mtDNA diversity in rabbit population from Sicily (Italy)

The European rabbit Oryctolagus cuniculus (O.c) lives all over the world and it represents an important resource for many predators. It has been classified as a Near-Threatened species in the Red List of Vertebrates of Italy. It is present in mediterranean basin as two known subspecies: O.c. cuniculus and O.c. algirus. The mediterranean geographic distribution of the two subspecies is still not well known. In particular, in Sicily, lacking of deep studies, is based on the body size and morphological characteristics; there wasn’t a complete description of the actual existing subspecies and previous studies only reported the morphological characteristics of the sicilian rabbit population. In this study, we analyzed genetic data, mitochondrial (mt) cytochrome b (cytb), from the rabbit population in Sicily in a phylogenetic framework. This is the first study concerning the genetics of the sicilian rabbit, to reconstruct intraspecific phylogeny by comparing cytb mtDNA sequences of 13 newly isolated O.cuniculus haplotypes from Sicily and 7 individuals from other countries (Canada, France, Mexico, North Italy, South Africa, Spain, Sweden). Our results show that the rabbit population from Sicily has a mitochondrial type (Lineage B) that has been previously shown to be associated with O. c. cuniculus and is similar to sequences from rabbits in North-Est Spain, Southern France, Sweden and South Africa

Genetic analysis of the critically endangered Trinidad Piping guan (Pipile pipile): Implications for phylogenetic placement and conservation strategies

Classified as critically endangered since 1994, the Trinidad Piping guan (Pipile pipile) is an endemic species estimated to number less than 200 individuals. Known to locals of Trinidad as the ‘Pawi’ this bird has been the subject of substantial hunting pressures and much of the species habitat has been destroyed through deforestation. Although officially protected since 1958, occasional recreational hunting of this elusive species still occurs. Due to difficulties locating and capturing the species, no genetic research has previously been performed using samples obtained from Trinidad. All previous research studies have been conducted using biological materials obtained from captive birds outside Trinidad and island data has never been obtained or compared. The genetic diversity of the remaining population was therefore examined through the investigation of mitochondrial haplotypes, pairwise comparison and SNP analysis. With the intention of assisting the protection of this endangered species by the location of remaining areas of habitation, methods of genetic identification were established for the Trinidad Piping guan utilising non-invasive feather samples. Species specific primers were created in the regions of the ND2 and cyt b genes of the mitochondrial genome to identify Pipile pipile. Species detection was further verified with the use of PCR-RFLP of the same gene regions digested with BsaXI, EcoRV and BsrDI. This combined approach allowed the separation of closely related taxa based on single inter-species SNPs. Confirmation of species identification was subsequently performed through the use of forensically informative nucleotide sequencing. The established methodologies were used in the current study to correct the classification of a UK breeding population of Piping guans thought to be Pipile pipile and to identify Trinidad field samples. These detection methods have implications for ecological studies through the location of populations from trace evidence collected in the field. In addition this method could be used to assist Trinidadian police forces in the identification of bushmeats or simply act as a deterrent to hunters. The sequence data obtained in the present study were also used to re-assess the phylogeny of Piping guans. As genetic sequence from a true island bird was previously unstudied, differences between phylogenies created using non-island and island bird data sets were examined. Combined analysis was performed on 1884bp of the ND2 and cyt b genes and placement of Trinidad Piping guan was found to differ from that which has been previously published.Funding from the University of Chester Gladstone Fellowship scheme and the World Pheasant Association

The role of healthy dog carriers of Babesia microti-like piroplasms

While in Europe Babesia canis has been traditionally held responsible for canine piroplasmosis, Babesia microti-like piroplasm (Bml) infection is being ever more observed in dogs, with the first clinical cases reported in northwestern Spain. This study examines the epidemiological role of healthy dogs living in endemic areas of Bml infection in Spain. The data obtained were used to describe the clinical status and map the geographical distribution of Bml infection in healthy dogs in northwestern SpainThe study was funded by the own sources of the Universidad Complutense of Madrid, SpainS

Identification and Characterization of Peak Activity, Environmental Variables, and Bacterial Pathogens in A. americanum L. at Ames Plantation, West Tennessee

