First application of a droplet digital PCR to detect Toxoplasma gondii in mussels.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/µL, 800 gc/µL, 80 gc/µL, 8 gc/µL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/µL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3-99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7-25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans.

Examining the Veterinary Electronic Antimicrobial Prescriptions for Dogs and Cats in the Campania Region, Italy: Corrective Strategies Are Imperative.

Companion animals are increasingly being recognised as important contributors to the spread of antimicrobial-resistant bacteria. The present work aimed to measure the antimicrobial drug prescribing in dogs and cats in the Campania Region, Italy by analysing the Veterinary Electronic Prescriptions (VEPs) between 2019 and 2020. The medical records associated with antimicrobial drug prescriptions were collected according to the drug administration (systemic or topical) and the rationale for the treatment chosen. In the period under investigation, 166,879 drugs were prescribed of which 129,116 (73.4%) were antimicrobial. A total of 83,965 (65%) antibiotics were prescribed to dogs, 40,477 (31.4%) to cats, and 4674 (3.6%) to other companion animals. In dogs, 90.5% of VEPs prescribed for systemic treatment included an antimicrobial Critically Important or Highly Important or Important for human medicine (WHO, 2018). The most widely prescribed class was fluoroquinolones. The antimicrobials prescribed were mainly metronidazole-spiramycin (29.7%), amoxicillin-clavulanic (19.6%), enrofloxacin and cephalexin in dogs (16.5%) and enrofloxacin (22.6%) and amoxicillin-clavulanic acid (21.4%) in cats. Based on the results, the widespread use of broad-spectrum antimicrobials and the use of molecules for which limitations should be observed according to the EMA guidelines has emerged.

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.

Cystic echinococcosis in wild boars (Sus scrofa) from southern Italy: Epidemiological survey and molecular characterization.

Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu lato (s.l.) is one of the most important parasitic zoonotic diseases in the world and it represents an important public health and socio-economic concern. In the Mediterranean basin, CE is widespread and it is endemic in Italy, with major prevalence in southern areas. Several studies have investigated CE in domestic pigs, however, such data in wild boars are scant. In the last decades the wild boar population in Italy has increased and this ungulate could play an important role in the spreading of CE in the wild. Here we report on the prevalence and fertility rate of hydatid cysts in wild boars that were shot during two hunting seasons (2016-2017) in the Campania region of southern Italy. For each animal, a detailed inspection of the carcass and organs (lungs, liver and spleen) was performed and when cysts were found, their number, morphology and fertility were determined by visual and microscopic examination. Cysts were classified morphologically as fertile, sterile, caseous and calcified. Protoscoleces and germinal layers were collected from individual cysts and DNA was extracted to identify different strains/genotypes of E. granulosus s.l. Out of a total of 2108 wild boars 93 (4.4%) were found positive for CE. Infected animals were 45 males and 48 females, aged between 1 and 8 years. The average number of cysts per wild boar was 1.3 (min 1 - max 13). The total number of cysts collected was 123, of which 118 (95.9%) in the liver, 4 (3.3%) in the lungs and 1 (0.8%) in the spleen. Of all analyzed cysts, 70 (56.9%) were fertile and 53 (43.1%) sterile/acephalous. The presence of fertile cysts in 19.4% of CE-positive animals is noteworthy. Overall, molecular diagnosis showed 19 wild boars infected with the pig strain (G7).

Official controls according to Integrated Regional Plan in Campania Region 2011/2014.

Due to EU law, European citizens enjoy one of the highest worldwide food safety standards. Along the entire food supply chain, mandatory controls are made to ensure that plant and animal products healthiness and food and feed safety are properly labeled and comply with the strictly EU rules. The process of review of European legislation began with the promulgation of the Regulation 178/2002/EC (Official Journal of the EC no. L31 of 01/02/2002) and was completed with the hygiene package. Regulation No. 178/2002/EC (the General Food Law) laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety.

Preliminary study on the inactivation of anisakid larvae in baccalà prepared according to traditional methods.

The European Food Safety Authority stated that many traditional marinating and cold smoking methods are not sufficient to kill A. simplex and asked to evaluate alternative treatments for killing viable parasites in fishery. Baccalà is a well-liked traditional product. The aim of study was to evaluate the effectiveness of the salting process on the inactivation of nematodes of the genus Anisakis in naturally infected Baccalà fillets. N. 19 fillets, subjected to a dual salting process (brine and dry salting) were analyzed. Visual inspection and chloropeptic digestion were performed. Larvae viability was evaluated, and parameters such as NaCl (%), moisture (%), WPS and aw were determined. In n. 17 samples parasites were found 123 parasites with a mean intensity of 7.23±4.78 and an mean abundance of 6.47±5.05. Visual examination has revealed 109 parasites. 61.8% of larvae were found in the ventral portions. The results show that salting process with a salt concentration of 18.6%, aw values of 0.7514 and 24.15% WPS in all parts of baccalà fillets, devitalise Anisakidae larvae in a 15-day period.

Trace elements in free-range hen eggs in the Campania region (Italy) analyzed by inductively coupled plasma mass spectrometry (ICP-MS).

Eggs from hens raised on rural or domestic farms are a good indicator of environmental contamination, as the hens are in close contact with the ground and the air and can therefore accumulate heavy metals and other toxic contaminants from the environment as well as from the diet. In this paper, we report the results of the determination of 19 trace elements (As, Be, Cd, Co, Cr, Cu, Hg, Mo, Mn, Ni, Pb, Sb, Se, Sn, Sr, Tl, U, V, Zn) in 39 hen egg samples collected from domestic poultry farms in the territory dubbed the "Land of fires" in the Campania region (Italy). This area is characterized by environmental problems caused by the illegal dumping of industrial or domestic waste in fields or by roadsides. In some cases, these wastes have been burned, thereby spreading persistent contaminants into the atmosphere. The content of trace elements in whole egg samples was determined by mass spectrometer after a microwave-assisted digestion procedure. Because European legislation does not indicate maximum values of these elements in this foodstuff, the results were compared with the content of trace elements reported in literature for eggs, in particular home-produced eggs, in various countries. In some cases (Cd, Cu, Ni, Mn), the content determined in this study was in line with those reported elsewhere, in other cases (Pb, Cr), lower values were found.

Polychlorodibenzodioxin and -furan and dioxin-like polychlorobiphenyl distribution in tissues and dairy products of dairy buffaloes.

A pilot study was performed on three different dairy buffalo herds exposed without exposure control conditions to Polychlorodibenzodioxins and -furans (PCDDs, PCDFs) and Dioxin-like Polychlorobiphenyls (DL-PCBs). This study dealt with the relationship between the contamination levels (pg WHO2005-TE/g fat) in individual raw milk and those in edible tissues and with the contamination transfer from farm bulk milk to dairy products. On a cumulative basis, kidney (41, 67, and 21 pg WHO-TE/g fat) resulted more in equilibrium with milk (48, 42, and 20) than did muscle (25, 31, and 9), while liver showed a large bioaccumulation (221, 304, and 75), with marked differences of the congener profile. Mozzarella cheese contamination (23, 42, and 29 pg WHO-TE/g fat) was higher than that of bulk milk (20, 36, and 21), which suggested a role of casein precipitation in congener transfer. The above information could improve the effectiveness of risk management during a "dioxin" crisis.