PCR-RFLP analysis of the ITS2 region to identify Schistosoma haematobium and S. bovis from Kenya.

Abstract

Abstract. Schistosoma haematobium, primarily a human parasite, and the closely related Schistosoma bovis from ruminants, are sympatric in many African countries such as Kenya. Because these two species 1) can inhabit the same Bulinus snails, 2) may be found in the same freshwater habitat, and 3) have morphologically similar cercariae, better means are needed to tell them apart. The second internal transcribed spacer (ITS2) region of the ribosomal gene complex (rDNA) of recent Kenyan isolates of both species was sequenced and found to be a 98% match. The S. bovis sequences were nearly identical (99%) to conspecific sequences from Niger; the S. haematobium sequences were nearly identical (99%) to conspecific sequences from Egypt, Mali, and Niger. Restriction fragment length polymorphism (RFLP) analysis of a 480 base pair (bp) PCR product containing the ITS2 region using two restriction enzymes, Taq1 and Sau3A1, yielded species-specific fragment patterns that allowed successful identification of a single S. haematobium cercaria. The protocol outlined here is useful in providing a rapid, one-day identification of S. haematobium (and likely S. bovis) cercariae (the infective larval stage) and/or other life cycle stages in a basic molecular biology laboratory. By helping to determine whether schistosome-infected bulinid snails in a particular body of water are transmitting a human or an animal schistosome, or both, this analysis will aid in disease control and in ongoing epidemiological studies.

Molecular confirmation of trematodes in the snail intermediate hosts from Ratchaburi Province, Thailand

“…In order to maintain the integrity of the DNA, it was stored in -20 °C . Specimens were amplified for detection of the region of ITS2 using the PCR method following the primer of Barber et al [28]. The forward primer was recorded as ITS3 (5'-GCA TCG ATG AAG AAC GCA GC-3') and the reverse primer was recorded as ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3').…”

Molecular Diagnosis of Schistosoma Infections in Urine Samples of School Children in Ghana

“…5,8,9 Until now their use in human epidemiological surveys has been limited with more studies exploring the potential of schistosome-specific PCR focusing more on the snail vector rather than its definitive host. 10,11 Recent developments in the simplification of DNA isolation procedures and PCR technology, especially real-time PCR, have made DNA amplification a worthwhile alternative for microscopy-based diagnostic methods. 8,[12][13][14] In this study, the prevalence and intensity of urinary schistosomiasis in Ghanian school children was assessed using real-time PCR.…”

Differentiation of Schistosoma Haematobium From Related Schistosomes by PCR Amplifying an Inter-Repeat Sequence

“…The importance of discriminating S. haematobium from other species later led to the development of molecular approaches, which have included isoenzyme profiling, 11 Southern blot analysis employing rDNA gene probes, 12 randomly amplified DNA, 13 and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the ITS2 region. 10 Amplification by simple PCR using defined primers, considered more suitable for large-scale screening, has not been accomplished for this purpose thus far.…”

“…9 Previously, the differential identification of snails infected by S. haematobium needed careful examination and testing of shed cercariae. Given that cercariae belonging to the S. haematobium-related species are not readily distinguishable morphologically from others within the group, a variety of approaches have been taken for differential identification, 10 with the standard method being infection of laboratory animals and subsequent parasite species identification based on the morphology of the adult worms. The importance of discriminating S. haematobium from other species later led to the development of molecular approaches, which have included isoenzyme profiling, 11 Southern blot analysis employing rDNA gene probes, 12 randomly amplified DNA, 13 and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the ITS2 region.…”