Analysis of bovine viral diarrhoea virus: Biobank and sequence database to support eradication in Scotland.

Samples from bovine viral diarrhoea virus (BVDV)-positive cattle were gathered by Scottish diagnostic laboratories and used to produce a Biobank of samples with associated location and identification data in support of the Scottish BVDV eradication scheme. The samples were subject to direct amplification and sequencing of the 5'-untranslated region (5'-UTR) to define the viral types and subtypes present. From 2693 samples collected prior to 2016, approximately 2300 sequences were obtained, representing 8 BVDV type 1 subtypes. No BVDV type 2 samples were detected. The samples came from all regions of the UK but 66 per cent were from Scotland. Analysis of the sequences showed great diversity in the 5'-UTR, with 1206 different sequences. Many samples carried virus with identical 5'-UTR sequences; often from single locations, but there were also examples of the same sequence being obtained from samples at several different locations. This work provides a resource that can be used to analyse the movement of BVDV strains both within Scotland and between Scotland and other nations, particularly in the latter stages of the Scottish eradication programme, and so inform the advice available to both livestock keepers and policymakers.

A field vaccine trial in Tanzania demonstrates partial protection against malignant catarrhal fever in cattle.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease of cattle that, in East Africa, results from transmission of the causative virus, alcelaphine herpesvirus 1 (AlHV-1), from wildebeest. A vaccine field trial involving an attenuated AlHV-1 virus vaccine was performed over two wildebeest calving seasons on the Simanjiro Plain of northern Tanzania. Each of the two phases of the field trial consisted of groups of 50 vaccinated and unvaccinated cattle, which were subsequently exposed to AlHV-1 challenge by herding toward wildebeest. Vaccination resulted in the induction of virus-specific and virus-neutralizing antibodies. Some cattle in the unvaccinated groups also developed virus-specific antibody responses but only after the start of the challenge phase of the trial. PCR of DNA from blood samples detected AlHV-1 infection in both groups of cattle but the frequency of infection was significantly lower in the vaccinated groups. Some infected animals showed clinical signs suggestive of MCF but few animals went on to develop fatal MCF, with similar numbers in vaccinated and unvaccinated groups. This study demonstrated a baseline level of MCF-seropositivity among cattle in northern Tanzania of 1% and showed that AlHV-1 virus-neutralizing antibodies could be induced in Tanzanian zebu shorthorn cross cattle by our attenuated vaccine, a correlate of protection in previous experimental trials. The vaccine reduced infection rates by 56% in cattle exposed to wildebeest but protection from fatal MCF could not be determined due to the low number of fatal cases.

Toll-like receptor gene expression in fresh and archived ovine pseudoafferent lymph DEC205+ dendritic cells.

Toll-like receptors (TLRs) are key regulators of the innate and adaptive immune response to bacterial, viral and fungal pathogens. To date, 10 human TLRs and 13 mouse TLRs have been identified and they exhibit tissue-specific mRNA/protein expression patterns. We recently cloned and characterized 10 ovine TLR genes. The present study was carried out to determine the expression profile of TLRs 1-10 in fresh and archived ovine pseudoafferent lymph (pAL) cells and pAL dendritic cells (pALDCs) using two-step quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with ovine specific primer/probe sets. Dendritic cells are important in the initiation and maintenance of immune responses and express a spectrum of pattern-recognition receptors (that includes the TLRs). Fresh and archived total pAL cells expressed all 10 ovine TLRs to a broadly similar extent and TLR1-10 mRNA expression was observed in DEC205(hi) pALDCs. In addition, there were changes in particular TLR transcript levels in DEC205(hi) pALDC in archived lymph samples at two time points after orf virus reinfection. The results show that frozen archived cells can be used for retrospective TLR gene expression analysis. Furthermore, changes in TLR gene expression in DEC205(hi) pALDC after orf virus reinfection in the skin of sheep suggests that more detailed analyses of TLR gene expression changes during disease processes are worthwhile. These data will be useful to inform future studies on the role of TLRs in disease pathogenesis and control.

Outbreak of wildebeest-associated malignant catarrhal fever in Ankole cattle.

During an outbreak of malignant catarrhal fever in a herd of Ankole cattle in a zoological collection, two adult cows and one adult bull from a herd of 15 died or were euthanased between July and November 2004. The clinical, gross postmortem and histological findings were typical of the disease in uk native domestic cattle. The diagnosis was confirmed by serology in two animals, and by pcr in all three; the pcr provided evidence of alcelaphine herpesvirus type 1 infection in all three animals and also of ovine herpesvirus type 2 infection in one.

Development of an enzyme-linked immunosorbent assay for the detection of antibodies against malignant catarrhal fever viruses in cattle serum.

