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.

Molecular identification of Mesocestoides sp. metacestodes in a captive gold-handed tamarin (Saguinus midas).

Mesocestoides spp. are tapeworms harbored in the intestine of many domestic and wild carnivores, birds of prey and, rarely, of humans. The life cycle is assumed to involve three hosts and the metacestode juvenile stage (tetrathyridium) may reproduce asexually, provoking severe systemic infections. In the present study, a case of a peritoneal infection by Mesocestoides sp. occurred in a captive gold-handed tamarin (Saguinus midas) is described. Phylogenetic positions based on CO1 and 12S loci were analysed to describe in details this unusual finding of Mesocestoides in a callitrichid monkey. The phylogenetic analyses has not clearly resolve relationships between the metacestode forms from the tamarin and any of the described Mesocestoides species available so far, including those described from non-human primates, thus supporting the hypothesis of a new taxon within the genus.

Indoor and outdoor winter activity of Culicoides biting midges, vectors of bluetongue virus, in Italy.

Indoor and outdoor winter activity of Culicoides spp. (Diptera: Ceratopogonidae) in central Italy was investigated in order to evaluate whether indoor activity might account for the overwintering of bluetongue virus, as has been hypothesized by some authors. Weekly Culicoides collections were performed at three farms over three consecutive winter seasons. At each farm, two black-light traps were operated simultaneously, indoors and outdoors. Culicoides were identified using both morphological and molecular means. The Culicoides obsoletus group accounted for 98.2% of sampled specimens. Within this group, C. obsoletus s.s. accounted for 56.8% and Culicoides scoticus for 43.2% of samples. Nulliparous, parous and engorged females were caught throughout the entire winter, both indoors and outdoors. At times, indoor catch sizes outnumbered outdoor collections. A significant inverse correlation was found between minimum temperature and the proportion of indoor Culicoides of the total midge catch, thus indicating that lower outdoor temperatures drive Culicoides midges indoors. High rates of engorged females were recorded indoors, possibly as the result of the propensity of C. obsoletus females to feed indoors. Higher proportions of parous females were found in indoor than in outdoor catches, indicating higher survival rates indoors and, consequently, higher vectorial capacities of midges sheltering indoors compared with those remaining outdoors.

Molecular detection and characterization of spotted fever group rickettsiae in ticks from Central Italy.

The aim of this study was to investigate the presence of rickettsial pathogens in ticks from Central Italy. A total of 113 ticks hailed from Latium and Tuscany regions were identified and tested by PCR to detect gltA, ompA, ompB genes of Rickettsia. Positive amplicons were sequenced and identified at species level. Ticks were analyzed individually or in pools. The percentage of positivity for SFG rickettsiae was 12.4%, expressed as minimum infection rate (MIR) assuming that one tick was positive in each positive pool. Rickettsia aeschlimannii was detected in Hyalomma marginatum, Rickettsia monacensis in Ixodes ricinus and Rickettsia massiliae and Rickettsia conorii in Rhipicephalus sanguineus sensu lato. These findings confirm the circulation of pathogenic rickettsiae in Latium and Tuscany regions. To our knowledge this is the first report of R. massiliae in Latium region.

Investigation on Opisthorchis felineus occurrence and life cycle in Italy.

