Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii and affects many individuals throughout the world. Infection may occur through congenital or acquired routes. The parasites enter the blood circulation and reach both the retina and the retinal pigment epithelium, where they may cause cell damage and cell death. Different routes of access are used by T. gondii to reach the retina through the retinal endothelium: by transmission inside leukocytes, as free parasites through a paracellular route, or after endothelial cell infection. A main feature of OT is the induction of an important inflammatory state, and the course of infection has been shown to be influenced by the host immunogenetics. On the other hand, there is evidence that the T. gondii phenotype also has an impact on the distribution of the pathology in different areas. Although considerable knowledge has been acquired on OT, a deeper knowledge of its mechanisms is necessary to provide new, more targeted treatment strategies. In particular, in addition to in vitro and in vivo experimental models, organotypic, ex vivo retinal explants may be useful in this direction.

Ocular toxoplasmosis is the most prevalent clinical manifestation of T. gondii infection, which causes irreversible retinal damage. Different experimental models have been developed to study this pathology. In the present study, a new, ex vivo model is proposed to contribute to the elucidation of disease mechanisms and to possible therapeutic solutions. Ex-vivo retinal explants, prepared from mouse retinas following established protocols, were incubated with T. gondii tachyzoites maintained in Vero cells. At different times, starting at 12 h up to 10 days of incubation, the explants were analyzed with immunofluorescence and Western blot to investigate their responses to parasite infection. T. gondii invasion of the retinal thickness was evident after 3 days in culture, where parasites could be detected around retinal cell nuclei. This was paralleled by putative cyst formation and microglial activation. At the same time, an evident increase in inflammatory and oxidative stress markers was detected in infected explants compared to controls. Cell death also appeared to occur in retinal explants after 3 days of T. gondii infection, and it was characterized by increased necroptotic but not apoptotic markers. The proposed model recapitulates the main characteristics of T. gondii retinal infection within 3 days of incubation and, therefore, allows for studying the very early events of the process. In addition, it requires only a limited number of animals and offers easy manipulation and accessibility for setting up different experimental conditions and assessing the effects of putative drugs for therapy.

• Both freeze-thaw and bead beating DNA extraction enable protozoa detection. • Extraction method performance varied by parasite type and sample matrix. • Results inform protocols for water quality and shellfish safety testing.

Coastal areas of Italy experience high anthropogenic pressure, with a population density estimated to be 360 people per km². This is correlated with the production of sewage or surface runoff of water contaminated with Toxoplasma gondii oocysts and other pathogens that can in turn enter the food chain and become a public health concern. Therefore, in the present study, we want to review the current knowledge on the presence of T. gondii in Italian coastal areas. The majority of research in Italy has been carried out on marine mammals, while fishes and mollusks have less often been screened. So far, T. gondii has been found in all sea animals tested, with genotypes II and III as well as atypical genotypes being the most commonly found when screened. However, more information is needed. We encourage routine screening of mollusks and fish that are sold in different food markets throughout the country in order to add information on the distribution of T. gondii and other parasites, preventing possible future outbreaks.