The Characterisation of Lactococcus garvieae Isolated in an Outbreak of Septicaemic Disease in Farmed Sea Bass (Dicentrarchus labrax, Linnaues 1758) in Italy.

In aquaculture, Lactococcus garvieae is a common fish pathogen that can cause significant economic losses in several fresh and saltwater species. Despite the extensive range of hosts, L. garvieae infection in sea bass (Dicentrarchus labrax) has rarely been reported. During the summer of 2023, an outbreak occurred in an inland farm in the Gulf of Follonica (Tuscany, Italy). Fish of various sizes were affected, showing apathy, inappetence, erratic swimming and eye lesions, while the mortality was low (2-3% per month). Anatomopathological examinations suggested a septicaemic infection characterised by melanosis, diffuse redness (skin and fins), paleness (gills and internal organs), haemorrhages and splenomegaly. Seventy swabs from the viscera of 14 subjects were collected and colonies similar to Streptococcus spp. grew from all the samples. Lactococcus garvieae was identified via the biochemical tests, API20STREP, MALDI-TOF, 16S rDNA and whole genome sequencing. Genetical characterisation revealed remarkable differences between this isolate and the strains previously isolated in Italian fish farms. Feed treatments with flumequine and erythromycin were ineffective. Considering the limited effects of antimicrobials, preventive measures, such as vaccination and biosecurity, should be implemented.

Tick-Borne Encephalitis, Lombardy, Italy.

Tick-borne encephalitis was limited to northeast portions of Italy. We report in Lombardy, a populous region in the northwest, a chamois displaying clinical signs of tickborne encephalitis virus that had multiple virus-positive ticks attached, as well as a symptomatic man. Further, we show serologic evidence of viral circulation in the area.

Is SARS-CoV-2 a Concern for Food Safety? A Very Low Prevalence from a Food Survey during the COVID-19 Pandemic in Northern Italy.

In 2019, SARS-CoV-2 was identified as the cause of an easily transmissible disease that was declared as a world pandemic. Foodborne transmission was never reported. However, early studies suggested that food could be involved in SARS-CoV-2 entry in the human gastrointestinal tract leading to possible infection, and highlighting the importance of further studies to inspect possible issues linked to food consumption. In this perspective, this work aimed at monitoring SARS-CoV-2 presence in some food and mains water samples in Northern Italy during the COVID-19 pandemic (2020-2022). A total of 1806 foods, 112 mains water samples, and 580 swabs on meat and dairy product surfaces were analyzed for SARS-CoV-2 RNA detection by Real-time PCR. All the analyzed samples were negative to viral RNA detection with the exception of one vegetable sample. Even if data on foodborne coronavirus transmission suggested a limited importance of this pathway, the impact of the current pandemic in Northern Italy deserved a rigorous investigation to rule out such possibility. Indeed, gaining insight on all SARS-CoV-2 possible transmission pathways, including the foodborne route, seemed of interest to maintain consumers' confidence and trust in food safety, and for the effective management of the current, and future, possible pandemics.

Preparation of Powdered Infant Formula: Could Product's Safety Be Improved?

The recent outbreak of Salmonella Agona linked to the consumption of infant formula (powdered formula) has rekindled the attention about the correct procedures for preparation and use of these products. International guidelines have already been published so far, particularly in association with Cronobacter sakazakii in early 2000s. FAO/WHO suggested to reconstitute formula with water at no less than 70°C. We therefore contaminated powdered formula with low levels of Salmonella spp and C sakazakii to evaluate the pathogens inactivation during the formula preparation using water at 70°C. In these conditions we observed a survival of both pathogens, indicating that the suggested recommendations may be not enough to guarantee the safety of this product. Higher temperatures are needed to reduce the biological risk, even if it may be not easily realized in actual domestic conditions. Moreover, the impact on the nutritional value of reconstituted formulas should be evaluated.

Listeria monocytogenes in Gorgonzola cheese: Study of the behaviour throughout the process and growth prediction during shelf life.

As reported on RASFF's portal, in the first 9months of 2016, a total of 13 "alerts/information for attention" were issued concerning the presence of Listeria monocytogenes in mould cheeses throughout Europe. This study analyzes the behaviour of L. monocytogenes in Gorgonzola cheese, a typical Italian soft blue-veined cheese, when contaminated at different time points. In the first challenge test, the pasteurized milk was contaminated and the complete cheese manufacture (cheesemaking, ripening) and shelf life was simulated. After a decrease during the first days of the cheesemaking, the pH remained constant for 35days (5weeks) and then it increased rapidly reaching the final values of 6.8±0.02 in the core and 5.8±0.4 on the rind. At the same time, the pathogen concentration decreased (about 2logCFU/g), although during the last week a rapid pathogen growth was observed after the rise in pH values. When the cheese was stored at thermal abuse condition (8-12°C), the pathogen concentration on the rind was 4.8±0.3 log CFU/g and after 66days (about 9weeks) no significant difference (p>0.05) was observed; whereas, a growth from 5.4±0.4 to 7.1±0.5logCFU/g was observed in the core. A second challenge test was performed using three batches of commercial slices of Gorgonzola cheese inoculated by L. monocytogenes and stored at 8°C. The maximum specific growth rates (µmax, 1/h) of L. monocytogenes estimated ranged from 0.007 to 0.061. The square root model was used to predict the µmax at others temperature and to establish the time necessary to reach the European critical legal limit of 2logCFU/g, in different storage scenarios. The predictions obtained in this study can be applied to any time-temperature profile, and in particular to the conditions to which the product is most likely to be subject in normal use, up to its final consumption. This study can be considered a valuable contribution also aimed at supporting the monitoring surveys carried out by officers of the Regional Veterinary Authority.

FLOQSwab™: Optimisation of Procedures for the Recovery of Microbiological Samples from Surfaces.

The FLOQSwab™ is a specimen collection device worldwide recognised for its superior performance in the clinical diagnostics. The aim of this work was to evaluate FLOQSwab™ for the recovery of microbiological samples from surfaces compared to the traditional swab (rayon tipped swab) as per ISO 18593:2004 standard. The FLOQSwab™, thanks to its innovative manufacturing technology, allows improving the efficiency of recovery and release of analyte. The study has been divided into two experiments. In the first experiment the two swabs were evaluated for their capacity to recover and release the analyte (three different bacterial loads of Escherichia coli). In the second experiment, the two swabs were evaluated for their capacity to recover three different bacterial loads of E. coli from two different surface materials (stainless steel and polypropylene). In all experiments the flocked swab demonstrated a higher recovery rate compared to the traditional rayon tipped swab. The data obtained from this preliminary study demonstrated that the FLOQSwab™ could be a good food surfaces collection device, which improves the recovery of the analyte and thus produces accurate results. Based on the outcomes of the study, a larger field study is in progress using the FLOQSwab™ for samples collection to improve both environmental monitoring and the efficacy of the hygiene controls for food safety.

Multiple-Strain Approach and Probabilistic Modeling of Consumer Habits in Quantitative Microbial Risk Assessment: A Quantitative Assessment of Exposure to Staphylococcal Enterotoxin A in Raw Milk.

Quantitative microbial risk assessment (QMRA) models are extensively applied to inform management of a broad range of food safety risks. Inevitably, QMRA modeling involves an element of simplification of the biological process of interest. Two features that are frequently simplified or disregarded are the pathogenicity of multiple strains of a single pathogen and consumer behavior at the household level. In this study, we developed a QMRA model with a multiple-strain approach and a consumer phase module (CPM) based on uncertainty distributions fitted from field data. We modeled exposure to staphylococcal enterotoxin A in raw milk in Lombardy; a specific enterotoxin production module was thus included. The model is adaptable and could be used to assess the risk related to other pathogens in raw milk as well as other staphylococcal enterotoxins. The multiplestrain approach, implemented as a multinomial process, allowed the inclusion of variability and uncertainty with regard to pathogenicity at the bacterial level. Data from 301 questionnaires submitted to raw milk consumers were used to obtain uncertainty distributions for the CPM. The distributions were modeled to be easily updatable with further data or evidence. The sources of uncertainty due to the multiple-strain approach and the CPM were identified, and their impact on the output was assessed by comparing specific scenarios to the baseline. When the distributions reflecting the uncertainty in consumer behavior were fixed to the 95th percentile, the risk of exposure increased up to 160 times. This reflects the importance of taking into consideration the diversity of consumers' habits at the household level and the impact that the lack of knowledge about variables in the CPM can have on the final QMRA estimates. The multiple-strain approach lends itself to use in other food matrices besides raw milk and allows the model to better capture the complexity of the real world and to be capable of geographical specificity.

