IDDM in rats induced by thymectomy and irradiation.

A diabetic syndrome closely resembling human IDDM has been induced in rats of specific pathogen-free origin by a combination of thymectomy and irradiation, with an overall incidence (10 wk postirradiation) in female rats of 34% for acute disease and 47% for islet lesions. Males were slightly more susceptible than females. Clinical features of the syndrome included hyperglycemia, insulinopenia, ketosis, and lipidemia, and corresponding islet pathology ranged from diffuse atrophy to focal atrophy and insulitis. Onset was usually acute, and the disease fatal unless early insulin therapy was initiated. Lymphocytic thyroiditis also was observed in a proportion of thymectomized and irradiated rats (49% in females) over the same period but with no apparent correlation to the occurrence of diabetes. A significant decrease in the incidence of disease was found in thymectomized and irradiated rats of conventional origin when compared with genetically identical specific pathogen-free rats, implicating a role for environmental factors in disease susceptibility. Diabetes inducement also was found to be strain related but not RT1u dependent. Both clinical signs and islet lesions were inhibited by early reconstitution of thymectomized and irradiated animals with syngeneic lymphoid cells from normal donors. Islet lesions and glucose intolerance could be transferred to syngeneic recipients by concanavalin A-activated lymphoid cells from acute diabetic donors. The close similarities between this experimental syndrome induced by immunological manipulation and the clinical condition in humans provide further evidence for an immune-mediated pathogenesis for IDDM.

Erythrocyte binding factors in the sera of the adult lamprey Geotria australis.

Adults of the Southern Hemisphere lamprey Geotria australis possess three major factors in their sera which bind to erythrocytes in vitro; in addition, an induced factor can also be demonstrated in the sera of animals immunized against human erythrocytes. These factors are a hemagglutinin, a hemolysin and a further binding factor together with a probable antibody. Furthermore, differences in agglutination patterns following immunization were noted between animals of different age groups. Old adults possessed a unique dilution pattern, containing a negative zone which occurred shortly after the initial dilutions, followed by a return to the normal agglutination pattern in the higher dilutions to a titer of 2(18). While the greatest agglutinating activity took place in sera taken from immunized animals, all animals possessed weak, low titer nonspecific direct agglutination together with a strong nonspecific binding activity which was revealed only by the use of an antiserum to lamprey plasma proteins. It is suggested that the interaction of these factors may account, in some measure, for the confusion concerning the nature and magnitude of the immune response in this phylogenetically important group of animals.

Induction of diabetes in PVG/c strain rats by manipulation of the immune system.

A combination of thymectomy and sublethal irradiation (Tx-X) consistently induced diabetes in female rats of the PVG/c strain. The incidence of diabetes varied from 10.7% to 53.4% in seven successive Tx-X groups (mean 29.7%). Both clinical and subclinical disease was observed with the majority of affected animals developing the former condition. This was acute in onset, rapidly fatal (1-4 days) and characterized by ketosis and lipidemia. Overtly diabetic rats had markedly raised plasma glucose concentrations compared to normal rats of the same strain and plasma immunoreactive insulin concentrations were correspondingly depressed in this group. Histopathological change within the islets of Langerhans correlated with clinical status and ranged from diffuse atrophy in the majority of the acutely diabetic rats to mild and focal lymphocytic insulitis in a proportion of the non-diabetic rats. Islet cell autoantibodies were demonstrated by indirect immunofluorescence in approximately 25% of clinically diabetic animals. The majority of diabetic rats were found to be responsive to insulin and the clinical signs could be reversed by daily parenteral insulin administration. These observations implicate the immune system in diabetes generation and are consistent with an immune mediated pathogenesis as the underlying cause of the islet cell destruction. This syndrome may thus be a potentially useful animal model for type 1 (insulin dependent) diabetes in man.

Preparation of diagnostic polyclonal and monoclonal antibodies against outer envelope proteins of Serpulina pilosicoli.

