Canine DNA Research Articles

Comparative analysis of whole exome sequencing kits for the canine genome.

As the public's interest in companion dogs grows, health issues in these animals are also emerging, necessitating the optimization of whole exome sequencing (WES) as a valuable method for disease prediction. While WES targeting the human genome is well established, WES targeting the canine genome is understudied, and there is a need to find effective analysis kits. We compared and analyzed the performance of three WES kits from Twist and Agilent using the canine genome as the target to perform genetic analysis of canine diseases effectively. The levels of total reads, the duplication rate, and variant calling in canine genomic DNA samples from seven healthy dogs (three beagles, one bichon fry, one maltese, one welsh corgi, and one mixed breed) without any interventions were examined through WES via Twist and Agilent kits. We found that while Twist had the lowest total read number, the number of reads in the SSXT series was significantly (P<0.05) greater. Twist showed low evenness and high standard deviation, but the SSXT series showed relatively high evenness. Compared with Twist, the SSXT series from a depth of 30× presented a significantly (P<0.05) greater target ratio. Among the four kits, the significantly lowest duplicate ratio was confirmed for SSXT (O/N) (30% lower than Twist). The most important performance of the kit, the number of variants detected, was 48,302 for Twist and 130,506 for SSXT (O/N). On the basis of the performance comparison results, SSXT (O/N) was found to have the best performance.

The canine DNA recovery project: preventing livestock injuries and fatalities

A new initiative aims to improve the accuracy and reliability of identifying dogs involved in livestock attacks using a novel DNA collection and analysis approach.

Development of a qPCR assay for the quantification of canine autosomal DNA recovered from livestock attacks

The absence of a standardised method to quantify canine DNA recovered from livestock attacks leaves forensic providers without an important quality control step to help support their decision making. Typically used to normalise the amount of DNA for STR amplification, modern forensic DNA quantification approaches use qPCR of target genes and can also include an Internal Positive Controls (IPC) to determine the presence of PCR inhibitors. The co-amplification of livestock DNA alongside canine DNA has meant that previously developed qPCR methods are not suitable for use so a standardised approach is needed. This research describes the development of a Taq-man multiplex qPCR assay that simultaneously quantifies the autosomal MC1R and Y-specific SRY gene to determine the concentration of canine DNA recovered from attacked livestock. Data suggests that the method is robust and reproducible with no significant difference in the standard curves produced from multiple runs or from different DNA standards derived from different canines. Assay sensitivity of between 15 and 31 pg is consistent with other forensic quantification assays and also in line with the sensitivity of the two tested canine STR kits, Canine Genotype 2.1 Kit and CaDNAP Panels 1 and 2. The assay is highly specific to canines when tested against 163 different dogs representing 33 different breeds and no cross-amplification of non-target species’ DNA was observed even from livestock DNA tested at 31.25ng/µl. This strongly suggests that any DNA detected on evidence collected from attacked livestock is canine. The assay also shows robust tolerance to common livestock inhibitors continuing to amplify when inhibitor-spiked DNA samples were tested. Both mixed and inhibited DNA samples underwent STR typing using two canine forensic STR kits with data showing the Canine Genotype 2.1 Kit displaying pronounced cross-amplification of livestock DNA and and/or extensive PCR inhibition leading to the complete loss of amplification when using this kit. Conversely the CaDNAP Panels 1 and 2 showed little cross-amplification of livestock DNA and improved inhibitor tolerance suggesting that this approach was better suited for the analysis of livestock attack samples. Findings are discussed and the impact of the observations on future work in this area are explored.

Development and validation of a novel 30-plex STR assay for canine individual identification and parentage testing

Domestic dogs are helpers in outdoor human work and companions for families; thus, individual canine identification and parentage testing are crucial in certain fields, including forensics and breeding programs. In this study, a six-dye fluorescent labeling multiplex amplification system containing 29 canine short tandem repeats (STRs) and the sex-determining marker DAmel was developed. The system was called the Tronfo Canine 30-plex STR Kit and was further validated according to the Scientific Working Group on DNA Analysis Methods and the Organization of Scientific Area Committees for Wildlife Forensics guidelines, including tests for PCR conditions, precision, species specificity, sensitivity, stability, repeatability and reproducibility, a population study, and a study of 16 paternity test cases. The results indicated that the novel canine STR assay was accurate, specific, reproducible, stable, and robust. Complete profiles were obtained with 31.25 pg of canine DNA. Additionally, 500 unrelated canine individuals were investigated using this novel system, and the combined power of discrimination and exclusion values were 0.999999999999999999 and 0.999996451039850, respectively. These results suggest that the Tronfo Canine 30-plex STR Kit is highly polymorphic, informative, and suitable for individual canine identification and parentage testing.

