Excystation Assays Research Articles

Cryptosporidium parvum inactivation from short durations of treatment with ozonated water produced by an electrolytic generation system.

Cryptosporidium is a waterborne pathogen that causes diarrhea in vertebrates and humans (mainly C. hominis and C. parvum). Ozone (O3) is a powerful disinfectant due to its high oxidative characteristics, and it is used to inactivate microorganisms in drinking water. As an alternative to the gas dissolution system for producing ozone from oxygen, a simpler electrolytic ozone generation system has recently been developed. In the present study, the efficacy of the ozonated water produced by this system in inactivating Cryptosporidium parasites (C. parvum) was evaluated at different current intensities (which change the ozone concentrations) and short exposure times (15-60s). Oocyst viability and integrity was assessed using vital dye staining, excystation assays, and scanning electron microscopy (SEM). SEM data revealed that oocyst walls were damaged by exposure to ozone molecules even at low concentrations (< 0.01mg/l for 1min) (current intensity 0.2 A), but that the excystation assay could not differentiate between deformed oocysts (dead) and partially excysted oocysts (alive). Exposure to ozonated water produced with a low current intensity (0.3 A) for 15 and 120s resulted in the inactivation of 96.2% (CT value < 0.003) and 99.4% (CT value < 0.020) of the oocysts, respectively. Thus, it was estimated that a CT value more than 0.020 was required to inactivate > 99% of the C. parvum oocysts. These results suggested that the electrolytic ozone generation system may be more effective than gas dissolution ozone generation; however, further studies using additional approaches are needed to obtain clearer evidence.

Potential anti-acanthamoebic effects through inhibition of CYP51 by novel quinazolinones

Acanthamoeba spp. are free living amoebae which can give rise to Acanthamoeba keratitis and granulomatous amoebic encephalitis. The surface of Acanthamoeba contains ergosterol which is an important target for drug development against eukaryotic microorganisms. A library of ten functionally diverse quinazolinone derivatives (Q1–Q10) were synthesised to assess their activity against Acanthamoeba castellanii T4. The in-vitro effectiveness of these quinazolinones were investigated against Acanthamoeba castellanii by amoebicidal, excystation, host cell cytopathogenicity, and NADPH-cytochrome c reductase assays. Furthermore, wound healing capability was assessed at different time durations. Maximum inhibition at 50 μg/mL was recorded for compounds Q5, Q6 and Q8, while the compound Q3 did not exhibit amoebicidal effects at tested concentrations. Moreover, LDH assay was conducted to assess the cytotoxicity of quinazolinones against HaCaT cell line. The results of wound healing assay revealed that all compounds are not cytotoxic and are likely to promote wound healing at 10 μg/mL. The excystation assays revealed that these compounds significantly inhibit the morphological transformation of A. castellanii. Compound Q3, Q7 and Q8 elevated the level of NADPH-cytochrome c reductase up to five folds. Sterol 14alpha-demethylase (CYP51) a reference enzyme in ergosterol pathway was used as a potential target for anti-amoebic drugs. In this study using i-Tasser, the protein structure of Acanthamoeba castellanii (AcCYP51) was developed in comparison with Naegleria fowleri protein (NfCYP51) structure. The sequence alignment of both proteins has shown 42.72% identity. Compounds Q1-Q10 were then molecularly docked with the predicted AcCYP51. Out of ten quinazolinones, three compounds (Q3, Q7 and Q8) showed good binding activity within 3 Å of TYR 114. The in-silico study confirmed that these compounds are the inhibitor of CYP51 target site. This report presents several potential lead compounds belonging to quinazolinone derivatives for drug discovery against Acanthamoeba infections.

Cationic Surfactant-Natural Clay Complex as a Novel Agent Against Acanthamoeba castellanii Belonging to the T4 Genotype.

