Disinfection of Cryptosporidium parvum Oocysts by Pulsed Light Treatment Evaluated in an In Vitro Cultivation Model

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.

Section: Methodsmentioning

confidence: 99%

“…For example, rodent models of infection are used to measure the infectivity of this parasite (3,23,29), but this approach is time-consuming, laborintensive, and expensive. In addition, it can determine infectivity of only a select number of Cryptosporidium species.…”

mentioning

confidence: 99%

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts

Brescia¹,

Griffin

Ware³

et al. 2009

“…The use of cell culture to study the intracellular development of Cryptosporidium was described over 2 decades ago (6). In addition to the study of Cryptosporidium biology, cell culture methods have been used to study Cryptosporidium inactivation by disinfectants (4,13,23,26,28), efficacy of chemotherapeutic agents (3,5,34,37), and detection of infectious oocysts in water samples (1,7,10,17). The developmental stages of the parasite in cell culture are detected using a variety of techniques, including immunofluorescent assay microscopy (27), flow cytometry (31), reverse transcription-PCR (22), and quantitative PCR (5,8,11,15).…”

mentioning

confidence: 99%

“…Mouse models have frequently been used to determine the infectivity of Cryptosporidium oocysts (4,9,16,20). However, animal infectivity assays are costly and not feasible for the analysis of environmental samples.…”

mentioning

confidence: 99%

Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

Sifuentes

Giovanni

2007

Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8-to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8-to 11-day-old and 11-to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

“…Mouse models have frequently been used to determine the infectivity of Cryptosporidium oocysts (2,10,18,20). However, animal infectivity determination is costly and labor-intensive and requires as long as 2 weeks to complete.…”

mentioning

confidence: 99%

Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

Giovanni

LeChevallier

2005

A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.Ongoing challenges for the water industry are Cryptosporidium detection and infectivity determination. Mouse models have frequently been used to determine the infectivity of Cryptosporidium oocysts (2,10,18,20). However, animal infectivity determination is costly and labor-intensive and requires as long as 2 weeks to complete. Furthermore, it is not feasible for the analysis of environmental samples. In an attempt to simplify infectivity determination, several in vitro methods have been developed, such as in vitro excystation and microscopic assays evaluating the uptake or exclusion of fluorogenic dyes (3, 4) and fluorescence in situ hybridization techniques (35). Various PCR strategies have also been developed to assess the viability of Cryptosporidium parvum oocysts, including the integration of in vitro excystation (5, 9, 36) and the use of reverse transcription-PCR for the detection of mRNA transcripts found only in viable oocysts (24,30).Strategies using in vitro cell culture of C. parvum are attractive alternatives to animal infectivity testing. In most of these assays, oocysts are stimulated to excyst by exposure to sodium hypochlorite (bleach), bile enzymes, and/or acidic pH (7,23,24,32) and are then inoculated onto in vitro-cultivated cell culture monolayers. Sporozoites released from oocysts invade and replicate within the intracellular environment of the cells. After incubation, parasites are detected within the cell cultures by a variety of techniques, including immunofluorescence microscopy (28), reverse transcription-PCR (24), and standard PCR (7). Quantification of cell culture infection for pharmaceutical and disinfection studies has been achieved with an enzyme-linked immunosorbent assay (38). Most-probablenumber format...