Adaptation of ray's fluid thioglycollate medium assay to detect and quantify planktonic stages of Perkinsus spp. parasites.

ABSTRACT The parasites Perkinsus spp. are relatively easily and inexpensively detected in host tissues, but available methods to detect free-living planktonic stages are technologically complex and expensive. As a result, few studies have been conducted to detect and quantify free-living stages during transmission. Here, we describe an adaptation of Ray fluid thioglycollate medium (RFFM) assay to detect and enumerate Perkinsus spp. parasites in environmental water samples. Recovery of in vitro cultured P. marinus was successful, but recovery rates were low. Filtration of water samples captured significantly more cultured P. marinus cells than centrifugation. Lipid supplementation during RFTM incubation enhanced recovery of cultured P. marinus, but not of naturally occurring Perkinsus spp. parasites. Comparisons between the modified RFFM filtration method and a more complex immunoassay revealed that the two methodologies were equally sensitive, indicating that the RFTM filtration method may be confidently applied to environmental water samples to determine relative concentrations of Perkinsus spp. parasites.

KEY WORDS: Perkinsus marinus, dermo, disease, oyster, RFTM, planktonic, filtration

INTRODUCTION

The protozoan parasite Perkinsus that are pathogenic to a variety of shellfish worldwide (Bower et al. 1994). Most notable in North America is Perkinsus marinus Levine, a protozoan endoparasite that causes dermo disease in the eastern oyster, Crassostrea virginica Gmelin. The parasite is common from Maine to Mexico (Krantz & Jordan 1996) and is responsible for mass oyster mortalities in Delaware Bay, Chesapeake Bay, and the Gulf of Mexico (Ford & Tripp 1996). It has long been known that direct transmission of P. marinus to C. virginica occurs via the water column (Ray 1954), but few attempts have been made to develop and apply methods to detect P. marinus in the water column. As a result, knowledge about planktonic stages of P. marinus and the processes that affect this important life cycle phase is limited. Applications of an immunoassay (Dungan & Roberson 1993) and the polymerase chain reaction (PCR) in combination with species-specific molecular genetic probes to quantify planktonic stages in environmental water samples have met with varying success (Roberson & Dungan 1994, Yarnall 1998, Ragone Calvo et al. 2003, Audemard et al. 2004). These methods are technologically complex, not readily available to many laboratories and expensive. This study describes the adaptation of Ray fluid thioglycollate medium (RFTM) assays for enumerating planktonic stages of Perkinsus spp. parasites from environmental water samples.

Since the discovery of P. marinus in the 1940s (Mackin et al. 1950), detection and enumeration methods have focused on categorizing infections within oyster tissue. These methods include histology, tissue smears, the RFTM assay, a polyclonal immunoassay and various molecular genetic probes. Tissue smears and histological methods that were initially used to diagnose infections (Mackin et al. 1950) were soon replaced by the RFTM assay (Ray 1952, 1966). During this assay, the parasite enlarges and develops a thick cell wall that stains blue-black with Lugol's iodine, facilitating its detection. The RFTM assay is easy to perform, inexpensive and sensitive when combined with NaOH digestion of the sample (Choi et al. 1989, Bushek et al. 1994). It is not species specific, but it appears to be genus-specific and remains the most commonly used method for detection of Perkinsus spp. infections in molluscan hosts (Bower & McGladdery 2003).

The RFTM assay has been adapted to quantify P. marinus in tissues (Choi et al. 1989), hemolymph (Gauthier & Fisher 1990), and entire individuals (Bushek et al. 1994, Fisher & Oliver 1996). Of particular relevance to quantifying P. marinus and other Perkinsus spp. in plankton is the discovery by Choi et al. (1989) that oyster tissues, bacteria and most other contaminants may be digested away with 2M NaOH leaving a suspension of parasites (actually only the cell walls) that may be stained and counted. Fisher and Oliver (1996) demonstrated that NaOH digestion does not affect the thick cell wall that P. marinus develops during RFTM incubation. Two assumptions, however, must be made to use the RFTM assay quantitatively. First, all life stages are assumed to enlarge during incubation and to stain with Lugol's iodine for detection (Ray 1952). Early comparisons between the RFTM method and histology revealed that all identifiable stages of P. marinus occurring within host oysters enlarged in RFTM (Ray 1954, Stein & Mackin 1957). Additional support for this assumption was provided when Ragone Calvo and Burreson (1994) were unable to detect any previously unknown stages of P. marinus with a polyclonal anti P. marinus antiserum applied to histological samples. The second assumption is that no proliferation occurs during incubation in RFTM. By monitoring P. marinus counts and enlargement throughout incubation in RFTM, Ray (1952) and Stein and Mackin (1957) provided evidence to support this assumption. In addition, extensive efforts to culture P. marinus continuously in RFTM were unsuccessful (Ray 1954, Prokop 1950, Mackin & Boswell 1956). Under these assumptions, NaOH digestion of RFTM incubated tissues is routinely used to quantitatively enumerate P. marinus in many laboratories.

We applied RFTM incubation and NaOH digestion to environmental water samples as a method to enumerate planktonic stages of P. marinus. After initial attempts yielded positive ([less than or equal to] 5 cells/ liter) results, a series of experiments was conducted to determine the efficiency of the method and to identify possible improvements. The goal was to develop a simple, inexpensive method to quantify P. marinus from environmental water samples using tools generally available to laboratories capable of performing standard RFTM assays.

METHODS AND RESULTS

Five experiments were conducted sequentially to examine various aspects of the assay. The first three experiments were performed with in vitro cultured P. marinus to determine the efficiency of and possible improvements to the method, whereas the latter two experiments examined naturally occurring P. marinus in environmental water samples. Because results of each experiment were used in designing subsequent experiments, methods and results are presented together for each experiment. All data were analyzed using SYSTAT statistical software (Wilkinson 1998).

Experiment 1

Centrifugation Versus Filtration

This experiment was designed to determine if there was a difference in recovery of parasite cells collected via centrifugation or filtration. Replicate samples (500 mL) of 1-[micro]m filtered seawater (FSW) were spiked with 1-mL aliquots of in vitro cultured P. marinus cells that had been maintained in ATCC 1886 medium (www.atcc.org) with minor modifications described in Bushek et al. (2000). After mixing the parasites in each sample, they were recovered as pellets via centrifugation (x650 g, x2603 g, or x5858 g for 15 min, n = 2 per treatment) or filtered (vacuum pressure = 175 mm Hg, n = 3) onto a Gelman GN-Metricel 0.45-[micro]m membrane. Pellets and filters were transferred to 15-mL conical centrifuge tubes containing 10 mL RFFM fortified with 10 [micro]L of penicillin-streptomycin (Sigma P-0781) to retard bacterial growth. After incubation at room temperature for one week in the dark, the samples were pelleted at x823 g for 15 min. Centrifuged samples were resuspended in 5 mL 0.2-[micro]m FSW, stained with Lugol's iodine and placed on a Gelman GN-Metricel Membrane filter paper (0.45 [micro]m pore size) under 100x magnification for enumeration (Fisher & Oliver 1996). Filtered samples were resuspended in 2M NaOH (Choi et al. 1989) to digest the filter (2 h at 60[degrees]C), then washed twice in 0.2-[micro]m FSW, resuspended in 5 mL 0.2-[micro]m FSW, stained with Lugol's iodine and enumerated with a Hausser counting chamber under 100x magnification. The enumeration of filtered samples differed from centrifuged samples because small portions of the filter that remained after digestion obscured the parasites when counted on a Gelman GN-Metricel Membrane filter. The Hausser counting chamber slowed processing because samples were no longer coplanar, but eliminated the problem of filter particulates obscuring parasites. Enumerating all parasites in these samples required focusing through layers of the medium.

In vitro cultured P. marinus were recovered and successfully enumerated from all centrifugation and filtration samples. The parasites were more difficult to detect than expected because, compared with parasites in oyster tissues, relatively little enlargement occurred during RFTM incubation, and staining with Lugol's iodine was weak. Although the initial concentration of parasites used to obtain initial aliquots had not been precisely determined, the percentage of cells recovered was much lower than expected, based on earlier cell counts from the in vitro cultures. Unfortunately, this oversight precluded an estimate of recovery efficiency. Nevertheless, because equal aliquots from the same parasite suspension were used, it was clear that significantly more parasites were consistently recovered with filtration than with centrifugation treatments (Tukey, P < 0.001, Fig. 1). Centrifugation recovery decreased with increasing centrifugal force; x650 g centrifugation resulted in a significantly higher parasite recovery than x5858 g centrifugation (Tukey, P = 0.018), but recovery at the intermediate velocity did not differ significantly from either extreme. Logistically, centrifugation was more time consuming than filtration, requiring 45 min to concentrate samples compared with 15 min for filtration. Because filtration was more efficient than centrifugation, subsequent experiments used the filtration method only.

