Short communication: Seroprevalence of paratuberculosis in Italian water buffaloes (Bubalus bubalis) in the region of Campania.

Paratuberculosis is a chronic enteric disease affecting virtually all ruminants, but only anecdotal information is currently available about the occurrence of this disease in water buffaloes (Bubalus bubalis). We carried out a survey study aimed at determining the prevalence of paratuberculosis in 2 provinces in the region of Campania, Italy, where about half of all Italian buffaloes are reared. From May 2017 to December 2018, we collected 201,175 individual serum samples from 995 buffalo herds. The sera were collected from animals over 24 mo old and were tested using a commercial ELISA test. The herd-level apparent prevalence result was 54.7%, and the animal-level apparent prevalence was 1.8%. The herd-level true prevalence was estimated using a Bayesian approach, demonstrating a high herd-level prevalence of paratuberculosis in water buffaloes from the Campania area. These findings suggest that the urgent adoption of paratuberculosis herd-control programs for water buffaloes in this area would be beneficial.

Serological and molecular survey of Q fever in the dog population of the Campania region, southern Italy.

Q fever is a re-emerging zoonosis whose epidemiological cycle in ruminants is well defined, while the role of other species (including pets) is still debated. In this study, the serological and molecular prevalence of Coxiella burnetii in a sample of dogs in the Campania region, southern Italy was evaluated. A seroprevalence of 5.97 % (16/268) was observed using a commercial multispecies ELISA, compared to only 2.7 % (5/197) at the molecular level. No risk factors correlated with higher levels of exposure except for the size of the animal (small dogs showed significantly higher seroprevalence). Positive samples were further evaluated for reactivity to phase I and II antigens using IFA and phase-specific ELISAs (for specific IgG detection). Two animals showed antibodies against both phases of infection, suggesting that Coxiella burnetii seroconversion in dogs follows similar dynamics to those observed in ruminants. One of the five samples that showed positive results in real-time PCR was confirmed at the PCR endpoint and showed similarity with other Coxiella spp. strains detected in tick and dog samples when sequenced. In this study, we demonstrated exposure to Coxiella burnetii for different categories of dogs in southern Italy, including pet dogs living indoors. Since reports of transmission of infection from pets to humans have been described in both rural and urban areas, careful surveillance of these species is also necessary. In the lack of additional information, comprehending the risk to humans requires monitoring of wild and domestic animal populations.

Detection of anti-HEV antibodies but no molecular positivity in dogs in the Campania region, southern Italy.

Paslahepevirus balayani genotypes 3 and 4 (HEV-3 and 4) have zoonotic potential and can be transmitted to humans and animals through the consumption of contaminated raw or undercooked meat. Although it has been demonstrated that dogs are susceptible to the infection and produce specific antibodies, the epidemiological role of this species is not yet well defined. This study aimed to evaluate the circulation of HEV at the serological and molecular level in the dog population of the Campania region, southern Italy. A total of 231 dogs were sampled, divided according to several variables (sex, age, origin, lifestyle, location, size, and breed), and tested for the presence of HEV antibodies using a commercial multi-species ELISA. A total of 197 blood samples and 170 stool samples were tested with two specific PCRs in order to detect viral RNA. A total of 19 out samples of 231 were seropositive, obtaining an exposure (8.2%) similar to that observed in other European countries. The univariate and multivariate analysis revealed a wide exposure to stray dogs and animals from the province of Salerno. All samples tested with molecular methods were negative. Defining the role of domestic carnivores continues to be a "one health" challenge, although it appears that they do not eliminate the virus and therefore do not pose a danger to humans. In the absence of other evidence, it is advisable to continue to carry out surveillance also for domestic animals, which, due to ethological characteristics or their position in the food chain, could be predisposed to being exposed to HEV.

Advanced/metastatic bladder cancer: current status and future directions.

In 2015 bladder cancer was the fourth most frequent malignancy and the eighth cause of death for cancer. At diagnosis, about 30% of bladder cancer (BC) patients present a muscle-invasive bladder cancer (MIBC) and 5% a metastatic bladder carcinoma (MBC). For fit MBC patients, combination chemotherapy (CC) is the standard of care for first-line treatment. CC includes both the treatment with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) either the classical or the dose-dense MVAC regimen, and the doublet therapy with cisplatin and gemcitabine (CG). Median progression free survival (PFS) was 7 months and median overall survival (OS) was 15 months. The present review provides an update on the management of MBC, with focus on target therapies, immune checkpoint inhibition, looking for prognostic and predictive factors.

