Search

Zoonotic viruses are thought to be the highest risk of epidemic diseases for next years. Among others, these also include rotavirus, agent of gastroenteritis in man and animals and hepatitis E virus (HEV), which causes acute hepatitis in humans and infect domestic and wild animals. Group A rotaviruses (RVA) infection is preferentially species-specific, heterologous rotavirus infections may also occur, including animal-tohuman virus transmission. Recently, a human RVA of rabbit origin was described in a child with gastroenteritis, and a bovine-like RVA was isolated from a laboratory rabbit . Hepatitis E is an emerging disease now recognized to be a zoonosis. Swine, wild boar, deer and rabbit are considered reservoirs of the zoonotic HEV genotypes 3 and 4. Recently HEV was detected in both farmed and wild rabbits in several areas of China, USA and France. The prevalence of serum antibodies against HEV ranges between 7% and 57% in young animals. Several evidences support a risk of zoonotic transmission of HEV from rabbits to humans, including experimental infection of non-human primates and swine with rabbit HEV and detection of HEV closely related to rabbit HEV in a man. HEV and RVA infections were studied in 21 farmed and 121 pet rabbits in Italy. Virus infection was investigated by serological analysis in sera and by detection of RNA in fecal samples. During 2013, a total of 121 rabbits attending veterinary examination were enrolled in this study. Sera and feces were collected from the same animals (except 7 fecal samples). Seventy-seven pets were asymptomatic, most others were affected by different pathologies. Between October 2013 and February 2014, 21 samples were collected from stud-mare rabbits affected by reproductive failure. Western blotting (WB). The capsid protein of an Italian gt3 swine HEV strain expressed in Sf9 cells from a recombinant baculovirus (BacΔ111ORF2HEV), the purified SA11 RV and crude extract of mock Sf9 (negative control) were subjected to WB. Sera were diluted 1:100. Immunocytochemistry. Sf9 cells infected with BacΔ111ORF2HEV and mock cells were fixed and incubated with rabbit sera (1:500). Replication foci were visualized by reaction with amino-ethylcarbazole. Reverse-transcription–polymerase chain reaction (RT–PCR) and DNA sequencing. RNA was extracted using Qiagen RNA-Easy-Mini kit. For both viruses, RT-PCRs were performed using the OneStep RT-PCR kit (Qiagen). For Rotavirus the NSP5 segment was amplified. For HEV an RT-PCR and a nested-PCR were conducted, amplifying a 300 bp ORF2 fragment. None of the 135 fecal samples, (114 pets and 21 farmed rabbits), showed presence of HEV genome. IgG against HEV were detected in 4 of 121 pet rabbits tested. Two seropositive animals were asymptomatic, one was affected by rhinitis. Rabbits showed no clinical signs of RVA infection. Twelve out of 52 sera recognized specific RVA proteins. Twelve samples resulted positive in RT-PCR, one was confirmed through sequence analyses. One animal was positive for both RVA and HEV IgG. None of the feces (farmed or pet rabbits) were positive for HEV genome. The seroprevalence in pet rabbits was 3.3% in this study, markedly lower than observed in farmed animals (36% in China and 55% in USA). The lower seroprevalence in our study might be explained with the different animal population; pets living in households have no contact with other animals, which reduces the possible HEV transmission through the fecal-oral route. The study of naturally occurring heterologous rotaviruses may help understand how rotavirus cross the host-species barrier and investigate the molecular determinants that control rotavirus host-species specificity and pathogenicity. Contact between pet rabbits and their owners, e.g. changing the litter, might favor animal-to-human transmission; similar risks occur in persons with occupational exposure to farmed rabbits. Possible foodborne transmission of rabbit HEV should also be evaluated.

