Salmonella Derby adaptation to swine and simultaneous attenuation for humans go through decay of Salmonella Pathogenicity Island I.

IMPORTANCE: This study integrated population data with in vitro assessment of virulence phenotypes to unveil that a considerable part of the global population of Salmonella Derby is evolving to enhance its host adaptation to the swine host and that this evolution is simultaneously increasing its attenuation for humans. The study shows that the fixation of deleterious mutations in SPI-1 has a role in this process. This evidence indicates that SPI-1 has a key role for S. Derby virulence in humans but not for its circulation in swine. The results show that genes generally considered essential for Salmonella pathogenesis do not play the same key role for all Salmonella serovars or lineages and/or all hosts. The study helps in understanding the molecular mechanisms underlying the ecology and host adaptation of Salmonella showing that the adaptation process can vary for different types of Salmonella and hosts.

Listeria monocytogenes Transmission from Donated Blood to Platelet Transfusion Recipient, Italy.

We report Listeria monocytogenes infection in a patient in Italy who was transfused with pooled platelet concentrate. Genomic analysis revealed that L. monocytogenes isolates from the donor blood unit, the transfused platelets, and the patient's blood culture were genetically closely related, confirming transfusion transmission. Additional surveillance and secondary bacterial screening could improve transfusion safety.

Different Roles of Wild Boars and Livestock in Salmonella Transmission to Humans in Italy.

Wild boar (Sus scrofa) is the most widely distributed large wildlife mammal worldwide. To investigate the transmission of Salmonella enterica amongst wild boars (Sus scrofa), humans, and livestock, we compared via pulsed-field gel electrophoresis and whole genome sequences the isolates of S. enterica serovar Typhimurium (biphasic and monophasic variants) and Enteritidis collected from wild boars, food-producing animals, and human patients in Emilia-Romagna region (Northern Italy) between 2017 and 2020. Specifically, we analysed 2175 isolates originated from human (1832), swine (117), bovine (128), poultry (76), and wild boar (22). The genomic analyses showed that wild boars shared most of their lineages of biphasic Typhimurium with bovines and most of Enteritidis with poultry, whilst we did not find any lineage shared with swine. Moreover, almost 17% of human biphasic Typhimurium and Enteritidis belonged to genomic clusters including wild boar isolates, but the inclusion of bovine and poultry isolates in the same clusters and the peculiar spatial distribution of the isolates suggested that human cases (and wild boar infections) likely originated from bovines and poultry. Consequently, wild boars appear not to play a significant role in infecting humans with these serovars, but seem to get infected themselves from livestock, probably through the environment.

Klebsiella pneumoniae carrying multiple alleles of antigen 43-encoding gene of Escherichia coli associated with biofilm formation.

A clinical strain of Klebsiella pneumoniae typed as sequence type 307 carrying three different alleles of the flu gene encoding the Escherichia coli virulence factor antigen 43 associated with biofilm formation was detected and characterized. The flu alleles are located in the chromosome inside putative integrative conjugative elements. The strain displays the phenotypes associated with Ag43, i.e. bi-phasic colony morphology and enhanced biofilm production. Furthermore, the strain produces low amount of capsule known to affect Ag43 function. Analysis of 1431 worldwide deposited genomes revealed that 3.7% Klebsiella pneumoniae carry one or two flu alleles.

Using Expert Elicitation for ranking hazards, promoters and animal-based measures for on-farm welfare assessment of indoor reared beef cattle: an Italian experience.

On-farm welfare assessment gives the opportunity to monitor and improve the quality of the animal life on the farm. In order to build the first Italian public standard for the welfare of indoor reared beef cattle, a list of 25 potential hazards and 22 potential promoters of beef cattle welfare was judged by a group of national experts by taking into account their negative or positive impacts on the welfare-state of the target population. In addition, the experts were asked to rank a list of 11 animal-based measures for identifying the most appropriate and important for measuring negative welfare outcomes in beef cattle. Based on experts' ratings, an "impact score" (ISoverall) was calculated for the proposed measures. Management hazards and promoters were ranked to have a greater impact on beef cattle welfare than housing factors. Keeping cattle in large (≥ 40 animals) and heterogeneous groups obtained the highest ISoverall among the proposed hazards (ISoverall = 5.54), followed by the presence of animals without free access to drinking water (ISoverall = 4.39) and the use of high-concentrate corn silage diets (concentrate > 80% and fiber < 6%) (ISoverall = 4.39). On the other hand, housing animals in small (≤ 20 animals) and homogeneous groups (ISoverall = 5.41), checking them at least twice a day (ISoverall = 4.62) and rearing cattle in loose housing systems with access to an outdoor area/pasture (ISoverall = 4.27) were ranked among the top 3 promoters. Concerning animal-based measures, experts scored lameness, severe respiratory diseases, body condition scoring and mortality rate to be measures most important for assessing serious welfare impairment.

