Autoimmune haemolysis in the dog: relationship between anaemia and the levels of red blood cell bound immunoglobulins and complement measured by an enzyme-linked antiglobulin test.

A direct enzyme-linked antiglobulin test (DELAT) was used to measure the levels of red blood cell (RBC) bound IgG, IgM, IgA and C3 in dogs with autoimmune haemolytic anaemia (AIHA). At presentation, one or more DELAT parameters was raised in each AIHA case, and the RBC were typically coated with immunoglobulin of more than one class, together with C3. There was no relationship between the levels of RBC-bound IgG, IgM or IgA and the severity of the anaemia, although a significant negative correlation (rs = -0.66, P < 0.02) was found between bound C3 and blood haemoglobin concentration. These results indicate that the level of sensitisation of erythrocytes with IgG alone is not a reliable predictor of the severity of haemolysis in different cases, and that the pathogenesis of AIHA can be complex, involving multiple immunoglobulin classes and complement in the destruction of RBC. A significant relationship (rs = 0.63, P < 0.02) was found between serum IgG concentration and haemoglobin levels, and it is suggested that this may be due to free IgG inhibiting the interaction of IgG-sensitised RBC with macrophages. Serial measurements from individual AIHA cases during treatment revealed that the levels of RBC-bound immunoglobulins fell simultaneously with improvements in anaemia. In one dog, a relapse was associated with increases in bound IgG and IgM. Transient relative reticulocytopenia at presentation was common, but was not related to the severity of the anaemia. However, in other cases there was a persistent failure to increase RBC production, which was associated with slower recovery.     

Serial haematology results in transfused and non-transfused dogs naturally infected with Babesia rossi

This prospective longitudinal study investigated the progression of haematological changes in 32 transfused and 54 non-transfused dogs naturally infected with Babesia rossi over the 1st 6 days following diagnosis and treatment. The effect of patient age on the results of complete blood counts was determined. Haematology data were analysed at presentation and at 24 hours, 3 days and 6 days after presentation. Dogs were treated with diminazene aceturate at diagnosis and a blood transfusion was given if deemed clinically required. Mildly to moderately regenerative normocytic normochromic anaemia was observed in all dogs throughout the study period. Transfused dogs more often had an inflammatory leukogram at presentation and at 24 hours, than dogs that were not transfused. In dogs with a left shift, a concurrent normal or decreased segmented neutrophil count was found more commonly than neutrophilia. Severe thrombocytopenia that resolved within a week was common. Blood transfusion alleviated the anaemia, but had no significant effect on white blood cell or platelet responses. Blood cell responses were not significantly influenced by age. In conclusion, the red blood cell and white blood cell responses were less than expected in dogs with babesiosis, given the degree of anaemia and inflammation present. The magnitude of thrombocytopenia and rapid return of the platelet count to normal suggested a possible immune-mediated mechanism for the thrombocytopenia.     

Antigen specificity in canine autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is the most common clinical manifestation of autoimmunity in the dog and generally presents as a profound, regenerative, Coombs' positive anaemia of acute or chronic onset. The disease pathogenesis involves formation of erythrocyte-specific autoantibodies of the IgG and IgM class that may fix complement resulting in intra- or extravascular haemolysis. Western blotting and immunoprecipitation studies using autoantibody eluted from the erythrocytes of dogs with AIHA have demonstrated specificity for erythrocyte glycophorins and the membrane anion-exchange molecule (band 3). Autoantibodies specific for the cytoskeletal molecule spectrin have been identified in serum by ELISA. The specificity of autoreactive T-cells has been examined in vitro using bulk cultures stimulated with a panel of autoantigens including intact erythrocyte membranes, purified glycophorin and spectrin fractions and a panel of overlapping 15-mer glycophorin peptides. Control responses to ConA and recall (vaccine antigens) and non-recall (KLH) antigens were measured in the same system. PBMC obtained from dogs that had recovered from AIHA consistently proliferated in response to erythrocyte membranes, with occasional responses to spectrin or glycophorin. PBMC from sone clinically normal dogs also responded to erythrocyte membranes. PBMC obtained from dogs closely related to AIHA cases gave the most consistent responses, including proliferation when stimulated by the glycophorin peptides. These data suggest that normal dogs harbour erythrocyte autoreactive lymphocytes, and that these cells may be primed in dogs recovered from AIHA or having genetic susceptibility to the disease.     

Canine multicentric lymphoma 1: Clinico-pathological presentation of the disease

The clinicopathological features of 90 cases of canine multicentric lymphoma are described. The majority of cases occurred in middle-aged dogs (five to eight years of age) with a mean age at presentation of 6–7 years. A marked sex predisposition was noted with male dogs out numbering females by 2:1. No significant breed predispositions were found. The most common presentation was a generalised lymphadenopathy but in approximately one-third of the cases this was accompanied by other signs including polyuria and polydipsia, facial oedema and upper respiratory tract obstruction. Approximately two-thirds of all the cases showed non-specific haematological abnormalities including thrombocytopenia, anaemia and white blood cell anomalies. All the cases were clinically staged according to the anatomical extent of the disease. The majority of the dogs were classified as stage 3 or above, denoting that the disease is invariably widespread or systemic upon initial presentation.     

Autoimmune haemolytic anaemia in dogs

The clinical, haematological and immunological findings in 24 dogs with Coombs' positive haemolytic anaemia are described; 33% were Old English Sheepdogs. Dogs with intravascular haemolysis had a shorter history of illnesses, more severe clinical signs including vomiting, jaundice and fever, and had a poor survival rate compared to dogs with extravascular haemolysis. The anaemia was severe and regenerative in 18 dogs, and was characterised by spherocytosis and microscopic red cell agglutination, with leukocytosis. Serum IgG levels were elevated in 20 dogs, and changes in IgM, IgA, C3 and C4 were found. Antinuclear antibody was also demonstrated in 13 dogs, of which 7 were Old English Sheepdogs. It is suggested that a distinct multisystem autoimmune syndrome exists within the local Old English Sheepdog population.     

Serum Acute Phase Protein Concentrations in Dogs with Autoimmune Hemolytic Anemia

Background: Serial monitoring of acute phase protein (APP) concentrations in canine autoimmune hemolytic anemia (AIHA) has not been reported.
Hypotheses: Acute canine AIHA is accompanied by an acute phase response (APR) characterized by increased C-reactive protein (CRP) and α1-acid glycoprotein (AAG) concentrations and decreased albumin concentrations.
Animals: Twenty-seven dogs with AIHA and 11 control dogs.
Methods: Prospective, cohort study. CRP, AAG, and albumin concentrations, white blood cell (WBC) count, and packed cell volume (PCV) were determined at admission (day 1), every 48 hours until death or discharge, and on days 30, 90, 180, and 365.
Results: Compared with controls, CRP and AAG concentrations were increased and albumin concentration was decreased in dogs with AIHA (days 1–7; P < .002) and normalized with disease stabilization (days 9–365; P > .05). APP concentrations on day 1 were not predictive of survival, duration of hospitalization, or number of blood transfusions ( P = .153–.940). PCV correlated with APP concentrations over time (CRP r =−.600, AAG r =−.665, albumin r = .533; P < .0001) as did WBC count (CRP r = .253, AAG r = .486, albumin r =−.246; P < .006). Day 1 CRP concentration was lower for dogs that received corticosteroids before referral (115.3 μg/mL) compared with dogs that did not (191.2 μg/mL; P = .02).
Conclusions: An APR occurs in canine AIHA. Initial APP concentrations are not predictive of acute survival, correlate with hematologic markers of remission, and normalize rapidly with disease stabilization.     

Use of an enzyme indirect antiglobulin test for the diagnosis of autoimmune haemolytic anaemia in the dog

Blood samples from 34 dogs with autoimmune haemolytic anaemia (AIHA) were studied using a variety of serodiagnostic tests. Enzyme enhancement of red cells was found to be necessary for an unequivocal laboratory diagnosis of AIHA because other techniques, the direct and the indirect Coombs' tests, often gave negative results. The most frequently encountered auto-antibody was an IgG, with a titre of 1024 or more.     

