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T W Molitor

Publications and source records attributed to T W Molitor.

At least 73 records · Page 4Linked to original sources
PubMed1992

Use of restriction endonuclease analysis and ribotyping to study epidemiology of Pasteurella multocida in closed swine herds.

One hundred and sixty-four clinical isolates of Pasteurella multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTP-labeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.

Animals↗
PubMed1992

Characterization of pseudorabies virus antibody responses in young swine after infection and vaccination by using an immunoglobulin M antibody capture enzyme-linked immunosorbent assay.

An immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MACELISA) was developed for the detection of pseudorabies virus (PRV)-specific IgM antibody in swine sera because false-positive reactions frequently occurred when sera from older swine were tested with an indirect IgM enzyme-linked immunosorbent assay. Monoclonal mouse anti-swine IgM was used as the capturing antibody, and rabbit anti-PRV hyperimmune gamma globulin was used as the indicating antibody. Sera from non-PRV-infected, experimentally infected, vaccinated and challenged, passively immune and challenged, and naturally infected swine were evaluated. The PRV MACELISA had a specificity of 95% and was as sensitive and reproducible as previously reported in direct assays. An antibody response was still detectable with the MACELISA 21 days after inoculation. The PRV MACELISA did not detect a consistent antibody response in sera from swine vaccinated with either killed-PRV or modified live-virus vaccines but did detect an antibody response in sera from passively immune pigs after challenge with virulent PRV. These results indicated that the PRV MACELISA may be useful for the rapid serodiagnosis of recent PRV infection in swine.

Animals↗
PubMed1992

Restriction of porcine parvovirus replication in nonpermissive cells.

Swine testicle (ST) cells and Madin-Darby canine kidney (MDCK) cells differ in their ability to support replication of porcine parvovirus (PPV). Viral replication events in ST cells, a permissive cell type, and MDCK cells, a nonpermissive cell type, were compared in an attempt to elucidate putative mechanisms of restrictive virus replication. Radiolabeled PPV bound to the cell surface of both cell types equally well and the binding was shown to be PPV specific, indicating that the restriction was not at the cell surface level. In contrast, profound differences in intracellular events in PPV replication were observed between these two cell types. Synthesis of viral DNA was limited in MDCK cells in that the percentage of cells with replicative-form DNA as determined by strand-specific probe in situ hybridization was approximately 100-fold lower in MDCK cells than in ST cells at the same multiplicity of infection. Northern (RNA) blot analysis, using oligonucleotide probes derived from both structural and nonstructural protein-coding regions of the PPV genome, revealed four PPV mRNA transcripts from infected ST cells. Comparatively, RNA species from the structural protein coding region were actively transcribed in MDCK cells, but synthesis of RNA species from the nonstructural protein coding region was negligible. Immunoprecipitation of viral polypeptides revealed the three characteristic structural polypeptides, VP1, VP2, and VP3, along with the nonstructural polypeptide, NS-1 from ST cells. In contrast, neither viral structural or nonstructural polypeptides nor progeny virions were produced from MDCK cells. The data suggest that mechanisms controlling permissiveness of cells to PPV infection are associated with the level of viral DNA replication, RNA transcription, and viral antigen expression but not absorption to the cell surface.

Animals↗
PubMed1992

Effects of transforming growth factor-beta on murine astrocyte glutamine synthetase activity. Implications in neuronal injury.

Cytokines have been implicated in the pathogenesis of a number of brain diseases in which neurological dysfunction has been attributed to a change in amino acid neurotransmitter metabolism. In the present in vitro study, we investigated the effects of cytokines on astrocyte glutamine synthetase (GS) activity and subsequently on N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity. Proinflammatory cytokines IL-1 alpha, IL-1 beta, and IL-6 at a concentration of 20 ng/ml did not affect GS activity; however, tumor necrosis factor-alpha inhibited this activity by 20% in mixed neuronal/astrocyte cultures. Treatment for 24 h with transforming growth factor (TGF)-beta 1 or -beta 2 inhibited up to 60% GS activity. TGF-beta 2 also inhibited GS in enriched astrocyte cultures with an ED50 of 10 pg/ml. Antibodies specific to TGF-beta 2 blocked this effect. Treatment of astrocytes with TGF-beta 2 (250 pg/ml) resulted in markedly dilated rough endoplasmic reticulum. Since astrocyte GS may play a protective role in NMDA receptor-mediated neurotoxicity, we treated mixed neuronal/astrocyte cultures with TGF-beta 2 (250 pg/ml) and found a threefold potentiation of NMDA receptor-mediated neurotoxicity. These data suggest that TGF-beta impairs astrocyte GS function and enhances neurotoxicity, thus providing insight into understanding one mechanism of cytokine-mediated central nervous system disease.

Animals↗
PubMed1992

Chronic morphine administration impairs cell-mediated immune responses in swine.

Swine were used as an experimental animal model to evaluate immunomodulating effects of the opiate drug, morphine, on antigen-specific humoral and cell-mediated immune responses. Morphine free base in peanut oil was administered at 4-day intervals for up to 42 days to maintain drug levels at or above 100 ng/ml. The effect of morphine administration on humoral immune responses was evaluated by immunization on day 7 of morphine treatment with a battery of antigens, including swine herpes virus (also known as pseudorabies virus, PRV), Brucella abortus and the Escherichia coli pilus antigens K88, K99, 987P and F41. Fourteen days later, swine were reimmunized with B. abortus and E. coli pilus antigens. Antibody titers to these antigens were evaluated on a weekly basis. Cell-mediated immunity was evaluated by measuring skin immune responses to the antigen 2,4-dintroflurobenozene (DNFB). In one experiment, swine were sensitized with DNFB on day 7 of morphine treatment at the same time as immunization with the other antigens. In a second series of experiments, swine were sensitized either 7 days before or 7 days after initiation of morphine treatment. Pigs were challenged with DNFB administered 27 days after the initiation of morphine treatment and skin responses were evaluated 24 h later. The ability of swine to resist PRV infection was evaluated by exposure to virulent virus on day 28 of morphine treatment. Chronic morphine administration did not impair the induction of the humoral immune responses to bacterial or viral antigens. In addition, morphine treatment did not alter the resistance of immunized swine to PRV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Morphine potentiates transforming growth factor-beta release from human peripheral blood mononuclear cell cultures.

Opiates modulate a variety of immune responses by peripheral blood mononuclear cells (PBMC). In the present study, we investigated the effect of morphine on the release of several cytokines upon stimulation with mitogens. An interleukin (IL)-4-dependent HT-2 cell proliferation assay was used to quantify transforming growth factor-beta (TGF-beta). IL-6 and tumor necrosis factor-alpha (TNF-alpha) were measured by enzyme-linked immunosorbent assays. Morphine (1 pM and 10 nM) alone did not significantly modulate the release of TGF-beta, IL-6 or TNF-alpha. Upon stimulation with lipopolysaccharide and phytohemagglutinin, PBMC released more (P less than .01) TGF-beta, IL-6 and TNF-alpha than unstimulated PBMC. Exposure of PBMC to morphine (1 pM) for 24 hr substantially amplified (P less than .05) the release of TGF-beta in the following 24 hr incubation period. Morphine did not alter the release of immunodetectable IL-6 or TNF-alpha from stimulated cells. The amplifying effect of morphine on the release of TGF-beta was mediated through a naloxone-sensitive mechanism. Given the fact that TGF-beta has a potent immunosuppressive effect, morphine-potentiated release of TGF-beta from PBMC may be involved in the immunomodulatory activity ascribed to morphine.

Adult↗
PubMed1991

Detection of tumor necrosis factor alpha from porcine alveolar macrophages using an L929 fibroblast bioassay.

Tumor necrosis factor plays a central role in the mediation of the pathophysiological sequelae of infection and inflammation in animals and humans. The elucidation of its role in respiratory disease of swine has not been investigated, due in part to the lack of a sensitive and specific quantitative assay for its presence in tissue and fluid samples. Here we describe the detection of porcine tumor necrosis factor utilizing L929 murine fibroblast cells and characterize various parameters affecting assay sensitivity. Plating cell density and length of exposure time to test supernatants were the most critical factors. Using standard assay conditions as described here, porcine tumor necrosis factor was detected in alveolar macrophage conditioned media diluted more than 400-fold. Specificity of the assay for porcine tumor necrosis factor was shown by inhibition of cytotoxicity with neutralizing polyclonal antibodies for human recombinant tumor necrosis factor. Furthermore, comparisons of bioactivity with tumor necrosis factor mRNA levels from lipopolysaccharide-stimulated porcine alveolar macrophages indicated that the L929 bioassay was specific for porcine tumor necrosis factor.

Animals↗
PubMed1991

Cocaine potentiates HIV-1 replication in human peripheral blood mononuclear cell cocultures. Involvement of transforming growth factor-beta.

Cocaine use is an important high risk behavior in the AIDS epidemic. In this study, we tested the hypothesis that cocaine potentiates the replication of HIV-1 in human PBMC. A coculture system was used in which PBMC from healthy donors were incubated in the absence (control) or presence of cocaine before activation with PHA. Cocultures were then constituted with PBMC infected with a clinical isolate of HIV-1. HIV-1 replication, which was assessed by the measurement of HIV-1 p24 antigen levels in coculture supernatants, was significantly increased in a dose-dependent manner by cocaine with maximal stimulation at a concentration of 10(-9) M (965 +/- 196 vs 303 +/- 80 pg p24 Ag/ml in control cocultures). Antibodies to transforming growth factor-beta (TGF-beta) blocked cocaine-enhanced HIV-1 replication and purified TGF-beta stimulated viral replication in a manner similar to that observed with cocaine. Augmentation of HIV-1 replication by TGF-beta was maximal at a concentration of 0.01 ng/ml; however, viral proliferation appeared to be inhibited by concentrations of TGF-beta of 1 ng/ml or greater. Taken together, these results indicate that cocaine augments the replication of HIV-1 in PHA-activated PBMC via a mechanism that appears to involve TGF-beta.

Cells, Cultured↗
PubMed1991

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus.

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.

Animals↗
PubMed1991

Enzymatic amplification of latent pseudorabies virus nucleic acid sequences.

To investigate various aspects of the latency of pseudorabies virus in swine (PRV, suid herpesvirus 1) we developed in vitro nucleic acid amplification methods based upon the polymerase chain reaction. Primers flanking a 156-bp region of the pseudorabies virus gp II gene were annealed to purified PRV DNA as well as DNA isolated from the trigeminal ganglia of swine latently infected with PRV and subjected to PCR amplification. Following amplification, 100 fg of PRV DNA was visualizable on stained gels and 1 fg (equivalent to 6 viral genome copies) was detectable when amplification was combined with blot hybridization. PRV-specific DNA sequences which remained undetectable by direct blot hybridization assays were amplified to levels visualizable on ethidium-bromide-stained gels in 5 of 5 experimental latently infected animals. In addition, oligonucleotide primers specific for a 223-bp region of the PRV immediate-early gene (IE 180) were capable of amplifying overlapping latency associated transcripts (LATs), via a cDNA intermediate, in 6 of 6 latently infected swine. These nucleic acid amplification methods should be applicable to the investigation of PRV latency, and gene expression during latency and reactivation, in which few cells harbor latent virus.

Animals↗
PubMed1991

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

Animals↗
PubMed1991

Cocaine-mediated suppression of superoxide production by human peripheral blood mononuclear cells.

Cocaine, like opiates, modulates a variety of immune functions. In the present study, we investigated the effect of cocaine on superoxide anion (O2-) production, an index of a microbicidal activity, by cultured human peripheral blood mononuclear cells. Release of O2- was measured by superoxide dismutase-inhibitable reduction of ferricytochrome C in response to phorbol myristate acetate. Peripheral blood mononuclear cells cultured in the presence of cocaine (1 microM) for 48 hr released less (P less than .05) O2- than did nontreated control cells (95.1 +/- 10.2 vs. 57.9 +/- 6.6 nmol/10(7) cells/60 min, respectively). This suppressive effect was dose-dependent. Antibodies to transforming growth factor-beta, a cytokine inhibitory of monocyte O2- production, abrogated (P less than .01) cocaine-mediated suppression, suggesting that transforming growth factor-beta is involved in the suppression. Also, naloxone blocked (P less than .01) the suppressive effects of both cocaine and transforming growth factor-beta on O2- production, suggesting that the suppressive mechanism is naloxone-sensitive.