On the optimal control of SIR model with Erlang-distributed infectious period: isolation strategies.

Mathematical models are formal and simplified representations of the knowledge related to a phenomenon. In classical epidemic models, a major simplification consists in assuming that the infectious period is exponentially distributed, then implying that the chance of recovery is independent on the time since infection. Here, we first attempt to investigate the consequences of relaxing this assumption on the performances of time-variant disease control strategies by using optimal control theory. In the framework of a basic susceptible-infected-removed (SIR) model, an Erlang distribution of the infectious period is considered and optimal isolation strategies are searched for. The objective functional to be minimized takes into account the cost of the isolation efforts per time unit and the sanitary costs due to the incidence of the epidemic outbreak. Applying the Pontryagin's minimum principle, we prove that the optimal control problem admits only bang-bang solutions with at most two switches. In particular, the optimal strategy could be postponing the starting intervention time with respect to the beginning of the outbreak. Finally, by means of numerical simulations, we show how the shape of the optimal solutions is affected by the different distributions of the infectious period, by the relative weight of the two cost components, and by the initial conditions.

Reduction trend of mcr-1 circulation in Emilia-Romagna Region, Italy.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2 mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018-2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018-2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Mutation of hilD in a Salmonella Derby lineage linked to swine adaptation and reduced risk to human health.

Salmonella enterica variants exhibit diverse host adaptation, outcome of infection, and associated risk to food safety. Analysis of the distribution of Salmonella enterica serovar Derby (S. Derby) subtypes in human and swine identified isolates with a distinct PFGE profile that were significantly under-represented in human infections, consistent with further host adaptation to swine. Here we show that isolates with this PFGE profile form a distinct phylogenetic sub-clade within S. Derby and exhibit a profound reduction in invasion of human epithelial cells, and a relatively small reduction in swine epithelial cells. A single missense mutation in hilD, that encodes the master-regulator of the Salmonella Pathogenicity Island 1 (SPI-1), was present in the adapted lineage. The missense mutation resulted in a loss of function of HilD that accounted for reduced invasion in human epithelial cells. The relatively small impact of the mutation on interaction with swine cells was consistent with an alternative mechanism of invasion in this pathogen-host combination.

Population Structure of Listeria monocytogenes in Emilia-Romagna (Italy) and Implications on Whole Genome Sequencing Surveillance of Listeriosis.

The population structure of human isolates of Listeria monocytogenes in Emilia-Romagna, Italy, from 2012 to 2018 was investigated with the aim of evaluating the presence of genomic clusters indicative of possible outbreaks, the proportion of cluster-associated vs. sporadic isolates and different methods and metrics of genomic analysis for use in routine surveillance. In the 2012-2018 period the notification rate of confirmed invasive cases in Emilia-Romagna was 0.91 per 100,000 population per year, more than twice the average rate of EU countries. Out of the total 283 cases, 268 (about 95%) isolates were typed through whole genome sequencing (WGS) for cluster detection with methods based on core-genome multi-locus sequence typing and single nucleotide polymorphisms. Between 66 and 72% of listeriosis cases belonged to genomic clusters which included up to 27 cases and lasted up to 5 years. This proportion of cluster-associated cases is higher than previously estimated in other European studies. Rarefaction analysis, performed by reducing both the number of consecutive years of surveillance considered and the proportion of isolates included in the analysis, suggested that the observed high proportion of cluster-associated cases can be ascribed to the long surveillance duration (7 years) and the high notification and typing rates of this study. Our findings show that a long temporal perspective and high surveillance intensity, intended as both exhaustiveness of the system to report cases and high WGS-typing rate, are critical for sensitive detection of possible outbreaks within a WGS-based surveillance of listeriosis. Furthermore, the power and complexity of WGS interpretation emerged from the integration of genomic and epidemiological information in the investigation of few past outbreaks within the study, indicating that the use of multiple approaches, including the analysis of the accessory genome, is needed to accurately elucidate the population dynamics of Listeria monocytogenes.

Is Yersinia bercovieri Surpassing Yersinia enterocolitica in Wild Boars (Sus scrofa)?

