A morphological and genetic description of pentastomid infective nymphs belonging to the family Sebekidae Sambon, 1922 in fish in Australian waters

Infective nymphal stages of the family Sebekidae Sambon, 1922 are reported from four species of fish in Australian waters for the first time. Infected fish were collected from locations in Western Australia, the Northern Territory and north Queensland. The infective nymphs of Alofia merki Giglioli in Sambon, 1922 and Sebekia purdieae Riley, Spratt et Winch, 1990 are reported and described for the first time. The remaining specimens were identified as belonging to the genus Sebekia Sambon, 1922 based on the combination of buccal cadre shape, shape and size of hooks, and overall body size, but could not be attributed to any of the other species of Sebekia already reported due to missing required morphological features. DNA sequences of members of the family Sebekidae are presented for the first time. The lack of knowledge on the pentastome fauna of wild crocodiles, and any potential intermediate hosts, in northern Australia, is also outlined.

Epidemiology and genetic architecture of blood pressure: a family based study of Generation Scotland

Hypertension is a major risk factor for cardiovascular disease and mortality, and a growing global public health concern, with up to one-third of the world’s population affected. Despite the vast amount of evidence for the benefits of blood pressure (BP) lowering accumulated to date, elevated BP is still the leading risk factor for disease and disability worldwide. It is well established that hypertension and BP are common complex traits, where multiple genetic and environmental factors contribute to BP variation. Furthermore, family and twin studies confirmed the genetic component of BP, with a heritability estimate in the range of 30-50%. Contemporary genomic tools enabling the genotyping of millions of genetic variants across the human genome in an efficient, reliable, and cost-effective manner, has transformed hypertension genetics research. This is accompanied by the presence of international consortia that have offered unprecedentedly large sample sizes for genome-wide association studies (GWASs). While GWAS for hypertension and BP have identified more than 60 loci, variants in these loci are associated with modest effects on BP and in aggregate can explain less than 3% of the variance in BP. The aims of this thesis are to study the genetic and environmental factors that influence BP and hypertension traits in the Scottish population, by performing several genetic epidemiological analyses. In the first part of this thesis, it aims to study the burden of hypertension in the Scottish population, along with assessing the familial aggregation and heritialbity of BP and hypertension traits. In the second part, it aims to validate the association of common SNPs reported in the large GWAS and to estimate the variance explained by these variants. In this thesis, comprehensive genetic epidemiology analyses were performed on Generation Scotland: Scottish Family Health Study (GS:SFHS), one of the largest population-based family design studies. The availability of clinical, biological samples, self-reported information, and medical records for study participants has allowed several assessments to be performed to evaluate factors that influence BP variation in the Scottish population. Of the 20,753 subjects genotyped in the study, a total of 18,470 individuals (grouped into 7,025 extended families) passed the stringent quality control (QC) criteria and were available for all subsequent analysis. Based on the BP-lowering treatment exposure sources, subjects were further classified into two groups. First, subjects with both a self-reported medications (SRMs) history and electronic-prescription records (EPRs; n =12,347); second, all the subjects with at least one medication history source (n =18,470). In the first group, the analysis showed a good concordance between SRMs and EPRs (kappa =71%), indicating that SRMs can be used as a surrogate to assess the exposure to BP-lowering medication in GS:SFHS participants. Although both sources suffer from some limitations, SRMs can be considered the best available source to estimate the drug exposure history in those without EPRs. The prevalence of hypertension was 40.8% with higher prevalence in men (46.3%) compared to women (35.8%). The prevalence of awareness, treatment and controlled hypertension as defined by the study definition were 25.3%, 31.2%, and 54.3%, respectively. These findings are lower than similar reported studies in other populations, with the exception of controlled hypertension prevalence, which can be considered better than other populations. Odds of hypertension were higher in men, obese or overweight individuals, people with a parental history of hypertension, and those living in the most deprived area of Scotland. On the other hand, deprivation was associated with higher odds of treatment, awareness and controlled hypertension, suggesting that people living in the most deprived area may have been receiving better quality of care, or have higher comorbidity levels requiring greater engagement with doctors. These findings highlight the need for further work to improve hypertension management in Scotland. The family design of GS:SFHS has allowed family-based analysis to be performed to assess the familial aggregation and heritability of BP and hypertension traits. The familial correlation of BP traits ranged from 0.07 to 0.20, and from 0.18 to 0.34 for parent-offspring pairs and sibling pairs, respectively. A higher correlation of BP traits was observed among first-degree relatives than other types of relative pairs. A variance-component model that was adjusted for sex, body mass index (BMI), age, and age-squared was used to estimate heritability of BP traits, which ranged from 24% to 32% with pulse pressure (PP) having the lowest estimates. The genetic correlation between BP traits showed a high correlation between systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) (G: 81% to 94%), but lower correlations with PP (G: 22% to 78%). The sibling recurrence risk ratio (λS) for hypertension and treatment were calculated as 1.60 and 2.04 respectively. These findings confirm the genetic components of BP traits in GS:SFHS, and justify further work to investigate genetic determinants of BP. Genetic variants reported in the recent large GWAS of BP traits were selected for genotyping in GS:SFHS using a custom designed TaqMan® OpenArray®. The genotyping plate included 44 single nucleotide polymorphisms (SNPs) that have been previously reported to be associated with BP or hypertension at genome-wide significance level. A linear mixed model that is adjusted for age, age-squared, sex, and BMI was used to test for the association between the genetic variants and BP traits. Of the 43 variants that passed the QC, 11 variants showed statistically significant association with at least one BP trait. The phenotypic variance explained by these variant for the four BP traits were 1.4%, 1.5%, 1.6%, and 0.8% for SBP, DBP, MAP, and PP, respectively. The association of genetic risk score (GRS) that were constructed from selected variants has showed a positive association with BP level and hypertension prevalence, with an average effect of one mmHg increase with each 0.80 unit increases in the GRS across the different BP traits. The impact of BP-lowering medication on the genetic association study for BP traits has been established, with typical practice of adding a fixed value (i.e. 15/10 mmHg) to the measured BP values to adjust for BP treatment. Using the subset of participants with the two treatment exposure sources (i.e. SRMs and EPRs), the influence of using either source to justify the addition of fixed values in SNP association signal was analysed. BP phenotypes derived from EPRs were considered the true phenotypes, and those derived from SRMs were considered less accurate, with some phenotypic noise. Comparing SNPs association signals between the four BP traits in the two model derived from the different adjustments showed that MAP was the least impacted by the phenotypic noise. This was suggested by identifying the same overlapped significant SNPs for the two models in the case of MAP, while other BP traits had some discrepancy between the two sources

