Evidence of new sequence types of methicillin-resistant Staphylococcus pseudintermedius in Italy.

Staphylococcus pseudintermedius (SP) and methicillin-resistant SP (MRSP) is one of the most important veterinary pathogens in the dog. Herein, from a total of 126 S. pseudintermediusstrains, 23 MRSP (18%) were identified. Multi-Locus Sequence Typing (MLST) revealed that most of MRSP strains belonged to ST71 (26%), which have been already reported in Italy and other countries. Interestingly, nine new sequence types (39%), from 1053 up to 1061, were described for the first time. Moreover, the isolated MRSP strains showed relevant antibiotic resistance profiles. This report highlights the circulation of new sequence types of MRSP in Italy and underlines the need of a global epidemiological surveillance to limit the increasing spread of multidrug-resistant MRSPstrains worldwide, since they may represent a considerable concern for dog's health.

Biological investigations of essential oils extracted from three Juniperus species and evaluation of their antimicrobial, antioxidant and cytotoxic activities.

AIMS: To conduct biological investigations and to evaluate the antimicrobial and antioxidant activities of the essential oils (EOs) extracted from Juniperus communis, J. scopulorum and J. horizontalis; to screen their mechanisms of action by conducting the cell membrane permeability assay (CMP); and to determine the possible cytotoxicity of the three EOs against human neuroblastoma cells (SH-SY5Y). METHODS AND RESULTS: The antifungal activity was tested against four phytopathogenic fungi (Monilinia fructicola, Aspergillus niger, Penicillium expansum and Botrytis cinerea). The antibacterial activity was evaluated against two Gram-positive (G+ve) (Bacillus megaterium and Clavibacter michiganensis) and three Gram-negative (G-ve) bacterial strains (Pseudomonas fluorescens, P. syringae pv. phaseolicola and Xanthomonas campestris). Results showed that the three tested EOs have antifungal activity against M. fructicola and P. expansum and effective antibacterial activity against P. syringae pv. phaseolicola and B. megaterium. Moreover, the three EOs were evaluated for their ability to inhibit the growth of SH-SY5Y cells with MTT assay. J. communis EO was the more effective with an IC50 of 53·7 µg ml-1 . The antioxidant capacity of the three EO did not differ as measured by the DPPH assay. CONCLUSIONS: The three tested juniper EOs showed promising antimicrobial and antioxidant activity and cytotoxic effects against human neuroblastoma cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: The outfindings from this research showed promising antimicrobial effects of the three oils against the majority of the tested phytopathogens with a potential to utilize them as natural alternatives to synthetic drugs, the cause of global environmental problems, pathogen resistance and difficulty to control many post-harvest plant diseases.

Reactive oxygen metabolites in alpha-herpesvirus-seropositive Mediterranean buffaloes (Bubalus bubalis): a preliminary study.

In the present study on Bubalus bubalis of the Campania Region (Italy) the serum levels of derivatives of reactive oxygen metabolites (d-ROMs), anti-ROM and oxidative stress index (Osi) were evaluated. These data were then related to the seropositive status of the animals against alpha-herpesviruses, precisely Bubaline herpesvirus 1 (BuHV-1) and Bovine herpesvirus 1 (BoHV-1). Clinically healthy Mediterranean buffaloes were selected for this study. The serum samples of these animals were taken, and d-ROMs, anti-ROM and Osi were measured using commercially available tests. The preliminary data demonstrated that animals seropositive to both BuHV-1 and BoHV-1 present more oxidative stress than seronegative animals, as revealed by a significant increase in d-ROMs. Our results provide, for the first time, insight into the reac- tive oxygen species (ROS) modulation induced by the herpesvirus in Bubalus bubalis.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy from the pediatric perspective.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disease caused by mutations of the AutoImmune REgulator gene. The clinical spectrum of the disease encompasses several autoimmune endocrine and non-endocrine manifestations, which may lead to acute metabolic alterations and eventually life-threatening events. The clinical diagnosis is defined by the presence of at least two components of the classic triad including chronic mucocoutaneous candidiasis (CMC), chronic hypoparathyroidism (CH), Addison's disease (AD). Other common features of the disease are hypergonadotropic hypogonadism, alopecia, vitiligo, autoimmune hepatitis, Type 1 diabetes, gastrointestinal dysfunction. APECED usually begins in childhood. CMC is the first manifestation to appear, usually before the age of 5 yr, followed by CH and then by AD. The clinical phenotype may evolve over several years and many components of the disease may not appear until the 4th or 5th decade of life. The phenotypical expression of the syndrome shows a wide variability even between siblings with the same genotype. In view of this heterogeneity, an early diagnosis of APECED can be very challenging often leading to a considerable diagnostic delay. Therefore, clinicians should be aware that the presence of even a minor component of APECED in children should prompt a careful investigation for other signs and symptoms of the disease, thus allowing an early diagnosis and prevention of severe and life-threatening events. Aim of this review is to focus on clinical presentation, diagnosis and management of the major components of APECED in children particularly focusing on endocrine features of the disease.