Yersiniosis was the fourth reported zoonosis in the European Union in 2018. As well-known, pigs are recognized important reservoirs of Yersinia enterocolitica. The study was focused on Y. enterocolitica detection in mesenteric lymph nodes and faeces of 305 wild boars, but Yersinia bercovieri was more common, being isolated from 108 animals (35.4%). Cold season (p = 1.17 × 10-5) and young age (p = 0.004) significantly increased Y. bercovieri detection. Y. enterocolitica 1A belonging to six serotypes (O:4.32-4.33; O:5; O:6.30-6.31; O:7.8-8-8.19; O:10-34; O:12.25-12.26) was isolated from 8.2% (25/305) of the animals. Cold season significantly affected (p = 0.037) Y. enterocolitica detection.

Enhanced West Nile Virus Circulation in the Emilia-Romagna and Lombardy Regions (Northern Italy) in 2018 Detected by Entomological Surveillance.

With several human cases reported annually since 2008 and the unapparent risk of infection of blood donors, the West Nile virus (WNV) is emerging as an important health issue in Europe. Italy, as well as other European countries, experienced a recrudescence of the virus circulation in 2018, which led to an increased number of human cases. An integrated surveillance plan was activated in the Emilia-Romagna and Lombardy regions (Northern Italy) since 2008 in order to monitor the intensity and timing of WNV circulation. A fundamental part of this plan consists in entomological surveillance. In 2018, the surveillance plan made it possible to collect 385,293 mosquitoes in 163 stations in the two Regions. In total 269,147 Culex mosquitoes were grouped into 2,337 pools and tested for WNV, which was detected in 232 pools. Circulation started in the central part of the Emilia-Romagna region in the middle of June, about one month before the previous seasons. Circulation suddenly expanded to the rest of the region and reached the Lombardy region in the middle of July. WNV circulated more intensively in the eastern part of the surveyed area, as confirmed by the highest number of human cases. A relationship between the number of mosquitoes collected and the virus incidence emerged, but the data obtained highlighted that the probability of detecting the virus in a given site was less than expected with a higher number of collected mosquitoes. A significant relationship was observed between the temperature recorded one week before the sampling and the number of collected mosquitoes, as well as between the estimated number of WNV-positive mosquitoes and the temperature recorded two weeks before the sampling. The two weeks delay in the influence of temperature on the positive mosquitoes is in line with the time of the virus extrinsic incubation in the mosquito. This finding confirms that temperature is one of the principal drivers in WNV mosquito infection. The surveillance system demonstrated the ability to detect the virus circulation early, particularly in areas where circulation was more intense. This allowed evaluating the effect of mosquito abundance and weather factors on virus circulation.

Geographical restriction of Hepatitis E virus circulation in wild boars (Sus scrofa) in Emilia-Romagna region, Northern Italy.

Hepatitis E virus (HEV) is a singlestrand RNA virus that causes an acute viral hepatitis in humans. Among its eight recognized genotypes, HEV-3 and HEV-4 are zoonotic, infecting humans, pigs and wild boars. Recently, HEV-3 has been also detected in red deer, which represents another reservoir of HEV. Consumption of raw pork products (mainly liver sausages), undercooked wild boar meat, raw wild boar liver and deer meat has been responsible for foodborne HEV human worldwide. From November 2018 to March 2019, liver samples collected from 97 wild boars hunted in Emilia-Romagna region (Northern Italy) were tested for HEV RNA. The hunting area included two territories for an extension of 33 km2, named A (about 13 km2,natural park, deciduous wood) and B (about 20 km2, cultivated fields in proximity of a river) areas. Distance between the two areas ranged between 8 to 10 km. A total of 73 wild boars were hunted in area A, and 24 in area B. HEV RNA was detected by Real-time RT- PCR in 23/73 liver samples of wild boars living in area A only (31.5% - 95% CI: 22.0-42.8%). The HEV sequences (n=13) clustered within genotype 3. The majority of positives belonged to animals < 12 months (12/25; 48%), followed by subadults (13-24 months) (7/16; 43.8%) and adults (4/32; 12.5%). This difference was found to be statistically significant (p=0.0024). In absence of pig farms, the restriction of HEV-positive animals to a well-defined territory of 13 km2 (Boschi di Carrega Regional Park) could hypothetically be related to the presence of red deer (Cervus elaphus), which lived in area A at the beginning of the hunting season. Further studies are needed to confirm or deny our hypothesis.

