Detection of Hepatitis E Virus Antibodies in Domestic and Wild Animal Species in Central Italy

Hepatitis E virus (HEV) is known for its zoonotic potential. Although several mammalian species have been indicated as possible viral reservoir, the host range of the infection is partially defined. In this work serum samples collected from wild brown hares, red deer, wild rabbits, cattle living in semi-wild state and wild boar-hunting dogs were tested by a multi-species ELISA assay. Only sera from red deer (5.6%), wild rabbit (38.5%) and wild-boar hunting dogs (14.3%) scored positive. The investigation indicated the circulation and the high endemicity of HEV in various animal species in Central Italy, and the importance that these species can play in the epidemiology of infection

Effect of 1,3-1,6 β-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica)

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3-1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection

EFFETTO NUTRACEUTICO DEI BETA-­‐GLUCANI SU API INFETTE DA DEFORMED WING VIRUS (DWV).

Il Deformed Wing Virus (DWV) o virus delle api deformi è responsabile di una infezione spesso in forma subclinica che è molto diffusa nell'Apis mellifera. Il virus appartiene alla famiglia Picornavirales e, come gli altri membri della famiglia, è caratterizzato da un virione nudo di piccole dimensioni racchiudente un genoma a ssRNA (+). Il virus infetta le forme larvali durante il loro sviluppo e l’infezione si manifesta con la comparsa di api neo-sfarfallate con gravi deformazioni a carico delle ali e con ridotte dimensioni del corpo. La malattia può portare al collasso della colonia ed in genere le forme più gravi sono associate ad infestazioni di Varroa destructor, un acaro che agisce sia come parassita che come amplificatore e vettore biologico del virus. Le api, così come altri invertebrati, hanno sviluppato un'ampia varietà di meccanismi di difesa innata contro i vari agenti patogeni, seppur in assenza di un sistema immunitario adattativo. In molti dei meccanismi immunodifensivi degli invertebrati sono coinvolte proteine secrete da cellule dell'emolinfa dotate di attività opsonizzante, chemiotattica, battericida e perossidasica. Tali meccanismi possono essere modulati dalla presenza di varie molecole definite immunomodulatori. Tra queste i ß-glucani, polisaccaridi ramificati non amidacei costituiti da molecole di glucosio unite insieme mediante legami glicosidici β(1,3) e β(1,6), possono rappresentare una alternativa all'utilizzo di sostanze di sintesi chimica. I ß-glucani sono costituenti della parte solubile della fibra vegetale, sono presenti in molti cereali ma anche nelle pareti cellulari di vari agenti patogeni. Al fine di comprendere se i ß-glucani possano avere una efficace azione immunomodulante sulle api, la molecola è stata integrata nell’ alimentazione di gruppi sperimentali di api in un modello di infezione naturale e sperimentale con DWV. Gli esperimenti condotti hanno permesso di quantificare il tasso di mortalità, di stimare il numero e il grado di attivazione delle cellule deputate all’attività immunitaria, di quantizzare il virus presente nei vari distretti delle api (testa/addome) e di valutarne la replicazione. I risultati ottenuti hanno dimostrato che la somministrazione di ß-glucani per via orale in api infette da DWV è associata a un significativo aumento del tasso di sopravvivenza, ad un incremento del numero di emociti, e ad una riduzione della carica virale. Tali risultati suggeriscono che questa molecola possa contribuire ad incrementare la difesa delle api verso agenti patogeni

Generazione di metodiche diagnostiche ed immunizzanti per il virus della Malattia Emorragica Epizootica (EHDV)