Regulatory T-cell modulation by green tea in chronic lymphocytic leukemia.

Regulatory T cells (Tregs) are considered to be key immunomodulatory cells of the immune system and are increased in chronic lymphocytic leukemia (CLL). Rai stage 0 identifies patients with early stage CLL for which there is no effective intervention at the present time and a "wait and see" policy is usually adopted. Some biological and clinical studies have reported that green tea constituents, such as epigallocatechin-gallate (EGCG), have antitumor effects on hematologic malignancies including CLL. We report data on a clinical trial in which green tea extracts were given orally to 12 patients with stage 0 CLL and 12 healthy subjects. Ten patients and 10 controls completed the 6-month scheduled therapy. Two patients and 2 controls stopped therapy within 1 month because of tachycardia and epigastralgia. Eight out 10 evaluable patients (80 percent) showed a reduction of lymphocytosis and absolute number of circulating Tregs, as well. One patient (10 percent) had a stabilization of lymphocytosis and a reduction of Tregs, and 1 patient (10 percent) showed an increase of both lymphocytosis and Tregs. Only the non-responding patient progressed after 5 months from the end of green tea administration and chemotherapy was given. Interestingly, both IL-10 and TGF-beta serum levels declined throughout the green tea intake period, in both patients and controls. These data seem to indicate that green tea is able to modulate circulating Tregs in CLL patients with early stage of the disease. This can result in the control of lymphocytosis as well as in the prevention of disease progression.

Prevalence and antimicrobial susceptibility of salmonella in European wild boar (Sus scrofa); Latium Region - Italy.

The prevalence of Salmonella spp. infection was determined in 499 wild boars harvested during the 2010-2011 and 2011-2012 hunting seasons in the Latium Region of Italy. We conducted a microbiological assessment on faeces collected at slaughter and we examined serum samples for the presence of antibodies to Salmonella spp. by ELISA assay. Out of 383 serum samples examined, 255 (66.5%) were positive for Salmonella spp. antibodies. Overall, 10.8% (54/499) of the animals were positive by microbiological assessment. The Salmonellae most frequently isolated were S. enterica subsp. salamae II (24%), S. enterica subsp. Diarizonae III b (12.9%), S. enterica subsp. houtenae IV (11.1%) and S. Fischerhuette (7.4%); less common Salmonella isolates included S. Veneziana (5.5%), S. Napoli (5.5%), S. Kottbus (5.5%), S. Thompson (5.5%), S. enterica subsp. arizonae III a (3.7%), S. Toulon (3.7%), S. Burgas (1.8%), S. Tennelhone (1.8%), S. Ferruch (1.8%), S. choleraesuis (1.8%), S. Paratyphi (1.8%), S. Stanleyville (1.8%), S. Typhimurium (1.8%) and S. enterica subsp. enterica 4,5,12:1:- (1.8%). These isolates were tested against 16 antimicrobial agents and exhibited resistance to sulphonamides (92.5%), sulphonamides and thrimetroprim (14.8%), colistin (14.8%), streptomycin (18.5%), gentamycin (5.5%), tetracycline (5.5%), ceftiofur (3.7%), cefazoline (1.8%), cefotaxime (1.8%), nalidixic acid (1.8%), amoxicillin and clavulanic acid (1.8%) and ampicillin (3.7%). Our data, the first collected on this species in Italy, suggest that European wild boars are frequent carriers of antimicrobial-resistant Salmonellae and are likely involved in the transmission of antimicrobial resistance throughout the environment.

A new clinical approach: use of blood-derived stem cells (BDSCs) for superficial digital flexor tendon injuries in horses.

