Abundance of questing ticks and molecular evidence for pathogens in ticks in three parks of Emilia-Romagna region of Northern Italy.

INTRODUCTION AND OBJECTIVE: Infectious and parasitic diseases transmitted by ticks, such as Lyme diseases, granulocytic anaplasmosis and piroplasmosis, have been frequently reported in Europe, with increasing attention to them as an emerging zoonotic problem. The presented study was performed to assess the distribution and the density of questing ticks in three regional parks of Emilia-Romagna region of Northern Italy, and to seek molecular evidence of potential human pathogens in tick populations. MATERIALS AND METHODS: In the period April-October 2010, 8,139 questing ticks were collected: 6,734 larvae, 1,344 nymphs and only a few adults - 28 females and 33 males. The abundance of Ixodes ricinus questing ticks was compared among different sampling sites and related to microclimate parameters. 1,544 out of 8,139 ticks were examined for the presence of pathogens: PCR was used to detect piroplasms DNA and Real time Taqman PCR for Anaplasma phagocytophilum and Borrelia burgdorferi s.l. RESULTS: The predominant species was I. ricinus (overall abundance 1,075.9/100 m(2) ); more rarely, Dermacentor marginatus (n = 37 - 0.45%), Scaphixodes frontalis (n = 13 - 0.16%), Hyalomma spp. (n = 6 - 0.07%) and Ixodes acuminatus (n = 3 - 0.04%) were also found. 28 out of 324 (8.6%) samples of ticks were PCR-positive for piroplasm DNA. 11 amplicons of 18S rRNA gene were identical to each other and had 100% identity with Babesia EU1 (Babesia venatorum) using BLAST analysis. Real time Taqman PCR gave positive results for A. phagocytophilum in 23 out of 292 samples (7.9%), and for B. burgdorferi s.l. in 78 out of 292 samples (26.7%). I. ricinus was the only species found positive for pathogens by molecular analysis; 16 tick samples were co-infected with at least 2 pathogens. DISCUSSION: The peak of nymph presence was in May, and the higher prevalence of pathogens occurred in April-June, most often in nymphs; therefore, spring season could represent the higher risk period for the transmission of pathogens. These data could provide guidelines for the preventions of tick-trasmitted diseases in this region.

Molecular evidence of Leishmania infantum in Ixodes ricinus ticks from dogs and cats, in Italy.

Leishmaniosis, caused by Leishmania infantum, is an endemic zoonosis in the Mediterranean basin. To date, phlebotomine sand flies are the only accepted biological vectors of Leishmania parasites to dogs and humans. The absence of the primary vector in autochthonous Leishmania outbreaks suggests a possible role of fleas or ticks as alternative vectors. In this study, 119 ticks were collected between August 2007-June 2008 and between March 2010-October 2010 from various animal species and humans living in Italian areas where canine leishmaniosis is endemic (i.e. rural areas of the North) and were tested for the presence of L. infantum DNA. Nine (7.5%) out of 119 ticks resulted PCR positive. All ticks were morphologically identified as Ixodes ricinus ticks, 3 from 1 cat, 6 from 4 dogs. To our knowledge, this is the first evidence of L. infantum DNA in ticks from cat, suggesting that the debate about the epidemiological role of ticks in canine leishmaniosis might be extended to feline leishmaniosis.

Human exposure to piroplasms in Central and Northern Italy.

TA serosurvey has been conducted in Northern and Central Italy to investigate the presence in humans of antibodies against zoonotic Babesia and Theileria species. The study focused on a total of 432 volunteers, of which 290 were persistently exposed to tick bites because of their jobs (forester employees, livestock keepers, veterinary practitioners, farmers and hunters) and 142 resident in the same area less frequently exposed. An indirect fluorescent antibody test (IFAT) for humans was used to detect antibodies to Babesia microti, IFAT tests for veterinary use were modified to detect reactivity to Babesia bovis, Babesia canis and Theileria equi. A laboratory-derived ELISA was employed to detect antibodies to Babesia divergens. Both reactive and 10 negative sera were analysed against plasmodial antigens to evaluate possible aspecificity. A high reactivity to piroplasm antigens was found, showing significant difference between the sera of the two groups of volunteers (24% vs 7.%; p<0.001). No cross-reactivity was observed, while each professional group showed reactivity that would fit with the professional risk exposure. In particular, a high reactivity to B. microti and B. divergens antigens was observed in foresters and hunters (32% and 12%, respectively). This is the first report on the human seroreactivity to piroplasms in Italy; it also provides additional epidemiological information on these tick-borne zoonoses in Europe. Our findings suggest the possible occurrence of piroplasm infections in Italy and alert physicians to consider these otherwise neglected parasitic diseases when dealing with any febrile illness, especially in subjects exposed to tick bites.

