梨形鞭毛蟲及隱孢子蟲分析方法評估與存在性調查及其囊體表面性質與過濾行為之探討

Abstract

梨形鞭毛蟲(Giardia)和隱孢子蟲(Cryptosporidium)是常見的腸道寄生性原生動物，人類感染此類寄生性原生動物的主要原因是接觸到宿主的糞便或是食用已遭原蟲污染的食物或飲水。近年來所爆發的數起梨形鞭毛蟲和隱孢子蟲的大型感染案例，則都是因為原蟲污染飲用水源，而淨水設備不能有效將其去除所引起。水體中梨形鞭毛蟲及隱孢子蟲的檢測方法包括三個步驟：水樣的收集與濃縮、原蟲與其他雜質顆粒分離以及原蟲的檢測。ICR原蟲分析法是美國所公告的第一個檢測梨形鞭毛蟲與隱孢子蟲之標準方法，但此法的原蟲回收率過低，一直為人所詬病，因此經改良的原蟲分析法- Method 1622及1623乃分別在1997與1999年由美國環保署提出。此二方法在水樣的收集與濃縮步驟中所用的方法，不但與ICR原蟲分析法不同，其在原蟲與雜質顆粒分離步驟也使用了磁性抗體法，因此Method 1622及1623可大幅改善原蟲回收率並降低偵測極限。由於國外已爆發多起由飲用水所引起之大型梨形鞭毛蟲症和隱孢子蟲症感染案例，因此藉由對飲用水體進行致病性原生動物及相關水質之含量檢測，以瞭解此二類原生動物在檢驗上與移除上的特性，進而採取必要之措施，以保障大眾健康，實有其必要性。 本論文首先乃探討原蟲分析過程中各種濃縮與純化方法之回收率，並檢測台灣地區大型水廠原水與清水以及山區簡易自來水中梨形鞭毛蟲與隱孢子蟲的含量以及相關之水質指標。此外水源區豢養動物之糞便檢體中原蟲存在情形以及比較兩種原種濃縮法應用在實際水樣採集時之優缺點，亦為本論文研究重點。而原蟲表面特性、原蟲過濾去除之碰撞效率、不同水體中原蟲含量與指標微生物之相關性以及本研究所選定用以評估水廠中原蟲去除率之取代物-小球藻(Chlorella)的表面特性分析與其碰撞效率，在本論文中亦有深入之討論。 本研究所使用的三種原蟲濃縮技術，以Envirochek及濾膜法的原蟲回收率最高。雖然濾管法最適合用於大水量的過濾，但此法易造成大量原蟲漏失。在流洗與離心過程，以Envirochek之震盪法及手指搓揉濾膜法原蟲回收率較高。而在原蟲純化步驟，IMS比Percoll-sucrose梯度澄清法的回收率高。在兩種原蟲的回收率比較上，可發現梨形鞭毛蟲回收率較隱孢子蟲高。 在探討IMS原蟲分離法於各種操作條件下之回收率實驗中可得知，純水中隱孢子蟲之回收率為82.6 ±18.2% (n = 52)，梨形鞭毛蟲的回收率為81.0 ±5.2% (n = 7)。當受測水樣之濁度升高時，對隱孢子蟲之回收率影響不大，但梨形鞭毛蟲之回收率則會明顯降低。增長IMS之反應時間以及提升磁性抗體之添加量都能夠有效提升原蟲回收率，而當IMS之反應在體積相差十倍之小離心管與Dynal公司特製之玻璃管中進行時，其原蟲回收率相差不大。 在本論文中，我們分別對24個取自11處水廠的原水與清水水樣以及26個取自25處簡易自來水之水樣進行原蟲存在性及其水質參數之分析。結果顯示，24個原水水樣中，梨形鞭毛蟲的平均含量為349.2 cysts/100 l，隱孢子蟲的平均含量為685.0 oocysts/100 l ; 24個水廠清水水樣中，梨形鞭毛蟲的平均含量為36.1 cysts/100 l，隱孢子蟲的平均含量為73.3 oocysts/100 l ;至於26個簡易自來水水樣，其梨形鞭毛蟲與隱孢子蟲的平均含量則分別為79.5 cysts/100 l與22.1 oocysts/100 l。統計結果顯示，此二類原蟲在水廠原水中之含量與濁度高低具有高度之相關性。在101個受測之糞便檢體中，有11個檢體對梨形鞭毛蟲呈陽性反應，有22個對隱孢子蟲呈陽性反應。此結果與水體中原蟲含量以及採樣地點進行比對後，可間接證明水源區之農業活動將會使水中原蟲含量升高。梨形鞭毛蟲與隱孢子蟲感染風險之估算結果顯示，飲用水若未經適當之處理，台灣地區將有爆發梨形鞭毛蟲症與隱孢子蟲症流行之虞。 以濾膜法及濾管法同時對台灣水廠之原水與清水進行檢測時可發現，雖然濾膜法比濾管法的原蟲回收率高，但是濾管法卻比濾膜法具較佳的偵測極限。當水樣濁度增加時，此二種分析方法的原蟲回收率皆會隨之下降，偵測極限值則會升高。 微生物的表面特性（表面電位與疏水性）將影響其在淨水程序中膠凝與吸附之機制。在pH值接近中性的狀況下，梨形鞭毛蟲孢囊與隱孢子蟲卵孢囊的界達電位值約分別為 -17 mV與 -38 mV。孢囊與卵孢囊在天然原水中之等電位點分別出現在 pH 2.2 及 pH 3.3。本研究中原蟲疏水性的測定方法乃使用MATH法。結果顯示，不但梨形鞭毛蟲與隱孢子蟲皆具疏水性，而且隱孢子蟲的疏水性較梨形鞭毛蟲強。此二類原蟲在pH值越低時，其疏水性越強。在本論文之原蟲過濾實驗中，乃以直徑2毫米的聚苯乙烯顆粒及玻璃顆粒作為填充濾材，用以探討原蟲過濾程序中之碰撞行為。實驗結果顯示，離子強度對於梨形鞭毛蟲與隱孢子蟲移除效率之提升很有幫助，而且原蟲移除效率會隨著pH值的增加而降低。此外，在相同的操作條件下隱孢子蟲的移除效率皆較梨形鞭毛蟲高。而梨形鞭毛蟲與隱孢子蟲在各操作條件下乾淨濾床碰撞係數之變化趨勢則與其移除效率的變化情形類似。 自然水體中的原蟲含量雖與部分的指標微生物具相關性，但此相關性會受水質型態及採集地點的影響。分析小球藻特性時可發現，於相同水質條件下，其所帶的負電位較隱孢子蟲少，因此在混凝過程隱孢子蟲將消耗較多的混凝劑。以MATH法檢測小球藻之疏水性時，其值雖然與隱孢子蟲之檢測值類似，但是在乾淨床碰撞實驗中，小球藻之碰撞效率值卻比隱孢子蟲所求得之值低，因此在水廠之過濾程序，隱孢子蟲應該會有較佳的移除率。

Protozoan parasites Giardia and Cryptosporidium have been recognized as common pathogenic protozoa of the gastrointestinal tract. Human infection with these pathogens is through either direct dermal contact or ingestion of contaminated food and/or water. Water is perhaps the major route for massive outbreaks of giardiasis and cryptosporidiosis. Because of many outbreaks of giardiasis and cryptosporidiosis in the last decade, there is an urgent need to understand the occurrence of protozoan parasites. Methods for detection of Giardia and Cryptosporidium in water samples involve three stages: sample collection and concentration; separation of protozoan parasites from other debris; and detection of parasites. The Information Collection Rule (ICR) protozoan method of USA, a fluorescent antibody procedure is the first standard method for detecting Giardia and Cryptosporidium in water samples. However, this method has been heavily scrutinized