[FIGURE 1 OMITTED]

Experiment 2

Recovery Efficiency

This experiment involved a series of treatments designed to measure recovery efficiency and identify potential sources of parasite loss. In addition, because enlargement and staining were lower than expected in Experiment 1, each treatment was duplicated with RFTM that was supplemented 1:100 (v/v) with lipid concentrate (+lipids). Nickens et al. (2002) reported that the addition of lipids may increase enlargement and cell wall development of in vitro cultured P. marinus during RFTM incubation. The lipids used for supplementation (GIBCO BRL 21900-030) were derived from cod liver oil and supplemented with cholesterol, Pluronic F-68, DL-[alpha]-tocopherol acetate and Tween 80. Treatment 1 (water + filter + RFTM) and treatment 2 (water + filter + RFTM + lipids) replicated the entire RFTM filtration methodology used in Experiment 1, except that samples consisted of only 200-mL FSW and were spiked with known concentrations of viable cultured P. marinus clones (4.04 x [10.sup.5] cells of isolate LA22 clone 8, 5.64 x [10.sup.5] cells of isolate LA25 clone 2, and 3.97 x [10.sup.5] cells of isolate LA25 clone 4; These isolates were derived from oysters collected from the Gulf coast of Louisiana, USA and provided by J LaPeyre). In treatments 3 (filter + RFTM) and 4 (filter + RFTM + lipids), parasites were added directly to the filter without applying a vacuum. These treatments tested the effects of handling the parasites (i.e., moving parasites from culture medium into seawater and capturing them on the filter). In treatments 5 (RFTM) and 6 (RFTM + lipids), parasites were added directly to RFTM to determine if the filter or NaOH digestion interfered with enlargement and detection of the parasites. Because no filter was used in treatments 5 and 6, there was no need to perform the NaOH digestion step.

The percentage of parasites recovered was calculated for each treatment then arcsine transformed to normalize the data for analysis of variance (Sokal & Rohlf 1981). The variables filter, water and lipids were examined for differences in recovery. A treatment involving water but no filter would have completed a 3-way ANOVA; however, this treatment was not possible to test without centrifugation, which would have confounded the experiment. The absence of this treatment resulted in lost degrees of freedom for the 3-way ANOVA. Therefore, the 3-way interaction between filter, water and lipids; and the 2-way interaction between filter and water, were removed as suggested for a fractional factorial design in which data cells are missing (Wilkinson 1998).

The recovery efficiency of the RFTM filtration method used in Experiment 1 (treatment 1) was surprisingly low: 2.71% [+ or -] 0.42 (Table 1). Lipid supplementation resulted in significantly higher recovery across all treatments (Table 2, Fig. 2). The addition of lipids increased the number of parasites detected and generally increased the intensity of staining, but did not increase enlargement of the cells. The ANOVA in Table 2 indicated a significant effect of adding the cultured cells to water before capturing them on a filter. Inspection of Figure 2 indicates that treatments 1 and 2, when parasites were first diluted in FSW, resulted in considerably lower recovery than their respective treatments (3 and 4) when parasites were added directly to filters. Recovery was intermediate when parasites were added directly to RFTM (Fig. 2). Because recovery for treatments 5 and 6 was not greater than recovery for other treatments, cell loss was not attributed to effects of NaOH digestion.

