Le malattie trasmesse da zecche sono un importante problema sia per la salute animale che per quella umana e negli ultimi decenni hanno aumentato notevolmente la loro diffusione, in seguito ai cambiamenti climatici, che hanno permesso la distribuzione delle zecche in aree prima non interessate. Per tale motivo si è deciso di effettuare un’indagine sulla diffusione delle zecche e sui patogeni da loro trasmessi, mediante campionamenti sia a livello ambientale, sia su animali e umani infestati in quattro siti di tre parchi dell’Emilia Romagna, dove non risultavano precedenti segnalazioni, nelle province di Bologna e Ravenna, da Aprile a Ottobre 2010. In totale sono state raccolte 8212 zecche. Dall’ambiente sono state campionate 6734 larve, 1344 ninfe, 61 adulti; dagli animali e da persone sono stati raccolti 68 adulti e 5 ninfe appartenenti a diverse specie di Ixodidae. Sono state condotte analisi sull’abbondanza delle zecche nelle diverse aree di raccolta, in funzione del periodo di campionamento, della temperatura e dell’umidità relativa misurata a 5 cm dal suolo al momento del campionamento e della vegetazione. Su tutti gli individui adulti e su pool di ninfe e di larve, per un totale di 393 campioni, sono state condotte analisi di tipo molecolare per la ricerca di piroplasmi, Anaplasma phagocytophilum e Borrelia burgdorferi s.l. Attraverso la PCR e il sequenziamento, è emerso che il 7,6% dei campioni era positivo per piroplasmi, tra i quali è stata riscontrata anche la presenza delle specie zoonosiche Babesia EU1 e B. divergens. La real-time PCR eseguita solo sui campioni costituiti da ninfe e adulti ha evidenziato una prevalenza del 9,2% per A. phagocytophilum e del 21,6% per B. burgdorferi s.l. Su questi patogeni sono state quindi condotte analisi di tipo filogenetico. In alcuni campioni sono state riscontrate coinfezioni con combinazioni di due patogeni contemporaneamente. / Tick-borne diseases are an important problem both for animals and for humans, which increased in the last years because of climatic changes that allowed the distribution of ticks in new areas. The aim of this study was to investigate the presence of ticks and pathogens transmitted, through samplings from environment and from animals and humans affected. The questing ticks were collected from 3 parks of Emilia Romagna region, for a total of 4 sites, in the provinces of Bologna and Ravenna from April to October 2010. A total of 8212 ticks were sampled. From environment were obtained 6734 larvae, 1344 nymphs and 61 adult ticks; from animals and humans 68 adults and 5 nymphs of different species of Ixodidae. Concerning the environmental sampling was analyzed the prevalence of ticks related to season, temperature and humidity measured at 5 cm above ground level at the moment of sampling and predominant vegetation. All the adult ticks, pools of nymphs and larvae, for a total of 393 samples, were investigated for the presence of piroplasms, Anaplasma phagocytophilum and Borrelia burgdorferi s.l. by molecular analyses. PCR and sequencing revealed that 7.5% of them was positive for piroplasms, among which were found two zoonotic species: Babesia EU1 and B. divergens. Real-time PCR on nymphs and adults showed a prevalence of 9.2% for A. phagocytophilum and 21.6% for B. burgdorferi s.l. This last was also analyzed by PCR-RFLP to identify the genospecies present. In some samples were found coinfections with combinations of two pathogens.

Le attività di ricerca della presente tesi di dottorato si sono focalizzata principalmente sulla parassitofauna dei pesci marini allevati in Grecia ed in Italia con particolare attenzione allo studio degli ectoparassiti di maggior rilievo sanitario in maricoltura ed alla ricerca di endoparassiti di potenziale interesse zoonosico, in particolare larve di nematodi Anisakidae del genere Anisakis. Nel corso del triennio sono stati sottoposti ad esami parassitologici 916 spigole (Dicentrarchus labrax) e 462 orate (Sparus aurata) prelevate presso diverse tipologie di allevamenti greci ed italiani. Per quanto concerne le spigole, la presenza di ectoparassiti è stata riscontrata nel 29,2% e nel 61,9% dei soggetti provenienti rispettivamente da impianti siti in Grecia ed in Italia, mentre le orate hanno presentato percentuali di positività rispettivamente del 87,5% e del 26,7%. Gli ectoparassiti dominanti sono risultati essere il monogeneo Diplectanum aequans nelle spigole ed il ciliato Cryptocaryon irritans e il monogeneo Furnestinia echeneis nelle orate, sebbene sia stato possibile studiare anche il coinvolgimento di altri ectoparassiti, quali il monogeneo Sparicotyle chrysophrii ed il dinoflagellato Amyloodinium ocellatum, nel determinismo di alcuni episodi morbosi. Le osservazioni istopatologiche hanno permesso di caratterizzare le lesioni causate dagli ectoparassiti a diverse intensità d’infestazione. Per quanto concerne la ricerca di parassiti zoonosici, con particolare riferimento agli stadi larvali di nematodi Anisakidae del genere Anisakis, si sono condotti esami parassitologici a livello di cavità viscerale e di muscolo laterale in tutti i soggetti provenienti da allevamenti in gabbia (626 soggetti, di cui 441 spigole e 185 orate). Tutti i soggetti esaminati sono risultati negativi, indicando come il rischio di infestazione da larve di nematodi anisakidi possa essere considerato trascurabile in spigole ed orate allevate in gabbia, come già dimostrato per il salmone atlantico (EFSA, 2010). / The research activities of this PhD thesis have been focused primarily on parasitofauna of marine fish reared in Greece and Italy, with particular reference to the study of ectoparasites showing main health significance in mariculture and to the search of potentially zoonotic endoparasites, namely larval stages of Anisakidae nematodes owing to Anisakis genus. During the three-years research period, 916 European sea bass and 462 sea bream, coming from different farming systems in Greece and Italy, have been subjected to parasitological examinations. With reference to sea bass, the presence of ectoparasites has been detected in 29,2% and 61,9% of fish coming from Greek and Italian farms respectively. Sea breams showed prevalence of ectoparasites of 87,5% and 26,7% (Greece and Italy respectively). Dominant ectoparasites were: the monogenean Diplectanum aequans in sea bass, and the ciliate Cryptocaryon irritans and the monogenean Furnestinia echeneis in sea bream, although it was observed also the involvement of other ectoparasites, such as the monogenean Sparicotyle chrysophrii and the dinoflagellate Amyloodinium ocellatum, in the etiology of some disease outbreaks. Histopathogical observations allowed to study and characterize the lesions caused by the ectoparasites found during the survey, with relation to infection intensity. Concerning the research activities aimed at assess the possible presence of zoonotic helminths, namely the larval stages of Anisakidae nematodes of Anisakis genus, parasitological exams have been carried out on visceral organs and lateral muscle of all the 626 samples coming from cages (441 sea bass and 185 sea bream). All the fish resulted negative for Anisakis larvae, pointing out that the risk of infestation by zoonotic anisakid nematodes may be considered negligible in caged sea bass and sea bream, as already assessed for Atlantic salmon (EFSA, 2010).