PREVALENCE, ISOLATION, AND GENETIC CHARACTERIZATION OF TOXOPLASMA GONDII IN CHICKEN

Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world because of its capability of infecting all warm-blooded animals. It has been reported that up to one third of the world population is infected with this parasite. Chickens are recognized as good indicators of the environmental T. gondii oocysts contamination because they obtain food from the ground. Thus, the prevalence of T. gondii in chicken provides more insight related to public health concern from T. gondii. Previous studies have shown a high isolation rate from free-range chickens raised in the United States. The objectives of this study were to evaluate the microbial safety and infection of T. gondii in free-range chickens available at the grocery stores and farms for the consumers to purchase and genotype T. gondii isolates. Chicken hearts were obtained from the local markets and also from the farms raising free- range chickens. Heart juice was obtained from cavities of each heart. Modified agglutination test (MAT) for detection of IgG antibodies was conducted with those heart juice samples with titer of 1:5, 1:25, and 1: 100. Each seropositive heart was pepsin digested and bioassayed into a group of two mice. Six weeks post inoculation (p.i.) mice were bled and euthanized to examine the infection of T. gondii. In addition, multiplex multilocus nested PCR-RFLP was performed to genetically characterize T. gondii isolates with eleven PCR-RFLP markers including SAG1, SAG2, altSAT2, SAG3, BTUB, GRA6, c22-8, c29-a, L358, PK1, and Apico. One hundred fifty from a total of 997 samples (15.0%) were found seropositive for T. gondii. No viable T. gondii was isolated from chicken hearts that were sampled. A total of four genotypes were identified, including one new genotype and three previously identified genotypes. The results suggest that T. gondii oocysts could present in the environment and infect the food animals. T. gondii prevalence in chicken hearts could reflect the environmental contamination of T. gondii and prevalence information can be used to manage T. gondii infection risk.

