One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Class 1 integron and Enterococcus spp. abundances in swine farms from the " Suckling piglets" to the "Fatteners" production category.

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e., "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment.

Tetracyclines in Processed Animal Proteins: A Monitoring Study on Their Occurrence and Antimicrobial Activity.

In 2013, the European Union (EU) lifted the feed ban restriction, authorizing the use of non-ruminant (NR) processed animal proteins (PAPs) as ingredient in aquafeed. A further relaxation is soon expected, and NR PAPs will be allowed in next future in poultry and pig feed, avoiding cannibalism. Other potential hazards linked to PAPs as raw material should be evaluated. Antibiotics administered along the lifecycle of animals may leave residue in tissues and bones and still be present in PAPs. This monitoring study aimed to determine tetracyclines (TCLs), known to cumulate in bones, in PAPs and their possible residual antibiotic activity (RAC). A sensitive Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) method for the quantification of TCLs in PAPs was developed and applied to 55 PAPs from EU manufactures. Most PAP samples (n = 40) contained TCLs (concentrations 25.59 ÷ 456.84 µg kg-1). Among samples containing more than 25 µg kg-1 for at least three TCLs, three PAPs were chosen for RAC test before and after TCLs extraction procedure applying an in vitro acidic digestion: in two out of those three samples, RAC was observed after in vitro digestion. TCLs were determined in the digested PAPs (concentrations 26.07 ÷ 64.55 µg kg-1). The detection of TCLs in PAPs should promptly target the risk assessments of this unconsidered way of exposure to antibiotic residues.

Ticks infesting humans and associated pathogens: a cross-sectional study in a 3-year period (2017-2019) in northwest Italy.

BACKGROUND: Tick-borne diseases are common throughout Europe. Ticks transmit pathogens to the host while feeding and together with mosquitoes, they are major vectors of infectious agents worldwide. In recent years, there has been a marked increase in the incidence of tick-bite events and tick-borne disease in northwest Italy, but information on the prevalence of tick-borne pathogens in ticks removed from humans remains scarce. To fill this gap, we report here the prevalence of tick bites and tick-borne pathogens documented for humans in Piedmont, northwest Italy, in the 3-year period 2017-2019. METHODS: Ticks attached to humans during 2017-2019 were collected from residents of urban and rural area by physicians and veterinarians working with local veterinary agencies. All ticks (n = 1290) were morphologically identified to the species level. A subset of ticks removed from children (age 0-18 years) and the elderly (> 70 years), both age groups considered to be at-risk populations, was screened by biomolecular analysis to detect pathogens (e.g. Rickettsia spp., Borrelia spp., Anaplasma spp.). Pathogen identity was confirmed by Sanger sequencing. RESULTS: Ticks were taxonomically assigned to ten species of six genera (Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus). Most belonged to the genus Ixodes: 1009 ticks (78.22%) were classified as Ixodes ricinus. A subset of 500 ticks collected from the two at-risk populations were subjected to PCR assay to determine the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. The overall prevalence of infection was 22.8% (n = 114; 95% confidence interval [CI]: 19.19-26.73%), meaning that at least one pathogen was detected: Rickettsia spp. (prevalence 15%, n = 76; 95% CI 12.17-18.65%); Borrelia spp. (prevalence 6.4%, n = 32; 95% CI 4.42-8.92%); and Anaplasma spp. (prevalence 1.2%, n = 6; 95% CI 0.44-2.6%). CONCLUSIONS: Our data underline the importance of surveillance in the epidemiology of tick-borne diseases and the implementation of strategies to control tick infestation and associated pathogens.

Mosquitoes of the Maculipennis complex in Northern Italy.

The correct identification of mosquito vectors is often hampered by the presence of morphologically indiscernible sibling species. The Maculipennis complex is one of these groups that include both malaria vectors of primary importance and species of low/negligible epidemiological relevance, of which distribution data in Italy are outdated. Our study was aimed at providing an updated distribution of Maculipennis complex in Northern Italy through the sampling and morphological/molecular identification of specimens from five regions. The most abundant species was Anopheles messeae (2032), followed by Anopheles maculipennis s.s. (418), Anopheles atroparvus (28) and Anopheles melanoon (13). Taking advantage of ITS2 barcoding, we were able to finely characterize tested mosquitoes, classifying all the Anopheles messeae specimens as Anopheles daciae, a taxon with debated rank to which we referred as species inquirenda (sp. inq.). The distribution of species was characterized by Ecological Niche Models (ENMs), fed by recorded points of presence. ENMs provided clues on the ecological preferences of the detected species, with An. daciae sp. inq. linked to stable breeding sites and An. maculipennis s.s. more associated to ephemeral breeding sites. We demonstrate that historical Anopheles malaria vectors are still present in Northern Italy.

Application of a risk-based standardized animal biomonitoring approach to contaminated sites.

Biomonitoring has been used to disclose the public health impact of contaminated sites. This study aimed at setting up good practices to apply biomonitoring targeting animal matrixes to design risk-based surveillance and exposure assessment plans. A nine-step protocol targeting farmed animals was devised and tested in three case study areas including (1) a waste dump, (2) a waste incinerator, and (3) a secondary aluminum smelter. Between 2010 and 2012, in each study area, the following 9-step best practices were applied: hazard identification, GIS project creation, risk area delimitation, control area selection, receptors (livestock) identification, farms and matrixes selection, sampling study design, on-farm secondary sources exclusion, and statistical and geostatistical analysis. Dairy farms and free-range laying hens were the primary targets: eggs from both risk and control areas and milk from risk areas were sampled and submitted for detection of selected tracking contaminants compatible with the putative sources. Comparison data (risk vs. control) of heavy metal concentrations in eggs were available only for case study 2, whereas egg comparison data of persistent organic pollutants were available for all the risk-control pairs. After taking into account potential secondary sources, no concern from metals was arisen, whereas high concentrations of persistent organic pollutants were detected in all risk areas; however, only for the aluminum smelter case study, the contamination was broad and higher in the risk area compared with the control one. The protocol has proved to be easily applicable and flexible to varying contexts and able to provide helpful data to inform risk management decisions.

A single nucleotide variant in the promoter region of the CCR5 gene increases susceptibility to arthritis encephalitis virus in goats.

BACKGROUND: The small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses that includes caprine arthritis encephalitis virus (CAEV) and Maedi-Visna virus (MVV). SRLVs affect the production and welfare of sheep and goats worldwide. There is currently no effective treatment. Their high mutation rate precludes vaccine development, making innovative control measures necessary. A variant of the chemokine (C-C motif) receptor 5 (CCR5) gene is reportedly involved in resistance to human immunodeficiency (HIV) infection in humans and to SRLV in sheep. The aim of this study was to analyse the genetic structure and variability of the CCR5 gene in goats and to carry out a cross-sectional study to investigate the role of CCR5 genetic variants in controlling susceptibility/resistance to CAEV. RESULTS: The variant g.1059 T located in the promoter region revealed an interesting association with high proviral loads (a 2.8-fold increased risk). A possible explanation could be an alteration of the transcriptional level. Overexpression of the CCR5 receptor on the cell surface may increase virus internalization and proviral load as a consequence. CONCLUSIONS: Our findings could be advantageously used to reduce the susceptibility of goat herds to CAEV by negatively selecting animals carrying the g.1059 T mutation. Eliminating animals predisposed to high proviral loads could also limit the development of clinical signs and the spread of the virus, since these animals are also highly efficient in shedding the virus.

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS.

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.

Phenotypical Variability in Bovine Spongiform Encephalopathy: Epidemiology, Pathogenesis, and Diagnosis of Classical and Atypical Forms.

After thirty years, bovine spongiform encephalopathy (BSE) still represents the biggest crisis in the field of food safety. Initially detected in the United Kingdom in 1986, BSE spread to many other countries all over the world, involving approximately 200,000 cattle. The origin of BSE is uncertain, but epidemiological studies suggest that the source was cattle feed prepared from prion-infected animal tissues. The implementation of the drastic measures, including the ban of meat and bone meal from livestock feed and the removal of specified risk material from the food chain, has eventually resulted in a significant decline of the epidemic. For many years, it was believed that the disease was caused by transmission of a single prion strain. However, since 2004 two types of BSE, with distinct phenotypical characteristics, have been detected in Italy and France. These atypical types are characterized by distinct Western Blot profiles of abnormal protease-resistant prion protein, named high-type (H-BSE) and low-type (L-BSE). At present, there is no comprehensive information about the origin of the atypical BSEs (sporadic vs. acquired), and data about the pathogenesis of both atypical forms are very limited as compared to the classical type (C-BSE). This chapter will provide a well-organized overview of what is known about classical and atypical BSE. It will review information on the main epidemiological features, pathogenesis, and the criteria for routine diagnosis based on rapid tests, histological, immunohistochemical, and Western blot analysis. Furthermore, a brief overview about the most recently in vitro techniques will be also provided.

Gravimetric quantitative determination of packaging residues in feed from former food.