AIMS: In this study, we present an innovative therapy using stem cells that were obtained from the peripheral blood of racehorses affected by uninduced superficial digital flexor tendon (SDFT) injuries. MAIN METHODS: Blood-derived stem cells (BDSCs) were generated from the blood samples of three horses in the presence of macrophage colony-stimulating factor (M-CSF). The racehorses received a single autologous BDSC treatment, which resulted in the successful repair of the tendons injuries. KEY FINDINGS: The results demonstrated that the BDSCs injection into the damaged tendon stimulated the regeneration of normal tissue. Furthermore, a relationship may exist between the speed and the quality of new tissue formation and the welfare and management of the treated animals. SIGNIFICANCE: This study demonstrates that stem cell technology offers new tools for tissue repair that in many cases is considered incurable, and provides additional evidence that BDScs injections increase the speed and quality of the regeneration process in different animal tissues.

Noonan-like syndrome with loose anagen hair associated with growth hormone insensitivity and atypical neurological manifestations.

Noonan-like syndrome with loose anagen hair (NS/LAH; OMIM 607721), recently related to the invariant c.4A>G missense change in SHOC2, is characterized by features reminiscent of Noonan syndrome. Ectodermal involvement, short stature associated with growth hormone (GH) deficiency (GHD), and cognitive deficits are common features. We report on a patient with molecularly confirmed NS/LAH exhibiting severe short stature associated with GH insensitivity (GHI), and chronic complex tics, a neurological feature never described before in this syndrome. IGF1 generation test revealed only a blunted increase in IGF1 after exogenous GH treatment, revealing mild GH insensitivity associated with proper STAT5 activation. Most common causes of secondary tics in childhood were excluded.

Effect of dietary mannan-oligosaccharides on in vivo performance, nutrient digestibility and caecal content characteristics of growing rabbits.

To evaluate the effect of mannan-oligosaccharides (MOS) on in vivo performance, nutrient digestibility, fermentation characteristics and caecal microbial populations of rabbits, 144 thirty-five days old hybrid Hyla were equally divided into three groups, one of which was fed the same diet without additives (control group), one with antibiotics (colistin sulphate, 144 mg/kg; tylosin, 100 mg/kg; oxytetracyclin, 1000 mg/kg) and one with MOS (1 g/kg of diet). Mortality rate, live weight, feed intake and feed conversion ratio were recorded up to 62 days of age. At 60 days nutrient digestibility was measured by acid insoluble ash method. The caecal content of 10 rabbits per group was collected at 62 days and analysed for volatile fatty acids production, ammonia content and microbial count. Rabbits from the control group had a significantly (p < 0.01) lower body weight at 62 days (1638.9 g vs. 1779.4 g and 1862.5 g, respectively for the control, MOS and antibiotic groups) while the antibiotic group showed a higher (p < 0.05) feed intake than the control group (127.9 g/day vs. 109.3 g/day). Rabbits from the MOS group had a higher apparent digestibility of cellulose (34.27% vs. 29.61% and 27.49%, respectively for the MOS, control and antibiotic groups) and, as a consequence a higher level of acetate in the caecal content (39.93 mmol/l vs. 34.21 mmol/l and 23.09 mmol/l, respectively for the MOS, control and antibiotic groups). Caecal microflora of the MOS group rabbits also had a higher fermentative activity in respect of protein source, as demonstrated by the higher productions of branched chain fatty acids. MOS and antibiotics significantly reduced the colonies of Coliformis (2.32 vs. 3.20 vs. 2.40 logCFU/g, respectively for the MOS, control and antibiotic groups, p < 0.01). Mannan-oligosaccharides at 1 g/kg of diet can be used as an alternative to antibiotics during the rabbit's growth period.

A multicancer-like syndrome in a dog characterized by p53 and cell cycle-checkpoint kinase 2 (CHK2) mutations and sirtuin gene (SIRT1) down-regulation.

