Uptake and transmission of Toxoplasma gondii oocysts by migratory, filter-feeding fish

https://doi.org/10.1016/j.vetpar.2010.01.002Get rights and content

Abstract

From bottlenose dolphins, to walruses, to sea otters, the parasitic protozoan Toxoplasma gondii is infecting marine mammals around the world. Whereas the terrestrial transmission pathways of T. gondii are well-described, the transmission pathway by which marine mammals are being infected is unknown. We hypothesize that migratory filter feeders, specifically northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax), are serving as biotic vectors for T. gondii within the marine environment. By filtering oocysts from seawater, these fishes could be transporting the oocysts from nearshore to pelagic environments. In this study, we experimentally exposed northern anchovies and Pacific sardines to T. gondii oocysts under laboratory conditions. Following exposure, the fishes’ alimentary canals were harvested and assayed for the presence of T. gondii by PCR. Fish exposed to as few as 1197 oocysts/L seawater tested positive for T. gondii by PCR. In total, the PCR assay detected T. gondii DNA in 66% (40/61) of the exposed fishes. Oocyst infectivity was confirmed by mouse bioassay: 30% (7/23) of mice developed toxoplasmosis when fed fish exposed to 100,000 oocysts/L. This study demonstrates that both northern anchovies and Pacific sardines can filter T. gondii oocysts out of seawater under experimental conditions. Our experiments with anchovies demonstrated that the oocysts persisted in the fish for at least 8 h post-exposure and our experiments with sardines demonstrated that the oocysts remained infectious inside the fish's alimentary canals.

Introduction

Toxoplasma gondii is a parasitic protozoan than can cause spontaneous abortion, congenital birth defects, severe illness, and death in warm-blooded animals (Dubey and Beatie, 1988). Terrestrially, T. gondii is spread primarily through the ingestion of oocysts, passed only in felid fecal matter, and through the carnivorism of infected tissues. In 1951, Ratcliffe and Worth first described toxoplasmosis in marine mammals, illustrating that T. gondii is not limited to the terrestrial animals (Ratcliffe and Worth, 1951). Subsequently, toxoplasmosis has been described in 33 different marine mammal species encompassing eight different families (Phocidea, Otariidae, Delphinidae, Balenopteridae, Trichechidae, Mustelidae, Ursidae, and Odobenidae) (Dabritz et al., 2007). The range of outcomes from T. gondii infection in marine mammals includes encephalitis, myocarditis, lymphadenitis, miscarriage, and death (Domingo et al., 1992, Dubey et al., 2003, Dubey et al., 2004, Honnold et al., 2005, Jardine and Dubey, 2002, Migaki et al., 1977).

To date, most of what is known about T. gondii in marine mammals stems from research on southern sea otters (Enhydra lutris) (Dubey et al., 2003). Southern sea otters are an endangered, federally protected species, and toxoplasmosis is considered a major cause of their mortality (Cole et al., 2000, Kreuder et al., 2003, Miller et al., 2004). Miller et al. (2002) observed that sea otters living in areas of high coastal freshwater runoff (100,001–1,000,000 acre-ft/year) supported a three-fold higher T. gondii infection rate compared to areas with low coastal runoff (0–10,000 acre-ft/year). Animals living in Morro Bay, CA, (35.361°N, 120.870°W) and Monterey Bay, CA, (36.790°N, 121.799°W), in particular, were considered to be at high risk of T. gondii exposure because of the areas’ high runoff rates (Kreuder et al., 2003, Miller et al., 2002, Miller et al., 2008). These studies hypothesized that freshwater run-off carries oocysts from the terrestrial environment to the marine environment where they are bioaccumulated by filter-feeding bivalves (Kreuder et al., 2003, Lindsay et al., 2001, Miller et al., 2002, Miller et al., 2004).

Bivalve bioaccumulation of parasitic oocysts under laboratory conditions has previously been described for T. gondii (Arkush et al., 2003, Lindsay et al., 2001, Lindsay et al., 2004). More recently, T. gondii oocysts have also been detected in one wild mussel maintained on a test platform in Monterey Bay, CA (Dabritz et al., 2007, Miller et al., 2008). However, oocyst accumulation by nearshore bivalves does not explain transmission to mammals that do not feed on bivalves. Belugas, for example, are not known to eat bivalves, yet they have been infected by this parasite (Dubey et al., 2003). Based upon the global prevalence of T. gondii infection in a variety of marine mammals, we propose that filter-feeding fish could serve as biotic vectors for T. gondii in the marine environment. Fish have previously been shown to be biotic vectors for organic material between salt marshes and coastal environments and have similarly transmitted marine toxins such as domoic acid (Lefebvre et al., 1999, Lefeuvre et al., 1999).

To test the ability of migratory filter-feeding fish to serve as vectors for T. gondii, we exposed northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax) to T. gondii oocysts under laboratory conditions. Both northern anchovies and Pacific sardines filter feed as larvae and adults, and are the primary prey of many marine fish, birds, and mammals (Cury et al., 2000, Kucas, 1986). Northern anchovies migrate horizontally from the nearshore (<30 km) to the pelagic environment (up to 480 km), and vertically from the epipelagic zone (sunlit) to the mesopelagic zone (twilight) (Whitehead et al., 1988). Pacific sardines also migrate from the nearshore to the pelagic environments, and migrate annually from Southern California to British Columbia (Haugen, 1973, Kucas, 1986, Pacific Fishery Management Council, 1988). Based on these factors, both fishes could potentially disseminate oocysts to a wide-variety of new hosts and habitats. We developed two novel DNA extraction techniques to detect the presence of oocysts in the fish's alimentary canals by PCR and we fed sucrose-purified alimentary canals to mice in a bioassay to determine if oocysts remained infectious post-filtration.

Section snippets

Purification and enumeration of T. gondii oocysts

Partially purified VEG strain oocysts isolated from the feces of laboratory-infected cats were kindly provided by Dr. Dubey (USDA) and used for the study. These oocysts were further purified using a cesium chloride gradient as previously described (Dumetre and Darde, 2004). The purified oocysts were resuspended in 2% H2SO4 and stored at 4 °C.

The oocysts were enumerated using two different methods. For the fish exposures, the purified oocysts were enumerated by hemacytometer counts (Baker, 1980).

PCR-based assay for bioaccumulation

For experiments E2 and E3, a sample was considered positive for T. gondii if the PCR gel had a 167 bp gel band. If there was not a 167 bp band, the sample was considered negative. Our initial assays with northern anchovies were performed using the lysis/bead bash protocol to extract DNA from the intestinal contents. Using this protocol, we were able to detect as few as five purified oocysts by PCR (Fig. 1). In a direct comparison test, we also examined the efficiency of DNA extraction using the

Discussion

Our findings present a novel pathway for how T. gondii may be transmitted throughout the marine environment. This study has demonstrated that both northern anchovies and Pacific sardines can filter T. gondii oocysts out of seawater under experimental conditions, that the oocysts persist in the alimentary canal for at least 8 h (anchovies) and that the oocysts remain infectious (sardines). This is the first study to demonstrate the ability of any marine vertebrate to serve as a vector for T.

Conflict of interest statement

There are no conflicts of interest.

Acknowledgements

The authors would like to acknowledge the technical assistance of Steve Vogel at the Monterey Bay Aquarium, Deke Wells of the Mello Boy, Eunice Varughese, David Erisman, Sharon Detmer, Diana Miller, Paula McCain, Katrina Pratt, and all of the CalPoly undergraduates who assisted with tissue processing. The United States Environmental Protection Agency through its Office of Research and Development collaborated in the research described here. It has been subjected to Agency review and approved

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