Aarti Kathrani

Polymorphisms in the TLR4 and TLR5 gene are significantly associated with inflammatory bowel disease in German shepherd dogs.

Inflammatory bowel disease (IBD) is considered to be the most common cause of vomiting and diarrhoea in dogs, and the German shepherd dog (GSD) is particularly susceptible. The exact aetiology of IBD is unknown, however associations have been identified between specific single-nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and human IBD. However, to date, no genetic studies have been undertaken in canine IBD. The aim of this study was to investigate whether polymorphisms in canine TLR 2, 4 and 5 genes are associated with IBD in GSDs. Mutational analysis of TLR2, TLR4 and TLR5 was performed in 10 unrelated GSDs with IBD. Four non-synonymous SNPs (T23C, G1039A, A1571T and G1807A) were identified in the TLR4 gene, and three non-synonymous SNPs (G22A, C100T and T1844C) were identified in the TLR5 gene. The non-synonymous SNPs identified in TLR4 and TLR5 were evaluated further in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 55 unrelated GSDs with IBD were compared to a control group consisting of 61 unrelated GSDs. The G22A SNP in TLR5 was significantly associated with IBD in GSDs, whereas the remaining two SNPs were found to be significantly protective for IBD. Furthermore, the two SNPs in TLR4 (A1571T and G1807A) were in complete linkage disequilibrium, and were also significantly associated with IBD. The TLR5 risk haplotype (ACC) without the two associated TLR4 SNP alleles was significantly associated with IBD, however the presence of the two TLR4 SNP risk alleles without the TLR5 risk haplotype was not statistically associated with IBD. Our study suggests that the three TLR5 SNPs and two TLR4 SNPs; A1571T and G1807A could play a role in the pathogenesis of IBD in GSDs. Further studies are required to confirm the functional importance of these polymorphisms in the pathogenesis of this disease.

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.

TLR5 Risk-Associated Haplotype for Canine Inflammatory Bowel Disease Confers Hyper-Responsiveness to Flagellin

Single nucleotide polymorphisms (SNP) in the TLR5 gene have been associated with human inflammatory bowel disease (IBD) and animal models of this disease. We recently demonstrated a significant association between three non-synonymous SNPs in the canine TLR5 gene and IBD in German shepherd dogs (GSDs). However, so far, no direct link between these SNPs and a disturbance in TLR5 function was shown. In the present study, we determined the functional significance of the canine TLR5 SNPs by transfecting the identified risk-protective and risk-associated haplotype into human embryonic kidney cells (HEK) and assessed nuclear factor-kappa B (NF-κB) activation and CXCL8 production after stimulation. In addition, a whole blood assay for TLR5 activation was developed using blood derived from carrier dogs of either haplotype. There was a significant increase in NF-kB activity when cells transfected with the risk-associated TLR5 haplotype were stimulated with flagellin compared to the cells expressing the risk-protective TLR5 haplotype. This difference in NFkB activation correlated with CXCL8 expression in the supernatant measured by ELISA. Furthermore, whole blood taken from carrier dogs of the risk-associated TLR5 haplotype produced significantly more TNF after stimulation with flagellin compared to that taken from carriers of the risk-protective haplotype. Thus, we show for the first time a direct functional impact of the canine IBD risk-associated TLR5 haplotype, which results in hyper-responsiveness to flagellin compared to the IBD risk-protective TLR5 haplotype. Our data potentially suggest that similarly to human IBD and experimental models, TLR5 may also play a role in canine IBD. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease.

Elevated canine pancreatic lipase immunoreactivity concentration in dogs with inflammatory bowel disease is associated with a negative outcome

