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ABSTRACT. We have examined the presence of hemoplasmas, hemotropic mycoplasmas, among domestic cats (Felis catus) in Japan by using a species specific PCR, and found 'Candidatus Mycoplasma turicensis', a recently recognized hemoplasma species. A total of 60 feline blood samples collected in 2004 and 2005 were subjected to PCR amplification for the detection of Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'. Six blood samples collected from domestic cats were found infected with the 'Candidatus M. turicensis'. All of them are also infected with other species of hemoplasmas, M. haemofelis and/or 'Candidatus M. haemominutum'. This is the first to demonstrate 'Candidatus M. turicensis' infections among cat population in Japan. KEY WORDS: feline, hemoplasma, 16S rRNA.

A method for detecting wild-type canine parvovirus (CPV) strains which contaminate vaccines for dogs has been developed by PCR. PCR primers which distinguish vaccine strains from the most common, recent strains of wild-type CPV in many countries, including Japan and the United States, were developed. This PCR is based on the differences in nucleotide sequences which determine the two antigenic types of this virus. CPV vaccine strains derived from antigenically old-type virus prevalent in former times were not detected by PCR with differential primers. Detection sensitivity of PCR was 100-to 10,000-fold higher than that of the culture method in Crandell feline kidney cells.

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Mycoplasma is a common contaminant in cell cultures. It is not easy to find mycoplasma contamination in cell cultures because most cases of mycoplasma contamination are not apparent from microscopic observation. Although various procedures are now available to detect mycoplasmas in cell cultures (4, 6), serological and culture methods are still indispensable for the identification of Mycoplasma species. Currently five Mycoplasma species, M. fermentans, M. hyorhinis, M. orale, M. arginini, and M. salivarium, are known to account for about 95% of the mycoplasma contamination in cell cultures (3). In the present paper, we show a rapid method to detect and identify these Mycoplasma species in cell cultures.This method depends on real-time polymerase chain reaction (PCR) with SYBR Green I and melting curve analysis. SYBR Green I is a non-specific fluorescence dye with a high affinity for double-stranded DNA (7). Universal primers for Mycoplasma species are the same as described previously (1), namely F1 (5'-ACAC-CATGGGAGYTGGTAAT-3') and R1 (5'-CTTCWTC-GACTTYCAGACCCAAGGCAT-3'), and they amplify the entire spacer region between the 16S and 23S rRNA genes of mycoplasmas. Real-time PCR was performed in a SmartCycler instrument (Cepheid, Sunnyvale, Calif., U.S.A.) with TaKaRa Ex Taq R-PCR version (Code #RR007A, TaKaRa Bio, Japan). The reaction mixture contained 1 µl of each primer (10 pmol/µl), 2.5 µl of 10ϫ reaction buffer, 20 nmol of each deoxynucleotide, 0.25 µl of 250 mM MgCl 2 , 0.2 µl (5 U/µl) TaKaRa Ex Taq HS DNA polymerase, 2.5 µl of 1:3,000 SYBR Green I (Catalogue #50513, BMA Corp., Rockland, Me., U.S.A.), and water to a volume of 23 µl. Finally, 2 µl of samples (cell culture fluid) as templates was added to this mixture. DNA extraction is usually not necessary in real-time PCR tests for detecting mycoplasmas in fresh cell cultures. Cell cultures grown for 3-5 days after passage are suitable for direct testing. Cell debris from frozen cell cultures may inhibit realtime PCR. Amplification was achieved with 40 cycles of denaturation at 94 C for 10 sec, renaturation at 55 C Abstract: A quantitative real-time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10 4 to 10 colony-forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures. Rapid Detection and Differentiation of the Major Mycoplasma Contaminants in Cell Cultures

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