Acanthamoeba is a protozoan pathogen that is widely distributed in the environment. Given the opportunity, it can cause a serious eye infection known as Acanthamoeba keratitis as well as a fatal brain infection known as granulomatous amoebic encephalitis. Inappropriate use of contact lenses can contribute to contracting Acanthamoeba keratitis, and contact lens disinfectants are not always effective in eradicating Acanthamoeba. Therefore, there is a need to develop novel antimicrobial agents with efficient antiamoebic properties. In this study, we tested octadecyltrimethylammonium (ODTMA)-clay (montmorillonite) complex as a novel antiamoebic agent. Using A. castellanii belonging to the T4 genotype of keratitis origin, amobicidal assays were performed to determine the effects of ODTMA-cay complex on the viability of parasites at various concentrations ranging from 10 to 100 μg. Adhesion and cytopathogenicity assays were performed to investigate ODTMA effects on A. castellanii-mediated binding and damage to human cells. Encystation and excystation assays were conducted to establish ODTMA-mediated inhibitory effects against the cyst stage of A. castellanii. Using cell survival assays, the results revealed that ODTMA-clay complex exhibited amobicidal activity against keratitis-causing A. castellanii in a dose-dependent manner. Pretreatment of A. castellanii with ODTMA-clay complex inhibited parasite adhesion to as well as parasite-mediated human cell damage. Using encystation and excystation assays, it was revealed that ODTMA-clay complex inhibited A. castellanii cysts at 100 μg (P<0.05). To the best of our knowledge, for the first time, it was shown that ODTMA-clay complex exhibited anti-Acanthamoebic activities. The possibility of adding ODTMA-clay in a contact lens cleaning solution to formulate effective disinfectants is discussed further.

Synthetic Dihydropyridines as Novel Antiacanthamoebic Agents.

Acanthamoeba is an opportunistic pathogen widely spread in the environment. Acanthamoeba causes excruciating keratitis which can lead to blindness. The lack of effective drugs and its ability to form highly resistant cyst are one of the foremost limitations against successful prognosis. Current treatment involves mixture of drugs at high doses but still recurrence of infection can occur due to ineffectiveness of drugs against the cyst form. Pyridine and its natural and synthetic derivatives are potential chemotherapeutic agents due to their diverse biological activities. To study the antiamoebic effects of four novel synthetic dihydropyridine (DHP) compounds against Acanthamoeba castellanii belonging to the T4 genotype. Furthermore, to evaluate their activity against amoeba-mediated host cells cytopathogenicity as well as their cytotoxicity against human cells. Dihydropyridines were synthesized by cyclic dimerization of alkylidene malononitrile derivatives. Four analogues of functionally diverse DHPs were tested against Acanthamoeba castellanii by using amoebicidal, encystation and excystation assays. Moreover, Lactate dehydrogenase assays were carried out to study cytopathogenicity and cytotoxicity against human cells. These compounds showed significant amoebicidal and cysticidal effects at 50 μM concentration, whereas, two of the DHP derivatives also significantly reduced Acanthamoebamediated host cell cytotoxicity. Moreover, these DHPs were found to have low cytotoxicity against human cells suggesting a good safety profile. The results suggest that DHPs have potential against Acanthamoeba especially against the more resistant cyst stage and can be assessed further for drug development.

Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and effects of conjugation with silver nanoparticles.

Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii. In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1H-tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti-Acanthamoeba effects at different concentrations ranging from 5 to 50μM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti-Acanthamoeba studies. Amoebicidal assay showed that at concentration 10μM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii. Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti-Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii. Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti-Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii. However, mechanistic and in vivo studies are needed to explore further translational values.

Repositioning of Guanabenz in Conjugation with Gold and Silver Nanoparticles against Pathogenic Amoebae Acanthamoeba castellanii and Naegleria fowleri.

