Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Source of C. parvum oocysts.The oocysts were obtained from experimental infection of a female Holstein calf, extracted from the feces using continuous flow centrifugation, purified by cesium chloride gradient centrifugation, and stored at 4°C in phosphate-buffered saline (PBS) (pH 7.4).

Processing of C. parvum spiked oyster tissue samples.One hundred C. virginica oysters were obtained from Oyster Ranchers Association Inc., Ridge, MD; placed into 208 liters of artificial seawater (salinity, 12 ppt); and left for 2 days to equilibrate. The recovery of oocysts was compared using four different oyster tissue processing methods (groups 1 to 4) (Fig. 1). For treatment group 1, the entire oyster tissue from 11 oysters, including hemolymph aspirated from the adductor muscle prior to shucking, was pooled, added to 60 ml of PBS, and homogenized by 5 pulses of 30 s each, using an Omni Mixer homogenizer (Omni International, Marietta, GA). The homogenate was divided evenly among 11 50-ml conical tubes. For group 2, hemolymph was collected from 11 oysters, which were then shucked and dissected to remove the gills and digestive diverticula. These tissues were added to the hemolymph along with 140 ml of PBS, homogenized, and divided evenly among 11 tubes. For group 3, only hemolymph was collected from 11 oysters, which was then pooled and distributed evenly among 11 tubes. For group 4, the meat was processed separately from the hemolymph. The whole meat from 11 oysters was pooled, added to 120 ml of PBS, and homogenized as described above, while hemolymph from those oysters was pooled and kept separately. Again, both homogenate and hemolymph were divided equally among 11 tubes.

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FIG. 1.

Diagram presenting four treatment methods of Eastern oyster (Crassostrea virginica) tissue spiked with Cryptosporidium parvum oocysts.

Ten of 11 tubes from each group were spiked with C. parvum oocysts, and the last tube was treated as an unspiked negative control. Each tube was spiked with ∼1.0 × 10³ oocysts except tubes from group 4, where meats and hemolymph were kept separate. For this group, each tube was spiked with ∼5 × 10² oocysts. All tubes were vortexed and stored overnight at 4°C. To verify the actual number of oocysts spiked into each sample, the same volume of oocyst suspension was added directly to three slide wells, allowed to dry, and fixed with methanol and then oocysts were counted using immunofluorescent antibodies (IFA). The average of the replicates was used as the actual number of oocysts spiked into each sample.

Ten milliliters of diethyl ether was added to each tube of oyster homogenate, resulting in a total volume of 30 ml. The tubes were vortexed for 30 s and then centrifuged (1,000 × g; 10 min) to pellet oocysts. The supernatant, including the tissue suspended between the aqueous and organic layers, was removed, and the pellet was washed twice, once with 20 ml of PBS and again with 20 ml of distilled water. For group 4, where hemolymph was not homogenized with the meat, the hemolymph was added to the distilled water during the second wash. The pellets were resuspended in 10 ml distilled water. For group 3, where only the hemolymph was collected, distilled water was added to the hemolymph to a 10-ml total volume.

Processing of oysters exposed in C. parvum-contaminated water.A 208-liter tank filled with artificial seawater at 12-ppt salinity and holding 55 oysters was spiked with 1.0 × 10⁵C. parvum oocysts per oyster for a total inoculum of 5.5 × 10⁶C. parvum oocysts. To induce filter feeding, 1 ml of Instant Algae shellfish food (Reed Mariculture Inc., Campbell, CA) was added to the tank. Oysters were allowed to filter the water for 24 h. The recovery of oocysts was compared using four different processing methods, with 10 oysters processed separately for each group (i.e., I to IV) (Fig. 2). For treatment group I, whole oyster tissue and hemolymph from each oyster were homogenized together after 1× PBS was added to a total volume of 20 ml. For treatment group II, the gills and digestive diverticula were added to the hemolymph along with 1× PBS to a total volume of 20 ml and then homogenized. For treatment group III, the hemolymph was drawn and each oyster was dissected to remove the gills and digestive diverticula. The meat and hemolymph were kept in separate tubes. After 1× PBS was added to the gills and digestive diverticula to a total volume of 20 ml, the meat was homogenized. The hemolymph samples were brought up to a 10-ml total volume with 1× PBS. For treatment group IV, oocyst recovery from meat and that from hemolymph were characterized separately in order to determine the distribution of oocysts between meat and hemolymph after natural uptake. Hemolymph was drawn from the oysters, and gills and digestive diverticula were dissected from the meat and stored separately. Gills and diverticula were homogenized after 1× PBS was added to a total volume of 20 ml. Hemolymph samples were brought up to a total volume of 10 ml with 1× PBS. To determine whether the distribution of oocysts between oyster meat and hemolymph is related to exposure time, 10 oysters were harvested from the tank 1 week after water contamination and processed as described for group IV.

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FIG. 2.

Diagram presenting four treatment methods applied to Eastern oysters (Crassostrea virginica) exposed to Cryptosporidium parvum in contaminated water.

Ten milliliters of diethyl ether was added to each tube of oyster homogenate, resulting in a total volume of 30 ml. The tubes were vortexed for 30 s and then centrifuged at 1,000 × g for 10 min to pellet C. parvum oocysts. The supernatant was removed, and the pellet was washed twice, once with 20 ml of PBS and again with 20 ml of distilled water. For group III, where hemolymph was not homogenized with the meat, the hemolymph was added to 10 ml of distilled water during the second wash. The pellets were resuspended in 10 ml distilled water. For group IV, immunomagnetic separation (IMS) was performed separately on the hemolymph and resuspended homogenate pellets.

IMS and IFA procedures.To recover C. parvum oocysts from the homogenate pellets or hemolymph, each 10-ml sample was processed by IMS according to the manufacturer's instructions (Dynabeads anti-Cryptosporidium and anti-Giardia kit; Dynal) except for the addition of a second wash step using 1 ml of 1× SL-Buffer A and a second elution that was combined with the first elution for 100 μl total eluant on each slide well. Negative controls consisted of 10 ml of distilled water, and for the positive control, 10 ml of distilled water was spiked with ∼1.0 × 10³C. parvum oocysts. Slides were fixed with methanol and air dried, and oocysts were enumerated by IFA using a commercially available test kit (MeriFluor Cryptosporidium/Giardia; Meridian Bioscience Inc.) per the manufacturer's instructions.

PCR detection of C. parvum oocysts from oyster homogenates.To determine the limit of detection using the optimized processing method for recovery, a nested PCR procedure was performed on DNA extracted from C. parvum oocysts recovered from spiked oyster homogenates. Five unexposed oysters were processed as described for treatment group 4 from the homogenate spiking experiment, except that IMS eluants were stored in microcentrifuge tubes instead of being fixed onto slides. Each oyster sample was spiked with 10, 100, 1,000, or 10,000 C. parvum oocysts. Tris-EDTA (pH 8) was added to the IMS eluants to a final volume of 500 μl. Twenty microliters of 10% sodium dodecyl sulfate and 5 μl 20-mg/ml proteinase K were added to each tube and then incubated overnight at 45°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation. To determine if PCR inhibitors were preventing detection of spiked homogenates, genomic DNA was added to the DNA extracted from the oyster sample spiked with 1.0 × 10⁴ oocysts just prior to PCR amplification. A 434-bp fragment of the 18S rRNA gene was amplified using methods modified from the work of Jellison et al. (17). Briefly, a 1,056-bp fragment was amplified using the forward primer KLJ1 (5′-CCACATCTAAGGAAGGCAGC-3′) and reverse primer KLJ2 (5′-ATGGATGCATCAGTGTAGCG-3′). For the secondary PCR, the forward primer CPB-DIAGF (5′-AAGCTCGTAGTTGGATTTCTG-3′) and the reverse primer CPB-DIAGR (5′-TAAGGTGCTGAAGGAGTAAGG-3′) were used to amplify the 434-bp fragment. The initial amplification reaction was performed with 15 μl of DNA template, and 3 μl of the primary amplification product was used as a template for the secondary PCR. Each PCR consisted of 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 60 s; an initial denaturation step consisting of incubation at 94°C for 4 min and a final extension step consisting of incubation at 72°C for 10 min were also included. PCR products were visualized on a 1% agarose gel stained with ethidium bromide.