Nell’uomo, l’epatite E è una malattia con caratteristiche tipicamente di epatite acuta con scarse probabilità di cronicizzazione. L’agente eziologico (HEV) è un virus a RNA, classificato nel genere Hepevirus. Nei mammiferi si distinguono almeno 4 diversi genotipi di HEV: 1 e 2 infettano solo l’uomo, 3 e 4 hanno caratteristiche zoonotiche e infettano uomo e animali (suino, cinghiale, cervo). Nei Paesi in via di sviluppo la malattia nell’uomo si manifesta con grandi epidemie causate dal consumo di acqua contaminata da feci ed è sostenuta da ceppi di genotipo 1 e 2. Nei Paesi industrializzati, i casi clinici di epatite E sono prevalentemente sporadici e generalmente legati a viaggi in Paesi endemici. Tuttavia, negli ultimi anni è stato descritto un numero crescente di casi autoctoni, causati da infezioni sostenute dai genotipi 3 e 4. Alcuni di essi sono stati messi in relazione al contatto diretto o indiretto con suini, cinghiali o cervi infetti da genotipo 3. In Europa la presenza e la diffusione dell’infezione di HEV di genotipo 3 è stata dimostrata sia in allevamenti suini sia in animali selvatici (cinghiali, cervi). Nel presente lavoro è stata valutata la presenza di HEV in una popolazione di cervi (Cervus elaphus) dell’Appennino tosco-emiliano: 200 sieri, raccolti tra il 2007 e il 2010 da altrettanti animali abbattuti nell’ambito di piani di controllo della specie, sono stati esaminati per la ricerca di anticorpi anti-HEV. Per la ricerca del genoma virale sono stati esaminati 91 sieri scelti in modo casuale. La ricerca degli anticorpi è stata condotta utilizzando un test ELISA basato sulla proteina ricombinate del capside di un ceppo italiano di HEV suino (genotipo 3), da noi espressa e purificata. Complessivamente, il 13,5% dei campioni di siero sono risultati positivi al test ELISA utilizzato. La presenza dell’RNA virale è stata rilevata in 10 dei 91 sieri selezionati (11%). L’esame di due sequenze ha confermato la presenza di HEV di genotipo 3. I risultati ottenuti dimostrano che l’infezione da HEV è presente anche nella popolazione di cervi dell’Appennino Tosco-Emiliano e confermano come anche questa specie possa rappresentare un potenziale serbatoio per ceppi di genotipo 3 con potenzialità zoonotiche. Considerando che la presenza di HEV è già stata segnalata in popolazioni di cinghiale, l’osservazione della diffusione di HEV anche nei cervi individua un potenziale ulteriore rischio di infezione per contatto diretto e/o indiretto di alcune categorie (cacciatori, veterinari, operatori faunistico-venatori).

L’epatite E è una malattia dell’uomo con caratteristiche di epatite acuta, causata da un virus a RNA, HEV. I ceppi di HEV che infettano i mammiferi, sono classificati in 4 genotipi, tra cui l’1 e il 2 infettano solo l’uomo, mentre i genotipi 3 e 4 infettano sia l’uomo sia gli animali. I ceppi zoonotici sono stati identificati principalmente nel suino, che è considerato il serbatoio asintomatico, e occasionalmente altre specie animali (cinghiale, cervo, coniglio). Nei paesi in via di sviluppo la malattia ha un ciclo interumano e causa diffuse epidemie causate dal consumo di acqua contaminata con feci. Nei paesi industrializzati vengono descritti casi sporadici autoctoni sostenuti da ceppi dei genotipi 3 e 4. In alcuni di questi casi è stata provata la trasmissione del virus (genotipo 3) attraverso il consumo di carne poco cotta di maiale o cervo. In Italia, il genotipo 3 è stato messo in evidenza nel fegato di suini macellati ed è stato associato ad alcuni casi umani. Nel presente studio, è stata investigata la presenza di HEV in prodotti a base di carne suina (salsicce di fegato), che nel nostro Paese sono spesso consumate crude. Nel 2012, 4 confezioni di salsicce di fegato, fresche e stagionate, sono state acquistate in un punto vendita. L’RNA totale è stato estratto da porzioni di salsiccia di circa 250 mg. Attraverso una real-time PCR quantitativa sono state ricercate porzioni di genoma di HEV e di Adenovirus suino (come indicatore di contaminazione fecale). Il genoma di HEV è stato ritrovato sia in salsicce fresche (10 positivi su 45 porzioni da 250 mg) sia stagionate (1 campione positivo su 23 analizzati). Sono state condotte anche RT-PCR convenzionali e le analisi di sequenza hanno rilevato la presenza di HEV di genotipo 3, vicino per identità nucleotidica a ceppi umani e suini circolanti in Europa. Sebbene non sia stato possibile dimostrare la vitalità del virus, né sia nota la dose infettante, i risultati confermano un possibile rischio associato al consumo di alcuni alimenti. Considerando che all’elevata prevalenza di HEV nei suini corrisponde un ridotto numero di casi nell’uomo, è possibile ipotizzare che HEV genotipo 3 abbia una bassa virulenza nell’uomo o necessiti di una dose infettante elevata. In tal senso, non essendo ancora disponibile un sistema efficace di crescita in vitro, l’utilizzo della biologia molecolare e in particolare della qRT-PCR si è rilevato molto utile per una stima quantitativa della presenza del virus.

