Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus)

Abstract

More than 68 billion chickens were produced globally in 2018, emphasising their major contribution to the production of protein for human consumption and the importance of their pathogens. Protozoan Eimeria spp. are the most economically significant parasites of chickens, incurring global costs of more than UK £10.4 billion per annum. Seven Eimeria spp. have long been recognised to infect chickens, with three additional cryptic operational taxonomic units (OTUs) first described more than 10 years ago. As the world's farmers attempt to reduce reliance on routine use of antimicrobials in livestock production, replacing drugs that target a wide range of microbes with precise species- and sometimes strain-specific vaccines, the breakthrough of cryptic genetic types can pose serious problems. Consideration of biological characteristics including oocyst morphology, pathology caused during infection and pre-patent periods, combined with gene-coding sequences predicted from draft genome sequence assemblies, suggest that all three of these cryptic Eimeria OTUs possess sufficient genetic and biological diversity to be considered as new and distinct species. The ability of these OTUs to compromise chicken bodyweight gain and escape immunity induced by current commercially available anticoccidial vaccines indicates that they could pose a notable threat to chicken health, welfare, and productivity. We suggest the names Eimeria lata n. sp., Eimeria nagambie n. sp. and Eimeria zaria n. sp. for OTUs x, y and z, respectively, reflecting their appearance (x) or the origins of the first isolates of these novel species (y, z).

Keywords: Chickens; Cryptic species; Eimeria; Food security; Genome sequencing; Operational taxonomic unit.

Graphical abstract

Fig. 1

Sporulated oocysts of the Eimeria Operational Taxonomic Unit (OTU) genotypes x, y, and z collected from domestic chickens (Gallus gallus domesticus). Photomicrographs of sporulated oocysts are shown for (A) OTUx, (B) OTUy and (C) OTUz. Composite line drawings are shown for (D) OTUx, (E) OTUy and (F) OTUz. RB, residual body; SB, stieda body; PG, polar granule. Scale bars = 10 µm.

Fig. 2

Dimensions of sporulated oocysts representing Eimeria collected from domestic chickens (Gallus gallus domesticus). (A) Oocysts from all seven Eimeria spp. that infect chickens and Operational Taxonomic Units (OTUs) x, y, and z. For close comparison subsets of (B) Eimeria maxima and OTUx, (C) Eimeria brunetti and OTUy, and (D) Eimeria acervulina, Eimeria mitis, and OTUz, are shown. Note: The scales used vary between panels to improve illustration of differences between oocysts with closely comparable dimensions.

Fig. 3

The effect of Eimeria Operational Taxonomic Unit (OTU) infection on broiler chicken bodyweight gain (BWG). Groups of eight 21 day old Ross 308 broiler chickens were infected by oral inoculation of (A) OTUx or (B) OTUz oocysts. Weight was measured at the time of infection and 10 days later. Data points indicate individual chickens, with the mean and S.D. presented for each group. Superscript letters identify groups that were statistically significantly different from each other within an experiment (P < 0.05). Note: The scales used on the Y axes vary between panels to improve visualisation of intra-group variation.

Fig. 4

Optimal Neighbour-Joining tree inferred using a 1154 bp alignment of the partial Eimeria 18S rDNA locus. Evolutionary distances were calculated using the Kimura 2-parameter model. The number of base substitutions per site between sequences are shown to the right of the sequence identifiers, presented as a/b (x), where a = maximum number within a recognised species/Operational Taxonomic Unit (OTU) group, b = minimum between recognised species/OTUs, and the figure in parentheses is the fold difference. Pairwise analyses were conducted using the Maximum Composite Likelihood method. Figures shown in blue indicate analysis when the long and short form Eimeria maxima sequences were pooled. Aus, Australia; Nig, Nigeria.

Fig. 5

Optimal Neighbour-Joining tree inferred using a 791 bp alignment of the partial Eimeria cytochrome C oxidase I (COI) locus. Evolutionary distances were calculated using the Kimura 2-parameter model. The number of base substitutions per site between sequences are shown to the right of the sequence identifiers, presented as a/b (x), where a = maximum number within a recognised species/Operational Taxonomic Unit (OTU) group, b = minimum between recognised species/OTUs, and the figure in parentheses is the fold difference. Pairwise analyses were conducted using the Maximum Composite Likelihood method. Aus, Australia; Nig, Nigeria.

Fig. 6

Optimal Neighbour-Joining tree inferred using a 4135 amino acid alignment of 56 concatenated Eimeria gene models rooted on Eimeria falciformis. Support for each node is presented, indicating outcomes from Maximum Likelihood (ML)/Neighbour-Joining (NJ)/Bayesian methods. For ML, the General Time Reversible (GTR) model was used with a gamma distribution. For NJ evolutionary distances were calculated using the Kimura 2-parameter model. For Bayesian inference analysis the GTR+G model was used, including four runs with 1,000,000 generations, a sample frequency of 10, and 25% burn-in.

Fig. 7

Dichotomous key discriminating Operational Taxonomic Units (OTUs) x, y and z from the seven recognised Eimeria spp. that infect chickens.