Statistical analysis.The statistical analysis was performed using STATA 8.0 (Stata Corporation, College Station, TX). A Kruskal-Wallis nonparametric test of equality was used to determine if there were any significant differences in recovery between any of the oyster processing method groups. Pairwise comparisons using a nonparametric two-sample Mann-Whitney test were used to determine which groups had significantly different oocyst recoveries. Results were considered to be significant at P < 0.05.

Recovery of C. parvum oocysts from spiked tissue samples. C. virginica homogenates were spiked with ∼1.0 × 10³ or ∼5.0 × 10²C. parvum oocysts, depending on processing treatment group. Based on direct IFA counts of the spiking suspension the actual numbers of oocysts spiked into the homogenates were on average 742 and 345 oocysts, respectively. The average numbers of oocysts recovered and the recovery percentages for each treatment group are presented in Table 1. There were significant differences in recovery among the treatment groups (Kruskal-Wallis, P = 0.0001). Pairwise comparisons using a two-sample Mann-Whitney test showed that oocyst recovery from treatment group 4 was significantly greater than the recovery from treatment group 3 (P = 0.0143), oocyst recovery from treatment group 3 was greater than recovery from group 2 (P = 0.0002), and oocyst recovery from treatment group 2 was greater than recovery from group 1 (P = 0.0036). The recovery efficiency of the IMS kit as determined by numbers of oocysts recovered from distilled water spiked with C. parvum oocysts was 90%. No oocysts were detected in any of the negative-control oysters by IFA.

Recovery of C. parvum oocysts from oysters exposed to C. parvum-contaminated water.After 24 h of exposure to C. parvum-contaminated water, all oysters tested positive for the oocysts by IFA, regardless of the processing method used to recover oocysts. The average number of oocysts observed by IFA for each treatment group is shown in Table 1. For treatment group IV, where hemolymph and tissue homogenates were processed and analyzed separately, the average recovery from hemolymph alone was 116 oocysts (14% of total recovery), while the average recovery from the tissue homogenate was 713 oocysts (86% of total recovery). A nonparametric Kruskal-Wallis test of equality showed that there were significant differences in oocyst recoveries depending on processing method used (P = 0.0404). Pairwise comparisons using a nonparametric two-sample Mann-Whitney test showed that recovery from treatment group I was greater than recovery from treatment group II (P = 0.0494) and treatment group IV (P = 0.0082) but not statistically different from recovery from treatment group III (P = 0.0696).

One week after exposure to C. parvum-contaminated water, average recoveries from oysters (n = 10) processed using the method for treatment group IV were 27 oocysts (i.e., 40% total recovery) from hemolymph samples and 40 oocysts from tissue homogenates (i.e., 60% of total recovery).

PCR detection of C. parvum oocysts from oyster homogenates.For oysters processed as described for treatment group 4 of the homogenate spiking experiment, as few as 10 spiked oocysts could be detected using a nested PCR procedure (Fig. 3). Cryptosporidium DNA was amplified for each of the spiked samples, including the DNA extract to which genomic DNA was added, indicating that PCR inhibition was not observed.

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FIG. 3.

Nested PCR detection of Cryptosporidium parvum oocysts spiked into Eastern oyster (Crassostrea virginica) tissue homogenate. Lanes: M, 100-bp molecular marker; 1, PCR negative control; 2, PCR positive control; 3, 10⁴ oocysts plus C. parvum genomic DNA; 4, 10⁴ oocysts; 5, 10³ oocysts; 6, 10² oocysts; 7, 10 oocysts.