Evaluation of alternative vaccination routes against paratuberculosis in goats

Abstract

Paratuberculosis is a chronic granulomatous enteritis, caused by Mycobacterium avium subspecies paratuberculosis (Map), that affects ruminants worldwide. Vaccination has been considered the most cost-effective method for the control of this disease in infected dairy herds. However, currently available vaccines do not provide complete protection and interfere with the diagnosis of both paratuberculosis and bovine tuberculosis, limiting its use. Because of that, efforts are being made for the development of new vaccines. The primary objective of this study was to evaluate the efficacy of two whole-cell inactivated experimental vaccines against paratuberculosis in goats, administered through the oral (OV) and intradermal (IDV) routes, and compare them with that of the commercial subcutaneous vaccine Gudair^® (SCV). Over an 11-month period, the effect of vaccination and a subsequent Map challenge on the specific peripheral immune responses and Map-DNA fecal shedding were recorded. At the end of the experiment, tissue bacterial load and lesion severity were assessed. The experimental vaccines did not induce specific humoral immune responses and only elicited mild and delayed cellular immune responses. Although the OV reduced lesion severity, neither this vaccine nor the IDV prototype was able to reduce fecal shedding or tissue bacterial load. Moreover, although the SCV did not confer sterile immunity, it outperformed both experimental vaccines in all these parameters.

Keywords: goat; immunization strategies; intradermal vaccine; oral vaccine; paratuberculosis.

Figure 1

Experimental design scheme. After a 2-week adaptation period, goat kids were divided into four groups and vaccinated through different routes [oral (OV), intradermal (IDV), and subcutaneous (SCV) or left unvaccinated (NV)]. Experimental (OV and IDV) and commercial vaccines (SCV—Gudair^®) were used. One month after vaccination, three or seven animals from each group were orally challenged with Map (INF). A total of eight groups were formed: OV (oral vaccine), OV-INF (oral vaccine, challenged), IDV (intradermal vaccine), IDV-INF (intradermal vaccine, challenged), SCV (subcutaneous vaccine), SCV-INF (subcutaneous vaccine, challenged), NV (non-vaccinated), and NV-INF (non-vaccinated, challenged). Squares indicate sampling time points (blood, feces, and tissues) from the beginning of the study to 12 months. *Fecal samples to assure the vaccine reached the intestine were taken from the orally vaccinated animals at 3, 7, and 14 dpv.

Figure 2

Kinetics of the peripheral immune response. (A) Dynamics of the specific anti-Map antibody response through the experiment, expressed as the mean Log₂ (OD₄₅₀ ratio) of each group. (B) Kinetics of the IFN-γ production by whole blood stimulated with PPDa. (C) Kinetics of the IFN-γ production by whole blood stimulated with PPDb, expressed as the mean avian and bovine Log₂ (OD index) of each group. (D) bTB interference, calculated by subtracting the OD of the PPDa from the PPDb stimulated samples, results are expressed as a mean, and the gray horizontal dotted line represents the cutoff ratio of the BOVIGAM^™ test (0.100). The experimental groups were as follows: OV (oral vaccine), OV-INF (oral vaccine, challenged), IDV (intradermal vaccine), IDV-INF (intradermal vaccine, challenged), SCV (subcutaneous vaccine), SCV-INF (subcutaneous vaccine, challenged), NV (non-vaccinated), and NV-INF (non-vaccinated, challenged). The vertically dotted red line represents the vaccine administration time-point, and the vertically dotted green line represents the challenge time-point. The results from the statistical analysis are shown in Supplementary Table S1.

Figure 3

Representative microscopic lesions and Map detection in tissue samples. (A) Focal lesion: multiple well-demarcated granulomas located in the interfollicular area of the Peyer’s patches (medial ileum, Goat 64, SCV-INF group); (B) Multifocal lesion: granulomas (arrowheads) can be seen in both the lymphoid tissue and lamina propria (medial jejunum Peyer’s patch, Goat 22, OV-INF group); (C) Diffuse lesion: the lamina propria is a diffuse granulomatous infiltrate affects the lamina propria (medial jejunum, Goat 42, IDV-INF group). (D) Multifocal to coalescing granulomatous foci present in the cortex of a jejunal lymph node (goat 83, NV-INF group). Representative images of Map-positive samples as detected through: (E) Ziehl–Neelsen staining. Some acid-fast staining can be observed in some of the macrophages present in the apex of the villi (arrowheads) (distal jejunum Peyer’s patch, Goat 82, NV-INF group). (F) Immunohistochemistry. Macrophages positive to Map immunostaining are observed in the diffuse granulomatous infiltrate affecting the lamina propria over the lymphoid tissue of a distal jejunum Peyer’s patch (distal jejunum Peyer’s patch, Goat 82, NV-INF group). Unless indicated otherwise, scale bars equal to 100 μm.