[FIGURE 2 OMITTED]

Experiment 3

Lipid Dose Effects

This experiment was designed to determine if a higher concentration of lipids would further enhance recovery of in vitro cultured P. marinus. Nine, 200-mL samples of FSW were spiked with known concentrations of viable cells from one of three discrete P. marinus isolates (10.2 x [10.sup.5] cells of ATCC 50768; 7.53 x [10.sup.5] cells of ATCC 50889 and 4.57 x [10.sup.5] cells of ATCC 50776). Lipid dose treatments included no lipids (control), 1:100 (v/v) lipids/RFTM, and 1:10 (v/v) lipids/RFTM. Lipids were added to RFTM prior to sample incubation. Three replicate samples were tested per treatment and a different P. marinus isolate was used for each replicate to ensure results were not isolate specific. Water samples were filtered and processed following the procedure outlined in Experiment 2, treatment 1. After recovery rates were calculated, a 1-way ANOVA and a control versus lipid contrast were used to examine lipid dose effects.

Results showed that both lipid dosage treatments recovered more parasites than the control (Fig. 3), but the increase was not significant (1-way ANOVA, P = 0.555). A contrast of control versus lipid treatments was also not significant (P = 0.346). Although not significant, recovery was lower for the higher lipid concentration. Recovery in the 1:100 treatment was similar to treatment 2 in Experiment 2. That is, variability within treatments was high and may be attributed to lipid addition, differences between the P. marinus isolates, or both. Additional experiments that include more isolates than were available at the time of these experiments are needed to resolve the source of variation.

[FIGURE 3 OMITTED]

Experiment 4

Environmental Water Sample Enumeration and Lipid Dose Effects

This experiment was essentially identical to Experiment 3, but the methods were applied to environmental water samples rather than in vitro cultured P. marinus. Three environmental water samples (500-mL each) were collected August 22, 1998 from Oyster Landing, North Inlet, South Carolina using two automated ISCO water samplers. Each sample was prefiltered through a 25-[micro]m nylon mesh, divided into three equal aliquots, and then processed as described for Experiment 3 using the same lipid treatments. Because P. marinus is the only species of Perkinsus that has been documented to occur in South Carolina (Reece et al. 1997, 2001), all cells positive per the RFTM assay were considered to be P. marinus. Counts were standardized to parasites per liter and then [log.sub.10]-transformed to normalize the distribution for a 1-way ANOVA (Sokal & Rohlf 1981). Tukey multiple comparisons were used to differentiate significance levels among treatments, and a control versus lipid contrast was calculated to test the effect of adding lipids.

Unambiguous positives were obtained from all environmental water samples. Cells were easily detected and enumerated, ranged in size from 10-35 [micro]m, and stained blue-black with Lugol's iodine. A 1-way ANOVA revealed a significant difference between the environmental water sample lipid dose treatments (P = 0.001). The 1:100 (v/v) lipid treatment recovered significantly more parasites than the 1:10 (v/v) lipid treatment (141 [+ or -] 19 cells [L.sup.-1] versus 3 [+ or -] 2 cells [L.sup.-1], Tukey P = 0.001), but not significantly more than the no-lipid control (89 [+ or -] 34 cells [L.sup.-1], Tukey P > 0.05). The control treatment also produced a recovery rate significantly greater than the 1:10 (v/v) lipid treatment (Tukey P = 0.003). A contrast comparing the control versus both lipid treatments was significant (P = 0.030); however, as a result of the low recovery in the 1:10 treatment the combined lipid effect was to decrease recovery compared with the control. These data indicate that 10% (v/v) lipid additions may inhibit recovery. Variability across the treatments was low compared with Experiments 2 and 3.

Experiment 5

Comparison of RFTM Filtration and Immunoassay Methods

Prior to this work, the only other attempts to enumerate planktonic stages of P. marinus used a polyclonal immunoassay with near genus-level specificity (Roberson & Dungan 1994, Ragone Calvo et al. 2003, but see Bushek et al. 2002). The present experiment was conducted to compare detection of planktonic P. marinus via the polyclonal immunoassay and the RFTM filtration methods. Paired environmental water samples (500-mL) were collected September 26, 1998 from Oyster Landing, North Inlet, South Carolina using two automated ISCO water samplers. The paired samples were processed using the RFTM filtration and the immunoassay methods. All samples were prefiltered through 25-[micro]m nylon mesh. RFTM samples were processed as described for Experiment 2, treatment 1. The immunoassay samples were processed using methods described by Bushek et al. (2002), which is a modification of the protocol described by Dungan and Roberson (1993). Briefly, the samples were concentrated at x100 g for 15 min and resuspended in sterile FSW with 10% buffered formalin. Particulates were then captured on a 1.0-[micro]m pore size black polycarbonate filter in a Swinnex-13 membrane filter unit, washed three times with phosphate buffered saline (PBS), blocked with a blocking buffer for 30 min and then washed with phosphate buffered saline containing Tween-20 (PBST). The primary antibody (rabbit anti P. marinus) was applied for 30 min then washed with PBST before the secondary antibody (goat antirabbit-FITC) was applied. After 30 min, the sample was washed with PBST, counterstained with Evan blue, washed with PBS, and finally mounted on slides to enumerate parasites via epifluorescence microscopy. Using this procedure, the nucleus of any Perkinsus spp. parasites present fluoresced bright green, making them distinguishable from other cells and debris. Counts from both the immunoassay and RFTM assays were standardized to parasite cells per liter and results were compared using a paired t-test.

