Brian Catchpole

Canine diabetes mellitus: can old dogs teach us new tricks?

BackgroundDiabetes is common in dogs, with an estimated prevalence of 0.32% in the UK. Clinical signs, as in man, include polydipsia, polyuria and weight loss, associated with hyperglycaemia and glucosuria. Diabetes typically occurs in dogs between 5 and 12 years of age, and is uncommon under 3 years of age. Breeds predisposed to diabetes include the Samoyed, Tibetan Terrier and Cairn Terrier, while others such as the Boxer and German Shepherd Dog seem less susceptible. These breed differences suggest a genetic component, and at least one dog leucocyte antigen haplotype (DLA DRB1*009, DQA1*001, DQB1*008) appears to be associated with susceptibility to diabetes.MethodsCanine diabetes can be classified into insulin deficiency diabetes (IDD), resulting from a congenital deficiency or acquired loss of pancreatic beta cells, or insulin resistance diabetes resulting mainly from hormonal antagonism of insulin function.ResultsThere is no evidence for a canine equivalent of human type 2 diabetes. Adult-onset IDD, requiring insulin therapy, is the most common form, with pancreatitis and/or immune-mediated beta cell destruction considered to be the major underlying causes of the disease.DiscussionAutoantibodies to insulin, recombinant canine GAD65 and/or canine islet antigen-2 have been identified in a proportion of newly diagnosed diabetic dogs, suggesting that autoimmunity is involved in the pathogenesis of disease in some patients.ConclusionThe late onset and slow progression of beta cell dysfunction in canine diabetes resembles latent autoimmune diabetes of the adult in man.

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.

Study of 253 dogs in the United Kingdom with diabetes mellitus

Clinical information and blood samples were collected from 253 dogs with naturally occurring diabetes mellitus. Over half of them were labrador retrievers, collies, Yorkshire terriers or crossbred dogs, and approximately 80 per cent of them were diagnosed between the ages of five and 12 years. The majority of the dogs were receiving insulin therapy once a day, but in the dogs receiving insulin injections twice a day there was a trend for lower serum fructosamine concentrations, suggesting better glycaemic control. The proportion of female dogs with diabetes was lower than in previous surveys. The disease was diagnosed more commonly in the winter months, a seasonal pattern also observed in human beings with diabetes, suggesting that similar environmental factors might be involved in the disease.

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.

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 Crohns 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.

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.

Evaluation of a continuous glucose monitoring system in cats with diabetes mellitus.

A continuous glucose monitoring system (CGMS) was evaluated in 14 cats with naturally occurring diabetes mellitus. The device measures interstitial fluid glucose continuously, by means of a sensor placed in the subcutaneous tissue. All cats tolerated the device well and a trace was obtained on 15/16 occasions. There was good correlation between the CGMS values and blood glucose concentration measured using a glucometer (r=0.932, P<0.01). Limitations to the use of the CGMS are its working glucose range of 2.2–22.2 mmol/l (40–400 mg/dl) and the need for calibration with a blood glucose measurement at least every 12 h. When compared to a traditional blood glucose curve, the CGMS is minimally invasive, reduces the number of venepunctures necessary to assess the kinetics of insulin therapy in a patient and provides a truly continuous glucose curve.

Genetics of canine diabetes mellitus: are the diabetes susceptibility genes identified in humans involved in breed susceptibility to diabetes mellitus in dogs?

Diabetes mellitus is a common endocrinopathy in companion animals, characterised by hyperglycaemia, glycosuria and weight loss, resulting from an absolute or relative deficiency in the pancreatic hormone insulin. There are breed differences in susceptibility to diabetes mellitus in dogs, with the Samoyed breed being overrepresented, while Boxers are relatively absent in the UK population of diabetic dogs, suggesting that genetic factors play an important role in determining susceptibility to the disease. A number of genes, linked with susceptibility to diabetes mellitus in humans, are associated with an increased risk of diabetes mellitus in dogs, some of which appear to be relatively breed-specific. Diabetes mellitus in dogs has been associated with major histocompatibility complex (MHC) class II genes (dog leucocyte antigen; DLA), with similar haplotypes and genotypes being identified in the most susceptible breeds. A region containing a variable number of tandem repeats (VNTR) and several polymorphisms have been identified in the canine insulin gene, with some alleles associated with susceptibility or resistance to diabetes mellitus in a breed-specific manner. Polymorphisms in the canine CTLA4 promoter and in other immune response genes are associated with susceptibility to diabetes mellitus in a number of pedigree breeds. Genome wide association studies are currently underway that should shed further light on the genetic factors responsible for the breed profile seen in the diabetic dog population.

Anti-insulin antibodies in dogs with naturally occurring diabetes mellitus

The presence of anti-insulin antibodies was determined by ELISA in serum samples from 30 diabetic dogs receiving bovine insulin therapy and 30 normoglycaemic dogs. Twenty of the diabetic dogs had significant reactivity to both bovine (heterologous) and porcine (homologous) insulin compared to control dogs. In contrast there was no significant difference between the two populations in reactivity to canine distemper virus (CDV) or canine thyroglobulin. The high degree of correlation between anti-bovine insulin and anti-porcine insulin antibodies suggested cross-reactivity which was confirmed by performing a competition ELISA, with antibody binding to bovine insulin inhibited by pre-incubating serum with porcine insulin. The insulin B-chain, rather than the A-chain was the most reactive component of the insulin molecule although in some cases, diabetics with antibody reactivity to whole insulin protein showed minimal reactivity to the individual subunits. The data suggest that treatment of diabetic dogs with bovine insulin can lead to anti-insulin antibody production. These antibodies cross-react with homologous insulin and recognise conformational as well as linear epitopes.