Studies on the mycoplasmal pneumonia in slaughter pigs. 2. Isolation of mycoplasmas from lung tissues and identification of isolates by nested-PCR technique

도축돈의 마이코플라즈마성 폐렴에 관한 연구 2. 폐조직에서의 균분리와 nested-PCR방법에 의한 동정

  • Lim, Young-Taek (Department of Animal Science, College of life Sciences, Dankook University) ;
  • Seok, Ho-Bong (Department of Animal Science, College of life Sciences, Dankook University)
  • 임영택 (단국대학교 생명자원과학부 동물자원 전공) ;
  • 석호봉 (단국대학교 생명자원과학부 동물자원 전공)
  • Accepted : 2002.04.24
  • Published : 2002.06.29

Abstract

We report that mycoplasma organisms from lung tissues of slaughter pigs were identified to genes fragments with references use of nested-PCR technique(nPCR). Seven strains of mycoplasma species were isolated from 70 lung tissues. The organisms were detected by in vitro amplification of 16S rRNA and 23S rRNA genes. Nucleotide sequences of the spacer between 16S and 23S in the ribosomal RNA operons of mycoplasma were identified by the analysis of products from the nested PCR. Four common PCR primers, MhF1, MhF2 MhR1 and MhR2, were designed by analysis between these sequences by first amplified with F1, R1 and second with F2, R2, respectively. Specific amplification of the spacer region for reference strains of M. hyopneumoniae, M. hyorhinis, M. flocculare were confirmed by first round of PCR in which the traduced fragments of 690bp, 460bp, 630bp. But amplications of second round was changed to 240bp, 210bp, 230bp, respectively. Three different strains (M. hyopneumoniae:4, M. hyorhinis:2, M. flocculare:1) were detected by the nested-PCR technique. The results suggest that the detection of swine mycoplasma by n-PCR can be analyzed the nucleotide sequences between rRNA operons and homology study.

References

  1. Ross RF. Mycoplasmal disease, In: Leman AD, Straw BE, Mengeling WL et al. Diseses of Swine, 7lh ed. Ames, Iowa: Iowa State University Press, 537-551, 1992
  2. Kobisch M, Friis NF. Swine mycoplasmoses. Rev. Sci. Tech. Off. Int. Epiz. 15(4): 1569-1605, 1996
  3. Weisburg WG, Tully JG, Rose DL et al. A phylo-genetic analysis of the Mycoplasmas : Basis for their classification. J. Bacteriol. 171:6455-6467, 1989
  4. Livingston CW Jr, Stair EL, Undeidahl, NR et al. Pathogenesis of mycoplasmal pneumoniae in swine. Am. J. Vet. Res. 33:2249-2258, 1972
  5. Amanfu W, Weng CN, Ross RF. et al. Diagnosis of mycoplasmal pneumoniae of swine : Sequential study by direct imunoflurence. Am. J. Vet. Res. 45:1349-1352, 1984
  6. Amstrong CH, Freeman MJ, Sands-Freeman L et al. Comparison of enzyme-linked immunosorbent assay and the indirect hemmagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae. Can. J. Comp. Med. 47:464-470, 1983
  7. Ross RF, Whittlestone P. Recovery, identification and serological response to porcine mycoplasmas. In : J. G. Tully, S. Razin, eds. Methods in mycoplasmology, Vol. 2. New York Academic Press, 1983
  8. Piffer IA. Ross RF. Effect of age on susceptibility of pigs to Mycoplasma hyopneumoniae. Am. J. Vet. Res. 45:478-481 1984
  9. Nicolet J, Paroz P, Bruggmann S et al. Tween 20 soluble proteins of Mycoplasma hyopneumoniae as antigen for an enzyme-linked immunosorbent assay. Res. Vet. Sci. 29:305-309, 1980
  10. Le Potier MF, Abiven P, Kobisch M. A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae. Ann. Rech. Vet. 23:239-247. 1994
  11. Harasawa R, Mizuasawa H, Nozawa K et al. Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions. Res. Microbiol. 144, 489-493, 1993a
  12. 이순신, 이동석, 한정희 등. Mycoplsma hyopneumomiae의 검출과 DNA fingerprinting에 관한 예비적 연구. 한국마이코플라스마학지,11(1):3-12, 2000
  13. Stemke GW, Phan R, Young TF et al. Differentiation of Mycoplsma hyopneumoniae, M. flocculare, and M. hyorhinis on the basis of amplification of a 16S rRNA gene sequence. Am. J. Vet. Res, 55:81-84, 1994
  14. Morton HE, Roberts RJ. Production of anti-mycoplasma (PPLO) in rabbits. PSEBM. 125:538-543 1966
  15. Harasawa R, Uemori T, Asada K et al. Sensitive detection of mycoplasmas in cell cultures by using two-step polymerase chain reaction, In 'Rapid Diagnosis of Mycoplasmas' Kahane I and Adoni A (eds) Plenum, New York, 227-232. 1993b