Canine DNA Research Articles

Correction of Clinical Manifestations of Canine Mucopolysaccharidosis I with Neonatal Retroviral Vector Gene Therapy

Mucopolysaccharidosis I (MPS I) (Hurler syndrome) is due to deficient alpha-L-iduronidase (IDUA) activity and is the most common of the MPS disorders. Neonatal MPS I dogs were injected intravenously (IV) with a gamma retroviral vector containing a complete long-terminal repeat (LTR) and an internal human alpha(1)-antitrypsin (hAAT) promoter upstream of the canine IDUA complementary DNA (cDNA). This resulted in stable serum IDUA activity of 366 +/- 344 units (U)/ml (28-fold normal) for up to 1.8 years, which likely derived primarily from secretion of IDUA by transduced liver cells. Retroviral vector (RV)-treated dogs had >18% of normal IDUA activity in organs and had decreased severity and/or incidence of hernias, chest deformities, joint disease, facial dysmorphia, corneal clouding, valvular heart disease, and aortic dilatation as compared with untreated MPS I dogs. The marked reduction that was observed in lysosomal storage in the brain of RV-treated dogs may have been due in part to expression from the LTR of the vector in cells in the brain. This possibility will be explored in future studies, because the potential for insertional mutagenesis has raised concerns about using vectors with an intact LTR. If proven safe, this gene therapy technique may be utilized in treating children with Hurler syndrome.

Large-Scale SNP Genotyping with Canine Buccal Swab DNA

The dog is an attractive model for genetic studies of complex disease. With drafts of the canine genome complete, a large number of single-nucleotide polymorphisms (SNPs) that are potentially useful for gene-mapping studies and empirical estimations of canine diversity and linkage disequilibrium (LD) are now available. Unfortunately, most canine SNPs remain uncharacterized, and the amount and quality of DNA available from population-based samples are limited. We assessed how these real-world challenges influence automated SNP genotyping methods such as Illumina's GoldenGate assay. We examined 384 SNPs on canine chromosome 9 and successfully genotyped a minimum of 217 and a maximum of 275 SNPs using buccal swab samples for 181 dogs (86 beagles, 76 border collies, and 15 Australian shepherds). Call rates per SNP and sample averaged 97%, with reproducibility within and between analyses averaging 98%. The majority of these SNPs were polymorphic across all 3 breeds. We observed extensive LD, albeit less than reported for surveys using fewer dogs, consistent between breeds. Analyses of population substructure indicated that beagles are distinct from border collies and Australian shepherds. These results demonstrate the suitability of amplified canine buccal samples for high-throughput multiplex genotyping and confirm extensive LD in the dog.

CHIC—The Canine Health Information Center

The Canine Health Information Center (CHIC) is a centralized canine health database, jointly sponsored by the Orthopedic Foundation for Animals (OFA) and the AKC Canine Health Foundation (AKC CHF). The CHIC mission is to provide a source of health information for owners, breeders, and scientists that will assist in breeding healthier dogs. Dogs are issued CHIC numbers when there are tested in accordance with the health screening protocol established by their parent club. All CHIC results are available in an online publicly accessible database, so the results can be widely utilized in making more informed breeding decisions. CHIC also operates a DNA bank which stores canine DNA samples along with corresponding genealogic information and phenotypic health data. These samples are made available to the research community at large for canine health research purposes to facilitate the sample collection process and provide optimized family groups.

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 × 10 0 cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.

Molecular characterization of the canine mitochondrial DNA control region for forensic applications.

The canine mitochondrial DNA (mtDNA) control region of 133 dogs living in the area around Innsbruck, Austria was sequenced. A total of 40 polymorphic sites were observed in the first hypervariable segment and 15 in the second, which resulted in the differentiation of 40 distinct haplotypes. We observed five nucleotide positions that were highly polymorphic within different haplogroups, and they represent good candidates for mtDNA screening. We found five point heteroplasmic positions; all located in HVS-I and a polythymine region in HVS-II, the latter often being associated with length heteroplasmy. In contrast to human mtDNA, the canine control region contains a hypervariable 10 nucleotide repeat region, which is located between the two hypervariable regions. In our population sample, we observed eight different repeat types, which we characterized by direct sequencing and fragment length analysis. The discrimination power of the canine mtDNA control region was 0.93, not taking the polymorphic repeat region into consideration.

Greyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents

Greyhound meningoencephalitis is currently classified as a breed-associated idiopathic central nervous system inflammatory disorder. The non-suppurative inflammatory response can be distinguished from the other breed-associated disorders based on histopathology and lesion topography, however the nature of the response primarily suggests a viral infection. In the present study PCR and RT-PCR technologies were employed on frozen cerebral tissue from confirmed cases of meningoencephalitis to target specific viruses and protozoa likely to be implicated and to exclude the presence of bacterial 16SrRNA. Secondly, degenerate primers were used to detect viruses of the herpesvirus and flavivirus families. In addition cerebral tissues were probed for West Nile Virus. Viral nucleic acid sequences to Borna disease virus, to louping ill, tick borne encephalitis, West Nile and other flaviviruses were not detected. Canine distemper virus was detected in one animal with 97% homology to strain A75/15. Degenerate PCR for herpesviruses detected viral amplification products in one animal with 90% homology to canine herpesvirus DNA polymerase gene. Protozoal amplification products were only detected in a single dog with pathological confirmation of a combination of lesions of greyhound meningoencephalitis and a protozoal encephalomyelitis. Neospora was confirmed with sequence homology to Austrian strain 1. Bacterial 16SrRNA was not detected. The present study supports previous observations that many of the known microbial causes of canine meningoencephalitis are not involved. Findings could reflect that the causal agent was not specifically targeted for detection, or that the agent is at undetectable levels or has been eliminated from brain tissue. The potential roles of genetics and of molecular mimicry also cannot be discounted.

Genomic expression patterns of mitral valve tissues from dogs with degenerative mitral valve disease

To evaluate global genome expression patterns of mitral valve tissues from dogs with degenerative mitral valve disease (DMVD). Anterior mitral valve leaflets of 4 dogs with severe DMVD and 4 healthy control dogs. Transcriptional activities of 23,851 canine DNA sequences were determined by use of an oligonucleotide microarray. Genome expression patterns of tissue from dogs with DMVD were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and by comparing it with gene expression from healthy control dogs. 229 transcripts were differentially expressed (>or= 2-fold change). In dogs with DMVD, expression of 159 transcripts was upregulated and expression of 70 transcripts was downregulated. Of the 229 transcripts, 152 genes could be specifically identified. These genes were grouped into 1 of 9 categories on the basis of their primary physiologic function. Grouping revealed that pathways involving cell signaling, inflammation, extracellular matrix, immune function, cell defense, and metabolism were generally upregulated. Inflammatory cytokines and the serotonin-transforming growth factor-beta pathway were identified as contributory to the pathophysiologic aspects of DMVD. Evaluation of global expression patterns provides a molecular portrait of mitral valve disease, yields insight into the pathophysiologic aspects of DMVD, and identifies intriguing genes and pathways for further study.

Relevant aspects for forensic STR analysis of canine DNA: Repeat-based nomenclature and sensitive PCR multiplexes

Eight polymorphic canine STR markers were co-amplified in two newly designed PCR multiplex reactions (FH2087Us, FH2611, PEZ2, PEZ6, FH2328l, PEZ15, FH2054, WILMS-TFs). The sequence structure of selected alleles of these markers was the basis for the implementation of a repeat-based nomenclature. The PCR multiplexes amplify short amplicon lengths which makes the assay sensitive to the analysis of degraded DNA.

Cloning and characterization of canine thyroglobulin complementary DNA

Canine thyroglobulin (cTg) is one of the thyroid autoantigens associated with hypothyroidism caused by autoimmune thyroiditis in dog. To identify canine-specific areas in cTg, we cloned, by reverse transcriptase PCR, and sequenced the complete cDNA of cTg. It revealed an open reading frame of 8289 nucleotides, which encode a polypeptide of 2762 amino acids that is 78.9 and 78.1% identical to bovine and human thyroglobulin, respectively. This complete cTg sequence may be useful to promote the understanding of the primary structure of cTg and, it will be informative data in the further search about antigenic epitopes associated with autoimmune thyroiditis and pathogenesis of cTg-associated thyroid diseases in dog.

A Proposal for Standardization in Forensic Canine DNA Typing: Allele Nomenclature of Six Canine‐Specific STR Loci

In this study a proposal for the allele nomenclature of six polymorphic short tandem repeat (STR) loci (PEZ3, PEZ6, PEZ8, PEZ10, FHC2161, and FHC2328) for canine genotyping (Canis lupus familiaris) is presented. The nomenclature is based on the sequence data of the polymorphic region of the microsatellite markers as recommended by the DNA commission of the International Society of Forensic Haemogenetics (ISFH) in 1994 for human DNA typing. To cover commonly and rarely occurring alleles, a selection of homozygous and heterozygous animals were analyzed and subjected to sequence studies. The alleles consisted of simple tri- and tetra-nucleotide repeat patterns as well as compound and highly complex repeat patterns. Several alleles revealing the same fragment size but different repeat structures were found. The allele designation described here was adopted to the number of repeats, including all variable regions within the amplified fragment. In a second step the most commonly occurring alleles were added to an allelic ladder for each marker allowing a reliable typing of all alleles differing in size. A total number of 142 unrelated dogs from surrounding municipal animal homes, private households, and canines in police duty were analyzed. The data were added to a population database providing allele frequencies for each marker.

Selective enhancement of the type 1 cytokine response by expression of a canine interleukin (IL)-12 fused heterodimeric DNA

Interleukin-12 (IL-12) is a heterodimeric cytokine that is a principal mediator of the innate immune response and modulator of acquired cell-mediated immunity. Administration of exogenous IL-12 can direct the host adaptive T cell response toward a type 1 phenotype. The co-administration of IL-12 with vaccine antigens has been shown to augment the vaccine-induced T H1 response and protection against intracellular pathogens. We show here that a canine IL-12 DNA, constructed by fusing the p35 and p40 subunit cDNAs with an interspacing linker, generated stable IL-12 transcripts when placed under control of a strong constitutive promoter. The protein expressed from this fused cDNA was fully functional in promoting a type 1 (IFN-γ) and suppressing a type 2 (IL-4) cytokine response following both in vitro transfection of a canine cell line and in vivo delivery to dogs. This DNA construct may be useful as an adjuvant for vaccines that target tumors or intracellular pathogens of the dog.

Immunogenicity of a multicomponent DNA vaccine against visceral leishmaniasis in dogs

Vaccination of dogs, the domestic reservoir of Leishmania chagasi, could not only decrease the burden of canine visceral leishmaniasis (VL), but could also indirectly reduce the incidence of human VL. Intramuscular vaccination of foxhounds with a Leishmania multicomponent (10 antigen) DNA vaccine resulted in antigen-induced lymphoproliferative and IFN-γ (but not IL-4) responses. This response was not augmented by co-administration of canine IL-12 or GM-CSF DNA adjuvants. The multicomponent DNA vaccine also induced a delayed type hypersensitivity (DTH) response to viable L. donovani promastigotes and led to a reduction of parasite burden in an in vitro intracellular infection model, and in the draining lymph node of dogs early after cutaneous challenge. Thus, the multicomponent DNA vaccine was effective in priming dogs for a parasite-specific type 1 cellular immune response, which was able to restrict parasite growth.

Canine DNA Subjected to Whole Genome Amplification is Suitable for a Wide Range of Molecular Applications

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.

Fifty-Four New Gene-Based Canine Microsatellite Markers

Fifty-four new markers were developed to fill in gaps in the current map of canine microsatellites and to complement existing markers that may not be sufficiently informative in highly inbred canine pedigrees. Canine genes contained on the radiation hybrid map were used to obtain the sequence of the human homolog. A BLAST search versus the canine whole genome shotgun (wgs) sequence resource was used to obtain the sequence of the canine genomic contigs containing the homolog of the corresponding human gene. Canine sequences that contained microsatellites and mapped back to the correct location in the human genome were used to design primers for amplification of the microsatellites from canine genomic DNA. Heterozygosities of the markers were tested by genotyping grandparental DNAs obtained from the Nestle Purina Reference family DNA distribution center plus DNAs from unrelated Bouviers and Irish wolfhounds. Canine map positions of markers on the July 2004 freeze of the canine genome assembly were determined by in silico PCR or BLAST.

Liposome–DNA complexes infused intravenously inhibit tumor angiogenesis and elicit antitumor activity in dogs with soft tissue sarcoma

Intravenous gene delivery using liposome-DNA complexes (LDC) has previously been shown to elicit antitumor activity, but only in rodent tumor models. Therefore, we conducted a study to determine in a large animal spontaneous tumor model whether intravenous infusions of LDC could target gene expression to cutaneous tumor tissues and whether repeated treatments had an effect on tumor growth or angiogenesis. A total of 13 dogs with cutaneous soft tissue sarcomas were enrolled in the study and were randomized to receive a series of 6 weekly infusions of LDC containing either canine endostatin DNA or DNA encoding an irrelevant gene (luciferase). Serial tumor biopsies were obtained to assess transgene expression, tumor microvessel density (MVD), and intratumoral leukocyte inflammatory responses. We found that intravenous infusion of LDC did not result in detectable gene expression in cutaneous tumor tissues. However, two of 13 treated dogs had objective tumor responses and eight dogs had stable disease during the treatment period. In addition, a significant decrease in tumor MVD was noted in six of 12 treated dogs at the completion of six treatments. These results suggest that intravenous infusions of LDC may elicit nonspecific antitumor activity and inhibit tumor angiogenesis.

TP53 EXPRESSING SQUAMOUS CELL CARCINOMA OF THE TONSIL IN A CAPTIVE POLAR WOLF (CANIS LUPUS ARCTOS)

The gross and histopathologic findings for a primary tonsillar squamous cell carcinoma in a captive 11-yr-old male polar wolf (Canis lupus arctos) are described. The carcinoma had metastasized to regional lymph nodes of the pharynx, the precardial mediastinum, and the lungs. Tumor suppressor protein TP53 was detected by immunohistochemistry in the nuclei of poorly differentiated, cytokeratin-positive cells of the primary neoplasm and the metastases. Canine oral papillomavirus DNA was not detectable by polymerase chain reaction (PCR).

Polymerase Chain Reaction Screening for DNA Viruses in Paraffin‐Embedded Brains from Dogs with Necrotizing Meningoencephalitis, Necrotizing Leukoencephalitis, and Granulomatous Meningoencephalitis

The objective of this investigation was to determine whether or not herpesvirus (herpes‐), adenovirus (adeno‐), or canine parvovirus DNA is present in the brains of dogs with necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE), and granulomatous meningoencephalitis (GME). Paraffin‐embedded brain specimens from 12 histopathologically confirmed dogs with NME, 3 with NLE, and 7 with GME were screened for viral DNA with degenerate herpes‐ and adenovirus polymerase chain reaction (PCR) and a canine parvovirus‐specific PCR. Positive‐control specimens included genomic viral DNA and paraffin‐embedded tissues from dogs with confirmed herpes‐, adeno‐, or canine parvovirus infections. Herpes‐, adeno‐, or canine parvovirus DNA was amplified by PCR from the corresponding positive‐control specimens. Negative controls included 7 dogs with various brain disorders and produced no viral amplicons. The 22 dogs with NME, NLE, and GME were negative for viral DNA. Additional studies testing for other viruses or inherited genetic mutations are warranted to gain insight into the etiologies of NME, NLE, and GME. We discuss potential etiologies and provide a clinical and histopathologic overview of these common canine encephalitides.

Genomic expression patterns of cardiac tissues from dogs with dilated cardiomyopathy

To evaluate global genome expression patterns of left ventricular tissues from dogs with dilated cardiomyopathy (DCM). Tissues obtained from the left ventricle of 2 Doberman Pinschers with end-stage DCM and 5 healthy control dogs. Transcriptional activities of 23,851 canine DNA sequences were determined by use of an oligonucleotide microarray. Genome expression patterns of DCM tissue were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with gene expression for tissues from 5 healthy control dogs. 478 transcripts were differentially expressed (> or = 2.5-fold change). In DCM tissue, expression of 173 transcripts was upregulated and expression of 305 transcripts was downregulated, compared with expression for control tissues. Of the 478 transcripts, 167 genes could be specifically identified. These genes were grouped into 1 of 8 categories on the basis of their primary physiologic function. Grouping revealed that pathways involving cellular energy production, signaling and communication, and cell structure were generally downregulated, whereas pathways involving cellular defense and stress responses were upregulated. Many previously unreported genes that may contribute to the pathophysiologic aspects of heart disease were identified. Evaluation of global expression patterns provides a molecular portrait of heart failure, yields insights into the pathophysiologic aspects of DCM, and identifies intriguing genes and pathways for further study.

A PCR Multiplex and Database for Forensic DNA Identification of Dogs

Animal-derived trace evidence is a common finding at crime scenes and may provide an important link between victim(s) and suspect(s). A database of 558 dogs of pure and mixed breeds is described and analyzed with two PCR multiplexes of 17 microsatellites. Summary statistics (number of alleles, expected and observed heterozygosity and power of exclusion) are compared between breeds. Marked population substructure in dog breeds indicates significant inbreeding, and the use of a conservative theta value is recommended in likelihood calculations for determining the significance of a DNA match. Evidence is presented that the informativeness of the canine microsatellites, despite inbreeding, is comparable to the human CODIS loci. Two cases utilizing canine DNA typing, State of Washington v. Kenneth Leuluaialii and George Tuilefano and Crown v. Daniel McGowan, illustrate the potential of canine microsatellite markers for forensic investigations.

Extracellular Signal-regulated Kinases 1/2 Control Claudin-2 Expression in Madin-Darby Canine Kidney Strain I and II Cells

The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.