The purpose of this study was to prepare specific sera for use in the rapid detection and identification of the intestinal spirochaete Serpulina pilosicoli. In Western blot analysis, with pig antiserum which was raised against whole cells of S. pilosicoli and absorbed with outer envelope protein extracts from S. hyodysenteriae and S. innocens, a prominent protein with Mr of c. 72 kDa was consistently identified in outer envelope preparations of S. pilosicoli strains. Immunogold labelling demonstrated that this was located on the outer surface of intact S. pilosicoli cells. Two monoclonal antibodies (MAbs), designated C12 and M96, were raised against the protein. Although C12 reacted with a protein band of c. 72 kDa, this was also present in preparations from strains of other Serpulina spp. examined. MAb M96 reacted with an 80-kDa protein which was present only in preparations made from strains of S. pilosicoli. This was used in Western blot analysis and in an immunodot-blot assay with outer envelope extracts to specifically identify S. pilosicoli strains isolated from man, pigs, dogs and poultry. An indirect immunofluorescence test with MAb M96 also was used to detect and identify whole S. pilosicoli cells. Therefore, both the cross-absorbed antiserum and MAb M96 are potentially useful reagents for the detection and identification of S. pilosicoli.

Humoral immunity in disseminated Aspergillus terreus infection in the dog.

Aspects of humoral immunity were studied in 17 dogs with disseminated aspergillosis (16 cases Aspergillus terreus, 1 case Aspergillus flavipes). All dogs had markedly raised serum IgG levels by single radial immunodiffusion (range 1500-6000 mg dl-1). Despite this, serum antibody to A. terreus was demonstrated in only 7/16 cases by agar gel diffusion, 9/16 cases by counter immunoelectrophoresis, 10/16 by ELISA and 11/16 by an indirect immunofluorescence assay. Serum antibody was also detected in 2/5 clinically normal relatives of 2 cases, indicating previous exposure or subclinical infection.

Measurement of protease thermostability, twitching motility and colony size of Bacteroides nodosus.

Total extracellular protease activity of Bacteroides nodosus in TAS liquid culture varied directly with cell mass and buffer concentration between 20 and 50 mM HEPES, MOPS and TES buffers, but not with Tris which gave anomalous high cell counts, nor with Na2Co3 which showed a decline of protease activity and cell mass. The stability of HEPES-buffered crude protease preparations were estimated on the basis of temperature or Ca2+ activity. Variation of the estimates for cellular twitching was greater than that for colony diameter in benign and virulent strains of B. nodosus. Surface translocation, quantified on the basis of colony diameter, reached a limit after 72 h incubation on modified TAS agar, ranging from 0.04 to 0.14 mm per h for six isolates tested.

Evidence for immunosuppression associated with Jembrana disease virus infection of cattle.

Jembrana disease virus (JDV) is a newly recognised bovine lentivirus causing an acute disease syndrome in Bali cattle (Bos javanicus) in Indonesia. We evaluated the effect of JDV infection on the antibody response to chicken ovalbumin (cOVA) and Brucella abortus Strain 19 in Bali cattle. In infected cattle the IgG and IgM response to cOVA was suppressed and delayed and the IgG response to B. abortus Strain 19 was delayed. The results indicate that the humoral immune response is suppressed and delayed in JDV infected cattle.

Characterisation of virulent and benign strains of Bacteroides nodosus.

The extracellular proteases of 395 isolates of B. nodosus from ovine, bovine and caprine foot lesions were classified as either thermostable or thermolabile. Stable protease was associated with one and unstable protease with four distinctive isoenzyme patterns, each pattern differentiated by the relative mobility of paired isoenzymes. Pathogenicity tests on 64 isolates showed a correlation between the production of stable protease and the production of virulent ovine footrot lesions. The mean values for total protease activity, twitching motility and colony diameter were significantly higher for virulent compared to benign isolates, but the range of values overlapped. SDS-PAGE whole-cell electrophoretic profiles of virulent isolates were similar to the profiles of some benign isolates.

Autoantibodies to pancreatic islet cells in canine diabetes mellitus.

Indirect immunofluorescence on normal canine pancreatic tissue fixed in Bouin's solution was used to detect islet cell antibodies in dogs with diabetes mellitus, other endocrine diseases, and pancreatitis. 18 of 25 dogs with diabetes mellitus alone, 2 of 8 dogs with diabetes mellitus and concurrent pancreatitis, and 2 of 2 dogs with diabetes mellitus and concurrent pancreatic exocrine insufficiency were positive for autoantibody. 2 of 12 dogs with hypoadrenocorticism, 3 of 6 dogs with hyperadrenocorticism, 6 of 28 dogs with hypothyroidism and one of 19 dogs with pancreatitis alone were also antibody positive. None of 20 healthy dogs or 20 dogs with disorders other than those of the pancreas or endocrine organs were antibody positive. Islet cell antibodies were demonstrated in dogs with diabetes mellitus and other endocrine disorders. The possibility of autoimmune involvement in the development of diabetes mellitus in the dog should be considered.

