Saudi Journal of Biological Sciences (2014) 21, 65–70

King Saud University Saudi Journal of Biological Sciences

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Development of -specific primers for identification of arabica, the intermediate host of mansoni in Saudi Arabia

Saleh A. Al-Quraishy a,*, Saad M. Bin Dajem b, Osama M. Mostafa b,c, Essam H. Ibrahim b,d, Ahmed Al-Qahtani e a Zoology Department, College of Science, King Saud University, Saudi Arabia b Biology Department, Faculty of Science, King Khaled University, Abha P.O. Box 9004, Saudi Arabia c Zoology Department, Faculty of Science, Ain Shams University, Abbassia 11566, Cairo, Egypt d Blood Products Quality Control and Research Department, National Organization for Research and Biological Control, Cairo, Egypt e Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia

Received 30 April 2013; revised 26 May 2013; accepted 1 October 2013 Available online 11 October 2013

KEYWORDS Abstract is mediated through the intermediate host Biomphalaria arabica Biomphalaria arabica; which lives in Saudi Arabia. Molecular characterization and identification of this intermediate host Schistosoma mansoni; are important for epidemiological studies of schistosomiasis. The present work aimed to determine RAPD–PCR; the molecular variations among the populations of B. arabica found in Southern part of Saudi Genetic variability; Arabia, and to develop species-specific primers for identification of these snails as a first step in Species-specific primer; the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infec- Saudi; tions in a single step. Five populations of Saudi B. arabica snails were collected from freshwater Genomic DNA inter/intra- bodies. Three populations were collected from Asser and two populations were collected from specific variation AL-Baha. Genomic DNA was extracted from snails and was amplified using five different RAPD–PCR primers. The banding patterns of amplified materials by primers P1 and P5 were identical in all populations. However, the rest primers displayed intra-specific differences among populations with variable degrees. Largest sizes of RAPD–PCR products were cloned into TA

* Corresponding author. Tel.: +966 (1) 4675754. E-mail address: [email protected] (S.A. Al-Quraishy). Peer review under responsibility of King Saud University.

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1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2013.10.001 66 S.A. Al-Quraishy et al.