A novel loop-mediated isothermal amplification (LAMP) assay to diagnose feline panleukopenia.

Protoparvovirus carnivoran1, known commonly as feline panleukopenia virus (FPV) is a highly contagious and environmentally stable parvovirus of domestic as well as wild felids. A rapid and robust diagnostic tool will aid in implementing prompt treatment and control measures. A loop-mediated isothermal amplification (LAMP) as a point-of-care diagnostic tool for diagnosing feline panleukopenia was standardised using faecal samples of cats. The assay will reduce the cost and time required to diagnose feline panleukopenia. A set of two outer primers (F3 and B3) and two inner primers (FIP and BIP) were designed to target the viral polypeptide (VP2) gene of FPV. Optimisation of the LAMP reaction was done at 60 °C for one hour after an initial denaturation at 95 °C for five minutes. Visualisation of the result based on the addition of SYBR Green 1 dye offered an easy and reliable diagnosis. The detection limit of the standardised LAMP assay was as low as 1.25 ng/μl of the target DNA. Species specificity of the LAMP primers revealed no amplification of the non-target DNA of any other species except that of the canine parvovirus DNA template. DNA extracted from 100 PCR-positive and 20 PCR-negative faecal samples were subjected to the standardised assay and compared with PCR. Analysis of the results revealed that the LAMP assay was 100% sensitive and 90% specific compared to PCR. The LAMP assay could be a reliable tool for the point-of-care diagnosis of feline panleukopenia in limited resource settings.

Assessment of Y chromosome copy number alterations in non-neoplastic and neoplastic leukocytes of male dogs

The loss of the Y chromosome (ChrY), also known as LOY, is a common genetic alteration observed in men. It occurs in non-neoplastic cells as an age-related change as well as in neoplastic cells of various cancer types. While well-documented in humans, LOY has not been extensively studied in non-human mammals. In this study, we developed simple digital PCR-based assays to assess the copy number of ChrY relative to the X chromosome (ChrX) and chromosome 8 (Chr8) to evaluate ChrY numerical alterations in male canine DNA specimens. Using these assays, we analyzed non-neoplastic leukocytes from 162 male dogs without hematopoietic neoplasia to investigate the occurrence of age-related LOY in non-neoplastic leukocytes. Additionally, we examined 101 tumor DNA specimens obtained from male dogs diagnosed with various types of lymphoma and leukemia to determine whether copy number alterations of the ChrY occur in canine hematopoietic cancers. Analysis of the 162 non-neoplastic leukocyte DNA specimens from male dogs of varying ages revealed a consistent ∼1:1 ChrY:ChrX ratio. This suggests that age-related LOY in non-neoplastic leukocytes is rare or absent in dogs. Conversely, a decreased or increased ChrY:ChrX ratio was detected in canine neoplastic leukocytes at varying frequencies across different canine hematopoietic malignancies (P = 0.01, Fisher's exact test). Notably, a higher incidence of LOY was observed in more aggressive cancer types. To determine if this relative LOY to ChrX was caused by changes in ChrY or ChrX, we further analyzed their relative copy numbers using Chr8 as a reference. Loss of ChrX relative to Chr8 was found in 21% (9/41) of B-cell lymphomas and 6% (1/18) of non-T-zone/high-grade T-cell lymphomas. In contrast, a subset (29%, 4/14) of T-cell chronic lymphocytic leukemia showed gain of ChrX relative to Chr8. Notably, no relative LOY to Chr8 was detected indolent hematopoietic cancers such as T-zone lymphoma (0/9) and chronic lymphocytic leukemia of B-cell (0/11) and T-cell origins (0/14). However, relative LOY to Chr8 was present in more aggressive canine hematopoietic cancers, with incidences of 24% (10/41) in B-cell lymphoma, 44% (8/18) in non-T-zone/high-grade T-cell lymphoma, and 75% (6/8) in acute leukemia. This study highlights both similarities and differences in LOY between human and canine non-neoplastic and neoplastic leukocytes. It underscores the need for further research into the role of ChrY in canine health and disease, as well as the significance of LOY across various species.

