Phylogenetic Analysis of Giardia lamblia Human Genotypes in Fars Province, Southern Iran.

BACKGROUND: This study is the first phylogenetic genotype analysis of Giardia lamblia in Iran. The main objective was to determine genotyping and identify the sub-assemblages of Giardia lamblia isolates involved in the transmission of giardiasis in Fars Province, south of Iran, in 2012.
METHODS: Forty G. lamblia isolates were collected from the patient's fecal samples with gastrointestinal discomfort referred to the health centers and hospitals in Shiraz, Fars Province, south of Iran. Purification of G. lamblia cysts from fecal samples and DNA extraction were performed using monolayer of sucrose density gradient and Phenol-Chloroform-Isoamylalcohol (PCI) respectively. Semi-nested PCR and sequence analysis were then performed using the primers (GDHeF, GDHiF, and GDHiR) which amplified a 432-bp fragment of Giardia glutamate dehydrogenase (gdh) gene. Phylogenetic analysis was carried out using a neighbor-joining tree composed of the nucleotide sequences of G. lamblia isolates obtained in this study and the known sequences isolates published in GenBank.
RESULTS: G. lamblia sub-assemblage AII was the most prevalent genotype with 80% of the cases and 20% of the cases belong to sub-assemblage BIII and BIV based on the DNA sequence of the gdh. G. lamblia isolates at Fars Province were widely distributed within assemblage A cluster (sub-assemblage AII) and the remaining isolates were dispersed throughout the assemblage B cluster (sub-assemblage BIII and BIV).
CONCLUSION: PCR Sequencing and phylogenetic analysis was a proper molecular method for genotyping and discriminating of the of G. lamblia sub-assemblages in fecal samples, using the glutamate dehydrogenase gene that suggests a human contamination origin of giardiasis.

Introduction

The taxonomy of Giardia has revealed six Giardia species: G. lamblia in mammals, including human; G. agilis in amphibians; G. ardeae and G. psittaci in birds; G. microti and G. muris in rodents (1). G. lamblia (synonym of G. intestinalis and G. duodenalis) is the most common intestinal protozoan parasite of a wide range of mammalian hosts and has a global distribution (2, 3). G. lamblia is a species complex with eight major genotypes or assemblages (A–H). Molecular characterizations based on multiple loci are used as the main tool to study genetic variation of different populations of Giardia (4, 5). Phylogenetic analysis using the small subunit ribosomal RNA (SSU-rDNA), glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes have been used to study genetic variability and relations within assemblages of G. lamblia from different hosts. The gdh locus has been used to successfully genotype isolates of G. lamblia from a range of vertebrate hosts and it allows the discrimination between the various subgenotypes of G. lamblia assemblages (6, 7). In this study, glutamate dehydrogenase marker was used in the molecular characterization methods for genotyping and subtyping the isolates of G. lamblia from patients.

G. lamblia is one of the most common intestinal parasites in Iran. The prevalence of giardiasis varies in different parts of Iran (5%–23%) (8). Molecular epidemiology of human giardiasis is still unclear in Iran. Previously, there was no study performed using phylogenetic technique for G. lamblia in Iran. Only little information was available on the G. lamblia genotypes. Three studies based on PCR-RFLP assay, targeting the gdh gene of G. lamblia isolates genotype in Tehran, Shiraz and Isfahan provinces showed that assemblage A was the most prevalent (9–11). In contrast, two studies based on PCR-RFLP assay, targeting the gdh gene of G. lamblia isolates in Tabriz and in Uremia showed that assemblage B was the most prevalent genotype (12, 13).

Molecular studies on G. duodenali isolates and discrimination of genotypes is a useful way to know the transmission route and effective prevention management of giardiasis. The objective of this study was to determine genotyping and identify the sub-assemblages of G. lamblia isolates involved in the transmission of giardiasis in Fars Province, south of Iran. Genotypic characterization of the gdh gene was performed by using genomic DNA directly extracted from human fecal samples to evaluate the potential transmission routes of G. lamblia in this area.

Materials and Methods

Sample collection

Forty G. lamblia isolates were collected from the patient’s fecal samples with gastrointestinal discomfort referred to the health centers and hospitals in Shiraz, Fars Province, South of Iran in 2012. Then, the samples were sent to the Research Laboratory of Intestinal Protozoa in the Department of Parasitology and Mycology in School of Medicine at Shiraz University of Medical Sciences for further examination. Informed consent was taken from the patients and the study was approved by Ethics Committee of the university.

