Removal of Toxoplasma gondii Oocysts from Sea Water by Eastern Oysters (Crassostrea virginica)
Abstract
SUMMARY. Toxoplasma gondii infections have been reported in a number of marine mammals. Presently it is not known how these animals acquire T. gondii from their aquatic environment. The eastern oyster, Crassostrea virginica, has been shown to remove Cryptosporidium oocysts from seawater and a similar phenomenon may be occurring with T. gondii oocysts and marine invertebrates. The present study was done to determine if eastern oysters could remove and retain T. gondii oocysts from seawater. Oocysts of the VEG strain of T. gondii (1 × 106 oocysts) were placed in seawater (32 ppt NaCl) containing live eastern oysters. The infected seawater was removed one day postinoculation (PI) and replaced with fresh seawater. Selected oysters were removed at 1, 3 and 6 days PL Hemolymph, gill washes, and oyster tissue were collected separately at each observation time. The oyster tissue was homogenized and all 3 samples fed separately to mice. Toxoplasma gondii positive mice were observed at each time period. The results indicate that T. gondii oocysts can be removed from seawater by eastern oysters and retain their infectivity. Contaminated raw oysters may serve as a source of T. gondii infection for marine mammals and humans.
Toxoplasma gondii is acquired by the ingestion of tissue cysts in raw or under cooked meat, by ingestion of oocysts, or by transplacental infection in mammalian species. Clinical and subclinial toxoplasmosis has been reported in marine mammals [1,2,4,5,11–15]. Cole et al. [2] suggested that marine mammals acquired T. gondii infection by ingesting invertebrates which were acting as phoretic agents for T. gondii oocysts. They believed that cat feces containing T. gondii oocysts could be entering the marine environment through storm run- off and were picked up by invertebrate food items of Southern sea otters. Oocysts of Cryptosporidium have been demonstrated in oysters [7,8,9] and mussels [3,10] from marine environments demonstrating the ability of coccidial oocysts to make in to these environments and to be obtained by potential phoretic agents.
MATERIALS AND METHODS
Eastern oysters, Crassostrea virginica, were obtained from commercial sources and acclimated in a 20 gallon aquarium containing artificial seawater (32 ppt NaCl). Two oysters were selected for processing and mouse feeding (see below) on day 0 and were used as preinfection controls (Table 1). Oocysts of the VEG strain of T. gondii. were recovered from the feces of experimentally infected cats, sporulated, and used in the present study. The seawater containing eastern oysters was inoculated with a total of 1 × 106 sporulated T. gondii oocysts. The aquarium box filter was removed to prevent possible removal of oocysts in the filter. The infected seawater was removed 24 hours postinoculation (PI) and replaced with fresh seawater. The box filter was then placed back on the aquarium.
| Day PI | # of oysters | Material examined | # of mice fed /# of mice positive |
|---|---|---|---|
| 0 | 2 | Hemolymph | 2/0 |
| 0 | 2 | Oyster tissue | 2/0 |
| 1 | 3 | Hemolymph | 3/0 |
| 1 | 3 | Gill washes | 3/0 |
| 1 | 3 | Oyster tissue | 3/3 |
| 3 | 2 | Hemolymph | 2/0 |
| 3 | 3 | Gill washes | 3/0 |
| 3 | 3 | Oyster tissue | 2/1 |
| 6 | 2 | Hemolymph | 3/0 |
| 6 | 2 | Gill washes | 3/1 |
| 6 | 2 | Oyster tissue | 3/0 |
Selected oysters were removed at 1 day, 3 days, and 6 days PI of sea water, and thoroughly washed in tap water, and processed for mouse feeding (Table 1). Hemolymph, gill washes, and oyster tissue were collected separately at each observation time from 2 to 3 oysters and pooled for mouse feeding. The oyster tissue was homogenized in a stomacher machine, and filtered through cheesecloth to remove particulate material. Collected material was pelleted by centrifugation and resuspended in approximately 2–3 ml of Hanks' balanced salt solution. The processed material was fed to female, 25–30 g, CD-I mice using an animal feeding needle. Mice were housed separately in groups of 2–3 mice based on material fed and day of feeding.
Mice were bled 72–79 days post feeding. Their sera were examined in the modified direct agglutination test [6] for antibodies to T. gondii at a 1:50 dilution. Mice were bled again then killed 92–98 days after feeding oyster material and their brains examined for T. gondii tissue cysts.
RESULTS AND DISCUSSION
No evidence of T. gondii infection was found in the 2 eastern oysters from the source lot of oysters. None of the mice fed oyster tissue developed acute toxoplasmosis or died. Toxoplasma gondii infection was found in mice fed homogenized oyster tissue and gill washes but not hemolymph (Table 1).
This study demonstrates that eastern oysters can remove T. gondii oocysts from seawater and that the oocysts retain their infectivity for mice. Contaminated raw oysters may serve as a source of T. gondii infection for marine mammals and humans.
ACKNOWLEDGMENTS
Supported in part by a Virginia Graduate Marine Science Consortium Sea Grant to GF, DSL, and SAS. DSL and KKP thank Dr. Ron Payer, USDA Beltsville for teaching them the experimental methods used to collect specimens from oysters used in the present study.
