Protozoa reduction through secondary wastewater treatment in two water reclamation facilities.

The State of Nevada, USA Administrative Code requires a 12-log enteric virus reduction/inactivation, 10-log Giardia cyst reduction, and 10-log Cryptosporidium oocyst reduction for Category A+ reclaimed water suitable for indirect potable reuse (IPR) based on raw wastewater to potable reuse water. Accurately demonstrating log10 reduction values (LRVs) through secondary biological treatment prior to an advanced water treatment train enables redundancy and resiliency for IPR projects while maintaining a high level of public confidence. LRVs for Cryptosporidium and Giardia resulting from secondary biological treatment are not fully established due to a wide range of performance variabilities resulting from different types of secondary biological treatment processes employed in water reclamation. A one-year investigation of two full-scale northern Nevada (e.g. ≤4 mgd; 1.5 × 107 L/day) water reclamation facilities (WRFs) was conducted to monitor Cryptosporidium oocysts and Giardia cysts in untreated wastewater and secondary effluent. This study aimed at establishing secondary treatment LRVs, monitor WRF performance and attempted to correlate performance to protozoan reduction. California's IPR regulations, in which Nevada IPR regulations were modeled after, were based on a maximum concentration of 5-logs (cysts/L) of Giardia and 4-logs (oocysts/L) of Cryptosporidium. The recovery-corrected Giardia and Cryptosporidium concentrations measured in untreated influent (20 samples each at each WRF) were below 5-log cysts/L at the 99th percentile (maximum 4.4-log cysts/L) and 4-log oocysts/L (maximum 2.7 log oocysts/L), respectively. Both secondary treatment WRFs produced secondary effluent that is consistently better than federal and the State of Nevada requirements and perform within an operating envelop for other secondary facilities. Given the results, it appears that a minimum conservative estimate for LRVs for well-operated secondary activated sludge treatment plants (at the 5th percentile) of 0.5 LRV credit for Cryptosporidium and 2.0 LRV for Giardia is warranted. These minimum LRVs are consistent with a conservative review of the available literature.

Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Further evaluation and validation of the VETSCAN IMAGYST: in-clinic feline and canine fecal parasite detection system integrated with a deep learning algorithm.

BACKGROUND: Fecal examinations in pet cats and dogs are key components of routine veterinary practice; however, their accuracy is influenced by diagnostic methodologies and the experience level of personnel performing the tests. The VETSCAN IMAGYST system was developed to provide simpler and easier fecal examinations which are less influenced by examiners' skills. This system consists of three components: a sample preparation device, an automated microscope scanner, and analysis software. The objectives of this study were to qualitatively evaluate the performance of the VETSCAN IMAGYST system on feline parasites (Ancylostoma and Toxocara cati) and protozoan parasites (Cystoisospora and Giardia) and to assess and compare the performance of the VETSCAN IMAGYST centrifugal flotation method to reference centrifugal and passive flotation methods. METHODS: To evaluate the diagnostic performance of the scanning and algorithmic components of the VETSCAN IMAGYST system, fecal slides were prepared by the VETSCAN IMAGYST centrifugal flotation technique with pre-screened fecal samples collected from dogs and cats and examined by both an algorithm and parasitologists. To assess the performance of the VETSCAN IMAGYST centrifugal flotation technique, diagnostic sensitivity and specificity were calculated and compared to those of conventional flotation techniques. RESULTS: The performance of the VETSCAN IMAGYST algorithm closely correlated with evaluations by parasitologists, with sensitivity of 75.8-100% and specificity of 93.1-100% across the targeted parasites. For samples with 50 eggs or less per slide, Lin's concordance correlation coefficients ranged from 0.70 to 0.95 across the targeted parasites. The results of the VETSCAN IMAGYST centrifugal flotation method correlated well with those of the conventional centrifugal flotation method across the targeted parasites: sensitivity of 65.7-100% and specificity of 97.6-100%. Similar results were observed for the conventional passive flotation method compared to the conventional centrifugal flotation method: sensitivity of 56.4-91.7% and specificity of 99.4-100%. CONCLUSIONS: The VETSCAN IMAGYST scanning and algorithmic systems with the VETSCAN IMAGYST fecal preparation technique demonstrated a similar qualitative performance to the parasitologists' examinations with conventional fecal flotation techniques. Given the deep learning nature of the VETSCAN IMAGYST system, its performance is expected to improve over time, enabling it to be utilized in veterinary clinics to perform fecal examinations accurately and efficiently.