Riassunto La malattia emorragica epizootica (EHD) è una malattia infettiva virale non contagiosa che colpisce ruminanti selvatici e domestici. La malattia è causata dal virus della malattia emorragica epizootica (EHDV), membro della famiglia Reoviridae, genere Orbivirus. EHDV è considerato essere un arbovirus in quanto viene trasmesso da insetti del genere Culicoides, i quali rappresentano i suoi vettori biologici. Negli ultimi anni sono stati segnalati due sierotipi di EHDV, EHDV-6 e EHDV-7, in diversi paesi del bacino del Mediterraneo, dove hanno causato infezione nei bovini con importanti perdite economiche, soprattutto dovute alla riduzione della produzione di latte. Questo evento, insieme al fatto che il virus viene trasmesso da un vettore già presente nella maggior parte dei paesi Europei, ha sollevato preoccupazioni per una possibile diffusione di EHDV in Europa, che potrebbe seguire quindi lo stesso percorso del virus della Bluetongue (BTV). Sulla base di questa eventualità, la disponibilità di saggi diagnostici e vaccini sicuri rappresenterebbe un aspetto fondamentale per programmi adeguati di sorveglianza e controllo. In questo studio, si segnala lo sviluppo di un saggio ELISA competitivo "Home-made"(cELISA) basato sulla proteina virale ricombinante VP7 (r-VP7) come antigene. La proteina, generata tramite il sistema di espressione del Baculovirus, è stata testata su un totale di 275 campioni di siero compresi i ceppi di riferimento EHDV, campioni di EHDV positivi e negativi precedentemente testati e campioni positivi per BTV e African Horse sickness virus (AHSV). Accuratezza, sensibilità e specificità del saggio sviluppato sono stati rispettivamente 93.1%, 84.1% e 96.9%, indicando che tale saggio può essere quindi utilizzato come valido strumento diagnostico. Il principale vantaggio del test sviluppato è stato l'uso dell’antigene r-VP7 senza complicate fasi di purificazione richiedenti tempo. Al fine di sviluppare un vaccino candidato sicuro sono state generate le EHDV-Virus-Like Particles (VLPs). I geni che codificano le quattro proteine capsidiche principali di un ceppo di campo di EHDV-6 sono stati isolati e clonati per generare due Baculovirus ricombinanti. La corretta espressione dei geni è stata valutata monitorando la sintesi di ciascun mRNA virale in cellule d’insetto infettate e successivamente confermata da Western Blot. Studi di microscopia elettronica hanno confermato la formazione e la purificazione delle VLPs assemblate. I risultati ottenuti in questo studio offrono strumenti diagnostici e di prevenzione interessanti per il futuro per la prevenzione e il controllo del virus della malattia emorragica epizootica, per non essere impreparati in caso di possibili incursioni future. Abstract Epizootic haemorrhagic disease (EHD) is an infectious non-contagious viral disease, affecting wild and domestic ruminants. The disease is caused by epizootic haemorrhagic disease virus (EHDV), which is a member of the Reoviridae family within the Orbivirus genus. EHDV is considered as an arbovirus since it is transmitted by insects of the genus Culicoides, which represent its biological vectors. In recent years two serotypes of EHDV namely EHDV-6 and EHDV-7 have been reported in several countries of the Mediterranean Basin where have caused infection in cattle causing important economic loss, especially due to the reduction of milk production. This event, along with the fact that the virus is transmitted by a vector that is already present on most European countries, has raised concern about a possible EHDV spreading in Europe by following the route of Bluetongue virus (BTV). On this eventuality, the availability of diagnostic assays and safe vaccines would represent a key aspect for adequate surveillance and control programs. In this study, we report the development of a “Home-made” competitive ELISA (cELISA) based on the recombinant viral protein VP7(r-VP7) as antigen. The protein, generated by the baculovirus espression system, was tested on a total of 275 serum samples including EHDV reference strains, previously tested positive and negative EHDV samples, and on BTV and African horse sickness virus (AHSV) positive samples. The accuracy, sensitivity and specificity of the developed assay were 93.1, 84.1 and 96.9%, respectively, indicating that it can used as a valid diagnostic tool. The main advantage of the developed test was the use of rVP7 without complicated and time-consuming purification steps. In order to develop a safe vaccine candidate EHDV-Virus-Like Particles (VLPs) were generated. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned to generate two recombinant baculoviruses. The correct expression of genes was evaluated by monitoring the synthesis of each viral mRNA in infected insect cells and subsequently confirmed by Western Blot analysis. Electron microscopy studies confirmed the formation and purification of assembled VLPs. The results obtained in this study offer interesting diagnostic and prevention tools for the prevention and control of EHDV, in order not to be unprepared in the event of his possible future incursion

