Membranoproliferative glomerulonephritis possibly associated with over-vaccination in a cocker spaniel

Membranoproliferative glomerulonephritis was observed in a seven-month-old male cocker spaniel dog. The clinical, microbiological, biochemical, radiographic and ultrasonographic examinations ruled out neoplasia, congenital disease and infectious disease. The anamnesis revealed that the owner had vaccinated the dog seven times, one vaccination per month, without veterinarian supervision. In both kidneys, severe thickening of the glomerular capillary walls was observed. Electron microscope examination revealed a large number of electron-dense deposits that were primarily in the glomerular subendothelial spaces and the basal membrane, which is compatible with antigen-antibody complexes. The immunohistochemical examination revealed that the antigen present in the glomeruli corresponded with the antigen present in the vaccine. We report a type III hypersensitivity nephropathy in a young dog, which was possibly caused by over-vaccination.     

Investigation of antibodies to extractable nuclear antigens in dogs.

Determination of antibodies to specific nuclear antigens, termed extractable nuclear antigen (ENA), was investigated in healthy dogs and in dogs with autoimmune, inflammatory, and neoplastic diseases. Using a counterimmunoelectrophoresis method, the dogs' sera were tested for antibodies against the nuclear antigens single-stranded DNA, Sm, Ro, La, ribonucleoprotein, Scl, and proliferating cell nuclear antigen. Antibodies to the Ro antigen were found in 1 dog with discoid lupus erythematosus, in 1 dog with pemphigus erythematosus, and in 1 dog with facial pyoderma and chronic superficial keratitis. In 15 dogs, antibodies were detected to ENA, but the precipitin lines were too weak to identify the specific ENA. These antibodies were found in some dogs with systemic lupus erythematosus, discoid lupus erythematosus, pemphigus erythematosus, dermatomyositis, vitiligo, lymphoma; in the dog with facial pyoderma and chronic superficial keratitis; and in 1 healthy dog. The highest percentage of dogs with antibodies to ENA in a large series (greater than 8) of this study was in dogs with systemic lupus erythematosus (4 of 13; 31%).     

Plasma von Willebrand factor concentration and thyroid function in dogs

Plasma von Willebrand factor antigen concentration was determined in 15 dogs with suspected hypothyroidism, in 1 dog with hyperthyroidism, and in 14 euthyroid dogs. The mean +/- SEM von Willebrand factor:antigen concentration in hypothyroid dogs (47.1% +/- 12.6%) was significantly decreased (P less than 0.0005), compared with that in euthyroid dogs (94.7 +/- 5.6%). Four hypothyroid dogs were given thyroxine for 1 month and all 4 had an increase in von Willebrand factor:antigen concentration. The plasma von Willebrand factor:antigen concentration was 200% in the hyperthyroid dog. Seemingly, reduced concentrations of plasma von Willebrand factor:antigen can be found in dogs in association with congenital von Willebrand disease or with von Willebrand disease acquired through hypothyroidism.     

Hemangiosarcoma in a geriatric Labrador retriever

A geriatric Labrador retriever dog was presented for acute collapse. The dog was conscious but lethargic, tachypneic, tachycardic with weak femoral pulses, occasional pulse deficits, and pale mucous membranes. Radiography, ultrasonography, quick assessment tests, and a complete blood (cell) count (CBC)/biochemistry panel indicated internal hemorrhage and potential problems with hemostasis. The dog was euthanized. A necropsy, histopathology, and immunohistochemistry for CD31 and Factor VIII-related antigen cell markers supported a diagnosis of splenic hemangiosarcoma.     

Babesia gibsoni rhoptry-associated protein 1 and its potential use as a diagnostic antigen

A cDNA encoding the rhoptry-associated protein 1 (RAP-1) homologue was obtained by immunoscreening an expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1740bp. Computer analysis suggested that the sequence contains an open reading frame of 1425bp encoding an expected protein with a molecular weight of 52kDa. Based on the sequence similarity, this putative protein was designated as the B. gibsoni RAP-1 (BgRAP-1). The BgRAP-1 gene was expressed in the Escherichia coli BL21 strain, and the recombinant BgRAP-1 was used as the antigen in the enzyme-linked immunosorbent assay (ELISA). The results can differentiate between the B. gibsoni-infected dog sera and the Babesia canis infected dog sera or the normal dog sera. Furthermore, the antibody response against the recombinant protein was maintained during the chronic stage of infection, indicating that the recombinant BgRAP-1 protein might be a useful diagnostic antigen for the detection of antibodies to B. gibsoni infection in dogs.     