Taraxacum koksaghyz Rodin as an alternative source of natural rubber and inulin; agronomic advances and genetic linkage map

126 p. El contenido del capítulo 3 está sujeto a confidencialidad.

Environmental and genetic effects on production traits of ewes originally from distinct environments.

Wool production and reproductive performance components of similar genotypes, brought from distinct production areas, were evaluated during five years trial at similar environments, such as, joining season and stocking rate on winter improved pasture. The least squares means revealed that the origin (breed) effect concentrated upon the Corriedale ewes wool production, whereas in Romney females it affected the reproductive performance.

Effects of fishery protection on biometry and genetic structure of two target sea cucumber species from the Mediterranean Sea

Sea cucumber fisheries are now occurring in most of the tropical areas of the world, having expanded from its origin in the central Indo-Pacific. Due to the overexploitation of these resources and the increasing demand from Asian countries, new target species from Mediterranean Sea and northeastern Atlantic Ocean are being caught. The fishery effects on biometry and genetic structure of two target species (Holothuria polii and H. tubulosa) from Turkey, were assessed. The heaviest and largest individuals of H. polii were found into the non-fishery area of Kusadasi, also showing the highest genetic diversity. Similar pattern was detected in H. tubulosa, but only the weight was significantly higher in the protected area. However, the observed differences on the fishery effects between species, could be explained considering the different percentage of catches (80% for H. polii and 20% for H. tubulosa).

West versus East Mediterranean Sea: origin and genetic differentiation of the sea cucumber Holothuria polii

We studied the genetic structure of the sea cucumber Holothuria (Roweothuria) polii (Delle Chiaje 1823) by analysing the mitochondrial DNA variation in two fragments of cytochrome oxidase I (COI) and 16S genes. Individuals were collected in seven locations along the Mediterranean Sea, which cover a wide range of the species distribution. We found high haplotype diversity for COI and moderate diversity for 16S, and low nucleotide diversity for both genes. Our results for the COI gene showed many recent and exclusive haplotypes with few mutational changes, suggesting recent or ongoing population expansion. The Western and Eastern Mediterranean populations exhibited slight but significant genetic differentiation (COI gene) with higher genetic diversity in the East. The most ancient haplotype was not present in the westernmost sampling location (SE Spain). The oldest expansion time was observed in Turkey, corresponding to mid-Pleistocene. Turkey had also the highest genetic diversity (number of total and exclusive haplotypes, polymorphisms, haplotype and nucleotide diversity). This suggests that this region could be the origin of the subsequent colonizations through the Mediterranean Sea, a hypothesis that should be assessed with nuclear markers in future research.

Drug resistance and genetic diversity of Mycobacterium tuberculosis in Luanda, Angola: a molecular epidemiological perspective

Poster presented at the 36th Annual Congress of the European Society of Mycobacteriology. Riga, Latvia, 28 June - 1 July 2015

Association of kinase insert domain-containing receptor (KDR) gene polymorphism/ haplotypes with recurrent spontaneous abortion and genetic structure

