Bioaccumulation and elimination of Cryptosporidium parvum oocysts in experimentally exposed Eastern oysters (Crassostrea virginica) held in static tank aquaria
Introduction
The protozoan parasite Cryptosporidium parvum is a pathogen commonly found in domestic ruminants across the globe, with higher prevalence in young ruminants such as calves. Oocysts are transmissible to humans and other animals/wildlife via direct contact with contaminated fecal material or by contact with or consumption of contaminated sources such as water and food (Dixon et al., 2011). Although human cryptosporidiosis is generally a self-limiting disease, oocyst shedding may persist for up to 38 days and infections can be fatal in immunocompromised individuals (Chappell et al., 1996, Peng et al., 1997, Graczyk et al., 1998).
Cryptosporidium oocysts have been detected in a variety of bivalve shellfish species from both clean and contaminated harvesting areas across the globe using immunofluorescence assays (Fayer et al., 1998, Fayer et al., 2002, Fayer, 2008, Freire-Santos et al., 2000, Gómez-Couso et al., 2003b, Giangaspero et al., 2005, Levesque et al., 2006, Graczyk et al., 2007a, Schets et al., 2007, Robertson and Gjerde, 2008, Leal et al., 2008, Lucy et al., 2010), with several studies confirming oocysts as zoonotic C. parvum by polymerase chain reaction (PCR) techniques (Fayer et al., 2003, Gómez-Couso et al., 2004, Gómez-Couso et al., 2006a, Gómez-Couso et al., 2006b, Miller et al., 2005, Molini et al., 2007, Leal et al., 2008, Leal et al., 2011; Giangaspero et al., 2009, Levesque et al., 2010). Contamination of bivalves occurs predominantly in coastal or estuarine environments with wastewater sewage discharges and agricultural run-off from farms (Sunnotel et al., 2007). It has been established that the number of zoonotic protozoa retained by bivalve shellfish is not correlated to bivalve size or the fecal coliform levels at the harvesting site (Gómez-Couso et al., 2003b, Graczyk et al., 2007a). Oysters are of particular public health concern as they are often consumed raw, and oocysts recovered from varying oyster species can remain viable after filtration for periods of 7–33 days or more (Freire-Santos et al., 2000, Robertson, 2007).
The ability for bivalve shellfish to capture, bioaccumulate, and eliminate Cryptosporidium oocysts remains poorly understood. Based on published data from experimentally and naturally contaminated bivalves, bioaccumulation of oocysts is a multifactorial process that is known to vary depending on the following: bivalve and/or pathogen physiology (such as size and species), exposure period, exposure dose, and factors that impact filtration rates such as salinity and temperature (Pile and Young, 1999, Freire-Santos et al., 2000; Rouillon and Navarro, 2003, Graczyk et al., 2006, Nappier et al., 2008, Nappier et al., 2010). Differential selection of various particles by size and/or shape has been documented, but most data suggest that undescribed qualitative factors also influence capture efficiencies and retention (Ward and Shumway, 2004, Espinosa et al., 2008). For example, some studies indicate that Cryptosporidium oocysts are readily ingested by Suminoe oysters (Crassostrea ariakensis), whereas cysts of another zoonotic protozoan parasite, Giardia duodenalis, are not (Graczyk et al., 2006). Particles that are selectively digested are excreted in feces, whereas particles that are rejected by the gills or the labial palp prior to digestion form the pseudofeces (Shumway et al., 1985).
Several studies on Asian brackish water clams (Corbicula japonica) concluded that ~ 90% of oocysts spiked into tank water were actually ingested by the clams, most of which was excreted in the feces less than 4 days post-water exposure (Izumi et al., 2004). Further studies on this species in depuration systems quantified the number of oocysts in clam tissue and feces over time and determined that oocyst excretion patterns are linear whether clams underwent single or multiple oocysts exposures (Izumi et al., 2006).
Similar work on oyster species that are of high economic importance in North America is needed, as the selectivity of oocysts, maximum concentration levels, and rates of oocyst excretion observed in freshwater clams likely varies from other shellfish species due to the aforementioned factors. It has been documented that non-native Suminoe oysters retain significantly more oocysts than Eastern oysters (Crassostrea virginica) under similar holding conditions (Fayer et al., 1997, Graczyk et al., 2006), and Suminoe oysters were still contaminated 1 week after oocysts were no longer detectable in Eastern oysters (Nappier et al., 2010). It is therefore possible that Eastern oysters pose less of a public health risk for contracting cryptosporidiosis compared to other oyster species cultured in North America.
Most available studies on oysters have focused on contamination with multiple pathogens simultaneously, which may alter the particle selection processes. Additionally, many detection studies in shellfish only examined specific target tissues (Fayer et al., 1998, Miller et al., 2005, Graczyk et al., 2006) as opposed to whole tissue homogenates. Assessing natural contamination events in whole, individual bivalves are more feasible and practical as oocysts can be found throughout the digestive system (gills and digestive diverticula) as well as within the hemolymph, flesh, and shell innerwater (Gómez-Couso et al., 2005, Miller et al., 2006, Li et al., 2006, Schets et al., 2007, Fayer, 2008). There is currently no gold standard by which oocysts are to be recovered from shellfish, and several published methods are currently available (Robertson, 2007). Some protocols employ the use of an immunomagnetic separation step that is routinely used for water samples prior to examination by immunofluorescence microscopy, however the efficacy of this protocol in varying bivalve species is unclear (Miller et al., 2005, Miller et al., 2006, Li et al., 2006, Schets et al., 2007, Robertson and Gjerde, 2008). Additionally, no detailed studies have been conducted to quantify Cryptosporidium oocyst retention and elimination in Eastern oysters over time, although Fayer et al. (1997) did examine oyster gills and hemolymph for oocysts by histology for up to 7 days after experimentally exposing samples to an inoculum equivalent to 630 oocysts/mL. Nappier et al. (2008) did quantify C. parvum bioaccumulation in Eastern oysters co-localized with Suminoe oysters (C. ariakensis) for 29 days, but oysters were simultaneously exposed to multitude of other protozoa and viruses, which may affect the differential selection process of some pathogens over others.