Valutazione dell'attivita antimicrobica del latte d'asina

Scopo: Lo scopo del presente lavoro di tesi è stato quello di verificare, attraverso indagini microbiologiche, l’attività antimicrobica del latte d’asina su alcuni microrganismi patogeni ed alteranti frequentemente riscontrati nel latte, al fine di descriverne eventuali azioni inibenti e/o favorenti la crescita. Materiali e metodi: Per le indagini microbiologiche sono stati utilizzati ceppi di referenza appartenenti alla ceppoteca del Dipartimento di Scienze Veterinarie dell'Università di Pisa, latte d’asina proveniente della Provincia di Arezzo (San Sepolcro), e latte di capra UHT. E’ stata testata l’attività antimicrobica del latte d’asina, sia crudo che termizzato, sui microrganismi dopo diversi tempi di stoccaggio dei campioni di latte a temperatura di refrigerazione (1, 3, 6 giorni). Inoltre, è stata testata la sopravvivenza degli stessi microrganismi in latte di capra addizionato di diverse percentuali di latte d’asina (1%, 2,5%, 5%, 10%), in previsione di un possibile impiego del latte d’asina stesso come additivo naturale ad attività battericida. Il contenuto di lisozima è stato determinato secondo la metodica del Lysoplate Assay. Risultati e discussione: I risultati ottenuti hanno evidenziato come l’attività antibatterica del latte d’asina (crudo e termizzato) si manifesti principalmente nei confronti dei batteri Gram-positivi (S. aureus ed E. faecalis). Le diverse percentuali di latte d’asina addizionate a latte caprino non hanno determinato variazioni nello sviluppo dei microrganismi testati. La concentrazione di lisozima riscontrata nei campioni di latte d’asina, sia crudo che termizzato, è stata mediamente pari a quella riscontrata in letteratura. Ad oggi quindi non sarebbe prudente riconoscere al latte d'asina un'incontrovertibile attività antibatterica, soprattutto in considerazione della notevole variabilità della concentrazione del lisozima riscontrabile nel latte stesso e attestata in letteratura. Purpose: The aim of the present thesis was to assess, by means of microbiological analysis, the antimicrobial activity of donkey milk against some pathogenic and spoilage microorganisms frequently detected in milk, in order to highlight a potential antibacterial and/or growth-promoting activity. Materials and methods: Reference strains belonging to the Department of Veterinary Science (Pisa University) collection were used for the trials. Donkey milk from Arezzo Province (San Sepolcro) and goat's milk UHT were employed. Antimicrobial activity of donkey milk (raw and thermized) against different microorganisms was evaluated after different times of storage at refrigeration conditions (1, 3, 6 days). Moreover, the survival growth rate of microorganisms in goat's milk supplemented with different percentages of donkey milk (1%, 2.5%, 5%, 10%) was evaluated for a possible employement of donkey milk as a natural additive with antimicrobial activity. The content of lysozyme was also determined according to Lysoplate Assay method. Results and discussion: Our results showed that antibacterial activity of donkey milk (raw and thermized) is mainly directed against Gram-positive bacteria (S. aureus and E. faecalis). The different percentages of donkey milk added to goat's milk did not affected the survival growth rate of tested microorganisms. The detected lysozyme concentration, both in raw and thermized milk, was in accordance with the present literature. To date, it would not be prudent to attribute to donkey milk an incontrovertible antibacterial activity, especially taking into account the considerable variability of lysozyme concentration in milk

Accumulation Evaluation of Potential Microplastic Particles in Mytilus galloprovincialis from the Goro Sacca (Adriatic Sea, Italy)