Opisthorchiasis is a fish borne parasitic infection caused by helminths of the genus Opisthorchis (Digenea, Opisthorchiidae), affecting humans and other fish-eating mammals. Despite Opisthorchis felineus was first described in Italy in 1884, no cases of human opisthorchiasis were reported in this country until 2004; from then on, 4 outbreaks due to this species have been recorded in Central Italy. Following the more relevant of these outbreaks, involving 34 people in August 2007, snails, fishes and fecal samples collected from the Bolsena and Bracciano lakes (Central Italy) were analyzed in order to define the cycle of O. felineus in the area and investigate its prevalence in the different hosts. Pools of 20-40 snails each (4983 specimens altogether) of the genus Bithynia were analyzed by PCR for parasite DNA detection. Eight hundred and ninety-four fish belonging to 12 species were collected from the two lakes and tested for metacercariae both by muscle compression and digestion techniques. Eighty-seven fecal samples of 5 putative definitive host species were collected very close to the two lakes and tested for parasite eggs detection by formalin-ethyl acetate concentration technique. Identification at the species level of metacercariae and eggs, respectively, from fish and stool was confirmed by PCR analysis and sequencing. O. felineus DNA was detected in 0.08% (overall minimum infection rate) of snails of the genus Bithynia from the two lakes. The tench, Tinca tinca, was the only fish found infested in both lakes (prevalence 88.5%). O. felineus eggs were found only in cat feces (prevalence 46.4%). The tench represents the only threat for the human consumption in the study area while Coregonus sp., the most economically important species for the local fishery and frequently consumed raw marinated, resulted to be not infected. The high prevalence recorded both in fish and in definitive host suggests a widespread and massive presence of the parasite in the area. Further studies are needed to better investigate the possible role of some cyprinids species as intermediate hosts, in order to check their safety for human consumption.

Biotic and abiotic factors influencing distribution and abundance of Culicoides obsoletus group (Diptera: Ceratopogonidae) in central Italy.

In the framework of a bluetongue surveillance program including clinical, serological, and entomological activities, Culicoides biting midges were light trapped weekly in two regions of central Italy, Lazio and Tuscany. In the period January 2002 through December 2005, 3,944 collections were carried out in 189 trap sites distributed in all the provinces of the two regions. Abundance data of C. obsoletus group were analyzed in relation to trap site altitude, distance from the sea, land use, and number of farmed animals. Species seasonality and overall temporal trend were also described. C. obsoletus was distributed over the whole study area, almost in all trapping sites and with high abundances. The species group was dominant among all captured Culicoides, with higher abundances recorded inland and in areas where land cover was partially or completely natural-wooded. Adults on the wing were caught all year round, with peaks in May-June and middle October. The observed trend through years recorded a peak during autumn 2002, in concomitance with a local epidemic of bluetongue.

Genetic variation within and between G1 and G3 genotypes of Echinococcus granulosus in Italy revealed by multilocus DNA sequencing.

Numerous studies have provided evidence that Echinococcus granulosus exists as a complex of different strains, that differ in a wide variety of criteria that have an impact on the epidemiology, pathology and control of cystic hydatid disease (CHD) and, to date, 10 distinct genotypes (G1-G10) have been identified. In Italy, sequence analysis of the mitochondrial cox1 and nad1 genes showed the occurrence of the G1 genotype, the common sheep strain, the G3 genotype, the buffalo strain and of one isolate identified as G2 genotype, the Tasmanian sheep strain. In the present work, we have analysed E. granulosus strains in Italy, by genotyping a large sample of isolates and by checking out the genetic differentiation within and among the G1 and G3 genotypes using an additional mitochondrial gene as marker, the rrnS gene. Sequencing of the rrnS gene revealed a significant genetic differentiation between isolates identified as belonging to the G1 and G3 genotypes, with fixed nucleotide substitutions. This study provides further evidence of the occurrence of the E. granulosus G3 buffalo strain in Italy, a strain previously thought to be confined to the Indian region.

Proliferative peritoneal and pleural cestodiasis in a cat caused by metacestodes of Mesocestoides sp. Anatomohistopathological findings and genetic identification.

A 10-year-old female cat was brought to Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana for post-mortem examination. The animal used to live, together with 26 other cats, in the big terrace of an apartment at the 8th floor in Rome; and was always fed with industrial pet food. Anamnesis referred balance troubles, vomit and convulsions, during a couple of days, followed by sudden death. At necropsy, the cat presented mucoid rhinitis, purulent tracheitis, small areas of pneumonia, dark spots in the liver, catarrhal-hemorrhagic gastritis, fibrinous enteritis and meningeal hyperemia. Thoracic and abdominal cavities were completely invaded by hundreds of larval stages of cestodes. The same parasites were also included in nodules in pancreatic, lung and kidney parenchyma. Microscopic examination of parasites allowed their identification as larval stages (metacestodes) of cestodes of the genus Mesocestoides. The molecular genotyping of the metacestodes indicates a close relationship with members of the genus Mesocestoides, although a significant variation was found with respect to the available sequences of other species of the genus.

