Reproducibility and Feasibility of Classification and National Guidelines for Histological Diagnosis of Canine Mammary Gland Tumours: A Multi-Institutional Ring Study

Histological diagnosis of Canine Mammary Tumours (CMTs) provides the basis for proper treatment and follow-up. Nowadays, its accuracy is poorly understood and variable interpretation of histological criteria leads to a lack of standardisation and impossibility to compare studies. This study aimed to quantify the reproducibility of histological diagnosis and grading in CMTs. A blinded ring test on 36 CMTs was performed by 15 veterinary pathologists with different levels of education, after discussion of critical points on the Davis-Thompson Foundation Classification and providing consensus guidelines. Kappa statistics were used to compare the interobserver variability. The overall concordance rate of diagnostic interpretations of WP on identification of hyperplasia-dysplasia/benign/malignant lesions showed a substantial agreement (average k ranging from 0.66 to 0.82, with a k-combined of 0.76). Instead, outcomes on ICD-O-3.2 morphological code /diagnosis of histotype had only a moderate agreement (average k ranging from 0.44 and 0.64, with a k-combined of 0.54). The results demonstrated that standardised classification and consensus guidelines can produce moderate to substantial agreement; however, further efforts are needed to increase this agreement in distinguishing benign versus malignant lesions and in histological grading. View Full-Tex

Pathological Prion Protein in the Tongues of Sheep Infected with Naturally Occurring Scrapie

Tongue involvement by prion spreading was shown to be a common outcome after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources in rodent models. It is also known that bovine spongiform encephalopathy, which is pathogenic for humans, is experimentally transmissible to sheep and can lead to a disease indistinguishable from scrapie. A recent European Food Safety Authority opinion recommended research into PrP(sc) accumulation in the tongues of ruminants. We report on the detection of PrP(sc) in the tongues of seven scrapie-infected sheep by immunohistochemistry and Western blotting

Reproducibility and Feasibility of Classification and National Guidelines for Histological Diagnosis of Canine Mammary Gland Tumours: A Multi-Institutional Ring Study

Histological diagnosis of Canine Mammary Tumours (CMTs) provides the basis for proper treatment and follow-up. Nowadays, its accuracy is poorly understood and variable interpretation of histological criteria leads to a lack of standardisation and impossibility to compare studies. This study aimed to quantify the reproducibility of histological diagnosis and grading in CMTs. A blinded ring test on 36 CMTs was performed by 15 veterinary pathologists with different levels of education, after discussion of critical points on the Davis-Thompson Foundation Classification and providing consensus guidelines. Kappa statistics were used to compare the interobserver variability. The overall concordance rate of diagnostic interpretations of WP on identification of hyperplasia-dysplasia/ benign/malignant lesions showed a substantial agreement (average k ranging from 0.66 to 0.82, with a k-combined of 0.76). Instead, outcomes on ICD-O-3.2 morphological code /diagnosis of histotype had only a moderate agreement (average k ranging from 0.44 and 0.64 , with a k-combined of 0.54). The results demonstrated that standardised classification and consensus guidelines can produce moderate to substantial agreement; however, further efforts are needed to increase this agreement in distinguishing benign versus malignant lesions and in histological grading

Neuropathological Characterization of Dolphin Morbillivirus Infection in Cetaceans Stranded in Italy

Simple Summary There is abundant literature reporting demyelination in dogs and pinnipeds affected by morbillivirus infection, but myelinopathy is poorly investigated in stranded cetaceans affected with the virus. Also, the neuropathogenesis of cetacean morbillivirus infection has not been fully clarified, leaving questions on cell tropism unanswered. A novel dolphin morbillivirus lineage of Atlantic origin circulating in Italian waters replaced the previous Mediterranean strain in late 2015; however, differences in virulence and pathogenesis between the two strains have not yet been documented. The aims of the present study were to: describe histopathological changes and immunohistochemical findings in the central nervous system of 31 cetaceans which tested positive on molecular investigations for the two dolphin morbillivirus strains; characterize by double indirect immunofluorescence staining the areas of myelin damage. The most frequently observed morbillivirus-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Demyelination was detected by means of a specific myelin biomarker. Inside and around the demyelinated areas there were morbillivirus antigen-bearing cells of mainly neuronal and microglial origin, associated with marked astro and microglia reactivity. Molecular and immunohistochemical analysis suggested a higher neurotropic affinity of the novel circulating strain. Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis

Pro-Inflammatory and Cytotoxic Effects of Polystyrene Microplastics on Human and Murine Intestinal Cell Lines

