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

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.

INTRODUCTION

Schistosomiasis, caused by parasitic helminth of the genus Schistosoma, is the second most prevalent tropical disease after malaria and a leading cause of severe morbidity in many parts of the world.[1] Freshwater pulmonate snails of the genus Bulinus act as intermediate hosts for Schistosoma haematobium and its closely related species. The snails occur commonly throughout much of Africa and adjacent regions. The disease poses a threat to about 600 million people in more than 76 countries including Nigeria.[2] S. haematobium, which causes urinary schistosomiasis in humans, afflicts about 150 million people in 53 countries in Africa and the Middle East.[3] In many 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.[45] Since these schistosome species often infect snails inhabiting the same bodies of water, examining cercariae or infected snails for estimating the transmission of S. haematobium is always confounded by the need to differentially identify S. haematobium.[678] Although cercariae belonging to these related species are not readily distinguishable morphologically from others within the group, quite a number of approaches have been taken for differential identification. The standard method being infection of laboratory animals and subsequent parasite species identification based on the morphology of the adult worms, but this method is laborious and time consuming.

Nevertheless, the importance of discriminating S. haematobium from other species later led to the development of molecular approaches such as isoenzyme profiling,[9] Southern blot analysis employing recombinant deoxyribonucleic acid (DNA) gene probes,[10] randomly amplified DNA[11] and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer 2 region.[12] In addition, a tandemly repeated DNA sequence termed Dra1 was recently identified in the genome of S. heamatobium.[13] Although Dra1 was not found in Schistosoma mansoni or Schistosoma japonicum even by dot hybridization, however, it was observed that this gene is also present in the genome of other schistosomes belonging to the S. haematobium group. The Dra1 repeat is a useful tool to determine whether or not a snail has been exposed to schistosomes; however, it will not inform as to which species of the parasite is responsible. This has limited the identification of S. haematobium infected snails by this PCR assay to areas where the other related schistosome species are absent or very rare.

In recent times, differentiating S. haematobium from S. bovis, S. mattheei, S. curassoni and S. intercalatum, but not from S. magrebowiei, was achieved through PCR.[14] 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. Thus in this study, we developed a new PCR assay using a pair of primers (ShND-1/ShND-2) to amplify a target sequence (GenBank accession number KF834975) from S. haematobium mitochondrion complete genome (GenBank accession number DQ157222).[15] The assay was able to successfully differentiate S. haematobium from S. magrebowiei, in addition to other schistosome species that are closely related to S. haematobium, in clinical samples, adult worms and field caught Bulinus snail intermediate hosts.

MATERIALS AND METHODS

Adult schistosomes

Adult worms of S. haematobium and other schistosome species belonging to S. haematobium group were collected from the Wolfson Wellcome Biomedical Laboratories, Zoology Department, Natural History Museum, London, UK in 2008 and were since kept in 70% ethanol at 4°C. The schistosome species used were as follows; S. haematobium from Loum, Cameroon; S. bovis from Iranga, Tanzania; S. curassoni from Dakar, Senegal; S. intercalatum from Edea, Cameroon; S. mattheei from Denwood, Zambia and S. magrebowiei from Lochinvar, Zambia.

Urine cell pellets from infected school children

A total of 20 positive urine samples collected from infected school-aged children in an endemic community in Ogun state, southwestern Nigeria were first screened for hematuria using hemastix followed by visual examination. Using a stereomicroscope, about 10 ml of each of the urine samples was examined for the presence of terminal spined ova of S. haematobium. The urine samples, including five from uninfected persons (control) were allowed to sediment, the supernatant was decanted and the sediment was centrifuged at 5000 g for 10 min. The supernatant was decanted again and cell pellets were washed three times with 25 ml phosphate-buffered saline (0.8% NaCl, 2.7 mM Kcl, 1.8 mM KH2 PO4 , 8 mM Na2 HPO4 pH 7.4) and stored immediately at −80°C until used.

