Exploration of the potential utility of the luciferase immunoprecipitation system (LIPS) assay for the detection of anti-leptospira antibodies in dogs.

Canine leptospirosis represents a diagnostic challenge to veterinarians, due to the variability in presenting clinical signs and interpretation of serology test results in dogs that have been vaccinated previously. None of the commercially available serological assays, including the microscopic agglutination test (MAT), have been verified to be capable of differentiating infected from vaccinated animals (DIVA). Recent work identified that half of primary practice attending dogs were up to date with their leptospirosis vaccination and would be expected to have circulating anti-leptospira antibodies (Taylor et al., 2022), indicating that this is a relevant issue for suspected leptospirosis cases in dogs in the UK. This study aimed to explore the utility of three leptospiral outer membrane proteins (OMPs: LipL32, LipL21 and LipL41) as potential DIVA targets in the luciferase immunoprecipitation system (LIPS) assay. N and C terminal nanoluciferase tagged recombinant proteins were generated for each OMP. Differences in reactivity between serum samples from MAT positive dogs (n = 29) and paired samples (n = 6 dogs) taken pre and 21 days post leptospirosis vaccination were assessed against these six constructs. Reactivity was greater towards the N terminal than the C terminal recombinant proteins for all three OMPs. None of the constructs appeared to demonstrate DIVA capability, although two (pNLF1-N-FLAG/LipL32 and pNLF1-N-FLAG/LipL21) were able to detect vaccine seroconversion. The findings of this work suggest that these particular OMP targets do not offer DIVA ability, however LipL32 and LipL21 may be suitable for use in immunoassays for vaccine trials or for detection of infections in humans, where there is no requirement for DIVA capability.

Spatio-temporal distribution and agroecological factors associated with canine leptospirosis in Great Britain.

Leptospirosis is an important global zoonotic disease that affects a wide range of mammalian species. Canine leptospirosis outbreaks have been reported after metereological events such as flooding (eg. in Brazil and the United States of America) suggesting an environmental association, but there has been no such study in Great Britain (GB). The distribution of cases across GB is also unreported. Objectives of this study were to: (1) assess the spatio-temporal variation of leptospirosis test submissions (2) explore associations between agroecological risk factors and distribution of different canine leptospirosis serogroups in GB, and (3) generate probability of presence maps for the different serogroups. Data analysed comprised laboratory submissions (n = 3986) to IDEXX laboratories between 1st January 2009 and 31st December 2018 for PCR or MAT leptospirosis testing. Spatial and seasonal scan statistics were used to investigate spatial and temporal clustering of positive tests, logistic regression was used to identify significant agroecological risk factors for positive tests, and the Maxent algorithm was used to model the environmental niche of four serogroups. There was an increased risk of a positive test result in the West Midlands of England (relative risk = 2.16) and between October and January (relative risk = 1.54). Logistic regression identified season and region to be significantly associated with a positive diagnosis,with higher odds of a positive test in Autumn (OR = 1.86 95 %CI 1.29-2.69) and Winter (OR = 1.51, 95 %CI 1.02-2.23) and in the East (OR = 2.20, 95 %CI = 1.31-3.71) and West Midlands (OR = 2.32, 95 %CI 1.45-3.71). The increased test-positive proportion in Autumn together with the increased odds of a positive diagnosis in Autumn suggests there may be a seasonal pattern to the canine leptospirosis in GB. The most important variable associated with higher leptospirosis presence in all ecological niche models was higher average annual temperature. The importance and retention of other variables differed between serogroups. Overall, a higher probability of leptospirosis presence was predicted in southern England and a low probability in Scotland and northern England. Although leptospirosis vaccine usage provides protection against the majority of serogroups identified here, one is not represented in the currently licensed vaccine formulations and therefore leptospirosis should remain a differential diagnosis in vaccinated dogs demonstrating consistent clinical signs of the disease.

Dog leucocyte antigen (DLA) class II haplotypes and risk of canine diabetes mellitus in specific dog breeds.