Experimental thyroid autoimmunity in the dog.

Thyroid autoimmunity was induced in 6 crossbred dogs by a single injection of thyroid extract in complete Freund's adjuvant alone, or followed by reimmunization with thyroid extract and incomplete Freund's adjuvant or peanut oil. Total thyroxine levels, autoantibodies to thyroglobulin and thyroid microsomal antigen and thyroid histopathological changes were sequentially monitored for up to 150 days. All dogs developed fluxuating levels of thyroid autoantibodies detectable by enzyme-linked immunosorbent-assay (ELISA). Histopathological damage was observed in one or more thyroid gland biopsies in 4 of the dogs, in 3 cases the lesions were of lymphocytic thyroiditis, in the fourth dog the lesion was a granulomatous reaction. Histopathological thyroid damage was observed in biopsies taken during periods of maximal autoantibody titer. Fixed immunoglobulin was not demonstrable in thyroid gland biopsies by direct immunofluorescence. In 5 dogs total thyroxine levels fell to below the normal range at some period during the study. However in only one dog did this correlate with a time when biopsy findings revealed histopathological thyroid damage. High levels of thyroid autoantibody may indicate histopathological thyroid damage in dogs, however lower levels of thyroid antibody may be found without evidence of histological damage or alteration in thyroid function.

Isolation and in vitro translation of messenger RNA encoding allergens of the cat flea, Ctenocephalides felis.

Polyadenylated [poly(A)+] mRNA was isolated from the cat flea, Ctenocephalides felis by oligothymidylic acid-cellulose spin column chromatography and translated in vitro using a cell-free rabbit reticulocyte system. The relative incorporation of 35S-methionine into trichloroacetic acid-precipitable translation products obtained using poly (A+) mRNA was 48.5-fold over control translations performed without added mRNA. SDS-PAGE analysis of the translation products in combination with autoradiography showed that many proteins with apparent molecular weights in the range 14-90 K were synthesized. Immunoprecipitation studies performed using flea-allergic dog sera showed that several of the synthesized proteins corresponded with flea allergens. The allergenicity of the lysates was also confirmed by skin testing.

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses.

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.

Effect on humoral tolerance (IgG and IgE) in dogs by oral administration of ovalbumin and Der pI.

The effect of oral administration of ovalbumin (OVA) or recombinant house dust mite allergen (Der p I) to dogs upon specific IgG and IgE reactions to subcutaneous immunization with these antigens was studied. Daily feeding of 10 x 10 g of OVA resulted in a non-responsiveness to subsequent parenteral immunization with OVA in two young dogs. The same two dogs were also immunized parenterally with Der p I and showed a pronounced IgG response against native Der p I, confirming that the non-responsiveness to OVA was antigen-specific. Thus, it has been demonstrated that it is possible to induce oral tolerance in dogs. Two other dogs of the same litter that received 2 x 10 mg of recombinant Der p I in a crude yeast lysate per os reacted to immunization with OVA with pronounced IgG and IgE production against OVA, further confirming the antigen-specificity of the OVA tolerance. However, tolerance to Der p I was not induced, as evidenced by a strong IgG response to immunization after per os application of the antigen, possibly because the oral dose was too small.

Immunologic study of systemic aspergillosis in German shepherd dogs.

Data are presented from a series of eight cases of disseminated canine aspergillosis (A. terreus) in German Shepherd dogs referred to Murdoch University Veterinary Hospital. Immunoglobulin determination revealed depression of serum IgA (cases 1 and 5) and IgM (case 2) levels and elevated levels of IgG in all cases. Total complement activity (CH50) and complement components tests, (C3, C4) were present in normal amounts in all cases. Using agar gel diffusion, serum antibody to A. terreus was found in only one case and aspergillus antigenaemia in two of the remainder. Lectin transformation of lymphocytes in two dogs was found to be depressed relative to normal controls in case 1 and initially in case 2. Two dogs failed to respond to the intradermal injection of A. terreus antigen.

Cyclosporin immunosuppression of sheep: pharmacokinetics and allograft survival.

