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Diagnosis of porcine cysticercosis using rat basophilic leukemia (RBL) IgE reporters and loop-mediated isothermal amplification (LAMP) technology

¹Justus Liebig University Giessen, Institute of Parasitology, Germany
²New England Biolabs, United States
³International Livestock Research Institute (ILRI), Kenya

Abstract

Cysticercosis is a neglected zoonotic disease caused by larval stages of Taenia solium, with a huge impact on public health (neurocysticercosis in the brain) and the livelihoods of small-scale pig farmers in many low-income countries. Diagnosis of porcine cysticercosis is mostly based on tongue palpation and carcass inspection, complemented by IgG-based serological analyses. These diagnostic methods often show cross-reactivity or are not sensitive enough in pigs with low infestation. Here, we describe the development of a pig Immunoglobulin E (IgE) reporter system and selection, cloning, and recombinant expression of candidate diagnostic allergens of T. solium. In addition, we adopted a molecular approach using loop-mediated isothermal amplification (LAMP) technology to distinguish cysticercosis caused by T. solium from the related, co-endemic T. hydatigena (non-zoonotic).
For developing the reporter system, Rat Basophilic Leukemia Neuropeptide Y-monomeric Red Fluorescent Protein expressing cells (RBL NPY-mRFP) were transiently transfected with a pig/rat high-affinity IgE receptor alpha chain (FcεRIα) chimeric construct followed by antibiotic selection for stable transfectants. Putative IgE-binding T. solium allergens were selected by a combination of published work on transcriptomic and proteomic data and allergenicity predictions. Chosen allergens were cloned into expression vectors and transiently transfected into HEK-2936E cells in suspension. In LAMP method, we targeted cytochrome c oxidase 1 (cox1) gene of T. solium and T. hydatigena, incubated LAMP reactions at 65°C for 30 min and recorded the post-amplification colour (yellow-positive, pink-negative).
Stable transfection of the chimeric pig-rat FcεRIα was confirmed at the mRNA level by RT-PCR. Five candidate T. solium oncospheral diagnostic allergens (E5LBB8, K0A0S9, Q2XNL7, Q9NI46, W8P1J2) were identified through bioinformatic analysis and three HEK293-6E transfected allergens showed expected protein band size in Western blot analysis upon recombinant expression. The LAMP assay was able to detect 10 pg/µL of ^{T. solium DNA} in pig serum, without cross-reactivity with T. hydatigena. We are now focusing on the detection of T. solium DNA through LAMP assay in field studies.
This study shows proof-of-principle for a serological reporter assay for a lab-based approach, while LAMP is expected to introduce a field-applicable point-of-care test for porcine cysticercosis.

Keywords: Allergen, cysticercosis, diagnosis, IgE, LAMP, RBL NPY-mRFP, Reporter system, Taenia hydatigena, Taenia solium

Contact Address: Md. Shahadat Hossain, Justus Liebig University Giessen, Institute of Parasitology, Schubertstrasse 81, 35392 Giessen, Germany, e-mail: shahadat.parabau.edu.bd