Penile cancer: prognostic and predictive factors in clinical decision-making.

Penile cancer (PC) is a typical tumor of non-industrialized countries. The incidence is 20-30 times higher in Africa and South America, considering the elevated prevalence of sexually transmitted diseases. Histologically, PC includes squamous cell carcinoma (SCPC), the most frequent, and nonsquamous carcinoma (NSCPC). Early diagnosis is the goal, whereas later diagnosis relates to poor functional outcomes and worse prognosis. The 5-year survival rate is 85% for patients with histologically regional negative lymph nodes, compared to 29%-40% for those with histologically regional positive lymph nodes. To date no new drugs are approved, and there are few new data about molecular mechanisms underlying tumorigenesis. The SCPC remains a rare tumor and the current therapeutic algorithm is based principally on retrospective analysis and less on prospective trials. In this review article, biomarkers of prognosis and efficacy of current treatments are summarized with a focus on those that have the potential to affect treatment decision-making in SCPC.

Feline herpesvirus-1 down-regulates MHC class I expression in an homologous cell system.

Cytotoxic T lymphocytes (CTLs) are an essential component of the immune defense against many virus infections. CTLs recognize viral peptides in the context of the major histocompatibility complex (MHC) class I molecules on the surface of infected cells. Many viruses have evolved mechanisms to interfere with MHC class I expression as a means of evading the host immune response. In the present research we have studied the effect of in vitro Feline Herpesvirus 1 (FeHV-1) infection on MHC class I expression. The results of this study demonstrate that FeHV-1 down regulates surface expression of MHC class I molecules on infected cells, presumably to evade cytotoxic T-cell recognition and, perhaps, attenuate induction of immunity. Sensitivity to UV irradiation and insensitivity to a viral DNA synthesis inhibitor, like phosphonacetic acid, revealed that immediate early or early viral gene(s) are responsible. Use of the protein translation inhibitor cycloheximide confirmed that an early gene is primarily responsible.

Bovine papillomavirus type 1 DNA and E5 oncoprotein expression in water buffalo fibropapillomas.

Papillomas and fibropapillomas may occur in the skin and in different organs in animals. Ten different genotypes of bovine papillomavirus (BPV) have been identified. BPV-1 through BPV-10 are all strictly species-specific, but BPV-1/2 may also infect other species such as equids, inducing fibroblastic tumors. BPV-1 and BPV-2 are associated with fibropapillomas in cattle; these tumors are formed by excessive proliferation of virus-infected dermal fibroblasts and epidermal keratinocytes. Nine water buffalo (Bubalus bubalis) were examined for the presence of multiple cutaneous and perivulvar tumors. Cutaneous and perivulvar fibropapillomatosis were confirmed histologically. Negative-stain transmission electron microscopic examination revealed papillomavirus-like particles in the fibropapillomas, and papillomaviral DNA was also detected by the polymerase chain reaction. The amplified long control region (LCR) DNA sequence was identical to that of BPV-1. The BPV-1 E5 oncoprotein was strongly expressed in the tumor cells thus confirming a causal role of the virus. This article represents the first report of cutaneous, perivulvar, and vulvar fibropapilloma associated with BPV-1 infection in the water buffalo and describes another example of cross-species infection by BPV-1.

Prognostic and predictive factors in testicular cancer.

OBJECTIVE: Testicular cancer is a relatively rare neoplasia, with an incidence of about 1,5% among male malignancies, usually in the third and fourth decade of life. Although several histological variants are known, with some histotypes affecting older patients (e.g., spermatocytic seminoma), there is a clear predominance (90-95%) of germ cell tumors among young adults patients1. Testicular Germ Cell Tumor (TGCT), undoubtedly the seminoma histological variant more than non-seminoma one, is definitely a highly curable disease, with a distinctive sensitivity to cisplatin-based therapy (and for seminomas to radiotherapy) and an outstanding cure rate of nearly 80% even for patients with advanced disease. So far, clinical and pathohistological features supported our efforts to choose the best treatment option for patients suffering from this malignancy, but we don't clearly enough know molecular and pathological features underlying different clinical behaviors, mostly in early-stage disease: by improving this knowledge, we should better "shape" therapeutic or surveillance programs for each patient, also in order to avoid unnecessary, if not harmful, treatments.

