Indian Journal of Animal Research

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Full Research Article

ITS- 1 gene based PCR as a diagnostic tool for phylogeographic characterization of Trypanosoma evansi in naturally infected equines in agro-climatic zones of Uttar Pradesh, India

Vijendra Kumar Pal1,*, Rajender Kumar2, Amit Singh1, Hemant Kumar Singh1
1Department of Veterinary Parasitology, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya-224 229, Uttar Pradesh, India.
2ICAR-National Research Centre on Equines, Hisar- 125 001, Haryana, India.

ABSTRACT

Background: Six representative ITS1-PCR amplified products were used for the amplifications of conserved regions of 18S and 5.8S rDNA to amplify Internal Transcribed Spacer1 (ITS1) gene of 540 bp of Typanosoma evansi isolates in equine from three agro-climatic zones of Eastern Uttar Pradesh, India.

Methods: Multiple sequence alignment was done by MAFFT online multiple sequence alignment tools by selecting the necessary parameters. The phylogenetic study of ITS1 gene revealed all the three isolates (T. evansi horse India Uttar Pradesh- North Eastern Plane Zone, T. evansi mule India Uttar Pradesh- Eastern Plane Zone and T. evansi horse India Uttar Pradesh- Vindhyan Zone) of T. evansi were clustered in single group.

Result: Multiple alignment of nucleotide sequence of ITS1 genes showed that T. evansi isolates from Eastern region of Uttar Pradesh, Indian had > 99% nucleotide homology with isolates of camel Iran (KX898420), camel Egypt (MW603779.1) and buffalo China (FJ712715.1).

INTRODUCTION

Equine trypanosomosis caused by Trypanosoma evansi, is an endemic disease in India, reported from different states of country comparatively higher in Northern India (Singh et al., 2010; Chaudhry et al., 2000; Sumbria et al., 2014). The parasite is transmitted mechanically through the blood sucking dipteran flies and can also be vertical, horizontaland iatrogenic (Desquesnes et al., 2001). The effective control of the disease requires molecular- based techniques which are more reliable than microscopy test (Desquesnes et al., 2013; Bhagwan et al., 2015; Sumbria et al., 2015; Diallo et al., 2018; Agrawal et al., 2024). The isolation of several genes from T. evansi such as invariable surface glycoprotein (ISG) genes, nuclear DNA (Deoxyribonucleic acid), kinetoplastic DNA, ribosomal DNA (Ijaz et al., 1998 and Maharana et al., 2019) and a region from rRNA (Ribosomal Ribonucleic acid) internal transcribed spacer 1 (ITS-1) (Agbo et al., 2001) are contributed major role in molecular diagnosis. The 18S rRNA gene has been found as superior target for phylogenetic study due to low substitution rate, conserved region, five different genotypes (A, B, C, Dand E) based on the presence of different clades (Qablan et al., 2013; Liu et al., 2016; Ketter-Ratzon et al., 2017) and occurrence in several copies (Qablan et al., 2013).

The detailed and systematic information is insufficient about to the phylogenetic characterization of Trypanosoma evansi infection in equine population in the Eastern Uttar Pradesh, India. Therefore the present study was undertaken to know the phylogeographic characterization of Trypanosoma evansi in naturally infected horses in agro-climatic zones of Eastern Uttar Pradesh, India.

MATERIALS AND METHODS

Study area

The present study was conducted in North Eastern Plain Zone, Eastern Plain Zone and Vindhyan Zone of Eastern region of Uttar Pradesh, India. It was carried out at Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kumarganj, Ayodhya, Uttar Pradesh and ICAR-National Research Centre on Equines (NRCE), Sirsa Road, Hisar, Haryana, India from February, 2020 to February, 2021. The climate of study area is defined as humid sub-tropical and has three major seasons viz. winter, summer and rainy season (Table 1).

Table 1: Salient features of three agro-climatic zones of Eastern region, Uttar Pradesh, India.



Extraction of genomic DNA

Blood samples were collected randomly from 524 equines from study area across different seasons. Total genomic DNA was extracted from microscopically positive whole blood using DNeasy Blood and Tissue Kit-50 (Qiagen India Pvt. Ltd., New Delhi). Out of positive samples, six representative DNA samples; two from each agro-climatic zone of study area were stored at -40°C for phylogenetic study (Fig 1).

Fig 1: Agarose gel electrophoresis of 540 bp of PCR product for phylogenetic analysis of six representative Trypanosoma evansi isolates.



Optimization and amplification of Internal transcribed spacer1 (ITS1) gene of T. evansi

Kin 1 (ITS1-R, 20 bp length) and Kin 2 (ITS1-F, 16 bp length) set of primers (540 bp) used for the amplifications of conserved regions of 18S and 5.8S rDNA to amplify Internal Transcribed Spacer1 (ITS1) gene of 540 bp of T. evansi (McLaughlin et al., 1996). ITS1-PCR reaction was manipulated in 25μl reaction mixture containing more than 40 nanogram (ng)/µl of genomic DNA samples. Each PCR cycle was consisted of denaturation at 95°C for 30 sec, annealing at 54°C for 30 sec and extension at 72°C for 30 sec. PCR products were purified from gel extraction (Fig 2) procedure by QIAquick gel extraction kit (Qiagen) and were eluted in 20 μl of NFW (Nuclease-free water) and stored at -40°C until further use.