Background: Recurrent spontaneous abortion is one of the diseases that can lead to physical, psychological, and, economical problems for both individuals and society. Recently a few numbers of genetic polymorphisms in kinase insert domain-containing receptor (KDR) gene are examined that can endanger the life of the fetus in pregnant women. Objective: The risk of KDR gene polymorphisms was investigated in Iranian women with idiopathic recurrent spontaneous abortion (RSA). Materials and Methods: A case controlled study was performed. One hundred idiopathic recurrent spontaneous abortion patients with at least two consecutive pregnancy losses before 20 weeks of gestational age with normal karyotypes were included in the study. Also, 100 healthy women with at least one natural pregnancy were studied as control group. Two functional SNPs located in KDR gene; rs1870377 (Q472H), and rs2305948 (V297I) as well as one tag SNP in the intron region (rs6838752) were genotyped by using PCR based restriction fragment length polymorphism (PCR-RFLP) technique. Haplotype frequency was determined for these three SNPs’ genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to study genetic variation. Results: Functional SNP (rs1870377) was highly linked to tag SNP (rs6838752) (D´ value=0. 214; χ2 = 16.44, p<0. 001). K means clustering showed that k = 8 as the best fit for the optimal number of genetic subgroups in our studied materials. This result was in agreement with Neighbor Joining cluster analysis. Conclusion: In our study, the allele and genotype frequencies were not associated with RSA between patient and control individuals. Inconsistent results in different populations with different allele frequencies among RSA patients and controls may be due to ethnic variation and used sample size.

Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Modifies the Host Epigenome

The apparent simplicity of viruses hides the complexity of their interactions with their hosts. Viruses are masters at circumventing host defenses and manipulating the cellular environment for their own benefit. The replication of the largest known family of single-stranded DNA viruses, Geminiviridae, is impaired by DNA methylation and Arabidopsis mutants affected in cytosine methylation are hypersusceptible to geminivirus infection. This implies that plants might use methylation as a defense against geminiviruses and that the viral genome is a target for plant DNA methyltransferases. We have found a novel counter-defense strategy used by geminiviruses, that reduces the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues. Furthermore, we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species. Additionally, we identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 downregulation. The presence of Rep, suppresses TGS of an Arabidopsis transgene and of host loci whose expression is strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for geminiviral replication, displays TGS suppressor activity through a mechanism distinct from the one thus far described for geminiviruses.

Use of flucinolone acetonide for patients with diabetic macular oedema:patient selection criteria and early outcomes in real world setting

Introduction: Fluocinolone acetonide slow release implant (Iluvien®) was approved in December 2013 in UK for treatment of eyes which are pseudophakic with DMO that is unresponsive to other available therapies. This approval was based on evidence from FAME trials which were conducted at a time when ranibizumab was not available. There is a paucity of data on implementation of guidance on selecting patients for this treatment modality and also on the real world outcome of fluocinolone therapy especially in those patients that have been unresponsive to ranibizumab therapy. Method: Retrospective study of consecutive patients treated with fluocinolone between January and August 2014 at three sites were included to evaluate selection criteria used, baseline characteristics and clinical outcomes at 3-month time point. Results: Twenty two pseudophakic eyes of 22 consecutive patients were included. Majority of patients had prior therapy with multiple intravitreal anti-VEGF injections. Four eyes had controlled glaucoma. At baseline mean VA and CRT were 50.7 letters and 631 μm respectively. After 3 months, 18 patients had improved CRT of which 15 of them also had improved VA. No adverse effects were noted. One additional patient required IOP lowering medication. Despite being unresponsive to multiple prior therapies including laser and anti-VEGF injections, switching to fluocinolone achieved treatment benefit. Conclusion: The patient level selection criteria proposed by NICE guidance on fluocinolone appeared to be implemented. This data from this study provides new evidence on early outcomes following fluocinolone therapy in eyes with DMO which had not responded to laser and other intravitreal agents.

