Antigenicity assessment of the Theileria equi merozoite antigen (EMA-2) expressed in Pichia pastoris in mice and horses.

Equine theileriosis is a severe equine disease caused by the protozoan Theileria equi, which is prevalent in tropical and subtropical areas. In this study, a recombinant equi merozoite antigen-2 (rEMA-2) of T. equi was used as an immunogen. Two groups of 10 mice each were divided into control and vaccinated groups. Sixty mares seronegative for theileriosis were divided in two groups, one vaccinated and another group as a control animal. Mice and mares of the vaccinated groups were inoculated with 150 µL of the vaccine containing 50 µg of rEMA-2 and 2 mL of the vaccine containing 200 µg of rEMA-2, respectively, at days 0 and 21. The immunogenicity of rEMA-2 was evaluated by ELISA and fluorescent antibody test (IFAT) using serum from vaccinated mice, mares and antigenicity in naturally infected horse. At every point throughout the ELISA study, there were significant differences between the vaccinated and control groups (p < 0.05). The vaccine induced 3- and 4-fold IgG increases in mice at the 14th and 28th day, respectively, compared to the control group. The horses' IgG dynamics showed a significant (p < 0.05) increase in the total IgG titer as early as day 7, which increased until day 28 at which time a more significant (p < 0.001) IgG titer was observed. In evaluating the isotypes, we observed a trend similar to that of total IgG, where IgG(T) (IgG3-5) were significantly (p < 0.05) more elevated than the other isotypes analyzed, followed by IgGb (IgG4-7) and IgGa (IgG1). Positive fluorescence was detected by IFAT, suggesting that the protein is immunogenic and conserves some epitopes identical to the native T. equi antigens present in the equine blood smear. Thus, our results suggest that rEMA-2 can be a promising vaccinal antigen.

Larvicidal activity of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus.

Effective control of gastrointestinal parasites is necessary in sheep production. The development of anthelmintics resistance is causing the available chemically based anthelmintics to become less effective. Biological control strategies present an alternative to this problem. In the current study, we tested the larvicidal effects of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus larvae. Bacterial suspensions [2 × 108 colony-forming units (CFU) g-1 of the feces] of B. thuringiensis var. israelensis and recombinant Escherichia coli expressing Cry11Aa toxin were added to naturally H. contortus egg-contaminated feces. The larvae were quantified, and significant reductions of 62 and 81% (P < 0·001) were, respectively observed, compared with the control group. A 30 mL bacterial suspension (1 × 108 CFU mL-1) of B. thuringiensis var. israelensis and recombinant E. coli expressing Cry11Aa toxin were then orally administered to lambs naturally infected with H. contortus. Twelve hours after administration, feces were collected and submitted to coprocultures. Significant larvae reductions (P < 0·001) of 79 and 90% were observed respectively compared with the control group. The results suggest that the Cry11Aa toxin of B. thuringiensis var. israelensis is a promising new class of biological anthelmintics for treating sheep against H. contortus.

Expressão heteróloga da EMA-2 (equi merozoite antigen) de Theileria equi em Pichia pastoris com potencial utilização em imunobiológicos / Heterologous expression of EMA-2 (equi merozoite antigen) of Theileria equi in Pichia pastoris with potential use in immunobiologics

A piroplasmose equina causada por Theileria equi acomete os equinos de forma endêmica no Brasil e em diversos outros países tropicais e subtropicais. Considerada uma das mais importantes doenças de equinos, causa danos à saúde animal e perdas econômicas. A proteína equi merozoite antigen (EMA-2) é uma das principais proteínas de superfície, expressa nos diversos estágios do ciclo do parasita, estimula resposta imune em animais infectados, tornando-se um possível candidato para utilização em diagnóstico. O gene EMA-2 foi clonado e expresso na levedura Pichia pastoris. A proteína EMA-2 recombinante (rEMA-2) foi caracterizada antigenicamente por Western Blot e por ELISA indireto, utilizando-se soro de equino positivo para theileriose. O resultado do ELISA demonstrou uma especificidade de 90,9% e sensibilidade de 83,3%, quando comparado ao padrão, sendo superior à imunofluorescência (80,6% de especificidade e 75,0% de sensibilidade), o que sugere que a rEMA-2 expressa em P. pastoris é um promissor antígeno para ser utilizado como ferramenta no imunodiagnóstico de theileriose equina.

Theileria equi, the causative agent of equine piroplasmosis, is endemic in Brazil and many other tropical and subtropical countries. It is considered one of the most important diseases of horses causing animal health problems and significant economic loss. The Protein equi merozoite antigen-2 (EMA-2) is a major surface protein that is expressed in different parasite cycle stages and induces immune response in infected animals, being a possible candidate to be used in diagnose. EMA-2 gene was cloned and expressed in the yeast Pichia pastoris, and the recombinant protein EMA-2 (rEMA-2) was characterized by Western Blot and indirect ELISA using equine positive sera. The ELISA results demonstrated a specificity of 90.9% and a sensitivity of 83.3% compared to the standard ELISA and being superior to immunofluorescence (80.6% of specificity and 75.0% of sensitivity) suggesting that the rEMA-2 expressed in P. pastoris is a promising antigen to be used as a tool in immunodiagnostic of theileriasis.