Molecular investigation on tick-borne pathogens of zoonotic concern in ticks from Emilia Romagna Region

The aim of this study was to assess the prevalence of tick-borne agents, such as piroplasms, Anaplasma phagocytophilum and Borrelia burgdorferi s.l, in ticks from parks of Emilia Romagna and from animals and humans from the same areas. The survey was carried out from 4 sites located in three parks of the provinces of Bologna and Ravenna: two sites were in the park “Gessi Bolognesi and Calanchi dell’Abbadessa” and one in the park “Monteveglio Abbey” (both in the province of Bologna) one site was located in the park “Carnè” (in the province of Ravenna), usually attended by people. In each site questing ticks were collected by flagging every 15 days from April to October 2010; feeding ticks were sampled from different animals (13 dogs, 13 horses, a cat and a cattle) and three humans by private vets and owners. Ticks were stored in alcohol 70% and identified by taxonomic keys. For molecular analyses, ticks were processed in pools of 10 larvae or 5 nymphs (from the same site and sampling time) or individually when adults. DNA was extracted by commercial kit (Nucleo Spin Tissue, Macherey NagelGmbH & Co. Germany), according to the manufacturer’s instructions, increasing the period of lysis of ticks from 10 to 30 minutes. A total of 393 samples obtained from 330 larvae, 1165 nymphs and 127 adults were tested for the presence of piroplasms by the amplification of 18S rRNA (Armstrong et al., 1998, Am J Trop Med Hyg, 58: 739–742). Only the samples of nymphs and adults (360) were analyzed for the presence of A. phagocytophilum and B. burgdorferi s.l. Taqman real-time PCR were performed for msp2/p44 gene of A. phagocytophilum (Drazenovich et al., 2006, Vector Borne Zoon Dis, 6: 83-90) and for 16S rRNA gene of B. burgdorferi (Barbour et al., 2009, Am J Trop Med Hyg, 81: 1120-1131) considering positive a Ct-value < 40. The PCR positive samples for piroplasms were purified with Nucleo-Spin Extract II (Macherey-Nagel) and sequenced by BMR Genomics (Padova). The PCR recognized piroplasms in 30 (7.6%) out of 393 samples. At sequence analysis, 17 samples (4.3%), 2 from feeding ticks and 15 from environment, were positive for Theileria buffeli/sergenti/orientalis group; 11 samples (3.4%) of questing ticks collected in all sites were positive for Babesia EU1 and 2 samples (0,6%) of questing ticks from Carnè park were positive for B. divergens/capreoli. All the positive questing ticks were Ixodes ricinus species, whereas the 2 positive feeding ticks were adults of Hyalomma marginatum marginatum, sampled from a dog and a horse. Thirty-three samples out of 360 (9.2%) were positive for A. phagocytophilum. Among these, all the 23 positive questing ticks were I. ricinus species collected from all sites but not in the park “Carnè”; the 10 positive feeding ticks included 6 I. ricinus from dogs (2), cat (1) and human (1), one Ixodes acuminatus, one Rhipicephalus turanicus, one R. bursa and one R. sanguineus collected from 3 dogs. Concerning B. burgdorferi s.l, 78 positive ticks (21.6%) out of 360 were obtained; only I. ricinus questing ticks from all the sampling sites resulted infected. Sixteen samples, from all sites, were co-infected with at least 2 pathogens.