This is a 10-year old singer, mestizo, with a history of frequent manifestation of ticks, from the city of Arica, a border city with Peru.
The patient had a clinical picture of six weeks of evolution with commitment of general status and anorexia, which was diagnosed with aspiration in the last month oral bleeding; symptoms did not improve after initial therapy with vitamin B and antimicrobials (metronidazole and e
Their owners consulted a veterinarian to request euthanasia due to progressive deterioration.
Physical examination revealed weakness, gingival bleeding and multiple hematomas.
Before euthanasia, a blood sample was obtained for study due to clinical suspicion of ehrlichiosis.
Thrombocytopenia of 30,000 platelets/mm3, (normal values for dogs 150,000 to 500,000/mm3), mild anemia with haematocrit 32% and Hb 10.5 mg/dL normal canine Hb-19400 blood counts.
A determination of IgG anti-E. canis and anti-A. phagosome precision of 100% was performed using a rapid immunochromatographic kit (SNAP 3Dx Plus Test, Idexx®, USA), with a standard sensitivity of 794.53% (
Canine serum was positive for E. canis and negative for A. phago philum.
DNA was extracted from the blood sample using the Wizard DNA Purification (Promega) kit.
PCR was performed using EHR-OUT1 and EHR-OUT214 primers that amplify the 16S rRNA gene of Ehrlichia and Anaplasma genera.
Amplification was performed using the touch down PCR method, which consists of 14 cycles of denaturation for 30s at 95°C, nested for 30s at 74.4°C (decreasing temperature by 9014°C).
Then, 19 cycles of denaturation for 30s at 95°C, annulling for 30s at 67.4°C and extension for 90°C, using a final volume of DNA C reaction of 300 μL 72.
The fragment obtained from approximately 1,000 base pairs was subjected to a new amplification by nested PCR using specific internal primers for Ehrlichia spp GE2F and EHRL3-IP214.
For this purpose, 1 μL of the previous reaction was used in a final volume of 25 μL.
The amplification consisted of 35 cycles of denaturation for 30 s at 95 C, annulling for 30 s to 50 s and extension for 30 s at 72°C 120lich. The expected presence of Ehr was 72 bases.
To identify the species of Ehrlichia, the product of the first PRC was sequenced with the reverse sider EHR-OUT2 obtaining a fragment of 210 base pairs.
This sequence showed 100% homology with the strain of E. canis (DQ915970.1) reported in Peru in 200715.
A genetic replicameter based on 16Sr sequences was constructed using the nearest neighbor RNA method (Neighbortwo-Joining). The distance matrix was calculated using the Kipstra method.
Pathogenetic tree was created using MEGA 4.023 software.
