Anke Hendricks

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.

Do dog owners perceive the clinical signs related to conformational inherited disorders as 'normal' for the breed? A potential constraint to improving canine welfare

Selection for brachycephalic (foreshortened muzzle) phenotypes in dogs is a major risk factor for brachycephalic obstructive airway syndrome (BOAS). Clinical signs include respiratory distress, exercise intolerance, upper respiratory noise and collapse. Efforts to combat BOAS may be constrained by a perception that it is ‘normal’ in brachycephalic dogs. This study aimed to quantify ownerperception of the clinical signs of BOAS as a veterinary problem. A questionnaire-based study was carried out over five months on the owners of dogs referred to the Queen Mother Hospital for Animals (QMHA) for all clinical services, except for Emergency and Critical Care. Owners reported the frequency of respiratory difficulty and characteristics of respiratory noise in their dogs in four scenarios, summarised as an ‘owner-reported breathing’ (ORB) score. Owners then reported whether their dog currently has, or has a history of, ‘breathing problems’. Dogs (n = 285) representing 68 breeds were included, 31 of which were classed as ‘affected’ by BOAS either following diagnostics, or by fitting case criteria based on their ORB score, skull morphology and presence of stenotic nares. The median ORB score given by affected dogs’ owners was 20/40 (range 8–30). Over half (58%) of owners of affected dogs reported that their dog did not have a breathing problem. This marked disparity between owners’ reports of frequent, severe clinical signs and their perceived lack of a ‘breathing problem’ in their dogs is of concern. Without appreciation of the welfare implications of BOAS, affected but undiagnosed dogs may be negatively affected indefinitely through lack of treatment. Furthermore, affected dogs may continue to be selected in breeding programmes, perpetuating this disorder.

Frequency of superantigen-producing Staphylococcus intermedius isolates from canine pyoderma and proliferation-inducing potential of superantigens in dogs

This preliminary study investigated the potential role of staphylococcal superantigens in the pathogenesis of canine pyoderma. The staphylococcal enterotoxins A (SEA), SEB, SEC and SED, and the toxic shock syndrome toxin-1 (TSST-1) were assayed in isolates from skins of dogs with pyoderma. Culture supernatants from 25 of 96 isolates were positive for multiple superantigens, with SEA and SEC being the most frequently detected. In in vitro stimulation of canine peripheral blood mononuclear cells and quantitative flow cytometry revealed that low concentrations of SEA and SEB were potent stimulators of blastogenesis of T cells.

Prostaglandin E2 is Variably Induced by Bacterial Superantigens in Bovine Mononuclear Cells and Has a Regulatory Role for the T Cell Proliferative Response

Signal transduction in antigen presenting cells via MHC class II molecules induces production of prostaglandin E2 (PGE2) known to possess immunoregulatory potential. Since Staphylococcus aureus superantigens (SAgs) utilize MHC class II molecules as primary ligands, we wanted to know whether PGE2 is induced after in vitro SAg stimulation of bovine blood mononuclear cells (boMNC), and whether this arachidonic acid metabolite modulates the preferential SAg-induced proliferative response of bovine CD8+ T cells. SEB as well as SEA induced maximal amounts of PGE2 on day 2 of culture (1-2.5 x 10(-8) mol/l per 2 x 10(5) boMNC). PGE2 production could be inhibited completely by indomethacin (10(-5) mol/l) causing enhanced proliferation of boCD4+ T cells (174%) as well as of boCD8+ T cells (122%) between day 4 and 6 of the in vitro culture, however, only in a subset of the tested animals. Notably, the striking preference of proliferation of boCD8+ over boCD4+ T cells following SAg stimulation remained largely unchanged after inhibition of endogenous PGE2 synthesis or after addition of exogenous PGE2. Higher concentrations of exogenously added PGE2 (> or = 10(-8) mol/l) inhibited the proliferation reaction, mainly due to an increased death rate of both CD4+ and CD8+ blasts. In contrast, lower PGE2 concentrations between 10(-8)-10(-9) mol/l even slightly enhanced the proliferation of both T cell subsets, depending on the individual cell donor. Summing up: These data show that SAgs, indeed, can induce PGE2 production in boMNC which can enhance or reduce the proliferative response of bovine CD4+ and CD8+ T cells.