L'epatite E è una malattia umana con caratteristiche di epatite acuta, causata da un ssRNA virus (HEV). Nel 1997, HEV è stato identificato per la prima volta nei suini (SwHEV). In seguito, diverse evidenze, tra cui la vicinanza genetica tra ceppi umani e suini, suggerirono la trasmissione zoonotica del virus. Nella presente tesi, l’identificazione di SwHEV è stata condotta mediante ricerca di porzioni di genoma virale attraverso RT-PCR. Dal 2011 al 2013, sono stati analizzati 343 campioni fecali (da 19 allevamenti) e 70 bili (da 2 macelli) prelevati da altrettanti suini, in diverse Regioni italiane. E’ stato inoltre condotto uno studio retrospettivo su 78 feci (da 3 allevamenti) raccolte nel 2000. Il virus è stato identificato nel 24,5% e 19,2% delle feci raccolte rispettivamente nel 2011-2013 e nel 2000. Nessuna bile è risultata positiva. Mediante sequenziamento del genoma intero di uno dei virus identificati, è stata condotta l’analisi filogenetica per valutarne il grado di correlazione con alti ceppi suini e umani. La presenza di HEV è stata valutata lungo la filiera di produzione suina, dal macello al punto vendita. Trentaquattro campioni di feci, fegato e muscolo sono stati raccolti in un macello da altrettanti suini sani (età:6-7 mesi). Quattordici feci e 2 fegati, sono risultati positivi per HEV. Sono state prelevate 129 salsicce sia allo stabilimento di trasformazione sia alla vendita, ma nessuna è risultata positiva. La presenza di HEV è stata valutata anche nelle salsicce di fegato, fresche e secche, acquistate presso una macelleria. Il genoma virale è stato rilevato nel 22,2% delle salsicce fresche e nel 4,3 % di quelle secche ma la vitalità del virus non è stata dimostrata. In conclusione, lo studio condotto ha confermato l’ampia circolazione di HEV nei suini e la possibile contaminazione dei prodotti carnei derivati, confermando la necessità di una continua sorveglianza. / Hepatitis E is an acute disease of humans, caused by a positive ss RNA virus, Hepatitis E virus (HEV). Several evidences, including genetic similarity between human and swine HEV strains from the same area, suggest zoonotic transmission. From 2011 to 2013, we conducted a survey for HEV in 19 swine farms and 2 slaughterhouses in different Italian regions, collecting 343 feces and 70 bile samples. Moreover, a retrospective study was conducted on 78 animals housed in 3 farms locate in Northern Italy and collected in 2000. Overall HEV was detected in 24.5% and 19.2% of fecal samples collected in 2011-2013 and in 2000, respectively. On the contrary, none of bile samples was positive. Furthermore, the genome of one SwHEV strain was completely sequenced to evaluate possible correlation with swine and human HEV strains. We investigated presence of HEV in a swine food chain from farm to finish products, enrolling a big Italian factory which trades pig meat across the country. Thirty-four feces, liver and muscle samples were collected in a slaughterhouse from 34 clinically healthy pigs, aged 6-7 month old. At slaughterhouse 14 fecal samples and 2 liver, resulted positive for HEV. Moreover, none of the sausages (129) collected at processing and point of sale were positive. We have also evaluated the presence of HEV and fecal cross-contamination in pork liver sausages bought at a grocery in Italy. The genome of HEV was detected in both raw (22.2%) and dry (4.3%) liver sausages, but the viability of the virus was not demonstrated. Finally, several cell lines were inoculated with HEV-positive fecal and pork liver samples to attempt HEV replication but virus growth was not established.

Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually all warm-blooded species, including humans, but cats are the only definitive hosts. Humans or animals acquire T. gondii infection by ingesting food or water contaminated with sporulated oocysts or by ingesting tissue cysts containing bradyzoites. Toxoplasmosis has the highest human incidence among zoonotic parasitic diseases, but it is still considered an underreported zoonosis. The importance of T. gondii primary infection in livestock is related to the ability of the parasite to produce tissue cysts in infected animals, which may represent important sources of infection for humans. Consumption of undercooked mutton and pork are considered important sources of human Toxoplasma gondii. The first aim of this thesis was to develop a rapid and sensitive in- house indirect ELISA for the detection of antibodies against T. gondii in sheep sera. ROC-curve analysis showed high discriminatory power (AUC=0.999) and high sensitivity (99.4%) and specificity (99.8%) of the method. The ELISA was used to test a batch of sheep sera (375) collected in the Forli-Cesena district. The overall prevalence was estimated at 41.9% demonstrating that T. gondii infection is widely distributed in sheep reared in Forli-Cesena district. Since the epidemiological impact of waterborne transmission route of T.gondii to humans is now thought to be more significant than previously believed, the second aim of the thesis was to evaluate PCR based methods for detecting T. gondii DNA in raw and finished drinking water samples collected in Scotland. Samples were tested using a quantitative PCR on 529 bp repetitive elements. Only one raw water sample (0.3%), out of the 358 examined, tested T. gondii positive demonstrating that there is no evidence that tap water is a source of Toxoplasma infection in Scotland.