Development of culture-based serological assays to diagnose Babesia divergens infections.

Babesioses are hematic tick-borne diseases that induce malaria-like disorders in domestic, wild animals, and humans. Although indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) commercial kits are available to test the presence of antibodies against most Babesia species, no kit exists to serologically diagnose the infections due to Babesia divergens, one of the most important zoonotic species. To fill this gap and to develop assays to detect animal and human infections, in vitro cultures (microaerophilous stationary phase system) of B. divergens were organized. Infected erythrocytes were adsorbed as corpuscular antigen (CA) on IFAT slides and ELISA microwells. The supernatant medium of the cultures (metabolic antigen, MA) was collected and employed in ELISA and western blot (WB) assays. B. divergens was also used to produce positive sera in Meriones unguiculatus and to infect a calf. Serological tests were set up with sera from experimentally/naturally infected animals, and possible cross-reactions were evaluated using heterologous sera from cattle positive to other piroplasms. Sera from clinically healthy people at risk of infection were also tested. As expected, assays based on the purified MAs from in vitro cultures proved more sensitive and specific than CA-IFAT and CA-ELISA. In fact, MA-ELISA provided satisfactory performances (even if 8.4%-15.7% cross-reactions were evidenced), and the WB developed proved totally sensitive and specific. WB indicated as immunodominant antigens two major protein bands at 33 and 37 kDa, which were also evidenced in 2.2% of the human sera tested, proving the parasite transmission to humans also in Italy.

Anaplasma phagocytophilum in ticks from parks in the Emilia-Romagna region of northern Italy.

The aim of this study was to assess the prevalence and phylogenetic characterisation of Anaplasma phagocytophilum in ticks in three parks of the Emilia-Romagna region of northern Italy, an area in which no survey of this agent had previously been conducted. A total of 360 tick samples were analysed; 292 were sourced from the environment and 68 from animals and humans. Real-time polymerase chain reaction revealed that 33 tick samples (9.2%) were positive for A. phagocytophilum. Ixodes ricinus was the only species found positive from the samples retrieved from the environment and was the most commonly infected among ticks removed from hosts. Sequence analysis of the 23S-5S rRNA gene performed on 23 samples revealed six variant sequences that differed by only a few nucleotides when compared to the GenBank sequences from humans, horse and small mammals. Msp4 gene sequences obtained from 7 samples were compared to those described in ruminants, especially roe deer (Capreolus capreolus) and goat isolates from different countries. The results of this study provided evidence of the circulation of A. phagocytophilum in the sites studied and indicated the possible involvement of wild ruminants. Additional studies that extend the sampling areas, or cover different sites, would contribute to a better understanding of the ecology and disease dynamics of A. phagocytophilum in northern Italy and would provide valuable information on zoonotic risks.

Angiostrongylus vasorum in 20 dogs in the province of Chieti, Italy.

After a case of Angiostrongylus vasorum (canine lungworm) was diagnosed in the province of Chieti (Italy) in early 2008, parasitological research was conducted to investigate the presence of the parasite in dogs in the area. A total of 178 dogs, 56 carcasses and 122 stool samples were examined between January and September 2008. The carcasses were examined for the presence of adult parasites in the right ventricle and pulmonary artery, and larval forms in the internal organ and brain tissues. The faeces were inspected for larval form L1 using three diagnostic methods that are currently used to test for endoparasites and larvae of bronchopulmonary strongyles. A total of 20 cases of canine angiostrongylosis were diagnosed (8.9%), with adult parasites being identified in 5 dogs, and L1 larvae in another 15. The anatomo-pathological examination of the carcasses of the dogs infested with adult nematodes revealed pneumonia, pleurisy, reddish foam in the trachea, effusion of serohaemorrhagic fluid in the thoracic cavity and enlarged mediastinic and mesenteric lymph nodes. Histological examination of the tissues showed serious, similar syndromes with lesions caused by colonisation of the kidneys, lymph nodes and brain by the parasites. Given the large number of cases confirmed in relation to the period of study (9 months), it is essential to include angiostrongylosis among the differential diagnoses made in clinical and post-mortem examinations of dogs in the province of Chieti and in the neighbouring areas.

Antimycotic effectiveness against dermatophytes: comparison of two in vitro tests.