BACKGROUND: Canine diabetes mellitus (DM) is a common endocrine disease in domestic dogs. A number of pathological mechanisms are thought to contribute to the aetiopathogenesis of relative or absolute insulin deficiency, including immune-mediated destruction of pancreatic beta cells. DM risk varies considerably between different dog breeds, suggesting that genetic factors are involved and contribute susceptibility or protection. Associations of particular dog leucocyte antigen (DLA) class II haplotypes with DM have been identified, but investigations to date have only considered all breeds pooled together. The aim of this study was to analyse an expanded data set so as to identify breed-specific diabetes-associated DLA haplotypes. METHODS: The 12 most highly represented breeds in the UK Canine Diabetes Register were selected for study. DLA-typing data from 646 diabetic dogs and 912 breed-matched non-diabetic controls were analysed to enable breed-specific analysis of the DLA. Dogs were genotyped for allelic variation at DLA-DRB1, -DQA1, -DQB1 loci using DNA sequence-based typing. Genotypes from all three loci were combined to reveal three-locus DLA class II haplotypes, which were evaluated for statistical associations with DM. This was performed for each breed individually and for all breeds pooled together. RESULTS: Five dog breeds were identified as having one or more DLA haplotype associated with DM susceptibility or protection. Four DM-associated haplotypes were identified in the Cocker Spaniel breed, of which one haplotype was shared with Border Terriers. In the three breeds known to be at highest risk of DM included in the study (Samoyed, Tibetan Terrier and Cairn Terrier), no DLA haplotypes were found to be associated with DM. CONCLUSIONS: Novel DLA associations with DM in specific dog breeds provide further evidence that immune response genes contribute susceptibility to this disease in some cases. It is also apparent that DLA may not be contributing obvious or strong risk for DM in some breeds, including the seven breeds analysed for which no associations were identified.

Inconsistent MHC class II association in Beagles experimentally infected with Leishmania infantum.

The clinical outcome of Leishmania infantum infection in dogs varies from subclinical infection to severe disease. Researchers attribute this variability in clinical manifestations to the ability of the immune response to limit pathogen multiplication and dissemination, which is, in part, likely determined by the immune response genes. The aim of this study was to test the hypothesis that MHC class II genes are associated with disease outcome of experimental L. infantum infection in Beagles. Dog leukocyte antigen (DLA) class II haplotypes were characterised by sequence-based typing of Beagle dogs experimentally infected with L. infantum during vaccine challenge studies. Variability of response to infection was determined by clinical score, serology and quantification of L. infantum DNA in the bone marrow over the study period. Dogs showed limited DLA diversity and the DLA profiles of dogs recruited for the different vaccine challenge studies differed. There were variable responses to infection, despite the apparent restriction in genetic diversity. One haplotype DLA-DRB1*001:02-DQA1*001:01-DQB1*002:01 was associated with increased anti-Leishmania antibodies in one infection model, but no DLA associations were found in other groups or with parasite load or clinical score. Examination of this particular DLA haplotype in a larger number of dogs is required to confirm whether an association exists with the immune or clinical responses to L. infantum infection.

The Canine POMC Gene, Obesity in Labrador Retrievers and Susceptibility to Diabetes Mellitus.

BACKGROUND: Diabetes mellitus (DM) in dogs is a common endocrinopathy with a complex genetic architecture. Disease susceptibility in several breeds is associated with polymorphisms in immune response genes, but in the Labrador retriever breed, no genetic associations with DM have been identified. A deletion in the pro-opiomelanocortin (POMC) gene in Labrador retrievers is associated with increased appetite and risk of obesity. HYPOTHESIS/OBJECTIVES: To characterize the POMC deletion in Labrador retrievers, to develop a simple genetic test for this mutation, and to test the hypothesis that the POMC gene deletion is associated with an increased risk of DM in this breed. ANIMALS: Sixty-one non-diabetic Labrador retrievers aged >6 years and 57 Labrador retrievers with DM. METHODS: Case-control genotyping study to compare the frequency of the POMC deletion in dogs with and without DM. After polymerase chain reaction (PCR) and sequencing to characterize the mutation, a PCR-based test was developed and validated using 2 different restriction fragment length polymorphism assays. RESULTS: A 14-base-pair deletion was confirmed and localized to exon 3 of the canine POMC gene. A PCR-based test for the deletion was successfully developed. There was no association between the presence of the POMC deletion mutation and DM in this population of Labrador retriever dogs (P = .31). CONCLUSIONS AND CLINICAL IMPORTANCE: This study adds to the existing scientific literature indicating that there is little evidence for a direct link between obesity and DM in dogs.

Feline hypersomatotropism and acromegaly tumorigenesis: a potential role for the AIP gene.

Acromegaly in humans is usually sporadic, however up to 20% of familial isolated pituitary adenomas are caused by germline sequence variants of the aryl-hydrocarbon-receptor interacting protein (AIP) gene. Feline acromegaly has similarities to human acromegalic families with AIP mutations. The aim of this study was to sequence the feline AIP gene, identify sequence variants and compare the AIP gene sequence between feline acromegalic and control cats, and in acromegalic siblings. The feline AIP gene was amplified through PCR using whole blood genomic DNA from 10 acromegalic and 10 control cats, and 3 sibling pairs affected by acromegaly. PCR products were sequenced and compared with the published predicted feline AIP gene. A single nonsynonymous SNP was identified in exon 1 (AIP:c.9T > G) of two acromegalic cats and none of the control cats, as well as both members of one sibling pair. The region of this SNP is considered essential for the interaction of the AIP protein with its receptor. This sequence variant has not previously been reported in humans. Two additional synonymous sequence variants were identified (AIP:c.481C > T and AIP:c.826C > T). This is the first molecular study to investigate a potential genetic cause of feline acromegaly and identified a nonsynonymous AIP single nucleotide polymorphism in 20% of the acromegalic cat population evaluated, as well as in one of the sibling pairs evaluated.

Immunotherapy for canine cancer--is it time to go back to the future?

Over the last 50 years, the significance of the immune system in the development and control of cancer has been much debated. However, recent discoveries provide evidence for a role of immunological mechanisms in the detection and destruction of cancer cells. Forty years ago veterinary oncologists were already investigating the feasibility of treating neoplasia by enhancing anticancer immunity. Unfortunately, this research was hindered by lack of a detailed understanding of cancer immunology, this limited the specificity and success of these early approaches. The great forward strides made in our understanding of onco-immunology in recent years have provided the impetus for a resurgence of interest in anticancer immunotherapy for canine patients. In this article both these initial trials and the exciting novel immunotherapeutics currently in development are reviewed.

Association between nucleotide oligomerisation domain two (Nod2) gene polymorphisms and canine inflammatory bowel disease.

The most important genetic associations that have been implicated to play a role in the etiology of Crohn's disease (CD) in humans are single nucleotide polymorphisms (SNPs) in nucleotide oligomerisation domain 2 (NOD2). The aim of this study was to investigate whether SNPs in the canine NOD2 gene are associated with inflammatory bowel disease (IBD) in German shepherd dogs (GSDs) and other canine breeds. A mutational analysis of the NOD2 gene was carried out in 10 randomly selected GSDs with IBD. The mutational analysis identified five non-synonymous SNPS, of which four in exon 3 of the NOD2 gene were evaluated in a case-control study using sequence based typing. Sequencing information from 55 GSDs with IBD were compared to a control group consisting of 61 GSDs. In addition, 85 dogs of other breeds with IBD and a breed-matched control group consisting of 162 dogs were also genotyped. All four SNPs were in complete linkage and, in the GSD population, were found to be in Hardy-Weinberg equilibrium. When the GSD case population was compared to the GSD control group, the heterozygote genotype for all four SNPs was more frequently found in the IBD population (p=0.03, OR=2.30, CI=1.07-4.94). However, these results were not mirrored in other canine breeds. Our study suggests that the four SNPs in exon 3 of NOD2 are significantly associated with IBD in GSDs when analyzed in an over-dominant model. However, these results were not mirrored in other canine breeds with IBD. This suggests that the etiology of this disease is complex and may involve the interaction of SNPs present in several genes or pathways to bring about the inflammatory changes seen in the intestine.

Recombinant canine IgE Fc and an IgE Fc-TRAIL fusion protein bind to neoplastic canine mast cells.

Screening for expression of the high affinity receptor for IgE by reverse transcriptase PCR, revealed that almost all canine mast cell tumors expressed FcɛRIα mRNA, supporting the rationale for developing anti-neoplastic treatments based on molecules that could target this receptor. Use of cytotoxic cytokines to trigger an apoptotic signal is one strategy for inducing cell death in malignant mast cells. The coding sequences for canine IgE and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were identified through genome analyses. Selected regions of the coding sequences for these genes were cloned and compared to the predicted genome sequences. The Fc region of canine IgE, death domain of canine TRAIL and an IgE Fc: TRAIL fusion construct were generated and epitope-tagged proteins expressed, using a eukaryotic expression system. Specific binding of recombinant canine IgE Fc-containing proteins to recombinant human FcɛRIα and to a canine mast cell tumor line expressing FcɛRIα (C2), but not one failing to express FcɛRIα (MCLA), was demonstrated. Specific binding of the IgE: TRAIL fusion protein was not abrogated by the TRAIL moiety. These results are proof of principle that canine IgE targeting to FcɛRIα can be used as a platform for selective delivery of therapies to FcɛRIα-expressing cells, potentially enhancing their therapeutic index and efficacy.

A polymorphism in the melanocortin 4 receptor gene (MC4R:c.92C>T) is associated with diabetes mellitus in overweight domestic shorthaired cats.

BACKGROUND: Feline diabetes mellitus (DM) shares many pathophysiologic features with human type 2 DM. Human genome-wide association studies have identified genes associated with obesity and DM, including melanocortin 4 receptor (MC4R), which plays an important role in energy balance and appetite regulation. HYPOTHESIS/OBJECTIVES: To identify single nucleotide polymorphisms (SNPs) in the feline MC4R gene and to determine whether any SNPs are associated with DM or overweight body condition in cats. ANIMALS: Two-hundred forty domestic shorthaired (DSH) cats were recruited for the study. Of these, 120 diabetics were selected (60 overweight, 60 lean), along with 120 nondiabetic controls (60 overweight and 60 lean). Males and females were equally represented. METHODS: A prospective case-control study was performed. Genomic DNA was extracted from blood samples and used as template for PCR amplification of the feline MC4R gene. The coding region of the gene was sequenced in 10 cats to identify polymorphisms. Subsequently, genotyping by restriction fragment length polymorphism (RFLP) analysis assessed MC4R:c.92C > T allele and genotype frequencies in each group of cats. RESULTS: No significant differences in MC4R:c.92C>T allele or genotype frequencies were identified between nondiabetic overweight and lean cats. In the overweight diabetic group, 55% were homozygous for the MC4R:c.92C allele, compared to 33% of the lean diabetics and 30% of the nondiabetics. The differences between the overweight diabetic and the nondiabetics were significant (P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: We identified a polymorphism in the coding sequence of feline MC4R that is associated with DM in overweight DSH cats, similar to the situation in humans.

Novel diabetes mellitus treatment: mature canine insulin production by canine striated muscle through gene therapy.

Muscle-targeted gene therapy using insulin genes has the potential to provide an inexpensive, low maintenance alternative or adjunctive treatment method for canine diabetes mellitus. A canine skeletal muscle cell line was established through primary culture, as well as through transdifferentiation of canine fibroblasts after infection with a myo-differentiation gene containing adenovirus vector. A novel mutant furin-cleavable canine preproinsulin gene insert (cppI4) was designed and created through de novo gene synthesis. Various cell lines, including the generated canine muscle cell line, were transfected with nonviral plasmids containing cppI4. Insulin and desmin immunostaining were used to prove insulin production by muscle cells and specific canine insulin ELISA to prove mature insulin secretion into the medium. The canine myoblast cultures proved positive on desmin immunostaining. All cells tolerated transfection with cppI4-containing plasmid, and double immunostaining for insulin and desmin proved present in the canine cells. Canine insulin ELISA assessment of medium of cppI4-transfected murine myoblasts and canine myoblast and fibroblast mixture proved presence of mature fully processed canine insulin, 24 and 48 h after transfection. The present study provides proof of principle that canine muscle cells can be induced to produce and secrete canine insulin on transfection with nonviral plasmid DNA containing a novel mutant canine preproinsulin gene that produces furin-cleavable canine preproinsulin. This technology could be developed to provide an alternative canine diabetes mellitus treatment option or to provide a constant source for background insulin, as well as C-peptide, alongside current treatment options.

Breed-independent toll-like receptor 5 polymorphisms show association with canine inflammatory bowel disease.

Inflammatory bowel disease (IBD) is thought to be the most common cause of vomiting and diarrhoea in dogs. Although IBD can occur in any canine breed, certain breeds are more susceptible. We have previously shown that polymorphisms in the TLR4 and TLR5 (toll-like receptor) genes are significantly associated with IBD in German Shepherd dogs (GSDs). In order to allow for the development of novel diagnostics and therapeutics suitable for all dogs suffering from IBD, it would be useful to determine if the described polymorphisms are also significantly associated with IBD in other breeds. Therefore, the aim of this study was to investigate whether polymorphisms in the canine TLR4 and TLR5 genes are associated with IBD in other non-GSD canine breeds. The significance of the previously identified non-synonymous single nucleotide polymorphisms (SNPs) in the TLR4 (T23C, G1039A, A1571T and G1807A) and TLR5 genes (G22A, C100T and T1844C) were evaluated in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 85 unrelated dogs with IBD consisting of 38 different breeds was compared with a breed-matched control group consisting of 162 unrelated dogs. Indeed, as in the GSD IBD population, the two TLR5 SNPs (C100T and T1844C) were found to be significantly protective for IBD in other breeds (P = 0.023 and P = 0.0195 respectively). Our study suggests that the two TLR5 SNPs, C100T and T1844C could play a role in canine IBD as these were found to be protective factors for this disease in 38 different canine breeds. Thus, targeting TLR5 in the canine system may represent a suitable way to develop new treatment for IBD in dogs.

Targeted knockdown of canine KIT (stem cell factor receptor) using RNA interference.

Canine mast cell tumours often express KIT mutations that result in constitutive activation of the c-kit receptor and which are associated with more aggressive disease. The aim of the current study was to determine whether small inhibitory RNA (SiRNA) molecules could specifically target canine KIT mRNA for knock-down. Canine beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and KIT sequences were cloned into the psiCHECK™-2 vector. SiRNA molecules, designed to target gene-specific sequences, were co-transfected with plasmid DNA into Chinese hamster ovary (CHO) cells. Renilla and firefly luciferase activity was measured using the Dual-GLO(®) Luciferase Assay (Promega). Using this reporter system, canine housekeeping gene-specific SiRNA molecules demonstrated knockdown of their targets (72.0% knockdown for B2M and 94.5% knockdown for GAPDH). An SiRNA molecule targeting exon 2 of canine KIT successfully knocked-down reporter gene expression of a KIT(26-407) construct (90.8% knockdown). An SiRNA molecule targeting a 48 base-pair in-tandem duplication mutation in KIT exon 11 selectively knocked down expression of the KIT(1569-1966mutant) construct (93.1% knockdown) but had no effect on the KIT(1569-1918wild-type) construct. The results show that RNA interference can be used to inhibit canine KIT mRNA expression and has the potential to selectively target the mutant version of KIT that is expressed by some malignant mast cells.

Evaluation of mucosal bacteria and histopathology, clinical disease activity and expression of Toll-like receptors in German shepherd dogs with chronic enteropathies.

The pathogenesis of chronic enteropathies in dogs likely involves an interaction between the intestinal immune system and luminal intestinal bacteria. German shepherd dogs (GSD) are particularly predisposed to chronic enteropathies. The present study sought to evaluate expression patterns of certain pattern recognition receptors of the innate immunity (Toll-like receptors, TLR), clinical disease activity and histopathological severity in GSD with chronic enteropathies. Mucosal biopsies were collected from the duodenum, colon and ileum of 13 affected GSD and 10 healthy greyhounds as controls. Dogs were objectively assessed using published scoring systems for clinical and histological severity of disease. Diversity of the duodenal microbiota was assessed by construction of 16S rRNA gene libraries. Expression of TLR2, TLR4, TLR5 and TLR9 in biopsies of the duodenum, ileum and colon was assessed by quantitative real-time PCR. TLR4 expression was increased in all intestinal segments in GSD, however, TLR5 expression was very low compared to the healthy dogs. The microbiota in the duodenum of GSDs was significantly different to that of the greyhounds, with an over-representation of 16S rRNA gene sequences belonging to the classes of Bacilli, and Erysipelotrichi, and to the orders of Lactobacillales, Actinomycetales and Erysipelotrichales. These findings could point to a distinct pathogenesis of chronic enteropathies in GSD, with differentially high and low expression of TLR4 and TLR5, respectively, and increased proportions of specific members of the Lactobacillales potentially playing a role.

CTLA4 promoter polymorphisms are associated with canine diabetes mellitus.

Canine diabetes mellitus (DM) shares many similarities with human type 1 diabetes (T1D). It is a complex genetic disorder, which shows marked differences in breed susceptibility, with Samoyed dogs being highly susceptible, whereas the Boxer breed is relatively resistant. A number of immune response genes, which have been associated with human T1D, have also been implicated in determining susceptibility to canine DM, suggesting an immune-mediated component to the disease pathogenesis. Single nucleotide polymorphisms (SNPs) in the CTLA4 gene have consistently and reproducibly been associated with human T1D and other autoimmune diseases but the canine CTLA4 gene has not previously been investigated for involvement in canine DM. SNPs of particular interest in the human association studies are those in the promoter region which affect CTLA4 expression levels, and that of exon 1 which results in a non-synonymous amino acid change. We performed a canine SNP discovery investigation of CTLA4 on a region of DNA containing exon 1 and 1.5 kb upstream sequence in order to identify promoter region SNPs. Confirmed SNPs were used in a genetic association study of a canine diabetic cohort showing that CTLA4 promoter polymorphisms were associated with diabetes in crossbreed dogs and in five Pedigree breeds-Samoyed, Miniature Schnauzer, West Highland White Terrier, Border Terrier and Labrador. Meta-analysis of these breeds showed 9 out of 15 SNPs were associated with DM and genotype and haplotype analyses also confirmed the allelic associations in these breeds.

Expression of Toll-like receptor 2 in duodenal biopsies from dogs with inflammatory bowel disease is associated with severity of disease.

There is growing evidence that aberrant innate immune responses towards the bacterial flora of the gut play a role in the pathogenesis of canine inflammatory bowel disease (IBD). Toll-like receptors (TLR) play an important role as primary sensors of invading pathogens and have gained significant attention in human IBD as differential expression and polymorphisms of certain TLR have been shown to occur in ulcerative colitis (UC) and Crohn's disease (CD). The aim of the current study was to evaluate the expression of two TLR important for recognition of commensals in the gut. TLR2 and TLR4 mRNA expression in duodenal biopsies from dogs with IBD was measured and correlated with clinical and histological disease severity. Endoscopic duodenal biopsies from 20 clinical cases and 7 healthy control dogs were used to extract mRNA. TLR2 and TLR4 mRNA expression was assessed using quantitative real-time PCR. TLR2 mRNA expression was significantly increased in the IBD dogs compared to controls, whereas TLR4 mRNA expression was similar in IBD and control cases. In addition, TLR2 mRNA expression was mildly correlated with clinical severity of disease, however, there was no correlation between TLR2 expression and histological severity of disease.

Association of canine anal furunculosis with TNFA is secondary to linkage disequilibrium with DLA-DRB1*.

