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Ready-to-eat (RTE) salads and berries are increasingly consumed in industrialized countries. These products can be contaminated by pathogenic parasites that have been responsible for foodborne outbreaks worldwide. In Italy, there are few data on contamination of RTE salads and berries with parasite transmission stages and this requires more-in-depth investigations. To estimate the prevalence of contamination with Cryptosporidium spp. and Giardia duodenalis in these fresh products, a total of 324 packages of local RTE mixed salads - belonging to three different industrial brands - and 324 packages of berries - blueberries from Peru, blackberries from Mexico, raspberries from Italy - were bought from supermarkets located in the Provinces of Bari and Foggia, Apulia, Italy. A pool size of nine packages was chosen and a total of 72 pools were processed in the whole year. After washing, the pellets were examined by microscopy (FLOTAC) and tested using conventional simplex PCR, targeting Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp., and sequencing. Several Cryptosporidium species and Giardia duodenalis assemblages, some of which are of potential zoonotic relevance, as well as Entamoeba spp., were identified in both matrices. By microscopy, Giardia-like cysts in local raspberries and Entamoeba-like cysts in imported blueberries were detected. Giardia duodenalis (Assemblages A, B and E) and Entamoeba histolytica were molecularly confirmed with overall prevalences of 4.6% (95% C.I. 3.0-6.8) and 1% (95% C.I. 0.3-2.1), respectively. Molecular methods identified Cryptosporidium ryanae, Cryptosporidium bovis, Cryptosporidium xiaoi, and Cryptosporidium ubiquitum in both matrices, with a prevalence of 5.1% (95% C.I. 3.3-7.3). A distinct seasonality in prevalence was observed for G. duodenalis, with most positives occurring in spring, whereas Cryptosporidium showed no significant seasonal variations. These results highlight that inadequate management of fresh produce, both locally produced and imported, along the food chain may have the potential for consequences on human health.

The human genome has been subjected to selective pressures to resist to infectious agents in spite of a heavy segregational load. With this regard, thalassaemia and glucose-6-phosphate dehydrogenase deficiency have been considered an efficient genetic protection against P. falciparum malaria in Sardinia, insular Italy. In this island, some multiple sclerosis (MS)-associated HLA haplotypes have the highest odds ratios in the same highestrate malarious areas of the island. Moreover, tumor necrosis factor (TNF) polymorphisms epidemiologically associated with both MS and malaria are ten-fold more frequent amongst Sardinians compared to other populations worldwide4. A possible association between MS and malaria in this island was never analysed experimentally. We studied the immunological response of mononuclear cells to P. falciparum and the killing effect of macrophages on parasites in Sardinian MS patients and in matched healthy controls (HC).

A field trial performed in-home conditions was conducted on 24 dogs naturally infected with Giardia, in order to compare the efficacy of fenbendazole and metronidazole. Animals were allocated in groups randomly in order to obtain two groups of 12 dogs each with similar parasitic loads of Giardia cysts: dogs in Group A were treated with fenbendazole (Panacur®, Intervet Italia Srl) administered at the dose of 50 mg/kg orally once a day for 5 consecutive days, dogs in Group B were treated with metronidazole (Flagyl®, Zambon Italia Srl) administered orally at the dose of 50 mg/kg, once a day for 5 consecutive days. All the dogs that were shedding Giardia cysts after the first treatment (Day 0) were retreated (either at Day 7 or at Day 14 or at Day 21) until a negative result was obtained with the same treatment. Additionally, all the dogs were re-examined at Day 50. All the dogs were tested for the presence of Giardia cysts using a fecal flotation method (FLOTAC). The percent efficacy of the treatments (A and B) was calculated at each sampling point (Days 7, 14, 21, and 50) as reduction in mean Giardia cysts. After the first therapy, on day 7, 4/12 (33.3%) dogs tested positive for Giardia cysts in the Group A and 5/12 (41.7%) in the Group B. Efficacies at (Days 7, 14, 21, and 50) of the treatments against Giardia infection were 80.9, 94, 100, and 97% in the Group A and 70.8, 99, 100, and 97.1% in the Group B. Statistically significant differences were not observed between the efficacy of Fenbendazole and Metronidazole against infection by G. duodenalis (P = 0.686). Molecular analysis revealed full homology (i.e., 100% with JN416550) with the canine specific assemblage D in six positive dogs. Different hypotheses might explain the re-appearance of the Giardia cysts in some dogs after treatment, e.g., re-infection from the home environment, the correct medication given by the owners, the diet, as well as treatment failure, but also biological issues related to the intermittent excretion of Giardia cysts.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.