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T W Molitor

Publications and source records attributed to T W Molitor.

101 records · Page 6Linked to original sources

Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum.

A radial immunodiffusion enzyme assay for the detection and quantitation of antibodies to pseudorabies virus in swine sera was developed and the methods were standardized. The assay combined the principle of radial immunodiffusion with enzyme-linked immunosorbent assay. Quantitation of pseudorabies virus antibody titers was determined by measuring the diameter of a colored circular zone after overnight incubation of antibody with antigen. The specificity and sensitivity of the radial immunodiffusion enzyme assay were compared with that of the standard virus-neutralization test, and the results were determined to be correlated highly (r = 0.694, P less than 0.0001). The assay also appeared to be highly reproducible and simple to perform.

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Farm studies of porcine parvovirus infection.

Epizootics of reproductive failure associated with porcine parvovirus infection were investigated on 38 farms. Mummification was common to all epizootics. The mean number of mummified fetuses per affected litter was 3.1. Mean number of pigs born alive for gilts and sows farrowing litters with mummified fetuses was 5.0, but for sows without mummified litters on the same farms it was 9.0. Mummified fetuses were observed in aborted litters on 8 (21.1%) farms. Gilts and sows that appeared pregnant but "lost" their swollen abdomens and failed to farrow were observed on 12 (39.5%) farms. Delayed return to estrus was evident on only 3 (7.9%) farms with a continuous farrowing program. On 21 farms with a batch farrowing program, only 2 (10%) epizootics involved more than 1 batch. Up to 100% of the pregnant swine in the group farrowing together produced mummified pigs, but less than 20% of the breeding herd was affected in 26 (68.4%) cases. The prevalence of disease was not related to herd size, housing, or farrowing management.

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Porcine parvovirus: virus purification and structural and antigenic properties of virion polypeptides.

Porcine parvovirus (PPV) was extensively purified from infected swine fetal homogenates by CaCl2 precipitation followed by CsCl density centrifugation. Two species of particles possessing PPV-specific hemagglutinating activity were observed banding at densities of 1.39 and 1.30 g/ml, representing full and empty 20-nm virion particles, respectively. Both classes of particles contained three major polypeptides. A, B, and C, with respective molecular weights of 83,000, 64,000, and 60,000. The amount of polypeptide A was similar in both species (approximately 10%); however, the B protein was most abundant in the 1.30-g/ml particles, whereas the C protein was the major polypeptide found in the 1.39-g/ml particles. Antisera generated to each sodium dodecyl sulfate-polyacrylamide gel-purified virion structural protein had reactivities qualitatively similar to those of conventional antisera raised against intact PPV in a variety of standard serological assays. The antisera generated against the individual sodium dodecyl sulfate-denatured PPV polypeptides were able to react with native, intact PPV virions and were capable of neutralizing virus infectivity.

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Role of the rat in the transmission of porcine parvovirus.

Rats experimentally inoculated with porcine parvovirus (PPV) shed virus in excreta from 3 to 21 days. Rats inoculated subcutaneously with PPV responded serologically with hemagglutination-inhibition titers (512-1,024). The PPV antigen was readily detected in lung and spleen 2 and 3 days after rats were inoculated and in liver and intestine, 4 days. The rats remained clinically healthy. Rats given PPV orally or in drinking water either with PPV-infected cell culture fluid or swine fetal homogenate failed to respond serologically to PPV, the exception being 2 of 4 rats exposed to swine fetal homogenate over a 5-day span. Pigs exposed to PPV-contaminated rat excreta, either by direct oral dosing or by contaminating the feed, failed to seroconvert. Pigs given (IM) PPV which had been isolated on cell culture from rat excreta did seroconvert. Results of these experiments indicated that rats became infected with PPV, but did so after systemic challenge exposure or prolonged oral exposure to highly infective swine fetal homogenate. Insufficient virus was shed by rats to cause susceptible pigs to seroconvert upon oral feeding--thus indicating that a minimal dose is necessary to ensure oral challenge. In a preliminary experiment, seronegative pigs given different doses of PPV orally showed a gradient level of serologic response and different rates of shedding.

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Effect of porcine parvovirus serostatus on the reproductive performance of mated gilts in an infected herd.

Gifts from 2 commercial swine operations (farm A and farm B) that were bred each week were tested for porcine parvovirus antibody. On farm A, 21.9% (weekly, 2% to 35%) of the 657 gilts tested over a 16-week period were seronegative within 1 week of mating. On farm B, 17.7% (weekly, o to 40%) of the 164 gilts tested over a 10 week period were seronegative within 1 week of mating. Eighty-one gilts from farm A which were seronegative at time of mating were retested at the end of the gestation. Of the 81 gilts, 4 1 had developed antibody titers. The litter size decreased by 1.3 live pigs/litter. The percentage of small litters (less than or equal to 6 born alive) in the group that seroconverted was 36.6% compared with 10% in the gilts that remained seronegative throughout gestation.

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Elevated concentrations in serum immunoglobulins due to infection by ovine progressive pneumonia virus.

