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Abstract Glioblastoma (GBM) is the most common malignant primary brain cancer that, despite recent advances in the understanding of its pathogenesis, remains incurable. GBM contains a subpopulation of cells with stem cell-like properties called cancer stem cells (CSCs). Several studies have demonstrated that CSCs are resistant to conventional chemotherapy and radiation thus representing important targets for novel anti-cancer therapies. Proton sensing receptors expressed by CSCs could represent important factors involved in the adaptation of tumours to the extracellular environment. Accordingly, the expression of acid-sensing ion channels (ASICs), proton-gated sodium channels mainly expressed in the neurons of peripheral (PNS) and central nervous system (CNS), has been demonstrated in several tumours and linked to an increase in cell migration and proliferation. In this paper we report that the ASIC3 isoform, usually absent in the CNS and present in the PNS, is enriched in human GBM CSCs while poorly expressed in the healthy human brain. We propose here a novel therapeutic strategy based on the pharmacological activation of ASIC3, which induces a significant GBM CSCs damage while being non-toxic for neurons. This approach might offer a promising and appealing new translational pathway for the treatment of glioblastoma.

Animals, including wildlife, are part of One-Health concept since many infectious diseases can affect both humans and animals. In this study, 126 red foxes (Vulpes vulpes) from Northern Italy in 2022–2023 were tested by molecular assays for Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), Circovirus canine (CanineCV), Canine distemper virus (CDV), and Leptospira spp. A total of 39 of 126 (30.9%) red foxes were infected with at least one pathogen and five of these were coinfected: 20/126 (15.9%) red foxes tested positive for PPVC-1, 3/126 (2.4%) for CAdV, 20/126 (15.9%) for CanineCV, and 2/126 (1.6%) for Leptospira spp. DNA. No foxes tested positive for CDV RNA. The pathogens identified were genetically analysed. New findings were reported such as a fox with multiple feline panleukopenia virus (FPV) and canine parvovirus type 2b (CPV-2b) infection associated with quasispecies dynamics, typical genetic characteristics of the identified CanineCV, and the first detection in red foxes of Leptospira ST198 related to L. interrogans serogroup Australis. Further studies are necessary to investigate the transmission between domestic animals and wildlife and to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments.

Feline immunodeficiency virus (FIV) is responsible for immunodeficiency syndrome in cats. Several viral subtypes have been identified, each with a variable geographical distribution. To date, the subtype B is known to be the genotype spread in Italy. In this study, the genetic diversity of FIV in northern Italy was assessed by detecting proviral DNA in the blood samples of 50 cats determined to be positive through an anti-FIV antibodies test. These cats were tested using six different PCR assays, and the identified viruses were sequenced and analyzed. Forty-eight cats were confirmed positive, and several FIV subtypes were characterized. As expected, the subtype B was the most commonly observed, and the subtype A was reported for the first time in Italy. Moreover, a new taxon possibly representing an additional FIV subtype was detected, and one virus belonging to subtype B potentially had a recombinant origin. The genetic variability between the FIV viruses that emerged in this study may lead to the potential diagnostic failure of single molecular tests. Therefore, a new diagnostic strategy, which adopts different molecular tests and sequencing, is recommended to monitor the evolution and spread of FIV.

Genomic analyses of spike glycoprotein genes of European bat SARS-related coronaviruses suggest that furin cleavage sites can be acquired in the bat reservoir via conserved molecular mechanisms, supporting a natural origin of SARS-CoV-2.

Abstract Background Canine parvovirus (CPV) is one of the most important pathogens of dogs. Despite vaccination, CPV infections are still ubiquitous in dogs, and the three antigenic variants 2a, 2b and 2c are variously distributed in the canine population worldwide. To date, no information is available on CPV variants circulating in some European countries. The aim of this study was to genetically characterise the CPV detected in ten dogs with clinical signs of acute gastroenteritis in Romania. The presence of Carnivore protoparvovirus 1 DNA was investigated in faecal samples using an end-point PCR targeting the complete VP2 gene and positive amplicons were sequenced and analysed. Results All ten dogs with acute gastroenteritis tested positive to Carnivore protoparvovirus 1 DNA in faecal samples. The identified viruses belonged to CPV-2c type, showed identical sequences of the VP2 gene and were characterised by distinctive amino acid residues in the deduced VP2 protein: 5-glicine (5Gly), 267-tirosine (267Tyr), 324-isoleucine (324Ile) and 370-arginine (370Arg). These distinctive amino acid residues have already been reported in CPV-2c widespread in Asia and occasionally detected in Italy and Nigeria. Conclusions Since CPV-2c with VP2 amino acid residues 5Gly, 267Tyr, 324Ile and 370Arg were never reported before 2013, it can be assumed that this virus is progressively expanding its spread in the world dog population. This study adds new data about the presence of this new virus in Europe and underline worrying questions about its potential impact on the health of the canine population.

