M.L. García-Leal; P.E. Brandão; M.K. Hagiwara; S.R.R. Lucas
School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
Mycoplasma haemofelis is an epicellular gram-negative hemoparasite that can induce anemia in domestic and wild felids and is one of the feline haemoplasmas species. The inability to culture this pathogen and the low sensitive of the cytological diagnosis, led to choose of the conventional PCR assay for the diagnosis of infected animals. The aim of this report is to describe a case where M. haemofelis was detected by a direct PCR assay, from an anticoagulated blood sample collected of one domestic cat with feline leukemia virus infection and splenic lymphoma The base of this assay was the amplification of 16S ribosomal RNA (rRNA) gene, using universal primers that amplify 1316 base pars (bp) for PCR and 675 bp for Nested-PCR. Initially, the DNA was extracted from the refrigerated blood sample, stored for 20 days, with an available commercial kit. The PCR amplification product was identified by ethidium bromide fluorescence after electrophoresis in a 1.5% agarose gel, then purified and sequenced. The homology of the sequence search was carried out with the databases of GenBank and was identified as M. haemofelis (FJ544859). After identification, with the same blood sample stored at 4°C for two months, a new extraction was performed, with the phenol chloroform method. The extracted DNA was stored at -20°C. Five months after the initial collect, the whole blood was used without DNA extraction, to compare PCR assay between the three samples: two different extraction methods and direct PCR (without DNA extraction).
The nucleotides sequence of M. haemofelis (EU442639) was used as a positive control in all the reactions. Ultrapure water with all PCR reagents was used as a negative control. Although the direct sample had suffered protein coagulation even in the first PCR, positive band of 675 bp was obtained in the Nested-PCR reaction. The obtained band was reliable for the diagnostic. These result indicated that the direct Nested-PCR was suitable for the diagnostic in this case, and the storage time of the sample did not interfere with M. haemofelis detection.