Canine DNA Research Articles

DNA methylation landscape of 16 canine somatic tissues by methylation-sensitive restriction enzyme-based next generation sequencing

DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps of 16 somatic tissues from two dogs. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (> 70%, 52.5–64.6% of all CpG sites analyzed) or unmethylated (< 30%, 22.5–28.0% of all CpG sites analyzed) which are methylation patterns similar to other species. The overall methylation status of CpG sites across the 32 methylomes were remarkably similar. However, the tissue types were clearly defined by principle component analysis and hierarchical clustering analysis with DNA methylome. We found 6416 CpG sites located closely at promoter region of genes and inverse correlation between DNA methylation and gene expression of these genes. Our study provides basic dataset for DNA methylation profiles in dogs.

The Common Bed Bug (Hemiptera: Cimicidae) Does Not Commonly Use Canines and Felines as a Host in Low-Income, High-Rise Apartments.

The common bed bug (Cimex lectularius L.) is a known pest and an obligate blood-feeding ectoparasite. Bed bugs can feed on warm-blooded animals including humans, bats, poultry, and rabbits, but no research has investigated the use of companion animals (canines and/or felines) as a blood source. This study investigates how long known host DNA could be detected in a bed bug and the prevalence of bed bugs feeding on companion animals. Laboratory-reared bed bugs were fed host blood to determine how long DNA of human, feline, canine, and rabbit blood could be detected up to 21 d postfeeding. Additionally, 228 bed bugs were collected from 12 apartments with pets (6: canine, 5: feline, and 1: canine and feline), characterized as engorged or unengorged, and then screened with host-specific primers to identify the bloodmeal. Host meals of human, rabbit, feline, and canine blood were detected up to 21 d after feeding laboratory strains. All bed bugs died after feeding on the canine blood, but DNA could be detected up to 21 d post feeding/death. Of the field-collected bed bugs analyzed, human DNA was amplified in 158 (69.3%) bed bugs, canine DNA amplified in 7 bed bugs (3.1%), and feline DNA amplified in 1 bed bug (0.4%). Results of this study suggest that bed bugs predominately feed on humans and rarely feed on companion animals when they cohabitate in low-income, high-rise apartments. Additionally, results from this study warrant future investigations into host use by bed bugs in different housing structures and socioeconomic environments.

Полиморфизм ДНК собак (Canis familiaris L.). III. VNTR- и STR-локусы. Их применение в собаководстве и криминалистике

The application of mini- and microsatellite polymorphisms of dog DNA, also referred to as VNTR- and STR-loci, respectively, in dog breeding and criminalistics is considered. Their use in dog breeding is shown to clarify pedigrees, establish paternity and purebred, as well as to differentiate breeds mainly in the form of microsatellite DNA polymorphism. In criminalistics, dogs can be both participants in crime scenes in the form of attacks on humans or pets, and some witnesses through whose DNA extracted from their fur or feces, by DNA identification of a particular dog, it may be help to get out to the perpetrator or at least to the crime scene, which is also a good help in its disclosure. At the same time, population studies of dogs, during which the prevalence of certain alleles of marker traits in the form of STR loci in different territories is established, contribute to making the right decisions. The databases on STR-polymorphism of dog DNA or their prototypes are briefly described. Attention is paid to the sources of forensic canine DNA, as well as methods of its extraction and preliminary evaluation of isolated preparations. The use of VNTR polymorphism was rather short-lived, and was quickly replaced by STR polymorphism. There are some trends in the introduction of new polymorphic traits in this area in the form of single-nucleotide polymorphism or SNPs, potentially providing more accurate information, including for DNA identification of individuals. The issues of universal DNA certification of dogs are discussed, which can contribute to improving the culture of keeping dogs and will allow the elimination of stray dogs in the future, which will be humanistic character and potentially reduce the number of aggressor dogs.

