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.

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.

Seroconversion against SU5 derived synthetic peptides in sheep experimentally infected with different SRLV genotypes.

Synthetic peptides were generated, corresponding to SU5 domain of envelope glycoprotein of Italian SRLV isolates It-561 and It-Pi1, belonging respectively to MVV- and CAEV-like genotypes. The peptides, encompassing an N-terminal variable and a C-terminal conserved antibody-binding site, were used in an ELISA assay to analyse the sera of two groups of sheep experimentally infected with these isolates. The kinetics and specificity of the humoral response to the homologous and heterologous antigen and the affinity maturation of the sera were evaluated. Seroconversion occurred between week 3 and 8. The response to SU5 antigen was mostly type-specific. The few broadly reacting sera may reflect the production of antibodies directed to the SU5 constant antibody-binding site. All sera underwent with time avidity maturation, resulting in the appearance of high affinity antibodies. This study suggests constant monitoring of the circulating viral variants to develop a panel of diagnostic peptides representative of local genotypes.

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.

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.

Natural vectors of dirofilariasis in rural and urban areas of the Tuscan region, central Italy.

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.

Evaluation of an ELISA to detect antibodies to maedi-visna virus in individual and pooled samples of milk from sheep.

An elisa was used to detect antibodies to maedi-visna virus in samples of serum and milk from individual sheep; the results obtained indicated that the elisa can be used to detect antibodies in milk. The assay was also applied to samples of bulk-tank milk; a standard curve was created and used to calculate the seroprevalence of maedi-visna in 11 flocks of sheep and the results were compared with the results obtained by applying the elisa to individual serum samples. There was good agreement between the seroprevalences calculated from the standard curve for bulk-tank milk and from the individual serum samples.

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses.

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.

Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections.

Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia. The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life. Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining detectable for long period. Transmembrane (TM) domain of envelope glycoprotein contains a well conserved motif known to form an immunodominant epitope in several lentiviruses. In this study a simple strategy was developed to express the entire CA and the TM epitope in a single fusion protein from equine, feline and small ruminant lentiviruses in prokaryotic system and evaluated the diagnostic utility of a purified preparation in an indirect ELISA for each of the three infections. Results demonstrate that, for FIV and SRLV infections, the combination of CA and TM fractions increases the sensitivity of diagnostic tests based only on CA. The corresponding CA/TM antigen from EIAV showed excellent agreement with Coggins test.

A new sensitive serological assay for detection of lentivirus infections in small ruminants.

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99. 8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.

Low risk of Lyme borreliosis in a protected area on the Tyrrhenian coast, in central Italy.

A comprehensive Lyme borreliosis risk assessment process was applied in S. Rossore Estate, on the Tyrrhenian coast, near Pisa, Italy. Host-seeking Ixodes ricinus nymphs peaked in May in oak-dominated deciduous wood (median, Q1-Q3, number of nymphs/50 m dragging = 4.5, 2.5-8), whereas host-seeking larvae peaked in August in the same habitat type (6.0, 4-17/50 m dragging). Prevalence of I. ricinus infestation was 88.9% in wild rodents (n = 11), 64.3% in fallow deer (n = 28) and 0.0% in wild boars (n = 5). Borrelia burgdorferi sensu lato was not isolated from rodents' organs, and from 80 I. ricinus nymphs and 50 adults. Moreover, PCR for B. burgdorferi sl carried out on 110 nymphs and 12 adult ticks also gave negative results. Forest workers were at higher risk of tick bite than other Estate employees (relative risk (RR): 1.7, p = 0.02). In spite of high levels of tick exposure, B. burgdorferi sl specific antibodies were not detected in sera from Estate personnel (n = 30) and sentinel animals (dogs, n = 23, fallow deer, n = 61).

Isolation of Brucella melitensis from alpine ibex.

Eleven alpine ibex (Capra ibex) and 27 chamois (Rupicapra rupicapra) from Gran Paradiso National Park (Italy) were examined in March 1996. A 7-yr-old ibex buck had thick-walled carpal joints and enlargement of the right testicle characterized by necrosis and fibrosis. Microscopically, testicular lesions were characterized by large areas of necrosis, fibrosis with irregular aggregates of macrophages and lymphocytes, and scattered foci of suppuration. Specimens of the carpal bursae and testicle were cultured in serum dextrose agar and serum dextrose antibiotic plates. A Gram-negative coccobacillus was isolated from the testicle and subsequently identified as Brucella melitensis biotype 2. This is the first recognized case of brucellosis in alpine ibex.

Spatial distribution and seasonality of ticks (Acarina: Ixodidae) in a protected area in the northern Apennines.

Infestation of small rodents by ixodid ticks and frequency of host-seeking ticks collected by dragging were studied at Orecchiella Natural Park (Northern Apennines) in 1994 and 1995. Levels of infestation of Apodemus spp. by immature Dermacentor marginatus were higher in beech wood (5.1 larvae and 1.3 nymphs per mouse) than in oak-chestnut wood and in coniferous wood. Larval D. marginatus peaked in mid summer, whereas nymphs peaked in late summer. Host-seeking Haemaphysalis punctata were mostly found on south-facing limestone rocks with scarce vegetation (7.8 larvae/km dragging). Conversely, D. marginatus larvae were most frequent in wooded areas (3.2 larvae/km dragging). Ixodes ricinus was rare in the Park, and Borrelia burgdorferi was not isolated from ear punches collected from 122 small rodents.

