Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex
The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1:5120 to 1:10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis.
References
[1]
Hale HH, Helmboldt CF, Plastridge WN, Stula EF (1962) Bovine mastitis caused by a Mycoplasma species. Cornell Vet 52: 582–591.
[2]
Pfutzner H, Sachse K (1996) Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Rev Sci Tech 15: 1477–1494.
[3]
Xin JQ, Li Y, Guo D, Song NH, Hu SP, et al. (2008) First isolation of Mycoplasma bovis from calf lung with pneumoniae in China. Chinese Journal of Preventive 30: 661–664.
[4]
Tschopp R, Bonnemain P, Nicolet J, Bumens A (2001) Epidemiological study of risk factors for Mycoplasma bovis infections in fattening calves. Schweiz Arch Tierheilkd 143: 461–467.
[5]
Nicholas RA, Ayling RD (2003) Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci 74: 105–112. doi: 10.1016/s0034-5288(02)00155-8
[6]
Caswel JL. Arehambauh M (2007) Mycoplasma bovis pneumonia in cattle. Anim Health Res Rev 8: 161–186. doi: 10.1017/s1466252307001351
[7]
Lysnyansky I, Rosengarten R, Yogev D (1996) Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. J Bacteriol 178: 5395–5401.
[8]
Nussbaum S, Lysnyansky I, Sachse K, Levisohn S, Yogev D (2002) Extended repertoire of genes encoding variable surface lipoproteins in Mycoplasma bovis strains. Infect Immun 70: 2220–2225. doi: 10.1128/iai.70.4.2220-2225.2002
[9]
Sachse K, Pfutzner H, Heller M, Hanel I (1993) Inhibition of Mycoplasma bovis cytadherence by a monoclonal antibody and various carbohydrate substances. Vet Microbiol 36: 307–316. doi: 10.1016/0378-1135(93)90097-q
[10]
Robino P, Alberti A, Pittau M, Chessa B, Miciletta M, et al. (2005) Genetic and antigenic characterization of the surface lipoprotein P48 of Mycoplasma bovis. Vet Microbiol 109: 201–209. doi: 10.1016/j.vetmic.2005.05.007
[11]
Scherm B, Gerlach GF, Runge M (2002) Analysis of heat shock protein 60 encoding genes of mycoplasmas and investigations concerning their role in immunity and infection. Vet Microbiol 89: 141–150. doi: 10.1016/s0378-1135(02)00158-x
[12]
Prysliak T, van der Merwe F, Perez-Casal J (2013) Vaccination with recombinant Mycoplasma bovis GAPDH results in a strong humoral immune response but does not protect feedlot cattle from an experimental challenge with M. bovis. Microb Pathog 12: 1–8. doi: 10.1016/j.micpath.2012.12.001
[13]
Jaffe JD, Berg HC, Church GM (2004) Proteogenomic mapping as a complementary method to perform genome annotation. Proteomics 4: 59–77. doi: 10.1002/pmic.200300511
[14]
Ueberle B, Frank R, Herrmann R (2002) The proteome of the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products. Proteomics 2: 754–764. doi: 10.1002/1615-9861(200206)2:6<754::aid-prot754>3.0.co;2-2
[15]
Jores J, Meens J, Buettner FF, Linz B, Naessens J, et al. (2009) Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species. Vet Immunol Immunopathol 131: 238–245. doi: 10.1016/j.vetimm.2009.04.016
[16]
Dallo SF, Kannan TR, Blaylock MW, Baseman JB (2002) Elongation factor Tu and E1 β subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae. Mol Microbiol 46: 1041–1051. doi: 10.1046/j.1365-2958.2002.03207.x
[17]
Su HC, Hutchison CA 3rd, Giddings MC (2007) Mapping phosphoproteins in Mycoplasma genitalium and Mycoplasma pneumoniae. BMC Microbiol 7: 47–63. doi: 10.1186/1471-2180-7-63
[18]
Pinto PM, Chemale G, de Castro LA, Costa AP, Kich JD, et al. (2007) Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins. Vet Microbiol 121: 83–93. doi: 10.1016/j.vetmic.2006.11.018
[19]
