Joint report of the Third International Workshop of Canine Immunogenetics. II. Analysis of the serological typing of cells.

Using a standardized microlymphocytotoxicity assay, seven international laboratories evaluated 144 anti-dog lymphocyte antigen (DLA) sera in 319 mixed breed and 152 Beagle dogs. The workshop confirmed the serological definitions for DLA-A2, A3, A9; DLA-B4, B5, B6, B13; DLA-C11(Cwl); and C12(Cw2). Two new specificities were assigned to the DLA-A locus (Aw14 and Aw15) in only the mixed breed dogs. A third specificity (Cw3), was assigned to the DLA-C locus. The antigen and gene frequencies of these alleles differed between the two groups of dogs, but the frequencies of the "blank" were similar in both groups. Future international collaborations will be necessary to definite more completely the polymorphisms of the major histocompatibility complex (MHC) of the dog. Those efforts will benefit from the standard serological test established in this workshop.

Sedative effect of medetomidine and its reversal by atipamezole in house musk shrews (Suncus murinus).

This study was undertaken to evaluate the sedative effect of medetomidine, an alpha 2-adrenoceptor agonist, and the counteractive effect of atipamezole, an antagonist to medetomidine, in house musk shrews (Suncus murinus). Two hundred, 300, 400, or 600 micrograms/kg of medetomidine was intraperitoneal injected into 89 house musk shrews. A sedative effect was produced in one to two minutes after injection. The dose-dependent prolongation of the sedative duration and the dose-dependent appearance of a hypothermic effect were demonstrated. With 200 micrograms/kg of medetomidine, the sedative effect obtained was not adequate in some of the animals. With 300 micrograms/kg and above, a stable sedative state was induced in all the animals. The duration of sedation in the house musk shrews was much longer (p < 0.01) in males than in females. This suggested the higher susceptibility of male house musk shrews to this drug. The sedative effect and hypothermia obtained with 400 micrograms/kg of medetomidine were completely counteracted by more than 2.0 mg/kg of atipamezole. With 0.5 and 1.0 mg/kg of atipamezole, only a partial antagonistic action was produced. Transient vomiting appeared in 4.5% of the house musk shrews at approximately one minute after injection of medetomidine. This side-effect had occurred before the sedative effect was obtained, and was not serious enough to be a problem. None of the 89 house musk shrews died in this experiment. The above results show that the combination of medetomidine and atipamezole is a highly effective and safe anesthetic treatment which permits easy handling of house musk shrews.

Detection of iron in a blackish lesion in a case of feline corneal sequestration.

Qualitative and quantitative analyses showed that there was iron (Fe) in a blackish lesion removed from a cat with severe corneal sequestration. Chromatic compound did not extract. The Fe content of the lesion was about 100 times higher than that of a normal cornea, as measured by X-ray fluorescence. Results of other chemical analyses indicated that the Fe in the blackish cornea was not derived from blood in the neovascularized limbus.

Immunosuppression in dogs by pretransfusions with ultraviolet (UV)-irradiated whole blood.

Ultraviolet (UV)-irradiated whole dog blood prevented the initiation of proliferative responses in allogeneic mixed lymphocyte reactions (MLR). When dogs were given 4 weekly intravenous transfusions of UV-irradiated allogeneic donor whole blood, peripheral blood lymphocytes (PBL) of the recipients responded less significantly to donor PBL in MLR for over one week after the final transfusion. Red blood cell (RBC)-crossmatching of these dogs was negative. Dogs treated with UV-irradiated blood did not produce anti-donor PBL antibody, or IgG, IgM and C3 determined by the indirect Coombs test. These dogs also had negligible delayed type hypersensitivity (DTH) responses to donor PBL at the end of the treatment period, recognized as slightly suppression of skin graft rejection. In contrast, six dogs receiving injections of untreated allogeneic whole blood evidenced high proliferation in MLR, agglutination in RBC-crossmatching, acute rejection of skin grafts and three of them produced C3 in the Coombs test.

Breed differences in the phenotype and gene frequencies in canine D blood group system.

The D system of canine blood groups was studied in 3,191 dogs of many different breeds. The frequencies of the D system phenotypes and genes were measured. These frequencies varied considerably between the breeds native to Japan. The frequency of the D1 phenotype was higher in breeds native to Japan than in those of non-Japanese origin. Conversely, non-Japanese breeds generally had the D2 phenotype. The dogs described as mongrel in Japan had D system frequencies intermediate between native Japanese and non-Japanese breeds. One of the most interesting findings was that in the Afghan bound the frequency of the D1 gene (0.3333) was the same as in the Shiba, though only the Shiba was native to the Japanese isles. Another Japanese breed was the Tosa, and its D1 gene frequency was 0.063, a value even lower than that for the non-Japanese Maltese (0.097).

Prophylactic efficacy of milbemycin oxime against multiple infection of dogs with Dirofilaria immitis.

