Meta-analysis of the Cetacea gut microbiome: Diversity, co-evolution, and interaction with the anthropogenic pathobiome.

Despite their critical roles in marine ecosystems, only few studies have addressed the gut microbiome (GM) of cetaceans in a comprehensive way. Being long-living apex predators with a carnivorous diet but evolved from herbivorous ancestors, cetaceans are an ideal model for studying GM-host evolutionary drivers of symbiosis and represent a valuable proxy of overall marine ecosystem health. Here, we investigated the GM of eight different cetacean species, including both Odontocetes (toothed whales) and Mysticetes (baleen whales), by means of 16S rRNA-targeted amplicon sequencing. We collected faecal samples from free-ranging cetaceans circulating within the Pelagos Sanctuary (North-western Mediterranean Sea) and we also included publicly available cetacean gut microbiome sequences. Overall, we show a clear GM trajectory related to host phylogeny and taxonomy (i.e., phylosymbiosis), with remarkable GM variations which may reflect adaptations to different diets between baleen and toothed whales. While most samples were found to be infected by protozoan parasites of potential anthropic origin, we report that this phenomenon did not lead to severe GM dysbiosis. This study underlines the importance of both host phylogeny and diet in shaping the GM of cetaceans, highlighting the role of neutral processes as well as environmental factors in the establishment of this GM-host symbiosis. Furthermore, the presence of potentially human-derived protozoan parasites in faeces of free-ranging cetaceans emphasizes the importance of these animals as bioindicators of anthropic impact on marine ecosystems.

Prevalence of Blastocystis sp. and other gastrointestinal pathogens among diarrheic COVID-19 patients in Italy.

Background: Gastrointestinal pathogens (GPs) contribute significantly to the burden of illness worldwide with diarrhoea being the most common among gastrointestinal symptoms (GSs). In the COVID-19 disease, diarrhoea, could be one of the initial presenting symptoms. However, no data on the potential correlation between diarrhoea-causing pathogens and SARS-CoV-2 infection are available. Therefore, we carried out a 2-years retrospective study aimed to evaluate the prevalence of "classic" GPs among SARS-CoV-2 infected and non-infected patients with diarrhoea in Italy. Methods: Results of SARS-CoV-2 research from nasopharyngeal and detection of GPs from stool swab samples by Allplex™ SARS-CoV-2 and GI Virus, Bacteria and Parasite Assay were analysed for all patients with diarrhoea referring to Policlinico Ospedaliero Universitario, Foggia, (Italy) from February 2022 to October 2023. Results: Out of the 833 involved patients, 81 (3.9%) were COVID-19 positive, while 752 (90.3%) were COVID-19 negative. Among COVID-19-positive patients, 37% (n = 30/81) were found positive for one or more GPs with a higher prevalence of protozoan parasites (18.5%) (Blastocystis ST1-ST4 subtypes, Dientamoeba fragilis genotype I), followed by bacteria (7.4%) (Campylobacter sp., Salmonella sp.). Viral pathogens were more frequent among COVID-19 negative patients (Adenovirus, Norovirus). Among GPs, Blastocystis ST3 subtype was the most prevalent registered in the 16% of patients (p = 0.0001). Conclusions: Based on obtained results, a likely interaction between the classic GPs and SARS-CoV-2 infection can be speculated, driven by protozoan parasites. Moreover, these results also provide baseline data to understand more deeply Blastocystis sp. role in this scenario of dysbiosis, particularly in those cases of SARS-CoV-2 co-infection.

Activity of a foam in preventing rebound of vancomycin-resistant Enterococcus faecium-containing droplets generated from the toilet bowl.

In hospital environments, droplets generated by urination within shared toilets may represent a route of dissemination for bacteria such as vancomycin-resistant Enterococcus faecium (VREfm), which contributes significantly to the burden of hospital-acquired infections. We investigated the potential activity of a foam in preventing the generation of droplets containing Enterococcus spp. during urination. A uniform layer of foam was deposited in the inner walls and at the bottom of an experimental toilet contaminated with suspensions of Enterococcus strains (including a VREfm strain). Human urination was simulated, and colonies of Enterococcus were recovered through a toilet lid where agar plates had been placed. Results showed that the foam was able to suppress production of droplets containing Enterococcus spp. generated by a liquid hitting inner toilet walls. Conversely, Enterococcus colonies were recovered in absence of foam. Moreover, the foam did not show antibacterial activity. We propose a new non-antimicrobial approach aimed at limiting transmission of multidrug-resistant bacteria, particularly in healthcare settings.

