Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Molecular detection of transcriptionally active ovine papillomaviruses in commercial equine semen.

Virological evaluation was performed on equine semen to detect the presence of papillomaviruses (PVs) using droplet digital polymerase chain reaction (ddPCR) as the aim of this study was to investigate whether the sperm from asymptomatic stallions harbors ovine papillomaviruses (OaPVs). Twenty-seven semen samples were analyzed, 18 of which were commercially acquired. The remaining nine samples comprising semen and peripheral blood, were collected from nine stallions with no apparent signs of PV-related diseases during clinical examination at the Didactic Veterinary University Hospital (DVUH) of Naples. OaPV was detected in 26 semen samples. OaPV1 was the most prevalent virus infecting equine semen. OaPV1 infected 21 semen samples (~80.8%) and showed a high number of DNA and RNA copies per microliter. qPCR was used to detect OaPV1 DNA in the 18 semen samples. ddPCR was used to detect and quantify the expression of OaPV2, OaPV3, and OaPV4. qPCR failed to detect DNA for these genotypes. Additionally, ddPCR was used to detect the transcriptionally active OaPV1 in six blood and semen samples from the same stallion. ddPCR failed to detect any nucleic acids in OaPVs in peripheral blood samples from the three stallions. In one semen sample, ddPCR detected OaPV1 DNA but failed to detect any nucleic acid in the remaining two semen samples, and peripheral blood from the same animals of the remaining 18 semen samples was not available, OaPV1 and OaPV4 were responsible for nine and five single infections, respectively. No single infections with either OaPV3 or OaPV4 were seen.

A pseudorabies outbreak in hunting dogs in Campania region (Italy): a case presentation and epidemiological survey.

BACKGROUND: Pseudorabies is an infection of domestic and wild pigs that has occasionally been reported in dogs with fatal encephalitis. Hunting dogs are predisposed to pseudorabies exposure due to incorrect practices (administration of raw infected meat) or close contact with infected wild boars. This study described an outbreak of pseudorabies in two hunting dogs in the Campania region, southern Italy. CASE PRESENTATION: Two hunting dogs were hospitalized after a hunting trip, with fever, itching, and self-inflicted lesions. Laboratory tests showed mild anemia and marked leukocytosis. Despite conservative therapy, both animals died 48 h after the presentation of symptoms. One of the carcasses was sent to the Department of Veterinary Medicine and Animal Production in Naples to confirm the suspicion of pseudorabies. DNA was extracted from different matrices and used as a template for real-time PCR to detect PRV. Several samples (brain, cerebellum, brainstem, lung, and liver) tested positive. Subsequent sequence analyses of glycoprotein E from DNA extracted from the brain stem revealed a sequence similarity to those described in previous cases of pseudorabies in dogs in Italy, France and Belgium. One month after the outbreak, blood samples were collected from 42 dogs belonging to the same hunting team and from 245 dogs (cohort population) living in the Campania region. All samples were tested with two commercial ELISAs to detect seroconversion against glycoproteins B and E. A seroprevalence of 19% was observed in the hunting team affected by the outbreak, while only 0.8% was observed in the regional dog population. CONCLUSIONS: The data reported in this study demonstrate potential exposure to PRV by dead-end hosts, particularly hunting dogs. The sequencing results indicated the homogeneity of PRV strains circulating in the different Italian regions.

Antibiotic Resistance of Bacteria Isolated from Clinical Samples and Organs of Rescued Loggerhead Sea Turtles (Caretta caretta) in Southern Italy.

Antimicrobial resistance affects all environments, endangering the health of numerous species, including wildlife. Increasing anthropic pressure promotes the acquisition and dissemination of antibiotic resistance by wild animals. Sea turtles, being particularly exposed, are considered sentinels and carriers of potential zoonotic pathogens and resistant strains. Therefore, this study examined the antibiotic resistance profiles of bacteria isolated from loggerhead sea turtles hospitalised in a rescue centre of Southern Italy over a 9-year period. Resistance to ceftazidime, doxycycline, enrofloxacin, flumequine, gentamicin, oxytetracycline and sulfamethoxazole-trimethoprim was evaluated for 138 strains isolated from the clinical samples or organs of 60 animals. Gram-negative families were the most isolated: Vibrionaceae were predominant, followed by Shewanellaceae, Pseudomonadaceae, Enterobacteriaceae and Morganellaceae. These last three families exhibited the highest proportion of resistance and multidrug-resistant strains. Among the three Gram-positive families isolated, Enterococcaceae were the most represented and resistant. The opportunistic behaviour of all the isolated species is particularly concerning for diseased sea turtles, especially considering their resistance to commonly utilised antibiotics. Actually, the multiple antibiotic resistance was higher when the sea turtles were previously treated. Taken together, these findings highlight the need to improve antimicrobial stewardship and monitor antibiotic resistance in wildlife, to preserve the health of endangered species, along with public and environmental health.

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.

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.

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.

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.

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.