Snail intermediate hosts

A total of 20 snails collected from Erepoto (n = 8) and Apojula (n = 12), two urinary schistosomiasis endemic areas of Lagos and Ogun states respectively, located in the southwestern region of Nigeria were examined. Recent screening of the snails for schistosomes Dra1 repeat by PCR confirmed that all of them were infected with either animal or human infective schistosomes, while PCR-RFLP and phylogenetic analysis of their sequenced partial Cox 1 gene showed that all the snails were Bulinus truncatus.[1617]

DNA extraction from adult schistosomes and snails

Genomic deoxyribonucleic acid (gDNA) was extracted from adult worms and snails using CTAB extraction buffer containing 2-Mercaptoethanol; hexadecyltrimethyl-ammonium bromide (CTAB); tris (hydroxymethyl) amino-methane; ethylenediaminetetraacetic acid, disodium salt solution; and sodium chloride. Each adult worm or snail tissue (homogenized) was placed in a sterile 1.5 ml eppendorf tube, 500 μl of CTAB solution was added followed by 10 μl of proteinase K solution (20 mg/ml) and was incubated at 55°C for 1 h, with occasional gentle mixing. DNA was extracted from the CTAB buffer by adding an equal volume of chloroform/isoamyl alcohol (24:1) to each tube. The organic and aqueous layers were gently mixed for 5 min and spun at 13,000 rpm for 20 min. The upper aqueous layer was removed into another sterile eppendorf tube and an equal volume of 100% ethanol was added, mixed and was incubated at −20°C overnight in order to enhance DNA precipitation. The solution was spun at 13,000 rpm for 20 min and the pellet was washed with 70% ethanol and was spun for another 20 min. The supernatant was removed and the pellet was dried at room temperature. When completely dried, the pellet was resuspended in 20 μl of nuclease-free water.

Extraction of gDNA from urine pellets

Urine cell pellets were digested with 1% sodium dodecyl sulfate and 50μg/ml proteinase K (Roche Diagnostics, Mannheim, Germany) at 48°C overnight. gDNA was extracted from the solution by adding an equal volume of chloroform/isoamyl alcohol (24:1) to each tube. The organic and aqueous layers were gently mixed for 5 min and spun at 13,000 rpm for 20 min. The upper aqueous layer was removed into another sterile tube and an equal volume of 100% ethanol was added, mixed and incubated at −20°C overnight in order to enhance DNA precipitation. The solution was spun at 13,000 rpm for 20 min and the pellet was washed with 70% ethanol and was spun for another 20 min. The supernatant was removed and the pellet was dried at room temperature. When completely dried, the pellet was re-suspended in 25 μl of water and stored at 4°C until used.

Primer design

Primers (ShND-1/ShND-2) were designed using Primer3 program,[18] whereas the G + C content of the target sequence was calculated using Oligonucleotide Properties Calculator program (http://www.basic.northwestern.edu/biotools/oligocalc.html). Primer size for both forward and reverse primers is 20 bp with annealing temperature of 55°C. The target sequence was taken from S. haematobium mitochondrion complete genome (GenBank accession number DQ157222).[15]

PCR assays

For sensitivity and specificity studies, PCR assays were carried out on all the extracted gDNA to amplify the target sequence using Bioneer AccuPower® PCR PreMix (Bioneer, Alameda, CA, USA) in a reaction volume of 20 μl containing 1 U of Top DNA polymerase, 250 μM of each dNTPs, 10 mM of Tris-Hcl (pH 9.0), 30 nM of Kcl, 1.5 mM of MgCl2, 25 pmol of each of the two primers (First BASE Laboratories Sdn Bhd, Selangor, Malaysia) and the template DNA. All PCR amplifications were performed with Hybaid thermal cycler (Hybaid, OMN-E Thermal Cycler) and a thermal profile involving 5 min at 95°C, followed by 35 cycles each of 1 min at 95°C, 1 min at 55°C, followed by 1 min at 72°C and a final elongation step at 72°C for 10 min. The amplified products were visualized on 1.5% agarose gel stained with ethidium bromide at a concentration of 0.5μg/ml. Photo documentation was performed with Gel Documentation and Analysis System (Clinx Science instruments, USA).