In this report the concomitant infection with canine parvovirus type 2 (CPV-2), Hepatozoon canis and Ehrlichia canis in two puppy dogs from Southern Italy is described. Dogs were referred to a veterinary university hospital for the acute onset of lethargy and gastrointestinal signs. A complete clinical and clinicopathological evaluation was carried out and the multiple infection was confirmed by microscopic detection of inclusion bodies in peripheral blood smear, rapid immunoenzymatic tests, indirect fluorescent antibody tests, and molecular assays. Sequence analysis revealed that the CPV-2 identified belonged to the 2c variant and had amino acid residues in the predicted VP2 protein typical of “Asian-like” strains widespread in Asia and occasionally reported in Romania, Nigeria and Italy, particularly in the region of Sicily. Numerous monocytes were infected by both H. canis gamonts and E. canis morulae, suggesting that this co-infection is not accidental and that E. canis preferably infects those cells parasitized by H. canis. The clinical presentation of these animals was severe but supportive cares associated with early etiological therapy allowed a good prognosis. Movement of puppies from geographic areas where vector-borne pathogens are endemic must be carefully evaluated and core vaccinations and ectoparasite prevention treatments must be rigorously adopted.

Abstract Background Urine neutrophil gelatinase‐associated lipocalin (NGAL) is a promising biomarker of acute kidney injury (AKI) in dogs. Objectives To evaluate the utility of urinary NGAL for characterizing AKI according to volume responsiveness, presence of inflammation and sepsis, and prognosis. Animals Dogs with AKI (n = 76) and healthy controls (n = 10). Methods Prospective study. Clinical and clinicopathologic data including absolute urine NGAL concentration (uNGAL) and NGAL normalized to urine creatinine concentration (uNGALC) were measured upon admission. Dogs were graded according to International Renal Interest Society (IRIS) AKI guidelines and compared based on AKI features: volume‐responsive (VR‐) AKI vs. intrinsic (I‐) AKI based on IRIS criteria; VR‐AKI and I‐AKI based on urine chemistry; inflammatory versus noninflammatory; septic versus nonseptic; and survivors versus nonsurvivors. Nonparametric statistics were calculated, and significance set at P

Dual functionalized liposomes were developed to cross the blood–brain barrier (BBB) and to release their cargo in a pathological matrix metalloproteinase (MMP)-rich microenvironment. Liposomes were surface-functionalized with a modified peptide deriving from the receptor-binding domain of apolipoprotein E (mApoE), known to promote cargo delivery to the brain across the BBB in vitro and in vivo; and with an MMP-sensitive moiety for an MMP-triggered drug release. Different MMP-sensitive peptides were functionalized at both ends with hydrophobic stearate tails to yield MMP-sensitive lipopeptides (MSLPs), which were assembled into mApoE liposomes. The resulting bi-functional liposomes (i) displayed a < 180 nm diameter with a negative ζ-potential; (ii) were able to cross an in vitro BBB model with an endothelial permeability of 3 ± 1 × 10−5 cm/min; (iii) when exposed to functional MMP2 or 9, efficiently released an encapsulated fluorescein dye; (iv) showed high biocompatibility when tested in neuronal cultures; and (v) when loaded with glibenclamide, a drug candidate with poor aqueous solubility, reduced the release of proinflammatory cytokines from activated microglial cells.

Canine adenovirus type 1 (CAdV-1) is the causative agent of a systemic and potentially fatal viral disease of domestic and wild canids. In Italy, CAdV-1 infection has also been occasionally described in dogs, but information on the epidemiology and its genomic features is still limited. A study was conducted on 291 dogs suspected of infectious gastrointestinal disease. Samples collected from dogs in southern Italy between 2017 and 2020 were analyzed. Virological and histopathological assays were carried out. The presence of CAdVs and other canine viral enteropathogens was investigated, and sequence and phylogenetic analyses were performed. CAdV-1 was detected in six (2.1%) dead stray dogs alone or in mixed infections with other viruses. Gross lesions and histopathological findings referred to CAdV infection were observed, also involving the central nervous system tissues. All inoculated samples were successfully isolated. Sequence analysis evidenced divergences with the circulating strains previously described in Italy and a closer relation with older CAdV-1 strains collected from other countries, suggesting a genetic heterogeneity of CAdV-1 in Italy. The evidence of the circulation of CAdV-1 and its genomic features allows us to have more in-depth knowledge of the epidemiology and evolution of the CAdV-1 genomic variants.

