Differentiating Schistosoma haematobium from related animal schistosomes by PCR amplifying inter-repeat sequences flanking newly selected repeated sequences

Abstract

In schistosomiasis elimination programs, successful discrimination of Schistosoma haematobium from the related animal Schistosoma parasites will be essential for accurate detection of human parasite transmission. Polymerase chain reaction assays employing primers from two newly selected repeated sequences, named Sh73 and Sh77, did not discriminate S. haematobium when amplifying Sh73-77 intra- or inter-repeats. However, amplification between Sh73 and the previously described DraI repeat exhibited discriminative banding patterns for S. haematobium and Schistosoma bovis (sensitivity 1 pg and 10 pg, respectively). It also enabled banding pattern discrimination of Schistosoma curassoni and Schistosoma intercalatum, but Schistosoma mattheei and Schistosoma margrebowiei did not yield amplicons. Similar inter-repeat amplification between Sh77 and DraI yielded amplicons with discriminative banding for S. haematobium, and S. bovis; however, S. mattheei was detected only at low sensitivity (1 ng). The Sh73/DraI assay detected snails infected with S. haematobium, S. bovis, or both, and should prove useful for screening snails where discrimination of S. haematobium from related schistosomes is required.

Figure 1.

Successful species discrimination using inter-repeat polymerase chain reaction (PCR) amplification with reverse primer from Schistosoma haematobium DraI repeat, and direct primer from newly selected Sh73 repeat (4297-73d, panel A), or direct primer from Sh77 repeat (4297-77d, panel B). The amplification assay at different concentrations of S. haematobium genomic DNA is shown (lane 1: 10 ng; lane 2: 1 ng; lane 3: 0.1 ng; lane 4: 0.01 ng; lane 5: 1 pg), Schistosoma bovis DNA (lane 6: 10 ng; lane 7: 1 ng), and Schistosoma mattheei DNA (lane 8: 10 ng; lane 9: 1 ng). Lanes 10–11: negative controls, M: DNA size marker. Arrows point to specific bands of S. haematobium (Sh) and Schistosoma bovis (Sb).

Figure 2.

Comparison of polymerase chain reaction (PCR) amplicon banding patterns and sensitivity for Schistosoma haematobium and Schistosoma bovis genomic DNA using the new inter-repeat approach (primers 4297-73d). The amplification reactions shown here used different concentrations of S. haematobium genomic DNA (lane 1: 10 ng; lane 2: 1 ng; lane 3: 0.1 ng; lane 4: 0.01 ng; lane 5: 1 pg; 6: 0.1 pg), or S. bovis genomic DNA (lane 7: 10 ng; lane 8: 1 ng; lane 9: 0.1 ng; lane 10: 0.01 ng; lane 11: 1 pg). Lane 13: No DNA (negative control) and M: DNA size marker. Arrows point to specific bands of S. haematobium (Sh) and S. bovis (Sb).

Figure 3.

Inter-repeat polymerase chain reaction (PCR) amplification using DraI reverse primer and 73 direct primers at different concentrations of Schistosoma haematobium genomic DNA (lane 1: 0.1 ng; pg). Lanes 8–11, represent PCR amplification of “mixed target DNA” composed of 1 pg S. haematobium genomic DNA with different added concentrations of Schistosoma bovis genomic DNA (lane 8: 1 ng; lane 9: 0.1 ng; lane 10: 10 pg; lane 11: 1 pg). Lanes 12–15: show negative PCR amplification results for extracts from different uninfected Bulinus snails. Lane 16: No DNA (negative control), and M: DNA size marker.

Figure 4.

Inter-repeat polymerase chain reaction (PCR) amplification using DraI reverse primer and 73 direct primer targeting different Bulinus snails (1–21) that were collected from a region co-endemic for Schistosoma haematobium and Schistosoma bovis. M: DNA size marker.

Figure 5.

Inter-repeat polymerase chain reaction (PCR) amplification using DraI reverse primer and 73 direct primer for different species of terminal spined schistosomes. Lane 1: Schistosoma curassoni, 10 ng; lane 2: Schistosoma margrebowiei 10 ng; lane 3: Schistosoma intercalatum 10 ng; lane 4: S mattheei, 10 ng; lane 5: Schistosoma bovis, 10 ng; lane 6: Schistosoma haematobium 1 ng; lane 7: S. haematobium 0.1ng; lane 8: no DNA. M: DNA size marker.