Oocyst Recovery Research Articles

Evaluation of molecular-based methods for the detection and quantification of Cryptosporidium spp. in wastewater

Cryptosporidium poses significant public health risks as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized wastewater surveillance methods for Cryptosporidium spp. challenges implementation design and the comparability between studies. Thus, this study compared and contrasted Cryptosporidium wastewater surveillance methods for concentrating wastewater oocysts, extracting oocyst DNA, and detecting Cryptosporidium genetic markers. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome Particles, with and without additional immunomagnetic separation purification (except for the Nanotrap Microbiome Particles). Oocyst DNA extraction by either the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit were evaluated and the impact of bead beating and freeze-thaw pretreatments on DNA recoveries was assessed. Genetic detection via qPCR assays targeting either the Cryptosporidium 18S rRNA gene or the Cryptosporidium oocyst wall protein gene were tested. Oocyst recovery percentages were highest for centrifugation (39–77 %), followed by the Nanotrap Microbiome Particles (24 %), electronegative filtration with a PBST elution (22 %), and Envirocheck HV capsule filtration (13 %). Immunomagnetic separation purification was found to be unsuitable due to interference from the wastewater matrix. Bead-beating pretreatment enhanced DNA recoveries from both the DNeasy Powersoil Pro kit (314 gc/μL DNA) and the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA recoveries to under 92 gc/μL DNA, likely through DNA degradation. Finally, while both qPCR assays were specific to Cryptosporidium spp., the 18S rRNA assay had a 5-fold lower detection limit and could detect a wider range of Cryptosporidium spp. than the Cryptosporidium oocyst wall protein assay.

Isolation and Establishment of eimeria tenella Populations from Local Broiler Chicken Farms

Coccidiosis is a major intestinal disease in the chicken production industry that is caused by one or more of the seven Eimeria species known to infect chickens. Development of effective diagnostic methods and controls requires comprehensive knowledge on the Eimeria species in the local population, particularly the pathogenic E. tenella. In this study, 35 faecal samples were collected from local chicken farms, and through microscopic observation, nine samples (26%) were found to contain Eimeria oocysts. Subsequently, two positive samples, namely NSN6 and SGR6, were selected and propagated via passage in coccidian-free chickens. Species identification analyses based on oocyst morphological characterisation suggested the presence of small-sized oocysts (E. acervulina and/or E. mitis) and medium-sized oocysts (E. necatrix, E. tenella, and/or E. praecox) in NSN6 while in SGR6, the large-sized oocysts (E. brunetti and/or E. maxima) were also present in addition to the small and medium-sized oocysts. Subsequently, species identification with PCR using species-specific primers was successful in determining the presence of specific Eimeria species, which are E. acervulina and E. tenella in NSN6, and E. acervulina, E. tenella, and E. maxima in SGR6. The E. tenella populations of NSN6 and SGR6, namely EtNSN6 and EtSGR6, respectively, were established via passage in coccidian-free chickens and oocyst recovery from the caeca. The purity of EtNSN6 and EtSGR6 populations were confirmed based on oocyst morphological characterisation and PCR. The established EtNSN6 and EtSGR6 populations would be useful as research models for local strains in the development of more effective control methods.

Detection of Cyclospora cayetanensis in produce irrigation and wash water using large-volume sampling techniques

The recent increase of reported cyclosporiasis outbreaks associated with fresh produce has highlighted the need for understanding environmental transmission of Cyclospora cayetanensis in agricultural settings and facilities. Conducting such environmental investigations necessitates robust sample collection and analytical methods to detect C. cayetanensis in water samples. This study evaluated three sample collection methods for recovery of C. cayetanensis oocysts from water samples during seeded recovery experiments. Two filtration-based methods, dead-end ultrafiltration (DEUF) and USEPA Method 1623.1, were evaluated for oocyst recovery from irrigation water. A non-filter-based method, continuous flow centrifugation (CFC), was evaluated separately for recovery from creek water and spent produce wash water. Median C. cayetanensis recovery efficiencies were 17% for DEUF and 16–22% for Method 1623.1. The DEUF method proved to be more robust than Method 1623.1, as the recovery efficiencies were less variable and the DEUF ultrafilters were capable of filtering larger volumes of high-turbidity water without clogging. Median C. cayetanensis recovery efficiencies for CFC were 28% for wash water and 63% for creek water, making it a viable option for processing water with high turbidity or organic matter. The data from this study demonstrate the capability of DEUF and CFC as filter-based and non-filter-based options, respectively, for the recovery of C. cayetanensis oocysts from environmental and agricultural waters.