Brain-eating amoebae cause devastating infections in the central nervous system of humans, resulting in a mortality rate of 95%. There are limited effective therapeutic options available clinically for treating granulomatous amoebic encephalitis and primary amoebic meningoencephalitis caused by Acanthamoeba castellanii (A.castellanii) and Naegleria fowleri (N.fowleri), respectively. Here, we report for the first time that guanabenz conjugated to gold and silver nanoparticles has significant antiamoebic activity against both A.castellanii and N.fowleri. Gold and silver conjugated guanabenz nanoparticles were synthesized by the one-phase reduction method and were characterized by ultraviolet-visible spectrophotometry and atomic force microscopy. Both metals were facilely stabilized by the coating of guanabenz, which was examined by surface plasmon resonance determination. The average size of gold nanoconjugated guanabenz was found to be 60 nm, whereas silver nanoparticles were produced in a larger size distribution with the average diameter of around 100 nm. Guanabenz and its noble metal nanoconjugates exhibited potent antiamoebic effects in the range of 2.5 to 100 μM against both amoebae. Nanoparticle conjugation enhanced the antiamoebic effects of guanabenz, as more potent activity was observed at a lower effective concentration (2.5 and 5 μM) compared to the drug alone. Moreover, encystation and excystation assays revealed that guanabenz inhibits the interconversion between the trophozoite and cyst forms of A.castellanii. Cysticdal effects against N.fowleri were also observed. Notably, pretreatment of A.castellanii with guanabenz and its nanoconjugates exhibited a significant reduction in the host cell cytopathogenicity from 65% to 38% and 2% in case of gold and silver nanoconjugates, respectively. Moreover, the cytotoxic evaluation of guanabenz and its nanoconjugates revealed negligible cytotoxicity against human cells. Guanabenz is already approved for hypertension and crosses the blood-brain barrier; the results of our current study suggest that guanabenz and its conjugated gold and silver nanoparticles can be repurposed as a potential drug for treating brain-eating amoebic infections.

Cobalt nanoparticles as novel nanotherapeutics against Acanthamoeba castellanii

BackgroundSpecies of Acanthamoeba are facultative pathogens which can cause sight threatening Acanthamoeba keratitis and a rare but deadly brain infection, granulomatous amoebic encephalitis. Due to conversion of Acanthamoeba trophozoites to resistant cyst stage, most drugs are found to be ineffective at preventing recurrence of infection. This study was designed to test the antiacanthamoebic effects of different cobalt nanoparticles (CoNPs) against trophozoites and cysts, as well as parasite-mediated host cell cytotoxicity.MethodsThree different varieties of CoNPs were synthesized by utilizing hydrothermal and ultrasonication methods and were thoroughly characterized by X-ray diffraction and field emission scanning electron microscopy. Amoebicidal, encystation, excystation, and host cell cytopathogenicity assays were conducted to study the antiacanthamoebic effects of CoNPs.ResultsThe results of the antimicrobial evaluation revealed that cobalt phosphate Co3(PO4)2 hexagonal microflakes, and 100 nm large cobalt hydroxide (Co(OH)2) nanoflakes showed potent amoebicidal activity at 100 and 10 µg/ml against Acanthamoeba castellanii as compared to granular cobalt oxide (Co3O4) of size 35–40 nm. Furthermore, encystation and excystation assays also showed consistent inhibition at 100 µg/ml. CoNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release without causing significant damage to human cells when treated alone.ConclusionsTo our knowledge, these findings determined, for the first time, the effects of composition, size and morphology of CoNPs against A. castellanii. Co3(PO4)2 hexagonal microflakes showed the most promising antiamoebic effects as compared to Co(OH)2 nanoflakes and granular Co3O4. The results reported in the present study hold potential for the development of antiamoebic nanomedicine.

Oleic acid-conjugated silver nanoparticles as efficient antiamoebic agent against Acanthamoeba castellanii.

