Association between anal sac gland carcinoma and dog leukocyte antigen-DQB1 in the English Cocker Spaniel.

Anal sac gland carcinomas occur frequently in English Cocker Spaniels and, to a lesser extent, in other spaniel breeds. The disease typically presents in dogs aged 8 years or older and frequently metastasises to the local lymph nodes. The association between anal sac gland carcinoma in English Cocker Spaniels and the major histocompatibility complex class II loci (the dog leukocyte antigen loci DLA-DRB1, -DQA1, -DQB1) was investigated in 42 cases and 75 controls. Based on a corrected error rate of 0.017 for each test, the allele distribution in DLA-DRB1 showed no significant difference between cases and controls (P value = 0.019), while a significant difference was obtained for DLA-DQA1 and -DQB1 alleles (P values are 0.010 and 3.3 × 10⁻5). The DLA-DQB1*00701 allele was the most common in both cases and controls, but it had a higher frequency among the former (0.89) than in the latter (0.61), while the second most common allele had a higher frequency in the controls (0.23) than in the cases (0.07). Haplotype distributions were also significantly different between the two groups (P value = 1.61 × 10⁻4). This is the second disease in English Cocker Spaniels for which the most common DLA-DQB1 allele in the breed has been shown to have a higher frequency in cases than controls, while the second most common allele in the breed (*02001) has a significantly higher frequency in the controls, compared with the cases.

Whole-Body Barometric Plethysmography Characterizes Upper Airway Obstruction in 3 Brachycephalic Breeds of Dogs.

BACKGROUND: A novel test using whole-body barometric plethysmography (WBBP) was developed recently to diagnose brachycephalic obstructive airway syndrome (BOAS) in unsedated French bulldogs. HYPOTHESIS/OBJECTIVES: The hypotheses of this study were: (1) respiratory characteristics are different between healthy nonbrachycephalic dogs and brachycephalic dogs; and among pugs, French bulldogs, and bulldogs; and (2) obesity and stenotic nares are risk factors for BOAS. The main objective was to establish a diagnostic test for BOAS in these 3 breeds. ANIMALS: A total of 266 brachycephalic dogs (100 pugs, 100 French bulldogs, and 66 bulldogs) and 28 nonbrachycephalic dogs. METHODS: Prospective study. Exercise tolerance tests with respiratory functional grading, and WBBP were performed on all dogs. Data from WBBP were associated with functional grades to train quadratic discriminant analysis tools to assign dogs to BOAS+ and BOAS- groups. A BOAS index (0-100%) was calculated for each dog. Receiver operating characteristic (ROC) curves were used to evaluate classification ability. RESULTS: Minute volume was decreased significantly in asymptomatic pugs (P = .009), French bulldogs (P = .026), and bulldogs (P < .0001) when compared to nonbrachycephalic controls. Respiratory characteristics were different among breeds and affected dogs had a significant increase in trace variation. The BOAS index predicted BOAS status for each breed with 94-97% (95% confidence interval [CI], 88.9-100%) accuracy (area under the ROC curve). Both obesity (P = .04) and stenotic nares (P = .004) were significantly associated with BOAS. CONCLUSIONS AND CLINICAL IMPORTANCE: The WBBP can be used as a clinical tool to diagnose BOAS noninvasively and objectively.

A cryptic deletion of 2q35 including part of the PAX3 gene detected by breakpoint mapping in a child with autism and a de novo 2;8 translocation.

We report a de novo, apparently balanced (2;8)(q35;q21.2) translocation in a boy with developmental delay and autism. Cross species (colour) paint (Rx) and SKY FISH, forward and reverse chromosome painting, and FISH with subtelomeric probes were used to examine the patient's karyotype, but further rearrangements were not detected. FISH with region specific clones mapping near 2q35 and 8q21.2 breakpoints and STS mapping performed on the isolated derivative chromosomes were used to refine the location of the breakpoints further. A cryptic deletion of between 4.23 and 4.41 Mb in extent and involving at least 13 complete genes or transcription units was found at the breakpoint on 2q35. The deletion includes the promoter and 5' untranslated region of the paired box 3 (PAX3) gene. The child has very mild dystopia canthorum which may be associated with the PAX3 haploinsufficiency. The 8q21.2 breakpoint is within MMP16 which encodes matrix metalloproteinase 16. We postulate that the cryptic deletion and rearrangement are responsible for the patient's phenotype and that a gene (or genes) responsible for autism lies at 2q35 or 8q21.2. The results present a step towards identifying genes predisposing to autism.

