MICROBIAL ECOLOGY OF THE CONTINENTAL AND COASTAL ENVIRONMENTS Assessment of Toxoplasma gondii levels in zebra mussel (Dreissena polymorpha) by real-time PCR: an organotropism study M. Palos Ladeiro & A. Bigot-Clivot & D. Aubert & I. Villena & A. Geffard Received: 23 July 2014 /Accepted: 2 March 2015 /Published online: 14 March 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Water quality is a public health concern that calls for relevant biomonitoring programs. Molecular tools such as polymerase chain reaction (PCR) are progressively becoming more sensitive and more specific than conventional tech- niques to detect pathogens in environmental samples such as water and organisms. The zebra mussel ( Dreissena polymorpha) has already been demonstrated to accumulate and concentrate various human waterborne pathogens. In this study, first, a spiking experiment to evaluate detection levels of Toxoplasma gondii DNA in zebra mussel organs using real- time PCR was conducted. Overall, lower DNA levels in the hemolymph, digestive gland, and remaining tissues (gonad and foot) were detected compared to mantle, muscle, and gills. Second, an in vivo experiment with 1000 T. gondii oocysts per mussel and per day for 21 consecutive days, followed by 14 days of depuration time in protozoa-free water was per- formed. T. gondii DNA was detected in all organs, but greatest concentrations were observed in hemolymph and mantle tis- sues compared to the others organs at the end of the depuration period. These results suggest that (i) the zebra mussel is a potential new tool for measuring T. gondii concentrations and (ii) real-time PCR is a suitable method for pathogen de- tection in complex matrices such as tissues. Keywords Freshwater . Mollusk . Protozoa . Molecular detection . Spiking experiment . Hemolymph Introduction Toxoplasma gondii is the main parasite responsible for zoono- ses worldwide (Tenter et al. 2000). As a coccidian parasite, it has a complex life cycle. Asexual division occurs within felids and warm-blooded organisms, whereas sexual division occurs only in felids. T. gondii is the causative agent of toxoplasmo- sis. It has three infectious stages: the tachyzoite, the bradyzoite in tissue cysts, and the sporozoite under the form of environment-resistant oocysts (Dubey 2004). In immunocom- petent people, toxoplasmosis is generally asymptomatic, but it can cause severe clinical symptoms in immunocompromised people. In the case of congenital toxoplasmosis, these symp- toms can be blindness, mental retardation, encephalitis, or systemic infection of the fetus (Jones and Dubey 2010). Analysis of water samples typically requires filtering of large quantities of water that are then concentrated and puri- fied prior to analysis for the pathogen. Analysis of the parasite can be conducted using mouse inoculation (Villena et al. 2004) or through infection experiments with cultured cells (Miller et al. 2008). One sensitive assay that was recently developed for rapid detection of T. gondii in water is loop- mediated isothermal amplification (LAMP) (Sotiriadou and Karanis 2008). Quantitative methods such as flow cytometry, microscopy (Shapiro et al. 2010), or molecular analysis can also be used for the analysis of this parasite (Skotarczark 2010). Of these quantitative methods, polymerase chain reac- tion (PCR) represents a simple, reproducible, and specific technology that is increasingly used to detect pathogens with- in a few hours (Yang and Rothman 2004). For quantitative purposes, real-time PCR can be an advantageous approach Responsible editor: Markus Hecker M. Palos Ladeiro : A. Bigot-Clivot (*) : A. Geffard Unité Stress Environnementaux et BIOsurveillance des milieux aquatiques, UMR-I 02 (SEBIO), Université de Reims Champagne-Ardenne, Reims, France e-mail: aurelie.bigot@univ-reims.fr M. Palos Ladeiro : D. Aubert : I. Villena Laboratoire de Parasitologie-Mycologie, EA 3800, SFR CAP-Santé FED 4231, Hôpital Maison Blanche, Reims, France Environ Sci Pollut Res (2015) 22:13693–13701 DOI 10.1007/s11356-015-4296-y