Genomic DNA was extracted from the EDTA-blood samples, conjunctival swabs and sand flies using the DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions.
Blood samples, conjunctival swabs and sand fly DNA samples were tested for L. infantum DNA using a TaqMan real-time quantitative PCR (qPCR) assay that targeted a 120-bp fragment of the kinetoplast minicircle DNA, as previously reported [13 (link)]. A positive (reference DNA sample from blood, conjunctival swab and sand fly DNA, respectively) and a negative (PCR grade water) control were included in each qPCR run.
The qPCR was performed only on samples collected on SDs 0 and 630 and, in addition, on DNA blood samples at all time points from: (i) all dogs that tested positive at the beginning of the study and became negative during the study; (ii) all dogs that tested positive by ELISA only; and (iii) all dogs that tested only as low positive on qPCRs performed on swab DNA samples on SD 630.
For E. canis, Anaplasma platys and A. phagocytophilum, specific PCR tests were performed on all DNA from blood samples on SD 0 and SD 630 collected from dogs with at least 1 year follow-up. To determine the presence of E. canis, a PCR targeting a 345-bp fragment of the 16S ribosomal RNA (rRNA) gene [14 (link)] of various species, including E. canis, Ehrlichia chaffeensis, Ehrlichia muris, Ehrlichia ruminantium, A. phagocytophilum, A. platys, Anaplasma marginale, Anaplasma centrale, Wolbachia pipentis, Neorickettsia sennetsu, Neorickettsia risticii and Neorickettsia helminthoeca, was performed according to previously described thermal-cycling conditions [15 (link)].
To identify the presence of Anaplasma DNA, we performed species-specific nested PCRs for A. platys and A. phagocytophilum DNA targeting a 678-bp and a 546-bp fragment of the 16S rRNA gene, respectively, according to previously described protocols [16 (link)].
A positive (E. canis and A. platys or A. phagocytophilum reference DNA sample) and a negative (PCR grade water) control were included in each PCR run. Amplification products were visualised on 1.5% agarose gels stained with ethidium bromide.
PCR products (from samples positive for E. canis and A. platys/phagocytophilum) were sent to a commercial service (CeMIA SA, Larissa, Greece) for purification and sequencing on both strands (Sanger sequencing). The results were assembled with Seqman 8.1 software (DNASTAR, Madison, WI, USA). Assembled sequences were aligned using the Basic Local Alignment Tool (BLAST) and compared with reference sequences using the MegAlign application of the Lasergene software package (DNASTAR).
Blood samples, conjunctival swabs and sand fly DNA samples were tested for L. infantum DNA using a TaqMan real-time quantitative PCR (qPCR) assay that targeted a 120-bp fragment of the kinetoplast minicircle DNA, as previously reported [13 (link)]. A positive (reference DNA sample from blood, conjunctival swab and sand fly DNA, respectively) and a negative (PCR grade water) control were included in each qPCR run.
The qPCR was performed only on samples collected on SDs 0 and 630 and, in addition, on DNA blood samples at all time points from: (i) all dogs that tested positive at the beginning of the study and became negative during the study; (ii) all dogs that tested positive by ELISA only; and (iii) all dogs that tested only as low positive on qPCRs performed on swab DNA samples on SD 630.
For E. canis, Anaplasma platys and A. phagocytophilum, specific PCR tests were performed on all DNA from blood samples on SD 0 and SD 630 collected from dogs with at least 1 year follow-up. To determine the presence of E. canis, a PCR targeting a 345-bp fragment of the 16S ribosomal RNA (rRNA) gene [14 (link)] of various species, including E. canis, Ehrlichia chaffeensis, Ehrlichia muris, Ehrlichia ruminantium, A. phagocytophilum, A. platys, Anaplasma marginale, Anaplasma centrale, Wolbachia pipentis, Neorickettsia sennetsu, Neorickettsia risticii and Neorickettsia helminthoeca, was performed according to previously described thermal-cycling conditions [15 (link)].
To identify the presence of Anaplasma DNA, we performed species-specific nested PCRs for A. platys and A. phagocytophilum DNA targeting a 678-bp and a 546-bp fragment of the 16S rRNA gene, respectively, according to previously described protocols [16 (link)].
A positive (E. canis and A. platys or A. phagocytophilum reference DNA sample) and a negative (PCR grade water) control were included in each PCR run. Amplification products were visualised on 1.5% agarose gels stained with ethidium bromide.
PCR products (from samples positive for E. canis and A. platys/phagocytophilum) were sent to a commercial service (CeMIA SA, Larissa, Greece) for purification and sequencing on both strands (Sanger sequencing). The results were assembled with Seqman 8.1 software (DNASTAR, Madison, WI, USA). Assembled sequences were aligned using the Basic Local Alignment Tool (BLAST) and compared with reference sequences using the MegAlign application of the Lasergene software package (DNASTAR).
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