The effects of levamisole poisoning on the haematological and biochemical parameters in dogs

This study was designed to evaluate possible organ and system disorders associated with experimentally induced levamisole poisoning in dogs. For this purpose, twelve clinically healthy dogs of different ages, sexes and breeds were used. They were divided into two equal groups (Group A and Group B) and given levamisole orally at a dose of 25 mg/kg of body weight daily for three days. The dogs in Group B were also injected with atropin sulphate (0.04 mg/kg of body weight) subcutaneously (sc) 1 hour after each administration of levamisole. Routine clinical examinations were made and some haematological, biochemical and blood gas parameters were established at various times after administration of levamisole. The dogs in Group A developed severe neurological signs, gastric haemorrhage, bloody vomiting, colic, anaemia and four dogs died. In Group B these signs were mild and only one dog died. Levamisole poisoning was characterised by a significant reduction in the total number of red blood cells (RBCs), concentration of haemoglobin (Hb) and packed cell volume (PCV), and by anaemia. Peripheral blood pH, actual bicarbonate of plasma (HCO3), actual base excess (BE), partial pressure of oxygen (pO2) and saturated oxygen (O2SAT) increased in both groups of animals and these dogs developed metabolic alkalosis 48 hours after the first administration of levamisole. The results of the study also show that levamisole poisoning in dogs causes a significant increase in the activity of serum alanine aminotransferase (ALT) and of alkaline phosphatase (AP) and in the concentration of urea in both Group A and Group B. In the study, atropin sulphate reduced the severity of the clinical signs and the number of deaths, but it was not alone sufficient to remedy levamisole poisoning in dogs.     

Aetiology and prevalence of canine anaemia in Zaria: a review of 2139 cases observed at the Veterinary Teaching Hospital of the Ahmadu Bello University, Zaria, Nigeria (1990-2003)

An investigation was conducted at the Ahmadu Bello University Veterinary Teaching Hospital (ABUVTH) between January, 1990 and September, 2003 to determine the aetiology and prevalence of canine anaemia in Zaria, Nigeria. Out of the 5278 mongrel dogs presented during the period 1990-2003, 2139 (40.5%) were found to be anaemic, with packed cell volume (PCV) values ranging from 7 to 36%. The clinical signs presented by these dogs include: Pale mucous membranes, weakness, depression, anorexia/inapettence and reduced activity. About 50 dogs (about 1%) with helminths and haemoparasitic infestations had high PCV values (37-40%) without clinical presentation of anaemia or disease. Most of the dogs with anaemia (n = 2016 or 94.2%) had parasitic infestations. About 1580 (about 74%) of the anaemic cases, attributed to parasitic infestations occurred between May and October. A few dogs (n = 55, 2.6%) had anaemia due to poor nutrition, while 68 (3.2%) had anaemia with unknown cause. The public health significance of the parasites reported in this study is discussed.     

Detection of Leishmania amastigotes in peripheral blood from four dogs--Short communication

The authors carried out microscopic examination of blood smears of 1438 dogs infected with Leishmania infantum. Unusual findings of leishmaniosis associated with circulating parasitised cells are described in four dogs. Most of the dogs presented severe illness, with lethargy, dysorexia, emaciation and alterations of the haematological pattern (anaemia, thrombocytopenia, neutrophilia and monocytosis). In three cases, leishmaniosis was associated with ehrlichiosis. On examination of peripheral blood smears, Leishmania sp. amastigotes were observed both in various circulating leukocytes (neutrophil, monocyte, macrophage) and free. In conclusion, parasites can rarely be detected in blood smears (in 0.28% of the animals examined); thus, the time-consuming microscopic search for amastigotes can make only a weak contribution to the conventional diagnosis of canine leishmaniosis.     

Immunopathogenesis of canine aural haematoma

Data are presented from 15 dogs with aural haematoma. The series included six Labrador retrievers and four golden retrlevers and the mean age was 8.0 ± 3.02 years. Five dogs had evidence of pruritic skin disease and five further cases had other concurrent disease. Haematoiogy and serum biochemistry were normal in 12 and 13 of the 15 dogs, respectively. Ail dogs were Coombs' negative and serum antinuclear antibody had negative or low titres in ail the 11 cases tested. Histopathological examination of biopsies from the affected ears revealed variable degrees of erosion of auricular cartilage with fibrovascular granulation tissue filling the cartilage defects. There was minimal perichondrai inflammation. The biopsies were studied by immunohistochemlstry for deposition of lmmunoglobulln G (IgG), immunogiobulln M (IgM) and complement C3. In one dog there was basement membrane zone deposition of IgG and in another there was focal interepithellai deposition of both igG and IgM. The findings of this study do not support an autoimmune pathogenesis for canine aural haematoma, but suggest that an early immunological event may underlie the observed cartilage erosion.     

