1. Latifah Ibrahim, Normaznah Yahaya, Amal Nasir Mustafa.
Polymorphism of The Trophozoite Antigen (TSA 417) Gene of Malaysia local Isolates of Giardia duodenalis and Comparison With The Pathogenic Reference Strain WB.
Latifah Ibrahim, Normaznah Yahaya, Amal Nasir Mustafa.
Biotechnology Unit, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia.
INTRODUCTION
Giardia duodenalis is a protozoan pathogen that infects the gastrointestinal tract of many species of animals and human. It can cause diarrhea and malabsorption in children and also infects AIDS patients with weak immune system. G. duodenalis undergoes antigenic variation by changing the expression of its variant specific surface proteins 9VSPs). Previous investigations showed that the antigenic surfaces of G. lamblia are basically variable among different isolates. The objective of the study was to detect the variation in the DNA sequence of the VSP gene of Malaysian isolates of G. duodenalis and to compare with the pathogenic reference strain WB
MATERIALS AND METHODS
RESULTS
Giardia isolates and strains
Six Malaysian isolates used in the studies were 110, 7304, 7306, 6304, M007 and These isolates were obtained from six Orang Asli, aborigines aditted to the hospital for the aborigines at Gombak, Selangor. Pathogenic human strain WB and G. muris of wild mouse origin, were obtained commercially from American Type Culture Collection (ATCC). All the isolates were maintained in in vitro culture.
Polymerase Chain Reaction (PCR)
The genomic DNA were subjected to PCR with specific primers LP3 and RP3. PCR amplification was carried out with 2.5 units of the Taq DNA polymerase (Fermentas AB, Lithuania) in a reaction mixture (100ul), containing dNTPs (200uM) and 2.5 mM MgCl2 and subjected to 30 cycles of 1 minute at 95C (denaturation), 30 seconds at 58C (annealing) and 1 minute at 72C (extension).
Gene Cloning using ‘Zero Blunt PCR Cloning Kit’
Cloning of PCR product was performed using a commercial kit (Zero Blunt PCR cloning Kit, Invitrogen).
DNA sequencing
Automated DNA sequencing was carried out using ABI PRISM 377 machine (Applied Biosystem INC).
DISCUSSION
(Figure 1) show the ectrophoresis of genomic DNA of the six local isolates. WB strain was used as standard DNA from all the isolates have good quality, because they were in high molecular weight that was more than 2642 bp compared with 1kb ladder Boehringer Mannhaeim).
Primer sets LP3/RP3 could detect Giardia specifically and distinguish the G. duodenalis type from other Giardia species. The primers were derived from the trophozoite surface antigen
( TSA 417 gene).
In this study PCR products of the expected size ( approximately 520 bp) were generated from all the 6 isolates. The primer of LP3/RP3 would gave 520 bp product with all Giardia sp tested.
Figure 2 showed the PCR product of positive product. The PCR products were then cloned using ‘Zero Blunt PCR Cloning Kit’ (Invitrogen), and subsequently sequenced using ABI PRISM 377 machine (Applied Biosystem Inc).
Comparison of DNA sequences of the 520 bp PCR products of all the isolates studied against the European Molecular Biology Laboratory (EMBL) and GeneBank nucleotide databases using the BLAST programme, revealed that all the PCR products belong to Trophozoite variable surface Specific protein TSA417 of Giardia duodenalis .
BLAST results of the local isolates of open reading frame were shown in Table 2. The results showed that ORF ( open reading frame) reading matched with Trophozoite variable surface Specific protein TSA41, from G. duodenalis parasites. Six genes showed E value of <10e-5. (e<10e-5) showed significant value). The highest E value is 3e-23 with score value of 197 and 93 percent identity.
A phylogeny tree was drawn using software from clustal-W (Pearson format) from EMBL-EBL (Figure 4) using the DNA sequence of the TSA 417 gene. The phylogenetic analysis reveals 3 clusters. . The first cluster comprised local isolates 110 and 6307; the second cluster consists of isolates 2002, 7304, which were similar to the pathogenic strain WB. The Third cluster consists of isolates 6307 which were similar with isolate M007. However none of the Malaysian isolate were similar with the published gene sequence TSA 417 from the GeneBank.
CONCLUSION
This study suggested that local isolates and were likely to be pathogenic, since they were in the similar cluster with the pathogenic reference strain from human (WB strain). However futher study is needed to confirm the pathogenicity of these isolates.