Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Canine memory T-cell subsets in health and disease.

A more complete understanding of canine T-lymphocyte immunity is necessary for improving diagnostic and therapeutic approaches to canine diseases, developing cell-based canine immunotherapeutics, and evaluating dogs as large mammal models for comparative immunology research. The aim of this study was to utilize CD45RA (indicating antigen inexperience) and CD62L (indicating lymph node homing capability), to quantify canine memory T-cell subsets in healthy dogs and dogs with various diseases. Peripheral blood mononuclear cells (PBMCs) were prospectively collected from dogs belonging to one of four groups:dermatologic inflammation (n = 9), solid tumors (n = 9), lymphoma (n = 9), and age-/weight-matched healthy control dogs (n = 15). Dogs receiving prednisone or any other immunomodulating medication within two weeks were excluded. Flow cytometry was performed and T-cell subsets were defined as CD4+ or CD8+, and naïve (TN), central memory (CM), effector memory (EM), or terminal effector memory re-expressing CD45RA (TEMRA). T-cell subset proportions were compared between each disease group and their healthy age-/weight-matched controls using a Mann-Whitney test. Significantly increased %CD8+ TN (P = 0.036) and decreased %CD8+ TEMRA (P = 0.045) were detected in dogs with dermatologic inflammation compared to healthy controls. Furthermore, %CD4+ TN positively correlated with Canine Atopic Dermatitis Extent and Severity Index (CADESI) score within the inflammation group (ρ = 0.817, P = 0.011). No significant differences between either cancer group and their healthy controls were detected. Taken together, these data indicate that dermatologic inflammation can alter proportions of peripheral blood T-cell subsets, possibly due to the migration of antigen-specific T-cells into tissues. Furthermore, these findings support the utility of CD45RA and CD62L in characterizing clinical canine immune responses.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Identification of major and minor chicken allergens in dogs.

BACKGROUND: Allergens targeted by serum-specific immunoglobulin E (sIgE) in dogs clinically allergic to chicken have not been reported. OBJECTIVES: To characterise the allergens targeted by sIgE in dogs sensitised and allergic to chicken. ANIMALS: Sera from three dogs not sensitised to chicken, from 10 chicken sensitised dogs and from 12 chicken allergic dogs. METHODS AND MATERIALS: Enzyme-linked immunosorbent assay (ELISA) and immunoblotting with a commercial chicken extract were utilized. The bands identified on immunoblotting were sequenced by mass spectrometry for allergen characterization. RESULTS: Using ELISA, we detected chicken-sIgE above the positive threshold in zero of three (0%) nonsensitised dogs, five of five (100%) chicken-sensitised dogs (a selection criterion), and in seven of 12 (58%) chicken-allergic dogs. Immunoblotting performed with the same extract revealed IgE-bound protein bands in 100% of all chicken-sensitised and -allergic dogs, respectively. To identify the allergens, we excised the corresponding bands on the electrophoretic gel, and submitted them for sequencing by mass spectrometry. We conclusively identified seven major allergens (serum albumin, pyruvate kinase M, enolase 3, creatine kinase M, lactate dehydrogenase A, glyceraldehyde-3-phosphate dehydrogenase and triose-phosphate isomerase) and one minor allergen (troponin C), which are relevant to dogs. CONCLUSIONS AND CLINICAL RELEVANCE: We identified herein seven major chicken allergens for dogs, several of which are known to be cross-reactive allergens for humans. Based on their degree of sequence identity, these allergens exhibit the theoretical potential to be cross-reactive between poultry and mammalian meats; six of these allergens already are known to be cross-reactive between chicken and fish species. Future studies should address the clinical relevance and cross-reactivity potential of these chicken allergens in dogs.

Feline allergic diseases: introduction and proposed nomenclature.

BACKGROUND: Feline allergic diseases present as challenging problems for clinicians, not least because of the number of reaction patterns of the feline skin, none of which are specific for allergy. Furthermore, there is some controversy over the nomenclature that should be used in their description. OBJECTIVES: To review the literature, assess the status of knowledge of the topic and the extent to which these diseases could be categorized as atopic in nature, and make recommendations concerning nomenclature. METHODS: Atopic diseases in humans and cats were researched. A comparison then was made of the essential features in the two species. RESULTS: There were sufficient similarities between human atopic diseases and the manifestations of feline diseases of presumed allergic aetiology to justify the use of "atopic" to describe some of the feline conditions affecting the skin, respiratory and gastrointestinal tract. However, none of the allergic skin diseases showed features consistent with atopic dermatitis as described in man and the dog. CONCLUSIONS AND CLINICAL IMPORTANCE: The term "Feline Atopic Syndrome" (FAS) is proposed to encompass allergic diseases of the skin, gastrointestinal tract and respiratory tract, and "Feline atopic skin syndrome" (FASS) proposed to describe allergic skin disease associated with environmental allergies. We are not aware of any adverse food reactions in cats that are attributable to causes other than immunological reactions against the food itself. We therefore propose an aetiological definition of "Food Allergy" (FA) to describe such cases.

