First 2-DE approach towards characterising the proteome and immunome of Besnoitia besnoiti in the tachyzoite stage.

ABSTRACT

Bovine besnoitiosis is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. It is considered to be a re-emergent disease in Europe and is also present in Africa and Asia. Due to the chronic and debilitating course of the disease, bovine besnoitiosis is responsible for severe economic losses. However, many aspects of the disease and parasite biology remain unknown. Proteomics studies could help to investigate relevant biological processes as well as host immune response associated with parasite infection. Both the proteome and immunome of the tachyzoite stage of B. besnoiti of the Bb-Spain1 isolate are described herein for the first time. Tachyzoite protein extracts were first separated by 2-DE SDS-PAGE using pH 3-10 NL IPG strips for Coomassie Brilliant Blue-stained gels and immunoblots. Eighty-five out of 265 spots visualised on Coomassie-stained gels were immunogenic when pooled serum from naturally infected cattle was used, and the distribution of immunogenic spots correlated with the 1-DE IDA pattern. Because most spots were found in the acidic range of the pH gradient, pH 3-6 L IPG strips were used next, and 58 out of 123 visualised spots proved to be immunogenic. Twenty-seven spots were identified by MALDI TOF/TOF to be 20 different proteins due to the presence of protein species. All proteins identified corresponded to highly conserved proteins among eukaryotes. Six proteins identified are related to energy metabolism, 3 are heat shock proteins, 4 proteins are related to host cell invasion processes, and 2 proteins are involved in cell redox homeostasis. A tryptophanyl tRNA synthetase, a putative gbp1p, nucleoredoxin, a putative receptor for activated C kinase, and a nuclear movement domain-containing protein were also identified. Among these proteins, fructose-1,6-bisphosphate aldolase, lactate dehydrogenase, pyruvate kinase, enolase, HSP60, HSP70, HSP90, actin and profilin proved to be immunogenic, and 5 were cross-reactive antigens between B. besnoiti and N. caninum. This first proteomic approach carried out in B. besnoiti should be followed by other studies to identify more specific parasite proteins.