OBJECTIVES To investigate whether elevated canine pancreatic lipase immunoreactivity (CPLI) concentrations in dogs with inflammatory bowel disease (IBD) is associated with a worse clinical outcome. METHODS Serum CPLI assays were performed on serum stored from cases diagnosed with IBD. Thirty-two dogs with CPLI results within the reference range were designated as the control group and 15 dogs had CPLI above the reference range. Clinical signs, age, serum lipase and amylase activities, serum albumin and cobalamin concentrations, abdominal ultrasound examination, histopathology on small intestinal biopsies, management of IBD and outcome were compared between the two groups. RESULTS No significant differences were found in clinical activity score (P=0.54), number of antibiotic-responsive disease cases (P=0.480), number of steroid-responsive disease cases (P=0.491), serum amylase activity (P=0.058), serum cobalamin concentration (P=0.61), serum albumin concentration (P=0.052), abdominal ultrasound score (P=0.23) and histopathology scores for IBD (P=0.74) between the two groups. Dogs with increased CPLI concentration were significantly older and had a higher serum lipase activity than dogs with a CPLI concentration within the normal reference range (P=0.001, P=0.001, respectively). Moreover, dogs with increased CPLI concentration responded poorly to steroid treatment (P=0.01) and were significantly more likely to be euthanased at follow-up (P=0.02). CLINICAL SIGNIFICANCE CPLI should be measured in cases of canine IBD as elevated CPLI was associated with a worse outcome.

Canine breeds at high risk of developing inflammatory bowel disease in the south-eastern UK

Genetics are an important factor in the development of human inflammatory bowel disease (IBD); however, there is very little information available regarding the role of genetics in canine IBD. The purpose of this study was to gather information about which canine breeds in the south-eastern UK are at a high risk for developing IBD. Determination of such breeds may help further genetic research in this complex disease. The computer medical records at the Queen Mother Hospital for Animals, Royal Veterinary College dating from August 1, 2003 to December 31, 2009 were retrospectively searched for cases diagnosed with IBD. Five hundred and forty-six dogs with IBD were identified, representing 86 different breeds. The comparison group consisted of all dogs from these same 86 breeds without IBD admitted to the hospital during the same period that amounted to 27,463 dogs. The breeds at significantly higher risk of developing IBD compared with mixed-breed dogs consisted of weimaraner (odds ratio [OR]=3.6797, 95 per cent confidence interval [CI]=2.0167 to 6.7141, P<0.0001), rottweiler (OR=2.9697, 95 per cent CI=1.7569 to 5.0196, P<0.0001), German shepherd dog (GSD) (OR=2.4101, 95 per cent CI=1.5826 to 3.36705, P<0.0001), border collie (OR=1.9936, 95 per cent CI=1.1655 to 3.4101, P=0.0118) and boxer (OR=1.6961, 95 per cent CI=1.0441 to 2.755, P=0.0328). This study demonstrates for the first time canine breeds in the south-eastern UK that are highly susceptible to developing IBD. Identification of such breeds may allow for a more focused investigation of genetic mutations associated with canine IBD.

CD11c+ Cells are Significantly Decreased in the Duodenum, Ileum and Colon of Dogs with Inflammatory Bowel Disease

CD11c serves as a marker for human and murine dendritic cells (DCs) and cells expressing this marker have been shown to have similar morphological and functional characteristics in the canine immune system. The aim of this study was to quantify CD11c(+) cells in the duodenum, ileum and colon of healthy dogs and dogs with inflammatory bowel disease (IBD). Endoscopic biopsies from the duodenum (n=12 cases), ileum (n=8 cases) and colon (n=12 cases) were obtained from dogs diagnosed with IBD. Intestinal tissue from 10 healthy beagle dogs was used as control. Immunofluorescence microscopy was carried out using an anti-canine CD11c monoclonal antibody. Labelled cells were recorded as cells per 120,000 μm(2). The canine chronic enteropathy clinical activity index (CCECAI) was calculated for all dogs with IBD. In addition, sections from all dogs with IBD were evaluated according to the guidelines of the World Small Animal Veterinary Association Gastrointestinal Standardization Group. The number of CD11c(+) cells in the duodenum, ileum and colon of dogs with IBD was significantly reduced compared with controls (P<0.01, P<0.01 and P<0.05, respectively). There was a significant negative correlation between the number of CD11c(+) cells in the colon of dogs with IBD and the CCECAI (P=0.044, r(2)=-0.558). Chronic inflammation in canine IBD appears to involve an imbalance in the intestinal DC population. Future studies will determine whether reduced expression of CD11c could be a useful marker for the diagnosis and monitoring of canine IBD.

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 Crohns 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.

