Isolation, identification and molecular characterization of Mycoplasma bovis in mastitic dairy cattle by PCR and culture methods

Mycoplasma bovis cattle isolation PCR mastitis

Mycoplasma bovis is well known as one of the major causative agents of mastitis in dairy cattle herds. The aim of this study was the identification of Mycoplasma bovis strains by PCR and traditional culture methods from a total number of 328 milk samples collected from cows with clinical mastitis symptoms from all over Iran. First step cultures in PPLO broth and agar showed 58 samples (17.69%) as positive. Out of 328 samples, 97 samples (29.57%) were positive for Mycoplasma genus according to the amplification of the 16SrRNA gene performed by PCR and from them, 31 (31.97%) samples were positive by PCR on the P48 gene. The purified P48 positive PCR products were sequenced and results were compared to M. bovis reference strain PG45 (CP002188). A phylogenic tree was created using Neighbor-joining method in MEGA6 software. The studied strain IB220 showed 100% identity with the reference strain of M. bovis and followed the same phylogenetic roots while studied strain IB216 showed 99.7% homology with the reference strain. Twelve selected geographical isolated strains were subjected to Gene Bank under accession numbers of KX772789 to KX772800. This is the first study of the molecular characterization of Mycoplasma bovis in dairy cattle with clinical mastitis from Iran.

Article Details
  • Section
  • Research Articles
Download data is not yet available.
Arcangioli MA, Chazel M, Sellal E, Botrel MA, Bezille P, Poumarat F, Calavas D, Le Grand D (2011) Prevalence of Mycoplasma bovis udder infection in dairy cattle: preliminary field investigation in southeast France. New Zealand vet j 59(2):75-78.
Baird SC, Carman J, Dinsmore RP, Walker RL, Collins JK (1999) Detection and identification of Mycoplasma from bovine mastitis infections using a nested polymerase chain reaction. J Vet Diagn Invest. 11:432-435.
Bashiruddin JB, Frey J, Königsson MH, Johansson KE, Hotzel H, Diller R, de Santis P, Botelho A, Ayling RD, Nicholas RA, Thiaucourt F, Sachse K (2005) Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial. The Veterinary Journal 169:268–275.
Behrens A, Heller M, Kirchhoff H, Yogev D, Rosengarten R (1994) A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis. Infect Immun 62:5075-5084.
Cai HY, Bell-Rogers P, Parker L (2005) Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples. J Vet Diagn Invest 17:537-545.
Eissa SI, Hassan AM, Hashem YM, Shaker MM (2012) Comparative Molecular Study of Mycoplasma bovis Isolates from Egyptian Buffaloes and Cows Suffered from Mastitis. Euro J of Biol Sci 4(4):114-120.
Fu P, Sun Zh, Zhang Y, Yu Z, Zhang H, Su D, Jiang F, Wu W (2014) Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein. Biomed-Central Veterinary Research 10:42.
Giacometti M, Nicolet J, Johansson KE, Naglic T, Degiorgis MP, Frey J (1999) Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16SrRNA gene. Zentralbl Veterinärmed 46:173–180.
Hala SH, Hotzel SH (2013) Investigation of phenotypic variations amongst Mycoplasma bovis field German isolates using SDS-PAGE. Beni-Suef Uni j basic applied and sci 2:103-107.
Hotzel H, Frey J, Bashiruddin J, Sachs K (2003) Detection and Differentiation of Ruminant Mycoplasmas, Methods in Molecular Biology. In: PCR Detection of Microbial Pathogens: Methods and Protocols. Humana Press Inc. Totowa: NJ 216. pp 231-245.
Kirk JH, Lauerman LH (1994) Mycoplasma mastitis in dairy cows. Compend Contin Educ Pract Vet 16:541-551.
Kojima A, Takahashi T, Kijima M, Ogikubo Y, Nishimura Y, Nishimura S, Harasawa R, Tamura Y (1997) Detection of mycoplasma in avian live virus vaccines by polymerase chain reaction. Biologicals 25:365-71
Li Y, Zheng H, Liu Y, Jiang Y, Xin J, Chen W, Song Z (2011) The Complete Genome Sequence of Mycoplasma bovis Strain Hubei-1. Plos One 6:6:e20999.
Lysnyansky I, Yogev D, Levisohn Sh (2008) Molecular characterization of the Mycoplasma bovis P68 gene, encoding a basic membrane protein with homology to P48 of Mycoplasma agalactiae. FEMS Microbiol Lett 279:234–242.
Nicholas RAJ, Ayling RD (2003) Mycoplasma bovis: disease, diagnosis and control. Res in Vet Sci 74:105-112.
Park A (2014) Cattle Health and Welfare Group, Mycoplasma bovis Information note. Animal and plant health agency. CHAWG December 2014 pp 1-4.
Pfützner H, Sachse K (1996) Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Rev Sci Tech 15:1477–1494.
Pinnow CC, Butler JA, Sachse K, Hotzel H, Timms LL, Rosenbusch RF (2001) Detection of Mycoplasma bovis in preservative-treated field milk samples. J Dairy Sci 84:1640–1645.
Pourbakhsh SA, Shokri GR Banani M, Elhamnia F, Ashtari A (2010) Detection of Mycoplasma synoviae infection in broiler breeder farms of Tehran province using PCR and culture methods. Arch of Razi Ins 65(2):75-81.
Radaelli E, Castiglioni V, Losa M, Benedetti V, Piccinini R, Nicholas RAJ, Scanziani E, Luini M, (2011) Outbreak of bovine clinical mastitis caused by Mycoplasma bovis in a North Italian herd. Res in Vet Sci 91:251–253.
Radostits OM, Gay CC, Blood DC, Hinch Cliff KW (2000) Diseases of the mammary gland. In: Veterinary Medicine: A textbook of the diseases of cattle, sheep, pigs, goats and horses. 9th ed, Saunders Press, London.
Robino P, Alberti A, Pittau M, Chessa B, Miciletta M, Nebbia P, Le Grand D, Rosati S (2005) Genetic and antigenic characterization of the surface lipoprotein P48 of Mycoplasma bovis. Vet Microbiol 109(3–4):201.
Rossetti BC, Frey J, Pilo P (2010) Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR. Molecular and Cellular Probes 24:321-323.
Sabramaniam S, Bergonier D, Poumarat F, Capaul S, Schlatter Y, Nicolet J, Frey J (1998) Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR. Molecular and Cellular Probes 12:161–169.
Thomas A, Dizier I, Linden A, Mainil J, Frey J, Vilei EM (2004) Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis. The Vet J 168:100–102.
Wawegama NK, Browning GF, Kanci A, Marc S, Markham M (2014) Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle. Clin and Vacc Immun 21(2):196–202.
Wise KS, Calcutt MJ, Foecking MF, Roske K, Madupu R, Methe BA (2011) Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523). Infect Immun 79: 982–983.
Most read articles by the same author(s)