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.

Geographical and seasonal distribution of the bluetongue virus vector, Culicoides imicola, in central Italy.

Following the first incursion of bluetongue virus (BTV) into Italy, the geographical and seasonal distribution of the biting midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae), the main vector of BTV and African horse sickness virus, was investigated in two regions of central Italy (Lazio and Tuscany). Surveillance of Culicoides was carried out between July 2001 and December 2002 using light traps: 1917 collections were made in 381 trap sites, well distributed across both regions. During the survey, bluetongue outbreaks were recorded in both regions. Culicoides imicola was found in 89 (23%) trap sites, distributed fairly continuously along the whole western coastline, between 41.2697 degrees N and 44.05724 degrees N. It was found only occasionally inland and usually in low abundance, with catches of more than 1000 specimens per night found in only two sample sites and 74% of catches numbering fewer than 10 specimens. Adults were caught from March to mid December, with peaks ranging from the end of August to mid November. The coastal distribution and the presence of only few sites with year-round records of adult vectors suggests that colonization may have occurred recently, by passive wind-dispersal from external source areas (Sardinia and Corsica). Alternatively, the species may occur in established, previously undetected, autochthonous populations that are limited from extension inland and northern-ward within Lazio and Tuscany by cool winter temperatures.

Helminth communities in eels Anguilla anguilla from Adriatic coastal lagoons in Italy.

The composition and diversity of the total and intestinal component and infra-communities were determined in eels Anguilla anguilla from three shallow lagoons on the Adriatic coast of Italy to determine whether the helminth communities would differ in composition and structure from those in eels from lagoons on the Tyrrhenian coast. The lagoons differed in respect of their management regimes and the extent of freshwater influx. Both freshwater and marine species of helminths were found in the eels in all three lagoons, but the freshwater component was richer in Valle Figheri. A suite of three digenean eel specialist species occurred in all three lagoons, of which any two members dominated each community. This conferred a high degree of similarity between the communities of the three lagoons. The same three species also dominated helminth communities in eels in lagoons along the Tyrrhenian coast of Italy, and compositional similarity levels were similar within and between western and eastern groups. Species richness was higher in the component communities of the eels of the Adriatic lagoons when compared to the Tyrrhenian ones, but diversity and dominance indices were of a similar order of magnitude and range. Intestinal helminth communities were richer and more diverse in two of the Adriatic lagoons because the proportion of eels with zero or one helminth species was, unusually, in the minority. It was nevertheless concluded that infracommunity structure was similar in eels from both western and eastern lagoons and that the hypothesis that it would differ in Adriatic lagoons could not be supported. The findings provide further evidence of the similarity in composition and structure of helminth communities in eels from coastal lagoons throughout Europe.

Composition and diversity of helminth communities in eels anguilla anguilla in the river tiber: long-term changes and comparison with insular europe

Most studies of helminth communities in the European eel Anguilla anguilla have been undertaken in the British Isles, and there are very few analyses of community composition and structure from continental Europe. To fill this gap and test the hypothesis that helminth communities in freshwater eels in the British Isles are not typical of those of continental Europe, helminth communities of eels in the River Tiber below Rome were analysed by season using data collected in 1980 and new data from 1996. The intestinal helminth communities in the Tiber eels were species poor and characterized by low diversity. Most eels harboured one or no parasite species and communities were heavily dominated by the acanthocephalan Acanthocephalus clavula. Intestinal helminth infracommunity richness and diversity did not differ between seasons within a year or between the same seasons in 1980 and 1996, although some changes in composition were apparent. Intestinal infracommunities from Tiber eels were very similar in characteristics to those analysed from the British Isles, and their temporal changes also showed close similarities to those reported from rivers in the UK. It seems likely therefore that conclusions derived from British studies can be applied to helminth communities of eels on the continent.