The status of tick-borne diseases (TBD) in the southeastern United States is uncertain due to a number of factors including, but not limited to emerging pathogens, misdiagnoses, and modifications to landscapes. Ehrlichiosis and rickettiosis are two of the most common TBDs; these are caused by Ehrlichia and Rickettsia bacteria that can be transmitted by a number of different tick species. The objectives of this study were to identify Amblyomma americanum (the Lone Star tick) peak activity and habitat preferences and characterize the potential role of A. americanum in tick-borne disease cycles in southwestern Tennessee. Using vegetation drags and CO2-baited traps, ticks were collected monthly from May to September 2012 from 100 sites on the Ames Plantation Research and Education Center (Ames). Using a one-way analysis of variance, we identified the peak activity of A. americanum for adults as being in May or June and of nymphs as being bimodal with a peak in June and again in August. Trapping data were analyzed in a contingency table; results indicated significant trapping differences in the number of nymphs and adults collected by the two trapping methods. Environmental and trapping data were correlated using an ANCOVA to evaluate trapping efficacy under different environmental stressors and to identify landscapes in which A. americanum adults and nymphs are notably more abundant. Of 925 adult A. americanum screened for Ehrlichia and Rickettsia bacteria, 1.8% (n = 17) and 38% (n = 353) were PCR positive, of which 8 ticks (0.8%) were positive with both pathogens. Using ArcGIS we displayed pathogen positive A. americanum locations; calculating Moran’s I for each pathogen indicated there was no significant clustering among pathogen positive locations. The identification of pathogens and co-infections within A. americanum from western Tennessee warrants further investigations to understand the role ticks and their environment have in the distribution of TBD

Overlapping Protein-Encoding Genes in Pseudomonas fluorescens Pf0-1

The annotated genome sequences of prokaryotes seldom include overlapping genes encoded opposite each other by the same stretch of DNA. However, antisense transcription is becoming recognized as a widespread phenomenon in eukaryotes, and examples have been linked to important biological processes. Pseudomonas fluorescens inhabits aquatic and terrestrial environments, and can be regarded as an environmental generalist. The genetic basis for this ecological success is not well understood. In a previous search for soil-induced genes in P. fluorescens Pf0-1, ten antisense genes were discovered. These were termed ‘cryptic’ genes, as they had escaped detection by gene-hunting algorithms, and lacked easily recognizable promoters. In this communication, we designate such genes as ‘non-predicted’ or ‘hidden’. Using reverse transcription PCR, we show that at each of six non-predicted gene loci chosen for study, transcription occurs from both ‘sense’ and ‘antisense’ DNA strands. Further, at least one of these hidden antisense genes, iiv14, encodes a protein, as does the sense transcript, both identified by poly-histidine tags on the C-terminus of the proteins. Mutational and complementation studies showed that this novel antisense gene was important for efficient colonization of soil, and multiple copies in the wildtype host improved the speed of soil colonization. Introduction of a stop codon early in the gene eliminated complementation, further implicating the protein in colonization of soil. We therefore designate iiv14 “cosA”. These data suggest that, as is the case with eukaryotes, some bacterial genomes are more densely coded than currently recognized

Doctor of Philosophy

dissertationCone snails (genus Conus) have attracted scientific interest for the great neuropharmacological potential of their venoms to treat chronic pain, which consist of a complex mixture of peptides known as conotoxins. For discovery purposes, we have carried out a survey of the venom-ducts of 22 Conus species using next generation high throughput RNAseq (NGS). In silico analyses of these data are complicated because paralogous conotoxin precursors display both highly conserved, as well as hyper varied regions. As a result, NGS-based discovery involves an inherent trade off between fidelity of transcript assembly and sensitivity towards novel discovery. On the one hand, overly lenient assembly parameters create a few, long, but misassembled chimeric transcripts, which lessen the true discovery potential of NGS. On the other hand, overly stringent assembly parameters can mistake sequencing artifacts as novel discoveries. Moreover, many new conotoxins likely remain undiscovered. This fact can complicate homology-based discovery efforts using tools such as BLAST because reference databases may lack homologous peptides, leading to false negative results. With these problems in mind, I developed a comprehensive pipeline for discovery of conotoxins and their modification enzymes from high throughput RNAseq data. My pipeline includes (1) simulation software for benchmarking purposes, (2) a ‘partial extension pipeline' that employs a novel kmerization tool called Taxonomer to rapidly cluster and taxonomically classify reads prior to assembly, and (3) a discovery engine that can identify novel conotoxins even when they lack significant homologs. Collectively, my pipeline maximizes the discovery potential of Conus RNAseq data, identifying on average ~ 30% more full length toxins per sample than any other than approach in use today

