Calzolari M, Bonilauri P, Bellini R, Albieri A, D efilippo G, Maioli G, Tamba M, Angelini P, Dottori M

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1 TAB RICERCHE EFFETTUATE Amadori M ARGOMENTI VARI Il benessere animale: un compromesso tra produttività economica e qualità della vita zootecnica Aree di azione e formazione continua per la prevenzione primaria in medicina veterinaria / a cura di G. Brambilla... [et al.]. - Roma : Istituto Superiore di Sanità, (ISTISAN rapporti ; 11 / 6) p ref bib [Nr. Estr. 4673] Bertocchi L, Tranquillo V, Donina D, Cinotti G, C ompiani R, Sgoifo_Rossi CA La soia "alternativa" fa bene alla lattifera Allev Magazine. - Vol. no 3 ( 2011). - p [Nr. Estr. 4629] Bompadre G, Cinotti S Gestione dei problemi comportamentali nell interazione uomo-cane = Managing behavioural problems in human-dog interactions Ann Ist Super Sanita'. - Vol. 47 no 4 ( 2011). - p bib ref [Nr. Estr. 4931] La gestione dei disturbi comportamentali nel cane richiede competenze professionali e specialistiche. Attualmente la figura professionale di riferimento è quella del medico veterinario comportamentalista. L approccio di tipo clinico al disturbo comportamentale riportato dal proprietario del cane permette al medico veterinario comportamentalista di formulare una diagnosi e di prescriverne la terapia comportamentale e/o farmacologica. L obiettivo della terapia comportamentale è di generare un cambiamento di tipo stabile nella percezione, e perciò nella emozione ad essa correlata, di uno stimolo, e nel comportamento abitualmente conseguente. Di fondamentale importanza è stabilire, sulla base dei sintomi, lo stato patologico del soggetto, utile a individuare l alterazione funzionale dei principali sistemi di neurotrasmettitori coinvolti, a cui far fronte mediante un adeguato trattamento farmacologico. Nell attuazione della terapia comportamentale il medico veterinario comportamentalista collabora, ove necessario, con un equipe di figure qualificate in ambito cinofilo. The management of dog behavioural problems requires the expertise of professionals such as the veterinary behaviourist. Clinical assessment of behavioural disorders allows the veterinary behaviourist to formulate a diagnosis and prescribe a behavioural and/or pharmacological therapy. The objective of such therapy is to produce a stable change in the perception of a stimulus and the resulting emotion, leading to the correction of the behavioural problem. It may be crucial to evaluate the subject s pathological state in response to the observed symptoms in order to identify the functional impairment of the pivotal neurotransmitter systems involved in the disorder. This allows selecting a suitable pharmacological treatment. In order to implement behavioural therapy, the veterinary behaviourist collaborates, where necessary, with a team of qualified canine trainers. Calzolari M, Bonilauri P, Bellini R, Albieri A, D efilippo G, Maioli G, Tamba M, Angelini P, Dottori M Four years of mosquito-based arbovirus surveys in Emilia-Romagna Region (Northern Italy) International Meeting on Emerging Diseases and Surveillance IMED : Vienna, Austria, February 4-7, 2011 : abstracts / [s.l. : s.n., 2011]. - p 183 [Nr. Estr. 4600] International Meeting on Emerging Diseases and Surveillance : Vienna, Austria : February 4-7, 2011) Background: In recent years human diseases due to mosquito-borne viruses were increasingly