High interannual variability in connectivity and genetic pool of a temperate clingfish matches oceanographic transport predictions

Adults of most marine benthic and demersal fish are site-attached, with the dispersal of their larval stages ensuring connectivity among populations. In this study we aimed to infer spatial and temporal variation in population connectivity and dispersal of a marine fish species, using genetic tools and comparing these with oceanographic transport. We focused on an intertidal rocky reef fish species, the shore clingfish Lepadogaster lepadogaster, along the southwest Iberian Peninsula, in 2011 and 2012. We predicted high levels of self-recruitment and distinct populations, due to short pelagic larval duration and because all its developmental stages have previously been found near adult habitats. Genetic analyses based on microsatellites countered our prediction and a biophysical dispersal model showed that oceanographic transport was a good explanation for the patterns observed. Adult sub-populations separated by up to 300 km of coastline displayed no genetic differentiation, revealing a single connected population with larvae potentially dispersing long distances over hundreds of km. Despite this, parentage analysis performed on recruits from one focal site within the Marine Park of Arrábida (Portugal), revealed self-recruitment levels of 2.5% and 7.7% in 2011 and 2012, respectively, suggesting that both long- and short-distance dispersal play an important role in the replenishment of these populations. Population differentiation and patterns of dispersal, which were highly variable between years, could be linked to the variability inherent in local oceanographic processes. Overall, our measures of connectivity based on genetic and oceanographic data highlight the relevance of long-distance dispersal in determining the degree of connectivity, even in species with short pelagic larval durations.

Prevalence and genetic diversity of Blastocystis in family units living in the United States

The human gut is host to a diversity of microorganisms including the single-celled microbial eukaryote Blastocystis. Although Blastocystis has a global distribution, there is dearth of information relating to its prevalence and diversity in many human populations. The mode of Blastocystis transmission to humans is also insufficiently characterised, however, it is speculated to vary between different populations. Here we investigated the incidence and genetic diversity of Blastocystis in a US population and also the possibility of Blastocystis human-human transmission between healthy individuals using family units (N = 50) living in Boulder, Colorado as our sample-set. Ten of the 139 (~ 7%) individuals in our dataset were positive for Blastocystis, nine of whom were adults and one individual belonging to the children/adolescents group. All positive cases were present in different family units. A number of different Blastocystis subtypes (species) were detected with no evidence of mixed infections. The prevalence of Blastocystis in this subset of the US population is comparatively low relative to other industrialised populations investigated to date; however, subtype diversity was largely consistent with that previously reported in studies of European populations. The distribution of Blastocystis within family units indicates that human-human transmission is unlikely to have occurred within families that participated in this study. It is not unexpected that given the world-wide variation in human living conditions and lifestyles between different populations, both the prevalence of Blastocystis and its mode of transmission to humans may vary considerably.

Applying Least Absolute Shrinkage Selection Operator and Akaike Information Criterion Analysis to Find the Best Multiple Linear Regression Models between Climate Indices and Components of Cow’s Milk

This study focuses on multiple linear regression models relating six climate indices (temperature humidity THI, environmental stress ESI, equivalent temperature index ETI, heat load HLI, modified HLI (HLI new), and respiratory rate predictor RRP) with three main components of cow’s milk (yield, fat, and protein) for cows in Iran. The least absolute shrinkage selection operator (LASSO) and the Akaike information criterion (AIC) techniques are applied to select the best model for milk predictands with the smallest number of climate predictors. Uncertainty estimation is employed by applying bootstrapping through resampling. Cross validation is used to avoid over-fitting. Climatic parameters are calculated from the NASA-MERRA global atmospheric reanalysis. Milk data for the months from April to September, 2002 to 2010 are used. The best linear regression models are found in spring between milk yield as the predictand and THI, ESI, ETI, HLI, and RRP as predictors with p-value < 0.001 and R2 (0.50, 0.49) respectively. In summer, milk yield with independent variables of THI, ETI, and ESI show the highest relation (p-value < 0.001) with R2 (0.69). For fat and protein the results are only marginal. This method is suggested for the impact studies of climate variability/change on agriculture and food science fields when short-time series or data with large uncertainty are available.