Molecular Methods for the Detection of Toxoplasma gondii Oocysts in Fresh Produce: An Extensive Review.

Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure.

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.

Evaluation of a modified method for the detection of Cryptosporidium oocysts on spinach leaves

Despite the infection risk associated with the consumption of contaminated food, techniques for recovering and detecting Cryptosporidium oocysts from fruit and vegetables are generally inadequate due to the variable recovery efficiencies and high reagent costs, such as those presented by ISO 18744:2016 “Microbiology of the food chain -Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits”. Although an improved method for recovering these parasites from Iceberg lettuce, which reported increased recovery efficiency as well as lower costs, has been published, it appears to have limitations for the recovery of Cryptosporidium from saponin-rich leaves such as spinach (Spinacia oleraceae), which have previously been implicated in Cryptosporidium parvum outbreaks. In this study, we refined the method to improve its use with these more challenging samples. The use of alkaline elution buffer (1 M glycine) of different pH values was evaluated for their effectiveness in removing C. parvum from spinach leaves. The refinement of Utaaker's method showed, from spinach leaves inoculated with 100 oocysts, an increased oocyst recovery rate with an overall mean recovery rate of 33.79% ± 2.82%. The emergence of parasitic foodborne illnesses and outbreaks associated with the consumption of fresh produce demonstrates the need for the development of an optimal recovery process for parasites from suspected foods. Results showed that refinement of existing protocols could improve the retrieval of Cryptosporidium oocysts from these more challenging leafy greens.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20–60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

Evaluation of the U.S. Food and Drug Administration validated molecular method for detection of Cyclospora cayetanensis oocysts on fresh and frozen berries

Outbreaks and sporadic cases of Cyclospora cayetanensis have been linked to consumption of berries. The efficacy of the U.S. Food and Drug Administration (FDA) method for detection of C. cayetanensis was evaluated in fresh berries (blackberries, strawberries, blueberries and mixed berries) and in frozen mixed berries. The protocol included seeding with C. cayetanensis oocysts, produce washing, DNA extraction and a dual TaqMan assay. As few as five oocysts were detected in every type of fresh berry analyzed. All berry samples seeded with 200 oocysts were positive and all unseeded berry samples were negative. No significant differences were observed among any of the berry types analyzed in detection rates, CT values and estimated oocyst recovery percentages. Mixed berries were seeded and frozen for up to seven weeks. As few as five oocysts were also detected. No significant differences were observed in C. cayetanensis CT values between fresh and frozen mixed berries at any seeding level. In conclusion, the FDA BAM Chapter 19B method for the detection of Cyclospora was robust, consistent, and showed high sensitivity in all types of berries analyzed. Evaluation of the FDA detection method in berries will provide reliable laboratory support for surveillance programs and for outbreak investigations.

Improved cell culture immunofluorescent assay for detection of infectious Cryptosporidium spp. in prefinished water

The New York City Department of Environmental Protection's (NYC) Hillview Reservoir is an uncovered prefinished water storage reservoir postultraviolet (post‐UV) light disinfection. As an additional tool to the detection of total (viable and nonviable) oocysts using Methods 1623/1623.1, the performance of an improved cell culture immunofluorescent assay (CC‐IFA) for the specific detection of infectious oocysts in this NYC reservoir matrix was evaluated. Mean CC‐IFA matrix spike recovery of infectious Cryptosporidium parvum and Cryptosporidium hominis oocysts was comparable to the mean Method 1623 recovery of total oocysts, ranging from 29 to 46%. Experiments using ≤5 C. parvum or ≤10 C. hominis viable oocyst spikes prepared using flow cytometry indicated that the reservoir matrix did not adversely affect CC‐IFA detection. Therefore, the improved CC‐IFA appears useful for the detection of environmentally relevant, low numbers of infectious oocysts in the reservoir matrix and for the refined assessment of Cryptosporidium risk.