cloning vector as a preparatory step for DNA sequence analysis. After sequencing, similarity searches of obtained DNA sequences revealed that there are no similar sequences submitted to gene- bank data bases and its associated banks. The results obtained will be helpful in the development of simultaneous identification of B. arabica snails and diagnosis of S. mansoni infection within it in a single step by an implementation of multiplex PCR. ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction et al., 1998; Spatz et al., 1999; Vidigal et al., 2000a). Vidigal et al. (2000a) characterized Biomphalaria snails using the According to the Health Statistical Year Book of Ministry of PCR–RFLP and found three variant molecular profiles for the Health in Saudi Arabia (2008) the prevalence rate of Biomphalaria amazonica in specimens from the same or differ- bilharziasis in the Kingdom of Saudi Arabia (KSA) is 2.78/ ent localities. Thereby, considerable attention has been given 100,000; 24.6% of the population suffers from urinary schisto- to the molecular identification. Such intraspecific variation somiasis while that of the intestinal schistosomiasis is 75% and was further studied by Vidigal et al. (2000b) and Dejong the combined infection represented 0.4%. Schistosomiasis et al. (2001). Nevertheless, Teodoro (2009), using higher num- cases of 55.5% were discovered among Saudi individuals and ber of snails, suggested that this diversity should be the result 44.5% were among non-Saudi individuals. According to of the occurrence of two species instead of one. Among the ge- geographical regions of KSA, urinary schistosomiasis was netic markers used so far in studies on Biomphalaria, the ITS- recorded in Jazan and Asser; intestinal schistosomiasis was rDNA and the mitochondrial DNA (mt rDNA) have been recorded in Ta’if, Al-Bahah, Asser and Bishah and co-infections shown to be particular useful for phylogenetic reconstruction were recorded in Asser. between closely related species (Vidigal et al., 2000b; DeJong There are 34 identified species of the genus Biomphalaria et al., 2001; Caldeira et al., 2010; Jannotti-Passos et al., (: ) in Africa and in the Neotropic region, 2010; Teodoro et al., 2010). In addition tRNA and rRNA out of which , , are used for Biomphalaria identification (Jannotti-Passos , Biomphalaria prona, Biomphalaria et al., 2010). pfeifferi, , , Genetic markers obtained by randomly amplified polymor- Biomphalaria choanomphala, Biomphalaria camerunensis, phic DNA (RAPD) have been widely used in studies on intra- Biomphalaria stanley and Biomphalaria arabica are regarded and inter-populational genetic variability of several organisms as intermediate hosts of the trematode Schistosoma mansoni, (Oliveira et al., 2008, 2010). RAPD has been used in studies of the aethiological agent of human intestinal schistosomiasis genetic variability of molluscan populations and have been (Arfaa, 1976; Malek, 1958). used in development of species-specific markers. It was found that the snail Biomphalaria arabica acts as the The present work aimed to determine the genetic variability intermediate host for S. mansoni in Saudi Arabia (Arfaa, among different populations of B. arabica snails and to devel- 1976); for Schistosoma haematobium, on the other hand, three op a species-specific primer for the identification of these snails species of snails namely, Bulinus truncates, Bulinus beccarii and as a primary step in simultaneous identification of B. arabica Bulinus wrighti, have been incriminated as intermediate hosts snails and diagnosis of S. mansoni infection within it by an (Arfaa et al., 1989). However, in spite of the importance of implementation of multiplex PCR. B. arabica in the epidemiology of intestinal schistosomiasis in Saudi Arabia, investigations on its genetic structure have not 2. Materials and methods been developed yet. The identification of Biomphalaria species and studies on 2.1. Snails population structure of the snails have provided important information on intestinal schistosomiasis occurrence and dis- Five populations of Saudi B. arabica snails were collected tribution in different areas and regions. For a long time mala- from freshwater bodies. Three populations were collected cologists have been looking for characters that could aid in the from Asser (A, B and C) and two populations were collected specific identification of these snails (Vidigal et al., 1998). Their from AL-Baha (D and E). Snails were kept in plastic trays; morphological identification is based mainly on the reports of each tray contained 25 snails at a time in 1.5 l of dechlorinat- Paraense (1975, 1981, 1984, 1988) which use shell and ed water and was supplied with lettuce leaves (fresh or boiled reproductive organ morphology. The high variability of these and dried) for snail feeding. Trays were loosely covered with characters, the environmental influence on shell size and shape, a glass plate to reduce evaporation, and maintained at room the small size of specimens and the similarity among some temperature of 27–29 C. Water in the trays was changed species, complicate specific identification especially for every two days. non-experts (Vidigal et al., 1998). Polymerase Chain Reaction and Restriction Fragment 2.2. DNA extraction Length Polymorphism (PCR–RFLP) of the internal tran- scribed spacer region (ITS) of ribosomal DNA has been suc- cessfully used in studies on Biomphalaria snails, separating Genomic DNA extractions from Saudi B. arabica were done the majority of the species, even very similar ones (Caldeira using DNA easy Blood & Tissue Kit from QIAGEN, Development of species-specific primers for identification of Biomphalaria arabica 67 according to the protocol of Vidigal et al. (2000). DNA was ex- nucleotide BLAST (blast) option of Internet (http:// tracted from 6 to 7 individuals per population. Purified geno- www.ch.embnet.org/software/bBLAST.html). mic DNA was kept at 70 C till used. 2.9. Diagnostic specific primer designing 2.3. Randomly amplified polymorphic DNA (RAPD) Depending on DNA sequence obtained from RAPD fragment RAPD analysis was performed on purified genomic DNAs sequence analysis, specific primers have been designed to target from Saudi B. arabica snails. The procedure was carried out the B. arabica genome using Gene Runner program. These using the Ready-To-Go RAPD Analysis Kit (GE Healthcare primers were as follows: GRP2FOR1 CAA TCC TAC AAA Limited, Amersham Place Little Chalfont, Buckinghamshire), AGT CTT GAA CTA AAA and GRP2REV1 CCC GTG following protocols recommended by the manufacturer, tak- TTT GGG ATA GGT TA. In addition to B. arabica, negative ing extreme care to prevent contamination and following control samples were included. These negative controls were the protocol. The primers used in this study were: P1: genomic DNA of B. truncates and B. beccari snails. Amplifica- 50-GGT GCG GGA A-3’, P2: 50-GTT TCG CTCC-30, tion was performed using QIAGEN Fast Cycling PCR Kit P3:50-GTA GAC CCG T-30, P4:50-AAC GCG CAAC-30 and (Qiagen). Each reaction was carried out in a final volume of P5: 50-CCC GTC AGC A-30. After amplification, 10 llof 25 ll with a final concentration containing 1.0· of reaction each PCR product and 100-base pair DNA ladder (Invitro- buffer which included the concentration of 50 ng genomic gen) were run in a 1.5% agarose gel and the banding pattern DNA, 2.0 mM Magnesium Chloride, 2 units Taq DNA Poly- of the randomly amplified DNA was visualized with ethidium merase, 0.4 mM dNTP-mixture and 10 pM of each primer. bromide (0.5 lg/ml). Banding pattern was photographed Reaction mixtures were overlaid with mineral oil and cycling using gel documentation system (Quantum-Capt, Viber Lour- procedure was performed using model 480 programmable mat). Migration distance was calculated and band sizes were thermal cycler. Cycling program was 94 C for 4 min, 40 cycles determined. of 94 C for 30 s, 55 C for 30 s and 72 C for 30 s, and final extension at 72 C for 7 min. The PCR products were run in 2.4. Similarity coefficient 1.3% gel. Banding pattern was photographed using gel docu- mentation system (Quantum-Capt, Viber Lourmat). Migration The genetic variability among Saudi B. arabica populations distance was calculated and band sizes were determined. was evaluated by analyzing the electrophoretic band patterns of RAPD–PCR obtained on the gels and by determining the 3. Results similarity coefficient as described by Dice (1945). 3.1. RAPD–PCR 2.5. Purification of PCR products