Sixty-seven serum samples were obtained from 2 sheep flocks. Agar gel immunodiffusion (AGID) was used to separate progressive pneumonia virus (PPV)-infected sheep from noninfected sheep by the presence of precipitating antibodies. Immunoglobulin (Ig), total protein, and albumin concentrations were then measured from all 67 sera to determine whether differences existed between PPV-infected and non-infected sheep. A significant difference (P less than 0.0005) was found in both total protein and Ig concentration between PPV-infected and noninfected sheep. This corresponding difference was absent in albumin measurements. The significant differences (P less than 0.0005) in Ig and total protein concentrations were then used to evaluate a field test for diagnosing progressive pneumonia. The possibility of using either total protein or Ig concentrations as a field test was found to be highly unlikely due to variation in individual values.

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Evaluation of the agar-gel immunodiffusion test for the detection of precipitating antibodies against progressive pneumonia virus of sheep.

Various cell cultures were evaluated for their ability to support progressive pneumonia virus infection in vitro. Ovine trachea cells supported progressive pneumonia virus infection for an extended time,were extremely durable and could be passaged up until 30 passages. Progressive pneumonia virus infected ovine trachea cells were then used for the production of antigen for agar-gel immunodiffusion. A method for concentrating antigen, diafiltration, was compared to dialysis against polyethylene glycol. Using diafiltration, the concentrated virus was easily quantitated, less viscous (and therefore easier to apply) and only produced one precipitation line. Agar-gel immunodiffusion was used to survey 401 animals from two sheep flocks. One flock (96 sheep) was free of progressive pneumonia while the other flock had 111 of 305 total animals positive for precipitating antibodies. The incidence of precipitating antibodies in sheep ranged from 23% for yearling ewes to 80% in ewes seven years old.

Age Factors↗

Characterization of transforming growth factor-beta 1 gene expression in porcine immune cells.

We have investigated the regulation of transforming growth factor beta 1 gene expression in a variety of porcine immune cell populations, including peripheral blood mononuclear cells (PBMC), peripheral blood monocytes, alveolar macrophages and lymphoid cells from various swine lymphoid tissues. Using porcine transforming growth factor beta 1 cDNA probes in Northern blot assays, messages of 2.5 and 3.5 kb TGF beta 1 mRNA were detected in the cells investigated. A variety of mitogenic and immunomodulatory substances were examined for their ability to induce TGF beta 1 mRNA expression. These include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS), dexamethasone (Dex), tumor necrosis factor (TNF) and interleukin (IL)-1 alpha. While low level constitutive expression of TGF beta 1 mRNA was detected from all cells investigated, PMA treatment of PBMC and alveolar macrophages resulted in a more than 10-fold increase in the steady-state level of TGF beta 1 mRNA within 2 hr of PMA addition. Also, the effect of opiate drugs, methadone (Md) and morphine (Mor), on TGF beta 1 gene expression was determined. Cells treated with opiates expressed the same levels of TGF beta 1 mRNA as untreated cells. Since TGF beta 1 biological activity can be induced by opiates, the regulation of TGF beta 1 gene expression likely involves mechanisms that do not cause changes in mRNA levels.

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Rapid and sensitive detection of foot-and-mouth disease virus in tissues by enzymatic RNA amplification of the polymerase gene.

Direct detection of foot-and-mouth disease (FMD) virus from infected bovine and porcine tissue was investigated using a modified polymerase chain reaction (PCR) technique. A high degree of conservation was found in the genomic region coding for the viral RNA polymerase among the seven FMD viral (FMDV) serotypes. An oligomeric primer pair and probe were constructed from consensus sequence data within this area. First strand cDNA was synthesized using random hexamers and Moloney MuLV reverse transcriptase. The oligomeric primers used for PCR of the random primed cDNA yielded a 454-base-pair target amplification product. The PCR product was sized by agarose gel electrophoresis and hybridized strongly with the consensus sequence oligomeric probe. The PCR product was further examined by digestion with NcoI, confirming the predicted internal restriction enzyme site. All seven serotypes of FMDV RNA were amplified in a few hours and the PCR product tested positive. The sensitivity of the enzymatic amplification for detection of FMDV was 10 TCID50 by gel electrophoresis and less than 1 TCID50 when combined with hybridization to a labeled probe. The technique was specific, as determined by examination of at least 12 other viruses, including enteroviruses and other agents of vesicular disease. In vitro enzymatic amplification of cDNA from FMDV RNA using the modified PCR technique is highly specific, rapid and at least as sensitive as presently used procedures for FMDV laboratory diagnosis.

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Comparison of enzyme-linked immunosorbent assay with dot immunobinding assay for detection of antibodies against Pasteurella multocida in turkeys.

A dot immunobinding assay (DIA) was developed to detect antibodies against Pasteurella multocida in turkey serum. Five coating antigens, namely, whole-cell (WC) antigen, sonicated cell lysate (SCL), crude capsular extract (CE), formalin extract (FE), and heat-stable antigen (HSA), were compared by enzyme-linked immunosorbent assay (ELISA) and DIA using reference antisera against P. multocida organisms. WC and SCL antigens showed higher sensitivity, whereas FE and HSA antigens were more specific coating antigens in both assays. The specificity of DIA was greater than ELISA by comparing the P/N ratios of HSA against serum prepared from heterologous serotype of P. multocida. The DIA had also several distinct advantages over the ELISA, which included reduction of the manipulation time and more uniform binding of coating antigens onto the nitrocellulose membranes compared with binding of coating antigens to microtiter plates for ELISA.

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