Multilocus genotyping of Giardia duodenalis

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.


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.


3. Amplification of gdh gene

432 bp

11


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.


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).


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


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


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


Multiple alignments of bg sequences from this study with reference sequences obtained from GenBank,


34 assemblage B isolates

20 Isolates

4 Isolates

11 patternsB3 genotype

2 Isolates

20

Phylogenetic tree of G. duodenalis

genotypes constructed by neighbour-joining


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


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


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


Multiple alignments of gdh sequences from this study with reference sequences obtained from GenBank,


24 assemblage B isolates B3 & B4 genotype22 patterns

2 Isolates

2 Isolates

23

Phylogenetic tree of G. duodenalis

genotypes constructed by neighbour-joining


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

Documents

Multilocus genotyping of Giardia duodenalis