Detection of bovine papillomavirus type 2 in the peripheral blood of cattle with urinary bladder tumours: possible biological role

Bovine papillomavirus type 2 (BPV-2) infection has been associated with urinary bladder tumours in adult cattle grazing on bracken fern-infested land. In this study, we investigated the simultaneous presence of BPV-2 in whole blood and urinary bladder tumours of adult cattle in an attempt to better understand the biological role of circulating BPV-2. Peripheral blood samples were collected from 78 cattle clinically suffering from a severe chronic enzootic haematuria. Circulating BPV-2 DNA was detected in 61 of them and in two blood samples from healthy cows. Fifty of the affected animals were slaughtered at public slaughterhouses and neoplastic proliferations in the urinary bladder were detected in all of them. BPV-2 DNA was amplified and sequenced in 78% of urinary bladder tumour samples and in 38.9% of normal samples as a control. Circulating episomal BPV-2 DNA was detected in 78.2% of the blood samples. Simultaneous presence of BPV-2 DNA in neoplastic bladder and blood samples was detected in 37 animals. Specific viral E5 mRNA and E5 oncoprotein were also detected in blood by RT-PCR and Western blot/immunocytochemistry, respectively. It is likely that BPV-2 can persist and be maintained in an active status in the bloodstream, in particular in the lymphocytes, as a reservoir of viral infection that, in the presence of co-carcinogens, may cause the development of urinary bladder tumours

Modulation of telomerase activity, bTERT and c-Myc induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin during Bovine Herpesvirus 1 infection in MDBK cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) influences infection of kidney cells (MDBK) with Bovine Herpesvirus 1 (BHV-1) through an increase in virus replication and an acceleration of BHV-1-induced apoptosis. Previously our group demonstrated that BHV-1, in the early stages of infection, significantly up-regulates telomerase activity in MDBK cells, while, in the late phases of infection, when BHV-1-induced apoptosis occurred, a down-regulation of telomerase activity was detected. Hence, herein, for the first time, we described the influences of TCDD on telomerase activity during virus infection. In kidney cells (MDBK) infected with BHV-1 and exposed to different doses of TCDD we explored telomerase activity by TRAP assay. Concomitantly, we examined protein levels of both bTERT and c-Myc by Western blot analysis. In all groups, TCDD induced an acceleration in down-regulation of telomerase activity. Particularly, TCDD drastically and significantly decreased telomerase activity when virus-induced apoptosis took place. This result was accompanied from an accelerated down-regulation of bTERT and c-Myc. Finally, in the presence of TCDD, we evidenced a dose-dependent overexpression of aryl hydrocarbon receptor. Hence, our data suggest that TCDD, through a significant acceleration in down-regulation of telomerase activity, bTERT and c-Myc, may contribute to accelerated BHV-1-induced apoptosis

SARS-CoV-2 exposure in hunting and stray dogs of southern Italy

: Evidence of exposure to the pandemic SARS-CoV-2 has been described in numerous animal species, including pets, which are predisposed to coming into contact with this virus due to their close relationship with owners. It has been accepted that dogs are poorly susceptible to this virus and that seroconversion, rather than shedding, occurs following infection, which can occur directly through contact with infected owners or indirectly through environmental contamination. In this study, the seroprevalence of SARS-CoV-2 was evaluated in apparently health hunting and stray dogs of Campania region, southern Italy (sampled in September 2023). A total of 5/112 (4.5%) animals tested seropositive using two different commercial ELISAs. Stray animals had greater exposure than hunting dogs. The feces and blood of each animal were tested with a real-time PCR targeting the nucleocapsid and ORF1ab coding sequences. No animal tested positive in molecular investigations, indicating a past exposure without active infection at the time of sampling

Combined effects of PI3K and SRC kinase inhibitors with imatinib on intracellular calcium levels, autophagy, and apoptosis in CML-PBL cells

Imatinib induces a complete cytogenetic regression in a large percentage of patients affected by chronic myeloid leukemia (CML) until mutations in the kinase domain of BCR-ABL appear. Alternative strategies for CML patients include the inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR ) pathway, which is constitutively activated in leukemia cells and seems important for the regulation of cell proliferation, viability, and autophagy. In this study, we verified the effect of imatinib mesylate (IM), alone or in association with LY294002 (LY) (a specific PI3K protein tyrosine kinase inhibitor) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) (a Src tyrosine kinase inhibitor), on viability, intracellular calcium mobilization, apoptosis, and autophagy, in order to verify possible mechanisms of interaction. Our data demonstrated that PP1 and LY interact synergistically with IM by inducing apoptosis and autophagy in Bcr/Abl+ leukemia cells and this mechanism is related to the stress of the endoplasmic reticulum (ER ). Our findings suggest a reasonable relationship between apoptotic and autophagic activity of tyrosine kinase inhibitors (TKIs) and the functionality of smooth ER Ca2+-AT Pase and inositol triphosphate receptors, independently of intracellular calcium levels. Therapeutic strategies combining imatinib with PI3K and/or Src kinase inhibitors warrant further investigations in Bcr/Abl+ malignancies, particularly in the cases of imatinib mesylate-resistant diseas