Comparative Phylogeography of Central African Duikers Using Non-invasive Sampling Methods

The present study sets out to assess patterns of evolutionary diversification in central African duikers (subfamily Cephalophinae). The sampling strategy consisted of collecting geo-referenced duiker feces across 43 sites and seven countries. However, several challenges related to the use of non-invasive samples needed to be addressed prior to large scale DNA amplification. First, the best storage method for obtaining DNA from fecal samples needed to be established. Our study revealed that while silica is best for nuclear microsatellite analyses, RNAlater is the best storage medium for maximal mitochondrial amplification. Moreover, extracting DNA as early as possible always provided the best results. Second, since it is impossible to determine the species identity of duiker feces solely based on their morphology, a simple and reliable molecular method was needed. A tree-based approach based on ~650 base pairs of the control region amplified from reference samples was found to be the most reliable method to recover the identity of unknown samples. Third, for fine scale analyses of population genetic structure, a set of twelve nuclear microsatellites were assembled from existing bovid data. These microsatellites markers were chosen because they are very polymorphic, cross amplify among targeted taxa, co-amplify with combined markers of the same multiplex, and are powerful enough for individual identification. Patterns of mitochondrial and nuclear microsatellite variation were used to test two important hypotheses of diversification in the tropics: the Pleistocene refugia and the riverine barrier hypotheses. Analyses of historical and contemporary population genetic structure were carried out on the three most abundant species in our sampling area: the bay duiker (C. dorsalis), the Peter’s duiker (C. callipygus), and the blue duiker (P. monticola) using mitochondrial and nuclear markers described above. These data show that (1) southwest Nigeria and southwest Cameroon comprise genetically distinct populations in C. callipygus and P. monticola species, (2) signatures of demographic expansion for all three taxa are broadly coincident with the location of hypothesized upland refugia in Gabon and Equatorial Guinea and (3) the Sanaga, Ogooué, and Sangha rivers may constitute a partial riverine barrier and/or act as fluvial refugia for duikers

Molecular cloning and characterization of the human p19INK4d gene promoter

Abstractp19INK4d, a member of the INK4 family of cyclin-dependent kinase (CDK) inhibitors, negatively regulates the cyclin D–CDK4/6 complexes, which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. To investigate the mechanism of transcriptional regulation of the p19INK4d gene, we characterized the 5′-flanking region of the human p19INK4d gene. The cap-site hunting method revealed that the transcription starts at −16 nucleotide (nt) upstream of the initiation codon. The 5′-flanking region of the human p19INK4d gene was ligated to a luciferase reporter gene and possessed functional promoter activity. Luciferase assay with a series of truncated 5′-flanking regions indicated that the region from −81 to −2 nt could drive the transcription of the p19INK4d gene. Several Sp1 and activating protein 2 binding sites are located within the region from −81 to −2 nt. Mutation of the second Sp1 binding site from −33 to −25 nt decreased the promoter activity. Collectively, it was demonstrated that the human p19INK4d gene is under the control of TATA-less promoter and the Sp1 binding site is involved in the transcription

