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Biomedical subjects

F C Thomas

Publications and source records attributed to F C Thomas.

At least 19 recordsLinked to original sources
PubMed1991

Dot immunoperoxidase assay using monoclonal antibody for detection of bluetongue virus antigens.

A rapid, simple dot immunoperoxidase assay (DIPA) is described for visual detection and identification of bluetongue virus (BTV) antigens in samples of infected cell culture fluid. The assay was performed using nitrocellulose (NC) paper and 'dipsticks'. Dots of samples were adsorbed to the NC surface and the remaining non-specific binding sites were blocked with skim milk solution. BTV was detected with either of two murine monoclonal antibodies (4H4, 5G12) to the major group specific antigens of BTV, and the complex was reacted with a peroxidase conjugated anti-mouse immunoglobulin G (heavy- and light-chain specific). Positive reactions were easily visualized as brown spots after enzyme degradation of substrate containing H2O2 and diaminobenzidine (DAB). The DIPA was specific in detecting BTV in samples of cell culture fluid from baby hamster kidney (BHK-21) cells infected with U.S.A. isolates of the five BTV serotypes (2, 10, 11, 13 and 17) known to exist in the U.S.A., and South African isolates of 17 BTV serotypes (1-12, 14-16, 18 and 20), but not with two North American isolates of epizootic hemorrhagic disease of deer virus (EHDV) representing serotypes 1 and 2. Attempts to detect BTV directly in infected sheep blood cells and chick embryo tissue suspensions by DIPA were unsuccessful. Of 55 cell culture fluid samples examined from BHK-21 or Vero cell monolayers inoculated with 55 clinical specimens, propagated initially in embryonating chicken egg (ECE) 11 proved positive and 44 were negative by DIPA. The results were in complete agreement with the conventional ECE and tissue culture isolation systems. The DIPA appears to have potential application, especially as a 'dipstick' kit, for rapid and inexpensive laboratory diagnosis of bluetongue virus infection.

Animals↗
PubMed1991

Gamma irradiation alters bluetongue virus protein antigen.

Bluetongue virus (BTV) antigen, prepared for a monoclonal antibody (MAb)-based competitive enzyme-linked immunosorbent assay (C-ELISA), was exposed to 1, 2, 3, 4, 5 and 6 Mrad of gamma irradiation. The major group-specific BTV protein (VP7) reactive with the Mab was altered at higher doses of radiation, as revealed by immunoblotting studies. As well, a reduction in immunoreactivity was noted when irradiated antigen was used in the ELISA.

Animals↗
PubMed1989

Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting blue-tongue virus antibodies in cattle and sheep.

The performances of a competitive enzyme-linked immunosorbent assay (ELISA) using a group specific monoclonal antibody against bluetongue virus, an indirect ELISA and the standard agar gel immunodiffusion (AGID) test were compared in the detection of serum antibody against bluetongue virus. Test sera consisted of 1300 bovine, 530 ovine and 160 carpine samples from bluetongue-free areas of Canada, 605 bovine and ovine field samples from the USA and Barbados and 464 samples from 79 cattle and sheep experimentally infected with 19 South African and five USA serotypes of bluetongue virus. The diagnostic specificity of the competitive ELISA, as determined for the bluetongue virus-free cattle sera was superior (99.92 per cent) to that of the indirect ELISA (99.85 per cent) and the AGID (99.0 per cent). The specificities of the competitive ELISA for sheep (99.63 per cent) and goats (100.0 per cent) sera were also higher than those of the AGID test. The performance of the ELISA tests was similar whether a gamma-ray-irradiated (2.0 Mrad) or a non-irradiated bluetongue virus antigen preparation was used. The competitive ELISA results for bovine field sera from endemic areas demonstrated a relatively low level of agreement (92.04 per cent) with AGID test results, with 9.7 per cent false negatives. The possible presence in these sera of antibody to cross-reacting antigens or to other orbiviruses, eg, epizootic haemorrhagic disease virus, which react in the AGID but not in the competitive ELISA may account for this lack of agreement.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The distribution of lipids and sterols in cell types from the marine sponge Pseudaxinyssa sp.