Hepatitis E is an infectious disease caused by a small RNA virus (Hepatitis E Virus, HEV), which can cause acute hepatitis in humans. Four mammalian HEV genotypes are known, among which g3 and g4 are considered zoonotic. In developed countries, hepatitis E cases were mostly associated with travelling in endemic areas. However, recently autochthonous cases have been increasingly reported in developed country, including Italy. In Italy, g3 strains have been detected in swine, in both farms and slaughterhouses, with prevalence that can reach 50% (2,3). Swine strains often share a high nucleotide identity with HuHEV. During the last 10 years, hepatitis E cases have been correlated with consumption of swine, wild boar and deer meat or organs (particularly liver) consumed raw or undercooked, worldwide (1). In this study, we investigated the presence of HEV in liver sausage, which is often consumed raw or undercooked in Italy. In 2012, 4 packages (300 gr) of pork liver sausages were bought at a butcher shop. Sausages were chopped in 45 slices (250 mg each) and spiked with Murine Norovirus (MNV). After RNA extraction, samples were analyzed by RT‐PCR for MNV detection (extraction process control). Samples positive for MNV were then analyzed for HEV and Porcine Adenovirus (PAdV as index virus of fecal contamination) by real‐time RT‐PCR and real‐time PCR, respectively. Positive samples were further analyzed by RT‐PCR with specific primers for two different genomic regions (ORF1 and ORF2). Two out of 45 samples (4.4%) were positive for HEV. Moreover both samples were positive for HEV ORF1, and one was also confirmed in ORF2. This result suggests different sensitivity of the two PCR protocols. Sequence analysis confirmed the presence of swine g3 HEV strains in both samples. One sausage sample was positive for HEV and PAdV, suggesting that also HEV presence could be related to fecal contamination of pork liver. Attempts to infect A549 cells with a homogenate from a HEV and PAdV positive liver sausage were unsuccessful. This study confirms that pig liver sausage can be contaminated with HEV, which might at least partially reflect fecal contamination of pork meat and liver during improper slaughtering practice. Further studies to investigate residual HEV infectivity in pork are needed

Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free-living red deer (Cervus elaphus) population in central Italy by detecting virus-specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti-HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, and is an unenveloped pos1tive sense single-stranded RNA wus. Swine HEV strains are genetically closely related to human strains from the same area, suggesting the occurrence of zoonotic transmission. Recently, human cases of hepatitiS E have been linked to the consumption of raw or undercooked meat or organs from deer, wild boars or p1gs. The disease is now considered an emerging food-borne transmitted zoonosis. During 2006, a pilot investigation was performed to determine the prevalence of HEV in pig farms located in Northern Italy., 274 faecal samples were collected from healthy fattening animals (3-4 and 8-9 months of age) and from healthy breeding animals (gilts and sows) from 6 different farms, and analyzed using a Nested-RT-PCR target1ng the open reading frame 2 (ORF2) region. Stool samples were suspended in water, and viral RNA extraction was performed using a commercial kit. Extracted viral RNA was subjected to RT -PCR amplification using degenerate primers conA 1conS1 for the first amplification, and degenerate primers conA2-conS2 for the nested PCR, yielding a final fragment of 145 bp. HEV RNA was detected in sixty-nine of the 274 (25.2%) examined samples. None of the six farms resulted negative and the prevalence within the farms ranged between 2% and 60.5% For the characterization of the strains, randomly selected positive samples were subjected to nucleotide sequencing, and aligned with those present in the NCBI Data Bank Sequence analysis showed that all stra1ns were Swine Hepatitis E belonging to Genotype 3. These preliminary results confirm that swine HEV is widespread in Italian swine farms.