Toxoplasma gondii Investigation of Home-Reared Pigs through Real-Time PCR and Digital Droplet PCR: A Very Low Prevalence.

Toxoplasma gondii is a widespread protozoon that can infect both animals and humans. The main route of human infection is the consumption of the raw or undercooked meat of several animal species, including pigs. Although T. gondii represents a public health concern, control during slaughter is not mandatory, leading to a lack of information on the impact on human contagion as well as poor data availability in domestic animals intended for human consumption. We studied the presence of T. gondii in home-reared pigs, an unconventional type of farming subjected to stringent breeding conditions dictated by Italian regulation. Thus, the diaphragms, livers and masseter muscles from 480 pigs in Napoli Province (Italy) were analyzed using real-time PCR and digital droplet PCR. The results showed four matrices that tested positive for T. gondii with very low protozoan loads (0.62%), belonging to three different animals. The low density of the animals (the maximum was four animals per farm) and the biosafety farming features decisively contributed to the bioexclusion of this pathogen. Comparing these results to intensive and extensive farm data, lower exposure to the parasite was revealed, suggesting that this farming method might mitigate the risk of human exposure through meat consumption.

SARS-CoV-2 Serological and Biomolecular Analyses among Companion Animals in Campania Region (2020-2021).

The first reports of SARS-CoV-2 among domestic and wild animals, together with the rapid emergence of new variants, have created serious concerns regarding a possible spillback from animal hosts, which could accelerate the evolution of new viral strains. The present study aimed to investigate the prevalence and the transmission of SARS-CoV-2 among both owned and stray pets. A total of 182 dogs and 313 cats were tested for SARS-CoV-2. Specimens collected among owned and stray pets were subjected to RT-PCR and serological examinations. No viral RNA was detected, while anti-N antibodies were observed in six animals (1.3%), one dog (0.8%) and five cats (1.7%). Animals' background revealed that owned cats, living with owners with COVID-19, showed significantly different prevalence compared to stray ones (p = 0.0067), while no difference was found among dogs. Among the seropositive pets, three owned cats also showed moderate neutralizing antibody titers. Pets and other species are susceptible to SARS-CoV-2 infection because of the spike affinity towards their ACE2 cellular receptor. Nevertheless, the risk of retransmission remains unclear since pet-to-human transmission has never been described. Due to the virus' high mutation rate, new reservoirs cannot be excluded; thus, it is reasonable to test pets, mostly if living in households affected by COVID-19.

Genotyping of Toxoplasma gondii in wild boar (Sus scrofa) in southern Italy: Epidemiological survey and associated risk for consumers.

Toxoplasma gondii is a widespread protozoan parasite (phylum Apicomplexa), which causes a zoonotic parasitic disease, known as toxoplasmosis. The aim of this study was to evaluate the occurrence and genotypes of T. gondii in wild boars of southern Italy and thus to assess the risk of infection for consumers. The boars were inspected during the hunting season within the regional project 'Wild Boar Emergency Plan in Campania', and molecular analyses were performed on 338 boars analysing a total number of 884 matrices (263 brains, 310 hearts and 311 masseter muscles). Toxoplasma gondii was detected in 134 out of 338 boars (39.6%). No significant statistical difference between genders was found (χ2  = 0.15 p = .70). The prevalence was 47.1%, 39.3% and 39.2% in piglets, yearlings and adults, respectively (χ2  = 0.41; p = .81). The highest prevalence of T. gondii was found in masseter muscles (74/311, 23.8%), followed by the heart (70/310, 22.6%) and brain (58/263, 22.0%), respectively. Microsatellite (MS) analysis of 11 samples revealed eleven T. gondii genotypes (nine atypical, one belonging to type II one to type III). Most of the genotypes found were thus atypical and may be virulent in humans. Hierarchical clustering analysis showed the presence of three distinct clusters, with the majority of atypical genotypes in the GII-GIII cluster. The high prevalence of infection in masseters highlights the potential risk for public health, considering that this muscle is commonly used to prepare raw meat products ('guanciale' and sausages), which may be a source of T. gondii infection in humans. Wild boars may act as an interface role between wildlife, livestock and humans. Our data highlight the urgent need to minimize the risk of infection for animals and humans by setting up a surveillance programme and preventive strategies in a One Health approach to wildlife species.

Parasite Load and STRs Genotyping of Toxoplasma gondii Isolates From Mediterranean Mussels (Mytilus galloprovincialis) in Southern Italy.

Toxoplasmosis is a zoonotic food-borne disease caused by Toxoplasma gondii, a land-derived protozoan parasite that infects a broad range of terrestrial and aquatic hosts. T. gondii may reach coastal waters via contaminated freshwater runoff and its oocysts may enter into the marine food web. Marine invertebrates as mussels being filter feeders are exposed and may concentrate T. gondii oocysts representing a potential source of infection for animals and humans. The present works investigated the prevalence, parasite burden and genotypes of T. gondii in the Mediterranean mussels (Mytilus galloprovincialis) from southern Italy. We sampled a total of 382 individual Mediterranean mussels from May to August 2018 from seven production sites in the Gulf of Naples (Campania region). An additional sample including 27 farmed Mediterranean mussels was obtained in February 2018 from a mollusk depuration plant in Corigliano Calabro (Calabria region). T. gondii DNA was detected in 43 out of 409 (10.5%) Mediterranean mussels from seven out of eight sampling sites. The number of T. gondii copies/g in the digestive gland ranged from 0.14 to 1.18. Fragment analysis of Short Tandem Repeats (STRs) at 5 microsatellite loci was performed from 10 T. gondii PCR positive samples revealing the presence of five distinct genotypes including one corresponding to type I and four atypical genotypes. These findings suggest potential implications of epidemiological importance for human and animal health because both type I and atypical genotypes could be highly pathogenic.

Detection of Hepatitis A Virus and Other Enteric Viruses in Shellfish Collected in the Gulf of Naples, Italy.

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015-2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV; 8.9%), norovirus GI (NoVGI; 10.8%) and GII (NoVGII; 39.7%), rotavirus (RV; 9.0%), astrovirus (AsV; 20.8%), sapovirus (SaV; 18.8%), aichivirus-1 (AiV-1; 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

Draft Genome Sequences of Seven Strains of Shiga Toxin-Producing Escherichia coli O111 with Variation in Their Sensitivity to Novobiocin.

Inclusion of novobiocin as a selective agent for enrichment media and selective agars inhibits the growth of some Shiga toxin-producing Escherichia coli (STEC) strains, particularly non-O157 STEC, which can yield false-negative detection results. Here, we report the draft genomic sequences of seven STEC O111 isolates with different sensitivities to novobiocin.

Draft Whole-Genome Sequences of Seven Listeria monocytogenes Strains with Variations in Virulence and Stress Responses.

Listeria monocytogenes is an important foodborne pathogen that causes listeriosis. Here, we report the draft genome sequences of seven L. monocytogenes strains isolated from food, environmental, and clinical sources. Sequence differences at the genome level may help in understanding why these strains displayed different virulence and stress response characteristics.

Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli.

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c, the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

Evaluation and Comparison of Four Protein Extraction Protocols for Mono- and Two-Dimensional Electrophoresis in Mytilus Galloprovincialis.

In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i) TRIzol reagent; ii) Lysis buffer; iii) phenylmethanesulfonyl fluoride; iv) trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis.