P C R - R F L P ANALYSIS
OF GLARDIA INTESTINALIS USING A GIARD/A-SPECIFIC G E N E ,
G L O R F - C 4
Y O N G T.*, H A N K.*, Y A N G H.* & P A R K S.*
Summary:
A cDNA clone encoding GLORF-C4 was isolated from the W B strain, an assemblage A Giardia intestinalis. Interestingly, GLORF- G4 has been previously reported as an assemblage B-specific gene. Using two primers based on GLORF-C4 of the G S strain, a prototype assemblage B, GLORFG4 gene was amplified from all the groups of G. intestinalis, and applied to detect the presence of cysts of G. intestinalis from faecal samples of cyst-passers. RFLP analysis of this PCR product successfully classified G. intestinalis into two distinct groups, assemblages A and B.
KEY WORDS : Giardia intestinalis. GLORF-C4, PCR-RFLP
Résumé : ANALYSE EN P C R - R F L P DU GÈNE SPÉCIFIQUE G L O R F - C 4 DE GLARDIA INTESTINALLS
Un clone d'ADN complémentaire codant pour le gène GIORF-C4 qui était considéré comme spécifique des souches de
l'assemblage B a été isolé de la souche WB (de l'assemblage A) de Giardia intestinali. Ce gène GLORF-C4 a été amplifié à partir de tous les groupes de G. intestinale en utilisant des
oligonucleotides spécifiques du gène GLORF-C4 de la souche GS, prototype des souches de l'assemblage 8, et utilisé pour détecter la présence de cystes de G. intestinalis dans les matières fécales de sujet excrétant des cystes. L'analyse en RPLF de ces produits d'amplification a permis la classification des différents groupes de G. intestinalis en deux catégories distinctes : celle de l'assemblage A et celle de l'assemblage B.
MOTS CLÉS : Giardia intestinalis, GLORF-C4, PCR-RFLP.
G
iardia intesinalis is a significant cause o f diar- - rheal outbreaks worldwide. O n the basis o f the s h a r e d m o r p h o l o g i c a l c h a r a c t e r i s t i c s , it includes a wide range o f organisms, which are ubi- quitous in various mammalian hosts. B e c a u s e o f its z o o n o t i c potential, classification o f this organism has b e e n an important issue, which may provide clues to monitor possible sources for the outbreaks as well as to identify the routes o f infections. For categorising G. intestinalis, several m o d e s o f classification have b e e n employed. T w o major lineages have b e e n defined for analysis o f human-derived G. intestinalis, assem- b l a g e s A and B (Mayrhofer et al, 1 9 9 5 ) , w h i c h cor- r e s p o n d either to the Polish' and the 'Belgian' g e n o - types ( H o m a n et al.. 1 9 9 2 ) , or to the groups I plus II and the group III plus IV (Andrews et al, 1 9 8 9 ; Monis et al, 1996), respectively. Alternatively, diverse G. intes-* Department of Parasitology and Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul 120-752, Korea.
** Correspondence : Prof. Soon-Jung Park
Department of Parasitology and Institute of Tropical Medicine, Yonsei University College of Medicine. 134 Shinchon-dong, Seo- daemun-gu. Seoul 120-752, Korea
Tel.: +82-2-361-5295 - Fax: +82-2-363-8676 E-mail : sjpark6l5@yumc.yonsei.ac.kr
tinalis has b e e n classified into three groups, 1, 2, and 3 (Nash, 1 9 8 5 ; Nash et al., 1 9 8 5 , 1 9 9 0 ) . It has b e e n suggested that groups 1 plus 2 and group 3 correspond to a s s e m b l a g e A and a s s e m b l a g e B , respectively (Lu et al., 1 9 9 8 ) . Analysis o f animal-derived G. intestinalis identified three additional a s s e m b l a g e s , a s s e m b l a g e C and D r e c o v e r e d from dogs (Monis et al, 1 9 9 8 ) , and a s s e m b l a g e E (a "Novel livestock" l i n e a g e ) defined by Ey et al. ( 1 9 9 7 ) . R e c e n t p h y l o g e n e t i c investigation using four h o u s e k e e p i n g g e n e s and allozyme analysis s h o w e d that G. intestinalis includes at least seven dif- ferent lineages, assemblage A to G (Monis et al, 1999).
In this report, w e found a cDNA c l o n e o f G. intesti- nalis showing a slight increase in transcription during encystation p r o c e s s . Despite o f the m i n o r increase in transcription o f this cDNA, it attracted our attention in that this cDNA c l o n e isolated from W B isolate, an a s s e m b l a g e A strain, turned out to b e GLORF-C4, pre- viously reported as the a s s e m b l a g e B-specific g e n e from the GS strain (Nash & Mowatt, 1 9 9 2 ) . Moreover, n o h o m o l o g o u s g e n e for this cDNA has yet b e e n identified in other organisms.
Finding o f GLORF-C4 in the W B isolate led us to inves- tigate whether this g e n e is prevalent in different groups o f G. intestinalis. W e also a s k e d if this unique g e n e could b e applied to detect and subgroup the G. intes- tinalis obtained from the faecal samples.
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/200209165
MATERIALS AND METHODS
CLONING O F G L O R F - C 4 F R O M T H E W B S T R A I N
F
o r t h e c D N A o f 2 5 0 b p s , s h o w i n g a slight increase in transcription during in vitro encysta- tion, the n u c l e o t i d e s e q u e n c e w a s c o m p a r e d with EMBL and G e n B a n k databases using the Blast program. T o find a full g e n e for this cDNA, w e also carried c D N A library s c r e e n i n g . F o r that p u r p o s e , G. lamblia cDNA library constructed b y Prof. Chung D. ( K y o u n g b o o k National University, K o r e a ) w a s employed. Using this A,ZAPII-based cDNA library (Stra- t a g e n e ) , c l o n e s hybridizing with the 3 2P - l a b e l e d cDNA w e r e purified until h o m o g e n e i t y and r e s c u e d as plas- mids as directed b y manufacturer. Identities o f the isolated cDNAs w e r e verified using an automatic DNA s e q u e n c e r ( P h a r m a c i a ) .
CELL C U L T I V A T I O N A N D D N A P R E P A R A T I O N G. intestinalis W B (ATCC#30957) and GS (ATCC#50581) strains w e r e axenically cultivated using TYI-S-33 media (Keister, 1 9 8 3 ) . An a x e n i c culture o f Y S - 2 7 strain (Chang et al., 1 9 9 5 ) was used as a s o u r c e for g e n o m i c DNA o f Entamoeba histolytica. Eshcerichia coli DH5a and Vibrio vulnificus (ATCC#29307) w e r e propagated in Luria broth with different saline concentrations ( 1 , 2 %, respectively). Acanthamoeba culbertsoni (ATCC#30171) was cultured in CGV medium (Willaert, 1 9 7 1 ) and used as a s o u r c e for g e n o m i c DNA.
DNAs o f the a b o v e strains w e r e extracted b y p h e n o l extraction as described (Park et al., 1 9 9 9 ) . G e n o m i c
DNA o f Toxoplasma gondii RH strain was a gift o f Prof.
Ahn M . (University o f Hanyang, K o r e a ) .
P C R A M P L I F I C A T I O N U S I N G P R O B E S F O R G. INTESTINALIS
G e n o m i c DNAs from G. intestinalis W B and GS strains w e r e e m p l o y e d as templates for PCR with diverse pri
m e r s . P C R a m p l i f i c a t i o n w a s p e r f o r m e d u s i n g a G e n e A m p PCR System 9 6 0 0 (Perkin-Elmer). T h e PCR solutions contained 10 mM KCl, 8 raM ( N H ^ S C ^ , 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl,, 0.01 % ( w / v ) gelatin, 100 LIM o f e a c h d e o x y r i b o n u c l e o t i d e , 20 p m o l o f e a c h primer, 1 Unit o f T a q p o l y m e r a s e ( T a k a r a ) and 5 0 to 2 0 0 ng o f template DNA. Primers u s e d in this e x p e r i ment are listed in T a b l e I .
For SSU-rDNA specific primers, RH4-RH11 (Hopkins et al., 1 9 9 7 ) and J W 1 - J W 2 (Weiss et al, 1 9 9 2 ) , the reac
tions w e r e p r o c e e d e d b y 3 5 cycles c o m p r i s e d with the following three steps : denaturation at 94°C for 3 0 s e c , hybridization at 5 9 ° C for 1 min, and polymerization at 72°C for 3 0 s e c . In the reaction mixtures, glycerol and dimethylsulfoxide w e r e included at final concentrations o f 10 % and 5 %, respectively. In c a s e o f f/m-specific primers ( 7 4 9 3 - 5 9 4 5 and 4 1 3 0 - 4 1 3 1 : B a r u c h et al, 1 9 9 6 ) , a total o f 35 PCR c y c l e s w e r e carried out during w h i c h DNAs w e r e denatured at 94°C for 3 0 s e c and the primers w e r e a n n e a l e d at 55°C for 1 min and e x t e n d e d at 72"C for 1 min. For GGL and G G R primers for the giardin gene, PCR was performed b y 25 cycles o f a m p l i f i c a t i o n m a d e o f 9 4 ° C for 1 min, 6 0HC for 1 min, and 72°C for 1 min. GLORF-C4 was amplified with C4-F and C4-R u n d e r the following PCR condition ( 3 5 cycles : 94°C, 3 0 s e c , 58°C, 1 min, 7 2HC , 1 m i n ) .
Gene P r i m e r s Nucleotide s e q u e n c e Size ( b p s ) Reference
GLORF-C4 C4-F 5 AGCTCATCTTCGTCCTCTA3' 443 This study
C4-R 5'CAATCTTGTTTGCATACGA3'
giardin GGL 5'AAGTGCGTCAACGAGCAGCT3' 171 Mahbubani et al. (1992)
GGR 5 'TTAGTGCTTTGTG ACCATCG A3 '
Urn 4131 5ATGCCTGCTCGTCGCCCCTTC3' 683 Baruch et al. (1996)
4130 5'CACTGGCCAAGCTTCTCGCAG3'
7493 5'GCAGAATGTGTACCTAGAGGGG3' 812 Baruch et al. (1996)
5945 5 TAGTCTCCGAGCTCCTTCTGG3'
SSU-rDNA RHU 5'CATCCGGTCGATCCTGCC3' 292 Hopkins et al. (1997)
RH4 5 AGTCGAACCCTGATTCTCCGCCAGG3
JW1 163 Weiss et al. (1992)
JW2 5GCGCACCAGGAATGTCTTGT3'
5TCACCTACGGATACCTTGTT3' Table I. - Oligonucelotide primers used in detection of Giardia intestinalis.
YONG T., HAN K., YANG H. & PARK S.
P C R - R F L P OF GlARDIA INTESTINALIS
P C R D E T E C T I O N O F G, INTESTINALIS F R O M F A E C A L S A M P L E S
Giardia-positive s a m p l e s identified b y light m i c r o s c o p y w e r e p r o c e s s e d t o purify g e n o m i c DNA b y s u c r o s e gradient centrifugation a n d s u b s e q u e n t p h e n o l e x t r a c t i o n , as previously d e s c r i b e d ( Y o n g et al., 2 0 0 0 ) . Using t h e s e g e n o m i c DNA as t e m p l a t e s , p r e s e n c e o f G. intestinalis w a s e x a m i n e d b y PCR reaction ( 3 5 cycles : 9 4 ° C , 3 0 s e c , 58°C, 1 min, 7 2 ° C , 1 m i n ) .
RESULTS
SEQUENCE OF G L O R F - C 4 IN W B STRAIN
S
e q u e n c e data o f GLORF-C4 g e n e o f t h e W B strain w e r e s u b m i t t e d t o EMBL d a t a b a s e s a n d o b t a i n e d the a c c e s s i o n n u m b e r A J 2 9 1 7 5 6 . T h e n u c l e o t i d e s e q u e n c e o f G L O R F - C 4 in W B w a s a l i g n e d with that in G S a n d found to h a v e 7 3 % identity (Fig. 1 ) . B o t h d e d u c e d a m i n o a c i d s e q u e n c e s s h o w 7 2 % identity (data n o t s h o w n ) . T h i s result is intriguing in that it d e m o n s t r a t e s a significant variation in t h e s a m e g e n e within t h e s a m e s p e c i e s . D u e t o this variation, G L O R F - C4 might h a v e b e e n s u g g e s t e d as a n a s s e m b l a g e - s p e cific g e n e , w h i c h s e e m e d t o b e p r e s e n t o n l y in t h e a s s e m b l a g e B , but n o t in t h e a s s e m b l a g e A (Nash &
Mowatt, 1 9 9 2 ) .
DETERMINATION OF SPECIFICITY OF G L O R F - C 4 For comparison, WB and GS strains were employed as prototypes for the assemblages A and B , respecti
vely. Two primers, C4-F and C4-R were used to amplify a partial sequence of this gene as a 443 bp DNA frag
ment from the two strains of G. intestinalis, as well as from the other organisms.
As a result of PCR on the genomic DNAs isolated from WB and GS, the DNA fragments of 443 bps were amplified. On the contrary, PCR with the genomic DNAs of other protozoa ( A c a n t b a m o e b a culbertsoni, Entamoeba histolytica, and Toxoplasma gondii) and the bacteria ( E s c h e r i c h i a coli and Vibrio vulnificus) failed to produce any PCR product (Fig. 2). This result clearly demonstrated the unique presence of GLORF-C4 in G. intestinalis.
COMPARISON OF G L O R F - C 4 WITH OTHER PROBES B a s e d o n this u n i q u e p r e v a l e n c e o f G L O R F - C 4 within G. intestinalis, it w a s c o m p a r e d with o t h e r g e n e s
GS ATGTTCAACCCGAGACACCCTGGAGGCGAATTCTTTGGAAGAAAGCACCACCGAAGGCAT WB ATGCACGAGCCAAGGCACGCCAACGGCGAGTTCTTTGGAAGGAGGCATCATGGAAGGCGT
C4-F
GS GCGCCAGATGGAAGATCTAGCTCATCTTCGTCCTCTAGCTCTGAGTGCGAGCTTGGCTAT WB CCACCGGATGAAAGCTCCAGCTCATCATCGTCCTCTAGCTGTGAGTGCGAAGCTGGCCAT GS GACGAAAGAAGATGCTGTAGGCAGGAGCCGGATGCTCATTGTTGCGATCGCGAGCATCGT WB GATGAAAGGGAGTGCTGTAGACAGGGCCCAGGCCCATACTGCTGCGACCGCGAGCGCCGC GS AAATTCTGGAAGCACCCGCCTGAATTCCCTCACTTTGGCAAGTGTAAGGGACGTCATGCG WB AAATTCTGGAAACACGGGCCCCCGTTCTCCCACTTTATTAAGGGCAGGGGACATCATGGA GS CTTCCTTTCCCAAGGATGGAGCACTGGCCTGGCCCCAAGCACTGTCCATTTCTATCTGAG WB CCCCACTGTTCAAAAGAAGAGCGCTGGACCGGTCCCAAACCCTGCCCATTTCTGTCATCC GS AAGGACATGAAGGACCTTCCTGTTACTGTTCACCATGGGTGCATGCCTATTGTTGCTCAC WB AAAGATATGAATGAGCTTCCTGTCACCGTTCATCACGGAAGCATGCCGATTGTCGCACGT GS GGAGTGGTCATGTTTCACATAGTATCCGAAACAAAGCCGTCCATAACCATCTCTAACACT WB GGAGTGGTCACGTTTCATATTGTATCAGAAATAAAGCCGTCCATAACAATCTCCAATACC GS GGCGCCATCAAGGTGGAGGCCAATGGAATCCAGCTTCTCAGTGCTTCTCTGAAGGAGAAG WB GGCGCCATCACTGTAGAGGCAGATGGAATCAAGCTTCTTAGCGCCTCCCTGAAGGAAAAT
C4-R
<
GS TACCCCGTCGATAAGGTCGTCGCATCGTATGCAAACAAGATTGCCACCATCCGTATTCCA WB TATCCTGCTGATAAGGTCGTTGCGTCGTATGCAAACAAGATTGTCGTTATCCGTATTCCT GS TGTGATGCGGCCTTCTCTGAGTCTCCTGTGGATATCCCTATCACGATCCAAAAGTGA WB TGCAACATAGTCACACCTGAGCCTTCTGTTGATAITCCTATTACGATTCAGAAGTAA
Fig. 1. - Aligned nucleotide sequences o f the GLORF-C4 gene of G intestinalis isolate WB (assemblage A genotype; EMBL accession number AJ291756) and its putative ortholog (GenBank accession number M90390) from isolate GS, an assemblage B genotype. The seg
ments corresponding to PCR primers C4-F and C4-R are indicated by arrows.