West Nile Virus Surveillance in 2013 via Mosquito Screening in Northern Italy and the Influence of Weather on Virus Circulation.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

Behaviour of Listeria Monocytogenes and Escherichia Coli O157:H7 During the Cheese Making of Traditional Raw-Milk Cheeses from Italian Alps.

The behaviour of Listeria monocytogenes and Escherichia coli O157:H7 was studied during the manufacture and ripening of two traditional Italian Alps cheeses. Each cheese type was manufactured in a pilot plan from raw cow milk (without the addition of starter cultures) artificially inoculated with L. monocytogenes and E. coli O157:H7 to a final concentration of about 4 log CFU/mL. The pathogens were enumerated throughout the cheese making and ripening processes to study their behaviour. When the milk was inoculated with 4 Log CFU/mL, the pathogens counts increased in the first time during the manufacturing process and then remained constant, until the end of ripening, or decreased significantly. Results indicate that the environment and nature of food borne pathogens affected the concentration of the bacteria during the manufacturing and ripening process. Thus, the presence of low cells numbers of L. monocytogenes and E. coli O157:H7 in milk destined for the production of raw milk cheeses characterized by a cooking of the curd less than 48°C can constitute a hazard for the consumer.

Waterborne norovirus outbreak during a summer excursion in Northern Italy.

In September 2011, an acute gastroenteritis outbreak affected 33 children in Northern Italy. Patients had drunk river water during an excursion. Identical GI.4 norovirus genomes were detected from one patient's stools and from the river water. Improper discharge of human sewage into the river may have caused this waterborne outbreak.

Synergistic effect of high hydrostatic pressure (HHP) and marination treatment on the inactivation of hepatitis a virus in mussels (Mytilus galloprovincialis).

Consumption of raw or insufficiently cooked mussels contaminated with hepatitis A virus (HAV) is a major cause of infection to humans. The origin of mussels commonly used for the preparation of marinated seafood salads is often unknown, since different producers worldwide undergo a precooking treatment at the original collection site with methods and parameters not always indicated. These treatments could be insufficient for the inactivation of HAV, which is characterized by a high temperature resistance. Both high hydrostatic pressure (HHP) and marinade treatments have been shown to affect HAV vitality. In this study, two treatments (HHP and marinating) were combined in order to assess a potential synergistic effect on the virus vitality. A kinetic test was conducted by subjecting the experimentally-contaminated mussels (HAV titre: 10(6)/ml TCID50) to marinating, and to different HHP treatment (4,000; 5,000; and 6,000 bar for 1, 5, and 9 min). Virus post-treatment vitality was assessed by its ability to grow on cell cultures and by quantitative real-time RT-PCR to evaluate virus resistance under such conditions. Marinating treatment alone (final pH 4.3, and NaCl 2 %) did not inactivate the virus. On the other hand, the use of HHP treatment alone on non-marinated HAV-contaminated mussels was effective only above 5,000 bar for 5 min. The results of the present study elucidate the synergistic effect of a combination between marination and HHP treatments on the inactivation of the virus.

Microbiological and Physico-Chemical Changes During Manufacture of an Italian Goat Cheese Made from Raw Milk.

The aim of this work was to study the microbiological and physico-chemical changes throughout three cheesemaking replicates of Italian Formaggelle di capra cheese made from raw goat milk. Therefore, during the process, three samples of milk, curd and cheese at 3, 7, 11, 14, 21 and 30 days of ripening old cheese were taken from three cheesemaking replicates. The average of total mesophilic bacteria and Enterobacteriaceae count in raw milk was 5.27±0.57 and 3.8±1.02 Log cfu/mL, respectively. Lactic acid bacteria was the predominant bacterial group during the process, and they developed in different ways in each of the media used (M17 and MRS agar). Variability of microbial concentrations was observed between three cheesemaking replicates. A correlation between the presence of higher levels of Enterobacteriaceae in milk and the presence of other contaminants bacteria such as Escherichia coli ß-glucuronidase-positive and coagulase-positive staphylococci was observed. In cheesemaking replicate n. 2, E. coli level was 5.07±0.03 Log cfu/mL and increased by about 1 log until the last week of ripening, when the level decreased to 5.69±0.2 Log cfu/mL. The milk used for the cheesemaking replicate n. 2 was found to be contaminated also by coagulase-positive staphylococci (3.18±0.06 Log cfu/mL), but the behaviour of this group appeared to be very variable. In this study a first step of process control and microbial groups study was performed and the cheesemaking process was registered in the website www.ars-alimentaria.it, the Italian site supported by the Italian Board of Health.

