Mitochondrial genomes of Australian chicken Eimeria support the presence of ten species with low genetic diversity among strains
Graphical abstract
Introduction
Protozoan parasites in the genus Eimeria cause the intestinal disease coccidiosis. This important global livestock disease has a significant economic impact on the poultry industry where high-density housing of large numbers of birds favours parasite transmission. Management of the disease is achieved by strict farm biosecurity as well as in-feed coccidiostats and or vaccination. Despite significant management efforts, coccidiosis outbreaks still occur due to widespread drug resistance and environmental persistence (Blake and Tomley, 2014). The disease has been further promoted by the expansion of floor reared, and free range husbandry techniques that increase flock exposure to infectious oocysts in the environment. Parasite elimination has not proved feasible; instead control is based on parasite suppression allowing birds to develop natural immunity. The annual global cost of coccidiosis, including production losses, prevention and treatment, has been estimated at over USD$3 billion (Dalloul and Lillehoj, 2006).
Seven species of Eimeria are recognized and found globally infecting chickens. In Australia a further three operational taxonomic units (OTU) were characterized by Cantacessi et al., 2008 (Cantacessi et al., 2008). Little is known about the prevalence or distribution of these OTU although all three cryptic species have now been identified in flocks from Africa (Clark et al., 2016, Fornace et al., 2013) and a recent global survey of Eimeria suggests they may be widespread in the southern hemisphere, but not the northern (Clark et al., 2016). It is unlikely that the OTU, or any of the chicken infecting Eimeria species, originated in Australia as chickens have only been present in the country since European settlement in the late 18th century.
Genetic characterisation of poultry Eimeria has historically been based on a limited number of relatively small (<1000 bp) gene regions (Barta et al., 1997, Cantacessi et al., 2008, Lew et al., 2003, Ogedengbe et al., 2011). With the advent of genome sequencing, paired with a reduction in sequencing costs, it has now become possible to compare much larger regions of DNA. Phylogenetic comparisons of Eimeria based on entire mitochondrial genomes have now been published (Hikosaka et al., 2011, Lin et al., 2011, Liu et al., 2012, Ogedengbe et al., 2014, Ogedengbe et al., 2013) with the interesting discovery that Eimeria necatrix and Eimeria tenella are more closely related to turkey Eimeria than they are to the remaining chicken Eimeria (Miska et al., 2010, Ogedengbe et al., 2014).
As with other apicomplexan parasites, the Eimeria mitochondrial genome has been reduced to roughly 6 kb in length and contains only 3 genes; cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and cytochrome b (Cyt b) plus numerous short fragments of small and large subunit ribosomal DNA (SSU and LSU rDNA) (Lin et al., 2011).
An underlying question for all parasites is to understand how genetic diversity influences epidemiology and pathogenicity and its implication in therapeutic and vaccination strategies as well as disease control. Experimental outcomes can be profoundly influenced by the choice of Eimeria species and strain, and a lack of standardization among experiments currently hampers meaningful comparisons.
The aim of this study was to characterise the mitochondrial genomes of multiple strains of all Australian species of chicken infecting Eimeria, including OTU-X, −Y and −Z, to assess how they align to global isolates, and to determine if genetic markers capable of distinguishing among strains could be identified. Having this information could assist with improved vaccine monitoring and quality control and provide the tools for molecular epidemiology to characterise different strains of Eimeria.
Section snippets
Samples and DNA extraction
Animal Science Queensland, within the Queensland Department of Agriculture and Fisheries housed oocysts from pure Australian strains of each of the seven characterized Eimeria species and three OTU (Table 1). Prior to DNA extraction oocysts were washed with distilled water, then suspended in 80 μl of phosphate-buffered saline (PBS, pH 7.2) and mechanically homogenized using 0.1 g of 1 mm glass beads for 5 min in a MiniBeadbeater-96 (Biospec Products, Bartlesville, OK, USA). DNA was extracted from
Results
Complete mitochondrial genomes were sequenced from 25 Eimeria isolates representing all 10 species known to occur in chickens throughout Australia, including OTU-X, −Y and −Z. Additional genome sequences were obtained for strains of E. zuernii and E. falciformis. The chicken Eimeria genomes vary in size from 6166 to 6419 nucleotides with the final alignment consisting of 6488 bases with an average GC content of 34.7%. Species-level comparisons among the chicken infecting Eimeria ranged from 2.2
Discussion
A comparison of the mtDNA amino acid Eimeria phylogeny to those constructed using ribosomal DNA 18S sequences (Chapman et al., 2013, Megía-Palma et al., 2015) shows general systematic congruence between the genomes. The genus Caryospora has a basal position in the phylogeny, the rabbit infecting species group together and the avian infecting Isospora sp. and primate infecting Cyclospora are nested within the genus Eimeria. A major difference between the nuclear DNA and mtDNA genome trees is
Conclusions
Australian species of chicken Eimeria align closely to their international counterparts. A phylogeny constructed from complete mitochondrial genome sequences places all three OTUs within the clade containing Eimeria species that only infect chickens. Within species mitochondrial genetic diversity was present but low among Australian isolates. Genetically different strains of the same species were frequently found in flocks. The advent of strain-specific markers will assist in future studies
Acknowledgements
We would like to thank Bioproperties Pty Ltd for access to pure strains of the three Eimeria OTUs and also pure Eimeriavax vaccine strains. Thanks also to Professor Robin B. Gasser for providing DNA of outgroup species E. zuernii and E. falciformis. We appreciate the anonymous reviewer feedback received as it helped to resolve the conundrum of outgroup choice. Funding for this project was provided by the Chicken Meat RIRDC (project PRJ-002473), the Poultry CRC (project Morgan 1-2-3) and the
References (37)
- et al.
