Introduction

Toxoplasma gondii is a zoonotic pathogen with up to 60% of acquired infections associated with foodborne transmission. Consumption of raw fresh produce (FP) contaminated with T. gondii oocysts is one infection route. The relative importance of FP as source for human T. gondii infection is underestimated as standardized detection method(s) are lacking. We developed a standard operating procedure (SOP) for molecular detection of T. gondii oocysts in leafy green salads, validated it by a ring trial (RT) and are currently applying it in a multicentre survey (MS) on ready-to-eat (RTE) salad at European level.

Methods

The SOP was implemented in 7 laboratories using video tutorials. A RT was organized to evaluate laboratory performance and efficiency of different steps of the procedure. An evidence-based sampling strategy was designed to conduct a MS.

Results

Implementation in the laboratories was successful and allowed identification and resolution of procedure limitations. The expected LoD (10 ocysts/30g salad) was reached. RT analysis confirmed robustness of the procedure and comparability of results among participants. The MS on two types of RTE mixed salads (baby leaves and cut-leaf mixes) in 10 European countries has started and preliminary analysis will be presented.

Conclusions

The application of a well-validated SOP is proving to be a useful tool to investigate the occurrence of T. gondii contamination in RTE-salad, which is needed to assess the associated potential risk for humans.

Acknowledgments: RT and survey participants. This work was done as part of TOXOSOURCES project, EU Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme. NMLU is granted by a UCM-Santander/2018 predoctoral fellowship.

Introduction

The parasitic protozoan Giardia duodenalis is responsible for human and animal giardiasis, a diarrheal disease with a worldwide distribution. Human disease ranges from asymptomatic, to acute and chronic diarrhea, eventually causing malnutrition and stunting in children, and long term post-infectious sequelae. Two Giardia genetic groups (Assemblages A and B) cause human infection. Differences at genomic level between and within Assemblages can account for variations in growth rate, infectivity, and pathogenicity. Extracellular vesicles (EVs) operate as cargoes from cell to cell, carrying proteins and nucleic acids, being implicated in physiological and pathological processes. Giardia also releases EVs. Aim of this work was to isolate EVs from assemblage A and B reference isolates to define and compare EVs proteomes in the attempt to find a link with differences in assemblage pathogenicity.

Methods

Released EVs and freely secreted protein were purified from trophozoites, grown in well-defined culture condition, by differential ultracentrifugation. EVs were morphologically and biochemically characterized by electron microscopy and dynamic light scatter (DLS). EVs and secretome was characterized by proteomic analysis.

Results

EVs with different sizes and shapes were released by both Giardia isolates, in different amount. EVs, but not the secretome, showed the presence of typical EVs markers. Unique Giardia proteins, such as virulence factor candidates and assemblage-specific proteins were also present.

Conclusions

Our results provide evidence that differences in amount and content of EVs occur between Assemblage A and B pointing on their role in determining difference in Giardia isolates pathogenicity and virulence.

Abstract Body

Giardia duodenalis is a widespread protozoan parasite causing the commonest parasitic diarrheal disease-affecting humans. Although G. duodenalis infections in humans are largely asymptomatic, they often result in severe and chronic diseases and can cause post-infectious sequelae. The armamentarium of currently approved drugs for symptomatic giardiasis is still limited and restricted to compounds with a broad antimicrobial activity, often associated with severe side effect and to increasing cases of treatment-refractory giardiasis, particularly with nitroimidazoles. This scenario is alarming and poses further challenges for an effective management of giardiasis.

Aim of this lecture is to provide an overview of the current knowledge concerning alternative non-pharmacological approaches that are under development as new promising treatment for giardiasis. Alteration of the intestinal microbiota has been observed in patients with giardiasis and microbiota composition is likely responsible of failure of Giardia infection in rodents model. Modulation of the gut microbiome by the use of probiotics (microbial cell preparations or components of microbial cells) is getting attention. Probiotics (bacteria and yeasts), alone or in support to drug treatment, promote shortening/reduction of the gastrointestinal illness as well as counteract intestinal damages caused by G. duodenalis. Pre-clinical evidence suggest that probiotics can act directly against the parasite and/or promote protective response in the gut. In addition, parasite endosymbiotic viruses such as the Giardia-specific Giardiavirus possess, can have a significant impact on the parasite growth and survival. The exploitation of parasite-specific viruses as biological weapon against parasitic diseases is getting momentum In the near future Giardiavirus might offer a drug-free, cutting-edge, safe and target-specific therapy for giardiasis.

Introduction

Cryptosporidium parvum is a major cause of gastrointestinal illness in ruminants and humans, and is highly prevalent in Europe. A few subtypes appear to predominate and cause outbreaks, yet the reasons for this are unclear. We performed the first large-scale comparative genomics study of human- and ruminant-derived C. parvum isolates, aiming at improving our understanding of the population structure and evolution of this pathogen in Europe.

Methods

We sequenced the genome of 116 parasite strains using NGS. Trimmed reads were mapped to a reference genome to obtain a set of filtered Single Nucleotide Polymorphisms (SNPs). We investigated the parasite population structure, and searched for recombination events and for genes under selective pressure.

Results

We identified >32,000 SNPs, mostly located in telomeric and subtelomeric regions. Phylogenetic analysis revealed three strongly supported lineages, with no correlation with host species, country of origin, or subtype. Pairwise SNP distances revealed 12 distinct clusters of highly similar strains, representing outbreaks in humans or localized foci of animal infections. Interestingly, all outbreak clusters belonged to only one of the lineages.

Conclusions

We identified three phylogenetically distinct lineages of C. parvum, one of which comprised all strains linked to outbreaks. Exploring the genomic differences between these lineages may allow identification of genetic determinants with capability to cause outbreaks.

Funding: This work was done as part of the PARADISE project, supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme