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 i n t e r n a l t r a n s c r i b e d s p a c e r 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.


INTRODUCTION
in developing diagnosis and a control strategy, generating knowledge on bionomics, epidemiology, immunogenomics and anticoccidial agent preference. Molecular techniques have been designated as reliable and specific tools to identify the species of Eimeria (Jenkins et al., 2006;Schnitzler et al., 1999). Internal transcribed spacer (ITS-1;Schnitzler et al., 1999), ITS-2 (Lien et al., 2007) and the 5.8S ribosomal ribonucleic acid (rRNA; Molloy et al., 1998) genomic regions are targeted for the identification of species of Eimeria. Hence, the present study attempted to explore the diversity of different isolates of E. tenella from Bangladesh using the ITS-1 gene.

Study area and selection of broiler farms
The study was carried out on 18 broiler farms of the Mymensingh district in Bangladesh. These farms were selected due to their ease of approachability and large flock size.

Collection of samples and isolation of oocysts
Approximately 250 g of litter sample slurry was collected from each farm area adjacent to feeder and water lines. Processing of litter samples and isolation-identification of oocysts were performed according to the protocol described by Alam et al. (2020). The floatation technique was applied to isolate and concentrate Eimerian oocysts using saturated sodium chloride (specific gravity 1.18-1.2) followed by microscopy. Sporulation of oocysts was triggered by culture at 28 • C in 2.5% potassium dichromate for 1-2 days. Finally, the concentrated oocysts were pelleted using repeated centrifugation and elimination of media.

Extraction of genomic DNA
For genomic DNA extraction, pelleted sporulated oocysts were washed with a 1 mM sodium hypochlorite solution for 10 min at 4 • C, followed by three-times rinsing with deionised water. To crack the oocyst wall and release the sporozoites, a specialised tissue homogeniser was used.
Genomic DNA was isolated according to the manufacturer's instructions using the QIAamp stool DNA isolation kit (Qiagen). The extracted DNA samples were measured to ascertain the concentration of DNA and kept at -20 • C until further use.

Amplification and sequencing of the ITS1 gene of E. tenella
Eimeria tenella species were identified by a single PCR assay using species-specific primers targeting the ITS-1 as previously described by Schnitzler et al. (1999). Forward (EtF: 5'-AATTTAGTCCATCGCAACCCT-3') and reverse (EtR: 5'-CGAGCGCTCTGCATACGACA-3') species-specific primer sequences were used in this study. Amplification of the ITS-1 sequences of genomic rDNA was carried out in 25 µl reaction volumes containing 2 µl of DNA template, 10 pmol of reverse and forward primers, 3.0 mM MgCl 2 , 2.0 µl 10 X PCR Buffer, 200 µM of each deoxyribose nucleotide triphosphate and 0.4 U Taq DNA polymerase. The thermal program of PCR was as follows: denaturation step at 95 • C for 5 min, 35 cycles of denaturation at 95 • C for 30 s, annealing at 58 • C or 65 • C for 30 s and extension at 72 • C for 1 min and a final extension at 72 • C for 3 min. The successfully amplified PCR products were visualised using 2% agarose gel to verify that they represented a single band. All positive samples were purified using Qiaquick kits (Qiagen) and direct cycle sequencing of PCR-amplified fragments was performed using a commercially available automated sequencer.

Phylogenetic analysis
The sequences of ITS-1 were aligned using the program Clustal W within MEGA v.6.0 (Tamura et al., 2013). Intra-population diversity parameters such as nucleotide diversity, genotype diversity and the average number of nucleotide differences were calculated using DnaSP version 5.1 (Rozas, 2009). 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 based on the Tamura-Nei model (Tamura et al., 2013). Confidence limits were assessed using the bootstrap procedure (1000 replicates) for neighbour-joining trees, and other settings were obtained using the default values in MEGA v.6.0 (Tamura et al., 2013). A 50% cut-off value was implemented for the consensus tree.

Species identification and genotyping of E. tenella
A total of 18 Eimeria field samples were isolated from 18 local commercial broiler production farms in Mymensingh district. To confirm the species identity of Eimeria samples, species-specific primers of E. tenella were used to amplify the ITS1 region. The results revealed that all the field isolates were confirmed to be E. tenella since they represented a specific single 271 bp band using the specific primer of E. tenella (Figure 1). Positive PCR products of each isolate were subjected to sequencing, and sequences were edited, aligned and the resulting aligned 263 bp sequences were analysed. The sequences were compared with those in the NCBI database using the Basic Local Alignment Search Tool (BLAST) for nucleotide. Species identification was determined from the best-scoring reference sequence of the BLAST output. Eighteen PCR positive samples that were sequenced for E. tenella showed 99.23%-100% homology with E. tenella DNA sequences deposited in the GenBank database.  Table 2). The overall nucleotide diversity and genotype diversity were 0.00507 and 0.8235, respectively, among the ITS-1 sequences of E. tenella.

Phylogenetic analysis of E. tenella
We constructed a phylogenetic tree with the seven genotype sequences produced in this study and 59 sequences of different  (Figure 2).

DISCUSSION
Coccidiosis in chickens caused by Eimeria spp. are prevalent worldwide including Bangladesh Awais et al., 2012;Franco, 1993;Islam et al., 2020;Jenkins et al., 2017). E. tenella is the most injurious coccidian agent causing haemorrhagic diarrhoea in chickens and significant loss to the global economy. Susceptibility to coccidiosis is high at the age of 15-50 days, and the estimated morbidity is 50%-70% (Clark et al., 2017;Fornace et al., 2013). To protect commercial and backyard chicken farms, a specific diagnosis of infection is mandatory.
Traditional morphometry of Eimeria oocyst is approved as the gold standard. Such diagnoses are also based on oocyst morphology, infection site, pre-patent period, sporulation time and need intensive labour, expertise and more time (Vrba et al., 2010). Moreover, the size and shapes of oocysts vary due to variation in metabolic activity of parasites or hosts based on infection duration and severity. Therefore, morphometric indices may lead to misdiagnosis (Olufemi et al., 2020).
In addition, the involvement of multiple species of coccidiosis is quite common in the field, and close resemblance of morphology and pathological features usually misguide diagnosis leading to neglecting the subclinical form (Kumar et al., 2014). To overcome the difficulties in morphometry-based diagnosis, molecular diagnostic tools are progres-sively utilised Kawahara et al., 2010;Schnitzler et al., 1999). Variation in the findings between non-molecular and molecular diagnoses of coccidian parasites has already been reported (Jatau et al., 2016;Olufemi et al., 2020), and greater sensitivity was recorded for PCR-based identification in detecting even all stages of parasites (Jatau et al., 2016). Several factors can be vital for effective PCRbased diagnosis. The thick oocyst wall can act as a barrier in generating the desired genomic DNA quality (Fernandez et al., 2003). In addition, PCR inhibitors released from Taq DNA polymerase can influence the PCR reaction (Haug et al., 2007). Sequences of the ITS-1 and ITS-2 genomic regions and 18S rDNA, the small subunit rRNA are widely used for the identification, ecological genetic studies and phylogenetic and evolutionary analyses at the F I G U R E 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