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

Phenoloxidase activity and haemolymph cytology in honeybees challenged with a virus suspension (deformed wings virus DWV) or phosphate buffered suspension (PBS)

The innate immune system of honeybees mainly consists in antimicrobial peptides, cellular immunity and melanisation. In order to investigate the immune response of honeybees to immune stressors, three stress degrees were tested. Newly emerged bees naturally DWV-infected were collected from a Varroa mite free apiary and divided into three experimental groups: naturally DWV infected bees. PBS injected bees. and artificially DWV super infected bees. Phenoloxidase activity and haemolymph cellular subtype count were investigated. Phenoloxidase activity was highest (P<0.05) in DWV-superinfected bees. and the haemocyte population differed within the three observed groups. Although. immune responses following DWV infection have still not been completely clarified. this investigation sheds light on the relation between cell immunity and the phenoloxidase activity of DWV naturally infected honeybees exposed to additional stress such as injury and viral superinfection

Effect of dietary supplementation of a mix of chestnut and quebracho tannins on intestinal morphology, bacterial load, Eimeria spp oocyst excretion and immune response after vaccination in rabbits

Among plant products, tannins exhibited a strong antioxidant activity and their employment was studied as food or feed addictive with positive effects. Nevertheless, their health-enhancing properties remain to be completely clarified, particularly in rabbits. The aim of the present study was to investigate the influence of a mix of chestnut and quebracho tannins as supplementation in rabbit diet on intestinal morphology, as well as bacterial loads and Eimeria spp coccidial oocyst excretion and humoral immune response after vaccination. Rabbits were fed four different diets: basal diet (negative control, group CN), basal diet with anticoccidial supplementation (positive control, group CP) and basal diet with tannins mix 0.3% (group T0.3) or 0.6% (group T0.6). Samples of intestine, liver, spleen thymus, mesenteric lymph nodes, kidney and heart were submitted to histopathologic investigations. Morphometric analyses were performed on portion of duodenum, jejunum and ileum. Intestinal contents were analyzed by flotation test for Eimeria spp. oocysts excretion and by microbiological assays for Clostridium perfringens and Escherichia coli. Rabbits belonging to the four experimental groups were vaccinated by means of a live attenuated myxomatosis virus and sera were collected individually at three time points to determine antibody response.The obtained results demonstratedthe absence of toxic effects of the tannin mix on examined organs, particularly on liver and kidney and any negative influence on the growth performance. A relevant anti-inflammatory effect on the intestinal tract, a reduction of Eimeria spp. oocysts excretion and a positive immunomodulatory and antibacterial effects were also observed. Diets supplemented with the tannins mix affected also the morphology of examined intestinal tracts, particularly duodenum and ileum

Next generation sequencing study on RNA viruses of Vespa velutina and Apis mellifera sharing the same foraging area

The predator Asian hornet (Vespa velutina) represents one of the major threats to honeybee survival. Viral spillover from bee to wasp has been supposed in several studies, and this work aims to identify and study the virome of both insect species living simultaneously in the same foraging area. Transcriptomic analysis was performed on V. velutina and Apis mellifera samples, and replicative form of detected viruses was carried out by strand‐specific RT‐PCR. Overall, 6 and 9 different viral types were reported in V. velutina and A. mellifera, respectively, and five of these viruses were recorded in both hosts. Varroa destructor virus‐1 and Cripavirus NB‐1/2011/HUN (now classified as Triato‐like virus) were the most represented viruses detected in both hosts, also in replicative form. In this investigation, Triato‐like virus, as well as Aphis gossypii virus and Nora virus, was detected for the first time in honeybees. Concerning V. velutina, we report for the first time the recently detected honeybee La Jolla virus. A general high homology rate between genomes of shared viruses between V. velutina and A. mellifera suggests the efficient transmission of the virus from bee to wasp. In conclusion, our findings highlight the presence of several known and newly reported RNA viruses infecting A. mellifera and V. velutina. This confirms the environment role as an important source of infection and indicates the possibility of spillover from prey to predator

In Vitro Antibacterial Activity of Manuka (Leptospermum scoparium J.R. et G. Forst) and winter Savory (Satureja montana L.) Essential Oils and Their Blends against Pathogenic E. coli Isolates from Pigs

Neonatal diarrhoea (ND), post-weaning diarrhoea (PWD) and oedema disease (OD) are among the most important diseases affecting pig farming due to economic losses. Among the main aetiological agents, strains of Escherichia coli are identified as the major responsible pathogens involved. Several strategies have been put in place to prevent these infections and, today, research is increasingly studying alternative methods to antibiotics to reduce the antibiotic resistance phenomenon. Essential oils (EOs) are among the alternative tools that are being investigated. In this study, the in vitro effectiveness of winter savory and manuka essential oils and their mixtures in different proportions against strains of E. coli isolated from episodes of disease in pigs was evaluated. The EOs alone demonstrated slight antibacterial effectiveness, whereas the blends, by virtue of their synergistic action, showed remarkable activity, especially the 70%–30% winter savory–manuka blend, showing itself as a potential tool for prevention and therapy

