Antibodies against a tumour-associated antigen in an AKR lymphoma conditioned to grow in BALB/c mice

An experimental model was used in which AKR lymphoma cells (L15) were conditioned to grow in BALB/c mice leading to tumour-bearing (progressor) and tumour-rejecting (regressor) animals. The behaviour of antibodies present in the sera of these animals was studied using as antigen L15 cells or a soluble tumour-associated antigen TEs. Both sera showed similar IIF and haemagglutinating activity. However differences were observed for the complement cytotoxicity assay. Regressor serum as well as a rabbit anti-tumour-associated antigen serum were strongly cytotoxic for the AKR lymphoma cells while progressor serum showed markedly lower activity. Specific antibodies against the tumour-associated antigen were purified. In both sera they were located mainly in IgG1 but also in IgG2. The purified antibodies agglutinated specifically sensitized sheep erythrocytes and reacted by indirect immunofluorescence with L15 cells but not with AKR thymocytes. It is suggested that two qualitatively different humoral immune responses are involved in the mechanisms leading to tumour enhancement or rejection.     

Immunochemical studies of oligosaccharides obtained from the lipopolysaccharide of Brucella ovis

Brucella ovis rough lipopolysaccharide (R-LPS) was studied with respect to its heterogeneity, chain length, sugar composition and immunological activity. R-LPS was mildly hydrolysed and oligosaccharides were recovered in the upper phase after partition with chloroform-methanol. Gel-filtration of the upper phase in a column of Bio-Gel P-2 yielded oligosaccharides of 2, 4, 6 and 7 monosaccharide units, 2-keto-deoxy-octulosonic acid (KDO), and monosaccharides. Strong acid hydrolysis followed by paper chromatography showed that the hexa- and heptasaccharides are both composed of glucose, KDO and an unidentified sugar while tetrasaccharide is composed of glucose, mannose and glucosamine. These three oligosaccharides were able to inhibit the LPS-antibody reaction in a solid phase radioimmunoassay, suggesting the oligosaccharides bear antigenic determinants of LPS.     

Immunochemical studies on a Brucella ovis specific protein antigen

A Brucella ovis surface protein antigen (P-II), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-II labeled with 125I, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains (B. abortus and B. melitensis).     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K = 0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K=0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

Immunodiffusion test antigen from bluetongue virus-infected newborn mouse brains

Three methods of extracting bluetongue virus (BTV)-infected newborn mouse brains to prepare immunodiffusion (ID) test antigen were used. The most readily readable and reproducible results were obtained with fluorocarbon-extracted brains homogenized in 8.5% sucrose. Mouse brain- and reference cell culture-derived antigens gave a line of identity with anti-BTV serum. Extracts of noninfected brains were nonreactive. ID tests on field-collected bovine sera, comparing the two types of antigen, resulted in only 73% agreement due to a greater sensitivity of cell culture-derived antigen. A 70.5% agreement resulted when comparing mouse brain-derived antigen in ID tests with complement fixation tests, the latter being least sensitive. ID test results with sera from experimental sheep gave 95.9% agreement between cell culture- and mouse brain-derived antigens. Between ID, which detects antibody to the BTV common or group antigen, and virus neutralization, which detects type-specific antibody, the agreement was 71.4% with postchallenge sera. Data from pre- and postinjection sera, however, indicate the possible activity in Texas of viruses other than International BTV Types 10, 11, 13, and 17.     

CAMP-reaction among skin isolates obtained from a dog with an acute squamous eczema

The primary culture of a clinical specimen obtained from a dog with an acute squamous eczema revealed 3 different bacterial cultures. Two of these cultures, a beta-hemolytic Staphylococcus aureus and a group B streptococcal culture, demonstrated synergistic hemolytic activities on this primary culture plate. The group B streptococcus had the serotype surface antigens Ib/c, protein antigen c in its c beta component.     

Protective effect by cell-free antigen obtained from culture supernatant of phase I Bordetella bronchiseptica.