Adult↗
PubMed1990

Serodiagnosis of equine monocytic ehrlichiosis in selected groups of horses in Minnesota.

Antibody titer to Ehrlichia risticii was determined, in 2,549 equine serum samples, using an indirect fluorescent antibody assay. During 1986, samples were obtained from the Minnesota State-Federal Equine Infectious Anemia Diagnostic Laboratory, the Minnesota Racing Laboratory, from horses admitted to the University of Minnesota Veterinary Teaching Hospital, and as a result of field investigations of horses with acute diarrhea. Results of the study revealed antibody prevalence of 33, 24, 47, and 25% for the respective groups. There was no statistical association between seropositive status and age, sex, breed, or clinical problem of horses referred to the teaching hospital. There was an increase in the total percentage of seropositive samples over the duration of the sample collection period, suggesting a seasonal exposure pattern, and E risticii was associated with clinical and subclinical infections in horses of Minnesota.

Animals↗
PubMed1990

Factors associated with circulation of pseudorabies virus within swine herds.

Data were collected from 104 Minnesota swine farms quarantined for pseudorabies virus (PRV) infection. Each herd was serologically evaluated for the presence of antibodies to PRV in finishing pigs. Herd management practices, swine housing design, and disease profiles were described for each farm. Multiple logistic regression analysis was used to determine which factors were associated with circulation of PRV in the finishing pigs of farrow-to-finish farms. Sixty-seven (64%) of the herds had no serologic evidence of PRV circulation in the finishing section, whereas 37 herds (36%) contained at least one PRV seropositive finishing pig. The odds of a given finishing herd being seropositive for PRV were 2.85 times higher if the finishing pigs were housed in confinement (P = 0.01), 2 times higher if Actinobacillus (Haemophilus) pleuropneumoniae was a clinical problem in the herd (P = 0.03), 1.36 times less for each year that passed since the herd quarantine was issued (P = 0.01), 1.74 times higher if clinical signs of PRV were reported (P = 0.04), and 1.52 times higher if animal protein was included in at least one of the rations (P = 0.08).

Animals↗
PubMed1990

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen.

Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.

Animals↗
PubMed1990

Pseudorabies virus latency: restricted transcription.

Cloned pseudorabies virus (PRV) sequences representing over 80% of the viral genome were radiolabeled and individually hybridized to nucleic acid in the trigeminal ganglia of acutely and latently infected swine. In acutely infected animals, all cloned probes hybridized to PRV RNA and DNA. In contrast, during latency transcription was found to be limited to a selected region, corresponding to the IE gene, analogous to that of two other alpha-herpesviruses. Viral DNA was not detected in latently infected neurons by in situ hybridization.

Animals↗
PubMed1990

Replication of two porcine parvovirus isolates at non-permissive temperatures.

Previous studies have shown that replication in vitro of the porcine parvovirus (PPV) isolate, KBSH, was restricted at 39 degrees C but not at 37 degrees C. In contrast, replication of the Kresse isolate was restricted at 37 degrees C but not at 39 degrees C. In this study, Kresse and KBSH isolates were passaged up to ten times in swine testicle (ST) cells at non-permissive temperatures, and at subsequent passage viral protein synthesis, viral DNA synthesis, and progeny virus were evaluated. KBSH became adapted for replication at 39 degrees C upon serial passages, displaying an appreciable increase in viral progeny, viral polypeptides, and viral DNA concentration. This finding was also observed with Kresse virus isolate continuously passaged at 37 degrees C. Neither isolate became adapted for replication at 32 degrees C. In an attempt to examine the effect of in vitro passage at non-permissive temperatures on pathogenicity in swine, KBSH passaged 10 times either at 37 degrees C or 39 degrees C was inoculated into swine fetuses. Two of four fetuses inoculated with 39 degrees C-passaged KBSH were dead and hemorrhagic or mummified. All four fetuses inoculated with 39 degrees C-KBSH contained viral antigen and viral DNA. In contrast, fetuses inoculated with 37 degrees C-passaged KBSH, or with cell culture fluid were normal in appearance. Viral antigen and viral DNA were not demonstrated in fetuses inoculated with 37 degrees C-KBSH or cell culture fluids. These findings suggest the possibility that the ability to replicate at 39 degrees C is associated with virulence in swine fetuses.

Animals↗