West Nile virus surveillance using sentinel birds: results of eleven years of testing in corvids in a region of northern Italy.

The natural transmission cycle of West Nile virus (WNV) involves birds as primary hosts and mosquitoes as vectors, but this virus can spread to mammals, human beings included. Asymptomatic infected donors pose a risk to the safety of blood transfusions and organ transplants, as WNV can be transmitted through these medical procedures. Since 2009, the region of Emilia-Romagna in northern Italy has been implementing an integrated surveillance system in order to detect WNV circulation in the environment at an early stage. Here we report the results of the two components of the surveillance system, the active testing of corvids and humans, and demonstrate that bird surveillance alone improves a surveillance system based solely on human case detection. As WNV risk reduction measures are applied on a provincial basis, we assessed the ability of this surveillance system component to detect virus circulation prior to the notification of the first human case for each province. Overall, 99 epidemic seasons were evaluated as a result of 11 years (2013-2023) of surveillance in the nine provinces of the region. In this period, 22,314 corvids were tested for WNV and 642 (2.9%) were found to be infected. WNV was generally first detected in birds in July, with sample prevalence peaks occurring between August and September. During the same period, 469 autochthonous human cases were notified, about 60% of which were reported in August. WNV was detected 79 times out of the 99 seasons considered. The virus was notified in birds 73 times (92.4%) and 60 times (75.9%) in humans. WNV was first or only notified in birds in 57 seasons (72.1%), while it was first or only notified in humans in 22 seasons (27.8%). Active surveillance in corvids generally allows the detection of WNV before the onset of human cases. Failure of virus detection occurred mainly in seasons where the number of birds tested was low. Our results show that active testing of a minimum of 3.8 corvids per 100 km2 provides a satisfactory timeliness in the virus detection, but for early detection of WNV it is crucial to test birds between mid-June and mid-August.

Eleven Years of Health Monitoring in Wild Boars (Sus scrofa) in the Emilia-Romagna Region (Italy).

In recent years, the growth of wild ungulates has increased the focus on their health monitoring. In particular, the health status of wild boars is relevant for the economic impact on the pig industry. The Emilia-Romagna region activated a wildlife monitoring plan to better evaluate the health status of the wild boar population. Between 2011 and 2021, samples of found dead and hunted wild boar have been examined for trichinellosis, tuberculosis, brucellosis, african swine fever, classical swine fever, Aujeszky's disease, swine vesicular disease, and swine influenza A. Trichinella britovi was identified in 0.001% of the examined wild boars; neither M. bovis nor M. tuberculosis were found in M. tuberculosis complex positive samples; 2.3% were positive for Brucella suis; 29.4% of the sera were positive for Aujeszky's disease virus; and 0.9% of the samples were positive for swine influenza A virus. With an uncertain population estimate, the number of animals tested, the number of positives, and the sampling method do not allow us to make many inferences but suggest the need to implement and strengthen the existing surveillance activity, as it seems to be the only viable alternative for safeguarding animal and human health.

Evaluation of Control Program Against Streptococcus agalactiae Infection in Dairy Herds During 2019-2021 in Emilia-Romagna Region, Northern Italy.

Streptococcus agalactiae (group B Streptococcus, GBS) is a causative agent of mastitis in dairy cattle, mainly causing a subclinical disease associated with a high somatic cell count (SCC), and a consequent decrease in production yield and quality of milk. GBS has been almost eradicated in many Northern European countries, but there are warnings of its re-emergence as a zoonotic threat. In Italy, only two regions carry out a GBS control program: Lombardy and Emilia-Romagna. In Emilia-Romagna, the program has been in place since 2019 and provides for the bacteriological culture of bulk-tank milk (BTM) of all dairy farms every 6 months and the voluntary application of herd eradication programs in the case of positive results. To assess the progress of the program in Emilia Romagna, in terms of herd-level prevalence and GBS transmission between herds, we analyzed the results of 17,056 BTM cultures from 2,831 dairy herds, sampled bi-annually in the period 2019-2021 (six rounds total). The impact of GBS infection on SCC and milk production was also evaluated. The results show a decreasing trend in both the incidence rate (from 3.0 to 1.5%) and apparent prevalence (from 8.9 to 5.2%) of GBS over the study period. By using a susceptible-infected-susceptible (SIS) model for the estimation of the transmission parameters, a basic reproductive number R0 of 1.4 was calculated, indicating an active spread of GBS in the dairy cattle population of the Emilia-Romagna region. GBS infected farms have a consistently higher BTM SCC than negative ones (+77,000 cells/ml), corresponding to a 0.4 kg/cow/day milk loss. Moreover, GBS infected herds resulted in almost three times more likelihood of having non-marketable milk by exceeding the legal SCC limit. This study demonstrates the need to maintain the current control program against GBS to lower its occurrence and prevent significant market losses to farmers.