Reduction of Escherichia Coli O157:H7 During Manufacture and Ripening of Italian Semi-Dry Salami.

In order to simulate a contamination at the processing plant, one batch of freshly-processed salami batter (20 kg) was inoculated (1% v:w) with 5 log colony forming unit (CFU)/g of a multi-strain cocktail of two strains of Escherichia coli O157:H7 (registered and wild strain). Another batch was inoculated (1% v:w) with sterile physiological saline solution and used to check the lactic acid bacteria (Lab) behaviour and the changes of physicochemical parameters (pH and aw ). Both batches were then processed to obtain a semi-dry salami (Hungarian-style): microbiological and physico-chemical properties were monitored during 94 days of ripening. During the manufacturing process, the levels of pathogen decreased of about 2.18 log CFU/g with respect to the initial inoculated levels. The behaviour of the indigenous bacteria such as Lab and the physico-chemical properties can help to determine the fate of pathogens throughout processing.

Study of Growth Potential of Listeria Monocytogenes in Low Fat Salami: An Innovative Italian Meat Product.

In the last years, consequently to EC Regulation no. 1924/2006 on nutrition and health claims made on foods, some Italian food businnes operators (FBOs) leaders in the meat sector, invested in research to develop innovative products such as low fat salami, containing up to 30% less fat than the traditional one. For FBOs it is essential to demonstrate for each production process whether the substrate allows the growth of L. monocytogenes and whether L. monocytogenes could reach or exceed the limit of 100 cfu g-1 at the end of the shelf life, as stated by EC Regulation no. 2073/2005. In the present study, the growth potential of L. monocytogenes during the shelf life of low fat salami packed in modified atmosphere was evaluated. The results show that the product is unable to support the growth of pathogen, even if the storage temperature is between 8 and 12°C.

Effect of intramammary infection in Bergamasca meat sheep on milk parameters and lamb growth.

Pooled milk samples from 115 Bergamasca meat sheep were collected aseptically five times from lambing to weaning to determine the prevalence of intramammary infection, somatic cell counts and milk quality parameters (protein, fat and lactose), and effects of infection on lamb weight gain. The global prevalence of subclinical intramammary infection was 51.2%. The Staphylococcus genus was responsible for the greatest prevalence (53.3% among infected udders). Staphylococcus aureus was isolated in 8.4% of infected milk samples. Infection status had significant effects on fat and protein percentage and on somatic cell count. Lamb growth was greatest for lambs of ewes with no infection and decreased as the number of infected samples increased. No significant differences were detected in the growth of lambs with dams infected by different bacterial species.

Major constituents, leptin, and non-protein nitrogen compounds in mares' colostrum and milk.

Five Haflinger mares were hand-milked at 0 h (pre-suckle) and 6 h (postsuckle), 12, 24, 48, 72 and 96 h after parturition. Total solids, protein, fat, lactose, calculated gross energy content, leptin and non-protein nitrogen components (urea, alpha-amino nitrogen, creatinine and allantoin) were determined. The levels of the major constituents differed significantly in pre-suckle colostrum from subsequent samples. Leptin levels were the highest in whole (9 ng x mL(-1) of immunoreactive human equivalent HE +/- 0.48 ng x mL(-1), SEM) and skimmed (7.8 ng HE x mL(-1) +/- 0.52 ng x mL(-1), SEM) pre-suckle colostrum, declined sharply at 6 hours postpartum, and more slowly subsequently. Mean urea concentration was constant at around 5.0 mM, while a-amino N increased over the observation period and creatinine and allantoin decreased. These findings provide a further indication that mares' milk can be regarded as a functional food.