INTRODUCTION: We have investigated SIRT1, p53 and cell cycle-checkpoint kinase 2 (CHK2) gene dysfunction in a dog with a multicancer syndrome-like in order to evaluate their potential role in the determinism of the disease and to establish a possible correlation between SIRT1 transcript level and p53 expression status. MATERIAL AND METHODS: Blood sample and tumour samples from a pure breed English Setter dog with different tumours were used for this study. Nucleotide sequence analysis was performed with a DNA autosequencer in order to examine p53 and CHK2 mutations. In addition, the expression level of SIRT1 was quantified by Southern Blot analysis of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RESULTS: Cytological examination revealed five different tumours: a cutaneous sebaceous epithelioma, a cutaneous mast cell tumour, a testicular Sertoli cell tumour, an oral malignant melanoma, and a cutaneous squamous cell carcinoma. Sequencing analysis revealed the presence of a nucleotide substitution, (CGG>CAG) exon 7 of the p53 gene in DNA from peripheral blood mononuclear cells (PBMCs) as well as in the melanoma; whereas the other four cancers showed the loss of the wild-type allele. Furthermore, CHK2 mutation at codon 311 has been identified in the melanoma and sebaceous epithelioma. In addition, SIRT1 cDNA expression decreased in all tumour samples compared to cDNA SIRT1expression level in peripheral blood mononuclear cells (PBMCs) in the same dog. CONCLUSIONS: These results suggest that the germ line mutation of the p53 gene at codon 248 might be, at least, one cause of the multicancer syndrome-like in our dog; furthermore, we show a possible correlation between SIRT1 transcript level and p53 mutations status. The regulatory role of SIRT1 in tumour suppressor pathways suggests that the net effect seen may represent both direct and indirect downstream regulation and it is likely to depend on the presence or absence of functional p53.

Natural compounds in anti-leukaemic therapy: a review.

Human leukemia results from multiple mutations that lead to abnormalities in the expressions and functions of genes that maintain the delicate balance between proliferation, differentiation and apoptosis. Continued research on the molecular aspects of leukemia cells has resulted in the developments of several potentially useful therapeutic agents. Discovery of new cellular and/or molecular pathways enabling innate or acquired resistance of cancers to current chemotherapeutics to be overcome is therefore of crucial importance if one wants to efficiently combat those cancers associated with dismal prognoses. In this concern, natural compounds are regarded as new chemical entities for the development of drugs against various pharmacological targets, including cancer, and, above all, leukemia.

Synthesis and cytotoxic activity of new ß-carboline derivatives.

On the basis of harmine and 1-methoxy-canthin-6-one chemical structures, a series of novel 1,4-disubstituted and 1,4,9-trisubstituted ß-carbolines and tetracyclic derivatives were designed and synthesized. Cytotoxic activities of these compounds in vitro were investigated in a human tumor cell line panel. Almost all compounds demonstrated interesting cytotoxic activities in particular against prostate cancer cells PC-3 with IC50 in the low micromolar range. Compound X was found to be the most potent one with IC50 value of 8.0 µM; this suggests further studies with models of prostate cancer.

Targeting regulatory T cells for anticancer therapy.

Regulatory T-cells (Tregs) comprise a group of either thymically derived or peripherally induced suppressor CD4+ cells involved in the control of effector T-cells against both self- and foreign-antigens. They are found increased in tumor tissues and are thought to be involved in pathogenesis of cancer by providing tumors with a mechanism to evade immune detection and destruction. Despite the fact that mechanisms of Tregs regulation are still in progress, efforts are made aiming to develop approaches to deplete or inhibit tumor-associated Tregs function. This could lead to restore antitumor immunity and emerging strategies for therapeutic vaccination, and immunotherapeutic targeting of Tregs with specific drugs are underway.

Circulating regulatory T cells in "clinical" monoclonal B-cell lymphocytosis.

Regulatory T-cells (Tregs) constitute a small subset of cells involved in antitumour immunity and are generally increased in patients with chronic lymphocytic leukemia (CLL). No data is available on Tregs in monoclonal B-cell lymphocytosis (MBL), a disease entity characterized by less than 5000/microL circulating clonal B-cells in absence of other features of lymphoproliferative disorders. We used multicolour flow cytometry to evaluate the number of circulating Tregs in 56 patients with "clinical" MBL, 74 patients with previously untreated CLL and 40 healthy subjects. MBL patients showed a lower absolute number of Tregs, compared to CLL patients, but slightly higher than controls. Moreover, the absolute cell number of Tregs directly correlated both with more advanced Rai/Binet clinical stages and peripheral blood B-cell lymphocytosis. Of note, the absolute number of Tregs was found lower in MBL patients than in CLL patients staged as 0/A Rai/Binet. The study showed that Treg increase gradually from normal subjects to "clinical" MBL patients and are significantly higher in CLL patients as compared to MBL patients. Moreover, a significant direct relationship was found between higher Treg values and a higher tumor burden expressed by B-lymphocytosis or more advanced clinical stages. In light of this data, MBL seems to be a preliminary phase preceding CLL. The progressive increase of Treg numbers might contribute both to the clinical evolution of MBL to overt CLL and to CLL progression.

Analysis of apoptosis induced by Caprine Herpesvirus 1 in vitro.

It is known that Caprine Herpesvirus 1 (CpHV-1) causes apoptosis in mitogen-stimulated as well as not stimulated caprine peripheral blood mononuclear cells (PBMC). Initial experiments in Madin Darby bovine kidney (MDBK) cells revealed that CpHV-1 infection induced apoptotic features like chromatin condensation and DNA laddering. Thus, to characterize in more detail this apoptotic process, activation of caspase-8, -9 and -3 in MDBK cells CpHV-1 infected was investigated and demonstrated. In addition, CpHV-1 infection resulted in disruption of mitochondrial membrane potential, cytochrome c release and alterations in the pro- and anti-apoptotic proteins of Bcl-2 family. Proteolytic cleavage of poly(ADP-ribose) polymerases (PARP), confirming the activation of downstream caspases, was also observed. Our data indicated that a "cross-talk" between the death-receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway occurred in CpHV-1-induced apoptosis in vitro.

Verbena officinalis essential oil and its component citral as apoptotic-inducing agent in chronic lymphocytic leukemia.

We evaluated the pro-apoptotic activity of Verbena officinalis essential oil and of its main component citral, on lymphocytes collected from normal blood donors and patients with chronic lymphocytic leukemia (CLL). The number of apoptotic cells was greater in CLL patients than in healthy subjects at all different times of incubation (4, 8 and 24 hours) for samples treated with Verbena officinalis essential oil (A) and citral (B) as well vs controls at different concentrations (0.1% and 0.01%). The greater pro-apoptotic ability was shown by both essential oil of Verbena officinalis and citral at lower concentrations (after 4 h A 0.1%: 17.8% vs 37.1%; A 0.01%: 15.8% vs 52%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%; after 8 h A 0.1%: 23% vs 38%; A 0.01%: 22.2% vs 55%; B 0.1%: 32% vs 42.2%; B 0.01%: 22% vs 54.3%; after 24 h A 0.1%: 5% vs 20.7%; A 0.01%: 25.8% vs 47.2%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%). Patients carrying deletion 17p13 (p53 mutation) showed a reduced ability to undergo apoptosis with respect to patients with other genomic aberrations or normal karyotype. The proapoptotic activity of Verbena officinalis essential oil and citral is thought to be due to a direct procaspase 3 activation. These data further support evidence that indicate natural compounds as a possible lead structure to develop new therapeutic agents.

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.

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.

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases Bovine Herpesvirus type-1 (BHV-1) replication in Madin-Darby Bovine Kidney (MDBK) cells in vitro.

Dioxin-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental toxin of current interest. In the last years, higher levels of TCDD than those permitted in UE [European Commission. 2002. European Commission Recommendation 2002/201/CE. Official Gazette, L 67/69] were detected in milk samples from cow, water buffalo, goat, and sheep raised on some areas of Campania Region (South Italy). Dioxin often causes immunosuppression and might render the animal liable to viral infections. In addition, viral infections are able to alter the pattern of dioxin distribution in different organs of the exposed animals. Bovine Herpesvirus type-1 (BHV-1) is a widespread pathogen, which causes infectious rhinotracheitis and infectious pustular vulvovaginitis in cattle. Herein, we have studied the effects of TCDD and BHV-1 infection, in Madin-Darby Bovine Kidney (MDBK) cells, alone as well as in association, so as cellular proliferation, apoptosis, and virus replication. We have observed an increase in cell viability of confluent monolayers at low TCDD concentrations. TCDD treated cells demonstrated increased viability compared to controls as evaluated by MTT test. TCDD exposure increased cell proliferation but induced no changes on apoptosis. Cells exposed to TCDD along with BHV-1 showed a dose-dependent increase in cytopathy, represented by ample syncytia formation with the elimination of the cellular sheets and increased viral titer. These results suggest that TCDD increases viral replication in MDBK cells while BHV-1 further decreases viability of TCDD exposed cells. Since very low concentrations (0.01 pg/ml) are sufficient to augment BHV-1 titer, TCDD may contribute to reactivate BHV-1 from latency, leading to recurrent disease and increase virus transmission.