An intact male, 8 years-old Labrador (Canis lupus familiaris) was referred to the Pathology Department of Firat University for postmortem examination from a farm in Bingol Province where in the eastern Turkey. The dog born in coastal region of Bursa Province, western Turkey and one year ago transferred to the Bingol. According to personal information, the animal had been inappetant, coughing, with acute severe respiratory distress and hematuria. The dog died suddenly and was submitted for necropsy. Systemic necropsy was performed and all tissues were examined grossly and major organs including heart, lungs, liver, spleen, kidneys, brain, eyes and testis were fixed in 10% neutral formalin, embedded in paraffin, sectioned at 5 microm thickness and stained with hematoxylin and eosin and examined with a light microscope.
For each organ, 10 microm-thick sections from paraffin-embedded tissues were cut and placed in 1.5 ml eppendorf tubes. To avoid cross-contamination of samples, the microtome blade was cleaned with xylene between each block. Sections were deparaffinized with 1 ml xylene for 10 min at 37  C. Subsequently, samples were centrifuged at 4000 rpm for 5 min and the supernatant was removed. This procedure was repeated once. After deparaffinization, rehydration in 100%, 90%, 80% and 70% ethanol followed. Thereafter 70% ethanol was removed and tissue lysis solution was added (Fermentas DNA Purification Kit). For DNA extraction, we chose a commercially available DNA extraction kit based on a simple salting out procedure. We used the manufacturers' protocol for paraffin-embedded tissues with a modification of the proteinase K step, namely, an overnight digestion and addition of 25 microl aliquots of proteinase K (20 mg/ml). The incubation was performed with constant agitation at 56  C. After extraction, the DNA pellet was air-dried and resuspended in 50 microl sterile distilled water and stored -20  C. DNA was also extracted from the adult worm as mentioned above for use as a positive control.
The PCR was performed using two sets of primers. Initially, we used one primer pair: 5'-CATCAGGTGATGATGTGATGAT-3' (forward) and 5'-TTGATTGGATTTTAACG TATCATTT-3' (reverse), which have been described by Mar et al., for the amplification of a 302 bp ITS-2 gene segment. A second set of primers was used: 5'-AGTGTAGAGGGTCAGCCTGAGTTA-3' (forward) and 5'-ACAGGCACTGACAATA CCAAT-3' (reverse), identical to those described in a previous study for the amplification of a 203 bp cytochrome oxidase subunit 1 (CO1) gene fragment of D. immitis. Positive control DNA was used for each PCR and distilled water was used as a negative control. The amplified products were separated by gel electrophoresis in 2.5% agarose gel with a Tris-boric acid-EDTA (TBE, pH 8.3) buffer at 90 V for 30 min. Following electrophoresis, the amplified products were visualized with ethidium bromide staining for 30 min at room temperature.
Subsequently, 203 bp bands were cut from the gel and amplified DNA fragments were purified by QIAquick Gel Extraction Kit (Qiagen, USA). The CO1 sequences were automatically obtained using a 377 ABI PRISM system (Applied Biosystems). Nucleotide sequence analysis was undertaken by BLAST algorithms and databases from the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov).