No abstract

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.

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.

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.

Extracellular Vesicles (EVs) are nano-sized double-lipid-membrane-bound structures, acting mainly as signalling mediators between distant cells and, in particular, modulating the immune response and inflammation of targeted cells. Milk and colostrum contain high amounts of EVs that could be exploited as alternative natural systems in antimicrobial fighting. The aim of this study is to evaluate cow colostrum-derived EVs (colosEVs) for their antimicrobial, anti-inflammatory and immunomodulating effects in vitro to assess their suitability as natural antimicrobial agents as a strategy to cope with the drug resistance problem. ColosEVs were evaluated on a model of neonatal calf diarrhoea caused by Escherichia coli infection, a livestock disease where antibiotic therapy often has poor results. Colostrum from Piedmontese cows was collected within 24 h of calving and colosEVs were immediately isolated. IPEC-J2 cell line was pre-treated with colosEVs for 48 h and then infected with EPEC/NTEC field strains for 2 h. Bacterial adherence and IPEC-J2 gene expression analysis (RT-qPCR) of CXCL8, DEFB1, DEFB4A, TLR4, TLR5, NFKB1, MYD88, CGAS, RIGI and STING were evaluated. The colosEVs pre-treatment significantly reduced the ability of EPEC/NTEC strains to adhere to cell surfaces (p = 0.006), suggesting a role of ColosEVs in modulating host–pathogen interactions. Moreover, our results showed a significant decrease in TLR5 (p < 0.05), CGAS (p < 0.05) and STING (p < 0.01) gene expression in cells that were pre-treated with ColosEVs and then infected, thus highlighting a potential antimicrobial activity of ColosEVs. This is the first preliminarily study investigating ColosEV immunomodulatory and anti-inflammatory effects on an in vitro model of neonatal calf diarrhoea, showing its potential as a therapeutic and prophylactic tool.