Полиморфизм ДНК собак (Canis familiaris) и его применение. IV. мтДНК

The structural organization of the mitochondrial genome of dogs is briefly considered, paying more attention to its variable areas, including the control region and its hypervariable parts HV1 and HV2, as well as a block of tandem decameric repeats and a homopolymer tract of cytosines and thymines. It is noted that the detected polymorphism of mitogenomes forming the clades with haplotypes, with few exceptions, are practically unrelated to dog breeds and their geographical place of residence. In general, the polymorphism of the mitochondrial DNA of dogs does not allow unambiguously identifying a specific individual and in criminalistics, in most cases, it is possible with one or another probability, depending on the variable areas taken into analysis, only to exclude suspected dogs that could lead to their owners from further investigation. Moreover, this requires the creation of population databases of canine mitogenomes, which can be used to calculate the probability of coincidence of haplotypes. Examples of individual cases of investigation of crimes in which polymorphism of DNA of dogs were used, including those that ended with a conviction, are given. Attention is drawn to the need for wider introduction into forensic practice of usage of canine DNA, which can obtained from traces left by dogs mainly in the form of saliva or wool (individual hairs).

Ribosomal DNA as target for the assessment of DNA degradation of human and canine DNA.

The assessment of DNA amount and DNA integrity can support forensic DNA analysis, in particular of problematic traces such as single telogen hairs where STR typing success is often hampered by low amounts and strong degradation of nuclear DNA. Common strategies consist of quantitative polymerase chain reaction (qPCR)-based analysis of the abundance of a short versus a long nuclear amplicon, the latter prone to DNA degradation. To increase sensitivity, commercial qPCR solutions rest on amplification of multi-copy DNA sequences. Here we show that ribosomal DNA (rDNA) sequences are well suited for the same purpose. Because rDNA sequences are present in high copy number in most eukaryotic species, qPCR strategies can easily be adapted to non-human species. In this paper, we establish qPCR-based assays for human or dog DNA, respectively, which allow for sensitive analysis of DNA amounts and DNA degradation. We show that the human system can be applied to DNA of single telogen hairs, where STR typing success correlates with measured amounts and integrity of the DNA. By adapting the system to dog rDNA sequences we found that single telogen dog hairs often displayed less DNA degradation than human telogen hairs, in most cases allowing for successful STR typing. Thus, qPCR-based analysis of rDNA represents a cost-effective, highly sensitive strategy to assess DNA amount and integrity that can be adapted to hairs or other traces from various animal species.

“No shit Sherlock”! Canine DNA and policing public space

PurposeThis paper argues that this animal surveillance has the potential for considerable function creep going far outside the scheme's original objectives and acts as a conduit for more problematic surveillance of humans. This results in social sorting of people with subsequent unforeseen consequences leading to discrimination and curtailment of freedoms for both animals and their owners. Ultimately this opens people up to further intrusive targeting by commercial interests and, more alarmingly, scrutiny from law enforcement agencies.Design/methodology/approachAn empirical study examining an initiative involving the collection of canine DNA sources data from publicly available Cabinet, Select Committee and Scrutiny Committee records from the London Borough of Barking and Dagenham (LBBD). It also draws on news media sources, publicity material from the company running the scheme and from this and other local authorities. Methods include analysis of documents, semiotic and discourse analysis.FindingsThis paper highlights the importance of animals to surveillance studies and examines the extent to which animals are a part of the surveillant assemblage in their own right. It also demonstrates how nonhuman animals extend the reach of the surveillant assemblage.Social implicationsThe scheme was called a badge of considerate dog ownership, yet it is one that can be franchised to tie up with diverse income streams being described as advantageous in the age of austerity. In 2017, it was reported that this scheme was to be rolled out in other areas and was moving from being voluntary to being mandatory with the enforcement of Public Space Protection Orders (PSPOs). These have been described as “geographically defined ASBOS” that have come into force under the Anti-social Behaviour and Policing Act (2014); they often work to criminalise activities that were not previously considered illegal.Originality/valueIn the theorising of surveillance, animals have been largely overlooked. Epidemiological studies proliferate, yet the role of animals in many aspects of everyday surveillance has been neglected.

Genome-wide DNA methylation analysis using MethylCap-seq in canine high-grade B-cell lymphoma.

