Clonal dynamics in western North American aspen (Populus tremuloides).

Clonality is a common phenomenon in plants, allowing genets to persist asexually for much longer periods of time than ramets. The relative frequency of sexual vs. asexual reproduction determines long-term dominance and persistence of clonal plants at the landscape scale. One of the most familiar and valued clonal plants in North America is aspen (Populus tremuloides). Previous researchers have suggested that aspen in xeric landscapes of the intermountain west represent genets of great chronological age, maintained via clonal expansion in the near absence of sexual reproduction. We synthesized microsatellite data from 1371 ramets in two large sampling grids in Utah. We found a surprisingly large number of distinct genets, some covering large spatial areas, but most represented by only one to a few individual ramets at a sampling scale of 50 m. In general, multi-ramet genets were spatially cohesive, although some genets appear to be fragmented remnants of much larger clones. We conclude that recent sexual reproduction in these landscapes is a stronger contributor to standing genetic variation at the population level than the accumulation of somatic mutations, and that even some of the spatially large clones may not be as ancient as previously supposed. Further, a striking majority of the largest genets in both study areas had three alleles at one or more loci, suggesting triploidy or aneuploidy. These genets tended to be spatially clustered but not closely related. Together, these findings substantially advance our understanding of clonal dynamics in western North American aspen, and set the stage for a broad range of future studies.

Influenza virus.

This unit contains several methods for infecting mice with influenza virus. It also includes protocols needed to propagate influenza virus in hen eggs, quantitate virus titers (in tissue culture medium and in influenza-infected mouse serum), and adopt human isolates of influenza for growth in mice. Methods for measuring the 50% mouse lethal dose are also included. Finally, protocols for generating anti-influenza cytotoxic T lymphocytes (CTL) from splenocyte precursors and harvesting pulmonary CTL following respiratory tract challenge of mice with influenza virus are provided.

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.

An array immunosensor for simultaneous detection of clinical analytes.

A fluorescence-based immunosensor has been developed for simultaneous analysis of multiple samples. A patterned array of recognition elements immobilized on the surface of a planar waveguide is used to "capture" analyte present in samples; bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This immunosensor was used to detect physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D-dimer, a marker of sepsis and thrombotic disorders, in spiked clinical samples.

Array biosensor: optical and fluidics systems.

Optical and fluidics systems have been developed as central components for an automated array biosensor. Disposable planar waveguides are patterned with immobilized capture antibodies using a physically isolated patterning (PIP) method. The PIP method enables simultaneous deposition of several antibodies and completely circumvents cross-immobilization problems encountered with other array deposition processes. A multi-channel fluidics cell allows numerous assays to be performed on the patterned waveguide. The sensing arrays are optically interrogated using a diode laser with a tailored output to optimize coupling to and maximize excitation uniformity within the waveguide. A patterned cladding is employed to optically isolate the waveguide from perturbations induced by the permanently attached flow cells. Compact optics image the evanescently excited fluorescence onto a large area, cooled CCD array. The image data is processed and automated signal analysis corrects for local background and noise variations.

Rapid detection of D-dimer using a fiber optic biosensor.

We describe a rapid and sensitive method for detection and quantification of D-dimer and other crosslinked fibrin degradation products (XL-FDPs), which are present in elevated concentrations in patients with sepsis and thrombotic disorders. The method utilizes a sandwich fluoroimmunoassay immobilized in the sensing region of an evanescent wave biosensor. Physiological concentrations of D-dimer and high molecular weight XL-FDP could be determined in buffer and plasma samples on calibrated fibers in 11 min. Samples from septic patients were assayed using ELISA and the fiber optic method; concentrations determined by fiber optic assay were strongly correlated with those determined by ELISA (r = 0.918); intra- and inter-assay errors were comparable to those from ELISAs. Given its accuracy and rapid response time, this fiber optic biosensor shows great potential for development as a diagnostic tool.

Oral immunization with a replication-deficient recombinant vaccinia virus protects mice against influenza.

Mice immunized with two intragastrically administered doses of a replication-deficient recombinant vaccinia virus containing the hemagglutinin and nucleoprotein genes from H1N1 influenza virus developed serum anti-H1 immunoglobulin G (IgG) antibody that completely protected the lungs from challenge with H1N1. Almost all of the mice given two intragastric doses also developed mucosal anti-H1 IgA antibody, and those with high anti-H1 IgA titers had completely protected noses. Intramuscular injection of the vaccine protected the lungs but not the noses from challenge. We also found that the vaccine enhanced recovery from infection caused by a shifted (H3N2) influenza virus, probably through the induction of nucleoprotein-specific cytotoxic T-lymphocyte activity. A replication-deficient, orally administered, enteric-coated, vaccinia virus-vectored vaccine might safely protect humans against influenza.

Characterization of monoclonal antibodies against a type SAT 2 foot-and-mouth disease virus.

