Introduction
Ascaridia galli (Schrank, 1788) is one of the most common nematodes affecting chickens and other birds that have a worldwide distribution. Ascaridia galli infection has been associated with retarded growth, weight loss, reductions in egg production, intestinal blockage and death in severe cases, causing economic losses to the poultry industry (Daş & Gauly, Reference Daş and Gauly2014). The prevalence of A. galli is very high (varies from 22% to 84%) in chickens from free range and organic production in some countries (Sherwin et al., Reference Sherwin, Nasr, Gale, Petek, Stafford, Turp and Coles2013; Thapa et al., Reference Thapa, Hinrichsen and Brenninkmeyer2015). Ascaridia galli may be a problem in birds that come into contact with the open environment with infectious eggs of A. galli (such as free range and organic farms). In a recent study, neither artificial nor natural infection with A. galli was found to influence external and internal egg quality, irrespective of infection intensity (Sharma et al., Reference Sharma, Hunt and Hine2018, Reference Sharma, Hunt, Hine and Ruhnke2019). In China, A. galli is also considered a predominant parasite in domestic chickens (Tian et al., Reference Tian, Liang, Wang, Sun, Liu, Yang, Hu, Yu and Zhao2015).
Due to its maternal inheritance, fast evolutionary rate and lack of recombination (Boore, Reference Boore1999), mitochondrial DNA (mtDNA) has been extensively used for studies on genetic diversity and phylogenetic analyses at various taxonomic levels of different organisms, including nematodes (Goswami et al., Reference Goswami, Chaudhary, Verma and Singh2015; Yong et al., Reference Yong, Song, Eamsobhana, Goh and Lim2015; Aguado et al., Reference Aguado, Grande, Gerth, Bleidorn and Noreña2016; Bastos Gomes et al., Reference Bastos Gomes, Miller, Vaughan, Jerry, Mccowan, Bradley and Hutson2017). For example, mitochondrial (mt) cytochrome c oxidase subunit 1 (cox1) sequences are useful genetic markers for the identification and differentiation of Dirofilaria immitis (Heidari et al., Reference Heidari, Kia, Arzamani, Sharifdini, Mobedi, Zarei and Kamranrashani2015). Furthermore, mtDNA is a useful and reliable marker for the identification of cryptic nematode species (Blouin, Reference Blouin2002). A recent study showed that mt cox1 sequences can provide a rich source of genetic markers to assess the genetic diversity and cryptic species of Dictyocaulus lungworms (Ács et al., Reference Ács, Hayward and Sugár2016). Trichuris infecting primates represents a complex of cryptic species, with some species being able to infect both humans and non-human primates based on mt genome datasets (Hawash et al., Reference Hawash, Andersen, Gasser, Stensvold and Nejsum2015). Although the mt cox1 gene has been studied in A. galli in South Africa (Malatji et al., Reference Malatji, Tsotetsi, Van Marle-Koster and Muchadeyi2016), no information is available about genetic variation and cryptic species among A. galli isolates using mt cox1 polymorphisms.
The objectives of the present study were to investigate the genetic variation in mt cox1 genes among A. galli isolates from domestic chickens in Hunan Province, China, to evaluate further the claim that A. galli may include cryptic species and to assess the utility of the cox1 gene as a potential genetic marker for the management of A. galli in chickens and other birds.
Materials and methods
All adult roundworms of A. galli (n = 26) were obtained from free-range adult domestic chickens, which were naturally infected with A. galli and that were from one flock of the same geographical origin in Hunan Province, China (table 1). These A. galli specimens were obtained from the gastrointestinal tracts of chickens in the slaughterhouse, washed in physiological saline, identified to species level primarily based on morphological characters, fixed in 70% (v/v) ethanol and stored at −20°C until use. Total genomic DNA was extracted from individual A. galli samples by sodium dodecyl sulphate/proteinase K treatment, column-purified (WizardTM DNA Clean-Up, Promega, Madison, Wisconsin, USA) and eluted into 30 μl water according to the manufacturer's recommendations.
Geographical origins in China of Ascaridia galli samples used in the present study, as well as their GenBank accession numbers for sequences of the pcox1 gene.
