Scopo primario delle attività di ricerca condotte nell’ambito di questo dottorato è stato quello di studiare le infezioni da protozoi e microsporidi enterici in specie ittiche d’allevamento e le micobatteriosi ittiche in ambienti d’allevamento e selvatici per ampliare le conoscenze su eziopatogenesi e diagnosi di queste patologie. Spigole, orate, rombi e cefali da diversi sistemi d’allevamento e di differenti età sono stati sottoposti ad indagini parassitologiche, molecolari ed istopatologiche per evidenziare, identificare e studiare protozoi Apicomplexa e Microsporidi a livello enterico, applicando diverse colorazioni istochimiche e, sui campioni positivi per microsporidi, la colorazione immunoistochimica con anticorpo anti-Encephalitozoon cuniculi. Su mugilidi selvatici e su spigole allevate sono stati condotti studi sui quadri istopatologici associati alle infezioni da Micobatteri atipici al fine di valutare la progressione delle lesioni. Le attività di ricerca hanno permesso di: individuare e descrivere in orate d’allevamento episodi d’infezione enterica da Enterospora nucleophila, microsporidio responsabile di sindromi emaciative in giovanili; rilevare massive infezioni da Cryptosporidium scophthalmi in rombi allevati e di infezioni da Cryptosporidium sp. in post-larve ed avannotti di orata; studiare infezioni da Mycobacterium spp. (M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum, M. nonchromogenicum) in mugilidi selvatici con lesioni granulomatose positive alla ZN, e da Photobacterium damselae subsp. piscicida in soggetti con lesioni granulomatose negative; approfondire le conoscenze sulla micobatteriosi nella spigola descrivendo la concomitante presenza di lesioni a diverso stadio in tutti gli organi associate a presenza di batteri alcool-acido resistenti identificati come M. abscessus, M. scrofulaceum e M. gordonae sia all’interno delle lesioni che liberi nei tessuti e nei vasi, ipotizzando una forma di generalizzazione ematogena. Il rilievo di infezioni da micobatteri atipici in pesci marini allevati e selvatici appare di rilievo anche per le loro implicazioni in Sanità Pubblica. / Main aim of the research activities carried out within this PhD was to study the infections due to enteric protozoa and Microsporidia in farmed marine fish and mycobacteriosis by atypical mycobacteria in wild and farmed marine fish in order to increase the knowledge about etiopathogenesis and diagnosis of these diseases. European sea bass, gilthead sea bream, turbots and mullets from different breeding systems and at different ages were subjected to parasitological, molecular and histopathological analyses using different histochemical methods; moreover immunohistochemical stain with antibody for Encephalitozoon cuniculi on positive samples for microsporidiosis was performed. Wild mullets and farmed European sea bass were subjected to studies on histopathological features associated to atypical mycobacteria infections in order to evaluate the progression of lesions. The research activities have allowed to: identify and describe in sea bream episodes of enteric infection by Enterospora nucleophila, microsporidium causing emaciative syndromes in juveniles; detect Cryptosporidium scophthalmi massive infections in farmed turbots and Cryptosporidium sp. infection in post-larvae and fry of sea bream; study infections due to Mycobacterium spp. (M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum, M. nonchromogenicum) in wild mullets with granulomatous lesions positive for ZN, and due to Photobacterium damselae subsp. piscicida in subjects with negative lesions; increase the knowledge on mycobacteriosis in sea bass describing in all organs the simultaneous presence of lesions at different stages associated with the presence of positive ZN bacteria identified as M. abscessus, M. scrofulaceum and M. gordonae, both within the lesions that free in tissues and vessels, allowing to hypothesize a form of hematogenous generalization. The finding of atypical mycobacteria in wild and farmed fish is also relevant for their implications for public health.

