Subgroups of canine antinuclear antibodies in relation to laboratory and clinical findings in immune-mediated disease

Background:  Autoimmune system diseases in dogs are commonly referred to as systemic lupus erythematosus (SLE), with a positive antinuclear antibody (ANA) test as a hallmark. In human patients, other systemic ANA-positive diseases with overlapping diagnostic features, referred to as SLE-related diseases, are described.  Objectives:  The objective of this study was to investigate whether different patterns of ANA reactivity represent different systemic autoimmune diseases in dogs. Methods:  Dogs with serum positive for ANA by indirect immunofluorescence (IIF-ANA, titer ≥1:100) (n=56) were identified retrospectively from the patient population at the Department of Small Animal Clinical Sciences, Swedish University of Agricultural Sciences. Dogs were grouped on the basis of ANA staining patterns, and the results of immunodiffusion tests. Clinical, hematologic, serum biochemical, radiologic, and pathologic examinations were described for each group.  Results:  Dogs with a chromosomal–positive, homogeneous ANA staining pattern (n=14) had clinical signs involving multiple organ systems; 8 dogs were anemic. Dogs with a speckled IIF-ANA staining pattern (n=42) primarily had clinical signs of musculoskeletal disorders, fatigue and fever. Precipitating antibodies by immunodiffusion were found only in dogs with a speckled IIF-ANA staining pattern and comprised 4 different subgroups based on antigen specificity. Conclusions: In dogs with homogeneous IIF-ANA staining, SLE is a probable diagnosis because of the diversity of clinical manifestations and autoantibody reactivity against chromosomal antigens. Dogs with a speckled IIF-ANA pattern may have SLE-related diseases, which, in turn, may be correlated with different immunodiffusion subgroups. These syndromes had overlapping clinicopathologic features, as described for human patients.     

Evaluation of platelet activation in canine immune-mediated haemolytic anaemia

Objectives : To establish whether heightened platelet activation is a common feature of canine immune-mediated haemolytic anaemia, and to evaluate the hypothesis that platelet activation plays a role in the pathogenesis of thromboembolism. Methods : Using whole-blood flow-cytometric analysis, the proportion of activated platelets and platelet-leucocyte aggregates in blood samples from 14 dogs with immune-mediated haemolytic anaemia and 14 healthy dogs was calculated. General linear models with binomial errors were used to compare groups. Results from the immune-mediated haemolytic anaemia-affected dogs were then correlated with established risk factors for thromboembolism in canine immune-mediated haemolytic anaemia, D-dimer concentration and antithrombin activity. Results : There was a strong correlation between platelet activation and severe thrombocytopenia, with heightened platelet activation being observed predominantly in severely thrombocytopenic dogs. Clinical Significance : Dogs with immune-mediated haemolytic anaemia, particularly those with concurrent severe thrombocytopenia, are likely to have heightened platelet activation, which may play a role in the pathogenesis of thromboembolism.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

PK-PD结合模型及其在兽用抗菌药研究中的应用

随着PK-PD结合模型的发展,其在兽用抗菌药中的应用研究也受到了高度重视。本文通过对PK-PD结合模型理论的阐述,根据抗菌药物参数,对各类抗菌药物给药方案的制订作一简要分析,以期对优化临床给药方案和促进抗菌药物的临床合理使用有所帮助。     

Use of therapeutic plasmapheresis in a case of canine immune-mediated hemolytic anemia

Objective – To investigate the clinical application and potential utility of plasmapheresis in canine immune-mediated hemolytic anemia.
Case Summary – A 7-year-old spayed female Maltese diagnosed with immune-mediated hemolytic anemia was initially treated with prednisone, cyclosporine, and received multiple transfusions of packed RBC. Because of the progression of clinical signs despite traditional medical therapy, plasmapheresis was initiated. Plasma immunoglobulin G and immunoglobulin M levels were measured before, during, and after treatment to help determine if there had been a significant decrease in immunoglobulin levels with plasmapheresis. Plasmapheresis was successfully performed over a 2.5-hour period in this dog with minimal complications. Hypocalcemia was identified as a known complication of circuit anticoagulation, and was corrected through calcium supplementation. Post-plasmapheresis there was a decrease in immunoglobulin G and immunoglobulin M levels, and the patient showed clinical improvement. Following discharge the dog had no known complications of therapy, and had complete resolution of the anemia.
New or Unique Information Provided – Plasmapheresis was performed successfully with minimal complications. Because transfusion requirements appeared to be reduced, and the procedure was well tolerated, there may be a place for this modality in severe cases to act as a bridge until medical therapy takes full effect. Because of the cost of performing this therapy, and the potential requirement for multiple treatments, it should be reserved for selected patients.     