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cell-associated herpesviruses of the subfamily gammaherpesvirinae. In this study, an ELISA (WC11-ELISA) was developed to detect antibody to malignant catarrhal fever virus (MCFV) in cattle serum and compared to the commercially produced competitive-inhibition ELISA (CI-ELISA). Crude lysate antigen from alcelaphine herpesvirus-1 strain WC11 was bound to 96-well microplates and used to capture antibodies to MCFV. Dilutions of test sera were added to wells containing bound MCF antigen and control wells containing uninfected cell lysates. A horseradish peroxidase-labelled rabbit-anti-bovine IgG conjugate detected antibodies to MCF, and the results were expressed as absorbance readings at 450 nm. Samples were selected blind from cattle sera which had been sent to the laboratory for diagnostic testing for MCFV antibodies and were tested in both the WC11-ELISA and the CI-ELISA. Good agreement between the WC11-ELISA and CI-ELISA test (k=0.86, n=95) results was found.

Structure of the BoLA-DRB3 gene and promoter.

The cattle major histocompatibility complex (MHC) class II DR gene product is a heterodimer encoded by the BoLA-DRA and -DRB3 genes. Several groups have isolated cDNA and genomic clones for these genes, but their full genomic organization has not been described. We used a combination of long-range polymerase chain reaction (PCR), cloning and sequencing to define the organization of the DRB3 gene on existing genomic clones and in genomic DNA. We estimate the size of the coding region to be 11.4 kbp. Sequencing of full-length PCR clones from two different haplotypes confirmed that they carried complete DRB3 genes and allowed the design of probes and primers to isolate and characterize the DRB3 promoter and 3' end. Fragments carrying the 5' end of the DRB3 gene and its promoter were identified on bacterial artificial chromosome (BAC) clones carrying the BoLA-DR genes. A 10-kbp promoter fragment was subcloned from one clone and a 1.7-kbp region including exon 1 and the promoter was sequenced. A 3-kbp fragment encoding exons 4-6 and the entire 3' untranslated region of the DRB3 gene was isolated from lambda clone A1 and sequenced. This provides us with improved characterization of the DRB3*0101 and DRB3*2002 alleles, and also subcloned 5' and 3' flanking regions of the polymorphic DRB3 gene for use in functional studies.

Establishment of a sequence-based typing system for BoLA-DRB3 exon 2.

A rapid, high-resolution sequence-based typing (SBT) system for BoLA-DRB3 exon 2 was developed. Amplification of the entire exon was achieved by a fully nested PCR with locus-specific primers and sequencing was performed directly on the PCR product. Heterozygous sequence data were obtained by automated sequence analysis of both alleles. Forward and reverse sequence data were assembled to improve identification of all heterozygous positions. Specific software (Haplofinder, Roslin Institute Software, Roslin, UK) was designed for allele assignment. Fifty-four females from a Holstein-Charolais resource herd cross, their 12 sires and five unrelated Holstein animals were used to establish the method. In parallel, these animals were typed by DRB3 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to confirm the results. Polymerase chain reaction-RFLP analysis defined 15 known types in the 71 animals, while SBT of the same animals showed 19 known alleles. Subsequently, 72 more animals from the same resource herd were typed by the established SBT method without PCR-RFLP typing. This SBT strategy and the Haplofinder software can be applied to the analysis of any polymorphic locus for which suitable locus-specific primers and allelic sequences are available.

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis.

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.

Sequence duplication at the 3' end of BoLA-DQB genes suggests multiple allelic lineages.

Full-length cDNAs encoding the DQB genes expressed by three BoLA class II haplotypes (DH8A, DH15B, and DH24A) were amplified by reverse-transcription polymerase chain reaction, cloned, and sequenced. The sequence data revealed that the DH8A haplotype expressed two DQB genes (DQB*1005 and DQB*1201) while the DH15B and DH24A haplotypes expressed the same single gene (DQB*0101). Comparison of the three alleles showed that the 3' untranslated (3'UT) sequence of the DQB*1201 allele contained a duplication of about 200 bp. This repeat was also found in other DQB alleles from cattle and sheep, but only in haplotypes with duplicated DQB genes. This 200-bp repeat and other features of the 3'UT may provide useful markers of DQB evolution, allowing us to distinguish and selectively amplify the different DQB loci.

Sequence and transfection of BoLA-DRB3 cDNAs.

Bovine MHC (BoLA-) DRB3 alleles encoded by the DH8A, DH22A and DH24A class II haplotypes were cloned from cDNA and characterized by sequence analysis. Comparison with other full-length DRB3 sequences suggested that DRB3 alleles may have evolved through multiple lineages. All three BoLA-DRB3 alleles were shown to express on the surface of transfected cells, and the transfectants were used to define or confirm the class II specificity of a panel of monoclonal antibodies.

