Unravelling major histocompatibility complex diversity in the Soay sheep of St Kilda

The major histocompatibility complex (MHC) is one of the most variable regions in the vertebrate genome. Many genes within the MHC play important roles in the development of an immune response, including the response to pathogens, by presenting pathogen fragments to T cells. Pathogen-mediated balancing selection is thought to be important in maintaining the high levels of allelic variation at these loci, though the precise mechanism remains unclear. The number of studies of MHC diversity in non-model organisms has increased dramatically in recent years as genotype data have become cheaper and easier to generate; however, key limitations in many such studies remain a lack of high quality MHC genotypes and associated phenotype data. Many studies focus on a single MHC locus, assuming that one locus will represent the full range of variation within each MHC haplotype. Alternatively, the products of different loci may co-amplify, preventing locus-specific genotypes and hence heterozygosity being accurately determined. Non-model systems are also often limited by small sample sizes and limited recording of associated host and pathogen measures, which, combined with high levels of allelic variation at MHC loci, can limit statistical power. Finally, few MHC studies control for the general effect of relatedness in explaining host traits before testing for MHC effects. With so many methodological impediments, it is challenging to identify robust associations between MHC variation and host phenotypes, such as parasite burden or fitness, and to draw conclusions about the mechanisms underpinning the maintenance of diversity at MHC loci. In this thesis, I address these problems by developing a SNP-based haplotyping system for a population of unmanaged Soay sheep (Ovis aries) on Hirta, St. Kilda, for which data is available on pedigree, phenotypic traits and fitness and its components over a 30- year study period. The ovine MHC consists of four classes of loci, within which loci are tightly clustered and show reduced recombination rates compared to the genome average. Although the mammalian MHC is usually highly variable, one would expect that the number of haplotypes within an MHC class in an island population of sheep with no immigration to be limited. The class IIa region of the ovine MHC includes the classical class II loci which are typically thought to be involved in the presentation of peptides derived from extracellular pathogens, including gastrointestinal helminths, in sheep and other mammals. In chapters 2 to 4, I describe the characterisation of class IIa haplotypic diversity in the Soay sheep using direct Sanger sequencing of PCR amplified fragments, which, in combination with cloning, revealed eight distinct haplotypes. With this knowledge of haplotypic diversity, and genotypes for a sample of Soay sheep typed on the Ovine Infinium HD chip (approximately 600K SNPs), I developed a panel of 13 SNPs which could be used to impute the class IIa haplotypes. This panel was genotyped by KASP (Kompetitive Allele Specific PCR) in 6034 samples and used to impute the class IIa haplotypes. After quality control measures, class IIa haplotypes were successfully imputed for 5349 individuals. Evidence of balancing selection was identified using the Ewens-Watterson test at different life history stages and within the standing population each year between 1985 and 2012, showing that allele frequencies were more even than would be expected under neutrality. However, there was no evidence of deviation from Hardy-Weinberg equilibrium identified at different life stages or in the standing population in any year. In chapter 5, I investigate associations between the MHC class IIa haplotypes and individual-level data on host phenotypes – body weight, plasma immunoglobulin levels (measured as anti-Teladorsagia circumcincta third larval stage IgA, IgE and IgG) and strongyle faecal egg counts (FEC). Associations were tested within mixed effects models which were used to account for repeated measures and control for fixed effects known to affect the response variables, as well as within an animal model framework to account for relatedness between individuals. Haplotype heterozygosity was unrelated to any of the traits investigated, suggesting a general heterozygote advantage is unlikely to be operating within the Soay sheep. Six of the eight class IIa haplotypes were associated with multiple traits in different age-sex classes, although many of these associations were removed after inclusion within animal models. The evidence of balancing selection and associations between class IIa haplotypes and phenotypes related to health offers a promising glimpse into the evolutionary mechanisms which may be operating to maintain diversity within this region

Developmental validation of a forensic profiling system for black rhinoceros (Diceros bicornis) and extraction of nuclear DNA from historic rhinoceros horns

