Cristina Bonoli

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.

Detection of Babesia EU1 in Ixodes ricinus ticks in northern Italy.

Babesia EU1, a potentially important emerging zoonotic pathogen, already detected in ticks and wild ruminants of different European Countries, was found in three pools of Ixodes ricinus nymphs in three different sites located in a single District of north-eastern Italy. Totally 356 ticks (60 pools) were collected from the environment during a surveillance activity in the year 2006. Babesia EU1 estimated individual tick prevalence in the area is 0.85%. The finding that also in northern Italy the tick population is carrying Babesia EU1 suggests a wide geographical spreading of this zoonotic pathogen in Europe.

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.

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.

Cortisol levels in cats' hair in presence or absence of Microsporum canis infection.

The purpose of this work was to perform a preliminary screening in the domestic cat to assess the concentration of cortisol in hairs by radioimmunoassay technique (RIA) in presence or absence of Microsporum canis infections. A total of 245 cats (7 with cutaneous lesions referable to dermatophytosis and 238 apparently healthy) coming from 14 shelters were examined. M. canis was isolated in 126 (51.4%) cats. The cortisol levels were significantly higher in cats with lesions or without lesions but with a high number of colonies in the plates (≥ 10 CFU) than in cats negative or with a lower number of colonies. The results obtained seem to highlight that chronic high levels of cortisol in cats could possibly promote the dermatophytes infections. Furthermore, in High-CFU asymptomatic cats, it could be present a state of infectious, and they, therefore, represents not a simple mechanical carrier.

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.

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle.

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.

Grains colonised by moulds: fungal identification and headspace analysis of produced volatile metabolites

Abstract The aim of this work was to verify if the headspace analysis of fungal volatile compounds produced by some species of Fusarium can be used as a marker of mould presence on maize. Eight samples of maize (four yellow maize from North Italy and four white maize from Hungary), naturally contaminated by Fusarium and positive for the presence of fumonisins, were analyzed to detect moisture content, Aw, volatile metabolites and an enumeration of viable moulds was performed by means of a colony count technique. Headspace samples were analysed using a gas-chromatograph equipped with a capillary column TR-WAX to detect volatile metabolites of moulds. Furthermore mac ro- and microscopic examination of the colonies was performed in order to distinguish, according to their morphology, the genera of the prevalent present moulds. Prevalent mould of eight samples was Fusarium, but other fungi, like Aspergillus, Penicillum and Mucora were observed. The metabo-ceae, lites produced by F. graminearum and F. moniliforme were Isobutyl-acetate, 3-Methyl-1-butanol and, only at 8 days, 3-Octanone. The incubation time can affect off flavour production in consequence of the presence of other moulds. Further studies on maize samples under different conditions are needed in order to establish the presence of moulds using the count technique and through the identification of volatile compounds.