Seasonal succession of tabanid species in equine infectious anaemia endemic areas of Italy.

Equine infectious anaemia (EIA) is a disease with an almost worldwide distribution, with several outbreaks having been reported recently in European countries. In Italy, two regions, Lazio and Abruzzo, are considered as endemic areas for this disease. In nature, the EIA virus is mechanically transmitted by biting flies such as tabanids (Diptera: Tabanidae), although few studies have investigated the epidemiological implications. In the present study, several sites characterized by different levels of EIA prevalence were sampled. In sites with high tabanid populations, a seasonal succession of tabanid species with a dual-peak corresponding to early active species (i.e. in June to July) and late active species (i.e. in August to September) was clearly observed. Moreover, a positive correlation was found between EIA prevalence and tabanid abundance and species richness, suggesting that tabanid diversity might extend the duration of the seasonal transmission period of EIA. Further observations are required to better assess how vector diversity influence EIA transmission.

Deep sequencing and variant analysis of an Italian pathogenic field strain of equine infectious anaemia virus.

Equine infectious anaemia virus (EIAV) is a lentivirus with an almost worldwide distribution that causes persistent infections in equids. Technical limitations have restricted genetic analysis of EIAV field isolates predominantly to gag sequences resulting in very little published information concerning the extent of inter-strain variation in pol, env and the three ancillary open reading frames (ORFs). Here, we describe the use of long-range PCR in conjunction with next-generation sequencing (NGS) for rapid molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from the 2006 Italian outbreak. These isolates were from foals believed to have been exposed to the same source material but with different clinical histories: one died 53 days post-infection (SA) while the other (DE) survived 5 months despite experiencing multiple febrile episodes. Nucleotide sequence identity between the isolates was 99.358% confirming infection with the same EIAV strain with most differences comprising single nucleotide polymorphisms in env and the second exon of rev. Although the synonymous:non-synonymous nucleotide substitution ratio was approximately 2:1 in gag and pol, the situation is reversed in env and ORF3 suggesting these sequences are subjected to host-mediated selective pressure. EIAV proviral quasispecies complexity in vivo has not been extensively investigated; however, analysis suggests it was relatively low in SA at the time of death. These results highlight advantages of NGS for molecular characterization of EIAV namely it avoids potential artefacts generated by traditional composite sequencing strategies and can provide information about viral quasispecies complexity.

An Acute Multispecies Episode of Sheep-Associated Malignant Catarrhal Fever in Captive Wild Animals in an Italian Zoo.

In July 2011, in a zoological garden in Rome, Italy, malignant catarrhal fever (MCF), a fatal, systemic disease of Artiodactyla, was suspected on the basis of neurological signs and gross lesions observed in a banteng, the first animal to die of this infection. An MCF type-specific PCR with subsequent sequencing of the PCR amplicon confirmed the aetiological agent as ovine herpesvirus-2 (OvHV-2). Biological samples were collected from the dead animals for gross, histological, bacteriological, virological and serological examinations. An epidemiological investigation was conducted to identify the source of the outbreak, as further deaths due to OvHV-2 still occurred after the removal of the acknowledged reservoirs, domestic sheep and goats. For this purpose, samples from other susceptible species and reservoir hosts were collected for virological and serological analysis. In conjunction, a retrospective sero-investigation was conducted on sera collected between 1999 and 2010 from some of the species involved in the present episode. In total, 11 animals belonging to four different species (banteng, Himalayan tahr, Nile lechwe and sika deer) died between July 2011 and October 2012. The severe gross and histological lesions were consistent with the disease, namely haemorrhages and congestion of several organs as well as lymphoid cell infiltrates and vasculitis of varying severity. The virological tests confirmed that all animals had died of sheep-associated MCF. The investigation indicated that the OvHV-2 infection could have been due to the arrival of sheep in the petting zoo, with cases commencing after first lambing and subsequent shedding of virus. This was also supported by the serological retrospective study that indicated limited previous MCF virus circulation. Further MCF cases that occurred even after the removal of the domestic sheep and goats were attributed to the mouflon. This episode confirms the importance of biosecurity measures in zoos, which house MCF susceptible species, especially those endangered.

Challenges and proposed solutions for more accurate serological diagnosis of equine infectious anaemia.

Serological diagnosis of equine infectious anaemia virus (EIAV) infections has depended mainly on the agar gel immunodiffusion test (AGIDT). This study documents the presence of EIAV genetic sequences in a number of persistently infected horses and mules whose serums were interpreted as negative/equivocal on AGIDT, but positive on more than one ELISA test and in immunoblot tests. Strategies designed to take advantage of the combined strengths of the ELISA and AGIDT are shown effective in a national surveillance program for EIA in Italy where 17 per cent (25/149) of the equids considered to be infected with EIAV on combined/comparative serological data had reactions in the AGIDT that were interpreted as negative or equivocal. These data document the benefits of using a three-tiered laboratory system for the diagnosis of EIA. Although the ELISA-first strategy introduces some confusing results, the discovery of up to 20 per cent more cases of EIA makes it compelling. In our opinion, it is better and more defensible to find two samples in 1000 with resolvable but falsely positive ELISA tests for EIA than to release two to three horses in 10,000 with falsely negative test results for EIA (the rates seen in the Italian surveillance presented here).