Evaluation of a fluorescence polarization assay for the detection of serum antibodies to Brucella abortus in water buffalo (Bubalus bubalis).

The fluorescence polarization assay (FPA) was evaluated for the serological diagnosis of brucellosis in water buffalo (Bubalus bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. Sera from 890 buffalos from the Campania Region - 526 positive sera and 364 negative sera according to the complement fixation test (CFT) - were evaluated in this study. All samples were tested with the Rose Bengal test (RBT), CFT, and FPA in parallel and in blind fashion. Sensitivities (Sn) were 84.5% and 92.6%, and specificities (Sp) were 93.1% and 91.2% for RBT and FPA, respectively, relative to CFT. Finally, receiver operating characteristic (ROC) analysis suggested a cut-off value of 117 millipolarization (mP) units. On the whole, these results suggested that FPA might replace RBT in the diagnosis of buffalo brucellosis for its better performance relative to CFT, its adjustable cut-off useful in different epidemiological situations, its reliability, ease of performance, and for its potential application in field and high-throughput laboratories.

Evaluation of molecular methods for the detection of Brucella species in water buffalo milk.

Brucellosis is a highly infectious disease affecting both animals and humans. The current standard tools for the diagnosis of this bacterial infection are serological and microbiological. In this study, we evaluated the feasibility of molecular assays as diagnostic tools for the detection of Brucella spp. in water buffalo milk. For this purpose, we first compared different DNA extraction protocols and PCR methods on artificially spiked milk samples. The most sensitive methods were then used to examine milk from serologically positive and negative water buffaloes. Molecular results were compared with serological and bacteriological test results. Milk samples from 53 Brucella seropositive buffaloes (by either rose Bengal or complement fixation test) were positive by ELISA, 37 were positive by culture, 33 were positive by PCR, and 35 were positive by real-time PCR. Of the 37 culture-positive samples, a total of 25 and 26 were positive by PCR and real-time PCR, respectively. Of the 16 culture-negative samples, 8 were positive by PCR and 9 by real-time PCR. Thus, although culture showed greater sensitivity than PCR, some animals found positive by serological methods and PCR tested negative by milk culture. The combined use of bacteriological and molecular tools increased the number of positive samples to 46. In conclusion, these results suggest that the simultaneous application of these 2 direct detection methods (culture and PCR) could be more useful than one test alone for the diagnosis of Brucella spp. in buffalo milk.

Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems.

Bacterial pathogens are a potential cause when a mare fails to conceive to a fertile stallion on a well-managed breeding farm on one or more cycles in the same season. Furthermore, emerging bacterial resistance to commonly used (topical) antibiotics has been demonstrated. In this study, a total of 586 uterine swabs from mares with fertility problems were evaluated and the bacterial isolates were identified and measured for resistance to 10 antibiotics most commonly used during bacterial equine infection. Forty-nine percent of the examined mares were positive at bacteriological investigations. Amongst 347 successful isolations, 31.7% were Streptococcus group C and 18.4% Escherichia (E.) coli, both considered frequently associated with fertility problems. Determination of the antibiotic susceptibility pattern of Streptococcus group C (110 organisms) revealed that only the amoxicillin/clavulanic acid was highly active with 82.7% of the isolates being inhibited. For E. coli, a major number of drugs displayed a high potency.

Health-related quality of life and psychosocial implications in testicular cancer survivors. A literature review.

OBJECTIVE: In this review, we focused our attention on Quality of Life (QoL) of testicular cancer survivors (TCSs), in general and in the most relevant areas. Several key findings have been highlighted in our review. MATERIALS AND METHODS: PubMed, MEDLINE and PsycINFO databases were consulted to find published studies, from 1980 to May 2017, that met our inclusion criteria. RESULTS: The majority of studies investigated older adult TCSs, while few studies on adolescent and young adult patients were available. Many studies indicate that health-related QoL (HRQoL) is similar among the TCSs and the general population. Even if QoL deteriorates so clear at the time of diagnosis and throughout treatment, afterward returns to normal levels, as defined by the matched controls. However, there are numerous chronic conditions consequent to diagnosis and treatment of testicular cancer that plague survivors and affect QoL, like Raynaud-like phenomena, peripheral neuropathy, fatigue, anxiety, sexual, fertility and body image problems. Even if these problems can have no effects on the measures of global QoL, they have an impact on the quality of life. Differences between TCSs with and without a partner bring to different outcomes in the adjustments to cancer. CONCLUSIONS: It is necessary to identify TCSs with higher risks of poorer QoL outcomes, to focus interventions on the areas with the greatest impairments. Further researches should consider the effects of testicular cancer on the impaired areas, collecting more data to better identify survivor's needs and consequent interventions, with a special focus on adolescent and young adult TCSs. Other works are requested on therapies, preventive and ameliorative, to reduce chronic side effects of testicular cancer treatment.

