Biomedical subjects

T W Molitor

Publications and source records attributed to T W Molitor.

At least 91 records · Page 5Linked to original sources

PubMed1990

In vitro exposure of preimplantation porcine embryos to porcine parvovirus.

Early porcine embryos at the four- to eight-cell stage can be infected with either the virulent (NADL-8) or avirulent KBSH strain of porcine parvovirus (PPV) by microinjection or by incubation of embryos with virus. Treatment of embryos by microinjection of virus or incubation in media with virus did not significantly inhibit in vitro development of the embryos when compared with untreated controls. RNA-DNA hybridization was used to identify the presence of virus associated with embryos. It was found that PPV-DNA was present in viable embryos after microinjection of embryos with KBSH and NADL-8 strains of PPV and after incubation of embryos with KBSH strain. The data indicated the presence of replicative virus associated with viable porcine embryos.

Journal Article↗

PubMed1990

The detection and differentiation of foot-and-mouth disease viruses using solid-phase nucleic acid hybridization.

Thirteen complementary DNA (cDNA) probes were used to detect the presence of foot-and-mouth disease virus (FMDV) RNA extracted from cell cultures. When labelled with 32P, these probes enabled the detection of 1 pg of FMDV-RNA, or 1 virus copy per cell. Two FMDV A12 probes that coded for the leader, structural protein VP1 region and part of the polymerase gene respectively, showed no hybridization with other closely related picornaviruses. Differentiation between FMDV serotypes A, O and C was possible, using cDNA probes from individual serotypes that corresponded to structural protein VP1.

Aphthovirus↗

PubMed1990

Persistence of porcine parvovirus in swine infected in utero and followed through maturity.

The potential of porcine parvovirus (PPV) to persistently infect swine exposed in utero was studied. Forty eight 80- to 95-day-old fetuses from 5 PPV seropositive sows were inoculated intramusculary with a virulent strain of PPV or with cell culture medium (controls). Blood samples were collected at birth prior to nursing and at monthly intervals thereafter and tested for antibodies to PPV. Virus-inoculated and control pigs were euthanized at either 1 week before birth (-1), at birth (0) and at weeks 2, 4, 6, 8, 10, 22, and 28 after birth. Presence of viral DNA and antigen was evaluated using slot blot DNA hybridization and indirect FA techniques, respectively. All inoculated fetuses (n = 26) and 7 control fetuses (n = 22) seroconverted in utero, and these pigs maintained antibody titers greater than log10 2 for the period of testing (0-38 weeks after birth). After passive antibody titers had reached subdetectable levels in control animals, animals remained seronegative through an additional 14 weeks of testing in spite of close contact with infected pigs. Virus antigen was not detected in any tissues examined from pigs euthanized at term. In contrast, PPV DNA was detected consistently from pigs at birth from various tissues, and from the lung of one pig at 6 weeks of age and from the lymph nodes of one pig euthanized at 28 weeks of age. The results indicate that pigs infected with PPV in utero may be persistently infected, however the likelihood of shedding to contact animals is minimal.

Animals↗

PubMed1990

Detection of porcine parvovirus using nonradioactive nucleic acid hybridization.

Nonradioactive slot blot hybridization assays were established for the detection of porcine parvovirus (PPV), using either a digoxigenin-labeled DNA probe or a biotinylated RNA probe. All probes were prepared from a 3.3-kb Pst1-EcoR1 DNA fragment of the NADL8 isolate of PPV. The sensitivity and specificity of the probes in a slot blot system were evaluated in comparison with a 32P-radiolabeled RNA probe. Using an anti-digoxigenin alkaline phosphatase detection system, at least 1 ng of viral replicative form (RF) DNA, or the equivalent of 100 plaque forming units (PFU) of infectious virus, could be detected by the digoxigenin-labeled DNA probe. When the biotinylated RNA probe and a strepavidin-alkaline phosphatase detection system were employed, 0.1 ng of RF DNA, or the equivalent of 10 PFU of infectious virus, were detected, comparable to the sensitivity of the 32P-radiolabeled RNA probe. Hybridization was not observed with control DNA samples extracted from swine testicle cells, porcine kidney (PK-15) cells, uninfected mixed swine fetal tissue, or from an unrelated DNA virus (pseudorabies virus) infected PK-15 cells. Different isolates of PPV, namely NADL8, NADL2, KBSH, and Kresse, reacted on an equimolar basis in sensitivity and specificity to the biotinlyated probe. Extraction of DNA directly on the filter membrane (direct filter hybridization) was employed in an attempt to reduce processing time by eliminating DNA extraction steps. Direct filter hybridization was indeed less time consuming; it was also comparable in sensitivity and specificity to those methods employing purified DNA.

Animals↗

PubMed1990

A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescein, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

Coexistence of neurotransmitters within single nerve fibers or terminals can be convincingly demonstrated by the use of multicolor immunofluorescence. The present study examined whether three-color immunocytochemical localization of coexisting neurotransmitters can be performed using the blue fluorophore AMCA. Spectrofluorometric examination of secondary antibodies conjugated with AMCA, fluorescein, and lissamine rhodamine showed that the peaks of excitation and emission were well separated and that dots of AMCA-conjugated IgG dried on slides were not visible when viewed using microscope filters for rhodamine and fluorescein. These findings suggest that AMCA might be suitable for three-color immunofluorescence. The usefulness of AMCA for triple labeling was tested directly by staining sections of rat brainstem and spinal cord for serotonin (5HT), substance P (SP), and either enkephalin (ENK) or prepro-thyrotropin-releasing hormone 160-169 (ppT), a marker peptide for thyrotropin-releasing hormone. Triple labeling for 5HT, SP, and ppT was observed in both brainstem and spinal cord but was only very rarely observed for 5HT,SP, and ENK. No evidence was found for artifactual triple labeling, although false negatives appeared to be possible in some circumstances. We conclude that AMCA can be combined with fluorescein and lissamine rhodamine for three-color immunofluorescent studies of coexisting neurotransmitters. In addition, the coexistence of 5HT with ENK appears to be much less common than the coexistence of 5HT with either SP or ppT.

Animals↗

PubMed1989

Temperature dependent replication of porcine parvovirus isolates.

The replication of four porcine parvovirus isolates, NADL-8, NADL-2, KBSH, and Kresse, in swine testes cells were compared at temperatures of 32, 37, and 39 degrees C. Replication of the Kresse isolate was restricted at 32 and 37 degrees C as evidenced by progeny virus, virus polypeptide and viral DNA synthesis, but not at 39 degrees C. In contrast, replication of KBSH was restricted at 39 degrees C, but not at 37 or 32 degrees C. Findings from this study support the contention that replication of KBSH and Kresse isolates are temperature dependent.

Animals↗

PubMed1989

Comparison of protective immunity and inflammatory responses of pigs following immunization with different Actinobacillus pleuropneumoniae preparations with and without adjuvants.

Three experiments were performed to evaluate the inflammatory response, the antibody response and protection from experimental challenge of various Actinobacillus pleuropneumoniae serotype 5 (Ap5) vaccines in swine. In the first experiment, subcutaneous injections of either a water-in-oil (W/O) emulsion or Freund's complete adjuvant (FCA) caused lesions at the site of injection, while intraperitoneal injection of the W/O emulsion caused no lesions. In the second experiment, intraperitoneal (IP) injection of a W/O emulsion containing unwashed Ap5 cells (6-h culture) and/or supernates from a 24-h culture resulted in severe peritoneal lesions, while W/O emulsion containing PBS-washed Ap5 cells resulted in minimal peritoneal lesions. Ap5 alone or W/O alone failed to cause peritoneal lesions. The third experiment compared the antibody response and protection from challenge of pigs immunized with either 6-h PBS-washed Ap5 cells emulsified in oil - IP, 6-hour Ap5 cells adjuvanted with dimethyl diodacyl ammonium bromide - IP, Ap5 antigen alone - IP, a commercial vaccine - subcutaneously or saline - IP. All groups, except the saline-treated group, responded with high antibody titers to Ap5 2 weeks following vaccination; however, titers from the W/O plus antigen group were significantly higher than the three other groups (P less than 0.05). Following intranasal challenge with Ap5, all animals responded with increased antibody titers. All pigs were euthanized 10 days after challenge and evaluated for pneumonia and the lungs cultured for bacteria. The lungs of all pigs, excepting the W/O plus antigen group, contained pneumonic lesions and A. pleuropneumoniae was cultured from these lesions. These results, along with results from other groups, suggest that intraperitoneal immunization using oil-adjuvanted vaccine may be an effective method for protecting pigs from pneumonia due to A. pleuropneumoniae. Its efficacy may be due to stimulation of local respiratory mucosal immunity.

