Anal Furunculosis Research Articles

The Intersection of Human and Veterinary Medicine-A Possible Direction towards the Improvement of Cell Therapy Protocols in the Treatment of Perianal Fistulas.

The effective treatment of perianal fistulizing Crohn's disease is still a challenge. Local administration of mesenchymal stromal cells (MSCs) is becoming a part of accepted treatment options. However, as a fledgling technique, it still can be optimized. A new trend in translational research, which is in line with "One Health" approach, bases on exploiting parallels between naturally occurring diseases affecting humans and companion animals. Canine anal furunculosis (AF) has been indicated as condition analogous to human perianal Crohn's disease (pCD). This narrative review provides the first comprehensive comparative analysis of these two diseases based on the published data. The paper also outlines the molecular mechanisms of action of MSCs which are likely to have a role in modulating the perianal fistula niche in humans, and refers them to the current knowledge on the immunomodulatory properties of canine MSCs. Generally, the pathogenesis of both diseases shares main determinants such as the presence of genetic predispositions, dysregulation of immune response and the relation to intestine microbiota. However, we also identified many aspects which should be further specified, such as determining the frequency of true fistulas formation in AF patients, elucidating the role of TNF and Th17 pathway in the pathogenesis of AF, or clarifying the role of epithelial-to-mesenchymal transition phenomenon in the formation of canine fistulae. Nevertheless, the available data support the hypothesis that the results from testing cell therapies in dogs with anal furunculosis have a significant translational value in optimizing MSC transplants procedures in pCD patients.

Dysbiosis in a canine model of human fistulizing Crohn’s disease

ABSTRACT Crohn’s disease (CD) is a chronic immune-mediated inflammatory condition caused by the loss of mucosal tolerance toward the commensal microbiota. On average, 29.5% and 42.7% CD patients experience perianal complications at 10 and 20 y after diagnosis, respectively. Perianal CD (pCD) result in high disease burden, diminished quality of life, and elevated health-care costs. Overall pCD are predictors of poor long-term outcomes. Animal models of gut inflammation have failed to fully recapitulate the human manifestations of fistulizing CD. Here, we evaluated dogs with spontaneous canine anal furunculosis (CAF), a disease with clinical similarities to pCD, as a surrogate model for understanding the microbial contribution of human pCD pathophysiology. By comparing the gut microbiomes between dogs suffering from CAF (CAF dogs) and healthy dogs, we show CAF-dog microbiomes are either very dissimilar (dysbiotic) or similar (healthy-like), yet unique, to healthy dog’s microbiomes. Compared to healthy or healthy-like CAF microbiomes, dysbiotic CAF microbiomes showed an increased abundance of Bacteroides vulgatus and Escherichia coli and a decreased abundance of Megamonas species and Prevotella copri. Our results mirror what have been reported in previous microbiome studies of patients with CD; particularly, CAF dogs exhibited two distinct microbiome composition: dysbiotic and healthy-like, with determinant bacterial taxa such as E. coli and P. copri that overlap what it has been found on their human counterpart. Thus, our results support the use of CAF dogs as a surrogate model to advance our understanding of microbial dynamics in pCD.

Anal sac diseases and perianal fistula

Disease of the anal sac is a common reason for presentation of dogs (and less commonly cats) to the veterinary surgeon. They are often just emptied by a veterinary surgeon or veterinary nurse, but this does not always lead to resolution of clinical signs. An understanding of the diseases affecting the anal sacs and possible treatment options can increase the likelihood of a positive outcome. This paper will look at these issues, together with mention of perianal fistula (anal furunculosis) a disease that often involves the anal sacs.

Treatment of perianal fistulas with human embryonic stem cell-derived mesenchymal stem cells: a canine model of human fistulizing Crohn's disease.

To evaluate the safety and efficacy of intralesional injection of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) in canine anal furunculosis dogs. Dogs naturally develop an immune-mediated disease called canine anal furunculosis, which shares many features with human fistulizing Crohn's disease. The hESC-MSCs were well tolerated and 1 month postinjection, accompanied by reduced serum levels of IL-2 and IL-6, two inflammatory cytokines associated with Crohn's disease. All six dogs were found to be completely free of fistulas at 3 months postinjection. However, at 6 months, two dogs had some fistula relapse. Results of this study provide the first evidence of the safety and therapeutic potential of hESC-MSCs in a large animal model.