Purification technique

Purification of G. lamblia cysts, which were to be used for DNA extraction, was done using the monolayer sucrose density gradient technique. Single step sucrose gradient with the specific gravity at 0.85 M was performed on positive stool samples in order to concentrate the cysts from fecal samples (14). Aliquots of the purified fecal samples were stored at 4 °C and −20 °C.

DNA extraction

The genomic DNA of G. lamblia was extracted using the Phenol-Chloroform-Isoamylalcohol (PCI) method with pretreatment by Triton X100 on purified fecal samples (14). DNA extracts were stored at −20 °C for PCR analysis.

PCR amplification of gdh gene

A semi-nested PCR was performed using three primers known as GDHeF, GDHiF, and GDHiR that amplify a 432-bp fragment of the gdh gene (7) with some slight modifications. PCR reaction mixtures consisted of 12.5 pmol of each primer, 200 μmol of each dNTP, 1.5 mM of MgCl2, were carried out in 25 μl volumes on a Corbett Research Thermal Cycler, Australia with the following amplification conditions: one cycle of 94 °C for 2 min, 56 °C for 1 min and 72 °C for 2 min, followed by 31 cycles of 94 °C for 30 sec, 56 °C for 20 sec and 72 °C for 45 sec and a final extension of 72 °C for 7 min. Both positive and negative controls were included in each round of PCR to validate results. One microliter of PCR product from the primary reaction was added to the secondary PCR containing primers GDHiF and GDHiR. Reactions were visualized in UV on 1.5% agarose gels stained with ethidium bromide.

Sequence analysis of gdh gene

Forty of the PCR products successfully amplified for the gdh locus were sent to First BASE Laboratories (http://www.base-asia.com) for commercial DNA sequencing reactions in both forward and reverse directions using primers GDHiF and GDHiR. Nucleotide sequences were analyzed using the computer program of sequencing scanner software. Multiple alignments and sequence alignment of these sequences were carried out using the BioEdit and the ClustalW MEGA4 programs. The gdh sequences of G. lamblia obtained in this study were aligned with previously published reference sequences of G. lamblia isolates from the GenBank database (Table 1). DNA sequencing of PCR products compared with already known G. lamblia sequences obtained from GenBank using ClustalW in the MEGA4. The accession numbers obtained for each reference assemblages from GenBank were L40509 (AI), L40510 (AII), AF069059 (BIII), L40508 (BIV), U60984 (C), U60986 (D), U47632 (E), AF069057 (F) and AF069058 (G) (5, 6, and 15). To determine similarities or homologies to known genes in NCBI GenBank sequence database, all obtained nucleotide sequences were submitted to BLAST searches. Sequence representatives for each of the assemblages identified in the studies were submitted to GenBank.

Table 1:

Giardia gdh gene sequences available in GenBank used in this study

Isolate	GenBank accession number	Sub-assemblage	Country	Reference
Ad-1	L40509	AI	Australia	(6)
Portland 1	M84604	AI	USA	(16)
Ad-1	AY178735	AI	Australia	Unpublished data
F22	EF507606	AI	Brazil	(17)
Ad-2	L40510	AII	Australia	(6)
Ad-113	AY178736	AII	Australia	Unpublished data
H32	EF507674	AII	Brazil	(8)
NLH20	AY826194	AII	Netherland	(5)
GH-125	AB195222	AII	Japan	(18)
TIG20	AB434776	AII	Iran	Unpublished data
T11	JF968202	AII	Iran	Unpublished data
16	JF917086	AII	Iran	Unpublished data
Bah-12	AF069059	BIII	Australia	(19)
FCQ-21	AY178756	BIII	Australia	Unpublished data
Cub-G89	EU594665	BIII	Cuba	(20)
Cub-G81	EU594667	BIII	Cuba	(20)
gd-ber9	DQ090540	BIII	Norway	(21)
gd-ber10	DQ090541	BIII	Norway	(21)
TIG12	AB434535	BIII	Iran	Unpublished data
A14	JF968198	BIII	Iran	Unpublished data
Ad-7	L40508	BIV	Australia	(6)
Ad-28	AY178738	BIV	Australia	Unpublished data
Ad-45	AY178739	BIV	Australia	Unpublished data
Ad-85	AY178755	BIV	Australia	Unpublished data
NLH13	AY826191	BIV	Netherland	(4)
H30	EF507672	BIV	Brazil	(17)
GH-156	AB182126	BIV	Japan	(22)
GH-158	AB188825	BIV	Japan	(23)
Ad-141	U60984	C	Australia	(15)
Ad-148	U60986	D	Australia	(15)
P-15	U47632	E	Australia	(24)
Ad-23	AF069057	F	Australia	(19)
Ad-157	AF069058	G	Australia	(19)
G. ardeae	AF069060		Australia	(19)

