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Clinical and Vaccine Immunology, April 2007, p. 420-425, Vol. 14, No. 4
1071-412X/07/$08.00+0     doi:10.1128/CVI.00439-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigens for Rapid Detection of Antibodies against Mycoplasma agalactiae in Sheep{triangledown} ,{dagger}

Marisa Fusco,1 Lucia Corona,1 Toniangelo Onni,1 Elisabetta Marras,2 Carla Longheu,1 Graziano Idini,1 and Sebastiana Tola1*

Istituto Zooprofilattico Sperimentale della Sardegna G. Pegreffi, 07100 Sassari, Italy,1 CRS4, Edificio 3 Loc Piscinamanna, 09010 Pula, Italy2

Received 17 November 2006/ Returned for modification 4 January 2007/ Accepted 22 January 2007

We developed a new recombinant enzyme-linked immunosorbent assay (rELISA) for serodiagnosis of contagious agalactia (CA), a disease caused by Mycoplasma agalactiae in sheep and goats. The assay is based on two M. agalactiae surface proteins, namely, P80 and P55. Identification of these immunodominant and common antigens was accomplished by examining the antibody response elicited in sheep during experimental infection and comparing it to the protein expression profiles of 75 M. agalactiae field strains. Our rELISA was tested with 343 sera, collected from sheep with a laboratory-confirmed diagnosis of CA (n = 223) and from healthy animals (n = 120). All sera had previously been tested by Western blotting (WB) for reactivity against M. agalactiae. In addition, our rELISA was compared with a commercial routine ELISA based on inactivated antigens (CHEKiT). Among the 223 samples that were WB positive for M. agalactiae, 209 (93.7%) tested positive for rP80-P55 with our ELISA, whereas only 164 (73.8%) tested positive with the CHEKiT ELISA. Among the 120 samples tested that were WB negative for M. agalactiae, 96.7% were confirmed as negative with our rELISA, while only 75.8% were confirmed as negative with the CHEKiT ELISA. A comparison of the results with receiver operating characteristic curves indicated that the differences observed between our rELISA and the CHEKiT ELISA are statistically significant. The use of recombinant peptides instead of inactivated antigens could significantly improve the discrimination of positive and negative animals, bringing significant advantages in controlling the import/export of live animals and helping in eradication of this economically detrimental disease.

* Corresponding author. Mailing address: Istituto Zooprofilattico Sperimentale, Via Duca degli Abruzzi 8, 07100 Sassari, Italy. Phone: 0792892339. Fax: 0792892324. E-mail: sebastiana.tola{at}izs-sardegna.it

{triangledown} Published ahead of print on 7 February 2007.

{dagger} Supplemental material for this article may be found at http://cvi.asm.org/.

Clinical and Vaccine Immunology, April 2007, p. 420-425, Vol. 14, No. 4
1071-412X/07/$08.00+0     doi:10.1128/CVI.00439-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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