Luisa De Martino

Prevalence of Antibodies to Selected Viral and Bacterial Pathogens in Wild Boar (Sus scrofa) in Campania Region, Italy

Serum samples were collected from wild boars (Sus scrofa) harvested during the 2005–2006 hunting season in Campania, southern Italy. Samples were tested for antibodies to Leptospira interrogan, Brucella spp., Salmonella spp., Aujeszky disease virus (ADV), porcine reproductive and respiratory stress syndrome virus (PRRSV), porcine parvovirus (PPV), classical swine fever virus (CSFV), and swine vesicular disease virus (SVDV). Of the 342 serum samples tested, 15 (4.4%) were seropositive to Brucella spp., nine (2.6%) were seropositive to L. interrogans, 66 (19.3%) were seropositive for Salmonella spp., 105 (30.7%) were seropositive for ADV, 27 (7.9%) were seropositive for PPV, and 129 (37.7%) were seropositive for PRRSV. All sera tested seronegative for SVDV and CSFV antibodies. These results, recorded for the first time in Campania, support the hypothesis that wild boar are reservoirs of certain infectious agents, but some infections in wild boars originate from their domestic counterparts.

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.

Effect of Salmonella typhimurium porins on biological activities of human polymorphonuclear leukocytes

The effect of Salmonella typhimurium porins on human polymorphonuclear leukocytes (PMNs) was studied. Labeled porins were shown to bind to the PMNs, and could be completely displaced by unlabeled porins. The binding caused modifications of membrane integrity and of the physico-chemical characteristics of the PMN surface, e.g. decreased oxidative burst, decreased hydrophobicity and altered cell morphology. The porins acted as both chemotaxins and chemotaxinogens. When PMNs were preincubated with porins their migration in the presence of commonly used chemoattractants (serum activated by zymosan or N-formyl-L-methionyl-L-leucyl-L-phenylalanine) was inhibited.

Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells. Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV. The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed.

Effect of low-nutrient seawater on morphology, chemical composition, and virulence of Salmonella typhimurium

The response of Salmonella typhimurium to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 32-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Lipid and carbohydrate content of bacterial cells rapidly declined within the first 16 days, while DNA and proteins exhibited a more gradual decline over the 32 days of starvation. In contrast, RNA content did not decrease appreciably upon nutrient starvation. Structural damage occurred especially after 16 days of starvation. After 32 days of nutrient deprivation, we recorded degenerative cellular forms, a coccoidal cell shape, a decrease in cellular volume, and the loss of the three-layered outer membrane. The morphological and structural alterations correlated with virulence in infected animals. We observed a decrease in virulence of S. typhimurium after 9, 16, and 32 days of starvation, reaching a maximal decrease after 32 days of nutrient deprivation. The decrease in virulence correlated to surface hydrophobicity alterations, adherence to eukaryotic cells, and phagocytosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induced autophagy in a bovine kidney cell line

The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK) cells where no signs of apoptosis were observed, but herein, analysis of MDBK cell morphology, in a large number of exposed cells, revealed some alterations, as expanded cytoplasm, an increase of intercellular spaces and many pyknotic nuclei. Hence, the aim of the current study was to elucidate the influences of dioxin on cell proliferation and cell death. We found that dioxin increased proliferation, as well as, activated cell death with autophagy, as we detected by increased amount of LC3-II, an autophagosome marker. Furthermore, formation of acidic vesicular organelles was observed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. These results were accompanied by down-regulation of telomerase activity, bTERT and c-Myc. Key tumor-suppressor protein p53 and expression of cell cycle inhibitor p21Waf1/Cip1 were activated after TCDD exposure. These changes occurred with activation of ATM phosphorylation in the presence of a decrease in Mdm2 protein levels. Taken together, these results support the idea that TCDD in MDBK cells, may exert its action, in part, by enhancing cell proliferation, but also by modulating the incidence of induced cell death with autophagy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells.

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

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.

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.

Bid cleavage, cytochrome c release and caspase activation in canine coronavirus-induced apoptosis

Abstract A previous study demonstrated that infection of a canine fibrosarcoma cell line (A-72 cells) by canine coronavirus (CCoV) resulted in apoptosis (Ruggieri et al., 2007). In this study, we investigated the cell death processes during infection and the underlying mechanisms. We found that CCoV-II triggers apoptosis in A-72 cells by activating initiator (caspase-8 and -9) and executioner (caspase-3 and -6) caspases. The proteolytic cleavage of poly(ADP-ribose) polymerases (PARPs) confirmed the activation of executioner caspases. Furthermore, CCoV-II infection resulted in truncated bid (tbid) translocation from the cytosolic to the mitochondrial fraction, the cytochrome c release from mitochondria, and alterations in the pro- and anti-apoptotic proteins of bcl-2 family. Our data indicated that, in this experimental model, both intrinsic and extrinsic pathways are involved. In addition, we demonstrated that the inhibition of apoptosis by caspase inhibitors did not affect CCoV replication, suggesting that apoptosis does not play a role in facilitating viral release.