Multicentric Molecular and Pathologic Study On Canine Adenovirus Type 1 in Red Foxes (Vulpes vulpes) in Three European Countries

Canine adenovirus type 1 (CAdV-1) is the agent of infectious canine hepatitis, a severe frequently fatal disease affecting primarily dogs (Canis lupus familiaris). The virus has been detected in many wild carnivore species. Our aim was to evaluate the prevalence and genetic and histopathologic features of CAdV-1 in wild red foxes (Vulpes vulpes). Kidney and liver samples were obtained from 86 subjects, coming from the UK (n\ubc21), Italy (n\ubc36), and Germany (n\ubc29). We used PCR, targeting the viral E3 gene and flanked regions, to detect the presence of the virus; viral E3, fiber, and E4 genes were sequenced and their sequences were compared with published sequences. Kidneys and liver from foxes in Italy and Great Britain (n\ubc57) were prepared for histologic and immunohistochemical examination for CAdV-1. Viral DNA was detected in 22% (19 of 86) kidney samples, with E3 and E4 genes showing reported and unreported single nucleotide changes. No pathologic changes or viral immunopositive signals were detected in the examined tissues. Our study suggests that red foxes could be considered potential shedders of CAdV-1, as they showed a relatively high prevalence without related pathologic changes in the organs examined

Hepatitis E virus infection in wild rabbit (Oryctolagus cuniculus) in Italy and in the UK: a serological, molecular, and pathological study

A novel animal strain of HEV demonstrating zoonotic potential rabbit HEV (rHEV) has been recently identified in farmed rabbits in China. To investigate the presence of rHEV in wild rabbit population, serum and tissue samples were taken from 65 rabbits, 35 Italian and 30 from UK. Sera were submitted to a double antigen sandwich ELISA, whereas hepatic tissues and other organs to molecular and pathological investigations. Sixteen serum samples (24.6%) scored positive for anti-HEV antibodies, and six samples (9%) of hepatic tissue were positive for HEV RT-PCR, while the other organs were negative. Sequencing and phylogenetic analysis of HEV RNA-positive samples indicated that while two Italian rabbits were infected with strains closely related to wild boar and swine strains (HEV-3), the other four (2 Italian and 2 English) were clustered within rHEV clade. Megalocytosis and multifocal areas of hepatocytes degeneration and necrosis with a pericentrilobular pattern were observed in rHEV-infected rabbits. In those infected by the strain analogous to HEV-3, the lesions were mainly localized in the periportal areas and were less severe. In both cases, inflammatory infiltrates were predominantly composed of CD3-positive lymphocytes and a reduced number of macrophages. By immunohistochemistry, only liver samples taken from HEV RNA-positive rabbits scored positive for viral antigen. Our results indicated that HEV infection is present in rabbit population with different clades and is endemic in the Italian and English wild rabbit population, suggesting the possibility that this species may be infected with rHEV or swine HEV-3 strains

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

Generation of virus like particles for epizootic hemorrhagic disease virus

Epizootic hemorrhagic disease virus (EHDV) is a distinct species within the genus Orbivirus, within the family Reoviridae. The epizootic hemorrhagic disease virus genome comprises ten segments of linear, double stranded (ds) RNA, which are packaged within each virus particle. The EHDV virion has a three layered capsid-structure, generated by four major viral proteins: VP2 and VP5 (outer capsid layer); VP7 (intermediate, core-surface layer) and VP3 (innermost, sub-core layer). Although EHDV infects cattle sporadically, several outbreaks have recently occurred in this species in five Mediterranean countries, indicating a potential threat to the European cattle industry. EHDV is transmitted by biting midges of the genus Culicoides, which can travel long distances through wind-born movements (particularly over water), increasing the potential for viral spread in new areas/countries. Expression systems to generate self-assembled virus like particles (VLPs) by simultaneous expression of the major capsid-proteins, have been established for several viruses (including bluetongue virus). This study has developed expression systems for production of EHDV VLPs, for use as non-infectious antigens in both vaccinology and serology studies, avoiding the risk of genetic reassortment between vaccine and field strains and facilitating large scale antigen production. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned into transfer vectors, to generate two recombinant baculoviruses. The expression of these viral genes was assessed in insect cells by monitoring the presence of specific viral mRNAs and by western blotting. Electron microscopy studies confirmed the formation and purification of assembled VLPs

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