Genetic characterization of equine influenza viruses isolated in Italy between 1999 and 2005.

During local respiratory disease outbreaks, occurring in 2003 and 2004 in horse training stables within race-tracks in Rome, and on a stud horse farm in Bari in 2005, four strains of equine influenza (EI) virus were isolated. All outbreaks occurred in flu-vaccinated horses. Here, we are reporting the results of the genetic characterization of these isolates, together with that of another EI virus strain isolated in 1999 from a dead foal presenting pulmonary lesions. Alignment and phylogenetic analyses were carried out using the haemagglutinin amino acid sequences. The Rome and Bari isolates were identified as members of the American lineage, closely related to other recent strains isolated in America as well as in Europe, including the latest recommended American lineage vaccine prototype A/eq/SouthAfrica/4/2003. In contrast, the Italian 1999 isolate was clustered within the European lineage. In Italy, the most recent outbreaks of EI have been caused by the currently circulating American-like strains, even in vaccinated populations, confirming that vaccines should contain an updated representative strain of this lineage. Presently, companies are still in the process of registering updated vaccines but no product is yet available on the market.

Extended phylogeny of equine arteritis virus: division into new subgroups.

To determine a conclusive phylogeny, equine arteritis viruses from Italy, Austria, Hungary, Sweden, South Africa and other parts of the world were analysed by reverse-transcription polymerase chain reaction amplification and direct sequencing. The nucleotide sequences corresponding to the variable part of the large glycoprotein GP5, specified by open reading frame 5, were compared and added to a previously published phylogenetic tree in which a clear division between 'European' and 'American' type viruses had been established. Adding the sequences determined in this study and new sequences retrieved from GenBank revealed additional diversity and new subgroups.

Active circulation of bluetongue vaccine virus serotype-2 among unvaccinated cattle in central Italy.

Several seroconversions occurring in 2002 among sentinel cattle during the bluetongue-vaccination campaign in Lazio and Tuscany (central Italy) led to the suspicion of vaccine-virus circulation. Therefore in 2003, 17 seroconverting sentinel herds were investigated for the characteristics of the virus involved. From these farms, 91 unvaccinated animals and 57 Culicoides pools were tested for the presence of the bluetongue vaccine virus (serotype-2) or other strains. The presence of vaccine virus serotype-2 was confirmed by PCR followed by restriction analysis in the whole blood of 17 unvaccinated sentinel cattle and 12 pools of Culicoides imicola or C. obsoletus. Of the 17 herds, five were positive only for vaccine virus serotype-2, four were positive for other strains and two for both the vaccine and other strains; the remaining premises were virologicaly negative. The vaccine virus serotype-2 also was detected in areas not included in the vaccination campaign.

Identification of Culicoides obsoletus (Diptera: Ceratopogonidae) as a vector of bluetongue virus in central Italy.

In 2001 and 2002, 235 outbreaks of bluetongue were observed in the Lazio and Tuscany regions of central Italy. During entomological surveillance Culicoides imicola, the main vector of bluetongue virus in the Mediterranean region, was detected in only 14 of 28 municipalities affected by outbreaks; Culicoides obsoletus was the most abundant species, contributing 83 per cent of individuals in catches, whereas C. imicola contributed only 2 per cent. In affected municipalities the maximum catch of C. obsoletus was 18,000 specimens, compared with 54 of C. imicola. In October 2002 bluetongue virus serotype 2 was isolated from a single pool of wild-caught, non-blood-engorged parous C. obsoletus inoculated on to BHK-21 cells. Its identity was confirmed by reverse transcriptase-PCR.

Association between the 2001-2003 bluetongue epidemic in Lazio and Tuscany (central Italy) and distribution and abundance of Culicoides imicola and C. obsoletus vectors.

During the epidemic of bluetongue (BT) in Lazio and Tuscany between 2001 and 2003, the distribution pattern of Culicoides imicola did not always correspond either geographically or seasonally, with virus circulation. Culicoides obsoletus was observed to be abundant, ubiquitous and active throughout the year. The geographical and seasonal distribution of BT virus (BTV), C. imicola and C. obsoletus was compared. The territory of the two regions was divided into 30 cells each measuring 1 600 km(2). The presence of C. obsoletus was recorded in every cell, while C. imicola was detected in 18 of the 30 cells, but was absent in 6 of the 21 cells that indicated the presence of BTV. The occurrence of seroconversions appeared to be positively correlated with maximum C. obsoletus catches. Seroconversions were recorded throughout the year, even when C. imicola was not active, whereas C. obsoletus was detected during the entire period. The occurrence of BTV circulation in areas and periods where C. imicola was absent, and the abundant and constant presence of adult C. obsoletus in all the cells, suggest the active role of the latter species in BTV circulation in central Italy.

