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First Genotype Characterization of Giardia Intestinalis Assemblage E from Goat Kids in Bangladesh

Mohammad Alamgir Hossain
Department of Pathology and Parasitology, Chittagong Veterinary and Animal Sciences University (CVASU), Bangladesh.

Sohana Akter Mina
Department of Genetic Engineering and Biotechnology, University of Chittagong, Bangladesh.

Lolo Wal Marzan
Department of Genetic Engineering and Biotechnology, University of Chittagong, Bangladesh.

Mohammad Asif Imran Khan Emon
Department of Genetic Engineering and Biotechnology, University of Chittagong, Bangladesh.

Rasel Das
Nanotechnology and Catalysis Research Center (NANOCEN), University of Malaya, Malaysia.

AMAM Zonaed Siddiki
Department of Pathology and Parasitology, Chittagong Veterinary and Animal Sciences University (CVASU), Bangladesh.

Abstract/ Executive Summary:

Giardiasis is an important protozoan disease caused by Giardia and manifested as life-threatening diarrhea in animals and humans worldwide. No study has been performed to find zoonotic epidemiology and diversity of Giardia in animals in Bangladesh. Hence, there exists a room to characterize Giardia protozoan parasite in animals to find out their roles as a source of human infection. To address this hurdle, fecal samples of 100 goat kids were genotyped by nested PCR amplification of β-giardin gene fragment followed by sequencing and analysis. The total prevalence of Giardia in goat kids was 3% (3/100) and the infection is more widespread in younger, Black Bengal breed and female goat kids. Further analysis of β-giardin gene locus has shown and clustered the Giardia in Assemblage E rather than Assemblages A and B. It suggests the low zoonotic transmission frequency from the goat kids and has least epidemiological significance to humans. Further study on this field is prerequisite in terms of broad geographical areas, age groups, sex and evaluates zoonotic significance along with genetic diversity in other host species as well.

Keywords: Genotypes; Giardia, Giardiasis; Nested PCR; Goat kids

Location: Chittagong, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Goat

Objectives:

1. To study molecular characterization of Giardia in goat kids

2. To elucidate their possible zoonotic hazards in this region

Methodology:

A total of 100 fresh fecal samples were collected from goat kids [1-6 months old] from a local veterinary clinic in Chittagong, Bangladesh. All were affected with diarrhea when admitted to the clinic. Feces were collected directly from the rectum of each goat into a plastic specimen cup followed by immediately capped, labeled properly. Feces were preserved in -20°C freezer until DNA extraction was carried out. Genomic DNA was extracted from fecal samples, using the QIAamps stool mini kit [Qiagen] according to manufacturer’s instructions and stored at -20°C. Extracted DNA concentration was measured by Thermo scientific Nano drop 2000 spectrophotometer. Nested PCR analysis was performed to identify β-giardin gene locus in G. intestinalis. Forward and reverse primers namely G7 and G759 have been used in primary PCR reaction to amplify about 753 bp target DNA fragments. In addition, secondary PCR reaction has performed by using forward primer G7n and reverse primer G759n in order to amplify about 511 bp target DNA fragments. All the reaction mixtures and cycling conditions were same as previously published standard protocols. In each experiment, positive control was carried out as standard Giardia genomic DNA along with sample run without DNA template as negative control element. A PCR thermal cycler [Applied Biosystem 2720 Thermal cycler ®] was used in both primary and secondary PCR reaction cycles and the products were analyzed by 1.5% agarose gel electrophoresis. The PCR products of representative strains from each restriction group were purified with the PCR Clean-Up kit [Promega®, USA] and were sequenced on an ABI sequencer [ABI Prism, 3130, USA]. After sequencing of the representative nested PCR products, the quality of the sequence was carefully assessed manually based on the corresponding electro-chromatogram and have submitted in GenBank NCBI database. The final nucleotide sequence data reported in this paper are available in the GenBank under the accession numbers JX122074, JX122075, and JX122076 Specific Assemblage of Giardia of positive isolates was determined by comparing the sequences using the Basic Local Alignment Search Tool. The variations of nucleotide position at different points were revealed using ClastalW2 multiple sequence alignment programme to classify genotype into subgroups. To compare nucleotide variability and genetic relationship of β-giardin gene sequences of goat kids in Bangladesh [Genbank: JX122074, JX122075 and JX12076] with other infected animals in different countries, a phylogenetic tree was constructed. During this study top 23 BLAST hits of significant E-value [0.00] of β-giardin gene sequences of Genbank accession no. JX122074, JX122075 and JX12076, respectively were retrieved. Regenerative sequences of hits have eliminated in order to get non-redundant β-giardin gene sequences. The sequences were retrieved from GenBank database and were initially aligned under default conditions by using MUSCLE program. Furthermore, identical conserved regions of sequences of scored in the alignment was used to generate the tree. The sequence alignment was performed under default conditions and the tree was constructed by the neighbor-joining method. The poorly aligned sequence and also the internal gaps residue were taken off from the alignments to make a precise evolutionary tree by using the Jalview program. Finally, the phylogenetic tree of β-giardin gene sequences was constructed by using [ClustalW2-Phylogeny] program.

Source of Information: J. Veterinar Sci Technolo 2014, Vol: 5(5), ISSN: 2157-7579

Article Link (Online Journal): http://dx.doi.org/10.4172/2157-7579.1000201

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It was observed that younger Black Bengal breed and female goat kids are more vulnerable to Giardia than older Jamunapari breed and male goat kids. Occurrence of Assemblage E during this study suggests that zoonotic transmission of Giardia spp. from goat kids could be of low epidemiological significance in absence of Assemblages A and B. However, further study on epidemiological investigation of humans, domestic and wild animals as well as water catchment areas and drinking water sources necessary to acquire better information about the prevalence, host affiliations and geographical distributions of the different Assemblages of G. intestinal in animals from different countries.

Knowledge Management: Journal