multilocus genotyping of giardia duodenalis
TRANSCRIPT
Multilocus genotyping of Giardia duodenalis
reveals human to human transmission pattern
in Tehran, Iran
Elham Razmjou
Saeideh Hashemi-Hafshejani, Ahmad Reza Meamar, Lameh Akhlaghi
Department of Medical Parasitolgy, School of Medicine,
Iran University of Medical Sciences, Tehran, Iran
Giardia duodenalis protozoan parasite lives in the small
intestine of human and a large number of domestic and wild
animals.
Giardia duodenalis is a species complex with at least eight
distinct assemblages nominated as A to H, which are similar in
morphology but are heterogeneous in genotype.
2
Introduction
Today, multilocus genotyping (MLG) has become a useful tool
for genotyping and subtyping of G. duodenalis.
Assemblages A and B are the main cause of infection in
humans, as well as a wide range of mammals.
3
Introduction
The purpose of this study was to identify G. duodenalis
assemblages, sub-assemblages, and genotypes based on
multilocus analysis of triose phosphate isomerase (tpi), β -giardin
(bg) and glutamate dehydrogenase (gdh) genes.
Better understanding of the genetic diversity of Giardia helps to
further explore the taxonomy and molecular epidemiology of this
parasite.
4
Introduction
5
Study Area
Materials and methods
6
Materials and methods
Patients and sample enrollments
Sixty two Giardia positive
samples were collected from
referred patients for routine stool
examination to the laboratory of
health centers and hospitals in
Tehran who invited to participate in
the study.
7
Materials & methods
Fecal samples preparation and DNA extraction
Infection with Giardia was confirmed in collected fecal samples by light
microscopy or Formal Ether sedimentation techniques. Giardia cysts were
isolated and concentrated with the sucrose flotation technique (Coklin et al.,
2007).
The genomic DNA of isolated cysts was extracted using the QIAamp DNA
Mini Kit (QIAGEN, Germany) following manufactures protocol with modifications
of Verweij et al. (Verweij et al., 2003).
Coklin, T., Farber, J., Parrington, L., Dixon, B., 2007. Prevalence and molecular characterization of Giardia duodenalisand Cryptosporidium spp. in dairy cattle in Ontario, Canada. Veterinary Parasitology 150, 297-305.
Verweij, J.J., Schinkel, J., Laeijendecker, D., van Rooyen, M.A., van Lieshout, L., Polderman, A.M., 2003. Real-time PCR for the detection of Giardia lamblia. Mol Cell Probes 17, 223–225.
8
*Sulaiman, I.M., Fayer, R., Bern, C., Gilman, R.H., Trout, J.M., Schantz, P.M., Das, P., Lal, A.A., Xiao, L., 2003. Triosephosphate Isomerase Gene Characterization and Potential Zoonotic Transmission of Giardia duodenalis. Emerging Infectious Diseases 9, 1444-1452.
A 530-bp fragment of tpi gene was amplified through a nested-PCR using
primer pairs, reaction mixture and conditions according to Sulaiman et al.
procedure (Sulaiman et al., 2003).
530 bp
Multilocus genotyping of isolates
1. Amplification of tpi gene
9
*Cacciò, S.M., De Giacomo, M., Pozio, E., 2002. Sequence analysis of the β-giardin gene and development of a polymerase chain reaction–
restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol. 32, 1023-1030.
*Lalle, M., Pozio, E., Capelli, G., Bruschi, F., Crotti, D., Caccio, S.M., 2005. Genetic heterogeneity at the β-giardin locus among human and animal
isolates of Giardiaduodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol. 35, 207-213.
A 511-bp fragment of bg gene was amplified through a nested-PCR using primer pairs,
reaction mixture and conditions according to Cacciò et al. (Cacciò et al., 2002) and
Lalle et al. (Lalle et al., 2005) procedure.
Multilocus genotyping of isolates
2. Amplification of bg gene
511 bp
10
*Read CM, M.P., Thompson RCA, 2004. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using
PCR-RFLP. Infect Genet Evol 4, 125–130.
A 432-bp fragment of gdh gene was amplified through a semi nested-PCR using primer
pairs, reaction mixture and conditions according to Read et al. (Read CM, 2004)
procedure.