Effect of desmopressin on immune-mediated haemorrhagic disorders due to canine monocytic ehrlichiosis: a preliminary study

To evaluate the possible use of desmopressin acetate (DDAVP) in haemorrhagic disorders consequent to canine monocytic ehrlichiosis (CME), three dogs infected by Ehrlichia canis, with a history of thrombocytopenia and recent bleeding, were studied. The dogs were administered desmopressin (1 μg/kg b.w. s.c.) every 24 h on three occasions. Blood samples were collected immediately before, and after 2 and 48 h the first DDAVP administration, to assess haematological, clinical chemistry and clotting time parameters. Spontaneous bleeding stopped within 1 h after the first DDAVP injection. Buccal mucosa bleeding time (BMBT) was shortened from 9.6 to 2.3 min within 2 h after the treatment. A statistically significant increase in platelet PLT count and fibrinogen, and a statistically significant decrease of PT and aPTT were observed after DDAVP administration. The haemorrhagic disorders caused by CME appear to be quickly corrected by DDAVP administration, giving the clinician the time necessary to administer appropriate chemotherapy.     

The canine shoulder: selected disorders and their management with physical therapy

The shoulder joint is the most mobile of all main limb joints. While its primary motion is in a sagittal plane, the shoulder has a significant amount of abduction and adduction, and internal and external rotation. Its stability is ensured by the joint capsule, by its specialized bands (medial and lateral glenohumeral ligaments), and by large tendons located inside (eg, tendon of origin of the biceps brachii muscle) or immediately outside the joint (eg, supraspinatus, infraspinatus, teres minor, and subscapularis). Sprains or strains of all supporting structures of the canine shoulder have now been reported and the shoulder pathology resembles the pathology of the human shoulder that includes strains and tears of the rotator cuff muscles, adhesive capsulitis, and calcific tendonitis.     

夏季犬饲养管理注意点

由于夏季天气炎热,温度高,湿度大,容易使犬产生热射病（中暑）和消化道疾病,给犬的饲养管理带来影响,严重时可直接影响犬的繁育。预防疾病,保证犬平稳渡过炎热的夏季,在犬的饲养管理上要有科学性和灵活性,做到饲养上精心、管理上细心,经常了解和观察犬的动态,及时发现人的异常情况,做到早预防、早发现、早报告、早治疗。具体注意以下几点：     

Determination of expression of cyclooxygenase-1 and -2 isozymes in canine tissues and their differential sensitivity to nonsteroidal anti-inflammatory drugs

OBJECTIVE: To evaluate cyclooxygenase isozyme distribution in tissues from dogs and determine the differential sensitivity of canine cyclooxygenase (COX)-1 and -2 isozymes to nonsteroidal anti-inflammatory drugs (NSAIDs). SAMPLE POPULATION: Canine tissue samples (stomach, duodenum, ileum, jejunum, colon, spleen, cerebral cortex, lung, ovary, kidney, and liver) were obtained from 2 dogs for northern and western blot analyses, and blood for whole blood COX assays was obtained from 15 dogs. PROCEDURE: 11 NSAIDs were evaluated to determine their COX-2 selectivity in whole blood assays. The concentrations of the drug needed to inhibit 50% of enzyme activity (IC50) were then calculated for comparison. Expression and tissue distribution of COX isozymes were determined by northern and western blot analysis. RESULTS: Aspirin, diclofenac, indomethacin, ketoprofen, meclofenamic acid, and piroxicam had little selectivity toward COX isozymes, whereas NS398, carprofen, tolfenamic acid, nimesulide, and etodolac had more than 5 times greater preference for inhibiting COX-2 than COX-1. All canine tissues examined, including those from the gastrointestinal tract, coexpressed COX-1 and -2 mRNA, although protein expression was observed only for COX-1. CONCLUSIONS AND CLINICAL RELEVANCE: Canine COX-2 was selectively inhibited by etodolac, nimesulide, and NS398; tolfenamic acid and carprofen also appeared to be preferential COX-2 inhibitors in dogs. The roles of COX-1 as a constitutive housekeeping enzyme and COX-2 as a proinflammatory inducible enzyme (as determined in humans) appear to apply to dogs; therefore, COX-2-selective inhibitors should prove useful in reducing the adverse effects associated with nonselective NSAIDs.     

Haemoplasma infection is not a common cause of canine immune-mediated haemolytic anaemia in the UK

Objective : The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum,” are commonly associated with immune-mediated haemolytic anaemia (IMHA) in UK dogs. Methods : Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non-anaemic dogs (n=113). DNA was extracted from 100 μl of blood and subjected to real-time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co-amplification of canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous internal control. Results : Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or “Candidatus M. haematoparvum” DNA. Clinical Significance : Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.     

