Impact of sample collection and processing on Cryptosporidium parvum oocyst infectivity

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Impact of sample collection and processing on Cryptosporidium parvum oocyst infectivity

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Title:
Impact of sample collection and processing on Cryptosporidium parvum oocyst infectivity
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Coulliette, Angela D.
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Tampa, Florida
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University of South Florida
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English
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viii, 89 leaves : ill. ; 29 cm.

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Cryptosporidium parvum ( lcsh )
Disinfection and disinfectants ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF ( FTS )

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Thesis (M.A.)--University of South Florida, 2001. Includes bibliographical references (leaves 79-84).

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University of South Florida
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Universtity of South Florida
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028255477 ( ALEPH )
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F51-00155 ( USFLDC DOI )
f51.155 ( USFLDC Handle )

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IMP ACT OF SAMPLE COLLECTION AND PROCESSING ON CRYPTOSPORIDIUM PARVUM OOCYST INFECTIVITY by ANGELA D. COULLIETTE -" A thesis submitted in partial fulfillment of the requirements for the degree of Master of Sci ence Co lleg e ofMarine Science University of South Florida May 2001 Major Profes so r : Joan B. Rose, Ph.D

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Examining Committee : Office of Graduate Studies University of South Florida Tampa Florida CERTIFICATE OF APPROVAL This is to certify that the thesis of ANGELA D COULLIETTE in the graduate degree program of Marine Science was approved on March 20, 2001 for the Master of Science degree Member : E. Member : Valene J Harwood, Ph D Memltr: John H Paul III, Ph. b

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Table of Contents List ofTables m List of Figures v Abstract VI Chapter One: Review ofRelated Literaure 1 Introduction 1 Cryptosporidium parvum Biology 2 Cryptosporidium Outbreaks 3 Cryptosporidium Occurrence 4 Methods for Recovery and Detection of Cryptosporidium from Water 7 Recovery Methods for Cryptosporidium parvum 8 Detection Methods for Cryptosporidium parvum 11 Viability Methods for Cryptosporidium parvum 12 Cell Cu lture 13 Research Objectives 15 Chapter Two : Materials and Methods 18 Cryptosporidium parvum Oocysts Purification 18 Sterling Diagnostic Laboratory 18 Pleasant Hill Farms 19 Cryptosporidium parvum Oocysts Treatment 20 Bleach Treatment for Cell Culture 20 Antibiotic Treatment for Cell Cu lt ure 21 No Treatment for Cell Culture 21 Cryptosporidium parvum Aged Treatment Experiment 21 Cell Culture Foci Detection Method-Most Probable Number Program 22 Culture Media 22 Cell Maintenance 22 Cell Infection 23 Antibody Labeling 24 Enumeration 24 Cr y ptosporidium parvum Age Study 25 Cryptosporidium parvum Age Range Study 25 Cryptosporidium parvum Immunomagnetic Separation (IMS) Study 25 Dynal kit 25

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Hach kit 28 Clyptosporidium parvum Aged IMS Experiment 29 EPA Information Collection Rule (ICR) 29 EPA Method 1623 30 Environmental Samples 31 Normalizing Data 32 Chapter Three: Results 34 Cryptosporidium parvum Oocyst Treatment Study 34 Sterling Diagnostic Laboratory (SDL) 34 Pleasant Hill Fann (PHF) 35 Cryptosporidium parvum Aged Treatment Study 38 Sterling Diagnostic Laboratory (SDL) 38 Pleasant Hill Farm (PHF) 41 Cryptosporidium parvum Age Study 43 Sterling Diagnostic Laboratory (SDL) 43 Pleasant Hill Farm (PHF) 46 Cryptosporidium parvum Age Range Study 48 Sterling Diagnostic Laboratory (SDL) 48 Pleasant Hill Farm (PHF) 50 Cryptosporidium parvum IMS Study 53 Dynal 53 Hach 56 Clyptosporidium parvum Aged IMS Study 57 Dynal 57 Hach 59 EPA Information Collection Rule (ICR) 60 EPA Method 1623 62 Finished Water 62 Surface Water 64 Envirom11ental Samples 66 Chapter Four: Discussion and Conclusions 67 Preparation and Treatment of Oyptosporidium parvum Oocysts 67 Methods for Recovery and of Cly ptosporidium parvum Oocysts 72 Environmental Samples 75 Final Conclusions 76 References 78 Appendix A : Parameters for Experiments 84 11

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List ofTables Table 1 Data for Treatments Effects on SDL C. parvwn Oocyst Infectivity 34 Table 2 Treated SDL and PHF C. parvum Oocysts and the % of Wells Contaminated 34 Table 3 Data for Different Treatment Effects on PHF C. parvum Oocysts Infectivity 37 Table 4 Data for Treatment Effects on Aged SDL C. parvum 39 Table 5 Treated Aged SDL and PHF C. parvum Oocysts and the % of Wells Contaminated 39 Table 6 Data for Treatment Effects on Aged PHF C. parvum 41 Table 7 SDL C. parvum Oocyst Age Study Data 45 Table 8. PHF C. parvum Oocyst Age Study Data 47 Table 9. Data for Age Range SDL Oocysts 49 Table 10. Data for PHF Age Range 51 Table 11. Data for Dynal IMS Processed C. parvum Oocysts (Lot# 909 142 1 ) 54 Table 12 Data for Dynal IMS Processed C. parvum Oocysts (Lot# 00619-25) 54 Table 13 Data for Hach IMS Processed C. parvum Oocysts (Lot# 90914 21) 56 Table 14 Data for Dynal IMS Processed Aged SDL C. parvum Oocysts 57 Tabl e 15 Data for Hach IMS Processed Aged C. p arv um Oocysts 59 Table 16 Data for EPA ICR Processed C. parvum Oocysts in Surface Water 60 Table 17 D ata for EPA Method 1623 Processed C. parvum Oocysts in Surface Water 62 Ill

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Table 18 Table 19 Data for EPA Method 1623 Processed C. parvwn Oocysts in Surface Water Ooc yst Detection by IFA and Cell Culture FDM-MPN Data for Envirorunental Samples I V 65 66

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List of Figures Figure 1. Treatment Effects on SDL C. parvum Oocyst Infectiv it y 33 Figure 2. Treatment Effects on PHF C. parvum Oocyst Infectivity 36 Figure 3. Treatment Effects on Aged SDL C. parvum Oocyst Infectivity 38 Figure 4 Treatment Effects on Aged PHF C. parvum Oocyst Infectivity 41 Figure 5. Effects of Aging on SDL C.parvum Oocyst Infectivity 44 Figure 6. Effects of Age on PHF C. parvum Oocyst Infectivity 46 Figure 7. Age Range Averages of Aging SDL C. parvum Oocyst Infectivity 48 Figure 8. Age Range Averages of Aging PHf C. parvum Oocysts Infectivity 50 Figure 9. Effects of the Capture / Disassociation Steps in Dynal IMS Kit on SD L C. parvum Oocyst Infecti vty 53 Figure 10. Effects of the Disassociation Step in the Hach IMS Kit on SDL C.parvum Oocyst Infectivity 55 11. Effects of the Disassociation Step in Dynal IMS Kit on Aged SDL C. parvum Oocyst Infectivity 57 Figure 12. Effec ts of the Disassociation Step in the Hach Kit on Aged SDL C. parvum Oocyst Infectivity 58 Figure 13. Effects of EPA ICR on C. parvum Oocyst Infectivity in Surface Water 60 Figure 1 4. Effects ofEPA Method 1623 on C. parvum Oocyst Infectivity in Finished Water 62 Figure 15. Effects of EPA Method 1623 on C. parvum Oocyst Infectivity in Surface Water 64 v

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IMP ACT OF SAMPLE COLLEC TION AND PROCESSING ON CR YPTOSPORIDIUM PAR VUM OOCYST INFECTIVITY b y ANGELA D. COULLIETTE An Abstract of a thesis submi tted in partial fulfillment of the requirements for the degree of Master of Science Co ll ege ofMarine Science University of South Florida May 2001 Major Professor: Joan B. Rose, Ph.D. VI

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Cryptosporidium parvum has become the focus for water testing due to numerous waterborne outbreaks and the potential danger to the public Oyptosporidium recovery and detection methods have been evaluated for recovery efficiencies, as well as providing insight on Cryptosporidium outbreaks and the occurrence of oocysts in surface and ground water. However, these methods do not determine infectivity and whether the oocyst preparation has an effect on the physical integrity and infectivity of the C. parvum oocyst sample processing has yet to be reported This research used the cell culture FDM-MPN assay to evaluate oocyst viability Since laboratory prepared oocysts are used for recovery efficiencies and other experiments, the preparations vendors follow to purify oocysts from feces treatments to sterilize oocysts prior to cell culture, and how oocysts age over time were assessed The effects of environmental monitoring techniques, such as IMS ICR, and Method 1623 on laboratory prepared C. parvum oocysts were then evaluated Two vendors that prepare oocysts for laboratory use were evaluated Sterling Diagnostic Laboratory (SDL) oocysts needed bleach as an anti-microbial agent for oocysts prior to cell culture treatment; otherwise the cell culture system became contaminated. The variability between SDL l ots was small after they were treated Pleasant Hill Farms (PHF) oocysts were less likely to be contaminated and therefore did not require treatment. Yet PHF had a large variability in infectious oocysts between lots Blea c h appeared to be the best choice as an anti-microbial agent and enhanced excystation in the cell culture system as compared to antibiotics and no treatment. SDL oocysts were significantly different in infectivity at 1 to 30 days compared to 60 to 90 days old PHF oocysts showed no significant differences in Vll

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infectivity between ages, which may be an artifact of the large variability of infectious oocysts Dynal and Hach immunomagnetic separation (IMS) kits had no effect on oocyst infectivity and when the acid disassociation step was eliminated, the beaded samples proved compatible with the cell culture FDM-MPN in seeded samples Seeded surface water was processed by EPA Method 1623 and no effects were observed on C. parvum oocyst infectivity Limited assessment of surface and treated wastewater in Tampa Bay showed no infectious C. parvum oocysts Sterling Diagnostic Laboratory (SDL) when bleach treated was the vendor of choice due to the small variability between oocyst lots. Bleach provided enhanced excystation of C. parvum and assured minimal contamination in the cell culture system, therefore was determined the most efficient treatment. C. parvum oocysts appeared to decrease in infectivity after 30 days in both ve ndors however a sig nificant difference was found between 1 to 30 and 60 to 90 day oocysts in SDL oocysts Using oocysts approximately 30 days old and no older than 60 days i s suggested for maximum infectivity in cell culture IMS kit s evaluated and surface water samp les processed by EPA Method 1623 had no effect s on C. parvum oocyst infectivity The limited surfac e water samples processed by EPA ICR and finishe d water sam ples processed by EPA Method 1 623 suggest that these recovery method s do not have an effect o n infecti v ity but further samples should be conducted Abstract Approved: Date Approved : -=---1-'--".._...=:::..:....:: ....__..=-,_,___ VIII

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Chapter One: Review of Related Literature Introduction Cryptosporidium parvum has become the focus of many studies on waterborne di sease and transmission in response to outbreaks endangering the immunocompetent and immunocomprornised populat io ns worldwide. Assessing the viability of the oocy st (environmental stage) ha s become an important topic Cell culture is an efficient tool that can us ed for C. parvum infection studies. The cell lines showing the greatest affinity for C. parvum infection, the most appropriate media and culture conditions and optimal infectivity conditions for in vitro infection of C. parvum oocysts have been reported (Upton et al1994a and 1994b) The Foci Detection Method-Most Probable Number Assay (FDM-MPN) is a n in vitro cell culture method that has been developed and utili z ed to determine the quantity of infectious oocysts in a sample (Slifko et al 1997 1999). While present techniques detect the presence of oocysts from raw and treated water, a standard procedure using cell culture to detect infectious and viable oocysts has not been accepted. There is currently a need to study the variability and the heterogeneity of the cell culture system for measuring and understanding the infectivity of oocyst preparations or populations 1

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Cryptosporidium parvum Biology Cryptosporidium parvum is an enteric protozoan parasite that is transmitted by the fecal-oral route. Humans have contracted Cryptosoridiosis by foodbome, waterborne, person-to-person and surface transmission pathways (Fayer et al. 2000). Th i s protozoan is classified in the phylum-Apicomplexa, classSporozoasida, subclass-Coccidiasida, orderEncoccidiorida, suborderEimeriorina, and familyCryptosporidiidae (Fayer and Ungar 1986) Cryptosporidium parvum is an obligate intestinal organism and has reportedly infected over 152 mammalian hosts including humans (Fayer et al. 2000). At least two genotypes been confirmed for C. parvum, a human genotype (GI) and a genotype which infects cattle, mice, goats, lambs, horses, and humans (Gil) (Peng et al. 1997). There are ten (1 0) reported species in the genus Cryptosporidium, which have reportedly crossed species barrier (Fayer et al. 2000) Two separate reports document several Cryptosporidium species crossing species barriers in patients with HN. They had been diagnosed having C. parvum genotype I and ll, C. felis, C. melagradis and a "new" dog genotype (Pieniazek et al. 1999, Morgan et al. 2000). The life cycle of C. parvum oocysts includes sexual and asexual reproductive stages (Current 1987). Thick walled oocysts (environmental stage) containing 4 sporo z oites are excreted in the feces of an infective individual. Upon ingestion by a suitable host, the oocysts undergo a process known as excystation in the small intestine. The acidic environment and warm temperature cause the oocyst to re l ease the infect i ous life stage, the sporozoite. The sporozoites attach to microvilli on enterocytes. The sprorozoites move into a parasitophorous vacuole within the cell membrane but outside 2

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the cell. Once inside these vacuoles, the sporozoites produce either a type I meronts (asexual) or type II meronts (sexual) Type I meronts have 6 to 8 merozoites that infect nearby cells and produce additional type I meronts. Type II meronts with 4 merozites develop into the sexual stages. The type II meront produces either a macrogamont with macrogametes inside or a microgamont with microgametes inside. The released microgametes fertilize the macrogametes to form zygotes. Approximately 80% of zygotes form thick-walled oocysts that are passed into the hosts feces, while the other 20% form thinned-walled oocysts that excyst within thereby repeating infection. The oocysts are the environmental stage of the organism and are excreted in the feces The oocyst has a two-layered outer wall, which provides protection once it is excreted Cryptosporidium Outbreaks Waterborne outbreaks ofCryptosporidiosis between 19 84 and 1 999 have been reported 9 times in drinking water and 12 times in recreational waters (Smith and Rose 1997, Sorvillo et al. 1992 Moore et al. 1993 MacKenzie et al. 1995, Bongard et al. 1994, Kramer et al. 1998, Wilberschied 1 995, Reagan et al. 1996, Hopkins et al. 1 997, CCN 1996a, CCN 1997, CCN 1998a, CCN 1998b CCN 1998c). The waterborne outbreaks associated with drinking water occurred in Bexar Co unty, Texas in 1984 with an estimated 2000 cases; Carrol County, Georgia in 1987 w ith an estimated 13,000 cases; Berks County, Pennsylvania in 1991 wi th an estimated 551 cases; Jackson County, Oregon in 1992 with an estima ted 15, 000 cases; Milwaukee County, Wisconsin in 1993 with an estimated 403,000 cases; Cook County, Minnesota in 1993 with 27 cases; Yakima County, Washin g ton in 1993 with 10 cases; Walla Walla County, Washington in 3

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1994 with 86 cases; and Clark County, Nevada in 1994 with 103 cases (Solo-Gabriele et al. 1996; Roefer 199 6; Fox et al. 1996; Rose 1997; Craun et al. 1 998). The Georgia, Oregon Wisconsin, and Nevada (presumed) water treatment plants processed raw water from surface water sources (lakes, rivers), in which contamination occurred from agriculture and/or environmental runoff. At the time of the outbreaks the raw water was treated by, but not limited to, the following steps: coagulation, flocculation, rapid mixing, se dimentation, dual-media filtration and post-disinfection steps (Solo-Gabriel et al. 1 996; Roe fer 19 96; Fox et al. 1996). One or more of the above steps were found sub-optimal during inv estigations following the outbreaks (Solo Gabriele et al. 1996; Roefer 1996; Fox et al. 1 996). The remaining five drinking water outbreaks in Minnesota Pe1msylvania, Texas, and Washington (two outbreaks) had groundwater s ources (we ll s). The wells were contaminated by surface water runoff or influence from human sewage / septic tank effluent (Solo-Gabriele et a l. 1 996). The treatment steps for the raw water at the time of the outbreaks consisted of minimal treatment or none at all. The treatment at the time of outbreaks in Minnesota was pressured filtration and chlorination, in Pennsylvania and Texas the treatment was chloi-ination, and in both Washington outbreak s there was no treatment (Solo-Gabriele et al. 1996). Cryptosporidium Occurrenc e Ground waters, surface waters, and open reservoirs are a ll sources of drinking water; any of which may be contaminated with Cryptosporidium. For groundwater, 4