Fig. 2. - PCR detection of GLOFR-C4 gene in two groups of Giardia intestinalis, other protozoa and bacteria.
Using two primers, C4-F and C4-R based on GLORF-C4 sequence of GS, GLORF-C4 DNA of 4 4 3 bps were amplified from the follo
wing genomic DNAs; lane 1, W B (assemblage A Giardia intesti
nalis); lane 2, GS (assemblage B Giardia intestinalis); lane 3, Acan- thamoeba cubertsoni; lane 4 . Entamoeba histolytica; lane 5.
Toxoplasma gondii; lane 6, Escherichia coli DH5a; lane 7, Vibrio vulnifucus; lane 8, DNA size marker, 100 bp ladder.
( M a h b u b a n i et al, 1 9 9 2 ; W e i s s et al., 1 9 9 2 ; B a r u c h et al., 1 9 9 6 ; H o p k i n s et al., 1 9 9 7 ) , w h i c h had b e e n reported as a p r o b e to detect G. intestinalis ( T a b l e I ) . U p o n PCR e m p l o y i n g t w o pairs o f the primers cor
r e s p o n d i n g SSU-rDNA region, J W 1 plus J W 2 (Weiss et al., 1 9 9 2 ) and R H U plus RH4 ( H o p k i n s et al., 1 9 9 7 ) , t h e DNA fragments o f the e x p e c t e d s i z e s , 1 6 3 and 292 b p s , w e r e o b t a i n e d (Fig. 3A). W h e n the primers c o m p l e m e n t a r y to the region o f giardian g e n e , G G L and G G R ( M a h b u b a n i et al., 1 9 9 2 ) w e r e used, a DNA fragment o f 171 b p s w a s g e n e r a t e d from b o t h isolates.
T h e s e three sets o f primers m e n t i o n e d a b o v e have b e e n applied to detect G. intestinalis from clinical and environmental samples.
PCR with t w o pairs o f tim-specific primers ( 4 1 3 0 plus 4 1 3 1 and 7 4 9 3 plus 5 9 4 5 ; B a r u c h et al., 1 9 9 6 ) resulted in production o f 6 8 3 and 8 1 2 b p DNA fragments, res
pectively (fig. 3A, lane 8 - 1 1 ) . W h e n t h e s e t w o DNA fragments w e r e digested with either Xho I (for 6 8 3 b p DNA) or Hind III (for 8 1 2 b p DNA), W B and GS s h o w e d different digestion patterns (fig. 3 B ) , thus pro
viding a tool to classify G. intestinalis.
W e also tested w h e t h e r the GLORF-C4 PCR fragment could b e used to categorise different groups o f G. intes
tinalis b y PCR-RFLP. Digestions o f the GLORF-C4 PCR fragments with various restriction e n d o n u c l e a s e s , such as Nco I, Apa I, Hind III, or Tru9 I, provided an straightforward w a y o f distinguishing the a s s e m b l a g e B from the a s s e m b l a g e A strain (data not s h o w n ) . Dif
ferential digestion patterns b e t w e e n W B and GS iso
lates with Trn9l or Nco I are p r e s e n t e d in Fig. 3 B .
Fig. 3. - (A) PCR detection of Giardia intestinalis WB (assemblage A) and GS (assemblage B ) strains using various primers, SSU-rDNA primers, giardin primers, tim primers, or GLOFR-C4 primers. W repre
sents WB whereas G indicates GS isolate. Using these isolates as the templates, PCR was performed using the following primers. Lane 1, DNA size marker. 100 bp ladder; lane 2. 3, JW1 and JW2 (for SSU-rDNA); lane 4, 5, RH11 and RH4 (for SSU-rDNA); lane 6, 7, GGL and GGR (for giardin); lane 8, 9. П30 and 4131 (for tim); lane 10.11, 5925 and 7493 (for tim); lane 12, 13, C4-F and C4-R (for GLORF-C4).
(B) Grouping of Giardia intestinalis using ftm-specific primers and GLORF-C4 primers. W represents WB whereas G indicates GS iso
late. Using these isolates as templates, reactions were carried out with the following primers and the resultant PCR products were digested with indicated restriction endonucleases. lane 1, DNA size marker, 100 bp ladder; lane 2, 3, 4130 and 4131 (Xhol); lane 4, 5, 5925 and 7493 (Hind III); lane 6, 7, C4-F and C4-R (Tni9 I ) ; lane 8, 9, C4-F and C4-R (Nco I).
