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Molecular Identification of Mecistocirrus Digitatus in Cattle from Mymensingh Region of Bangladesh

Shanaz Parvin
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Anita Rani Dey
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Shirin Akter
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Anisuzzaman
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Hasanuzzaman Talukder
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Mohammad Zahangir Alam
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Gastrointestinal nematode infections of livestock are ranked in the top twenty diseases affecting small-holder farmers’ livestock. Mecistocirrus digitatus is one of the most prevalent parasitic nematode among the trichostrongylids causing severe health hazards leading to production losses in cattle worldwide. This study was conducted to explore the existence and genetic diversity of M. digitatus parasite populations from cattle characterizing second internal transcribed spacer (ITS-2) gene of nuclear ribosomal DNA (rDNA). A total of 23 adult Mecistocirrus parasites were collected from abomasa of slaughtered cattle from Mymensingh district of Bangladesh. After the extraction of DNA from adult parasites, ITS-2 of nuclear rDNA gene was amplified and sequenced. The edited and aligned sequences were employed for analysis to determine sequence variation and genetic diversity. All the sequences were found to have high identical ratio with M. digitatus of a published sequence and sequence identities ranged from 97.9% to 100%. Genetic analysis revealed 3 distinct ITS-2 genotypes among the M. digitatus isolates. The nucleotide and genotype diversities were 0.00089 and 0.170, respectively for ITS-2 sequences. Phylogenetic analysis (neighbour joining, maximum likelihood and maximum parsimony) of ITS-2 sequences indicated the existence of a single cluster within M. digitatus population in the study area. In conclusion, our study could confirm M. digitatus in the analyzed parasite isolates by amplifying and sequencing ITS-2 gene. Most of the isolates from our present study presented identical genotypes indicating that low genetically diversified parasites are circulating in Mymensingh region of Bangladesh. The findings of our study creates a basis for further molecular epidemiological surveys applying more M. digitatus parasite isolates from different regions of Bangladesh.

Keywords: Mecistocirrus digitatus, Genotyping, Internal transcribed spacer-2, Cattle, Bangladesh

Location: Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Cattle

Objectives:

The purpose of molecular identification and characterization of M. digitatus based on the ITS-2 sequences and to provides some imperative information/data on genetic diversity of this GIN isolates in the study area.

Methodology:

Parasite materials and isolation of DNA: Recovery of adult worms was carried out according to standard procedures as described by MAFF (1986). Briefly, after slaughtering of cattle, the abomasum was separated from the other stomach parts and ligated at both ends. The abomasum was then taken directly to the laboratory and the contents were poured into a glass beaker. Both the abomasum and its contents were carefully examined and individual adult male worms were collected. The collected parasites were then washed extensively in physiological saline. The male parasites were identified based on the morphological features such as the bursa having a small, symmetrical dorsal lobe, shorter ventroventral ray than lateroventral and anteolateral rays with 3.8-7 mm long slender spicules united together for almost their whole length (Soulsby, 1986). Only adult male worms were utilized for DNA amplification by PCR in order to avoid the danger of temperamental DNA enhancement from the eggs of female worms (Gharamah et al., 2012). The parasites were preserved in absolute alcohol, until DNA was extracted. Total genomic DNA was isolated from 24 individual worms using QIAamp DNA Mini Kit (Qiagen, Germany) according to manufacturer’s instructions. Briefly, one adult parasite was taken into an eppendorf tube, tissues were disintegrated, lysed and proteins were digested by proteinase K and washed extensively. Trapped DNA was eluted by elution buffer. Extracted DNA was measured by a Nanodrop spectrophotometer and stored at −20 oC until further use. PCR amplification and sequencing: Molecular characterization of M. digitatus using ITS-2 gene was performed. ITS-2 region (~320 bp) was amplified using previously published primers; forward 5′- ACGTCTGGTTCAGGGTTGTT-3′ and reverse-5′- TTAGTTTCTTTTCCTCGCCT-3′ (Stevenson et al., 1995). PCR was performed in a 50 μl reaction for 30 cycles (Initial denaturation of 95 °C for 5 min followed by denaturation at 95 °C for 1 min, primer annealing at 55 °C for 60 s and extension at 72 °C for 60 s), followed by a final elongation of the PCR product for 5 min. at 72 °C. A negative and positive control containing distilled water and DNA of Haemonchus contortus, respectively, were included during PCR to ensure reliability, validity and to check for possible contaminations of the amplification reactions. PCR products (5µl) were visualized on agarose gel (1.5%) with EZ-Vision® IN-Gel (Amresco, USA). Sequencing of PCR products of ITS-2 gene: All PCR products were sequenced directly using appropriate forward and reverse primers. The PCR products were column purified (Wizard PCR-Preps, Promega,) and then subjected to sequencing directly (BigDye Terminator v.3.1 cycle sequencing kit, Applied Biosystems in an automated sequencer (PRISM3730, ABI using respective forward and reverse primers (in separate reactions). Forward and reverse sequences were aligned and edited using the BioEdit software (Hall, 1999). The sequences were aligned using MEGA v.10.1.8 software (Tamura et al., 2013) and deposited in GenBank under the accession numbers: LC594625-LC594627. Molecular data analysis: The sequences of ITS-2 were aligned using the program Clustal W within MEGA v.10.1.8 (Tamura et al., 2013). Pairwise comparisons were performed with previously published sequences, and identities (%) were calculated using the program BioEdit (Hall, 1999). Phylogenetic analysis was performed using neighbour joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) methods based on the Tamura-Nei model (Tamura et al., 2013). Confidence limits were assessed using the bootstrap procedure (1000 replicates) for constructing NJ, MP and ML trees, and other settings were obtained using the default values in MEGA v.10.1.8 (Tamura et al., 2013). A 50% cut-off value was implemented for the consensus tree. A hierarchical analysis of molecular variance (AMOVA) was performed to estimate the genetic diversity within and among populations (isolates) using the Arlequin 3.1 package (Excoffier et al., 2005). In addition, sequences retrieved from GenBank were used for comparisons.

Source of Information: J Bangladesh Agril Univ 19(1): 67–72, 2021 ISSN 1810-3030 (Print) 2408-8684 (Online)

Article Link (Online Journal): https://doi.org/10.5455/JBAU.6175

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

All the sequences were found to have high identical ratio with M. digitatus of a published sequence and sequence identities ranged from 97.9% to 100%. Genetic analysis revealed 3 distinct ITS-2 genotypes among the M. digitatus isolates. The nucleotide and genotype diversities were 0.00089 and 0.170, respectively for ITS-2 sequences. Phylogenetic analysis (neighbour joining, maximum likelihood and maximum parsimony) of ITS-2 sequences indicated the existence of a single cluster within M. digitatus population in the study area. In conclusion, our study could confirm M. digitatus in the analyzed parasite isolates by amplifying and sequencing ITS-2 gene. Most of the isolates from our present study presented identical genotypes indicating that low genetically diversified parasites are circulating in Mymensingh region of Bangladesh. The findings of our study creates a basis for further molecular epidemiological surveys applying more M. digitatus parasite isolates from different regions of Bangladesh.

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