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Epidemiological study on Cryptosporidiosis in Taiwan
論文的第一部份為探討台灣地區家畜排出隱孢子蟲卵囊之流行狀況，使用modified Ziehl-Neelsen carbolfuchsin stain以及免疫螢光染色（IFA）進行家畜包含牛、羊、牧場狗及牧場老鼠之糞便檢察。調查結果得知各種動物糞便中隱孢子蟲的檢出率，分別為牛37.6﹪（173/460）、羊35.8﹪（44/123）、牧場老鼠35.7﹪（15/24）、牧場狗37.5﹪（3/8）。
論文的第二部份為首次於台灣地區所採集到的牛隻糞便檢體分離出之卵囊中確認其基因型（genotype）。以隱孢子蟲18S rRNA基因的特異性引子對進行PCR分析，定序增幅出的基因片段，首次確認牛隻糞便檢體中之卵囊屬於C. parvum bovine genotype。
第三部份以modified Ziehl-Neelsen carbolfuchsin stain、免疫螢光染色（IFA）套組及PCR，進行對於台灣地區牧場工作人員及其家屬以及蘭嶼地區居民之糞便進行檢查，以了解排出隱孢子蟲卵囊之流行狀況。調查結果發現，台灣本島取得之糞便檢體之檢出率為29.2%（7/24），蘭嶼地區為27.1%（78/288）。以隱孢子蟲特異性引子進行PCR分析對，增幅出1750 bp之隱孢子蟲18S rRNA的DNA片斷。
經由調查發現，台灣的家畜排出隱孢子蟲卵囊的情形十分普遍，人與家畜有較高的感染率；研究發現自牛隻糞便檢出之卵囊屬於C. parvum bovine genotype致病原為人畜共通致病因子的隱孢子蟲卵囊，因此台灣地區人畜共通隱孢子蟲症傳染爆發之可能性不容忽略。因其傳播途經跟人的生活環境中受動物糞便污染有關。
Cryptosporidium are protozoan parasites that infect a wide range of vertebrates. Species within this genus cause human cryptosporidiosis, in which diarrhea is a major symptom. Significant morbidity and mortality, especially in immunocompromised individuals, had been reported in both the developing and developed countries. Transmission is through the ingestion of the transmissive stages, that is, the oocysts. This fecal-oral route of transmission could be manifested through person to person contact, zoonotic, waterborne, foodborne and airborne transmission of the oocysts.
The objective of this thesis is to elucidate the epidemiological status of Cryptosporidium infection in various animals and humans in Taiwan proper, and also to examine the risk of potential waterborne cryptosporidiosis.
The first part of this thesis describes the prevalence of Cryptosporidium oocyst shedding in domestic animals in Taiwan. We conducted fecal survey on Cryptosporidium in livestock including cattle, goats, farm dogs and farm rats using modified Ziehl-Neelsen stain method and immunofluorescence assay (IFA). We observed that 37.6 % (173/460) of cattle, 35.8 % (44/123) of goats, 37.5 % (3/8) of farm dogs and 62.5 % (15/24) of farm rats examined were shedding Cryptosporidium oocysts, respectively.
The second part of this thesis describes the first genotyping of Cryptosporidium oocyst from cattle feces in Taiwan. Using Cryptosporidium specific primer pairs directed against the 18S rRNA gene in a PCR (Polymerase Chain Reaction) amplification, followed by sequencing of the amplicon, we confirmed that one of the C. parvum-like oocysts isolated from cattle feces is that of the C. parvum bovine genotype.
The third part of this thesis describes the prevalence of Cryptosporidium oocyst shedding by humans in Taiwan proper and Lan-yu (Ochid) island. A stool survey for Cryptosporidium oocyst among dairy farmers and their family members on Taiwan proper and residents of Lan-yu island was carried out using modified Ziehl-Neelsen staining method, IFA method and PCR analyses. In Taiwan proper, 29.2 % (7/24) of the dairy farming community members were positive for Cryptosporidium oocyst shedding. In the Lan-yu island, 27.1% (78/288) of the inhabitants examined were positive for Cryptosporidium oocyst shedding. We confirmed that the oocysts found in the stool samples of humans in Taiwan proper were that of the genus Cryptosporidium by PCR analyses.
The fourth part of this thesis describes the detection of Cryptosporidium oocyst and Giardia cyst in faucet water sample from cattle and goat farms in Taiwan. Water examination for the presence of the two aforementioned protozoa was conducted using PTFE membrane filtration method followed by immunomagnetic separation (IMS) and immunostaining with fluorescinated monoclonal antibodies. Of the 55 different water samples from various water sources examined, 2 were found to contain both Cryptosporidium oocyst and Giardia cyst, and 1 with only Cryptosporidium oocyst.
In conclusion, Cryptosporidium oocyst shedding in livestock in Taiwan is ubiquitous in both domestic animals and humans in Taiwan proper. Since the Cryptosporidium detected in cattle feces belongs to C. parvum bovine genotype, its zoonotic potential in causing a human outbreak of cryptosporidiosis should not be ignored. Transmission of Cryptosporidium oocyst from animal to human through the contamination of the environment by animal feces should be considered.
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