Sviluppo e valutazione di un test di RT real time PCR per la identificazione di bovini persistentemente infetti da BVDV

BVDV, un RNA virus appartenente alla famiglia Flaviridae genere Pestivirus, è l'agente eziologico della Diarrea virale bovina/Malattia delle mucose. BVDV è responsabile di un complesso sistema di patologie che determina perdite rilevanti a carico degli allevamenti bovini. La precoce individuazione ed eliminazione dei soggetti persistentemente infetti (PI), principali serbatoi ed eliminatori del virus, gioca un ruolo chiave per il controllo di questo patogeno. Questa tesi si è posta l'obiettivo di sviluppare un nuovo protocollo di Real Time RT PCR per l'individuazione dei bovini PI valutando la sensibilità del test, i costi e i tempi di lavorazione su differenti campioni biologici. Sono stati testati campioni ematici, biopsie auricolari e bulbi piliferi provenienti da 10 bovini PI e da 10 bovini non PI.. La Real Tume RT PCR ha rilevato la presenza dell'RNA virale in tutte le tipologie di campione provenienti dai soggetti PI, nessuna fluorescenza è stata riscontrata nei campioni provenienti dai non PI. I dati offrono interessanti informazioni per la scelta del miglior metodo (semplice, economico, rapido, a basso stress per gli animali, applicabile su larga scala) per la raccolta di campioni biologici in corso di programmi di eradicazione sistematica della BVD. Particolarmente interessante è l'utilizzo dei bulbi piliferi per real-time RT-PCR che associa una sensibilità paragonabile al test eseguito sul siero di sangue ad una maggiore facilità di campionamento

Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria

Impaired Visual Inhibition in Amnestic Mild Cognitive Impairment

Objective.The pathophysiology of amnestic mild cognitive impairment (aMCI) and Alzheimer disease (AD) is still a matter of debate. Visual system might be precociously altered, especially for its cholinergic connections. We thus studied patients with aMCI compared to AD with paired-pulse flash-visual evoked potentials (paired-F-VEPs), a putative marker of cholinergic function. Methods. We enrolled 12 adult patients with aMCI and 12 with AD. 14 normal age- and sex-matched subjects acted as controls (HS). Stimuli were single flashes, with interspersed random flash pairs at critical interstimulus intervals (ISIs, 16.5 to 125 ms) with closed eyes. The "single" (unconditioned) F-VEP was split into a "main complex" (50 to 200 ms after the flash) and a "late response" (200 to 400 ms). As for paired stimulation, the "test" F-VEP emerged from electronic subtraction of the "single" F-VEP from the "paired"-F-VEP. Results. In the single F-VEP, P2 latency was prolonged in patients (aMCI and AD) compared to HS (p < .05). As to the paired F-VEPs, in aMCI the "late response" normal inhibition was abolished at ISIs 50-62.5 ms (p <= .016), compared to AD and controls. No changes were detected for the "main complex". Conclusions. Paired-F-VEPs demonstrate a defective neural inhibition in the visual system of patients with aMCI at critical intervals. It may represent a compensatory mechanism against neuronal loss, the failure of which may be involved in AD development. Paired-F-VEPs may warrant inclusion in future preclinical/clinical studies, to evaluate its potential role in the pathophysiology and management of aMCI

A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV- 1), an important pathogen of cattle worldwide. The assay was based on conserved 5’UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step

Characterization of D-17 Canine Osteosarcoma Cell Line and Evaluation of Its Ability to Response to Infective Stressor Used as Alternative Anticancer Therapy

Osteosarcoma (OSA) is a rare cancer both in human and dog although the incidence rate in dogs is 27 times higher than in human. Many studies employed D-17 as cell line for in vitro test to evaluate conventional anticancer therapies; however, little is known about D-17 cell line. The aim of our study was to evaluate the basal level of gene expression of pivotal molecules in the innate immune response and cell cycle regulation and to establish the ability of this cell line to react to Salmonella typhimurium (ST) infective stressor. IL15, IL10, iNOS, TLR5, CD14, PTEN and IL18 were expressed in an inconsistent manner among experiments. The other genes under study were expressed in all samples. ST showed ability to penetrate D-17 causing pro-inflammatory response. Our results outline the expression in D-17 of important genes involved in innate immune response. These results provide important data on D-17 basal gene expression profile useful for in vitro preliminary evaluation of new therapeutic approaches

Emergency management of the individuals potentially contaminated by radioactive material and management of the hospitalization path