The cox1 gene was amplified with primers JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (5′-TAAAGAAAGAACATAATGAAAATG-3′) (Bowles & McManus, Reference Bowles and McManus1994). Polymerase chain reactions (PCRs) (25 μl) were performed in 3.0 μl of magnesium chloride (25 mm), 0.25 μl of each primer (50 pmol/μl), 2.5 μl 10 × rTaq buffer (100 mm Tris-hydrochloride and 500 mm potassium chloride), 2 μl of deoxy-ribonucleoside triphosphate mixture (2.5 mm each), 0.25 μl of rTaq (5 U/μl) DNA polymerase (TaKaRa Biotechnology, Dalian, China), 2 μl of DNA sample and 14.75 μl water in a thermocycler (Biometra, Göttingen, German). The cycling conditions were 94°C for 5 min (initial denaturation), followed by 35 cycles of 94°C for 30 s (denaturation), 55°C for 30 s (annealing), 72°C for 1 min (extension) and then 72°C for 5 min (final extension). Negative control (without DNA template) was included in each amplification run. Each amplicon (5 μl) was examined by 1% (w/v) agarose gel electrophoresis to validate amplification efficiency. PCR products were sent to Life Technology (Beijing, China) for sequencing from both directions.
Sequences of the mt cox1 gene were separately aligned using the software MAFFT 7.263 (Katoh & Standley, Reference Katoh and Standley2016). The level of sequence differences (D) among A. galli isolates were calculated by pairwise comparisons using the formula D = 1 − (M/L), where M is the number of alignment positions at which the two sequences have a base in common, and L is the total number of alignment positions over which the two sequences are compared (Chilton et al., Reference Chilton, Gasser and Beveridge1995).
To study the phylogenetic relationships with representative roundworm species, A. galli (JX624728), Ascaridia columbae (JX624729), Ascaris suum (HQ704901), Baylisascaris ailuri (NC_015925), Contracaecum rudolphii B (FJ905109), Baylisascaris procyonis (NC_016200), Baylisascaris schroederi (NC_015927), Baylisascaris transfuga (NC_015924), Heterakis beramporia (KU529972), Heterakis gallinae (KU529973), Toxascaris leonina (NC_023504), Toxocara canis (NC_010690), Toxocara cati (NC_010773), Toxascaris leonina (KC902750) and Toxocara malaysiensis (NC_010527) were considered into the present study. Sequences of a portion of the cox1 (pcox1) with consensus lengths (441 bp) were aligned using the MAFFT 7.263 program. Maximum likelihood (ML) analyses were performed in PhyML 3.0 (Guindon et al., Reference Guindon, Dufayard, Lefort, Anisimova, Hordijk and Gascuel2010) using the subtree pruning and regrafting method with a BioNJ starting tree, and the GTR + I + G model with its parameter for the DNA dataset determined for the ML analysis using JModeltest (Posada, Reference Posada2008) based on the Akaike information criterion. Bootstrap support (Bp) for ML trees was calculated using 100 bootstrap replicates. Phylograms were drawn using FigTree v.1.31 (http://tree.bio.ed.ac.uk/software/figtree/).
Results and discussion
Genomic DNA was isolated from 26 individual adult A. galli samples. To examine sequence difference in the mt cox1 gene sequences and to assess the magnitude of genetic diversity in these sequences within A. galli, amplicons of pcox1 (approximately 450 bp) were amplified individually and subjected to agarose gel electrophoresis. The mt cox1 sequences were deposited in GenBank under the accession numbers KX266841–KX266860 and OM004022–OM004027 (table 1).
The sequences of pcox1 (441 bp) were obtained from the 26 samples, and the A + T contents of the sequences were 65.6–66.9%, consistent with that of a previous study from mt cox3, nad1 and nad4 genes among A. galli samples in China (Li et al., Reference Li, Liu, Wang, Song, Lin, Zou, Liu, Xu and Zhu2013). A substantial level of nucleotide difference was detected among the mt cox1 of A. galli samples. The intra-specific sequence variations among different populations of A. galli isolates were 0–7.7% for pcox1. The inter-specific sequence differences among other members of the infraorder Ascaridomorpha were 11.4–18.9%. Comparison of the pcox1 sequences among four Baylisascaris species revealed a sequence difference of 2.7–6.1% (Xie et al., Reference Xie, Zhang and Niu2011a, Reference Xie, Zhang and Wangb). In addition, sequence diversity (7.9–12.9%) was also detected in five Toxocara species by analysis of mt cox1 gene sequences (Li et al., Reference Li, Lin, Song, Sani, Wu and Zhu2008). Hence, the present findings provide additional genetic evidence for the existence of cryptic species within A. galli.