Avian metapneumovirus (AMPV) is an enveloped negative sense single stranded RNA virus which is a major endemic respiratory pathogen of global domestic poultry. Since reverse genetic (RG) techniques have been applied to this pathogen several reports have investigated the effects of single and multiple genomic mutations and gene deletions or insertions on viral biology. The vectoring abilities of subtype A strains have been also investigated. The aim of this study was to develop a new RG system in order to extend this type of studies to the B subtype, gain a better understanding of the viral capacity to accept and express heterologous extra sequences. Initially was performed a nucleotide and amino acid comparison between subtypes A and B, to establish whether subtype A RG components could be partially or fully substituted. Eventually a cDNA copy of subtype B genome was generated and the virus rescued using subtype A and B RG components. The vectoring abilities of subtype B was firstly investigated to accept and express both spike (S1) and nucleocapsid (N) genes of infectious bronchitis virus (IBV). After viruses have been recovered by RG, all recombinants were found to be highly stable during passage in vitro and subsequently tested as candidate vaccines by eye-drop inoculation of one-day-old chickens. When chicks were challenged with IBV, negligible protection and replication results were observed. To improve the ability of the recombinant to induce protective immunity, the sequence of subtype B cDNA was modified in order to increase the replication in the respiratory tract. The new recombinants were rescued and challenge in vivo, showing an increase of the protection. Other strategies were adopted to improve the protection, as increasing the expression of the foreign genes and multiple genes insertions, but in most of the cases the virus rescue proved to be not possible.

Into the family Papillomaviridae, four different genera Delta Epsilon Xi and Dyoxi include the fifteen Bovine Papillomaviruses (BPVs) that have been characterized so far in cattle, even though it has been estimated that type number may exceed 20. Current classification system assumes that the host species in which a PV was firstly detected, is the original host, and the identified types are therefore named after it. Papillomaviruses (PVs) are indeed generally considered to be highly specific for their hosts; however, within the Delta-PVs, BPV types 1, 2 and 13 are well recognized to infect multiple species. Our results deepen the knowledge on circulation of BPV types in Italy and further contribute to a better understanding on their intra-genus variability. Besides observing that BPVs can contribute to the spread of other epitheliotropic viruses, we confirmed the host-jumping ability of the Delta PVs. The presence of co-infections between BPVs belonging to Delta and Xi genera in the healthy skin and mucosa of chamois and deer strongly suggest the reservoir role of wild ruminants belonging to the Bovidae and Cervidae families for domestic ruminant PVs. Furthermore, we identified the Eqsarc1 variant in healthy subjects and in non-equid species, suggesting an “equine adaptation” of the virus variant.

The public awareness that chemical substances are present ubiquitously in the environment, can be assumed through the diet and can exhibit various health effects, is very high in Europe and Italy. National and international institutions are called to provide figures on the magnitude, frequency, and duration of the population exposure to chemicals, including both natural or anthropogenic substances, voluntarily added to consumers’ good or accidentally entering the production chains. This thesis focuses broadly on how human population exposure to chemicals can be estimated, with particular attention to the methodological approaches and specific focus on dietary exposure assessment and biomonitoring. From the results obtained in the different studies collected in this thesis, it has been pointed out that when selecting the approach to use for the estimate of the exposure to chemicals, several different aspects must be taken into account: the nature of the chemical substance, the population of interest, clarify if the objective is to assess chronic or acute exposure, and finally, take into account the quality and quantity of data available in order to specify and quantify the uncertainty of the estimate. / La consapevolezza che le sostanze chimiche sono presenti in modo ubiquitario nell'ambiente, che possono essere assunte attraverso la dieta e che sono in grado di causare svariati effetti negativi sulla salute è molto alta nelle popolazione Europea ed Italiana. Le istituzioni nazionali e internazionali, sono chiamati a fornire stime dell’entità, della frequenza e della durata dell'esposizione della popolazione alle sostanze chimiche, includendo sostanze chimiche sia naturali che di origine antropica, sia volontariamente aggiunte che accidentalmente presenti nella catena di produzione. Questa tesi si concentra sostanzialmente su come può essere stimata l’esposizione della popolazione umana, con particolare attenzione agli approcci metodologici e con un focus specifico sulla valutazione dell'esposizione alimentare e biomonitoraggio. Dai risultati ottenuti nei diversi studi raccolti in questa tesi, si evince che quando si seleziona il metodo da utilizzare per la stima di esposizione ad uno o più sostanze chimiche, diversi aspetti devono essere presi in considerazione: la natura della sostanza chimica, la popolazione inclusa nello studio, è necessario chiarire se l'obiettivo è quello di stimare l'esposizione cronica o acuta, infine, deve essere valutata la qualità e la quantità dei dati disponibili al fine di specificare e quantificare l’incertezza delle stime.

