Response to Esteve-Gassent et al.: flaB sequences obtained from Texas PCR products are identical to the positive control strain Borrelia burgdorferi B31

Abstract

Feria-Arroyo et al. had reported previously that, based on PCR analysis, 45% of Ixodes scapularis ticks collected in Texas and Mexico were infected with the Lyme disease spirochete Borrelia burgdorferi (Parasit. Vectors 2014, 7:199). However, our analyses of their initial data (Parasit. Vectors 2014, 7:467) and a recent response by Esteve-Gassent et al. (Parasit. Vectors 2015, 8:129) provide evidence that the positive PCR results obtained from both ribosomal RNA intergenic sequences and the flagellin gene flaB are highly likely due to contamination by the B. burgdorferi B31 positive control strain.

Fig. 1

Alignment of flaB sequences. The flaB Texas specimen PCR product sequences obtained by Esteve-Gassent et al. [4] with the corresponding flaB regions of the B. burgdorferi B31 Genome (AE000783 and CP009656), the variant B31 flaB sequence X15661, the B. burgdorferi N40 Genome (CP002228), and B. burgdorferi 297 flaB (AB035616) are shown. The Texas specimen sequences are identical to the B31 sequence except for one nt difference at nt 64 in sample MMWMA69-70 and apparent sequence errors near the ends of other sequences. The location of the cytosine (C) difference reported by Esteve-Gassent et al. [4] is at nt 67 of the X15661 sequence. The GenBank accession numbers for the Esteve-Gassent et al. sequences are as follows (in parentheses): BWTX12-16 (KM875668.1); BW17 (KM875669.1); BWTX24 (KM875670.1); GEWMA9 (KM875671.1); GEWMA12 (KM875672.1); LPWMA14-15 (KM875673.1); MMWMA69-70 (KM875674.1); MMWMA80 (KM875675.1)

Fig. 2

Comparison of phylogenetic trees. a Our reconstruction of the phylogenetic tree of flaB nucleotide sequences from 8 Texas specimens, the B31 genome, and the variant B31 flaB X15661. The tree was prepared using sequences trimmed to equal length by removing the first 4 positions and the last 2 positions of the aligned sequences in Fig. 1. The tree is consistent with 100 % identity between 7 of 8 Texas specimens with the B31 sequence. MMWMA69-10 and B31_X15661 each have one nucleotide difference. b Fig. three-a in the Esteve-Gassent et al. response [4], in which reverse complement sequences for B. burgdorferi N40 and BWTX12-16, as well as sequence ends that were not trimmed to the same length, were apparently utilized. Note the difference in the scale bars