Michael W. Ware

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

ABSTRACT Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay.

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.

Autofluorescence of Toxoplasma gondii and Related Coccidian Oocysts

This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.

Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water by Using U.S. Environmental Protection Agency Method 1623

ABSTRACT U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the methods recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.

Comparison of filters for concentrating microbial indicators and pathogens in lake-water samples

ABSTRACT Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

Low pressure ultraviolet studies for inactivation of Giardia muris cysts

Aims: The research was initiated to confirm earlier ultraviolet (u.v.) light inactivation studies performed on Giardia cysts using excystation as the viability indicator. Following this, a comparison of in vitro excystation and animal infectivity was performed for assessing cyst viability after exposure to low‐pressure u.v. irradiation.

Predictive models for Escherichia coli concentrations at inland lake beaches and relationship of model variables to pathogen detection.

ABSTRACT Predictive models, based on environmental and water quality variables, have been used to improve the timeliness and accuracy of recreational water quality assessments, but their effectiveness has not been studied in inland waters. Sampling at eight inland recreational lakes in Ohio was done in order to investigate using predictive models for Escherichia coli and to understand the links between E. coli concentrations, predictive variables, and pathogens. Based upon results from 21 beach sites, models were developed for 13 sites, and the most predictive variables were rainfall, wind direction and speed, turbidity, and water temperature. Models were not developed at sites where the E. coli standard was seldom exceeded. Models were validated at nine sites during an independent year. At three sites, the model resulted in increased correct responses, sensitivities, and specificities compared to use of the previous days E. coli concentration (the current method). Drought conditions during the validation year precluded being able to adequately assess model performance at most of the other sites. Cryptosporidium, adenovirus, eaeA (E. coli), ipaH (Shigella), and spvC (Salmonella) were found in at least 20% of samples collected for pathogens at five sites. The presence or absence of the three bacterial genes was related to some of the model variables but was not consistently related to E. coli concentrations. Predictive models were not effective at all inland lake sites; however, their use at two lakes with high swimmer densities will provide better estimates of public health risk than current methods and will be a valuable resource for beach managers and the public.

Determining UV inactivation of Toxoplasma gondii oocysts by using cell culture and a mouse bioassay.

ABSTRACT The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV-irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque (TOP) assay, and a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay. The results from the animal bioassay show that 1- and 3-log10 inactivation is achieved with 4 mJ/cm2 UV and 10 mJ/cm2 low-pressure UV, respectively. TOP assay results, but not RT-qPCR results, correlate well with bioassay results. In conclusion, a 3-log10 inactivation of T. gondii oocysts is achieved by 10-mJ/cm2 low-pressure UV, and the in vitro TOP assay is a promising alternative to the mouse bioassay.

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation

Aims:  To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters.

Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry‐Based Analysis of Giardia lamblia and Giardia muris

IARDIA is the protozoan parasite that is the etiologic agentof giardiasis. This illness is the most common parasitic dis-ease and is estimated to infect at least 100,000 people each year inthe United States (Furness, Beach, and Roberts 2000). Symptomsof giardiasis range from asymptomatic to severe abdominal pain,chronic diarrhea, andin rarecases,death, withyoung children andimmunocompromised individuals being at the greatest risk of ser-ious illness (Adam 1991; Upcroft and Upcroft 2001). Infectiontypically occurs through the fecal-oral route and has been docu-mented to be associated with many waterborne disease outbreaksworldwide. A majority of these outbreaks occurred due to con-tamination of the drinking water supplies with untreated sewage.There are at least six species of Giardia including Giardia lam-blia (also known as Giardia duodenalis or Giardia intestinalis),which can infect a wide range of hosts including humans (Caccioetal.2005).Thecurrentdetectionmethodemployedtomonitorthepresence of Giardia cysts in surface and drinking water relies pri-marily on microscopic techniquesthat detect thepresenceof Giar-dia cysts in the sample, but the method is not species specific nordoes it determine cyst viability (U.S. Environmental ProtectionAgency 1999). More recently, a PCR-based genotyping tool hasbeen developed and used to identify the different Giardia spp.present in environmental water (Sulaiman et al. 2004). This tech-nique however can be prone to contamination and thus other al-ternative approaches are currently being explored. In particular,matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) has been used to identify andclassify bacteria (Donohue et al. 2006) and parasites (Magnuson,Owens,andKelty2000;Mouraetal.2003),althoughthisapproachhas not been applied to study Giardia spp.This manuscript describes a MALDI-TOF MS-based approachthat is used to characterize the mass spectral fingerprints of intactG. lambliaandGiardiamuriscysts.Thisstudy identified commonmass spectral peaks shared by the two species as well as peaksspecific to G. lamblia and others specific to G. muris, which areuseful in differentiating the two organisms. Additional analysesrevealed that the mass spectral profiles of intact cysts consistedpartly of peaks representing trophozoite-derived proteins, basedon comparison with purified trophozoites. These results suggestthe potential application of intact cell MALDI-TOF MS as an al-ternative high throughput approach for species identification ofGiardia spp.MATERIALS AND METHODSCyst propagation and purification. Giardia lamblia (H3strain; assemblage B) and G. muris (obtained from Drs. ErikHewlett and John Andrews, Case Western Reserve School ofMedicine, Cleveland, OH) cysts were propagated using Mongo-lian gerbils or CF-1 mice, respectively (Jackson Laboratories, BarHarbor, ME). Feces from infected rodents were collected andcysts were harvested and purified using a sucrose/percoll (Sigma,St. Louis, MO) gradient (Hayes et al. 2003). Purified cysts used inall experiments were 7 days from the time of collection.In vitro excystation and trophozoite purification. The invitro excystation of cysts was performed as described (Rice andSchaefer 1981) and efficiency of excystation was determinedmicroscopically to routinely be 90% excysted. Following ex-cystation, trophozoite/cyst mixtures were labeled with FITC-con-jugated anti-Giardia antibody (Giardi-a-Glo