The organization that has to face the radiological emergencies in a nuclear disaster or terroristic attack must be carefully planned. In the hospitals with a level one Emergency Department, it is possible to draw a pathway for the contaminated patients, based on internal resources and on the recommendations of the nuclear medicine associations (as AIMN – Associazione Italiana di Medicina Nucleare; SNM – Society of Nuclear Medicine). The "Santi Antonio e Biagio e Cesare Arrigo" hospital in Alessandria is a modern hospital and is the reference of an extended area, for its many highly specialized departments. In this background, and in a hospital with a level one Emergency Department, there are three qualified departments, able to play a primary role in the emergency management of the individuals potentially contaminated by nuclear materials: Emergency Department, Nuclear Medicine Unit and Health Physics Unit. Therefore the Alessandria Hospital is suitable to admit patients potentially contaminated by radioactive material, to determ i n e the level of contamination, to decontaminate them in the emergency decontamination area, to hospitalise and treat them in the nuclear medicine rooms

Molecular Characterization of CF33 Canine Cell Line and Evaluation of Its Ability to Respond against Infective Stressors in Sight of Anticancer Approaches

Spontaneous mammary tumors are the most frequent neoplasms in bitches and show similarities with human breast cancer in risk factors, clinical course, and histopathology. The poor prognosis of some cancer subtypes, both in human and dog, demands more effective therapeutic approaches. A possible strategy is the new anticancer therapy based on immune response modulation through bacteria or their derivatives on canine mammary carcinoma cell lines. The aim of the present study was to analyze the CF33 cell line in terms of basal expression of immune innate genes, CXCR4 expression, and interaction with infectious stressors. Our results highlight that CF33 maintains gene expression parameters typical of mammary cancer, and provides the basal gene expression of CF33, which is characterized by overexpression of CXCR4, CD44, RAD51, LY96, and a non-continuous expression of TP53 and PTEN. No mutations appeared in the CXCR4 gene until the 58th passage; this may represent important information for studying the CXCR4 pathway as a therapeutic target. Moreover, the CF33 cell line was shown to be able to interact with Salmonella Typhimurium (ST) (an infective stressor), indicating that these cells could be used as an in vitro model for developing innovative therapeutic approaches involving bacteria

Emergency management of the individuals potentially contaminated by radioactive material and management of the hospitalization path

The organization that has to face the radiological emergencies in a nuclear disaster or terroristic attack must be carefully planned. In the hospitals with a level one Emergency Department, it is possible to draw a pathway for the contaminated patients, based on internal resources and on the recommendations of the nuclear medicine associations (as AIMN &ndash; Associazione Italiana di Medicina Nucleare; SNM &ndash; Society of Nuclear Medicine). The &ldquo;Santi Antonio e Biagio e Cesare Arrigo&rdquo; hospital in Alessandria is a modern hospital and is the reference of an extended area, for its many highly specialized departments. In this background, and in a hospital with a level one Emergency Department, there are three qualified departments, able to play a primary role in the emergency management of the individuals potentially contaminated by nuclear materials: Emergency Department, Nuclear Medicine Unit and Health Physics Unit. Therefore the Alessandria Hospital is suitable to admit patients potentially contaminated by radioactive material, to determ i n e the level of contamination, to decontaminate them in the emergency decontamination area, to hospitalise and treat them in the nuclear medicine rooms

Antimicrobial and Immunomodulatory Potential of Cow Colostrum Extracellular Vesicles (ColosEVs) in an Intestinal In Vitro Model

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Is 2,3,5-pyrroletricarboxylic acid in hair a better risk indicator for melanoma than traditional epidemiologic measures for skin phenotype?

International audienceThis study aims to assess type of melanin as a risk indicator for skin tumors, in a sample of melanoma cases and controls within a larger multicenter study (Helios 2), held in Europe and South America in 2001-2002. In each case and control, the melanin content in hair was assessed by three methods: 1) the amount of 2,3,5-pyrroletricarboxylic acid (PTCA); 2) the absorbance ratio with ultraviolet spectroscopy; and 3) the spectra of near-infrared spectroscopy. Statistical analysis was performed in a Bayesian setting, defining priors for confounders and effect modifiers from the larger study data set. Subjects with values of PTCA of less than 85 ng/mg carried an increased risk (26 vs. seven discordant pairs: odds ratio = 4.4, 95% confidence interval: 1.52, 14.54), adjusted by hair color, eye color, and number of nevi (n = > or =40). The absorbance ratio showed a weaker and nonsignificant odds ratio of 1.5. After correction by misclassification, near-infrared spectroscopy was associated with an odds ratio of 2.3 (95% confidence interval: 1.36, 4.22). The amount of PTCA is thus a strong and independent risk indicator for melanoma. Incorporating PTCA determination into epidemiologic studies is therefore recommended