The RFTM and immunoassay methodologies for enumeration of Perkinsus spp. parasites were not significantly different (Paired t-test for means P = 0.411, Table 3). This indicated that both methods were equally sensitive.

DISCUSSION

In contrast to Yarnall et al. (2000), we successfully detected and enumerated P. marinus in samples collected from enzootic waters using standard RFTM methods. Yarnall (1998) reveals that Yarnall et al. (2000) used centrifugation to concentrate their samples in which their PCR assays did not yield enough DNA for quantitation. Interestingly, a nearby contemporaneous study using a flow cytometric immunoassay estimated planktonic P. marinus concentrations of 200-400 parasites [L.sup.-1] (Ragone Calvo et al. 2003). Our comparison of detection via RFTM versus the immunoassay indicated that both methods were equally sensitive when applied to environmental water samples. In this study, centrifugation was less efficient than filtration, which may account for the lack of detection by Yarnall et al. (2000) when results from Ragone Calvo et al. (2003) indicated that the parasite was abundant. A more recent study using improved molecular genetic tools was able to quantify P. marinus with PCR methods from the same area as the Yarnall et al. (2000) study (Audemard et al. 2004).

It should be noted that the RFTM and immunoassay methods are genus-specific and may detect other species if present. Hence, as species-specific molecular methods become more widely available (e.g., Audemard et al. 2004) they may be necessary where multiple species occur. It may also be valuable to compare species-specific and genus-specific methods in samples collected over space and time. Such experiments will provide insight into the inter and intragenus cross-reactivity of the methods as well as the co-occurrence of Perkinsus species.

The low recovery of in vitro cultured parasites in this study was disconcerting, but may not be relevant to enumerating Perkinsus spp. abundances from environmental samples. Compared with parasites obtained from infected hosts, it was clear that cultured cells did not enlarge or stain well after incubation in RFTM. This difference indicates that in vitro cultured P. marinus cells are physiologically different from cells harvested from infected hosts. Bushek et al. (1997) found that in vitro cultured cells appeared to have a reduced ability to survive injection into oysters, and Ford et al. (2002) clearly demonstrated a reduction in virulence following in vitro culture using the same culture methods used in the present study. Our attempts to identify steps in the methodology that account for cell loss were largely unsuccessful, leading us to the conclusion that cell loss is largely a result of handling the cells, possibly from the shock experienced by cells when transferred from culture medium to FSW. This may partly explain the loss of virulence reported by Ford et al. (2002), as they similarly transferred parasites from culture medium to seawater and then into hosts. Interestingly, enlargement, staining and recovery of cultured P. marinus improved with the addition of lipids (1:100 v/v); albeit not always significantly, indicating potential deficiencies in the culture medium. Regardless, the RFTM assay and an independent polyclonal immunoassay performed similarly with environmental samples indicating approximately equal detection efficiencies.

Based on the earlier results and discussion, the RFTM filtration method is a simple, inexpensive method for enumerating Perkinsus spp. parasites in environmental water samples. Several additional refinements of the method have resulted in a simpler and less time-consuming RFTM filtration method (Fig. 4). Specifically, washing and resuspension of the pelleted, NaOH-hydrolyzed samples in deionized water rather than FSW was convenient, efficient and did not affect sample storage (Gauthier & Fisher 1990). In addition, stained samples may be read on a Hausser counting chamber at 40x magnification instead of 100x magnification because the natural cells enlarge more during RFTM incubation than in vitro cultured cells, stain characteristically and are easily observed under 40x magnification. This dramatically decreased the time required to scan the counting chamber. Lipids were not added to the final protocol because it was not clear from the results of Experiments 3 and 4 that lipids actually improved the overall recovery of planktonic parasites because of the high variability and reduced recovery in 1:10 (v/v) lipids/RFTM samples.