Actinobacillus↗

PubMed1989

Use of in situ hybridization for the detection of foot-and-mouth disease virus in cell culture.

Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.

Animals↗

PubMed1988

Porcine parvovirus: replication in and inhibition of selected cellular functions of swine alveolar macrophages and peripheral blood lymphocytes.

The ability of four isolates of Porcine Parvovirus (NADL-8, NADL-2, KBSH, and Kresse) to replicate in and affect the functions of swine peripheral blood lymphocytes and alveolar macrophages was studied in vitro. V-strand and C-strand viral DNA was present in both concanavalin A- and non-treated lymphocytes as well as alveolar macrophages following infection with all four isolates. Indirect fluorescent antibody assays on swine testis cells, inoculated with cell lysates of NADL-8-infected peripheral blood lymphocytes (both concanavalin A- and non-treated) and alveolar macrophages, indicated that these immune cells supported the production of progeny virus. The quantity of viral DNA and progeny virus was dependent upon the multiplicity of infection and length of time following infection. Infection of lymphocytes and alveolar macrophages with PPV was associated with a decrease in cell viability. Peripheral blood mononuclear cells and alveolar macrophages infected with any of the four isolates demonstrated reduced lymphocyte blastogenesis and non-Fc-mediated alveolar macrophage phagocytosis, respectively.

Animals↗

PubMed1987

Inhibition of porcine parvovirus replication by empty virus particles.

The influence of empty porcine parvovirus (PPV) particles on viral replication was examined in cell cultures and in swine. Following extensive purification, homogeneous preparations of full and empty PPV preparations were obtained and used for in vitro and in vivo analyses. In the first in vitro experiment, swine testes cells were infected with mixtures of various ratios of empty and full (E/F) particles. The production of both intracellular and extracellular virus was markedly inhibited in the presence of empty particles. This inhibition was dependent upon the concentration of empty particles present in the mixture. In the second in vitro study, various concentrations of empty particles were added prior to full virus infection. Again, marked inhibition of progeny virus production was evident and related to the concentrations of empty particles added. Based on the results of in vitro studies, the influence of empty particles on PPV infection in swine was tested by infecting mid-term and late-term gestation swine fetuses with various E/F particle ratios. Both mid-term and late-term fetuses exposed to 0:1, 1:1 and 5:1 E/F ratios displayed gross pathological evidence of PPV infection whereas fetuses exposed to E/F ratios of 30:1 or greater were grossly normal in appearance. However, fetuses infected with 30:1, 50:1 and 300:1 E/F ratios showed evidence of virus in their tissues by DNA hybridization. Regardless of the E/F ratios, late-term infected fetuses responded with high antibody titers ranging from 1024 to 4096. The results from these studies suggested that empty particles interfered with viral replication in both cell culture and in animals.

Animals↗

PubMed1987

Pathogenicity of a skin isolate of porcine parvovirus in swine fetuses.