A cautionary tale of low-pass sequencing and imputation with respect to haplotype accuracy

BackgroundLow-pass whole-genome sequencing and imputation offer significant cost savings, enabling substantial increases in sample size and statistical power. This approach is particularly promising in livestock breeding, providing an affordable means of screening individuals for deleterious alleles or calculating genomic breeding values. Consequently, it may also be of value in companion animal genomics to support pedigree breeding. We sought to evaluate in dogs the impact of low coverage sequencing and reference-guided imputation on genotype concordance and association analyses.ResultsDNA isolated from saliva of 30 Labrador retrievers was sequenced at low (0.9X and 3.8X) and high (43.5X) coverage, and down-sampled from 43.5X to 9.6X and 17.4X. Genotype imputation was performed using a diverse reference panel (1021 dogs), and two subsets of the former panel (256 dogs each) where one had an excess of Labrador retrievers relative to other breeds. We observed little difference in imputed genotype concordance between reference panels. Association analyses for a locus acting as a disease proxy were performed using single-marker (GEMMA) and haplotype-based (XP-EHH) tests. GEMMA results were highly correlated (r ≥ 0.97) between 43.5X and ≥ 3.8X depths of coverage, while for 0.9X the correlation was lower (r ≤ 0.8). XP-EHH results were less well correlated, with r ranging from 0.58 (0.9X) to 0.88 (17.4X). Across a random sample of 10,000 genomic regions averaging 17 kb in size, we observed a median of three haplotypes per dog across the sequencing depths, with 5% of the regions returning more than eight haplotypes. Inspection of one such region revealed genotype and phasing inconsistencies across sequencing depths.ConclusionsWe demonstrate that saliva-derived canine DNA is suitable for whole-genome sequencing, highlighting the feasibility of client-based sampling. Low-pass sequencing and imputation require caution as incorrect allele assignments result when the subject possesses alleles that are absent in the reference panel. Larger panels have the capacity for greater allelic diversity, which should reduce the potential for imputation error. Although low-pass sequencing can accurately impute allele dosage, we highlight issues with phasing accuracy that impact haplotype-based analyses. Consequently, if accurately phased genotypes are required for analyses, we advocate sequencing at high depth (> 20X).

The employment of real-time polymerase chain reaction for analysis of canine meat in meatball products for halal authentication analysis.

Meatballs are a popular meat-based food consumed widely in Indonesian society. However, the issue of unethical substitution of halal meatballs with non-halal meats, particularly pork and canine meat (CM), has emerged. The existence of non-halal meats, including CM, in food products is prohibited in Islam, necessitating the development of reliable analytical techniques for their identification. In this study, we designed species-specific primers (SSPs) targeting the D-loop region of mitochondrial DNA for CM meatball product identification. The study was commenced by creating specific primers for canine DNA using Integrated DNA Technologies software and subsequently performing DNA isolation. The designed primers were then subjected to comprehensive evaluation using RT-PCR, including specification, linearity, limit of detection, efficiency, and repeatability. The results indicated that the primer D-Loop 443 (forward: 5'-GGG ACA TCT CGA TGG ACTA ATG-3', reverse: 5'-GCG GTC ATA GAT GAG TGA TAG C-3') designed and validated in silico using primer-basic local alignment search tool nucleotide (BLAST) program from NCBI accurately identified canine DNA when the optimal annealing temperature was set at 57.5oC. The real-time PCR technique utilizing the D-loop 443 primer exhibited the ability to amplify canine DNA down to a minimum quantity of 100 pg, with an efficiency value of 91.8%, a correlation coefficient (R) of 0.990, and a precision value (RSD) of 0.30%. The SSP-based RT-PCR method developed is a versatile and efficient tool for detecting CM in meatballs. Its implementation helps maintain consumer trust and addresses concerns regarding the substitution of halal meats with non-halal alternatives.