Seven antimycotic drugs (econazole, enilconazole, fluconazole, griseofulvin, itraconazole, ketoconazole, and miconazole) were tested against 36 dermatophyte strains (19 M. canis, 7 T. mentagrophytes, 5 M. gypseum, 2 M. cookei, 1 T. rubrum, 1 T. ajelloi, and 1 T. terrestre) isolated from animals, humans, and the environment. Two in vitro methods were compared: a micro-dilution test based on the CLSI M38-A method, and a disk-diffusion test. Fluconazole was not effective in vitro against the dermatophytes. Econazole and enilconazole were the most effective. Thirteen strains were griseofulvin-resistant. The correlation between the two methods was statistically significant for enilconazole, griseofulvin, itraconazole, and miconazole.

Tick reservoirs for piroplasms in central and northern Italy.

Ticks, collected in central and northern Italy from pets, livestock, wild animals and the environment (n=2107), were identified by microscopy and processed by molecular diagnostics to determine the species that act as a reservoir for piroplasms. A total of 11 ixodid tick species were identified, with five of them proving to be piroplasm positive. Molecular diagnostics identified Theileria equi and eight Babesia species in 52 adult specimens, mostly (n=50) removed from piroplasm-free vertebrate hosts. Ixodes ricinus hosted the highest number of species, although the highest infection rate was recorded in Hyalomma marginatum (9.1%), followed by I. ricinus (5.1%), Dermacentor marginatus (5%), Rhipicephalus turanicus (3.1%) and R. sanguineus (1.2%). Novel tick/pathogen associations were detected, suggesting that certain tick species (such as Hy. marginatum, R. sanguineus and I. ricinus) are vector of more piroplasm species than previously thought. Trans-stadial maintenance of the piroplasms was observed in each positive tick species; vertical transmission of B. canis canis was demonstrated in R. sanguineus. Finally, the detection of Babesia sp., B. microti-like species and B. rodhaini, phylogenetically related to zoonotic species, suggests that the human population could be at risk of infection in the studied area.

Wild ungulates as Babesia hosts in northern and central Italy.

Babesia and Theileria species were investigated in wild ungulates of Northern and Central Italy. Of 355 blood samples examined, 108 (30.4%) were positive to molecular diagnostics (polymerase chain reaction [PCR] with specific primers and sequencing). The sequence analysis showed that the roe deer is a susceptible host for several piroplasms belonging both to Babesia (31%) and Theileria (14.2%) species, whereas fallow deer and wild boar harbor only Theileria species (49% and 2.6%, respectively). Strains related to B. divergens are highly present (28.3%) in the roe deer, which, however, also harbors Babesia MO1 type and Babesia microti-like organisms. Babesia EU1 type is described for the first time in a roe deer in Italy. The finding in roe deer of Babesia species involved in human babesiosis is of concern for public health, mainly because ecological changes in progress cause the increase of both the deer species and the vector tick populations.

Larvae of Elaphostrongylus cervi in a population of free-living red deer in Italy.

Hunter-killed red deer (n = 68; Cervus elaphus) harvested from the Italian provinces of Bologna (Emilia Romagna) and Pistoia (Tuscany) (44 degrees 00'N 11 degrees 00'E) from October 2001 to January 2002 were examined for protostrongylid larvae. Twenty-eight animals (41%) had protostrongylid larvae in feces, lungs, and inguinal and iliac lymph nodes. Of these 28 animals, 20 were adults (71%), four were yearlings (14%), and four were calves (14%). Shape, length, width, and the location to the nematodes were consistent with Elaphostrongylus cervi, which has not been previously reported in Italy.

The inhibition of Candida albicans by selected essential oils and their major components.

Many volatile oils are known to possess antifungal properties and are potentially applicable as antimycotic agents. By studying the efficacy of essential oils against different pathogenic mycetes, we have evaluated the in-vitro inhibiting activity of some essential oils and their main constituents against a strain of Candida albicans. Sixteen commercial essential oils and forty-two pure constituents (alcohols, aldehydes, ketons, phenols and hydrocarbons), were tested by using a semisolid agar antifungal susceptibility (SAAS) method. Gas chromatography/mass spectroscopy analyses of the oils tested were performed. The essential oils of Origanum vulgare, Satureja montana, Mentha piperita, Cinnamomum verum, Cymbopogon flexuosus showed maximum inhibitory activity (MIC = 500 ppm) after 7 days. According to the results of the examination of pure constituents, beta-phellandrene proved to be the most interesting component among cyclic monoterpenic hydrocarbons as it showed a strong activity (MIC = 50 ppm). The most active of phenols was carvacrol (MIC 100 ppm). The open-chain alcohol 1-decanol was the most active of alcohols at 50 ppm. Finally, among aldehydes, a strong activity was shown by trans-cynnamaldehyde (MIC 50 ppm).