Detection of canine distemper virus antigen in canine serum and its application to diagnosis

Canine distemper virus (CDV) antigen was detected in the serum of dogs by an ELISA and the results of this assay were compared with an anti-CDV immunoglobulin M (IgM) antibody test. In paired sera from 26 naturally infected dogs, the antigen-positive rate was 26.9 per cent at the first examination and 11.5 per cent at the second examination two to three weeks later. The antigen was detected in three of the 10 dogs which were negative for anti-CDV IgM antibody at the first examination. It could also be detected in the serum of between eight and two of 40 specific pathogen-free dogs vaccinated against CDV, for up to four weeks after they were vaccinated.     

Clinical experience with butyl-2-cyanoacrylate adhesive in the management of canine and feline corneal disease

PURPOSE: To examine and evaluate clinical indications and postoperative outcome in a series of small animal patients in which corneal disease was managed by the application of butyl 2-cyanoacrylate adhesive. MATERIALS AND METHODS: In this retrospective study all small animal patients were identified that presented to the Royal Veterinary College, University of London over a 2-year period, in which corneal disease was managed by the application of butyl 2-cyanoacrylate. Indications for application, complicating factors prior to gluing, glue retention time, postoperative comfort, and extent of subsequent corneal reaction and scarring were noted for each case. Long-term follow-up data concerning visual and cosmetic outcome were obtained from owners and referring veterinarians. RESULTS: Thirty-seven patients (28 dogs and 9 cats), in which 39 eyes were treated, were identified. Indications for corneal gluing in this series included stromal ulceration (26/39 eyes); descemetocele (4/39 eyes); corneal laceration/foreign body (5/39 eyes); lamellar keratectomy (3/39 eyes) and superficial ulceration (1/39 eyes). At least one factor responsible for initiation, persistence or progression of the ulcer was identified in 66.7% of eyes prior to corneal gluing. These included keratomalacia; confirmed bacterial keratitis; corneal edema related to endothelial disease and keratoconjunctivitis sicca. Cyanoacrylate was generally well tolerated by patients with only 8/34 eyes demonstrating transient blepharospasm and increased lacrimation postoperatively. Retention time of cyanoacrylate varied widely from < 1 week to approximately 6 months, but was < 2 months in the majority (89%) of eyes. Exaggerated corneal vascularization was an infrequent postoperative complication, noted in only six canine eyes, and did not appear to be related to initial corneal disease, glue retention time or breed. CONCLUSIONS: Butyl 2-cyanoacrylate offers a convenient, economical and effective alternative to other treatment modalities, such as conjunctival grafts, in the management of corneal defects in canine and feline patients.     

Binding of canine anaphylactic antibodies on human basophils: application to canine allergy diagnosis