The sponge Pseudaxinyssa sp., unique in sterol and fatty acid composition, was cellularly dissected into fractions enriched in each of the major cell types present in the sponge: microbial symbionts (cyanobacteria), small sponge cells (pinacocytes and choanocytes), and large sponge cells (archeocytes and cyanophytes). Three phototrophic microbial symbionts were also isolated from the cell fractions and grown in culture. An unsymmetrical distribution of fatty acids and sterols was observed for the sponge cells: small cells contained larger quantities of long chain fatty acids (greater than C24) and smaller quantities of sterols than were present in the larger sponge cells. Moreover, the rare sterols 24-isopropylcholesterol predominated in the smaller sponge cells, whereas its 22-dehydro analog predominated in the larger sponge cells. Long chain fatty acids and sterols were not detected in the cultured microbial symbionts. This constitutes the first report of lipid variability according to cell type for this most primitive group of Metazoa.

Animals↗
PubMed1988

Nontransmission of bluetongue virus by embryos from bluetongue virus-infected sheep.

Donor sheep were infected either by bites of bluetongue virus (BTV)-infected (serotype 11, "Texas Station strain") Culicoides variipennis or by inoculation with 100,000 median chicken embryo intravascular lethal doses of BTV (serotype 11) from a suspension made from infected C variipennis. Fourteen embryos from 4 BTV-infected ewes bred by rams not infected with BTV were transferred to 8 BTV-seronegative recipient ewes, and 35 embryos and 4 unfertilized eggs from 14 BTV-infected ewes bred by BTV-infected rams were transferred to 19 BTV-seronegative recipient ewes. Eleven pregnancies and 12 lambs resulted. None of the recipients or lambs seroconverted, and BTV was not isolated from the pregnant recipient ewes or their lambs at slaughter 30 days after parturition.

Animals↗
PubMed1987

Embryo transfer as a means of controlling the transmission of viral infections. IX. The in vitro exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus.

When zona pellucida-intact porcine embryos were exposed to 10(7) plaque-forming units (pfu)/ml of swine vesicular disease virus (SVDV) and then washed, infectious virus could be isolated from all of the embryos. Culturing the embryos for 24 or 48 h or treating the embryos with pronase, trypsin, or antiserum after virus exposure and washing reduced the number of embryos carrying virus and lessened the amount of virus on each of the embryos. None of the treatments, however, was capable of disinfecting every embryo.

Journal Article↗
PubMed1987

Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus.

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.

Journal Article↗
PubMed1987

Embryo transfer as a means of controlling the transmission of viral infections. XI. The in vitro exposure of bovine and porcine embryos to vesicular stomatitis virus.

Infectious virus was isolated from both porcine and bovine zona pellucida-intact embryos that had been exposed to the Indiana strain of vesicular stomatitis virus (VSV) and then washed. The amount of virus isolated from embryos depended on their initial exposure level. Porcine embryos always retained more virus than bovine embryos. When embryos were cultured for 24 h after viral exposure and washing, the number of embryos carrying VSV and the amount of virus on each of the embryos was reduced. Trypsin (0.25%) was also found to be effective in inactivating/removing the VSV from embryos, suggesting that most, if not all, of the virus was bound to the zona pellucida.

Journal Article↗
PubMed1987

Blocking dot-ELISA, using a monoclonal antibody for detection of antibodies to bluetongue virus in bovine and ovine sera.