The primary aims of this study were to compare the bioaccumulation and elimination of C. parvum oocysts by Eastern oysters when placed under acute (1 day) or chronic (7 days) exposure periods in a high salinity static tank system using a processing method modified for detecting oocysts from individual oysters without the use of immunomagnetic separation techniques. Secondary aims were to quantitate the number of oocysts retained by oysters over time under these exposure conditions (chronic vs. acute) as well as the number of oocysts present in fecal material (consisting of pseudofeces, feces, and settled debris) and tank water.
Section snippets
Source of C. parvum oocysts
Oocysts were isolated by fecal flotation methods following a previously published protocol (Budu-Amoako et al., 2012), from cattle fecal samples submitted to the Atlantic Veterinary College for diagnostic testing. Subsamples of oocysts were stained by direct immunofluorescence antibody (IFA) as described by Budu-Amoako et al. (2012), and species identity was confirmed by sequencing the 18S rRNA gene in both directions (Genome Quebec Innovation Centre at McGill University, Montreal, Quebec).
Recovery efficiency testing for spiked oocyst doses in oysters and fecal material
The mean recovery efficiency of oocysts from individual spiked oysters after processing varied between 72% (10 oocysts/oyster) to 86% (5000 oocysts/oyster) (Table 1). Similarly, the recovery efficiency of oocysts from spiked oyster fecal pellets ranged from 67% (10 oocysts/10 mL settled fecal material) to 84% (5000 oocysts/mL) (Table 1).
Oocysts recovered from oyster fecal material and water samples in exposure trials
When under chronic exposure over 7 days, approximately 41% and 43% of the total tank inoculum was recovered from oysters from both the low and high exposure groups,
Oocyst recovery methods
The methods described in this study showed recovery efficiencies for C. parvum oocysts that are higher than other published studies (Graczyk et al., 1999, Fayer et al., 2002, Robertson and Gjerde, 2008). This method is cost-effective as it eliminates the use of immunomagnetic separation, which has been shown to either improve or reduce oocyst recovery in different studies (MacRae et al., 2005, Miller et al., 2006, Schets et al., 2007, Robertson and Gjerde, 2008). This technique also enabled the
Acknowledgments
We wish to thank Mathew Saab and Cynthia Mitchell for their technical assistance in the laboratory, as well as the UPEI Biosafety Committee and AVC animal housing facility for their aid in procuring a biocontainment level 2 certified room for conducting tank experiments. We would also like to acknowledge the AVC diagnostic parasitology lab for providing us with C. parvum oocysts.
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2023, Food and Waterborne ParasitologyApplication of next generation sequencing for detection of protozoan pathogens in shellfish
2020, Food and Waterborne ParasitologyCitation Excerpt :Preliminary findings suggested a potential reduction of oyster DNA amplification by this digest solution treatment. To further evaluate this effect, hemolymph (oyster circulatory fluid) was collected through aspiration with a needle and treated using a modified pepsin-HCl digestion protocol as described for whole oyster tissue homogenates (Willis et al., 2014). Both pepsin-HCl treated and untreated hemolymph and homogenate samples were spiked with 5 μl of parasite stock DNA solutions (50 uL of eluted DNA from 10,000 oocysts) and subjected to nucleic acid extraction, multiplex PCR and gel electrophoresis as described above.
Bayesian risk assessment model of human cryptosporidiosis cases following consumption of raw Eastern oysters (Crassostrea virginica) contaminated with Cryptosporidium oocysts in the Hillsborough River system in Prince Edward Island, Canada
2020, Food and Waterborne ParasitologyCitation Excerpt :The 30-day relay period is part of the scenarios explained in the following sections. It was chosen to assess the residual contamination at 30 days of relay because these periods have been shown to be enough to eliminate fecal coliforms but not Cryptosporidium oocysts (Gómez-Couso et al., 2003; Schijven et al., 2013), which can remain viable after filtration periods of 7–33 days or more and still be infectious to humans (Freire-Santos et al., 2000; Robertson, 2007; Willis et al., 2014). The depuration count data and Poisson regression were used to assess the depuration rate, and estimate the depuration reduction rates at days 14 (oo.red.14), 21 (oo.red.21), and 30 (oo.red.30), as presented in Table 2.
Comparison of Cryptosporidium oocyst recovery methods for their applicability for monitoring of consumer-ready fresh shellfish
2019, International Journal of Food MicrobiologyCitation Excerpt :For each level of contamination, the test was carried out in 6 replicates. Cryptosporidium oocysts were extracted and separated from shellfish homogenate portions using the following methods: i) the method of Robertson and Gjerde (2008) utilising pepsin digestion of shellfish in conjunction with immunomagnetic separation (IMS) of oocysts (method A), ii) the method employing of pepsin-HCl treatment without the application of IMS described by Willis et al. (2014) (method B), and iii) a strainer method with direct oocyst extraction and separation from shellfish tissue debris using IMS (method C; Miller et al., 2005a). The percentage of recovered Cryptosporidium oocysts was determined based on the number of FITC-C-mAb stained oocysts recovered from each sample in comparison to the number of oocysts present in seeding suspensions.