Phylogenetic analysis of gdh gene

Phylogenetic analysis was performed using Neighbor-joining (NJ) method in the MEGA4 program. The evolutionary distance-based analysis was conducted using the Kimura 2-parameters model to estimates the distance among assemblages identified in this study. For each calculation, branch reliability was assessed using bootstrap analysis (1000 replicates). A neighbor-joining (NJ) tree composed of the 40-nucleotide sequences of G. lamblia isolates obtained in this study and 33 sequences of G. lamblia isolates of the gdh gene in the GenBank database (Table 1) was reconstructed. The homologous nucleotide sequence of G. ardeae (GenBank accession number AF069060) was used as outgroup.

Results

The Giardia gdh locus with 432 bp was successfully amplified by semi-nested PCR. Fig. 1 shows a representative gel electrophoresis photograph of the PCR products amplified with gdh primers.

Fig. 1:

Electrophoresis of PCR products with gdh primers (expected size of 432 bp) Lane 1 = Negative Control (primers + buffer) // Lane 2 = Positive control

Lane 3–14 = G. lamblia isolates from patients // Lane M = 100 bp DNA marker (Fermentas, Canada)

Sequence analysis of the gdh gene

Sequence analysis was performed on 40 PCR products of G. lamblia based on gdh amplification both directions using GDHeF, GDHiF and GDHiR primers.

The genotyping results indicated the presence two main genotypes A and B of G. lamblia. Based on the DNAsequences of the glutamate dehydrogenase, 32 isolates (80%) were detected as assemblage A and 8 isolates (20%) were identical to assemblage B. After performing a BLAST search, analysis revealed all 32 isolates assemblage A sequences were 100% similar to sub-assemblage AII (GenBank Accession number L40510). From 8 isolates as assembalge B, three isolates were 99% similar to sub-assemblage BIII (GenBank Accession number AF069059), one isolate was 99% similar to sub-assemblage BIV (GenBank Accession number L40508) and four isolates were found between sub-assemblage BIII and BIV (Table 2). Comparison of gdh gene nucleotide substitutions between G. lamblia isolates obtained from this study and GenBank database published reference has shown in Fig. 2.

Table 2:

Genotyping characterization of 40 G. lamblia patients isolates from Fars Province, Iran

Patient isolates	Sub-assemblages
32 G. lamblia isolates (GIS Group) (80%):	AII (L40510)
GIS1, GIS4, GIS7, GIS8, GIS9, GIS10, GIS11, GIS12, GIS13, GIS14, GIS15, GIS16, GIS17, GIS18, GIS19, GIS20, GIS21, GIS22, GIS24, GIS26, GIS27, GIS28, GIS29, GIS32, GIS33, GIS34, GIS35, GIS36, GIS37, GIS38, GIS39 and GIS40
3 G. lamblia isolates (7.5%):	BIII (AF069059)
GIS3, GIS23 and GIS25
1 G. lamblia isolates (2.5%):	BIV (L40508)
GIS5
4 G. lamblia isolates (10%):	Between BIII & BIV
GIS30, GIS31, GIS2 and GIS6

Accession numbers for reference sequences obtained from GenBank are given in parentheses

Fig. 2:

Sequence analysis for gdh gene nucleotide substitutions of G. lamblia isolates obtained from this study compared with reference sequences

Phylogenetic analysis of gdh DNA sequences was determined to further clarify the relationship of the different genotypes to each other. Fig. 3 shows the Phylogenetic tree based construction on the neighbor-joining method of the gdh sequences from G. lamblia of patients isolates determined in this study and GenBank reference isolates and other G. lamblia assemblages previously published (Table 1).

Fig. 3:

Phylogenetic relationships of G. lamblia isolates in this study and reference isolates and other G. lamblia assemblages previously published in GenBank (Table 1) inferred by Neighbor-Joining analysis of Kimura’s distances calculated based on the nucleotide sequences of gdh with the MEGA4 program. Bootstrap values #50% are not shown. Accession numbers for reference sequences obtained from GenBank are given in parentheses. The sequence of Giardia ardeae was used as an out-group

▲ is the indicator for Iranian isolates.