Nucleotide sequencing and alignment

Gel slices each containing the target fragment from two infected snails and two positive urine specimens were excised from agarose gel and purified with Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol and were selected for subsequent sequence analysis. Both the forward and reverse strands of the purified PCR products were sequenced using a dilution of the original PCR primers. Sequencing was performed by Inqaba Biotech (Pretoria, South Africa) on Applied Bio systems ABI 3500 × L Genetic Analyzer platform using Sanger sequencing approach. The sequence has since been submitted to the GenBank database and was given accession number KF834975.

RESULTS

Assay sensitivity and specificity

Sensitivity of the assay was determined by PCR amplification of different concentrations of S. haematobium gDNA serially diluted from 10 ng to 0.1 pg. Figure 1 shows that up to 1 pg (lane 6) of S. haematobium gDNA could be detected. For assay specificity, different concentrations of gDNA from S. haematobium and its closely related schistosome species were subjected to amplification by the PCR assay and their products examined by agarose gel electrophoresis. The results [Figure 2] show that even 0.1 ng of S. haematobium DNA was clearly detectable by the PCR, whereas there was no amplification signal with 10 ng and 1 ng of DNA from all other species including S. magrebowiei.

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

Detection of S. haematobium infection in snails and urine

Previous PCR screening of the 20 snails for schistosomes Dra1 repeat confirmed that all the snails were infected with either human or animal infective schistosome species, whereas PCR-RFLP and phylogenetic analysis of their sequenced partial Cox 1 genes confirmed that the snails were B. truncatus[1617] to establish the schistosome species present in these snails and urine specimens, the gDNA was subjected to ShND-1/ShND-2 PCR amplification. Results showed that eight snails were actually infected with human infective S. haematobium [Figure 3]. All the 20 urine samples were also positive for S. haematobium infection, whereas the five negative controls were negative.

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)

Sequence analysis and alignment

A nucleotide BLAST search was carried out at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to ascertain what the sequences were. All the 4 sequences (2 forward and 2 reverse) showed great similarity with the same sequence as given by: > gi | 102549914 | gb | DQ157222.2|:3371-4078 S. haematobium mitochondrion, complete genome 100% coverage and identity. When a protein-protein blast search was carried out for non-redundant protein sequences using Blastp (protein-protein BLAST), they all showed great similarity with the same sequences as given by >gi | 107735927 | gb | YP_626524.1 | 452-467 S. haematobium nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 3 gene.

DISCUSSION

PCR primers ShND-1/ShND-2 were designed to amplify a portion of S. haematobium mitochondrion complete genome (GenBank accession number DQ157222.2) for differential identification of S. haematobium DNA content from other schistosomes in its group. On the basis of the obtained results, a differential identification of S. haematobium from its closely related schistosome species; S. bovis, S. mattheei, S. curassoni, S. intercalatum and S. magrebowiei was achieved. The nucleotide analysis performed on the obtained sequences indicated amplification of a 1117 bp DNA sequence (GenBank accession number KF834975) encoding S. haematobium NADH dehydrogenase subunit 3 gene (GenBank accession number YP_626524.1). The PCR assay is therefore capable of identifying snails infected with S. haematobium from other snails carrying other closely related animal schistosome species in areas where they are found alongside S. haematobium.

Even though differentiation of S. haematobium from S. bovis, S. mattheei, S. curassoni and S. intercalatum, other than S. magrebowiei, had been reported earlier,[14] specific identification of these species from one another, as we have been able to achieve in this study, will facilitate better study of their distributions. It also will help to better define the effects of competition between these species in sympatric endemic areas. These results will also allow extension of large-scale PCR monitoring approaches for snails infected with S. haematobium,[19] which had been previously limited to areas where cross-reacting Dra1 containing animal schistosome species were absent or very rare. However, further validation of the assay is required for field studies since only a few field-collected snails were examined in this study. Such validation should include a whole range of infected and uninfected snails with different signal intensities so as to assess the range of sensitivity among field-collected snails.