Sequence of the cDNA encoding ovine tumor necrosis factor-alpha: problems with cloning by inverse PCR.

We have cloned and sequenced the ovine tumor necrosis factor-alpha (TNF-alpha)-encoding cDNA, using gene amplification by polymerase chain reaction (PCR) technology, to aid studies of assorted diseases in this species. We used primers selected from published TnfA sequences of other species on a cDNA template prepared from lipopolysaccharide-stimulated ovine alveolar macrophages, to generate a product representing the central region of the molecule. We then used a novel method based on 'inverse PCR' to generate a product containing the 5' and 3' ends of the molecule. Here, we present the complete sequence of the ovine TNF-alpha cDNA and compare it with other published TNF sequences. The cloned cDNA has a leader sequence of 156 bp followed by a protein-coding sequence of 702 bp and a 3'-untranslated region of 800 bp. The protein product of the gene is a protein of Mr = 25,586, 79% homologous to human TNF-alpha. An mRNA produced by alveolar macrophages, which hybridises to the cloned gene, is induced greatly, with a peak induction time of approx. 135 min, in response to stimulation by lipopolysaccharide and to plating on plastic. We also discuss the resolution of some artefacts of the inverse PCR technique.

Cloning of a cDNA encoding the ovine interleukin-2 receptor 55-kDa protein, CD25.

A 1.3-kb cDNA that encodes the entire 825-bp coding region of ovine CD25, the interleukin-2 receptor 55-kDa protein, has been isolated. Comparison of the deduced amino acid sequence with CD25 proteins from other species shows the ovine sequence to have the greatest homology with that of the bovine species.

Nucleotide sequence of the canine rod-opsin-encoding gene.

The major parts of two canine rod-specific opsin (Ops) transcripts have been cloned by polymerase chain reaction from retinal mRNA. Both transcripts are derived from the same gene. The 5' leader sequence of the transcripts was cloned from canine peripheral blood DNA. The transcripts code for a protein of 348 amino acids (aa), M(r) 38,962 (prior to any protein modification). The aa sequence suggests that in common with other sequenced Ops, canine rod Ops contains seven transmembrane domains, and residues believed essential for retinal pigment binding and for palmitate binding are conserved in the canine protein. Northern blotting using the central part of the ops gene as probe suggested that mature transcripts of three different sizes (about 1900, 2600 and 5500 bases) were found in retina. Of these, the 2600-base transcript was the most abundant. RACE cloning of the 3' end of ops showed that at least two of these size classes originate from differential transcript termination.

Sequence of the sheep interleukin-10-encoding cDNA.

The ovine interleukin-10 (oIL-10)-encoding cDNA has been cloned and sequenced using gene amplification by the polymerase chain reaction (PCR). We present the complete coding sequence of the ovine IL-10 gene, as well as the predicted amino acid (aa) sequence. The oIL10 DNA coding sequence is 531 nucleotides long and the mature protein product is predicted to be 18,367 Da, consisting of 158 aa, excluding a 19-aa N-terminal hydrophobic signal peptide. The oIL-10 protein is > 77% identical to pig and human IL-10, > 71% identical to rodent IL-10 and > 68% identical to viral IL-10.

A possible novel interaction between the 3'-end of 18 S ribosomal RNA and the 5'-leader sequence of many eukaryotic messenger RNAs.

A novel interaction between the 5'-untranslated region of eukaryotic messenger RNAs and non-contiguous sequences in the 18 S ribosomal RNA is proposed. The small ribosomal RNA contains, at its 3'-terminus, a heavily conserved hairpin structure. It is suggested that mRNA 5'-leader sequence stabilises this structure by interacting with other conserved nucleotides which flank it. Sequences closely related to the required sequence (A-U-C-C-A-C-C) occur quite commonly in eukaryotic mRNAs and are often found immediately upstream from the AUG-codon. This interaction may have a role in the events which lead up to the initiation of protein synthesis.

cGMP phosphodiesterase-alpha mutation causes progressive retinal atrophy in the Cardigan Welsh corgi dog.

PURPOSE: To screen the alpha-subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6A) as a potential candidate gene for progressive retinal atrophy (PRA) in the Cardigan Welsh corgi dog. METHODS: Single-strand conformation polymorphism (SSCP) analysis was used to screen short introns of the canine PDE6A gene for informative polymorphisms in members of an extended pedigree of PRA-affected Cardigan Welsh corgis. After initial demonstration of linkage of a polymorphism in the PDE6A gene with the disease locus, the complete coding region of the PDE6A gene of a PRA-affected Cardigan Welsh corgi was cloned in overlapping fragments and sequenced. SSCP-based and direct DNA sequencing tests were developed to detect the presence of a PDE6A gene mutation that segregated with disease status in the extended pedigree of PRA-affected Cardigan Welsh corgis. Genomic DNA sequencing was developed as a diagnostic test to establish the genotype of Cardigan Welsh corgis in the pet population. RESULTS: A polymorphism within intron 18 of the canine PDE6A gene was invariably present in the homozygous state in PRA-affected Cardigan Welsh corgis. The entire PDE6A gene was cloned from one PRA-affected dog and the gene structure and intron sizes established and compared with those of an unaffected animal. Intron sizes were identical in affected and normal dogs. Sequencing of exons and splice junctions in the affected animal revealed a 1-bp deletion in codon 616. Analysis of PRA-affected anti obligate carrier Cardigan Welsh corgis showed that this mutation cosegregated with disease status. CONCLUSIONS: A single base deletion at codon 616 in the PDE6A gene cosegregated with PRA status with zero discordance in Cardigan Welsh corgis with PRA. A lod score of 4.816 with a recombination fraction (theta) of zero strongly suggests that this mutation is responsible for PRA in the breed. The mutation is predicted to lead to a frame shift resulting in a string of 28 altered codons followed by a premature stop codon. The authors suggest that this type of PRA be given the name rod-cone dysplasia 3 (rcd3).

Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester.

The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15 microliters of sample, is rapid, and is able to detect virus at titres below 10(3)/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection.

Expression of maedi-visna virus major core protein, p25: development of a sensitive p25 antigen detection assay.

The gene for the major core protein, p25, of maedi-visna virus (MVV) was cloned using a PCR (polymerase chain reaction) strategy employing primers designed for the insertion of the gene directly into yeast Ty-VLP expression vectors. In this system p25 is expressed as a fusion protein which self-assembles into virus-like particles (VLPs) due to interaction of the Ty A fusion partner. High levels (50-60 mg/l) of p25 fusion protein were produced, and p25 was recovered in soluble and highly pure form following cleavage from the Ty particle by Factor Xa protease digestion. The p25 protein produced in yeast is antigenically authentic, as defined by its reactivity with p25-specific antisera and its ability to elicit antibodies reactive with native viral p25 protein; although the cleaved, soluble form of p25 was found to be considerably more antigenic than the hybrid Ty-p25 VLP. Using this reagent anti-p25 monoclonal and polyclonal antibodies were generated. These sera and the p25 protein have been used to develop a sensitive MVV p25 detection assay. These reagents and assays will facilitate further studies of viral replication and immune response to the virus.

A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.

The use of flow cytometry to detect transfected gene products.

Flow cytometry and fluorescence-activated cell sorting (FACS) are techniques of great power used to screen cells rapidly for expression of particular gene products. These techniques have been of general utility in identifying and selecting populations of cells of defined characteristics from body fluids and other natural sources, More recently they have received extensive attention as methods for screening cell-surface expressed gene products in transfected cells. These methods rely on the indirect coupling of detector molecules, usually fluorochromes, to specific molecules on the target cells. This may occur through conjugation of the fluorochrome to the ligand of a receptor, or, as is more generally the case, through the use of fluorochromeconjugated antibodies specific for the transfected gene product. Cells displaying specific surface fluorescence following exposure to a flurochrome conjugate may subsequently be positively selected (or excluded) by FACS. Since cells are sorted individually, FACS is an ideal technique for picking up very rare events and for finding very minor subpopulations. In theory at least, the experimenter may recover a single cell of the desired phenotype from a relatively large population. However, the examination of single cells.

A temporal study of RNAs produced in maedi-visna virus infection of choroid plexus cells.

We have examined the types and cellular distribution of transcripts of maedi-visna virus during infection of choroid plexus fibroblasts. Early in infection (19 hours post infection) only small spliced transcripts are found in the cytoplasm. Little virus specific RNA is detected in the nucleus at this stage. Later in infection structural gene transcripts are detectable in the cytoplasm as well as the nucleus. We have measured the half life of each type of transcript in total cellular RNA late in infection by two different methods (northern blotting or S1 protection assays after alpha-amanitin treatment, or specific hybridisation after pulse chase). Both of these methods suggest half lives for unspliced viral RNAs of 8-12 hours. Although the gel based methods were unsuitable for the accurate determination of half life for the shorter transcripts, they did confirm a long half life for these transcripts: pulse chase measurements suggested that this was similar to that seen for the unspliced transcripts. These observations are consistent with the virus exerting temporal control on protein synthesis by a method analogous to that of the HIV Rev protein.

Identification of a region of the alcelaphine herpesvirus-1 genome associated with virulence for rabbits.

The gammaherpesvirus Alcelaphine Herpesvirus 1 (AHV-1) causes the fatal lymphoproliferative disease known as malignant catarrhal fever (MCF), in susceptible hosts. The virulent C500 isolate of AHV-1 became attenuated for the laboratory model, the rabbit, as a result of serial passage in cells of bovine origin. This work describes the identification of a region of the central unique sequence of the C500 genome, located close to the terminal repeat units of the molecule, which is altered on attenuation. The virulent C500 genome contains two copies of a sequence of approximately 2 kbp, contained within a 7 kbp region of the unique DNA located adjacent to the terminal repeats at the left end of the molecule. In the genome of the attenuated virus, there are also two copies of the 2 kbp sequence but they are located at the ends of the attenuated genome unique region, adjacent to the terminally repeated sequences. One open reading frame (ORF), designated putative polypeptide 5, was altered on attenuation such that the 3' sequence was lost. The location of this ORF, coupled with the loss of its 3' sequence, suggests that this ORF may encode a gene involved in the virulent mechanisms of this virus, in a manner similar to that of the transforming proteins of Herpesvirus saimiri (HSV).

The survival and growth of ovine afferent lymph dendritic cells in culture depends on tumour necrosis factor-alpha and is enhanced by granulocyte-macrophage colony-stimulating factor but inhibited by interferon-gamma.

An in vitro culture system is described which allows an analysis of the signals responsible for the survival, growth and functional maturation of afferent lymph dendritic cells (ALDC), a subpopulation of migrating dermal dendritic cells involved in antigen carriage and presentation to T-cells. Purified ALDC survived and grew for up to 30 days in lymph node conditioned medium and survived 14 days in recombinant ovine (rov) TNF-alpha whereas none were detected after 24 h in rov GM-CSF, rov IFN-gamma or rh M-CSF. However, when rov GM-CSF was added to cultures along with rov TNF-alpha, increased numbers of ALDC compared with input numbers (growth) were recorded on Days 14 and 21. In contrast, when 50-200 units ml-1 of rov IFN-gamma were added to cultures of ALDC along with TNF-alpha or rov TNF-alpha plus rov GM-CSF, cell survival and growth was inhibited. Antibody blocking studies confirmed the cytokine specificity of these effects. ALDC cultured in rov TNF-alpha or rov TNF-alpha plus rov GM-CSF retained MHC Class-II and ov CD-1 antigen expression and accessory function for autologous ov CD-4 T-cell proliferation, although at reduced levels compared with freshly isolated cells. Neither fresh nor cultured ALDC expressed coagulation factor XIIIa.

Confirmation of the rod cGMP phosphodiesterase beta subunit (PDE beta) nonsense mutation in affected rcd-1 Irish setters in the UK and development of a diagnostic test.

Rod/cone dysplasia type one (rcd-1) is an early onset inherited retinal dystrophy segregating in the Irish setter breed. It is classed as one of the autosomal recessive canine generalised Progressive Retinal Atrophies (PRA). The disease results in complete loss of photoreceptors by approximately one year of age. Levels of retinal cGMP are markedly elevated and of abnormal distribution in rod photoreceptors. Rod phosphodiesterase activity is absent and mRNA encoding the beta subunit (PDE beta) of the holoenzyme is uniquely reduced in predegenerate retinae. Using retinae from normal, unrelated adult dogs we have PCR-amplified and sequenced the cDNA for PDE beta. The cDNA is almost identical to that recently described for the Irish setter in the USA apart from two translationally silent single nucleotide changes. Using carrier and affected setters from a UK breeding colony we have screened genomic DNA and can confirm the G to A transition in rcd-1 affected dogs at position 2420, creating an amber mutation in codon 807. However, PRA-affected Tibetan terriers and miniature longhaired dachshunds are normal at this locus, underlining the genetic heterogeneity of this disease group. In addition we have developed a rapid, PCR-based diagnostic test for this mutation that will differentiate normal dogs from asymptomatic carriers.

Isolation of canine retinal arrestin cDNA and exclusion of three candidate genes for Swedish Briard retinal dystrophy.

PURPOSE: Mutations of genes encoding various retina-specific proteins are known to cause a wide spectrum of inherited retinal dystrophies in different species. In the canine, several types of genetic retinal dystrophies have been described affecting primarily the photoreceptors and/or the retinal pigment epithelium. We are performing a systematic analysis of canine candidate genes for such diseases to identify the one mutated in the retinal dystrophy in Swedish Briard dogs. METHODS: We isolated and characterised the full length cDNA of canine retinal arrestin by the method of rapid amplification of cDNA ends (RACE). RESULTS: The full length cDNA isolated by us is 1,575 base pairs (bp) long and contains a 1,218 bp-long open reading frame. CONCLUSIONS: The homology of the canine arrestin protein is highest with the human analogue (88.9%) and lowest with mouse arrestin (85.3%). The most obvious sequence differences among the different arrestins are in the extreme carboxyl terminus. PCR-SSCP (single strand conformation polymorphism) analysis and direct sequencing of retinal cDNA didn't provide any evidence that mutations in the canine arrestin gene are responsible for the retinal dystrophy seen in the Swedish strain of Briard dogs. Similar data were obtained for the genes encoding rhodopsin and the beta-subunit of photoreceptor-specific phosphodiesterase by segregation analysis.

Genomic screening for fucosidosis in English Springer Spaniels.

OBJECTIVE: To develop a robust molecular genetic test for alpha-L-fucosidosis in English Springer Spaniels and to screen dogs from the United Kingdom and United States for the mutant allele. ANIMALS: 35 English-bred English Springer Spaniels, 60 American-bred English Springer Spaniels, and 1 affected dog and its parents from a family of English Springer Spaniels in Colorado. PROCEDURE: Polymerase chain reaction analysis was used to amplify the mutated region in the gene encoding alpha-L-fucosidase. High guanine-cytosine (GC) content of the region required use of an amplification buffer with high pH. Mutant and normal alleles were separated by polyacrylamide gel electrophoresis. Molecular genetic test results were compared with enzyme data. RESULTS: A 262-bp PCR product was amplified from normal dogs and compared with a 248-bp product from affected dogs. Carriers had 1 copy of each allele, distinguishable by the 14-bp size difference. Two carriers among the English-bred dogs were identified by use of enzyme and genomic DNA analyses. The molecular defect in dogs from Colorado was proven to be the same as that in British and Australian dogs. None of the other 60 American-bred dogs carried the mutant allele. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR method that can be used to identify dogs affected with or carriers of the autosomal recessive disease fucosidosis was established. Amplification was achieved within a GC-rich region, using a method that may be useful in overcoming amplification problems in GC-rich areas within other genes. Using this test, fucosidosis can be controlled and ultimately eradicated from the English Springer Spaniel population.