Publication fees of this manuscript have been sponsored by Bayer HealthCare – Animal Health division, in the framework of the 11th CVBD World Forum Symposium. Abbreviations CCelsius degreeBpbase pairsDNAdeoxyribonucleic acidEDTAethylenediamine tetraacetic acidIFATimmunofluorescence antibody testIgGImmunoglobulins G em k /em k agreementminminutes em VZ185 n /em numberOROdds RatioPBSPhosphate buffered salinePCRPolymerase Chain ReactionVBPsVector-Borne PathogensLmicroliters Footnotes Competing interests The authors declare that they have no competing interests. Authors contributions MGP and LSG conceived the research study. them [2C10]. In addition, the comparison of vector-borne pathogens from cats and from their ectoparasites (fleas and ticks) has not been fully explored . The aims of this investigation which was carried out in two regions (Calabria and Sicily) of southern Italy were: (i) to evaluate the flea and tick species collected from outdoor domestic cats and determine if they harbor VBPs; (ii) to evaluate exposure of outdoor cats to VBPs by means of antibody and molecular screening;?and (iii) to compare the VBPs DNA from feline blood and from your ectoparasites (fleas and ticks) collected from them. Methods The present study integrates data already published on 132 ticks collected from a large number of cats (and antigens by the immunofluorescence antibody test (IFAT) using commercial packages (Fuller Laboratories Fullerton, California, USA). The manufacturers protocol was followed for all those serological tests using a cut-off dilution of 1 1:64 for and and IgG antibodies was investigated using (strain MHOM/IT/80/IPT1) antigen slides manufactured by the . Morphometric identification of fleas and ticks was made through a stereomicroscope before DNA extraction for polymerase chain reaction (PCR) assays [13, 14]. Afterwards, fleas from each cat were extracted and processed VZ185 and only for cats transporting more than one single flea, pools were carried out. Specifically, a number spanning from two to five fleas collected from each cat was pooled for molecular investigations. Conversely, ticks were in any case extracted and processed individually. DNA extraction from 300?l of blood was performed using High Pure PCR Template preparation kit (Roche, VZ185 Mannheim, Germany). DNA was eluted in 100?L of elution buffer and stored at -20?C until used. DNA Smcb extraction from individual ticks, fleas and flea pools was carried out using High Pure PCR Template preparation kit (Roche, Mannheim, Germany) according to the manufacturers tissue protocol with some modifications. Briefly, all ectoparasites were washed twice in sterile PBS answer for 5? min shaking it slowly, then overnight at 4?C. Each flea was manually cut by a sterile lancet in four pieces and then suspended in 200?l of Tissue Lysis Buffer of the same kit. DNA was eluted in 50?l of elution buffer and stored at -20?C for later analysis. Real-time PCR technology was applied as explained elsewhere , to identify specific DNA target for spp., VZ185 piroplasmids (spp. and spp.), spp., spp., spp. VZ185 and from ticks and feline blood samples while only the last four pathogens were investigated on fleas due to economical restrictions. All positive PCR results for each ectoparasite or cat were sequenced according to the Big-Dye Terminator Cycle Sequencing Ready reaction Kit (AB, Life Technologies) using the same primers. Sequences obtained were compared with GenBank database (www.ncbi.nlm.nih.gov/BLAST). All positive PCR results for hemoplasmas or were not sequenced. Instead, species-specific real time PCRs were performed as explained by Martinez et al.  to discriminate among feline hemoplasmas species ((Mycoplasma haemominutum (Mycoplasma turicencis (real time PCR . For each pathogen investigated, Kappa agreement test (GraphPad InStat) was used to establish agreement between serological and molecular results in cats, between molecular results in cats, ticks or fleas and between serological results in cats and molecular results in ticks or fleas. The Kappa values were evaluated as follows: no agreement (antigens (Table?1). Table 1 Serological results of investigated pathogens in 42 cats infested by ectoparasites (((3 engorged), 17 19 (16 engorged), ten (four engorged), two engorged spp. were identified. The number of ectoparasites collected from individual cats ranged from one.