The effectiveness of systemic antimicrobial treatment in canine superficial and deep pyoderma: a systematic review

AIM To identify and evaluate existing evidence for the effectiveness of systemic antimicrobial treatments for naturally occurring superficial and deep canine pyoderma. METHOD Electronic searches of PubMed, MEDLINE and CAB Direct were carried out (25 May 2011) without date or language restrictions. Proceedings of ESVD/ECVD, AAVD/ACVD, NAVDF and WCVD annual congresses were searched. Unpublished studies were sought via the Veterinary Dermatology discussion list and Veterinary Information Network. RESULTS Seventeen full-length, peer-reviewed controlled trials reporting clinical outcomes of systemic antimicrobial treatment for canine pyoderma were identified. Outcomes specific to superficial or deep pyoderma were reported in nine and five studies, respectively. Five studies reported outcomes only for nondifferentiated pyoderma depth. Heterogeneity of study designs and outcome measures made meta-analysis inappropriate. A good level of evidence was identified supporting the high efficacy of subcutaneously injected cefovecin in superficial pyoderma and for oral amoxicillin-clavulanic acid in deep pyoderma. A fair level of evidence was identified for moderate to high efficacy of oral amoxicillin-clavulanic acid, clindamycin, cefadroxil, trimethoprim-sulphamethoxazole and sulfadimethoxine-ormetoprim in superficial pyoderma and oral pradofloxacin, oral cefadroxil and subcutaneously injected cefovecin in deep pyoderma. Eleven trials reported observations of adverse effects in treated pyoderma cases by intervention group; four dogs were withdrawn owing to the severity of adverse effects. CONCLUSIONS There is a need for greater numbers of adequately sized, blinded, randomized controlled trials evaluating systemic antimicrobial interventions for canine pyoderma. Improved differentiation between superficial and deep pyoderma in outcome reporting, outcome measure standardization and association of outcomes with causative bacterial species and their resistance patterns are required.

Impact of Facial Conformation on Canine Health: Brachycephalic Obstructive Airway Syndrome

The domestic dog may be the most morphologically diverse terrestrial mammalian species known to man; pedigree dogs are artificially selected for extreme aesthetics dictated by formal Breed Standards, and breed-related disorders linked to conformation are ubiquitous and diverse. Brachycephaly–foreshortening of the facial skeleton–is a discrete mutation that has been selected for in many popular dog breeds e.g. the Bulldog, Pug, and French Bulldog. A chronic, debilitating respiratory syndrome, whereby soft tissue blocks the airways, predominantly affects dogs with this conformation, and thus is labelled Brachycephalic Obstructive Airway Syndrome (BOAS). Despite the name of the syndrome, scientific evidence quantitatively linking brachycephaly with BOAS is lacking, but it could aid efforts to select for healthier conformations. Here we show, in (1) an exploratory study of 700 dogs of diverse breeds and conformations, and (2) a confirmatory study of 154 brachycephalic dogs, that BOAS risk increases sharply in a non-linear manner as relative muzzle length shortens. BOAS only occurred in dogs whose muzzles comprised less than half their cranial lengths. Thicker neck girths also increased BOAS risk in both populations: a risk factor for human sleep apnoea and not previously realised in dogs; and obesity was found to further increase BOAS risk. This study provides evidence that breeding for brachycephaly leads to an increased risk of BOAS in dogs, with risk increasing as the morphology becomes more exaggerated. As such, dog breeders and buyers should be aware of this risk when selecting dogs, and breeding organisations should actively discourage exaggeration of this high-risk conformation in breed standards and the show ring.

How Long and Low Can You Go? Effect of Conformation on the Risk of Thoracolumbar Intervertebral Disc Extrusion in Domestic Dogs