for its low recoveries. Method 1622 and 1623, improved concentration procedures by adopting the immunomagnetic separation (IMS), were expected to have higher recovery and lower detection limit. In this study, we studied recovery efficiencies of various filtration techniques accompanied purification methods for (oo)cysts, the occurrence of both parasites in Taiwan, the surface characteristics of (oo)cysts, and the removal mechanism in filtration. The reliabilities of indicator microorganisms to predict the occurrence of Giardia and Cryptosporidium and the microbial surrogate to determine the (oo)cysts removal at water treatment processes were discussed. Out of the three concentration techniques studied, the Envirochek capsule filtration followed by the membrane filtration consistently gave the highest recovery efficiencies for both protozoan parasites. Although the cartridge filtration is the most suitable technique for handling large quantities of water, it also brought the greatest loss of protozoan parasites. For elution and centrifugation, both membrane filter eluted with hand-kneading and Envirochek capsule with wrist-action shaking attained higher recovery efficiencies for (oo)cysts. IMS, the purification procedure in Method 1623, had higher recovery efficiency for both Giardia and Cryptosporidium than the Percoll-sucrose density gradient purification which was the flotation procedure of the ICR protozoan method. In general, Giardia cysts attained higher recovery efficiency than Cryptosporidium oocysts. The recovery efficiencies of Method 1623, due to the use of Envirochek capsule filtration combined with the IMS, were higher for both Giardia and Cryptosporidium, than the ICR protozoan method. IMS method was evaluated on the basis of recovery efficiencies of Giardia cysts and Cryptosporidium oocysts at various IMS operation conditions. The average recoveries for different concentrations of Cryptosporidium and Giardia in deionized water were 82.6 ±18.2% (n = 52) and 81.0 ±5.2% (n = 7), respectively. No significant change in Cryptosporidium recovery was observed by altering the debris ratio of water samples, while increased debris in water lowered the Giardia recovery. Prolonging the reaction time and increasing the amount of immunomagnetic beads improved the recovery for both parasites. IMS recoveries of (oo)cysts seeded in eppendorf with small reaction volume were similar with those seeded in glass tubes with ten times the reaction volume. Forty-eight raw and treated water samples collected from eleven large water treatment plants and twenty-six samples from small water systems were checked for the occurrence of Giardia and Cryptosporidium along with some water quality parameters. The indirect immunofluorescence assay (IFA) was used for the detection of cysts and oocysts in water samples. The average concentrations in 24 raw water samples were 349.2 cysts/100 liters for Giardia and 685.0 oocysts/100 liters for Cryptosporidium. The average concentrations in treated water samples were 36.1 cysts/100 liters and 73.3 oocysts/100 liters for Giardia and Cryptosporidium, respectively. For small water systems, the average concentrations in water samples were 79.5 cysts/100 liters for Giardia and 22.1 oocysts/100 liters for Cryptosporidium. The concentrations of these