Multilocus genotyping of isolates
3. Amplification of gdh gene
432 bp
11
Multilocus genotyping of isolates
4. Sequences and phylogenetic analysis
The nested-PCR products for each locus were excised
purified from agarose gel by using Gel Extraction Kit (QIAGEN, Germany)
sequenced in both directions (Macrogen, Korea)
viewed and read by CHROMAS
(Technelysium Pty Ltd.,Queensland, Australia)
12
The sequencing results were aligned with DNASIS MAX
(version 3.0; Hitachi, Yokohama, Japan).
The sequences were blasted (http://blast.ncbi.nlm.nih.gov)
to compare similarity of the sequences of the samples with
the sequences in GenBank database.
Phylogenetic analysis was performed in MEGA 7
(www.megasoftware.net) using neighbour-joining
(NJ) algorithms with evolutionary distances
calculated by Kimura-2 parameter method
(Kimura, 1980) and 1000 bootstrap value.
Multilocus genotyping of isolates
4. Sequences and phylogenetic analysis
13
Results
Assemblages identification
Multilocus sequence analysis
Assemblages
A B Discordant
26 (41.9%) 27 (43.5%) 9 (14.5%)
Discordant Assemblages
Isolate tpi bg gdh
IGT3 A B -
IGT4 B B A
IGT5 A B -
IGT16 A B A
IGT38 - B A
IGT93 B A -
IGT165 A B -
IGT213 - A B
IGR386 - B A
Multilocus sequence analysis of 62 Giardia-positive samples by tpi, bg and gdh genes
14
Gene Assemblages
A B Total
tpi 29 (53.7%) 25 (46.3%) 54 (87.1%)
bg 28 (45.2%) 34 (54.8%) 62 (100%)
gdh 28 (53.8%) 24 (46.2%) 52 (83.9%)
The sequencing analysis of tpi, bg and gdh gene
Results
Assemblages identification
Isolates Accession no. Nucleotide position from the
start of the gene
Assemblage A 12 103 373 419 510
A2 U57897 - C T G T
A2 KJ888993 - - - - -
A1 KR051228 - T C - -
A1 L02120 - T C - -
A1 GU564274 - T C - -
A2
IGT2,5,12,16, 25, 26, 37,
39,40,41,7H, 143,
165,IGR81, 287,IGA340
- - - - -
A2 IGT3 - - - - G
A2 IGT8, 9, 11, 13, 14, 19,
20, 21, 22, 30,31
A - - - -
A2 IGT29 - - - A -
15
Genotyping of assemblage A
Multiple alignments of tpi sequences from this study with reference sequences obtained from GenBank,
representing genotypes of assemblages A.
29 assemblage A
isolates
16 Isolates
11 Isolates
genotype A2
4 patterns
Isolates Accession no. Nucleotide position from the start of the gene
Assemblage B 12 65 139 142 184 278 367 508
B3 AY368165 G C C C G G T C
B4 L02116 A T T T A A C T
B3 IGT1, 15, 17, 18 A - T - - A C -
B3 IGT4 A T - - - A C -
B3 IGT7,23,24,27, 28,33,34,
35, 93
A - - - - A C -
B3 IGT10 A - - - - A C T
B3 IGT32, 36, 52, 101,
164,182,IGA305,IGR519
- - - - - A C -
B4 IGT110 A T T - - A C -
B4 IGR197 A - T T - A C -
16
Genotyping of assemblage B
Multiple alignments of tpi sequences from this study with reference sequences obtained from GenBank,
representing genotypes of assemblages B.
25 assemblage
B isolates
4 Isolates
9 Isolates
7 patterns
B3 & B4
genotypes8 Isolates
17
Phylogenetic tree
of G. duodenalis genotypes
constructed by neighbour-joining
analysis, based on the nucleotide
sequences of tpi sequences retrieved
from this study (LC183913-LC183966)
compared with reference sequences
of known assemblages from Genbank.
Bootstrap values obtained from 1000
replicates are indicated on branches in
percentage. The evolutionary
distances were computed using the
Kimura 2-parameter method (Kimura,
1980) and are in the units of the
number of base substitutions per site.
There were a total of 520 positions in
the final dataset. Evolutionary
analyses were conducted in MEGA7
(Kumar et al., 2016).