About three quarters of human emerging infectious diseases are caused by zoonotic pathogens, and many of them are spread by vectors such as mosquitoes. Mathematical models nowadays represent very powerful tools to make investigations and predictions for biological dynamical systems, providing helpful insights that can be extremely valuable for several aims. In this thesis, we will focus on a particular mosquito-borne zoonosis, West Nile virus (WNV), a flavivirus of emerging public health relevance in Europe and North America, and its main European vector, Culex pipiens mosquitoes. As the transmission of mosquito-borne diseases is largely driven by the abundance of the vector, to design appropriate control strategies it is crucial to understand the population dynamics of existing vector populations and evaluate how it depends on biotic and environmental factors. This thesis presents some new mathematical models that provide insights on several aspects of mosquito population dynamics by using different statistical and computational approaches, including for instance Linear Models and Markov chain Monte Carlo technique. Specifically, they aim to study the effect of biotic and abiotic factors on Cx. pipiens dynamics by using adult mosquito trapping data, gathered over several years in Northern Italy, to feed theoretical models. Furthermore, the effects of host competition and vector feeding preferences on the dynamics of a vector-borne infection (such as WNV) are investigated through a more theoretical study.

Context: West Nile virus (WNV) is an arthropod-borne virus considered a One Health challenge because of its increasing impact on human and animal health. It is one the most widely distributed viruses of the encephalitic Flaviviruses. It may cause severe neurological symptoms in humans and animals and is recognized as a serious public health problem also because of its impact on blood transfusion and organ transplantation. First identified in Africa in 1937, it was later introduced and spread in Italy, where in many regions it is now endemic, due to the increasingly favorable climatic and environmental conditions. Aim: The main objectives of this study, based on an interdisciplinary One Health approach, were: (1) to characterize the geographical distribution within specific host and vector populations in Africa; (2) to describe its phylogeographical patterns between Africa and Europe; (3) to define the genetic structure and epidemiology of Italian WNV strains, giving an insight of the viral circulation dynamics in the Italian territory. Methodology: Ecological and epidemiological studies were combined with molecular and phylogenetic analyses, carrying out field sampling activities, cellular culture, viral infection, immunofluorescent assay, multiplexed RT-PCR, sequencing, data analysis, and novel technique design. These activities were carried out both in Italy and in Senegal. Results: Our study evidences: (i) the circulation of several WNV lineages [Lineage 1 (L1), 2 (L2), 7 (L7), and 8 (L8)] in the African Continent; (ii) the presence of diverse competent mosquito vectors in Africa, mainly belonging to the Culex genus; (iii) the lack of vector competence studies for several other mosquito species found naturally infected with WNV in Africa; (iv) the need of more vector competence studies on ticks; (v) the circulation of WNV among humans, animals and vectors in at least 28 African countries; (vi) the lack of knowledge on the epidemiological situation of WNV for 19 African countries, and (vii) the importance of carrying out specific serological surveys in order to avoid possible bias on WNV circulation in Africa (objective 1). Furthermore, a new set of WNV L1 and L2 genome-specific primers for tiled-amplicon sequencing have been designed and a consistent dataset of 64 WNV L2 and 31 WNV L1 Italian genomes and of 3 WNV L2 and 7 WNV L1 Senegalese genome sequences from samples collected in Italy and Senegal between 2006 and 2022 has been produced. Twenty more WNV L1 and L2 Senegalese sequences obtained from samples collected in Senegal between 1985 and 2018 have been shared by the Institut Pasteur Dakar of Senegal and added to the dataset. This allowed the conduction of phylogenetic and phylogeographic analyses, evidencing: (viii) the presence of a strong viral connection between Africa and Europe, with intercontinental circulation supported by birds crossing international boundaries while migrating through the African-Eurasian