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.

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.

Short communication: Detection of human Torque teno virus in the milk of water buffaloes (Bubalus bubalis).

Forty-four raw milk and 15 serum samples from 44 healthy water buffaloes reared in Caserta, southern Italy, the most important region in Europe for buffalo breeding, were examined to evaluate the presence of Torque teno viruses (TTV) using molecular tools. Furthermore, 8 pooled pasteurized milk samples (from dairy factories having excellent sanitary conditions) and 6 Mozzarella cheese samples were also tested. Four of the cheese samples were commercial Mozzarella cheese; the remaining 2 were prepared with TTV-containing milk. Human TTV were detected and confirmed by sequencing in 7 samples of milk (approximately 16%). No TTV were found in serum, pooled pasteurized milk, or Mozzarella cheese samples. The samples of Mozzarella cheese prepared with TTV-containing milk did not show any presence of TTV, which provides evidence that standard methodological procedures to prepare Mozzarella cheese seem to affect viral structure, making this food fit for human consumption. The 7 TTV species from water buffaloes were identified as genotypes corresponding to the tth31 (3 cases), sle 1981, sle 2031, and NLC030 (2 cases each) human isolates. Although cross-species infection may occur, detection of TTV DNA in milk but not in serum led us to believe that its presence could be due to human contamination rather than a true infection. Finally, the mode of transmission of TTV has not been determined. Contaminated of the food chain with TTV may be a potential risk for human health, representing one of the multiple routes of infection.

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.

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.

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein.

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.

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.

Viral and Antibody Prevalence of Hepatitis E in European Wild Boars (Sus scrofa) and Hunters at Zoonotic Risk in the Latium Region.

Hepatitis E virus (HEV) is a member of the genus Hepevirus within the family Hepeviridae. Hepatitis E is recognized as a zoonosis, and swine and wild boars (Sus scrofa) are known reservoirs of HEV infection. The aim of this study was to investigate the presence of HEV in wild boars and hunters exposed to infection in central Italy (Latium region). During the hunting season, blood samples were collected from 228 wild boars and 20 hunters. The seroprevalence of HEV infection was determined using a commercial enzyme-linked immunosorbent assay, previously validated for use in man, pigs and wild boars. The estimated HEV seroprevalence in wild boars and in hunters was 40.7% (93/228; 95% confidence interval [CI] 34.4-47.1%) and 25% (5/20; 95% CI 6.1-43.9%), respectively. Liver samples were collected from the boars and HEV RNA was detected by nested reverse transcriptase polymerase chain reaction. Fifty-five of 164 tested wild boar liver samples (33.5%; 95% CI 26.2-40.7%) and three of 20 (15.0%; 95% CI 1.3-28.7%) tested human serum samples were positive for HEV RNA. Phylogenetic analysis of the nucleotide sequences obtained from PCR products indicated that the HEV strains present in wild boars and the human population all belonged to genotype 3, supporting the zoonotic role of wild boars in the spread of HEV infection.

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.

Interactions between bovine endothelial cells and Pasteurella multocida: association and invasion.

We investigated the association and the invasion of a bovine aortic endothelial cell (BAEC) line by Pasteurella multocida to study the potential role of internalized bacteria and possible intracellular survival during Pasteurella infections. Our data indicate that P. multocida is able to adhere to and to invade BAECs. The density of the bacterial population plays a defined role for an optimal mechanism of interaction between bacteria and cells, as does the incubation period of association and invasion. The optimal bacteria/cells ratio was found to be 100/1, while the optimal infection time was approximately 4 h of incubation. Bacterial internalization was dependent on microfilament and microtubule stability. The invasion ability of P. multocida in the presence of cytochalasin D was reduced by 60%; in the presence of colchicine it was reduced by 97% and in the presence of nocodazole it was reduced by 95%. Our data show that internalized P. multocida did not induce mortality of invaded endothelial cells. Some Pasteurella cells were able to survive and undergo exocytosis.

Role of Pasteurella multocida, Pasteurella haemolytica and Salmonella typhimurium porins on inducible nitric oxide release by murine macrophages.