Hemolytic Transfusion Reactions in a Dog With an Alloantibody to a Common Antigen

Alloantibodies to high-frequency red cell antigens, defined as inherited traits occurring in 92% to 99% or more of the general population, are recognized as a cause of hemolytic transfusion reactions in humans. Here we describe a dog (dog erythrocyte antigen [DEA] 1.2-and DEA 4-positive) sensitized by prior blood transfusion, for which a compatible blood donor could not be found; transfusion of DEA 1.1-negative blood resulted in hemolytic transfusion reactions. Patient serum from days 1 (before first transfusion) and 16 was available for further testing; using 4 dogs with different blood types as potential donors, the major crossmatches were compatible using serum from day 1. However the crossmatches were all incompatible with serum from day 16, indicating that the patient was sensitized to an antigen after the first transfusion. The presence of an alloantibody against DEA 1.1 was not ruled out in this patient, but the incompatibility reactions of patient serum with red cells from donors negative for DEA 1.1 indicated that an alloantibody against a red cell antigen other than DEA 1.1 or any other known DEA for which typing reagents were available (DEA 3, 5, and 7) was present. Subsequently, red cells from 1 of the patient's siblings (DEA 1.2-, 4-, and 7-positive) were found not to agglutinate when incubated with patient's serum from day 16, ruling out the presence of an anti-DEA 7 antibody, and suggesting that an alloantibody against a common red cell antigen missing in the patient and sibling was responsible for the blood incompatibility reactions. Failure to obtain a compatible crossmatch with several universal donors in a dog previously transfused should raise a suspicion that an alloantibody to a common red cell antigen may exist and that a sibling may be a source of compatible blood.     

A case of T-cell chronic lymphocytic leukemia progressing to Richter syndrome with central nervous system involvement in a dog

An 8-year-old neutered Beagle dog was presented with polyuria and polydipsia. Routine clinicopathologic testing showed a significant lymphocytosis and proteinuria. Lymphocytes were of small to intermediate in size with a mature morphology. Infectious disease screening was negative. PCR for antigen receptor gene rearrangements showed a clonal T-cell receptor (TCR) rearrangement consistent with T-cell chronic lymphocytic leukemia (CLL). Bone marrow cytology showed <30% lymphocytes, while the proportion in splenic fine-needle aspirate cytology was considered increased. The dog was initially monitored but started on prednisolone and chlorambucil therapy 2 months later due to worsening clinical signs and progressive lymphocytosis. After an additional 2 weeks, the dog developed multifocal spinal pain and single-node lymphadenomegaly. Cytology of the lymph node showed a monomorphic population of large lymphoblasts consistent with lymphoma. Cytology of a cerebrospinal fluid sample also showed large lymphoblasts. PCR for antigen receptor gene rearrangement at both sites showed a clonal TCR rearrangement of the same molecular size as in the initial leukemic cells. The dog was diagnosed with a transformation of the CLL to Richter syndrome (RS) with involvement of the central nervous system (CNS). Therapy was started with L-asparaginase and an increased dose of prednisolone; however, the dog was euthanized due to progressive clinical signs. To our knowledge, this is the first report of canine RS with direct involvement of the CNS.     

Systemic lupus erythematosus in a colony of dogs

A colony of dogs was obtained by the mating of a female German Shepherd Dog crossbred and a male Belgian Shepherd Dog crossbred, both with systemic lupus erythematosus (SLE). The colony also contained 16 dogs representing F1, F2, and F3 generations. Ten colony dogs had circulating antinuclear antibodies, and 5 of the 10 had clinical signs of SLE. Two F3-generation females had signs of severe SLE. Two dogs had antibodies to extractable nuclear antigen, notably 1 dog had antibodies to Smith (Sm) antigen and 1 had antibodies to Sjogren syndrome A (SSA) antigen. Thymulin (serum thymic factor associated with zinc) titers were generally low in the descendants, but fluctuations were detected within the same dog. In vitro response of lymphocytes from these colony dogs to concanavalin A was maximal for lower mitogenic concentrations, compared with response of lymphocytes from 10 healthy dogs. The suppressive lymphocyte activity in 6 autoimmune colony dogs was diminished in comparison with the activity in 5 nonautoimmune colony dogs and 6 healthy dogs.     

