A total of 2681 Ixodidae ticks grouped into 1578 homogeneous pools were included (Table 1). The analyzed samples originated from 1454 privately-owned dogs from 78 Italian NUTS3 provinces (hereinafter NUTS3, Nomenclature of Territorial Units for Statistics, level 3), (mean = 18.64 dogs/province, standard deviation = 24.75) and 1389 municipalities (LAU2, Local Administrative Units, level 2).
DNA of protozoa belonging to the genera Babesia and Theileria was detected in 435 pools (MIR = 27.57%; 95% CI = 25.42–29.82) from 395 dogs.
A significantly higher prevalence was found in I. ricinus (χ2 = 5.5, p<0.05) and in ticks of the R. sanguineus group (χ2 = 4.1, p<0.05) compared to other tick species as well as in adult ticks (χ2 = 9.99, p<0.05) and engorged females (χ2 = 15.82, p<0.05). Dogs living in urban environments were at a lower risk of carrying a Babesia/Theileria-infected tick (χ2 = 109.04, p<0.05; odds ratio (OR) = 0.31; 95% CI = 0.24–0.39%) compared to dogs living in rural and forest habitats; housing (indoor, garden, kennel) did not influence the risk of being parasitized by an infected tick (p>0.05). Geographical distribution at the NUTS3 level of Babesia/Theileria-infected ticks is reported in Figure 1. Piroplasms were detected in 53 provinces (53/78 = 67.95%, 95% CI = 56.96–77.25%) (Figure 1a) with significant differences among the provinces (p<0.05). Considering NUTS3 provinces where at least 20 dogs were sampled, piroplasms were detected with MIR values ranging from 0% (95% CI = 0.00–17.59%) to 61.90% (95% CI = 40.88–79.25%) (Supplementary material S1, Figure 1b). Regular antiparasitic treatment significantly reduced the risk of being parasitized by Babesia/Theileria-positive ticks (χ2 = 144.97, p<0.05; OR = 0.24; 95% CI = 0.19–0.31%). Although dogs treated with collars (χ2 = 53.60, p<0.05; OR = 6.99; 95% CI = 3.89–12.55%) and spot-on products (χ2 = 119.29, p<0.05; OR = 7.75; 95% CI = 5.18–11.59%) were more likely to be parasitized than those treated with oral formulations. Sequencing determined the presence of at least 9 species of the genus Babesia and 5 species belonging to the genus Theileria, as reported in Table 2. For 37 PCR-positive samples, sequencing was not possible due to low-quality DNA. The zoonotic B. venatorum was the most prevalent species (MIR = 7.54%; 95% CI = 6.34–8.95%), followed by unspecified Babesia spp. (MIR = 4.37%; 95% CI = 3.47–5.50%) and B. capreoli (MIR = 3.55%; 95% CI = 2.74–4.58%). Other zoonotic isolates belonged to the B. microti group, which were reported with MIR = 2.41% (95% CI = 2.41%; 1.76–3.29%). For 4 tick-pools, it was possible to specifically determine the presence of B. microti “Munich-type” (MIR = 0.25%; 95% CI = 0.1–0.65%). Piroplasms with the domestic dog as their primary reservoir host were reported with a lower prevalence (B. canis MIR = 0.38%, 95% CI = 0.17–0.83%; B. vogeli MIR = 0.63%, 95% CI = 0.34–1.16%). The geographical distribution of zoonotic and dog-related piroplasms is reported in Figure 2.
Genomic DNA of Gram-negative bacteria of the genera Anaplasma and Ehrlichia was detected in 165 tick- pools (MIR = 10.46%; 95% CI = 9.26–11.79%) from 160 dogs.
A significantly higher prevalence was found in I. ricinus (χ2 = 93.53, p<0.05; OR = 5.33; 95% CI = 3.70–7.67%), while ticks of the genus Rhipicephalus were significantly less infectedχ2 = 94.43, p<0.05; OR = 0.19; 95% CI = 0.13–0.27%). Engorged I. ricinus females were significantly more infected than other developmental stages (χ2 = 15.16, p<0.05; OR = 2.39; 95% CI = 1.48–3.53%). A higher infection prevalence was found in tick-pools of dogs from forest environments compared to dogs living in only urban or rural environments (χ2 = 4.63, p<0.05; OR = 5.27; 95% CI = 3.66–7.59). Housing and use of antiparasitic treatment had no effect on the risk of being parasitized by infected ticks (p>0.05). Geographical distribution at NUTS3 level of Anaplasma/Ehrlichia-infected ticks is reported in Figure 1. Anaplasma/Ehrlichia DNA was detected in 46 of the 78 provinces sampled (P = 58.97, 95% CI = 47.89–69.22%) (Figure 1c) with significant differences between the NUTS3 provinces (p<0.05). Considering NUTS3 where at least 20 dogs were sampled, Anaplasma/Ehrlichia DNA was detected with MIR values ranging from 0% (95% CI = 0.00–15.46%) to 22.73% (95% CI = 10.12–43.44%) (Supplementary material S2, Figure 1d). The zoonotic A. phagocytophilum was identified by sequencing in 80 tick-pools (MIR = 5.07%, 95% CI = 4.09–6.27%) from 35 provinces, while A. platys and E. canis, which causecyclic canine thrombocytopenia and canine monocytic ehrlichiosis, were detected in 13 (MIR = 0.82%; 95% CI = 0.48–1.4%) and 21 (MIR = 1.33%; 95% CI = 0.87–2.03%) pools respectively. A. ovis was detected in 3 tick-pools from Catania province (Sicily, Southern Italy) (MIR = 0.19%, 95% CI = 0.06–0.56%). Uncultured Anaplasma spp. was amplified from 36 pools (MIR = 2.28%, 95% CI = 1.65–3.14%) and uncultured Ehrlichia spp. from 12 pools (MIR = 0.76%, 95% CI = 0.43–1.32%), including 1 isolate from northeastern Italy of Candidatus E. walkerii [GenBank: AY098730], previously identified in I. ricinus ticks attached to asymptomatic human patients from the same part of Italy . Table 2 reports the overall sequencing results for Anaplasma/Ehrlichia related to tick species. Figure 3 shows the geographical distribution of zoonotic and canine-related Anaplasmataceae (A. platys and E. canis)..
B. burgdorferi s.l.
B. burgdorferi s.l. DNA was detected in 10 tick pools (MIR = 0.63%, 95% CI = 0.34–1.16%) from 10 different dogs. All infected pools were comprised of adult individuals (n = 8 non-engorged adults and n = 2 engorged females). Infected pools belonged to the genus Ixodes (I. ricinus n = 4, I. hexagonous n = 1) and to the R. sanguineus group, with no statistically significant differences among genera or species due to the small number of positive samples. All dogs with B. burgdorferi s.l. positive ticks were housed indoors with access to a garden. Seven dogs regularly attended rural and forest environments, while 3 lived exclusively in an urban setting. Antiparasitic treatment was reported for 6 dogs, but active in only 2 dogs. Sequencing identified n = 6 B. burgdorferi s.l. and n = 4 B. afzelii (Table 2). Geographical distribution at NUTS3 level of B. burgdorferi s.l. is reported in Figure 1 (cf also Supplementary material S3). B. burgdorferi s.l. was detected in 11.54% of the sampled NUTS3 provinces (95% CI = 6.19–20.50%).