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Rose (1991) reported 5.6% occurrence rate ofoocysts; while Hancock et al (1998, 2000) reported 11% occurrence of oocysts in 1998 and 2000 studies. LeChevallier and his colleges (1991) studied surface waters for Cryptosporidium oocysts and reported that 87% of samples from 66 treatment plants in 14 U.S states and I Canadian province were positive for the parasite with a geometric mean of2. 7 oocysts/L (0.07 184 oocysts / L range). In a follow up study, 51.5% ofthe samples from 72 treatment plants in 15 U S states and 2 Canadian provinces were positive for Oyptosporidium oocysts with a geometric mean of2.4 oocysts / L (0.06565.1 oocysts /L range) (LeChevallier et al 1995). The samples had observable sporozoites detected in 54% of the oocysts (LeChevallier et al 1995) The difference in oocyst occurrence between the 1991 and 199 5 studies was hypothesized to be due to cyclic variations (environmental or seasonal) (LeCevallier et al. 1995). Based on numerous studies, the occurrence of Cryp to spor idium oocysts in s urface waters ranged from 9.1 to 1 00 % occurrence in pristine to non-pri s tine watersheds (Rose 1997). The geometric means in non-pristine watersheds ranged from 0.01 to 5800 oocysts / L (0 58 to 1920 oocysts / L range) and in pristine watersheds was 0.003 to 0.29 oocysts / L (Rose 1997). Marine waters are often influenced b y wastewa ter discharges located within a clos e proximity of recreational beaches and shellfish beds (Rose et al. 1991, Gomez Bautista et al. 2000). Johnson et al. (1995) reported that marine waters at a sewage outfall had 0 02 to 0.44 oocyst/L. Infectious oocysts were detect e d by immunofluorescence, animal infectivity and PCR-RFLP assay in environmental san1ples of blue mussels and cockles at a dose of 5 x 103 I mussel or cockle; which is potentially dangerous to the immunocompetent in Gallaecia (northwest Spain) (Gomez-Bautist e t al. 5

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2000). Infectious C. parvum oocysts have been recovered from freshwater clams, marine eastern oysters, and mussels (Gomez-Bautist et al. 2000). Oocysts will filter through the shellfish within 48 hours so a depuration time of 72 hours would allow for the consumption of non-contaminated shellfish (Gomez-Bautist et al. 2000). Treated drinking waters samples were also evaluated for Cryptosporidium oocyst in the 1991 and 1995 LeCevallier studies Twenty-seven percent of the drinking water samples in the 1991 study contained Cryptosporidium oocysts [82 samples from 66 treatment plants (14 U.S states and 1 Canadian province)] with a geometric mean of 0.015 oocysts/L (0.001 0.48 oocysts/L range) (LeChevallier et al 1991) All oocysts appeared non-viable based on detectable sporozoites and no documented illnesses were associated with the systems evaluated (LeCevallier et al. 1991). In the 1995 study, 13.4% of 72 treatment plants in 15 U.S. states and 2 Canadian provinces were positive for Cryptosporidium oocysts with a geometric mean of3.3 oocysts/L (0.029 57 oocysts / L range) (LeChevallier et al. 1995). Sporozoites were observed in 35% of the oocysts during the 1995 study (LeChevallier et al. 1995). Other reports have shown that treated drinking waters sampled (158) 3.8 to 33.3 % were positive for Cryptosporidium with a range of0.001 to 0.48 oocysts / L (Rose et al. 1997). These findings suggest that Cryptosporidium oocysts will be present in drinking water despite treatment plants efforts. A few questions needing to be answered are determining which of the species are present, and if C. parvum oocysts are the dominant species are they infectious ? Open reservoirs consist of a protected surface water storage area where treated water is sent and stored until needed. LeChevallier et al (1997) evaluated the occurrence of Cryptosporidium in water samples from the inlet and effluent taps of six open 6

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reservoirs. Fifteen percent of the samples were positive for Cryptosporidium oocysts at the inlet with a geometric mean of 1.2 oocysts / 1 OOL (0. 71 2.4 oocysts/ 1 OOL range) while th e effluent had 25% occurrence with a geometric mean of 8.1 oocysts / 1 OOL (1. 7-31 oocysts / 1 OOL) (LeChevallier et al. 1997). This indicates that despite the reservoirs being protected from human fecal matter, contamination occurs from agricultural and environmental sources. However, the percentages of oocysts present in the effluent samp l es are similar to the numbers found in treated drinking water stud ies (LeChevallier et al. 199 5, Rose et al. 1997). Methods for Recovery and Detection of Cryptosporidium from Water Over the last severa l decades numerous protocols have been developed for recovery and detection of oocysts from water. In addition, s tudies on disinfection and oocyst viability have been undertaken All of these research s tudi es and development of m ethods have relied upon laboratory preparations of oocysts. Factors such as oocyst age, animal source, oocyst collection and purification techniques for preparation have not been evaula ted The importance of these factors has been highlighted for all research protocols and it has been suggested that a common and consistent method for oocyst preparation be u sed. When Cryp to sporidum parvum emerged as a problematic waterborne pathogen, methods were rapidly developed in an effort to identify contaminated waters. Techniques to detect C. parvwn in drinking, ground, waste, and other waters were developed from methods derived from Giardia investigations (Jakubowski 2000). Methods utili z in g the organisms' s i ze, shape, weight density, antigens and DNA h ave emerged as techniques 7

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to detect this pathogen. While most methods are able to recover, detect or parti ally characteri z e C. parvUJn, they have not been able to determine the infectious nature of the oocyst. Recovery Methods for Cryptosporidium parvum Filtration is the most common method for recovering waterborne parasite such as Cryptosporidium. Two of the more common filters utilized in U.S. Environmenta l Protection Agency (EPA) protozoan detection methods are the 1 .0um nominal porosity yam-wound polypropylene cartridge filter and the 1.0-um absolute polyethersulphone membrane capsule (EPA 1995, EPA 1999). The yam-wound filter was the choice for the USEP A Information Collection Rule (ICR), while the filter capsule ha s been described in USEPA Method 1622 and 1623 (EPA 1 995, EPA 1999). The capture-recovery procedure for C. parvum oocysts oocysts using the USEPA ICR method is as follows: (1) water is passed through the yam-wound filters using water pumps or attachment directly to tap at a given flow rate (2) the filter is then cut apart, eluted with detergents and mechanically extracted with a stomacher, (3) th e eluent is concentrated by ce ntrifugation, ( 4) Percoll-Sucrose density gradients are used to clarify the sample, (5) the sample is stained using monoclonal antibodies tag ge d with FITC known as immunoflourescence assay (IFA) staining, and (6) epifluorescence microscopy (200X) of the characteristic Cryptosporidium apple-green fluorescence approximately 4 to 6 urn with confirmation under differential interference contrast (DIC) microsco p y (EPA 1995). 8

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USEP A ICR was implemented in May of 1996 for a year of sampling (Pontius et al. 1996, EPA 1994) in effort to monitor waterborne parasites and to de ve lop regulations. Clancy et al (1994) conducted a blind study of those laboratories involved in the year of ICR sampling to evaluate the efficiency and accuracy ofthe method. From the 16 l a boratories studied, low recoveries, poor precision and false negatives and positives resulted when using the USEPA ICR (Clancy et al. 1994). The water industry was concerned with the poor results and made improvements to the ICR protocol. A supplemental survey was conducted over a yea r time period to evaluate the improvements and also to evaluate the newl y developed EPA Method 1622. The survey revealed s imilar results as the last study despite the improvements. However, EPA Method 1622 had much better results (Connell et al 2000). Method 1622 is for the detection of Cryptosporidium, while Method 1623 incorporates Giardia as well. This research focused on Cryptosporidium, but actually utilized Method 1623 developments. EPA Method 1623 follows these basic steps: ( 1) sample collection using the filter capsule, (2) captured material is eluted from the filter using detergents and mechanical extraction with a wrist shaker, (3) the sample is concentrated with centrifugation, (4) oocysts are captured by iron beads coated with antibodies using an immunomagnetic separation (IMS) kit, (5) the samples are stained on well slides with fluore scen tly labeled monoclonal antibodies (FA) and DAPI ( 4' ,6-d ian1idino-2-phenylindol e ), and ( 6) microscopic evaluation by epifluorescence (200X) of the characteristic Cryptosporidium apple-green fluorescence approximately 4 to 6 urn, as well as the observation of 1 to 4 nuclei appearing blue with confinnation under DIC (EPA 1999). 9

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lmmunomagnetic separation (IMS) uses iron-tagged beads with antibodies and a magnetic field to isolate and concentrate an organism from an environmental sample (Jakubowski 1996). IMS advantages include removing more debris than earlier methods and allowing larger volumes to be processed IMS recoveries :r:anged from 25.4% in backwash water, 57.3% in raw water, to 94.3% in deionized water (DiGiovanni et al. 1999). However, these recoveries were not statistically higher than flotation techniques (DiGiovanni et al. 1999) IMS recoveries were shown to be similar for < I month (fresh) and 2 to 6 month (aged) old oocysts (Rochelle et al. 1999) and for 10 to 16 days (fresh) and 6 to 12 weeks (aged) old oocysts (Bukhari et al. 1998) Several commercial kits are available which have different bead sizes, magnet shapes, and disassociation steps; however the kits accepted by the EPA for incorporation into environmental monitoring procedure are Dynal and ImmuCell (EPA 1999). IMS has proven to be an efficient method in reducing background debris but consideration must be taken when choosing a kit due to varying recovery efficiencies The magnet shape and rotary action, bead size and antibody and the existence of a separation step all relate to recovery rates of the IMS kits (Bukari et al. 1998 Rochelle et al. 1999) USEP A Method 1622 proved to be a significant improvement in the detection of Cryptosporidium (Clancy et al1999). USEPA Method 1622 had higher recoveries, w hich ranged from 1.1 ( 1.7) to 35.3 ( 13) % in six performance evaluation trials (Clancy et al. 1999). EPA Method 1623 had reproducible results no false positives or negatives reporte d, and was faster and easier than USEPA ICR (Clancy et al. 1999 ). USEP A Method 1622 improvements over USEP A ICR may be attributed to sample collection and purification procedures (Connell et al. 2000) 10

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Detection Methods for Cryptosporidium parvwn Microscopic based methods are most commonly used for detection of C. parvum oocysts The immunofluorescence assay (IF A) method involves incubating the sample with antibodies directed against the antigenic components of C. parvum outer oocyst wall, and then using a secondary antibody that is fluorescently labeled (Jakubowski 1996). This method allows for detection and enumeration of the parasite. Antibody based methods, such as IF A, depending on the antibody specificity, can cross-react with other microorganisms (Fayer et al. 2000). Therefore, criteria of shape, size, and internal features are also used for identification. DAPI ( 4 ,6-diamido-2-phenylindole) is a general stain specific to DNA, while used to stain oocysts' 4 nuclei of the sporozoites inside Cryptosporidium oocyst, can cause a high level of background staining (Coming 1996) Viable and non viable oocysts will allow DAPI through their membrane, therefore DAPI detects presence of intact organisms and the potential for viability, but does not n ecessari ly reflect infectivity (Korich et al. 1997) Polymerase chain reaction (PCR) and reverse transcriptas e PCR (RT-PCR) are molecular methods which amplify DNA and RNA in an effort to detect low concentrations of Cryptospordium oocysts in water samples (Jakubowski 1996). Current molecular methods rely on t argeti ng undefined ge nomic fragments, small subunit ( 18S) rRNA gene, or the heat shock protein (hsp70) (Ko z wich et al. 2000) These molecular methods allow detection to the species level and genotype level, which aids in s pecifically identifying C. parvum, the pathogenic species for humans (DiGiovanni et al. 1999, Rochelle et al. 1997, Kozwich et al. 2000) 11

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Viability Methods for Cryptosporidium parvum Excystation is the release of the 4 internal sporozoites from the oocyst. Excystation naturally occurs when Cryptosporidium oocyst passes through the stomach and enters the intestinal track. This can be accomplished in-vitro by incubating the oocysts in excystation fluids (bile salts and trypsin at 37C) to excyst the sporozoites, the infectious life stage. In-vitro excystation is measured microscopically by counting oocysts (empty and full) and sporo z oites. Therefore, high numbers of oocysts are required for this infectivity assay (1.0 x 105 or more). This method is fairly inexpensive and is not too labor intensive except for the microscopic counting, however, purified and concentrated oocyst samples are needed. Fluorogenic dyes involve the staining of organisms' internal material to distinguish viability (Jakubowski 1996). The exclusion or inclusion of vital dyes depends on the physiological condition of the oocyst. Propidium iodide (PI) is frequently used to determine the viability of Cryptosporidium oocysts. Damaged or impaired membranes incorporate the stain which intercalates with nucleic acids forming a bright red fluorescent complex (Corning 1996 Sauch et al. 1991). Syto-9 stains dead oocysts green with viable oocysts having a green halo, while Syto-59 stains dead oocysts red and live oocysts remain unstained (Korich et al. 1997) Animal infectivity has been used for many years for the determination of C. parvum infectivity. The most common animal used is the neonatal mouse (Lindsay 1997). Animal infectivity assays have significant limitations including: (1) animals of young age required(< 5 days), (2) large numbers of animals are required, (3) the assay 12

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has a high cost, (4) and there is reportedly a high variability in results (Korich et al. 1997). RT -PCR has been proposed to detect infectious Cryptosporidium based upon the amplification of m.RNA. Kozwich (2000) has developed a novel integrated detection method with the Xtra Bind Capture System, which eliminates the tedious steps previously used for isolation and purification of C. parvum This system uses a solid phase material to extract and purify double stranded RNA, combined with a lateral flow detection protocol for positive results implied with the appearance of a colored line (Kozwich et al. 2000). This new RT-PCR method allows for larger volumes to be evaluated without the problems associated from debris or particulate inhibition (Kozwich et al. 2000) previously encountered using earlier methodologies. Cell Culture In an effort to study the biology of Cryptosporidium, researchers investigated cell culture as a tool for in vitro applications. Upton et al (1994a, 1994b) examined 11 cell lines and found that the human ileocaecal adenocarcinoma (HCT-8) cell line was superior in supporting the parasites' growth. RPM! 1640 medium was used for the metabolic needs of Cryptosporidium in a suitable atmosphere of 19.6% oxygen, 5.8% C02 73.7% N2, and 0.9% other gases. This environment can be provided by a 5% C02 / 95% air incubator (37C) (Upton 1994a) Cell culture supports life stages including meronts (type I and II), merozoites macrogametes, microgametes, and microgamonts but does not allow for the production of oocysts. 13

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Slifko et al (1997, 1999) first investigated an in vitro cell culture assay using HCT -8 cells to study the infectivity of Cryptosporidium oocysts from environmental samples The foci detection method-most probable number (FDM-MPN) assay is based on a dilution series (5or 10-fold) of oocysts which are prepared in 8-well chamber slides and then plated in replicate on a monolayer ofHCT-8 cells Excystation occurs directly on the monolayer with sporozoites infecting the HCT-8 cells The infected cells are incubated for 48 hours, as this allows for auto-reinfection and subsequent developmental stages (Slifko et al. 1997). The life stages (sporozoites, trophozoites, meronts (type I and II), merozoites, macrogametocyte, microgametocyte, and microgamete) are labeled by an indirect-antibody assay consisting of a primary antibody, anti-sporozoite IgG serum antibody prepared in rats; followed by a secondary fluorescein isothiocyanate antibody (FITC) which labels the Anti-Rat IgG antibody (Slifko et al. 1997). The life stages are viewed under epifluorescence microscopy and confirmed under Nomarski's differential contrast m i croscopy (Slifko et al. 1997). The presence or absence of foci clustering is noted for each dilution, which can be enumerated by the computer MPN program (Klee 1999) The FDM-MPN assay has proven to be an efficient tool in determining the infectivity of Cryptosporidium and can be used in conjunction with disinfection and survival studies or in conjunction with the U.S. EPAs' mandated monitoring requirements. DiGiovanni et al (1999) developed an assay combining cell culture with the genetic detection of Cryptosporidium parvum heat shock protein (hsp70) 70 gene. Heat shock proteins play a major role during oocyst infection of host cells. This makes hsp70 an ideal target due to the large quantities ofhsp mRNA produced during reproduction 14

