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J Med Microbiol 55 (2006), 1735-1740; DOI: 10.1099/jmm.0.46785-0
© 2006 Society for General Microbiology
ISSN 1473-5644

Humoral and cellular immune responses in sheep immunized with a 22 kilodalton exported protein of Mycobacterium avium subspecies paratuberculosis

Rachael C. Rigden1,{dagger}, Dakshina M. Jandhyala1,{dagger}, Chris Dupont1,{ddagger}, Dianna Crosbie-Caird1, Nicolas Lopez-Villalobos1, Norihiro Maeda2, Brigitte Gicquel2 and Alan Murray1,2

1 Institute of Veterinary, Animal and Biomedical Science, Massey University, Private Bag 11 222, Palmerston North, New Zealand

2 Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

Correspondence
Rachael C. Rigden
r.c.rigden{at}massey.ac.nz

Received 15 June 2006
Accepted 21 August 2006

An immunogenic 22 kilodalton exported Mycobacterium avium subspecies paratuberculosis (MAP) lipoprotein (P22) was previously identified, and found to belong to the LppX/LprAFG family of mycobacterial lipoproteins. N-terminal polyhistidine-tagged P22 was produced and purified from Escherichia coli. Antibody recognition of P22, and interferon-gamma (IFN-{gamma}) responses in vitro using blood from a sheep vaccinated with Neoparasec, confirmed its immunogenicity. To evaluate the immunogenicity of P22 in vivo, five sheep were immunized with a single dose containing 0.8 mg recombinant P22 protein in adjuvant. Blood was collected at 4, 13 and 29 weeks post-immunization (p.i.) and tested for anti-P22 antibodies and P22-specific IFN-{gamma} production. P22-specific antibodies were detected by Western blot analysis in all five Neoparasec-immunized sheep at the three time points. Three out of five P22-immunized sheep produced P22-specific antibodies for up to 13 weeks p.i., and two gave a response at 29 weeks p.i. Recombinant P22 was able to stimulate significant IFN-{gamma} production in blood of P22-immunized sheep at 13 and 29 weeks p.i. Recombinant P22 also elicited an IFN-{gamma} response in blood of sheep immunized with Neoparasec.

Abbreviations: Ab, antibody; Ag, antigen; dH2O, deionized water; HRP, horseradish peroxidase; IFN-{gamma}, interferon-gamma; JD, Johne's disease; MAP, Mycobacterium avium subspecies paratuberculosis; MTB, Mycobacterium tuberculosis; P22, 22kDa exported MAP protein; p.i., post-immunization; PPDA, avian purified protein derivative; RT, room temperature.

{dagger}These authors contributed equally to this work.

{ddagger}Present address: Department of Biology, University of Waterloo, 200 University Avenue W., Waterloo, ON N2L 3G1, Canada.




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