Bovines immunized with Babesia bigemina surface-exposed peptides containing conserved B-cell epitopes elicit an improved Th1 response when supplemented with live, probiotic yeasts.
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1
Universidad Autónoma de Querétaro, CA Salud Animal y Microbiología Ambiental, Mexico
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2
Phileo Lesaffre Animal Care, Mexico
Babesia bigemina is one of the causative agents of Bovine babesiosis in temperate regions of the world, including the Americas. It has been shown that a Th1 response correlates with protection in infected cattle. Immunodominant antigens of B. bigemina are highly variable, however conserved, subdominant epitopes are present in exposed antigens and can be used as vaccine candidates. The aim of this work was to characterize the cellular immune response of bovines immunized with conserved peptides from Babesia bigemina antigens and fed with a supplemented diet with live probiotic yeast. A total of twenty peptides ranging from 12 to 20 amino acids-long and containing conserved, predicted B-cell epitopes from seven vaccine candidate antigens of Babesia bigemina were chemically synthetized as dendrimers, pooled and used as vaccine antigen. Twenty-four bovines from a tick-free area and free of antibodies against Babesia bigemina were randomly distributed in four groups (n=6). Treatments evaluated were: 1) PBS and adjuvant and diet, 2) peptides with adjuvant and diet, 3) peptides with adjuvant and supplemented diet with live yeast Procreatin 7 (P7), and 4) peptides with adjuvant and supplemented diet with live yeast Actisaf F53. A twenty-one day adaptation period was included prior to the first immunization and all groups of cattle were given their respective diet every day and for the rest of the experiment. All bovines were immunized subcutaneously every three weeks for a total of four times with 1.5 mg of antigen emulsified with a commercial adjuvant. Ten days after the last immunization, blood samples were collected and used to evaluate lymphocyte proliferation and cytokine production. A total of 3 x 105 peripheral blood mononuclear cells (PBMC) were purified, incubated in vitro in 96-well culture plates at 37C in 5% CO2 and stimulated either with the pooled peptides or with Babesia bigemina crude antigen; this was done by triplicate. After 72 h, lymphocyte proliferation was determined by addition of CellTiter® to each well for 2 h at 37C. The results were read at 490 nm in an ELISA reader. In a simultaneous experiment, purified PMBCs were cultured in vitro at 37C in 5% CO2 and incubated with the peptides for 72h. After this time, the supernatant was removed and kept at -80C until used. Cytokine production was determined by evaluating the supernatants of triplicate cultures by commercial ELISA tests for INF-g, IL-2, IL-12, IL-4, IL-5, and IL-6. Statistical analysis was carried out by an Analysis of Variance (ANOVA) test and complemented with a Tukey’s test. Results of the lymphocyte proliferation assay using crude antigen, indicated that group 2 (immunized bovines without yeast inclusion) compared with group 1 (non-immunized bovines and without yeast inclusion) had a higher cellular response (p<0.05). However, group 3 (immunized bovines supplemented with Procreatin 7) had a higher cellular response compared with group 1 (p<0.001). When the lymphoproliferation assay was carried out using the pooled peptides as antigen, it showed that groups 3 and 4 had a higher lymphocyte proliferation response than that of group 2 (and 1) (p<0.05). When cytokine expression was determined, statistical difference among groups was not found with IFN-γ production (p>0.05). However, group 3 had the highest IFN-γ production followed by group 4, group 2 and finally group 1. For IL-12 expression, group 2 (immunized bovines without yeast inclusion) compared with the rest of the groups had a higher production (p<0.05). When IL-2 was evaluated, there were statistical differences among groups 1 and 3 and groups 1 and 4 (p<0.05). For IL-4, IL-5, and IL-6 the results showed that there was not a statistical difference among groups. All these results together show that the selected B. bigemina peptides induce a specific cellular immune response, characterized by a higher expression of Th1 cytokines, this profile correlates with protection in bovine babesiosis. The immune response was improved by the use of probiotics in immunized bovines.
Acknowledgements
Financed by Conacyt, Phileo Lesaffre, and Promep-Redes.
Keywords:
Babesia bigemina,
Th1 response,
probiotic yeasts,
Cellular Immune Response,
B-Cell epitopes
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Oral Presentation
Topic:
Veterinary and Comparative Immunology
Citation:
MOSQUEDA
J,
SOLORIO
S,
GARCÍA
A,
OLVERA-RAMÍREZ
A,
AGUILAR-TIPACAMÚ
G,
CANTO
G and
Ruiz
MH
(2015). Bovines immunized with Babesia bigemina surface-exposed peptides containing conserved B-cell epitopes elicit an improved Th1 response when supplemented with live, probiotic yeasts..
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00356
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Received:
16 May 2015;
Published Online:
15 Sep 2015.
*
Correspondence:
Dr. JUAN MOSQUEDA, Universidad Autónoma de Querétaro, CA Salud Animal y Microbiología Ambiental, Querétaro, Querétaro, 76140, Mexico, joel.mosqueda@uaq.mx