Detection of Giardia and Cryptosporidium in environmental matrices with immunomagnetic separation: two or three acid dissociations.

This study evaluated the technology of detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in environmental matrices obtained after water treatment on a bench scale. Calcium carbonate flocculation with immunomagnetic separation was the selected method to quantify the protozoa, and the importance of the number of acid dissociations in the immunomagnetic separation was assessed. When adding the third acid dissociation, an increase of 71% ± 6 in floated residue and 31.9% ± 28.7 in filter backwash water in cyst recovery was observed, while in oocyst recovery, a non-significant increase was detected. In the filtered water, this increased dissociation was important in the protozoa recovery with increases greater than 33%. The results showed that there is a strong interaction of these target organisms with the magnetic microspheres, since protozoa were still recovered in the third acid dissociation and some of them were still adhered to the magnetic microspheres.

Lesions caused by Sarcocystis spp., parasites of opossums (Didelphis aurita), in acute and chronic infections in birds (Melopsittacus undulatus).

Protozoa of the genus Sarcocystis are obligatory heterogenous parasites with both definitive and intermediate hosts. Opossums (Didelphis aurita) can shed multiple species of Sarcocystis with birds as the intermediate host. The pathologies of Sarcocystis species in birds have not been thoroughly elucidated. Therefore, the aim of the present study to determine the main lesions that can occur in acute and chronic infections in intermediate hosts, when they ingest infective sporocysts that are shed in the opossum's feces, using budgerigars as a model. To this end, 12 budgerigars, Melopsittacus undulatus, were divided into two groups that received an inoculum with 60 and 120 sporocysts. Birds that died or were euthanized were necropsied, and the lung, tongue, liver, brain, heart, and skeletal striated muscles were collected and fixed in 10% formalin for histopathological analysis. The infectivity varied according to the sample and infective dose. Acute histopathological lesions were characterized by evidence of slightly degenerated hepatocyte cords that permeated the region of the blood vessel and hepatic sinusoids. Pulmonary tissue lesions were also observed in the parabronchial region with the presence of inflammatory infiltrates associated with areas of edema and atelectasis. In chronic infections, few mature cysts were observed in the chest, and many mature cysts in the thigh and tongue muscles. Thus, it was possible to conclude that lesions are highly characteristic in acute infection and, in chronic infections, cysts were present but without major lesions. In this case, the preferred organs of parasitism were the thigh and the tongue.

First report on parasites of European beavers in the Slovak Republic.

European beaver (Castor fiber L. 1758) is the biggest rodent living in Europe. It is a semi-aquatic animal known for building dams and burrows. European beaver is a potential host for a wide range of parasites and other infectious diseases. In Slovakia, there is an increasing number of beavers but the data about their parasitic fauna are missing. Our work is the first documentation about the beaver's parasitofauna in Slovakia. In a 1-year study, we collected and examined 19 beaver fecal samples from the vicinity of beaver burrows inhabiting three particular localities at the Danube, Topla, and Laborec rivers in Slovakia. In these fecal samples, 4 different species of intestinal endoparasites were detected as follows: oocysts of Cryptosporidium, cysts of Giardia, eggs of Stichorchis subtriquetrus, and eggs and larvae of Travassosius rufus. Parasites were confirmed only in samples collected at river Topla. Based on our results, we can conclude that European beaver can be an important source of parasitic contamination of surface waters especially in the localities shared by people.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

A smartphone microscopic method for simultaneous detection of (oo)cysts of Cryptosporidium and Giardia.