Investigation into factors influencing performance of the canine antiglobulin test

Antiglobulin (Coombs') reagents were assessed for their ability to detect immunoglobulin and complement attached to red cells. Polyspecific and monospecific reagents were prepared using a number of immunisation protocols. Performance of these antisera against control red cells was compared, in a direct Coombs' test, with samples from cases of canine autoimmune haemolytic anaemia (AIHA). A combined reagent containing two monospecific antisera (anti-IgG + anti-C3) gave optimum results. Positive control red cells were required to standardise canine Coombs' reagents for the laboratory diagnosis of AIHA. The optimum incubation temperature for the canine Coombs' test was shown to be 37 degrees C.     

Phase II open‐label study of single‐agent hydroxyurea for treatment of mast cell tumours in dogs*

This prospective study evaluated the efficacy and safety of hydroxyurea (HU) in dogs with measurable mast cell tumours (MCTs). Dogs were treated with HU at 60 mg kg^?1per os q24h for 14 days then 30 mg kg^?1 q24h thereafter or until MCT recurrence. Forty‐six dogs were enrolled. The overall response rate was 28%. Two dogs had a complete response (CR) for 256 and 448 days, respectively. Eleven dogs had a partial response for a median duration of 46 days (range, 28–189 days). Grade 2 to 4 neutropenia occurred in eight dogs and grade 4 thrombocytopenia in two. Grade 3–4 anaemia occurred in seven dogs; overall, there was a significant decrease in haematocrit after treatment with HU. The median drop in haematocrit was 10%. This study demonstrated that HU has activity in the treatment of MCTs with mild anaemia being the primary adverse event.     

Canine lymphoid leukaemia and lymphoma with bone marrow involvement: A review of 24 cases

Eleven cases of canine lymphoid leukaemia (10 acute and one chronic) were compared with 13 cases of canine lymphoma with bone marrow abnormalities, with regard to presentation, treatment and survival. Breed, age, and sex were similar for the two groups of dogs, but there was a marked difference in presenting signs, haematological parameters and bone marrow analysis, with the leukaemic dogs showing more severe changes. The clinicopathological features of these cases are reported in this paper and discussed with regard to prognosis. The mean survival time was significantly lower (43 days) in dogs treated for acute lymphoblastic leukaemia than for dogs treated for lymphoma with bone marrow abnormalities (351 days).     

Detection of platelet-bound antibodies in beagle dogs after artificial infection with Ehrlichia canis

Six dogs were infected with Ehrlichia canis by intravenous injection of heavily infected DH82 cells. All dogs developed typical signs of canine monocytic ehrlichiosis. Using flow cytometric technology, platelet-bound IgG (PBIgG) were detected in 5 of the 6 dogs after experimental infection with E. canis over a period of 3-10 days post infection (PI). The first detection of PBIgG was made as early as day 3 PI in 2 out of 6 dogs, and on day 5 PI in 1 dog. On day 7 PI, PBIgG was detected in 2 dogs, and on day 10 PI in 3 out of 6 dogs. This is the first report documenting the presence of PBIgG following E. canis infection in dogs. This finding further supports the theory that the thrombocytopenia seen in canine monocytic ehrlichiosis has an immunological component and that exposure to an infectious agent, in this case the rickettsia E. canis, can trigger autoimmune mechanisms. Due to the heterogeneous appearance of PBIgG among the infected dogs it was concluded that other non-immunological mechanisms are probably also involved in the pathogenesis of the thrombocytopenia seen in canine monocytic ehrlichiosis.     

Isotype determination of circulating autoantibodies in canine autoimmune subepidermal blistering dermatoses

Abstract The three most common canine autoimmune blistering skin diseases (AISBD), bullous pemphigoid (BP), mucous membrane pemphigoid (MMP) and epidermolysis bullosa acquisita (EBA) have recently been separated based on clinical, histological and immunological grounds. The objectives of this study were to determine the isotype profiles of circulating autoantibodies in these dermatoses. Serum was collected from 5 dogs with BP, 15 with MMP and 11 with EBA. All sera were tested using an indirect immunofluorescence method using salt-split canine gingiva as substrate. Anti-basement membrane IgG autoantibodies were detected in all patients. Among the IgG autoantibodies, IgG1 and IgG4 were encountered most frequently, while IgG2 and IgG3 were uncovered in some dogs. IgE autoantibodies were detected more often than IgA or IgM autoantibodies in any of the three entities. The predominance of IgG1, IgG4 and IgE autoantibody isotypes in dogs with AISBD is very similar to the situation found in humans with the homologous diseases.     