Clinical signs and diagnosis of feline atopic syndrome: detailed guidelines for a correct diagnosis.

BACKGROUND: Feline atopic syndrome (FAS) describes a spectrum of hypersensitivity disorders characterised by highly diverse clinical presentations including skin, gastrointestinal and respiratory systems. Among these disorders is feline atopic skin syndrome (FASS), in which hypersensitivity is typically associated with environmental allergens, although food allergy may coexist. Involvement of other organ systems (e.g. asthma) also may occur. Because of its highly heterogeneous clinical presentation, diagnosis of FASS can be challenging. OBJECTIVES: A subgroup of the International Committee on Allergic Diseases of Animals was tasked to summarise the most current information on the clinical presentations of FASS and to develop diagnostic guidelines. METHODS AND MATERIALS: Online citation databases and abstracts from international meetings were searched for publications related to feline allergic conditions. These were combined with expert opinion where necessary. RESULTS: A total of 107 publications relevant to this review were identified. Compilation of these data enabled development of a detailed description of the clinical features of FASS and development of guidelines focusing on systematic elimination of other skin conditions with similar clinical characteristics. As allergen tests are frequently used by dermatologists to support a clinical diagnosis of FASS, a brief review of these methodologies was also performed. CONCLUSIONS AND CLINICAL IMPORTANCE: In a similar way to atopic dermatitis in dogs, FASS is a clinical diagnosis based on the presence of compatible clinical signs and exclusion of other diseases with similar clinical features. Elimination or exclusion of fleas/flea allergy, other parasites, infections and food allergy is mandatory before reaching a diagnosis of FASS.

Serum IgE and IgG responses to dietary antigens in dogs with and without cutaneous adverse food reactions.

BACKGROUND: It is suspected that many canine cutaneous adverse food reactions (CAFR) are true immunological hypersensitivities; however, few specific dietary allergens have been identified. OBJECTIVE: To compare serum immunoglobulin (Ig)E and IgG reactivity to specific food antigens in privately owned dogs with and without CAFR. ANIMALS: Eighteen adult dogs with nonseasonal pruritus recruited from a hospital population. METHODS AND MATERIALS: Dogs were fed an extensively hydrolysed poultry-based diet exclusively for 12 weeks. Serum was collected at the beginning of the trial. Canine atopic dermatitis extent and severity index and pruritus Visual Analog Scale scoring were performed at the beginning and end of the trial. Immunoblotting was performed to identify IgE and/or IgG binding to specific proteins in beef, egg, milk, chicken, pork, soy and wheat extracts. RESULTS: A CAFR (defined as an unequivocal relapse of pruritus after dietary challenge) was diagnosed in 10 dogs, with 60% relapsing when fed chicken-based diets. Binding of subjects' IgG to almost all proteins in all extracts was seen regardless of reported dietary history. Few proteins were exclusively or predominantly bound by IgE in CAFR dogs. Exceptions included a 42 kDa band (chicken), a 52 kDa band (beef), a 46 kDa band (beef and milk) and a poorly defined high molecular weight protein or proteins (beef and milk). CONCLUSION: This study demonstrated three protein bands and a poorly defined band predominantly recognized by sera from dogs with CAFR relative to non-CAFR dog sera. Almost all proteins were bound by IgG in all dogs, suggesting prior exposure to unreported foods.

Residual antibacterial activity of canine hair treated with five mousse products against Staphylococcus pseudintermedius in vitro.

BACKGROUND: Topical therapy alone can be effective in the treatment of canine pyoderma. Topical products are commercially available as shampoos, sprays, wipes and mousses. To date, no studies have evaluated the efficacy of commercially available mousse products in the treatment of canine pyoderma. OBJECTIVE: To determine the residual antibacterial activity of canine hairs treated with mousse products containing different active ingredients. ANIMALS: Fifteen client-owned dogs with no history of dermatological disease. METHODS AND MATERIALS: Dogs were treated once with five mousse products [(i) 2% chlorhexidine and 1% ketoconazole, (ii) 2% chlorhexidine and 2% miconazole, (iii) 3% chlorhexidine and 0.5% climbazole, (iv) 2% salicylic acid 10% ethyl lactate and (v) phytosphingosine HCl 0.05%; control]. Hair samples were collected from each treatment area before application, one hour after application and on days 2, 4, 7, 10 and 14 post-treatment. Collected hairs were weighed and plated on Mueller-Hinton agar plates streaked with a Staphylococcus pseudintermedius isolate showing no antimicrobial resistance. Plates were incubated for 24 h and bacterial growth inhibition zones around the hairs were measured. RESULTS: Mousses 1, 2 and 3 created significant inhibition zones up to Day 10 when compared to pre-treatment samples. On Day 14, only mousse 3 produced a significant zone of inhibition when compared to the pre-treatment sample. Mousses 4 and 5 showed no statistical difference between any of the samples. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest that three of the mousse products had residual activity in inhibiting S. pseudintermedius growth in vitro for at least 10 days.

Quantitation of anti-Pythium insidiosum antibodies before and after administration of an immunotherapeutic product to healthy dogs.

OBJECTIVE To evaluate the effect of an immunotherapeutic product on concentrations of anti-Pythium insidiosum antibodies in dogs. ANIMALS 7 healthy hound-crossbreds. PROCEDURES Antibody concentrations were evaluated before (day 0) and after administration of the immunotherapeutic product. The immunotherapeutic product was administered on days 0, 7, and 21. Serum was obtained on days 0, 7, 14, 21, 28, 35, 42, 49, and 56. Anti-P insidiosum antibody concentrations were measured and reported as the percentage positivity relative to results for a strongly positive control serum. RESULTS Mean ± SD percentage positivity before administration of the immunotherapeutic product was 7.45 ± 3.02%. There was no significant change in anti-P insidiosum antibody concentrations after administration of the product, with percentage positivity values in all dogs remaining within the range expected for healthy dogs (3% to 15%). CONCLUSIONS AND CLINICAL RELEVANCE Administration of the immunotherapeutic product to healthy dogs in accordance with the manufacturer's suggested protocol did not induce a significant change in anti-P insidiosum antibody concentrations. These results suggested that administration of the immunotherapeutic product may not interfere with postadministration serologic monitoring. However, further investigations will be required to determine whether there is a similar effect in naturally infected dogs.

Characterization, distribution, antimicrobial resistance and resistance risk factors in staphylococci isolated from cats from 2001 to 2014.

Relatively few studies have been published describing the patterns of staphylococcal isolation and antimicrobial resistance over time in cats. The objective of this retrospective study was to determine the frequency, location, characteristics and antimicrobial resistance profiles of staphylococci isolated by the Louisiana Animal Disease Diagnostic Laboratory between the years 2001 and 2014. All feline staphylococcal isolates were classified phenotypically. Isolates corresponding to known or possibly pathogenic species (Staphylococcus intermedius group (SIG) and Staphylococcus aureus (SA)) as well as Staphylococcus epidermidis (SE) and non-speciated coagulase-negative staphylococci (CNS) were further evaluated to determine antimicrobial resistance patterns. A total of 519 staphylococci were isolated. The largest percentage of isolates was CNS, representing 39.3% of the total, while SIG, SE, SA and non-speciated coagulase positive staphylococci (CPS) represented 18.1%, 10.2%, 8.3% and 7.3%, respectively. Methicillin resistance (MR) was identified in 57.1% of SA and 20.5% of SIG. Resistance to 3 or more antimicrobial classes (multidrug resistance; MDR) was demonstrated in 54.5% of SA and 23.9% of SIG. The prevalence of MDR increased over time in both SIG and SA, while the prevalence of MR increased over time in SIG. An increase in mean antimicrobial resistance score over time was seen in SIG. This study demonstrates a high and increasing prevalence of MDR in SIG and SA, as well as increasing prevalence of MR in SIG isolated from cats.

Survey of cytokines in normal canine tears by multiplex analysis: A pilot study.

