Semiquantitative test for Dirofilaria immitis as a predictor of thromboembolic complications associated with heartworm treatment in dogs.

A semiquantitative heartworm test of antigen concentration was evaluated as a predictor of thromboembolism after adulticide treatment. Seventeen dogs with naturally acquired infections of Dirofilaria immitis (heartworms) were studied before and after thiacetarsamide treatment, using physical examinations, arterial blood gas analyses, thoracic radiography, and pulmonary hemodynamic and arteriographic tests. Eight dogs were considered to have a low burden of heartworms and 9 had a high burden. Dogs with a high worm burden had more severe pulmonary thromboembolism with pulmonary hypertension, dilated pulmonary arteries, flow obstruction of the caudal pulmonary arteries, and parenchymal lesions in the caudal lung lobes. Dogs with a low worm burden had minimal changes. Within each group of dogs, the severity of thromboembolism was less in some dogs in which all heartworms were not killed. Six of the 9 dogs with a high burden of heartworms had surviving heartworms, and 1 of these dogs had 38 live heartworms. Only 4 of the 8 dogs with a low worm burden had complete heartworm mortality, but only 1 dog had more than 3 surviving heartworms. We concluded that dogs with a high worm burden were more likely to have pulmonary thromboembolism after thiacetarsamide treatment and that dogs with a low worm burden were more likely to have minimal changes. A semiquantitative heartworm test of antigen concentration is recommended as part of the pretreatment evaluation of dogs infected with heartworms.

A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay.

We have developed an antibody detection enzyme-linked immunosorbent assay (ELISA) for the identification of animals infected by feline immunodeficiency virus (FIV). The ELISA solid-phase antigen consists of recombinant FIV gag proteins expressed in bacteria. The proteins are purified from bacterial lysates as insoluble inclusion bodies. In the case of bacterially expressed p24gag, it is shown that all of the linear, sequential epitopes presented by viral p24 during infection are retained. Purified preparations can be substituted for solid-phase whole virus in the IDEXX PetChektm immunoassay. The antibody ELISA duplicates the sensitivity and specificity of the whole virus based PetChek plate assay.

Enzyme-linked immunosorbent assay methods for detection of feline leukemia virus and feline immunodeficiency virus.

Enzyme-linked immunosorbent assays have been widely used for diagnosis of FeLV and feline immunodeficiency virus (FIV) infections. Various ELISA kits for FeLV are available from several manufacturers. Although these tests are configured in a variety of formats, they are all direct antigen-detection systems for the viral core protein p27. On the other hand, ELISA for FIV exposure detects specific feline antibody to FIV. Basic immunoassay principles and the application of ELISA technology used in FeLV and FIV ELISA kits are described.

Immunoassay for detection of feline immunodeficiency virus core antigen.

The feline immunodeficiency virus (FIV) is a recently identified feline lentivirus that has been found at significant levels in domestic cat populations worldwide. A microdilution plate format, monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of the FIV group-associated antigen (gag) designated p24. Assays of serially diluted samples containing disrupted virus showed that the assay had a sensitivity limit of approximately 0.2 ng/ml for FIV p24. The assay was approximately eightfold more sensitive than the assay for viral reverse transcriptase activity when it was tested with diluted tissue culture samples. A qualitative confirmation assay by standard antibody inhibition techniques was coupled to the screening test methodology. The test was used to detect and confirm the presence of virus in cultured feline lymphocytes from infected animals.

Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase HPLC and the aminoterminal sequences of p10 and p24 were determined. Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 35[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of 50K (Pr50gag). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosidase H and glycopeptidase F revealed that p130 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.

Development and evaluation of immunoassay for detection of antibodies to the feline T-lymphotropic lentivirus (feline immunodeficiency virus).

The feline T-cell lymphotropic lentivirus (feline immunodeficiency virus) is a recently described feline-specific retrovirus that can produce chronic immunodeficiency-like disorders in cats. A microdilution plate format enzyme-linked immunosorbent assay has been developed to detect the presence of antibody to the virus in feline serum or plasma. Temporal studies performed with experimentally infected animals show that seroconversion can be demonstrated 3 to 4 weeks after exposure to the virus. Results of a serosurvey (n = 1,556 samples) indicate that infection is fairly common in both clinic (5.2%) and sick cat (15.2%) populations. Western blot (immunoblot) and sodium dodecyl sulfate radioimmunoprecipitation assays were developed to confirm microdilution plate test results and to identify peptides specific for the feline immunodeficiency virus. All microdilution plate test positive results and selected negative results were confirmed by one or both of these procedures. These data demonstrate that this microassay plate enzyme-linked immunosorbent assay is a very sensitive and specific test for detection of antibody to the feline immunodeficiency virus.