APPLICATION OF C4-F
AND C4-R TO FAECAL SAMPLES
C4-F a n d C4-R p r i m e r s w e r e e m p l o y e d to d e t e c t G. intestinalis in the faecal s a m p l e s o b t a i n e d from Korea, China, and Laos ( T a b l e ID. PCR reactions using t h e s e g e n o m i c DNAs as templates p r o d u c e d the s a m e 443 b p DNA fragment (data not s h o w n ) . Eight o f the fourteen s a m p l e s have b e e n previously g r o u p e d b y a different method, sequencing the 292 b p region o f SSU- rDNA ( Y o n g et al., 2 0 0 0 ) .
Y O N G T . , H A N К . , Y A N G H . & P A R K S.
PCR-RFLP OF GlARDTA INTESTINALIS
Sample n u m b e r Sampling site Grouping by 16S rDNAa Grouping by GLORF-C4
KP Seoul, Korea Assemblage A Assemblage A
KL Seoul, Korea Assemblage A Assemblage A
KC2 Seoul, Korea Assemblage A Assemblage A
CA1 Anhui, China Assemblage A Assemblage A
CA9 Anhui. China Assemblage B Assemblage B
CA12 Anhui, China ndb Assemblage A
CA 13 Anhui, China Assemblage A Assemblage A
CA14 Anhui, China Assemblage A Assemblage A
C A P Anhui, China Assemblage B Assemblage B
RD2 Savannakhet, Laos nd Assemblage B
RD25 Savannakhet, Laos nd Assemblage B
RD37 Savannakhet, Laos nd Assemblage B
RH i Savannakhet, Laos nd Assemblage B
RE19 Savannakhet, Laos nd Assemblage B
Tru9 I digestion o f GLORF-C4 PCR products from human faecal s a m p l e s s h o w e d that three s a m p l e s iso
lated from K o r e a b e l o n g to the a s s e m b l a g e A, as shown two products o f 303, and 140 bps. For the faecal s a m p l e s from China, both patterns o f digestion w e r e demonstrated, two as an a s s e m b l a g e B type (an intact 4 4 3 b p DNA fragment) and four as an a s s e m b l a g e A type. Five s a m p l e s from Laos w e r e turned out to b e a s s e m b l a g e B isolates (Fig. 4 and T a b l e II). PCR-RFLP r e s u l t s o f G L O F R - C 4 w e r e s u m m a r i s e d with t h e sequencing data o f SSU-rDNA for comparison, demons
trating that both types o f analyses resulted in the s a m e groupings.
1 2 3 4 5 6 7 8 9 1 0 1 1
Fig. 4. - PCR-RFLP analysis of GLORF-C4 isolated from human faecal samples. The GLORF-C4 DNA fragments amplified from various iso
lates were digested with Tru9 I endonuclease. Lane 1, 100 bp ladder; lane 2, WB (assemblage A): lane 3. GS (assemblage B ) ; lane 4-6, the Korean sample. KP. KL. KC2; lane 7-9, the Chinese samples.
CA13. CA1 I. C A P : lane 10-11. the Laos samples. RD2S. and RE19.
DISCUSSION
C
lassification o f G. intesitnalis has b e c o m e more important question as this organism attracted worldwide attentions as sources for outbreaks. Elucidation o f the contaminating sources and monitoring and surveys o f the possible sources are the prerequisites for prevention o f the outbreaks by this protozoan pathogen.
In this note, w e designed a set o f primers specific for GLORF-C4, which was s h o w n to b e unique to G. intes- tinalis. T h e y w e r e c o m p a r e d side b y side with other probes, k n o w n to detect and classify G. intestinalis.
C o m p a r e d with t w o pairs o f SSU-rDNA primers and a pair o f primers for giardin, C4-F and C4-R primers pro
vide a better w a y to classify G. intestinalis. In c a s e o f the former primers, s e q u e n c i n g o f the PCR products is required to identify the groups o f G. intestinalis.
With r/w-specific primers, classification o f Giardia iso
lates was feasible with PCR-RFLP. Actually, PCR-RFLP analysis o f tint fragment has an advantage over that o f GLORF-C4 in that the former method allows the sub- grouping o f G. intestinalis a s s e m b l a g e A into two sub- a s s e m b l a g e s A-l and A-2 ( B a r u c h et al., 1996). O n the o t h e r h a n d , w e f o u n d that d i g e s t i o n p a t t e r n s o f GLORF-C4 o f W B isolate ( a s s e m b l a g e A - l ) was exactly s a m e as those o f K l isolate, w h i c h has b e e n reported as an a s s e m b l a g e A-2 (Park et al., 1 9 9 9 ) (data not s h o w n ) . Therefore, sub-grouping o f a s s e m b l a g e A is not possible with PCR-RFLP analysis o f GLORF-C4.
Despite o f this ability, PCR-RFLP analysis o f tint frag
ments n e e d s two PCRs to identify the groups o f the isolates. O n the o t h e r hand, PCR-RFLP analysis o f GLORF-C4 could b e suggested as an improved, c o n v e nient method in w h i c h a single PCR product treated with either Nco I or Tni9 I can provide a c o m p l e -
a : Data from Yong et al. (2000).
b : nd. not done.
Table II. - Summary' of the faecal samples used in RFLP analysis.
YONG 1., MAIN K „ Y A N G H . & P A R K S.
mentary, confirming result in aspect o f the grouping of G. intestinalis.
Lastly, w e also applied our primers to the faecal samples from three different countries and found them useful to detect and classify Giardia isolates. D u e to the small number o f the faecal samples analysed, o u r data is pre
liminary to m a k e any conclusion o n distribution o f two assemblages in these countries. However, a suggestion can b e proposed based o n t w o facts : D a proposition made in SSU-rDNA analysis ( Y o n g et al, 2 0 0 0 ) , which G. intestinalis o f a s s e m b l a g e B is p h y l o g e n e t i c a l l y grouped closer to Giardia isolates obtained from animals, 2) grouping o f Laos isolates only to the assemblage B . Comparing with China and Korea, there may b e a higher probability o f cross-transmissions b e t w e e n humans and animals in Laos. T o confirm this, more investigation should b e performed including a large number o f human faecal samples as well as animal-originated samples.
ACKNOWLEDGEMENTS
W
e are grateful to Prof. Dong-II Chung o f K y o u n g b o o k National University, K o r e a for kindly providing us G. intestinalis cDNA library. W e also appreciate it J o n g w e o n Lee a n d Prof.Kyu-Ho L e e for their critical c o m m e n t s o n this manus
cript. This study w a s supported b y the Y o n s e i Medical University R e s e a r c h Fund o f 1 9 9 9 (Project#1999-01).
REFERENCES
ANDREWS R.H., ADAMS M . , BOREHAM P . F . , MAYRHOEF G. &
MELONI B . P . Giardia intestinalis : electrophoretic evidence for species complex. International Journal for Parasito
logy, 1 9 8 9 , 19, 1 8 3 - 1 9 0 .
BARUCH A.C., ISAAC-RENTON J . & ADAM R.D. The molecular epi
demiology o f Giardia lamblia : A s e q u e n c e - b a s e d approach. Journal of Infectious Disease, 1 9 9 6 , 174, 2 3 3 - 2 3 6 . CHANG J . K . , IM K . & SOH C. T. Axenization of Entamoeba his
tolytica, a Korean strain Y S - 2 7 . Korean Journal of Parasi
tology, 1 9 9 5 , 33, 3 8 7 - 3 9 0 .
E Y P . L . , MANSOURI M . , KULDA J . , NOHYNKOVA E . , MONIS P.T., ANDREWS R.H. & MAYRHOKER G. Genetic analysis of Giardia from hoofed farm animals reveals artiodactyls-specific and potentially zoonotic genotypes. Journal of Eukaryotic Microbiology, 1 9 9 7 , 44, 6 2 6 - 6 3 5 .
HOMAN W . L . , VAN ENCKEVORT F . H . J . , LIMPER L., VAN EYS G.J.M., SCHOONE G.J., KASPRZAK W . , MAJEWSKA A.C. & VAN KNAPEN F .
Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes.
Parasitology Research, 1 9 9 2 , 78, 3 1 6 - 3 2 3 .
HOPKINS R.M., MELONI B . P . , GROTH D.M., WETHERALL J . D . , REY-
NOLDSON J.A. & THOMPSON R.C.A. Ribosomal RNA sequen
cing reveals differences between the genotypes of Giardia isolates recovered from humans and animals in Germany.