Prevalence and genetic diversity of Blastocystis sp. among autochthonous and immigrant patients in Italy.

The prevalence of Blastocystis sp., its genetic diversity and the distribution of circulating subtypes (STs) were molecularly investigated in a cohort of autochthonous and immigrant patients with gastrointestinal symptoms hospitalized over the period February 2022-June 2023 at the Policlinico Ospedaliero-Universitario "Riuniti", Foggia, in Southern Italy. The population variables, including patient geographical origin, gender and age classes were reported. Out of the 927 investigated patients, 36 (3.9%) were positive for Blastocystis sp. A statistically significant association with African origin and age classes >18 years old was found. ST1 (allele 4), ST2 (alleles 9, 13), ST3 (alleles 34, 36) and ST4 (allele 92) were the subtypes detected with a different distribution between autochthonous and immigrant patients. Co-infections with enteric protozoa such as Giardia duodenalis and Dientamoeba fragilis, pathogenic bacteria as Clostridioides difficile, Campylobacter jejuni and Aeromonas sp. and viral infections such as Norovirus were found in 33% of cases. This is the first study of Blastocystis sp., its circulating subtypes and allele variability among patients with different geographical origin in an area of Southern Italy, in the Central Mediterranean, characterized by high immigrant pressure. These results provide baseline data to better investigate a potential interaction between Blastocystis sp. and other risk factors in patients with gastrointestinal symptoms.

Dolphins and sea turtles may host zoonotic parasites and pathogenic bacteria as indicators of anthropic pressure in the Gulf of Taranto (Northern Ionian Sea, Central-Eastern Mediterranean Sea).

The occurrence of protozoan parasites Giardia duodenalis and Cryptosporidium spp. such as the pathogenic bacteria Salmonella spp. and Escherichia coli was molecularly investigated in the following free ranging species of striped dolphins (Stenella coeruleoalba), Risso's dolphins (Grampus griseus) as well as loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles living in the Gulf of Taranto (Mediterranean Sea). Out of forty-one investigated individuals belonging to the 4 species, 13 (31.7%) were positive to one or more pathogens and zoonotic G. duodenalis assemblage A, Cryptosporidium parvum and S. enterica were identified in striped dolphins, loggerhead and green sea turtles. In this work, the presence of these opportunistic pathogens has been investigated in fecal samples of free ranging dolphin and sea turtle species for the first time. Moreover, this is the first record of C. parvum in loggerhead sea turtles. These results may provide baseline data for the potential role of cetaceans and sea turtles as potential sentinel species for zoonotic and terrestrial pathogens in the marine environment.

Genome-Wide Expression Analysis of Glyoxalase I Genes Under Hyperosmotic Stress and Existence of a Stress-Responsive Mitochondrial Glyoxalase I Activity in Durum Wheat (Triticum durum Desf.).

Glyoxalase I (GLYI) catalyzes the rate-limiting step of the glyoxalase pathway that, in the presence of GSH, detoxifies the cytotoxic molecule methylglyoxal (MG) into the non-toxic D-lactate. In plants, MG levels rise under various abiotic stresses, so GLYI may play a crucial role in providing stress tolerance. In this study, a comprehensive genome database analysis was performed in durum wheat (Triticum durum Desf.), identifying 27 candidate GLYI genes (TdGLYI). However, further analyses of phylogenetic relationships and conserved GLYI binding sites indicated that only nine genes encode for putative functionally active TdGLYI enzymes, whose distribution was predicted in three different subcellular compartments, namely cytoplasm, plastids and mitochondria. Expression profile by qRT-PCR analysis revealed that most of the putative active TdGLYI genes were up-regulated by salt and osmotic stress in roots and shoots from 4-day-old seedlings, although a different behavior was observed between the two types of stress and tissue. Accordingly, in the same tissues, hyperosmotic stress induced an increase (up to about 40%) of both GLYI activity and MG content as well as a decrease of GSH (up to about -60%) and an increase of GSSG content (up to about 7-fold) with a consequent strong decrease of the GSH/GSSG ratio (up to about -95%). Interestingly, in this study, we reported the first demonstration of the existence of GLYI activity in highly purified mitochondrial fraction. In particular, GLYI activity was measured in mitochondria from durum wheat (DWM), showing hyperbolic kinetics with Km and Vmax values equal to 92 ± 0.2 µM and 0.519 ± 0.004 µmol min-1 mg-1 of proteins, respectively. DWM-GLYI resulted inhibited in a competitive manner by GSH (Ki = 6.5 ± 0.7 mM), activated by Zn2+ and increased, up to about 35 and 55%, under salt and osmotic stress, respectively. In the whole, this study provides basis about the physiological significance of GLYI in durum wheat, by highlighting the role of this enzyme in the early response of seedlings to hyperosmotic stress. Finally, our results strongly suggest the existence of a complete mitochondrial GLYI pathway in durum wheat actively involved in MG detoxification under hyperosmotic stress.

Occurrence of the protozoan parasites Toxoplasma gondii and Cyclospora cayetanensis in the invasive Atlantic blue crab Callinectes sapidus from the Lesina Lagoon (SE Italy).

The occurrence of the protozoan parasites Toxoplasma gondii and Cyclospora cayetanensis was molecularly investigated in the hemolymph, gills, stomach, hepatopancreas and gonads of the eleven invasive Atlantic blue crab Callinectes sapidus from the Lesina Lagoon (Mediterranean Sea). Out of 11 blue crabs, 6 (54.5%) and 4 (36.4%) were found positive to T. gondii and C. cayetanenis, respectively; parasites were found only in the six females analysed, while the remaining five males resulted negative. Moreover, out of 55 tissues samples, 7 (12.7%) and 8 (14.5%) were positive to T. gondii and C. cayetanensis, respectively with hemolymph and gills being the most infected tissues. This is the first report of the presence of protozoan pathogens in wild crab species collected from a Mediterranean lagoon. The present results may provide a baseline reference on microbial infection in the species for invaded Mediterranean waters, and on the potential health risks related with its consumption if eaten raw.

Detection of Zoonotic Cryptosporidium ubiquitum in Alpine Wild Ruminants.

Cryptosporidium is a widespread apicomplexan protozoan of major zoonotic importance, characterized by a wide host range, and with relevant economic implications and potential negative effects on livestock and wildlife population dynamics. Considering the recent strong demographic increase of alpine ungulates, in this study, carried out in the Italian Northwestern Alps, we investigated the occurrence of Cryptosporidium spp. in these species and their potential involvement in environmental contamination with Cryptosporidium spp. oocysts. The immune-enzymatic approach revealed a Cryptosporidium prevalence of 1.7% (5/293), 0.5% (1/196) and 3.4% (4/119) in alpine chamois (Rupicapra rupicapra), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), respectively. Positive samples were subjected to polymerase chain reaction (PCR) amplification for the COWP and gp60 genes. The presence of Cryptosporidium was confirmed in one chamois and four roe deer. Sequences obtained clustered within Cryptosporidium ubiquitum, currently recognized as an emerging zoonotic species. This finding represents the first detection of zoonotic C. ubiquitum in chamois and in the Alpine environment. Despite the low observed prevalences, environmental contamination by oocysts could play a role as a potential source of infections for humans and livestock.

Wild whale faecal samples as a proxy of anthropogenic impact.