Nononcogenic hormone-independent alveoli produced by carcinogens in cultured mouse mammary glands.

Treatment of mouse mammary glands with a high concentration of 7,12-dimethylbenzo(a)anthracene in whole organ culture was reported by Banerjee et al. to transform foci of lobuloalveoli to a hormone-independent state, and to give rise to mammary hyperplastic outgrowths and adenocarcinomas in vivo. In the present study using the identical system, mammary glands of BALB/c mice were exposed to 7,12-dimethylbenzo(a)anthracene or N-2-fluorenylacetamide at low concentrations that bring about maximal incidences of the hormone-independent hyperplastic lobuloalveolar lesions with minimal cytotoxicity. After morphological development of the lobuloalveoli in culture, the glands were enzymatically dissociated into cells and inoculated into gland-free inguinal mammary fat pads of syngeneic mice bearing pituitary gland implants during the initial 8 weeks. After 11 months, fragments of the resultant mammary outgrowths from each mouse were implanted into the gland-free inguinal mammary fat pads of 3 syngeneic mice (not bearing pituitary gland supplements) and were permitted to grow for another 11 months. Mammary outgrowths from the primary and secondary implants were neither neoplastic, anaplastic, nor dysplastic. Also, no hyperplasia in any mammary outgrowth could be attributed to the action of either carcinogen, especially when outgrowths were compared with contralateral outgrowths that arose from the control glands exposed to dimethyl sulfoxide (solvent of the carcinogens) in culture and/or with untreated thoracic mammary glands of the same hosts. One interpretation of these findings is that the hormone-independent, hyperplastic alveolar lesions may not be an appropriate in vitro marker of oncogenic transformation by chemical carcinogens in culture. The great variety of procarcinogens and activated carcinogens that bring about this lesion in vitro and its morphological similarity to presumptive mammary preneoplastic lesions in vivo weigh against this interpretation. A second hypothesis is that high concentrations of procarcinogens, despite their considerable cytotoxicity, complete a multistep process of oncogenic transformation in surviving mammary epithelium, whereas low concentrations optimized to produce the lesions in maximal number do not.

Epidermal growth factor requirement for development of cultured mammary gland.

The mouse mammary gland in serum-free whole organ culture can be manipulated hormonally to undergo one complete physiological cycle consisting of lobuloalveolar development, functional differentiation and regression, mimicking processes that occur in vivo. A second cycle has not previously been achieved in vitro. The present study has identified a specific requirement for epidermal growth factor (EGF) in the morphological development of mammary lobuloalveoli, allowing two complete cycles of development and regression in culture.

Sialic acid: a specific role in hematopoietic spleen colony formation.

Vibrio cholerae neuraminidase (VCN) treatment of donor bone marrow cells results in a reduction in the number of hematopoietic colonies (CFUs) formed in the spleens of lethally irradiated mice. Treatment of marrow cells with sodium periodate under mild conditions, known to preferentially oxidize sialic acid, also reduced CFUs while subsequent potassium borohydride reduction restored CFUs to 80% of control levels. Innoculum viability as measured by in vitro incorporation of tritiated precursors into proteins, nucleic acids, and oligosaccharides was unaffected by VCN treatment. The ability of bone marrow cells in culture to respond to the hormone erythropoietin, as measured by the incorporation of 59Fe into cyclohexanone-extractable heme, was also not affected by neuraminidase, making a cytotoxic effect of the VCN preparation unlikely. Incubation of VCN-treated marrow with either beta-galactosidase or trypsin had no effect on the VCN-induced reduction in CFUs. These results are consistent with the idea that membrane sialic acid plays a direct and specific role in the implantation and development of CFUs.

Surface molecules of hematopoietic stem cells: requirement for sialic acid in spleen colony formation.

Enzymatic treatment was used to test the function of some surface peptides and carbohydrates in hematopoietic spleen colony formation. Proteases and most glycosidases had no effect on spleen colony formation, whereas treatment with Vibrio cholerae neuraminidase reduced colonies by one-half. Intact sialic acid (residues appear to play an important role in colony formation.