Modulation of apoptosis by caprine herpesvirus 1 infection in a neuronal cell line

Caprine herpesvirus type 1 (CpHV-1), like other members of the alpha subfamily of herpesviruses, establishes latent infections in trigeminal ganglion neurons. Our groups previously demonstrated that CpHV-1 induces apoptosis in goat peripheral blood mononuclear cells and in an epithelial bovine cell line, but the ability of CpHV-1 to induce apoptosis in neuronal cells remains unexplored. In this report, the susceptibility of Neuro 2A cells to infection by CpHV-1 was examined. Following infection of cultured cells with CpHV-1, expression of cell death genes was evaluated using real-time PCR and Western blot assays. Analysis of virus-infected cells revealed activation of caspase-8, a marker for the extrinsic pathway of apoptosis, and caspase-9, a marker for the intrinsic pathway of apoptosis at 12 and 24 h post-infection. Significant increase in the levels of cleaved caspase-3 was also observed at the acme of cytopathic effect at 24 h post-infection. In particular, at 3 and 6 h post-infection, several proapototic genes were under-expressed. At 12 h post-infection several proapototic genes such as caspases, TNF, Cd70, and Traf1 were over expressed while Bcl2a1a, Fadd, and TNF genes were underexpressed. In conclusion, the simultaneous activation of caspase-8 and caspase-9 suggests that CpHV-1 can trigger the death-receptor pathway and the mitochondrial pathway separately and in parallel. Our findings are significant because this is the first published study showing the effect of CpHV-1 infection in neuronal cells in terms of gene expression and apoptosis modulation

Detection of selected pathogens in reproductive tissues of wild boars in the Campania region, southern Italy

: Monitoring disease among wildlife is critical to preserving health in both domestic animals and wildlife, and it becomes much more critical when the diseases cause significant economic damage to the livestock industry or threaten public health. Given the continuous increase in populations and its role as a reservoir for several infections, wild boar (Sus scrofa) requires special attention regarding disease surveillance and monitoring. In this study, we investigated the molecular prevalence of selected pathogens in the wild boar population of Campania, southern Italy. The prevalence of pathogens causing reproductive problems in pigs (Sus domesticus), including porcine parvovirus (PPV), porcine circovirus types 2 and 3 (PCV-2 and PCV-3), pseudorabies virus (PRV), Coxiella burnetii, and Brucella suis, was evaluated by testing the reproductive organs collected from 63 wild boars with polymerase chain reaction. The most common pathogens were PPV (44.4%) and two porcine circoviruses (14.3%). PRV and C. burnetii, on the other hand, showed a significantly lower prevalence (1.6%). No reproductive organs tested were positive for B. suis. Risk factor analysis revealed a correlation between age and PCV-2 positivity, with animals less than 12 months old having significantly higher prevalence rates.Our findings suggest that wild boars hunted in the Campania region harbour several infections potentially transmissible to other mammals' reproductive tracts. Furthermore, our results emphasized the importance of strict adherence to biosecurity protocols on domestic swine farms, especially on free-range farms, to avoid interactions between domestic and wild animals

Role of Caprine Herpesvirus – 1 (CpHV-1) in the innate immuno-evasion Interferon mediate.

