Abbasi et al. Parasites & Vectors (2017) 10:364 DOI 10.1186/s13071-017-2281-7 RESEARCH Open Access The substructure of three repetitive DNA regions of Schistosoma haematobium group species as a potential marker for species recognition and interbreeding detection Ibrahim Abbasi1,2, Bonnie L. Webster3,4,5, Charles H. King6, David Rollinson3,4,5 and Joseph Hamburger1* Abstract Background: Schistosoma haematobium is the causative agent of human urogenital schistosomiasis affecting ~112 million people in Africa and the Middle East. The parasite is transmitted by snails of the genus Bulinus, which also transmit other closely related human and animal schistosomes. The accurate discrimination of S. haematobium from species infecting animals will aid effective control and elimination programs. Previously we have shown the utility of different repetitive nuclear DNA sequences (DraI, sh73bp, and sh77bp) for the identification of S. haematobium- group species and inter-repeat sequences for discriminating S. haematobium from S. bovis. Results: In this current study we clarify the structural arrangement and association between the three repetitive sequences (DraI, sh73bp, and sh77bp) in both S. haematobium and S. bovis, with a unique repeat linker being found in S. haematobium (Sh64bp repeat linker) and in S. bovis (Sb30bp repeat linker). Sequence data showed that the 3′- end of the repeat linker was connected to the DraI repetitive sequence array, and at the 5′-end of the repeat linker sh73bp and sh77bp were arranged in an alternating manner. Species-specific oligonucleotides were designed targeting the species-specific repeat linkers and used in a reverse line blot (RLB) hybridization assay enabling differentiation between S. haematobium and S. bovis. The assay was used to discriminate natural infections in wild caught Bulinus globosus. Conclusion: This research enabled the characterisation of species-specific DNA regions that enabled the design of species-specific oligonucleotides that can be used to rapidly differentiate between S. haematobium and S. bovis and also have the potential to aid the detection of natural hybridization between these two species. Keywords: Schistosoma haematobium, Schistosoma bovis, Hybridisation, DraI, Inter-repeat linkers, Reverse line blot (RLB) Background in humans is caused by infection with Schistosoma hae- Schistosomiasis is a parasitic disease prevalent in tropical matobium, affecting ~112 million people in Africa and the and subtropical regions caused by blood flukes of the Middle East, and another 436 million individuals are con- genus Schistosoma [1]. In 2012, about 42 million people sidered at risk of infection [1, 2]. The parasites reside in were treated with the anti-schistosomal drug praziquantel, the blood vessels surrounding the bladder and eggs are although in 2014 it was estimated that at least 258 million released in the urine of humans. Infection takes place in people required treatment [2]. Urogenital schistosomiasis freshwater that contains Bulinus species that serve as the snail intermediate hosts for the parasite [1]. * Correspondence: [email protected] Schistosoma haematobium is a member of a group of 1Department of Microbiology and Molecular Genetics, The Institute for closely related schistosomes known as the S. haemato- Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious bium-group schistosomes. The other species of this and Tropical Diseases, The Hebrew University - Hadassah Medical School, The Hebrew University of Jerusalem, 91120 Jerusalem, Israel group all cause intestinal schistosomiasis. Schistosoma Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abbasi et al. Parasites & Vectors (2017) 10:364 Page 2 of 9 intercalatum and S. guineensis infect humans in isolated diagnostic PCR for the discrimination of S. haemato- foci in central Africa, but how many people are infected bium from S. bovis [7, 9]. and where transmission occurs is still unknown. Schisto- In this study we further elucidated the arrangement of soma margrebowiei, S. leiperi, S. mattheei, S. curassoni the three repetitive DNA sequences, DraI, sh73bp, and and S. bovis are parasites of domestic livestock and wild sh77bp and their inter-repeat sequences, to further en- ungulates, mainly in Africa, with major veterinary and able the differential identification of S. haematobium economic impact, but these species are not widely and S. bovis. The differences in the repeat arrangements researched [3, 4]. All S. haematobium group species were used to develop a new detection approach based utilize Bulinus spp. for transmission. As the infective on direct PCR and reverse line blot analysis that can be cercariae that emerge from the snail