[FIGURE 4 OMITTED]

Direct water borne transmission of P. marinus was discovered more than 50 y ago (Ray 1954), yet little is known about the free-living stages of this or any other Perkinsus species that afflict various molluscs around the globe. As these protozoan pathogens continue to plague shellfisheries, the need to better understand their dispersal and transmission dynamics continues to grow. A thorough understanding of these dynamics can be achieved only through investigations exploring the timing and fate of free-living planktonic stages during transmission. The experiments discussed in this study outline a simple method to detect and enumerate planktonic stages of Perkinsus spp. The method adapts the well-known and extensively applied RFTM method that is known to detect all species of the genus Perkinsus. Hence, the method should be readily accessible to most laboratories that already use RFTM assays. Whereas the RFTM filtration method provides a simple and inexpensive methodology for enumerating P. marinus in environmental water samples, the absolute efficiency of the method remains questionable. Nonetheless, the RFTM filtration method may be used to indicate relative differences in parasite numbers.

ACKNOWLEDGMENTS

The authors thank K Hudson for assistance with culturing P. marinus isolates. M Chintala and J LaPeyre provided some of the isolates and C Dungan provided several discerning comments in developing these experiments. Funding for this research was provided by NSF-RUI Project #DEB-950957, and South Carolina Sea Grant Consortium ODRP Project R/OD-1.

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REBECCA ELLIN (1) AND DAVID BUSHEK (2) *

(1) North Carolina Coastal Reserve Program, 400 Commerce Avenue, Morehead City, North Carolina 28557; (2) Haskin Shellfish Research Laboratory, 6959 Miller Avenue, Port Norris, New Jersey 08349

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Recovery efficiency of the RFTM filtration method. Recovery rates
for Experiment 2, Treatment 1. The recovery rate for each replicate
is indicated.

  P. marinus         P. marinus            P. marinus
   Isolate              Added               Recovered

LA22 - 8          40.4 x [10.sup.4]      1.4 x [10.sup.4]
LA25 - 2          56.4 x [10.sup.4]      1.5 x [10.sup.4]
LA25 - 4          39.7 x [10.sup.4]      8.0 x [10.sup.3]

  Mean            45.5 x [10.sup.4]      1.2 x [10.sup.4]
[+ or -] (SEM)        [+ or -]              [+ or -]
                 (54.4 x [10.sup.2])    (2.2 x [10.sup.])

  P. marinus           Percent
   Isolate            Recovery

LA22 - 8                3.467
LA25 - 2                2.661
LA25 - 4                2.031

  Mean                  2.71%
[+ or -] (SEM)     [+ or -] (0.42)

TABLE 2.

ANOVA results for Experiment 2. Asterisk and bold indicate
significant effects at [alpha] = 0.05. df = degrees of freedom,
SS = sum of squares, MS = mean squares.

Source of Variation   df    SS      MS     F-Ratio   P-Value

Water# *               1   0.254   0.254    5.707    0.034#
Lipids# *              1   0.229   0.229    5.137    0.043#
Filter                 1   0.154   0.154    3.462    0.087
Water x Lipids         1   0.025   0.025    0.558    0.469
Lipids x Filter        1   0.002   0.002    0.055    0.818
Error                 12   0.534   0.045

Note: Bold indicate significant effects at [alpha] = 0.05. df =
degrees of freedom, SS = sum of squares, MS = mean squares
indicated with #.

TABLE 3.

Comparison of RFTM filtration method and immunoassay method.
Each replicate represents paired water samples taken at the same
time with automated ISCO water samplers. The number of
Perkinsus spp. parasites detected is reported for each method. The
methods were not significantly different ([alpha] = 0.05).

     Replicate               RFTM             Immunoassay

         1                    13                   20
         2                    33                   30
         3                    23                   10
         4                     7                    0
Mean [+ or -] (SEM)   19 [+ or -] (5.71)   15 [+ or -] (6.45)