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Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of -lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

FAECAL EVALUATION OF PDG IN A FEMALE OKAPI (NOME SCIENTIFICO) TO DEFINE OESTRUS CYCLE AND FERTILE WINDOW FOR REPRODUCTION PROGRAM OF CONSERVATION Rebecca Andreini1,3, Annalisa Zaccaroni1, Renato-Piccinini,2 Giuseppe Piscopo3,4 1 DIMEVET Bologna University 2 Parco Zoo Falconara Marittima 3 Delfini Bizantini Research Association 4 Veterinary Medicine Naples University The okapi (Okapia Johnstoni) number is declining rapidly in their native habitat and the species is listed as endangered in the IUCN red list. To increase conservation efforts and to improve the welfare of okapis under human care, the evaluation of endocrine profile is an essential part of the scientific research. The need to use a non-invasive matrix for the endocrine analysis in wild life species is linked to the difficulty in obtaining blood samples and to the necessity to not stress the animals with restraining and medical procedure that could affect their welfare. This study aims to evaluate the faecal concentration of pregnanediol-3-glucoronide (PDG) in faeces of a young female hosted in Falconara Marittima zoo, Italy. Faeces have been collected twice a week, immediately after the emission, by zoo staff from March to April 2019. Samples have been labelled and stored at – 20 °C till analysis. For hormones extraction 200 μg of faeces have been weighted and added with 2 ml of ethanol, vortexed at 100 rpm for 60 minutes, centrifuged at 2500 rpm for 20 minutes and dried. For PDG detection an ELISA kit has been used, following test procedure. As far as we know few information are available regarding oestrous cycle and reproduction of the okapi, so a comparison of our results with bibliographic data was difficult. Despite this, we analysed our results in order to define the different phases of the oestrous cycle and to estimate their length with the goal to identify the fertile window of the subject. The results will allow scientists to have a further knowledge of the physiology of this species and will represent an essential step to define best strategies for the management of the subjects under human care and to establish programmes to strengthen the effort in assisted reproduction.

Evolution has conserved "economic" systems that perform many functions, faster or better, with less. For example, three to five leukocyte types protect from thousands of pathogens. To achieve so much with so little, biological systems combine their limited elements, creating complex structures. Yet, the prevalent research paradigm is reductionist. Focusing on infectious diseases, reductionist and non-reductionist views are here described. The literature indicates that reductionism is associated with information loss and errors, while non-reductionist operations can extract more information from the same data. When designed to capture one-to-many/many-to-one interactions-including the use of arrows that connect pairs of consecutive observations-non-reductionist (spatial-temporal) constructs eliminate data variability from all dimensions, except along one line, while arrows describe the directionality of temporal changes that occur along the line. To validate the patterns detected by non-reductionist operations, reductionist procedures are needed. Integrated (non-reductionist and reductionist) methods can (i) distinguish data subsets that differ immunologically and statistically; (ii) differentiate false-negative from -positive errors; (iii) discriminate disease stages; (iv) capture in vivo, multilevel interactions that consider the patient, the microbe, and antibiotic-mediated responses; and (v) assess dynamics. Integrated methods provide repeatable and biologically interpretable information. © 2017 Rivas, Leitner, Jankowski, Hoogesteijn, Iandiorio, Chatzipanagiotou, Ioannidis, Blum, Piccinini, Antoniades, Fazio, Apidianakis, Fair and Van Regenmortel.

Present work reports about the monitoring of heavy metals in different species housed in zoos. Starting from 2009 a collaboration with Falconara and Pistoia zoos started to monitor the level of heavy metals in animals that died because of natural causes or pathologies. Tissue samples were collected during necropsies, when all information concerning health status of the animal were also registered. Samples were analysed with ICP-OES technique and data were correlated to health status, age and any other information available on the animal. Obtained data underline a reduced exposure to heavy metal, that is not strange given the position of the zoo far from main street or any other potential source of pollution. Some particular cases are also revised. The project can be considered as a starting point for the definition of background values for heavy metals in different wildlife species, which can help in identifying possible risky exposure in free wildlife.