Incorporating Student-Driven Research into the Undergraduate Forensic Science Curriculum: A Student-Led Study Comparing Dog Breed and Identity Determinations Utilizing Commercial Canine DNA Kits and Short Tandem Repeat Genotyping

Incorporating Student-Driven Research into the Undergraduate Forensic Science Curriculum: A Student-Led Study Comparing Dog Breed and Identity Determinations Utilizing Commercial Canine DNA Kits and Short Tandem Repeat Genotyping

The LASSIE MPS panel: Predicting externally visible traits in dogs for forensic purposes

Predicting the outward appearance of dogs via their DNA, also known as Canine DNA Phenotyping, is a young, emerging field of research in forensic genetics. The few previous studies published in this respect were restricted to the consecutive analysis of single DNA markers, a process that is time- and sample-consuming and therefore not a viable option for limited forensic specimens. Here, we report on the development and evaluation of a Massively Parallel Sequencing (MPS) based molecular genetic assay, the LASSIE MPS Panel. This panel aims to predict externally visible as well as skeletal traits, which include coat color, coat pattern, coat structure, tail morphology, skull shape, ear shape, eye color and body size from DNA using 44 genetic markers in a single molecular genetic assay. A biostatistical naïve Bayes classification approach was applied to identify the most informative marker combinations for predicting phenotypes. Overall, the predictive performance was characterized by a very high classification success for some of the trait categories, and high to moderate success for others. The performance of the developed predictive framework was further evaluated using blind samples from three randomly selected dog individuals, whose appearance was well predicted.

Exome sequencing and SDH (A, B) immunohistochemistry of canine Pheochromocytomas

Pheochromocytomas (PCs) are tumors originating from the chromaffin cells of the adrenal medulla. In people, there are highly correlated to inherited gene mutations in the succinate dehydrogenase (SDH) pathway; however, to date, little work has been done on the genetic basis of these tumors in animals. Out of the total of 2.203 Gb of canine DNA sequenced, 88.35% of bases mapped to exons and 11.65% mapped to introns. Out of 26 genes of interest containing 404 exons, 278 exons were sequenced 68.81%. Sequencing was considered successful when the average read depth was 3x and the entire exon was covered. Coverage ranged from 30% to 100%. Both SDHA and SDHB had exon mapped 46.6% and 62.5% respectively. Additionally, out of 45 known canine variants, exome technique able to detect 36 variants (80%). We performed SDHA and SDHB immunohistochemistry on 35 canine formalin-fixed, paraffin embedded. Interestingly, we had loss of immunoreactivity for both SDHA and SDHB in four samples, suggesting a mutation in SDHx including SDHA. Out of 35 samples, 6 had immunoreactivity for SDHA and 25 lacked immunoreactivities for SDHB. 29 out of the 35 (82%) may have an SDH family mutation other than SDHA. Exome sequencing and immunohistochemistry are able to predict malignant behavior and likelihood of reduction of PCC/PGLs in humans. This can be used to determine whether there are similar mutations in the pseudo-hypoxic, kinase signaling, and other genes of interest exist in dogs, as well as finding novel genes involved in canine Pheochromocytomas oncogenesis.

LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples.

Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O. lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O. lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites.

Taq man multiplex real-time pcr for identification of porcine and canine dna in halal food

The efficient and timely detection of prohibited animals in Halal food is very important. This study aimed to establish a rapid detection method for porcine and canine DNA in a food product. Porcine and Canine DNA were Contaminated in halal food products using multiplex Real-Time PCR shown positive specificity only in Porcine and Canine meat but shown negative results in chicken, cow, and fish. The results from porcine showed that the lowest level is at 0.001% and 0.1% in raw meat and cooked meat heated at 120 degrees celsius for 15 min, and 0.1% in diluted canine DNA respectively. The method for detection of the contamination of porcine and canine DNA in Halal food products with multiplex real-time PCR technique is suitable and high sensitivity for detecting the DNA of prohibited animals in meat products and raw materials, such as detecting contaminated porcine DNA in sweet jelly and soup cubes.

Hepadnavirus DNA Is Detected in Canine Blood Samples in Hong Kong but Not in Liver Biopsies of Chronic Hepatitis or Hepatocellular Carcinoma.