The cell-free antigen (CFA), with highly hemagglutination activity, obtained from the culture supernatant of Bordetella bronchiseptica was compounded with oil adjuvant to make a component vaccine (CFAV). In the immunization trial in mice, the offsprings whose mothers were immunized with CFAV escaped from death when challenged intrapleurally with virulent strain of B. bronchiseptica. The protective indices (difference of LD50 dose of the challenge strain between immunized and control groups) of the offsprings from CFAV-immunized mothers were over 3.0 in common logarithm value. Moreover, about 90% of the offsprings from CFAV-immunized mothers were negative in nasal turbinate atrophy, while over 80% of them from non-immunized mothers showed obvious turbinate atrophy when challenged intranasally with virulent strain. On the one hand, remarkable differences in the number of bacteria recovered from nostrils were observed between both test groups. It was concluded that CFAV is a very effective vaccine against B. bronchiseptica infection in animals.     

Progression of an orbital T-cell rich B-cell lymphoma to a B-cell lymphoma in a dog

An 11-year-old Shetland Sheepdog was presented for exophthalmos caused by a locally extensive, poorly defined mass located behind the right eye. The primary orbital mass was identified by light microscopy and immunohistochemistry as a T-cell rich B-cell lymphoma (TCRBCL) composed predominantly of BLA.36-positive large neoplastic lymphoid cells admixed with fewer CD3- and CD79a-positive small lymphocytes. The dog was treated for lymphoma, but 6 months after presentation it was euthanatized for suspected hepatic and gastrointestinal metastasis. Gross findings revealed an enlarged liver with multiple well-demarcated, randomly distributed 0.1-1.5-cm white nodules, five firm white submucosal jejunal nodules, and ileocecal, mediastinal, and hilar lymphadenopathy. Metastatic liver lesions consisted of sheets of monomorphic large neoplastic lymphoid cells that effaced and expanded portal and centrilobular zones. These cells were morphologically similar to the large neoplastic cells of the original orbital tumor and were CD3-negative and variably BLA.36-positive, consistent with B-cell lineage. Similar cells comprised the jejunal nodules and effaced the lymph nodes. The progression of TCRBCL to a diffuse B-cell lymphoma in this case is consistent with reported human cases and has not been previously reported in the dog.     

A case of intravascular lymphoma with mixed lineage antigen expression in a cat

A 3-year-old neutered male British shorthair cat was presented to the Clinic for Small Animal Medicine, Internal Medicine and Clinical Pathology, at the Justus-Liebig-University Giessen with a short history of lethargy, severe dyspnoea, fever, anaemia, thrombocytopenia and hyperbilirubinaemia. In blood smears and lymph node aspirates, large pleomorphic lymphatic blasts were detected. On necropsy splenic infarcts and moderate hydrothorax as well as moderate ascites could be observed. Histological examination revealed large numbers of lymphoid blasts within the lumen of the vessels of the lung, lymph nodes, the liver, the chorioid coat of the eyes, in meningeal vessels, arteries and veins of the brain, and within the larger splenic vessels. Neoplastic cells immunohistochemically stained positive for CD45R and CD3. Based on these findings, an intravascular lymphoma was diagnosed.     

Generalized thymic lymphoma in a 9-month-old ARK/J mouse

Necropsy on a 9-month-old ARK/J mouse, presenting with a large thymic mass as well as generalized hypertrophic lymphadenopathy, revealed a generalized thymic lymphoma on histopathological examination. This strain of mouse is genetically predisposed to developing this type of neoplasm.     

Immunochemical analyses of serum antibodies from pig herds in a Salmonella non-endemic region.