Genetics of canine anal furunculosis in the German shepherd dog

Canine anal furunculosis (AF) is characterised by ulceration and fistulation of perianal tissue and is a disease that particularly affects German shepherd dogs (GSDs). There are some similarities between AF and perianal Crohn's disease (CD) in man. An immune-mediated aetiopathogenesis for AF has been suggested due to tissue pathology, a major histocompatibility complex (MHC) association and clinical response to ciclosporin. Genome-wide association studies (GWAS) can be conducted in dogs with fewer markers and individuals than would be required in a human study. A discovery GWAS was performed on 21 affected and 25 control GSDs from the UK. No SNPs reached genome-wide significance levels at this stage. However, 127 nominally associated SNPs were genotyped in further 76 cases and 191 controls from the UK and Finland. Sequencing of these regions was undertaken to discover novel genetic variation. Association testing of these variants in the UK and Finnish cohorts revealed nine significantly associated SNPs, six of which cause non-synonymous changes in protein sequence. The ADAMTS16 and CTNND2 gene regions were most significantly associated with disease. Members of the butyrophilin protein family, important in intestinal inflammatory regulation, were also associated with disease, but their independence from the MHC region remains to be established. The CTNND2 gene region is also interesting as this locus was implicated in human ulcerative colitis and CD, albeit at a different candidate gene: DAP. We suggest that this represents a common association between inflammatory bowel disease-related conditions in both species and believe that future studies will strengthen this link.

Expanded dog leukocyte antigen (DLA) single nucleotide polymorphism (SNP) genotyping reveals spurious class II associations

The dog leukocyte antigen (DLA) system contains many of the functional genes of the immune system, thereby making it a candidate region for involvement in immune-mediated disorders. A number of studies have identified associations between specific DLA class II haplotypes and canine immune hemolytic anemia, thyroiditis, immune polyarthritis, type I diabetes mellitus, hypoadrenocorticism, systemic lupus erythematosus-related disease complex, necrotizing meningoencephalitis (NME) and anal furunculosis. These studies have relied on sequencing approximately 300 bases of exon 2 of each of the DLA class II genes: DLA-DRB1, DLA-DQA1 and DLA-DQB1. In the present study, an association (odds ratio=4.29) was identified by this method between Weimaraner dogs with hypertrophic osteodystrophy (HOD) and DLA-DRB1∗01501. To fine map the association with HOD, a genotyping assay of 126 coding single nucleotide polymorphisms (SNPs) from across the entire DLA, spanning a region of 2.5 Mb (3,320,000-5,830,000) on CFA12, was developed and tested on Weimaraners with HOD, as well as two additional breeds with diseases associated with DLA class II: Nova Scotia duck tolling retrievers with hypoadrenocorticism and Pug dogs with NME. No significant associations were found between Weimaraners with HOD or Nova Scotia duck tolling retrievers with hypoadrenocorticism and SNPs spanning the DLA region. In contrast, significant associations were found with NME in Pug dogs, although the associated region extended beyond the class II genes. By including a larger number of genes from a larger genomic region, a SNP genotyping assay was generated that provides coverage of the extended DLA region and may be useful in identifying and fine mapping DLA associations in dogs.

Lack of effects on lymphocyte function from chronic topical ocular cyclosporine medication: a prospective study

Topical cyclosporine has been widely used in the treatment of canine keratoconjunctivitis sicca without apparent documented clinical side effects. Thus the finding of reduced lymphocyte proliferation in animals treated with the drug at a concentration of 2% was both surprising and concerning. This study aimed to repeat the previous study and to compare the systemic effects of 2% cyclosporine in corn oil and 0.2% topical cyclosporine ointment (Optimmune, Intervet-Schering Plough, Welwyn, UK). Twenty dogs treated with Optimmune or with topical 2% cyclosporine in corn oil where previous treatment with Optimmune had failed were included in this study. Blood samples were taken at the time of first evaluation and at 1, 3 and 6 months of treatment to provide a biochemical and hematological health evaluation of the dogs and at each examination to measure circulating levels of cyclosporine and to obtain a lymphocyte population with which to determine a mitogen stimulation index (MSI) on treatment with phytohaemagglutinin-P (PHA) and conconavlin A (con-A). Levels of circulating cyclosporine were measured with an enzyme-multiplied immunoassay method and also the more sensitive quantification technique of mass spectroscopy (MS). No blood samples contained over 15 ng/ml cyclosporine, the lower limit of detection using the radioimmunoassay or the enzyme-multiplied immunoassay technique. Positive control samples taken from dogs treated with oral cyclosporine for anal furunculosis showed measurable levels in blood, demonstrating that the technique worked. Mean MSI values at 0, 1, 3 and 6 months of treatment were 10.2, 11.4, 11.6, and 10.5 for dogs treated with 0.2% cyclosporine and 10.4, 11.9, 11.7, and 12.9 for dogs treated with 2% cyclosporine. Mitogen stimulation index values were not statistically different between the first examination and any subsequent examination time-point. The findings of the study contradict those of the previous studies. No change in lymphocyte stimulation index was noted, neither were significant blood levels of cyclosporine documented after topical administration of either 0.2% or 2% cyclosporine. This study shows that topical cyclosporine is safe to use in the canine eye in line with the drug's safety record in this therapeutic regime over the past 20 years since its first use.

