Leaf morphology and ozone sensitivity of two open pollinated genotypes of black cherry (Prunus serotina) seedlings.

Two black cherry families differing in sensitivity to ozone (O(3)) were used to test the hypothesis that leaf morphology is related to foliar sensitivity to O(3). Two uninjured sections of leaf tissue were sampled from a single leaf collected from each of 12 open-grown O(3)-tolerant and 12 O(3)-sensitive seedlings. Standard histological techniques and light microscopy were used to examine 11 morphological characteristics. The O(3)-sensitive genotype had significantly greater stomatal density, thinner palisade mesophyll layer and thicker spongy mesophyll layer, lower ratio of palisade to spongy mesophyll, greater leaf weight and leaf area. In addition, total leaf thickness, guard cell length, and specific leaf mass were also slightly greater for the O(3)-sensitive genotype.

Foliar injury, leaf gas exchange and biomass responses of black cherry (Prunus serotina Ehrh.) half-sibling families to ozone exposure.

Open pollinated families of black cherry seedlings were studied to determine genotypic differences in foliar ozone injury and leaf gas exchange in 1994 and growth response following three growing seasons. An O(3)-sensitive half-sibling family (R-12) and an O(3)-tolerant half-sibling family (MO-7) planted in natural soil were studied along with generic nursery stock (NS) seedlings. Ozone exposure treatments were provided through open top chambers and consisted of 50, 75, and 97% of ambient ozone, and open plots from May 9 to August 26, 1994. Ambient ozone concentrations reached an hourly peak of 88 ppb with 7-hour averages ranging from 39 to 46 ppb. Seedlings in the 50 and 75% of ambient chambers were never exposed to greater than 80 ppb O(3). Visible foliar ozone injury (stipple) was significantly higher for R-12 seedlings than MO-7 seedlings and increased with increasing ozone exposures. For the chamber treatments averaged over all families, there was no significant difference in stomatal conductance and net photosynthetic rates, but there was a significant decrease in root biomass, and a significant decrease in root/shoot ratio between the 50 and 97% of ambient chambers. Stomatal conductance and net photosynthetic rates were significantly different between families with R-12 seedlings generally greater than MO-7 seedlings. The R-12 seedlings had a 7.5 mmol m(-2) increase in ozone uptake compared to MO-7, and at the same cumulative O(3) exposure R-12 exhibited 40.9% stippled leaf area, whereas MO-7 had 9.2% stippled leaf area. Significant differences were observed in stem volume growth and total final biomass between the open-top chambers and open plots. Although R-12 had the most severe foliar ozone injury, this family had significantly greater stem volume growth and total final biomass than MO-7 and NS seedlings. Root:shoot ratio was not significantly different between MO-7 and R-12 seedlings.

Responses of pregnant women to potential preterm labor symptoms.

OBJECTIVE: To examine the knowledge that healthy pregnant women have of appropriate actions to take in response to hypothetical symptoms of preterm labor. DESIGN: This was a descriptive, correlational study using a convenience sample. SETTING: Subjects were recruited from the private practices of obstetricians and nurse-midwives. PARTICIPANTS: Three hundred twenty pregnant women who were between 20 and 32 weeks gestation were asked to complete a 17-item demographic information sheet and an 18-item Health Pregnancy Questionnaire while waiting for prenatal visits. Questionnaires from 269 women were appropriate for analysis. RESULTS: Most respondents could select appropriate action responses to items that identified obvious symptoms of being in preterm labor. In response to three questions that posed hypothetical preterm labor symptoms that were more subtle or were similar to normally occurring discomforts of pregnancy, between 26% and 35% of the women selected a choice that would have delayed entry into care. With a 95% confidence interval, significant positive relationships were found between selecting best responses and having experienced a previous preterm labor and maternal age. CONCLUSION: This study supports the need for all pregnant women to receive information on identification of preterm labor symptoms and appropriate actions to take with regard to these symptoms.

Light environment alters ozone uptake per net photosynthetic rate in black cherry trees.

Foliar ozone uptake rates of different-sized black cherry (Prunus serotina Ehrh.) trees were compared within a deciduous forest and adjacent openings in north-central Pennsylvania during one growing season. Study trees included open-grown seedlings and saplings, forest understory seedlings and saplings, and sunlit and shaded portions of mature canopy tree crowns. Instantaneous ozone uptake rates were highest in high-light environments primarily because of higher stomatal conductances. Low ozone uptake rates of seedlings and saplings in the forest understory could be attributed partially to lower average ambient ozone concentrations compared to the canopy and open environments. Among the tree size and light combinations tested, ozone uptake rates were highest in open-grown seedlings and lowest in forest-grown seedlings. Despite lower ozone uptake rates of foliage in shaded environments, ozone uptake per net photosynthesis of foliage in shaded environments was significantly higher than that of foliage in sunlit environments because of weaker coupling between net photosynthesis and stomatal conductance in shaded environments. The potential for greater ozone injury in shaded environments as a result of greater ozone uptake per net photosynthesis is consistent with previous reports of greater ozone injury in shaded foliage than in sunlit foliage.

Size-mediated foliar response to ozone in black cherry trees.