A quantitative comparison of West Nile virus incidence from 2013 to 2018 in Emilia-Romagna, Italy.

BACKGROUND: West Nile virus (WNV) transmission was much greater in 2018 than in previous seasons in Europe. Focusing on Emilia-Romagna region (northern Italy), we analyzed detailed entomological and epidemiological data collected in 2013-2018 to quantitatively assess environmental drivers of transmission and explore hypotheses to better understand why the 2018 epidemiological season was substantially different than the previous seasons. In particular, in 2018 WNV was detected at least two weeks before the observed circulation in 2013-2017 and in a larger number of mosquito pools. Transmission resulted in 100 neuroinvasive human cases in the region, more than the total number of cases recorded between 2013 and 2017. METHODOLOGY: We used temperature-driven mathematical models calibrated through a Bayesian approach to simulate mosquito population dynamics and WNV infection rates in the avian population. We then estimated the human transmission risk as the probability, for a person living in the study area, of being bitten by an infectious mosquito in a given week. Finally, we translated such risk into reported WNV human infections. PRINCIPAL FINDINGS: The estimated prevalence of WNV in the mosquito and avian populations were significantly higher in 2018 with respect to 2013-2017 seasons, especially in the eastern part of the region. Furthermore, peak avian prevalence was estimated to have occurred earlier, corresponding to a steeper decline towards the end of summer. The high mosquito prevalence resulted in a much greater predicted risk for human transmission in 2018, which was estimated to be up to eight times higher than previous seasons. We hypothesized, on the basis of our modelling results, that such greater WNV circulation might be partially explained by exceptionally high spring temperatures, which have likely helped to amplify WNV transmission at the beginning of the 2018 season.

When resolution does matter: Modelling indirect contacts in dairy farms at different levels of detail.

Animal exchanges are considered the major pathway for between-farm transmission of many livestock infectious diseases. Yet, vehicles and operators visiting several farms during routine activities can also contribute to disease spread. Indeed, if contaminated, they can act as mechanical vectors of fomites, generating indirect contacts between visited farms. While data on animal exchanges is often available in national databases, information about the daily itineraries of trucks and operators is rare because difficult to obtain. Thus, some unavoidable approximations have been frequently introduced in the description of indirect contacts in epidemic models. Here, we showed that the level of detail in such description can significantly affect the predictions on disease dynamics. Our analyses focused on the potential spread of a disease in a dairy farm system subject of a comprehensive data collection campaign on calf transportations. We developed two temporal multilayer networks to model between-farm contacts generated by either animal exchanges (direct contacts) and connections operated by trucks moving calves (indirect contacts). The complete model used the full knowledge of the daily trucks' itineraries, while the partial informed one used only a subset of such available information. To account for various conditions of pathogen survival ability and effectiveness of cleaning operations, we performed a sensitivity analysis on trucks' contamination period. An accurate description of indirect contacts was crucial both to correctly predict the final size of epidemics and to identify the seed farms responsible for generating the most severe outbreaks. The importance of detailed information emerged even more clearly in the case of short contamination periods. Our conclusions could be extended to between-farm contacts generated by other vehicles and operators. Overcoming these information gaps would be decisive for a deeper understanding of epidemic spread in livestock and to develop effective control plans.

Optimal control of epidemic size and duration with limited resources.

The total number of infections (epidemic size) and the time needed for the infection to go extinct (epidemic duration) represent two of the main indicators for the severity of infectious disease epidemics in human and livestock. However, few attempts have been made to address the problem of minimizing at the same time the epidemic size and duration from a theoretical point of view by using optimal control theory. Here, we investigate the multi-objective optimal control problem aiming to minimize, through either vaccination or isolation, a suitable combination of epidemic size and duration when both maximum control effort and total amount of resources available during the entire epidemic period are limited. Application of Pontryagin's Maximum Principle to a Susceptible-Infected-Removed epidemic model, shows that, when the resources are not sufficient to maintain the maximum control effort for the entire duration of the epidemic, the optimal vaccination control admits only bang-bang solutions with one or two switches, while the optimal isolation control admits only bang-bang solutions with one switch. We also find that, especially when the maximum control effort is low, there may exist a trade-off between the minimization of the two objectives. Consideration of this conflict among objectives can be crucial in successfully tackling real-world problems, where different stakeholders with potentially different objectives are involved. Finally, the particular case of the minimum time optimal control problem with limited resources is discussed.

Limited Exchange of Salmonella Among Domestic Pigs and Wild Boars in Italy.