The gel showed many PCR product bands in each population The largest sizes of PCR products amplified by each primer (Fig. 1i–v). It was noted that B. arabica collected from differ- were chosen to be purified from gels using QIAquick Gel ent localities showed almost the same band pattern with minor Extraction Kit from Qiagen. differences when using different RAPD primers. Primer 1 re- vealed 9 bands in each population with size ranged from 186 2.6. Cloning of RAPD fragments to 570 bp, the banding pattern was identical in all populations. Also, nine bands appeared in each population by applying pri- Cloning of purified PCR products was done using TA cloning mer 2 with size ranged from 224 to 574 bp. However, popula- kit (Invitrogen), following protocols recommended by the tion (B) lacked band of size 386 bp. Primer 3 revealed manufacturer. Recombinant plasmids were extracted using moderate variations among populations, since a band of size QIAprep Spin Miniprep Kit (Qiagen Inc., Valencia, CA, 280 bp lacked in population (A), a band of size 790 was absent USA, Cat No. 27104). in population (B), and a band of size 300 was absent in popu- lation (C). Primer 4 displayed great variations among popula- 2.7. Testing of insert presence tions, since every population appeared with a characteristic banding pattern. However, all populations shared in bands Two microliters of extracted plasmid was digested with 140, 177, 300, 345 and 410 bp. Primer 5 revealed 9 bands in 10 units of EcoR1 (Gibco BRL) at 37 C for 2 h, run in each population with size ranged from 105 to 772 bp, the 1.5% agarose gel and recombinant plasmids shown to have banding pattern was identical in all populations. The similarity the insert were isolated and sent for DNA sequence analysis. coefficients among populations as revealed by primers P3, P4 and P5 are summarized in Table 1. 2.8. DNA sequence analysis of recombinant plasmid and sequence similarity search 3.2. Cloning of RAPD fragments

DNA sequence analysis of insert present in recombinant vector Largest PCR fragments of all investigated populations were was done using the automated sequencer ABI PRISM (model used for cloning. Ligation and transformation steps were suc- 310). The sequencing primers used were M13 primer and cessful. Checking of insert presence in each cloning reaction Reverse primer. Results obtained from sequence analysis were using miniprep and restriction enzyme digestion revealed the subjected to a similarity search using standard nucleotide- presence of each corresponding insert. 68 S.A. Al-Quraishy et al.

Figure 1 RAPD–PCR products using (i) primer 1, (ii) primer 2, (iii) primer 3, (iv) primer 4 and (v) primer 5. Lane M: 100 bp DNA ladder, Lane 1: C1a DNA from E. coli; Lane 2: BL21 (DE3) DNA from E. coli; Lane 3–7: snails DNA from A–E Samples.

Table 1 The similarity coefficients among B. arabica populations as revealed by RAPD–PCR using primers P3, P4 and P5. Similarity coefficient P3 P4 P5 A BCDEABCDEABCDE A 1 0.94 1 1 1 1 0.83 0.83 0.83 0.92 1 0.62 0.55 0.60 0.57 B 0.94 1 0.94 0.94 0.94 0.83 1 0.83 0.83 0.92 0.62 1 0.78 0.77 0.73 C 1 0.94 1 1 1 0.83 0.83 1 0.83 0.92 0.55 0.78 1 0.90 0.85 D 1 0.94 1 1 1 0.83 0.83 0.83 1 0.92 0.60 0.77 0.90 1 0.95 E 1 0.94 1 1 1 0.92 0.92 0.92 0.92 1 0.57 0.73 0.85 0.95 1

AAGAGCCCGTGTTTGGGATAGGTTAAAGGCTAGATGGGTCGATTCTTGTT ATCGATTAAAACACACTCATATATTTAGTAAATTCTACATACCCTGGAGTT ATGGGCTGTGCCAATTTTACTCACGTGTCAAGCTGTGTGTATGTTTACAGT CTCAGTGTAGCTTAGTGTGTCAGATTTTCCTTTTACATGCTATGTAAACTA GTCCTCTTAGTACAGGATGCACTTCCATGAAAGCTGTTTAGAGTTAAGTTT TTAGTTCAAGACTTTTGTAGGATTGTATAGACTCTAGTTGACGGGCTCT

Figure 2 A DNA sequence of B. arabica from cloned RAPD–PCR band.