Abstract Background Canine parvovirus type 2 (CPV-2) is the most important enteric virus infecting canids. It is a rapidly evolving virus; after its emergence in the 1970s, new antigenic variants (called CPV-2a, 2b and 2c) emerged and replaced the original antigenic type. The three antigenic variants are globally distributed with different frequencies and levels of genetic variability. This study focused on VP2 gene sequence analysis and the phylodynamics of CPV-2 which were detected in 123 dogs showing clinical signs of gastroenteritis collected in Italy from 1994 to 2017. Results For the most part, the sick dogs were young, and a third of them (32.5%) had been vaccinated. No statistical association was found between the CPV-2 antigenic variants, and sex, age, breed and vaccination status. Sequence analysis showed that all three antigenic types circulated in Italy; the CPV-2a type was the prominent genotype, followed by CPV-2c and CPV-2b, with notable differences regarding regional bases and significant fluctuations over time. Nucleotide sequence data showed high genetic heterogeneity with 67 nucleotide sequence types (ntSTs) identified, corresponding to 21 amino acid sequence types (aaSTs). The aaSTs and ntSTs obtained were distributed differently among the three CPV-2 antigenic variants: CPV-2a grouped 12/21 (57.1%) aaSTs and 41/67 (61.2%) ntSTs; CPV-2b grouped 5/21 (23.8%) aaSTs and 6/67 (8.9%) ntSTs, and CPV-2c grouped 4/21 (19.1%) aaSTs and 20/67 (29.9%) ntSTs. Canine parvovirus 2a was characterised by the highest genetic variability while CPV-2c was characterised by notable stability with a predominant amino acid profile during the entire sampling time. Canine parvovirus 2b re-emerged in recent years, showing a new and distinctive amino acid profile of the VP2 protein. Conclusions The findings of the present study provided new insights regarding the phylodynamics and evolution of CPV-2 in Italy, pointing out notable differences at the local level in the distribution of the CPV-2 variants and the selection of genetic subtypes. The evolution of CPV-2 has raised questions regarding the efficacy of vaccination; therefore, continuous monitoring regarding the evolution and spread of new CPV-2 variants should be a key aim of ongoing research.

A growing number of studies suggest that the lower urinary tract of humans and dogs can harbor a urinary microbiota. Nevertheless, a certain concern has developed that the microbiota reported could be due to unaccounted contamination, especially in low-biomass samples. The aim of this study was to investigate the bacterial community which populates the urine of healthy cats using two approaches: a culture-dependent approach which consisted of the expanded quantitative urine culture (EQUC) techniques capable of identifying live bacteria not growing in standard urine cultures, and a culture-independent approach which consisted of 16S ribosomal RNA next generation sequencing (16S rRNA NGS) capable of identifying bacterial DNA and exploring microbial diversity with high resolution. To avoid confounding factors of possible bacterial contamination, the urine was sampled using ultrasound-guided cystocentesis, and several sample controls and negative controls were analyzed. The urine sampled from the 10 cats included in the study showed no bacterial growth in the EQUC procedure. Although several reads were successfully originated using 16S rRNA NGS, a comparable pattern was observed between urine samples and the negative control, and no taxa were statistically accepted as non-contaminant. Taken together, the results obtained allowed stating that no viable bacteria were present in the urine of healthy cats without lower urinary tract disease and urinary tract infections, and that the bacterial DNA detected was of contaminant origin.

Abstract A 29 nucleotide deletion in open reading frame 8 (ORF8) is the most obvious genetic change in severe acute respiratory syndrome coronavirus (SARS-CoV) during its emergence in humans. In spite of intense study, it remains unclear whether the deletion actually reflects adaptation to humans. Here we engineered full, partially deleted (−29 nt), and fully deleted ORF8 into a SARS-CoV infectious cDNA clone, strain Frankfurt-1. Replication of the resulting viruses was compared in primate cell cultures as well as Rhinolophus bat cells made permissive for SARS-CoV replication by lentiviral transduction of the human angiotensin-converting enzyme 2 receptor. Cells from cotton rat, goat, and sheep provided control scenarios that represent host systems in which SARS-CoV is neither endemic nor epidemic. Independent of the cell system, the truncation of ORF8 (29 nt deletion) decreased replication up to 23-fold. The effect was independent of the type I interferon response. The 29 nt deletion in SARS-CoV is a deleterious mutation acquired along the initial human-to-human transmission chain. The resulting loss of fitness may be due to a founder effect, which has rarely been documented in processes of viral emergence. These results have important implications for the retrospective assessment of the threat posed by SARS.

Abstract Background Cats are susceptible to feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants 2a, 2b and 2c. Detection of FPV and CPV variants in apparently healthy cats and their persistence in white blood cells (WBC) and other tissues when neutralising antibodies are simultaneously present, suggest that parvovirus may persist long-term in the tissues of cats post-infection without causing clinical signs. The aim of this study was to screen a population of 54 cats from Sardinia (Italy) for the presence of both FPV and CPV DNA within buffy coat samples using polymerase chain reaction (PCR). The DNA viral load, genetic diversity, phylogeny and antibody titres against parvoviruses were investigated in the positive cats. Results Carnivore protoparvovirus 1 DNA was detected in nine cats (16.7%). Viral DNA was reassembled to FPV in four cats and to CPV (CPV-2b and 2c) in four cats; one subject showed an unusually high genetic complexity with mixed infection involving FPV and CPV-2c. Antibodies against parvovirus were detected in all subjects which tested positive to DNA parvoviruses. Conclusions The identification of FPV and CPV DNA in the WBC of asymptomatic cats, despite the presence of specific antibodies against parvoviruses, and the high genetic heterogeneity detected in one sample, confirmed the relevant epidemiological role of cats in parvovirus infection.

In this study, internal organs (tongue, intestine, and spleen) of 23 free-ranging Italian wolves (Canis lupus italicus) found dead between 2017 and 2019 were tested for Carnivore protoparvovirus 1, Canine adenovirus (CAdV), and Canine circovirus (CanineCV) using real-time PCR assays. Genetic characterisation of the identified viruses was carried out by amplification, sequencing, and analysis of the complete viral genome or informative viral genes. All the wolves tested positive for at least one of the DNA viruses screened, and 11/23 were coinfected. Carnivore protoparvoviruses were the most frequently detected viruses (21/23), followed by CanineCV (11/23) and CAdV (4/23). From the analysis of the partial VP2 gene of 13 carnivore protoparvoviruses, 12 were canine parvovirus type 2b, closely related to the strains detected in dogs and wild carnivores from Italy, and one was a feline panleukopenia-like virus. Of the four CAdV identified, two were CAdV-1 and two were CAdV-2. The complete genome of seven CanineCVs was sequenced and related to the CanineCV identified in dogs, wolves, and foxes worldwide. Close correlations emerged between the viruses identified in wolves and those circulating in domestic dogs. Further studies are needed to investigate if these pathogens may be potentially cross-transmitted between the two species.

Leptospirosis is an infectious disease that causes serious illness in dogs. For this reason, epidemiological and clinical studies focusing on disease characterization are widely advocated. The aim of this study was to characterize the leptospires identified in dogs with confirmed symptomatic acute leptospirosis. Leptospira spp. DNA detected in urine, blood, or both samples from nine infected dogs was analyzed using the multi-locus sequence typing (MLST) technique. Leptospires from two dogs were successfully typed: one was identified as belonging to Sequence Type (ST) 17 and one to ST198, both within the L. interrogans species, serogroups Icterohaemorrhagiae and Australis, respectively. Based on the results of routine serologic tests, antibodies reactive toward these serogroups are commonly revealed in dogs in Italy. This study provides the first molecular analysis that identifies infecting Leptospira directly on DNA from biological samples of dogs, showing that serogroup Australis can lead to a severe clinical presentation of leptospirosis in infected dogs.

Canine distemper virus (CDV), canine adenovirus (CAdV) and canine parvovirus type 2 (CPV-2) cause disease in dogs (Canis familiaris). These, or closely related viruses, may also infect wild carnivores. The aim of this study was to investigate exposure to CDV, CAdV and CPV-2 among fox populations in Norway. Arctic foxes (n = 178) from High-Arctic Svalbard were investigated for antibodies against CDV. Arctic foxes (n = 301) from Svalbard and red foxes from Low-Arctic (n = 326) and sub-Arctic (n = 74) regions in Finnmark County, Norway, were investigated for antibodies against CAdV and for the presence of carnivore protoparvovirus DNA in spleen and mesenteric lymph nodes using polymerase chain reaction. Seroprevalence against CDV in Arctic foxes decreased from 25% (1995/96) to 6% (2001/02), whereas the seroprevalence against CAdV increased from 25–40% during the seasons 1995/96 to 2001/02 to 68% for the last study year (2002/03). In red foxes, the seroprevalence against CAdV varied between 31% and 67% for the seasons 2004/05 to 2007/08, increasing to 80% for the last study year. Carnivore protoparvovirus DNA was not detected in any of the 301 Arctic foxes and the 265 red foxes investigated. These results show that CDV and CAdV are enzootic in the Arctic fox population (Svalbard), and that CAdV is enzootic in both the Low-Arctic and sub-Arctic red fox populations (Finnmark). Further studies are needed to better understand the infection biology and the impact of CDV and CAdV in these fox populations, and if viruses may be shared between foxes and other carnivores, including dogs.