DNA methylation is a comprehensively studied epigenetic modification and plays crucial roles in cancer development. In the present study, MethylCap-seq was used to characterize the genome-wide DNA methylation patterns in canine high-grade B-cell lymphoma (cHGBL). Canine methylated DNA fragments were captured and the MEDIUM-HIGH and LOW fraction of methylated DNA was obtained based on variation in CpG methylation density. In the MEDIUM-HIGH and LOW fraction, 2144 and 1987 cHGBL-specific hypermethylated genes, respectively, were identified. Functional analysis highlighted pathways strongly related to oncogenesis. The relevant signaling pathways associated with neuronal system were also revealed, echoing recent novel findings that neurogenesis plays key roles in tumor establishment. In addition, 14 genes were hypermethylated in all the cHGBL cases but not in the healthy dogs. These genes might be potential signatures for tracing cHGBL, and some of them have been reported to play roles in various types of cancers. Further, the distinct methylation pattern of cHGBL showed a concordance with the clinical outcome, suggesting that aberrant epigenetic changes may influence tumor behavior. In summary, our study characterized genome-wide DNA methylation patterns using MethylCap-seq in cHGBL; the findings suggest that specific DNA hypermethylation holds promise for dissecting tumorigenesis and uncovering biomarkers for monitoring the progression of cHGBL.

IL-18 immunoadjuvanted xenogeneic canine MMP-7 DNA vaccine overcomes immune tolerance and supresses the growth of murine mammary tumor

The development of the tumorigenesis and angiogenesis through proteolytic cleavage of extracellular matrix protein and basement membranes is promoted by Matrix metelloproteinases-7 (MMP-7). Consequently, MMP-7 is presumed as potential target for mammary cancer immunotherapy. However, MMP-7 is an endogenous tumor associated antigen (TAA); therefore, immunization is challenging. In current study, a potent anti-tumor immune response has been elicited through recombinant bivalent plasmid pVIVO2.IL18.cMMP7 which subside the highly metastatic 4 T1 cell line induced mammary tumors and efficiently negate the existing challenge of using MMP-7 as immunotherapeutic target. Balb/c mice were immunized with canine MMP-7 (cMMP-7) using interleukine-18 (IL-18), as an immunoadjuvant, to explore the potential of the combination regarding elicitation of a potent anti-tumor immune response. Mice vaccinated with pVIVO2.IL18.cMMP7 DNA plasmid reduced the tumor growth significantly along with augmentation of the immune response to fight against tumor antigen as depicted by substantial enrichment of CD4+ and CD8+ population in splenocytes, infiltration of immune system cells in tumor tissue and enhanced survival time of mice. Further, splenocyte supernatant examination of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were remarkably up-regulated demonstrating the stimulation of cell-mediated immune response. Thus the current observations vividly portray that administration of xenogeneic MMP-7 DNA vaccine bypasses the tolerance barrier.

Accelerated denaturation bubble-mediated strand exchange amplification for rapid and accurate detection of canine parvovirus.

Canine parvovirus (CPV), a strong infectious canine pathogen, has been recognized as a threat to canine health worldwide since the 1970s. Although convenient detection methods have been developed, such as the colloidal gold test strip, most of these methods are based on antibody detection, which is relatively ineffective for detecting pathogens during the incubation period. For institutions and businesses with many dogs, e.g., dog training centers and kennels, more sensitive detection methods are required to prevent the swift spread of CPV. Thus, we developed accelerated denaturation bubble-mediated strand exchange amplification (ASEA) for CPV detection, and it is a rapid, convenient, and cost-effective method. ASEA was able to distinguish CPV genomic DNA in a mixture that included canine genomic DNA as well as nucleic acids sourced from nine other common pathogens, with detection of target DNA as low as 8.0 × 10-18 M within 16.6 min. Coupled with the thermal lysis method modified by us that only requires 3 min to perform, the entire detection procedure can be completed within approximately 20 min and only requires a simple heating block and an ordinary fluorescence PCR instrument. Moreover, ASEA exhibited greater sensitivity than colloidal gold test strips in actual specimen detection. This technique is rapid, easy to perform, and highly sensitive, and therefore, this approach has the potential to rapidly detect CPV in institutions with large populations of dogs.