Duplicated DQ haplotypes increase the complexity of restriction element usage in cattle.

The MHC of cattle encodes two distinct isotypes of class II molecules, DR and DQ. Unlike humans, cattle lack the DP locus and about half the common haplotypes express duplicated DQ genes. The number and frequency of DQA and DQB alleles means that most cattle are heterozygous. If inter- and/or intrahaplotype pairing of DQA and DQB molecules occurs, cattle carrying DQ-duplicated haplotypes may express more restriction elements than would be predicted by the number of expressed alleles. We are investigating whether duplicated haplotypes cause differences in immune response, particularly in terms of generating protective immunity. We have analyzed the Ag-presenting function of DQ molecules in two heterozygous animals, one of which carries a duplicated haplotype. We compared the class II isotype specificity of T cell clones recognizing a putative vaccinal peptide from foot-and-mouth disease virus (FMDV15). We show for the first time that bovine T cells can recognize Ag in the context of DQ molecules. We also present evidence that interhaplotype pairings of DQA and DQB molecules form functional restriction elements. Both animals showed distinct biases to usage of particular restriction elements. Mainly DQ-restricted clones were derived from the animal with duplicated DQ genes, whereas the majority of clones from the animal with a single DQ gene pair were DR restricted. Furthermore, haplotype bias was observed with both animals. These experiments show that understanding of class II chain pairing in addition to knowledge of the genotype may be important in vaccine design where effective epitope selection is essential.

Association of bovine DRB3 alleles with immune response to FMDV peptides and protection against viral challenge.

We have analysed the influence of bovine MHC (BoLA) polymorphism on the immune response and degree of protection induced by peptide vaccines against foot-and-mouth disease (FMD) in cattle. The peptides used for animal immunisation were: A (VP1(138-156)), AT (peptide A linked to VP1(21-40)) and ACT (peptide A, linked to VP1(196-209) and VP1(21-40)). Sixteen different DRB3 types were found among the 46 cattle analysed by PCR-RFLP typing. No absolute correlation was observed, for any type, with the serum neutralising titres (SNT) values and the protection induced. However, among the most common haplotypes present, associations were observed between expression of different types with the levels of SNT and/or protection induced by peptides A and ACT. Thus, types DRB3.2*1, 3 and 7 were associated with increased levels of protection. In contrast, types DRB3.2*12 and 18 were associated non-protection, and DRB3.2*12 was also associated with low SNT titres. Overall, the results indicate that the polymorphism in BoLA class II molecules affects both the immune response and protection induced by potential FMD peptide vaccines.

Comparative organization and function of the major histocompatibility complex of domesticated cattle.

This review focuses on recent advances in research on the bovine major histocompatibility complex (BoLA), with specific reference to the genetic organization, polymorphism and function of the class II genes. The BoLA region is unlike the MHC of humans and mice in that a large inversion has moved several class II genes, including the TAP/LMP cluster, close to the centromere of bovine chromosome 23. Therefore, close linkage of MHC genes and other genes associated with the MHC in humans and mice does not appear to be required for normal immunological function. In cattle, polymorphism in the class IIa genes influences both the magnitude and the epitope specificity of antigen-specific T-cell responses to foot-and-mouth disease virus peptides. Disease association studies have demonstrated that BoLA alleles affect the subclinical progression of bovine leukemia virus (BLV) infection. This association is strongly correlated with the presence of specific amino acid motifs within the DRB3 antigen-binding domain. In addition to the practical significance of these findings, the association between BoLA and BLV provides a unique model to study host resistance to retrovirus infection in a non-inbred species. These studies contribute to our understanding of the evolution of the MHC in mammals, to the development of broadly effective vaccines, and to breeding strategies aimed at improving resistance to infectious diseases.

Using student, teacher and practice supervisor feedback to improve the quality of nurse education: how should we collect it and what should we do with it?

Many colleges and universities are committed to gathering feedback as a means of improving course quality. Typically student's views are sought and few institutions seek systematic feedback from both students and members of staff. We report on a pilot study employing student, teacher and practice supervisor feedback on a pre-registration, Diploma in Higher Education (Nursing) course. The paper discusses how we collected the feedback and how the gathered information will influence future planning and decision making.

A novel expression vector for transfection of bovine MHC class I genes.

A vector is described for the expression of genomic or cDNA copies of bovine major histocompatibility complex (MHC) class I genes in transfected mouse Ltk- cells. Class I gene fragments are amplified by the polymerase chain reaction, using primers in conserved parts of exon 2 and the 3'-untranslated region of the gene. Amplified class I gene fragments can then be subcloned into the expression vector, pBoLA-21, which contains the necessary 5'- and 3'-sequences for correct expression. The vector was tested by subcloning and expressing genomic and cDNA clones.