This study describes the sequence analysis of the H gene of 7 Canine distemper virus (CDV) strains identified in dogs in Italy between years 2002-2012. The phylogenetic analysis showed that the CDV strains belonged to 2 clusters: 6 viruses were identified as Arctic‑like lineage and 1 as Europe 1 lineage. These data show a considerable prevalence of Arctic‑like‑CDVs in the analysed dogs. The dogs and the 3 viruses more recently identified showed 4 distinctive amino acid mutations compared to all other Arctic CDVs.

This communication describes the coinfection with feline panleukopenia virus (FPV), feline herpesvirus 1 (FeHV-1), feline calicivirus (FCV) and feline coronavirus (FCoV) in a 1 year‑old domestic cat living in a feline shelter. The cat was referred to veterinary hospital with clinical signs related to diffuse gastro-intestinal inflammation, it had developed a severe pneumopathy with fibrinous exudation in all body cavities and died 8 days after initial presentation. Pathological findings and biomolecular diagnostic test results were compatible with an initial FPV infection that, in consequence of the lymphoid depletion, has fostered coinfection or reactivation of chronic-latent infections with FeHV-1, FCV, and FCoV. In the reported case, the simultaneous presence of different viruses exacerbated the clinical status of the host, resulting in multiple organ damage and leading it to its death.

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 μg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

In questo studio si riporta un caso di coinfezione da parvovirus canino (CPV) di tipo 2a e da virus della panleucopenia felina (FPV) in un gatto di 3 mesi, con la presenza di una variante di parvovirus avente caratteristiche genetiche intermedie tra CPV e FPV. Il riscontro di una variante virale caratterizzata da epitopi specifici sia per il FPV che per il CPV avvalora l'importanza del meccanismo evolutivo per mutazioni multistep nella produzione di nuove varianti e nell'emergenza di nuovi virus. Questo tipo di adattamento progressivo è già stato riscontrato durante l'emergenza del CPV. Sulla base dei risultati ottenuti, è possibile ipotizzare che il CPV abbia presumibilmente iniziato un nuovo processo di riadattamento nell'ospite felino, confermando l'importanza del salto d'ospite nell'emergenza di nuovi virus.

In this study we reported a case of co-infection with canine parvovirus (CPV) type 2a and feline panleukopenia virus (FPV) in a 3-month-old male kitten, with the presence of a parvovirus variant which is a true intermediate between CPV and FPV. The report of a viral variant which contained FPV- and CPV-specific epitopes stresses the importance of the mechanism of multistep mutation in the production of new variants and in the emergence of new viruses. This type of multistep adaptation has already been documented during the emergence of CPV and on the basis of our results, it was hypothesized that CPV had presumably started a new process of readaptation in the feline host, confirming the importance of viral host switching as a mechanism for the emergence of new viruses.

A documented case of canine granulocytic anaplasmosis coupled with the molecular characterization of the etiological agent is reported for the first time in Northern Italy. The patient showed nonspecific clinical signs such as fever and weight loss. The most relevant clinicopathological findings were thrombocytopenia, hypoalbuminemia, and normal azotemic proteinuria consistent with glomerular diseases. Blood smear examination revealed the presence of intracytoplasmatic inclusions in neutrophils associated with high positive serology for Anaplasma phagocytophilum. PCR analysis and sequencing of the amplicon confirm serological diagnosis of A. phagocytophilum. Phylogenetic analysis evidenced that the detected bacterial strain belongs to the A. phagocytophilum Europe 1 lineage. Data indicates that A. phagocytophilum circulates in natural environments of Emilia-Romagna region (Northern Italy) and its prevalence in dogs could be underestimated because the clinical signs are frequently nonspecific and a certain diagnosis requires the combination of clinicopathological and molecular assays. Pets living in this area should be regularly monitored and treated for ectoparasites to minimize health risks for humans and pets. Also, surveillance of A. phagocytophilum should be improved in Northern Italy and canine anaplasmosis should be considered in differential diagnosis of persistent proteinuria.

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.