PAGE 25

(Rochelle et al. 1997). RT-PCR detects all hsp rnRNA, which can be variable depending on the parasites robustness, whi l e ce ll culture PCR (CC-PCR) method targets the sing l e copy ofhsp70 per C. parvum genome (DiGiovanni et al. 1 999) Other targets in molecular based assays associated with C. parvum have been the small subunit (18S) rRNA gene and other genomic fragments with unknown functions (Kozwich et al. 2000). Research Objectives Cell culture has provided a great tool for analyzing the infectivity of Cryptosporidium parvum oocysts. Sterility is important in cell culture so as to not introduce foreign microbes particularly bacteria and fungi, which can cause contamination. Purification of oocysts from cal f feces and treatment of oocysts before inoculation into the host cell monolayer are two vital sterilization points. Cryptosporidium oocysts are usually cultivated in calves of age 2-9 days. The oocysts are collected from the infected calf feces and purified using either cesium chloride gradients or diethyl ether and sucrose gradients prior to storage in PBS with antibiotics. After the purification step, the oocysts are treated with antimicrobial agents, such as a n tibiotics and sodium hypochlorite (bleach), before inoculation int o the host cell monlayer. These purification methods, in combination w ith treatment procedures, may hav e detrimental impacts on the biological or physiological integrity of the C. parvum oocysts, which may alter the outcome of cell culture infectivity studies Much of the research involving Cryptosporidium parvum viability has focused on occurrence in water and disinfection of the parasite by water treatment. Minimal literature is available regarding the infectivity of the C. parvum oocyst as i t ages in the 15

PAGE 26

environment. Current articles have data from experiments where oocysts of varying ages, different purification procedures, and various treatments processes were used without consideration of how these factors may effect results. Significant improvements have been made in technological advances regarding sample collection and processing techniques for waterborne parasites, such as Cryptosporidium parvum. However, issues regarding the possible effects of these new techniqu e s on the parasites' pathnog e nicity or biological and physiological integrity remain questionable. EPA Information Collection Rule (ICR) and EPA Method 1623 are widely used for the collection and processing of C. parvum and other waterborne parasites. While both EPA ICR and EPA Method 1623 have the ability to detect C. parvum, they do not have the ability to determine whether the oocysts are infectious. The infectivity of C. parvum can be determined by the incorporation of cell culture into ICR and EPA Method 1623 in place o f the immunofluorescence assay (IF A) used However, we must first determine whether ICR or EPA Method 1623 alters the infectivity of C. parvum. This work focused on Cryptosporidium parvum oocyst infectivity in reference to age, purification processes, pretreatment for cell culture, and water recovery processing. The specific objectives were : 1 Determine the effect of purification and pretreatment methods on oocyst viability 2 Compare the effect of vendors' purification procedures on oocysts as they age 3. Determine the effect of age and storage on the level of viable Cryptospordium oocysts in a population. 16

PAGE 27

4. Evaluate two IMS kits and their effects on oocyst infectivity. 5. Determine whether the acidic separation step used in IMS is necessary or is detrimental to the oocysts 6 Evaluate the effect of EPA Method 1623 processes on Cryptosporidium oocyst infectivity. 17

PAGE 28

Chapter Two: Materials and Materials Cryptosporidium parvwn Oocyst Purification Sterling Diagnostic Laboratory (SDL) Purification of Cryptosporidium parvum oocysts (Harley Moon isolate; Ames, Iowa) for this research was conducted at the Sterling Diagnostic Laboratory (SDL) at the University of Arizona The initial debris was removed by mixing the fecal material with an antib iotic so lution, which was strained through a steel mesh. The remaining strained fecal suspension was left in a tall cylinder for 1 hour for the heavier material to fa ll and the upper layer was saved as it contained most of the oocysts. A sucrose solution was adjusted to a specific gravity (1.2 g/mL) and Tween 20 was added afterwards and mixed thoroughly. The fecal and oocyst suspension (-5 mL) was mixed with 30 ml of the sucrose / Tween 20 solution. The suspension mixture was centrifuged for 30 minutes at 1500 g and the top layer containing the oocysts was collected If stored before the cesium chloride gradient, the final oocyst suspension was placed in an antibiotic solution at 4C A stock so lution of cesium chloride (CsCl) was prepared to a specific gravity of 1 8 g/ml. Using TRIS and EDT A, three buffer solut ions of various concentrations were prepared and carefully lay ered in a 15 mL centrifuge tube according to specific gravities The oocyst suspension prepared from the sucrose gradient was rinsed twice by 18

PAGE 29

centrifugation and then added to the top of the TRIS/EDT A layers The 15 ml centrifuge tube was concentrated by centrifugation. The purified oocysts were concentrated in the interface between layer 2 and layer 3. The recovered oocysts were dialyzed overnight to remove the CsCl and then stored and shipped in an antibiotic solution of 0 001% Tween 20 with 100 units of penicillin, 1 OOug of streptomycin and 1 OOug of gentomycin at 4C Pleasant Hill Farm (PHF) Purification of Cryptosporidium parvum oocysts (Harley Moon isolate; Ames Iowa) was conducted at Pleasant Hill farms (PHF) in Troy, Idaho The initial debris was removed by mixing the fecal material with an antibiotic solution. The fecal suspension was strained through a steel mesh, and then left in a tall cylinder for 1 hour allowing the heavier material to fall, and the upper layer was saved as it contained most of the oocysts. Diethyl ether was a necessary step to absolve the fat from the oocyst suspension. The fecal suspension and diethyl either were mixed together in equal volumes Two distinct layers formed and the oocysts remained in the lower aqueous layer. This lower aque ous layer containing the oocysts was removed washed by centrifugation, and stored in an antibiotic solution at 4 C if not immediately processed by the sheathers sucrose gradient. The sheathers sucrose gradient was the same as described above for SDL but in the sucrose solution phenol was present. Once the oocysts had been recovered they were stored and shipped in phosphate buffered saline containing 1000 units of penicillin and 1000 units of streptomycin at 4 C 19

PAGE 30

Cryptosporidium parvum Oocysts Treatment Bleach Treatment for Cell Culture Both SDL and PHF (see Appendex A for lot#, age, raw data) laboratory prepared Cryptosporidium parvum oocysts (Harley Moon isolate; Ames, Iowa) were treated as follows. In a 1.5 1.7 mL polypropylene microcentrifuge tube the following was added: 890 ul of MQ water, 100 ul of cold commercial bleach (Clorox, 5.25% sodium hypochlorite) and 10 ul of C. parvum oocysts (1 x 1071InL). This mixture was vortexed (Type 16700 Maxi Mix 1 mixer, Thermoclyne) for approximately one minute and then l eft to sit at room temperature for 8 minutes after which time the sample was concentrated by centrifugation (10,500 xg for 4 minutes) (Spectrafuge 16M Midwest Scientific) The supematent was aspirated and 900 ul of IX PBS was added to the pellet. The tube was vortexed for a few seconds to thoroughly rins e the oocysts. This washing procedure using centrifugation was repeated twice more and after the last washing, the supematent was aspirated and 1000 ul of growth media (described pg. 26) was added The oocysts were vortexed for two minutes and then counted on a hemacytometer (Brightline 0 1 mm deep) After being diluted using tenfold dilutions, the samples were a s sayed following the FDM-MPN, described on pg. 26-28 (Slifko et al. 1997, 1999). Replicate bleach treated samples were assaye d from d ifferent lot numbers each on a different day. If a sample contaminated more than 25% of the cells (i e more than 6 of the 24 tota l inoculated wells for one MPN), the data were not included or calculated with the MPN program (See Appendix A for oocyst lot# and age) 20

PAGE 31

Antibiotic Treatment for C e ll Culture In a 2.0 mL microcentrifuge tube the following was added : 980 ul of growth media and 10 ul of an antibiotics solution consisting of 20 uL of 1 O,OOOu/mL of Pennicillin G 1 Omg/mL of Streptomyocin and 25mg/mL of Amphotericin B ., and 10 uL of C. parvum oocysts (1 x 1 05/ mL) This antibiotic solution (20 ul) was also added in e ach dilution tube before the ten-fold dilutions were carried out. The oocyst dilutions were then inoculated onto cells and the FDM-MPN was followed (pg. 26-28 Slifko et al. 1997 1999) Replicate antibiotic treated samples were assayed from different lot numbers each on a different day (See Appendix A for oocyst lot # and age) No Treatment for Cell Cultur e Oocysts with "no pretreatment" were diluted and inoculated directly on the cell monola yer with no disinfection and the MPN format was followed, previously described pg. 2 6-2 8 (Slifko et al. 1997 1999). Replicate no treated samples were assayed from diffe rent lot numbers each on a different day (S e e Appendix A for oocyst lot# and age) Cryptosporidium parvum Aged Treatment Experiment Four experiments were run with aged oocyst preparations. This included two lots from each vendor aged between 1 6 6 and 2 00 days. These oocysts had been received purified based on the protocols of the vendor and stored at 4 C The oocysts then underwent the antibiotic, bleach and no pretreatment procedures as previously described, 21

PAGE 32

prior to inoculation onto the cells Two samples were processed for each purification method (See Appendix A for oocyst lot# and age) Cell Culture Foci Detection Method Most Probable Number Assay The following cell culture methodology is described in detail in Slifko, T.R et al ( 1997, 1999). Culture Media Human illeocecal adenocarcinoma cells (HCT-8 cells) (ATCC CCL-224 American Type Culture Collection) were maintained with maintenance media which consisted of 475 mL RPMI 1640 with L glutamine (MediaTech Cellgro Herdon, Va.) and was supplemented with 25 mL fetal bovine serum (FBS), 50 mL of Opti-MEM 5 mL of mM L-glutamine, and 5 mL 1 M HEPES During oocyst infection, th e FBS was increased to 10% (growth media). The culture media was stored in sterili ze d bottles at 4 C Cell Maintenance The HCT -8 cells were maintained and passaged every 3 to 4 days The tissues culture flasks were incubated in 75-cm 2 tissue culture flasks in a 5% C02 incubator at 37C and 100 % humidity. Passaging the c e lls involved trypsinization with 5 mL of phosphate-buffered saline (PBS) EDTA and 5 mL of0. 25% trypsin. The HCT-8 cells had to be incubated for 5 -to 8-minutes in the 5 % C02 incubator covered with the trypsin 22

PAGE 33

solution to assist in the disruption of the monolayer. The cells were concentrated by a 5minute centrifugation (200 X g) and resuspended in 5 mL of maintenance medium (pg.26). The cell solution was placed in 25 mL of maint e nance media in the 75 cm2 tissue culture flasks Each tissue culture fla sk was split 1:5 every passage Lab Tech II well chamber slides (Nalge Nunc, Naperville, Ill.) were plated with 5 x 105 cells per well, 48 hour s prior to oocyst infection and grown to 70% to 95% confluence Cell Infection Maintenance media (described pg.26) was aspirated off the cell mono layers in the chamber slides and rinsed once with 1X PBS to wash off any dead cells. Oocyst dilutions (5-or 10-fold dilution) in growth media after bleach, antibiotic or no treatment (described pg. 26) were added to the wells and incubated for 48 hours in the 37C in the C02 atmosphere. Each of the six slides was labeled with one of the following: 105 104 103 10 2 101 and 10 (denoting oocyst numbers). Aliquots ( 150 ul) of the growth media/oocyst dilutions were inoculated into 6 of the 8 wells in the appropriately labeled slide for that dilution. The two remaining wells were left empty, but often one well woul d be infected with dead oocysts (boiled for 30 minutes) as a negative control. Positive controls, consisting of fresh young oocysts not processed by the examined procedure, were also conducted following the same FDM-MPN bleach treatment, dilution series, and inoculation The chamber slides were placed in the 5% C02 incubator for 48 hours. 23

PAGE 34

Antibody Labeling The media was aspirated off the cell mono layers and 100 % methanol was added to the wells to fix the cells The methanol was aspirated from the wells after 1 0 minutes and the chambers were removed from the slides. The slides were placed in pyrex labelin g dishes with blocking buffer consisting (30 rnL of 1X PBS, 3 rnL of 0.02% Tween 20, 600 ul of Tween-20, and 600 ul of goat serum). The slides were rocked (rocker platform, Bellco Biotechnology, Vineland N.J.) for at least one-half hour before the blockin g buffer was removed Primary antibody (30 rnL of 1X PBS and 10-30 ul of Rat IgG anti-C. parvum sporozoites, depending on the concentration of antibodies in that lot) was added to the slides and rocked for 1 hour. The slides were then rinsed three times with 1X PBS by rocking the tray 10 times and draining the tray. Secondary antibody (30 mL lX PBS and 93.3 uL of Anti-Rat IgG FITC conjugate) (Waterborne) was added to the slides, the trays were covered with aluminum foil, and rocked for at least one hour. The slides were then rinsed three times with 1X PBS by rockin g the tray 10 times and draining the tray. One drop of mounting medium (DABCO) was dropped between the wells of the slides. Coverslips were placed on top of the wells, excess DABCO was wiped off, and the coverslips were sea l ed with fingernai l polish. Enumeration Wells were examined for the presence of foci under 200X epiflourescent microscopy (480nrn excitation and 550 nrn emission) and 1000X for confirn1ation. Wells were noted as positive with three or more life stages as a result of secondary infection and clustering Negative wells were noted if there were less than three life stages and no 24

PAGE 35

clustering. These data were then entered into information collection rule (ICR) ge neral purpose most probable number calculator, version 1.00 (Klee I999) The program uses the number of replicates, dilu tions, volumes inoculated, and positive or negatives results of each well to generate a MPN with confidence intervals. Generally, a 6-well replicate, 5 dilution MPN was used Oyptosporidium parvwn Age Study Cryptosporidium parvum oocysts from SDL and PHF were bleach treated as previously described (pg. 24-25) and their infectivity was assessed approximately every I4 days for 78 to 107 days u sing the FDM-MPN assay, prev io usly described (pg 26-28) (Slifko et al 1997 1999) (See Appendix A for oocyst lot# and age) Cryptosporidium parvum Age Range Study Data from the Age Study was arranged into groups at days I to 30, 3I to 60, and 61 to 90+. The data were analy z ed using Bartlett's and Scheffe's statistical tests to detennine any significant differences (See Appendix A for oocyst lot# and age) Oyptosporidium parvum Immunomagnetic Separation (IMS) Study Dynal Cryptosporidium parvum oocysts were shipped overnight from the SDL with ice packs to the University of South Florida (USF) laboratory and stored at 4C. Oocysts were processed though the Dynal IMS protocol. First, a IX dilution of SL-buffer A (Dynal) was prepared from 1 OX SL-buffer A for every I mL of IX SL-buffer A n eeded. 25

PAGE 36

This dilution of IX SL-buffer A was set aside for later. The following mixture was placed into a flat sided Dynal Ll 0 tube and rotated (Glas-Co! ; Terre Haute USA) for at least 1 hour a t room temperature : 1000 ul of 1 OX SL-buffer A, 1000 ul of 1 OX SL-buffer B, 100 ul of C. parvum oocysts (1 x 107/mL), and 100 ul ofDynaBeads (Dynal A.S, Oslo, Norway). Control samples consisted of Cryptosporidium parvum oocysts and culture media, which were not processed by the Dynal IMS method. Non -B eaded Samples After rotatin g for 1 hour the sa mple s were placed in th e magnetic particle concentrator (Dynal MPC-1) with the flat side of the tube toward the magnet. The tube was gently rocked back and forth for at least one minute and then the supematent was decanted offwith the flat-sid e d tube remaining in the MPC-1 magnet. The oocyst and bead complex were attracted to the flat side of the tube next to the magnet. The tube was removed from magnet and the sample was resuspended in 1 mL of 1 X SL-buffer A. The sample was mixed gently and transferred to a 1.5 to 1. 7 mL microcentriufuge tube. The sample was placed in the magnetic particle concentrator (MPC-M) with the magnetic strip in place. The sample was rocked in the MPC-M for at least one minute The supe matent was then aspirated and discarded while the oocyst-bead complex remained ; and the magnetic strip was removed. Next, 50 ul of 0.1 hydrochloric acid (HCl) was added to the sample and vortexed for a few seconds. The tube was allowed to stand for 5 minutes at room temperature an d then vortexed again for a few seconds The sample was pla ced back in to MPC-M with the magnetic strip in place and left undisturbed for approximately 10 seconds. The supematent containing the oocysts was transferred to 26

PAGE 37

another 1 5 1 7 mL microcentrifuge tube, which contained 50 ul of 0.1 sodium hydroxide (NaOH), while the beads were attracted to the magnet. The sample was vortexed for a few seconds and pelleted by centrifugation. T he liquid was aspirated from the pellet. The pellet then followed bleach treatment as previously described (pg. 24-25) and infectivity was assessed by the FDM-MPN format (pg 26-28; Slifko et al. 1997, 1999). Beaded After rotating for 1 hour, the samples were placed in the magnetic particle concentrator (Dynal MPC-1) with the flat side of the tube toward the magnet. The tube was gently rocked back and forth for at least one minute, and then the supematent was decanted off and discarded with the flat-sided tube remaining in the MPC-1 magnet. The oocyst and bead complex were attracted to the flat side of the tube next to the magnet. The tube was removed from magnet and the sample was resuspended in 1 mL of IX SL buffer A. The sample was mixed gently and transferred to a 1.5 to 1.7 mL microcentriufuge tube. The sample was placed in the magnetic particle concentrator (MPC-M) with the magnetic strip in p l ace. The sample was rocked in the MPC-M for at least one minute. The supernatent was then aspirated and collected (contained the oocysts separated from the beads) and the magnetic strip was removed. The pellet then followed bleach treatment as previously described (pg. 24-25) and infectivity was assessed by the FDM-MPN format (pg. 26-28; Slitko et al. 1997, 1999). (See Appendix A for oocyst lot# and age) 27