West Nile and Usutu Viruses' Surveillance in Birds of the Province of Ferrara, Italy, from 2015 to 2019.

West Nile (WNV) and Usutu (USUV) viruses are mosquito-borne flaviviruses. Thanks to their importance as zoonotic diseases, a regional plan for surveillance of Arboviruses was implemented in Emilia-Romagna in 2009. The province of Ferrara belongs to the Emilia-Romagna region, and it is an endemic territory for these viruses, with favorable ecological conditions for abundance of mosquitoes and wild birds. From 2015 to 2019, we collected 1842 dead-found birds at a wildlife rehabilitation center, which were analysed by three different PCRs for the detection of WNV and USUV genomes. August was characterized by the highest infection rate for both viruses. Columbiformes scored the highest USUV prevalence (8%), while Galliformes and Strigiformes reported the highest prevalence for WNV (13%). Among Passeriformes (the most populated Order), Turdus merula was the most abundant species and scored the highest prevalence for both viruses. To optimize passive surveillance plans, monitoring should be focused on the summer and towards the avian species more prone to infection by both viruses.

Economic evaluation of whole genome sequencing for pathogen identification and surveillance - results of case studies in Europe and the Americas 2016 to 2019.

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

A Review of Risk Factors of African Swine Fever Incursion in Pig Farming within the European Union Scenario.

African swine fever (ASF) is a notifiable viral disease of pigs and wild boars that could lead to serious economic losses for the entire European pork industry. As no effective treatment or vaccination is available, disease prevention and control rely on strictly enforced biosecurity measures tailored to the specific risk factors of ASF introduction within domestic pig populations. Here, we present a review addressing the risk factors associated with different European pig farming systems in the context of the actual epidemiological scenario. A list of keywords was combined into a Boolean query, "African swine fever" AND ("Risk factors" OR "Transmission" OR "Spread" OR "Pig farming" OR "Pigs" OR "Wild boars"); was run on 4 databases; and resulted in 52 documents of interest being reviewed. Based on our review, each farming system has its own peculiar risk factors: commercial farms, where best practices are already in place, may suffer from unintentional breaches in biosecurity, while backyard and outdoor farms may suffer from poor ASF awareness, sociocultural factors, and contact with wild boars. In the literature selected for our review, human-related activities and behaviours are presented as the main risks, but we also stress the need to implement biosecurity measures also tailored to risks factors that are specific for the different pig farming practices in the European Union (EU).

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.

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.

Processing-Dependent and Clonal Contamination Patterns of Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis.

The quantitative and qualitative patterns of environmental contamination by Listeria monocytogenes were investigated in the production chain of dry-cured Parma ham. Standard arrays of surfaces were sampled in processing facilities during a single visit per plant in the three compartments of the food chain, i.e., ham production (19 plants) and postproduction, which was divided into deboning (43 plants) and slicing (25 plants) steps. The numbers of sampled surfaces were 384 in ham production, with 25 positive for L. monocytogenes, and 1,084 in postproduction, with 83 positives. Statistical analysis of the prevalence of contaminated surfaces showed that in ham production, contamination was higher at the beginning of processing and declined significantly toward the end, while in postproduction, prevalence rose toward the end of processing. Prevalence was higher in the deboning facilities than in slicing facilities and was dependent on the type of surface (floor/drainage > clothing > equipment). The qualitative pattern of contamination was investigated through an analysis of the survey isolates and a set of isolates derived from routine monitoring, including longitudinal isolations. Pulsed-field gel electrophoresis (PFGE) and whole-genome single-nucleotide polymorphism (SNP) analysis revealed a remarkable clonality of L. monocytogenes within plants, with the detection of 16 plant-specific clones out of 17 establishments with multiple isolates. Repeated detections of clonal isolates >6 months apart were also observed. Six was the maximum number of between-isolate differences in core SNPs observed within these clones. Based on the same six-SNP threshold, three clusters of clonal isolates, shared by six establishments, were also identified. The spread of L. monocytogenes within and between plants, as indicated by its clonal behavior, is a matter of concern for the hygienic management of establishments.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Napoli Strain SN310, Cause of a Multischool Outbreak in Milan, Italy, in 2014.