Securing poultry production from the ever-present Eimeria challenge
Trends Parasitol.
(2014) - et al.
Genetic characterization of three unique operational taxonomic units of Eimeria from chickens in Australia based on nuclear spacer ribosomal DNA
Vet. Parasitol.
(2008) - et al.
A selective review of advances in coccidiosis research
Adv. Parasitol.
(2013) - et al.
Cryptic Eimeria genotypes are common across the southern but not northern hemisphere
Int. J. Parasitol.
(2016) - et al.
A molecular survey of Eimeria in chickens across Australia
Vet. Parasitol.
(2015) - et al.
Concatenated mitochondrial DNA of the coccidian parasite Eimeria tenella
Mitochondrion
(2011) - et al.
Inter- and intra-strain variation and PCR detection of the internal transcribed spacer 1 (ITS-1) sequences of Australian isolates of Eimeria species from chickens
Vet. Parasitol.
(2003) - et al.
Characterization of the complete mitochondrial genomes of five Eimeria species from domestic chickens
Gene
(2011) - et al.
The complete mitochondrial genomes of five Eimeria species infecting domestic rabbits
Exp. Parasitol.
(2015) - et al.
DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)
Int. J. Parasitol.
(2011)
Epidemiological aspects of the use of live anticoccidial vaccines for chickens
Int. J. Parasitol.
Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences
J. Parasitol.
Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18S rDNA sequences
J. Eukaryot. Microbiol.
GenBank
Nucleic Acids Res.
Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development
Proc. Natl. Acad. Sci.
The complete mitochondrial genome of the foodborne parasitic pathogen cyclospora cayetanensis
PLoS One
Poultry coccidiosis: recent advancements in control measures and vaccine development
Expert Rev. Vaccines
Occurrence of Eimeria species parasites on small-scale commercial chicken farms in Africa and indication of economic profitability
PLoS One
Cited by (18)
First detection and characterisation of Eimeria zaria in European chickens
2023, Veterinary ParasitologyExploiting digital droplet PCR and Next Generation Sequencing technologies to determine the relative abundance of individual Eimeria species in a DNA sample
2021, Veterinary ParasitologyCitation Excerpt :Initially, the nuclear 18S ribosomal RNA gene (Barta et al., 1997) and associated internal transcribed spacer-1 and -2 (ITS-1 and ITS-2) were targeted because of the wide availability of reference sequences for these loci (Morgan et al., 2009; Schnitzler et al., 1998). More recently, genetic loci from the mitochondrial genome, such as cytochrome c oxidase subunit I (mtCOI; Ogedengbe et al., 2011a) and cytochrome c oxidase subunit III (mtCOIII; Cunha et al., 2009; Morgan and Godwin, 2017), have been exploited. Both of these protein-coding genes demonstrate appropriate interspecific variation for use as a genotyping target (Hebert et al., 2003; Ogedengbe et al., 2018), without the complications associated with the nuclear ribosomal DNA arrays (El-Sherry et al., 2013).
Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus)
2021, International Journal for ParasitologyCitation Excerpt :Gene models used for phylogenetic comparison are available at the Mendeley data repository under the accession https://doi.org/10.17632/nphkwt6h3j.1. Specific sequences are available for the 18S rDNA (GenBank accession number LT964974, LR877466) and COI (HG992976, LR877720) loci, in addition to the complete mitochondrial genome published by others (KX094955, KX094956; (Morgan and Godwin, 2017)). Etymology: this species has been named in recognition of the location of the phenotyped isolate, collected in Zaria, Kaduna state, Nigeria.