The study assessed Salmonella carriage in wild boars (Sus scrofa) and compared their isolates with those recovered from the domestic swine population of the same area of northern Italy (Emilia-Romagna), characterized by intensive pig farming and rather high density of wild boars. A total of 189 wild boars hunted during twelve months (2017-2018) were tested for Salmonella in mesenteric lymph nodes (MLN) and faecal samples. Antimicrobial resistance of recovered strains was tested against 14 antimicrobials. Salmonella was detected in 33/189 wild boars (17.5%), specifically from 30/189 MLN (15.9%) and 6/189 faecal samples (3.2%). Three animals were positive in both samples. Thirteen Salmonella serovars were identified, i.e. Typhimurium (the most common), Bovismorbificans, Coeln, Derby, Enteritidis, Gaminara, Hessarek, Houtenae IV, Kottbus, Napoli, Stanleyville, Thompson and Veneziana. Salmonella carriage was higher in warm than in cold months (P = 0.0013). Pregnancy status was never associated with Salmonella carriage, with significant difference in the recovery of the pathogen between non-pregnant and pregnant females (P = 0.003). Only one resistance pattern to streptomycin and tetracycline was found in 15 isolates (41.7%) belonging to Typhimurium (14/14; 100%) and Kottbus (1/3; 33.3%) serovars. Overlap with isolates from farmed pigs was limited at serotype level (Typhimurium, Derby, Enteritis, Bovismorbificans, Kottbus) and absent at PFGE level, and also antimicrobial resistance patterns were substantially different. This evidence indicates a substantial segregation of the two animal populations with regard to infectious contacts, possibly suggesting that biosecurity measures in place at industrial farm level limit the exchange of Salmonella.

Diagnostic value of composite milk sample vs single quarter milk sample for the detection of Staphylococcus aureus intra-mammary infections in cattle.

Staphylococcus aureus (S. aureus) is one of the most important causes of mastitis in dairy cattle. Control and eradication programs of S. aureus intra-mammary infections (IMI) are based on different factors included the correct detection and management of the infected cows. The present study aimed at evaluating the efficacy of composite milk samples (CMS) analysis, compared to quarter milk samples (QMS) analysis, for the bacteriological detection of S. aureus intra-mammary infections. During 2016, 661 CMS (hygienically collected) and 2644 QMS (aseptically collected) were obtained from 661 cows in 5 herds. All the samples were submitted to S. aureus bacteriological culture and somatic cell count (SCC) analysis. QMS bacteriological analysis on blood agar plates was able to detect 236 cows excreting S. aureus, while the bacteriological analysis of CMS, using selective agar, identified 229 positive cows. The concordance was 95% with an excellent Cohen's κ (0.89). Relative sensitivity and specificity of CMS vs QMS, considered as the reference test, were 91.5% ± 2.1 and 96.9% ± 1.3 (CI 95%), respectively. In addition, the relative sensitivity of CMS improved as the number of infected quarters per cow and the number of colony forming units (cfu) per sample increased. The predictive value of CMS results was better when paired with SCC data, in particular CMS showed better negative predictive value when SCC was <200,000 cells/mL and better positive predictive value when SCC was>200,000 cells/mL. The probability for a cow to be S. aureus positive was 56.4% in case of SCC > 200,000 cells/mL, while it was 18.6% in case of SCC < 200,000 cells/mL. The average SCC in CMS was significantly higher in positive cows and the value rose as the number of infected quarters per cow increased. Given the intermittent excretion of S. aureus in milk from dairy cows, it could be more advantageous to carry out several serial CMS, rather than few QMS, being CMS an easier to collect and less expensive milk sampling method. Thus, bacteriological examination of CMS, combined with SCC data of the same sample, could be extremely useful for the success of S. aureus IMI control plans, because repeated CMS are easier to be performed and could be more easily proposed to the farmers.

High-resolution diffusion pattern of human infections by Salmonella enterica serovar Napoli in Northern Italy explained through phylogeography.