We report the draft genome sequence of Salmonella enterica subsp. enterica serovar Napoli strain SN310, isolated from a stool sample of an affected pupil during a multischool outbreak in 2014 in Milan, Italy. This represents the first reported draft genome sequence of the emerging serovar Napoli.

Differential single nucleotide polymorphism-based analysis of an outbreak caused by Salmonella enterica serovar Manhattan reveals epidemiological details missed by standard pulsed-field gel electrophoresis.

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n=15) and food, feed, animal, and environmental sources (n=24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system.

A screen for and validation of prodrug antimicrobials.

The rise of resistant pathogens and chronic infections tolerant to antibiotics presents an unmet need for novel antimicrobial compounds. Identifying broad-spectrum leads is challenging due to the effective penetration barrier of Gram-negative bacteria, formed by an outer membrane restricting amphipathic compounds, and multidrug resistance (MDR) pumps. In chronic infections, pathogens are shielded from the immune system by biofilms or host cells, and dormant persisters tolerant to antibiotics are responsible for recalcitrance to chemotherapy with conventional antibiotics. We reasoned that the dual need for broad-spectrum and sterilizing compounds could be met by developing prodrugs that are activated by bacterium-specific enzymes and that these generally reactive compounds could kill persisters and accumulate over time due to irreversible binding to targets. We report the development of a screen for prodrugs, based on identifying compounds that nonspecifically inhibit reduction of the viability dye alamarBlue, and then eliminate generally toxic compounds by testing for cytotoxicity. A large pilot of 55,000 compounds against Escherichia coli produced 20 hits, 3 of which were further examined. One compound, ADC111, is an analog of a known nitrofuran prodrug nitrofurantoin, and its activity depends on the presence of activating enzymes nitroreductases. ADC112 is an analog of another known antimicrobial tilbroquinol with unknown mechanism of action, and ADC113 does not belong to an approved class. All three compounds had a good spectrum and showed good to excellent activity against persister cells in biofilm and stationary cultures. These results suggest that screening for overlooked prodrugs may present a viable platform for antimicrobial discovery.

Diarylacylhydrazones: Clostridium-selective antibacterials with activity against stationary-phase cells.

Current antibiotics for treating Clostridium difficile infections (CDI), that is, metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This Letter reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and Clostridium perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure-activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Manhattan Strain 111113, from an Outbreak of Human Infections in Northern Italy.

We announce the draft genome sequence of Salmonella enterica subsp. enterica serovar Manhattan strain 111113, isolated from a patient during an outbreak in northern Italy. The genome, which was obtained with Illumina MiSeq technology, is composed of 21 contigs for a total of 4,684,342 bp, with a G+C content of 52.17%.

Berberine-INF55 (5-nitro-2-phenylindole) hybrid antimicrobials: effects of varying the relative orientation of the berberine and INF55 components.