The phylogenetic tree shows that assemblage A was the most frequent, corresponding with 32 cases (80%). All isolates in cluster A were distributed in sub-assemblage AII. The second cluster was contained the human assemblage B isolates. Assemblage B was found in 8 cases (20%) of the studied samples. Resulting in assemblage B revealed 2 major lineages designated as assemblages BIII and BIV. Third and fourth were the dog clusters of either assemblage C or D. The fifth cluster was contained the cattle assemblage E. The sixth was contained the cat assemblage F cluster. The seventh was contained the rodent assemblage G cluster. Finally, the eighth cluster was contained the G. ardeae (Fig. 3). Phylogenetic analysis of the gdh gene of G. lamblia isolates from patients provided strong bootstrap support (100%) for the placement of genotypes A and B in separate clusters, indicating the differentiation of the assemblages A and B. Phylogenetic analysis has shown Fars isolates were widely distributed within assemblage A cluster (sub-assemblage AII) and the remaining Fars isolates were dispersed throughout the assemblage B cluster (sub-assemblage BIII and BIV). No samples were joined in clusters corresponding to assemblages C, D, E and F and G of G. lamblia, also in sub-assemblage AI.

Discussion

Recent progressions on the identification of Giardia assemblages and sub-assemblages have increased tremendously in our understanding of the biology and host-parasite relationship of G. lamblia. In this study, 40 known G. lamblia isolates from patients were used to determine the sub-assemblages that occur in Fars province, Iran. All 40 G. lamblia isolates can be divided into four main groups based on the results obtained from this study. These groups are (i) sub-assemblage AII, (ii) sub-assemblage BIII, (iii) sub-assemblage BIV and (iv) sub-assemblage BIII/BIV as shown in Table 2. The sequences of gdh for the entire 40 G. lamblia patient isolates were compared to the sequences of known G. lamblia assemblages published by GenBank (Table 1).

In our study, three G. lamblia patient isolates were associated with sub-assemblage BIII and one isolate was associated with sub-assemblage BIV. Four isolates was situated between sub-assemblage BIII and BIV (Table 2 and Fig. 3). Additionally, Sequencing and phylogenetic analysis of gdh gene sequences from this study and a large set of those which were available from the literature worldwide showed the sequences of the 32 isolates associated with sub-assemblage AII were highly genetically homogenous with 100% homology to published sequences.

While the other eight isolates associated with sub-assemblages, BIII and BIV showed variability within the study area.

The results of this study are similar to previous studies (9–11) in which G. lamblia cysts isolated from human feces were analyzed with PCR-restriction fragment length polymorphism (RFLP) assay, based on the detection of glutamate dehydrogenase (gdh) genes. In Tehran, the capital of Iran a majority of 87% of the G. lamblia isolates used in the studies were from sub assemblage AII, whereas 7.8% of the isolates were from sub-assemblage BIII (9). Moreover, In Fars Province, 74.41% isolates were typed as assemblage AII, 17.44% as assemblage BIII, 3.49% as assemblage BIV and 4.66% isolates as mixed assemblages AII and BIV (10). In Isfahan Province in the center of Iran, PCRRFLP, genotype A group II was detected in 59.7% isolates compared to genotype B that showed 34.32% samples as genotype B Group III and 2.98% sample as genotype B group IV (11). In contrast with our study, assemblage B was predominant in Tabriz and Uremia cities in the northwest of Iran when the gdh gene was targeted by PCR RFLP. In Tabriz analysis, PCR-RFLP revealed that 28 samples (33.3%) were in sub-assemblage AII and 44.4% and 22.2% belonged to sub-assemblages BIII and BIV, respectively (12). In Uremia, 93.3% of isolates were found to be related to the genotype BIII and 6.7% were related to the subgroup BIV (13).

Current study had focused on the genetic characterization of G. lamblia at the gdh gene using DNA samples from patients’ stool samples in Fars Province, Iran. Previous surveys performed on DNA extracted directly from fecal samples in various countries have confirmed that only G. lamblia assemblages A and B are associated with human infections (25). Genotypic analysis showed that only combination of assemblages AII and B are able to infect humans in this area of Iran, suggesting a human contamination origin. Most of our patients with sub-assemblage AII infection had acquired their parasites through the anthroponotic route, mainly involved human-to-human transmission. Zoonotic transmission may also occur. Reservoir sources of zoonotic transmission were found to be a number of livestock animal species like dairy cattle, companion animals like cats, dogs and wildlife animals (26). Unfortunately, due to limited funding, no animal sample was included in this study, so current result was not enough to demonstrate the role of anthroponotic and zoonotic aspects of giardiasis infections. Further molecular characterization of species, genotypes and subtyping analysis of specimens from both humans and animals and comparisons of various genetic loci are required for identification of the transmission of giardiasis between animals and humans and, understanding of giardiasis transmission.