3.3. DNA sequence analysis of inserts and sequence similarity Figs. 2 and 3. Results obtained from sequence analysis were search subjected to a similarity search using standard nucleotide- nucleotide BLAST (blast) option of Internet. These se- DNA sequence analysis of insert present in recombinant vec- quences showed no similarity with all submitted sequences tor was done using the automated sequencer. We obtained in the common gene bank databases and its associated several DNA sequences. These sequences are shown in banks. Development of species-specific primers for identification of Biomphalaria arabica 69

AAGAGCCCGTCAACTAGAGTCTATACAATCCTACAAAAGTCTTGAACTAA AAACTTAACTCAAAACAGCTTTCATGGAAGTGCATCCTGTACTAAGAGGA CTAGTTTACATAGCATGTAAAAGGAAAATCTGACACACTAAGCTACACTG AGACTGTAAACATACACACAGCTTGACACGTGAGTAAAATTGGCACAGCC CATAACTCCAGGGTATGTAGAATTTACTAAATATATGAGTGTGTTTTAATC GATAACAAGAATCGACCCATCTAGCCTTTAACCTATCCCAAACACGGGCT CT

Figure 3 Another DNA sequence of B. arabica from cloned RAPD–PCR band.

Figure 5 A map of Saudi Arabia showing the two locations of Figure 4 Amplification of different genomic DNAs from Biom- sample collection, star indicates Asser region and triangle indicates phalaria arabica, Bulinus truncates and Bulinus beccari snails using AL-Baha region. specific primers. Lane M: 100 bp DNA ladder, Lane CR: control reagent, Lanes 1–5: amplification products from B. arabica from different localities (A–E), Lane 6: Bulinus truncates and Lane 7: the same snail species collected from two different climates Bulinus beccari. was studied using RAPD–PCR patterns and analysis of the RAPD–PCR products. The RAPD–PCR banding patterns revealed by primers 1 3.4. Diagnostic specific primer designing and 2 were identical in all of the five populations of Saudi B. arabica snails collected from different freshwater bodies. How- Specific primers for the detection of B. arabica were designed. ever, the rest primers displayed intra-specific differences Using these primers in specific PCR resulted in expected PCR among populations with variable degrees. These results sup- products at 272 bp single band (Fig. 4). port the previous hypotheses of Abdel-Hamid et al. (1999), Knight et al. (1999), Oliveira et al. (2008) and Oliveira et al. 4. Discussion (2010) who demonstrate that RAPD–PCR is a suitable and efficient methodological approach to the analysis of genetic In the current study we collected snails from different localities variability of schistosomiasis vectors. and province. Asser Province in southwestern Saudi Arabia DNA sequence analysis of cloned RAPD–PCR products lies in the high mountains of Asser, at an altitude of about showed no sequence similarity with any submitted genes or se- 2250 meters above sea level, and approximately 200 km from quences in the gene bank and other associated banks. Most of the northern border of Yemen (Fig. 5). It has the lowest mean the sequences found in the gene banks concentrate on rRNA annual temperature compared with any of the southern urban (Vidigal et al., 1998; Caldeira et al., 1998, 2002). The applica- areas, a low atmospheric pressure of oxygen and a high annual tion of molecular approaches continues to contribute novel in- rainfall with rain falling mainly in winter and spring. Al Baha sights into the biology, including genomics of molluscs. To city lies in the south-west of the kingdom of Saudi Arabia be- date, several mitochondrial genomes of mollusks have been se- tween the holy Makkah and Asser (Fig. 5). It is the smallest of quenced, but the nuclear genome of a representative of the the Kingdom’s provinces (11,000 square km), at an altitude Phylum Mollusca remains to be fully characterized (Adema of about 2395 m above sea level. It is surrounded by a number et al., 2006). of cities, including Taif on the north, Beesha on the east, Abha In conclusion, the molecular biological techniques used in on the south and the Red Sea coast city of Al Qunfuda on the our studies are very useful for the generation of new knowl- west. The climate in Al Baha is greatly affected by its varying edge concerning the systematic and population genetics of B. geographic features. The climate in Al-Baha is mild with tem- arabica snails. In addition, the results obtained will be helpful peratures ranging between 12 and 23 C. Due to its altitude, Al in the development of simultaneous identification of B. arabica Baha’s climate is moderate in summer and cold in winter. snails and diagnosis of S. mansoni infection within it in a single Rainfall is higher in the mountainous region with falls in the step by an implementation of multiplex PCR. This will lead to range of 229–581 mm. The average throughout the whole re- the implementation of the molecular tools in schistosomiasis gion is 100–250 mm annually. The genetic variability among research in Saudi Arabia for the first time. 70 S.A. Al-Quraishy et al.

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