In 2012 a US multistate outbreak of listeriosis was linked to ricotta salata imported from Italy, made from pasteurized sheep's milk. Sampling activities were conducted in Italy to trace the source ...of Listeria monocytogenes contamination. The cheese that caused the outbreak was produced in a plant in Apulia that processed semi-finished cheeses supplied by five plants in Sardinia. During an 'emergency sampling', 179 (23·6%) out of 758 end-products tested positive for L. monocytogenes, with concentrations from <10 c.f.u./g to 1·1 × 10⁶ c.f.u./g. Positive processing environment samples were found in two out of four processing plants. A 'follow-up sampling' was conducted 8 months later, when environmental samples from three out of six plants tested positive for L. monocytogenes and for Listeria spp. PFGE subtyping showed 100% similarity between US clinical strains and isolates from ricotta salata, confirming the origin of the outbreak. The persistence of strains in environmental niches of processing plants was demonstrated, and is probably the cause of product contamination. Two PFGE profiles from clinical cases of listeriosis in Italy in 2011, stored in the MSS-TESSy database, were found to have 100% similarity to one PFGE profile from a US clinical case associated with the consumption of ricotta salata, according to the US epidemiological investigation (sample C, pulsotype 17). However, they had 87% similarity to the only PFGE profile found both in the US clinical case and in 14 ricotta cheese samples collected during the emergency sampling (sample B, pulsotype 1). Sharing of molecular data and availability of common characterization protocols were key elements that connected the detection of the US outbreak to the investigation of the food source in Italy. Simultaneous surveillance systems at both food and human levels are a necessity for the efficient rapid discovery of the source of an outbreak of L. monocytogenes.
Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (
Culicoides
spp.) and is caused by Bluetongue virus (BTV). BTV is the type species ...of the
Orbivirus
genus within the
Reoviridae
family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in
Spodoptera frugiperda
(Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.
Abstract
Background
In literature, there are no standardized laboratory methods to detect formed biomass by colorimetric analysis. The purpose of this study was to compare three staining methods and ...two different wavelengths for determination of biofilm formation of Listeria monocytogenes (Lm) strains.
Methods
Three strains of Lm isolated from different origin were tested using 96 well polistirene plates at 12 °C and 30 °C, after incubation the wells were subjected to washing, detaching and staining with crystal violet (CV) at 0.2% and 2% (Panreac EU) in 95% ethanol and with Gram's crystal violet solution (Merck KGaA, Germany). The absorbance at 492nm and 540nm wavelengths was read using a spectrophotometer (SIRIO S, Seac, Firenze, Italia).
Results
The strains incubated at 12 °C displayed production of biofilm when stained with CV 2% and with Gram's crystal violet solution, both at 492 and 540 nm (with better evidence at 540 nm). If CV 0.2% was used to stain and reading at both optical densities there was evidence of weak or no biofilm production.
At 30 °C, the biofilm production was displayed at both temperature and with all the stains. For all the strains and for all the conditions tested, the absorbance was greater but not proportional using the Gram's crystal violet solution, versus the CV 0,2% and CV 2%, and absorbance was higher at 540nm versus at 492nm.
Conclusions
Results confirmed the lack of reproducibility of each of the method used to detect and quantify the biomass produced during a biofilm formation test in vitro and the absence of ratio between the different results obtained using different CV concentration and wavelengths for reading.
Key messages
Biofilm production at 12 °C could not be adequately detected staining the wells with CV 0,2%. Absorbance could be influenced by the solvent in the stain used (ethanol, methanol or phenol or mixtures). To obtain data for assessment of biomass formation, being the method characterized by poor reproducibility, the laboratory should use at least the same stain and wavelength.
Background
In 2019, case fatality from Listeria monocytogenes (Lm) was higher than the previous years confirming Lm as a major public health problem. Food is the main source of Lm infection and ...cross-contamination from the food-processing environment (FPEs) is the most common route of contamination. The aim of this work was to characterize FPEs Lm strains collected from the National Reference Laboratory (NRL) for Lm in order to evaluate circulating clonal complexes (CCs) and their environmental adaptation.
Methods
NRL Lm database actually consists of 798 FPEs sequenced strains. In silico MLST was evaluated. Sequences related to prevalent CCs were further investigated using Pasteur's cgMLST scheme and detecting metal and detergent resistance genes and stress survival islet (SSI).
Results
MLST identified 25 CCs and 4 singleton. Nine isolates gave inexact match with existing alleles and were not classified. CC9 (17.4%) and CC121 (14.2%) were the prevalent clones. CC9 cgMLST analysis revealed a relatedness among strains isolated from the same establishment type (meat or dairy), even if sampled in different regions. Indeed, CC121 cgMLST clustering analysis was strictly associated to the point of sampling. Regarding stress adaptation genes, SSI1 and brcABC genes were detected in 100% and 2.9% of the CC9 strains, respectively. Meanwhile, SSI2 and transposon Tn6188 were detected, respectively, in 100% and 97.3% of CC121 strains.
Conclusions
Results confirmed, in Italian FPEs, prevalence of hypovirulent Lm strains, as previously observed in Italian food. Tested isolates highlighted adaptation to low pH and high salt concentration (SSI1 genes for CC9), to alkaline and oxidative stress (SSI2 genes for CC121) and benzalkonium chloride tolerance (Tn6188 in CC121) resulting in a best survival under stress conditions. Interestingly, CC9 clustering denoted allelic proximity among strains isolated in different Italian regions paying attention to clones spreading at national level.
Key messages
Listeria monocytogenes CC9 and CC121 prevalence in Italian establishment confirms environment is the main route of food contamination at production level.
Prevalent food-processing environmental Italian Listeria monocytogenes strains denotes adaptation to environmental stress and clones spreading at national level.
Background
Studies checking antimicrobial resistance (AMR) of Listeria monocytogenes (Lm) are ongoing, coupled with disease surveillance. Lm resistant strains are reported and are increasing. The aim ...of the work was to investigate AMR of clinical strains of Lm collected from nine regions (mostly in Lombardy and Marche regions) from 2008 to 2020.
Methods
The dataset consisted of 233 Lm human strains collected from nine regions during the period 2008-2020. All the strains were tested using Sensititre™ Haemophilus Plate (Thermo Scientific, Milano, Italy). Results were expressed according to EUCAST breakpoints for Lm, S. pneumoniae and E. faecalis. Resistant strains were sequenced using Illumina platform, ABRicate was used to predict antimicrobial resistance genes and plasmids.
Results
Most of the strains were detected in Lombardy and Marche. The MIC results showed that 11 out of 233 strains were resistant, one to ampicillin, four to tetracycline and six to clarithromycin. Analysing the sequences, all the resistant strains showed to have a common antimicrobial resistant pattern (mprF, norB, FosX, (MLS)lin, lin and lmo0919). Genomic determinants for ampicillin and clarithromycin resistance were not detected. Indeed, only in 3 out of 4 resistant strains tetM was found. Meanwhile, plasmids were detected in 7 strains.
Conclusions
The distribution of the strains considered in this work did not reflect the real epidemiological situation in Italy, because their origin was mostly from two regions. The emergence of AMR also for Lm needs to be considered. Moreover, official breakpoints for Lm are not defined and to date limited to five antimicrobials excluding clarithromycin and tetracycline, where the pathogen showed resistance. Finally, the incongruence between phenotypic and genomic data suggests that the mechanisms involved in the resistance is not fully known especially for ampicillin. Further investigations are needed to improve the knowledge on Lm AMR.
Key messages
Monitoring AMR of Listeria monocytogenes is crucial, only effective drug in clinical cases should be used.
Further investigations are needed to explore the mechanisms involved in antimicrobial resistance of Listeria monocytogenes.
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). In this study, we followed over a 31-month period the natural transmission of JSRV in adult sheep ...and in their offspring. We established groups derived from flocks with either a high or low incidence of OPA and monitored virus transmission, clinical disease and macroscopic/microscopic lung lesions at necropsy. Results obtained show that (i) JSRV infection can occur perinatally or in the first few months of life in lambs and in adult sheep; (ii) only a minority of JSRV-infected animals develop clinical disease during their commercial lifespan; and (iii) JSRV is more readily detectable in peripheral blood leucocytes and lymphoid organs than in the lungs. These data support a model of opportunistic JSRV infection and tumorigenic conversion of type II pneumocytes/Clara cells in the lungs, while lymphoreticular cells serve as the principal virus reservoir.
OBJECTIVE
: Perfectionism, poor self-esteem and stress have all been described as important risk factors for eating disorders. The purpose of this study was to assess whether a stressful situation is ...significantly correlated to and associated with significantly higher levels of perfectionism, stress, quantifiable measures of eating disorders, and with significantly lower levels of self-esteem in a non-clinical sample.
METHOD
: Thirty-five female university students completed the Multidimensional Perfectionism Scale, the Rosenberg Self-Esteem Scale, the Perceived Stress Scale, and the Eating Disorder Inventory two times; once on an average university day and once on the day of an exam. Descriptive statistics and t-tests were calculated to verify whether a stressful situation was associated with a significant difference in levels of perfectionism, self-esteem, stress, and measures of eating disorders. Bivariate correlations were calculated for both the stress and non-stress situation, to observe how the dimensions of perfectionism, self-esteem, and stress were associated with measures of eating disorders.
RESULTS
: During the stress situation, the study participants had, on average, significantly higher levels of concern over mistakes, body dissatisfaction, drive for thinness, and perceived stress. Bivariate correlations revealed that during the stress situation perceived stress, cognitive variables and measures of eating disorders showed significant correlations with each other that were absent in the non stress situation.
DISCUSSION
: The results of the present study suggest that the dimensions of pathological perfectionism, low self-esteem, and perceived stress are related to an increase in dieting thoughts and dissatisfaction with body aspect in non-clinical women during a performance that could potentially challenge the perception of their self-esteem. The stressful situation can be interpreted as an experience of invalidation, which could explain the connection between cognitive constructs and behaviours related to eating disorders.
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