Fig 2: Agarose gel slice containing 540 bp of PCR product of Trypanosoma evansi isolates for extraction of genomic DNA.



Sequence and phylogenetic analysis of ITS1-T. evansi gene

PCR products were sequenced by outsource sequencing agency (Genosys Informatics, Azadpur, Delhi, India). Best homologous sequences were selected by searching NCBI BLAST (National Centre for Biotechnology, Basic Local Alignment search tool) database on the basis of E- value and pasted in separate file in FASTA format. Multiple sequence alignment was done by MAFFT (Multiple alignments using fast Fourier transform). Phylogenetic analyses of aligned sequences of six isolates were done independently with the sequences of the ITS-1 gene available in GenBank database by utilizing the MEGA soft software version 6.0. Phylogenetic tree was constructed by maximum likelihood method by setting the boot strap value and algorithm for T. evansi-Tamura-Nei-TN93 model (Tamura et al., 2011).

RESULTS AND DISCUSSION

The phylogenetic study of ITS1 gene revealed all the three isolates (T. evansi horse India Uttar Pradesh- North Eastern Plane Zone, T. evansi mule India Uttar Pradesh- Eastern Plane Zone and T. evansi horse India Uttar Pradesh- Vindhyan Zone) of T. evansi were clustered in single group (Fig 3).

Fig 3: Phylogenetic tree based on Internal Transcribed Spacer1 (ITS1) gene of rypanosoma evansi isolates.



ITS 1 T. evansi horse India Uttar Pradesh -North Eastern Plane Zone and ITS 1 T. evansi horse India Uttar Pradesh-Vindhyan Zone clone were more associated with T. evansi isolates cattle from India- Assam (KY114581.1), T. evansi isolates donkey India- Gujarat (KR858269.1),T. evansi camel Iran (KX898420) and T. evansi camel Egypt (MW603779.1) whereas ITS 1 T. evansi mule India Uttar Pradesh- Eastern Plane Zone clone was more related to T. evansi isolates from T. evansi camel Iran (KX898420.1), T. evansi camel Egypt (MW603779.1), T. evansi camel Egypt (MW603778.1) and T. evansi buffalo China (FJ712715.1). This result may helpful to know  about genetic variability among different isolates of T. evansi from other parts of India and PCR primers may be designed from conserved regions for development of sensitive molecular diagnostic.

ITS of the rDNA are most appreciated target for molecular charecterization and resolution of taxonomic identities of trypanosome belonging to subgenus, species and type (Cupolillo et al., 1995; McLaughlin et al., 1996). Each transcribed unit of the rDNA locus is composed of 18S, 5.8S and 28S rRNA genes, as well as various ITS (ITS1 and ITS2) flanked by non-transcribed spacers (Hernandez et al., 1993). McLaughlin et al., (1996) were designed a pair of Kin primer (Kin 1 and Kin 2) especially for kinetoplastid species. These primers especially target to the conserved domain of the 18S and 5.8S rDNA to amplify the ITS1 gene amplify which is situated between 18S and 5.8S rRNA genes. The present investigation of sequencing and molecular characterization based on the ITS1 region of rDNA was carried out to evaluate genetic mutability among Indian equine T. evansi isolates from three agro-climatic zone of Eastern region of Uttar Pradesh, India. The phylogenetic tree shows also the mutative relationship of the sequences in which the length of the branch was corresponding to the assessed genetic distance between the sequences. The length of the branch is very small between ITS 1 T. evansi horse India Uttar Pradesh -North eastern plane zone and ITS 1 T. evansi horse India Uttar Pradesh -Vindhyan zone from T. evansi camel Iran (KX898420) and T. evansi camel Egypt (MW603779.1) having nucleotide identity > 99%. ITS 1 mule isolate from Eastern Plain Zone of Eastern region, Uttar Pradesh was more closely related to T. evansi camel Iran (KX898420.1), T. evansi camel Egypt (MW603779.1 and MW603778.1) and T. evansi buffalo China (FJ712715.1) having nucleotide identity > 99%. Similar finding was reported by Kumar et al., (2019).

CONCLUSION

T. evansi isolates from Eastern region of Uttar Pradesh, Indian had > 99% nucleotide homology with isolates of camel Iran (KX898420), camel Egypt (MW603779.1) and buffalo China (FJ712715.1).

ACKNOWLEDGEMENT

Thanks to all the members of Indian Council of Agricultural Research- National Research Centre on Equines (NRCE), Hisar, Haryana, India for providing lab facilities, their contribution and support.  We would like to thank to the Vice-chancellorandUAT, Kumarganj, Ayodhya, India for providing facilities to do the present work.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

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