Genetic diversity of Giardia isolates from patients in Chandigarh region: India

Objective The aim of study was to characterize Giardia isolates genetically among patients in Chandigarh region, India. For this, nested PCR targeting fragment of the glutamate dehydrogenase (GLUD1 earlier named as GDH) gene was used. Phylogenetic analysis was done by constructing neighbor-joining tree made out of the nucleotide sequences of G. intestinalis isolates obtained in this study and with the known sequences published in GenBank. Results Out of 40 samples, GLUD1 gene was amplified in 33 samples (82.5%). The product of GLUD1 gene was successfully sequenced only in 32 samples. In these samples, assemblage B was found in 27 (84.37%) samples whereas 5 (15.6%) samples had assemblage A. Among assemblage B most of them were of BIII. Therefore, genotyping of Giardia would be helpful in conducting epidemiological studies.


Introduction
Giardia intestinalis is well known intestinal parasite of humans and mammals. Giardia causes approximately 280 million cases of giardiasis worldwide annually [1]. Most of these cases are associated with lower socioeconomic status. In the year 2004, giardiasis was included in WHO 'Neglected Diseases Initiative' because of its high prevalence in communities with low socio-economic status.
Giardiasis is acquired due to ingestion of cysts of Giardia in water or food [2]. Giardia intestinalis is composed of eight major genotypes or assemblages (A-H) [2]. Genotype A and B are common among humans having variable distribution frequency in different geographical locations and these assemblages mainly considered as zoonotic assemblages as they are able to infect both men and animals [3]. These assemblages are further divided into subassemblages on the basis of either digestion by restriction enzyme or sequence analysis. Assemblages A are classified as AI, AII,AIII and AIV. Subassemblages AI and AII were commonly found in humans while AI, AIII and AIV are subassemblages of animals. Zoonotic potential is linked with only subassemblage AI [4]. Assemblage B, catogorised into four sub-assemblages BI, BII, BIII and BIV. As per literature subassemblages BIII and BIV were reported in humans while other two are specific for animals [4,5]. The BIII sub-assemblage is closer to subassemblages BI and BII and therefore has zoonotic potential. The GLUD1 (earlier known as GDH) locus has been utilized for genetic characterization of G. intestinalis isolates in vertebrates [4] hosts and is able to categorize them into sub genotypes/subassemblages. The present work was aimed to determine assemblages and subassemblages of Giardia isolates involved in its transmission by using glutamate dehydrogenase (GLUD1) marker.

Polymerase chain reaction amplification
The two-step PCR was employed for the amplification of GLUD1 gene (432 bp) by using previously published primers given by Read et al. [6]. The conditions and primers for both primary and secondary reactions are given as Additional file 1: Table S1. The first set of PCR reaction comprised of 2.0 μL of DNA template, 12.5 μL 2 × Go Taq Green Mix, 1 μL of each primer (10 μM), 1 μL of Bovine Serum Albumin (BSA) whereas in case of secondary PCR, DNA template was replaced by the product of primary reaction. For the negative control, nuclease-free water and for the positive control, DNA of Giardia strain, Portland 1 was used for each PCR reaction. All the precautions were taken to prevent contamination.

PCR product purification and sequencing
All PCR-positive samples were sequenced using secondary primers. PCR products were sequenced in both forward and reverse directions. By using BLAST, nucleotide similarity of sequenced amplicons was searched in GenBank (http://www.ncbi.nlm.nih.gov/blast ). CLUSTAL X was used to determine multiple sequence alignments. Neighbor-joining distance trees were prepared using MEGAX software (https ://www.megas oftwa re.net/) (Fig. 1). Bootstrap values were based on 1000 replicas. All the sequences obtained during the study were submitted to the GenBank (Accession number: MT584168-MT584199).
The direct links which are publicly available are as follows: Out of 40 samples, the GLUD1 gene was amplified in 33 samples (82.5%) (Table1, Fig. 2). The possible reason for this occurrence indicated the less parasitic load in these isolated samples. The product of GLUD1 gene was successfully sequenced only in 32 samples. In these samples, assemblage B was found in 27 (84.37%) among which 18 (66.66%) were sub-genotype BIII and 9 (33.33%) were sub-genotype BIV samples whereas 5 (15.6%) samples had assemblage A. Four of them were AI subgenotype and only1 belonged to sub-genotype AII.

Discussion
Giardia intestinalis is the most common and frequent intestinal parasitic agent of gastroenteritis mainly in the developing countries [7]. The present study provides data on genetic diversity of Giardia isolates from patients in the Chandigarh region. As per results, assemblage A and B are common among this population, which is in concordance to other studies [8,9]. In our study, assemblage B was the predominant genotype observed followed by assemblage A. However, similar observations were previously reported and observed worldwide which showed that assemblage B was predominant in comparison to assemblage A [9][10][11][12]. There are studies which showed the presence of assemblage B in Rhesus macaques (Macaca mulatta) and potable water resources of Northern India [13,14]. But other studies have reported the assemblage A as the predominant genotype in other regions [15]. Due to geographical variations, differences were observed in the prevalence of various genotypes and the detection of these variations would be helpful in designing effective therapeutic approaches.

Conclusion
The results showed that PCR sequencing and phylogenetic analysis is an excellent molecular technique for genotyping of Giardia intestinalis. Detection of Giardia intestinalis assemblages and sub-assemblageswould be helpful in conducting epidemiological studies.

Limitation of study
Present study involves only single locus for genotyping and also the sample size is less so it is difficult to interpret zoonotic potential of these isolates. Therefore, multi-locus typing data is required to differentiate between Giardia isolates.