Rectal/urinary toxicity after hypofractionated vs. conventional radiotherapy in high risk prostate cancer: systematic review and meta analysis.

OBJECTIVE: The aim of our report was to review the literature concerning the toxicity of radiation therapy in patients treated for high-risk prostate cancer, and to evaluate the differences in toxicity between conventional fractionation and hypofractionated treatments, in view of different techniques used in high-risk prostate cancer patients. MATERIALS AND METHODS: PubMed database has been explored for studies concerning acute and late urinary/gastrointestinal toxicity in high-risk prostate cancer patients treated with radiotherapy. Prospective studies, concerning potential relationship between acute/late genitourinary (GU)/gastrointestinal (GI) toxicity and prostate radiotherapy in patients with high-risk prostate cancer, were included in the final analysis. Data collected from single arm, phase II non-randomized and randomized studies have been evaluated to perform odds ratio for toxicity risk. Furthermore, meta-analysis randomized prospective trials were considered suitable because they had recruited high-risk prostate cancer patients who didn't undergo surgery, with available data on ≥ G2 toxicity frequency. RESULTS: The initial search provided 606 results, but only 35 manuscripts met all eligibility requirements and were included in this report. In order to perform odds ratio we observed a decrease in late gastrointestinal toxicity for patients treated with hypofractionated schemes compared to CV treated ones. Among patients who underwent conventional treatment, SIB seemed to decrease acute genitourinary side effects; SIB-Hypo treated patients suffered less toxicity than patients treated with hypofractionated- sequential boost schemes. Hypo-SIB schemes would seem less toxic in terms of acute gastrointestinal and late genitourinary side effects than CV-SIB. Therefore, our focus shifted to 6 clinical trials evaluating genitourinary and gastrointestinal toxicity in patients who had been randomized to receive conventional fractionation or hypofractionated treatment, in both cases with IMRT technology. Our meta-analysis of these randomized trials involving patients with high-risk prostate cancer showed a statistically significant increase in late genitourinary toxicity for hypo-treated patients; no difference was observed in acute genitourinary/gastrointestinal toxicity, and in late gastrointestinal toxicity. CONCLUSIONS: Our analysis doesn't want to establish a definitive truth; very few trials assessed only high risk-class patients. Our purpose is to stimulate further randomized prospective trials focusing both on the effectiveness and toxicity profile (toxicity/effectiveness ratio), taking into account the use of different technologies and doses.

Bovine herpesvirus type 1 (BHV-1) up-regulates telomerase activity in MDBK cells.

The proliferative capacity of mammalian cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA synthesis. The critical role of telomerase activation in tumor progression and maintenance has been well established in studies of cancer and of oncogenic transformation in cell culture. Experimental data suggest that telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase also promotes disease. Evidence regarding the role of telomerase in the pathogenesis of several viruses including human immunodeficiency virus has led to an increased interest in the role of telomerase activity in other virus infections. In this research we evaluated the telomerase modulating activity of Bovine herpesvirus 1 (BHV-1) in MDBK cells. MDBK cells were infected at different multiplicity of infection with BHV-1 Cooper strain and telomerase activity at different times post-infection was measured by the TRAP assay. Our data indicate that BHV-1 significantly up-regulates telomerase activity at 3 and 6h post-infection decreasing after the 24h post-infection. Our data, showed that the effect was mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an immediate early gene is primarily responsible.

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells.

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.

The involvement of oxidative stress in bovine herpesvirus type 4-mediated apoptosis.

Bovine herpesvirus type 4 (BHV-4) belongs to the gamma-2-herpesviruses of the Gammaherpesvirinae subfamily. BHV-4 has a worldwide distribution and has been isolated in a variety of clinical diseases as well as from healthy cattle. In this report we demonstrate that BHV-4 induces apoptosis in MDBK cells. In the early phases of apoptosis, cells show an increase in the intracellular level of reactive oxygen species, which is indicative of oxidative stress. This precedes DNA fragmentation, a hallmark typical of apoptosis. Cells were protected from apoptosis only by certain antioxidants (butylated hydroxyanisole and ebselen), whereas N-acetylcysteine turned out to be ineffective. Antioxidants that protected cells from apoptosis prevented oxidative stress but failed to block virus growth. These observations suggest that oxidative stress may be a crucial event in the sequence leading to apoptotic cell death but apoptosis is not required for the multiplication of BHV-4.