Isolates Accession no. Nucleotide position from the start of the gene
Assemblage A 34 61 73 130 142 160 190 214 250 274 289 292 317 325 355 373 391 398 421 424 463 481
A2 AY072723 C A G G C A T G T T G T C T A C G G C G T G
A1 X85958 - - - - - - - - - - - - - - - - - - - - C -
A3 AY072724 - - - - - - - - - - - - T C - - - - - - - -
A4 AY545642 - - - - - - - - - - - - - - - - - A - - C -
A3 IGT2,11,12,13,14,20,21,22,25,26,30,37,39,40, 117,143,IGR81,287
- - - - - - - - - - - - T C - - - - - - - -
A2 IGT5,8, 19,29,31,41 - - - - - - - - - - - - - - - - - - - - - -
A2 IGT9, IGA340 - - - - - - - - - - - - - C - - - - - - - -
A2 IGT213 - - - - - G - - - - - - - C - - - - - - - -
A3 IGT93 T - A - - - C - - C A C - C - - A - - C - C
A3 IGT7H - G - A T G - T C C A - T C C T A - T C - -
18
Genotyping of assemblage A
Multiple alignments of bg sequences from this study with reference sequences obtained from GenBank,
representing genotypes of assemblages A.
28 assemblage A isolates
18 Isolates
6 Isolates
A2 & A3 genotypes6 patterns
2 Isolates
Isolates Accession no. Nucleotide position from the start of the gene
Assemblage B 67 85 160 211 241 250 289 292 307 334 340 358 395 398 407 421 451 466 467 478 502
B3 AY072727 C A G C C C A C C A C G G G G T G C G G C
B4 AY072728 - G - T - - - - - - - - - - - C - T - - T
B3
IGT1,3,4,6,7,10,17,18,23,24,27,28,32, 35,152,164,IGR12, 197,519,IGA458
- - - - - - - - - - - - - - - - - - - - -
B3 IGT5 T - - - - - - - - - - - - - - - - - A - -
B3 IGT33 - - - - - T - - - - - - - - - - - - - - -
B3 IGT15,16,34,36 - - - T - - - - - - - - - - - - - - - - -
B3 IGT52 - G - - - - - - - - - - - - - - - - - A -
B3 IGT110 T - - T - - - - - - - - - - - - A - - - -
B3 IGT165 T - - T T T - T - - - A - - - C - - - - -
B3 IGT182 - - - T T T G T - G T A - - - C - - - - -
B3 IGA305 T - - T - - - - - - - - - - - - A - - - -
B3 IGT38, IGR101 T - - T - - - - - - - - - - - - - - - - -
B3 IGR386 T - - C - - - - - - - - - - - - - - - - -
19
Genotyping of assemblage B
Multiple alignments of bg sequences from this study with reference sequences obtained from GenBank,
representing genotypes of assemblages B.
34 assemblage B isolates
20 Isolates
4 Isolates
11 patternsB3 genotype
2 Isolates
20
Phylogenetic tree of G. duodenalis
genotypes constructed by neighbour-joining
analysis, based on the nucleotide
sequences of bg sequences retrieved from
this study (LC183967-LC184028) compared
with reference sequences of known
assemblages from Genbank. Bootstrap
values obtained from 1000 replicates are
indicated on branches in percentage. The
evolutionary distances were computed
using the Kimura 2-parameter method
(Kimura, 1980) and are in the units of the
number of base substitutions per site. There
were a total of 507 positions in the final
dataset. Evolutionary analyses were
conducted in MEGA7 (Kumar et al., 2016).
Isolates Accession no.
