Use of an 18s rRNA metagenomics approach as a method of detection of multiple infections in field blood samples collected on FTA cards from cattle

West, D

Abstract

Parasitic disease causes major health problems in humans and animals globally. Diseases in tropical and remote areas, sometimes known as neglected tropical diseases, pose some of the greater problems due to the difficulty in collecting and analysing samples. Collecting samples from remote and tropical areas is difficult, especially for epidemiological studies because of the problems associated with transport and storage of samples. The use of technologies such as FTA cards, that fix and store nucleic acids direct from tissues, have transformed the collection of field samples for PCR. However, samples from FTA cards have yet to be used for multiple diseases identification. The majority of studies look to identify a single disease or, using several techniques, laboriously identify a small number of diseases from a single sample.
With the use of Next Generation Sequencing, this study looked to break new ground by identifying multiple pathogenic DNA from FTA card samples taken from cattle by amplifying the variable the V9 region of the 18s sequence using primers designed to target multiple eukaryotic sequences. A number of protocols were modified and optimised in order to elute a sufficient quality and quantity of DNA to use for NGS. Due to the age of the FTA cards, the samples were more difficult to elute from. The results of the NGS showed that multiple diseases were successfully identified to a genus level, sequencing expected pathogens such as Theileria and Eimeria. Also identifying Leptomonas, a relative of the Trypanosomatidae family. There were also a large number of bacteria and fungus identified, which were unexpected and were as a result of the age of the FTA cards used. Some of these bacteria are known to be detritivores and could play a role in breaking down substances on the cards, shortening their shelf life. The age of the cards was a major limitation as it made it difficult to elute DNA. The success of this study will allow for many samples to be collected from remote areas and tested via FTA cards. The next step will be to conduct a similar experiment on a larger scale with experimentally infected FTA cards and then move forward onto testing FTA card samples collected from humans.

• Thesis Type: Dissertation
• Acceptance Date: Jun 29, 2018
• Deposit Date: Sep 25, 2018
• Publicly Available Date: Sep 25, 2018