Valorisation of former foodstuff products (FFP) in feed is part of a long-term strategy for sustainability. An approach to valorise FFP outside the waste value chain is their use as an alternative source of feed materials, with a subsequent optimisation of the environmental impact of products. In the current practice of food production, food packaging is provided to ensure the maintenance of food quality and safety during transport and storage. One of the problems of reusing FFP is how to deal with packaging materials or remains that can become residues in the feed. The aim of this study is to propose a fast and sensitive gravimetric method, fit for routine official controls, for the determination of packaging residues in feed. The developed method can briefly be summarised as: (1) visual selection of the undesired ingredients which can be identified as remnants of packaging materials; (2) weighing of the selected materials; (3) defatting; (4) dehydration; (5) final weighing; and (6) reporting of weight and percentage. Moreover, the method has been validated through the determination of some of the parameters listed in Council Regulation 2004/882/EC (i.e., specificity, limit of quantification (LOQ), recovery, repeatability, within-laboratory reproducibility and measurement uncertainty).

Estimating canine cancer incidence: findings from a population-based tumour registry in northwestern Italy.

BACKGROUND: Canine cancer registry data can be put to good use in epidemiological studies. Quantitative comparison of tumour types may reveal unusual cancer frequencies, providing directions for research and generation of hypotheses of cancer causation in a specific area, and suggest leads for identifying risk factors. Here we report canine cancer incidence rates calculated from a population-based registry in an area without any known specific environmental hazard. RESULTS: In its 90 months of operation from 2001 to 2008 (the observation period in this study), the population-based Piedmont Canine Cancer Registry collected data on 1175 tumours confirmed by histopathological diagnosis. The incidence rate was 804 per 100,000 dog-years for malignant tumours and 897 per 100,000 dog-years for benign tumours. Higher rates for all cancers were observed in purebred dogs, particularly in Yorkshire terrier and Boxer. The most prevalent malignant neoplasms were cutaneous mastocytoma and hemangiopericytoma, and mammary gland complex carcinoma and simplex carcinoma. CONCLUSIONS: The Piedmont canine cancer registry is one of few of its kind whose operations have been consistently supported by long-term public funding. The registry-based cancer incidence rates were estimated with particular attention to the validity of data collection, thus minimizing the potential for bias. The findings on cancer incidence rates may provide a reliable reference for comparison studies. Researches conducted on dogs, used as sentinels for community exposure to environmental carcinogens, can be useful to detect excess risks in the incidence of malignant tumours in the human population.

Control of Mosquito-Borne Diseases in Northwestern Italy: Preparedness from One Season to the Next.

INTRODUCTION: Mosquito-borne diseases (MBDs) are spreading worldwide due to globalization and climate change, representing a threat for both humans and animals. Of great concern are the infections caused by viruses belonging to the Flavivirus genus as West Nile virus (WNV) and Usutu virus (USUV) transmitted by Culex sp. or Dengue virus and Zika virus (ZIKV), transmitted by Aedes sp. This work describes the surveillance protocol enforced in Piedmont (Northwestern Italy) to control MBDs spread, focusing on the activities performed on mosquitoes during the 2015 vector season. MATERIALS AND METHODS: From July to October, mosquitoes were fortnightly sampled in 50 selected sites according to risk factors with CDC dry ice-baited traps and BG-Sentinel traps baited with BG-Lure and dry ice. Adults were counted, identified to species level, pooled, and screened for flaviviruses using different reverse transcription-PCR protocols and sequencing. Finally, phylogenetic analysis was performed on a dataset including 2014 and 2015 WNV sequences and reference sequences retrieved from GenBank. RESULTS AND DISCUSSION: A total of 17,000 mosquitoes, grouped in 730 pools, were tested. Five pools of Culex pipiens were positive for WNV Lineage 2 in Novara, Alessandria, Vercelli, and Torino Provinces. One pool of C. pipiens and one pool of Anopheles maculipennis s.l. were positive for USUV in Vercelli and Alessandria Provinces. In Vercelli Province one pool of C. pipiens resulted positive both for WNV and USUV. Control measures were quickly implemented. Phylogenetic analyses showed that the WNV Lin 2 sequences from Piedmont region cluster with those circulating in Northeastern Italy in the previous years. Given the positive trend in WNV activity compared to 2014 and the emergence caused by other flavivirus as ZIKV, the level of attention for the 2016 vector season may be increased and this surveillance protocol could represent an important tool for public health authorities.

Detection of West Nile and Usutu Viruses in Italian Free Areas: Entomological Surveillance in Piemonte and Liguria Regions, 2014.

West Nile virus and Usutu virus have established in different parts of Italy over the past 10 years. Piemonte and Liguria Regions (Northwestern Italy) are known to be nonendemic areas, despite the presence of competent vectors and environmental conditions conducive to maintaining infection. This work evidences for the first time, through an entomological surveillance implemented on the basis of risk factor approach, the presence of West Nile and Usutu viruses in Piemonte and Liguria Regions (Northwestern Italy).

Identification of single nucleotide polymorphisms in Toll-like receptor candidate genes associated with tuberculosis infection in water buffalo (Bubalus bubalis).

BACKGROUND: Toll-like receptors play a key role in innate immunity by recognizing pathogens and activating appropriate responses. Pathogens express several signal molecules (pathogen-associated molecular patterns, PAMPs) essential for survival and pathogenicity. Recognition of PAMPs triggers an array of anti-microbial immune responses through the induction of various inflammatory cytokines. The objective of this work was to perform a case-control study to characterize the distribution of polymorphisms in three candidate genes (toll-like receptor 2, toll-like receptor 4, toll-like receptor 9) and to test their role as potential risk factors for tuberculosis infection in water buffalo (Bubalus bubalis). RESULTS: The case-control study included 184 subjects, 59 of which resulted positive to both intradermal TB test and Mycobacterium bovis isolation (cases) and 125 resulted negative to at least three consecutive intradermal TB tests. The statistical analysis indicated that two polymorphisms exhibited significant differences in allelic frequencies between cases and controls. Indeed, the TT genotype at TLR9 2340 C > T locus resulted significantly associated with susceptibility to bovine tuberculosis (P = 0.030, OR = 3.31, 95% CI = 1.05-10.40). One polymorphism resulted significantly associated with resistance to the disease, and included the CC genotype, at the TLR4 672 A > C locus (P = 0.01, OR = 0.26, 95% CI = 0.08-0.80). Haplotype reconstruction of the TLR2 gene revealed one haplotype (CTTACCAGCGGCCAGTCCC) associated with disease resistance (P = 0.04, OR = 0.51, 95% CI = 0.27-0.96), including the allelic variant associated with disease resistance. CONCLUSIONS: The work describes novel mutations in bubaline TLR2, TLR4 and TLR9 genes and presents their association with M. bovis infection. These results will enhance our ability to determine the risk of developing the disease by improving the knowledge of the immune mechanisms involved in host response to mycobacterial infection, and will allow the creation of multiple layers of disease resistance in herds by selective breeding.

Data on milk dioxin contamination linked with the location of fodder croplands allow to hypothesize the origin of the pollution source in an Italian valley.

Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs) have similar toxic, endocrine-disrupting, and carcinogenic activity. They are classified as persistent organic pollutants accumulating in the environment and the tissues of living organisms. High concentrations of PCDD/F and dl-PCB have been detected in bovine milk collected in a Piedmont valley (Northwestern Italy) since 2004. This geographic study describes the local distribution of pollution from PCDD/Fs and dl-PCBs. Since their presence in animal products could be traced back to the ingestion of contaminated fodder, dioxin levels in cow milk were related to the distribution of fodder cropland parcels. Specifically, the aim of the study was to determine, through an exploratory approach, whether the contamination was consistent with one common point source of contamination or different scattered sources. Data for PCDD/F and dl-PCB concentrations in the bulk milk from 27 herds, sampled over a 4-year period (2004-2007), were matched to the georeferenced land parcels the dairy farmers used for growing fodder. Isopleth maps of dioxin concentrations were estimated with ordinary kriging. The highest level of pollution for both PCDD/Fs and dl-PCBs was geographically juxtaposed: in both instances, the location of the local steel plant was within this extremely highly polluted area. The study results support the hypothesis for one common point source of contamination in the valley. The exploratory spatial analysis applied in this research may provide a valuable, novel approach to straightforward identification of a highly likely source of dioxin contamination of dairy products (even in the absence of top soil contamination data).

Molecular characterization of flaviviruses from field-collected mosquitoes in northwestern Italy, 2011-2012.