PAGE 38

Hach Cryptosporidium parvum oocysts were s hipped overnight from the SDL with ice packs to the USF laboratory and stored at 4 C Oocysts were processed through the HACH protocol. Briefly oocyst preparations were added to a polypropylene centrifuge: 4000 ul ofProNetic Wash Buffer, 100 ul of C. parvum oocysts, and 2000 ul ofCrypto Antibody Beads (HACH, Loveland, Colorado) Samples were rotated (Glas-Col; Terre Haute USA) for at least 30 minutes at room temperature. The oocyst-bead complex was separated into two tubes and placed in the ProNetic Three Tube Magnet. The sample was rocked for at least four times; the bead-oocyst concentrate was kept and then the supematent was aspirated off and discarded. The sample was split into two equal parts. One part was applied directly to the bleach treatment (described pg. 24-25) (beaded sample) and the FDM-MPN format (previously described pg 26-27; Slifko et al. 1997, 1999). The other portion was processed though the disassociation step (non-beaded sample). This took place by resuspending the bead-oocyst complex in 2 drops of the ProNetic Detachment Reagent which contained hydrochloric acid and demineralized water. The sample was vortexed for two minutes and then captured by the ProNetic Micro Tube Magnet. The freed oocyst were collected in the s upematent. The sample was transferred to a microcentrifuge tube containing 2 drops ofProNetic Neutralizing Reagent (sodium hydroxide demineralized water) vortexed for a few seconds, and pelleted by centrifugation. The supematent containing the oocysts was then subjected to the bleach treatment (described pg 24-25) and the FDM-MPN format (previously 28

PAGE 39

described pg. 26-28; Slifko et al. 1997 1999). Control samples consisted of Cryptosporidium parvum oocysts and culture media, which were not processed by the Dynal IMS method. Cryptosporidium parvum Aged IMS Experimen t Two experiments were conducted with aged oocysts being processed by Dynal and Hach IMS kits, which included one sample for each beaded and non-beaded for each kit. The oocysts were stored at 4C and then processed by IMS with and without disassociation. The samples then underwent the FDM-MPN assay as previously described (pg 26-28; Slifko et al. 1997, 1999). EPA Information Collection Rule (ICR) Surface water (1 OL) collected from Lake Starvation in Hillsborough County was seeded with Cryptosporidium parvum oocysts ( 1 x 107 /mL) from SDL (preparation described pg. 22-23). The seeded water was stirred with a paddle for approximately two minutes T h e water was then pumped through a yam-wound filter (Filterite) of 1 urn nominal porosity at 7.57 gallons/minute The sample was then processed according to EPA ICR (EPA 1995) The filter yam material was removed from the polystyrene core and placed in stomacher bags that could hold up to 3,500 mL liquid vo lum e The yam fibers were washed with 1 .75 L of eluting solution for two five-minute intervals The eluting solution conta ined 100 mL of 1% SDS, 100 mL of 1% Tween 80, 100 mL of 1 OX PBS, and 0 1 mL of anti foam adjusted to ph 7.4 and to a final volume of 1 L with DI water. The sample was collected into a 250 mL conical cen trifu ge bottle and fresh 29

PAGE 40

eluting solution was added to the fibers and washed again. After the second interval of y.rashing, the fibers were squeezed to remove excess water and then thrown away. The sample was pelleted by centrifugation for 20 minutes at I ,050 x g The pellet was vortexed and transferred to a 15 mL polypropylene centrifuge tube and stored overnight in IX PBS at 4 The next day immunomagnetic separation (IMS) using the Dynal kit replaced the flotation purification step. The magnetic beads were not removed; the Dynal IMS procedure for beaded samples was previously described on page 29-30. The concentrate was then bleach treated and introduced into the FDM-MPN (described pg. 24-25 and pg. 26-28 respectively) (Slifko et al. I997, Slifko et al. I999). Control samples consisted of Cryptosporidium parvum oocysts and culture media, which were not processed by the EPA ICR and Dynal IMS methods. EPA Method I623 Finished (tap) water samples (SOL) were seeded with SDL prepared (pg. 22-23) Cryptosporidium parvum oocysts at the USF. The 50 L finished water samples were stirred for approximately two minutes with a paddle. The water samples were then processed according to EPA Method 1623 protocol (EPA I999) The water samples were filtered thorough the Envirochek Pall Gellman filter capsule (I urn absolute porosity) at a flow rate of2 L/minute using a Teel model IP579F pump. The capsule was vented after the entire volume had been filtered and eluting solution was added to the filter capsule; effort was made to cover the entire filter. The eluting solution consisted of I 000 IX PBS, IOO rnL ofLaureth-12 solution, IO mL ofTris buffer, and 2 mL ofO.SM EDTA The filter was placed on the mechanical wrist shaker at 600 rpm for two 5-minute 30

PAGE 41

intervals lying at a 12 o'clock position. Between intervals fresh eluting solution was and th e other was collected in a 250 mL conical centrifuge bottle The samples once eluted from the filters, were centrifuged at 11 OOX g for 15 minutes. The supematent was aspirated and the pellet was transferred into a 15 mL polypropylene centrifuge tube with IX PBS and stored overnight at 4. The next day, the pellet was processed by Dynal IMS kit following the beaded sample protocol (described pg.29-30). However, the Dynabead s IgG iron bead antiCryptosporidium anibodies were used to capture the oocysts (Dynal A.S, Oslo, Norway). The concentrate was then bleach treated and introduced into the FDM-MPN (described pg. 24-25 and pg. 26-28 respectively) (Slifko et al. 1997, Slifko et al. 1999). Control samples consis t ed of Cryptosporidium parvum oocysts and culture media which were not processed by the EPA Method 1 623 and Dynal IMS methods. Environmental Samples Water samples (15 to 20 L) from four sites were processed through a modified ICR (EPN814B-95-003, June 1995) method with the Dynal IMS kit replacing the floatation pmification step, with bleach treatment (pg. 24-25) and the incorporation into the FDM -MPN assay after purification (Slifko et al. 1997, Slifko et al. 1 999) (procedur e pg. 26-28). The sites included Lake Starvation (surface water site), effluent from Howard F. Curren wastewater treatm e nt plant at the outfall into the bay (marine water site), seco nd ary treated (nitrofied) water from Howard F. Curren wastewater treatment plant and tertiary treated (denit r ified/chlorinated/ and dechlorinated) water from Howard F. Curren wastewater treatment plant. 3 1

PAGE 42

Normalizing Data Samples inoculated into cell monolayers were not equivalent and the different oocyst concentrations were reflected in the calculated MPN. Therefore a hemacytometer count was taken before oocysts were inoculated into the cell monolayer. Once the MPN was generated by the Most Probable Number calculator computer program (Klee 1999), normali zation was completed to compare samples. The hemacytometer counts taken are each divided by the highest hemacytometer count for that experiment. The highest hemacytometer count had a ratio of 1.0 while the others were < 1.00. The generated MPNs were then divided by its particular normali zed hemacytometer count. This process "normalized" the MPNs for comparison by equalizing the initial oocyst concentration 32

PAGE 43

Chapter Three: Results Cryptosporidium parvum Oocyst Treatment Study This study concentrates on the effects of disinfection treatments on young (<35 days) Cryptosporidium parvum oocyst infectivity. Results provided insight on the disinfection treatments as anti-microbial agents and excystation reagents. Sterling Diagnostic Laboratory (SDL) Cryptosporididiumpan,um SDLprepared oocysts, Lot# 90105-12 at age 15 days were subjected to bleach treatment (pg. 24), antibiotic treatment (pg. 25), and no treatment (pg.25). As replicate tests, Lot# 90803-07 at ages 12 and 15 days underwent these treatments on subsequent days. Oocysts treated by bleach resulted in 4.41 x 103 infectious oocysts MPN/ ml (Lot# 90105-12), 3.89 x 102 infectious oocysts MPN/ ml (Lot# 90803-07, 12 days), and 1.02 x 102 infectious oocysts MPN/ml (Lot# 90803-07, 15 days) (Figure 1, Table 1, CI included in Table 1). Contamination from the bleach treated oocysts ranged from 0 to 13% ofthe wells (Table 2). Oocysts treated by antibiotics resulted in 6.22 x 102 infectious oocysts MPN/ml (Lot# 90803-07, 12 days) (Figure 1, Table 1, CI included in Table 1 ). The replicate samples treated with antibiotics had more than 50% and 42% contamination in the wells (Lot# 90105-12 50%; Lot# 90803-07, 15 days, 42%), therefore data was not calculated (Table 2). Oocysts with no treatment 33

PAGE 44

resulted in 58%, 50%, and 46% contamination of the we ll s in Lot# 90105-12, Lot# 90803-07 ( 12 days), and Lot# 90803-07 ( 15 days), respectively (Table 2). Figure 1. Treatment Effects on SDL C. parvU1n Oocyst Infectivity ...J E ';::2 tn 0 0 0 tn :;, 0 :;:; 0 .!!! s::: z a.. :1E 1 00E+04 1.00E+03 1.00E+02 1 00E+01 Trial #1 (lot# 90105-12 15 days) Trial #2 (Lot# 90803-07, 12 days) Trial #3 {lot# 90803-07 15 days) o B leach T reatme nt Antibiotic Treatment 34

PAGE 45

Table 1. Data for Treatments Effec ts on SDL C. parvum Oocyst Infectivity Oocyst lot# /age Treatment llemacytometer Normalized MPN/mL1 (days) (CI =co nfidence intervals) 90105-12 / 15 Bleached 9.00 X 104 4.41 X 10 j (1.59 X 103 -1.02 X 1 04 ) 90803-07 I 12 Bleached 5 63 X 104 3 89 X 10..: (1.42 X 1021.16 X 103 ) 90803-07/ 15 Bleach ed 8.13 X 104 1.02 X 10:l (2 .92 x 101-2.34x 102 ) 90105 -12/ 15 Antibiotics 8.QQ X 104 90803-07 I 12 Antibiotics 8 50 X 104 6.22 X lQ.l (1.84x 102-1.37x 103 ) 90803-07 I 15 Antibiotics 4.88 X 104 90 1 05-12/ 1 5 None 7 75 X 104 90803-07 / 12 None 7 .7 5 X 104 90803 07 / 15 None 6.50 X 104 Ta bl e 2. Contami nation i n SDL and PHF C. parvum Oocysts (Percent of Wells Con t aminated) SDL Lot# 90105 12 Lot# 90803-07 Lot# 90803-07 15 days 12 days 15 days Bleach 0% 13% 13% Antibiotics 50% 13% 42 % No Treatment 58% 50% 46 % PIIF Lot# 99-3 Lot# 99-16 Lot# 99-16 29 days 2 days 3 days Bleach 0% 0% 0% Antibiotics 0% 0% 0 % No Treatment 0% 8% 8% Pleasant Hill Farm (PHF) Thre e lots ofPHF prepared C. parvum oocysts (Lot# 99-3 age 29 days ; Lot # 99-16, age 2 days; and Lot# 991 6, age 3 d ays) were su bjected to bl each treatment (pg 24), 1 Unab l e to assay due t o contaminated ho s t ceJI monolayer 35

PAGE 46

antibiotic treatment (pg 25), and no treatment (pg.25). Oocysts treated by bleach t : esulted in 2.17 x 104 infectious ooocysts MPN / ml (Lot# 99-3), 1.98 x 103 infectious ooocysts MPN / ml (Lot# 99-16, 2 days), and 3.13 x 103 infectious ooocysts MPN / ml (Lot# 99-16, 3 days) (Figure 2, Table 3, CI included in Table 3). Bleach treatment of oocysts resulted in 0% contamination of the wells for the three replicates (Table 2). Oo cys ts treated by antibiotics resulted in 5 65 x 102 infectious oocysts MPN / ml (Lot# 99-3), 2.65 x 102 infectious ooc ys ts MPN/ml (Lot# 99-16, 2 days), and 1.06 x 102 infectious oocysts MPN / ml (Lot # 99-16, 3 days) (Figure 2, Table 3, CI included in Table 3). Antibiotic treatment of oocysts result ed in 0% contamination of the wells for the three replicates (Table 2). Oocysts with no treatment resulted in 1.48 x 103 infectious oocysts MPN / ml (Lot# 99-3) 4.98 x 102 infectious oocysts MPN / ml (Lot# 99-16,2 days), and 3.70 x 101 infectious oocysts MPN / ml (Lot# 99-16, 3 days) (Figure 2, Table 3, CI included in Table 3) Contamination in the wells from untreated oocysts ranged from 0 to 8% of the wells for the three replicates (Table 2). 36

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Figure 2. Treatment Effects on PHF C. parvum Oocyst Infectiv ity E 1 00E+05 ,----=-----------------------------, en (i) 1.00E+04 >. (.) 0 0 1 00E+03 en :::l 0 :;::: 1 00E+02 (.) c z a. 1 00E+01 1 .00E+OO +-__.....__-== (29 days) (2 days) EJ B leached Antibiotics o No Tre atmen t 37 (3 days)

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Table 3. Data for Different Treatment Effects on PHF C. parvum Oocysts Infectivity Oocyst lot# /age Treatment llernacytonneter Normalized MPN/rnL (days) (confidence intervals) 99-3/29 Bleached 6 50 X 104 2.17 X 104 (8.19 X 103-5.45 X 104 ) 99-16/2 Bleached 4.95 X 10 ) 1.98 X (7.43 X 102-5.36 X 103 ) 99-16/3 Bleached 8.63 X 104 3 .13 X 103 (1.06 X 1036.96 X 103 ) 99-3/29 Antibiotics 1.40 X 10) 5.65 X 10:l (2.03 X 102 1.63 X 103 ) 99-16/2 Antibiotics 7.75 X 104 2.65 X 102 (9.52 X 1016 .15 X 102 ) 99-16/ 3 Antibiotics 8.13 X 104 1.06 X 10J (3. 83 X 1022 90 X 103 ) 99-3/29 None 1.10 X 105 1.48 X 103 (5.02 X 1023.83 X 103 ) 99-16/2 None 7 .00 X 104 4.98 X 102 (1.36 X 102 1.24 X 103 ) 99-16 / 3 None 6 88 X 104 3.70 X 107 (1.09 X 102 8.78 X 102 ) CJyptosporidium parvum Aged Treatment Study This study concentrates on the effects of disinfection treatments on aged ( > 166 days) Cryptosporidium parvum oocyst infectivity. Results provided insight on the disinfection treatments as anti-microbial agents and excystation reagents. Sterling Diagnostic Laboratory(SDL) Cryptosporididium parvum SDL prepared oocysts Lot# 90803-07 at age 174 days were su bj ected to bleach treatment (pg. 24) antibiotic treatment (pg. 25), and no 38

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treatment (pg.25). Subsequent tests were run with Lot # 90706 -11 a t age 200 days Oocysts at 174 days of age treated by bleach resu lt ed in 6.96 x 102 i nfectious oocysts MPN/ m l oocysts treated by antibiotics resulted in 1.27 x 103 infectious oocys t s MPN/ m l and oocysts w ith no treatment resulted in I.06 x 103 infect i o u s oocysts MPN/ml (Figure 3, Tabl e 4, CI i ncluded in Tab le 4). Contamination of Lot# 90803-07 oocysts was 0% in the wells for a ll treatments (Table 5) Similar leve l s of i n fectiv ity were found with Lot# 90706-1I oocysts, which resu lted in 2 .67 x I 03 infectio u s oocysts MPN/ml, 4 .42 x I 02 infectious oocysts MPN/ml, and 8.02 x 102 infec t ious oocysts MPN/ml for b leach, antibiotic, and no treatment, respectively (Figure 3, Table 4, CI in cluded i n Tab l e 4). Contamination for Lot# 90706-11 oocysts were 16% in the bleached treated oocysts, 3% in the antibiotic treated oocysts, and 0% in the no treatment oocysts (Tab l e 5). Figure 3 Treatment Effects on Aged SDL C. parvum Oocyst Infectivity 1. 00E+05 ..J E 1 00E+04 1/j 1/j >. () 0 1 0 0E+03 0 1/j ;:::, 0 .. 1 0 0E+02 () c:: z Q.. 1. 0 0E+01 Trial # 1{Lot# 90803 09 1 7 4 d a ys) Trial #2 {Lot# 90 7 061 1 200 d ays) GJ Bleach Treatrrent Antibiotic Treatrrent o No Treatrrent 39