2 reported in Emilia-Romagna region (Italy) - chikungunya virus in 2007, West Nile virus (WNV) in 2008 and Usutu virus (USUV) in suggesting the need for an investigation to determine the presence and geographic distribution of arboviruses. For this purpose, a mosquito based survey was started in late summer 2007; this plan was implemented to cover the whole season in 2008, the survey continued in 2009 and fi nally in 2010 network of evenly distributed, regularly working sampling stations were activated over the whole Region. Methods and Materials: Mosquitoes trapped by modifi ed CDC traps baited by CO2 were pooled according to date, location and species, with a maximum number of 200 specimens per pool. The pools were grinded and centrifuged and an aliquot of supernatant was collected, RNA was extracted and retro-transcribed then analyzed by different genus- PCRs targeting fl aviviruses, alphaviruses and orthobunyaviruses and specifi c-pcrs targeting WNV and USUV. The obtained amplicons were sequenced. Results: A total of 678,236 mosquitoes, grouped in 5,796 pools, were analyzed from 2007 to The most tested species resulted Culex pipiens (86.4 % of total mosquitoes), followed by Aedes caspius (9.2 %), Ae. vexans (3.4 %) and Ae. albopictus (0.6 %). The survey allowed the detections of WNV in 32 pools (2 in 2008, 27 in 2009 and 3 in 2010), USUV in 147 pools (56 in 2009, 91 in 2010) and Tahyna virus in 3 pools (one per year from 2008). Furthermore sequence data showed the presence in tested mosquitoes of RNA of mosquito-only flaviviruses. Conclusion: The inquiry conducted on sampled mosquitoes pointed out important epidemiological data on temporal and geographic diffusion of the detected viruses in Emilia-Romagna and their spreads in mosquitoes. These results confi rmed that, if mosquito trapping effort is intensive, detection of viruses in mosquitoes might precede detection of virus activity by other surveillance tools The results obtained by the described survey are encouraging and show how this mosquito-based surveillance could constitute the foundation a public health alert system targeting mosquito borne arboviruses. Calzolari M, Donati L, Bellini R, Defilippo F, Ba lzani A, Galli E, Zani G, Luppi A, Bonilauri P, Dottori M Field evaluation of six mosquito traps Malattie emergenti trasmesse da vettori: il rischio di zanzare AEDES : 9-10 Maggio 2011 Cervia / [s.l. : s.n., 2011]. - 1 p [Nr. Estr. 4916] Malattie emergenti trasmesse da vettori: il rischio di zanzare AEDES : Cervia : 9-10 Maggio 2011) Candotti P, Rota_Nodari, Diegoli C Proposta di un piano d'azione per la soppressione del suino in allevamento = Proposal of an on farm killing action plan for pigs Atti Convegno SIPAS. - Vol. 37 ( 2011). - p bib ref [Nr. Estr. 4632] Meeting Annuale della Societa' Italiana di Patologia ed Allevamento dei Suini (SIPAS) (37 : Piacenza : marzo 2011) Vengono prese in esame le caratteristiche delle diverse tecniche utilizzabili per la soppressione del suino in allevamento. Sulla base dell'economicità epraticabilità, lo stordimento mediante pistola a proiettile captivo penetrante (suini di peso >kg) e il trauma cranico da percussione (suini <kg) seguiti da jugulazione vengono ritenute le tecniche migliori effettuabili in campo. Viene proposto anche un algoritmo decisionale. The Authors review different methods for the on farm euthanasia of pigs. Captive bolt stunning (pigs weighting >kg) and blunt trauma (pigs <kg) followed by exanguation are identified as the most suitable techniques to apply in field. A decision algorithm for euthanasia is also proposed. Defilippo F, Bonilauri P, Massirio I, Calzolari M, Maioli G, Dottori M Prima segnalazione di Symphoromyia immaculata (Meigen, 1804) in Emilia Romagna Ig Aliment Disinfestazione Ig Ambient. - Vol. 28 no 2 ( 2011). - p [Nr. Estr. 4917]