Restricted dog leucocyte antigen (DLA) class II haplotypes and genotypes in Beagles

Beagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restriction within this breed and the impact this might have on vaccine responses are rarely considered. This study was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breeding colony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were determined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagles had significantly different DLA haplotype frequencies in comparison with pet Beagles and both groups showed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result in selective antigen presentation and vaccine responses that are not necessarily representative of those seen in other dog breeds.     

A 38-kDa protein from Babesia gibsoni and its antibody response in an experimentally infected dog

A cDNA encoding the Babesia bovis 12D3 antigen homologue was obtained by immunoscreening the expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1406 bp. Computer analysis suggested that the sequence contains an open reading frame of 1052 bp encoding an expected protein with a molecular weight of 36kDa. Based on homology analysis, this putative protein was designated as the B. gibsoni 12D3 antigen (Bg12D3). The Bg12D3 gene was expressed in the Escherichia coli BL21 strain, and the chronically infected dog serum reacted with the recombinant protein. The antiserum against the recombinant Bg12D3 protein can recognize a 38-kDa native protein, which is consistent with its expected size. Moreover, the purified recombinant proteins were used as the antigen to detect the antibody response in an experimentally infected dog by the enzyme-linked immunosorbent assay (ELISA). Our results indicated that the Bg12D3 protein was recognized by the host immune system and that it induced an antibody response in chronic B. gibsoni infection. These results allowed us to identify a new member of the 12D3 antigens and its characteristic immune response in canine B. gibsoni infection.     

Immunopathology of the eye: Purification of canine retinal “S” antigen

Canine retinal S antigen has been purified to study the retinal progressive atrophy of the dog. The purified antigen will be used to detect, by the ELISA technique, specific autoantibody in dogs with ocular diseases.     

The effect of ex vivo refrigerated storage and cell preservation solution (Cyto-Chex II) on CD11b expression and oxidative burst activity of dog neutrophils

The expression of CD11b and oxidative burst activity of dog neutrophils undergoing ex vivo refrigerated storage was studied using flow-cytometry . Additionally, the effect of a proprietary cell stabilization reagent (Cyto-Chex) on the expression of CD11b and oxidative burst activity was studied. Expression of CD11b was very high (>90% positive) on dog neutr ophils isolated from peripheral blood. Dog neutrophils showed a rapid and sustained increase in CD11b antigen density (P<0.01) during refrigerated storage, this increase was prevented by treatment with Cyto-Chex but was not completely blocked on the first day. There were no significant differences in mean antigen density between any days in the non-preserved group or between Days 1 to 4 in the Cyto-Chex treated group. The non-treated group showed significantly greater mean antigen density at all time points when compared to the preservative treated group (P<0.0001). Treatment with Cyto-Chex did not interfere with measurement of oxidative burst function on the first 2 days. Alterations of both resting oxidative activity and stimulated response were observed over time in both treated and untreated blood samples. Cyto-Chex treated samples showed a dramatic, significant decline in stimulated response after the third day of storage (P<0.001), while non-treated cells showed steadily increasing, but non-significant differences in stimulated response. Cyto-Chex was demonstrated to be a useful reagent for stabilization of dog neutrophil membrane antigens during storage, however this reagent is not recommended for preservation of cells for functional assays.     