AIM: S. aureus is one of the most important causative agents of bovine mastitis and the clinical outcome can vary in relation to the strains involved in the mammary infection. The aim of this study, funded by Lombardy Region, was to develop a diagnostic protocol for the identification of genetic subtypes of S. aureus responsible for bovine mastitis, with particular virulence and diffusiveness. MATERIAL AND METHODS: S. aureus strains associated with intra-mammary infections were isolated from 8 "control" (< 4% of infected cows) and 8 "case" (> 28% of infected cows) herds for a total of 2015 cows included in the study. Milk samples were cultured with standard methods and the DNA was extracted from S. aureus strains using a protocol described in literature (Cremonesi et al., 2006). Twenty-four and 626 strains isolated from single quarters of "control" and "case" herds respectively, were analysed by both RS-PCR (Fournier et al., 2008) and multiplex PCR. The first method is based on the amplification of the 16S-23S rRNA intergenic spacer region, the multiplex PCR targets the genes encoding for enterotoxins and other virulence genes such as leukocidins and leukotoxins. A subset of 55 strains representative of "control" and "case" isolates was also analysed by ribotyping (automatic Riboprinter), by Identibac array tube system and by protein profiling using MALDI-TOF MS. RESULTS: The RS-PCR analysis of the 650 strains revealed 10 different profiles. In 5 out of 8 "case" herds this method identified the genotype GTB and GTB-variant indicated in literature (Graber et al., 2009) as characteristic of highly diffusive and pathogenic strains, while GTK, GTR_VI and a new genotype named GTbM circulated in the remaining 3 herds. Seven out of the 8 "control" herds, revealed only one S. aureus profile with a predominance of the GTS genotype. Except for 4 out of the 55 representative strains analysed, ribotyping gave a response similar to RS-PCR. Moreover, MALDI-TOF and Identibac analyses proved to be able to integrate the molecular profiling of S. aureus isolated in field. CONCLUSION: Our results confirmed that RS-PCR could be used as a rapid test for molecular typing of S. aureus strains isolated from bovine mastitis and could identify genetic subtypes with particular virulence. Ribotyping, MALDI-TOF and Identibac analyses showed their capability for molecular strains characterization, integrating RS-PCR data. Multivariate analysis by combining data from different techniques could be used to discover a simple panel of markers associated to S. aureus virulence and diffusiveness. BIBLIOGRAFY Cremonesi P., et al. (2006). J. Dairy Sci., 89:163-169; Fournier C., et al. (2008). Res. Vet. Sci., 85: 439-448; Graber HU., et al. (2009). J. Dairy Sci., 92(4) 1442-1451

Methicillin-resistant Staphylococcus aureus (MRSA) has been reported in human medicine as a cause of nosocomial and community-associated infections. In veterinary medicine, the microorganism has been identified in a wide range of animals and diseases, including dairy cows mastitis. Therefore, MRSA is considered an emerging threat with a high zoonotic potential. AIM - In the present study, we investigated the diffusion of MRSA in Italian dairy herds and the prevalence of infected animals at herd level. MATERIALS AND METHODS Quarter milk samples were aseptically taken from all lactating cows of 83 S. aureus infected herds and bacteriological analysis was performed by culture on blood-agar plates. The hemolytic, Gram-positiv and coagulase-positive colonies of growth were identified as S. aureus. At least four isolates morphologically representative for each herd were tested by oxacillin disk-diffusion method to detect methicillin-resistance. DNA of oxacillin resistant isolates was extracted and the presence of mecA gene was investigated by PCR. RESULTS - All oxacillin-resistant isolates were confirmed as MRSA by mecA positivity. Prevalence of S. aureus infected cows and frequency of MRSA isolation were registered at herd level. Overall prevalence of mammary infections by S. aureus ranged 0,7 - 62%: in 28 herds (33.7%) the prevalence was higher than 25%, while in 27 herds (32.5%) it was lower than 4.7%. Methicillin-resistant strains were detected in 11 farms (13,25%), all belonging to the low-prevalence group. In 5 out of such 11 herds, all S. aureus isolates were MRSA, while in the remaining 6 herds both MRSA and methicillin-susceptible S. aureus (MSSA) were present. CONCLUSIONS - Our data confirmed the potential risk of subclinical mastitis caused by MRSA and suggest a low diffusiveness of these strains in infected herds. This characteristic could reduce the zoonotic potential of bovine MRSA, but the real prevalence in dairy farms could be underestimated, since bacteriological analysis of milk is usually performed only when a clear problem of mammary infections affects the herd.

Chronic inflammation and reactive oxygen species (ROS) production induced by crystalline silica are involved in the development of silicosis and lung cancer pathogenesis. ROS can generate lipid peroxydation of cell membranes that can produce methylglyoxal (MG), a strong cell proliferation inhibitor and apoptosis inducer. MG is naturally removed by glyoxalase I (GI) and glyoxalase II (GII) through a glutathione (GSH) dependent mechanism. Therefore mRNA expression of glyoxalases is correlated to MG concentration and oxidative stress.evaluate oxidative stress induced by crystalline silica by glyoxalases mRNA expression and methylglyoxal concentrationIn bronchial epithelial cell culture (BEAS-2B), exposed to 50 microg/cm2 crystalline silica (Min-U-Sil 5), for 2, 6, 12, and 24 hours, GI and GII mRNA levels and MG intracellular concentration were measured respectively by Real-Time PCR and HPLC.Crystalline silica exposure induced a significant reduction in mRNA expression of glyoxalases and an increase of MG intracellular concentration.The results suggest a possible use of MG and mRNA expression of GI and GII as crystalline silica induced oxidative stress indicators.