BACKGROUND: Food and water-borne illness caused by ingestion of (oo)cysts of Cryptosporidium and Giardia is one of the major health problems globally. Several methods are available to detect Giardia cyst and Cryptosporidium oocyst in food and water. Most of the available methods require a good laboratory facility and well-trained manpower and are therefore costly. There is a need of affordable and reliable method that can be easily implemented in resource limited settings. METHODOLOGY/PRINCIPLE FINDINGS: We developed a smartphone based microscopic assay method to screen (oo)cysts of Cryptosporidium and Giardia contamination of vegetable and water samples. The method consisting of a ball lens of 1 mm diameter, white LED as illumination source and Lugols's iodine staining provided magnification and contrast capable of distinguishing (oo)cysts of Cryptosporidium and Giardia. The analytical performance of the method was tested by spike recovery experiments. The spike recovery experiments performed on cabbage, carrot, cucumber, radish, tomatoes, and water resulted in 26.8±10.3, 40.1±8.5, 44.4±7.3, 47.6±11.3, 49.2 ±10.9, and 30.2±7.9% recovery for Cryptosporidium, respectively and 10.2±4.0, 14.1±7.3, 24.2±12.1, 23.2±13.7, 17.1±13.9, and 37.6±2.4% recovery for Giardia, respectively. The spike recovery results are comparable with data obtained using commercial brightfield and fluorescence microscope methods. Finally, we tested the smartphone microscope system for detecting (oo)cysts on 7 types of vegetable (n = 196) and river water (n = 18) samples. Forty-two percent vegetable and thirty-nine percent water samples were found to be contaminated with Cryptosporidium oocyst. Similarly, thirty-one percent vegetable and thirty-three percent water samples were contaminated with Giardia cyst. CONCLUSIONS: The newly developed smartphone microscopic method showed comparable performance to commercial microscopic methods. The new method can be a low-cost and easy to implement alternative method for simultaneous detection of (oo)cysts in vegetable and water samples in resource limited settings.

Morphological and molecular characterization of Cystoisospora laidlawi oocysts (Apicomplexa: Eimeriidae) in farmed American mink (Neovison vison) in Denmark.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5 µm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221 bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

Toxoplasmagondii oocysts, Giardia cysts and Cryptosporidium oocysts in outdoor swimming pools in Brazil.

The accidental ingestion of treated recreational water is an important transmission route of waterborne protozoa worldwide. The present study aimed to provide the first evaluation of swimming pools in Brazil, analysing the presence of pathogenic protozoa (Toxoplasma gondii, Cryptosporidium spp. and Giardia spp.) by parasitological and molecular methods. A total of 57 samples were collected from 21 public swimming pools, either directly from the pool or filter backwash water and concentrated using the membrane filtration technique. Giardia cysts and Cryptosporidium oocysts were visualized by direct immunofluorescence assay after purification by immunomagnetic separation. Toxoplasma gondii oocysts were detected by autofluorescence visualization using the supernatant discarded during the purification step as a sample. Positive samples were submitted to molecular analysis. The molecular markers were used: SSU-rRNA, tpi, gdh and bg, for Giardia DNA amplification, and 18S rRNA gene fragment amplification was used for the Cryptosporidium oocysts. The 529-bp repeat element (REP529) fragment and the 35-fold repetitive B1 gene were employed as a target for T. gondii. Amplified products were submitted to sequencing and phylogenetic analysis. Giardia cysts were detected in 19.0% and Cryptosporidium oocysts in 9.5% of swimming pools. In one swimming pool (4.7%), both protozoa were detected on at least one occasion. Structures similar to T. gondii oocysts were detected in 33.3% of the samples, ranging from one to 23 per slide. Giardia was confirmed by DNA amplification in three swimming pools; Giardia duodenalis Assemblage A was identified by the phylogenetic positioning of the ß-giardin gene. Toxoplasma gondii DNA was detected in 14.2% of swimming pools. The present study represents the first report of the occurrence of T. gondii oocysts in swimming pools. Recreational activity in swimming pools contaminated by chlorine-resistant protozoa can represent a high risk of infection for bathers and swimmers.

Presence of eimerid oocysts in faeces of a quarantined dog in Iceland is explained by coprophagic behaviour prior to its importation. Case report.

BACKGROUND: All dogs imported into Iceland must undergo mandatory quarantine in a special station before introduction into the country. A faecal sample is collected from the first stool passed by the dog in this station and subsequently examined for the presence of intestinal parasite stages. CASE PRESENTATION: In May 2019 unsporulated oocysts were detected in faeces from a 7-year-old household dog that had been imported from Sweden. Most of the oocysts studied strongly resembled those of Eimeria canis Wenyon, 1923. As this species is not valid, the purpose of the present article was to identify the correct species and examine their possible origin. Studies confirmed the presence of two distinct unsporulated oocyst morphotypes in the faeces; measurements and photomicrographs confirmed their identification as Eimeria magna Pérard, 1925 and Eimeria stiedai (Lindemann, 1865) Kisskalt and Hartmann, 1907, both common parasites of European rabbits, Oryctolagus cuniculus (L., 1758). When the owner of the dog was questioned about the food administrated to the dog prior to its import to Iceland, it turned out that it had exclusively been fed dry dog food pellets. However, the owner also reported that on the morning prior to transportation to Iceland, the dog was allowed to move freely in a grassland area where rabbits are common and heaps of their faeces are present. Furthermore, the owner confirmed that the dog consumed rabbit faeces that morning. CONCLUSION: It is believed that this coprophagic behaviour can explain the detection of rabbit eimerids in the dog's faeces, and that such behaviour must be taken into consideration by veterinarians and other diagnostic personnel when they detect atypical cysts or eggs during coprological examinations.

Detection of Cryptosporidium oocysts and Giardia cysts in vegetables from street markets from the Qinghai Tibetan Plateau Area in China.

Cryptosporidium and Giardia are well-known parasitic protozoans responsible for waterborne and foodborne diarrhoeal diseases. However, data are not available on market vegetables contaminated with Cryptosporidium and Giardia in China. In the present study, 642 different vegetable samples were collected from Xining City street vendors in the Qinghai Province to study the Cryptosporidium and Giardia contamination rates via PCR and sequence analyses. Cryptosporidium spp. and Giardia duodenalis were detected in 16 (2.5%) and 73 (11.4%) samples, respectively. Two species of Cryptosporidium, C. parvum (n = 11) and C. andersoni (n = 5), were identified. G. duodenalis assemblage B was identified in almost all positive samples (n = 72), except one sample that contained G. duodenalis assemblage E. We report on the rate of Cryptosporidium and Giardia contamination in vegetables for the first time from the Qinghai Tibetan Plateau Area (QTPA) in China.

Detection of Toxoplasma gondii oocysts in fresh vegetables and berry fruits.

BACKGROUND: Toxoplasma gondii is the third most important contributor to health burden caused by food-borne illness. Ingestion of tissue cysts from undercooked meat is an important source of horizontal transmission to humans. However, there is an increasing awareness of the consumption of fresh fruit and vegetables, as a possible source for oocyst transmission, since this stage of the parasite can persist and remain infective in soil and water for long time. Herein, we outline findings related with detection of T. gondii oocysts in vegetables and berry fruits, which are usually raw consumed. The procedure includes the estimation of the number of oocysts. METHODS: Food samples were collected from local producers and supermarket suppliers. Toxoplasma gondii oocysts were concentrated after washing the samples by applying high resolution water filtration and immunomagnetic separation (method 1623.1: EPA 816-R-12-001-Jan 2012), in order to (i) remove potential Cryptosporidium spp. oocysts and Giardia spp. cysts present in the samples; and (ii) select T. gondii oocysts. Toxoplasma gondii oocyst detection and an estimation of their numbers was performed by conventional PCR and real time qPCR, using specific primers for a 183-bp sequence of the T. gondii repetitive DNA region. All PCR-positive DNA samples were purified and sequenced. Restriction enzyme digestion with EcoRV endonuclease confirmed the presence of the T. gondii DNA fragment. In addition, the presence of the parasite was observed by fluorescent microscopy, taking advantage of the oocysts autofluorescence under UV light. RESULTS: Forty percent of the analysed samples (95% CI: 25.5-56.5%) presented the expected PCR and digested DNA fragments. These fragments were confirmed by sequencing. Microscopic autofluorescence supported the presence of T. gondii-like oocysts. The estimated mean (± SE) oocyst concentration was 23.5 ± 12.1 oocysts/g, with a range of 0.6-179.9 oocysts/g. CONCLUSIONS: Our findings provide relevant evidence of contamination of fresh vegetables and berry fruits with T. gondii oocysts.

Investigations from Northern Greece on mussels cultivated in areas proximal to wastewaters discharges, as a potential source for human infection with Giardia and Cryptosporidium.