Pleural mesothelioma in a nine-month-old dog

This paper reports on an unusual case of pleural epitheloid mesothelioma in a nine-month-old male, mixed breed dog. The dog was presented in-extremis and, on post mortem examination, multiple, exophytic, frequently pedunculated, yellowish-red, soft to firm masses ranging from 3 mm to 6 cm in diameter were diffusely distributed over, and attached to, the pericardial and parietal pleural surfaces. Microscopically, these masses consisted of round to partially polygonalshaped, anaplastic cells with minimal cytoplasm and hyperchromatic nuclei covering papillomatous projections or as part of more densely cellular masses. A supporting fibrovascular stroma and mitotic figures were also evident. Constituent tumour cells were labeled positively with antibodies against both vimentin and cytokeratin. In contrast, the same cells exhibited equivocal labeling with an antibody directed against calretinin antigen and did not label with antibodies against carcinoembryonic antigen (CEA) and milk fat globule-related antigen (MFGRA). Such tumours are rare in dogs, particularly in such a young animal.     

Diagnosis of systemic cryptococcosis by fecal cytology in a dog

A 3-year-old Boxer was presented with progressive diarrhea, vomiting, and lethargy of 5-months duration. The dog had watery black feces, a mature neutrophilia, and microcytic anemia. Cytologic evaluation of a direct fecal smear stained with Wright's-Giemsa revealed numerous encapsulated, narrow-based, budding organisms consistent with Cryptococcus sp. Pyogranulomatous inflammation and Cryptococcus organisms also were observed in ultrasound-guided fine-needle aspirates of the small intestine and mesenteric lymph nodes, and in histologic sections of colonic biopsies obtained by endoscopy. Multifocal chorioretinitis by fundic examination was consistent with systemic mycosis, and the reciprocal antigen titer (1600) on a cryptococcal antigen latex agglutination test for Cryptococcus neoformans was markedly increased. Using immunohistochemistry, the organism was identified further as C neoformans var. grubii (C neoformans var. neoformans serotype A). After 3 weeks of antifungal treatment, ultrasound examination revealed urinary bladder wall thickening, and Cryptococcus organisms were found in a urine sediment preparation. After 4 months of treatment, the dog was clinically normal and had no abnormal findings on CBC, serum biochemistry, urinalysis, or fecal cytology; however, the antigen titer remained unchanged, mesenteric lymphadenomegaly and jejunal wall thickening were still evident, and cytologic evaluation of fine-needles aspirates of the jejunal wall revealed budding Cryptococcus organisms. Intestinal involvement in dogs with cryptococcosis is rare, and diagnosis by fecal cytology has not been documented previously.     

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink.     

Effects of doxycycline on early infections of Dirofilaria immitis in dogs

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.     

A survey of canine echinococcosis in Gobi Altai Province of Mongolia by coproantigen detection

Few studies have been carried out for the prevalence of canine echinococcosis in Mongolia. This study was designed to elucidate a preliminary information of the prevalence from feces collected in the field. Sixty-seven fecal samples from dogs and 2 red foxes in Altai town were collected and examined for Echinococcus coproantigen and eggs. Coproantigen detection was performed by a sandwich ELISA using a monoclonal antibody EmA9 raised against Echinococcus multilocularis somatic antigen. Of the dog samples examined, 17 (25.4%) were positive by the ELISA. One out of two foxes was positive, too. Taeniid egg-positive feces were recognized in 12 dog feces. Only 6 samples were both coproantigen and egg positive. Eggs of Ancylostoma sp., Trichuris sp.; and Capillaria sp.; were also registered.     

Preliminary report on the pulmonary pathology associated with subcutaneous injections ofDirofilaria immitis antigen

Thickening of alveolar septa was seen in a dog given subcutaneousD. immitis antigen, whereas control animals had normal lung structure at autopsy.     

Acquired myasthenia gravis and cholangiocellular carcinoma in a dog

Acquired myasthenia gravis and cholangiocellular carcinoma were diagnosed in a 7-year-old English Setter referred because of forelimb lameness, exercise-induced weakness, and fever. Three months earlier, the dog had had a pleuropulmonary infection caused by a Fusobacterium sp. The concurrent development of myasthenia gravis and cholangiocellular carcinoma in this dog may be explained by a paraneoplastic syndrome, although it is unproven. The cholangiocellular carcinoma may have possessed an acetylcholine receptor-like antigen on the tumor surface, which induced autoantibodies to cross-react with acetylcholine receptors at the neuromuscular junction.