Luigi Bertolotti

Borrelia lusitaniae in Immature Ixodes ricinus (Acari: Ixodidae) Feeding on Common Wall Lizards in Tuscany, Central Italy

Abstract Lizards and small rodents were live captured in Tuscany, central Italy, from May through August 2005. Prevalence of infestation by larval Ixodes ricinus L. (Acari: Ixodidae) and mean numbers of larvae per host were not significantly different for common wall lizards, Podarcis muralis Laurenti, and Apodemus spp. mice, whereas infestation levels by nymphs were significantly greater on lizards. Borrelia lusitaniae, which was previously shown to be dominant in host-seeking I. ricinus in the same study area, was detected by polymerase chain reaction (PCR) in 19.8% (95% confidence interval: 14.4, 26.0) of larval ticks and in 52.9% (27.8, 77.0) of nymphs that were collected from lizards. Moreover, 18.8% (7.2, 36.4) and 25.0 (3.2, 65.1) of lizards’ tail biopsies and blood samples, respectively, were positive for B. lusitaniae. Conversely, attached ticks and ear biopsies from Apodemus spp. mice were PCR negative. Passerine birds belonging to 10 species were live captured in March 2005, and Borrelia valaisiana was detected in 57.1% (18.4, 90.1) of I. ricinus nymphs feeding on Eurasian blackbirds, Turdus merula L. Results of this study suggest that lizards play an important role as reservoirs for B. lusitanae and may affect the dominance of this genospecies in the Mediterranean area.

Small Ruminant Lentivirus genotype E is widespread in Sarda Goat

The highly divergent SRLV genotype E has recently been characterized in Italy as a low pathogenic caprine lentivirus in the Roccaverano breed. The availability of a genotype specific diagnostic test based on a comparative assay, using a combination of genotype specific recombinant antigens allows a wide serosurvey in other goat populations. The island of Sardinia still has the highest small ruminant population of any Italian region and crossbreeding has been limited to goats, mainly with the Maltese breed. A serological survey was carried out on sheep flocks and goat herds, using individual sera as well as a bulk milk-adapted procedure. Genotype E was identified in more than 50% of goat herds and none of the sheep flocks thus supporting the idea that this genotype is specifically associated with the goat species. The full-length proviral sequence of a Sardinian isolate revealed and confirmed the deletion of dUTPase subunit and the absence of both vpr gene and the 71bp repeat of the LTR. Genetic similarity of this isolate with the prototype strain Roccaverano was not more than 84%, supporting the designation of two subtypes within genotype E. Nevertheless, in vitro properties of the Sardinian strain were different from those of the Roccaverano strain in terms of ability to infect synovial membrane and produce syncitia. Remarkable differences in the HV1 and HV2 of the env gene were recorded, with the Sardinian isolate displaying sequence motif more similar to arthritic strains. Data presented suggest diffusion of genotype E is wider than previously thought.

Borrelia lusitaniae and Spotted Fever Group Rickettsiae in Ixodes ricinus (Acari: Ixodidae) in Tuscany, Central Italy

Abstract Prevalence of infection by Borrelia burgdorferi s.l. and spotted fever group (SFG) rickettsiae was estimated in host-seeking ticks in an area in Tuscany, central Italy, where Lyme borreliosis was reported in a forestry worker. B. burgdorferi s.l. was identified by polymerase chain reaction in 16.7% (95% CI = 10.3, 24.8) of Ixodes ricinus (L.) nymphs and 39.6% (95% CI = 26.5, 54.0) of adults. Borrelia lusitaniae accounted for 82.9% of positive samples, followed by Borrelia garinii (9.8%), Borrelia afzelii (2.4%), and Borrelia burgdorferi s.s. (2.4%). One Rhipicephalus spp. adult was infected with B. garinii (prevalence = 8.3%; 95% CI = 0.21, 38.5). Prevalence of infection by SFG rickettsiae was 38.5% (95% CI = 26.7, 51.4) in I. ricinus nymphs, 34.6% (95% CI = 22.0, 49.1) in I. ricinus adults, and 50% (95% CI = 21.1, 78.9) in Rhipicephalus spp. adults. Phylogenetic analysis showed the similarity of B. lusitaniae strains that were identified in this study and of a strain that was previously isolated from a human patient in Portugal. Results of this study confirm the dominance of B. lusitaniae in areas in the Mediterranean basin and the infection by SFG rickettsiae in I. ricinus.

Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus Ticks Collected on Wild Boars in Tuscany, Italy

ABSTRACT During the hunting season 2007–2008, 494 Dermacentor marginatus (Sulzer) ticks were collected from 109 hunter-killed wild boars, Sus scrofa, in Luccas province, Tuscany, Italy. Rickettsia slovaca, the causative agent of tick-borne lymphadenopathy (TIBOLA), was detected in 32.1% of ticks tested (n = 112) by using polymerase chain reaction primers targeting gltA, ompA, and ompB rickettsial genes. Moreover, Rickettsia raoultii was found for the first time in Italy, with 1.8% infection prevalence. This study confirms the risk posed to humans by ticks and tick-borne pathogens in the study area, where cases of spotted fever rickettsiosis (TIBOLA) are reported.

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.

Temporal Variations in the Usefulness of Normalized Difference Vegetation Index as a Predictor for Ixodes ricinus (Acari: Ixodidae) in a Borrelia lusitaniae Focus in Tuscany, Central Italy

Abstract Host-seeking ticks were collected during monthly dragging sessions from November 2004 through October 2006 in Tuscany, central Italy. Normalized difference vegetation index (NDVI), which was calculated from Landsat ETM+ 7 remote sensing data recorded in August 2001, was significantly correlated with numbers of host-seeking immature Ixodes ricinus L. (Acari: Ixodidae) during periods of relatively low rainfall such as summer 2005 (Spearman’s ρ = 0.78, P < 0.001 for nymphs in July) and to a lower extent in spring–summer 2006. In spring 2005, when rainfall was relatively high, the correlation was weak and not statistically significant. Generalized estimating equations (GEEs), taking into account repeated sampling of the same dragging sites, were used to model the effects of NDVI and season on counts of host-seeking I. ricinus nymphs. Seasonal variations of the effect of NDVI yielded a significant NDVI-by-season interaction in the first year of the study (November 2004–October 2005), but not in the second year (November 2005–October 2006) when there was a 2.5-fold increase of the number of nymphs per 100-m dragging for every 0.1 unit increase in NDVI (95% confidence interval = 1.6, 3.0). Risk maps that were obtained based on GEE results confirmed that the predicted number of I. ricinus nymphs per 100 m was relatively homogeneous through the study area during the 2005 spring peak of activity. Conversely, in 2006, the predicted abundance of nymphs was greater in moist bottomland habitat (characterized by high NDVI) than in dry, typically Mediterranean, upland habitat.

Expression and antigenic characterization of bubaline herpesvirus 1 (BuHV1) glycoprotein E and its potential application in the epidemiology and control of alphaherpesvirus infections in Mediterranean water buffalo

Bubaline herpesvirus 1 (BuHV1) is a member of ruminant alphaherpesviruses antigenically related to bovine herpesvirus 1 (BoHV1). The impact of BuHV1 infection in infectious bovine rhinotracheitis control program is difficult to establish, due to the lack of specific diagnostic test. The ectodomain of glycoprotein E of BuHV1 was expressed as recombinant secreted protein and used in indirect ELISA as well as in a discriminatory test using the BoHV1 counterpart. A panel of monoclonal antibodies was produced against BuHV1; 6 out of 7 anti-gE monoclonal antibodies specifically recognized the BuHV1 gE. Results indicated BuHV1 gE as a sensitive marker of infection compared to seroneutralization (SN) test or blocking ELISA. When BoHV1 and BuHV1 gEs were immobilized in different wells of the same ELISA microplate, bovine and water buffalo sera were more reactive against the respective infecting virus. About one third of seropositive buffaloes with no history of contact with cattle and having higher SN titres, reacted in BoHV1 gE blocking ELISA, possibly because of steric hindrance. Since in two occasions BuHV1 was also isolated from water buffalo scoring gB+/gE+ BoHV1 blocking ELISA, we conclude that the combination of the two blocking ELISAs is not suitable to differentiate between BoHV1 and BuHV1.