OBJECTIVES: The purpose of this pilot study was to use a multiplexed assay to measure cytokines in normal stimulated canine tears. METHODS: 25 healthy dogs were included in the study. Stimulated tears were collected in capillary tubes from the right (OD) and left (OS) eyes and stored at -80 °C until batch sample analysis was performed. The samples were analyzed utilizing Luminex® canine-validated multiplex beads on a Bio-Rad multiplex analyzer for IL-2, IL-6, IL-7, IL-8, IL-10, TNF-α, and IFN-γ. Based upon previous human studies, tears were initially evaluated at a 1:10 dilution. Eight random samples were later re-analyzed without dilution. RESULTS: Diluting the samples 1:10 rendered all analytes undetectable except IL-8. A repeat analysis of eight randomly selected undiluted samples still demonstrated very low cytokine levels except for IL-8 (16/16 eyes; 2254 ±â€¯1677 pg/ml OD, 1095 ±â€¯786.8 pg/ml OS); and IFN-γ (15/16 eyes; 13.37 ±â€¯13.08 pg/ml OD,16.08 ±â€¯19.4 pg/ml OS). CONCLUSION: This pilot study is the first to analyze cytokines in canine tears. This study demonstrated that IL-8 is consistently detected in both diluted and undiluted samples, but undiluted samples may be superior to 1:10 diluted samples for evaluation of other cytokines in canine tears.

Determination of irritant threshold concentrations of multiple tree, grass, weed and mould allergens for intradermal testing of horses residing in the southern USA.

BACKGROUND: Appropriate allergen threshold concentrations (TCs) for intradermal testing (IDT) have not been established in horses for many pollen and mould allergens. OBJECTIVES: To determine the TCs in non-allergic horses and describe the frequency of late phase reactions for 26 allergens, including trees, grasses, weeds and moulds in horses residing in the southern Unites States. ANIMALS: Twenty four clinically normal horses in the southern United States. METHODS: Threshold concentrations for different allergens were determined using IDT subjective measurements at 30 minutes. Delayed reactions were evaluated at 4 and 24 h. RESULTS: Threshold concentrations (all PNU/mL) were established for eight tree allergens (black willow 1,000, box elder 1,000, live oak 1,000, pecan 2,000, white ash 4,000, red oak 4,000, red mulberry 2,000 and green ash 2,000); two grass allergens (Johnson grass 250 PNU/mL and Kentucky blue grass 500 PNU/mL); two weeds (carelessweed 1,000 PNU/mL, great ragweed 500 PNU/mL) and one mould (Curvularia 8,000 PNU/mL). The TC was not determined due to excessive reactivity at the lowest concentration tested (1,000 PNU/mL) for bahia and perennial rye grass. Eleven other allergens did not meet the criteria to establish a TC when evaluated at 30 min due to lack of positive reactions. Multiple allergens caused positive reactions in ≥10% of horses at 4 h. Reactions at 24 h were rare with the exception of one horse. CONCLUSIONS AND CLINICAL IMPORTANCE: This study identified intradermal TC for multiple pollen and mould allergens in horses. These values may prove useful for optimizing allergen concentrations for IDT of allergic horses.

Sepsis and multi-organ dysfunction associated with postgrooming furunculosis in a dog.

BACKGROUND: Postgrooming furunculosis, as previously described in the dog, is a type of pyoderma that typically responds to routine antimicrobial therapy. Systemic clinical signs are common but are usually mild. ANIMAL: A 3-year-old spayed female great dane was presented with marked dorsal furunculosis of 24 h duration. The clinical signs and blood analyses were consistent with sepsis, systemic inflammatory response syndrome and disseminated intravascular coagulopathy (DIC). METHODS AND RESULTS: Skin culture revealed multi-drug resistant Pseudomonas aeruginosa infection. The dog did not respond to aggressive medical therapy including intravenous antibiotic therapy, fresh frozen plasma and haemodynamic support, and was humanely euthanized. Postmortem findings were consistent with postgrooming furunculosis with associated sepsis and DIC affecting multiple organs including the lungs and brain. CONCLUSIONS AND CLINICAL IMPORTANCE: Postgrooming furunculosis can progress to sepsis and multiple organ dysfunction in the dog and can lead to death.

Dorsal skin necrosis secondary to a solar-induced thermal burn in a brown-coated dachshund.

A 5-year-old neutered male brown dachshund dog was presented for a large dorsal cutaneous burn that occurred following direct sunlight exposure outdoors in high ambient temperatures. Although burns are quite common in dogs, full-thickness solar-induced radiation burns are less common and have not been previously reported in animals without a black hair coat.