In a large comparative survey of Danish and Swedish slaughter pig herds performed prior to this work, it was unexpectedly found that some Swedish herds harbored seropositive pigs. Serum samples from the Swedish herds had moderate responses in the Salmonella mix-ELISA (detecting serogroup B and C1 infections) compared to the Danish herds classifying some of them as seropositive using a cut-off value at 40 OD%. In Sweden, extensive Salmonella control is carried out by bacteriological screening of feces and lymph nodes, and the overall prevalence has been proven to be below 0.1%. The serological positive results were therefore unexpected; hence the reactivities of the Swedish sera were studied by a number of immunochemical analyses (Western blot, indirect ELISA, inhibition ELISA, avidity ELISA) and compared to sera from Danish pig herds with verified Salmonella infections ("the reference sera").In Western blot, the Swedish sera had high binding reactivities against Salmonella Typhimurium LPS of different molecular weights, and gave binding patterns similar to that of the reference sera. Pre-incubation with free S. Typhimurium LPS or PS (the polysaccharide part of LPS) was able to inhibit the reactivity of the Swedish sera in the mix-ELISA. Reactivities against other related bacterial LPS such as Citrobacter freundii LPS and Yersinia enterocolitica O:3 LPS were observed in the Swedish sera, but these LPS antigens were unable to inhibit the reactivities in the mix-ELISA as efficiently as S. Typhimurium LPS. Furthermore, the Swedish sera did not bind Salmonella LPS of another serogroup (S. Meleagridis LPS, serogroup E1) or rough Salmonella LPS, both lacking the specific O-antigenic parts of S. Typhimurium LPS. The avidity of the Swedish sera was much lower than the avidity of the reference sera, which could indicate the presence of transient low-dose infections or stimulation by inactivated bacteria in feed. The results obtained in this investigation strongly indicate that the Swedish sera contain antibodies directed against the O-antigenic part of LPS from S. Typhimurium or possibly on as yet unknown bacterium.     

Neoplastic angioendotheliomatosis in a dog: an angiotropic lymphoma

Neoplastic angioendotheliomatosis was diagnosed in a nine-year-old, intact male German Shorthaired Pointer exhibiting progressive neurological signs over a six-month period. At necropsy, there was multifocal asymmetric, hemorrhagic necrosis within the cerebral hemispheres, centrum semiovale, caudate nucleus, and internal capsule. Histologically, there was extensive intravascular proliferation of pleomorphic mononuclear cells within the brain and multiple parenchymatous organs. Transmission electron microscopy demonstrated the absence of intercellular junctions between neoplastic cells. These cells were not attached to the vascular lining endothelium; Weibel-Palade bodies and a basement membrane were lacking. By indirect immunofluorescence, positive cytoplasmic staining of intravascular neoplastic cells for IgG was demonstrated. Peroxidase-antiperoxidase technique for Factor VIII related antigen was negative. As in man, this rare neoplastic disorder appears to be a lymphoma, apparently of the B cell line.     

Development of a serologic assay for cysticercosis, using an antigen isolated from Taenia spp cyst fluid

An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (ThFAS) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of ThFAS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of ThFAS were shown to react nonspecifically. Antibodies against the less than 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the less than 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.     

Immunoreactivity of fractionated antigens obtained from autoclaved extracts of an arthritogenic isolate of Erysipelothrix rhusiopathiae

The immunoreactive antigens in heat-extracted (autoclaved) preparations of an arthritogenic strain of Erysipelothrix rhusiopathiae (isolate VRS 229, serotype 1a) have been identified by gel diffusion precipitin (GDP) tests and a novel application of the enzyme linked immunosorbent assay (ELISA) procedure. Antigens precipitated by ethanol treatment of autoclaved extracts of this strain were resolved into 4 major peaks (A,B,C and D) after gel permeation chromatography on Sephacryl S200. Peak A was confirmed as a protein peak (Lowry positive) which was excluded from the gel. This peak was identified to be ELISA-reactive when assayed with serum from pigs infected with other isolates corresponding to serotypes 1a, 1b and 2. However, it did not form precipitin lines in GDP tests. Peak B was Lowry-positive and also contained carbohydrates. It was not as reactive in ELISA tests but rapidly formed precipitin lines with serum from pigs infected with the homologous isolate, but only erratically with serums from pigs infected with other serotype 1a and 1b isolates, and not with serotype 2 isolates. Peaks C and D were high in carbohydrate and phosphate content respectively but were both non-reactive in GDP tests and only slightly so by ELISA. Since serotypes 1 and 2 are the most predominant among isolates from infected pigs it is likely that the commonly recognised A antigen is a useful ELISA reagent for the diagnosis of E. rhusiopathiae infection; B antigen on the other hand, would probably be of limited diagnostic value.     

Efficacy of feline panleucopenia vaccine to prevent infection with an isolate of CPV2b obtained from a cat.

Cats vaccinated against FPLV were protected against infection with a feline isolate of CPV2b. Nonvaccinated cats developed a lymphopenia and excreted virus which infected susceptible in-contact cats.