Hybrid antimicrobials containing an antibacterial linked to a multidrug resistance (MDR) pump inhibitor make up a promising new class of agents for countering efflux-mediated bacterial drug resistance. This study explores the effects of varying the relative orientation of the antibacterial and efflux pump inhibitor components in three isomeric hybrids (SS14, SS14-M, and SS14-P) which link the antibacterial alkaloid and known substrate for the NorA MDR pump berberine to different positions on INF55 (5-nitro-2-phenylindole), an inhibitor of NorA. The MICs for all three hybrids against wild-type, NorA-knockout, and NorA-overexpressing Staphylococcus aureus cells were found to be similar (9.4 to 40.2 microM), indicating that these compounds are not effectively effluxed by NorA. The three hybrids were also found to have similar curing effects in a Caenorhabditis elegans live infection model. Each hybrid was shown to accumulate in S. aureus cells to a greater extent than either berberine or berberine in the presence of INF55, and the uptake kinetics of SS14 were found to differ from those of SS14-M and SS14-P. The effects on the uptake and efflux of the NorA substrate ethidium bromide into S. aureus cells in the presence or absence of the hybrids were used to confirm MDR inhibition by the hybrids. MDR-inhibitory activity was confirmed for SS14-M and SS14-P but not for SS14. Molecular dynamics simulations revealed that SS14 prefers to adopt a conformation that is not prevalent in either SS14-M or SS14-P, which may explain why some properties of SS14 diverge from those of its two isomers. In summary, subtle repositioning of the pump-blocking INF55 moiety in berberine-INF55 hybrids was found to have a minimal effect on their antibacterial activities but to significantly alter their effects on MDR pumps.

High-throughput screen for novel antimicrobials using a whole animal infection model.

The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen. Here, we describe an automated, high-throughput screen of 37,200 compounds and natural product extracts for those that enhance survival of C. elegans infected with E. faecalis. Using a robot to dispense live, infected animals into 384-well plates and automated microscopy and image analysis, we identified 28 compounds and extracts not previously reported to have antimicrobial properties, including six structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use.

Antibacterial activity of berberine-NorA pump inhibitor hybrids with a methylene ether linking group.

Conjugation of the NorA substrate berberine and the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group gave the 13-substituted berberine-NorA inhibitor hybrid, 3. A series of simpler arylmethyl ether hybrid structures were also synthesized. The hybrid 3 showed excellent antibacterial activity (MIC Staphylococcus aureus, 1.7 microM), which was over 382-fold more active than the parent antibacterial berberine, against this bacterium. This compound was also shown to block the NorA efflux pump in S. aureus.

Structure-activity relationships of 2-aryl-1H-indole inhibitors of the NorA efflux pump in Staphylococcus aureus.

The synthesis of 22 2-aryl-1H-indoles, including 12 new compounds, has been achieved via Pd- or Rh-mediated methodologies, or selective electrophilic substitution. All three methods were based on elaborations from simple indole precursors. SAR studies on these indoles and 2-phenyl-1H-indole in Staphylococcus aureus as NorA efflux pump inhibitors indicated 5-nitro-2-(3-methoxycarbonyl)phenyl-1H-indole was a slightly more potent inhibitor than the lead INF55. A promising new antibacterial lead compound against S. aureus (2-phenyl-1H-indol-5-yl)-methanol, was also found.

Protective effect of cyanidin 3-O-beta-D-glucoside on ochratoxin A-mediated damage in the rat.

The aim of the present study was to verify whether the oral administration of cyanidin 3-O-beta-D-glucoside (C3G) might counteract damage induced by chronic exposure (28 d) to ochratoxin A (OTA) in rats and if its effect may be mediated by haeme oxygenase-1 (HO-1). Forty male Sprague-Dawley rats, individually caged, were divided into four groups of ten animals. A control group received a commercial diet, group C3G received the control diet supplemented with C3G (1 g/kg feed), group OTA received the control diet supplemented with 200 parts per billion of OTA, and group OTA+C3G received the OTA group diet supplemented with C3G (1 g/kg feed). After 4 weeks of treatment animals were killed and the liver, kidneys and brain of each rat were collected and homogenised to evaluate non-proteic thiol groups (RSH), lipid hydroperoxide (LOOH) levels, HO-1 expression and DNA fragmentation. Rats of the OTA group showed a significant (P < 0.001) decrease in RSH content of kidney and liver and a significant (P < 0.001) increase of LOOH in all the examined tissues compared with the control group. In the OTA+C3G group both RSH content and LOOH levels were similar to those observed in the control group, demonstrating that C3G was able to counteract the effects of OTA. A significant (P < 0.001) induction of HO-1 was evident in kidney and liver of both OTA and C3G groups. DNA damage occurred in all the examined tissues of the OTA group, whereas C3G was able to prevent it. The present study confirmed that the effects of OTA are mediated by oxidative stress and demonstrated that C3G efficiently counteracted deleterious effects of OTA because of its antioxidant and HO-1-inducing properties.