A chronically immunosuppressed sheep model was established using a regimen of cyclosporin A (CsA; 2-3mg/kg twice daily) and ketoconazole (10mg/kg twice daily). Blood CsA concentrations reached a steady-state after 17 days of treatment. The clearance of CsA decreased from a mean (95% CI) of 9.47 (6.2-12.7)ml/min/kg after a single (first) dose (3mg/kg i.v.) to 1.62 (1.38-1.86)ml/min/kg after 18 days of CsA (3mg/kg i.v. twice daily) co-administration with ketoconazole. These data indicated that the combination of CsA and ketoconazole could be used to give stable high concentrations of CsA in the sheep. Using this regimen in the sheep, the long-term survival of skin allografts was monitored as an indicator of effective immunosuppression. CsA in blood was measured daily and CsA dose adjusted to various target concentration ranges. Provided that the trough concentration of blood CsA was maintained between 1500-2500 mg/l, long-term healthy skin allografts were maintained on the sheep without significant adverse effects on haematological or biochemical parameters.

Enzyme-linked immunosorbent assay for the detection of antibodies to infectious bronchitis virus.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis (IB) virus is described. The immune response of chickens following vaccination with IB virus was monitored using this test, and the titers were compared with those obtained by serum neutralization. The ELISA appears to be suitable for IB serology.

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.

Serum immunoglobulin A concentrations in normal and diseased dogs.

The normal level of serum IgA in Western Australian dogs was defined by single radial immunodiffusion using sera from 100 healthy randomly selected adult crossbred animals. Serum IgA values of 185 animals from six breeds were also determined. The mean and variance of serum IgA of these groups were similar to the crossbred dogs with the exception of German shepherd dogs where these values were statistically greater. In addition, 210 dogs with a range of chronic diseases (autoimmune, hypersensitivity, pyoderma, neoplasia, demodecosis, disseminated aspergillosis) were assayed and low values recorded in five cases. In all disease groups the mean serum IgA value was significantly greater than in the crossbred group and the variance significantly greater in most of these groups. The German shepherd group were the only normal dogs with a mean and variance similar to those of the clinical series suggesting that this breed may have a primary defect in IgA metabolism.

Factors affecting the composition of mare uterine fluid.

The influx of protein and polymorphonuclear leucocytes (PMN) into the uterine lumen was examined at different intervals after intrauterine infusion of fluids. The intrauterine infusion of both phosphate buffered saline (PBS) and a solution of lipopolysaccharide (LPS) derived from Escherichia coli resulted in a biphasic influx of protein in the uterine flushings peaking three and six hours after infusion. LPS infusion caused an additional influx of protein at 24 hours. The initial influx of protein preceded a biphasic influx of PMN which peaked six and 24 hours after both infusions. Uterine flushings obtained following PBS and LPS infusion contained both serum-derived and uterine-specific proteins. To investigate whether the influxes were a general response to reproductive mucosal stimulation, several regions of the reproductive tract were subjected to physical manipulation. Results suggested that these influxes were initiated chiefly by stimulation of the cervical and, or, uterine region.

Survey of thyroglobulin autoantibodies in dogs.

An enzyme-linked immunosorbent assay was used to detect autoantibodies to thyroglobulin in dogs with and without clinical evidence of thyroid disease. Autoantibodies were found in 59% of 34 clinically hypothyroid dogs, 43% of 65 dogs with nonthyroidal endocrine diseases, 47% of 64 healthy dogs closely related to antibody-positive canine hospital patients, and 13% of 1,057 canine hospital patients without endocrine disorders. Dogs with bacterial skin disease and alopecia of unknown causes had an increased prevalence of autoantibodies compared with the prevalence of autoantibodies in healthy dogs and dogs with other diseases. Three breeds of dogs, Great Danes, Irish Setters, and Old English Sheepdogs, had an increased occurrence of autoantibodies compared with that in other breeds and dogs of mixed breeding. A familial tendency to thyroid autoimmunity was demonstrated in a group of Great Dane dogs. The occurrence of autoantibodies was not influenced by age or sex. Thyroid function, as assessed by total thyroxine estimation, was depressed in hospitalized dogs compared with the thyroid function in healthy dogs, irrespective of thyroglobulin-antibody status. Two of 11 autoantibody-positive dogs monitored for an 18-month period developed low thyroxine concentration and depressed thyroid responsiveness to exogeneous thyrotropin stimulation.