Induction of B cell responses upon experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis

Abstract

The objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis would invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5(dim) and CD5(bright) markers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25(+) and CD45RO(+) B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. avium subsp. paratuberculosis demonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. avium subsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. avium subsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation.

Fig. 1.

Percentages of B cells from PBMCs isolated from control noninfected calves (⧫) and calves infected with Mycobacterium avium subsp. paratuberculosis by the following methods: oral (▪), intraperitoneal (▴), oral with dexamethasone administration (○), and oral with mucosal scrapings from a cow with clinical disease (*). Data are for a 12-month study with nonstimulated PBMCs (A) and PBMCs stimulated with a whole-cell sonicate of Mycobacterium avium subsp. paratuberculosis (MPS) (B). Data are expressed as means ± SEM. Significant differences between control and infection groups for given time points are represented by asterisks (*, P < 0.01; **, P < 0.05).

Fig. 2.

Percentages of CD5^dim B cells from PBMCs isolated from control noninfected calves (⧫) and calves infected with Mycobacterium avium subsp. paratuberculosis by the following methods: oral (▪), intraperitoneal (▴), oral with dexamethasone administration (○), and oral with mucosal scrapings from a cow with clinical disease (*). Data are for a 12-month study with nonstimulated PBMCs (A) and PBMCs stimulated with MPS (B). Data are expressed as means ± SEM. Significant differences between control and infection groups for given time points are represented by asterisks (*, P < 0.01; **, P < 0.05).

Fig. 3.

Percentages of CD5^bright B cells from PBMCs isolated from control noninfected calves (⧫) and calves infected with Mycobacterium avium subsp. paratuberculosis by the following methods: oral (▪), intraperitoneal (▴), oral with dexamethasone administration (○), and oral with mucosal scrapings from a cow with clinical disease (*). Data are for a 12-month study with nonstimulated PBMCs (A) and PBMCs stimulated with MPS (B). Data are expressed as means ± SEM. Significant differences between control and infection groups for given time points are represented by asterisks (*, P < 0.01; **, P < 0.05).

Fig. 4.

Comparison of in vitro stimulation with medium control (NS) and a whole-cell sonicate of Mycobacterium avium subsp. paratuberculosis (MPS) on the percentage of CD5^bright B cells from PBMCs isolated from calves at 1 month of infection (A) and 12 months of infection (B). Data are expressed as means ± SEM. Significant differences between NS and MPS-stimulated PBMCs within a treatment group are represented by asterisks (*, P < 0.01; **, P < 0.05).

Fig. 5.

Percentages of CD25⁺ B cells from PBMCs isolated from control noninfected calves and calves infected with Mycobacterium avium subsp. paratuberculosis by the following methods: oral, intraperitoneal, oral with dexamethasone administration, and oral with mucosal scrapings from a cow with clinical disease. Data are for a 12-month study with nonstimulated PBMCs (A) and PBMCs stimulated with MPS (B). Data are expressed as means ± SEM. Significant differences between the control and infection groups for given time points are represented by asterisks (*, P < 0.01; **, P < 0.05).

Fig. 6.

Percentages of CD45RO⁺ B cells from PBMCs isolated from control noninfected calves and calves infected with Mycobacterium avium subsp. paratuberculosis by the following methods: oral, intraperitoneal, oral with dexamethasone administration, and oral with mucosal scrapings from a cow with clinical disease. The data are for a 12-month study with nonstimulated PBMCs (A) and PBMCs stimulated with MPS (B). Data are expressed as means ± SEM. Significant differences between the control and infection groups for given time points are represented by asterisks (*, P < 0.01; **, P < 0.05).

Fig. 7.

Immunoblotting of serum from a representative calf for each treatment group. (A) Control; (B) oral; (C) intraperitoneal; (D) oral with dexamethasone; (E) oral with mucosal scrapings from a cow with clinical disease. Sera were blotted against a whole-cell sonicate preparation of Mycobacterium avium subsp. paratuberculosis strain K-10. Lanes 1 to 17, days −3, −2, 7, 14, and 21 and months 1 through 12, respectively.

Fig. 8.

Scattergram of EvELISA S/P ratios demonstrating antibody reactivity of sera from 4 calves in the i.p. treatment group compared to baseline values for one control noninfected calf. Values over the time course of the study are shown, and each symbol represents a single calf across time.