Towards improving East Coast fever (ECF) diagnosis: Validation of loop-mediated isothermal amplification (LAMP) assays for rapid detection of Theileria parva, the causative agent of ECF in cattle

Abstract

East Coast fever (ECF) is a serious tick-borne parasitic disease of cattle and is a major constraint to the livestock sector in east, central and southern Africa. ECF is caused by a protozoan parasite, Theileria parva, transmitted by the tick, Rhipicephalus appendiculatus. Several serological and molecular assays have been developed, for use in epidemiological studies, monitoring control programmes, and for research. Amongst the plethora of molecular assays for T. parva is a p104 gene nested-PCR (p104-nPCR) assay, a validated ‘gold-standard’ PCR assay. However, p104-nPCR requires sophisticated equipment and 1-2 days to perform. Two simple, rapid molecular assays for T. parva, PIM-LAMP and p150-LAMP, have been reported. Based on isothermal LAMP technology, they require minimal instrumentation and are very rapid, with potential for use in the field and in resource-poor laboratories in Africa. However, these LAMP assays have not been fully validated for routine use. The main objective of this study was to validate the LAMP assays and compare their performance with the p104-nPCR assay. Comparisons were made using purified DNA from whole blood samples from experimentally infected cattle, cattle in ECF-endemic areas of Uganda and Tanzania, and from cattle from a Kenyan ranch that is ECF-free.Results for experimentally-infected cattle showed that p104-nPCR detected T. parva in 11/12 (92%) of cattle at day 14 post-infection while the PIM-LAMP assays was at 42%. However, the p150-LAMP was inconsistent, giving false positive and negative results and lacked reproducibility. For the field samples, 4/50 (8%) of samples from Uganda and 14/50 (28%) of samples from Tanzania were consistently positive with the p104-nPCR and the PIM-LAMP assays. The p150-LAMP assay produced inconsistent results and the results were not reproducible. All 48 samples from the ranch tested negative in both the p104-nPCR and PIM-LAMP assays, whereas the p150-LAMP detected assay 27/48 (56%) as positive. Owing to the questionable performance of the LAMP assays, an additional study to develop new LAMP primers was undertaken. Bioinformatics analysis of three target genes (p104, PIM and p150) was done to identify sequences that are conserved between different strains of T. parva. Four sets of new LAMP primers were designed with LAMP Designer V1.13 Software. The LAMP primer sets were tested on genomic DNA purified from a T. parva-infected cell line. None of the primers gave amplification products despite testing several reaction conditions, illustrating some of the difficulties developing new LAMP assays. The study tested different blood DNA extraction methods to identify a simple, rapid and cost-effective method. Infected cattle blood was serially diluted and extracted either with a commercial kit, with direct heating in PBS or with heating in 10% Chelex-100. All DNA extracts were subjected to the three assays. Results obtained with the p104-nPCR assay and those from the PIM/p150-LAMP were consistent and reproducible in all cases suggesting that the expensive DNA extraction kit or methods can be replaced.These results support the p104-nPCR assay as the method of choice to diagnose ECF cases due to its consistency and reproducibility. However, based on the simplicity and rapidity of the LAMP assays, it is suggested that the PIM-LAMP assay could be considered for initial screening for infections caused by T. parva in the field prior to confirmation by laboratory based PCR diagnosis