Soehnlen MK, Aydin A, Lengerich EJ, Houser BA, Fenton GD, et al. (2011) Blinded, controlled field trial of two commercially available Mycoplasma bovis bacterin vaccines in veal calves. Vaccine 29: 5347–5354. doi: 10.1016/j.vaccine.2011.05.092
[20]
Maunsell FP, Woolums AR, Francoz D, Rosenbusch RF, Step DL, et al. (2011) Mycoplasma bovis infections in cattle. J Vet Intern Med 25: 772–783. doi: 10.1111/j.1939-1676.2011.0750.x
[21]
Maunsell FP, Donovan GA (2009) Mycoplasma bovis infections in young calves. Vet Clin North Am Food Anim Pract 25: 139–177. doi: 10.1016/j.cvfa.2008.10.011
[22]
Zhao P, He Y, Chu YF, Gao PC, Zhang X, et al. (2012) Identification of novel immunogenic proteins in Mycoplasma capricolum subsp. Capripneumoniae strain M1601. J Vet Med Sci 74: 1109–1115. doi: 10.1292/jvms.12-0095
[23]
Thomas A, Leprince P, Dizier I, Ball H, Gevaert K, et al. Identification by two-dimensional electrophoresis of a new adhesin expressed by a low-passaged strain of Mycoplasma bovis. Res Microbiol 156: 713–718. doi: 10.1016/j.resmic.2005.02.008
[24]
Wasinger VC, Pollack JD, Humphery-Smith I (2000) The proteome of Mycoplasma genitalium. Chaps-soluble component. Eur J Biochem 267: 1571–1582. doi: 10.1046/j.1432-1327.2000.01183.x
[25]
Alonso JM, Prieto M, Parra F (2002) Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp. mycoides SC. Vet Microbiol 89: 277–289. doi: 10.1016/s0378-1135(02)00258-4
Perez-Casal J, Prysliak T (2007) Detection of antibodies against the Mycoplasma bovis glyceraldehyde-3-phosphate dehydrogenase protein in beef cattle. Microb Pathog 43: 189–197. doi: 10.1016/j.micpath.2007.05.009
[28]
Sun XM, Ji YS, Elashrama SA, Lu ZM, Liu XY, et al. (2012) Identification of antigenic proteins of Toxoplasma gondii RH strain recognized by human immunoglobulin G using immunoproteomics. J proteomics 77: 423–432. doi: 10.1016/j.jprot.2012.09.018
[29]
Yang YL, Wang L, Yin JG, Wang XL, Cheng SP, et al. (2011) Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine. Mol Immunol 49: 175–184. doi: 10.1016/j.molimm.2011.08.009
[30]
Bashiruddin JB, Frey J, Konigsson MH, Johansson KE, Hotzel H, et al. (2005) Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial. Vet J 169: 268–275. doi: 10.1016/j.tvjl.2004.01.018
[31]
Hou X, Fu P, Zhang HY, Zhang YW, Wu WX (2012) Development of loop-mediated isothermal amplification for rapid detection of Mycoplasma bovis. Journal of Agricultural Biotechnology 20: 218–224.
[32]
Tenk M, Bálint A, Stipkovits L, Biró J, Dencso L (2006) Detection of Mycoplasma bovis with an improved pcr assay. Acta Vet Hung 54: 427–435. doi: 10.1556/avet.54.2006.4.1
[33]
Regula J, Ueberle B, Boguth G, Gorg A, Schnolzer M, et al. (2000) Towards a two-dimensional proteome map of Mycoplasma pneumoniae. Electrophoresis 21: 3765–3780. doi: 10.1002/1522-2683(200011)21:17<3765::aid-elps3765>3.0.co;2-6
[34]
G？rg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4: 3665–3685. doi: 10.1002/pmic.200590007
[35]
Webster KA, Giles M, Dawson C (1997) A competitive ELISA for the serodiagnosis of hypodermosis. Vet Parasitol 68: 155–164. doi: 10.1016/s0304-4017(96)01062-x
[36]
Tiwari S, Kumar A, Thavaselvam D, Mangalgi S, Rathod V, et al. (2013) Development and comparative evaluation of a plate enzyme-linked immunosorbent assay based on recombinant outer membrane antigens Omp28 and Omp31 for diagnosis of human brucellosis. Clin Vaccine Immunol 20: 1217–1222. doi: 10.1128/cvi.00111-13
[37]
Landis JR, Koch GG (1977) The measurement of observer agreement for categorical data. Biometrics 33: 159–174. doi: 10.2307/2529310