flyways; (ix) the WNV L1 Western-Mediterranean cluster probable spread from Senegal, where the virus was first reported in 1979, to Italy, where the lineage first appeared in Europe in 1998, and to France in 2000, and the presence of back re-introductory events from Italy, Spain, and France to North and West Africa from the 2010s; and (x) the first African introduction of WNV L2 in Europe in Hungary in 2004, possibly from South African countries (objective 2). Our study also gives an insight of the dynamics of the viral circulation in Italy, demonstrating: (xi) the endemic presence of WNV L1 and L2 in part of Italy supported by resident wild birds and vector competent mosquitoes mainly belonging to the Culex genus; (xii) the current existence of two diverse WNV L1 strains circulating in Italy, one in the North-East, and one circulating intra-regionally in the Campania region; (xiii) suggested characteristic silent periods observed for WNV L1 in the country, with unnoticed circulation lasting sometimes for more than 10 years; (xiv) the 2022 WNV L1 increasing incidence of neurological disease cases in humans; (xv) the presence of genetically stable WNV L2 strains in Italy with continuous circulation throughout the time; (xvi) the presence of overwintering mechanisms supported by bird-to bird, rodent-to bird, or mosquito-to bird transmission routes; (xvii) the existence of WNV L1 and L2 co-infections in birds and mosquitoes; (xviii) the existence of a continuous transmission of the two strains between Western Mediterranean countries, supported by short distance migratory birds; and (xix) the crucial importance of the surveillance system other than the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases in Italy (objective 3). Conclusion: Our work points out the existence of high genetic diversity of WNV strains in Africa, the spread of L1 and L2 strains from Africa to Europe, and the existence of continuous transmission episodes among several Western-Mediterranean countries, with few recently suspected back introductory events from Europe to Africa. The progressive increase of the WNV L2 circulation both temporally and spatially in the Mediterranean countries and the WNV L1 re-appearance in Europe, both associated with a significant impact on humans and animal health, other than the strong WNV incidence in Italy and its endemization in part of its territory, evidence a solid WNV epidemic risk for Italy and a persistent threat for WNV spread into new areas. To predict and control future epidemics, it is crucial to constantly monitor the circulation and evolution of WNV in Europe and Africa, and to implement coordinated surveillance plans in both Continents, even in areas which are not currently affected.

In recent years, the world has faced major health challenges, from the rise of antibiotic resistance to the emergence of new pathogens with pandemic potential. This highlights the importance of considering human health as inextricably intertwined with that of other animals and the environment in which they live. This paradigm is known as “One Health”, which is the integration of environmental sciences, veterinary science, and medicine. Within this perspective, viruses, the most abundant biological entities on Earth, play a central role in connecting different organisms, deeply influencing the health of their hosts. Despite their great importance, most viruses are still poorly understood, mainly because of the technical and economic limitations posed by isolation, cultivation, and single colony sequencing. However, recently developed genomic technologies offer a cheaper and more sensitive alternative to study viruses, allowing a better integration of data from various sources and making it possible to explore how they circulate among different hosts and environments. In this thesis, I hypotesise that, by combining different classes of genomic methods with One Health practices, it is possible to reveal much more of the entire picture of viral diversity and evolution that by simply using them in a separated way. I show that this is the case for each one of the scientific questions addressed in this work, which are organised in three main chapters: - In the second chapter I analysed 22 metatranscriptomes from tick samples from different parts of Italy, obtaining a set of 91 viral contigs for which I reconstructed the phylogeny, with the aim to identify the presence of possible pathogens and characterise the unexplored viral diversity in the