Giuseppe Iovane

Methicillin-Resistant Staphylococci Isolated from Healthy Horses and Horse Personnel in Italy

Methicillin-resistant staphylococci (MRS) were isolated from nasal swabs of 56 of 159 (35.2%; 95% confidence interval [CI]: 27.9–43.2%) healthy horses. Two nasal swabs were collected from each horse; 43 of 159 (27%; 95% CI: 20.5–34.8%) of the cohort were colonized by MRS strains in 1 nostril, while in the remaining 13 of 159 (8.2%; 95% CI: 4.6–13.9%), different or identical MRS strains were isolated in both nostrils. Of the 29 humans in close contact with the horses tested, 4 (13.8%; 95% CI: 4.5–32.6%) were found to be carriers of MRS. All isolates were coagulase negative with the exception of 2 coagulase-positive MRS strains, Staphylococcus aureus and Staphylococcus pseudintermedius, both isolated from horses. To assay the methicillin resistance, a susceptibility test to oxacillin with standardized disk diffusion method, a PBP-2a latex agglutination test, and a methicillin resistance gene (mecA) polymerase chain reaction assay were performed. Pulsed-field gel electrophoresis patterns of isolates from horses and humans in close contact with the horses revealed similarity. The results suggest evidence of transmission between animals, from animals to humans, and vice versa.

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.

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.

Involvement of FOXO Transcription Factors, TRAIL-FasL/Fas, and Sirtuin Proteins Family in Canine Coronavirus Type II-Induced Apoptosis

n our previous study, we have shown that canine coronavirus type II (CCoV-II) activates both extrinsic and intrinsic apoptotic pathway in a canine fibrosarcoma cell line (A-72 cells). Herein we investigated the role of Sirtuin and Forkhead box O (FOXO) families in this experimental model using Nortern Blot and Western Blot analysis. Our results demonstrated that mitochondrial SIRT3 and SIRT4 protein expression increased from 12 and 24 h post infection (p.i.) onwards, respectively, whereas the nuclear SIRT1 expression increased during the first 12 h p.i. followed by a decrease after 36 h p.i., reaching the same level of control at 48 h p.i. Sirtuins interact with/and regulate the activity of FOXO family proteins, and we herein observed that FOXO3A and FOXO1 expression increased significantly and stably from 12 h p.i. onwards. In addition, CCoV-II induces a remarkable increase in the expression of TNF-related apoptosis-inducing ligand (TRAIL), while we observed a slight up-regulation of FasL/Fas at 36 p.i. with a decrease of both proteins at the end of infection. Furthermore, we found that virus infection increased both bax translocation into mitochondria and decreased bcl-2 expression in cytosol in a time-dependent manner. These data suggest that FOXO transcription factors mediate pro-apoptotic effects of CCoV-II, in part due to activation of extrinsic apoptosis pathway, while some Sirtuin family members (such as SIRT3 and SIRT4) may be involved in intrinsic apoptotic pathway. Moreover, these results propose that TRAIL is an important mediator of cell death induced by CCoV-II during in vitro infection.

INTEGRIN RECEPTORS PLAY A ROLE IN THE INTERNALIN B- DEPENDENT ENTRY OF Listeria monocytogenes INTO HOST CELLS

Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host’s E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (ΔinlA) carrying a deletion in the gene coding for inlA. The ΔinlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-β3- and anti-β1-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-β2- and anti-β4-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the β3- and β1-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes ΔinlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that β3- and β1-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells.

Expression of iron-related proteins during infection by bovine herpes virus type-1.

Bovine herpesvirus 1 (BHV‐1), a dsDNA animal virus, is an economically important pathogen of cattle and the aetiological agent of many types of disease. The efficient replication of a DNA virus is strictly dependent on iron since this metal plays a crucial role in the catalytic center of viral ribonucleotide reductase. Consequently, iron metabolism is an important area for virus/host interaction and a large body of evidence suggests that viral infection is potentially influenced by the iron status of the host. The aim of the present study was to address the effects of BHV‐1 on iron metabolism in Madin‐Darby bovine kidney (MDBK) cells at different times of post‐infection. For this purpose, cell viability, iron regulatory proteins (IRPs) activity and levels, transferrin receptor 1 (TfR‐1), ferritin expression and LIP were evaluated. Our data demonstrate that a productive BHV‐1 infection in MDBK cells determines an overall decrease of IRPs RNA‐binding activity without affecting their expression. As consequence of this modulation, an increased ferritin mRNA translation and a decreased TfR‐1 mRNA translation were also observed. Moreover, the LIP level was decreased following viral infection. These results are consistent with the hypothesis that by reducing the iron up‐take and by enhancing the sequestration of free iron, animal cells will limit the iron availability for virus proliferation. Therefore, the results presented herein support the view that iron metabolism could be critical for the interaction between DNA viruses, such as BHV‐1, and mammalian cells. Delineation of the interplay among pathogen and host may provide new antimicrobial agents. J. Cell. Biochem. 104: 213–223, 2008.

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis

In order to determine the ability of bovine herpesvirus type 1 (BHV‐1) to suppress apoptosis, we examined the effects of BHV‐1 infection on sorbitol‐induced apoptosis on Madin–Darby bovine kidney (MDBK) cells. BHV‐1 suppresses sorbitol‐induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV‐1), indicating that BHV‐1 has one or more anti‐apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis‐inhibiting and ‐promoting factors were analyzed on BHV‐1 infected cells during the process of sorbitol‐induced apoptosis. Our results revealed that the expression of bcl‐2 and bcl‐xL decreased after 5 and 3 h p.i., respectively; while bax and procaspase‐3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV‐1 has one or more genes encoding apoptosis‐inhibiting factors which interfere with the involvement of bcl‐2 gene family members and apoptotic pathway, depending upon caspase‐3, triggered by sorbitol. J. Cell. Biochem. 89: 373–380, 2003.

Characterization of enterotoxigenic E. coli (ETEC), Shiga-toxin producing E. coli (STEC) and necrotoxigenic E. coli (NTEC) isolated from diarrhoeic Mediterranean water buffalo calves (Bubalus bubalis).

Abstract Two hundred and twenty Escherichia coli isolates from 314 Mediterranean water buffalo calves less than 4weeks old affected by severe diarrhoea with a lethal outcome were characterized for the presence of the virulence factors LT, ST, Stx1, Stx2, haemolysins, intimin, CNF1, CNF2, CDT-I, CDT-II, CDT-III, CDT-IV, and F17-related fimbriae (F17a, F17b, F17c, F17d). The prevalence of ETEC, STEC and NTEC were 1.8%, 6.8% and 20.9%, respectively. The ETEC isolates were all LT-positive and ST-negative. The STEC isolates were all Stx and intimin-positive, with Stx1 (80%) more frequent than Stx2 (27%). The NTEC isolates were all CNF and Hly-positive, with CNF2 (83%) more frequent than CNF1 (22%). Susceptibility assays to 11 antimicrobials displayed high rates of resistance (>30%) to antimicrobials tested. These data show that the most prevalent strains in diarrhoeic water buffalo calves were NTEC, mostly CNF2 and HlyA-positive, with strong associations CNF2/CDT-III and CNF2/F17c.

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. J. Cell. Biochem. 103: 221–233, 2008.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Promotes BHV-1 Infection in Mammalian Cells by Interfering with Iron Homeostasis Regulation

Mammalian cells require iron to satisfy metabolic needs or to accomplish specialized functions, and DNA viruses, like bovine herpesvirus 1 (BHV-1), require an iron-replete host to efficiently replicate, so that iron bioavailability is an important component of viral virulence. Cellular iron metabolism is coordinately controlled by the Iron Regulatory Proteins (IRP1 and IRP2), whose activity is affected by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a current and persistent environmental contaminant. Considering that TCDD enhances BHV-1 replication, herein we analyzed the effects of TCDD on iron metabolism during BHV-1 infection in MDBK cells, and presented evidences of a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Moreover, an up-regulation of transferrin receptor 1 (TfR1) and a concomitant down-regulation of ferritin were observed. This scenario led to an expansion of the labile iron pool (LIP) and induces a significant enhance of viral titer, as confirmed by increased levels of BHV-1 infected cell protein 0 (bICP0), the major transcriptional regulatory protein of BHV-1. Taken together, our data suggest that TCDD increases the free intracellular iron availability thereby promoting the onset of BHV-1 infection and rendering bovine cells more vulnerable to the virus.