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.

Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mam-How to cite this paper: Zoccola, R. , .95025 424 Advances in Microbiology mals (C.Re.Di.Ma) performed the first inter-laboratory ring trial with the aim to standardize a diagnostic biomolecular approach for DMV in Italy. Viral isolation is usually considered the "gold standard" for the definitive diagnosis of most pathogens, but it is not often feasible in DMV diagnosis, due to the poor preservation of virus-targeted tissues in stranded cetacean carcasses, as well as to the lack of appropriate sensitivity of cell lines towards DMV variability. Therefore direct viral detection on tissues by means of reverse transcription-PCR (RT-PCR) represents a valuable option for DMV infection's diagnosis. For detecting DMV in cetacean die-offs occurred in the Mediterranean basin since 2013, C.Re.Di.Ma developed an RT-PCR based method targeting to a 287 bp fragment of DMV nucleoprotein (N) gene. With the purpose to evaluate its performances in terms of accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility), it was submitted to a ring trial. So, 12 Public Laboratories belonging to the Italian dead stranded marine mammals diagnostic network were asked to analyze a panel of 40 samples (positive and negative for DMV, using different dilutions of a viral suspension obtained from a cell culture supernatant of a DMV strain) with the aforementioned technique. Furthermore, we also aimed at comparing the accuracy of other 7 molecular methods routinely applied for DMV detection in Italy. For this purpose, the second panel of identical 40 DMV +ve and −ve samples was provided to Laboratories that routinely used DMV detection methods other than those developed by C.Re.Di.Ma, in order to be analyzed simultaneously with the method they usually applied. The C.Re.Di.Ma technique showed high accuracy [mean Se = 97.8% (95% CI 84.2% -99.3%), mean Sp = 98.1% (95% CI 72.5% -99.9%)] and very good precision [k combined equal to 0.91 (95% CI 0.87 -0.95)]. In conclusion, this study highlighted a satisfactory reliability of most of the molecular methods used in Italy for DMV detection.

Swinepox virus (SWPV) is responsible for sporadic acute poxvirus infections in swine worldwide, causing a pathognomonic eruptive proliferative dermatitis. Beside direct and congenital transmission, the pig louse Haematopinus suis acts as a mechanical vector and favors virus infection through skin lesions. Infections are generally described in domestic pigs, while only a few cases have been reported in wild boars, in Austria and Germany. In September 2022, SWPV infection was suspected at post-mortem examination of a wild boar piglet with characteristic lesions in Liguria, Northwest Italy. The piglet was heavily parasitized by swine lice (H. suis). SWPV was then confirmed by histological and molecular analyses. Possible viral co-infections were also investigated (African swine fever virus, classical swine fever virus, parvovirus, circovirus, Aujeszky’s disease virus and hepatitis E virus). This article describes gross and histopathologic features of SWPV infection, differential diagnosis, and potential vector-borne transmission to domestic pigs, presenting a brief review of the literature on the topic. SWPV infection is reported in wild boars in Italy for the first time. The finding of SWPV in a wild boar in an area with a very limited pig population may suggest the existence of a “wildlife cycle” in the area. Further investigations are needed to understand the real risk of transmission of SWPV to domestic pigs as well as the role of other arthropod vectors.

Canine Soft Tissue Sarcoma (STS) cell line A-72 has been largely employed for antiviral and antiproliferative studies. However, there are few information on their characteristics. Our aim was to evaluate A-72 expression level of genes and proteins involved in the innate immune response and cell cycle, their ability to respond to infective stressors and their possible use as a cellular model for anti-cancer studies in human and animal medicine. For this purpose, we evaluated the basal expression of immune-related, cell cycle and DNA repair genes on this cell line and tumoral tissues. A-72 ability to respond to a wild-type strain of Salmonella typhimurium was assessed. S. typhimurium showed ability to penetrate A-72 causing pro-inflammatory response accompanied by a decrease of cell viability. IL10 and IL18 genes were not expressed in A-72 while CXCL8, NOS2, CXCR4 and PTEN were highly expressed in all samples and TP53 was slightly expressed, as shown in human STS. Our results outline the ability of A-72 to respond to a bacterial agent by modifying the expression of important genes involved in innate immune response and provide a useful model for in vitro evaluation of new therapeutic approaches that could be translated into the human oncology.