Phylogenetic analysis of Eimeria tenella isolated from the litter of different chicken farms in Mymensingh, Bangladesh

Abstract

Background: Eimeria tenella is the most pathogenic intracellular protozoan parasite of seven Eimeria species causing chicken coccidiosis around the world. This species is particularly responsible for caecal coccidiosis leading to serious morbidity-mortality and financial loss in poultry production.

Methods: The present study explored the genetic diversity of E. tenella. Litter slurry was collected from 18 broiler farms located in Mymensingh district, Bangladesh. Litter samples were processed for oocyst isolation-identification using parasitological techniques followed by genomic DNA extraction from sporulated oocysts. For molecular analysis, the internal transcribed spacer 1 gene of E. tenella was amplified using species-specific primers and sequenced. After editing and alignment, 263 bp sequences were used for analysis.

Results: Genetic analysis showed seven distinct genotypes and detected six single nucleotide polymorphisms among the 18 E. tenella isolates. The nucleotide and genotype diversity were 0.00507 and 0.8235, respectively. A phylogenetic tree was constructed with 66 sequences (seven studied genotypes and 59 reference sequences from GenBank database). The neighbour-joining tree represented that the studied E. tenella isolates were grouped with reference E. tenella isolates with strong nodal support (100%) and the nucleotide sequences of E. tenella, E. necatrix, E. acervulina, E. brunetti, E. maxima, E. mitis and E. praecox formed separate clusters without any geographical boundaries.

Conclusions: This is the first study on the genetic analysis of E. tenella from Mymensingh district, Bangladesh. These findings will provide baseline data on the species conformation and genetic variations of E. tenella. Further extensive investigation will be needed to reveal the population genetic structure of this parasite and thus will facilitate the planning of effective control strategies.

Keywords: Bangladesh; Eimeria tenella; ITS1; chicken; phylogenetic analysis.

FIGURE 1

Agarose gel electrophoresis of the PCR products. Amplification of ∼271 bp i n t e r n a l t r a n s c r i b e d s p a c e 1 (I T S 1) gene for Eimeria tenella represented on 1.5% agarose gel; M, 100 bp marker; 1–3, I T S 1 gene product from samples; P, positive PCR control; N, negative PCR control

FIGURE 2

Neighbour‐joining phylogeny of 66 ITS1 gene sequences of E. tenella. The analysis involved 66 nucleotide sequences (seven studied genotype sequences and 59 reference sequences, retrieved from the GenBank database). The sequences were aligned and constructed a neighbour‐joining tree. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Red bullets indicate studied genotype sequences