3 Defilippo F, Caimi M, Calzolari M, Maioli G, Bon ilauri P, Parco V, Balzani A, Zini N, Dottori M Three years of arborival surveiillance on mosquitoes from Ticino River Park (Northern Italy) 6th European Mosquito Control Workshop : September 2011 Budapest, Hungary : abstract book / [s.l. : s. n., 2011]. - p 103 [Nr. Estr. 4914] European Mosquito Control Association Workshop (6th : Budapest, Hungary : September 2011) Kruse H, Magnino S Prevention and control of zoonoses : the World Health Organization perspective L epidemiologia veterinaria nel contesto di one world, one health : VI workshop nazionale di epidemiologia veterinaria : Orvieto 1-2 dicembre 2011 : riassunti / a cura di F. Baldinelli... [et al.]. - Roma : Istituto Superiore di Sanità, c (ISTISAN congressi ; 11/C8) p 7-8 [Nr. Estr. 4949] Workshop Nazionale di epidemiologia veterinaria (6. : Orvieto : 1-2 dicembre 2011) Maioli G, Bonilauri P, Bianchi A, Sacchi C, Lu ppi A, Defelippo F, Calzolari M, Dottori M Surveillance su TBEV in ticks collected on hunted wild animals in Lombardia region (Italy) : preliminary results 5th annual meeting Epizone : "Science on alert" : April, 2011, Arnhem, The Netherlands : posters / [Wageningen : Epizone, 2011]. - p 168 [Nr. Estr. 4800] Annual meeting Epizone (5th : Arnhem, The Netherlands : April, 2011) Massi P, Tosi G, Graziani R, Rossi PE Impiego di fitasi di origine vegetale come alternativa alle fitasi batteriche nell'alimentazione della gallina ovaiola Zootec Int. - Vol. 2 ( 2011). - p [Nr. Estr. 4672] Pistone D, Pajoro M, Fabbi M, Bandi C, Conti D, S assi W, Magnani P, Montagna M Risultati preliminari su patogeni batterici in zecche (Acarina : Ixodidae) raccolte su uccelli migratori in Nord Italia XVI Convegno italiano di ornitologia : Settembre 2011 Cervia : programma e riassunti dei contributi accettati come presentazioni orali e come poster / [s.l. : s.n., 2011]. - p [Nr. Estr. 4903] Convegno italiano di ornitologia (16 : Cervia : Settembre 2011) Gli uccelli migratori durante i loro spostamenti annuali possono veicolare ectoparassiti, come le zecche. Sfruttando questi ospiti. le zecche non solo riescono a coprire lunghe distanze, influenzando la propria distribuzione geografica e dinamica di popolazione, ma possono anche diffondere i patogeni, di cui sono vettori. Le larve e le ninfe di zecche (Ixodidae e Argasidae) compiono il pasto di sangue anche sugli uccelli. Infatti, durante la migrazione autunnale del 2010, 134 ospiti sono stati parassitati da larve e ninfe di Ixodes ricinus. Nel dettaglio, le zecche sono state rinvenute su 9 specie differenti di uccelli: Turdus. philomelos. T. merula. T iliacus. Erithacus rubecula, Sviria Parus inraor. Fringilla coelebs. Arahus trivialls. Coccothraustes coccothraustes. Il Tordo bottaccio (T philomelos) è risultata la specie più parassitata (84 soggetti con una media di 2.15 zecche/individuo), seguito dal pettirosso (E. rubecula).con 16 soggetti con media di Le zecche

4 sono state conservate in etanolo 967 per i successivi studi molecolari e identificate morfologicamente. Il presente lavoro, svolto presso la Fondazione Europea il Nibbio di Arosio (CO). è finalizzato: all' identificazione morfologica e molecolare (DNA harcoding) delle zecche rinvenute sugli uccelli migratori: all' individuazione delle aree di provenienza delle zecche mediante analisi di similarità genetica: a valutare mediante PCR la presenza di batteri patogeni. Dalle prime indagini molecolari su un campione di 40 zecche prelevate da 21 uccelli emerge una positività a batteri del genere Borrelia (10%) e Rickettsia (15%). Per identificare i patogeni a livello specifico e valutarne l'eventuale rischio si procederà al sequenziamento dei prodotti di PCR. Sulla base dei risultati preliminari riferiti al campionamento del 2010 si prevede di implementare i dati con la stagione 2011 e estendere lo screening a Francisella tularensis e Coxiella burneti, rispettivamente agenti di Tularemia e Febbre Q. Ricchi M, Barbieri G, Arrigoni N High resolution melting with unlabelled probe approach for the analysis of short sequence tandem repeats loci to sub-type Mycobacterlurn avium subsp. paratuberculosis Molecular diagnostics: from single-cells to next generation sequencing : 5th international qpcr Symposium & Industrial Exhibition & Application Workshops : 28th March - 1st April 2011, Freising-Weihenstephan, Germany : qpcr 2011 event proceedings / editor, Michael W. Pfaffl. - [Munich : Technical University Munich (TUM), 2011]. - p bib ref [Nr. Estr. 4797] International qpcr Symposium & Industrial Exhibition & Application Workshops (5th : Freising-Weihenstephan, Germany : 28th March - 1st April 2011) Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of paratuberculosis in ruminants and other species. Many methods have been proposed to sub-type Map: multiplex PCR for IS900 integration loci, IS900 restriction fragment length polymorphism, amplified fragment length polymorphism, Pulsed Field Gel Electrophoresis and genotyping microarrays (1, 2). However, at the moment, methods based on the amplification of Micro- and Mini- satellites tandem-repeats loci are considered the emerging techniques for Map typing (1). In this regard, Short Sequence Tandem Repeats loci (SSR1, SSR2, SSR8 and SSR9) showed the highest discriminatory power; however, to identify these loci, the direct sequencing of the amplicons is required. Our aim was developing alternative methods to the sequencing. We focused on SSR8, which ìs constituted by triplets ranging from three to six repetitions. To identify each allele, we developed a method based on an asymmetric qpcr, followed by High Resolution Melting (HRM) analysis with unlabelled probes. In order to improve the efficiency of asymmetric PCR reaction, we designed primers ac,cordingly to the Linear-After-The-Exponential (LATE) PCR strategy, while to avoid any elongation during the amplification, the unlabelled probe was blocked adding C6-amino group at its 3' end. All PCR reactions were carried out on StepOne Plus system (Applied Biosystems, Milan, Italy) in a fina! volume of 25 ml using LCGreen Plus+ Melting Dye (Idaho technology Inc, Salt Lake City, USA) and Right-Taq (EuroClone, Pero, Italy). HMR analysis was carried out according to Palais (3), without ROX normalisation and smoothing the graphs with running average method. For the development of the technique we used three Map strains containing, respectively, three, four and five repetitions; for the allele with six repetitions, due to its rarity, we synthesised an artificial amplicon. HMR analysis showed two melting domains for each sample: one relative to the amplicon omoduplex (DNA double strand) and another to the heteroduplex DNA single strand /probe. The omoduplex domain did not allow any differentiation between the various alleles, while the heteroduplex domain showed almost 3 C between each allele, permitting an unbiased identification. These data were confirmed analysing 40 Map field strains. The method herein proposed is robust, reproducible, cost effective and faster than direct sequencing and could be usefully applied to resolve locus SSR8. Ricchi M, Barbieri G, Cammi G, Garbarino CA, Ar rigoni N High-resolution melting for analysis of short sequence repeats in Mycobacterium avium

5 subsp. paratuberculosis FEMS Microbiol Lett. - Vol. 323 no 2 ( 2011). - p bib ref [Nr. Estr. 4798] Analysis of micro- and minisatellite loci is widely used in sub-typing of Mycobacterium avium subsp. paratuberculosis. Microsatellite (short sequence repeat, SSR) loci have shown highest discriminatory power, but direct sequencing of amplicons is required for correct assignment of the repeat number. We developed an alternative method to sequencing, focusing on the SSR8 locus (constituted by GGT triplets from three to six repeats). The approach is based on asymmetric quantitative PCR, followed by high-resolution melting analysis with unlabelled probes (UP-HRM). Data showed perfect concordance between direct sequencing and UP-HRM, which is faster, simpler and more cost effective. Rovetta A, Stacchiotti M, Ferrone A, Lavazza A, Sb erveglieri R, Rezzani R, Aleo MF Cisplatin-induced atrophy in C2C12 myotubes: a multidisciplinary microscopic study 10th MCM2011 Multinational Congress on Microscopy : September 4-9, 2011 Urbino, (Italy) : proceedings / [s.n. : s.n, 2011]. - p bib ref [Nr. Estr. 4862] Multinational Congress on Microscopy (10th : Urbino, (Italy) : September 4-9, 2011) Stacchiotti A, Li_Volti G, Lavazza A, Schena I, Al eo MF, Rodella LF, Rezzani R Different role of Schisandrin B on mercury-induced renal damage in vivo and in vitro Toxicol. - Vol. 286 ( 2011). - p bib ref 61 [Nr. Estr. 4706] Mercuric chloride (HgCl2) causes acute oxidant renal failure that affects mainly proximal tubules. Schisandrin B (Sch B), an active lignan from the fruit of Schisandra chinensis, has been successfully used to treat gentamicin nephrotoxicity, but its role against mercury damage is still largely unknown. Here we analysed in vivo and in vitro the efficacy of Sch B supplementation against HgCl2 nephrotoxicity, focusing on histopathology, stress proteins, oxidative (cytochrome c oxidase) and nitrosactive markers (enos, nnos). Wistar rats were treated with Sch B (10 mg/kg/day p.o.) or vehicle (olive oil) for 9 days, then coadministered with a single HgCl2 nephrotoxic dose (3.5 mg/kg i.p.) and killed after 24 h. The tubular and mitochondrial damage induced by mercury was limited by Sch B coadministration in vivo. Remarkably, after Sch B and mercury challenge, HSP25, HSP72, GRP75 were reduced in the renal cortex, cytochrome c oxidase increased and enos and nnos were restored in glomeruli. In contrast, NRK-52E proximal tubular cells treated with Sch B 6.25 µm plus HgCl2 20 µm did not show any amelioration on viability and oxidative stress in respect to HgCl2 20 µm alone. Moreover, after Sch B plus mercury in vitro treatment, HSP72 staining persisted while HSP25 further increased. Thus, in our experimental conditions, Sch B cotreatment afforded better protection against mercury poisoning in vivo than in vitro. This discrepancy might be partly attributable to Sch B influence on glomerular perfusion as corroborated by the recovery of vasoactive markers like macular and endothelial nitric oxide isoforms. Stacchiotti A, Rovetta F, Cadei M, Grigolato PG, Lavazza A, Rezzani R, Aleo MF ER stress is an important player in the cross-talk between cisplatin-induced autophagy or apoptosis in renal NRK52E cells Toxicol Lett. - Vol. 205S ( 2011). - p S177 [Nr. Estr. 4941] Cisplatin (cispt) is a potent chemotherapeutic but its use is limited by severe nephrotoxicity. Cytoprotective autophagy and apoptosis are involved in cispt nephrotoxicity but the cross-talk

6 between their pathways is still object of debate. The cellular site driving the switch between survival or cell death is actually unknown. We studied autophagy and apoptosis in a renal in vitro model (NRK-52E cell line) exposed from 2 h up to 72h to 10 or 50_M cispt and investigated the molecular mechanisms of cell response in these different stress conditions (moderate or severe). Furthermore, to best address the potential involvement of endoplasmic reticulum in the cell commitment to autophagy or apoptosis, we considered specific ER stress markers, such as the pro-survival chaperones GRP94 and GRP78 and the pro-apoptotic factors GADD153/CHOP and GADD34. Methods: Autophagy was assessed by transmission electron microscopy, LC3/Atg8 immunoblotting and immunofluorescence, while apoptosis by cytofluorimetry and nuclear morphology analysis. ER markers expression was evaluated by semiquantitative RT-PCR and/or immunoblotting. Results: 10_M cispt caused a significant increase of autophagy at 2 h, preserving NRK-52E cell viability; this protective effect was supported by the increase of apoptosis after co-administration of the autophagy inhibitor 3-methyladenine. Differently, 50_M dose early induced apoptosis leading irreversibly to cell death. GRP94 and GRP78 were over-expressed after 24 h by 10_M cispt but down-regulated by 50_M. The proapoptotic transcription factors CHOP and GADD34 had opposite trends, being up-regulated only after 50_McisPt treatment. CisPtinduced dose-dependent effects on UPR pathways well correlate with the cell commitment to survival or death.