Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy).

Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates.

Short communication: In vitro antimicrobial susceptibility of Mycoplasma bovis isolates identified in milk from dairy cattle in Belgium, Germany, and Italy.

The objective of this study was to assess the in vitro antimicrobial susceptibility of 73 isolates of Mycoplasma bovis isolated from milk of dairy cattle herds of Belgium, Germany, and Italy. Minimal inhibitory concentration (MIC) values were determined by the microbroth dilution method for the following antimicrobials: erythromycin, spiramycin, tilmicosin, tylosin, lincomycin, enrofloxacin, doxycycline, oxytetracycline, florfenicol, and tiamulin. Macrolides, florfenicol, oxytetracycline, and enrofloxacin, were chosen because they represent antimicrobials families commonly used in several countries for treatment of M. bovis, and their MIC values in cattle population are reported in several studies, allowing a comparison with previous data. Doxycycline and tiamulin were selected to assess the susceptibility of M. bovis to new antimicrobials, because they are not registered in the European Union for the treatment of dairy cattle. Among the agents of the different antimicrobial classes, the macrolides showed the highest concentration to inhibit 90% of isolates (MIC90), all above the highest concentration tested: >8µg/mL for erythromycin, >16µg/mL for spiramycin, and >32µg/mL for tilmicosin and tylosin. Also the MIC90 of lincomycin was above the highest concentration tested (>32µg/mL), but the distribution of the MIC values was almost perfectly bimodal: 41 isolates had a MIC ≤0.5µg/mL and 30 isolates >32µg/mL. Oxytetracycline had a 2-fold higher concentration to inhibit 50% of isolates (2 vs. 0.5µg/mL) and 1-fold higher MIC90 (4 vs. 2µg/mL) than doxycycline. Enrofloxacin and florfenicol had both a MIC90 of 2µg/mL, whereas tiamulin had a MIC90 of 0.5µg/mL. Significant differences on the MIC values were found among the 3 countries for several antimicrobials: compared with Germany, Belgium and Italy showed significantly higher MIC for lincomycin, spiramycin, and tylosin, and lower for oxytetracycline and florfenicol. The Belgian isolates showed the lowest MIC for enrofloxacin compared with Germany and Italy. The MIC results obtained in our study suggest the presence of a high level of resistance of M. bovis isolates originating from milk to macrolides in all countries involved in this study. On the contrary, a low level of resistance was found against the antimicrobials that are not used in cattle, such as tiamulin and doxycycline, highlighting a possible link between antimicrobial treatments and development of resistance in the studied M. bovis population.

Short communication: comparing real-time PCR and bacteriological cultures for Streptococcus agalactiae and Staphylococcus aureus in bulk-tank milk samples.