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Table 4. Data for Treatment Effects on Aged SD L C. parvum Oocyst lot# /age Treatment llemacytometer Normalized MPN/mL (days) (confidence intervals) 90803-07/174 Bleached 2.63 X 10:> 6.96 X 10l (2.20 X 102 1.52 X 103 ) 90706-11/200 Bleached 4.88 X 10:> 2.67 X 10j (6.50 X 1028.51 X 103 ) 90803-07/174 Antibiotics 2.44 X 10:> 1.27 X 10j (3.66 X 102-3.10 X 103 ) 90706-11/ 200 Antibiotics 4 57 X 10:> 4.42 X 10 l (1.60 X 102 -1.03 X 103 ) 90803-07 / 17 4 None 2.34x 10:> 1.06 X 10 j (3.10 X 102-3.40 X 103 ) 90706-11/ 200 None 3.76 X 10:> 8.02 X 10l (2 .63 X 102 1.78 X 103 ) Table 5. Contamination for Aged SDL and PHF C. parvum Oocysts (Percent of Wells Contaminated) SDL Lot# 90803-09 Lot# 90706-11 174 days 200 days Bleach 0% 16% Antibiotics 0% 3% No Treatment 0% 0% PIIF Lot# 99-16 Lot# 99-14 166 days 185 days Bleach 0% 8% Antibiotics 3% 0% No Treatment 0% N/A 40

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Pleasant Hill Farms (PHF) Cryptosporididium parvum PHF prepared oocysts Lot# 99 16 at 1 66 days resulted in 1 18 x 103 infectious oocysts MPN/ ml and 2. 71 x 1 03 infectious oocysts MPN / ml for bleach and antibiotic treatments (Figure 4, Table 6 CI included in Table 6) The no treatment samples were not assayed, as for the shipment of age sample oocyst stock was limited in volume. The contamination in Lot# 99-16 for bleach and antibiotic treated oocysts was 8% and 0% of the wells (Table 6). Lot# 99-14 at 185 days had 1.25 x 103 infectious oocysts MPN/ ml 7.88 x 102 infectious oocysts MPN/ ml, and 1.60 x 103 infectious oocysts MPN/ ml for bleach, antibiotic, and no treatment, respectively (Figure 4, Table 6, CI included Table 6). The contamination in the wells for bleach, antibiotics and no treatments was 0%, 3%, and 0% of the wells, respectively (Table 6). 41

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Figure 4. Treatment Effects on Aged P HF C. parvum O ocyst Infectivity ....J E 1 00E+{) 3 tJ> tJ> (.) 0 0 (/) 1 00E+{)2 :::::s 0 :;:; (.) Q) -c z 1 00E+{) 1 a.. ::!: Trial # 1 ( Lot# 99-16 1 66 days) Trial #2 (Lot# 99 -14, 1 85 days ) o Bleach Treatment Antibiotic Treatment 0 No Treatment Table 6 Data for Treatment Effects on Aged PHF C. parvum Oocyst lot# /ag e Treatm ent llemac yt om eter Norma l ize d MPN/mL ( d ays) (confide n ce intervals) 99-I6/ I66 Bleached 8.25x I04 l.I8 X IOj (3 69 X I 02 2.82 X I 03 ) 99 14/ 185 Bleached 4.1 2 X 1 04 1.25 X 1 03 (3.83 X 1 022 .74 X 103 ) 99 16/ 166 An t ibiotics 1.31 X 10:> 2 .71 X 10 j (7.86 X 1026 60 X 1 03 ) 99-14/ 185 Antibiotics 4 04 X 104 7.88 X lOl (2.41 X 1 02 1.72 X 103 ) 99-14 / 1 85 None 9.25 X 104 1.60 X 10j (5.74 X 102-4.62 X I 03 ) 42

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Treatment Study Summary SDL C. parvum oocysts required bleach treatment in younger (<35 days) oocysts to prevent contamination while the aged SDL oocysts (> 166 days) resulted in no contamination regardless of treatment. No comparison could be made betw een treatments in the young SDL oocysts due to percent contamination in the antibiotic and no treatments PHF young and old C. parvum oocysts resulted in minimal contamination if any, with all three treatments. The varying results between vendors can be attributed to the differing preparation techniques and antibiotic storage concentrations The difference in percent contamination between the young and aged SDL oocysts is related to microbial die-off in the aged oocysts. Cryptosporidium parvum Age Study This study focused on the change in Cryptosporidium parvum oocyst infectivity over time Results provided insight on the age of maximum infectivity in the oocysts and if variability existed within or between lots of oocysts. C. parvum oocysts were bleach treated before inoculated into the cell culture system. Sterling Diagnostic Laboratories (SDL) Figure 5 and Table 7 show the change in infectivity of C. parvum oocysts over time for three separate lots of oocysts. Lot# 90105-12 began with oocyst at an age of 15 days with 3.37 x 104 infectious oocysts MPN I mL and continued with similar infectivies throughout the study until 62 days of age with 1 07 x 104 infectious oocysts MPN I mL. The infectivity of this same lot (Lot# 901 05-12) dropped slightly at 78 days to 4.11 x 103 infectious oocysts MPN I mL, but at th e last time point of 107 days 1.78 x 104 infectious 43

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oocysts MPN/ mL were found. The slight decrease then increase between 62, 78, and 107 days in Lot# 90105-12 was within the estimated variability ofthe FDM-MPN assay. Lot# 90601-06 age study began with oocysts at an age of 10 days with 1.87 x 104 infectious oocysts MPN/ mL and continued with similar infectivity to 37 days with 1.07 x 104 infectious oocysts MPN/ mL. Lot# 90601 06 had a significant decrease in infectivity at 76 days with 9.20 x 101 infectious oocysts MPN/ mL, however, at 94 days 4 06 x 104 infectious oocysts MPN/ mL were found. Lot# 0020 1-10 age study began with oocysts at an age of 7 days with 1 .17 x 105 infectious oocysts MPN/ mL and continued with similar infection to 64 days with 4.26 x 104 infectious oocysts MPN/ mL. Lot# 00201-10 had a slight decrease in infectivity at 78 days with 2.90 x 103 infectious oocysts MPN I mL. 44

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Figure 5. Effects of Aging on SDL C.parvum Oocyst Infectivity 1 00E+06 1 00E+05 ...J VI 1 00E+04 VI >. ... .... A \ ..... .... () 0 0 VI 1 00E+03 :I 2 t) Q) 1 00E+02 .E z Q. :E 1 00E+01 1 .00E+OO 0 20 40 Lot# 90105-12 ..... 60 Age (days) Lot# 90601-06 45 --80 100 120 Lot# 00201-10

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Table 7. SD L C. parvum O ocyst Age Study Data Lot #9 0105-12 Lot #90601-06 Lot #00201-10 Age (days) MPN/mL Age (days) MPN/mL Age (days) MPN/mL 15 3.37 X 104 10 1.8 7 X 104 7 1.17 X 10:> 22 4 .9 4 X 104 23 2.54 X 104 13 6.75 X 1 04 28 9.82 X 104 37 1.0 7 X 104 20 5 80 X 1 04 35 4 09 X 104 76 9.20 X 101 26 9.41 X 104 41 9.41 X lOT 94 4.06 X 104 34 8.02 X 104 49 3.69 X 104 48 6.47 X 104 58 4.31 X 104 56 2.47 X 104 62 1.07 X 104 64 4 26 X 104 78 4.11 X 103 78 2.90 X 10 j 92 1.17 X 103 10 7 1.78 X 103 Ple asant Hill Farms (PHF) Figure 6 and Table 8 show the infecivity of C. parvum oocysts from the PHF vendor over tim e from t hr ee given lots. Lot# 99-3 began with oocysts at an age of29 days with an infectivity of3.52 x 104 infectious oocysts MPN/ mL infectious oocysts / mL and cont in ued with simi l ar infection up to 34 days of age with 2 2 7 x 104 infectious oocys ts MPN/ mL. Infectivity did n ot decrease for Lot# 99 3 until 44 days when the l eve l dropped to 3.3 x 103 infec tiou s oocysts MPN / mL and continued to s t ay at this infecivity range throughout the rest of th e st ud y up to age 94. Lot# 99-9 age study began with oocysts at an age of 11 days with an infectivity of2.58 x 104 infectious oocysts MPN / mL and an increase in infecti v ity at 26 days was noted (2.02 x 105 infectious oocysts MPN/ mL). The in fec tivit y for Lot # 99-9 did not decrease until 7 9 days of age r e sulting in 2 23 x 1 03 infe ct iou s oocysts MPN / mL. Lot # 00-5 age s tud y b egan w ith 46

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oocys t s a t an age of 4 days with 6.9 1 x 104 infec tio us oocysts MP N / mL and continued with similar infec ti on up to 31 days with 2.85 x 1 04 infecti ous oocysts MPN/mL, and a slig h t increase at 61 days of age with 1 02 x 1 05 infectious oocysts MPN/ mL. The infectivity for Lot# 00-5 decreased at 75 days to 9 96 x 1 03 i nfectious oocysts MPN/ mL. Figure 6 Effects of Age on PHF C. parvum Oocyst Infectivity 1.0 0E+06 ..J 1. 00E+05 E 1/) 1 00E+04 0 0 1 0 0E+0 3 0 u c 1. 00 E+02 z a.. :E 1. 00E+01 1. 00E+OO ... 0 ... A .& 2 0 4 0 Lot# 99 3 ... 6 0 80 1 0 0 A ge (da ys ) Lo t# 99-9 .A. Lot# 00-5 47 120 140

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Table 8. PHF C. parvum Oocyst Age Study Data Lot #99-9 Lot #99-3 Lot #00-5 Age (days) MPN/mL Age (days) MPN/mL A2e (days) MPN/mL 11 2 58 X 104 29 3.51 X 104 4 6.91 X 104 26 2.02 X 10 34 2 27 X 104 10 1.26 X 10' 40 9.91 X 104 44 3.30 X 103 17 7 87 X 104 79 2.23 X 10J 58 1.68 X 103 23 1.64 X 10 73 2.95 X 103 31 2 85 X 104 120 7.23 X 103 45 4.49 X 104 53 9.12 X 104 61 1.02 X 10 75 9.96 X 10J Cryptosporidium parvum Age Range Study Data from the age study was statistically analyzed to determine if C. parvum oocysts in a particular age group were more or less infective than another age group St e rling Diagnostic Laboratory (SDL) The same data from the age study were grouped into age ranges to statica ll y compare the changes in infectivities (Figure 7 Table 9). The oocyst group of 1 to 30 days of age averaged 6.24 x 104 i nfectious oocysts MPN /mL (.46 x 1 04 ) (n=9), the oocyst group of 31 to 60 days of age averaged 3.89 x 104 infectious oocysts MPN / mL (.47 x 1 04 ) (n=8), the oocyst group of 61 to 90+ days of age averaged in 3.58 x 104 infectious oocysts MPN / mL (.07 x 104 ) (n=5), and the oocyst group of 61 to 80+ days of age averaged in 1.30 x 104 infectious oocysts MPN/ mL (.80 x 105 ) (n=7) There was a significant differ e nce found between the age groups 1 to 30 days and 60 to 90+ da y s by Bartletts and Scheffe's statistical tests. 48

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Figure 7 Age Range Averages of Aging SDL C. parvum Oocyst Infectivity 1.00E+05 ...1 E iii ... 1.00E+04 1/1 (.) 0 1 00E+03 0 1/1 ::s 0 :+: (.) 1 00E+02 C1) .... c:: z 1.00E+01 a.. ::::!: 1.00E+OO 1 to 30 49 T 31 to 60 Age (days) 61 to 90

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Tab l e 9. Data for Age Range SDL Oocysts Age L ot# Age N ormal ize d Ave r age Standard Range ( d ays) M P N (Infectio u s Deviatio n (Infect i o u s Oocysts/mL) Oocysts/mL) 1 to 30 90105-12 15 3.37 X 104 90601-06 10 1.87 X 104 00201-10 7 1.11 x 1 00201 -10 13 6 .75 X 1 04 0020 1 1 0 20 5 80 X 1 04 90105-12 22 4.94 X 90105-12 28 9.82 X 104 9060 1 -06 23 2 54 X 104 00201-10 26 9.41 X 1 04 6 24 X 1 04 .46 X 104 31 t o 60 90105 -12 35 4 .09 X 90601-06 37 1.07 X 10'1 00201-10 34 8.02 X 104 90105-12 41 9.41 X 10 j 90105-12 49 3 .69x 1 04 90105-12 58 4.31 X 1 04 00201-10 48 6.47 X 1 04 00201-10 56 2.47 X 104 3.89 X 1 04 .47 X 1 04 6 1 t o 9 0 + 90105 -12 62 1.07 X 104 90105-12 78 4 .11 X 10j 90105-12 92 1.17 X 1 O J 90105 -12 107 1.78 X lOJ 90601-06 7 6 9 .20x 1 0 90601-06 94 4 06 X 1 04 00201-10 64 4 26 X 1 04 0020 1 1 0 78 2.90 X 10j 1.30 X 104 1.80 X 104 Pleasant Hill Farms (PHF) The same groupings for PHF oocysts were evaluated statically to compare the changes in infectivities (Figure 8 Table 1 0) The oocyst group of 1 to 30, 31 to 60 61 to 90+ days of age averaged 1.93 x 105 infectious oocys t s MPN /mL ( 3 74 x 1 05 ) (n = 13), 4.56 x 104 infec t iou s oocysts MPN / mL (.82 x 1 04 ) (n=8) and 3.63 x 104 i nfectious 50

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oocysts M PN/ m L ( 4 .07 x 1 05 ) (n=7), re sp ec t ive ly. Th e r e was n o s i gnificant d ifference f o u nd b e t ween the age groups b y Bart l et t s an d S c heff e s s tat is ti ca l test s Figure 8. Age Range Ave r ages of Aging PHF C. parvum Oocysts Infe ctivity 1 00E+06 1 00E+05 ...J E Cl) i 1 00E+0 4 u 0 0 Cl) 1 .00E+ 03 ::I 0 :;: u .E 1 00E+02 z a.. == 1.00E+01 1 .00E+OO 1 to 30 51 31 to 60 Age (days) 61 to 90

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Table 10. Data for PHF Age Range Age Lot# Age Normalized Average Standard Range (days) MPN (Infectious D ev i ation (Infectious Oocysts/mL) Oocysts / mL) 1 to 30 99-9 11 2.58x104 00-5 4 6 .91 X 1 04 00-5 10 1.26 X 10:> 00-5 17 7 .87x 104 99-16 2 3.20 X 103 99-16 5 5.06 X 103 99-11 12 1.63 X 10 99-11 18 1.53 X 10:> 00-6 3 6 .75 X 104 99-3 29 3.51x104 99-9 26 2.02x10' 00 5 23 1.64 X 10' 99-11 25 1.42 X 10 1.93 X 105 .74 X 105 31 to 60 99-9 40 9.91 X 104 99-3 34 2.27 X 104 00-5 31 2.85 X 104 99-3 44 3 30 X 103 99-3 58 1.68x103 00-5 45 4.49 X 104 00 -5 53 9.12 X 104 99-16 47 7.32x104 4 56x 104 82 X 104 61 to 90+ 99-3 73 2.95 X 103 99-3 120 7.23 X 103 99 9 79 2.23x103 99-9 97 7.23 X 104 00-5 61 1.02 X 10' 00-5 75 9.96 X 105 99-16 65 5.71 X 104 3 .63 X 104 07 X 104 Age Study and Age R ange Summary SDL C. parvwn oocysts had less lot to lot variability than PHF. SDL oocysts also h a d an apparent increase in infectivity around 30 days and an apparent drop in infectivity after 60 days which was confirmed with Bartl e tts and Scheffe's statistical tests The 52

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high variability between l ots with PHF oocysts led to no significant differences between age ranges based upon the same statistical tests. Cryptosporidium parvum IMS Study This study concentrated on the effects of the acid disassociation step in immunomagnetic separation (IMS) on young ( <35 days) Cryptosporidium parvum oocyst infectivity. This provided insight on whether removing the bead was a necessary step and if this step altered oocyst infectivity. Ooocyst were bleach treated before inoculation into the cell culture system. Controls were C. parvum oocysts and cu ltur e media inoculated into the cell culture system without being processed by IMS. Dynal Experiments were conducted with the Dynal IMS system using oocysts less than 2 months of age They (Figure 9, Tab le 11, and Table 12) illustrate the infectious oocysts for each sample processed with Lot# 90914-21 at an age of31 days and Lot# 00619 25 at an age of 19 days. Data between the two trials were not normalized to each other due to contributing variability factors (age of oocysts, lot number, etc.) Cryptosporidium parvum oocysts from Lot# 90914-21 resulted in an average of2.83 x 105 infectious oocysts MPN / mL ( 2. 78 x 1 05 ) for the controls (n = 2) 6.12 x 104 infectious oocysts MPN/ mL ( 2.75 x 104 ) for the non-beaded samples (n=4), and 4.39 x 1 04 infectious oocysts MPN / mL ( 7.24 x 1 03 ) for the beaded samples (n=3). Cryptosporidium parvum oocysts from Lot# 006 1 9-25 resulted in an average of 5.45 x 53