Nécrose de la peau dorsale secondaire à une brûlure thermique induite par l'exposition au soleil chez un Dachsund à pelage brun. Un chien Dachsund brun stérilisé âgé de 5 ans a été présenté pour une grande brûlure cutanée dorsale qui s'était produite après une exposition directe au soleil à l'extérieur durant des températures élevées. Même si les brûlures sont assez courantes chez les chiens, les brûlures du troisième degré causées par l'exposition au soleil sont moins communes et n'ont pas déjà été signalées chez des animaux sans un pelage à poil noir.(Traduit par Isabelle Vallières).

Atopic dermatitis in the domestic dog.

Dogs may develop a syndrome of spontaneous, inflammatory, pruritic dermatitis that shares many features with human atopic dermatitis, including a young age of onset, characteristic lesion distribution, immunoglobulin E sensitization to common environmental allergen sources, and evidence of epidermal barrier dysfunction. There are also several important differences between canine and human atopic dermatitis. Although dogs may suffer from multiple-organ hypersensitivity syndromes, there is no evidence that this species experiences the progressive evolution from cutaneous to respiratory allergy characteristic of the human atopic march. Despite the presence of epidermal barrier derangement, there is no significant association between canine atopic dermatitis and mutations in filaggrin. Finally, treatment of canine disease relies much less heavily on topical therapy than does its human counterpart, while allergy testing and allergen-specific immunotherapy provide an often essential component of effective clinical management of affected dogs.

Review: Lymphocytes, cytokines, chemokines and the T-helper 1-T-helper 2 balance in canine atopic dermatitis.

BACKGROUND: The development of atopic dermatitis (AD) and other cutaneous hypersensitivities involves the activation and differentiation of allergen-specific lymphocytes. Although hypersensitivity is often considered to be a 'T-helper 2-polarized' lymphocyte response, recent evidence suggests that clinical disease is associated with the development of multiple lymphocyte phenotypes. OBJECTIVES: The purpose of this paper is to review recent advances in the understanding of the roles of lymphocytes, cytokines and noncytokine factors in the pathogenesis of canine AD. METHODS: Citation databases, abstracts and proceedings from international meetings published between 2001 and 2013 were reviewed in this update. Where necessary, older articles were included for background information. RESULTS: The development of canine AD is associated with changes in both cutaneous and circulating lymphocyte populations. These lymphocyte responses are characterized by the production of a complex variety of cytokines, including not only T-helper 2 but also T-helper 1, T-helper 17 and regulatory T-cell responses. In addition, microarray gene expression analysis has enabled the identification of a number of noncytokine factors that appear to be associated with atopic inflammation. These include the calcium-binding protein S100A8, serum amyloid A and a number of protease inhibitors, as well as genes involved in epidermal barrier formation, innate immunity receptors, cell cycle proteins and apoptosis. CONCLUSIONS: The development of AD in dogs is characterized by the development of a delicate balance between a variety of T-cell phenotypes and inflammatory mediators, including cytokines, chemokines and noncytokine factors.

Review: Innate immunity, lipid metabolism and nutrition in canine atopic dermatitis.

BACKGROUND: The pathogenesis of canine atopic dermatitis (AD) involves dysfunction of the adaptive immune system. Recent evidence suggests that nonantigen-specific inflammatory elements may play a role in the development and perpetuation of canine AD. OBJECTIVES: The objective of this review is to provide an update on recent advances in the understanding of the role of innate immune cells, keratinocytes, lipid metabolism and nutrition in the pathogenesis of AD in dogs. METHODS: Citation databases, abstracts and proceedings from international meetings published between 2001 and 2013 are reviewed in this update. Where necessary, older articles are included for background information. RESULTS: Members of the innate immune system (including dendritic cells, Langerhans cells and mast cells) and keratinocytes interact with each other and with environmental antigens during both induction and effector phases of atopic inflammation. The responses of these cells and associated noncellular factors (such as complement and protease-activated receptors) to environmental stimuli influence the entire future course of the immune response to a given agent. Abnormalities in lipid metabolism may also influence the pathogenesis of canine AD via the production of inflammatory mediators and by alteration of epidermal barrier function and antigen presentation. However, a lack of fully controlled studies precludes definitive interpretation of these data. CONCLUSIONS AND CLINICAL IMPORTANCE: Evidence indicates that the cells and noncellular components of the innate immune system and the epidermis may play critical roles during both the sensitization and the effector phases of canine AD. Derangements in lipid metabolism may be involved in the pathogenesis of AD in dogs, but additional controlled studies are required in this area.