Abstract Using the technique of human basophil passive sensitization, as employed for human allergy díagnosis, we checked the ability of canine anaphylactic antibodies to sensitize human basophils. Therefore, by sensitizing human basophils with sera taken from dogs allergic to house dust mite, we demonstrated basophil activation as measured by alcian blue staining. Basophil activation was inhibited by heating dog sera at 56 °C for 6 h and by a human myeloma IgE. Basophil activation was also shown by histamine and leukotriene (LTC4) release. These results indicate canine anaphylactic antibodies bind to human basophil IgE receptors and also that they are IgE. The three methods described here for measuring basophil activation may lead to díagnostic methods applicable to canine allergy díagnosis. Resumen Mediante el método de la sensibilización pasiva de basófilos humanos como se utiliza para el díagnóstico de la alergia humana, evaluamos la capacidad de los anticuerpos anafilácticos caninos de sensibilizar basófilos humanos. Asi, sensibilizando basófilos humanos con suero extraido de perros con alergia al ácaro del polvo, demostramos la activación de basófilos medíante la tinción de Azul de Alcián. Se inhibió calentando suero canino a 56 °C durante 6 h. y por IgE de mieloma humano. La activación de los basófilos se mostró también por la liberación de histamina y leucotrieno (LTC4). Estos resultados indican que los anticuerpos caninos anafilácticos se unen a los receptores de IgE en basófilos humanos y también que son IgE. Los tres métodos descritos aqui para medir la activación de basófilos pueden llevar a métodos de díagnóstico aplicables al díagnóstico de la alergia canina. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Union de anticuerpos anafilácticos caninos a basófilos humanos: aplicacion al díagnóstico de alergia canina). Veterinary Dermatology 1996; 7 : 185–91.] Résumé Utilisant une technique de sensibilisation passive de basophiles humains, employee pour le díagnostic allergologique chez l'homme, nous avons testé la capacité des anticorps anaphylactiques canins à sensibiliser des basophiles humains. Ainsi, par sensibilisation de basophiles humains avec des sérums provenant de chiens allergiques aux acariens de la poussière de maison, nous avons démontré l'activation des basophiles mesurée par coloration au bleu alcian. Celle-ci est inhibée par des sérums canins chauffés à 56 °C pendant 6 heures et par un myélome IgE humain. L'activation des basophiles a été aussi démontrée par libération d'histamine et de leucotriénes (LTC4). Ces résultats prouvent la présence d'anticorps anaphylactiques canins fixés à des récepteurs IgE de basophiles humains et que ceux-ci sont des IgE. Les trois méthodes décrites ici pour mesurer l'activation des basophiles peuvent être utilisées pour le díagnostic allergologique chez le chien. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Fixation d'anticorps anaphylactiques canins sur des basophiles humains). Veterinary Dermatology 1996; 7 : 185–91.] Zusammenfassung Mit der Technik der passiven Basophilensensibilisierung beim Menschen, wie man sie für die Allergiedíagnose beim Menschen anwendet, untersuchten wir die Möglichkeit, menschliche Basophile durch kanine anaphylaktische Antikörper zu sensibilisieren. Dazu wurden humane Basophile mit Sera von Hunden sensibilisiert, die allergisch auf Hausstaubmilben reagierten. Dabei demonstrierten wir eine Basophilenaktivierung, die durch Elsässerblau-Färbung gemessen werden konnte. Der Vorgang wurde verhindert durch Erhitzen der Hundesera auf 56 °C für 6 Stunden und durch humanes Myelom-IgE. Basophilenaktivierung wurde auch durch Histamin-und Leukotrien(LTC4)-Ausschüttung gezeigt. Diese Ergebnisse zeigen, daß kanine anaphylaktische Antikörper sich an humane basophile IgE-Rezeptoren binden und auch IgEs darstellen. Die drei hier beschriebenen Methoden zur Messung der Basophilenaktivierung können zu einer díagnostischen Methode führen, die für die Diagnostik kaniner Allergie anwendbar ist. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Die Bindung von anaphylaktischen Antikörpern des Hundes an Basophile Zellen des Menschen: Anwendung für die Allergiedíagnose beim Hund). Veterinary Dermatology 1996; 7 : 185–91.]     

Mechanisms of platelet destruction in immune-mediated thrombocytopenia: in vitro studies with canine platelets exposed to heterologous and isologous antiplatelet antibodies

Normal canine platelets coated with heterologous or isologous antiplatelet antibody were interacted with viable canine neutrophils in vitro. Platelet phagocytosis was assessed by detecting nitroblue tetrazolium (NBT) reduction, measuring uptake of opsonised 51Cr-labelled platelets, and electron microscopy. The NBT reduction and uptake of 51Cr-labelled opsonised platelets were markedly increased. Electron microscopy revealed phagocytosis of antibody-coated platelets and their degradation intracellularly. Exposure of canine platelets to rabbit anti-canine platelet antibody in vitro produced morphological changes in platelets and caused serotonin release. Serotonin was not released in the absence of antiplatelet antibody or in the presence of normal rabbit gamma-globulin. Morphological changes in the platelets included disappearance of alpha and dense granules and exaggeration of the open canalicular system. These observations indicate that circulating platelets may be vulnerable to an antiplatelet antibody and that antibody-mediated phagocytosis of platelets is an important mechanism in the pathogenesis of immune-mediated thrombocytopenia.     

核酸探针检测犬冠状病毒方法的建立和初步应用

以^32P标记犬冠状病毒特异性RT—PCR及产物制成核酸探针，与CCV NL-18株、犬瘟热病毒、犬细小病毒、犬副流感病毒、犬腺病毒、狂犬病病毒反转录产物、正常CRFK细胞以及做10～10000倍稀释的CCV NL-18株反转录产物杂交，进行核酸探针的敏感性和特异性试验。结果表明，该探针仅与CCV HLl8株反转录产物呈阳性杂交，与对照病毒和正常细胞反转录产物均呈阴性反应。初步应用试验结果，该探针可与国内CCV分离病毒YSl、CI1株杂交，且可从8份腹泻犬粪便中检出5份CCV阳性病料。本研究为在我国开展犬冠状病毒感染的分子流行病学调查和临床诊断提供了一种敏感、特异的检测方法。