Phylogenetic analysis of G. lamblia isolates from this study and previously published G. lamblia assemblages revealed two different clusters A and B. In cluster A there were two subclusters AI and AII. Within AI subcluster, three G. lamblia isolates Portland 1 (GenBank accession number: M84604-USA), F22 (EF507606-Brazil), and Ad-1 (AY178735-Australia) matched with reference isolate Ad-1(L40509-Australia). We did not have any sample of this study in this subcluster. Within subcluster AII, 32 homolog isolates of this study completely matched (GIS Group) with reference isolate Ad-2 (GenBank accession number: L40510-Australia). Moreover, other isolates Ad-113 (AY178736-Australia), TIG20 (AB434776-Iran), T11 (JF968202-Iran), 16 (JF917086-Iran), H32 (EF507674-Brazil), NLH20 (AY826194-Netherland) and GH-125 (AB195222-Japan) were in this subcluster. In cluster B there were two sub-clusters BIII and BIV with some subgroups that reveal variation in this assemblage. Within the BIII subcluster, reference isolate Bah12 (GenBank accession number: AF069059-Australia) and Fcq-21 (AY178756-Australia) matched in a subgroup. Isolates cub-G89 (EU594665-Cuba), cub-G81 (EU594667-Cuba), isolates gd-ber9 (DQ090540-Norway), gd-ber10 (DQ090541-Norway), and A14 (JF968198-Iran) did not match with Bah12. Moreover, the isolate GIS3 (this study) and TIG12 (AB434535-Iran) (in a subgroup) and 2 other isolates from this study GIS23 and GIS25 had variant with Bah12. In BIV sub cluster, Ad-28 (GenBank accession number: AY178738-Australia), Ad-45 (AY178739-Australia), H30 (EF507672-Brazil) and NLH13 (AY826191-Netherland) matched with reference isolate Ad7 (L40508-Australia). Isolate Ad-85 (AY178755-Australia) was found very close to this subgroup. Another two samples GH-158 (AB188825-Japan) and GH-156 (AB182126-Japan) (in a subgroup) and 5 of our samples GIS5, GIS31, GIS30 and GIS2 and GIS6 (in a subgroup), set far distance from reference isolate Ad7 in this subcluster. Analysis of the cluster B indicated genetic heterogeneity in assemblage B. On the other hand, separate samples from Japan, Cuba, Norway, and Iran having different sub category may suggest that in a country and in different countries in the world have different strains. Some isolates from this study (GIS23, GIS25, GIS2, GIS6, GIS30 and GIS31) that were very far distanced from the reference isolates in cluster B could be the new strains that need more investigations in future studies (Table 1 and Fig. 3).

Geographic distribution of each Giardia assemblage in humans varies in different parts of the world. Our results with predominance of assemblage A was same to studies from Brazil (27), Colombia (28), France (29), Iran (9), Italy (30), Mexico (31–33), New Zealand (34), Portugal (3), Thailand (35), United States (36) and Yemen (37). Some studies from Argentina (38), Australia (39, 40), Bangladesh (41), Belgium (42), Colombia (43), Egypt (44, 45), India (46), Malaysia (47), Morocco (48), Netherland (4), Nicaragua (49), Norway (23, 50), The Philippines (51), Thailand (52), United Kingdom (2, 53) and Sweden (54) with a predominance of assemblage B, differed from our study. Predominant G. lamblia sub-assemblage AII has also been shown in Australia (6), Bangladesh (41), Belgium (42), England (2), France (55), Iran (13), Mexico (33), Nicaragua (49), Peru (56) and The Philippines (51).

Conclusion

Human giardiasis is one of the most common intestinal parasitic diseases in Iran, and its molecular epidemiology needs more investigations. PCR sequencing and phylogenetic analysis using the glutamate dehydrogenizes gene are proper molecular methods for G. lamblia genotyping and could discriminate G. lamblia sub-assemblages in fecal samples. The high prevalence of sub-assemblage AII in this study and other similar investigations conducted in Iran reemphasized the importance of applying all the health recommendations. Health education and improvement of sanitation conditions during drinking water and food supply are the most important basic strategies for the control and prevention of anthroponotic transmission of giardiasis in this community. The results could use in the future studies for clinical management and prevention policies.