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

Abstract

Background: 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.

Results: Loop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In this study we have developed a panel of species-specific LAMP assays targeting the seven Eimeria species that infect the chicken. Each assay has been shown to be genuinely species-specific with the capacity to detect between one and ten eimerian genomes, equivalent to less than a single mature schizont. Development of a simple protocol for template DNA preparation from tissue collected post mortem with no requirement for specialist laboratory equipment supports the use of these assays in routine diagnosis of eimerian infection. Preliminary field testing supports this hypothesis.

Conclusions: Development of a panel of sensitive species-specific LAMP assays introduces a valuable new cost-effective tool for use in poultry husbandry.

Figure 1

Eimeria LAMP assay species-specificity. Electrophoresis of LAMP products amplified from a DNA panel representing Eimeria acervulina (lane 1), E. brunetti (2), E. maxima (3), E. mitis (4), E. necatrix (5), E. praecox (6), E. tenella (7) and the host (chicken, 8) as a background control. Panels represent LAMP assays specific for E. acervulina (a), E. brunetti (b), E. maxima (c), E. mitis (d), E. necatrix (e), E. praecox (f) and E. tenella (g).

Figure 2

Eimeria species-specific LAMP assay sensitivity using Eimeria tenella as an example. Neg = no template negative control.

Figure 3

LAMP diagnosis of eimerian infection from three field sample examples. D = duodenal sample, J/I = jejuno-ileal sample, C = caecal sample. (a) Eimeria acervulina LAMP assay (b) Eimeria maxima LAMP assay.

Figure 4

The importance of Chelex 100 during field sample DNA preparation. Eimeria acervulina LAMP of all three duodenal samples found to be positive for E. acervulina by lesion scoring and multiplex PCR. Samples prepared in the absence (-) or presence (+) of Chelex 100.