Optical mapping compendium of structural variants across global cattle breeds.

Structural variants (SV) have been linked to important bovine disease phenotypes, but due to the difficulty of their accurate detection with standard sequencing approaches, their role in shaping important traits across cattle breeds is largely unexplored. Optical mapping is an alternative approach for mapping SVs that has been shown to have higher sensitivity than DNA sequencing approaches. The aim of this project was to use optical mapping to develop a high-quality database of structural variation across cattle breeds from different geographical regions, to enable further study of SVs in cattle. To do this we generated 100X Bionano optical mapping data for 18 cattle of nine different ancestries, three continents and both cattle sub-species. In total we identified 13,457 SVs, of which 1,200 putatively overlap coding regions. This resource provides a high-quality set of optical mapping-based SV calls that can be used across studies, from validating DNA sequencing-based SV calls to prioritising candidate functional variants in genetic association studies and expanding our understanding of the role of SVs in cattle evolution.

A cattle graph genome incorporating global breed diversity.

Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds, especially those from Africa for which genomic resources are particularly limited, despite their disproportionate importance to the continent's economies. To mitigate this issue, we have generated assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into a graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1 Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research.

Use of a Pan-Vbeta primer permits the amplification and sequencing of TCRbeta chains expressed by bovine T-cell clones following a single semi-nested PCR reaction.

We report in this study the design and validation of a Pan-Vbeta primer that in combination with Cbeta-specific primers enables the amplification, in a single semi-nested PCR, of TCRbeta chains expressed by bovine T-cell clones irrespective of the expressed Vbeta sequence. Using the Pan-Vbeta primer we examined the TCRbeta chains expressed by 16 Theileria parva-specific clones that had not been previously analysed. TCRbeta chain sequence was obtained from 15 of the clones following direct sequencing of the PCR product, whilst the other clone appeared to express 2 different TCRbeta chains which were characterised following sub-cloning of the PCR product. We have also successfully used the Pan-Vbeta primer to amplify the TCRbeta chains expressed by 19 T-cell clones, on which previous analysis using Vbeta-subfamily-specific primers had failed to do. Sequencing of these TCRbeta chains has identified members of 2 novel bovine Vbeta subfamilies-Vbeta5 and VbetaX. This method offers a simple and rapid method of analyzing the TCRbeta chains of bovine T-cell clones that has many potential applications in the investigation of bovine T-cell responses.

Dissection of the clonal composition of bovine alphabeta T cell responses using T cell receptor Vbeta subfamily-specific PCR and heteroduplex analysis.

Although techniques that permit analysis of the clonal composition of T cell populations have been used extensively to provide a better understanding of the mechanisms that influence efficacy of T cell responses in humans and mice, such methods are lacking for other animal species. In this paper we report the establishment and validation of a panel of Vbeta subfamily-specific semi-nested PCR assays, and a CDR3beta heteroduplex technique for analysing the clonal diversity of bovine alphabeta T cell responses. Development of these methods was based on available sequence data for 48 functional Vbeta genes classified within 17 subfamilies. These techniques were used to determine the clonal composition of parasite-reactive CD8(+) T cells obtained from two animals immunised with the protozoan parasite Theileria parva. Analyses of uncloned T cell lines as well as large panels of cloned T cells derived from each of these lines confirmed the specificity and sensitivity of the assays. Specific PCR products were obtained from 96% of the T cell clones examined, indicating that the currently identified Vbeta genes represent most of the functional Vbeta subfamilies in cattle. Heteroduplex analyses, coupled with sequencing of PCR products, identified over 20 clonal expansions within each of the T cell lines, distributed over a large number of Vbeta subfamilies, although a limited number of clonotypes numerically dominated the response in both animals. The development and validation of these methods provides for the first time a generic set of molecular tools that can be used to perform detailed analysis of the TCR diversity and clonal composition of bovine T cell responses.

Analysis of T-cell receptor BV gene sequences in cattle reveals extensive duplication within the BV9 and BV20 subgroups.

We investigated the repertoire of functional T-cell receptor beta-chain variable genes (TRBV genes) in cattle by analysing the nucleotide sequences and predicted amino acid sequences of a set of cDNA clones isolated from lymph node T cells. Thirty-nine distinct TRBV sequences were identified, bringing the total number of recognised bovine TRBV gene segments to more than 40. Sixteen TRBV subgroups were defined based on their sequence homology to each other and to human TRBV genes. All of the main phylogenetic lineages of BV gene subgroups described in humans and mice were represented. Eight of the subgroups were found to contain more than one member. The most striking feature of the results was the large number of sequences (more than half of the sequenced clones) in the BV9 and BV20 subgroups, which were found to contain 12 and 8 distinct sequences, respectively. In contrast, the corresponding human TRBV subfamilies contain a single member. The results indicate that, as in humans, there has been extensive gene duplication within the TRBV locus during evolution. However, duplication of different BV subgroups in cattle has resulted in a TRBV gene repertoire distinct from that found in other species.

Lumboperitoneal shunting as a cause of visual loss in benign intracranial hypertension.

The causes of visual loss in benign intracranial hypertension are related to long standing papilloedema, ischaemic optic neuropathy or haemorrhage into a subretinal neovascular membrane. Decompression procedures generally preserve or improve visual acuity but surgical treatment with subtemporal decompression may lead to visual impairment. Such a deficit has been recorded in the past as occurring with ventriculography. Postulated mechanisms have included brain herniation, spasm of vessels supplying the visual cortices or retinal vascular disturbance. To our knowledge treatment with lumboperitoneal shunting has not previously been reported as leading to further significant visual loss in this condition. This report describes such an occurrence in a patient. Retinal vascular disturbance is postulated on the basis of several normal CT scans, normal CSF pressure measured after surgery and visual evoked responses suggesting retinal or optic nerve damage.

Developmental anomalies affecting the fourth ventricular outflow region: a report of four cases.

Developmental anomalies affecting the hind brain, cerebellum and spinal cord with associated malformations of the skull base and cervical spine, show a wide degree of variability and overlap so that cases are often difficult to classify into a fixed category such as the Arnold-Chiari malformation Type 1 or Type 2, or the Dandy-Walker syndrome. The 4 cases presented here illustrate this problem and offer support for the hypothesis that these conditions may be merely the varying expression of a common pathological process. The CT brain scan may prove to be a valuable means of allowing early recognition of these anomalies so that effective surgical treatment can be given at an early stage in their natural history, possibly thereby preventing the progression or development of central cord cavitation with its significant and permanent disabilities.

Posttraumatic vascular malformations of the orbit.

The close association of the external and internal carotid circulations in the orbital cavity is well recognized. Two cases are presented in which local trauma to the orbit apparently resulted in the formation of an arteriovenous fistula. These lesions progressively expanded, producing classical features of proptosis, chemosis, and bruit. The investigation by selective angiograms and the surgical management of these malformations are discussed. A further case is presented in which an early lesion was noted in angiography after a head injury, with subsequent disappearance of the lesion some weeks later.

Posttraumatic vascular malformations of the orbit.

The close association of the external and internal carotid circulations in the orbital cavity is well recognized. Two cases are presented in which local trauma to the orbit apparently resulted in the formation of an arteriovenous fistula. These lesions progressively expanded, producing classical features of proptosis, chemosis, and bruit. The investigation by selective angiograms and the surgical management of these malformations are discussed. A further case is presented in which an early lesion was noted in angiography after a head injury, with subsequent disappearance of the lesion some weeks later.