The pathogenic properties of a skin isolate of porcine parvovirus (PPV), designated Kresse isolate, were compared with NADL-8 isolate, a prototype isolate of PPV, by in utero inoculation of mid-term and late-term gestation swine fetuses. Fetuses from pregnant sows of mid-gestation were inoculated with either NADL-8 or Kresse virus. Both isolates were highly pathogenic to mid-gestation fetuses. In contrast, dramatic differences in pathogenicity between these 2 isolates were observed in fetuses inoculated late in gestation. Such fetuses from each of 4 sows were inoculated with NADL-8 or Kresse virus isolate and sacrificed at 10, 18, 21, or 23 days postinoculation (PI). NADL-8-inoculated fetuses were grossly normal. The pathogenic effects of Kresse isolate were evident by gross pathology in fetuses collected at 18, 21, and 23 days PI, but not at 10 days PI. Hemagglutination (HA) and fluorescent antibody (FA) methods were used to identify virus in various tissues of late-gestation fetuses collected at 10 and 21 days PI. At 10 days PI, HA antigens were detected only in livers of NADL-8-inoculated fetuses, but in all tissues examined of Kresse-inoculated fetuses, including the brain. PPV specific fluorescence was demonstrated in tissues of fetuses inoculated with NADL-8 and Kresse virus. The major difference was that virus antigen was found in the brains of fetuses inoculated with Kresse virus, but not in NADL-8 infected fetuses. At 21 days PI, HA antigen was not detected in any of the tissues of fetuses inoculated with NADL-8 virus, with PPV specific fluorescence by FA being found only in the kidney. However, fetuses inoculated with Kresse virus displayed HA antigen in liver and PPV-specific fluorescence in all tissues tested including the brain. Both isolates induced similar antibody responses, 1:128 to 1:256 at 10 days and 1:512 to 1:1024 at 21 days PI. In addition, immunoglobulin G (IgG) deposits were demonstrated in kidneys and skin of fetuses inoculated with Kresse virus and IgM in brain, but not in tissues from fetuses inoculated with NADL-8 virus.

Animals↗

PubMed1985

Potentiating effect of adjuvants on humoral immunity to porcine parvovirus vaccines in guinea pigs.

Fourteen different adjuvants, given either in single or combined form with another compound were compared in guinea pigs for their ability to potentiate humoral immunity to porcine parvovirus (PPV) antigen after 2 vaccinations. Two injections were given, the second 3 weeks following the initial vaccination. Antibody concentrations to PPV in sera from injected animals were measured over a 5-week period by the hemagglutination inhibition test. At the conclusion of the experiment, guinea pigs injected with the following adjuvants and PPV antigen: CP-20 961 (Avridin), 50% aluminum hydroxide gel, ethylene maleic anhydride (EMA), oil and water emulsion (O/W) and dimethyl-dioctadecyl-ammonium bromide (DDA) immunologically responded with high geometric mean HI titers (380, 224 and 427, 602, 512, 1202 respectively), whereas guinea pigs receiving Emulsan, sodium dodecyl sulfate (SDS), L-121, combinations of Emulsan/aluminum hydroxide, SDS/aluminum hydroxide and B. pertussis/aluminum hydroxide responded with low mean titers (54, 64, 18, 27, 11, 64, 14, 20 respectively). Guinea pigs injected with antigen without adjuvant responded weakly with geometric mean titers of 3.3 and 16 for the 2 groups tested. Prior to booster injection, guinea pigs immunized with 13 of the preparations had low (less than 4) or undetectable antibody titers. Antibody titers from guinea pigs receiving DDA adjuvant continued to rise throughout the duration of the experiment and at the conclusion had the highest mean titers of the groups tested (1202). The 2 groups immunized with 50% aluminum hydroxide gel had high mean titers (224, 427), but in both instances there was a wide range of titers within a group evidenced by high standard deviations. In contrast, guinea pigs receiving either DDA, CP-20 961, O/W or EMA had antibody titers within a narrow range and small standard deviation. The significance of aluminum hydroxide gel concentration on immunogenicity is discussed.

Adjuvants, Immunologic↗

PubMed1985

KBSH parvovirus: comparison with porcine parvovirus.

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.

Animals↗

PubMed1985

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.

Animals↗

PubMed1984

Pathogenicity of a modified-live pseudorabies vaccine virus in lambs.

Subcutaneous injections of modified-live pseudorabies virus (PRV) vaccine into lambs caused clinical signs and death within 1 week after injection in 4 of 5 inoculated lambs. The clinical signs included depression, high fever, muscle fasciculations and convulsions, occasionally followed by death within 48 hours of the initial clinical signs. Histologic examinations and virus isolation procedures demonstrated PRV in the CNS of infected lambs. Sera from sick lambs remained negative for PRV antibodies. Two subsequent serial passages of the vaccine virus in lambs resulted in similar clinical signs and death in 6 of 10 inoculated lambs. Again, PRV was isolated from tissues of sick lambs, and the histopathologic findings were characteristic of the disease. Affected lambs remained seronegative to PRV, as did lambs that remained clinically normal after inoculation. There was no evidence of PRV transmission to uninoculated lambs and pigs housed with the infected lambs.

Animals↗

PubMed1984

Porcine parvovirus DNA: characterization of the genomic and replicative form DNA of two virus isolates.

The genomic and replicative form (RF) DNA of porcine parvovirus (PPV) have been characterized. PPV isolate NADL-8 was found to have a 5000-base single-stranded genome, and a unique strand was encapsidated in virus particles. The RF DNA of isolate NADL-8 was found to be an infectious 5000-base pair (bp) molecule. Select restriction endonuclease sites were mapped along the RF DNA of PPV (NADL-8), and oriented with respect to the viral genomic DNA. The RF DNA of a second isolate of PPV, the less pathogenic, cell culture-adapted NADL-2 virus, was also analyzed. DNA preparations isolated from NADL-2 virus-infected cells contained two viral RF DNA species: a 5000-bp infectious molecule very similar if not identical to the NADL-8 virus RF DNA, and a noninfectious 4700-bp DNA molecule. The 4700-bp RF DNA molecule found in NADL-2 DNA preparations was similar in all respects to the 5000-bp RF DNA, except for a 300-bp deletion in the region encoding the viral capsid proteins. The presence of this defective variant in PPV (NADL-2) virus preparations is discussed in relation to the reduced pathogenic potential of the NADL-2 virus as compared to the NADL-8 virus.

Animals↗

PubMed1984

Antibody responses of guinea-pigs, rabbits and pigs to inactivated porcine parvovirus vaccines.

Antibody responses were compared in guinea-pigs, rabbits and pigs following vaccination with inactivated porcine parvovirus (PPV) vaccines. Mean PPV hemagglutination inhibition (HI) antibody titers of 52, 56 and 36 at 1 week after first vaccination and 896, 640 and 512 at 2 weeks after second vaccination were detected in guinea-pigs, rabbits and pigs, respectively. PPV vaccines prepared with greater concentrations of virus, as determined by hemagglutination (HA) units, and of aluminum hydroxide gel adjuvant, induced higher HI antibody titers in guinea-pigs. Optimal concentrations for inducing consistently high antibody titers consisted of vaccine virus with a HA titer of 256/0.1 ml and gel adjuvant at a final concentration of 50%. A second vaccination at 4 weeks compared to 2 or 3 weeks after first vaccination resulted in higher mean HI titers. These data provide preliminary information on the use of guinea-pigs or rabbits as laboratory animal models for testing the potency of PPV vaccines.

Animals↗

PubMed1984

In utero infection of swine fetuses with infectious bovine rhinotracheitis virus (bovine herpesvirus-1).

The pathogenesis of infectious bovine rhinotracheitis (IBR) virus (bovine herpesvirus-1) was studied in porcine fetuses after in utero inoculation. Laparotomies were performed on 8 seronegative pregnant sows at 34 to 86 days of gestation, and all fetuses in 1 uterine horn of each sow were exposed to IBR virus via inoculation into the amniotic sacs. Fetuses in the other horn served as controls. Clinical signs of infection were not observed in the sows, except for 2 sows that aborted at postinoculation days (PID) 11 and 15. Fetuses of the remaining 6 sows were collected at slaughter on PID 15 to 28. Fetuses were examined for gross abnormalities, presence of IBR virus in tissues, and the formation of neutralizing antibodies to IBR virus. Of 33 inoculated fetuses from 6 sows, 10 were mummified, 11 were hemorrhagic and/or edematous, and 12 were alive. Necrotic lesions were observed on the skin and in the liver of dead and live fetuses. Virus was recovered from 29 of 33 inoculated fetuses. Infectious bovine rhinotracheitis virus was isolated from fetal skin, liver, lungs, kidney, spleen, stomach contents, brain, amniotic fluid, and placenta. Virus was isolated from 4 of 11 fetuses recovered from 1 aborting sow. Antibodies to IBR virus were not detected in sera from the sows. However, antibodies were detected in 6 of 15 fetuses inoculated at 63 to 86 days of gestation and collected at slaughter at 86 to 112 days of gestation. The youngest fetus with detectable IBR antibody was estimated to be 74 days of gestation by measuring crown-rump length of the fetus.

Abortion, Veterinary↗