This paper is the first to describe characterization of monoclonal antibodies (MAbs) against a South African Territories 2 (SAT 2) foot-and-mouth disease virus (isolate Rho 1/48). Twelve MAbs which neutralized homologous virus were characterized in indirect and sandwich ELISA using purified Rho 1/48 virus particles, subunits, trypsin-treated, and chemically denatured virus. All the MAbs inhibited haemagglutination by parental virus. Binding of the MAbs to 73 SAT 2 field isolates was measured in a sandwich ELISA and defined four distinct antigenic regions. Preliminary characterization of escape mutants selected with some of the MAbs using virus neutralization tests, ELISA, and amino acid sequencing is included. MAbs 2, 25, 40, 48 and 64, reacted with a linear epitope on the VP1 loop region. An amino acid change at position 149 (valine to glutamic acid) was detected in mutants selected by MAb 2 and 40 and this eliminated binding and neutralization by all the other MAb. This epitope was conformation-dependent and was conserved in all 73 isolates of SAT 2 examined. Escape mutants isolated with MAb 41 and 44, had changes at positions 156 (glycine to aspartic acid), or 158 (serine to leucine) respectively. These MAbs bound with Rho 1/48 only out of 73 field strain viruses studies and the reactions of MAbs from the other groups was unaltered. MAb 27, 28 and 37 reacted with a conformation-dependent epitope on VP1 which was not conserved in field isolates. All mutants selected by these MAbs had a single amino acid substitution at position 149 (valine to alanine). The same change was always found in field isolates which did not bind MAbs from this group. MAb 11 reacted with a linear epitope associated with amino acids 147 or 148 on VP1 and showed similar binding characteristics to a conformation dependent MAb 7, no amino acid residue changes were found within VP1 for monoclonal antibody 7 mutants.

Non-pathogenic strains of Aujeszky's disease virus are not reactivated in swine after corticosteroid treatment

The Bartha-K and NIA-4 strain of Aujeszky's disease virus (ADV) were readily isolated from oropharyngeal swabs up to 7 days after intranasal vaccination of young piglets. Neither strain could be reisolated 14 days after starting treatment with 10 mg of the corticosteroid isoflupredone acetate per kg of body weight, administered intramuscularly for 4 consecutive days when pigs were 7-9 months of age. Similar treatment with corticosteroid pigs infected with two virulent ADV strains resulted in the reactivation of infection and recovery of ADV from oropharyngeal swabs. Serum neutralizing antibodies were present in all pigles vaccinated twice (2 week interval) intranasally with the attenuated ADV strains, 4 weeks after primary vaccination. However, these antibodies were no longer detectable in some pigs at 12(NIA-4) and 20(Bartha-K) weeks of age even in undilluted sera. Neutralizing antibodies resulting from infection virulent ADV were always detectable, were higher in titer than those produced by the vaccine strains and did not vary in a clear pattern after corticosteroid treatment. These results indicated that the Bartha-K and NIA-4 strains undergo little or no latency in swine and confirm the latency of virulent strains of ADV

Non-pathogenic strains of Aujeszky's disease virus are not reactivated in swine after corticosteroid treatment.

1. The Bartha-K and NIA-4 strains of Aujeszky's disease virus (ADV) were readily isolated from oropharyngeal swabs up to 7 days after intranasal vaccination of young piglets. 2. Neither strain could be reisolated 14 days after starting treatment with 10 mg of the corticosteroid isoflupredone acetate per kg of body weight, administered intramuscularly for 4 consecutive days when pigs were 7-9 months of age. 3. Similar treatment with corticosteroid pigs infected with two virulent ADV strains resulted in the reactivation of infection and recovery of ADV from oropharyngeal swabs. 4. Serum neutralizing antibodies were present in all piglets vaccinated twice (2 week interval) intranasally with the attenuated ADV strains, 4 weeks after primary vaccination. However, these antibodies were no longer detectable in some pigs at 12 (NIA-4) and 20 (Bartha-K) weeks of age even in undiluted sera. 5. Neutralizing antibodies resulting from infection with virulent ADV were always detectable, were higher in titer than those produced by the vaccine strains and did not vary in a clear pattern after corticosteroid treatment. 6. These results indicate that the Bartha-K and NIA-4 strains undergo little or no latency in swine and confirm the latency of virulent strains of ADV.

Experimental vaccination of rabbits with non-pathogenic strains of Aujeszky's disease virus.

1. The Bartha K and NIA-4 strains of Aujeszky's disease virus (ADV) were non-pathogenic for rabbits vaccinated once or twice by nasal instillation or intramuscular injection. Neutralizing antibodies were detected in 68% of the rabbits two weeks after primary vaccination and in all rabbits at challenge. 2. Challenge doses of virulent ADV greater than 10(5.0) median tissue culture infective doses (TCID50) resulted in the death of most vaccinated and all unvaccinated rabbits with typical signs of Aujeszky's disease within 4 days. ADV was recovered from brain and lung suspensions of vaccinated and unvaccinated rabbits who had died as a result of the challenge. 3. When the challenge dose was reduced to approximately 10(3.0) TCID50, rabbits vaccinated twice survived while all unvaccinated controls died within 3 days.