Acanthamoeba castellanii belonging to the T4 genotype is an opportunistic pathogen which is associated with blinding eye keratitis and rare but fatal central nervous system infection. A. castellanii pose serious challenges in antimicrobial chemotherapy due to its ability to convert into resistant, hardy shell-protected cyst form that leads to infection recurrence. The fatty acid composition of A. castellanii trophozoites is known to be most abundant in oleic acid which chemically is an unsaturated cis-9-Octadecanoic acid and naturally found in animal and vegetable fats and oils. This study was designed to evaluate antiacanthamoebic effects of oleic acid against trophozoites, cysts as well as parasite-mediated host cell cytotoxicity. Moreover, oleic acid-conjugated silver nanoparticles (AgNPs) were also synthesized and tested against A. castellanii. Oleic acid-AgNPs were synthesized by chemical reduction method and characterized by ultraviolet-visible spectrophotometry, atomic force microscopy, dynamic light scattering analysis, and Fourier transform infrared spectroscopy. Viability, growth inhibition, encystation, and excystation assays were performed with 10 and 5μM concentration of oleic acid alone and oleic acid-conjugated AgNPs. Bioassays revealed that oleic acid alone and oleic acid-conjugated AgNPs exhibited significant antiamoebic properties, whereas nanoparticle conjugation further enhanced the efficacy of oleic acid. Phenotype differentiation assays also showed significant inhibition of encystation and excystation at 5μM. Furthermore, oleic acid and oleic acid-conjugated AgNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release. These findings for the first time suggest that oleic acid-conjugated AgNPs exhibit antiacanthamoebic activity that hold potential for therapeutic applications against A. castellanii.

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts.

Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 μg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 μg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 μg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.

Quantitative dielectrophoretic tracking for characterization and separation of persistent subpopulations of Cryptosporidium parvum

Microbial persistence to antibiotics is attributed to subpopulations with phenotypic variations that cause a spread of susceptibility levels, leading to the recurrence of infections and stability of biofilms. Herein, persistent oocyst subpopulations identified by animal infectivity and excystation assays during the disinfection of Cryptosporidium parvum, a water-borne pathogen capable of causing enteric infections at ultra-low doses, are separated and characterized by quantitative dielectrophoretic tracking over a wide frequency range (10 kHz-10 MHz). To enable the simultaneous and facile dielectrophoretic tracking of individual oocysts, insulator constrictions in a microfluidic channel are utilized to spatially modulate the localized field over the extent needed for defining oocyst trajectories and for obtaining high-resolution displacement versus time measurements under both, positive and negative dielectrophoresis. In this manner, by obviating the need for averaging dielectrophoretic data over a large collection region, the force response is more sensitive to differences in electrophysiology from sub-population fractions. Hence, the electrophysiology of sensitive and persistent oocysts after heat and silver nanoparticle treatments can be quantified by correlating the force response at low frequencies (<100 kHz) to the integrity of the oocyst wall and at high frequencies (0.4-1 MHz) to the sporozoites in the oocyst. This label-free method can characterize heterogeneous microbial samples with subpopulations of phenotypically different alterations, for quantifying the intensity of alteration and fraction with a particular alteration type.

Excystation Signals Do Not Isolate Gregarine Gene Pools: Experimental Excystation of Blabericola migrator Among 11 Species of Cockroaches

An experimental excystation assay was used to test the potential species isolating effects of excystation signaling among gregarines. Oocysts of a single gregarine species, Blabericola migrator , were tested for activation, excystation, and sporozoite motility by using intestinal extracts from 11 species of cockroaches representing a cohesive phylogeny of 7 genera, 3 subfamilies, and 2 families of Blattodea. Sporozoite activation, excystation, and motility were observed for all excystation assay replications using intestinal fluid from blaberid hosts, but delayed activation or excystation was observed for all assay replications using intestinal fluid from hosts in the family Blattidae. The results illustrate a trend toward a generalized excystation signal among gregarines that is conserved across the host clade at a subfamily or family level but that is unlikely to play a significant role as a species-isolating mechanism among sibling gregarine species.