Comparison of Cryptosporidium oocyst recovery methods for their applicability for monitoring of consumer-ready fresh shellfish

Growing demand for fresh, unprocessed food favours the emergence of Cryptosporidium infections in humans. Mainly it is food of plant origin or unpasteurized milk which have been involved in food-borne outbreaks of cryptosporidiosis. So far consumption of shellfish contaminated with Cryptosporidium were not associated with human infections although such as possibility exists. In this study an attempt was undertaken to evaluate the analytical performance of three commonly used methods for recovery of Cryptosporidium oocysts from shellfish: i) pepsin digestion of shellfish in conjunction with immunomagnetic separation (IMS) of oocysts (method A), ii) pepsin-HCl treatment of shellfish homogenate without IMS (method B), and iii) a strainer method with direct oocyst extraction and separation from shellfish tissue using IMS (method C). Each method's performance was assessed according to the ISO standard requirements by testing shellfish homogenates seeded with different numbers of C. parvum oocysts. Two groups of parameters were compared, encompassing precision (coefficient of variation (CV)) and accuracy of measurements. These were described by linear regression models allowing calculation of the methods' limits of detection (LOD) and quantification (LOQ). In addition, oocyst recovery efficiencies from shellfish were calculated for each method. All three compared methods allowed for at least 66% recovery of Cryptosporidium oocysts from the tested samples. The best recovery (83.3–100%) in the whole range of tested suspensions was obtained for method C. The accuracy of method B was better (linearity of r2 = 0.9996 in the full measurement range) than that of method A (r2 = 0.968). Method C showed the best accuracy (r2 = 1) and precision (CV 0.2–14.1). Compared to other methods it was also characterised by the best LOD and LOQ, attaining ≅4 and ≅12 oocysts per 3 g of tested shellfish sample respectively. Despite a lack of the ability of method A to give the proportional results in oocysts recovery (non-linearity of the method) compared to the reference values, it achieved the highest LOD and LOQ values among the tested methods. As demonstrated here, the most efficient method for extraction of Cryptosporidium oocysts from shellfish tissues was method C employing sample homogenisation and separation of oocysts from tissue debris using IMS. Used alone this method does not in fact allow for identification of Cryptosporidium species but delivers quantitative results concerning the level of food contamination by parasites.

Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for detection of C. cayetanensis.

Discovery Recovery Methods and Detection of food borne parasites

In 2013, more than 640 individuals acquired Cyclospora infection in the US. Illness was associated with consumption of imported salad mixes and cilantro that were commercially processed and rinsed. To date, there is no protocol that would allow testing of large volumes of water to detect small number of parasites in produce wash‐waters. Cryptosporidium parvum and Cyclospora cayetanensis are protozoan parasites causing gastrointestinal illness in humans and animals both immunocompetent and immunocompromised hosts. Cryptosporidium parvum are spherical, 4–5μm in size, it can be acquired when ingesting foods and drinking, or accidentally ingesting, water contaminated with feces containing Cryptosporidium oocysts. The illnesses are characterized by acute diarrhea, stomach cramps, nausea, vomiting, and weight loss. Cyclospora cayetanensis are spherical as well, it is 8–10μm in size, it can also be acquired by person‐to‐person transmission and when in contact with infected animals; it is also spread via the fecal‐oral route (the host must ingest something that had been contaminated with feces that contain infectious oocysts). For oocysts to be termed infectious, the oocyst has to be sporulated (produce or form spores). The related disease Cyclosporiasis is acquired by ingestion of water or foods contaminated with human feces containing Cyclospora oocysts. Water samples were collected from a wash site in the southern area of Georgia every other day. The objective of this study was to compare two methods for oocyst recovery and detection from carrot wash‐water. The carrots were grated and washed with the water sample. The carrot wash‐water was spiked with ten and fifty oocysts of Cryptosporidium and Cyclospora. Two types of produce wash solutions fresh water and chlorinated water used by the fresh produce industry were evaluated ten liters of wash water were flocculated or filtered and then concentrated by centrifuging. Cryptosporidium oocysts from the concentrated water sample was treated with immunomagnetic separation (IMS) and detected by immunofluorescence assay (IFA). Cyclospora oocysts were concentrated by discontinuous sucrose gradients and examined by Nomarski microscopy. All samples were further examined by nested Polymerase Chain Reaction (PCR) specific for each parasite. Though both methods effectively recovered parasites, our trial also suggests that filtration and flocculation does not completely remove Cryptosporidium and Cyclospora oocysts in the wash water. In conclusion, these methods can significantly contribute to environmental testing during outbreak investigations and surveillance studies.Support or Funding InformationNSF #1067896

