Identification of Aichivirus in a Pet Rat (Rattus norvegicus) in Italy.

We investigated the occurrence of eight potential zoonotic viruses in 91 exotic companion mammals from pet shops in southern Italy via real-time PCR and end-point PCR. The animals were screened for aichivirus, sapovirus, astrovirus, hepatitis A, noroviruses (GI and GII), rotavirus, circovirus, and SARS-CoV-2. Among the nine species of exotic pets studied, only one rat tested positive for aichivirus. The high sequence similarity to a murine kobuvirus-1 strain previously identified in China suggests that the virus may have been introduced into Italy through the importation of animals from Asia. Since exotic companion mammals live in close contact with humans, continuous sanitary monitoring is crucial to prevent the spread of new pathogens among domestic animals and humans. Further investigations on detecting and typing zoonotic viruses are needed to identify emerging and re-emerging viruses to safeguard public health.

Lightning deaths in three outdoor dogs: A case study.

This case report presents findings in three German Shepherd placed outdoor, dead after a night of thunderstorm.

Ten different viral agents infecting and co-infecting children with acute gastroenteritis in Southern Italy: Role of known pathogens and emerging viruses during and after COVID-19 pandemic.

Acute gastroenteritis (AGE) represents a world public health relevant problem especially in children. Enteric viruses are the pathogens mainly involved in the episodes of AGE, causing about 70.00% of the cases. Apart from well-known rotavirus (RVA), adenovirus (AdV) and norovirus (NoV), there are various emerging viral pathogens potentially associated with AGE episodes. In this study, the presence of ten different enteric viruses was investigated in 152 fecal samples collected from children hospitalized for gastroenteritis. Real time PCR results showed that 49.3% of them were positive for viral detection with the following prevalence: norovirus GII 19.7%, AdV 15.8%, RVA 10.5%, human parechovirus (HPeV) 5.3%, enterovirus (EV) 3.3%, sapovirus (SaV) 2.6%. Salivirus (SalV), norovirus GI and astrovirus (AstV) 1.3% each, aichivirus (AiV) found in only one patient. In 38.2% of feces only one virus was detected, while co-infections were identified in 11.8% of the cases. Among young patients, 105 were ≤5 years old and 56.0% tested positive for viral detection, while 47 were >5 years old with 40.0% of them infected. Results obtained confirm a complex plethora of viruses potentially implicated in gastroenteritis in children, with some of them previously known for other etiologies but detectable in fecal samples. Subsequent studies should investigate the role of these viruses in causing gastroenteritis and explore the possibility that other symptoms may be ascribed to multiple infections.

A Tiny Viral Protein, SARS-CoV-2-ORF7b: Functional Molecular Mechanisms.

This study presents the interaction with the human host metabolism of SARS-CoV-2 ORF7b protein (43 aa), using a protein-protein interaction network analysis. After pruning, we selected from BioGRID the 51 most significant proteins among 2753 proven interactions and 1708 interactors specific to ORF7b. We used these proteins as functional seeds, and we obtained a significant network of 551 nodes via STRING. We performed topological analysis and calculated topological distributions by Cytoscape. By following a hub-and-spoke network architectural model, we were able to identify seven proteins that ranked high as hubs and an additional seven as bottlenecks. Through this interaction model, we identified significant GO-processes (5057 terms in 15 categories) induced in human metabolism by ORF7b. We discovered high statistical significance processes of dysregulated molecular cell mechanisms caused by acting ORF7b. We detected disease-related human proteins and their involvement in metabolic roles, how they relate in a distorted way to signaling and/or functional systems, in particular intra- and inter-cellular signaling systems, and the molecular mechanisms that supervise programmed cell death, with mechanisms similar to that of cancer metastasis diffusion. A cluster analysis showed 10 compact and significant functional clusters, where two of them overlap in a Giant Connected Component core of 206 total nodes. These two clusters contain most of the high-rank nodes. ORF7b acts through these two clusters, inducing most of the metabolic dysregulation. We conducted a co-regulation and transcriptional analysis by hub and bottleneck proteins. This analysis allowed us to define the transcription factors and miRNAs that control the high-ranking proteins and the dysregulated processes within the limits of the poor knowledge that these sectors still impose.

Targeting ATP2B1 impairs PI3K/Akt/FOXO signaling and reduces SARS-COV-2 infection and replication.

ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of intracellular Ca2+ content have been suggested to impair SARS-CoV-2 replication. Here, we demonstrate that a nontoxic caloxin-derivative compound (PI-7) reduces intracellular Ca2+ levels and impairs SARS-CoV-2 infection. Furthermore, a rare homozygous intronic variant of ATP2B1 is shown to be associated with the severity of COVID-19. The mechanism of action during SARS-CoV-2 infection involves the PI3K/Akt signaling pathway activation, inactivation of FOXO3 transcription factor function, and subsequent transcriptional inhibition of the membrane and reticulum Ca2+ pumps ATP2B1 and ATP2A1, respectively. The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 lacks toxicity in vitro, its prophylactic use as a therapeutic agent against COVID-19 is envisioned here.

An Update on Myocarditis in Forensic Pathology.

In forensic medicine, myocarditis is a complicated topic in the context of sudden death and medical malpractice. A good knowledge of the etiopathology, histopathology, and available literature are both indispensable and essential for the correct management and evaluation of the causal link. Some agents, which are rarely lethal for humans, are not necessarily related to death from myocarditis, even if an infection in other organs such as the gastrointestinal tract is documented. The diagnosis of the causes of death is often difficult and confusing. In some cases, the hypothetical diagnosis of myocarditis as the cause of death is formulated by deduction, causing error and misleading the correct temporal evaluation of pathological events. We reviewed the literature realizing that histomorphological data are scarce and often poorly documented. Only after COVID-19 have the histomorphological aspects of myocarditis been better documented. This is due to poor autopsy practice and poor accuracy in identifying the specific histotype of myocarditis with identification of the responsible agent. We believe that four points are essential for a better understanding and complete diagnosis of the disease: (1) clinical classification of myocarditis; (2) etiological classification of myocarditis; (3) pathophysiology of viral and bacterial infections with host response; and (4) histopathological diagnosis with precise identification of the histotype and pathogen. In the review we provide histological images from authoritative scientific references with the aim of providing useful information and food for thought to readers.

First description of Rhodococcus equi infection in common bottlenose dolphin (Tursiops truncatus).

Rhodococcus equi is a terrestrial bacterium and a common pathogen in foals (Equus caballus), in which causes pneumonia. This report describes for the first time the infection caused by R. equi in a common bottlenose dolphin (Tursiops truncatus) stranded in the Calabrian coast, Italy. The post mortem examination of the animal revealed lesions in lung and colon. The animal was also positive to dolphin morbillivirus. The histological study showed lesions attributable to R. equi infection, such as pyogranulomatous bacterial pneumonia and chronic granulomatous colitis. Whole genome sequencing of the isolated strain confirmed its identification as R. equi.

Morphometric, histopathological and molecular findings of Macracanthorhynchus hirudinaceus infection in wild boar (Sus scrofa) from continental Italy.

Although Macracanthorhynchus hirudinaceus is a neglected acanthocephalan of suids occasionally responsible for severe infections in humans, the spread of wild boar (Sus scrofa) populations in Europe could promote the circulation. Herein, we report the first morphometric, histological and molecular characterization of a severe M. hirudinaceus infection in a boar from continental Italy. The boar's intestine displayed granulomatous enteritis due to 24 helminths (14 females, 10 males), identified as adults of M. hirudinaceus by a combined morphometric/molecular approach. The phylogenetic analysis of the cox1 gene revealed a close relationship of the M. hirudinaceus sequence type found herein with those from Hungary and insular Italy. The high haplotype diversity and low nucleotide diversity of M. hirudinaceus specimens would suggest its rapid demographic expansion in the Mediterranean basin. More research is needed to assess the presence of M. hirudinaceus in susceptible beetle species and the role of boars in the epidemiology of infection.

The Label-Free Detection and Identification of SARS-CoV-2 Using Surface-Enhanced Raman Spectroscopy and Principal Component Analysis.

The World Health Organization (WHO) declared in a May 2023 announcement that the COVID-19 illness is no longer categorized as a Public Health Emergency of International Concern (PHEIC); nevertheless, it is still considered an actual threat to world health, social welfare and economic stability. Consequently, the development of a convenient, reliable and affordable approach for detecting and identifying SARS-CoV-2 and its emerging new variants is crucial. The fingerprint and signal amplification characteristics of surface-enhanced Raman spectroscopy (SERS) could serve as an assay scheme for SARS-CoV-2. Here, we report a machine learning-based label-free SERS technique for the rapid and accurate detection and identification of SARS-CoV-2. The SERS spectra collected from samples of four types of coronaviruses on gold nanoparticles film, fabricated using a Langmuir-Blodgett self-assembly, can provide more spectroscopic signatures of the viruses and exhibit low limits of detection (<100 TCID50/mL or even <10 TCID50/mL). Furthermore, the key Raman bands of the SERS spectra were systematically captured by principal component analysis (PCA), which effectively distinguished SARS-CoV-2 and its variant from other coronaviruses. These results demonstrate that the combined use of SERS technology and PCA analysis has great potential for the rapid analysis and discrimination of multiple viruses and even newly emerging viruses without the need for a virus-specific probe.