Primary immune-mediated thrombocytopenia in 30 dogs (1997-2003)

During a 6-year period, primary (idiopathic) immune-mediated thrombocytopenia was retrospectively evaluated in 30 dogs. Ages of the dogs ranged from 3 months to 10 years (median 4 years); female dogs were markedly overrepresented with 73%. Clinical examination revealed hemorrhages in 70% of the dogs. Platelet counts ranged from 0 to 111,000/microL (median 8000/microL); 77% of the dogs had platelet counts <30,000/microL. Seventeen dogs were anemic (hematocrit 9% to 36%; median 31%). Immunosuppressive therapy was performed in all but one dog. The recurrence rate of 19 dogs that were followed over an extended period (112 to 1684 days; median 340 days) was 26%. Twenty-nine (97%) dogs survived 14 days after initial presentation, and 27 (93%) dogs survived at least the following 15 to 1684 days (median 220 days).     

Immune-mediated disease in the old English sheepdog.

Immunological data are reported from 19 cases of immune-mediated disease recorded in the old English sheepdog breed in Western Australia between 1978 and 1989. The conditions included autoimmune haemolytic anaemia (seven), idiopathic thrombocytopenia (one), Evans' syndrome (five), multiple myeloma (two), systemic lupus erythematosus (one), discoid lupus erythematosus (one) and hypothyroidism (two). The most consistent serological findings were raised serum IgG (60 per cent), depressed serum IgM (60 per cent) and the presence of multiple autoantibodies (anti-red blood cell 78 per cent, antinuclear antibody 44 per cent, rheumatoid factor 19 per cent). An underlying, breed-related disorder of immune regulation may account for these observations.     

Clinicopathological changes and effect of imidocarb therapy in dogs experimentally infected with Babesia canis

In this study one spleen-intact dog (A) and two splenectomised dogs (BSE, CSE) were infected with Babesia canis. All animals developed an acute disease characterised by fever, haemoglobinuria and anaemia, the latter being more severe in the splenectomised dogs. Fever and parasitised red blood cells were detected for three days after imidocarb treatment in the splenectomised animals. Haematological abnormalities included regenerative anaemia, thrombocytopenia and leukopenia (due to neutropenia and lymphopenia) in the acute phase, soon followed by leukocytosis, neutrophilia and left shift a few days later. Acute hepatopathy was detected in all dogs with elevated ALT activity, which was more seriously altered in the splenectomised dogs. Diffuse changes in liver structure and hepatomegaly were seen by ultrasonography. Liver biopsy and histology revealed acute, non-purulent hepatitis in the splenectomised dogs. Both splenectomised dogs were successfully cured after collection of 400 ml highly parasitised blood, proving that large-amount antigen production is possible with rescuing the experimental animals. Whole blood transfusion, imidocarb and supportive care with infusions, antipyretics, glucocorticoids and diuretics were applied. The spleen-intact dog clinically recovered after receiving supportive treatment, with no imidocarb therapy. Microbial infections developed in both splenectomised animals (BSE: haemobartonellosis, CSE: osteomyelitis caused by Escherichia coli), probably as a consequence of immunosuppression after splenectomy and glucocorticoid therapy.     

Clinicopathological features of seven cases of canine myelofibrosis and the possible relationship between the histological findings and prognosis.

Seven dogs with non-regenerative anaemia were diagnosed as having myelofibrosis on the basis of the presence of collagen and increased deposits of reticulin fibre in the haemopoietic spaces of bone marrow core biopsies. A scoring system was used to assess the cellularity of the marrow and the amounts of collagen, reticulin and haemosiderin present. These scores, together with the haematological findings, were compared with the dogs' responses to treatment and their outcome. Treatment consisted of blood transfusions, where required, and anabolic steroids and corticosteroids. Three dogs deteriorated and were euthanased within three months of diagnosis, but the other four recovered fully. There was no correlation between the collagen and reticulin scores, or the degree of anaemia and the outcome, but the four dogs which recovered all had a macrocytosis when first examined. There was no evidence of an underlying lymphoproliferative or myeloproliferative disease in any of the seven cases.