ERA Collection:

Genetics of bovine vaccination

2011-08-01T09:53:20Z

Title: Genetics of bovine vaccination Authors: Leach, Richard Jonathan Abstract: Infectious disease is an important issue for animal breeders, farmers and governments. Solutions to control infectious disease are needed and research focused on the genetic loci determining variation in immune-related traits has the potential to deliver solutions. The primary aim of this thesis is to discover regions of the bovine genome which influence the immune response post immunisation. To accomplish this two types of immunising agents, a Foot-and-Mouth Disease Virus (FMDV) peptide (FMDV15) and a commercial vaccine for Bovine Respiratory Syncytial Virus (BRSV), were used to immunise the second generation (F2 and backcrosses) of the Roslin Bovine Genome (RoBoGen) herd, a Charolais Holstein cross population. The FMDV15 peptide consisted of two sections of the VP1 protein located on the FMDV capsid, together encompassing the major neutralising antibody sites that are known to be immunogenic. Protection against FMDV is generally believed to relate to the levels of neutralising antibody and has been correlated with IgG1 and IgG2 levels as well as interferon- . In addition it has been shown that T cell responses also play a role in protection against FMDV. Thus all of these were used as phenotypic measurements post immunisation to the FMDV15 peptide. The BRSV vaccine used was an attenuated live vaccine. Protective mechanisms against BRSV infection include IgA, IgG1, IgG2 and IgM BRSV-specific antibodies and antibody titres particularly those of the IgG isotypes are considered to be correlates of protection. Thus, IgG1 and IgG2 antibody levels were measured post vaccination with the BRSV vaccine. All phenotypes were measured across time, and allowed analysis of the primary and secondary adaptive immune responses. Both agents caused considerable variation in the phenotypes measured post immunisation, with significant responses detected two weeks post immunisation. REstricted Maximum Likelihood (REML) analysis attributed much of this variation to sire, highlighting the heritable component, and environmental effects. Significant positive correlations were detected across time within each trait for both the FMDV and BRSV responses. The FMDV and BRSV antibody levels also correlated with each other at later time points, suggesting that there may be animals which are genetically predisposed to be high or low responders in general. Initially a linkage mappingapproach was followed using 165 microsatellite markers, which detected 77 QTL in response to the FMDV peptide and 27 QTL in response to the BRSV vaccine. There were some overlapping QTL, for example QTL which spanned the Major Histocompatibility Complex. Further analysis was conducted by developing a Perl scripted program which genotyped the RoBoGen herd in two ways; 1) Single Nucleotide Polymorphism(s) (SNP) were genotyped within the confidence intervals of the previously discovered QTL and 2) SNP were genotyped via a candidate gene approach. Association study methodology, accounting for relationship stratification via principal components of the genetic relationship matrix, was used to detect significant SNP, in response to both the FMDV peptide and the BRSV vaccine. Twenty significant SNP associations were discovered across 19 traits, with some SNP located in genes with known biological relevance to an immune response, such as the Toll-Like Receptors (TLR), TLR4 and TLR8. This thesis has detected regions of the genome which are significantly associated with the immune responses elicited by two different agents, suggesting similar pathway(s)/gene(s) may be used in defence of multiple pathogens. Once regions of significance were detected, further analysis using SNP markers identified significant, non-synonymous SNP that were associated with the immunising agents. The novel markers discovered in this study may aid breeding for resistance to disease via marker assisted selection. In addition, they may also have highlighted new targets for vaccinologists to develop ‘next generation’ vaccines.

Functional analysis of the non-coding RNAs of murine gammaherpesvirus 68

2011-02-15T10:37:07Z

Title: Functional analysis of the non-coding RNAs of murine gammaherpesvirus 68 Authors: Choudhury, Nila Roy Abstract: Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data.

Animal sentinel surveillance: evaluating domestic dogs as sentinels for zoonotic pathogen surveillance