Toxoplasma gondii infection in Australian felines

Toxoplasma gondii is a significant human and animal parasite with worldwide occurrence. The population structure and prevalence in definitive hosts and intermediate hosts has been well studied in a number of regions including North and South America and Europe. While Toxoplasma gondii has been previously reported in a number of mammalian species in Australia, there is a surprising lack of information regarding the population structure of T.gondii in Australia and current rates of exposure in domestic companion animal hosts. Members of the felidae family are the only known definitive host of T. gondii in which sexual reproduction of the parasite occurs.Sexual reproduction results in shedding of environmentally resillient oocysts in faeces. All strains of T. gondii can be traced back to a feline host. As such, an understanding of T. gondii infection among feline hosts is an important first step to understanding general prevalence and population structure of the parasite in a particular area. The seroprevalence of T. gondii in cats worldwide is estimated to be 30-40%. In addition to being a definitive host of T. gondii, domestic cats are susceptible to clinical disease due to T.gondii. Although uncommon, feline toxoplasmosis occurs in immunosuppressed cats, congenitally infected kittens and occasionally in otherwise clinically healthy individuals. Little is known about whether parasite genotype is associated with severity of disease in naturally occurring toxoplasmosis of domestic cats. The first aim of this research was to expand knowledge of feline T. gondii infections in Australia by determining the seroprevalence of T. gondii in owned Australian cats and identifying riskfactors for infection. The second aim was to determine whether the genotype of T. gondii is a significant determinant of whether cats develop clinical toxoplasmosis. To estimate seroprevalence of T. gondii in Australian owned cats, Toxoplasma specific IgG ELISAs were performed on sera from 425 owned domestic Australian cats. A multivariate 12 analysis of the results from a questionnaire given to the owners was used to evaluate lifestyle factors which could contribute to increased likelihood of infection. Of the 425 cats tested in this study 38% (n=162) were seropositive. The prevalence in different geographic regions ranged from 16-71%. Cats fed raw beef or raw kangaroo in their diet or cats that hunted rodents were significantly more likely to be seropositive To identify the genotypes associated with latent and active infections in Australian cats, tissue samples were collected from cats undergoing routine post mortem examination at the University Veterinary Teaching Hospital (n=28). Serology to detect T. gondii specific IgG was performed after collection of heart blood from cats of unknown T. gondii serostatus. Cases were chosen based on the likelihood of a cat being exposed to the parasite. Results of a PCR targeting the B1 gene to detect T. gondii DNA were positive in tissue samples from 11 of 17 (65%) seropositive cats tested including four with clinical toxoplasmosis and seven with latent infections, as determined by serology, histologic findings and immunohistochemistry. Three of the four cats with clinical toxoplasmosis were immunosuppressed. T. gondii type II (ToxoDB genotype #3) was determined in four cats with clinical toxoplasmosis and three cats with latent toxoplasmosis using PCR-RFLP at 12 loci (SAG1, 5’SAG2 and 3’SAG2, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) and direct sequencing of the multicopy B1 gene. Novel T. gondii B1 gene polymorphisms were detected in two strains (at nucleotide posititions 233, 366 and 595) and a B1 gene polymorphism unique to Australia was identified in another (guanine/adenine at nucleotide position 378). One cat was coinfected with two or more type-II like strains at 3’SAG2. The results of this study suggest that the infecting T. gondii genotype is not a determinant of clinical disease in cats naturally infected with T. gondii and type II strains are the most prevalent in Australia. 13 The ToxoDB#3 genotype has been previously identified in Australian hosts. Given the high prevalence in the present study, it appears that this genotype may be endemic to Australian hosts and this discovery warrants further investigation of a larger sample set using high resolution techniques such as those presented in this thesis to confirm this. Naturally infected cats with active and latent infection were both infected with the same strain, indicating the infecting strain is not the major determinant of disease progression and that other host factors may be of greater importance. Throughout this program of research, epidemiological aspects of feline T. gondii infections in Australia were elucidated further. Key findings include a seroprevalence of 38% among owned Australian cats, the identification of a cat which was infected with more than one strain of T. gondii, the discovery that infections with the same ToxoDB#3 genotype can cause both clinical and latent infection in cats and the confirmation of diversity at the B1 locus in felines, which has been previously found in Australian wildlife species. This is the first survey of feline infections in owned Australian cats to evaluate lifestyle factors which contribute to the likelihood of exposure and it is also the first study to genetically characterise infecting strains in cats with clinical toxoplasmosis. Further studies are needed to fully understand the population structure and also the prevalence of T. gondii in Australia as current data is lacking in other domestic and wildlife species