Caprine Herpesvirus-1 (CpHV-1) is a member of Varicellovirus genus within Herpesvirus family. CpHV-1 is the responsible of a disease that causes respiratory symptoms, balanopostitis, vulvovaginitis and abortion in adult goats. It also affects young goats with a systemic disease [1]. Type I (IFN-a/ ) and type II (IFN g) interferons, are the first weapons of the host to fight against viral infections. Type I IFNs induce the expression of more than 100 Interferon Stimulated Genes (ISGs) to establish an antiviral state that limits viral replication and dissemination. [2] Type II IFN is produced by activated immune cells and leads to the production of a different subset of ISGs via a distinct signaling pathway. Many viruses are able to subvert both type I and type II IFN-mediated antiviral responses. Herpesviruses are able to evade the IFN response by targeting different transcriptions factors of the interferon (IFN) signaling pathway [3]. There are no reports in literature about the role of CpHV-1 in IFN antagonism. Aim of the work: Herein, we describe a work to address the possible role of CpHV-1 as modulator of the innate immune response interferon mediate. Materials and methods. To investigate whether CpHV-1 interferes with type I interferon production, we performed an IFN- Beta reporter assay, using a reporter plasmid that carries the IFN-Beta promoter driving the expression of a firefly luciferase gene. A renilla-luciferase reporter plasmid was used as control. For the activation of the pathway we used the constitutively active N-terminal (2CARD) domain of RIG-I. To evaluate the potential CpHV-1-mediated inhibition of IFN a/b signaling, we performed an ISRE54 reporter assay using a construct having an ISRE54 promoter driving the expression of firefly luciferase. A renilla-luciferase reporter plasmid was used as internal control. Results. Our results show that CpHV-1 infection strongly suppressed the activation of IFN-β promoter induced by RIGI 2 CARD domain. Moreover cells mock infected and treated with type I IFN showed a significant increase in luciferase expression, as expected compared with the cells that were not treated with type I IFN and were not infected. The cells infected with CpHV-1 and treated with type I IFN showed significantly reduced luciferase expression driven by the ISRE54 promoter. These results showed that CpHV-1 is a strong inhibitor of type I Interferon production and signaling pathways. [1] Tempesta M, Pratelli A, Greco G, Martella V, Buonavoglia C, 1999. Detection of caprine herpes virus 1 in the sacral ganglia of latently infected goats by polimerasi chain reactionj J Clin Microbiol 37, 1598-99. [2] Garcia-Sastre A, Biron CA (2006) Type 1 interferons and the virus-host relationship: a lesson in detente. Science 312: 879-882. [3] Afroz S, Brownlie R, Fodje M, van Drunen Littel-van den Hurk S. VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling. J Virol. 2016 Apr 29; 90(10): 4889-904

Combined addition of superoxide dismutase, catalase and glutathione peroxidase improves quality of cooled stored stallion semen.

During cold storage stallion spermatozoa experience undergo oxidative stress, which can impair sperm function and fertilizing capacity. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are the main endogenous enzymatic antioxidants in stallion seminal plasma, and counteract reactive oxygen species. Semen dilution reduces the endogenous antioxidant concentrations. The aim of this study was to investigate whether addition of 15 IU/mL each of SOD, CAT, and GPX to diluted stallion semen would ameliorate a reactive oxygen-mediated decrease in semen quality during 72 h of storage at 5 °C. Ejaculates (n = 7) were divided in two aliquots and diluted in INRA 96 without (control) or with addition of antioxidants. Semen analysis was performed at the time of dilution and every 24 h during chilled storage. Antioxidant supplementation completely inhibited the storage-dependent increase in activated caspase 3 (P < 0.05). Concomitantly, the antioxidant-supplemented samples had a greater percentage of viable, motile and rapidly moving sperm than control samples after 72 h storage (P < 0.05). The DNA damage, as evaluated by TUNEL assay and SCSA, increased with storage time (P < 0.05). Antioxidant supplementation did not prevent, but did significantly reduce the increase in DNA strand breakage. The results indicate part of the intrinsic apoptotic pathway leading to effector caspase activation was inhibited, although an activation of molecules with endonuclease activity still occurred. In conclusion, adding equal concentrations of SOD, CAT and GPX to a semen extender suppressed caspase-3 activation and improved preservation of stallion sperm motility and viability during 72 h of storage at 5 °C

Evaluation of the phase-specific antibody response in water buffalo (Bubalus bubalis) after two doses of an inactivated phase I Coxiella burnetii vaccine

: The control and management of Q fever outbreaks in ruminants are currently based on vaccination. Although buffalo (Bubalus bubalis) are intensively farmed in several countries and represent a reservoir for Coxiellosis, no evidence has been described regarding the efficacy of vaccination in this species. This work aimed to evaluate the humoral response, using appropriate phase-specific ELISAs, and the effects on abortion rate in buffalo by a field study. A total of 15 seropositive and 20 seronegative animals were vaccinated twice, three weeks apart, with a commercial phase I vaccine, and phase-specific antibodies were determined in the course of vaccination. Although anti-phase II antibody reactivity predominated after vaccination compared to phase I, both anti-phase I- and -phase II-antibody-reactivity significantly increased after the first (p = 0.001) and again after the second vaccination (p = 0.05). Seroconversion did not significantly depend on age or natural infection status. Once the vaccination cycle was completed, the herd study observed a reduced rate of abortion and placenta retention. Our data demonstrated that the vaccine principally induced a similar antibody response as in goats and sheep. These preliminary data appeared to support vaccination in buffalo, even in seropositive animals, although further studies are needed to better define the dynamics concerning seroconversion in this species