Chronic hepatitis and hepatocellular carcinoma (HCC) caused by the hepadnavirus hepatitis B virus (HBV) are significant causes of human mortality. A hepatitis-B-like virus infecting cats, domestic cat hepadnavirus (DCH), was reported in 2018. DCH DNA is hepatotropic and detectable in feline blood or serum (3.2 to 12.3%). Detection of HBV DNA has been reported in sera from 10% of free-roaming dogs in Brazil, whereas 6.3% of sera from dogs in Italy tested positive for DCH DNA by real-time quantitative PCR (qPCR). If DCH, HBV, or another hepadnavirus is hepatotropic in dogs, a role for such a virus in the etiology of canine idiopathic chronic hepatitis (CH) or HCC warrants investigation. This study investigated whether DCH DNA could be detected via qPCR in blood from dogs in Hong Kong and also whether liver biopsies from dogs with confirmed idiopathic CH or HCC contained hepadnaviral DNA using two panhepadnavirus conventional PCRs (cPCR) and a DCH-specific cPCR. DCH DNA was amplified from 2 of 501 (0.4%) canine whole-blood DNA samples. A second sample taken 6 or 7 months later from each dog tested negative in DCH qPCR. DNA extracted from 101 liver biopsies from dogs in Hong Kong or the USA, diagnosed by board-certified pathologists as idiopathic CH (n = 47) or HCC (n = 54), tested negative for DCH DNA and also tested negative using panhepadnavirus cPCRs. This study confirms that DCH DNA can be detected in canine blood by qPCR, although at a much lower prevalence than that reported previously. We identified no evidence to support a pathogenic role for a hepadnavirus in canine idiopathic CH or HCC.

The assessment of the efficacy of STRs panels recommended by the ISAG for canine pedigrees analysis for forensic casework

Abstract Canine DNA is widely used in forensic investigations, particularly in cases of dog attacks on humans. Nowadays, STR markers are employed worldwide in forensic laboratories to test human and animal genotypes. In the study we analysed the effectiveness of 18 STR panel as previously recommended by ISAG and the same panel with three additional markers – 21 STR, which has been recommended by ISAG as the core panel for dog identification since 2016. We calculated the PD, PID for these sets of panels and estimated RMP based on the DNA profile obtained during an investigation of a woman bitten by a dog. The high combined CPD value for 18 and 21 STRs showed values close to 1.0. The CPID value for theses panels was 5.2 × 10−10 to 6.4 × 10−14. Statistical analysis estimated the random DNA match, in the case of the woman bitten by a dog, with a probability of 4.3×1019 and 2.8×1022, using 18 and 21 STR panels respectively, and that the canine DNA profile from the crime scene originated from the suspected dog and not from another random dog. Our results show that both STR panels can be used effectively for individual identification and forensic casework.

Detection of Trypansoma cruzi in Kissing Bugs (Hemiptera: Reduviidae: Triatominae) Collected Across Oklahoma.

Trypanosoma cruzi is the causative agent of Chagas disease in humans and dogs in the Americas. Transmission predominantly occurs via the feces of infected kissing bugs (Hemiptera: family Reduviidae; subfamily Triatominae) contaminating bite site wounds or mucous membranes. To better understand Chagas disease entomologic risk in Oklahoma, kissing bugs collected from within the state were tested for T. cruzi DNA. Data including county of insect collection, species and instar, and specific locations where specimens were found were collated. Triatomines were also tested by PCR to potentially identify DNA of vertebrate species on which specimens had recently fed. In total, 110 kissing bugs from 22 counties were tested. All triatomines were identified as Triatoma sanguisuga nymphs or adults, with the exception of one possible T. lecticularia adult. Trypanosoma cruzi DNA was detected in 22 (20%) triatomines from 12 counties spanning the state. The majority of T. cruzi PCR positive kissing bugs were found inside homes or associated structures (i.e., garages, porches). Vertebrate DNA was identified in 27 (24.5%) triatomines, with human DNA detected in 25 (92.6%) of these specimens, and canine and raccoon DNA detected in one specimen each (3.7%). Two specimens tested positive for both T. cruzi and human DNA and one specimen tested positive for both T. cruzi and raccoon DNA. Results from this study indicate that kissing bugs carrying T. cruzi are widespread in Oklahoma, that positive kissing bugs infest homes and associated structures, and that human-vector, canine-vector, and wildlife-vector contact all occur within the state.

Forensic DNA phenotyping: Canis familiaris breed classification and skeletal phenotype prediction using functionally significant skeletal SNPs and indels.