Journal of Parasitology, 1 9 9 7 , 83, 4 4 - 5 1 .
KEISTER D . B . Axenic cultivation of Giardia lamblia in TYI-S- 3 3 medium supplemented with bile. Transaction of the Royal Society of Tropical Medicine and Hygiene, 1 9 8 3 , 7 7 ,
4 8 7 - 4 8 8 .
Lu S.Q., BARUCH A.C. & ADAMS R.D. Molecular comparison of Giardia lamblia isolates. International Journal for Parasitology, 1 9 9 8 , 2 8 , 1 3 4 1 - 1 3 4 5 .
MAHBUBANI M.H., BEJ A.K., PERLIN M., SCHAFER III F.W., JAKU- BOWSKI W . & ATLAS R.M. Differentiation of Giardia duo- denalis from other Giardia spp. by using polymerase chain reaction and gene probes. Journal of Clinical Microbio
logy, 1 9 9 2 , 30, 7 4 - 7 8 .
MAYRHOEFER G . , ANDREW R.H., E Y P . L. & CHILTON N . B . Divi
sion of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 2 7 loci and comparison with Giardia muris.
Parasitology, 1 9 9 5 , 111, 1 1 - 1 7 .
MONIS P.T., ANDREWS R.H., MAYRHOFER G . & E Y P . L . Molecular
systematics of the parasitic protozoan Giardia intestinalis.
Molecular Biology and Evolution, 1 9 9 9 , 16, 1 1 3 5 - 1 1 4 4 . MONIS P . T . , ANDREWS R.H., MAYRHOFER G . , MACKRILLJ., KULDAJ.,
Isaac-Renton J.L. & EY P.L. Novel lineages of Giardia intes
tinalis identified by genetic analysis of organisms isolated from dogs in Australia. Parasitology, 1 9 9 8 , 116, 7 - 1 9 .
MONIS P.T., MAYRHOFER G . , ANDREWS R.H., HOMAN W.L., LIMPER L.
& EY P.L. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasito
logy, 1996, 112, 1-12.
NASH T.E. Comparison of different isolates of Giardia. Microe- cology and Therapy, 1 9 8 5 , 15, 1 2 1 - 1 3 2 .
NASH T . E . , CONRAD J.T. & MERRITT J.W. Variant specific epi
topes of Giardia lamblia. Molecular Biochemical Parasi
tology, 1 9 9 0 , 42, 1 2 5 - 1 3 2 .
NASH T . E . , MCCUTCHAN T., KEISTER D., DAME J . B . , CONRAD J . T .
& GILLIN F.D. Restriction-endonuclease analysis of DNA from 1 5 Giardia isolates obtained from humans and ani
mals. Journal of Infectious Disease, 1 9 8 5 , 152, 6 4 - 7 3 . NASH T.E. & MOWATT M.R. Identification and characterization
of a Giardia lamblia group-specific gene. Experimental Parasitology, 1 9 9 2 , 75, 3 6 9 - 3 7 8 .
PARK S . J . , YONG T.S., YANG H.W., LEE D.H. & LEE K. Axenic
cultivation and characterization of Giardia lamblia isolated from humans in Korea. Korean Journal of Parasitology,
1 9 9 9 , 37, 1 2 1 - 1 2 5 .
WEISS J . B . , VAN KEULEN H. & NASH T . E . Classification of sub
groups of Giardia lamblia based upon ribosomal RNA gene sequence using polymerase chain reaction. Molecular and Biochemical Parasitology, 1 9 9 2 , 54, 7 3 - 8 6 .
WILLAKRT E . Isoelement et culture in vitro des amibes genre Naegleria. Annales de la Société Belge de Médecine Tropi
cale, 1 9 7 1 , 51, 7 0 1 - 7 0 8 .
YONG T.S., PARK S . J . , HWANG U . W . , YANG H . W . , LEE K , MIN D.Y., RIM H.J., WANG Y. & FENG Z. Ribosomal DNA sequen
cing for genotyping of Giardia lamblia recovered from humans in China and Korea. Journal of Parasitology, 2 0 0 0 , 86. 8 8 7 - 8 9 1 .
Reçu le 1 0 mai 2 0 0 1 Accepté le 2 0 novembre 2 0 0 1
