Identification of conserved peptides containing B-cell epitopes of Babesia bovis AMA-1 and their potential as diagnostics candidates.

The apical membrane antigen 1 (AMA-1) is a protein of the micronemes that is present in all organisms of the phylum Apicomplexa; it has been shown that AMA-1 plays an essential role for parasite invasion to target cells. It has been reported that AMA-1 is conserved among different isolates of Babesia; however, it is unknown whether the protein contains conserved B-cell epitopes and whether these epitopes are recognized by antibodies from cattle in endemic areas. In this research, using an in silico analysis, four peptides were designed containing exposed and conserved linear B-cell epitopes from the extracellular region of Babesia bovis AMA-1. The selected peptides were chemically synthesized, and then each peptide was emulsified and used to immunize two bovines per peptide. The antibodies produced against these peptides were able to recognize intra-erythrocytic parasites in an IFAT, except peptide 4, which was insoluble. The synthetic peptides were covalently fixed to the wells of an ELISA plate and incubated with sera from B. bovis naturally infected cattle. Peptides P2AMA and P3AMA were recognized by the sera of naturally infected cattle from different regions of Mexico. Statistical analysis showed that the ELISA test for peptides P2AMA and P3AMA had a concordance of 91.2% and 61.1% compared to the IFAT, a sensitivity of 94.56% and 71.74%, and a specificity of 76.19% and 14.2%, respectively. The presence of antibodies in bovine sera from endemic areas that bind to the identified peptides indicates that AMA-1 from B. bovis has conserved B-cell epitopes involved in the immune response under natural conditions. However, to propose their use as vaccine or diagnostics candidates, a further characterization of the humoral immune response elicited in cattle by these peptides is needed.

Babesia bovis RON2 contains conserved B-cell epitopes that induce an invasion-blocking humoral immune response in immunized cattle.

BACKGROUND: Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis. RESULTS: The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas. CONCLUSIONS: The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

Fluctuación poblacional de Haematobia irritans (Diptera: Muscidae) en un hato bovino en Tecomán, Colima, México / Population fluctuation of Haematobia irritans (Diptera: Muscidae) in a cattle herd in Tecoman, Colima, Mexico

The objective of the work was to study H. irritans population fluctuation in a cattle herd in Tecoman, Colima, Mexico. The cattle herd was visited weekly from March 2003 to May 2004; on each occasion, ten animals were randomly selected to estimate infestation by direct visualization method. Temperature and relative humidity (RH) were recorded daily. Population fluctuation was described and periods and population peaks were identified. Pearson's correlation analysis was calculated (P < 0.05) between temperature and RH, with fly average number per animal for each population period. H. irritans infestation had fluctuations, but with presence of flies all the year; the highest infestation was observed in summer, where two population peaks were present, with 236 and 120 flies per animal, respectively; the activity in autumn and winter also showed fluctuations and the population had an increment in spring, where a population peak could be observed, as well as at the beginning of the study (156 flies/animal) and at the end (323 flies/animal). In the first population period, a significant correlation coefficient (0.93) was found with temperature, similar situation was observed in the fourth period (-0.57); no significant coefficients were found in other population periods for the climatic factors in study.

El objetivo del trabajo fue estudiar la fluctuación poblacional de H. irritans en un hato de ganado bovino en el municipio de Tecomán, Colima, México. El hato fue visitado semanalmente de marzo de 2003 a mayo de 2004; en cada ocasión se seleccionaron al azar diez animales para estimar la infestación mediante el método de visualización directa. Se registró la temperatura y la humedad relativa (HR) diariamente. Se describió la fluctuación poblacional presentada y se identificaron los periodos y picos poblacionales. Se realizó un análisis de correlación de Pearson (P < 0.05) entre la temperatura y la HR, con los valores promedio del número de moscas por animal para cada uno de los periodos poblacionales. La infestación por H. irritans fue fluctuante, pero con presencia de moscas durante todo el año; los momentos de mayor infestación se observaron en verano, al presentarse dos picos poblacionales, con 236 y 120 moscas/animal; la actividad en otoño e invierno también fluctuó; se incrementó en primavera, durante la cual se observó un pico poblacional, tanto al inicio del estudio (156 moscas/animal) como al final (323 moscas/animal). Para el primer periodo poblacional se encontró un coeficiente de correlación significativo (0.93) con la temperatura, algo similar ocurrió para el cuarto periodo (-0.57); no fue posible detectar coeficientes significativos en otros periodos poblacionales para los factores climáticos en estudio.