Fingerprint of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia, using RAPD-PCR

Abstract

In the time schistosomisis control programs are implemented in many countries, schistosomiasis continues to spread throughout the world. Among these control strategies is the vector control. Within this context, analysis of the genetic variability of the intermediate host snails is important because it allows identification of specific sequences of the genome of this mollusk related to determine their fingerprint. We investigated Biomphalaria arabica, which is found in Saudi Arabia, the intermediate host of Schistosoma mansoni infection. Genetic fingerprint was studied by RAPD-PCR using our own different random primers as well as published primers. The electrophoretic patterns resulting from amplification showed specific polymorphic markers of B. arabica. This information will be helpful in the identification of the snails and demonstrating that RAPD-PCR is an appropriate and efficient methodological approach for establishment of genetic barcode development.

Introduction

Schistosomiasis is a water-based disease which is considered the second most important parasitic infection after malaria in terms of public health and economic impact. The signs following infection are rashes or itchy skin. Two months after infection, fever, chills, cough and muscle aches may occur, as the parasites mature. Untreated infections can result in blood in urine and stools, and enlarged liver and spleen. Schistosomiasis infection in humans, the definitive hosts, is caused by three main species of flatworm, namely Schistosoma haematobium, Schistosoma japonicum, and Schistosoma mansoni. Schistosomiais is prevalent in tropical and sub-tropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Of the 207 million people with schistosomiasis, 85% live in Africa. Schistosomiasis is endemic in 76 countries, most of which are in Africa. Other regions affected are the Americas (Brazil, Suriname and Venezuela, as well as several Caribbean islands); the Eastern Mediterranean (Islamic Republic of Iran, Iraq, Saudi Arabia, Syrian Arab Republic and Yemen; and eastern Asia (Cambodia, China, Indonesia, Japan, Lao People's Democratic Republic and the Philippines (WHO, 2010).

According to the Health Statistical Year Book of the Ministry of Health in Saudi Arabia (2008) the prevalence rate of bilharziasis in the Kingdom of Saudi Arabia (KSA) was 2.78/100,000; 24.6% of infected persons suffer from urinary schistosomiasis while 75% suffer from intestinal schistosomiasis; 0.4% have combined infections. Infection was 55.5% among Saudi individuals in comparison to 44.5% among non-Saudi individuals. According to geographical regions of KSA, urinary schistosomiasis was recorded in Jazan and Asser; intestinal schistosomiasis was recorded in Ta'if, Al-Bahah, Asser and Bishah and co-infections were recorded in Asser. Biomphalaria arabica snail acts as the intermediate host for S. mansoni in Saudi Arabia. For S. haematobium, on the other hand, three species of snails namely, Bulinus truncates, Bulinus beccarii and Bulinus wrighti, have been incriminated as intermediate hosts (Arfaa, 1976).

Various molecular approaches have been investigated for the identification and characterization of fresh water snail including: the examination of variation in ribosomal RNA (rRNA) genes, as determined by conventional restriction fragment length polymorphism (RFLP) analysis and by PCR-RFLP of the internal transcribed spacer (ITS); the use of randomly amplified polymorphic DNA (RAPDs) and sequence analysis of mitochondrial genes (El-Khayat et al., 2008, Hanelt et al., 2008, Ittiprasert et al., 2010, Lockyer et al., 2007, Pepe et al., 2009, Rollinson et al., 1998). The rRNA gene complex is common to all eukaryotes and is well suited for taxonomic studies as it contains regions which evolve at different rates, thus permitting analysis of relationships over a wide taxonomic level. In general the spacer regions are less conserved than the coding regions (DeJong et al., 2004, Jannotti-Passos et al., 2010, Johnston et al., 1993, Kane and Rollinson, 1994, Rollinson and Kane, 1991, Simpson et al., 1984, Stothard et al., 1996). Among the genetic markers used so far in studies on Biomphalaria, the nuclear ribosomal DNA (rDNA) internal transcribed spacers (ITS) have been shown to be particularly useful for species identification and phylogenetic reconstruction (Campbell et al., 2000, Pointier et al., 2005, Vidigal et al., 2000a).

Unlike conventional PCR-based analyses, RAPD approaches use single oligonucleotide primers of arbitrary sequence (between 5 and 20 bases) to initiate DNA strand synthesis under conditions of low stringency at a number of complementary binding sites scattered throughout the genome (Oliveira et al., 2008, Oliveira et al., 2010, Theron et al., 2004, Welsh and McClelland, 1990, Williams et al., 1999). Discrete amplification products from where primer sites are orientated in an inverted repeat and are within an amplifiable distance of each other. Several amplification fragments may be generated within a single RAPD reaction and, when visualized by either agarose or polyacrylamide electrophoresis separation, give rise to a RAPD profile.

Species of planorbid snails belonging to the genus Biomphalaria, among which the vectors of S. mansoni, have received great attention in molecular systematic and phylogeography studies (Campbell et al., 2000, DeJong et al., 2001, Mavárez et al., 2002, Pointier et al., 2005, Vidigal et al., 2000b). These studies have largely supported the pivotal body of work on the taxonomy of Neotropical Biomphalaria carried out by malacologist Lobato Paraense (Paraense, 2001) and, additionally, uncovered interesting patterns of intraspecific variability (Mavárez et al., 2002, Vidigal et al., 2004).

The present work aimed to develop a DNA fingerprint for B. arabica, the intermediate host of S. mansoni in Saudi Arabia, using RAPD-PCR.