two parasites exhibit a significant correlation with each other and also with the turbidity of the raw water. Among the 101 fecal specimens, eleven were positive for Giardia and 22 were positive for Cryptosporidium. It was also found that the occurrence of these two pathogens in the source water was directly linked to the surrounding farming activities. Risk assessment for adverse human effects arising from the presence of cysts and oocysts in water indicated the possibility of waterborne transmission of Giardia and Cryptosporidium infection in Taiwan if water is not adequately treated. In this study, we also assessed the performances of the two concentration methods for water samples collected from Taiwan waterworks. The membrane filtration method was characterized by higher recovery efficiency, however, higher detection limit. Inconsistent results were obtained from two methods in respect to the occurrences of both parasites, and the relationships of parasite concentrations with indicator microorganisms. It was discovered that turbidity reduced the recovery efficiency, and raised the detection limits for both parasites regardless of the method used. Surface characteristics of microorganisms such as surface charge and hydrophobicity are involved in interfacial interaction of cells such as flocculation and adhesion. The calculated zeta potentials of Giardia cysts and Cryptosporidium oocysts in raw waters at neutral pH were in average -17 mV and -38 mV, respectively. The isoelectric points of cysts and oocysts were determined as pH 2.2 and pH 3.3, respectively. The hydrophobicities of cysts and oocysts were determined from the microbial adherence to hydrocarbons (MATH) test. Although the initial removal rates of oocysts were larger than those of cysts in all trials, both demonstrated marked hydrophobicity. Hydrophobicity maxima of cysts and oocysts were observed at the lowest pH value measured. Filtration experiments of Giardia cysts and Cryptosporidium oocysts with 2mm-Φglass beads and 2mm-Φpolystyrene beads were conducted to investigate the behavior of (oo)cyst during filtration. Experimental results indicated that ionic strength enhanced the removal efficiencies for Giardia cysts and Cryptosporidium oocysts significantly. The removal efficiency of the two filters for Giardia decreased slightly from pH 2.4 to pH 8.7 and decreased significantly in pH up to pH 8.7, while that for Cryptosporidium slightly rippled beyond pH 8.7, and with the decreased in pH up to pH 8.7. The experimental collision efficiencies for cysts and oocysts corresponded to the (oo)cysts removal efficiencies in all trials, and all the experimental collision efficiencies for oocysts were higher than those for cysts. The occurrence of protozoa in raw waters related to some indicator microorganisms, although the correlation might vary with sampling sites and sampling methods. The zeta potentials of Cryptosporidium in tap and raw waters were more negative than those of Chlorella. Although the hydrophobicities of Chlorella were similar with Cryptosporidium oocysts, the experimental collision efficiencies for Cryptosporidium oocysts were higher than Chlorella. It indicated that Cryptosporidium would consume more coagulants during coagulation, with higher removal rate in the filtration process than Chlorella.