The occurrence of protozoan parasite, bacterial communities, organic pollutants and heavy metals was investigated in free-ranging species of fin (Balaenoptera physalus, n. 2) and sperm (Physeter macrocephalus, n. 2) whales from the Pelagos Sanctuary, Corsican-Ligurian Provencal Basin (Northern-Western Mediterranean Sea). Out of four faecal samples investigated, two from fin whales and one from sperm whale were found positive to Blastocystis sp. A higher number of sequences related to Synergistetes and Spirochaetae were found in sperm whales if compared with fin whales. Moreover, As, Co and Hg were found exclusively in sperm whale faecal samples, while Pb was found only in fin whale faecal samples. The concentration of both PAH and PCB was always below the limit of detection. This is the first report in which the presence of these opportunistic pathogens, bacteria and chemical pollutants have been investigated in faecal samples of free-ranging whale species and the first record of Blastocystis in fin and sperm whales. Thus, this study may provide baseline data on new anthropozoonotic parasite, bacterial records and heavy metals in free-ranging fin and sperm whales, probably as a result of an increasing anthropogenic activity. This survey calls for more integrated research to perform regular monitoring programs supported by national and/or international authorities responsible for preservation of these still vulnerable and threatened whale species in the Mediterranean Sea.

First multicenter coprological survey on helminth parasite communities of free-living loggerhead sea turtles Caretta caretta (Linnaeus, 1758) from the Adriatic Sea and Northern Ionian Sea.

The prevalence of endoparasite infections in 83 free-living specimens of Caretta caretta, classified as vulnerable species, from the Adriatic Sea and Northern Ionian Sea was investigated by coprological examination. Thirty-seven (44.6%) turtles were found to be infected with helminths. The helminth infections found were: Rhytidodes gelatinosus and Sulcascaris sulcata (18.1% each), Hapalotrema mistroides (13.2%), Cymatocarpus solearis (9.6%), Eniodotrema megachondrus (7.2%), Kathlania/Tonaudia sp. (3.6%), Neospirorchis sp., Octangium sagitta and Plesiochorus cymbiformis (1.2% each). There were no significant differences in the total prevalence of helminth infections between sexes, size classes, and seasonal periods. Conversely, the prevalence of helminth infections was significantly higher (P < 0.01) in accidentally caught turtles than in stranded turtles. Highly significant differences in prevalence of helminthiases were also seen among marine sampling areas. This report provides important baseline information about the helminth fauna of free-living C. caretta in the examined geographical region. This is also the first report of O. sagitta infection in C. caretta thus broadening the host range of the parasite.

Asia and Europe: So Distant So Close? The Case of Lipoptena fortisetosa in Italy.

In Europe, 5 Lipoptena species have been recorded, including Lipoptena fortisetosa. This species, native to Asian countries, was described as a parasite of sika deer and its appearance in Europe dates back to more than 50 years ago. Lipoptena fortisetosa has been recently reported in Italy, sharing its hosts with Lipoptena cervi. A morpho-molecular approach was developed to determine the phylogenetic interrelationship of Italian and Asian CO1 haplotypes sequenced from Lipoptena fly individuals collected in Italy, and their DNA sequences were compared with conspecifics available in GenBank; morphological key-characters (terminalia) of L. fortisetosa were compared with the original description. Two haplotypes were recorded from Italy and assigned to L. cervi and L. fortisetosa, respectively. The latter was part of the monophyletic clade L. fortisetosa, along with 2 Central European and 2 Korean haplotypes (100% identical to one of the Korean haplotypes); moreover, Italian L. fortisetosa female terminalia were consistent with the original description of Asian individuals. Pending more in-depth investigations, this study provides a first answer to the hypothesis of the recent colonization of Italy by L. fortisetosa from Asia as we did not detect any obvious and stable morphological and molecular differences in specimens from the 2 geographical areas. The presence of the sika deer in Europe was retraced and the possible route traveled by the parasite from Asia and the eco-biological factors that may have enhanced its settlement are discussed.

Efficiency of the Q3 lab-on-chip Real Time-PCR platform for detecting protozoan pathogens in bivalve mollusks.