An alternative method to recover Toxoplasma gondii from greenery and fruits

Toxoplasma gondii oocysts are an important form of contamination with a high dispersion in the environment, but their detection is still a challenge. This study evaluated the recovery of oocysts from strawberries and crisphead lettuce. Samples (250 g of strawberries or one head of lettuce) were experimentally inoculated with 10, 102, 103 and 104 T. gondii oocysts, by two separate processes, spot dripping and immersion. Then, 50 g of each sample was washed, filtered through a cellulose ester membrane, and concentrated by centrifugation. Three aliquots were taken for DNA extraction in a direct way, after freeze–thaw (FT) cycles or ultrasound (US), followed by PCR (B22-B23 and Tox4-Tox5 primers). The T. gondii DNA was amplified with the primers B22-B23 in all samples contaminated by dripping and when DNA extraction was carried out after FT or US. These techniques may be useful in epidemiological surveillance in the control of this zoonosis.

A new immunomagnetic bead separation–surfactant extraction treatment protocol for rapid and sensitive quantitative PCR detection of Cryptosporidium parvum DNA

The Cryptosporidium oocyst is encased in a robust wall that is extremely resistant to detrimental environmental factors such as chlorine used to disinfect potable water. Therefore, extracting oocyst DNA is not a trivial undertaking. Standard procedures used to extract DNA from oocysts, such as freeze–thaw (F/T) methods and DNA purification kits, are time-consuming and expensive and are difficult to implement in routine clinical practice. Therefore, we developed a surfactant extraction treatment (SET) that efficiently extracts DNA from the oocyst. Immunomagnetic separation (IMS) combined with quantitative real-time polymerase chain reaction (qPCR) detects pathogenic microorganisms with high sensitivity. The objective of the present study was to evaluate SET for its ability to simplify qPCR detection of 18S rDNA directly from immunomagnetic bead–oocyst conjugates. DNA extracted directly from the conjugates using SET did not affect DNA amplification in the qPCR assay. Further, the rate of DNA amplification using IMS–SET was greater than that using F/T combined with the DNA purification kit. The rate of recovery of oocysts from surface water samples spiked with oocysts did not differ significantly from previously published values. These data demonstrate that the new IMS–SET protocol using qPCR can simplify the recovery and detection of Cryptosporidium oocysts.

Optimization and validation of methods for isolation and real-time PCR identification of protozoan oocysts on leafy green vegetables and berry fruits