Reoccurrence of West Nile virus lineage 1 after 2-year decline: first equine outbreak in Campania region.

West Nile virus (WNV) is the most widespread arbovirus worldwide, responsible for severe neurological symptoms in humans as well as in horses and birds. The main reservoir and amplifier of the virus are birds, and migratory birds seem to have a key role in the introduction and spread of WNV during their migratory routes. WNV lineage 1 (L1) has been missing in Italy for almost 10 years, only to reappear in 2020 in two dead raptor birds in southern Italy. The present study reports the first equine outbreak in the Campania region. A 7-year-old horse died because of worsening neurological signs and underwent necropsy and biomolecular analyses. WNV-L1 was detected by real-time RT-PCR in the heart, brain, gut, liver, and spleen. Next Generation Sequence and phylogenetic analysis revealed that the strain responsible for the outbreak showed a nucleotide identity of over 98% with the strain found in Accipiter gentilis 2 years earlier in the same area, belonging to the WNV-L1 Western-Mediterranean sub-cluster. These results underline that WNV-L1, after reintroduction in 2020, has probably silently circulated during a 2-year eclipse, with no positive sample revealed by both serological and biomolecular examinations in horses, birds, and mosquitoes. The climate changes that have occurred in the last decades are evolving the epidemiology of WNV, with introductions or re-introductions of the virus in areas that were previously considered low risk. Thereby, the virus may easily amplify and establish itself to reappear with sporadic evident cases in susceptible hosts after several months or even years.

Prevalence of feline leukemia virus and feline immunodeficiency virus in cats from southern Italy: a 10-year cross-sectional study.

Introduction: Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are well-known retroviruses causing important infections in domestic cats worldwide. The goal of this study was to determine the prevalence of FeLV and FIV infections in cat living indoor and outdoor in southern Italy. Methods: The survey was conducted on 1322 stray and owned cats from the regions of Campania, Basilicata, and Calabria. It was carried out over a 10-year period to obtain a more realistic picture of the prevalence of these retroviral diseases in the country. FIV and FeLV status was determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (SNAP Combo Plus FeLV/FIV, IDEXX). Risk factors were analysed by logistic regression. Results and Discussion: The results showed that 101/1322 (7.64%) cats were positive for FeLV antigen and 110/1322 (8.32%) cats were positive for FIV antibody. Twenty-six of the 1322 cats (1.97%) were positive for both FIV and FeLV infection. Our results are similar to those published in recent studies in Europe. A statistically significant association (p < 0.05) was found between year, province, region, lifestyle and risk of FeLV infection. FIV positivity was instead statistically associated only with year and lifestyle.

Do Organochlorine Contaminants Modulate the Parasitic Infection Degree in Mediterranean Trout (Salmo trutta)?

We investigated the occurrence of organochlorine pollutants (OCs) in the muscle of brown trout and evaluated their potential modulation of parasite infection. The toxicological risk for consumer health was assessed, too. Trout were collected from the Sila National Park (Calabria region, South of Italy). The highest concentrations emerged for the sum of the 6 non-dioxin-like (ndl) indicator polychlorinated biphenyls (Σ6ndl-PCBs), followed by the 1,1,1-trichloro-2,2-di(4-chlorophenyl)-ethane (DDT), dioxin-like PCBs, hexachlorobenzene (HCB), and dieldrin. Measured on lipid weight (LW), the mean value of Σ6ndl-PCBs amounted to 201.9 ng g-1, that of ΣDDTs (the sum of DDT-related compounds) to 100.2 ng g-1, with the major contribution of the DDT-metabolite p,p'-DDE which was detected in all sample units (97.6 ng g-1 on average). Among dioxin-like congeners, PCB 118 showed the highest mean concentration (21.96 ng g-1 LW) and was detected in all sample units. Regression analysis of intestinal parasites on OC concentration was performed, controlling for two potential confounding factors, namely sex and sexual stage. The results evidenced the existence of interactions between the dual stressors in the host-parasite system in the wild. A negative and statistically significant correlation was estimated, suggesting that OCs may decrease parasite infection degree. Regarding the toxicological risk evaluation, OC concentrations were consistently below the current European Maximum Residue Limits.