Demand for rhinoceros horn in the late 20th century led to the population decline of black rhinoceros (Diceros bicornis) by more than 96 %, and the species remains critically endangered. Trade in rhinoceros and their products is prohibited under CITES legislation. Despite this, there has been a recent increase in rhinoceros horn demand for use in traditional Asian medicine (TAM) to treat a range of illnesses including cancer, childhood fevers and as a hangover cure. Consequently, the value of rhinoceros horn in illegal trade has risen dramatically, alongside a dramatic increase in poaching. Large numbers of horns exist in historic collections, and since 2011 there has been numerous thefts in both rhinoceros range states and throughout Europe. Wildlife DNA forensic analysis can be used to individually identify rhinoceros horn, matching a seized horn to a crime scene. This can provide valuable evidence in criminal proceedings. This study therefore aimed to validate a profiling system that could be used for D. bicornis horns and to determine an optimal DNA extraction method for historic horns. A developmental validation of a marker panel consisting of 15 short tandem repeat (STR) and one sexing locus was carried out to determine its reliability and efficacy in singleplex reactions. The markers were not found to deviate from patterns of Mendelian inheritance, and species specificity studies found that only two loci (WR7C and ZF1) amplified human and dog DNA. Alleles could be scored at DNA concentrations of less than 1 ng/μL for all loci except WR7C. The marker panel was found to be highly reproducible with a maximum d-value of 0.545, mean heterozygous balance of 1.30 and stutter ratio of 0.35. The marker panel was also robust to reductions in annealing temperature (TA) and increasing cycle number (up to 43 cycles), although increasing TA reduced amplification success. The markers were then combined into two multiplexes and allele frequency data was generated for 52 D. bicornis michaeli, and two markers (BlRh37D and IR12) were rejected for violating assumptions of Hardy-Weinberg equilibrium and linkage equilibrium. The average probability of identity (PIave) ranged between 1.06 x 10-11 and 1.16 x 10-7 with varying levels of relatedness and population structure. The marker panel was determined suitable for use in forensic casework analysis under the defined conditions, and limitations were described. Previous studies have shown that nuclear DNA (nDNA) can be extracted from modern horns, but how effective those methods would be on historic horns was unknown. Optimisation of a number of aspects of the extraction method was carried out, including sample preparation, chemical breakdown with dithiothreitol (DTT), mass of starting material and extraction kit. Mechanical breakdown of rhinoceros horn material using a mixer mill was determined unnecessary, whilst the addition of DTT was found to improve digestion. Irrespective of extraction kit, 20 mg of horn material yielded sufficient quality and quantity nDNA for forensic profiling. Qiagen extraction kits using the QIAamp Mini columns were found to yield superior DNA quantity compared with QIAamp MinElute columns. Whilst KingFisher Cell and Tissue DNA Kits yielded equivalent extract quantity, mitochondrial DNA contamination between samples was detected. The Qiagen method using QIAamp MinElute columns was therefore considered the most robust extraction method for historic rhinoceros horns. This work describes the conditions and limitations of a forensic profiling system for D. bicornis and an effective extraction methodology for historic rhinoceros horns. This enables this marker panel to be used with confidence in forensic casework analysis of both modern and historic horns.McDowall, Ia

Contemporary selection on MHC genes in a free-living ruminant population

Genes within the major histocompatibility complex (MHC) are the most variable identified in vertebrates. Pathogen‐mediated selection is believed to be the main force maintaining MHC diversity. However, relatively few studies have demonstrated contemporary selection on MHC genes. Here, we examine associations between MHC variation and several fitness measurements including total fitness and five fitness components, in 3400 wild Soay sheep (Ovis aries) monitored between 1989 and 2012. In terms of total fitness, measured as lifetime breeding success of all individuals born, we found haplotypes named C and D were associated with decreased and increased male total fitness respectively. In terms of fitness components, juvenile survival was associated with haplotype divergence while individual haplotypes (C, D and F) were associated with adult fitness components. Consistent with the increased male total fitness, the rarest haplotype D has increased in frequency throughout the study period more than expected under neutral expectations. Our results demonstrate that contemporary natural selection is acting on MHC class II genes in Soay sheep and that the mode of selection on specific fitness components can be different mode from selection on total fitness

Non-volant mammal inventory of Cusuco National Park, northwest Honduras: reporting the presence of Jaguar, Panthera onca (Linnaeus, 1758), and demonstrating the effects of zonal protection on mammalian abundance