country. This analysis not only clarified the molecular epidemiology of well-known pathogens such as Tick-borne encephalitis virus, but also allowed the discovery of at least 10 novel viral species. - In the third chapter, I investigated the origin and spread of West Nile virus, an emerging pathogen causing neurological disease worldwide. The goal was to expand the current knowledge of this virus by increasing the number of sequenced genomes and to reconstruct how the virus moved between Africa and Europe. Phylogeographic and phylodynamic methods showed that this pathogen originated in Africa and repeatedly invaded the European continent, revealing the dynamics of its evolution through space and time. - In the last chapter, I contributed to obtaining a set of DNA phages assembled from human microbiomes and viromes by manual curation and comparative genomics and developed a new approach to study their evolution in relation to their bacterial hosts. I found that the strength of co-phylogenetic patterns between viruses and their hosts is generally weak, suggesting that their ecological relationships emerge on short evolutionary timescales. Taken together, these results show how the integration of viral metagenomics and phylogenomics in One Health is essential to answer fundamental questions about the diversity of viruses and how they spread and evolve. Furthermore, the methods and protocols developed in these studies can be applied to similar cases, allowing a systematic exploration of many other datasets to expand our knowledge of the virosphere. This information can be used to implement containment strategies, public health policies, therapies, and biotechnologies.

Attività antropiche e pressioni di mercato stanno rapidamente riducendo la biodiversità. Per questa ragione, conservare il patrimonio ecosistemico, tassonomico e genetico risulta fondamentale al fine di garantire potenziale adattativo alle specie, e, in ultima analisi, un futuro sostenibile per il pianeta. Al fine di minimizzare la perdita di biodiversità, numerosi metodi sono stati proposti per priorizzare ecosistemi, specie e popolazioni. Il presente lavoro di tesi fornisce in primo luogo una revisione di tali approcci, proponendo un albero decisionale volto a favorirne un corretto utilizzo. Secondariamente, la variabilità genomica neutrale del bufalo d’acqua (Bubalus bubalis L.) è investigata per mezzo di un pannello di marcatori SNP a media densità, rivelando due centri di domesticazione (India Nord-occidentale, Cina-Indocina) e possibili rotte di migrazione per gli ecotipi ‘river’ e ‘swamp’. L’adattamento locale ad East Coast Fever, patologia endemica delle popolazioni bovine in Africa Sub-sahariana, è stato inoltre studiato in bovini autoctoni Ugandesi (Bos taurus L.) combinando tecniche di modellizzazione delle nicchie ecologiche e di genomica del paesaggio. L’approccio ha portato ad indentificare PRKG1 e SLA2 come possibili geni di adattamento. I risultati sono discussi alla luce delle possibili implicazioni nella conservazione del bufalo e nella gestione delle risorse genetiche animali Ugandesi. / Biodiversity is quickly disappearing due to human impact on the biosphere, and to market pressure. Consequently, the protection of both wild and domestic species needs to become a priority in order to preserve their evolutionary potential and, ultimately, guarantee a sustainable future for coming human generations. To date, tens of methods have been proposed to prioritize biodiversity for conservation purposes. Here, an ontology for priority setting in conservation biology is provided with the aim of supporting the selection of the most opportune methodologies given specific conservation goals. Further, two case studies are presented characterizing neutral and adaptive genomic diversity in water buffalo (Bubalus bubalis L.) and indigenous Ugandan cattle (Bos taurus L.), respectively. In particular, two independent domestication centres (North-western India and Indochina) and separate migration routes are suggested for the ‘river’ and ‘swamp’ water buffalo types. In the case of indigenous Ugandan cattle, the integration of species distribution modelling and landscape genomics techniques allowed the identification of PRKG1 and SLA2 as candidate genes for local adaptation to East Coast Fever, a vector-borne disease affecting bovine populations of Sub-Saharan Africa. Results are discussed for their implications in water buffalo conservation and Ugandan cattle adaptive management.