Leafy green vegetables and berry fruits have vastly different physical and biochemical characteristics, are typically consumed raw with minimal washing, and are potential transmission vehicles for food-borne disease caused by protozoan parasites such as Cryptosporidium, Cyclospora and Toxoplasma. Validation of a generic oocyst isolation and detection method applicable to each leafy green and berry type is required to provide reliable laboratory support for surveillance programs and as necessary for disease outbreak investigations. The objectives of the current study were to optimize and validate the performance of these methods for the isolation of protozoan oocysts from several types of leafy greens and berries. Eimeria papillata oocysts were used as a surrogate for coccidia of public health concern to spike produce samples. An artificial stomacher or orbital shaker was used, followed by centrifugation, to isolate and concentrate oocysts, respectively, and a qPCR melt curve analysis (qPCR MCA) was used for detection and identification. Processing methods, wash buffers and storage conditions were evaluated and optimized for five types of berries (blackberries, blueberries, cranberries, raspberries and strawberries), five types of herb (cilantro, dill, mint, parsley, thyme) and green onions. Blackberries, cranberries, raspberries and strawberries were most effectively washed by orbital shaking with an elution solution, while glycine buffer was more effective for blueberries. Stomaching with a glycine buffer was optimal for oocyst recovery in leafy herbs with soft stems, while aromatic woody-stemmed herbs such as thyme required orbital shaking to minimize the release of PCR inhibitors. Oocyst recovery from green onions was highest when processed by orbital shaking with elution solution. Oocyst recovery rates ranged from 4.1–12% for berries and 5.1–15.5% for herbs and green onions. As few as 3 oocysts per gram of fruit, or 5 oocysts per gram of herbs or green onions could reliably be detected using the optimized isolation methods and qPCR MCA.

Cryptosporidium in Wastewater: Occurrence, Removal and Inactivation

This study focused on one pathogen, Cryptosporidium parvum, and its occurrence in wastewater. In order to conduct an occurrence study, it was firstly necessary to develop methods for recovery of Cryptosporidium oocysts from wastewater matrices. Due to the differences in matrix composition from raw sewage to tertiary effluents, different methods for recovery and enumeration of oocysts were developed based on matrix quality.A single method was developed for raw sewage and primary influents; a second method for secondary and tertiary effluents; and a third method for biosolids. These methods were used in a survey of Cryptosporidium occurrence at 10 wastewater plants in the U.S. over a 15-month period. To determine if oocysts found in wastewater samples represented a public health risk, cell culture methods were employed to examine infectivity of recovered oocysts.This title belongs to WERF Research Report SeriesISBN: 9781843397182 (Print)ISBN: 9781780404417 (eBook)

Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR

Waterborne transmission of Toxoplasma gondii is a potential public health risk and there are currently no agreed optimised methods for the recovery, processing and detection of T. gondii oocysts in water samples. In this study modified methods of T. gondii oocyst recovery and DNA extraction were applied to 1427 samples collected from 147 public water supplies throughout Scotland. T. gondii DNA was detected, using real time PCR (qPCR) targeting the 529bp repeat element, in 8.79% of interpretable samples (124 out of 1411 samples). The samples which were positive for T. gondii DNA originated from a third of the sampled water sources. The samples which were positive by qPCR and some of the negative samples were reanalysed using ITS1 nested PCR (nPCR) and results compared. The 529bp qPCR was the more sensitive technique and a full analysis of assay performance, by Bayesian analysis using a Markov Chain Monte Carlo method, was completed which demonstrated the efficacy of this method for the detection of T. gondii in water samples.

Real scale environmental monitoring of zoonotic protozoa and helminth eggs in biosolid samples in Brazil.

Biosolid is the product of the activated sludge treatment system and its final disposition is subject of ongoing discussion as this residue can therefore harbor a great number and variety of pathogens. This study was aimed to (1) monitor the presence of Giardia and Cryptosporidium in biosolid samples from a treatment plant in Campinas, SP, Brazil, (2) observe Giardia cyst wall morphological integrity in treated samples using scanning electron microscopy (SEM) and (3) verify the presence and viability of helminth eggs. Cysts were present in 33.3% of the samples, whereas oocysts were detected in 8.3%. Viable Ascaris sp. Toxocara sp. and similar to Trichuris sp. eggs were found through the use of Mexican Official Norm. Results demonstrate the difficulties inherent in working with biosolid as factors such as temperature, ionic strength and pH influenced the recovery of cysts and oocysts. Pores and ruptures were not observed in cyst wall visualized by SEM following 45days of exposure to sunlight, only minimal morphological changes. These observations emphasize both the importance of adequate treatment of sewage sludge and the need to develop appropriate techniques for the detection of Giardia and Cryptosporidium in this type of sample. This is the first time that a study was done in a real scale for biosolid samples in determining the presence of pathogenic protozoa as Giardia and Cryptosporidium in Brazil, and also observed minimal cyst wall damage after sunlight treatment.

