Point of care colourimetric and lateral flow LAMP assay for the detection of Haemonchus contortus in ruminant faecal samples.

Rojesh Khangembam,Mariann Tóth,Róbert Farkas,Marián Várady,Alistair Antonopoulos,Nóra Vass,Levente Czeglédi

doi:10.1051/parasite/2021078

Abstract

In this study, we present an optimised colourimetric and a lateral flow LAMP assay for the detection of Haemonchus contortus in small ruminant faecal samples. Using a previously published LAMP primer set, we made use of commercially available colourimetric LAMP and lateral flow kits and combined this into an optimised diagnostic assay which was then tested on field faecal samples from Eastern and South-Eastern Hungary as well as a pure H. contortus egg faecal sample from Košice, Slovakia. Both assays showed no conflicts in visual detection of the results. Additionally, we modified and tested several centrifuge-free DNA extraction methods and one bead-beating egg lysis DNA extraction method to develop a true point of care protocol, as the source of the starting DNA is the main rate-limiting step in farm-level molecular diagnosis. Out of the various methods trialed, promising results were obtained with the magnetic bead extraction method. Sample solutions from the Fill-FLOTAC® technique were also utilised, which demonstrated that it could be efficiently adapted for field-level egg concentration to extract DNA. This proof of concept study showed that isothermal amplification technologies with a colourimetric detection or when combined with a lateral flow assay could be an important step for a true point of care molecular diagnostic assay for H. contortus.

Highlights

Haemonchus contortus (Rudolphi, 1803) Cobb, 1898 is a haematophagous gastrointestinal nematode (GIN) parasite of small ruminants [3]
We present a proof of concept study on point of care (POC) Colourimetric/Lateral Flow (LF)-Loop-mediated Isothermal Amplification (LAMP) assay for the detection of H. contortus with the potential to be multiplexed in the future that could offer confirmatory speciation diagnosis
A positive result was successfully detected in sheep faecal samples from Farm I and Farm III. gDNA extracted from the suspected H. contortus adult worms, obtained from the abomasum at post mortem from a farmed deer (Farm DF), showed positive results while the DNA samples of a few unidentified worms collected from the large intestines of the same deer were shown to be negative by the assay

Summary

Introduction

Haemonchus contortus (Rudolphi, 1803) Cobb, 1898 is a haematophagous gastrointestinal nematode (GIN) parasite of small ruminants [3]. Haemonchus contortus is one of the most pathogenic GIN parasites and has the potential to cause high mortality rates and significant production losses if left untreated or unmanaged [25, 34]. Haemonchosis is responsible for significant production losses due to reduced milk output, growth rates, and meat yield [3, 25]. Haemonchus contortus shows remarkable fecundity, with a single female able to shed upwards of 15,000 eggs per day [15], has an establishment rate of ~60% [12], and has one of the shortest patency periods of any GIN (~15 days) [15]

Methods

Results

Conclusion