Changes in time course of blood partial pressures of oxygen (PtcO2) and carbon dioxide (PtcCO2) before, during and after challenge with ultrasonically nebulized distilled water (UNDW) were evaluated in 22 children with mild asthma in basal conditions, and after 8 weeks of therapy with inhaled nedocromil sodium at a daily dosage of 8 or 16 mg. PtcO2 and PtcCO2 were followed using transcutaneous O2 and CO2 monitoring system. All asthmatic subjects presented a significant decrease in PtcO2 and/or PtcCO2 (20% basal value) during or after challenge. After therapy, the decrease in PtcO2 and PtcCO2 was normalized in the group treated with 16 mg/day, whereas only a partial yet significant reduction in the decrease of O2 and CO2 was observed in the group assuming 8 mg/-day. These data indicate that inhaled nedocromil is effective in treating bronchial hyperresponsiveness in childhood and that the dose required to achieve this effect is of 16 mg/day.

Naphthalene, the most volatile polycyclic aromatic hydrocarbon (PAH), was recently classified as possible human carcinogen by International Agencies for Research on Cancer Humans may be exposed to naphthalene from a wide variety of sources, including occupation, environment, personal habits. We assessed urinary excretion of 1-naphthol (1-NAF), biomarker of naphthalene exposure, in non-occupationally exposed subjects.Urinary 1-NAF, 1-hydroxypyrene (1-OHP), biomarker of exposure to pyrene and cotinine, biomarker of smoking habits, were measured in 104 adults (53 men, 51 women).1-NAF concentrations overlapped in males and females (median: men 0.35 Microg/g creat; women: 0.46 microg/g creat). Median concentration of 1-NAF was 6-fold higher in smokers compared to nonsmokers (respectively, 7.7 microg/g creatinine vs 1.3 microg/g creatinine). Between smokers, urinary cotinine was positively correlated to 1-naphthol (rho: 0.69; p0.01) and 1-OHP (rho: 0.53; p0.01). Higher 1-OHP concentrations were found in smokers (median: smokers 0.16 microg/g creatinine, not-smokers 0.05 microg/g creatinine;).In our study population, we found that 1-NAF excretion is much higher as compared to 1-OHP excretion. This is due to the ubiquitous presence of naphthalene in the environment. Smoking considerably increase the exposure to naftalene.

Background: To extract more information, the properties of infectious disease data, including hidden relationships, could be considered. Here, blood leukocyte data were explored to elucidate whether hidden information, if uncovered, could forecast mortality. Methods: Three sets of individuals (n = 132) were investigated, from whom blood leukocyte profiles and microbial tests were conducted (i) cross-sectional analyses performedat admission (before bacteriological tests were completed) from two groups of hospital patients, randomly selected at different time periods, who met septic criteria [confirmed infection and at least three systemic inflammatory response syndrome (SIRS) criteria] but lacked chronic conditions (study I, n = 36; and study II, n = 69); (ii) a similar group, tested over 3 days (n = 7); and (iii) non-infected, SIRS-negative individuals, tested once (n = 20). The data were analyzed by (i) a method that creates complex data combinations, which, based on graphic patterns, partitions the data into subsets and (ii) an approach that does not partition the data. Admission data from SIRS+/infection+ patients were related to 30-day, in-hospital mortality. Results: The non-partitioning approach was not informative: In both study I and study II, the leukocyte data intervals of non-survivors and survivors overlapped. In contrast, the combinatorial method distinguished two subsets that, later, showed twofold (or larger) differences in mortality. While the two subsets did not differ ingender, age, microbial species, or antimicrobial resistance, they revealed different immune profiles. Non-infected, SIRS-negative individuals did not express the high-mortality profile. Longitudinal data from septicpatients displayed the pattern associated with the highest mortality within the first 24 h post-admission. Suggesting inflammation coexisted with immunosuppression, one high-mortality sub-subset displayed high neutrophil/lymphocyte ratio values and low lymphocyte percents. A second high-mortality subset showed monocyte-mediated deficiencies. Numerous within- and between-subset comparisons revealed statistically significantly different immune profiles. Conclusion: While the analysis of non-partitioned data can result in information loss, complex (combinatorial) data structures can uncover hidden patterns, which guide data partitioning into subsets that differ in mortality rates and immune profiles. Such information can facilitate diagnostics, monitoring of disease dynamics, and evaluation of subset-specific, patient-specific therapies. © 2016 Chatzipanagiotou, Ioannidis, Trikka-Graphakos, Charalampaki, Sereti, Piccinini, Higgins, Buranda, Durvasula, Hoogesteijn, Tegos and Rivas.