Anal furunculosis (AF) is a chronic inflammatory disease of perianal tissues that particularly affects German Shepherd dogs (GSD). An immune-mediated aetiopathogenesis is suggested by T-cell infiltration, upregulated cytokine gene expression, clinical response to ciclosporin therapy and a strong genetic association with the DLA-DRB1*00101 allele. Given the close proximity of TNFA and DLA-DRB1 in the canine major histocompatibility complex (MHC), together with the strong linkage disequilibrium (LD) observed across this region, the primary disease association could be with either locus. We have investigated whether there may be an association of AF with TNFA gene polymorphism in GSDs. Cohorts of AF-affected and AF-unaffected GSDs of known dog leucocyte antigen (DLA) class II profile were genotyped for 10 single nucleotide polymorphisms (SNPs) in the canine TNFA locus using Sequenom iPLEX technology. Seven discrete TNFA haplotypes were identified in GSDs for combinations of these SNPs. TNFA haplotype frequencies were compared in cases and controls. The TNFA haplotype 3 (ATCGTTACGG), was at significantly increased frequency in cases (29% vs 15%, OR 2.5, 95% CI 1.4-4.8; P = 0.003). All seven discrete TNFA SNP haplotypes were examined for their association with DLA-DRB1/DQA1/DQB1 established haplotypes. TNFA haplotype 3 was preferentially associated with both DLA-DRB1*00101(3A)- and DLA-DRB1*00102(3B)-positive haplotypes. The DLA-DRB1* 00101/TNFA-3A haplotype was significantly associated with AF (19.3% vs 5.8%; OR 3.7, 95% CI: 1.5-8.9; P = 0.003), whereas the DLA-DRB1*00102/TNFA-3B haplotype was not (P = NS). These findings suggest that susceptibility to AF in GSDs is primarily associated with DLA-DRB1*00101 and any association with the TNFA locus is secondary and is likely to be because of LD.

Analysis of NOD1, NOD2, TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9 genes in anal furunculosis of German shepherd dogs.

Anal furunculosis (AF) primarily affects German shepherd dogs (GSD) and is characterised by inflammation and ulceration of the perianal tissues with development of cutaneous sinuses or rectocutaneous fistulae. Investigation of pattern recognition receptor (PRR) function has suggested that defective responses might occur in AF-affected GSD. The aim of the current study was to investigate whether canine PRR genes are involved in determining susceptibility to AF in this breed. Chromosomal location and coding sequences for NOD1, NOD2, TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9 were determined and microsatellite markers identified for each gene. Microsatellite genotyping of 100 control GSD and 47 AF-affected GSD showed restricted allelic variation for AHT H91 (associated with TLR5) and REN216 NO5 (associated with both TLR1 and TLR6) compared with non-GSD dogs. Genotyping of single nucleotide polymorphisms identified in canine TLR1, TLR5, TLR6 and NOD2 genes failed to show any significant associations between PRR polymorphisms and AF. The highly restricted PRR genotypes seen in GSD are likely to have resulted from selective breeding and might influence innate immune responses in this breed.

Susceptibility of the C2 canine mastocytoma cell line to the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which preferentially induces apoptosis in cells that have undergone malignant transformation. In humans, non-neoplastic cells are normally protected from the effects of TRAIL by expressing decoy receptors, lacking death domains. In contrast, neoplastic cells tend to downregulate their decoy receptor expression, increasing their susceptibility to the pro-apoptotic effects of TRAIL, via the functional TRAIL receptors. The aim of the current study was to investigate the effect of TRAIL on the canine C2 mastocytoma cell line to determine whether this agent might be a suitable treatment for mast cell tumors in dogs. C2 and MDCK cells were cultured with recombinant human TRAIL. Apoptosis was assessed using a Caspase 3 and 7 chemiluminescence assay and flow cytometry following Annexin V:FITC labelling. Cell metabolism was assessed using a colorimetric MTT-based assay. C2 cells demonstrated greater sensitivity to TRAIL-induced apoptosis compared to MDCK cells by all assessment methods. The dog genome assembly was searched for orthologs of TRAIL and its receptors using published sequences from other species for reference. Although a canine ortholog for TRAIL was identified, only one TRAIL receptor ortholog (TNFRSF11B) could be found. C2, but not MDCK, cells expressed mRNA for TNFRSF11B, detected by RT-PCR. In other species, TNFRSF11B is a decoy receptor, as even though it has a death domain it is secreted due to its lack of a transmembrane domain. The effect of TRAIL on the C2 cell line suggests that this cytokine might be suitable for treatment of mast cell tumors in dogs.