Experimental vaccination of rabbits with non-pathogenic strains of Aujeszky's disease virus

1. The Bartha K and NIA-4 strains of Aujeszky's disease virus (ADV) were non-pathogenic for rabbits vaccinated once or twice by nasal instillation or intramuscular injection. Neutralizing antibodies were detected in 68% of the rabbits two weeks after primary vaccination and in al rabbits at challenge. 2. Challenge doses of virulent ADV greater than 10**5.0 median tissue culture infective doses (TCID50) resulted in the death of most vaccinated and all unvaccinated rabbits with typical signs of Aujeszky's disease withing 4 days. ADV was recovered from braim and lung suspensions of vaccinated and unvaccinated rabbits who had died as a result of the challenge. 3. When the challenge dose was reduced to approximately 10**3.0 TCID50, rabbits vaccinated twice survived while al unvaccinated controls died within 3 days

Transmission of bovine leukaemia virus in milk.

Nineteen calves born to dams free of bovine leukaemia virus (BLV) did not possess maternally derived precipitating antibody to BLV in their sera after the ingestion of colostrum. Eight of these calves remained serologically negative after being fed milk from BLV-free cows while three (27.3%) of 11 similar calves that had been fed milk from BLV-infected cows developed antibody. Forty-four of 47 calves born to BLV-infected dams acquired maternal antibody to BLV after ingesting colostrum. Two (8.7%) of the 23 calves fed milk from BLV-free cows developed antibody to BLV probably as a result of transplacental or colostrum infection whereas four (16.7%) of the 24 calves fed milk from BLV-infected cows developed antibody. It is concluded that milk transmission of BLV is responsible in part for the high rates of infection encountered in our dairy herds and that calves lacking specific maternal antibody are more susceptible to BLV infection through the ingestion of milk than are calves with maternal antibody.

Transmission of bovine leukemia virus through milk ingestion.

Foi utilizada a prova de imunodifusao para detectar a presenca de anticorpos contra a glicoproteina maior (gp 51) do virus da leucemia bovina (VLB), para avaliar o desenvolvimento da infeccao em bezerros alimentados com leite de vacas livres e infectadas com o virus da leucemia. Os bezerros recem-nascidos receberam o colostro das maes durante cinco dias e depois tres litros de leite, diariamente, por periodos que variaram entre 11 e 49 dias. Todos os animais em experimentacao foram sangrados mensalmente ate os oito meses de idade, para acompanhar o aparecimento dos anticorpos como evidencia da infeccao. O VLB foi transmitido a um dos quatro bezerros (25,0%) sem anticorpos maternos, depois da ingestao de leite de vacas infectadas. Como testemunhos foram utilizados tres bezerros sem anticorpos maternos e 10 com anticorpos maternos que nao adquiriram a infeccao pelo VLB, apos a ingestao de leite de vacas nao-infectadas, por periodos similares. Concluiu-se que o VLB e eliminado no leite de vacas infectadas e se constitui numa fonte de infeccao para bezerros recem-nascidos

Enzootic bovine leukosis virus in Brazil.

A sero-epidemiological survey for antibodies to the glycoprotein of enzootic bovine leukosis virus showed that the infection is widely disseminated in the State of Rio de Janeiro, Brazil. Sero from 1,290 females and 154 males from 12 dairy herds were tested by the agar gel precipitin test. Seven hundred and one females (54.3%) and 68 males (44.2%) were found to have specific antibodies. These antibodies were demonstrated in all 7 age groups tested. The older age groups contained the highest percentage of reactors. The results are briefly discussed in relation to management practices and environmental conditions.

Prevalencia de anticorpos contra o virus da papilomatose bovina no Estado do Rio de Janeiro. / Bovine papilloma virus antibodies in the State of Rio de Janeiro

Soros de 1174 bovinos, de 10 rebanhos leiteiros do Estado do Rio de Janeiro, foram testados para anticorpos contra o virus de papillomatose bovina, utilizando-se a microprova de imunodifusao em agar. A especificidade das reacoes foi confirmada quando grupos de particulas virais foram observados nas linhas de precipitacao, pela microscopia eletronica. Anticorpos de origem materna foram demonstrados em 18,3% dos bezerros, menores de tres meses de idade, enquanto que anticorpos so foram demonstrados em 2,0% dos bezerros, entre quatro e seis meses de idade. Anticorpos tambem foram demonstrados em 21,5% dos bezerros, entre sete e 12 meses de idade; em 25% dos animais, entre 13 e 18 meses de idade; em 21,2% dis animais, entre 19 e 30 meses de idade; em 23,7% dos animais, entre 31 e 48 meses de idade e em 23,1% dos animais, maiores de 49 meses de idade