Metabolic Profiling in Tuberous Roots of Ranunculus asiaticus L. as Influenced by Vernalization Procedure.

Ranunculus asiaticus L. is an ornamental geophyte. In commercial practice, it is mainly propagated by rehydrated tuberous roots. Vernalization before planting is a common practice to overcome the natural dormancy of tuberous roots; however, little is known about the mechanisms underlying the plant's response to low temperatures. We investigated the influence of three preparation procedures of tuberous roots, only rehydration (control, C), and rehydration plus vernalization at 3.5 °C for 2 weeks (V2) and for 4 weeks (V4), on plant growth, leaf photosynthesis, flowering, and metabolism in plants of two hybrids, MBO (early flowering, pale orange flower) and MDR (medium earliness, bright orange flower), grown in pots in an unheated greenhouse. We reported the responses observed in the aerial part in a previous article in this journal. In this paper, we show changes in the underground organs in carbohydrate, amino acids, polyphenols, and protein levels throughout the growing cycle in the different plant stages: pre-planting, vegetative growth, and flowering. The metabolic profile revealed that the two hybrids had different responses to the root preparation procedure. In particular, MBO synthesized GABA and alanine after 2 weeks and sucrose after 4 weeks of vernalization. In contrast, MDR was more sensitive to vernalization; in fact, a higher synthesis of polyphenols was observed. However, both hybrids synthesized metabolites that could withstand exposure to low temperatures.

First molecular detection of Babesia vulpes and Babesia capreoli in wild boars from southern Italy.

Introduction: Following the increase of wild boar (Sus scrofa) populations in Europe, a potential risk of emerging infections by vector-borne pathogens may occur. Despite this, the circulation of piroplasmid species in these ungulates is still a neglected topic, particularly in the Mediterranean basin. Therefore, this study aimed to investigate the presence of Babesia/Theileria spp. in wild boars from southern Italy to assess the epidemiological role of these ungulates in the circulation of piroplasmids. Methods: By using a citizen science approach among hunters and veterinarians, wild boar spleen samples were collected in the Campania region (southern Italy) between 2016 and 2022. A combined semi-nested PCR/sequencing analysis targeting the V4 hyper-variable region of 18S rRNA was run to detect Babesia/Theileria spp. DNA. Results: Out of 243 boars, 15 (i.e., 6.2, 95% CI: 3.4-9.9) tested positive to Babesia/Theileria spp., Babesia vulpes (n = 13, 5.3, 95% CI: 3.1-8.9) the most prevalent, followed by Babesia capreoli (n = 2, 0.8, 95% CI: 0.2-2.9). Three different B. vulpes sequence types were identified (i.e., ST1, ST2, ST3), with the most representative as ST1 (60%), and a single B. capreoli sequence type. No statistically significant difference (p > 0.05) were found between the presence of the pathogens and boar age, sex, province and sample collection year. Discussion: Data demonstrate for the first time the occurrence of B. vulpes and B. capreoli in wild boars, which may play a role in the biological cycle of piroplasmids. We emphasize the importance of monitoring these ungulates to prevent potential foci of infection. The engagement of hunters in epidemiological scientifically based surveys can constitute a technically sound control strategy of piroplasmids in a One Health perspective.

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy.

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 raw wastewater samples collected from six wastewater treatment plants (WWTPs) and 57 mollusk samples obtained from eight harvesting sites in Campania, Italy. The monitoring period spanned from October 2021 to April 2022, and the results were compared and correlated with the epidemiological situation. In sewage, the ORF1b region of SARS-CoV-2 was detected using RT-qPCR, while in mollusks, three targets-RdRp, ORF1b, and E-were identified via RT-dPCR. Results showed a 92.3% rate of positive wastewater samples with increased genomic copies (g.c.)/(day*inhabitant) in December-January and March-April 2022. In the entire observation period, 54.4% of mollusks tested positive for at least one SARS-CoV-2 target, and the rate of positive samples showed a trend similar to that of the wastewater samples. The lower SARS-CoV-2 positivity rate in bivalve mollusks compared to sewages is a direct consequence of the seawater dilution effect. Our data confirm that both sample types can be used as sentinels to detect SARS-CoV-2 in the environment and suggest their potential use in obtaining complementary information on SARS-CoV-2.

A comparative study on the persistence and viability of SARS-CoV-2 wild-type and omicron variant on artificially contaminated surfaces: the role of fomites.