The aim of this study was to verify whether Pasteurella haemolytica, P. multocida and Salmonella typhimurium porins could affect the inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release by murine resident peritoneal macrophages in vitro. We also compared their effect with that elicited by P. haemolytica, P. multocida and S. typhimurium lipopolysaccharide (LPS) whose biological activity is well known. Variations in NO release and iNOS mRNA expression due to variable concentrations of porins were recorded and compared. We also investigated the synergism between bacterial products and interferon gamma (IFN-gamma). With this aim cells were incubated with porins together with murine rIFN-gamma prior to assessing the presence of NO in the supernatant and mRNA analysis. Porins in themselves were not able to induce NO release by resident peritoneal macrophages. Incubation of macrophages with IFN-gamma in the presence of porins increased NO release, whereas incubation in the presence of the arginine analog N(G)-monomethyl-L-arginine (NMA) inhibited NO release. The greatest NO release was obtained using porins at a concentration of 5 microg/mL. Porins, together with IFN-gamma, were also able to upregulate the mRNA expression of iNOS. Our findings suggest that gram-negative porins are able to modulate inflammatory and immunological responses by affecting the release of NO and the expression of iNOS gene in activated macrophages.

Modification of membrane fluidity and depolarization by some anthracyclines in different cell lines.

One of the major problems in cancer treatment is the progressive desensitization of cancer cells to chemotherapeutic drugs. Several hypotheses have been advanced to explain this property of cancer cells. In recent years different calcium channel blockers and other chemosensitizing agents like synthetic progestins have been used to revert drug resistance. In our experiments we evaluated the effects of Doxorubicin and Idarubicin on membrane fluidity and depolarization using normal lymphocytes, chronic lymphatic leukemia lymphocytes, normal breast and hormone dependent breast cancer cells and cardiomyocytes. The drugs were used alone or in combination with Verapamil and Medroxyprogesterone acetate. We showed that MPA enhances DOXO and IDA biochemical effects, acting not only on the membrane lipid bilayer, but also on ion channels. VERA instead does not seem to act through the same mechanism.

Medroxyprogesterone acetate increases anthracyclines uptake in chronic lymphatic leukemia cells: role of nitric oxide and lipid peroxidation.

Anthracyclines are one of the most used drugs in the therapy of several malignant tumors. Unfortunately, its use is still limited by their cardio-toxicity and by the presence of cancer cells resistant to these drugs. In the present study we evaluated the ability of a chemo-sensitizer agent, MPA (Medroxyprogesterone Acetate), to modify anthracyclines intranuclear uptake in normal leukocytes (NL) and in chronic lymphatic leukemia leukocytes (CLL). Moreover we evaluated the role of lipid peroxidation and nitric oxide (NO) production on antracyclines activity and on their combination with MPA. Our data show that MPA significantly increases anthracyclines uptake only in CLL cells and decreases anthracyclines induced lipid peroxidation.

Effect of cis-diamminedichloroplatinum (II) on rat thymocyte membrane potential.

The effect of cis-diamminedichloroplatinum (II) (cisDDP) on rat thymocytes has been investigated. Changes of anisotropy constant and depolarization of the cell membrane have been determined. The effect of cisDDP on the anisotropy constant has been observed on treated animal thymocytes but not on treated isolated cell membranes. CisDDP inhibits KCl-induced depolarization in isolated rat thymocytes. This inhibiting effect is still present at a concentration as low as 5 microM and it is dose-dependent with a 9.6 microM ED50. CisDDP also boosts the inhibition of the polarization caused by 10 microM tetrodotoxin (TTX) and 10 microM doxorubicin (DOXO). The replacement of Na+ by choline and the inactivation of Ca2+ by EGTA, in the incubation medium, reduces the inhibiting effect of cisDDP on the KCl-induced depolarization. These results suggest that cisDDP induced alterations could be due to the interference of cisDDP with the activity of the ionic channels pertaining to Na+ and Ca2+. This interference is stronger for Na+ channels.

Evaluation of buffalo colostrum quality by estimation of enzyme activity levels.

A study to evaluate the value and potential use of colostral enzymes as markers for the evaluation of buffalo colostrum quality was conducted. The enzymes gamma-glutamyltransferase (GGT), lactic dehydrogenase (LDH), and alkaline phosphatase (ALP) in buffalo's colostrum were measured spectrophotometrically, and their activities were correlated with the gamma-globulin content. Gamma-globulin concentration was determined following the electrophoretic separation of the colostral proteins and quantified with a densitometer. Colostrum was obtained from 15 dams, soon after calving. Means, standard deviations, correlation coefficients, and degree of significance were calculated using the general linear model procedure of the Statistical Analysis Systems program. The activity of GGT in the colostrum was the highest, followed by LDH and ALP. A significant correlation (r = 0.86; P < 0.001) was seen between GGT and gamma-globulin concentration in the colostrum, supporting the suggestion of using this enzyme as a marker for the evaluation of colostrum quality.

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.

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.