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Year 2010 - Volume 30, Number 5
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Detection of Mycoplasma mycoides cluster by indirect immunoperoxidase (IPI) and PCR-REA in the ear canal of bovines, 30(5):465-469
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ABSTRACT.- Santos S.B., Nascimento E.R., Faccini J.L.H., Barreto M.L., Almeida J.F., Pereira V.L.A. & Campos C.A.M. 2010. [Detection of Mycoplasma mycoides cluster by indirect immunoperoxidase (IPI) and PCR-REA in the ear canal of bovines.] Detecção do Grupo Mycoplasma mycoides por imunoperoxidase indireta (IPI) e PCR-REA em conduto auditivo de bovinos. Pesquisa Veterinária Brasileira 30(5):465-469. Departamento de Saúde Coletiva Veterinária e Saúde Pública, Universidade Federal Fluminense, Rua Vital Brazil Filho 64, Niterói, RJ 24230-340, Brazil. E-mail: elmiro@vm.uff.br
Mycoplasma mycoides cluster (MMC) was diagnosed by polimerase chain reaction-restriction endonuclease analysis (PCR-REA) and indirect immunoperoxidase (IPI), both, carried out in flushing from external ear canal, collected from bovine at slaughter time in the State of Rio de Janeiro, southeastern, Brazil. A total of 60 bovines were randomly selected. Sterile syringes (60mL) loaded with buffer solution (PBS, pH 7.2) were used for the ear canal flushing. The obtained samples were stored in glycerol (1:2) and frozen at -20oC until use. These specimens were diluted up to 10-5, inoculated in liquid and solid modified Hayflick´s media and incubated at 37oC for 2-3 days. The plates were kept in a microaerophilia condition and examined every two days under a stereomicroscope for the presence of typical colonies “fried-egg”. In this study, 35 strains selected in agreement with their biochemistry and physiologic proprieties, were used. From the 60 cultivated samples, 48 (80.00%) were positive for Mycoplasma spp. Under IPI the prevalence obtained for MMC was 20.0% (12/60) while by PCR-REA it was 41.7% (25/60). The IPI typing of these isolates resulted in 58.3% (7/12) for M.mycoides mycoides LC and 41.7% (5/12) for M. capricolum. PCR-REA for MMC was confirmed by the amplicon size of 785bp, compatible with this group. The Kappa value for the association between these two tests was 0.14 (p>0.05). After restriction analysis with AluI in all MMC strains the fragments size obtained were of 81, 98, 186 and 236bp, but not of 370bp that is compatible with Mycoides mycoides mycoides SC of bovine type. The presence of mycoplasmas species in the ear canal of asymptomatic bovines represent a risk of subsequent propagation of Mycoplasma spp. among bovine herds in Brazil. |
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