3. Results
3.1 Cloning and phylogenetic analysis B. bigemina Argentina strain was used to obtain genomic and cDNA for PCR with primers BbigAMA-1 F and R to amplify the AMA-1 gene.
The sequences had an identical length from both templates and of the estimated size according to primers' design (not shown). The nucleotide sequencing analysis presented a 1988-bp long gene (gene bank accession number: AB481200) and encoded a 595-amino acid peptide (gene bank accession number: BAH22706) (Fig. 1) with a computer-calculated molecular weight of 66.64-KDa. The protein has a signal peptide of 30 amino acids (Signal 3).
The nucleotide sequence had 98.2 % identity with the sequence reported in the B. bigemina genome sequence at Sanger institute. The …show more content…
Finally, the antiserum raised against rtBbigAMA1 reacted positively with merozoites of B. bigemina extracellular (Fig. 4A) or intracellular (Fig.4B), B. bovis (Fig. 4C), and B. microti (Fig. 4D) in IFAT. The green fluorescence of the BbigAMA1 was observed by IFAT in extracellular and intracellular stages of B. bigemina merozoites.
3.4. In vitro growth inhibition and invasion inhibition assays B. bigemina growth was significantly inhibited by anti-rtAMA-1 mouse serum (P< 0.01). Parasites in the treated cultures had a parasitemia of 0.56 % on day 3 while control cultures treated with anti-GST serum and normal bovine serum had 4 % and 6.5 %, respectively (Fig. 5A). The effect of anti-rtAMA-1 mice serum on merozoite invasion was evaluated. Extracellular merozoites were significantly higher (P< 0.01) with anti-rtAMA-1 mice serum than the anti-GST and the control sera (Fig. 5B). The invasion inhibition was 57.8 %, 67.8 %, and 81.2
% at 1, 6, and 24 hours post-invasion, respectively (Fig. 5C). 3.4. Vaccination and challenge infection To test the efficacy of rtBbigAMA1 as a potential vaccine, all mice were vaccinated with rtBbigAMA1 and GST. Fifteen days next to last immunization, mice were intraperitoneally infected with 1 × 107 B. microti-infected
3.1 Cloning and phylogenetic analysis B. bigemina Argentina strain was used to obtain genomic and cDNA for PCR with primers BbigAMA-1 F and R to amplify the AMA-1 gene.
The sequences had an identical length from both templates and of the estimated size according to primers' design (not shown). The nucleotide sequencing analysis presented a 1988-bp long gene (gene bank accession number: AB481200) and encoded a 595-amino acid peptide (gene bank accession number: BAH22706) (Fig. 1) with a computer-calculated molecular weight of 66.64-KDa. The protein has a signal peptide of 30 amino acids (Signal 3).
The nucleotide sequence had 98.2 % identity with the sequence reported in the B. bigemina genome sequence at Sanger institute. The …show more content…
Finally, the antiserum raised against rtBbigAMA1 reacted positively with merozoites of B. bigemina extracellular (Fig. 4A) or intracellular (Fig.4B), B. bovis (Fig. 4C), and B. microti (Fig. 4D) in IFAT. The green fluorescence of the BbigAMA1 was observed by IFAT in extracellular and intracellular stages of B. bigemina merozoites.
3.4. In vitro growth inhibition and invasion inhibition assays B. bigemina growth was significantly inhibited by anti-rtAMA-1 mouse serum (P< 0.01). Parasites in the treated cultures had a parasitemia of 0.56 % on day 3 while control cultures treated with anti-GST serum and normal bovine serum had 4 % and 6.5 %, respectively (Fig. 5A). The effect of anti-rtAMA-1 mice serum on merozoite invasion was evaluated. Extracellular merozoites were significantly higher (P< 0.01) with anti-rtAMA-1 mice serum than the anti-GST and the control sera (Fig. 5B). The invasion inhibition was 57.8 %, 67.8 %, and 81.2
% at 1, 6, and 24 hours post-invasion, respectively (Fig. 5C). 3.4. Vaccination and challenge infection To test the efficacy of rtBbigAMA1 as a potential vaccine, all mice were vaccinated with rtBbigAMA1 and GST. Fifteen days next to last immunization, mice were intraperitoneally infected with 1 × 107 B. microti-infected