Bioaccumulation and elimination of Cryptosporidium parvum oocysts in experimentally exposed Eastern oysters (Crassostrea virginica) held in static tank aquaria

https://doi.org/10.1016/j.ijfoodmicro.2013.11.033Get rights and content

Highlights

  • Bioaccumulation of oocysts in oysters is exponential in the first 24 h.

  • Contamination in oysters persists after 6 days of static tank depuration.

  • 33–34% of inoculated oocysts were recovered in oyster fecal material from chronic exposures.

Abstract

A variety of human enteropathogens, including viruses, bacteria, and parasites, have been shown to bioaccumulate in suspension-feeding bivalve shellfish. Cryptosporidium parvum is a zoonotic protozoan parasite that has been detected in many shellfish species within both fecally contaminated and clean oyster growing areas across the globe. For this study, C. parvum oocysts (1000 and 10,000) were spiked into 10 L of water in static tank systems housing Crassostrea virginica. Oysters were either held in the contaminated aquaria for 7 days of exposure or were exposed for 24 h and subsequently placed in a clean static tank system for the remainder of the trial. Individual oysters, fecal material, and tank water were analyzed for oocysts up to 7 days post-exposure via direct immunofluorescence. Oysters held under chronic exposure conditions gradually accumulated oocysts (1.5 or 34.4 oocysts/oyster/day for low or high dose exposure groups, respectively) between days 1 and 7, with an exponential uptake in oocysts observed within the first 24 h post-exposure (mean uptake of 29.6 or 241.9 oocysts/oyster, respectively). Oysters that were transferred to clean water after 24 h were capable of slowly depurating oocysts, following a linear trend. During chronic exposure trials 48–49% of the total spiked inoculum was recovered from oyster tissue, whereas 4.8–5.9% and 38–40% was recovered from tank water and from fecal material at day 7, respectively. In acute exposure trials, 30–31% of the total tank inoculum was found in oysters, suggesting that chronically exposed oysters were likely re-filtering some oocysts. Examinations of oyster fecal material from acute exposures revealed that 72–82% of oocysts recovered were already excreted at the time of oyster transfer (day 1), with only 18–28% being excreted during the static depuration phase. These data support that although most C. parvum oocysts are removed by C. virginica oysters within 24 h, elimination after this point occurs slowly. Additionally, chronic exposures demonstrate that wild or cultured oysters in saline environments that are frequently exposed to sources of Cryptosporidium may be unable to eliminate the parasites at a rate that balances initial uptake.

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.

References (60)

  • X. Li et al.

    Cryptosporidium oocysts in mussels (Mytilus edulis) from Normandy (France)

    Int. J. Food Microbiol.

    (2006)
  • D. Love et al.

    Removal of Escherichia coli, Enterococcus fecalis, coliphage MS2, poliovirus, and hepatitis A virus from oysters (Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) by depuration

    Int. J. Food Microbiol.

    (2010)
  • M. MacRae et al.

    The detection of Cryptosporidium parvum and Escherichia coli 0157 in UK bivalve shellfish

    J. Microbiol. Methods

    (2005)
  • W.A. Miller et al.

    New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast

    Int. J. Parasitol.

    (2005)
  • W.A. Miller et al.

    Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus)

    J. Microbiol. Methods

    (2006)
  • U. Molini et al.

    Temporal occurrence of Cryptosporidium in the manila clam Ruditapes philippinarum in Northern Adriatic Italian lagoons

    J. Food Prot.

    (2007)
  • L. Robertson

    The potential for marine bivalve shellfish to act as transmission vehicles for outbreaks of protozoan infections in humans: a review

    Int. J. Food Microbiol.

    (2007)
  • G. Rouillon et al.

    Differential utilization of species of phytoplankton by the mussel Mytilus edulis

    Acta. Oecol.

    (2003)
  • F.M. Schets et al.

    Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, the Netherlands

    Int. J. Food Microbiol.

    (2007)
  • S.E. Shumway et al.

    Particle selection, ingestion, and absorption in filter feeding bivalves

    J. Exp. Mar. Biol. Ecol.

    (1985)
  • A. Tamburrini et al.

    Long term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis)

    Int. J. Parasitol.

    (1999)
  • C.L. Chappell et al.

    Cryptosporidium parvum: intensity of infection and oocysts excretion patterns in healthy volunteers

    J. Infect. Dis.

    (1996)
  • M.V. Collins et al.

    The effect of high-pressure processing on infectivity of Cryptosporidium parvum oocysts recovered from experimentally exposed Eastern oysters (Crassostrea virginica)

    J. Eukaryot. Microbiol.

    (2005)
  • M.V. Collins et al.

    The effects of E-beam irradiation and microwave energy on Eastern oysters (Crassostrea virginica) experimentally infected with Cryptosporidium parvum

    J. Eukaryot. Microbiol.

    (2005)
  • L.A. Comeau et al.

    Comparison of Eastern oyster (Crassostrea virginica) and blue mussel (Mytilus edulis) filtration rates at low temperatures

  • E.P. Espinosa et al.

    Particle selection in the ribbed mussel Geukensia demissa and the Eastern oyster Crassostrea virginica: effect of microalgae growth stage

    Estuar. Coast. Shelf Sci.

    (2008)
  • E.W.C. Farias et al.

    Detection of Cryptosporidium spp. oocysts in raw sewage and creek water in the city of Sao Paulo, Brazil

    Braz. J. Microbiol.

    (2002)
  • R. Fayer

    Chapter 1: general biology

  • R. Fayer et al.

    Potential role of the Eastern oyster, Crassostrea virginica, in the epidemiology of Cryptosporidium parvum

    Appl. Environ. Microbiol.

    (1997)
  • R. Fayer et al.

    Survival of infectious Cryptosporidium parvum oocysts in seawater and eastern oysters (Crassostrea virginica) in the Chesapeake Bay

    Appl. Environ. Microbiol.

    (1998)
  • Cited by (19)

    • Application of next generation sequencing for detection of protozoan pathogens in shellfish

      2020, Food and Waterborne Parasitology
      Citation 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 Parasitology
      Citation 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 Microbiology
      Citation 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.

    View all citing articles on Scopus
    View full text