We generated mouse monoclonal antibodies to determine B cell epitopes of the recombinant Hsp70 protein and focused on linear epitopes. Subsequently, epitope-specific antibody responses, induced by vaccination of cattle and goats with recombinant MAP Hsp70, were analyzed to assess whether these this website antibodies recognized the same linear epitopes. Lastly, the monoclonal antibodies were used to study if these antibodies recognized native MAP Hsp70 protein in lesional tissue in naturally infected animals and if they interact with intact bacteria. Two Balb/c mice,

obtained from Charles River (Someren, the Netherlands), were used for the generation of MAP Hsp70 specific monoclonal antibodies. Animals were kept under standard housing and care conditions at the Central Animal Facilities of Utrecht University (Utrecht, the Netherlands). Thirty female goat kids (Saanen breed dairy Galunisertib mw goats, age 14 ± 3 days at the start of the experiment) were used. The kids were raised using conventional procedures and feeds, and were checked daily for general health. They were randomly assigned to one of the four experimental groups. Goat kids in groups 1 (n = 7) and 2 (n = 8) (uninfected controls) were housed separately from goat kids in groups 3 (n = 7) and 4 (n = 8) (MAP infected). Goat kids assigned to groups 2 and 4 were immunized once at the start of the experiment (day 0). The immunization consisted of the administration of 200 μg of recombinant

MAP Hsp70 in 1 mL phosphate buffered saline (PBS) containing 10 mg/mL dimethyl dioctadecyl ammonium bromide (DDA) adjuvant (Sigma Aldrich, USA) in the final preparation, subcutaneously in the lower neck region. Goat kids assigned to groups 3 and 4 were infected orally with 3 oral doses, at days 0, 2 and 4, of 2 × 109 cfu of MAP strain G195, originally isolated from a goat with clinical signs of paratuberculosis, grown on Middlebrook 7H10 supplemented with OADC and Mycobactin J (a generous gift from D. Bakker, CVI, Lelystad, the Netherlands). The cfu of the infection dose was determined by colony counts of serial dilutions on 7H10 agar plates. Blood samples

were taken from the vena jugularis on a weekly basis for a period of 3 months. Serum was stored at −20 °C, until further use. Goats were euthanized at the end of the experiment Rebamipide and tissue samples from ileum, jejunum, the ileocecal and a jejunal mesenteric lymph node were analyzed using MAP specific IS900 PCR [16], bacterial culture on mycobactin J supplemented HEY medium (BD Biosciences, Belgium) and histopathology. Sera from cattle subjected to a Hsp70 vaccination – challenge experiment, published previously [9], were used to characterize MAP Hsp70 specific antibody responses. In short, 4 groups of 10 female calves aged 29 ± 9 days, randomly assigned to one of 4 experimental groups, were used in that study. Treatment of the groups was identical to the goat kids described in Section 2.1.3.