Salmonella enterica serovar Napoli (serovar Napoli) is an emerging cause of human salmonellosis in Northern Italy. No specific reservoirs of serovar Napoli have been identified in Italy, so far. However, the environment, especially surface waters, has been hypothesized as an important source of infection based on the observation that genotypically different clusters of serovar Napoli are detected in different geographical macro-areas. To further support the hypothesis of a spatially-restricted pattern of serovar Napoli diffusion, a spatial segregation of serovar Napoli lineages should be observed also at smaller geographical scale. However, classical genotyping techniques used for Salmonella, such as pulsed-field gel electrophoresis (PFGE), did not possess enough discriminatory power to highlight spatial clustering of serovar Napoli within the macro-areas. To this purpose, we performed phylogeographical analyses based on genome-wide single nucleotide polymorphisms to test whether spatio-temporal evolution patterns of serovar Napoli in Northern Italy could be recognized with high geographical resolution, i.e. at local level. Specifically, we analyzed the local spread of the main PFGE clonal group, responsible for more than 60% of human infections in the study area, that did not show any geographical differentiation by PFGE within Northern Italy, i.e. the macro-area considered in the study. Both discrete and continuous phylogeography highlighted the existence of two main geographically-restricted clades: a Southern clade corresponding to the Po Valley and a Northern clade corresponding to the Pre-Alps area. Furthermore, the phylogeographical analyses suggested that the most probable site of origin of the clone was in an area of the Po Valley at the confluence of the Po and Ticino rivers, one of the most important Italian wetlands. These findings provide further support to the hypothesis that environmental transmission may play an important role in the ecology of serovar Napoli.

Salmonella Detection and Counting on Pig Carcasses and Cutting Lines in Italian Slaughterhouses.

During 2014-2015, 300 pig carcasses before chilling and 85 food contact surfaces (FCSs) at cutting lines were tested for Salmonella in three slaughterhouses (namely A, B, and C) of northern Italy. In slaughterhouses A and B, four carcass sites of 100 cm2 each (from both the exterior and interior side) were swabbed with a single sponge. In abattoir C, four 100 cm2 sites of the exterior and the interior sides were swabbed with two independent sponges. The population average prevalence of Salmonella-positive carcasses (which takes into account the structure of the study design, with multiple samples collected in a single day) in slaughterhouses A and B was 12.3%, while in slaughterhouse C it was 11.2%. Presence of Salmonella on exterior and interior sides of carcasses showed a low level of concordance (only 3/12 of the contaminated carcasses were positive on both sides). No significant difference was found for FCSs contamination in the three slaughterhouses, with a population average prevalence of Salmonella-positive FCSs of 19.9%. In addition, we found that the clustering due to the day of sampling account for more than 36% and 60% of the overall prevalence variation on carcasses and FCSs, respectively. Eight serovars were identified, with Salmonella Derby as the most common type. The counting of Salmonella on carcasses showed large variability. It was low (<0.0075 most probable number [MPN]/cm2) in 46.6% of the carcasses and as high as 2.7 MPN/cm2 in 4.7%. Specifically, we found that counts on carcasses fit with "heavy tailed" distributions (lognormal and Weibull with a small shape parameter), suggesting not negligible probability of episodes of high Salmonella contamination. The mean values of contamination obtained with the two distributions ranged from 0.235 to 0.435 MPN/cm2.

Rise and fall of outbreak-specific clone inside endemic pulsotype of Salmonella 4,[5],12:i:-; insights from high-resolution molecular surveillance in Emilia-Romagna, Italy, 2012 to 2015.

Background and aimEpidemiology of human non-typhoid salmonellosis is characterised by recurrent emergence of new clones of the pathogen over time. Some clonal lines of Salmonella have shaped epidemiology of the disease at global level, as happened for serotype Enteritidis or, more recently, for Salmonella 4,[5],12:i:-, a monophasic variant of serotype Typhimurium. The same clonal behaviour is recognisable at sub-serotype level where single outbreaks or more generalised epidemics are attributable to defined clones. The aim of this study was to understand the dynamics of a clone of Salmonella 4,[5],12:i:- over a 3-year period (2012-15) in a province of Northern Italy where the clone caused a large outbreak in 2013. Furthermore, the role of candidate outbreak sources was investigated and the accuracy of multilocus variable-number tandem repeat analysis (MLVA) was evaluated. Methods: we retrospectively investigated the outbreak through whole genome sequencing (WGS) and further monitored the outbreak clone for 2 years after its conclusion. Results: The study showed the transient nature of the clone in the population, possibly as a consequence of its occasional expansion in a food-processing facility. We demonstrated that important weaknesses characterise conventional typing methods applied to clonal pathogens such as Salmonella 4,[5],12:i:-, namely lack of accuracy for MLVA and inadequate resolution power for PFGE to be reliably used for clone tracking. Conclusions: The study provided evidence for the remarkable prevention potential of whole genome sequencing used as a routine tool in systems that integrate human, food and animal surveillance.