For more than 30 yr, a control plan for Streptococcus agalactiae and Staphylococcus aureus has been carried out in more than 1,500 dairy herds of the province of Brescia (northern Italy). From 2010 to 2011, the apparent prevalence of Strep. agalactiae has been relatively stable around 10%, but the apparent prevalence of Staph. aureus has been greater than 40% with an increasing trend. The aim of this paper was to estimate and compare the diagnostic accuracy of 3 assays for the detection of Strep. agalactiae and Staph. aureus in bulk-tank milk samples (BTMS) in field conditions. The assays were a qualitative and a quantitative bacteriological culture (BC) for each pathogen and a homemade multiplex real-time PCR (rt-PCR). Because a gold standard was not available, the sensitivities (Se) and specificities (Sp) were evaluated using a Bayesian latent class approach. In 2012 we collected one BTMS from 165 dairy herds that were found positive for Strep. agalactiae in the previous 2-yr campaigns of eradication plan. In most cases, BTMS collected in these herds were positive for Staph. aureus as well, confirming the wide spread of this pathogen. At the same time we also collected composite milk samples from all the 8,624 lactating cows to evaluate the within-herd prevalence of Strep. agalactiae. Streptococcus agalactiae samples were cultured using a selective medium Tallium Kristalviolette Tossin, whereas for Staph. aureus, we used Baird Parker modified medium with added Rabbit Plasma Fibrinogen ISO-Formulation. In parallel, BTMS were tested using the rt-PCR. Regarding Strep. agalactiae, the posterior median of Se and Sp of the 2 BC was similar [qualitative BC: Se=98%, posterior credible interval (95%PCI): 94-100%, and Sp=99%, 95%PCI: 96-100%; quantitative BC: Se=99%, 95%PCI: 96-100%, and Sp=99%, 95%PCI: 95-100%] and higher than those of the rt-PCR (at 40 cycle threshold, Se=92%, 95%PCI: 85-97%; Sp=94%, 95%PCI: 88-98%). Also in case of Staph. aureus, the posterior medians of BC were generally higher than those of rt-PCR. In fact, although the Se of BC was slightly lower (rt-PCR at 40 cycle threshold, median Se=99%, 95%PCI: 97-100%, and qualitative BC, median Se=94%, 95%PCI: 87-99%), the Sp was much higher (rt-PCR at 40 cycle threshold, median Sp=67%, 95%PCI: 38-97%; qualitative BC, median Sp=95%; 95%PCI: 76-100%). Our study confirms that BC and rt-PCR are reliable diagnostic tools to detect Strep. agalactiae and Staph. aureus, and rt-PCR results should be confirmed by BC carried out on BTMS and possibly on composite milk samples.

Isolation of Mycobacterium caprae (Lechtal genotype) from red deer (Cervus elaphus) in Italy.

During tuberculosis (TB) surveillance, 53 hunted red deer (Cervus elaphus) were collected to determine whether TB was present in free-ranging animals from an Italian alpine area. Samples (lungs, liver, intestine, and lymph nodes) were cultured and analyzed by real-time PCR assay carried out directly on tissue. Mycobacterium caprae was isolated from small granulomatous, tuberculosis-like lesions in the liver of a 12-yr-old female. Identification of suspect colonies was done by PCR restriction fragment length polymorphism analysis of the gyrb gene, and genotyping was performed by spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat analysis. The isolated strain was genetically identical to strains isolated in the study area in 2001 from dairy cows imported from Austria and in 2010 from an indigenous cow. The genotype, called "Lechtal," is the most frequently detected in the TB outbreaks in Austria and Germany. The possibility that red deer act as a maintenance host of M. caprae between TB outbreaks could be not excluded. Despite the high red deer population density, the detection of only one infected red deer could suggest that the wildlife management measures applied in the study area (prohibition of artificial feeding and secure removal of offal from hunted animals) may reduce the risk of TB spreading.

Swine vesicular disease in northern Italy: diffusion through densely populated pig areas.

At the end of 2006, a recrudescence of swine vesicular disease (SVD) was recorded in Italy and the disease spread widely throughout the northern regions. Lombardy, a densely populated pig area, was most affected and the presence of the disease caused heavy economic losses to the entire pig industry. Although SVD is considered only moderately contagious, the epidemic in the north was characterised by a rapid spread of the condition. Numerous difficulties were encountered in eradicating it. Over the past decade, there has been a significant increase in the population of pigs in Lombardy, concentrated mainly in a few areas which were the most severely affected during the 2006 to 2007 SVD epidemic. Increases in both the pig population and animal movements, combined with weak biosecurity measures, increased the spread rate of the disease and hampered eradication activities.

Anatomy of the episodic buffer: a voxel-based morphometry study in patients with dementia.

In 2000 Baddeley proposed the existence of a new component of working memory, the episodic buffer, which should contribute to the on-line maintenance of integrated memory traces. The author assumed that this component should be critical for immediate recall of a short story that exceeds the capacity of the phonological store. Accordingly, patients with Alzheimer's dementia (AD) should suffer of a deficit of the episodic buffer when immediate recall of a short story is impossible. On the other hand, the episodic buffer should be somewhat preserved in such patients when some IR can occur (Baddeley and Wilson, 2002). We adopted this logic for a voxel-based morphometry study. We compared the distribution of grey-matter density of two such groups of AD patients with and of a group of age-matched controls. We found that both AD groups had a significant atrophy of the left mid-hippocampus; on the other hand, the anterior part of the hippocampus was significantly more atrophic in patients who were also impaired on the immediate prose recall task. Six out of ten patients with no immediate recall were spared at "central executive" tasks. Taken together our findings suggest that the left anterior hippocampus contributes to the episodic buffer of the revised working memory model. We also suggest that the episodic buffer is somewhat independent from the central executive component of working memory.