cannot be identified used for schistosome species-specific identification. The easily by morphological examination there is a need to potential use of this new molecular diagnostic tool is provide reliable molecular markers to differentiate spe- discussed in relation to the monitoring of schistosomia- cies. This is particularly important in endemic areas sis transmission and also to help elucidate the natural targeted for transmission control wherever the S. hae- and on-going hybridization of S. haematobium and S. matobium-group species co-exist [4, 5]. Transmission of bovis in sympatric areas of West Africa [18–20]. S. bovis (a common cattle schistosome) most commonly overlaps with that of S. haematobium, and these two species are reported to be co-prevalent in many parts of Methods Africa and the Middle East [6]. The other S. haemato- Origin of the schistosome and snails samples bium-group species also co-exist with S. haematobium For assay development, adult schistosome worms, S. in specific foci in various parts of Africa [6]. haematobium (Mauritius NHM2695) and S. bovis The need to identify which S. haematobium group (Senegal NHM196), were provided by the Schistosomia- species are transmitted by different Bulinus snails has sis Collection at the Natural History Museum, London led to the development of several DNA-based molecular [21]. For assay testing, Bulinus snails were collected from methods for species-specific identification [7–9]. Mo- Katchetu pond, an endemic area near the Mombasa- lecular methods have included: Southern blot analysis Nairobi highway in Katchetu village, Mazeras, Kenya. This [10], random DNA amplification [11], PCR-RFLP ana- pond has been known to contain significant numbers of lysis of the ITS2 region of the ribosomal gene [12] and Bulinus globosus snails infected with schistosomes. Goats, direct PCR amplification using species-specific primers cattle, and humans frequently visit the pond for daily targeting multi-copy gene regions such as the mitochon- activities. Snails were collected by scooping, morphologic- drial cytochrome oxidase subunit 1 [13] and amplifica- ally identified as Bulinus spp. and immediately preserved tion of genomic DNA repetitive fragments [7, 9]. A in 100% ethanol for molecular identification of Schisto- commonly used repetitive DNA segment identified in S. soma infections. haematobium is the DraI repeat [14]. This tandemly ar- ranged repeat sequence has been utilized in Schistosoma detection in Bulinus snails to identify patent and pre- DNA extraction patent infections and has enabled the screening of large DNA was extracted from the adult worms by digestion populations of snails to evaluate the level of transmission in 0.5 ml of lysis buffer (0.1 M EDTA, pH 8.0, 0.1 M [15]. The DraI repeat has also been used for the molecu- Tris-HCl pH 7.5, 0.2 M NaCl, 1% SDS, 0.2% 2- lar diagnosis of S. haematobium infection by amplifica- mercaptoethanol and 100 μg Proteinase K) at 60 °C for tion of parasite DNA in human urine samples [16]. The 2 h followed by routine phenol extraction and ethanol DraI repeat offers high detection sensitivity but lacks precipitation method. Snails were removed from their specificity due to cross-amplification with the other S. shells and their whole bodies macerated in 0.5 ml of lysis haematobium-group species [14], hampering its diagnos- buffer (0.1 M EDTA, pH 8.0, 0.1 M Tris-HCl pH 7.5, tic utility in sympatric areas and hybrid zones. Further 0.2 M NaCl, 1% SDS, 0.2% 2-mercaptoethanol and studies have focused on finding DNA-based methodolo- 100 μg Proteinase K) and DNA extracted as above. The gies that are specific for the different S. haematobium- extracted DNA was suspended in TE buffer (1 mM Tris- group species [9, 17]. Further repeat DNA sequences, HCl, 0.1 mM EDTA). the sh73bp and sh77bp, were targeted with an aim to find a species-specific DNA diagnostic marker for S. Amplification and sequencing of the inter repeat region haematobium. An inter-repeat PCR, utilizing the DraI (sh73bp-DraI) reverse primer and sh73bp forward primer produced a PCR reactions were carried out in a total volume of differential DNA banding pattern for S. haematobium 25 μl using ready-mix PCR tubes (Syntezza, Jerusalem, (compared to that of S. bovis) providing a new one-step Israel). Each reaction contained 20 pmoles of each Abbasi et al. Parasites & Vectors (2017) 10:364 Page 3 of 9 forward (73 bp; 5′-CCT TGG TCA CGT GAT TTT C- Reverse line blot analysis 3′) and reverse primer (DraI; 5′-TCA CAA CGA TAC The technique was performed as previously described GAC CAA CC-3′) and 5 μlofS. haematobium or S. [22, 23], involving two main steps, as follows: bovis genomic DNA or snail-extracted DNA.