Identification of p53 gene alterations in canine mammary tumours using polymerase chain reaction and direct sequence analysis

Mammary tumours are mentioned as the most common tumours in female dogs and approximately half of them are detected malignant. p53 gene mutations are demonstrated to be the most common genetic alteration in canine mammary tumours. The present study was conducted to evaluate exon-1 of p53 gene mutations in tissue samples of canine mammary tumours by PCR and direct sequence analysis. After histopathological confirmation of the tissue sections by haematoxylin and eosin staining (10/26), deparaffinised samples were used for DNA extraction by silica gel method. Subsequently, p53 exon 1 was amplified through PCR assay using specific oligo nucleotide primers designed according to the canine DNA sequence available online. Microscopically, 10 out of 26 suspected tissue samples were recognised as malignant mammary gland tumours with various grades of malignancy. Surprisingly, one insertion of mutation was found in exon 1 of all examined samples corresponding to a sequence comprising 27 amino acids, between amino acids 30 to 57 in the p53 protein. Taken together, it seems that alteration of exon 1 p53 gene may lead to malignancy beha­viour, poor prognosis and short survival time in dogs with mammary carcinomas.

FVIII Protein Is Not Detectable in Human PBMCs or Livers from Dogs with an Intron-22 Inversion Mutation: Implications for FVIII Immunogenicity and Tolerance

A clinically relevant question is whether partial FVIII proteins are expressed in tissues from patients with severe hemophilia A (HA) due to an intron-22 inversion mutation. If so, this could in principle confer central immune tolerance to the expressed FVIII regions, thereby lowering the risk of anti-FVIII immune responses. In 2013, Pandey et al. reported detection of FVIII proteins in peripheral blood mononuclear cells (PBMCs), B cells and liver tissues from an intron-22 inversion subject and non-HA controls. They concluded that partial FVIII proteins were translated from inverted mRNA encoding F8 exons 1-22, and also from the F8B transcript, which contains F8 exons 23-26 (Nature Medicine 19(10), 1318-24). In 2014, the Montgomery and Ginsburg labs reported that FVIII protein is expressed principally, and possibly exclusively, in endothelial cells (ECs). These studies utilized mouse models with EC-directed deletion of F8 exons 17-18, or of the FVIII transporter protein LMAN1, respectively. The F8-EC-knockout mice had a severe hemophilia A (HA) phenotype. Earlier studies from several labs, including Pandey et al., relying primarily on antibody staining, had reported FVIII expression in additional cell types, including PBMCs and hepatocytes. The present study tests the hypothesis that partial FVIII proteins are expressed from inverted mRNA encoding F8 exons 1-22 and/or from F8B mRNA. A panel of FVIII-specific polyclonal and monoclonal antibodies (mAbs) was tested for FVIII specificity by staining permeabilized PBMCs, monocytes, T cells, blood outgrowth ECs, monocyte-derived macrophages and dendritic cells, HUVECs and B-cell lines. Positive and negative controls included FVIII-expressing BHK cells and non-engineered BHK cells. Specificity was further tested by immunoprecipitation (IP) of cell lysates followed by SDS-PAGE, Western blots and mass spectrometry to define proteins pulled down by the anti-FVIII antibodies. In addition, immunohistochemistry (IHC) staining was carried out for liver sections from HA dogs with an intron-22 inversion mutation and from otherwise normal (non-HA) control dogs. IP followed by SDS-PAGE, Westerns and/or mass spec revealed that a significant fraction of the anti-FVIII antibodies bound to other proteins besides FVIII in the tissues and cells examined. A cocktail of 4 anti-FVIII mAbs that were validated using (+) and (-) control samples was used to stain isolated and cultured cells and tissues. Cross-recognition of canine FVIII was confirmed by IP + Western blots. IHC using the mAb cocktail followed by HRP-conjugated secondary antibodies produced variable staining of liver tissues from HA and non-HA dogs, regardless of tissue preparation methods. However, use of fluorescent-labeled secondary mAbs produced signals well above background in ECs of control dog livers but not in the HA livers. Multiple confocal images were selected randomly, and average MFI values of 10 non-HA liver sections were well above those of 10 HA sections (p = 0.006). A second staining method, the Duolink Proximity Ligation Assay, was employed as an independent test to detect interactions between FVIII and VWF. Again, only normal control liver sections showed fluorescence above background (p =0.0004). F8B mRNA was not detected in canine liver and was not expected based on the canine intron-22 DNA sequence. We conclude that if protein is expressed from inverted mRNA encoding F8 exons 1-22, it is below the detection limit of these assays. Permeabilized human cells were tested for FVIII expression by staining using the mAb cocktail, or validated anti-FVIII-C2 mAbs to detect the putative protein encoded by F8B, followed by fluorescent detection. Cells were also stimulated with histamine followed by a chromogenic FVIII activity assay of supernatants. Results indicated that if FVIII or F8B protein are expressed in non-ECs, they are below the detection limit of these assays. We also note that if the putative F8B-encoded protein confers tolerance, immune responses of HA patients to the FVIII C2 domain would be quite rare, which is not the case. Although neonatal thymic expression of partial FVIII proteins may occur, immune tolerance to self-antigens requires repeated exposures of the immune system to the antigens. We conclude it is unlikely that HA patients with an intron-22 inversion mutation have universally acquired central tolerance to partial proteins encoded by inverted F8 mRNA or F8B. Disclosures Ragni: Shire/Takeda: Consultancy, Other: Study drug; Sangamo: Research Funding; OPKO: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding; Bayer: Consultancy; ICER: Consultancy; Biomarin: Consultancy, Research Funding; Alnylam/Sanofi: Consultancy, Research Funding; Spark Therapeutics: Consultancy, Research Funding. Pratt:Bloodworks NW: Patents &amp; Royalties: inventor on patents related to FVIII immunogenicity; Grifols, Inc: Research Funding.