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.

Interleukin-2 and interferon-gamma mRNA expression in canine anal furunculosis lesions and the effect of ciclosporin therapy

German Shepherd Dogs have an increased incidence of anal furunculosis (AF), which is a disease characterised by inflammation and ulceration of the perianal tissues. Ciclosporin, an immunosuppressive drug, has been successfully used to treat AF, suggesting that the pathogenesis of disease is likely to have an immune-mediated component. Previous research has shown that the cytokine mRNA profile in AF lesions is consistent with T cell-mediated inflammation. The aims of the current study were to quantify IL-2 and IFNγ mRNA expression in AF biopsies taken before and after treatment with ciclosporin and to compare cytokine expression with lesion severity. Twenty-two dogs with AF were recruited into the study and lesional biopsies were taken prior to ciclosporin therapy. Lesion severity was graded using a visual analogue scale. All dogs were evaluated after 4 weeks of ciclosporin therapy and, in 10 dogs with persistent disease, residual lesions were resected. RNA was extracted from AF-lesional tissue and control perianal tissue samples ( n = 10), which was used as the template for RT-PCR. Analysis of IL-2 and IFNγ mRNA expression was performed using real-time PCR. IL-2 and IFNγ mRNA was consistently detected in pre-treatment AF biopsies and, when quantified, this was significantly increased compared to control tissue ( P < 0.05). However, no correlation was seen between lesion severity and pre-treatment cytokine mRNA expression. In the ten paired pre- and post-treatment samples, IL-2 mRNA expression was significantly reduced in residual disease tissue following ciclosporin therapy ( P = 0.013). Treatment with ciclosporin seemed to result in decreased expression of IFNγ mRNA in AF lesions but this was not statistically significant. In six of the 10 dogs with persistent disease, T cell cytokine mRNA could still be detected in the tissues, suggesting that there was inadequate immunosuppression. The absence of a correlation between T cell cytokine expression and the severity of disease suggests that tissue destruction observed in AF might be a consequence of other inflammatory mediators or downstream effects of T cell activation.

Pattern-recognition receptor mRNA expression and function in canine monocyte/macrophages and relevance to canine anal furunuclosis

Pattern-recognition receptors (PRRs) are important components of the innate immune system, enabling early detection of infection. Defective PRR function has been implicated in several infectious and immune-mediated diseases of human beings, including Crohn's disease (CD). Anal furunculosis (AF) is an immune-mediated disease which primarily occurs in German shepherd dogs (GSD) and could result from a similar type of PRR dysfunction. The aim of the current study was to investigate canine PRR responses in vitro and to test the hypothesis that these were altered in AF-affected GSD. The pattern-recognition receptors TLR1, TLR2, TLR4, TLR6, TLR9, NOD1 (nucleotide-binding oligomerisation domain) and NOD2 were evaluated in the DH82 canine monocyte/macrophage cell line. These cells were found to express mRNA for all the selected PRRs with TLR2 mRNA the most and TLR5 mRNA the least abundant. A similar pattern of expression was found in canine blood-derived monocyte/macrophages. Stimulation of DH82 cells and blood-derived monocyte/macrophages using specific PRR-ligands, resulted in expression of pro-inflammatory cytokine mRNA. Quantification of TNFα mRNA and protein secretion from stimulated cells demonstrated variable responses with lipopolysaccharide (TLR4 ligand) and PAM 3CSK4 (TLR1/2 ligand) proving to be the most potent and CpG DNA (TLR9 ligand) the least potent. Comparing PRR responses in blood-derived monocyte/macrophages from healthy blood-donor dogs with those from AF-affected GSD showed a deficiency in the latter in response to LD-MDP (NOD2 ligand) at the mRNA level but not at the protein level. It is possible that dysfunctional NOD2 responses by cells of the monocyte/macrophage lineage are involved in the pathogenesis of AF.