In order to examine the prophylactic effects of milbemycin oxime (MO) against Dirofilaria immitis infection, experiments were carried out under multiple infection with D. immitis. Ten filaria-free beagles of age 4 to 8 months were each inoculated with a total number of 480 larvae 12 times at intervals of 15 days over a period of 6 months, and MO was given monthly for the 6 months at a dose of 0.25 mg/kg. The infection rate in the medicated group of dogs was nil, this suggesting complete protection of the infection, while in the non-medicated control group it ranged from 6.5 to 14.8% (mean, 11.4%).

Usefulness of computed tomography after myelography for surgery on dogs with cervical intervertebral disc protrusion.

Computed tomography after myelography (CTM) was performed pre- and postoperatively on four dogs diagnosed as having cervical intervertebral disc protrusion. The surgery was performed by ventral slot technique in all the cases. The direction of the ventral slot was precisely adjusted according to the location of the protruded discs as seen on CTM. Postoperative values for the transversal area of the spinal cord were greater than those measured preoperatively, suggesting effective decompression of the cord. The prognosis for these patients was excellent. In view of these results, it was considered that preoperative confirmation of the positional relationship between the spinal cord and the protruded disc by CTM was quite useful in planning the surgical technique for disc disease in the dog.

Autoantibodies and red blood cell membrane proteins in a case of canine autoimmune hemolytic anemia.

Autoantibodies and erythrocyte membrane proteins were analyzed in a case of a dog with autoimmune hemolytic anemia (AIHA). In crisis phase, antiglobulin test was positive. The eluate from the erythrocytes of the dog with AIHA gave agglutination against autologus erythrocytes. Immunoglobulin subclasses in the eluate were revealed to be IgG and IgA by the double diffusion test. Comparing the SDS polyacrylamide gel electrophoretic patterns of erythrocyte membrane proteins between the crisis and remission phases, there was a change in the protein on protein 4.1 region. However, there were no changes in blood group typings in two phases.

Xenotransplantation and high tumorigenicity of feline tumors in SCID mice.

Nine feline tumors resected at surgery were subcutaneously xenotransplanted into SCID mice. The primary take rate of the benign and malignant tumors was 50% (1/2) and 100% (7/7) respectively. Six of the eight primary tumor xenografts transplanted and grown in the first recipient were successfully transplanted serially. One feline mammary adenocarcinoma xenograft metastasized to the lung of a SCID mouse. The primary take rate of feline tumors to SCID mice was high and the established xenografts appear to be valuable tools for investigating growth and metastasis mechanisms, and various therapies for feline neoplasms.

Regression of hypertrophic osteopathy following removal of intrathoracic neoplasia derived from vagus nerve in a dog.

Surgical removal of an intrathoracic tumor derived from a vagus nerve was undergone in a dog with hypertrophic osteopathy. The tumor was pathologically diagnosed as malignant schwanoma. Soft tissue swelling, lameness, and itchiness in four limbs disappeared within 7 days after surgery. The proliferated periosteal osteophytes of the four limbs was progressively reduced with time by follow-up radiography on the 58th day after surgery. On the 710th day after surgery, these osteophytes were greatly decreased as osteopathy, malignant schwanoma.

Serum hemolytic activity of Babesia gibsoni-infected dogs: the difference in the activity between self and nonself red blood cells.

The serum hemolytic activity of Babesia gibsoni-infected dogs varied when assayed with nonself red blood cells from different dogs, whereas it did not vary when assayed with red blood cells, irrespective of self or nonself, from a particular dog throughout the experiment. The variety in activity determined with nonself red blood cells was not related to the type of red blood cell by DEA, D and J systems. Serum hemolytic activity with self red blood cells was different in the course of infection from that with nonself red blood cells, especially in the late stage of infection, when the activity with self red blood cells decreased more rapidly than that with nonself red blood cells. The results indicate that the serum hemolytic activity of B. gibsoni-infected dogs determined with self red blood cells probably reflects the in vivo activity, suggesting that the rapid decrease in activity in the late stage of infection is a way of acquired resistances for the host to recover from hemolytic anemia in the infection. The facts that the hemolytic activity increased by heating the serum at 56 degrees C, that the osmotic fragility of red blood cells remained almost on the same during the course of infection and that Coombs' test for red blood cells of the infected animal was negative suggest that the immune-mediated hemolytic anemia is not a possible mechanism for the progressive and severe anemia in B. gibsoni-infection. The present results support the previous notion that the increased serum hemolytic activity is at least one of the causes of anemia in canine B. gibsoni-infection.

Xenotransplantation of canine tumors into severe combined immunodeficient (SCID) mice.

Canine tumors resected at surgery or autopsy were subcutaneously xenotransplanted into SCID mice. Thirty of the seventy-three tumors (41.1%) grew primarily in SCID mice. The primary take rate of mammary tumors and skin and subcutaneous tumors was 55.9% (19/34) and 28.6% (8/28), respectively. One thyroid carcinoma, one oral leiomyosarcoma and one osteosarcoma also underwent successful primary growth. Thirteen of the forty-one benign tumors (31.7%) and 17 of the thirty-two malignant ones (53.1%) were successfully transplantable. The histological features of the primary xenografts and their original tumors were similar to each other in all the cases. Three xenografts of the four malignant tumors with metastasis in the canine patients were shown to grow also in SCID mice and two mammary tumor xenografts out of the three metastasized to the lungs of SCID mice as well. All the 30 primary tumor xenografts grown in the first recipient were successfully transplantable to the second generation of the SCID mice. Xenotransplantation using SCID mice appears to be a valuable tool for investigating the biological characteristics of canine neoplasmas.

Renal transplantation in cats with chronic renal failure.

Renal transplantation was performed on 6 cats with chronic renal failure. Clinical signs and the blood chemistry findings (BUN, Cr, IP) improved one week after renal transplantation. Renal anemia also improved 3-4 weeks after surgery. Two cases had the same complications, hydronephrosis and hydroureter within 1 or 2 weeks of surgery, evidenced by marked elevations of BUN and Cr, suggesting that ureteral obstruction at the site of ureterocystostomy is the main complication of renal transplantation in cats. As a new resolution for this problem, we transplanted the ureter with the intact ureteral opening of the bladder from the donor in the remaining 4 cats. Ureteral obstruction was not observed in them, thus the procedure was considered to be useful for avoiding this complication. Two cats died of pneumonia and other complications within 3 months. There were no acute rejections or side effects from the immunosuppressant during the observation period in the remaining four cats.

Changes in reticuloendothelial function in dogs with endotoxin-induced shock.

A shock model was experimentally produced by intravenous injection of a lethal dose (3 mg/kg) of endotoxin under general anesthesia induced by pentobarbital sodium using 7 beagles. The effect of this endotoxic shock on the reticuloendothelial function was investigated. The blood endotoxin concentration peaked immediately after administration and decreased subsequently. However, the value still remained on an increased level (1,051 pg/ml) even at 360 min after endotoxin treatment. The lipid emulsion test as an index of reticuloendothelial phagocytotic activity and the arterial ketone body ratio as an index of the energy charge in the liver decreased after endotoxin treatment and failed to recover during the experiment. Fibronectin, one of opsonic proteins, tended to decrease after injection of the endotoxin and was significantly (p < 0.01) low at 180 and 360 min compared with the value before injection of the endotoxin. These results suggested the depression of the reticuloendothelial function during endotoxin-induced shock.

Evaluation of extravascular thermal volume in the lung in dogs with endotoxin-induced shock by double indicator dilution method using heat and sodium ions.

Accuracy assessment was undertaken under varying hemodynamic conditions for a lung water volume measurement device which is based on the principle of a double indicator dilution method using heat and sodium ions. Changes in extravascular thermal volume were investigated in dogs with endotoxin-induced shock. The isoproterenol- or propranolol-induced changes in hemodynamics had no effect on the measurement. This confirmed the high accuracy of this measuring method. The measurement revealed a tendency for the extravascular thermal volume to gradually increase (p < 0.05) during endotoxin shock. This confirmed the gradual progression of pulmonary edema during endotoxin shock.

Application of oriented poly-L-lactide screws for experimental Salter-Harris type 4 fracture in distal femoral condyle of the dog.

The clinical usefulness of biodegradable oriented poly-L-lactide (PLLA) screws for experimental Salter-Harris type 4 fracture in the distal femoral condyle of dogs was evaluated. Bony union of the osteotomized fragment of the condyle was almost completed radiographically and histologically within 1 to 2 months after surgery, suggesting that PLLA screws maintained strength long enough to fix the fragment until bone healing. At 4 to 6 months after surgery, minute fissures were histologically confirmed on the surface of the screw thread, suggesting the early stage of biodegradation and absorption of the polymer. During the observational period, no significant difference between the treated femur and the contralateral non-treated femur in either total femoral length or maximum condyle width was observed, indicating no growth disturbance in the treated femur. From these results it was concluded that the PLLA screw might be an ideal implant for the reduction and fixation of epiphyseal plate fractures such as Salter-Harris type 3 or type 4 fractures.

Clinical evaluation of uniaxially oriented poly-L-lactide rod for fixation of experimental femoral diaphyseal fracture in immature cats.

Transverse diaphyseal fractures of the femur were experimentally made in immature cats, and were fixed by an intramedullary pinning technique using an uniaxially oriented poly-L-lactide (PLLA) rod, a biodegradable polymer. The healing process was evaluated radiographically and histologically. Formation of bony callus was completed in 8 weeks, and cortical bony union followed. The remodeling process was then observed form 12 to 16 weeks. The healing process was almost the same as when a metallic implant was used. Abundant periosteal callus formation may be attributable to the lower elasticity of the PLLA rod compared with metallic implants. Since no other abnormalities such as growth deformities were detected, it was concluded that the combined use of a uniaxially oriented PLLA rod and an external splint is clinically useful for the repair of diaphyseal fractures in immature cats.