Marine bivalves are usually cultivated in shallow, estuarine waters where there is a high concentration of nutrients. Many micro-pollutants, including the protozoan parasites Giardia duodenalis and Cryptosporidium spp., which also occur in such environments, may be concentrated in shellfish tissues during their feeding process. Shellfish can thus be considered as vehicles for foodborne infections, as they are usually consumed lightly cooked or raw. Therefore, the main objective of this study was to investigate the presence of both parasites in Mediterranean mussels, Mytilus galloprovincialis that are cultivated in Thermaikos Gulf, North Greece, which is fed by four rivers that are contaminated with both protozoa. Moreover, the occurrence of these protozoa was monitored in treated wastewaters from 3 treatment plants that discharge into the gulf. In order to identify potential sources of contamination and to estimate the risk for human infection, an attempt was made to genotype Giardia and Cryptosporidium in positive samples. Immunofluorescence was used for detection and molecular techniques were used for both detection and genotyping of the parasites. In total, 120 mussel samples, coming from 10 farms, were examined for the presence of both protozoa over the 6-month farming period. None of them were found positive by immunofluorescence microscopy for the presence of parasites. Only in 3 mussel samples, PCR targeting the GP60 gene detected Cryptosporidium spp. DNA, but sequencing was not successful. Thirteen out of 18 monthly samples collected from the 3 wastewater treatment plants, revealed the presence of Giardia duodenalis cysts belonging to sub-assemblage AII, at relatively low counts (up to 11.2 cysts/L). Cryptosporidium oocysts (up to 0.9 oocysts/L) were also detected in 4 out of 8 samples, although sequencing was not successful at any of the target genes. At the studied location and under the sampling conditions described, mussels tested were not found to be harboring Giardia cysts and the presence of Cryptosporidium was found only in few cases (by PCR detection only). Our results suggest that the likelihood that mussels from these locations act as vehicles of human infection for Giardia and Cryptosporidium seems low.

Cystoisospora felis infection in a captive jaguar cub (Panthera onca) in Michoacán, México.

Cystoisospora felis (Wenyon 1923) was identified in a 3-month-old, captive jaguar (Panthera onca) presenting with signs of gastrointestinal distress. The cub was fed beef, chicken, and commercial diet. Examination of fresh feces detected large (47.8 µm × 35 µm) unsporulated oocysts. Sporulated oocysts contained 2 sporocysts, each with 4 sporozoites. Oocyst morphometrics agreed with published features of C. felis described from domestic felines. Partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was PCR-amplified and sequenced; the resulting sequence showed 100% identity to a C. felis isolate from a domestic cat. This is the first molecularly confirmed report of C. felis infecting and producing clinically evident, enteric disease in a jaguar cub.

Cryptosporidium Diagnostic Assays: Microscopy.

Stained microscopy of fecal smears was the cornerstone of Cryptosporidium diagnosis for many years, and still provides a low-cost method for detecting oocysts. The development and commercialization of improved enzyme immunosorbent assays (EIA) for coproantigen detection provided an automatable method for mass testing, and rapid diagnostics when incorporated onto a cartridge format. Similarly, immunochromatographic lateral flow assays (ICLF) enable rapid diagnostics. Nevertheless, it is important that positive reactions by EIA or ICLF are confirmed. Here we describe microscopical methods using tinctorial stains for the diagnosis of acute cryptosporidiosis, and using immunofluorescent reagents for diagnosis or for confirmation of EIA or ICLF positive reactions.

High-Throughput Screening of Drugs Against the Growth of Cryptosporidium parvum In Vitro by qRT-PCR.

An effective method to quantify the parasite loads is the key to the evaluation of anti-cryptosporidial drug efficacy in vitro. However, high-throughput screening (HTS) of drugs against Cryptosporidium parvum in vitro was impractical by the labor-intensive traditional assays. Here we describe a simplified quantitative RT-PCR assay suitable for HTS of compounds and for evaluating drug efficacy against the growth of C. parvum in vitro.

In Vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds.

Hollow fiber technology is a powerful tool for the culture of difficult-to-grow cells. Cryptosporidium parvum has a multistage sexual and asexual life cycle that has proved difficult to culture by conventional in vitro culture methods. Here, we describe a method utilizing a hollow fiber bioreactor for the continuous in vitro growth of C. parvum that produces sexual and asexual stages. The method enables the evaluation of potential therapeutic compounds under conditions that mirror the dynamic conditions found in the gut facilitating preliminary pharmacokinetic and pharmacodynamic data to be obtained.

In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers.

Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite's natural environment in the small intestine. We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.