Efficacy of Wash Solutions in Recovering Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from Basil

Parasitic diseases can be acquired by ingestion of contaminated raw or minimally processed fresh produce (herbs and fruits). The sensitivity of methods used to detect parasites on fresh produce depends in part on the efficacy of wash solutions in removing them from suspect samples. In this study, six wash solutions (sterile E-Pure water, 3% levulinic acid-3% sodium dodecyl sulfate, 1 M glycine, 0.1 M phosphate-buffered saline, 0.1% Alconox, and 1% HCl-pepsin) were evaluated for their effectiveness in removing Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil. One hundred or 1,000 oocysts of these parasites were inoculated onto the adaxial surfaces of 25 g of basil leaves, placed in stomacher bags, and stored for 1 h at 21°C or 24 h at 4°C. Leaves were hand washed in each wash solution for 1 min. DNA was extracted from the wash solutions and amplified using PCR for the detection of all parasites. Oocysts inoculated at a concentration of 1,000 oocysts per 25 g of basil were detected in all wash solutions. At an inoculum concentration of 100 oocysts per 25 g, oocysts were detected in 18.5 to 92.6% of the wash solutions. The lowest variability in recovering oocysts from basil inoculated with 100 oocysts was observed in 1% HCl-pepsin wash solution. Oocyst recovery rates were higher at 1 h than at 24 h postinoculation. Unlike most bacteria, parasites cannot be enriched; therefore, an optimal recovery process for oocysts from suspected foods is critical. The observations in this study provide guidance concerning the selection of wash solutions giving the highest retrieval of parasite oocysts.

Effects of sub-zero storage temperatures on endoparasites in canine and equine feces

Fecal samples from wild and domestic carnivores are routinely frozen for three days at −80°C to kill eggs of Echinococcus spp., following recommendations from the World Health Organization (WHO) and World Organization for Animal Health (OIE). This is done to decrease the risk of zoonotic infection with these pathogenic cestodes. In addition, it is often necessary to freeze fecal samples collected for research prior to batch processing by a limited number of personnel, especially large numbers of samples or those collected in remote locations. The effect of freezing on the recovery of endoparasite eggs, cysts and oocysts from fecal samples is not well documented, even in hosts for which veterinary diagnostic submissions are relatively common. In this study, fecal samples from naturally infected dogs and horses were split into four treatment groups: fresh; −80°C for 3 days; −20°C for 30 days; and −80°C for 3 days followed by −20°C for an additional 30 days. Temperatures and times were chosen to simulate diagnostic and research protocols currently in place. Helminth eggs and sporocysts of Sarcocystis spp. were counted using a quantitative double centrifugation sucrose fecal flotation (modified Stoll egg count). Repeated measures ANOVA was used to detect differences in egg/sporocyst counts between the treatment groups for Sarcocystis spp. sporocysts, taeniid eggs (Taenia and/or Echinococcus spp.), ascarid eggs (Parascaris equorum, Toxocara canis, Toxascaris leonina), and strongylid type eggs (Uncinaria stenocephala, and equine strongylids, most likely cyathostomins). Counts for P. equorum and strongylid type eggs (both equine and canine) decreased significantly following freezing. Post-freezing, some samples that had been positive on fresh examination became negative for Parascaris and strongylid eggs. This study suggests that protocols requiring freezing artificially lowers recovery of eggs of Parascaris and strongylid nematodes in fecal surveys; however, recovery rates for eggs of other helminth species as well as sporocysts of Sarcocystis spp. were relatively robust compared to the freshly tested fecal samples. This is reassuring for large scale fecal surveys needed for fecal egg count reduction analyses following treatment, and for surveillance in wildlife and remote human and animal populations.