High-calcium mineral water as a calcium supplementing measure for post-thyroidectomy hypocalcemia.

AIM: The aim of this study was to test the possibility of enhancing blood calcium levels in totally thyroidectomized patients by supplementation with 1 L/d carbonate-bicarbonate-high-calcium mineral water. METHODS: This study enrolled 95 outpatients, totally thyroidectomized four months earlier, and hence treated with oral calcium and vitamin-D. At recruitment, ionized blood calcium was either below (Group A; N. 55) or above (Group B; N. 40, randomly divided in Group B1 [N. 20] and Group B2 [N.20]) the lower limit of the normal range (1.12 mmol/L). For one month, Group A was treated with 1 L/d high-calcium (483 mg/L) mineral water and continued the usual therapy with Ca and vitamin-D. In contrast, Group B1 and Group B2 substituted their Ca and vitamin-D therapy with 1 L/d high-calcium mineral water (Group B1) or 1 L/d of placebo mineral water (Ca:80 mg/L) (Group B2). RESULTS: After one month, a significant 7.5% increase in blood ionized-calcium levels was observed in Group A, no change in Group B1 and a significant drop below normality in Group B2 (Group B2 vs Group B1, P<0.001). Thereafter, 1 L/d of the high-calcium mineral water, given to Group B2 instead of placebo for an additional month, significantly enhanced ionized-calcium levels above the lower limit of normality (Group B2 vs Group B1, NS). CONCLUSION: These experiments show that calcium supplementation as 1 L/d of a high-calcium mineral water may efficaciously enhance blood calcium levels in thyroidectomized patients. This complementary treatment might at least in part contribute to the prevention and/or treatment of hypocalcemia and substitute vitamin-D and calcium therapies after thyroidectomy.

Swine vesicular disease surveillance and eradication activities in Italy.

Swine vesicular disease (SVD) was first observed in Italy in 1966, and was initially diagnosed as foot and mouth disease (FMD). The causative agent of SVD was classified as an Enterovirus within the family Picornaviridae. It was included in the list of diseases notifiable to the World Organisation for Animal Health (OIE) because of the similarity of its lesions to those produced by FMD; however SVD is often mild in nature and may infect pigs subclinically. During the last decade SVD has been persistently reported in Italy, and surveillance and eradication activities are in place. The central and northern parts of Italy have been designated SVD free since 1997, while the southern regions have not achieved disease-free status. However, occasional outbreaks of SVD have occurred in central and northern Italy and have been eradicated using rigorous control measures. Most recent SVD outbreaks in Italy have been subclinical; SVD can rarely be diagnosed now on the basis of clinical signs and it is necessary to use laboratory diagnosis. This paper examines the epidemiology of SVD in Italy, and considers the measures adopted in Europe for SVD control on the basis of current knowledge of the disease.

[Personality characteristics, defence mechanisms and binge eating in obese patients]. / Caratteristiche di personalità, meccanismi di difesa e binge in un gruppo di pazienti obesi.