Effect of Pasteurella haemolytica outer membrane proteins on bovine neutrophils.

The major outer membrane proteins (OMPs) isolated from Pasteurella haemolytica induce alterations of the biological activity of bovine polymorphonuclear leukocytes (PMNs). A dose-dependent reduction of the capacity of adherence to nylon wool in vitro was observed. OMPs also acted as chemotaxins at concentrations between 5 and 20 microg/ml. Concentrations lower than 5 microg/ml did not give considerable results. Preincubation with 5 microg/ml of OMPs led to modifications in the values of the phagocytic index and of intracellular killing, which were found to be diminished with respect to controls.

Role of Pasteurella multocida porin on cytokine expression and release by murine splenocytes.

The aim of this study was to verify whether Pasteurella multocida porin can affect the expression and release of IL-1alpha, IL-6, TNF-alpha, IL-4, IFN-gamma, IL-10 and IL-12 by murine splenocytes in vitro. P. multocida porin and lipopolysaccharide (LPS) were able to induce the release of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 in a dose-dependent fashion. The greatest release of these cytokines was obtained using P. multocida porin at a concentration of 5 microg ml(-1) and LPS at a concentration of 1 microg ml(-1). The time-courses of release showed that P. multocida LPS was able to stimulate the production of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 earlier than porin and at a greater rate. No effect was observed on IL-4 and IL-10 release under the same experimental conditions. P. multocida porin and LPS were also able to up-regulate the mRNA expression of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 p40. Our findings suggest that P. multocida porin is able to modulate inflammatory and immunological responses by affecting the release of several cytokines and the expression of their genes.

Natural caprine herpesvirus 1 (CpHV-1) infection in kids.

Twenty-seven kids aged 5-7 days from a flock of 200 goats in which a high rate of abortion occurred died over a 2-month period. All showed hyperthermia, abdominal pain and anorexia. Two of the kids were examined post mortem. Ulcerative and necrotic lesions affected the whole intestine, and macroscopical changes were also observed in the lungs, urinary bladder and liver. Histologically, a severe necrotizing enteritis as well as thickening of the alveolar septa and necrotic bronchiolo-alveolitis were detected. Prominent microscopical lesions were also present in the liver, urinary bladder, spleen, thymus, mesenteric lymph nodes and kidney. Macrophages containing eosinophilic intranuclear inclusion bodies appeared to be the main inflammatory cell in all the organs examined. Ultrastructurally, herpesvirus particles were evident. Characteristic morphological features of type-A capsids (empty), type-C capsids (large core), and type-B capsids were observed. A virus was isolated in cell culture from all organs examined in the two kids. Cytopathogenic effects (rounding cells, syncytia, vacuoles, cell lysis) and acidophilic intranuclear inclusions typical of herpesvirus infection were observed. The virus was identified as caprine herpesvirus 1 (CpHV-1) by the polymerase chain reaction (PCR) technique and by serum neutralization

Modulation of anthracycline activity in canine mammary tumour cells in vitro by medroxyprogesterone acetate.

Failure of chemotherapy with anthracyclines as a result of drug resistance and toxicity is a major problem in the clinical management of neoplasia. The aim of the present study was to evaluate the activity of medroxyprogesterone acetate (MPA) as a chemosensitiser on anthracycline cytotoxicity. The study investigated whether such an effect could be related to an increase in lipid peroxidation, nitric oxide production, membrane fluidity and intracellular anthracycline concentration. The results showed that anthracyclines decreased nitric oxide production but increased membrane viscosity (polarisation constant) and lipid hydroperoxide formation in canine mammary tumour cells. Moreover, it was found that both drug-induced cytotoxicity and membrane viscosity increased in the presence of MPA. Conversely, lipid hydroperoxides decreased in MPA-supplemented cells. Medroxyprogesterone acetate did not show any effect on nitric oxide production. The two anthracyclines used (doxorubicin and idarubicin) showed differential intranuclear accumulation in canine mammary tumour cells, and MPA significantly modified intracellular concentration of anthracyclines.