Francesco Tolari

Natural Vectors of Dirofilariasis in Rural and Urban Areas of the Tuscan Region, Central Italy

Abstract Entomological investigations by means of dog- and human-baited traps were carried out in summers 2000–2002 in urban and rural areas of the Tuscan region in central Italy. The aim of the study was to define the mosquito species involved in the transmission of Dirofilaria nematodes and to assess the risk that their presence might represent for animal and human health. Nocturnal fieldwork on host-seeking activity and feeding preferences was followed by microscopic identification of the mosquito species attracted and by molecular identification of Dirofilaria parasites in mosquitoes. In total, 3,611 mosquito females belonging to 12 species, largely represented by Culex pipiens L. and Aedes caspius (Pallas), were caught. Some females of each species collected fed on the dogs, indicating their possible role as an intermediate host, but filarial DNA was found only in Cx. pipiens, Anopheles maculipennis s.l. (Meigen), and Coquillettidia richiardii (Ficalbi). In rural environments, the DNA evidence indicated the presence of infective larvae of Dirofilaria immitis, whereas in urban areas, infective larvae of Dirofilaria repens were present. The role of Cx. pipiens as a vector for heartworm disease and subcutaneous infections in natural and artificial environments was confirmed, whereas Ae. caspius seemed refractory to the infection. The different role of the collected species is discussed. The vector competence of An. maculipennis and Cq. richiardii needs further investigation, because the importance of these species poorly represented, and the role of species such as Aedes albopictus (Skuse), characterized by a dominant diurnal activity pattern, has to be evaluated.

In situ PCR-associated immunohistochemistry identifies cell types harbouring the Maedi-Visna virus genome in tissue sections of sheep infected naturally

Maedi-Visna virus (MVV) is a non-oncogenic ovine lentivirus whose main targets are the lung, mammary gland, central nervous system and joints. Cells of the monocyte-macrophage lineage are the major viral target in vivo; other cell types are infected as well, as indicated by several studies, largely based on the examination of animals infected experimentally or on the in vitro infection of cultured cells. Aim of this study was to investigate the cell types harbouring the viral genome in lungs and mammary glands of animals infected naturally by using in situ PCR-associated immunohistochemistry. Several types of cells were infected: in the lung type I and II pneumocytes, interstitial and alveolar macrophages, endothelial cells and fibroblast-like cells. Epithelial cells, macrophages, endothelial cells and fibroblast-like cells were infected also in the mammary gland. These results indicate that the in situ PCR, a powerful technique which combines the high sensitivity of the conventional PCR with the ability to localise the cellular targets within a tissue, can be improved further by its association with the immunohistochemistry. This can be especially advantageous when the presence and localisation of the target sequence are investigated in the context of a tissue with its complex cellular organisation.

Hepatitis E Virus Genotype 3 in Humans and Swine, Bolivia

We determined the seroprevalence of hepatitis E virus (HEV) in persons in 2 rural communities in southeastern Bolivia and the presence of HEV in human and swine fecal samples. HEV seroprevalence was 6.3%, and HEV genotype 3 strains with high sequence homology were detected.

Presence of hepatitis E RNA in mussels used as bio-monitors of viral marine pollution

Mussels (Mytilus galloprovincialis), collected from a harvesting area approved by European Community Regulation, were transplanted to four polluted sites located in the Northwestern Mediterranean area (Tuscany). They were used as bio-monitors to test the quality of the marine water pollution. At different times after the transplantation, mussels were withdrawn and tested for presence of phages and enteric viruses by molecular tests. 52.4% of the transplanted mussel samples were positive for at least one enteric virus. Hepatitis A virus (HAV) was identified in each site (17/37; 45.9%). Three samples were positive for hepatitis E virus (HEV) (8.1%) and two (5.4%) for norovirus (NoV) genogroup I. Coliphages and RYC 2056 phages were detected in all sites, while HSP 40 phages were detected in three sites. Results demonstrate the ability of transplanted mussels in accumulating and retaining different species of enteric microorganisms. Their utility as bio-monitor organisms enables testing for viral marine pollution.

Infectivity of DWV Associated to Flower Pollen: Experimental Evidence of a Horizontal Transmission Route

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.

Characterization of new Small Ruminant Lentivirus subtype B3 suggests animal trade within the Mediterranean Basin.

Small ruminant lentiviruses (SRLVs) represent a group of viruses infecting sheep and goats worldwide. Despite the high heterogeneity of genotype A strains, which cluster into as many as ten subtypes, genotype B was believed to be less complex and has, so far, been subdivided into only two subtypes. Here, we describe two novel full-length proviral sequences isolated from Sarda sheep in two Italian regions. Genome sequence as well as the main linear epitopes clearly placed this cluster into genotype B. However, owing to long-standing segregation of this sheep breed, the genetic distances that are clearly >15 % with respect to B1 and B2 subtypes suggest the designation of a novel subtype, B3. Moreover the close relationship with a gag sequence obtained from a Turkish sheep adds new evidence to historical data that suggest an anthropochorous dissemination of hosts (small ruminants) and their pathogens (SRLV) during the colonization of the Mediterranean from the Middle East.

Serologic and molecular survey for hepatitis E virus in wild boar (Sus scrofa) in Central Italy.

The aim of this study was to further investigate the role of wild boar (Sus scrofa) as a reservoir for hepatitis E virus (HEV). Sixty-four blood and faecal samples collected from wild boar hunted in Central Italy in 2011–2012 were examined by indirect enzyme-linked immunosorbent assay and RT-PCR analysis. Positive RT-PCR samples were further examined by nucleotide sequence determination and subsequent phylogenetic analysis. Thirty-six sera (56.2%) were positive for HEV-specific antibodies, and six (9.4%) faecal samples scored RT-PCR-positive results. Four animals were positive by both enzyme-linked immunosorbent assay and RT-PCR. Phylogenetic analysis showed that the detected wild boar–derived HEV sequences clustered within genotype 3, with similarity to sequences of human origin collected in a nearby area in 2012. Our data confirm that HEV is endemic in the wild boar population in the research area and that these wild animals could play an important role in the epidemiology of HEV infection.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

Detection of hepatitis E virus in Italian pig herds

HEPATITIS E is a public health concern in many developing countries, where it is primarily transmitted by the faecal-oral route through contaminated water and food (Emerson and Purcell 2003). The disease is caused by a small, non-enveloped single-stranded RNA virus classified as the separate genus Hepevirus. Although hepatitis E disease occurs only sporadically in countries with good health care systems, the seroprevalence in healthy individuals can be high (Emerson and Purcell 2003). Porcine hepatitis E virus (HEV) is not pathogenic to general pig populations, but there is evidence that the virus may be a zoonotic agent and that animal reservoirs may exist. Experimental interspecies transmission of HEV between non-human primates and pigs has been demonstrated (Meng and others 1998), and seroepidemiological studies have shown that pig handlers are at higher risk of HEV infection than the general population (Meng and others 2002). In Japan, studies have supported the possibility of zoonotic transmission, as consumption of undercooked pig organs or meat and, in one case, of deer meat, was closely linked to cases of hepatitis E in human beings (Tei and others 2003, Yazaki and others 2003). The first porcine strain of HEV was characterised in the USA in 1997 (Meng and others 1997). Since then, several other porcine strains have been described worldwide. In the past few years, sporadic cases of autochthonous hepatitis E in human beings have been reported in several European countries, including Italy (Zanetti and others 1999). In many of these cases, the infecting HEV strain showed a high degree of homology with porcine strains of HEV detected in the same country (van der Poel and others 2001, Clemente-Casares and others 2003, Banks and others 2004a). In recent years in Europe, HEV in pig herds has been reported only in Spain (Clemente-Casares and others 2003), the UK (Banks and others 2004b) and the Netherlands (van der Poel and others 2001). This short communication describes the detection, by a nested reverse transcriptase-PCR (nested RT-PCR), of HEV in two Italian pig farms, and the phylogenetic analysis of the viral strains. Thirty-four faecal and 22 serum samples were collected from five different farrow-to-finish farms located in northcentral Italy. Samples were collected from healthy pigs between two and five months of age. Faecal samples represented pools of faeces from animals of the same age group. Total RNA was extracted from 140 μl of faecal suspension or serum using a QIAmp viral RNA kit (Qiagen), according to the manufacturer’s instructions. Template cDNA was reverse transcribed using random hexamers, according to standard protocols. A 145 base pair (bp) fragment of the open reading frame 2 of HEV was amplified from the prepared cDNA by nested PCR, as described by Erker and others (1999). A faecal suspension from a human patient with hepatitis E was used as a positive control. Nested RT-PCR products were visualised on 2 per cent agarose gel, and bands of the correct size were excised and purified with the QIAquick Gel Extraction Kit (Qiagen). Nucleotide sequencing was performed using the ABI PRISM BigDye Terminator kit, version 2·0 (Applied Biosystems). Sequences were assembled with SEQMAN (DNASTAR), and alignment was performed using the ClustalX algorithm. The HEV genome was detected in two faecal pools (5·9 per cent) collected at two different farms, but all the serum samples were negative. The positive faecal pools were obtained from groups of pigs aged 4·5 and 2·5 months, respectively. Phylogenetic analysis of the two viral sequences (113 bp), termed HEVBO/01 and HEVPI/01, was performed by the neighbour-joining method using PHYLIP 3·6. Bootstrap confidence values (500 replicates) were calculated by using the Seqboot and Consense programs. A phylogenetic tree (Fig 1) was created with the Treeview software using an avian HEV isolate as out group. Phylogenetic analysis of the viral sequences showed that the two Italian strains, HEVBO/01 and HEVPI/01, belonged to genotype 3, as did other porcine and human HEV strains indigenous to Europe. However, they differed significantly from each other, being only 84 per cent identical (18 nucleotide changes). The two Italian strains clustered with strains from countries where HEV is considered non-endemic. In particular, HEVPI/01 was related (with 90 per cent identity) to a human strain (AY540113) detected in a sporadic case of acute autochthonous hepatitis E in Spain (Buti and others 2004). In conclusion, this report represents the first description of HEV in Italian pig herds, and confirms the presence of the virus in apparently healthy pigs. These findings are important, because of the potential risk of transmission of porcine HEV to human beings, either by contact with infected pigs or by ingestion of contaminated undercooked meat. However, further studies are needed to address the true zoonotic potential of HEV in pigs. Studies are in progress to evaluate the prevalence of the infection in Italian pigs. The study of a high number of viral strains will be necessary to assess intraspecies and interspecies HEV homologies and to understand whether zoonotic transmission of HEV may occur in Italy.

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.