The zoonotic protozoan parasites Toxoplasma gondii, Cryptosporidium parvum and Giardia duodenalis have been recorded worldwide in economically important edible shellfish, and are thus likely to represent a significant public health risk. Therefore, an innovative, user-friendly diagnostic tool is required in order to improve food safety control. The Q3 system is a miniaturized platform whose efficiency and applicability were investigated and compared with results obtained using standard Real-Time PCR. Tanks of saltwater containing acclimated Mytilus galloprovincialis, Ruditapes philippinarum and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 103, 104 and 105). We then collected 30 specimens for each shellfish species from each group at 24 h and 72 h post-contamination. After DNA extraction, we tested all samples by Real-Time-PCR and Q3, and evaluated the sensitivity, specificity, predictive values, repeatability and concordance between the two systems. Concordance between Real-Time-PCR and Q3 was very good (p < 0.01), especially for Toxoplasma in M. galloprovincialis at both 24 h and 72 h after contamination, and in O. edulis at 72 h. The ability of Q3 to detect all the investigated pathogens was similar to that of Real-Time-PCR, and Q3 was efficient in detecting Toxoplasma in both M. galloprovincialis and O. edulis. This is the first study concerning the use of lab-on-chip technology in a food matrix, and in edible marine mollusks in particular.

Molecular epidemiology of Blastocystis sp. in dogs housed in Italian rescue shelters.

Blastocystis is a ubiquitous protozoan with a wide range of hosts. In humans, its presence has been associated with gastrointestinal disorders, although its role as a pathogen still needs to be elucidated. Until now, 17 Blastocystis subtypes (STs) have been identified, with ST1-ST4 the most commonly found in humans. Among domestic animals, the same STs reported in humans have been detected in dogs. An epidemiological survey on dog kennels was carried out to evaluate the prevalence of Blastocystis and the STs involved. Overall, 99 faecal samples were collected from the rescue shelters. Blastocystis detection was performed through conventional barcoding PCR targeting the 1800-bp SSU-rDNA, followed by sequencing and phylogenetic analysis. Blastocystis DNA was found in 21 faecal samples (21.2%), and all samples were successfully sequenced and identified as ST3 in a unique monophyletic group. The presence of Blastocystis was reported for the first time in dogs from Italy, with the identification of ST3, the subtype most commonly found in humans.

Toxoplasma gondii and Other Zoonotic Protozoans in Mediterranean Mussel ( Mytilus galloprovincialis) and Blue Mussel ( Mytilus edulis): A Food Safety Concern?

Mediterranean mussels ( Mytilus galloprovincialis) and blue mussels ( Mytilus edulis) are among the most consumed fishery products, but they are frequent vehicles of foodborne infection worldwide. In this study, we investigated the occurrence and seasonality of zoonotic protozoans in mussels farmed or sold at retail outlets in Italy. We collected and tested 1,440 M. galloprovincialis and 180 M. edulis. Pooled samples were molecularly tested for Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii and then sequenced. Sixty-two (45.9%; 95% confidence interval, 37.5 to 54.3%) mussel pools tested positive for one or more of the investigated pathogens. Both Mytilus species and samples from all the investigated areas harbored pathogens. Mussels were statistically more contaminated by Cryptosporidium spp., followed by T. gondii and G. duodenalis assemblage A, and M. galloprovincialis was more contaminated than M. edulis ( P < 0.01). Contamination was more likely in mussels at retail outlets ( P < 0.05) than in those from farms and in mussels collected in spring ( P < 0.01) than in other seasons. This is the first report of T. gondii found in M. galloprovincialis in Italy and in M. edulis in Europe. The detection of zoonotic protozoans in a widely consumed food source indicates the need for a more detailed microbiological risk analysis, especially considering that bivalve mollusks are often consumed raw worldwide.

Toxoplasma gondii Lineages Circulating in Slaughtered Industrial Pigs and Potential Risk for Consumers.