AIMS: This study aimed to investigate defence mechanisms and personality characteristics in obese subjects. In particular, we compared the use of defence mechanisms in two groups: obese persons vs. normal weight subjects. We also compared the defence mechanisms and personality characteristics of two groups of obese subjects: those with Binge Eating Disorder vs. those without this disorder. Finally, we investigated the presence of possible differences linked to gender or to age of onset of obesity. METHODS: 93 obese subjects and 68 normal weight subjects were administered a test battery composed of the following self-complete questionnaires (in the Italian version): Binge Eating Scale, Response Evaluation Measure-71, Eating Disorder Inventory-2 and Minnesota Multiphasic Personality Inventory). RESULTS: Obese subjects appear to use specific defence mechanisms. A gender effect was found on the use of defence mechanisms, on the psychological characteristics associated to an Eating Disorder and on personality features. Obese subjects with Binge Eating Disorder showed a marked tendency to manifest anxiety and bulimic behaviour. Obesity with onset in adolescence was associated with the possibility of developing drug dependence. CONCLUSION: Specific defence characteristics and personality features in obese subjects should be taken into account in designing a slimming program.

Classical swine fever in wild boar in the Lombardy region of Italy from 1997 to 2002.

In 1997, outbreaks of classical swine fever occurred in wild boar and domestic pigs in the Italian province of Varese (Lombardy region). Statutory animal health measures were imposed, and a control plan aimed at preventing the virus from spreading outside the infected hunting zone of 108 km2 was implemented. However, in 1998, virologically positive wild boar were found outside this zone, including within Swiss territory. The infected zone was then enlarged to include the three other hunting areas of the northern province of Varese and the neighbouring Swiss territory. Italian and Swiss veterinary services initiated a joint policy to control the disease by hunting young wild boar and conserving older, immune animals. The percentage of virologically positive animals gradually decreased, the last being found in July 2000. The seroprevalence initially increased, but in 2000 and 2001 it decreased from 42.2 per cent to 8.8 per cent, indicating that the wild boar population had had no further contact with the virus.

Radical addition to isonitriles: a route to polyfunctionalized alkenes through a novel three-component radical cascade reaction

The reaction of aromatic disulfides, alkynes, and isonitriles under photolytic conditions affords polyfunctionalized alkenes--beta-arylthio-substituted acrylamides or acrylonitriles--in fair yields through a novel three-component radical cascade reaction. The procedure entails addition of a sulfanyl radical to the alkyne followed by attack of the resulting vinyl radical to the isonitrile. A fast reaction, e.g., scavenging by a nitro derivative or beta-fragmentation, is necessary in order to trap the final imidoyl radical, since addition of vinyl radicals to isonitriles seems to be a reversible process. The stereochemistry of the reaction is discussed, particularly with respect to the stereochemical outcome of related hydrogen abstraction reactions by the same vinyl radicals. The lower or even inverted preference for either geometrical isomer observed in our cases with respect to that encountered in hydrogen abstraction reactions is explained in terms of transition-state interactions and/or isomerization of the final imidoyl radical. The latter possibility is supported by semiempirical calculations, which show that the spin distribution in the imidoyl radical can allow rotation of the adjacent carbon-carbon double bond prior to beta-fragmentation.

Cascade radical reaction of 2-alkynyl-substituted aryl radicals with aryl isothiocyanates: a novel entry to benzothieno[2,3-b]quinolines through alpha-(Arylsulfanyl)imidoyl radicals.

The novel cascade radical reaction of 2-(phenylalkynyl)aryl radicals with 4-Y-phenyl isothiocyanates (Y = H, OMe, Me, Cl, CN) provides a useful one-pot protocol for the production of 8-Y-substituted (12) and/or 9-Y-substituted benzothieno[2,3-b]quinolines (11). The whole process entails primary formation of an alpha-(2-alkynylarylsulfanyl)imidoyl radical that undergoes smooth 5-exo-dig cyclization onto the alkynyl triple bond. The derived 1-phenylvinyl radical then exhibits six-membered cyclization onto the isothiocyanate ring, to give 11, and/or five-membered ipso-cyclization to an azaspiro intermediate, whose eventual rearrangement affords 12. The overall findings clearly showed that the relative proportion of the outcoming isomeric benzothienoquinolines 11 and 12 can be markedly affected by the nature of the original isothiocyanate substituent. Moreover, the findings also furnished the first chemical evidence that enhancing the electrophilic power of the employed radical can properly enhance the reactivity of aryl radicals toward isothiocyanates.