Neotropical cloud forests are one of the most biodiverse yet threatened ecosystems with deforestation and hunting creating major problems. We report a non-volant mammal species inventory for Cusuco National Park, Honduras, spanning 2006–2016. Data were collected using 4 methods: tracks and signs, camera trapping, direct sightings and live trapping. Recorded species of conservation importance included Central American red brocket (Mazama temama (Kerr, 1792)), Baird’s tapir (Tapirus bairdii (Gill, 1865)), margay (Leopardus wiedii (Schinz, 1821)), and jaguar (Panthera onca (Linnaeus, 1758)); the latter recorded for the first time. Cusuco National Park is a site of strategic geographical importance within the Central American jaguar corridor. In total, 43 species were recorded from 26 families. We demonstrate the impact of zonal protection measures with higher mammal abundance recorded in the park’s highly protected core compared to the less protected buffer zone. We advise continued monitoring to provide robust time-series of population trends

Seroprevalence of HCV, HBV and HIV in two inner-city London Emergency Departments.

Summary: In this paper we build on work investigating the feasibility of human immunodeficiency virus (HIV) testing in emergency departments (EDs), estimating the prevalence of hepatitis B, C and HIV infections among persons attending two inner-London EDs, identifying factors associated with testing positive in an ED. We also undertook molecular characterisation to look at the diversity of the viruses circulating in these individuals, and the presence of clinically significant mutations which impact on treatment and control. Blood-borne virus (BBV) testing in non-traditional settings is feasible, with emergency departments (ED) potentially effective at reaching vulnerable and underserved populations. We investigated the feasibility of BBV testing within two inner-London EDs. Residual samples from biochemistry for adults (⩾18 years) attending The Royal Free London Hospital (RFLH) or the University College London Hospital (UCLH) ED between January and June 2015 were tested for human immunodeficiency virus (HIV)Ag/Ab, anti-hepatitis C (HCV) and HBsAg. PCR and sequence analysis were conducted on reactive samples. Sero-prevalence among persons attending RFH and UCLH with residual samples (1287 and 1546), respectively, were 1.1% and 1.0% for HBsAg, 1.6% and 2.3% for anti-HCV, 0.9% and 1.6% for HCV RNA, and 1.3% and 2.2% for HIV. For RFH, HBsAg positivity was more likely among persons of black vs. white ethnicity (odds ratio 9.08; 95% confidence interval 2.72–30), with anti-HCV positivity less likely among females (0.15, 95% CI 0.04–0.50). For UCLH, HBsAg positivity was more likely among non-white ethnicity (13.34, 95% CI 2.20–80.86 (Asian); 8.03, 95% CI 1.12–57.61 (black); and 8.11, 95% CI 1.13–58.18 (other/mixed)). Anti-HCV positivity was more likely among 36–55 year olds vs. ⩾56 years (7.69, 95% CI 2.24–26.41), and less likely among females (0.24, 95% CI 0.09–0.65). Persons positive for HIV-markers were more likely to be of black vs. white ethnicity (4.51, 95% CI 1.63–12.45), and less likely to have one ED attendance (0.39, 95% CI 0.17–0.88), or female (0.12, 95% CI 0.04–0.42). These results indicate that BBV-testing in EDs is feasible, providing a basis for further studies to explore provider and patient acceptability, referral into care and cost-effectiveness

Extinctions, genetic erosion and conservation options for the black rhinoceros (Diceros bicornis)

The black rhinoceros is again on the verge of extinction due to unsustainable poaching in its native range. Despite a wide historic distribution, the black rhinoceros was traditionally thought of as depauperate in genetic variation, and with very little known about its evolutionary history. This knowledge gap has hampered conservation efforts because hunting has dramatically reduced the species’ once continuous distribution, leaving five surviving gene pools of unknown genetic affinity. Here we examined the range-wide genetic structure of historic and modern populations using the largest and most geographically representative sample of black rhinoceroses ever assembled. Using both mitochondrial and nuclear datasets, we described a staggering loss of 69% of the species’ mitochondrial genetic variation, including the most ancestral lineages that are now absent from modern populations. Genetically unique populations in countries such as Nigeria, Cameroon, Chad, Eritrea, Ethiopia, Somalia, Mozambique, Malawi and Angola no longer exist. We found that the historic range of the West African subspecies (D. b. longipes), declared extinct in 2011, extends into southern Kenya, where a handful of individuals survive in the Masai Mara. We also identify conservation units that will help maintain evolutionary potential. Our results suggest a complete re-evaluation of current conservation management paradigms for the black rhinoceros