A modified solid phase blocking enzyme immunosorbent assay (ELISA), using a monoclonal antibody (McAb) against the group specific bluetongue virus (BTV) antigen is described for detection of anti-BTV antibodies in cattle and sheep sera. Dots of an optimal dilution of BTV antigens were adsorbed to nitrocellulose (NC) paper (hence dot-ELISA) and then the remaining adsorptive sites were saturated with gelatin. After exposure to bovine or ovine test serum the NC strips were reacted with the McAb. The presence of McAb was detected with a peroxidase-conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in test sera, BTV antigen sites were reactive with McAb as indicated by a brown colored dot after enzyme degradation of hydrogen peroxide in the presence of diamino benzidine (DAB) or amino ethylcarbazole (AEC). In the presence of sufficient anti-BTV antibody no color reaction was observed. The blocking (B) dot-ELISA was superior to the agar gel immunodiffusion (AGID) in detecting anti-BTV antibodies in bovine and ovine sera early after experimental infection with BTV type 10. In 5 of 7 animals inoculated by combined intravenous and subcutaneous routes, anti-BTV antibodies in sera were detectable as early as 7 days post infection (DPI), all of which were AGID negative. Comparable B-dot-ELISA and AGID results were found in 23 paired sera (pre and 20 DPI) from cattle experimentally infected with different types of BTV and in 100 AGID negative sera from Ontario dairy and Alberta beef cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Diagnostic efficacy of two rapid tests for detection of respiratory syncytial virus antigen.

With the availability of ribavirin therapy for serious respiratory syncytial virus (RSV) infections, rapid diagnostic tests for the detection of RSV antigen are increasingly important. Efficacies of a commercially available enzyme immunoassay (EIA) (Abbott Laboratories, North Chicago, Ill.) and a fluorescent-antibody assay (FA) were evaluated in a study involving 135 specimens from children with respiratory symptoms. A nasal wash specimen was cultured immediately on RSV-sensitive A549 cells; the nasal wash was also used for EIA. FA was performed on a nasopharyngeal swab specimen with bovine anti-RSV and anti-bovine immunoglobulin G antisera (Burroughs Wellcome Co., Research Triangle Park, N.C.). A total of 39 specimens (28%) were tissue culture positive, including 35 EIA-positive and 37 FA-positive samples (sensitivities, 90 and 95%, respectively). All 96 tissue culture-negative specimens were EIA negative (specificity, 100%); 94 of these 96 specimens were FA negative (specificity, 98%). Positive and negative predictive values for the tests were as follows: 100 and 96% for EIA, respectively, and 95 and 98% for FA, respectively. Other viruses, including influenza A virus, adenovirus, enterovirus, and herpes simplex virus, were isolated in nine cases. One adenovirus-positive specimen had a false-positive RSV FA result; all nine specimens were RSV EIA negative. Both tests performed well in our study and provide cost-effective alternatives to tissue culture. The RSV EIA, in particular, uses standard serologic techniques and equipment and does not require expertise in virology. More widespread availability of rapid diagnostic tests for RSV will hopefully result in early and appropriate use of antiviral therapy in patients at risk for serious RSV infections.

Antigens, Viral↗
PubMed1987

Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood.

An indirect (I) enzyme-linked immunosorbent assay (ELISA) and a competitive (C) ELISA, using a group-specific monoclonal antibody against bluetongue virus (BTV), are described for the detection of antibodies to BTV in cattle and sheep sera. The performance of these assays in detecting anti-BTV antibody in sequential serum samples and eluates from whole blood (WB) dried on filter paper from three calves and four sheep experimentally infected with type 10 BTV was evaluated. The C-ELISA was superior to the I-ELISA in the detection of anti-BTV antibody in the sera and WB samples from both cattle and sheep early after infection with BTV. BTV antibodies were demonstrable by C-ELISA in all the bovine and ovine sera and WB eluates by 9 days postinfection; whereas the I-ELISA results for sheep sera and WB eluates were similar, anti-BTV antibody was not detected in bovine serum and WB eluates until 26 and 14 days postinfection, respectively. While both ELISAs proved reliable, under the present test conditions involving detection of early postinfection reactions of experimentally infected animals, the C-ELISA was always as sensitive or more sensitive than the standard agar gel immunodiffusion test, the modified complement fixation test, and the plaque neutralization tests in the detection of anti-BTV antibodies. Unlike observations with the immunodiffusion test, no reaction was seen between BTV antigen and bovine epizootic hemorrhagic disease virus antiserum in either ELISA. The results suggest that either ELISA may be suitable for routine diagnostic testing and may have the potential to replace other tests for detection of anti-BTV group-specific antibodies and that the C-ELISA may have the most potential.