Microplastics (MPs; <5 mm) are present throughout the marine environment and are recognized as an emerging threat to aquatic ecosystems. Filter feeding organisms, such as mussels, are considered as bioindicators of MP pollution and are useful to evaluate the potential risks of MPs to human health. The work presented shows data on potential MPs found in Mytilus galloprovincialis samples collected from the Adriatic Sea during two sampling sections (1st sampling: December 2019 and 2nd sampling: May 2020). The mussels were subjected to digestion with H2O2 individually and filtered and the MP elements found were observed using a stereomicroscope and ultimately categorized by shape, size class and color, with the aid of a digital camera and data acquisition software. The highest MP concentrations were observed in the mussels collected in December 2019 (1.11 microplastic items per gram wet weight of mussels’ tissue), highlighting the possible influence of the following two main factors: greater river discharges following adverse weather events and higher river water pollution due to industrial activities. Indeed, the second sampling was performed after the Italian lockdown, due to the COVID-19 emergency. MP fibers (50–80%) were the most abundant type of MPs identified, followed by fragments (10–40%), granules (1.5–2.5%), non-categorized shape (1–2%) and foam (<1%). The color black (50–70%) and sizes smaller than 500 µm were the most dominant characteristics recorded both in the 1st sampling (50–70%) and the 2nd survey (30–50%). These data could be overestimated, due to the lack of polymer identification. The results of this study provide further data on the importance of bivalves as environmental bioindicators with regard to the pollution of MPs in the Adriatic Sea, supporting their instrumental role as environmental bioindicators for MP pollution

DOES THE ADDITION OF DONKEY MILK INHIBIT THE REPLICATION OF PATHOGEN MICROORGANISMS IN GOAT MILK AT REFRIGERATED CONDITION?

Currently, donkey milk is receiving an increasing attention from consumers and research community because of its several beneficial aspects, such as a poor allergenic nature and a remarkable antimicrobial compound content. In this study, we evaluated the growth rate of Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 7644TM, Campylobacter jejuni ATCC 33291, and Pseudomonas aeruginosa ATCC 27853 at refrigeration conditions (4±2 °C) in goat milk added with different percentages of donkey milk (1, 2.5, 5, 10% v/v) along 6 days of storage; furthermore, donkey milk and goat milk samples were employed as controls. Lysozyme content of donkey milk was determined and ranged from 1 mg.mL−1 up to 2 mg.mL−1. An inhibited growth rate in donkey milk samples was observed during storage for S. aureus, L. monocytogenes, and C. jejuni, with a growth decrease of 0.61 log CFU.mL−1, 5.55 log CFU.mL−1, and 1.72 log CFU.mL−1, respectively. These data confirm the antibacterial activity of donkey milk against Gram-positive and Gramnegative microorganisms; however, microbial growth rates in milk mixtures and goat milk were comparable, with no significant increase in antibacterial activity due to donkey milk addition. Considering a potential employment of donkey milk in dairy products, further studies should be performed in order to detect the optimal balance of milk mixtures in terms of caseins and antimicrobial molecules amounts

A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV- 1), an important pathogen of cattle worldwide. The assay was based on conserved 5’UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step

Outbreak of porcine epidemic diarrhoea virus (PEDV) in Abruzzi region, central-Italy

Here we report and characterize a porcine epidemic diarrhea (PED) outbreak which occurred in a swine fattening farm in the province of Teramo, Abruzzi region (central Italy), in January 2016. PED virus (PEDV) identification was determined by real-time RT-PCR performed on RNAs purified from fecal samples collected from two symptomatic pigs. Whole genome sequence (PEDV 1842/2016) was also obtained by next generation sequencing straight from RNA purified from one fecal sample. Genome comparison with extant global PEDV strains revealed a high nucleotide identity with recently reported European and American S-INDEL PEDVs. Efficient sequencing, share of genomic data combined with the implementation of epidemiological tools would be the ideal approach for study and analysis of transboundary infectious diseases as PED

Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of epizootic haemorrhagic disease virus (EHDV) antibodies

Epizootic haemorrhagic disease (EHD) is a vector-borne infectious viral disease of domestic and wild ruminants. EHD could spread from infected northern African countries in free territories like the EU; therefore, the availability of diagnostic assays would represent key components for adequate surveillance and control programs. In this study, the gene encoding the VP7 protein of EHD virus (EHDV) was expressed into a baculovirus-infected insect cell system. With this unpurified protein we developed a home-made competitive ELISA (cELISA) and a total number of 275 serum samples, originating from domestic and wild ruminants, were tested. 74/275 were previously shown to be positive for EHDV antibodies by a commercially available ELISA kit. A â\u80\u9cvery goodâ\u80\u9d agreement was demonstrated when compared to a commercial ELISA kit (Cohen's kappa value = 0.832). Samples which caused disagreement between the two assays originated from wildlife which highlights the need for further validation by using serum samples from wild animals