BACKGROUND: The genus Flavivirus comprises several mosquito-borne species, including the zoonotic pathogens West Nile and Usutu virus, circulating in animals and humans in Italy since 1998. Due to its ecological and geographical features, Piedmont is considered a risk area for flavivirus transmission. Here we report the results of a flavivirus survey (detection and genetic characterization) of mosquitoes collected in Piedmont in 2012 and the genetic characterization of three strains detected in 2011. METHODS: Pools of 1-203 mosquitoes, upon RNA extraction with TRIzol, were screened by a PCR assay for a 263 bp fragment of the Flavivirus NS5 gene. All positive samples were tested with a specific PCR for the E protein gene of Usutu virus and a generic Flavivirus RT-nested-PCR for a larger tract of the NS5 gene before sequencing. Phylogenetic trees were built with both NS5 fragments of representative Flavivirus species. DNA extracts of part of the positive pools were tested to detect sequences integrated in the host genome. RESULTS: Thirty-four mosquito pools resulted positive for flaviviruses, and twenty-five flavivirus sequences underwent phylogenetic analysis for the short NS5 fragment. Among the 19 sequences correlating with the insect-specific flavivirus group, ten samples, retrieved from Aedes albopictus, clustered within Aedes flavivirus, while the other nine aggregated in a separate clade composed of strains from various mosquito species (mainly Aedes vexans) from Piedmont and the Czech Republic. Six out of these nine also presented a DNA form of the sequence. The remaining sequences belonged to the mosquito-borne group: four, all from Culex pipiens, correlated to Italian Usutu virus strains, whereas two, from Ochlerotatus caspius, were highly similar to Marisma mosquito virus (MMV). CONCLUSIONS: Our findings confirm the circulation of Usutu virus and of the potentially zoonotic Marisma mosquito virus in Piedmont. This is the first detection of Aedes flavivirus in Piedmont. Finally, further evidence for the integration of Flavivirus nucleic acid into the host genome has been shown. These results underline the importance of continuing intense mosquito-based surveillance in Piedmont, supported by a mosquito control program in areas at high risk for human exposure.

[Environmental micropollutants in livestock products from Susa Valley, Piedmont]. / La contaminazione ambientale da microinquinanti degli allevamenti della Val di Susa.

OBJECTIVE: to describe the contamination due to dioxins and dioxin-like PCBs of cattle dairy and meat products in cattle herds from Susa Valley (Piedmont) since the second half of 2004. DESIGN: descriptive study based on a biomonitoring campaign on bovine bulk milk and muscle. SETTING AND PARTICIPANTS: the official samplings have been carried out between 2004 and 2010: the dataset refers to 99 mass milk samples and 24 muscle samples from 51 cattle herds based in 23 municipalities of the valley. MAIN OUTCOME MEASURES: counts and frequencies of samples exceeding either the maximum levels or the action levels set by the European legislation; moreover relative distributions of congeners (profiles) were considered. RESULTS: DL-PCBs showed median concentration values double when compared with dioxins values and a couple of congeners (2,3,4,7,8-PeCDF, 118 DL-PCB) were particularly abundant. More than 60% of both the milk samples and herds exceeded the action levels with the involvement of 10 municipalities most of which located nearby a local steel smelter. The DL-PCB values in milk were associated with the distance of each herd from the steel plant. However, the temporal trend highlights a clear decrease of the contamination over the 2004-2010 period, when the plant was renewed and the production levels where negatively impacted by the global economical crisis. CONCLUSION: the findings of the monitoring activity allowed the understanding of the origin and the spatio-temporal evolution of the contamination: this knowledge may be helpful in orienting future controls and the risk mitigation strategies.

A relevant long-term impact of the circulation of a potentially contaminated vaccine on the distribution of scrapie in Italy. Results from a retrospective cohort study.

A sudden increase in the incidence of scrapie in Italy in 1997 was subsequently linked to the use of a potentially infected vaccine against contagious agalactia. The relative risk for the exposed farms ranged between 6 and 40. The aim of this study was to assess the long-term impact of exposure to the potentially scrapie-contaminated vaccine on the Italian classical scrapie epidemic. We carried out a retrospective cohort study, fitting mixed-effects Poisson regression models, dividing national geographic areas into exposure categories on the basis of the vaccine circulation levels. We took into account the sensitivity of the surveillance system applied in the different areas. The population attributable fraction (PAF) was used to assess the impact on the total population of farms associated with the effect of circulation of the vaccine. The provinces where the vaccine was more often sold were noted to have a higher level of disease when compared to those provinces where the vaccine was sold less often (incidence rate ratio [IRR]: 2.7; 95% confidence interval [CI]: 1.1-6.5). The population attributable fraction was high (68.4%). Standardization techniques allowed to account for the potential of geographical variability in the sensitivity of the Italian surveillance system. Although the number of the directly exposed farms was limited, an important long-term impact of the vaccine circulation could be quantified in terms of secondary outbreaks likely due to the exchange of animals from directly exposed flocks.