Dogs and ticks
Fifty-eight ticks removed from 26 apparently healthy dogs and two sick dogs living in an outdoor shelter in an endemic area of canine leishmaniosis (Grosseto, Central Italy) were studied between March to May 2006 (Group 1). In addition, a total of 70 ticks were removed from 13 dogs with clinical signs and laboratory findings compatible with tick-borne diseases from April to November 2007 from veterinary clinics throughout Italy  (Group 2). After collection, the ticks were placed in a tube with 75% ethanol, and sent to laboratory San Marco where they were immediately stored at −20°C. Later, the ticks were dried at room temperature and morphologically identified . The mean number of ticks collected per dog and standard deviations were 3.1 ±3.3 and the range of ticks per dog was from 1 to 15. K3EDTA blood and sera were collected from all dogs studied from both groups and stored until use at −20°C. Different breeds and mixed-breed of dogs were studied in both groups.
The mean age ± SD of dogs in Group 1 was 5.05 ± 2.13 years. The information on age was available only for 17 dogs. Sixteen were females and 12 were males. All but one female dog were neutered. The mean age ± SD of dogs in Group 2 was 5.7 ± 4.7 years. The information about age was available only for 9 dogs. Six were females and three were males. Gender was not available for all dogs.
The ticks stored in 75% ethanol were dried, washed with PBS and left overnight in PBS at 4°C to eliminate ethanol. The DNA was isolated from individual ticks by using the High Pure PCR template preparation kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions with some modifications. The ticks were mechanically crushed with a sterile micropestle, suspended in 200 μL of tissue lysis buffer and 40 μL of proteinase K (100 μg/mL) and incubated overnight at 65°C. The final elution volume was 100 μL for each sample.
DNA extraction from canine whole blood was performed by using the High Pure PCR template preparation kit (Roche, Mannheim, Germany). 200 μL of whole blood was mixed with 200 μL of binding buffer with proteinase K (100 μg/mL) and incubated for 1 h at 72°C. The DNA extraction was then carried out according to the manufacturer’s instructions. The final elution volume was 50 μL for each sample.
Cultured L. infantum (MHOM/FR/78/LEM75) zymodeme MON-1, Leishmania tropica (MCAN/MA/90/LEM2007) zymodeme MON-102 and Leishmania braziliensis (MCAN/BR/81/RICO) zymodeme MON-43 reference strains were studied. In vitro-cultivated L. infantum promastigotes in the logarithmic phase of growth were washed three times by centrifugation for 5 min at 1500 g in sterile PBS and counted in a hemocytometer by using a light microscope. Genomic DNA was extracted from 200 μL of 73X106 promastigotes/mL as previously described .
Conventional PCR for tick mitochondrial 16 S rRNA gene
The efficiency of tick DNA extraction was evaluated by amplification of the tick mitochondrial 16 S rRNA gene (ribosomal DNA [rDNA]) using tick-specific primers in a conventional PCR: TQ16S-1 F 5′CTGCTCAATGATTTTTTAAATTGCTGTGG 3′ and TQ16S-2R 5′ ACGCTGTTATCCCTAGAG 3′, as previously described .
Each reaction was carried out in 50 μL volume containing 0.5 μmol/μL of each oligonucleotide primer, 2.5 mM of each dNTP (5PRIME GmbH, Hamburg, Germany), 5 μL of 10× PCR buffer, 1 U of Taq DNA polymerase (5PRIME GmbH, Hamburg, Germany) and 5 μL of the DNA.
PCR was performed in a thermocycler (Applied Biosystem, Europe) with 1 cycle of denaturation (8 min, 94°C), followed by 10 cycles of denaturation (1 min, 92°C) annealing (1 min, 48°C) and extension (1 min 30 s, 72°C) then 32 cycles of denaturation (1 min 92°C), annealing (1 min, 54°C), extension (1 min 30 s, 72°C) and a final extension step (10 min, 72°C) as previously described . DNA electrophoresis was carried out in 2.2% FlashGel System (Lonza, Rockland, ME USA), and DNA fragments were visualized.
Leishmania infantum real-time PCR and sequencing
Real-time PCR for L. infantum DNA detection was performed in all the tick and canine samples.
We used primer and probe sequences specific for the L. infantum kinetoplast mini-circle (TIBMOLBiol, Genova, Italy) as described . Real-time PCR was performed by using LightCycler FastStart DNA MasterPLUS Hybridization Probes (Roche, Mannheim, Germany) using LightCycler version 3.5.17 instrument (Roche, Mannheim, Germany). Positive and negative controls were used in all runs . Leishmania tropica and L. braziliensis DNA were not detected by qPCR hybridization probes while L. infantum DNA was detected.
One positive tick sample was sequenced. The kDNA real-time PCR product was sequenced at TIB MOL Biol (Genova, Italy). Sequences were compared with sequences deposited in GenBank using BLAST hosted by the National Center for Biotechnology Information, National Institutes of Health, USA (http://www.ncbi.nlm.nih.gov).
Canine serum anti-leishmanial antibodies were determined by ELISA, using sonicated crude L. infantum antigen as previously described . Briefly, dog sera were diluted to 1:400 and incubated in L. infantum antigen-coated plates (20 μg/mL) for 1 hour at 37°C. The plates were then washed with 0.05% Tween 20 in phosphate-buffered saline (PBS) and incubated with Protein A conjugated to horseradish peroxidase (1:10,000 dilution; Sigma) for 1 hour at 37°C. Plates were washed again with 0.05% PBS-Tween 20. The plates were developed by adding the substrate solution ortho-phenylene-diamine (SIGMA FAST OPD, Sigma). The reaction was stopped with 50 μl of 3MH2SO4. Absorbance values were read at 492 nm in an automatic microELISA reader (PROGRAMMABLE MPT READER DV 990BV4, N.T. Laboratory, Italy). All determinations included the serum from a sick dog with a confirmed infection as positive control (calibrator) and serum from a healthy dog as a negative control. The result was quantified as units (U) related to a positive canine serum used as a calibrator and arbitrarily set at 100 U. The cutoff was established at 14 U (mean + 4 SD of values from 32 dogs from non-endemic areas) and the results above this value were considered positive.
Differences among groups were analyzed by means of Chi-square with Yates correction if needed and Fisher’s exact test. A p < 0.05 was considered significant.