An 8-month-old female cat was presented with a history of muscle weakness, anorexia and lethargy. At physical examination the cat was anaemic, thin and depressed. According to the owner, the onset of the disease coincided with previous flea infestation. The cat was pyrexic (temperature of 39,6ºC, reference range 38–39ºC). Routine hematology revealed macrocytic-normochromic anaemia (Packed Cell Volume 20,4%, Haemoglobin 6,4 g/dl, red blood cell 3.08 x 10
6/uL, Mean corpuscular volume 66,2 fl, mean corpuscular haemoglobin concentration 31,3 g/dL). Cytological examination of the Romanowsky stained smear on referral demonstrated microscopical evidence of hemoplasmosis.
PCR assay was performed for the present case on referral day. DNA extraction was performed from whole blood by use of a DNA extraction protocol (EZ DNA isolation kit, Dr. Zeydanli, Ankara, Turkey) with regard to the manufacturer's instructions.
PCR assay was performed as previously described 7-9, by use of primers targetting the rRNA gene, producing a 170 base pair (bp) product from M. haemofelis and a 193 bp amplicon from “Candidatus M. haemominutum” with 5'- ACG AAA GTC TGA TGG AGC AAT A–3' forward primer and 5'- ACG CCC AAT AAA TCC GRA TAA T–3' reverse primer, Dr. Zeydanli, Ankara, Turkey).
The PCR amplification was carried out in 25 µl reaction mixtures containing 31 pmol of each primer, 200 µM (Amresco) deoxynucleotide triphosphates (dNTPs), 0.025 units Taq DNA polymerase (Gene Mark), 3,5 mM MgCl2, 1x PZR Buffer (Gene Mark) and 2 µl sample DNA.
For PCR assay optimization PCR machine (Biometra T-gradient) was used, within the following reaction conditions; 30 seconds at 94 ºC, followed by 45 cycles of 30 seconds at 57 ºC, and 45 seconds at 72ºC. Prior to the first cycle, initial denaturation for 5 minutes at 94 ºC and after the last cycle 10 minutes extension at 72ºC processes were applied. M. haemofelis and “Candidatus M. haemominutum” DNAs were used as positive controls. A reagent negative control (sterile pure water) was included in each PCR run for monitorizing contamination. Reaction products including 0,5 µg/ml were electrophoresed within 2.5% agarose gel containing ethidium bromide and visualised by UVP gel documentation system. DNA identification for M. haemofelis and “Candidatus M. haemominutum” included comparison of the size of of the PCR product within the size of known positive control DNA and with a 100 bp DNA ladder.
Based on cytology, haematological analysis and PCR assay a diagnosis of “Candidatus M. haemominutum” infection was made.
Commercial immunocromatographic rapid test kit (Speed FeLV test kit and Speed FIV test kit, Bioveto, Maya Vet. Ltd. Co., Ankara) was used in an attempt to diagnose FeLV and FIV infections. No sample was negative for FeLV P27 antigen or FIV antibody.
Therapy included enrofloxacine at a subcutaneously dosage of 5mg/kg once a day for 10 days. Fipronil was administered topically at a dosage of 7,5 mg/kg for flea control. At the third day of admission the clinical signs considered to account for haemoplasma worsened and the cat was found dead by the owner. Necropsy was discussed with the owner, who declined further research.