Effect of vinegar on the viability of Giardia duodenalis cysts

The inactivation of Giardia duodenalis cysts by vinegar was investigated. Experiments were carried out in 100 ml volume of vinegar (acetic acid 4%), undiluted or diluted in distilled water in ratios of 1:1, 1:15.6, and 1:62.5 (vol/vol), which were inoculated with 5 × 10 5 cysts obtained from human feces. Experiments were performed at room temperature (21 ± 1 °C) and at 4 °C. After contact times of 1.5 min, 10, 30, and 60 min, the cysts were recovered from the treatment fluid and subjected to an in vitro excystation assay to determine their viability. The relative viability, which was calculated in relation to controls (maximum excystation percentage), was significantly affected ( p < 0.1) by the vinegar concentration, contact time, and temperature. At 21 ± 1 °C, no cysts remained viable after being treated with undiluted vinegar for 60 min, while the treatment with 1:1, 1:15.6, and 1:62.5 vinegar–water mixtures decreased the relative viability to 1.8%, 19.4%, and 56.4%, respectively. The relative viability after corresponding treatments at 4 °C also decreased, but 23.6% to 48.8% remained viable after 60 min, and thus complete inactivation was not obtained with any treatment at that temperature.

Host Specificity of Five Species of Eugregarinida Among Six Species of Cockroaches (Insecta: Blattodea)

Eugregarines are typically distinguished using characters, such as epimerite morphology, trophozoite morphometrics, timing of reproductive association, gametocyst morphology, and method of dehiscence. In some cases, new gregarine species have been recognized based solely on host associations, whereas other gregarine species have been reported from several host taxa. We used reciprocal cross-species experimental infections and in vitro excystation assays to evaluate the host specificity of 5 species of eugregarines among 6 species of cockroaches: Protomagalhaensia granulosae Peregrine, 1970, and Gregarina cubensis Peregrine, 1970, described from Blaberus discoidalis, (death's head cockroach); Leidyana migrator Clopton, 1995, described from Gromphadorhina portentosa (Madagascar hissing cockroach); and Protomagalhaensia wolfi (Geus, 1969) Hays and Clopton, 2006, and Leidyana haasi (Gues, 1969) Hays and Clopton, 2006 infecting Nauphoeta cinerea (lobster cockroach). Eugregarines have not been reported from the other species of cockroaches used in this study: Blaberus giganteus (giant death's head cockroach), Henschoutedenia flexivitta (giant lobster cockroach), and Diploptera punctata (Pacific beetle mimic). Experimental infections were produced in all homologous host–parasite combinations. With the exception of 1 host–gregarine combination, no infection was produced in heterologous reciprocal combinations. Excystation occurred in all instances when oocysts were placed in homologous host gut homogenate but excystation was never observed in heterologous host gut homogenate.

