Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples.

Toxoplasmosis is a zoonotic disease caused by the protozoan parasite Toxoplasma gondii. Infection in humans has usually been related to the consumption of raw, undercooked or cured meat. The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR)-based assay for the detection and quantification of T. gondii in meat samples. To optimize the ddPCR, T.gondii reference DNA aliquots at five known concentrations: 8000 cg/µl, 800 cg/µl, 80 cg/µl, 8 cg/µl were used. Moreover, results obtained by ddPCR and quantitative PCR (qPCR) were compared using 80 known samples (40 positive and 40 negative), as well as 171 unknown diaphragm tissue samples collected at slaughterhouses. The ddPCR showed a sensitivity of 97.5% and a specificity of 100%, with a detection limit of 8 genomic copy/µl of T. gondii. A nearly perfect agreement (κ = 0.85) was found between results obtained by ddPCR and qPCR for both positive and negative known samples analysed. On the 171 diaphragm tissue samples from field, 7.6% resulted positive by ddPCR and only 1.2% by qPCR. Therefore, this innovative method could be very useful for the detection of T. gondii in meat samples, aiming to prevent human infections.

Towards an Integrated Approach for Monitoring Toxoplasmosis in Southern Italy.

Toxoplasmosis is a widespread worldwide zoonotic infection caused by the intracellular protozoan Toxoplasma gondii. This protozoan infection is considered one of the most important food-borne parasitic zoonoses globally. Beyond its impact on public health, toxoplasmosis has also important veterinary implications, because it causes miscarriage or congenital malformations in livestock with negative economic impacts. An integrated monitoring programme aimed to deepen the epidemiological data on toxoplasmosis and to identify the risk factors that may favour T. gondii infections in animals and humans was conducted in an endemic area of southern Italy. The monitoring activities were based on the following tasks: (i) parasitological analysis and risk factors for T. gondii in livestock (sheep, goat, cattle and water buffalo) farms; (ii) serological and molecular monitoring at slaughterhouse in meat-producing livestock; (iii) analysis of hospital discharge records (HDRs); (iv) outreach activities (information, dissemination and health education) to farmers, vet practitioners and school-age children. The present study confirmed a very high seroprevalence of T. gondii infection in livestock farms (e.g., up to 93.1% in sheep farms) in southern Italy and highlighted the potentially significant public health risk in this area.