Toxoplasma gondii is a cosmopolitan zoonotic protozoan parasite, and the consumption of raw or undercooked pig meat is one of the most important sources of T. gondii infection. Three predominant lineages, types I, II, and III, are widespread in Europe. Although still poorly understood, a relationship between each type and the severity of illness represents a public health issue. To gain further knowledge of the genotypes in circulation and of the potential risk for consumers, one heart sample and one diaphragm sample (206 total) were taken from each of 103 pig carcasses at an abattoir in Italy. Then, we used 529-bp repetitive element PCR and a B1 real-time PCR high-resolution melting assay coupled with sequencing to detect and genotype T. gondii isolates. T. gondii DNA was detected in 14 pigs (13.6%, 95% confidence interval = 7 to 20.2%), and types I (3.9%), II (5.8%), and III (3.9%) were identified. We found that heart tissue had a significantly higher PCR positivity rate for T. gondii than did diaphragm tissue. This is Europe's largest study on genotyping of T. gondii from pigs, and it demonstrates that all three main lineages are present in carcasses of industrially reared pigs in Italy. There is a potential risk to consumers of infection with any or all of the three lineages, and the related clinical consequences should be taken into account. This study suggests that monitoring of T. gondii types in meat is essential, especially in meat that is traditionally eaten raw or that is minimally processed.

Toxoplasma gondii infection and biosecurity levels in fattening pigs and sows: serological and molecular epidemiology in the intensive pig industry (Lombardy, Northern Italy).

Toxoplasma gondii is a widespread protozoan affecting animals and humans. One of the major routes of human infection is through the consumption of raw or under-cooked meat, particularly of certain animal species, including pigs. Although T. gondii represents an important public health issue, its control at slaughter is not mandatory. Consequently, available information on T. gondii infection in domestic animals destined for human consumption is scarce. Thus, an epidemiologic survey was designed to update information on T. gondii infection in pigs from intensive production. Fattening pigs and sows from conventional farms were sampled. Sera were tested with a commercial ELISA for anti-T. gondii antibodies, whereas molecular analysis by 529 bp repetitive element PCR and B1 real-time PCR with subsequent genotyping was performed on heart samples. Statistical analysis was carried out to detect farm management features and sanitary procedures enhancing the risk of infection. At the farm level, 63.6% (7/11) of farms housing sows and 6.7% (1/15) housing fattening pigs scored positive, with individual prevalences of 8.6% (13/151) in sows and 0.5% (1/219) in fattening pigs. T. gondii DNA was detected in eight sows and one fattening pig, and sequencing revealed the presence of all three genotypes (types I, II, and III). Furthermore, the decrease in the biosecurity level enhanced the risk of infection within a farm. The present survey outcomes confirm the spread of T. gondii infection in pig farms in an area of intensive swine production. The application of stricter sanitary procedures may contribute to a further reduction of infection.

Helminth infections in faecal samples of Apennine wolf (Canis lupus italicus) and Marsican brown bear (Ursus arctos marsicanus) in two protected national parks of central Italy

This article reports the results of a copromicroscopic and molecular investigation carried out on faecal samples of wolves (n=37) and brown bears (n=80) collected in two protected national parks of central Italy (Abruzzo Region). Twenty-three (62.2%) samples from wolves were positive for parasite eggs. Eight (34.78%) samples scored positive for single infections, i.e. E. aerophilus (21.74%), Ancylostoma/Uncinaria (4.34%), Trichuris vulpis (4.34%), T. canis (4.34%). Polyspecific infections were found in 15 samples (65.21%), these being the most frequent association: E. aerophilus and Ancylostoma/Uncinaria. Thirty-seven (46.25%) out of the 80 faecal samples from bears were positive for parasite eggs. Fourteen (37.83%) samples were positive for B. transfuga, and six (16.21%) of them also contained Ancylostoma/Uncinaria, one (2.7%) E. aerophilus and one (2.7%) both E. aerophilus and Ancylostoma/Uncinaria. Of the other samples, 19 (51.35%) were positive for Ancylostoma/Uncinaria, two (5.4%) for E. aerophilus and two (5.4%) for both. Molecular analysis found the roundworm and capillariid eggs found in wolves and bear samples to be Toxocara canis, Baylisascaris transfuga and Eucoleus aerophilus (syn. Capillaria aerophila). Considering the high prevalence of zoonotic intestinal helminths detected in this study, it is important to improve the knowledge and awareness of the general public and park operators regarding the potential health risk associated with infections in wildlife.

Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy.

To investigate the prevalence of protozoan contamination by Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Cyclospora cayetanensis, in 'ready to eat' (RTE) salads on sale in Italy, 648 packages were purchased from industrial and local brands. Nine individual packages from each brand were collected per month, pooled and subjected to microscopy and molecular analyses. Microscopic examination of 864 slides detected Cryptosporidium spp. but also Blastocystis hominis and Dientamoeba fragilis. Molecular tools identified G. duodenalis assemblage A, Cryptosporidium parvum and Cryptosporidium ubiquitum, T. gondii Type I and C. cayetanensis. B. hominis and D. fragilis were also molecularly confirmed. The overall prevalence of each protozoan species was 0.6% for G. duodenalis, 0.8% for T. gondii, 0.9% for Cryptosporidium spp., and 1.3% for C. cayetanensis, while prevalence for B. hominis was 0.5% and for D. fragilis 0.2%. Microscopy and/or molecular tools revealed that 4.2% of the samples were contaminated by at least one protozoan species, and 0.6% of samples presented contamination by two protozoan species, with a number of oocysts ranging from 62 to 554 per g of vegetable matter for T. gondii, and 46 to 1.580 for C. cayetanensis. This is Europe's first large-scale study on the presence of protozoans in packaged salads, and shows that RTE sanitation processes do not guarantee a product free from protozoans of fecal origin.

Wound Myiasis Caused by Sarcophaga (Liopygia) Argyrostoma (Robineau-Desvoidy) (Diptera: Sarcophagidae): Additional Evidences of the Morphological Identification Dilemma and Molecular Investigation.

In Mediterranean countries, Sarcophaga (Liopygia) crassipalpis, Sarcophaga (L.) argyrostoma, and Sarcophaga (L.) cultellata share the same ecological niche and can be responsible of myiasis. In this study, the main morphological characters of a larva found in a hospitalized woman were described and illustrated by light and SEM microscopy and the features discussed. Then, a fragment within the mitochondrial encoded cytochrome c oxidase subunit I (coxI) gene of ~735 bp was amplified and sequenced. The molecular investigation was necessary to confirm the species Sarcophaga (Liopygia) argyrostoma (99% of identity). Our findings showed that morphological descriptions of larvae of three Mediterranean species of Liopygia available in several papers might not be clear enough to allow for comparison and correct identification. Until results of reliable comparative studies of larvae of all three species will be available, the use of molecular tools is crucial, to avoid misleading or incomplete identification, and in particular when a myiasis becomes a legal issue.

Occurrence of Toxoplasma gondii in Carcasses of Pigs Reared in Intensive Systems in Northern Italy.

To evaluate the occurrence of Toxoplasma gondii and to genetically characterize its isolates in carcasses of industrial fattening pigs, blood, diaphragm, and heart samples were collected from 375 carcasses of pigs slaughtered to be processed for Parma ham production. Pigs had been bred on approved farms (n = 75) located in the so-called Food Valley in Italy. Sera were examined for immunoglobulin G antibodies to T. gondii by modified agglutination test (MAT). Both heart and diaphragm samples from seropositive carcasses were processed for the presence of T. gondii DNA (B1 locus) by real-time PCR and high resolution melting (HRM) assay. Anti-Toxoplasma antibodies were detected in 2.1% of pig carcasses, with titers from 1:10 to 1:320. T. gondii DNA was detected in all (eight) seropositive carcasses and in 11 (5 heart and 6 diaphragm samples) of 16 samples; that is, it was detected in heart tissue in two subjects, in diaphragm tissue in three subjects, and in both muscle tissues in three subjects. Toxoplasma genotypes were determined in seven of eight carcasses: type III was identified in four carcasses, type II in two, and both III and II in one carcass. The serological findings and the molecular detection of T. gondii strains suggest that cured meat products obtained from industrially bred pigs may be potential sources of toxoplasmosis for humans. Our results provide novel, important information regarding the seroprevalence and molecular prevalence of T. gondii in intensively reared pigs within this specific region of Italy, particularly because Parma ham from this region is known and consumed worldwide. On-farm preventive measures combined with slaughterhouse monitoring of carcasses of pigs bred for cured meat production should never be overlooked to prevent the introduction of T. gondii into the food chain and to ensure safety for consumers of these products.