Application of a direct flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.

Immunohistochemical detection of tumor suppressor gene p53 protein in feline injection site-associated sarcomas.

Sarcomas associated with injection sites are a rare but important problem in cats. Immunohistochemical detection of p53 protein may correlate to mutation of the p53 tumor suppressor gene, a gene known to be important in oncogenesis. The expression of nuclear p53 protein in 40 feline injection site-assocated sarcomas was examined by immunohistochemical staining. In 42.5% (17/40), tumor cell nuclei were stained darkly; in 20% (8/40), tumor cell nuclei were stained palely; and in 37.5% (15/40), tumor cell nuclei were unstained. Immunohistochemical detection of p53 protein in a proportion of injection site-associated sarcomas suggests that mutation of the p53 gene may play a role in the pathogenesis of these tumors.

Immunohistochemical detection of canine distemper virus in haired skin, nasal mucosa, and footpad epithelium: a method for antemortem diagnosis of infection.

A reliable antemortem diagnostic method is needed for determining infection with canine distemper virus (CDV). The utility of immunohistochemical detection of CDV antigen was examined was examined for samples of nasal and footpad epithelium and haired skin in dogs with and without detectable CDV antigen in the lung and/or brain. Tissues from 57 dogs at risk of CDV infection were tested. Viral antigen was found in the lung and/or brain of 28 dogs. Among these dogs, viral antigen was demonstrated in the epithelial cells of the nasal mucosa in 24 of 27 dogs, in the footpad epithelium in 24 of 26 dogs, and in the haired skin of the dorsal neck in 26 of 27 dogs. Among the 29 dogs without CDV antigen in either the lung or brain, 1 dog had positive staining for viral antigen in the skin and nasal mucosa. Biopsies of haired skin of the dorsal neck, which is relatively simple to sample, can be used for immunohistochemical testing for acute and subacute infection with CDV.

Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.

Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats.

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.

Evaluation of methods for dehydration of bovine colostrum for total replacement of normal colostrum in calves.

Different methods for the dry preservation of colostrum to be used as a total replacement for bovine colostrum were evaluated. Pooled colostrum from the first and second postpartum milkings from multiparous dairy cows containing immunoglobulin in excess of 40 g/L was freeze-dried, microwave vacuum evaporated, and spray-dried. Spray-drying produced a dried colostrum in which immunoglobulin quantity and function were preserved and was the most cost-effective. Other dehydration methods, although effectively conserving immunoglobulins, were too slow and costly to be used to produce a bovine colostrum replacer. Newborn, colostrum-deprived, dairy calves fed spray-dried colostrum containing 126 grams of immunoglobulin reconstituted in three liters of water as their sole source of immunoglobulin achieved normal mean serum immunoglobulin concentrations. Spray-dried colostrum with high concentrations of immunoglobulin may be produced economically and used as an effective and convenient colostrum replacer in newborn calves.

The detection of bovine respiratory syncytial virus in formalin fixed bovine lung with commercially available monoclonal antibodies and avidin biotin complex immunohistochemistry.

Eight commercially available monoclonal antibodies directed against respiratory syncytial virus antigens were tested for ability to detect bovine respiratory syncytial virus (BRSV) antigen in formalin fixed, paraffin embedded bovine lung using avidin-biotin complex immunohistochemical staining. Monoclonal antibodies from clone 18B2 purchased from Biosoft, Paris, France and those from clone 8G12 purchased from the Department of Veterinary Sciences, University of Nebraska, Lincoln, Nebraska stained BRSV antigen in infected bovine lung with acceptable background staining of uninfected tissues. This method offers advantages over other techniques for BRSV diagnosis in that fresh tissue is not required and all reagents may be purchased commercially.

Effects of morphine and caffeine on adenosine release from rat cerebral cortex: is caffeine a morphine antagonist.

Morphine (1 mg/kg) enhances the efflux of labelled adenosine and its derivatives from the rat cerebral cortex. Caffeine (40 mg/kg) does not alter either basal purine efflux rates or the enhanced efflux elicited by morphine. The antagonism by caffeine of morphine-evoked inhibition of acetylcholine (ACh) release from the cerebral cortex is unlikely therefore to the the result of an action of caffeine on the opiate receptor but is instead due to an antagonism of the inhibitory effect of released adenosine on ACh release.

The effect of morphine on purine and acetylcholine release from rat cerebral cortex: evidence for a purinergic component in morphine's action.

Morphine enhances the release of adenosine and its metabolites from the rat cerebral cortex and inhibits the release of acetylcholine. Naloxone antagoinizes the effects of morphine on both purine and acetylcholine release. The adenosine antagonists, caffeine and theophylline, reduce morphine's effects on acetylcholine release, and at the same time increase the spontaneous release of acetylcholine. It is suggested that morphine, acting at a naloxone-sensitive site, enhances the level of extracellular adenosine, which in turn inhibits the release of acetylcholine, and that some of morphine's actions are mediated by a purinergic step.

Morphine enhances adenosine release from the in vivo rat cerebral cortex.

Morphine (1 and 5 mg/kg), administered intravenously, increased the rate of efflux of purines from intact rat cerebral cortices prelabelled with 3H-adenosine. Naloxone antagonized morphine's action. It is suggested that the depressant actions of morphine on transmission in the central nervous system may be related to this enhancement of extracellular levels of adenosine and the adenine nucleotides.