Development and Evaluation of a Semi-Nested PCR Method Based on the 18S ribosomal RNA Gene for the Detection of Babesia aktasi Infections in Goats

Abstract

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10^-8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity.

Keywords: 18S ribosomal RNA gene; Babesia aktasi; goats; semi-nested PCR; specific primers.

Figure 1

Schematic representation of the DNA-binding regions for the primers Ba600F, Ba1420R, and Ba1019R1 selected for the detection of B. aktasi by semi-nested PCR assay. The primer selection was based on multiple alignment results using the B. aktasi 18S rRNA gene sequence (MN559399) and various Babesia sequences registered in GenBank.

Figure 2

Specificity of semi-nested PCR. Agarose gel electrophoresis of semi-nested PCR products (438 bp) from Babesia- and Theileria-positive reference controls and field DNA samples using B. aktasi-specific primers. M, 100 bp marker; lines 1 and 2, negative controls (1, PCR-grade water; 2, genomic DNA obtained from a one-month-old goat not infected with Babesia, Theileria, or Anaplasma species); lines 3–11, standard positive-control DNA samples (3, B. aktasi; 4, B. ovis; 5, B. motasi; 6, B. crassa; 7, B. divergens; 8, B. venatorum; 9, B. capreoli; 10, T. ovis; 11, T. annulata); lines 12–14, field DNA samples collected from apparently healthy goats infected with B. aktasi.

Figure 3

Multiple sequence alignment of three B. aktasi sequences (Seq1–3) obtained from the semi-nested PCR assay aligned with B. aktasi reference sequences (MN559399 and OM864353) available in GenBank.

Figure 4

Sensitivity of semi-nested PCR. Agarose gel image of the PCR amplicons from DNA isolated from 10-fold serial dilutions (10⁻¹ to 10⁻¹⁰) of infected blood containing 2% parasitemia. M, 100 bp marker; lanes 1–2, standard negative controls (1, PCR-grade water; 2, genomic DNA obtained from a one-month-old goat not infected with Babesia, Theileria, or Anaplasma species); 3, positive control (B. aktasi, GenBank accession number MN559399); 4, B. aktasi DNA isolated from 2% parasitic blood; lanes 5–14, DNA samples isolated from 10-fold dilution series ranging from 10⁻¹ to 10⁻¹⁰.