This review highlights a novel application of breed identification and prediction of skeletal traits in forensic investigations using canine DNA evidence. Currently, genotyping methods used for canine breed classification involve the application of highly polymorphic short tandem repeats in addition to larger commercially available SNP arrays. Both applications face technical challenges. An additional approach to breed identification could be through genotyping SNPs and indels that characterise the array of skeletal differences displayed across domestic dog populations. Research has shown that a small number of genetic variants of large effect drive differences in skeletal phenotypes among domestic dog breeds. This feature makes functionally significant canine skeletal variants a cost-effective target for forensic investigators to classify individuals according to their breed. Further analysis of these skeletal variants would enable the prediction of external appearance. To date, functionally significant genes with genetic variants associated with differences in size, bulk, skull shape, ear shape, limb length, digit type, and tail morphology have been uncovered. Recommendations of a cost-effective genotyping method that can be readily designed and applied by forensic investigators have been given. Further advances to improve the field of canine skeletal forensic DNA phenotyping include the refinement of phenotyping methods, further biological validation of the skeletal genetic variants and establishing a publicly available database for storage of allele frequencies of the skeletal genetic variants in the wider domestic dog population.

Data analysis workflow for the detection of canine vector-borne pathogens using 16\xa0S rRNA Next-Generation Sequencing

BackgroundVector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP.ResultsWe tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample.ConclusionsBloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

Towards Forensic DNA Phenotyping for Predicting Visible Traits in Dogs.

The popularity of dogs as human companions explains why these pets regularly come into focus in forensic cases such as bite attacks or accidents. Canine evidence, e.g., dog hairs, can also act as a link between the victim and suspect in a crime case due to the close contact between dogs and their owners. In line with human DNA identification, dog individualization from crime scene evidence is mainly based on the analysis of short tandem repeat (STR) markers. However, when the DNA profile does not match a reference, additional information regarding the appearance of the dog may provide substantial intelligence value. Key features of the dog’s appearance, such as the body size and coat colour are well-recognizable and easy to describe even to non-dog experts, including most investigating officers and eyewitnesses. Therefore, it is reasonable to complement eyewitnesses’ testimonies with externally visible traits predicted from associated canine DNA samples. Here, the feasibility and suitability of canine DNA phenotyping is explored from scratch in the form of a proof of concept study. To predict the overall appearance of an unknown dog from its DNA as accurately as possible, the following six traits were chosen: (1) coat colour, (2) coat pattern, (3) coat structure, (4) body size, (5) ear shape, and (6) tail length. A total of 21 genetic markers known for high predicting values for these traits were selected from previously published datasets, comprising 15 SNPs and six INDELS. Three of them belonged to SINE insertions. The experiments were designed in three phases. In the first two stages, the performance of the markers was tested on DNA samples from dogs with well-documented physical characteristics from different breeds. The final blind test, including dogs with initially withheld appearance information, showed that the majority of the selected markers allowed to develop composite sketches, providing a realistic impression of the tested dogs. We regard this study as the first attempt to evaluate the possibilities and limitations of forensic canine DNA phenotyping.

Prevalence, geographic distribution, and impact on lifespan of a dilated cardiomyopathy-associated RNA-binding motif protein 20 variant in genotyped dogs

IntroductionThe study objectives were to determine the prevalence and geographic distribution of a dilated cardiomyopathy (DCM)-associated RNA-binding motif protein 20 (RBM20) variant in canine DNA samples submitted for testing and to evaluate the influence of the genotype on cardiac phenotype and lifespan. AnimalsSamples from 2136 dogs including 1834 Standard Schnauzers (SSNZ), 266 Giant Schnauzers (GSNZ), and 36 dogs of other breeds. MethodsThe University of Missouri Canine Genetics Laboratory's sample-accession spreadsheet and Orthopedic Foundation for Animals' database were retrospectively reviewed for samples submitted for RBM20 genotyping from November, 2013, through May, 2018. Data analyzed included breed, date of birth, RBM20 genotype (homozygous wild-type, heterozygous variant [HET], or homozygous variant [HOM]), geographic origin of submission, pedigree, cardiac phenotype, and date of death or current age if alive. Results and DiscussionThe RBM20 variant was only detected in SSNZ and GSNZ. A total of 389 SSNZ were variant-positive (prevalence = 21.2%), with 361 HET (19.7%) and 28 HOM (1.5%). Of the HOM SSNZ, DCM was confirmed in 26 of 28 (92.9%), with the remainder lost to follow-up. The median lifespan of HOM SSNZ (3.06 years) was significantly shorter than that for HET (15.11 years) and wild-type (15.18 years) SSNZ. Twenty-six GSNZ were variant-positive (prevalence = 9.8%), with 23 HET (8.6%) and three HOM (1.1%). Nine GSNZ belonged to one family, including the three HOM GSNZ that all had DCM. ConclusionsThe HOM genotype is associated with DCM and premature death in SSNZ and GSNZ.