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103 infectious oocysts MPN/ mL ( 6 25 x 1 01 ) for the contro l s (n=2), 1.56 x 104 in fectious oocysts MPN / mL ( 2.62 x 103 ) for the non-beaded samp les (n=4), and 1.23 x 104 infectious oocysts MPN/ mL ( 1 24 x 1 03 ) for the beaded samples (n=5) While there was a slight variability between the replicate controls (Figure 9), the confidence intervals (error bars) overlap between the two control samples and Control2 had similar infectivity to all processed samples. Figure 9. Effects of the Capture/Disassocia tion Steps in Dynal IMS Kit on SDL C. parvum Oocyst Infectivty Trial#1 (lot#90914-21,31 days) Tri a l #2 (lot# 00619-25, 19 days) ::; E VI -VI >-(J 0 0 VI ::l 0 :;:; (J c -z a.. ::!: 54

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Table 11. Data for Dynal IMS Processed C. parvum Oocysts (Lot# 90914-21) Normalized MPN Average Standard (Infectious Oocyst/mL) Deviation Control! 4.80 X 10 (1.65 X lOS 1.13 X 106 ) Control2 8.64 X 104 2.83 X 10 > .78 X lOS (2.57 X 104 1.91 X lOS) Non-Beaded Sample# 1 4.05 X 104 (1.46 X 104 -1.17 X lOS) Non-Beaded Sample #2 4.63 X 104 (1.63 X 104 1.72 X lOS) Non-Beaded Sample #3 5.68 X (2.14 X 104 1.53 X lOS) Non-Beaded Sample #4 1.01 X 10> (3 82 X 104 -2.54 X lOS) 6.12 X .75 X 104 Beaded Sample #1 4 .61 X 104 (1.68 X 104 1.27 X lOS) Beaded Sample #2 4.99 X 104 (1.88 X 104 1.34 X lOs) Beaded Sample #3 3.59 x to (1.35 X 1049.01 X 104 ) 4 39 X .24 X 103 Table 12. Data for Dynal IMS Processed C. parvum Oocysts (Lot# 00619-25) Normalized MPN Average Standard (Infectious OocysUmL) Deviation Control! 2 77 X 103 (1.04 X 1037.46 X 103 ) Control2 2 68 x 10 j 5.45 X 10 j .25 X 101 (1.02 X 103-6.80 X 103 ) Non-Beaded Sample # 1 l.76x 10 J (6.31 x l02-5.08x 103 ) Non-Beaded Sample #2 3 .06 X 103 ( 1.11 X 1 03 -8.41 X 1 03 ) Non-Beaded Sample #3 7.72 X 10 j (2 .78 X 103 1.79 X 104 ) Non-Beaded Sample #4 3 06 x 10 j 1.56 X 10" .62 X 103 (1.10 X 103-8.84 X 103 ) Beaded Sample #1 2.51 X lOj (9.45 X 102-6.78 X 103 ) Beaded Sample #2 3.89xl03 (1.47 X 103-9. 78 X 103 ) Beaded Sample #3 3 39 X 10 j (1.22 X 103 -1.00 X 104 ) Beaded Sample #4 1.62x10J (4.68 X 1023 .39 X 103 ) Beaded Sample #5 8.67 X lOl 1.23 X 10" 1.24 X 103 (2.65 X 102 1.90 X 103 ) 55

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Hach A single trial was conducted with th e Hach IMS system using oocysts less than 2 months of age (Lot# 90914-21,30 days) (Figure 10, Table 13). The beaded and non-bea ded samples show similar infectivities to the control samples. Cryptosporidium parvum oocysts from Lot# 90914-21 resulted in an average of9.62 x 104 infectious oocysts MPN/mL ( 3 35 x 1 04 ) for the controls (n=2), 8.83 x 104 infectious oocysts MPN/mL ( 1.09 x 105 ) for the non-beaded samples (n=4), and 5.96 x 104 infectious oocysts MPN/ mL ( 3 .11 x 104 ) for the beaded samples (n=3). Figure 10 Effects of the Disassociation Step in the Hach IMS Kit on SDL C.parvum Oocyst Infectivity C/) 1 00E+03 ::J 0 .. (.) 1 00E+02 s::: -z c.. 1.00E+0 1 1.00E+OO Control1 Control 2 Beaded 1 Beaded 2 Beaded 3 Beaded 4 Non Non-NonNon-Beaded 1 Beaded 2 Beaded 3 Beaded 4 56

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_ Table 13. Data for Hach IMS Processe d C. parvum Oocysts (Lot# 909 14 21) Normalized MPN Average Standard (Infectious Oocyst/mL) Deviation Control! 7.24 x lOq (2 .28 X 104 -1.59 X lOS) Control2 1.20 x 10 ) 9.62 X 104 35 X 104 (3.45 X 104-3.03 X lOS) Non-Beaded Sample #1 5.92 X IOq (1.73 X 104 1.33 X lOS) Non-Beaded Sample #2 2.49 X 10 ) (8.96 X 1045.79 X 10 S ) Non-Beaded Sample #3 1.4 0 X 1 04 (5 .30 X 103 -3.53 x 1 04 ) Non-Beaded Sample # 4 3.07 X 104 8.83x104 1.0 9 X 10 s ( 1.10 X I 04 -7.15 X I 04 ) Beaded Sample # 1 1.00 X 1 0) (3.61 X 104-2.33 X lOS ) Beade d Sample # 2 6 .6 4 X IOq (2 .51 X 1 04 1.6 7 X lOS) Beade d Sample #3 4.06 X IOq (1.28 X 104 1.53 X 10S) Beaded Sample # 4 3.08x 104 5 96 X 104 .1 1 X 104 (8 92 X 1037 .43 X 1 04 ) Cryptosporidium parvum Aged IMS Study This study concentrated on the effect s of t he acid disassociation step in immunomagnetic separation (IMS) on age d ( > 102 days) Cryptosporidium parvum oocyst infectivity This prov i ded in sight on whether removing the bead was a necessary s t ep an d if this step altered oocyst infectivity. Ooocyst were bleach treate d before inocul atio n into the cell culture system. Contro l s were C. parvum oocysts and cu ltur e media inoculat ed into the cell cu l ture system without being proces sed by IMS Dynal Cryp t osporidium parvum oocysts older than two months (Lot# 909142 1 10 2 days) were processed by the Dynal IMS kit, and the resu lt s are shown in Figure 11 and 57

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Table 14. The control resulted in 2 06 x 103 infectious ooc y st s MPN / rnL, while the beaded and non-beaded samples resulted in 6.45 x 103 infectious oocysts MPN/mL and 2.17 x 103 infectious oocysts MPN/ rnL, respectively (CI in Table 14). Figure 11. Effects of the Disassociation Step in Dynal IMS Kit on Aged SDL C. parvum Oocyst Infectivit y 1 00E+05 :J E 1.00E+04 +:I Ill >. (.) 0 1.00E+03 0 T T T .l.. l. .L Ill :::l 0 .. (.) Q) -c: 1 00E+02 -z 1.00E+01 0.. 1 .00E+OO I Control Beaded Non-Beaded Lot#90914-21 ,102days Table 14. Data for Dynal IMS Processed Aged SDL C. parvum Oocysts Normalized MPN Confidence Interval (Infectious Oocyst/mL) (Infectious Oocyst/mL) Control 2.06 x to> (7.76 X 5 .56 X !OJ) Beaded Sample 6.45 X 103 (2.45 X 103 1.64 X 1 04 ) Non-Beaded Sample 2.17 X 1 OJ (2. 17 x IOJ ) 58

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Hach Cryptosporidium parvum oocysts older than two months (Lot# 90914-21, 102 day s) were processed by the Hach IMS kit, and the results are shown in Figure 12 and Table 15. The control resulted in 3.41 x 103 infectious oocysts MPN /mL, while the beaded and non-beaded samples resulted in 2.85 x 103 infectious oocysts MPN/mL and 1.82 x 104 infectious oocysts MPN/mL, respe ctive ly (CI in Table 15) Figure 12. Effects of the Disassociat ion Step in the Hach Kit on Aged SDL C. parvum Oocyst Infectivity 1. 00E+05 -g 1 00E+04 +:I 0 8 1 00E+03 1/1 ::I 0 :;:; 1 00E+02 1:: z 1 00E+01 1.00E+OO ..,.... l f Control T l I l .. Beaded Non-Beaded Lot# 90914-21, 102 days 59

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. Table 15. Data for Hach IMS Proce sse d Aged C. parvum Oocy sts Normalized MPN Confidence Interval (Infectious Oocyst/mL) Control 3.41 X I OJ (9 .19 X 102 -1.28 X 1 OJ) Beaded Sample 2 .85 x lOJ ( 1.0 8 X lOJ -7.15 X 10') Non-Beaded Sample 1.82 X 104 (5.92 X 1 03 -4 .0 0 X 104 ) IMS Study Summary The IMS studies u sing Dynal and Hach kits ill u s trated that th e acid disassociation step did not ha ve an effec t on young (<35 days) C. parvum oocyst infectivity. Although the aged oocysts ( > 102 days) res ulted in similar re s ults mor e samples should be examined. Beaded samp l es were compat ibl e with ce ll c ulture indicating that th e aci dic disassociation may not be necessary. EPA Information Collection Ru l e (I C R) This study examined the ICR collecting, processing and eluting steps and the effects the steps may have on you n g (<35 days) Clyptosporidium parvum oocyst infectivi ty. The contr o l s consisted of oocysts, whic h were not proc esse d by any s t eps of the ICR method. Figure 13 and Table 1 6 show the effects ofEPA ICR on SDL prepared Cryptosporidium parvum oocysts Lo t # 00104-12 a t a n age of 34 da ys. The control r es ult ed in 3.5 1 x 104 in fec ti ous oocysts MPN / mL whi l e th e two samples resulted in 8.38 60

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x 103 infectious oocysts MPN/mL and 1 .34 x 104 infectious oocysts MPN/mL having a standard dev i ation of 3 52 x 103 (n = 2) (CI included in Tab l e 16) Figure 13. Effects ofEPA ICR on C. parvum Oocyst Infectivity in Surface Wate r E rr o r B a rs= C J 1.00E+06 ::J E 1.00E+05 ':;:J 1/) >-0 1.00E+04 0 0 T I T ...... T -F ...... 1/) ::l 1.00E+03 0 :;:; 0 1 00E+02 c ! -z a.. 1 00E+01 ::!!: I"' 1.00E+OO Control Sample #1 Sample #2 (Lot# 00104-12 34 days) Table 16 Data for EPA ICR Processed C. parvum Oocysts in Surface Water Normalized MPN/mL Confidence Intervals Standard (Infectious Oo cys ts/ mL) (Infectiou s Oocysts / mL) De viat ion Control 3 .51 X 10 (8.24 X 1 OJ-1.20 X 1 N I A Sample #I 1.24 X 10' (1.97 X 1 OJ1.96 X 1 0_:} Sample #2 1.90 X 104 (3.89 X lOJ3.00 X 10 ) 52 X 10 j 61

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EPA Method 1623 This study examined EPA Method 1623 collecting, processing and eluting steps a nd the effects the ste ps may have on young ( <35 days) Cryptosporidium parvum oocyst infectivity. The controls consisted of oocysts, which were not processed by any steps of the ICR method Finished Water The results for finished water samples processed by EPA Method 1623 using SDL prepared Cryptosporidium parvum oocysts (Lot# 00918-26) at an age of33 days are shown in Figure 14 and Table 17. The control resulted in 2.97 x 106 infectious oocysts MPN/mL, while the two samples resulted in 5 .18 x 105 infectious oocysts MPN/mL and 6.22 x 104 infectious oocysts MPN/mL having a standard dev iatio n of 3 7 x 105 (n=2) (CI in Table 17). 62

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Figure 14 Effects ofEPA Method 1623 on C. parvum Oocyst Infectivity in Finished Water 1 00E+07 T ::J' 1 00E+06 J.. T <:JIUI uao;:, vi E -c;; -1 00E+05 T (.) .... 0 1.00E+04 0 Ill ::1 0 1.00E+03 :;::; (.) .! :. 1.00E+02 z c. 1 .00E+0 1 1 .00E+OO Control Sample #1 Sample #2 Lot# 00918-26 33 days Table 17 Data for EPA Method 1623 Processed C. parvum Oocysts in Surface Water Normalized Confidence Intervals Standard MPN/mL (Infectious Oocysts / mL) Deviation (Infectious Oocysts / mL) (lot# 00918-26, 33 days) Control 2 97 X 106 (l.OOx 106-6.63 x 10) N / A Sample #1 5 .18 X 105 (1.24 X 10:,1.68 X 106 ) Sample #2 6.22 X 104 (2 .0 3 X 104 1.37 X 10:,) .37 X 10 63

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Surface Water Surface water samp l es (10L) from Lake Starvation in Hillsborough County were seeded with Cryptosporidium parvum ooc ysts. The results of EPA Method 1623 processed Cryptosporidium parvum oocysts Lot# 00104-10 at an age of 32 days and Lot # 00918-26 at an age of25 days in surface water are shown in Figure 15 and Table 18. The two sets of experiments were not normalized to each other due to contributing variability factors (age of oocysts, lot number, etc.). Lot# 00104-10 resulted in 3 .51 x 104 infectious oocysts MPN / mL for the control, while the two samples resulted in 2 92 x 104 infectious oocysts MPN / mL and 1 72 x 104 infectious oocysts MPN / rnL having a standard deviation of .49 x 103 (n=2). Lot# 00918-26 resulted in 1.36 x 105 infectiou s oocysts MPN/rnL for the control, while the two samp les resulted in 4 .14 x 104 infectiou s oocysts MPN / rnL and 5.48 x 104 infectious oocysts MPN /mL having a standard deviation of.46 x 103 (n =2 ) All confide nce in tervals (CI) are in Table 18. 64

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Figure 15. Effects of EPA Method 1623 on C. parvum Oocyst Infectivity in Surface Water Error Bar = C l 1.00E+{)6 1 .00E+{)S ::J E Ui ... 0 1 00E+{)4 0 0 II) 1 .00E+{)3 :I 0 :;:; 0 s::: 1.00E+{)2 -z a.. ::!: 1 .QQE+{)1 1 .00E+{)O Control Sample #1 Sample #2 Control Sample #1 Sample#2 (Trial #1 00104-10, 32 days) (Trial #2 00918-26, 25 days) 65

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Table 18. Data for EPA Method 1623 Processed C. parvum Oocysts in Surface Water Normalized Confidence Intervals Standard MPN/mL (Infectious Oocyst s/ mL) Deviation (Infectious ooc ys ts/ mL) Lot#001 04-10, 32 days Control 3.51 X 104 (8.24 X 1 cY-1.20 X 1 0:>) NIA Sample #1 2.92 X 104 (1.10 X 104-7. 86 X 104 ) Sample #2 1.72 X 104 (4.86 X 10 J 4 .31 X 104 ) .49x 10j Lot# 00918-26, 25 days Control 1.36 X 10:> (4.89 X 104-3.16 X 10') N/A Sample #1 4.14 X 104 (1.49 X 1049.63 X 104 ) Sample #2 5.48 X 104 (2.07 X 104 1.38 X 10') .46 X 10j EPA ICR and EPA Method 1623 Summary The results for surface water seeded with C. parvum oocysts processed by EPA ICR were inconclusive. Finished water seeded with C. parvum oocy s ts processed by EPA Method 1623 also led to inconclusive results. However, surface water processed by EPA Method 1623 indicated no effect on young, laboratory prepared C. parvum oocyst infectivity. Environmental Samples With the previous studies indicating minimal effects on C. parvum oocyst infectivity, environmental samples were proce ss ed by these methods. Environmental samples were taken from the following locations: reclaimed effluent from North Dale Water Treatment Plant in Hillsborough County, Florida; surface water from Lake Starvation in Hillsborough County, Florida; boil site in Tampa Bay from the Howard F. Curren Wastewater Treatment Plant in Hillsborough County, Florida; 66