Mitochondrial gene sequences may provide reliable genetic markers in examining the taxonomic status of nematodes (Blouin, Reference Blouin2002). In the present study, all of the A. galli isolates grouped together with moderate statistical support (Bp = 92), indicating that all of the Ascaridia isolates were A. galli, and also resolved three distinct clades that, at present, do not seem to be geographically isolated (fig. 1a). In addition, phylogenetic analysis of the mt cox1 sequences also provided further support that AGCS22, AGCS23 and A. galli samples (JX624728) represent close but distinct taxa (fig. 1b). The differences among the three Ascaridia isolates are about the same (looking at branch lengths) as between B. ailuri and B. transfuga (fig. 1b). Taken together, the molecular evidence presented here supports the hypothesis that the gene pools of A. galli from domestic chickens have been isolated for a substantial period of time and that they represent a complex of cryptic species.
Phylogenetic relationship among Ascaridia galli isolates in China with other nematodes inferred by ML using the cox1 dataset. Bp values are indicated at nodes. The scale bars show the number of subsitutions per site. All of the A. galli isolates in the present study were grouped together with moderate statistical support (Bp = 92), indicating that all of the Ascaridia isolates were A. galli, and also resolved three distinct clades that, at present, do not seem to be geographically isolated (a). Phylogenetic analysis of the mt cox1 sequences provided further support that AGCS22, AGCS23 and A. galli samples (JX624728) represent close but distinct taxa (b). The differences among the three Ascaridia isolates are about the same (looking at branch lengths) as between Baylisascaris ailuri and Baylisascaris transfuga (b). The representative roundworm species were used as follows: A. galli (JX624728), Ascaridia columbae (JX624729), Ascaris suum (HQ704901), B. ailuri (NC_015925), Contracaecum rudolphii B (FJ905109), Baylisascaris procyonis (NC_016200), Baylisascaris schroederi (NC_015927), B. transfuga (NC_015924), Heterakis beramporia (KU529972), Heterakis gallinae (KU529973), Toxascaris leonina (NC_023504), Toxocara canis (NC_010690), Toxocara cati (NC_010773), Toxascaris leonina (KC902750) and Toxocara malaysiensis (NC_010527).
The present study provides additional genetic support for the existence of cryptic species within A. galli, but we believe it is still necessary to carry out more experimental research. Future studies could (1) examine population structure using a larger number of samples from different hosts and geographical locations, (2) implement the analysis of molecular variance and gene flow among different provinces in China; (3) characterize the complete mt genomes of these A. galli samples.
In conclusion, genetic diversity among A. galli isolates from Hunan Province, China, were revealed by sequence analyses of the mt cox1 gene. These results provide additional genetic evidence for the existence of cryptic species within A. galli. The results of the present study have implications for studying molecular epidemiology and population genetics of A. galli, and provide an additional genetic marker for the management of A. galli in chickens and other birds.
Author contributions
Y.Z. and S.-F.L. performed the experiments; Y.Z. and J.L. participated in the data analysis; Y.Z., S.-F.L. and J.L. edited the manuscript. All authors read and approved the final manuscript.
Financial support
This work was supported by a grant from the Scientific and Technological Research Projects of Henan Province, China (grant number 172102410051), funding from the Jiangsu Key Laboratory of Zoonosis (R1706) and a grant from the Youth Foundation of Xinyang Agriculture and Forestry University (grant number QN2021012).
Conflicts of interest
None.
Ethical standards
All experiments were supervised by the Animal Ethics Committee of Hunan Agricultural University (no. 43321503) and performed in accordance with the regulations and guidelines of this committee.