Risk of anal furunculosis in German Shepherd dogs is associated with the major histocompatibility complex

Anal furunculosis (AF) is a chronic, progressive inflammatory disease of the perianal tissues most frequently affecting middle-aged or older German Shepherd dogs (GSD). Because this breed accounts for over 80% of all reported cases, there is likely to be a genetic association with disease susceptibility. Although there are some similarities with perianal fistulation that occurs in human Crohn's disease, the aetiology and pathogenesis of AF are still poorly understood. Recent research has suggested an immune-mediated aetiology, and evidence for this has been further provided by clinical responses to the immunosuppressive drug cyclosporin. The aim of the current study was to investigate canine major histocompatibility complex immune response genes. Dog leucocyte antigen class II alleles and haplotypes were characterised by sequence-based typing of 107 GSD affected with AF and 196 breed-matched controls collected in the UK. A highly significant association of DLA-DRB1*00101 with the presence of AF was observed (OR = 5.01, CI = 2.7-9.3, P < 0.00000001). This association was confirmed in a second cohort of GSD collected in Finland. Homozygosity for this allele is associated with an earlier disease onset.

Preoperative Immunosuppressive Therapy and Surgery as a Treatment for Anal Furunculosis

To evaluate the efficacy of combining preoperative immunosuppressive therapy with surgical excision for treatment of anal furunculosis (AF) in dogs. Retrospective study. Dogs (n=25) with stages 1-4 AF. Preoperative immunosuppressive therapy was either cyclosporine A (CyA) alone or in combination with ketoconazole (Group 1; n=18), or azathioprine combined with prednisolone (Group 2; n=7). Surgical excision of residual draining tracts, cryptectomy, and anal sacculectomy were performed. Only dogs with postoperative clinical follow-up exceeding 9 months were retained for the study. Both immunosuppressive protocols were effective in reducing progression of AF. Subsequent draining tracts excision, cryptectomy, and anal sacculectomy were bilateral (12 dogs) or unilateral (13 dogs of which 4 had bilateral anal sacculectomy). Postoperative recovery was uneventful, except for 2 dogs that had wound breakdown. Recurrence was not observed in any of the dogs that had bilateral surgical excision and or in 9 dogs that had unilateral excision. Preoperative immunosuppressive therapy, combined with bilateral surgical resection of affected tissue consistently, resulted in resolution of AF. Four dogs that had recurrence had unilateral excision despite initial bilateral involvement, suggesting that all diseased tissue should be excised. These preliminary results suggest that immunosuppressive therapy before surgical excision for AF yields minimizes recurrence in dogs.

Matrix metalloproteinase mRNA expression in canine anal furunculosis lesions

Although the aetiology of anal furunculosis (AF) in dogs is poorly understood, there is evidence for an underlying immune dysfunction. This is illustrated by the presence of a T helper type 1 cytokine mRNA profile in AF lesions and the clinical response to ciclosporin therapy. Expression of MMPs 2, 9 and 13 were evaluated in AF lesional biopsies by real-time quantitative RT-PCR. There was significantly increased expression of both MMP-9 and MMP-13 mRNA in AF biopsies compared to controls ( p < 0.001) but no significant difference in MMP-2 mRNA expression. Since MMP-9 and MMP-13 are primarily produced by macrophages, these data suggest that ulceration could be the result of aberrant activation of this cell type in the tissues. It is feasible that such pathological macrophage activity occurs in response to interferon-gamma secreted by T helper type 1 cells. This could explain why the lesions resolve following treatment with the immunosuppressive drug ciclosporin.