2011-02-15T09:39:18Z

Title: Animal sentinel surveillance: evaluating domestic dogs as sentinels for zoonotic pathogen surveillance Authors: Halliday, J.E.B. Abstract: The capacity of zoonotic pathogens to infect multiple hosts creates surveillance challenges but also provides opportunities to gather data from animal species that can be used to understand risks to human health. This thesis presents a conceptual and practical assessment of the utility of domestic dog serosurveillance for the detection and surveillance of two pathogens, influenza A and Leptospira spp. The first chapter gives a theoretical framework that can be used to explore the attributes of animal sentinels and assess their utility in different contexts. In subsequent chapters, this framework is applied in a practical assessment of the utility of a domestic dog serosurveillance approach for the detection and surveillance influenza A and Leptospira spp. at two sites in Africa. Two cross-sectional surveys of the avian and mammal populations at a site in Northern Cameroon were conducted in early 2006 to determine if H5N1 influenza A viruses had circulated in this area and in which species that presence could be detected. Serological and molecular evidence of extensive H5 virus circulation in the domestic duck population was identified. 47% of domestic ducks at the Maga site were cELISA positive for anti-influenza A antibodies and 20% were HI test positive against an H5N1 antigen. There was also evidence of exposure to H5 subtype viruses in the local dog and pig populations. At the Kibera site in Nairobi, a cohort study was established to carry out surveillance of influenza A and Leptospira spp. in the domestic dog population and cross-sectional surveys of the domestic poultry and rodent populations were completed. There was no indication of influenza A circulation in any of the animal species surveyed, indicating low risk of zoonotic influenza A infection in the human population of Kibera. In contrast, there was extensive molecular and serological evidence of the presence of Leptospira spp. in both the rodent and dog populations. 18% of 236 trapped rodents were PCR positive for kidney carriage of pathogenic leptospires and the estimated seroprevalence of anti- Leptospira antibodies in the dog population ranged from 5-36% during the course of the study, indicating high potential risk of leptospirosis infection in the human population. The results indicate that dog serosurveillance can be used as useful tool for the determination of broad-scale patterns of pathogen presence and relative levels of population exposure. However, there are limitations of the data that can be gathered from animal sentinels and the complexities introduced particularly by incomplete understanding of diagnostic test performance must be recognized. Animal sentinel surveillance may be of most use for addressing fundamental questions of what pathogens are present where. In the developing world particularly where disease burden data are still lacking, dog sentinel serosurveillance can provide essential baseline data that can be used to target future research and resource allocation.

Assessment of genetic markers for the improvement of beef quality and consistency

2013-04-10T10:57:27Z

Title: Assessment of genetic markers for the improvement of beef quality and consistency Authors: Gill, Jennifer Abstract: The overall aim of this thesis was to investigate the genetic control of beef quality in a commercial population of Aberdeen Angus-sired cattle with a view to trait improvement. The population studied included 500 Angus-cross animals, all with purebred Aberdeen Angus sires, from a selection of farms throughout Scotland. A number of carcass-related weight traits and taste panel assessed sensory traits were measured on these animals. A population of 265 Charolais cross cattle (all with purebred sires) was then used to explore the extrapolation of results across breeds. The first aim of this thesis was to investigate heritabilities for important carcass and meat quality traits and to assess the quality of a number of taste-panel derived meat quality traits by calculating three consistency statistics. Consistency statistics (parameter range 0 to 1) for the taste panel traits were moderately high, particularly for panel member consistency and reproducibility, with values ranging from 0.48 to 0.81 and 0.43 to 0.73, respectively. Estimated heritabilities were low for most of the sensory taste-panel-evaluated traits, where the maximum value was 0.16 for overall liking, but were higher for carcass traits where carcass weight heritability was 0.7. To perform these analyses it was first necessary to confirm paternity using a number of genetic markers. Therefore, a comparison of the power of both microsatellite and SNP markers for paternity exclusion was carried out to determine the more effective method. Results indicated that approximately three times as many SNP markers than microsatellite markers were required for parentage exclusion, and a panel of 15 microsatellite markers was used to assign paternity before subsequent data analysis was carried out. The remaining aims of this thesis centred on exploring genetic markers for carcass and meat quality. Firstly, the Angus animals were genotyped for the del11 myostatin mutation which was found to be segregating at a relatively low frequency (0.04) and was shown to be associated with a 17.4 kg increase in carcass weight (P < 0.05) in the heterozygous animals when compared to the homozygous wild-type animals. By analysing the haplotype associated with the mutant allele, it was determined that there have been at least two separate introductions of the mutant allele into the Aberdeen Angus breed. A number of SNPs were also tested for their effects on the carcass and meat quality traits in the Angus animals. The SNPs fell into two groups: eight that have been incorporated into commercially available tests and a further 28 from alternative candidate genes that have effects in different breeds and species. In total, 17 SNPs significantly affected at least one of the traits measured. Of these significant associations, a number have been seen previously, such as the association between calpain and tenderness (P = 0.01) and growth hormone and eye muscle area (P = 0.05), and some of which were novel, such as the association between growth hormone receptor and steak odour (P = 0.02) and corticotrophin releasing hormone and gristle distance from fat (P = 0.004). A further six SNPs, identified by resequencing of the malic enzyme 1 (ME1) and small heterodimer partner (SHP) genes, were tested for their effects on the traits measured in this thesis. Five of the SNPs, including one which caused a non-synonymous amino acid change, had a significant effect on at least one of the traits tested including fat class (P = 0.002), eye muscle area (P = 0.01), sirloin weight before maturation (P = 0.03), sirloin steak tail length (P = 0.004) and juiciness (P = 0.004) where the effect sizes were 1.79 units, 565 mm2, 0.36 kg, 17.12 mm and 0.23 taste panel units, respectively. To assess the effect of the genotyped SNPs on intramuscular fat (IMF), a simple method of visible IMF quantification in the sirloin steak was developed using digital photographs and an image analysis program. Results showed that two SNPs in the calpain gene, known to be linked with an increase in meat tenderness, were associated with an increase in visible IMF% and the del11 mutation was associated with a reduction in visible IMF%. The heritabilities, SNP association validations and novel SNP-trait associations identified in this thesis provide tools for use in breeding programs, possibly via marker assisted selection to improve meat quality traits. However, the results seem breed-specific, as most of the significant effects were not replicated in the Charolais population.