Genetic diversity of equine arteritis virus.

Equine arteritis viruses (EAV) from Europe and America were compared by phylogenetic analysis of 43 isolates obtained over four decades. An additional 22 virus sequences were retrieved from GenBank. Fragments of the glycoprotein G(L) and the replicase genes were amplified by RT-PCR, prior to sequencing and construction of phylogenetic trees. The trees revealed many distinctive lineages, consistent with prolonged diversification within geographically separated host populations. Two large groups and five subgroups were distinguished. Group I consisted mainly of viruses from North America, whilst group II consisted mainly of European isolates. In most instances, where the geographic origin of the viruses appeared to be at variance with the phylogenetically predicted relationships, the horses from which the viruses were recovered had been transported between Europe and America or vice versa. Analysis of the replicase gene revealed similar phylogenetic relationships although not all of the groups were as clearly defined. Virus strains CH1 (Switzerland, 1964) and S1 (Sweden, 1989) represented separate 'outgroups' based on analysis of both genomic regions. The results of this study confirm the value of the G(L) gene of EAV for estimating virus genetic diversity and as a useful tool for tracing routes by which EAV is spread. In addition, computer-assisted predictions of antigenic sites on the G(L) protein revealed considerable variability among the isolates, especially with respect to regions associated with neutralization domains.

Bovine virus diarrhoea (BVD) control programme in an area in the Rome province (Italy).

A BVD control programme based on the identification and removal of persistently infected (PI) animals is being undertaken in an area in the Rome province, where BVD outbreaks had been previously detected. It involves 174 mainly dairy herds, from which blood samples of all bovines older than 1 year are obtained through the national brucellosis and leukosis eradication programme. Samples sufficient to detect the presence of seropositive animals at a prevalence of 5% or more are initially screened for antibodies against BVD virus (BVDV) using an immunoenzymatic assay. Upon identification of seroreagents additional blood samples are tested from the 6-12-month age category not included in the initial samples. Animals are considered immunotolerant if BVDV is demonstrated twice at a minimum 30-day interval. When no seropositive animals are detected during the first serological screening the herd is declared BVD-free if a second testing, preferably carried on the same animals previously tested, confirms the seronegative status of the herd. At present 147 farms have been tested, of which 63 (42.9%) are negative with respect to antibodies against BVDV. Of the 84 remaining herds in which one or more seropositives are detected, 13 are classified as recently infected. In eight of these recently infected herds, 22 PI animals have been identified.

Isolation of encephalomyocarditis virus from dormice (Myoxus glis) in Italy.

Two isolates of encephalomyocarditis (EMC) virus (ZRC 276RA/90 and ZRC 292RA/90) were isolated from two dormice (Myoxus glis) in Tuscany, Italy. The two isolates were lethal for laboratory mice and caused a rapid cytopathic effect characterized by rounded and wrinkled cells in both baby hamster kidney cells (BHK21) and African green monkey kidney cells (Vero). We found neutralizing antibodies against EMC virus in 408 (77%) of 529 domestic pigs (Sus scrofa scrofa) and in 165 (49%) of 338 wild boars (S. scrofa ferus majori) in Tuscany.

Seroprevalence of antibodies to hantaviruses and leptospires in selected Italian population groups.

A study of hantaviral and leptospiral antibodies in selected population groups was performed. Among high risk subjects in the Rome area, Hantaan antibody was found in mammalogists (10%) and dialysis patients (6%), while none of the trappers, oarsmen, river policemen and firemen studied tested positive for antibodies to hantaviruses. In occupationally-exposed subjects (farmers, rangers, lumbermen, hunters) from rural and densely forested areas of northern Italian regions, the prevalence of Hantaan antibody ranged from 3.3% to 8.8%. In the positive cases the comparative antibody titration using different hantaviruses showed a predominance of Hantaan virus (titer 1:128) compared to Puumala virus (titer 1:32); no reactivity was observed with Seoul or Prospect Hill viruses. In Rome, leptospiral antibodies were found in trappers (21%) and oarsmen (5%) at a titer of 1:50 or more, with a predominance for the L. icterohaemorrhagiae serotype (85%). In the Alpine areas the leptospiral antibody prevalence was 12% and L. icterohaemorrhagiae and L. bratislava were the predominant serotypes. The presence of hantavirus infections, suspected after the first epidemiological survey conducted in central Italy, is now supported by the new data obtained in northern Italian regions. Furthermore, the recent observation of one case of Hemorrhagic Fever with Renal Syndrome (HFRS) in the Udine area, not far from the Yugoslavian border, strongly confirms the presence of one or more hantaviruses in Italy.