Gene expression profiles associated with beef cattle resistance to Rhipicephalus ticks

Marima, Jacqueline Keena (2017-03)

Thesis (MScAgric)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Tick resistance is a complex trait influenced by numerous environmental, physiological and genetic factors. The length of the association between cattle breeds and tick species may play a vital role in the potency of the immune responses generated by the host post-infestation. The genetically determined components of host resistance, which may have evolved due to long periods of evolution of breeds in the presence of specific tick species, are regarded the most important factors of host resistance to ticks. The isolation and characterisation of genes associated with natural host resistance may provide a low-cost, environmentally sound and sustainable chemical-free alternative for tick control through gene introgression and improved accuracy of selection in breeding programs. This study examined the tick burdens and associated gene expression profiles in two ancient (Nguni – R. decoloratus and Brahman – R. microplus) and four modern (Nguni – R. microplus, Brahman – R. decoloratus, Angus – R. decoloratus and Angus – R. microplus) host-tick associations following artificial infestation. Approximately 100 unfed tick larvae of a single species were used to infest each animal, thereafter tick counts were enumerated 18-days post-infestation. Skin biopsies, from which RNA was extracted for use in the gene expression analyses, were collected pre-infestation from non-parasitized sites and 12-hours post-infestation at visible tick-bite sites. The panel of genes analysed comprised of cytokines (TLR5, TLR7, TLR9, TRAF6, CD14), chemokines and their receptors (CCR1, CCL2, CCL6), toll-like receptors (IL-1β, CXCL8, IL-10, TNF) and other candidate genes (BDA20, OGN, TBP, LUM, B2M) whose expression was normalized against RN18S1 (or β-actin-like). Custom 96-well RT2 Profiler PCR arrays, fitted with primers designed and optimised by Qiagen, were used for real-time PCR analyses using RT2 SYBR® Green dye and an ABI 7500 Standard real-time PCR cycler. The effects of breed, tick species and breed by tick species interaction on tick count were analysed using XLSTAT (2016) and SAS Enterprise Guide (2016). The fold regulation/change values were generated via the online RT2 Profiler PCR Array Data Analysis Web-portal (SABioscience - Qiagen), using the ΔΔCT method. The effects of breed, tick species and breed by tick species interaction on the differential gene expression of each gene were analysed using XLSTAT and SAS (2016). The expression levels of LUM, B2M, TRAF6 and TPB showed significant breed variations. The Nguni and Angus differed for TBP and TRAF6, while the Brahman and Angus differed for LUM and B2M. LUM and B2M displayed significantly higher expression levels in the Brahman and Nguni cattle. Significant breed, tick species and breed by tick species interaction effects were detected from the tick count data, with the Brahman carrying less ticks than both the Angus and Nguni cattle, while the R. microplus resulted in heavier tick burdens than the R. decoloratus ticks. In both experiments, there was a lack of evidence of any breed by tick species interaction which would implicate the effect of length of association between breeds and tick species in the host response to tick challenge in respect with gene expression and tick burden.

AFRIKAANSE OPSOMMING: Bosluis weerstand is ‘n komplekse eienskap wat beïnvloed word deur verskeie omgewings-, fisiologiese- en genetiesefaktore. Die lengte van die assosiasie tussen rasse en bosluis spesies mag ‘n essensiële rol speel in die sterkte van die immuun reaksie gegenereer deur die gasheer na besmetting. Die geneties bepaalde komponente van gasheer weerstand, wat kon ontwikkel het as gevolg van lang periodes van evolusie van rasse in the teenwoordigheid van spesifieke bosluis spesies, word beskou as die belangrikste faktore van gasheer weerstand tot bosluise. Die isolasie en karakterisering van gene geassosieer met natuurlike gasheer weerstand kan ‘n lae koste, omgewingvriendelike en volhoubare chemiese-vrye alternatief lewer vir bosluis beheer deur geen introgressie en verbeterde akkuraatheid van seleksie in teelprogramme. Hierdie studie het die bosluis lading en geassosieerde geenuitdrukking profiele na kunsmatige besmetting ontleed in twee antieke (Nguni – R. decoloratus en Brahman – R. microplus) en vier moderne (Nguni – R. microplus, Brahman – R. decoloratus, Angus – R. decoloratus en Angus – R. microplus) gasheer-bosluis assosiasies. Ongeveer 100 ongevoerde bosluis larwe van ‘n enkele spesie was gebruik om elke dier te besmet, waarna bosluis tellings 18 dae na besmetting geneem is. Vel biopsies, waaruit RNS geïsoleer is vir gebruik in die geenuitdrukking ontledings, was gekollekteer voor infestasie van af areas vry van parasiet besmetting en 12 ure na besmetting vanaf areas met sigbare bosluis bytplekke. Die paneel gene wat ontleed is het bestaan uit sitokiene (TLR5, TLR7, TLR9, TRAF6, CD14), chemokiene en hulle reseptore (CCR1, CCL2, CCL6), tol-agtige reseptore (IL-1β, CXCL8, IL-10, TNF) en ander kandidaat gene (BDA20, OGN, TBP, LUM, B2M) wat se uitdrukking genormaliseer was teen RN18S1 (of β-aktien-agtige). Pasgemaakte 96-well RT2 Profiler PKR arrays, toegerus met primers ontwerp en geoptimaliseerd deur Qiagen, was gebruik vir ware tyd PKR ontledings met die gebruik van RT2 SYBR® Groen kleurstof en ‘n ABI 7500 Standaard ware-tyd PKR cycler. Die effek van ras, bosluis spesie en ras by bosluis spesie interaksie op bosluis telling was ontleed deur gebruik te maak van XLSTAT (2016) en SAS Enterprise Guide (2016). Die vou regulasies/veranderingswaardes was gegenereer via die aanlyn RT2 Profiler PCR Array Data Ontledings Webportaal (SABioscience - Qiagen), deur gebruik te maak van die ΔΔCT metode. Die effek van ras, bosluis spesie en ras by bosluis spesie interaksie op die differensiële geen uitdrukking van elke geen was geontleed deur gebruik te maak van XLSTAT and SAS Enterprise Guide (2016). Die uitdrukkingsvlak van LUM, B2M, TRAF6 en TPB het beduidende ras variasie getoon. Die Nguni en Angus het verskil vir TBP en TRAF, terwyl die Brahman en Angus verskil het vir LUM en B2M. LUM and B2M het beduidende hoër uitdrukkingsvlakke in die Brahman en Nguni beeste getoon. Beduidende ras, bosluis spesie en ras by bosluis spesie interaksie effekte was waargeneem van die bosluis telling data, met die Brahman wat minder bosluise dra as beide die Angus and Nguni beeste, terwyl die R. microplus gelei het tot swaarder bosluis ladings as die R. decoloratus bosluise. In beide eksperimente was daar geen bewys van enige ras by bosluis spesie interaksie nie. Dit kan aandui dat die lengte van assosiasie tussen rasse en bosluis spesies geen effek op gasheer reaksie tot ‘n bosluis uitdaging ten opsigte van geen uitdrukking en bosluis lading kan hê nie.

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