Indirect transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been investigated but it is still not completely understood. The present study aimed to compare the persistence and viability of the lineage B.1 and omicron BA.1 subvariant in five daily-use materials to evaluate the role of fomites as a possible source of infection. Artificial contamination was performed in the first set of materials, ethylene vinyl acetate (EVA), cardboard, polystyrene, aluminium, and plastic. Further surfaces using BA.1 (glass, plexiglass, cotton, polyester, and tetrapak) were conducted. The persistence, viability of Vero E6 cell cultures and the residual infectivity of the two lineages were evaluated over 5 days. The results showed different stabilities between the tested matrices. In cotton and polyester, the RNA was undetectable in 24 and 48h post-contamination (p.c.), respectively, and the virus was not viable within 30 min, while in the other surfaces, both lineages, RNA was detectable until 120h p.c. A rapid decay of the viral load was revealed on cardboard, mostly for the omicron variant. Furthermore, on all the materials, longer stability of BA.1 was demonstrated, but showing a less intense CPE than the wild-type. EVA was the material that was able to better sustain virus stability as the virus developed CPE up to 72h p.c. In conclusion, the potential spread of SARS-CoV-2 through fomites is conceivable, albeit it is difficult to establish the real capacity to infect people. Nevertheless, thise information is fundamental to adopting the appropriate measures to mitigate the spread of SARS-CoV-2 and its variants.

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.

Salt-Induced Stress Impacts the Phytochemical Composition and Aromatic Profile of Three Types of Basil in a Genotype-Dependent Mode.

Basil (Ocimum basilicum L.) is among the most widely used aromatic plants of Lamiaceae, often grown in areas where salinity is an adverse factor. Most studies on the effect of salinity on basil focused on the influence of salt stress on productive traits, while few reported on how it affects the phytochemical composition and the aroma profile. Three basil cultivars (Dark Opal, Italiano Classico, and Purple Ruffles) were grown hydroponically for 34 days with two nutrient solutions that differed in NaCl concentration [no NaCl (Control) and 60 mM NaCl]. Yield, secondary metabolite concentration (ß-carotene and lutein), antioxidant activity [1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reduction antioxidant power (FRAP)], and aroma profile based on composition of volatile organic compounds (VOCs) were appraised in response to salinity applications. Salt stress significantly reduced fresh yield in Italiano Classico and Dark Opal by 43.34 and 31.69%, respectively, while no effect was observed in Purple Ruffles. Furthermore, the salt-stress treatment increased ß-carotene and lutein concentrations, DPPH, and FRAP activities, and the total nitrogen content of the latter cultivar. CG-MS analysis revealed significant differences in VOCs composition of the basil cultivars, with Italiano Classico and Dark Opal characterized by the predominance of linalool (average 37.52%), which, however, was negatively affected by salinity. In Purple Ruffles, the predominant VOC compound, estragole (79.50%), was not affected by the deleterious effects of NaCl-induced stress.

Evaluation of an Immunization Protocol Using Bovine Alphaherpesvirus 1 gE-Deleted Marker Vaccines against Bubaline Alphaherpesvirus 1 in Water Buffaloes.

European regulations on the control of infectious diseases provide measures to control Bovine alphaherpesvirus 1 (BoHV-1) infection in both cattle and buffalo. Owing to the reported serological cross-reactivity between BoHV-1 and Bubaline alphaherpesvirus 1 (BuHV-1), we hypothesized a new immunization protocol using BoHV-1 gE-deleted marker vaccines could protect water buffalo against BuHV-1. Five water buffaloes devoid of BoHV-1/BuHV-1-neutralizing antibodies were immunized with two commercial BoHV-1 gE-deleted marker vaccines at 0, 30, 210, and 240 post-vaccination days (PVDs). Five additional water buffaloes were used as controls. At 270 PVD (0 post-challenge days (PCDs), all animals were challenged intranasally with wild-type (wt) BuHV-1. The vaccinated animals produced humoral immunity (HI) as early as PVD 30 whereas, in control animals, antibodies were detected on PCD 10. After challenge infection, HI significantly increased in vaccinated animals compared to that in controls. Real-time PCR for gB revealed viral shedding in vaccinated animals from PCDs 2 to 10. In contrast, positive results were observed from PCDs 2 to 15 in the unvaccinated control group. Although the findings indicated the possible protection capabilities of the tested protocol, these findings did not support its protective roles in water buffaloes against wt-BuHV-1.