Differentiating Schistosoma haematobium from Schistosoma magrebowiei and other closely related schistosomes by polymerase chain reaction amplification of a species specific mitochondrial gene

Abstract

Introduction: Schistosoma haematobium infection afflicts about 150 million people in 53 countries in Africa and the Middle East. In many endemic areas, S. haematobium is sympatric with Schistosoma bovis, Schistosoma mattheei, Schistosoma curassoni, Schistosoma intercalatum and Schistosoma magrebowiei, its closely related species. In addition, they also develop in the same intermediate snail hosts. Since these schistosome species often infect snails inhabiting the same bodies of water, examining cercariae or infected snails for estimating transmission of S. haematobium is always confounded by the need to differentially identify S. haematobium from these other species. Recently, differentiating S. haematobium by polymerase chain reaction (PCR) from S. bovis, S. mattheei, S. curassoni and S. intercalatum, but not from S. magrebowiei was reported. However, to be able to evaluate residual S. haematobium transmission after control interventions in areas where S. haematobium may be sympatric with S. magrebowiei, a differential tool for accurate monitoring of infected snails is needed.

Materials and methods: Thus in this study, we developed a new PCR assay using a pair of primers, ShND-1/ShND-2, to amplify a target sequence of 1117 bp (GenBank accession number KF834975) from S. haematobium mitochondrion complete genome (GenBank accession number DQ157222). Sensitivity of the assay was determined by PCR amplification of different concentrations of S. haematobium gDNA serially diluted from 10ng to 0.1pg. For assay specificity, different concentrations of gDNA from S. haematobium and the other schistosome species, 20 positive urine samples and five controls as well as 20 infected snails were subjected to PCR amplification, while some of the PCR products were sequenced.

Results: The assay detected up to 1pg of S. haematobium gDNA, while a differential identification of S. haematobium DNA content from other closely related species was achieved when applied to urine and naturally infected snails. When a protein-protein blast search was carried out using Blastp, the amplified sequence was found to encode a protein that shows a 100% similarity with S. haematobium nicotinamide adenine dinucleotide dehydrogenase subunit 3 (GenBank accession number YP_626524.1).

Conclusion: The PCR assay was sensitive, specific and was able to successfully differentiate S. haematobium from S. magrebowiei, in addition to its other closely related animal infective schistosome species.

Keywords: Differential identification; Schistosoma haematobium; Schistosoma magrebowiei; nicotinamide adenine dinucleotide subunit 3 gene; polymerase chain reaction assay.

Figure 1

Detection sensitivity by ShND-1/ShND-2 polymerase chain reaction using different concentrations of Schistosoma haematobium genomic deoxyribonucleic acid: Lane 1: Deoxyribonucleic acid (DNA) size marker (Bioneer 25/100 bp Mixed DNA Ladder); lane 2: 10 ng; lane 3: 1 ng; lane 4: 0.1 ng; lane 5: 10 pg; lane 6: 1 pg; lane 7: 0.1 pg; lane 8: Negative control (no DNA)

Figure 2

Detection specificity by ShND-1/ShND-2 polymerase chain reaction. Lane 1: Deoxyribonucleic acid (DNA) size marker (Bioneer 25/100 bp Mixed DNA Ladder); lanes 2 and 3: 1 ng and 0.1 ng of Schistosoma haematobium DNA; lanes 4 and 5: 10 ng and 1 ng of Schistosoma magrebowiei DNA; lanes 6 and 7: 10 ng and 1 ng of Schistosoma bovis DNA; lanes 8 and 9: 10 ng and 1 ng of Schistosoma mattheei DNA; lanes 10 and 11: 10 ng and 1 ng of Schistosoma curassoni DNA; lanes 12 and 13: 10 ng and 1 ng of Schistosoma intercalatum DNA; lane 14: Negative control

Figure 3

Agarose gel stained with ethidium bromide showing the infection status of 10 Bulinus truncatus snails. Lane 1: Deoxyribonucleic acid (DNA) size marker (Solis BioDyne 100 bp DNA Ladder); lanes 2, 3 and 12: Uninfected snails; lanes 4-11: Snails naturally infected with Schistosoma haematobium; lane 13: Negative control (no DNA)