Maria Paola Tampieri

Prevalence and diagnosis of Babesia and Theileria infections in horses in Italy: a preliminary study.

Babesia caballi and Theileria equi are the causative agents of equine piroplasmosis. In this preliminary epidemiological study, 412 horses reared in central and northern Italy were sampled and three diagnostic methods compared, namely, the microscopy, the indirect fluorescent antibody test (IFAT) and a PCR. Possible risk factors (such as area, season, breed, activity, sex, age, and grazing) associated with serological positivity were evaluated. A seroprevalence of 68.4% was found: 12.4% of the animals had anti-T. equi antibodies, 17.9% anti-B. caballi antibodies and 38.1% had antibodies against both species. Of the seropositive samples, 3.1% and 9.4% were positive to microscopy and PCR, respectively; 31.5% of the horses were IFAT-negative but 1.4% and 2.4% of the corresponding blood samples were positive to microscopy and PCR, respectively. Molecular techniques revealed that the species present were closely related to T. equi, Theileria sergenti, Theileria buffeli and the Babesia microti-like piroplasm provisionally named Theileria annae. Grazing was found to be a pronounced risk factor for equine piroplasmosis.

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.

Effect of Selected Essential Oils and Pure Compounds on Saprolegnia parasitica

Saprolegnia parasitica Coker is the main agent of saprolegniosis, one of the most important causes of economic losses in the fish farming industry, affecting all developmental stages. In this work the activity of some essential oils are compared with the activity of their isolated chemical constituents (alcohols, aldehydes, ketones, phenols, and hydrocarbons) to evaluate molecular classes active against S. parasitica. This suggests potential new products to use against saprolegniosis. Sixteen commercial essential oils and 37 pure constituents were tested against a strain of Saprolegnia parasitica from cutaneous lesions of Perca fluviatilis Linnaeus, using a continuously agitated broth technique that allows both the Minimum Inhibitory Concentration (MIC) and the Minimum Lethal Concentration (MLC) to be evaluated. It was possible to evaluate the efficacy of each essential oil against S. parasitica in relation to its specific composition and the efficacy of its main constituents. Carvacrol (MIC of 50 ppm and a MLC of 100 ppm) and p-cymene (MIC and MLC = 200 ppm) are the main constituents of Origanum vulgare L. and Thymus vulgaris L. (MIC = 100 ppm, MLC = 200 ppm) and are the primarly responsible for the efficacy of these two essential oils against S. parasitica. The structure of the constituents was also evaluated in relation to their activities against S. parasitica. Besides monoterpenic aromatic compounds, the acyclic mono- and sesquiterpenic primary alcohols and aldehydes seem to be the most active compounds. The open chain alcohol 1-decanol [CH3-(CH2)8-CH2-OH] proved to be the most active agent with a MLC = 50 ppm.

Prevalence of Anaplasma phagocytophilum in fallow deer (Dama dama) and feeding ticks from an Italy preserve

Up to date, information concerning the Anaplasma phagocytophilum infection in fallow deer is scant, therefore, to verify its prevalence in these ungulates serological and PCR screenings were performed on blood of 72 fallow deer hunted in a Central-Northern Italian preserve. Molecular analyses were also performed on 90 ticks removed from the animals. A. phagocytophilum infection in fallow deer was confirmed in 20 out 72 by IFA assay and in 11 out 72 by PCR. The sequence obtained revealed a complete genetic homology among the blood samples and strong degrees of homology with other European isolates. Considering the 90 ticks collected we found that 7.3% of Ixodes ricinus harboured A. phagocytophilum specific DNA. The data obtained confirmed that fallow deer can be a competent host for A. phagocytophilum and, therefore, that may represent a biological reservoir playing an important role in the epidemiological scenarios of the infection, in the geographical areas where is widespread.

A survey of 120 isolates of Malassezia (Pityrosporum) pachydermatis

The morphological, cultural and biochemical characteristics of 120 isolates of Malassezia (Pityrosporum) pachydermatis, isolated from chronic otitis externa in the dog, are discussed.

Mosquitoes as vectors of Setaria labiatopapillosa

An infected bovine-baited trap was utilised in summer 1994 to catch possible intermediate hosts of S. labiatopapillosa in northeastern Italy. Collections were made for 21 nights from 8.00 p.m. to 6.00 a.m. every 2 h and, after 12 September, every 30 min. Among the 16,159 mosquitoes sampled, 11,052 were freshly blood-fed. Most of the unfed females and a representative sample of those which had fed were identified as follows: Culex pipiens, Aedes caspius, A. vexans, Culiseta annulata, Anopheles maculipennis s.l., A. claviger and Coquillettidia richiardii. In spite of having the highest relative density, C. pipiens is the species which fed the least frequently and showed lower susceptibility and efficiency. The 2 Aedes species appear to act as vectors, particularly A. caspius, which proved to be the most efficient vector (K.I. = 0.8). A. claviger and A. maculipennis contribute to S. labiatopapillosa transmission, but their scarce presence reduces their epidemiological relevance. The other species identified showed a complete refractoriness to the infection. The risks for veterinary and medical health are discussed.

Epidemiological survey on Cryptosporidium in an Equine Perinatology Unit

The present study aims to evaluate the prevalence, pattern of spread and risk factors for the transmission of cryptosporidiosis in foals and mares hospitalized in a University Equine Perinatology Unit, where a new subtype family of Cryptosporidium horse genotype was described by Caffara et al. (2013). Mares (36) and foals (37) hospitalized during the 2012 foaling season were included. Multiple sampling from each animal was performed (a total of 305 stool samples were collected). One hundred and eleven environmental samples (gauze swabs) were also collected before and after the breeding season. Fourteen foals were found positive for Cryptosporidium spp. by PCR in at least one sample; a total of 35 foal stool specimens were confirmed for the presence of the protozoa. Instead none of the stool specimens from mares were found positive. PCR-RFLP analysis shows Cryptosporidium parvum in 5 stool samples and Cryptosporidium horse genotype in 21. In 9 specimens, from 4 different foals, the profile was suggestive for a mixed infection. The subtyping at gp60 locus showed 2 strains as members of the subtype family IId and six of the subfamily IIa of C. parvum. Twenty isolates were identified as Cryptosporidium horse genotype subtype VIaA15G4. Five gauze swabs collected from the walls of the boxes where the animals were hosted out of 111 environmental samples examined were PCR positive for Cryptosporidium spp. Cryptosporidium parvum was detected in one sample collected before the foaling season, while Cryptosporidium horse genotype profile was observed in 4 wall samples collected at the end of the 2012 foaling season. The prevalence observed in foals (37.8%) was higher than that reported in other studies. These features and the diffusion of the same genotype point out as the EPU, where critically ill foals are hospitalized, can support the spread of cryptosporidiosis. Therefore, the manual tasks and the activities carried out in these facilities are of great importance, as they might favor the diffusion of the infection.

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.

New insights into the epidemiology of bovine piroplasmoses in Italy

Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques. Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively. Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.