Plastic is a polymer extremely resistant to degradation that can remain for up to hundreds or thousands of years, leading to the accumulation of massive amounts of plastic waste throughout the planet’s ecosystems. Due to exposure to various environmental factors, plastic breaks down into smaller particles named microplastics (1–5000 μm) and nanoplastics (<1 μm). Microplastics (MPs) are ubiquitous pollutants but, still, little is known about their effects on human and animal health. Herein, our aim is to investigate cytotoxicity, oxidative stress, inflammation and correlated gene modulation following exposure to polystyrene microplastics (PS-MPs) in HRT-18 and CMT-93 epithelial cell lines. After 6, 24 and 48 h PS-MPs treatment, cell viability (MTT) and oxidative stress (SOD) assays were performed; subsequently, expression changes and cytokines release were investigated by Real-Time PCR and Magnetic-beads panel Multiplex Assay, respectively. For each exposure time, a significantly increased cytotoxicity was observed in both cell lines, whereas SOD activity increased only in CMT-93 cells. Furthermore, Magnetic-beads Multiplex Assay revealed an increased release of IL-8 in HRT-18 cells’ medium, also confirmed by gene expression analysis. Results obtained suggest the presence of a pro-inflammatory pattern induced by PS-MPs treatment that could be related to the observed increase in cytotoxicity

Equus caballus Papillomavirus Type-9 (EcPV9): First Detection in Asymptomatic Italian Horses

Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9-L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9-L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2-L1 was found in 7 out of the 11 EcPV9-L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals

Detection of Equus Caballus Papillomavirus Type-2 in Asymptomatic Italian Horses

Equine Papillomavirus 2 (EcPV2) is responsible for squamous cell carcinomas (eSCCs) of external genitalia of both male and female horses. However, few studies report the EcPV2 prevalence among healthy horses. Currently, the lack of these data does not permit identifying at-risk populations and, thus, developing screening protocols aimed at the early detection of the infection, as for humans. The aim of our study was to estimate the genoprevalence of EcPV2 in clinically healthy horses in Italy and to evaluate their innate immune response. For this purpose, penile and vulvar swabs of 234 healthy horses were collected through sampling with sterile cytobrushes. Nucleic acids were isolated and EcPV2-L1 presence (DNA) and gene expression (RNA) were checked by RT-qPCR. Our results showed EcPV2-L1 DNA presence in 30.3% of the samples and L1 expression in 48% of the positive samples. No statistically significant differences were found in genoprevalence in relation to sex, age, and origin, while, concerning breeds, the Thoroughbred had the highest risk of infection. Concerning specifically the mares, 40.2% of them resulted in being positive for EcPV2; our findings show a major positivity in pluriparous (p = 0.0111) and mares subjected to natural reproduction (p = 0.0037). Moreover, samples expressing L1 showed an increased expression of IL1B (p = 0.0139) and IL12p40 (p = 0.0133) and a decreased expression of RANKL (p = 0.0229) and TGFB (p = 0.0177). This finding suggests the presence of an effective immune response, which could explain the low incidence of SCCs in positive horses, despite a high EcPV2 genoprevalence (30%)

Decrease in pathology and progression of scrapie after immunisation with synthetic prion protein peptides in hamsters

TLRs trigger innate immunity that recognizes conserved motifs of invading pathogens, resulting in cellular activation and release of inflammatory factors. The influence of TLR activation on resistance to HIV-1 infection has not been investigated in HIV-1 exposed seronegative (ESN) individuals. PBMCs isolated from heterosexually ESN individuals were stimulated with agonists specific for TLR3 (poly I:C), TLR4 (LPS), TLR7 (imiquimod), and TLR7/8 (ssRNA40). We evaluated expression of factors involved in TLR signaling cascades, production of downstream effector immune mediators, and TLR-expression in CD4+ and CD14+ cells. Results were compared with those obtained in healthy controls (HCs). ESN individuals showed: 1) comparable percentages of CD14+/TLR4+ and CD4+/TLR8+ CD14+/TLR8+ cells; 2) higher responsiveness to poly I:C, LPS, imiquimod, and ssRNA40 stimulation, associated with significantly increased production of IL-1beta, IL-6, TNF-alpha, and CCL3; 3) augmented expression of mRNA specific for other targets (CCL2, CSF3, CSF2, IL-1alpha, IL-8, IL-10, IL-12, cyclooxygenase 2) demonstrated by broader TLRs pathway expression analyses; and 4) increased MyD88/MyD88s(short) ratio, mainly following TLR7/8 stimulation. We also compared TLR-agonist-stimulated cytokine/chemokine production in CD14+ PBMCs and observed decreased IFN-beta production in ESN individuals compared with HCs upon TLR7/8-agonist stimulation. These data suggest that TLR stimulation in ESN individuals results in a more robust release of immunologic factors that can influence the induction of stronger adaptive antiviral immune responses and might represent a virus-exposure-induced innate immune protective phenotype against HIV-1