크립토스포리디움의 활성/감염성 판별법을 이용한 오존 및 자외선 소독능 평가

크립토스포리디움은 염소내성이 매우 강해 일반적인 표준정수처리공정의 소독으로는 제거가 불가능하다. 따라서 본 연구에서는 오존 및 UV를 이용한 단위소독공정에서 DAPI/PI 및 in vitro excystation을 이용하여 크립토스포리디움 불활성화를 평가하였으며, 또한 오존을 이용한 고도산화처리 파일럿에서는 세포배양법을 이용하여 크립토스포리디움 불활성화를 평가하였다. 오존 소독연구는 50 mL 용량의 piston type batch reactor에서 용존오존을 자동적으로 측정해주는 flow injection analysis (FIA) 시스템을 이용하여 실험한 결과, 1 log 제거에 필요한 CT값은 <TEX>$25^{\circ}C$</TEX>에서 DAPI/PI 및 in vitro excystation에 의해 각각 약 1.8, 2.2 <TEX>$mg/L{\cdot}min$</TEX>으로 나타났으며, 2 log 제거에 필요한 CT값은 각각 약 3.2, 3.8 <TEX>$mg/L{\cdot}min$</TEX>으로 나타났다. 또한 <TEX>$5^{\circ}C$</TEX>에서 크립토스포리디움 1 log 제거에 필요한 CT값은 DAPI/PI 방법에 의해 약 9.1 <TEX>$mg/L{\cdot}min$</TEX>으로 나타났으며, 2 log제거에 필요한 CT값은 14.8 <TEX>$mg/L{\cdot}min$</TEX>로 나타나, 같은 소독효과를 나타내기 위해서 저온에서는 상온에서보다 요존 요구량이 약 <TEX>$4{\sim}5$</TEX>배 정도 증가하여야 함을 확인하였다. 40 L규모의 오존 반응조를 이용한 파일럿 실험에서는 정수처리공정상 모래여과를 거친 물에 살아있는 크립토스포리디움을 접종한 것을 시료로 하여 연속적으로 흐르게 한 다음, 오존량을 변화시키고 체류시간은 5분으로 고정하여 불활성화를 평가하였다. 실험결과, 8 <TEX>$mg/L{\cdot}min$</TEX>의 CT값에서 DAPI/PI 및 excystation과 같은 생사판별법을 이용하였을 경우에는 약 0.2 log정도의 불활성화를 나타내었으며, 세포감염시험법을 이용하였을 경우에는 약 1.2 log정도의 불활성화를 나타냈다. 오존에 의한 크립토스포리디움의 소독능 평가에 단위공정 및 파일럿 실험 모두 2가지 생사판별법(DAPI/PI와 excystation) 사이에는 큰 차이를 나타내지 않았으나, 생사판별법과 세포감염시험법 사이에는 현저한 차이를 나타내었는데, 이는 세포감염시험법으로 측정하는 sporozoite 및 merozoite로의 분화과정이 생사판별법이 근거한 세포벽의 구조와 기능 유지 보다 더 오존 소독에 더 민감함을 알 수 있었다. 파일럿 실험에서의 CT값이 piston batch reactor에서의 CT값 보다 낮게 나타난 것은 파일럿 실험에서 수작업으로 인한 용존 오존 측정이 정밀하지 못하여 IOD가 농도에 반영되지 않았고, 반응조 규모(50 mL vs 40 L) 및 형태(회분식 vs 연속식)의 차이에 기인하는 것으로 여겨진다. 한편, UV를 이용한 단위공정에서는 크립토스포리디움 1, 2 log 제거에 필요한 IT값은 <TEX>$25^{\circ}C$</TEX>에서 각각 DAPI/PI 방법에 의해 약 25, 50 <TEX>$mWs/cm^2$</TEX>로 나타났으며, <TEX>$5^{\circ}C$</TEX>에서의 크립토스포리디움 1, 2 log제거에 필요한 IT값은 약 40, 80 <TEX>$mWs/cm^2$</TEX>로 나타났다. 온도 <TEX>$20^{\circ}C$</TEX> 감소 시 약 60% 정도의 IT값이 더 필요한데, 이것은 저온에서는 약한 자외선을 발산하는 저압저출력 UV 램프의 특성 때문인 것으로 사료되었다. In the ozone disinfection unit process of a piston type batch reactor with continuous ozone analysis using a flow injection analysis (FIA) system, the CT values for 1 log inactivation of Cryptosporidium parvum by viability assays of DAPI/PI and excystation were <TEX>$1.8{\sim}2.2\;mg/L{\cdot}min$</TEX> at <TEX>$25^{\circ}C$</TEX> and <TEX>$9.1mg/L{\cdot}min$</TEX> at <TEX>$5^{\circ}C$</TEX>, respectively. At the low temperature, ozone requirement rises <TEX>$4{\sim}5$</TEX> times higher in order to achieve the same level of disinfection at room temperature. In a 40 L scale pilot plant with continuous flow and constant 5 minutes retention time, disinfection effects were evaluated using excystation, DAPI/PI, and cell infection method at the same time. About 0.2 log inactivation of Cryptosporidium by DAPI/PI and excystation assay, and 1.2 log inactivation by cell infectivity assay were estimated, respectively, at the CT value of about <TEX>$8mg/L{\cdot}min$</TEX>. The difference between DAPI/PI and excystation assay was not significant in evaluating CT values of Cryptosporidium by ozone in both experiment of the piston and the pilot reactors. However, there was significant difference between viability assay based on the intact cell wall structure and function and infectivity assay based on the developing oocysts to sporozoites and merozoites in the pilot study. The stage of development should be more sensitive to ozone oxidation than cell wall intactness of oocysts. The difference of CT values estimated by viability assay between two studies may partly come from underestimation of the residual ozone concentration due to the manual monitoring in the pilot study, or the difference of the reactor scale (50 mL vs 40 L) and types (batch vs continuous). Adequate If value to disinfect 1 and 2 log scale of Cryptosporidium in UV irradiation process was 25 <TEX>$mWs/cm^2$</TEX> and 50 <TEX>$mWs/cm^2$</TEX>, respectively, at <TEX>$25^{\circ}C$</TEX> by DAPI/PI. At <TEX>$5^{\circ}C$</TEX>, 40 <TEX>$mWs/cm^2$</TEX> was required for disinfecting 1 log Cryptosporidium, and 80 <TEX>$mWs/cm^2$</TEX> for disinfecting 2 log Cryptosporidium. It was thought that about 60% increase of If value requirement to compensate for the <TEX>$20^{\circ}C$</TEX> decrease in temperature was due to the low voltage low output lamp letting weaker UV rays occur at lower temperatures.

Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4',6'-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.

Effect of Organic Acids and Hydrogen Peroxide on Cryptosporidium parvum Viability in Fruit Juices

Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 106 treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number–based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.

Decrease in Cryptosporidium parvum oocyst infectivity in vitro by using the membrane filter dissolution method for recovering oocysts from water samples.

Exposure of Cryptosporidium parvum oocysts to solutions used for cellulose acetate membrane (CAM) dissolution filtration reduced their infectivity in HCT-8 cells. Ethanol (95% [vol/vol] and 70% [vol/vol]) alone and short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.

The effect of temperature on the efficacy of ozonation for inactivating Cryptosporidium parvum oocysts

Examination of the effects of water temperature on the inactivation of Cryptosporidium parvum oocysts with ozone, ozonation experiments were conducted in a semi-batch mode with a wide temperature range of 3-30 degrees C. Inactivation was assessed in terms of mice infectivity and in vitro excystation. The temperature dependency of the CT products by a reduction in infectivity of 2 log10 could be described successfully by the Arrhenius equation, 1/CT = 1.086 x 10(18)e-12520/K where CT is the integrated ozone concentration over the contact time (mg/min/L) and K is the Kelvin temperature of water. As for the reduction in viability assessed by the excystation assay, protocol B, the obtained regression equation, 1/CT = 1.802 x 10(18)e-12640/K, was almost identical to that observed for the infectivity. Thus, the CT products required for a 2 log10 reduction in both infectivity and viability increased by an average factor of 4.2 for every 10 degrees C decrease in water temperature. Additionally, our findings suggested that the viability, as determined by protocol B, could substitute for animal infectivity in evaluating the effects of environmental factors on the efficacy of ozonation.

Effects of ozonation and chlorination on viability and infectivity of Cryptosporidium parvum oocysts

Experimental studies on ozonation and chlorination were conducted to determine capacity for inactivating Cryptosporidium parvum oocysts in batch modes at pH 7, 20°C. In both experiments, the log reduction of animal infectivity was linear and clearly decreased as disinfectant CT product increased. However, the curve of reduction in viability determined by both in vitro excystation assay and DAPI/PI permeability assay exhibited a shoulder. The CT products of ozone per 1 log reduction in infectivity were 3 mg middot min/L for 0.5 mg/L and 1.5 mg · min/L for 0.3 mg/L, while viability determined by in vitro excystation was reduced by only 0.2 logs for the CT product of 3 mg · min/L. In the chlorination experiment, thereduction of animal infectivity was up to 3 logs for the CT product of 2,700 mg middot; min/L, while reduction of viability was smaller at 0.16 logs in in vitro excystation and 0.04 logs in DAPI/PI permeability (in PI exclusion) for the same CT product. The CT product of free chlorine per 1 log reduction in infectivity was estimated to be in the range of 800 to 900 mg · min/L.

Intact Cryptosporidium parvum oocysts isolated after in vitro excystation are infectious to neonatal mice

In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.