TY - JOUR
T1 - Detection and quantification of Babesia bovis and Babesia bigemina using different target genes
AU - Giglioti, Rodrigo
AU - Filho, Anibal Eugênio Vercesi
AU - Domingos, Ana Gonçalves
AU - da Silva, Sérgio Silva
AU - Cunha, Rodrigo Casquero
AU - Ibelli, Adriana Mércia Guaratini
AU - Okino, Cintia Hiromi
AU - de Sena Oliveira, Márcia Cristina
N1 - Funding Information:
This study was supported by the São Paulo State Research Support Foundation (FAPESP, Grant # 2019/22675-6) and the Institute of Animal Science (APTA-SAA). AMGI is recipient of a productivity fellowship from National Council for Scientific and Technological Development (CNPq).
Publisher Copyright:
© 2023
PY - 2024/3
Y1 - 2024/3
N2 - Molecular assays have been widely used for the detection and quantification of bovine babesiosis due to their high sensitivity and specificity. However, variations in the sensitivity of pathogen detection may occur depending on the selected target gene. Thus, this study aimed to compare the detection sensitivity (DS) of Babesia bovis and B. bigemina infection levels in artificially and naturally infected cattle using quantitative PCR (qPCR) and six target genes. For B. bovis, the merozoite surface antigen genes 2b and 2c (msa-2b and msa-2c), and the mitochondrial cytochrome b gene (cybmt) were used. For B. bigemina, the genes encoding the proteins associated with rhoptry 1c (rap-1c), rap-1a, and cybmt were used. Six bovines, free of babesiosis, were artificially infected with 1 × 10−8 red blood cells infected (iRBC) with B. bovis (n = 3) or 1 × 10−6 B. bigemina iRBC (n = 3). The animals were evaluated daily until parasitemia was confirmed (≥ 2.0%). The quantity of iRBC present in each animal was determined by examining blood smears. Blood samples were then subjected to DNA extraction, serial dilution, and qPCR analysis to determine the DS of each target gene. In addition, 30 calves naturally infected by Babesia spp. were also evaluated using the same six target genes. Regarding the artificial infection, B. bovis cybmt showed 25-fold higher sensitivity than the msa-2b and msa-2c genes, while the B. bigemina cybmt exhibited 5-fold and 25-fold higher sensitivity than the rap-1a and rap-1c genes, respectively. The rap-1a gene was found to be 5 times more sensitive than the rap-1c gene, while the B. bovis msa-2b and msa-2c genes exhibited similar DS. The positive frequencies of naturally infected calves for the target cybmt, msa-2b, and msa-2c genes (B. bovis) were: 100%, 33.3% and 50%, while cybmt, rap-1a, and rap-1c genes (B. bigemina) were 90%, 83.3%, and 63.3%, respectively. This study may contribute to the selection of suitable genes for molecular monitoring of bovine babesiosis. Mitochondrial genes could be considered as an alternative to improve the sensitivity of B. bovis and B. bigemina detection using qPCR.
AB - Molecular assays have been widely used for the detection and quantification of bovine babesiosis due to their high sensitivity and specificity. However, variations in the sensitivity of pathogen detection may occur depending on the selected target gene. Thus, this study aimed to compare the detection sensitivity (DS) of Babesia bovis and B. bigemina infection levels in artificially and naturally infected cattle using quantitative PCR (qPCR) and six target genes. For B. bovis, the merozoite surface antigen genes 2b and 2c (msa-2b and msa-2c), and the mitochondrial cytochrome b gene (cybmt) were used. For B. bigemina, the genes encoding the proteins associated with rhoptry 1c (rap-1c), rap-1a, and cybmt were used. Six bovines, free of babesiosis, were artificially infected with 1 × 10−8 red blood cells infected (iRBC) with B. bovis (n = 3) or 1 × 10−6 B. bigemina iRBC (n = 3). The animals were evaluated daily until parasitemia was confirmed (≥ 2.0%). The quantity of iRBC present in each animal was determined by examining blood smears. Blood samples were then subjected to DNA extraction, serial dilution, and qPCR analysis to determine the DS of each target gene. In addition, 30 calves naturally infected by Babesia spp. were also evaluated using the same six target genes. Regarding the artificial infection, B. bovis cybmt showed 25-fold higher sensitivity than the msa-2b and msa-2c genes, while the B. bigemina cybmt exhibited 5-fold and 25-fold higher sensitivity than the rap-1a and rap-1c genes, respectively. The rap-1a gene was found to be 5 times more sensitive than the rap-1c gene, while the B. bovis msa-2b and msa-2c genes exhibited similar DS. The positive frequencies of naturally infected calves for the target cybmt, msa-2b, and msa-2c genes (B. bovis) were: 100%, 33.3% and 50%, while cybmt, rap-1a, and rap-1c genes (B. bigemina) were 90%, 83.3%, and 63.3%, respectively. This study may contribute to the selection of suitable genes for molecular monitoring of bovine babesiosis. Mitochondrial genes could be considered as an alternative to improve the sensitivity of B. bovis and B. bigemina detection using qPCR.
KW - Babesiosis
KW - Detection
KW - qPCR
KW - Quantification
KW - sensitivity
UR - http://www.scopus.com/inward/record.url?scp=85182225661&partnerID=8YFLogxK
U2 - 10.1016/j.rvsc.2023.105122
DO - 10.1016/j.rvsc.2023.105122
M3 - Article
C2 - 38194893
AN - SCOPUS:85182225661
SN - 0034-5288
VL - 168
JO - Research In Veterinary Science
JF - Research In Veterinary Science
M1 - 105122
ER -