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Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of <em>Eimeria</em> that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Research Institute of Biopharmacy and Veterinary Drugs

Although the validity of the coccidian species, Eimeria mivati, has been questioned by many researchers for a long time there has not been any molecular analysis that would help resolve this issue. Here we report on the discovery of the two types of small ribosomal subunit (18S) gene within the Eimeria mitis genome that correspond to the known 18S sequences of E. mitis and E. mivati, and this is in conflict with the existence of E. mivati as an independent species. We have carried out five single oocyst isolations to obtain five single-oocyst-derived strains of E. mitis and these were analyzed by the sequencing of 18S and mitochondrial cytochrome c oxidase subunit I genes. The two types of 18S gene were found to be present in each strain in roughly equal ratios. This indicates that if the strains carrying only one or the other 18S type exist, they will likely cross-breed and still represent a single species. However, the more probable explanation is that all strains of E. mitis contain two types of 18S gene and that the occasional detection of only one or the other type by sequencing might be caused by insufficient sampling. This is also the first report of the two types of 18S gene in Eimeria, which has already been described in some other apicomplexan species, most notably Plasmodium. We also found that these two types of ribosomal RNA differ significantly in their secondary structure. The biological significance of the two 18S gene variants in E. mitis is not known, however, we hypothesize that these variants might be used in different stages of the parasite's life-cycle as it is in other apicomplexan species investigated so far.

The discovery of the two types of small subunit ribosomal RNA gene in Eimeria mitis contests the existence of E. mivati as an independent species.

Although oocyst morphology was always considered as a reliable parameter for coccidian species discrimination we describe strain variation of turkey coccidia, Eimeria adenoeides, which remarkably exceeds the variation observed in any other Eimeria species. Two strains have been isolated - the first strain maintains the typical oocyst morphology attributed to this species - large and ellipsoidal - while the second strain has small and ovoid oocysts, never described before for this species. Other biological parameters including pathogenicity were found to be similar. Cross-protection between these 2 strains in 2 immunization and challenge experiments was confirmed. Sequencing and analysis of 18S and ITS1 ribosomal DNA revealed a close relationship according to 18S and a relatively distant relationship according to ITS1. Analysis of 18S and ITS1 sequences from commercial turkey coccidiosis vaccines Immucox®-T and Coccivac®-T revealed that each vaccine contains a different strain of E. adenoeides and that these strains have 18S and ITS1 sequences homologous to the sequences of the strains we have isolated and described. These findings show that diagnostics of turkey coccidia according to oocyst morphology have to be carried out with caution or abolished entirely. Novel PCR-based molecular tools will be necessary for fast and reliable species discrimination.

Description of the two strains of turkey coccidia Eimeria adenoeides with remarkable morphological variability.

Eimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and may under-diagnose subclinical disease. Recognition of the tight economic margins prevailing in modern poultry production and the impact of avian coccidiosis on poverty in many parts of the world has highlighted a requirement for a panel of straightforward and sensitive, but cost-effective, Eimeria species-specific diagnostic assays.

Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens.

Coccidiosis is a disease caused by apicomplexan parasites of the genus Eimeria, which has a significant economic impact on poultry production. Multiple species infecting the turkey have been described; however, due to the general lack of unambiguous description, their identification and taxonomy is debatable. In this work, a systematic approach was taken to isolate, characterise and compare coccidian species in the turkey. Individual species were tracked according to their unique 18S ribosomal DNA sequence. The single-oocyst isolation technique and passaging of mixed species field isolates in selectively immunised birds enabled the derivation of pure species. Six distinct strains representing five eimerian species that infect the turkey were obtained. It appears highly probable that these species represent all species described in the past with the exception of Eimeria subrotunda. The species were analysed using both traditional methods and DNA sequencing. For each strain the oocyst morphology, prepatent period, gross pathology, pathogenicity, host specificity and endogenous cycle were studied. Antigenic similarity was investigated in multiple cross-immunity experiments. For identification and quantification of each individual species or strain, quantitative real-time PCR markers were also developed. Parallel characterisation of pure strains allowed comprehensive comparison with the original descriptions and assignment of correct species names. The species Eimeria meleagridis, Eimeria dispersa, Eimeria gallopavonis, Eimeria meleagrimitis and Eimeria innocua were identified. Comparison of our data with those of previous studies indicates that Eimeria adenoeides is most probably a synonym for either E. meleagridis or E. gallopavonis, or a description based on a mixture of these species, and thus nomen dubium. The species E. dispersa and E. innocua were also found to infect Bobwhite Quail. Phylogenetic reconstruction based on 18S rDNA and cytochrome c oxidase subunit I gene (COI) sequences showed that these two species form a distinct clade unrelated to other turkey coccidia and point to a polyphyletic origin of the species infecting the turkey.

Coccidia of turkey: from isolation, characterisation and comparison to molecular phylogeny and molecular diagnostics.

Protozoan parasites of the Eimeria genus have undergone extensive speciation and are now represented by a myriad of species that are specialised to different hosts. These species are highly host-specific and usually parasitise single host species, with only few reported exceptions. Doubts regarding the strict host specificity were frequent in the original literature describing coccidia parasitising domestic turkeys. The availability of pure characterised lines of turkey and chicken Eimeria species along with the recently developed quantitative PCR identification of these species allowed to investigate the issue of host specificity using well-controlled cross-transmission experiments. Seven species of gallinaceous birds (Gallus gallus, Meleagris gallopavo, Alectoris rufa, Perdix perdix, Phasianus colchicus, Numida meleagris and Colinus virginianus) were inoculated with six species and strains of turkey Eimeria and six species of chicken coccidia and production of oocysts was monitored. Turkey Eimeria species E. dispersa, E. innocua and E. meleagridis could complete their development in the hosts from different genera or even different families. Comparison of phylogenetic positions of these Eimeria species according to 18S rDNA and COI showed that the phylogeny cannot explain the observed patterns of host specificity. These findings suggest that the adaptation of Eimeria parasites to foreign hosts is possible and might play a significant role in the evolution and diversification of this genus.

Vladimir Vrba

BIOPHARM

Research Institute of Biopharmacy and Veterinary Drugs

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens