Evaluation of Babesia bigemina 200 kDa recombinant antigen in enzyme-linked immunosorbent assay

Abstract

A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.

Spherical Body Protein 4 Is a New Serological Antigen for Global Detection of Babesia bovis Infection in Cattle

“…The Texas strain of B. bovis and the Argentina strain of B. bigemina were continuously cultured with bovine erythrocytes (RBC) by using a microaerophilous stationary-phase culturing system (1,15,20). The cultured parasite was harvested when the parasitemia reached 8 to 10%.…”

“…Positive serum samples were collected from cattle experimentally infected with B. bovis (n ϭ 25) or B. bigemina (n ϭ 30) (National Institute of Animal Health, Tsukuba, Ibaraki, Japan) or with Theileria orientalis (n ϭ 6) (Obihiro University of Agriculture and Veterinary Medicine, Japan), while nonBabesia-infected control sera (n ϭ 50) were obtained from healthy cattle that had been bred at Obihiro University of Agriculture and Veterinary Medicine, Washington State University (Pullman, WA), and Texas A&M University (College Station, TX) (19). Field bovine sera were collected from Brazil (n ϭ 108), Ghana (n ϭ 80), China (n ϭ 100), Thailand (n ϭ 100), Mongolia (n ϭ 81), South Korea (n ϭ 100), and Hokkaido, Japan (n ϭ 100) (1,5,18).…”

Genetic diversity of Theileria orientalis in tick vectors detected in Hokkaido and Okinawa, Japan

“…The veracity of expressed protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent Coomassie Brilliant Blue (CBB) staining. In a Western blot assay (Altangerel et al, 2009), antigenic differences among five rMPSPs were evaluated using a bovine serum sample prepared from cattle (Taiki farm) that was infected only with genotype 2 of T. orientalis according to the type-specific PCR assays described above .…”

“…After restriction digestion, PCR fragments were cloned into similarly digested pGEX4T-1 expression vectors (Amersham Pharmacia Biotech, Madison, CA, USA). Expression and purification of the glutathione-S-transferase (GST)-fusion recombinant MPSPs (rMPSPs) was performed as per our previous descriptions (Altangerel et al, 2009;Terkawi et al, 2011). The veracity of expressed protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent Coomassie Brilliant Blue (CBB) staining.…”

Principales marcadores moleculares utilizados para la identificación de Babesia bovis y Babesia bigemina

“…En la literatura científica, desde la década de los noventa se han reportado secuencias nucleotídicas de cepas circulantes de B. bovis y B. bigemina en diferentes regiones, reportándose genotipos idénticos en Asia, África, América, Europa y Oceanía (8,9); sin embargo, se han reportado cepas con variaciones genéticas asociadas a modificaciones endógenas y exógenas inducidas por tratamientos antiparasitarios, mecanismos de respuesta inmune del hospedador y procesos evolutivos que han generado mecanismos de resistencia y patogenicidad que garantizan su permanencia en el tiempo (7).…”