The role of surgery in the management of canine anal furunculosis. A review of the literature and a retrospective evaluation of treatment by surgical resection in 51 dogs

AIMS: To retrospectively evaluate the outcome of surgical management of anal furunculosis (AF) in 51 dogs. To compare the outcome of surgery with those of current medical protocols by way of a review of the literature. METHODS: Dogs referred for treatment of AF (n=51) were treated by en bloc surgical resection of diseased tissue and primary wound closure. This technique was combined with bilateral anal sacculectomy in all cases in which the anal sacs had not been previously removed. Immunomodulatory therapies were not used, with the exception of post-operative metronidazole antibiosis for 7–10 days. Follow-up was performed by the author using a telephone questionnaire. RESULTS: Forty-eight dogs were eligible for post-operative follow-up 1.5 to 36 (mean 17.4, median 18) months after surgery. Lesion recurrence, faecal incontinence and stricture formation occurred in 2%, 4% and 13% of dogs, respectively. The percentage of dogs considered by their owners to have an acceptable level of faecal continence and an improved quality of life was 94%. CONCLUSIONS AND CLINICAL RELEVANCE: The aetiopathogenesis of canine AF remains unclear. Whilst recent advances in medical management by the use of various immunomodulatory medications (such as cyclosporine) hold promise, this approach has yet to be refined with respect to affordability, long-term efficacy and morbidity. With meticulous surgical dissection and reconstruction techniques (anoplasty), excellent success rates can be achieved following a single surgical procedure with minimal complications. Surgery remains a viable treatment option, alone or in combination with immunomodulatory medications, until a more thorough understanding of this debilitating disease is achieved.

Cyclosporine treatment of anal furunculosis in 26 dogs

To evaluate the effect of cyclosporine on anal furunculosis lesions in 26 dogs. Lesions were graded as mild in 11 dogs, moderate in eight and severe in seven. Each dog was treated with approximately 4 mg/kg cyclosporine orally every 12 hours until the lesions resolved or showed no further improvement. Residual lesions were resected surgically. Eighteen dogs (69 per cent) experienced complete resolution, seven (27 per cent) improved but had residual lesions and one (4 per cent) showed no improvement. The mean duration of treatment until resolution or no further improvement was 8.8 weeks (range four to 24 weeks). Nine dogs (35 per cent) experienced recurrence. Six were from the group that had shown complete resolution and three were from the group that had surgery. Fifteen dogs (58 per cent) developed side effects to cyclosporine, although none required treatment to be discontinued. Mean duration of follow-up was 6.8 months (range one to 20 months). Cyclosporine was effective at resolving or reducing anal furunculosis lesions in 25 of 26 dogs (96 per cent). However, residual or recurrent lesions remain a potential problem, and surgical resection or long-term cyclosporine treatment may be necessary in some dogs.

Expression of cytokine mRNA in canine anal furunculosis lesions

The pattern of expression of cytokine MRNA in the lesions of anal furunculosis was evaluated in tissue biopsies from 15 dogs, and compared with the pattern in control skin samples...

Evaluation of once daily treatment with cyclosporine for anal furunculosis in dogs

Twenty.four dogs with anal furunculosis were treated with cyclosporine once daily for 13 weeks at dosages of 1.5, 3.0, 5.0 or 7.5 mg/kg, and re.examined after six and 12 months....

43. Expression of cytokine mRNA in canine anal furunculosis lesions

43. Expression of cytokine mRNA in canine anal furunculosis lesions

Association between anal furunculosis and colitis in the dog: preliminary observations.

Treatment of anal furunculosis in dogs is often unsatisfactory and may be associated with significant recurrence and complications. This may be compounded by the simultaneous presence of colitis in affected animals. Clinical signs associated with colitis and anal furunculosis may be similar, including faecal tenesmus, dyschezia and haematochezia. To examine the incidence of concurrent anal furunculosis and colitis, colonic biopsies were collected from 18 dogs referred for treatment of anal furunculosis. Nine dogs (50 per cent) had a histopathological diagnosis of colitis. Clinical signs more indicative of colitis than anal furunculosis (increased frequency of defecation, mucus in faeces and diarrhoea) were not observed more frequently in dogs with confirmed colitis compared with those with furunculosis alone. Therefore, while an association between colitis and anal furunculosis may exist, clinical signs alone cannot be used as an indicator of the presence of colitis in cases of anal furunculosis. The authors recommend that colonic biopsies should be undertaken in all dogs presented with anal furunculosis. Whether specific treatment of colitis in dogs with histopathological evidence of colitis improves the outcome of treatment for anal furunculosis awaits further study.