Nucleotide position from the start of the gene
Assemblage A 326 367 385
A2 L40510 A C T
A1 M84604 - T C
A2 IGT2,8,9,11-14,19-22, 25, 26, 29, 30, 37-41, 7H, 143, IGA340, IGR81, 287, 386
- - -
A2 IGT4 G - -
21
Genotyping of assemblage A
Multiple alignments of gdh sequences from this study with reference
sequences obtained from GenBank, representing genotypes of
assemblages A.28 assemblage A
isolates
27 Isolates
1 Isolates
genotype A2
2 patterns
Isolates Accession no. Nucleotide position from the start of the gene
Assemblage B 13 17 18 43 61 73 121 123 124 139 169 193 196 211 229 283 304 310 325 361 367 385 400 430
B3 AF069059 C C T C C C T C G G G T C T C C C C C C G C T T
B3 DQ090541 - - - - T - C - - - - - - C - T - - - -
B4 EU594666 - - - - - T C - - - - C - C - - T - T - A - - C
B4 L40508 - - - - - T - - - - - C - C - - T - T - A - - -
B3 IGT1,17 T - - - - T - - - - - C - C - - - T - - - - - -
B3 IGT7 T - - - - T - - - - - C - C - - - - - - - - - -
B4 IGT10 T G C - - T - - - - - C - C - T T - - - A - - -
B3 IGT15 T - - - - T C - - A A C - C - - - - - - A - - C
B4 IGT18 T - - - - T C - - - - C - C - - T - - - A - - C
B3 IGT23,24 T - - - - T - - - - - - - - - - - - - - - - - C
B3 IGT27 T - - - - T - - - - - - - - - - - - - - - - - -
B3 IGT28 T - - - - T - - - - - - - - - - - - - - A - - C
B3 IGT32 T - - - - - C - - - - C T - - T - - - - A - - C
B3 IGT34 T - - - - T C - - - - C - C T - - - - T A - - C
B3 IGT35 T - - - - T C - - - - C - C - - - - - - A - - C
B3 IGT36 T - - - - T C - A - - C - C - - - - - - A - - C
B3 IGR12 T - - - - - - - - - - - - - - - - - - - A - - C
B3 IGT52 T - - - - - - - - - - - - - - - - - - - - - - C
B4 IGR101 T - - - - T - - - - - - - C - T T - - - A - - C
B4 IGT110 T - - - - T C - - - - C - C - - T - - - A - T C
B4 IGT164 T - - - - T - - - - - C - C - - T - - - - - - C
B3 IGT182 T - - - - - - - - - - - - - - T - - - - - - C C
B3 IGR197 T - - - - - - - - - - - - - - T - T - - - - - C
B4 IGT213 T - - - - T C T - - - C - C - - T - - A - - C
B4 IGA305 T - - T - T C - - - - C - C - - T - T T A - - C
B3 IGR519 T - - - - - C - - - - - - - - - - - - - A - - C
22
Genotyping of assemblage B
Multiple alignments of gdh sequences from this study with reference sequences obtained from GenBank,
representing genotypes of assemblages B.
24 assemblage B isolates B3 & B4 genotype22 patterns
2 Isolates
2 Isolates
23
Phylogenetic tree of G. duodenalis
genotypes constructed by neighbour-joining
analysis, based on the nucleotide
sequences of Gdh sequences retrieved
from this study (LC184423-LC184474)
compared with reference sequences of
known assemblages from Genbank.
Bootstrap values obtained from 1000
replicates are indicated on branches in
percentage. The evolutionary distances
were computed using the Kimura 2-
parameter method (Kimura, 1980) and are
in the units of the number of base
substitutions per site. There were a total of
519 positions in the final dataset.
Evolutionary analyses were conducted in
MEGA7 (Kumar et al., 2016).
24
Assemblages MLG Genotype No. of isolates (isolate code)
tpi bg gdh
A
AII-1 A2 A2 A2 5
AII-8 A2 A3 A2 20
B
BIII-1 B3 B3 B3 15
BIII-2 B3 B3 B4 5
BIII-3 B4 B3 B3 1 (IGR197)
BIV-2 B4 B3 B4 1 (IGT110)
A+B
BIII-AII B3 B3 A2 1 (IGT4)
AII-BIII A2 B3 A2 1 (IGT16)
Multilocus genotyping
Assemblages, multilocus genotypes (MLG) and genotypes in Giardia
duodenalis assemblage A and B isolates.
25
22
25
Conclusion
Multilocus sequencing results of tpi, bg and gdh genes showed
Giardia cyst passer in Tehran are infected with assemblages A and B,
that is in line with other reported all over the world.
This is similar to the results of previous studies in Giardia- cyst
positive samples in Tehran, that were performed with one locus.
According to our knowledge this is the first study performed in Giardia
cyst passer in Iran, with multilocus genotyping (MLG).
26
Conclusion
The present study confirmed that the MLG was an important tool in
determining the assemblages and sub-assemblages for isolates of
assemblage A, but less important in determining the sub-
assemblages for isolates of assemblage B.
The finding of assemblage B and genotypes A2 and A3 that belong to
the sub-assemblages AII suggested human-to-human transmission
mode pattern in Tehran, Iran.
Conclusion
27
Saideh Hashemi-Hafshejani
This work represents a part of the MSc dissertation
of Saideh Hashemi-Hafshejani.
28