Characterization of enzootic nasal tumor virus capsid antigen.

The RT-PCR was carried out on tumor tissue from sheep with enzootic nasal tumor (ENT), using primers designed from conserved amino acid regions of related type D retroviruses. A 591 bp PCR fragment, corresponding to 90% of the capsid antigen was cloned, sequenced and expressed in E. coli. Alignment with ovine pulmonary carcinoma (OPC) virus showed 93% nucleotide and 96% amino acid homology. No amplification occurred when DNA from ovine fetal cell line was used as template. The recombinant protein, highly expressed in prokaryotic system, reacted in immunoblot with mouse antiserum to Mason Pfizer monkey virus (MPMV) p27, as well as sera from OPC and ENT diseased animals. Preliminary application of this antigen in ELISA suggested its potential use to detect seropositive animals in infected flocks.

Development and application of an antibody ELISA for the marker protein of ovine pulmonary carcinoma.

Ovine pulmonary carcinoma (OPC) is a contagious pulmonary neoplasia with a suspected retroviral etiology. The major core protein (P27) of the putative OPC virus cross-reacts with antibodies to P27 of the Mason-Pfizer monkey virus (MPMV), a type-D retrovirus. This serological reactivity serves as the only accepted biological marker for OPC. In order to make a useful reagent for the detection of the OPC marker for serodiagnosis and epidemiological studies, the MPMV-P27 coding region was cloned and expressed in Escherichia coli. Gel purified recombinant MPMV-P27 protein was used to develop an immunoassay. This recombinant enzyme-linked immunosorbent assay (ELISA) was then used to screen 223 sera from US sheep and 176 sera from Italian sheep. In this study, we found: (1) a high prevalence of infection with the putative OPC retrovirus in sheep with chronic pneumonia; (2) a subclinical infection with OPC virus may be more common in US sheep than indicated by the rare recorded occurrence of pulmonary carcinoma; (3) an apparent association between ovine lentivirus (OLV) and OPC infection.

Genetic and antigenic characterization of caev (caprine arthritis-encephalitis virus) recombinant transmembrane protein.

The env gene fragment of an Italian strain of Caprine Arthritis Encephalitis virus (CAEV) coding for the hydrophilic region of transmembrane protein was amplified, cloned and expressed in prokaryotic system as fusion protein with glutathione-S-transferase. Sequence analysis revealed 63 to 66% amino acid homology, when compared with three ovine lentiviruses and 83% when compared with one caprine lentivirus. The recombinant transmembrane protein was efficiently expressed, purified under denaturing conditions and used as antigen in western blotting and ELISA. Sera from clinically diseased goats strongly reacted in western blotting and naturally infected animals seroconverted between 20 and 33 weeks of age. An indirect ELISA performed with this antigen showed improved sensitivity in comparison with agar gel immunodiffusion test. Our results confirm that transmembrane protein is an important immunological marker in CAEV infection and its use as antigen may enhance the validity of serological diagnosis of Caprine Arthritis Encephalitis.

[Seroepidemiologic investigations in the Alpine ibex (Capra i. ibex) of Piz Albris in the canton of Grigioni (Switzerland)]. / Indagini sieroepidemiologiche su stambecchi (Capra i. ibex) del Piz Albris nel Cantone dei Grigioni (Svizzera).

Sero-epidemiological investigations in wild animals may allow to assess distribution of selected pathogens that sometimes seem to be involved in sanitary interrelationships between wild and domestic ungulates sharing the same areas. Serological studies were carried out to investigate the prevalence of antibody against 8 pathogens in Alpine ibex of Albris colony (Grisons, Switzerland). Investigated sera came from 89 animals shot by gamekeepers in 1990-1991. Antibody against smooth Brucella, Coxiella burnetti, Leptospira interrogans, Borrelia Burgdorferi, Mycobacterium paratuberculosis, BHV-1 and ovine-caprine lentiviruses were not detected in the tested sera. However, 31% of sera analysed were found to be positive for Chlamydia psittaci. Three sera showed high antibody titres ( > or = 1/128) suggestive of active infection in the animals. Any influence of Chlamydia psittaci in reproductive performance of free-ranging alpine ibex should be investigated through isolation of the agent. Results are discussed with reference to methods used and with epidemiological picture in Switzerland and were compared with results of serological investigations carried out in ibex in Italy and France.

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks.

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N'-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.

Isolation and reactivation of bovid herpesvirus 1 in goats.

A virus isolate, recovered from the brain of a goat which died following signs of nervous disease, was identified as BHV-1. Differentiation with the antigenically related caprine herpesvirus (BHV-6) was achieved by restriction endonuclease fingerprinting. Eighteen months after the last clinical episode, BHV-1 infection was reactivated in a seropositive goat from the same herd following dexamethasone treatment.