[Epidemiologic study of hepatitis C virus infections in the population monitored by the former Local Health Unit 10]. / Studio epidemiologico dell'infezione da HCV nella popolazione del comprensorio dell'ex USL 10.

During the first six months of 1994 serum samples from 726 patients were assayed for anti-HCV antibodies of which 114 where found to be seropositive. After excluding those belonging to those categories known to be "at risk", the 93 remaining patients were evaluated from a clinical and chemico-clinical point of view. The distribution of seropositivity compared to age showed that around 70% of this sample were aged between 51 and 80 years old. In clinical terms 30% of patients were asymptomatic, while over 40% presented chronic hepatitis and 16% suffered from cirrhosis. Mean levels of bilirubinemia, SGOT, SGPT, AFP and gamma-GT were generally above normal. In particular, over 90% of transaminase values were found to belong to WHO hepatotoxic classes 0-2; only a few cases showed a very high level of hepatic toxicity, while over 25% showed normal hepatic function.

Effect of bromocriptine on neurophysin secretion in pregnant women in labour.

In order to determine whether dopamine plays a role in the control of neuropituitary function in pregnant women during labour, blood levels of nicotine (NSN)- and estrogen (ESN)-stimulated neurophysins were measured in 119 women treated orally with placebo (n = 59, control group) or 5 mg bromocriptine, a potent dopaminergic receptor agonist (n = 60, experimental group). Serum samples were taken before drug ingestion (basal sample) and at delivery. The serum basal concentrations of NSN and ESN were similar in both groups of pregnant women in labour. At delivery, serum ESN levels were similar in all women regardless of the treatment, whereas NSN concentrations were significantly lower in the bromocriptine-treated women than in those who were given placebo. In additional experiments the effect of 5 mg bromocriptine on the serum concentrations of NSN and ESN was tested for 6 hours after drug ingestion in 10 healthy, non-pregnant women and in 8 women in the 3rd trimester of pregnancy. Bromocriptine did not modify the circulating levels of NSN and ESN in either of these 2 groups of women. Since NSN and ESN are thought to be associated with vasopressin and oxytocin, respectively, these results indicate that in non-pregnant women and in pregnant women during late pregnancy dopaminergic stimulation with a dopaminergic receptor agonist does not inhibit the release of either vasopressin or oxytocin during rest. In contrast, dopaminergic receptor stimulation appears to play an inhibitory role in the regulation of vasopressin, but not oxytocin secretion in pregnant women in labour.

[Urinary levels of dehydroepiandrosterone in a group of male patients with functional hyperprolactinemia]. / Livelli di deidroepiandrosterone urinario in un gruppo di pazienti di sesso maschile portatori di iperrprolattinemia di tipo funzionale.

On 22 male patients diagnosed as "functional hyperprolactinemia" (the Prolactin (PRL) basal value, was higher than the basal PRL means +/- 2 DS of a control group) we have measured the urinary excretion of Dehydroepiandrosterone (DHEA) mainly produced by adrenal cortex. Our results haven't shown no difference in the urinary excretion of DHEA values in hyperprolactinemic patients has been documented.

[Study of GH in patients suffering from sexual impotence and abnormal glucose tolerance test (author's transl)]. / Comportamento del GH in un gruppo di pazienti con intolleranza glucidica ed impotenza erettiva.

The correlation between dopamine-serotonin systems and sexual behaviour and the influence of these two amines on GH secretion is well known. We evaluated GH responses (maximum increase (delta) after OGTT and mean increase (delta M) after insulin test) in a group of 32 males suffering from erective impotence with impotence with abnormal reaction to a glucose tolerance test. Results were compared with those obtained in a group of 13 normal controls. No significant difference in basal values and dynamic responses between the two groups was present. Our data suggest that GH doesn't decrease the tolerance to glucose in these patients. The abnormal values observed during a glucose tolerance test may be due to some agent involved in the interactions between limbic system, ventral lateral and ventral medial hypothalamic nuclei and dopamine-serotonin systems. No influence by this system on GH secretion is evident.