Intervertebral disc extrusion (IVDE) is a common neurological disorder in certain dog breeds, resulting in spinal cord compression and injury that can cause pain and neurological deficits. Most disc extrusions are reported in chondrodystrophic breeds (e.g. Dachshunds, Basset Hounds, Pekingese), where selection for ‘long and low’ morphologies is linked with intervertebral discs abnormalities that predispose dogs to IVDE. The aim of this study was to quantify the relationship between relative thoracolumbar vertebral column length and IVDE risk in diverse breeds. A 14 month cross-sectional study of dogs entering a UK small animal referral hospital for diverse disorders including IVDE was carried out. Dogs were measured on breed-defining morphometrics, including back length (BL) and height at the withers (HW). Of 700 dogs recruited from this referral population, measured and clinically examined, 79 were diagnosed with thoracolumbar IVDE following diagnostic imaging ± surgery. The BL:HW ratio was positively associated with IVDE risk, indicating that relatively longer dogs were at increased risk, e.g. the probability of IVDE was 0.30 for Miniature Dachshunds when BL:HW ratio equalled 1.1, compared to 0.68 when BL:HW ratio equalled 1.5. Additionally, both being overweight and skeletally smaller significantly increased IVDE risk. Therefore, selection for longer backs and miniaturisation should be discouraged in high-risk breeds to reduce IVDE risk. In higher risk individuals, maintaining a lean body shape is particularly important to reduce the risk of IVDE. Results are reported as probabilities to aid decision-making regarding breed standards and screening programmes reflecting the degree of risk acceptable to stakeholders.

Intradermal test reactivity to Malassezia pachydermatis in atopic dogs

THE commensal yeast Malassezia pachydermatis is now recognised as a common cause of otitis and dermatitis in dogs (Gustafson 1955, Dufait 1983, Larsson and others 1988, Mason and Evans 1991). The factors which favour proliferation of the yeast are poorly understood. Concurrent skin diseases are often recognised and, among these, atopic disease is frequently seen in dogs with Malassezia dermatitis (Mason and Evans 1991, Bond and others 1996). Groups of atopic dogs have been shown to have elevated skin populations of the yeast when compared with groups of healthy dogs (Bond and others 1994, White and others 1998), although Mpachydermatis populations in the skin of individual atopic dogs can be either elevated or within normal limits. In the USA, immediate intradermal test reactivity has been observed in atopic dogs following the injection of Mpachydermatis extracts at concentrations which cause no reaction in healthy dogs, suggesting that type I hypersensitivity responses to yeast allergens could contribute to the clinical signs in some cases of canine atopic disease (Morris and oth-

Cornification defect in the Golden retriever: clinical, histopathological, ultrastructural and genetic characterisation

Veterinarians have recognised a nonpruritic skin disease in the Golden retriever breed characterised by excessive scaling of large, variably pigmented flakes of skin in otherwise healthy dogs. This prospective case series describes clinical, histopathological, ultrastructural and genetic features of this cornification defect in 17 affected dogs. The condition affects young dogs of either sex and is characterised by symmetrical, predominantly ventro-lateral scaling and hyperpigmentation of the trunk. Histopathological and ultrastructural changes of the stratum corneum are suggestive of delayed degradation of corneodesmosomes. A genetic aetiology is proposed and a single-trait autosomal recessive mode of inheritance discussed.

Superantigen-dependent accelerated death of bovine neutrophilic granulocytes in vitro is mediated by blood mononuclear cells.

While classical interactions of bacterial superantigens (SAgs) with antigen presenting cells and T cells have been studied intensively, the potential interactions of SAgs with granulocytes (PMNs) have gained much less attention. We investigated if in the bovine system SAgs have any direct or indirect influence on the fate of granulocytes, which are among those cells primarily responsible for the elimination of superantigen-producing bacteria. The tested SAgs (SEA, SEB) had no apparent direct effect on PMN viability (neutrophils and eosinophils). However, in the presence of blood mononuclear cells (MNCs), SAgs led to an accelerated death of neutrophils but not of eosinophils. Compared to medium controls, in SAg-stimulated cultures only about 20-50% of the neutrophils survived after 24 hours in vitro. Accelerated death of neutrophils required the presence of at least 10% MNC and started between 2.5-24 h after initiation of the co-culture between MNC and PMN. Minimal effective SEA concentrations ranged between 10-100 pg/l (SEB 0.1-10 ng/l). The effect could be mimicked by culture supernatants of SAg-stimulated MNCs, suggesting that direct cell-cell interactions are not required for the killing. In the human system, where we tested the role of TNF-alpha, an antibody specific for this cytokine was not able to abolish the death of human neutrophils. Brefeldin A, an inhibitor of golgi transport and cytokine secretion, which blocked the SAg-induced activation of bovine MNC did not abolish the killing of neutrophils. Blocking of nitric oxide generation or PGE2 synthesis also could not alter the SAg-induced killing of bovine neutrophils. The observed indirect negative effects of SAgs on neutrophils may provide new insights in mechanisms by which superantigens modulate the hosts immune response.