Detection of canine parvovirus types 2b and 2c in canine faecal samples contaminating urban thoroughfares in Brazil.

Canine parvovirus type 2 (CPV-2) is a highly contagious virus that causes acute gastroenteritis in dogs all over the world. Because of its stability in the environment, CPV-2 can remain infective for a long time, especially if protected in organic matter. To demonstrate CPV-2's potential as an environmental hazard for nonimmunized susceptible hosts, we investigated 50 faecal samples collected from public areas in a municipality of Paraná state, Brazil. Seven samples tested positive for CPV by a PCR assay targeting the partial VP2 gene, with three strains being confirmed as CPV-2b variant and one as CPV-2c variant by sequence analysis. These findings were supported by phylogenetic analysis, and the species identity of faecal samples source was confirmed by canine mitochondrial DNA amplification and sequencing. Our results demonstrate the presence of CPV in canine faeces contaminating urban thoroughfares and reinforce the importance of environmental control to reduce the potential exposure risks to susceptible hosts.

Forensic characterization and statistical considerations of the CaDNAP 13-STR panel in 1,184 domestic dogs from Germany, Austria, and Switzerland

Crime scene samples originating from domestic dogs such as hair, blood, or saliva can be probative as possible transfer evidence in human crime and in dog attack cases. In the majority of such cases canine DNA identification using short tandem repeat (STR) analysis is the method of choice, which demands, among others, a systematic survey of allele frequency data in the relevant dog populations. A set of 13 highly polymorphic canine STR markers was used to analyze samples of 1,184 dogs (including 967 purebred dogs) from the so-called DACH countries (Germany, Austria, Switzerland). This CaDNAP 13-STR panel has previously been validated for canine identification in a forensic context. Here, we present robust estimates of allele frequencies, which are essential to assess the weight of the evidence by estimating the probability of a matching DNA profile within the dog population under question, e.g. in the form of a random match probability (RMP). The geographical provenance of the tested dogs showed a negligible influence on the observed genotype variation. Therefore, we combined the STR data from all three countries into a single dog population sample (DPS). In contrast, pronounced genetic differentiation between dog breeds was found by principal component analysis and sub-structure analysis with the STRUCTURE software. These findings entailed the need to account for the effects of DPS breed composition on allele frequency estimates. A possible strategy, which was favored here, relies on collecting a DPS that is guided by the breed composition of the relevant dog population. In total, dogs from 166 different breeds were included in our DPS, 64 of them including at least 5 individuals (n = 771 dogs). Sampling reflected the abundance of breeds in the DACH countries with the following being the most common ones: German Shepherds (population frequency: 14.3%), Dachshunds (5.9%), Labrador Retrievers (3.9%), and Golden Retrievers (3.2%). The pedigree listing of the purebred dogs in our DPS ranked German Shepherds (DPS frequency 8.5%) first, followed by Labrador Retrievers (3.9%), Golden Retrievers (3%), and Dachshunds (2.5%). RMP values based on overall allele frequencies and accounting for substructure using FST between breeds ranged between 10-13 and 10-14 and represent a conservative approach of RMP assessment.

A DNA telomerase vaccine for canine cancer immunotherapy.

Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cells and low to absent in normal cells. Given that immune tolerance to telomerase is easily broken both naturally and experimentally, telomerase is an attractive tumor associated antigen for cancer immunotherapy. Indeed, therapeutic trials using human telomerase peptides have been performed. We have developed an immunogenic yet catalytically inactive human telomerase DNA construct that is in clinical trials with patients presenting solid tumors. Paralleling this human construct, we have developed a canine telomerase DNA vaccine, called pDUV5. When administered intradermally to mice combined with electrogene transfer, pDUV5 induced canine TERT specific cytotoxic T-cells as measured by IFN-γ ELISpot assay. Intradermal vaccination of healthy dogs with 400 μg of pDUV5 generated strong, broad and long lasting TERT specific cellular immune responses. In vitro immunization with cTERT peptides revealed the maintenance of cTERT specific T-cells in PBMCs from tumor bearing dogs showing that this repertoire was not depleted. This study highlights the potential of pDUV5 as a cancer vaccine and supports its evaluation for the treatment of spontaneous canine tumors.

Novel Parvovirus Related to Primate Bufaviruses in Dogs

A novel protoparvovirus species, related genetically to human bufaviruses, was identified in dogs with respiratory signs. The canine bufavirus was distantly related to the well-known canine protoparvovirus, canine parvovirus type 2, sharing low amino acid identities in the nonstructural protein 1 (40.6%) and in the capsid protein 1 (33.4%). By screening collections of fecal, nasal, and oropharyngeal samples obtained from juvenile dogs (<1 year of age), canine bufavirus DNA appeared as a common component of canine virome. The virus was common in the stool samples of dogs with or without enteric disease and in the nasal and oropharyngeal swab samples of dogs with respiratory signs. However, the virus was not detected in nasal and oropharyngeal swab samples from animals without clinical signs.

Development of a tick-borne pathogen QPCR panel for detection of Anaplasma, Ehrlichia, Rickettsia, and Lyme disease Borrelia in animals

Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Lyme disease associated Borrelia spp. are the most common tick-borne pathogens reported to infect human beings worldwide and other animals, such as dogs and horses. In the present study, we developed a broad-coverage SYBR Green QPCR panel consisting of four individual assays for the detection and partial differentiation of the aforementioned pathogens. All assays were optimized to the same thermocycling condition and had a detection limit of 10 copies per reaction. The assays remained sensitive when used to test canine and equine blood DNA samples spiked with known amounts of synthetic DNA (gBlock) control template. The assays were specific, as evidenced by lack of cross reaction to non-target gBlock or other pathogens commonly tested in veterinary diagnostic labs. With appropriate Ct cutoff values for positive samples and negative controls and the melting temperature (TM) ranges established in the present study, the QPCR panel is suitable for accurate, convenient and rapid screening and confirmation of tick-borne pathogens in animals.

A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs

Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format has many advantages. By joining two qPCR protocols into one, more results can be obtained in the same amount of time with reduced costs and embedded quality control. Reagents are preloaded and stored on the plate, reducing the operator’s hands-on time to set up a reaction, as well as decreasing manipulation steps, which reduces the risk of mistakes or contamination. Thus, the ready-to-use duplex format turns qPCR into a robust, easy-to-use tool, which could help increase the availability of qPCR for CVL diagnosis.

GENETIC CHARACTERIZATION OF CANINE PARVOVIRUS IN SYMPATRIC FREE-RANGING WILD CARNIVORES IN PORTUGAL.

Since its emergence in the 1970s, canine parvovirus (CPV) has been reported in domestic and nondomestic carnivores worldwide with severe implications on their health and survival. Here, we aim to better understand CPV circulation in multihost-pathogens systems by characterizing CPV DNA or viruses in 227 free-ranging wild carnivores of 12 species from Portugal. Collected samples during 1995-2011 were analyzed by PCR and sequence analysis. The canine parvovirus DNA was detected in 4 (2%) animals of two species, namely in wolves (Canis lupus; 3/63, 5%, 95% confidence interval=1.6-3.15) and in a stone marten (Martes foina; 1/36, 3%, 95% confidence interval=0.5-14.2). Viruses in two wolves had VP2 residue 426 as aspartic acid (so-called CPV-2b) and the third had VP2 residue 426 as asparagine (CPV-2a), while the virus in the stone marten uniquely had VP2 residue 426 as glutamic acid (CPV-2c). The comparative analysis of the full-length VP2 gene of our isolates showed other nonsynonymous mutations. The phylogenetic analysis demonstrated that the sequences from wolves clustered together, showing a close relationship with European domestic dogs (Canis lupus familiaris) and wolf strains while the viral sequence from the stone marten grouped with other viruses contained the glutamic acid VP2 426 along with raccoon (Procyon lotor), bobcat (Lynx rufus), and domestic dog strains. This study confirmed that wild carnivores in Portugal are infected by CPV variants, strongly suggesting viral transmission between the wild and domestic populations and suggesting a need for a better understanding of the epidemiology of the disease and its management in wild populations.

Canis mtDNA HV1 database: a web-based tool for collecting and surveying Canis mtDNA HV1 haplotype in public database

BackgroundCanine and wolf mitochondrial DNA haplotypes, which can be used for forensic or phylogenetic analyses, have been defined in various schemes depending on the region analyzed. In recent studies, the 582 bp fragment of the HV1 region is most commonly used. 317 different canine HV1 haplotypes have been reported in the rapidly growing public database GenBank. These reported haplotypes contain several inconsistencies in their haplotype information. To overcome this issue, we have developed a Canis mtDNA HV1 database. This database collects data on the HV1 582 bp region in dog mitochondrial DNA from the GenBank to screen and correct the inconsistencies. It also supports users in detection of new novel mutation profiles and assignment of new haplotypes.DescriptionThe Canis mtDNA HV1 database (CHD) contains 5567 nucleotide entries originating from 15 subspecies in the species Canis lupus. Of these entries, 3646 were haplotypes and grouped into 804 distinct sequences. 319 sequences were recognized as previously assigned haplotypes, while the remaining 485 sequences had new mutation profiles and were marked as new haplotype candidates awaiting further analysis for haplotype assignment. Of the 3646 nucleotide entries, only 414 were annotated with correct haplotype information, while 3232 had insufficient or lacked haplotype information and were corrected or modified before storing in the CHD.The CHD can be accessed at http://chd.vnbiology.com. It provides sequences, haplotype information, and a web-based tool for mtDNA HV1 haplotyping. The CHD is updated monthly and supplies all data for download.ConclusionsThe Canis mtDNA HV1 database contains information about canine mitochondrial DNA HV1 sequences with reconciled annotation. It serves as a tool for detection of inconsistencies in GenBank and helps identifying new HV1 haplotypes. Thus, it supports the scientific community in naming new HV1 haplotypes and to reconcile existing annotation of HV1 582 bp sequences.

Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA.

Canine parvovirus-2 (CPV-2), which is ubiquitously distributed worldwide, causes severe and often fatal gastroenteritis in dogs. Accurate, differential and rapid diagnosis of canine parvoviral enteritis remains a challenge for clinicians. A recently developed isothermal amplification technique, polymerase spiral reaction (PSR), was optimized for the first time for a viral pathogen with reference recombinant plasmid standards from different CPV-2 antigenic variants (CPV-2, CPV-2a, CPV-2b and CPV-2c) and subsequently validated using clinical samples. Addition of chromogenic substrate SYBR Green I after the completion of the reaction resulted in bright green fluorescence in positive samples, while negative samples and a no-template control remained orange. These results were further substantiated through visualization of a laddering pattern of the PSR-amplified product in an agarose gel in positive cases and the absence of this pattern in no-template control and negative samples. The PSR assay was found to be highly specific, as it did not react with other putative canine pathogens (canine adenovirus 1 and canine distemper virus). The sensitivity of the newly developed PSR technique was compared with that of conventional PCR, real-time PCR and LAMP, using a serial tenfold dilution of canine parvovirus DNA. The detection limit of PSR was found to be at the femtogram level, which is comparable with that of real-time PCR and LAMP, which are ten times more sensitive than conventional PCR. The assay was validated using 90 clinical samples, of which 54 were found positive, while only 45 samples were positive in conventional PCR. This novel assay, which is fully compliant with the 'ASSURED' concept for disease diagnosis, provides a simple, rapid, specific, sensitive and cost-effective method for diagnosis of canine parvoviral enteritis in veterinary clinics.