Animals↗
PubMed1986

Attempts to establish congenital bluetongue virus infections in calves.

Three bovine fetuses were inoculated in utero with approximately 10(3) plaque forming units of type 11 bluetongue virus. The gestational ages of the fetuses at the time of inoculation were 106, 113 and 122 days. They were spontaneously aborted 104, 65 and 109 days later, respectively, and the first and third of these fetuses were recovered. There was no grossly normal cerebral tissue, the meninges formed fluid filled sacs, and the cerebellums were reduced in size. Bluetongue virus was not isolated from the fetuses but the older one had neutralizing antibody. The three dams developed neutralizing antibody to bluetongue virus. The present work supports the observation by others that early fetal infections with bluetongue virus normally result in severe central nervous system damage and not in clinically normal, persistently infected calves.

Abortion, Veterinary↗
PubMed1985

Embryo transfer as a means of controlling viral infections. VI. Bluetongue virus-free calves from infectious semen.

Four Holstein heifers were superovulated and inseminated with infectious semen from a bull experimentally infected with type 17 bluetongue virus (BTV). A total of 20 embryos were collected at donor slaughter and transferred to 16 recipients. Ten recipients became pregnant of which one subsequently aborted, one gave birth to twins which died at birth, one was killed at term because of dystocia, and 7 gave birth to live calves one of which died perinatally. All animals were tested for BTV antibodies at the time of slaughter which was at least 30 days post partum for surviving heifers and calves. Two of the four donor heifers were retrospectively determined to have been infected by the semen (viremia demonstrated) and their embryos accounted for 9 of the 10 pregnancies including the six surviving calves. None of the recipients or calves developed BTV antibody by the termination of the experiment. This study suggests that BTV-free calves can be readily obtained from the use of BTV-positive semen.

Journal Article↗
PubMed1984

Comparison of some storage and isolation methods to recover bluetongue virus from bovine blood.

Bovine blood containing bluetongue virus was stored as whole blood with EDTA (4 degrees C), as washed cellular components (4 degrees C), and as washed cellular components with 10% DMSO (-70 degrees C). Periodic isolation attempts were made over a period of 330 days in four cell lines and embryonating chicken eggs (intravenous inoculation). Bluetongue virus was successfully isolated in all systems from most samples throughout the test period. There appeared to be more variation amongst days of attempted isolation within systems than between systems.

Animals↗
PubMed1983

Direct determination of vasa recta blood flow in the rat renal papilla.

Blood flow in vasa recta capillaries of the exposed renal papilla of young antidiuretic rats (n = 18) was determined by an adaptation of the video-photometric technique of Intaglietta. The erythrocyte velocity and capillary diameter in vasa recta (n = 97) were measured at the same location by means of fluorescence video microscopy, with fluorescein-labeled bovine gamma-globulin as a plasma marker. A factor relating erythrocyte velocity to mean cross-sectional blood velocity was determined in vitro to permit the calculation of single vasa recta blood flows from the measured indices, erythrocyte velocity and capillary diameter. Mean blood flow in descending vasa recta was 8.83 +/- 0.96 (SE) nl/min, significantly greater than that in ascending vasa recta, 4.82 +/- 0.34 nl/min. The total numbers of ascending and descending vasa recta at the base of the exposed papilla were also determined. Over 1500 vasa recta were identified as ascending vasa recta or descending vasa recta in electron micrographs of three papillas. At this level in the papilla (2 mm from the tip), there were four ascending vasa recta for each descending vas rectum. From the total numbers of ascending vasa recta and descending vas rectum, single vessel blood flows were converted to total blood flow. Total blood outflow in all ascending vasa recta, 11.3 microliter/min, substantially exceeded total blood inflow in all descending vasa recta, 5.2 microliter/min. The difference between outflow and inflow (6.1 microliter/min) represents an estimate of water by the papillary microcirculation, and is more than adequate to accommodate the known rate of water reabsorption from the collecting ducts of the exposed papilla.

Animals↗