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water after the nitrification stage from the Howard F. Curren Wastewater Treatment Plant in Hillsborough County, Florida; and water after dechlorination from the Howard F Curren Wastewater Treatment Plant in Hillsborough County, Florida No oocysts were detected in any of the samples except in the post-nitrification sample where 30 oocysts / 100 vol were found by IF A microscopy and at least 60 noninfectious oocysts MPN/ mL by cell culture (Table 19). The oocysts detected by cell culture had no int erna l structures, only the apple-green sphere was visualized. Table 19. Oocyst Detection by IF A and Cell Culture Data for Environmental Samples Sample Site Salinity/pH Volume IFA Cell Culture Filtered ( oocysts / 1 OOL) (oocysts / 100L) Reclaimed 400 + liters < 0.50 < 1.00 Effluent Lake Starvation 100+ liters < 2 00 < 4 00 Boil Site 22.5/7.7 20 liters < 10.0 < 20.0 Nitrified 0 / 7.6 20 liters 30 0 60 0 Chlorinated 0 1 8.3 20 liters < 10.0 < 20 0 did not measure Environmental Sample s Summary Cell culture proved to be compatible with EPA ICR, therefore allowing environmenta l samples to be analy ze d by an infectivity assay However, it was found that beads should be remo ve d as to not block any possible oocysts. It appears that IFA is more sensitive but i f an equivalent pellet vo lume were processed by the FDM-MPN the sens itivit y should be similar. 67

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Chapter Four: Discussion and Conclusions Preparation and Treatment of Oocysts Studies on the infectivity of C. parvum oocysts are now possible with advent of cell culture However the standard preparation and treatment of oocysts for use in cell culture and variability issues regarding the age and the lot of oocysts had not been previously developed and studied. This research examined the variability of oocyst preparations from two vendors who use different purification methodologies, and three different treatment procedures for oocysts prior to cell culture Sterlin g Diagnostic Laboratory (SDL) and Pleasant Hill Farms (PHF) we r e the two vendors who supplied C. parvum oocysts for this research. Animal diet fecal collec t ion and storage conditions are important factors regarding the quality of oocysts. The fecal collection (i .e. preparation, purification of the oocyst) procedures of disCOJ:!tinuous sucrose and cesium chloride gradients for SDL and diethyl ether followed b y sheathers sucrose gradients for PHF are related to selected calves diet of oral electrolytes and milk, respectively. SDL uses oral electrolytes so as to decrease the amount of lipid s in the fecal material, while PHF uses a milk diet, which reportedly supports a high e r oocyst yield. SDL stores the C. parvum oocysts in 0 001 % tween 20 with 100 U ofpenicillin, 100 ug of streptomycin, and 100 ug ofgentamycin, while PHF stores the oocysts in PBS containing 1000 U of penicillin and 1000 U of streptomycin. 68

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These factors influenced how the vendors' C. parvum oocysts responded to each treatment. Sterling Diagnostic Laboratory prepared oocysts were found to contain bacterial and fungal contamination. While bleach treatment successfully eliminated microorganisms' contamination of the cell monolayer, antibiotic and no treatment resulted in 42 to 58% contamination of wells. The antibiotics that the oocysts are shipped in did not eliminate bacterial and fungal contamination sufficiently. The contamination in Pleasant Hill Farms (PHF) prepared oocyst samples was minimal and their preparation was adequate, such that no treatment was needed prior to inoculation into cell culture. The higher concentration of antibiotics in the storage solution, the preparation u si ng diethyl ether, or both may play a role in microbial "cleanliness" of the PHF oocysts. SDL samples were successfu l only with th e bleach tr ea tment therefore no comparisons can be made to the other treatments. It was only after storage of 174 days that no contamination occurred for SDL oocysts in the antibiotic and no treatments Treatment of oocysts prior to inoculation may not be desired for an experiment, therefore PHF 5>ocysts could be used at any age, while SDL oocysts should be stored for at least 1 7 4 days (based upon this stu dy) in the antibiotic solution at 4 oc to assure no con tamination in the cell culture system. Bleach (10% vol / vol) has been commonly used in laboratories as a treatment for oocysts prior to cell culture inoculation (Upton et al. 1994, Griffiths et al. 1 994, Villacorta et al. 1996, Yang et al. 1 996, Slifko eta!. 1997 and 1999, and D en and Cliver 1998) and was developed primarily as a anti-microbial tool (Upton et al. 1994) as well as to maximize excystation. The summary of excystation studies showe d that sodiu m 69

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hypochlorite (bleach) resulted in comparatively higher percentages of excystation ( -40% to 70%) than other conditions (Rose et al. 1990) However, bleach treatment has been criticized as being too "harsh" in that it may lead to an underestimation of viability Research has shown that bleach treatment separates the inner and outer oocysts walls rendering the oocyst vulnerable to excystation when warmed to 37C (Upton 1997). While premature excystation could occur, Fayer et al (1995) showed that oocysts left in undiluted bleach for 120 minutes were still infectious based on animal infectivity, however this study was not quantitative Antagonistic or synergistic effects could occur when using bleach and the cell culture system along with other factors affecting the oocysts (i.e. age, preparation) Environn1ental oocysts are assumed to be somewhat aged and less hardy than the fresh laboratory prepared oocysts due to the impacts of sunlight, physical action, and biological predation. The aged laboratory oocysts for both vendors were subjected to the three treatments for insight on age degradation relating to environmental oocysts These samples experienced minimal contamination and had similar infectivities, regardless of prep3._!"ation or treatment. C. parvum oocysts are of fecal origin and naturally some bacteria will survive during the purification procedure and the storage in antibiotic solutions However, as already stated a low percentage of contamination was found after C. parvum oocysts were stored The decreased contamination could be attributed to the die-off of fecal bacteria. When assessing the best treatment for oocysts introduced into the FDM-MPN assay, maximum excystation is desired. Oocysts from the two vendors showed a slight increase with bleach treatment compared to the antibiotic and no treatments, with some 70

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lot to lot variation in PHF Lot# 99-3. For example, bleach treated oocysts had at least 1.00 log10 more infectious oocysts MPN/mL (Lot# 99-3, 29 days) than the oocysts tre a ted by antibiotics and nothing This was not statistically significant when considering other preparations and a g es Also this trend was not apparent in other PHF oocysts (2, 3 174 and 200 days). SDL oocysts required bleach treatment for successful cell culture evaluation. For both e x cystation and disinfection, bleach treatment provided the most efficient procedure for the FDM-MPN. No antagonistic effects with bleach preparation or age of oocysts were shown Bleach treatment can therefore be supported as a standard treatment to eliminate indigenous bacteria and fungus found in oocyst preparations prior to inoculation into cell culture for all studies conducted in this research. C. parvum is a coccidian parasite but unlike its nearest relatives (Emeria, Toxoplasma, etc ) the oocyst does not need to undergo sporulation and is immediately infe c tiou s upon excystation. However it has been suggested that a slight maturation in the environment is needed for the population to maximize infectivity and then a decrease in infectivity occurs over time. Campbell et al (1992) used two fluorogenic dyes 4 ,6diami?ino-2-phenylindole (DAPI) and propidium idodide (PI) as a measure of viability through the inclusion (DAPI) or exclusion (PI) of the dyes in the oocysts. The study s uggested that the two populations of oocysts could be found in any given preparation (Campbell et al. 1992) One group of oocysts was found to excyst and stain (takes up the DAPI) without prestimula t ion; and the second group needed an acid trigger before becoming DAPI penneable (Campbell et al. 1992). In this case, the treatment was exposur e to acidified HBSS for 1 hour at 37 C (Campbell et al. 1992) The permeability of the oocyst will depend on the isolate therefore affecting the oocysts ability to include 71

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or exclude DAPIIPI and its ability to withstand environmental pressures (Campbell et al. 1 992). Both oocyst populations would presumably excyst in-vivo when ingested due to the natural acidic trigger within the digestive system (Campbell et al. 1992). Therefore, to simu lat e this environment it was suggested that several steps were necessary ; (Campbell et al. 1992). The sample should be incubated in a dilute acid for at least 1 hour and then incubated with bile salt or sodi um deoxycholate in sodium hydrogen carbonate for 4 hours, both at 37C (Campbell et al. 1992). This second population of oocysts may mature with time and thus not require this acid treatment in order to excyst. Slifko et al (1999) found the maximum oocyst infectivity at ages between 10 to 30 days and that fresh oocysts ( < 7 days old) had a significantly lower infectivity (MPN / mL) compared to those 1 0 days or older. A steady drop in infectivity followed over the next 30 days (Slifko et al. 1999). Unpu blished data by Rochell e et al (1999) also supported a decrease in oocyst infectivity after 60 days, and this was confirmed in this pretreatment study. The research described here showed a peak in infectivity with C. parvwn oocysts at days in SDL and PHF oocysts with all lots. However, Sterling Diagnostic Laboratory (SDL) oocysts infectivity was more consistent from lot to lot as the Clyptosporidium parvum oocysts aged than the PHF oocysts. SDL C. parvum oocysts had a significant difference between 1 to 30 days old oocysts compared to 60 to 90 + day old oocysts by Bartlett's and Scheffe's statistical tests. PHF oocysts did not illustrate any significant difference between age groups This could be attributed to the high variability between oocyst lots. 72

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PHF prepared oocysts have proved to contain less microbes that will cause contamination in the cell culture system, however the variability between lots could make comparison of data difficult. SDL prepared oocysts avoid contamination when bleach treated or stored over 174 days and also have an understood aging process. The Cryptosporidium parvum oocysts prepared by SDL illustrate minimal variability between lots; one advantage when comparing data from experiment to another. Methods for Oocysts Recovery New methods are now available for recovery and detection of oocysts from water. These include new types of filters such as the polysulphone capsule filter and new concentration methods, such as IMS. Each of these recovery procedures involves concentration steps that may affect oocyst viability. IMS has a s tep in which an acidic solution is required to remo v e the beads from the oocysts. The Dynal IMS kit uses 0.1 N HCl while the Hach IMS kit provided a proprietor's acidic reagent which contained 0.1 N HCl. DiGiovanni et al ( 1999) found that 0.1 N HCl caused a significant drop in using ICCPCR, however this research and research by Rochelle et al (1999) did not. Thi s research found that IMS (Dynal and Hach) had no impact on the infectivity of C. parvum oocysts whether the beads we re attached o r not nor was impacted by age. IMS is primarily used for environmental samples. It efficiently recovers the specific target and is able to provide a very c lear sample free from most algal cells and other debris that would otherwise interfere with microscopic identification of the C. parvum oocysts. Dynal IMS has been reported to recover all oocysts regardless of viability 73

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(Campbell, correspondence), assuring confidence that if oocysts were damaged during processing from EPA ICR or EPA Method 1623 they would be recovered and analyzed in cell culture Despite the opportunity to exclude the bead disassociation step, as beads did not interfere with the FDM-MPN cell culture system, it was best to remove beads in environmental samples The high concentration of IMS beads may have interfered detection of C. parvwn oocysts, which are present in low concentrations in environmental samples. The eluting solutions for EPA ICR and Method 1623 are necessary to recover the oocysts from the filter fibers. Lauryl sulfate (SDS) and Laureth-12 are the primary detergent reagents in the eluting solutions for EPA ICR and Method 1623, respectively. The differences between the two reagents are that lauryl sulfate is an anionic solution with a positive charge, while Laureth-12 is a non-ionic solution. The Laureth-12 eluting solution was specifically developed for the Envirochek filter used in EPA Method 1623, which showed higher recoveries than the original Gelman buffer (Matheson et al. 1998) The IMS study represented the effect of the capture / disassociation step in EPA 1623 and showed that this step does not alter the infectivity of C. parvum oocysts. Thus, in the EPA ICR and EPA Method 1623 studies one could ascertain what effect the elution and concentration steps would have on the oocysts Evaluating EPA ICR and EPA Method 1623 effects on oocyst infectivity involved seeding with a high oocyst concentration to allow for sufficient dilutions used in the FDM-MPN assay. By dividing the original MPN by the hemacytometer, the percent infectivity was calculated to determine these concentration methods altered the infectivity of the oocysts compared to the controls. SDL oocysts used in this research had an overall 74

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percent infectivity ranging from .09% to 24%, while PHF oocysts ranged from 013% to 9%. Previous research in our laboratory has shown that PHF oocysts are usually more infectious, however most of this described research used SDL oocysts The percent infectivties for PHF oocysts ranged from 3 1% to 12.2% in another study (Slifko et al 1999). The data for EPA ICR and EPA Methods 1623 was adjusted from infectious oocysts MPN /mL to percent infectivity for evaluation. The FDM-MPN was originall y designed for survival and disinfection studies. Therefore, high concentrations of oocysts are used for dilution series and for determination of log losses The concentration of oocysts used in this research were adjusted to have at least 1 x 105 oocysts / mL for an accurate hemacytometer count of oocysts before inoculat i on (Linquist et al. 1999). EPA ICR preliminary samples indicate no effects from the elution and concentration steps. The percent infectivity for the control had a confidence limit from 1.19% to 17.3% (mean=5.1 %) while sample 1 and sample 2 overlapped this percent recovery with upper limits of2. 8% (mean = 1.2%) and 4.35% (mean = l.9%) respectively. results indicate that EPA ICR had no effect C. parvwn oocyst infectivity, however, with the low number of samples processed mor e samples should be examined EPA Method 1623 surface water control samples had confidence limits ranging from 1.3% to 19.3% (mean = 5 7%) The surface water samples processed by Method 1623 (n = 4) all had similar percent infectivities In the finished water study, control oocysts had resulted in a percent infectivity ranging from 27% to 179% (mean=80%) for the control. The first finished water sample ranged from 3 35% to 45.4% (mean=l.4%), therefore overlapping the controls percent infectivity. However, the second finished 75

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water sample resulted in 0.55% to 3.7% infectivities (mean=l.7%). This drop shown in the second sample could be due to the eluting solutions or concentration techniques. EPA Method 1623 uses a Laureth-12 detergent for elution and speculations of oocysts excysting while in the detergent have been raised. More research is needed in this a r ea of study as this study represents a preliminary investigation of the complete method. Environmental Samples This work did not focus on environmental sampling however with the variability and storage conditions defmed this is now possible. The samples taken around Hillsborough County in Florida did not contain any infectious Cryptosporidium oocysts Only the treated-nitrified waste water samples contained oocysts which were non infectious according to cell culture (pg 61) The species cannot be determined due to the non-species specific fluorescent antibodies used. Xiao et al (2001) reported that the dominant Cryptosporidium oocysts recovered from surface waters were C. parvum by a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) This recent finding places the FDM-MPN as a useful tool in assessing the infectivity of environmental oocysts. Chauret et al (1998) conducted a novel study to evaluate oocyst survival. The viability of oocysts stored in river water (0 to 0.5C) containing bacterial contamination of 5 x 104 CFU / mL was compared to oocysts stored in tap water. Viability was assessed through modified excystation using HBSS for 1 hour at 37 C followed by a 4 hour incubation in a PBS solution containing trypsin and taurocholic acid. The number of 76

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viable oocysts in the natural waters dropped by 0.5 log10 within the first 10 days but leveled off for the next 40 days, while oocysts in tap water had a similar number of viable oocysts throughout the measured 50 days Although results indicate that a percentage died within the first 1 0 days, the remaining oocysts were as resistant as the oocysts stored in tap water. Most of the chemical and physical stressors did not have an affect on C. parvum survival, however biological antagonism posed the largest influence on oocyst survival in natural waters (Chauret et al. 1998) While this study presents some interesting findings the methods to determine infectivity may have overestimated the true infectivity. Cell culture FDM-MPN would allow enumeration of the infectious oocysts, not just the potential for infectivity and this research would suggest that even under best circumstances (optimal storage 4 C, with antibiotics) that die off becomes greater after 30 days of age. Some research has sugges ted that s torage in feces prior to purification in preferable. Cell culture can now be used as an efficient tool to evaluate the effects of envirorunental monitoring methods on the infectivity of Cryptosporidium parvum oocysts Final Conclusions Preparation procedures may have an effect on the variability between lots of C. parvum oocysts, as shown by PHF oocyst lots compared to SDL oocyst lots Bleach treatment appears to be the most efficient treatment method regardless of purification proces s and age of C. parvum oocysts 77

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PHF and SDL prepared C. parvum oocysts decrease in infectivity after 60 days IMS acid disassociation step in Dynal and Hach kits does not appear to affect the infectivity of C. parvum oocysts EPA ICR preliminary results indicate that the elution and concentration steps do not affect the infectivity of C. parvum oocysts in surface water samples EPA Method 1623 preliminary results indicate that the elution and concentration steps do not affect the infectivity of C. parvum oocysts in surface water samples but may affect oocysts in finished water samples Cell culture is compatible with monitoring techniques such as EPA ICR and EPA Method 1623, and may serve a s an infectivity assay for the detection of infectiou s environmental C. parvum oocysts The cell culture FDM-MPN assay has proven to be a useful tool and is compatible w ith current techniques This research has further demonstrated applicability of combining recovery procedures w i th the FDM-MPN to allow for the detection of infectious of C. parvum oocysts providing a protocol that would benefit the government water industry, and most of all, the public. 78

