Detection and quantification of Babesia bovis and Babesia bigemina using different target genes

Rodrigo Giglioti; Anibal Eugênio Vercesi Filho; Ana Gonçalves Domingos; Sérgio Silva da Silva; Rodrigo Casquero Cunha; Adriana Mércia Guaratini Ibelli; Cintia Hiromi Okino; Márcia Cristina de Sena Oliveira

doi:10.1016/j.rvsc.2023.105122

Detection and quantification of Babesia bovis and Babesia bigemina using different target genes

Res Vet Sci. 2024 Mar:168:105122. doi: 10.1016/j.rvsc.2023.105122. Epub 2023 Dec 26.

Authors

Rodrigo Giglioti¹, Anibal Eugênio Vercesi Filho², Ana Gonçalves Domingos³, Sérgio Silva da Silva⁴, Rodrigo Casquero Cunha⁵, Adriana Mércia Guaratini Ibelli⁶, Cintia Hiromi Okino⁶, Márcia Cristina de Sena Oliveira⁶

Affiliations

¹ Instituto de Zootecnia, Rua Heitor Penteado, n. 56, Nova Odessa, São Paulo 13380-011, Brazil. Electronic address: rodrigo.giglioti@sp.gov.br.
² Instituto de Zootecnia, Rua Heitor Penteado, n. 56, Nova Odessa, São Paulo 13380-011, Brazil.
³ Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal.
⁴ C.R.O. Animal Science, Estrada Colônia São Domingos, Colônia, Turuçú, Rio Grande do Sul, Brazil.
⁵ Laboratório de Biologia Molecular Veterinária, Faculdade de Veterinária, Universidade Federal de Pelotas, Capão do Leão, Rio Grande do Sul, Brazil.
⁶ Embrapa Pecuária Sudeste, São Carlos, São Paulo, Brazil.

PMID: 38194893
DOI: 10.1016/j.rvsc.2023.105122

Abstract

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^-6B. 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.

Keywords: Babesiosis; Detection; Quantification; qPCR; sensitivity.

MeSH terms

Animals
Babesia bovis* / genetics
Babesia* / genetics
Babesiosis* / diagnosis
Cattle
Cattle Diseases* / diagnosis
Protozoan Proteins / genetics

Substances

Protozoan Proteins