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L’infezione da virus dell’ epatite E (HEV) nei suini e nell’uomo è stata segnalata in diversi Paesi. Nei suini, il virus causa infezioni asintomatiche, mentre nell’uomo è responsabile di epidemie di epatite ad andamento acuto nei Paesi a clima tropicale o subtropicale con condizioni igieniche scadenti, di casi sporadici in quelli sviluppati. HEV è stato isolato anche in diversi animali e l’analisi nucleotidica degli isolati virali di origine animale ha mostrato un elevato grado di omologia con i ceppi di HEV umani isolati nelle stesse aree geografiche, avvalorando l’ipotesi che l'infezione da HEV sia una zoonosi. In America del Sud HEV suino è stato isolato per la prima volta in suini argentini nel 2006, mentre solo dal 1998 esistono dati sull’ infezione da HEV nell’uomo in Bolivia. In questa indagine è stato eseguito uno studio di sieroprevalenza in due comunità rurali boliviane e i risultati sono stati confrontati con quelli dello studio di sieroprevalenza sopra menzionato condotto in altre zone rurali della Bolivia. Inoltre, mediante Nested RT-PCR, è stata verificata la presenza di HEV nella popolazione umana e suina. La sieroprevalenza per anticorpi IgG anti-HEV è risultata pari al 6,2%, molto simile a quella evidenziata nello studio precedente. La prevalenza maggiore (24%) si è osservata nei soggetti di età compresa tra 41 e 50 anni, confermando che l’ infezione da HEV è maggiore fra i giovani-adulti. La ricerca di anticorpi anti HEV di classe IgM eseguita su 52 sieri ha fornito 4 risultati positivi. Il genoma virale è stato identificato in uno dei 22 pool di feci umane e l'esame virologico di 30 campioni individuali fecali e 7 individuali di siero ha fornito rispettivamente risultati positivi in 4/30 e 1/7. La Nested RT-PCR eseguita sui 22 pool di feci suine ha dato esito positivo in 7 pool. L’analisi delle sequenze genomiche di tutti gli amplificati ha consentito di stabilire che gli isolati umani appartenevano allo stesso genotipo III di quelli suini e presentavano con questi una elevata omologia aminoacidica (92%). / Hepatitis E Virus (HEV) infection of swine and human has been reported in various countries. In humans the virus causes hepatitis E epidemics in areas with tropical or subtropical climate and poor sanitary conditions, and sporadic cases in developed countries. HEV infection in swine is quite spread and asymptomatic. HEV infections have been reported in different animals and animal strains were found to be closely related to human HEV strains from the same area, suggesting that hepatitis E may be a zoonosis. In South America, HEV of swine origin has been isolated for the first time in 2006 in Argentina, while human infection in Bolivia has been reported only in 1998 based on a serological investigation carried on in rural communities. In this study we have investigated the presence of HEV infection in two rural Bolivian communities and the results were compared with those of the aforementioned study. Anti-HEV IgG antibodies were detected in 6,2% of the sera examined. The highest seroprevalence (24%) was detected in the individuals aged from 41 to 50 years, confirming that the infection is more prevalent in young-adult individuals. Anti-IgM antibodies were detecte in 4 serum individuals out of 52 examined. HEV has been detected by means of Nested RT-PCR in 7 out of 22 pools of swine feces examined, in 1 pool out of 22 of human feces, 7 individual samples of human feces out of 30 and in 1 sample of human serum out of 7. Phylogenetic analysis demonstrated that human and swine HEV isolates could be included all into the genotype III and shared high degree of homology.

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.