Comparison of genetic and immunological responses to tick infestation between three breeds of sheep in South Africa

Abstract

The study investigated genetic parameters and immunological responses to tick infestation in three South African sheep breeds (Namaqua Afrikaner [NA], Dorper and SA Mutton Merino [SAMM]). The study aimed to estimate genetic and crossbreeding parameters for tick count (TC) and weaning weight (WW), to examine the histology of tick attachment and control sites, to select reference genes for normalizing gene expression data in this study, to compare cytokines gene expression at tick attachment and control sites and finally to compare cutaneous hypersensitivity reactions to unfed larvae extracts (ULE) of Rhipicephalus evertsi evertsi between NA, Dorper and SAMM sheep. Genetic parameters for WW and TC were estimated using data of lambs maintained on the Nortier Research Farm from 2010 to 2015. Firstly, data of purebred commercial Dorper and SAMM lambs were combined with data of their reciprocal crosses to assess breed effects and the possible effect of non-additive genetic variation on WW and TC. In the second analysis, data of purebred commercial Dorper lambs were combined with data of the unimproved, indigenous NA and the NA x Dorper cross. In Analysis 1 the coefficient of variations (CV) were 24%, 95% and 50% for WW, untransformed total TC and square root transformed total TC, respectively. Genotype affected WW but not TC. A heterosis estimate of approximately 4% was derived for WW. A single-trait h² estimate for TC was 0.11±0.09. A model analysing across-genotype h² yielded a slightly lower h² estimate of 0.08 ± 0.07. In Analysis 2 the CV were 27% and 55% for WW and square root transformed total TC, respectively. WW and TC were affected by genotype. WW exhibited heterosis amounting to 8.5% while the corresponding value for TC amounted to -23%. The single-trait h² estimate for TC was 0.06±0.05. A model analysing across-genotype h² yielded a substantially higher h² estimate of 0.27±0.07. These results suggest that genetic variation in TC was primarily associated with differences among genetic groups while differences between individual animals within genetic groups were not as important. Heterosis estimates for WW were variable between two analyses, but within ranges reported in the literature. This study established significant variation in TC between sheep genotypes when the indigenous NA breed formed part of the analysis. The NA x Dorper cross resembled the improved Dorper breed for WW but the unimproved, resistant NA for TC and exhibited worthwhile levels of heterosis for both traits. Indigenous ovine genetic resources may be instrumental in providing genetic material for adaptive traits in environments susceptible to high levels of tick infestation. Further research is required to elucidate the role that adapted indigenous ovine genetic resources may play in an integrated tick management strategy under conditions characterised by high levels of tick challenge. A histological study was conducted to assess histological features at tick attachment and control sites in pure breeds. Skin biopsies were examined using routine histological techniques for immunological cell infiltration and skin reactions. Marked variation in immunological responses to tick attachment within and between sheep breeds was observed. There were differences between the attachment and control sites in most of the skin changes (defects) except for four skin defects in the NA. However, all breeds had similar frequencies of skin defects at tick attachment sites. Tick attachment sites were more likely to be infiltrated by cells within as well as across breeds. The NA and SAMM breeds tended to demonstrate greater cellular infiltrations of specific leukocytes at tick attachment sites compared to Dorpers. Basophils, mast cells and eosinophils were increasingly recruited at tick attachment site in NA ewes compared to the Dorper and, occasionally, the SAMM breeds. These results suggest the importance of these cells in sheep resistance to tick infestation. Tick genera influenced the recruitment of neutrophils to tick attachment sites. Tick gender, sampling site as well as tick engorgement level did not affect the number of immunological cells. Further studies should be done with one tick species at a time to better comprehend the species-specific impact of tick attachment to animals belonging to divergent sheep breeds. Five genes (18S, GAPDH, YWHAZ, B2M and SDHA) were tested for their stability. SDHA, YWHAZ and B2M were the most suitable reference genes recommended by geNorm analysis for normalizing gene expression data in sheep skin. These findings will assist in normalizing data in gene expression studies at tick attachment and control sites of the NA, Dorper and SAMM breeds. This study suggested that no reference gene is stably expressed in different experimental conditions. The expression of IL-1β, IL-8, CCL2 and CCL26 was quantified in real-time qPCR. IL-1β and IL-8 were more highly expressed at tick attachment than at control sites. NA ewes expressed IL-1β more at tick attachment sites than Dorpers. The NA breed was also more likely to upregulate the expression of the CCL2, CCL26 and IL-8 genes at tick attachment sites compared to control sites than the other breeds. This indicates that IL-1 β, CCL26 and IL-8 may play a part in resistance or susceptibility of sheep to tick infestation. The differences in expression of the two chemokines between the resistant NA and more susceptible SAMM and Dorper imply that the NA breed could be able to overcome the anti-chemokine activity of tick saliva. ULE of R. evertsi evertsi induced hypersensitivity reactions in all the breeds. The indigenous NA displayed stronger reactions, immediate and delayed, than the commercial breeds. The results suggest that cell-mediated immune responses are invoked to fight against tick infestation in the NA. The hypersensitivity reaction may be used as a phenotypic marker to select animals or breeds that are more resistant to tick infestation. It is well-known that challenge-based research for promoting resistance to pathogens is under scrutiny from an ethical and welfare perspective. The methods employed here could be refined to enable routine evaluation of valuable animals without resorting to more invasive strategies, such as allowing adequate natural challenge to accrue over time in selection candidates. Overall, the component studies reported in the thesis increased the present understanding of ovine tick-host interactions and factors contibuting to breed differences in tick loads. URI