Evaluation of novel oral vaccine candidates and validation of a caprine model of Johne's disease

Abstract

Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn's disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne's Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne's disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 10(9) CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318) do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329) reduced fecal shedding and tissue colonization.

Keywords: Mycobacterium avium subsp paratuberculosis; diagnostic tests; goats; mutant vaccines; vaccine efficacy.

Figure 1

(A–C) Note how the severity of microscopic lesions increases along with the lesion score. Arrowheads indicate foci of granulomatous inflammatory infiltrate. (A) Representative Hematoxylin and Eosin stained section of non-challenged Sham vaccinated control ileum sample (100X). Lesion score = 0. (B) Hematoxylin and Eosin stained section of Silirum® vaccinated and challenged ileum sample (100X). Inset shows Kinouyan's acid-fast stain with a single acid-fast bacillus in center from area labeled with a small star (600X). Lesion score = 3.54. (C) Hematoxylin and Eosin stained section of higher lesion score in a 319 vaccine (group 5) ileum sample (100X). Inset shows Kinouyan's acid-fast stain with multiple acid-fast bacilli from area labeled by a small star (600X). Lesion score = 10.42.

Figure 2

Necropsy lesion scores for the challenged control and experimental groups at 13 months post-challenge. All negative controls (group 1, non-challenged kids) had lesion scores of 0. (Error bars represent standard error of the mean).

Figure 3

Monthly fecal culture results on Herrold's Egg Yolk medium with and without mycobactin J throughout the study. No positive samples were detected in the non-challenged control group. (Error bars represent standard error of the mean, −1 is baseline bleeding, 0 is challenge date and 1–13 represents months post-challenge).

Figure 4

Monthly fecal AgPath-ID™ PCR results throughout the study. No positive samples were detected in the non-challenged control group. (−1 is baseline bleeding, 0 is challenge date and 1–13 represents months post-challenge).

Figure 5

New York Animal Health Diagnostic Laboratory (NYAHDL) AGID test results over time. The NYAHDL AGID is an agar gel immunodiffusion test for measuring serum antibody to Mycobacterium avium subsp. paratuberculosis. Note that no animals in the Silirum® vaccine group and only 1 animal in the 329 vaccine group (group 7) became AGID positive. (−1 is baseline bleeding, 0 is challenge date and 1–13 represents months post-challenge).

Figure 6

Parachek® ELISA optical density (OD) readings over time. Note the almost immediate increase in ELISA OD for the Silirum® vaccinated group, the progressive increase in ELISA OD in most vaccine groups beginning around 5 months post-challenge and the leveling off of the Silirum® vaccinated group OD values at 9–13 months post-challenge. (Error bars represent standard error of the mean, −1 is baseline bleeding, 0 is challenge date and 1–13 represents months post-challenge).

Figure 7

(A–C) PPD skin test results over time. Note the strong skin responses to M. avium (A) and Johnin (C) PPD in multiple groups, and the generally lower responses to M. bovis (B) PPD with the exception of the Silirum® and 316 vaccines. (Error bars represent standard error of the mean, −1 is baseline bleeding, 0 is challenge date and 1–13 represents months post-challenge).