Lack of Correlation between Mucosal Immunoglobulin A-positive Plasma Cell Numbers and TLR5 Genotypes in German Shepherd Dogs with Idiopathic Chronic Enteropathy

It has been suggested previously that a deficiency in mucosal immunoglobulin (Ig) A production could be involved in the pathogenesis of chronic enteropathy in German shepherd dogs (GSDs). Recent research has shown that single nucleotide polymorphisms in the gene encoding Toll-like receptor (TLR)-5 are associated with an increased risk of development of chronic idiopathic enteropathy in this breed. IgA is essential for mucosal immunity and studies in mice have linked the interaction of TLR5 with its ligand flagellin to class switching of B cells into IgA-producing plasma cells. We hypothesized that dogs carrying the risk-associated (RA) genotypes for G22A and C100T genes of TLR5 would have a different number of IgA plasma cells in the duodenal and colonic mucosa compared with dogs carrying the risk-protective (RP) genotypes. Thirty-one GSDs were diagnosed with idiopathic chronic enteropathy by clinical exclusion diagnosis and histopathological confirmation. Immunohistochemistry was performed using goat anti-dog IgA primary antibody. Two sections of duodenum, and colon if available, were examined from each animal. Twelve images were captured from each section and IgA-positive cells were counted and expressed per 10,000 μm(2). TLR5 genotypes for the G22A and C100T genes were determined by polymerase chain reaction on blood samples. Numbers of IgA-positive cells in the duodenum and colon were slightly higher than those published previously for GSDs with or without chronic enteropathy (mean in the crypt area of the duodenum 52.6 ± 16.2; mean in the tip of the duodenal villus 51.12 ± 3.83; mean in the base of the duodenal villus 55.02 ± 3.3; mean in the crypt area of the colon 67.4 ± 4.3). There was no correlation between numbers of IgA-positive cells in duodenum or colon between dogs carrying the RA versus the RP alleles of TLR genes. Further studies are needed to assess the production of secretory IgA and its relationship to TLR5 genotypes.

Gall bladder mucoceles in Border terriers

Background Gall bladder mucoceles (GBM) are a leading cause of biliary disease in dogs with several breeds, including the Shetland Sheepdog, American Cocker Spaniel, Chihuahua, Pomeranian, and Miniature Schnauzer apparently predisposed. Objective To determine risk factors, clinical features, and response to treatment of GBM in Border terriers (BT). Animals Medical records of 99 dogs (including 51 BT) with an ultrasonographic (±histopathologic) diagnosis of GBM from three referral centers in the United Kingdom were collected. A control group of 87 similar‐aged BT with no ultrasonographic evidence of gall bladder disease was selected for comparison. Method Retrospective case‐control study. Odds ratios were calculated to establish breed predisposition. Signalment, presence of endocrine disease, clinicopathologic results, and outcome were compared between the BT, other breeds, and control BTs. Results The odds of identifying a GBM in a BT in this hospital population was 85 times that of all other breeds (95% confidence interval 56.9‐126.8). BT had similar clinical signs and clinicopathologic changes to other breeds with GBM. There was no evidence that endocrinopathies were associated with GBM in BT. Clinical Significance A robust breed predisposition to GBM is established for the BT.

Alterations in serum amino acid concentrations in dogs with protein-losing enteropathy

Background Certain amino acids are decreased in humans with inflammatory bowel disease (IBD) and supplementation with the same amino acids has shown beneficial effects in animal models of IBD. Currently, the amino acid status of dogs with protein‐losing enteropathy (PLE) is unknown. Hypothesis/Objective To determine if serum amino acid concentrations are abnormal in dogs with PLE and correlated with clinical and laboratory variables and outcome. Animals Thirty client‐owned dogs diagnosed with PLE and 12 apparently healthy dogs seen at Bristol Veterinary School. Methods Retrospective study using stored residual serum from fasted dogs with PLE, collected at the time of diagnostic investigation and from apparently healthy dogs. Serum was analyzed for 30 amino acids using an automated high‐performance liquid chromatography amino acid analyzer. Results Serum tryptophan concentrations were significantly decreased in dogs with PLE (median, 22 nmol/mL; range, 1–80 nmol/mL) compared with apparently healthy control dogs (median, 77.5 nmol/mL; range, 42–135 nmol/mL, P < .001). There were no significant differences in the remaining 29 serum amino acids between dogs with PLE and apparently healthy. Serum tryptophan concentrations were also significantly correlated with serum albumin concentrations in dogs with PLE (P = .001, R 2 = 0.506). Conclusions and Clinical Importance Decreased serum tryptophan concentration might play a role in the pathogenesis of canine PLE or be a consequence of the disease.