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References Arrowood, M.J. and Sterling, C.R. 1987. Isolation of Cryptosporidium Oocysts and Sporo zo ites Using Discontinuous Sucrose and Isopycnic Percell Gradients Journal of Parasitology. 73(2):314-319. Bongard et al, 1994. MMWR.43:561-563. Bukhari, Z and Smith, H.V. 1995 Effect ofThree Concentration Techniques on Viability of Cryptosporidium parvum Oocysts Recovered from Bovine Feces Journal of Clinical Microbiology. (33)10:2592-2595 Bukhari, Z., R.M. McCuin, C.R. Fricker, J L. Clancy. 1998. Irnmunomagnetic Separation of Cryptosporidium parvum from Source Water Samples of Various Turbidities AEM (64)11 :4495-4499 CCN, 1996a. Anonymous Cryptosporidium Capsule Newsletter (CCN). 2(2) :1112 CCN, 1997. Anonymous Cryptosporidium Capsule Newsletter (CCN). 2(11):7-8. CCN, 1998a. Anonymous. Cryptosporidium Capsule Newsletter (CCN). Outbreak in Texas-sewage Leak Suspected as a Source of Groundwater Contamination. 3(10):1-2 CCN, 1998b. Anonymous. Cryptosporidium Capsule Newsletter (CCN). 4(1) :1-2. CCN, 1998c. Anonymou s Oyptosporidium Capsule Newsletter (CCN) 3(5):4-3 Campbell, A., L.J Robertson, H.V. Smith. 1992. Viability of Cryptosporidium parvum Oocysts : Correlation of In Vitro Excystation with Inclusion or Exclusion of Fluorogenic Vital D yes AEM 58(11):3488-3493. Chauret, C., K Nolan, P. Chen, S. Springthorpe, S Sattar. 1998. Aging of Cryptosporidium parvum Oocy s ts in River Water and Their Susceptibility to Disinfection by Chlorine and Monochloramine. Canadian Journal of Microbiology 44:1154 1160. Clancy, J.L., Z. Bukhari, R.M. McCuin, Z. Matheson, C.R. Fricker. 1999a. USEPA Method 1622 J ournal AWWA (91)9 : 60-68. 79

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Clancy, J.L. and Hansen, J. 1999b. Use ofProtozoan Monitoring Data Journal AWWA. (91)5:51-65. Clancy, J.L., W.D. Gollnitz, and Z. Tabib. 1994. Commercial Labs: How Accurate Are They? Journal AWWA (86)5 : 89-97. Corning, 1996. Staining Microorganisms for Epifluorescent Detection and Enumeration. Coming Incorporated Separations Division. Corning Star Corporation. USA. Connell, K., C. C. Rodgers H.L. Shank-Givens, J. Scheller, M.L. Pope, and K. Miller. 2000. Building a Better Protozoa Data Set. Joumal A WW A. 92( 1 0):3043 Craun, G.F., S.A. Hubbs, F Frost, R.L. Calderson, and S.H. Via. 1998. Waterborne Outbreaks ofCryptosporidiosis. Journal AWWA (90)9 : 81-91. Current, W.L., 1987. The Biology of Cryptosporidium. 2ih Interscience Conference on Antimicrobial Agents and Chemotherapy New York, New York. DiGiovanni, G.D., F.H. Hashemi, N.J. Shaw, F .A. Abrams, M.W. LeChevallier, and M. Abbaszadegan. 1999. Detection of Infectious Cryptosporidium parvum Oocysts in Surface Water and Filter Backwash Samples by Imrnunomagnetic Separation and Integrated Cell Culture-PCR AEM (65)8:3427-3432 Deng, M.Q. and Cliver, D.O., 1998. Cryptosporidium parvum Development in the BS C-1 Cell Line. Journal of Parasitology 84(1):8 15. EPA, 1995. EPN814-B-95-003 EPA Protozoan Method for Detecting Giardia Cysts and Cryptosporidium Oocysts in Water by a F luor escent Antibody Procedure. USEPA. Office of Ground Water and Drinking Water. June 1995. EPA, 1997. EPA 821R -97-0023. Method 1622: Cryptosporidium in Water by Filtration/IMS/F A. USEP A. Office of Water. December 1997. Draft. EPA, 1999. EPA-821-R 99-006 Method 1623 : Cryptosporidium and Giardia in Water by Filtration/IMS/FA. USEPA. Office ofWater. April 1999 Fayer, R. and Ungar BLP, 1986. Cryptosporidiwn spp. And Cryptosporidiosis. Microbiological Review. 50:458-483 Fayer, R. 1995. Effect of Sodium Hypochlorite Exposure on Infectivity of Cryptosporidium parvum Oocysts for Neonatal BALB / c Mice. AEM. 6 1(2):844846 80

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Fayer, R, U. Morgan, and S.J. Upton. 2000. Epidemiology of Cryptosporidium: Transmission, Detection, and Identification. International Journal for Parasitology. (30) 1305-1322. Fox, KR. and Lytle, D.A. 1996. Milwaukee's Crypto Outbreak : Investigation and Recommendations Journal A WWA. (88)9 : 87-94. Griffiths, J.K., R. Moore, S. Dooley, G.T. Keusch, and S. Tzipori. 1994. Cryptosporidium parvum Infection of Caco 2 Cell Monolayers Induces an Apical Monolayer Defect, Selectively Increases Transmonolayer Permeability, and Causes Epithelial Cell Death. Infection and Immunity 62(10):4506-4514. Hancock, C.M., J.B. Rose, and M. Callahan. 1998. Crypto and Giardia in US Groundwater. Journal AWWA (90)3:58-61. Hopkins et al, 1997. Journal of Florida Medical Association. 84:44 1-445 Jakubowski,W., S. Boutros, W Faber, R Fayer, W. Ghlorse, M. LeCheavllier, J Rose S. Schwab, A. Singh, and M. Stewart. 1996. Environmental Methods for Cryptosporidium Journal AWWA. 88(9):107-121. Jakubowski, W. 2000. The Development and Status ofTissue Culture Assay Methods for Crypto s poridium in Wat e r WQTC Measuring Disinfection of Cryptosporidium : Cell Culture and Other In-Vitro Methods Workshop (S5) Salt Lake City, Utah. November 5 -9, 2000 Johnson, D.C., KA. Reynolds C.P. Gerba, I.L. Pepper, and J.B. Rose. 1995. Detection of Giardia and Cryptosporidium in Marine Waters Water Science and T e chnology. 31 (56):439 442. Klee, A.J., 1999. ICR Most Probable Number Calculato r Ver s ion 1.00 [online]. Risk Reduction Laboratory, U.S Environmental Protection A g ency Cincirmati Ohio http: / /www .epa gov / microbes Korich, D.G. J.L. Clancy, C. Fricker, M.M. Marshall, and H.V. Smith. 1997. Comparison of Crypto s poridium Viability/Infectivity Methods. WQTC Proce e dings. AWWA WQTC Water Technology Confemce Proceedings November 9-12 1997 Denver, Colorado Kozwich, D. K.A. Johansen, K. Landau, C.A. Roehl, S. Woronoff, and P.A. Roehl. 2000 Development of a Novel Rapid Integrated Crypto s poridium parvum Detection Assay AEM. 66(7) : 2711-2717. Kramer et al, 1998. Clinical Infectious Diseases. 26:27 -33. 8 1

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LeChevallier, M.W., W.O. Norton, and R.G. Lee. 1991. Giardia and Cryptosporidia spp In Filtered Drinking Water Supplies. Journal AWWA 57(9):2617. LeChevallier, M.W. and Norton, W.D., 1995. Giardia and Cryptosporidium in Raw and Finished Water. Journal A WWA. 87:54-68. LeChevallier, M.W., W.D. Norton, and T.B. Atherholt. 1997. Protozoa in Open Reservoirs. Journal AWWA. 89(9):84 -96 Lindsay, D. 1997. Laboratory Models of Cryptsporidium. Cryptosporidium and Cryptosporidiosis Fayer, R. (editor) CRC Press. pg. 209-223. Linquist, A., A. Dufor, L.J. Wymer, and F.W. Schaefer Ill. 1999. Criteria for Evaluation of Proposed Proto z oan Detection Methods. Journal of Microbiological Methods.37:33-43 MacKenzie, W et al, 1994. A Massive Outbreak in Milwaukee of Cryptosporidium Infection Transmitted Through the Public Water Supply. New England Journal of Medicine 331:161-167. Matheson, Z., T.M. Hargy, R.M. McCuin, J.L. Clancy, and C.R. Fricker. 1998. An Eva lu a tion of the Gelman Enviroch ek Capsule for th e Simulutaneous Concentration of Cryptosporidium and Giardia From Water. Journal of Applied Microbiology.85:755-761. Medema, G.J. et al, 1997. Survival of Cry ptosporidium parvum Escherichia coli, Faecal Ent erococcii and Clostridium p erfrige ns in River Water: Influence of Temperature and Autochthonous Microorgansims Water Science and Technology. (35) 11-12 : 249-252 Moore, A. et al, 1993. Surveillance for Waterborne Disease Outbreaks-United States 1991-1992 MMWR (#SS-5). Center for Disease Control and Prevention. Atlanta Georgia. 42 : 1-22. Morgan, U.M. et al, 2000. Molecular Characterization of Cryptosporidium Isolates Obtained From HIV -infected Individuals Living in Swit z erland, Kenya, and the USA. Journal of Clinica l Microbiology (38)1180-1183 Moulton-Hancock, C.M., J.B. Rose G.J. Vasconcelos, S.I. Harris, P .T. Klonicki, and G.D Sturbaum. 2000. Giardia and Cryptosporidium Occurrence in Groundwater Journal AWWA. 92(9):117-123. Peinazek, N.J., F.J. Borney-Liinares, S.B. Slemehda, A.J. da Silva, I.N.S. Moura, M.J. Arrowood, 0. Ditrich, and D.G. Addiss. 1999. New Cryptosporidium genotypes in HIV infected Persons. Emerging Infectious Diseases (5)444-449. 82

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Riggs, M.W. and Perryman, L.E., 1987. Infectivity and Neutralization of Cryptosporidium parvum Sporozoites. Infection and Immunity. 55: 2081. Pontius, F.W. and Clancy, J.L., 1999. ICR Crypto Data: Worthwhile or Worthless? Journal AWWA. 91(9) : 14-22. Reagan, J.M.R. et al, 1996. Outbreak ofCryptosporidiosis at a Day Camp-Florida, July August 1995 Infect Med. 13:726-728. Rochelle, P.A., R. de Leon, A. Johnson, M. Stewart, R.L. Wolfe. 1999. Evaluation of Irnmunomagnetic Separation for Recovery of Infectious Cryptosporidium parvum Oocysts from Environmental Samples. AEM. (65)2:841-845. Roefer, P .A., J T. Monscvitz, and D.J. Rexing. 1996. The Las Vegas Cryptosporidiosis Outbreak. JA WWA. (88)9:95-1 06. Rose,J.B, 1990. Occurrence and Control of Cryptosporidium in Drinking Water. Drinking Water Microbiology. McFeters (editor). Springer -Ve lag New York pg 294-321. Rose, J.B., C P. Gerba, and W. Jakubowski. 1991. Survey of Potable Water Supplies for Cryptosporidum and Giardia. Environmenta l Scienc e and Technology (25)8 : 1393. Rose, J.B. 1997. Environmental Ecology of Cryptosporidium and Public Health Implications. Annual Review of Public Health. (18)135-161. Robertson, L., A.T. Campbell, and H.V. Smith. 1992. Survival of Cryptosporidium parvum Oocysts Under Various Environmental Pressures AEM (158) 3494-3500. Sauch, J.F. et al, 1991. Propidium Iodide as an Indicator of Giardia Cyst Viability. AEM. 57 : 3243-3247. Slifko, T.R., D. Freidman, J.B. Rose, and W. Jakubowski. 1997. An In Vitro Method for D etecti ng Infectious Cryptosporidium Oocysts with Cell Culture. AEM. (63)9 : 3669-3675. Slifko, T.R., D.E. Huffman, J.B. Rose 1999. A Most Probable -Numbe r Assay for Enumeration of Infectious Crptosporidium parvum Oocysts AEM (65)9:39363941. Smith, H.V. and Rose, J B., 1997. Waterborne Cryptosporidiosis : Current Status. Parasitology Today. 14(1):14-22. Solo-Gabriele, H. and Neumeister, S. 1996. US Outbreaks of Cryptosporidiosis JAWWA (88)9:77-86 83

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Sorvillo, F.J. et al, 1992. Swimming Associated Cryp t osporidiosis. Amer ican Journal of Public Health. 82:742-744. Upton, S.J., M. Tilley, and D.B. Brillhart. 1994a. Comparative Development of Cryptosporidium parvum in MDBK and HCT-8 Cells Under Select Atmospheres. Biomedical Lett e rs. 49:265-271. Upton, S.J., M Tilley, and D.B. Brillhart. 1994b. Comparative Development of Cryptosporidium parvum (Apicomplexa) in 11 Continuous Host Cell Lines. FEMS Microbiology Letters 118:223-236. Upton, S.J., 1997. In Vitro Cultivation. Cryptosporidium and Crvotosporidiosis. Fayer R (editor). CRC Press 181-207 Villacorta, I., D Graaf, G. Charlier, and J.E. Peeters. 1996 Complete Development of Cryptosporidium parvum in MDBK Cells FEMS Microbiology Letters (142)129-132 Wilberscheid, 1995. Kansas Medicine 96:67-68 Xiao, L., A Singh, J. Limor, T.K. Graczyk, S. Gradus, and A. Lal. 2001. Molecular Charateri z ation of Cryptosporidium Oocy s ts in Samples of Raw Surface Water and Wastewater. AEM 67(3):1097-1101. Yang, H., M.C. Healey, C. Du and J. Zhang. 1996. Complete Development of Cryptosporidium parvum in Bovine Fallopian Tube Epithelial Ce lls. Infection and Immunity 64(1 ):349 -354. 8 4

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Appendices 85

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Appendix A: Parameters for Experiments Experiment Purification Treatment Lot# Oocyst Age Preparation SDL (pg. 1 8) Bleach (pg. 20) 90105-12 15 and Treatment Study (pg. 20) 90803 -0 7 12 90803-07 15 Antibiotics (pg. 21) 90105-12 15 90803-07 12 90803-07 15 No Treatment (pg. 21) 90105-12 15 90803-07 12 90803-07 15 PHF(pg.19) Bleach 99-3 29 99-16 2 99-16 3 Antibiotic 99-3 29 99-16 2 99-16 3 No Treatment 99 3 29 99-16 2 99 16 3 Aged SDL Bleach 90803-07 174 Purification and Treatment Study (pg. 21) 90706-11 200 -Antibiotics 90803-07 174 90706-11 200 No Treatment 90803-07 174 90706-11 200 PHF Bleach 99-16 166 99 14 185 Antibiotic 99-16 166 99 -14 185 No Treatment 99 14 185 86

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Appendices A: (Continued) Experiment Purification Treatment Lot# Oocyst Age Age Study SDL Bleach 90105-12 15 (pg. 25) 22 28 35 41 49 58 62 78 92 107 90601-06 10 23 37 76 94 00201-10 7 13 20 26 34 48 56 64 78 PHF 99-9 11 26 40 79 94 99-3 29 34 44 58 73 120 87

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Appendices A: (Continued) Experiment Purification Treatment Lot# Oocyst Age Age Study PHF Bleach 00-5 4 10 17 23 31 45 53 61 75 Dynal IMS SDL Bleach, Control 1 90914-21 35 (pg. 25) Bleached, Control2 Bleached, Non-Beaded 1 Bleached, Non-Beaded 2 Bleached, Non-Beaded 3 Bleach ed, Non-Beaded 4 Bleached, Beaded 1 Bleached, Beaded 2 Bleached, Beaded 3 Bleach, Control 1 00619-25 19 Bleached, Control 2 Bleached, Non-Beaded 1 Bleached, Non-Beaded 2 Bleached, Non-Beaded 3 Bleached, Non-Beaded 4 Bleached, Beaded 1 Bleached, Beaded 2 Bleached, Beaded 3 Bleached, Beaded 4 Bleached, Beaded 5 Hach IMS Bleach, Control 1 90914-21 30 (pg. 28) Bleached, Control 2 Bleached, Non-Beaded 1 Bleached Non-Beaded 2 Bleached, Non-Beaded 3 Bleached, Non-Beaded 4 Bleached, Beaded 1 Bleached, Beaded 2 Bleached, Beaded 3 88

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Appendices A : (Continued) Experiment Purification Treatment Lot# Oocyst A2e EPAICR SDL (pg.x) Bleached, Control 00104-12 34 (pg 29) Bleached Sample 1 Bl eached, Sample 2 EPA 1623 Bleached Control 00918-26 33 (pg 30) Bleached Sample 1 Bleached, Sample 2 89


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