| Enteric protozoan disease is an important public health problem in the world and is also one of the most difficult problems that human faced. These diseases can cause major outbreaks and exert a significant public health impact. Cryptosporidium (an essential examination index in the "Standards for Drinking Water Quality" in China), Giardia (an essential examination index in the "Standards for Drinking Water Quality" in China), Cyclospora, and Microspordia are the pathogens of cryptosporidiosis (one of the six diarrheal diseases by WHO), giardiosis cyclosporiasis (American FoodNet monitoring disease) and microsporidiosis, which are the emerging zoonotic enteric parasites, causing a generally self-limited clinical illness characterized by chronic diarrhea, abdominal cramps malnutrition in humans and animals. Oocysts or cysts are the infection stage of the four protozoa, and people are infected through eating contaminated food or drinking contaminated water. Characterizing the molecular biology of these infectious stages will assist in the clinical diagnosis and treatment of enteric diseases and help to understand their spread, trace their transmission sources, and identify new species or genotype. In this study, we develop a novel multiplex PCR method to detect Cryptosporidium, Giardia, Microspordia and Cyclospora infections in fecal samples obtained from humans and animals living in Shanghai. We also used this method to characterize the population genetic structure of these pathogens in these samples and in a sample of HIV/AIDS patients in Guangxi. The main results are as follows:1. Infection and genotype characteristic of Cryptosporidium, Giardia and Enterocytozoon in diarrheal outpatients. Approximately,30% of the 252 clinical diarrheal patients from a hospital in Shanghai (76/252) tested positive for at least one intestinal parasite. The positive rate of Microsporidia was 13.49%(34/252), while the positive rate of Cryptosporidium was 6.75%(17/252). No Cyclospora were found. Eight patients were co-infected with C. andersoni and E. bieneusi, and one patient was co-infected with Cryptosporidium and Microsporidia. All the Cryptosporidium and Microsporidia isolates were C. andersoni and E. bieneusi. All the Giardia isolates were assemblage C, except one assemblage B. Meanwhile, patients infected with C. andersoni and E. bieneusi showed higher detection rates in winter than in spring. There were no significant sex-or age-associated differences in detection rates of the three parasites. This is the first report of an epidemic of C. andersoni in humans in which the Giardia assemblage C was also isolated. Hence, C. andersoni could be the fourth major Cryptosporidium species infecting humans in addition to C. hominis, C. parvum and C. meleagridis. Moreover, Giardia assemblage C may be a new genotype that can infect humans (alongside assemblages A and B). Hence, our work reveals the public health importance of C. andersoni and the Giardia assemblage C.2. Infection and genotype characteristic of Cryptosporidium, Giardia and Enterocytozoon. in HIV/AIDS patients. We identified only two Cryptosporidium samples in 285 HIV/AIDS patients:C. andersoni and C. hominis. Subtyping analysis of the gp60 gene revealed that the C. homonis strain belonged to I bA19G2. The Giardia positive rate was 2.8%(8/285), seven of zoonotic assemblage B and one of assemblage C. A total of 33 HIV/AIDS patients (11.58%) were infected with Microsporidia Based on ITS genotype analysis, three new genotypes with one case each were identified:GX25, GX456 and GX458. Other positive samples clustered into four genotypes:PigEBITS7 (7 cases), TypeⅣ /K (8 cases), D (11 cases), and Ebpc (4 cases). Phylogenetic analyses showed that all the Microsporidia samples in the study belonged to one group. Statistical analyses showed that the Microsporidia positive rate of rural patients was significantly higher than that of other occupation groups. Those who drank unboiled water had a significant higher Microsporidia positive rate than those that drank other water sources.3. Giardia infection and its genotype characteristic in pets and zoo animals. We used molecular tools and morphological observations to identify pathogens in 84 fecal samples from a pet hospital and a zoo. The detection rate of PCR was significantly greater (15.48%; 13/84) than the morphological method (5.85%; 5/84). The glutamate dehydrogenase gene had a highest amplification rate (92.31%), followed by the beta-giardia gene (69.23%) gene and the triose phosphateisomerase gene (53.85%). We found four new nucleotide sequences and four assemblages (assemblages C, D, E and F) in these samples. We also observed’assemblage swamping’between assemblages D and C, and between F and D. Our results show that PCR is more sensitive than morphological methods for screening fecal samples. As different genes amplify at different rates, multi-locus genotyping should improve detection rates above that offered by amplifying single genes. We found two sequences with 100%homology to human-derived isolates at amino acid level, suggesting the potential for zoonotic transmission.4. Multilocus sequence typing of C. andersoni and E. bieneusi. Sequence analysis showed that all the MS land MS3 loci of C. andersoni isolates were A4 subtypes. No subpopulation structure was found among the 18 samples that successfully amplified at the five loci, though this result needs confirmation by further study.5. Development of a multiplex PCR method to detect Cryptosporidium, Giardia, Microsporidia and Cyclospora. We firstly in the word developed a novel multiplex PCR method that successfully detected each of of Cryptosporidium, Giardia, Microsporidia, and Cyclospora. The detection copy reached 10/L. The coincidence rates were close to 90% and far greater than the single gene PCR amplification method. Specificity and sensitivity experiments using clinical diaerrheal samples and animal samples revealed that our multiplex PCR method exhibited good specificity, high sensitivity and strong stability. |
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