexico,Poland,China,Nepal and Peru.However,it is not enough or reliable to carry out the evolutionary analysis only by single gene sequence.Multilocus sequence typing analysis should be conducted for further understanding.The positive C.cayetanensis isolates(n=76)by multilocus sequence analysis(MLST)showed that five microsatellites(CYC3,CYC13,CYC15,CYC21 and CYC22)loci were successfully amplified of 49,59,57,52,and 56,respectively.And,a total of 45 isolates were successfully amplified at all five microsatellite loci.Microsatellite CYC21 showed higher polymorphism and resolution than the other loci.The 45 isolates of the human Cyclospora formed a total of 29 multilocus genotypes(MLG26-MLG54).There were 79 C.cayetanensis isolates(including 34 isolates of known reference sequence)were formed 54 multilocus genotypes(MLG1 to MLG54),29 haplotypes,and 64 polymorphic sites.The genetic structure analysis of the C.cayetanensis showed that significant but incomplete linkage disequilibrium,and clonal population genetic structure.And,recombinant analysis showed that there were few recombination events(Rm = 5).Three main clades were generated based on the molecular phylogenetic analysis of the 54 contigs.The same three sub population groups(K = 3)were formed based on the sequence analysis of five microsatellite loci allelic profile data using STRUCTURE software,which the ?K reached a peak at K = 3.The MLST analysis providing an important newly perspective on population genetic analysis.The optimization of elution method for Cryptosporidium parvum oocysts on vegetables/fruits surface showed that the oscillating frequency was 100/min,the oscillating time was 1h(reversal every 15 min),and the elution solution was 1% Alconox?,which had the highest overall oocysts recovery rate,And,the recovery rate was 71.40%(31.42×104/44.00×104)when the first elution solution and second elution solution were summarized and count by microscopy.The present recovery is higher than that reported based on the immunomagnetic methods,and lower than that detected by flow cytometry.In the comparative study of the quality and quantity of extracted Cryptosporidium parvum oocysts genomic DNA in liquid solutions by three commercial kits,there were no significant difference between soil DNA extraction kit and stool DNA extraction kit.However,the time saving by soil DNA kit,and cost saving by stool DNA extraction kit.In summary,the present study optimized the elution method of protozoa on the surface of vegetables/fruits,which provided the technical support for future investigation and research.The detection of parasitic intestinal protozoa(Cryptosporidium,Giardia duodenalis,Cyclospora and Microsporidia)on vegetables/fruits surface in Henan area(Zhengzhou and Kaifeng)demonstrated that the overall infection rate was 3.8%(42/1099).Among them,the Enterocytozoon bieneusi infection rate was the highest in 3.5%(39/1099),and only small number of C.cayetanensis(n = 2),Cryptosporidium parvum(n = 1),and no Giardia duodenalis were identified.However,the infection rate of Microsporidia on the vegetable/fruit surface in Poland was 11.3%(9/80),which was significant higher than that of this study(P < 0.01).It is worth noting that the E.bieneusi genotype Ebp C identified on the surface of vegetables/fruits had been reported in humans in China and US,showing that it has strong human pathogenicity.In the present study,C.cayetanensis and C.parvum were identified in lettuce,Romaine lettuce,and Chinese chive surface,respectively.Vegetables/fruit surfaces carrying infection of Cryptosporidium spp.and Cyclospora spp.,which are also common reported in South Korea,Ghana and Ethiopia,etc.This is the first reports for the vegetables/fruits surface carrying status of parasitic protozoan in China,including E.bieneusi,C.cayetanensis and C.parvum.In conclusion,the overall infection rate of C.cayetanensis was low in hospital patients in Henan,China.MLST analysis indicated that the C.cayetanensis isolates in human had linkage disequilibrium and clonal population structure.As well as,this study optimized the elution methods of protozoa on the vegetable/fruit surface,and provided technical support for the subsequent investigation and research.Meanwhile,the carrying infection status of Enterocytozoon bieneusi,Cyclospora cayetanensis and Cryptosporidium parvum on the surface of common vegetables/fruits were first reported in China.However,the MLST data from other sources(human and animals,soil,vegetables and water source)should be obtained in depth for determining the genotypes and population evolution,and transmission ways. |
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