Local ozone concentration and visible foliar injury were measured over the 1994 growing season on open-grown black cherry (Prunus serotina Ehrh.) trees of varying size (age) within forest stands and adjacent openings at a site in north-central Pennsylvania. Relationships were determined between visible ozone injury and ozone exposure, as well as calculated between injury and ozone uptake expressed as the product of stomatal conductance and ozone concentration. In addition, simultaneous measurements of visible symptoms and leaf gas exchange were also conducted to determine the correlation between visible and physiological injury and ozone exposure. By September, the amount of leaf area affected by visible foliar ozone injury was greatest for seedlings (46%), followed by canopy trees (20%) and saplings (15%). A large amount of variability in foliar ozone symptom expression was observed among trees within a size class. Sum40 and Sum60 (ozone concentration > 40 and > 60 nl liter(-1)) cumulative exposure statistics were the most meaningful indices for interpretation of foliar injury response. Seedlings were apparently more sensitive to ozone injury than larger trees because their higher rates of stomatal conductance resulted in higher rates of ozone uptake. Seedlings also had higher rates of early leaf abscission than larger trees with an average of nearly 30% of the leaves on a shoot abscised by 1 September compared to approximately 5% for larger trees. However, per unit ozone uptake into the leaf, larger trees exhibited larger amounts of foliar injury. The amount of visible foliar injury was negatively correlated (r(2) = 0.82) with net photosynthetic rates, but was not related to stomatal conductance. Net photosynthesis and stomatal conductance thus became uncoupled at high levels of visible foliar injury.

Vertical gradients of ozone and carbon dioxide within a deciduous forest in Central Pennsylvania.

Ambient concentrations of ozone (O(3)) and carbon dioxide (CO(2)) were measured at locations from the forest floor to the top of the canopy in a deciduous forest at the Moshannon State Forest in northcentral Pennsylvania. O(3) concentrations were measured from May-September for three years (1993-1995) while CO(2) concentrations were measured only during July and August of 1994. O(3) concentrations increased steadily during the day at all locations, peaking during the middle to late afternoon hours. O(3) concentrations then steadily declined to their lowest point, just before dawn. Vertical O(3) concentration gradients varied seasonally and among years. However, O(3) concentrations were highest within the forest canopy and lowest at the forest floor, with an average difference of approximately 13%. Differences in O(3) concentrations between the canopy and forest floor were greatest at night. O(3) concentrations were slightly higher at locations within the canopy than above the canopy. CO(2) concentrations were consistenly higher near the forest floor and were higher above the canopy than within the canopy. CO(2) concentrations were higher at night than during the day at all locations, especially near the forest floor.

Predicting Ozone Uptake from Meteorological and Environmental Variables.

Predictions of foliar ozone uptake rates of seedling and canopy black cherry trees (Prunus serotina Ehrh.) were made using concurrent measurements of ambient ozone concentration and other environmental and meteorological data during two growing seasons in north-central Pennsylvania.

Physiology, morphology, and ozone uptake of leaves of black cherry seedlings, saplings, and canopy trees.

Patterns of ozone uptake were related to physiological, morphological, and phenological characteristics of different-sized black cherry trees (Prunus serotina Ehrh.) at a site in central Pennsylvania. Calculated ozone uptake differed among open-grown seedlings, forest gap saplings, and canopy trees and between leaves in the upper and lower crown of saplings and canopy trees. On an instantaneous basis, seedling leaves had the greatest ozone uptake rates of all tree size classes due to greater stomatal conductance and higher concentrations of ozone in their local environment. A pattern of higher stomatal conductance of seedlings was consistent with higher incident photosynthetically-active radiation, stomatal density, and predawn xylem water potentials for seedlings relative to larger trees. However, seedlings displayed an indeterminate pattern of shoot growth, with the majority of their leaves produced after shoot growth had ceased for canopy and sapling trees. Full leaf expansion occurred by mid-June for sapling and canopy trees. Because many of their leaves were exposed to ozone for only part of the growing season, seedlings had a lower relative exposure over the course of the growing season, and subsequently lower cumulative uptake, of ozone than canopy trees and a level of uptake similar to upper canopy leaves of saplings. Visible injury symptoms were not always correlated with patterns in ozone uptake. Visible symptoms were more apparent on seedling leaves in concurrence with their high instantaneous uptake rates. However, visible injury was more prevalent on leaves in the lower versus upper crown of canopy trees and saplings, even though lower crown leaves had less ozone uptake. Lower crown leaves may be more sensitive to ozone per unit uptake than upper crown leaves because of their morphology. In addition, the lower net carbon uptake of lower crown leaves may limit repair and anti-oxidant defense processes.

Adaptation of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the determination of virus-neutralizing antibodies using the virus-neutralization assay.

The tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] has been widely used for bioassays. Herein is described the use of the MTT dye with a virus-neutralization (VN) assay to titer infectious bursal disease virus (IBDV)-neutralizing antibodies. A standard VN assay using chicken embryo fibroblasts (CEFs) and IBDV was used for the assessment of IBDV-neutralizing antibodies. The percent of CEF killing due to IBDV was quantitated using MTT, and the absorbance (A) data were used to calculate the VN antibody titer. This method of calculation offers the expression of VN titer in terms of units of activity per unit of volume.

Virus isolation accuracy: an evaluation of eight laboratories involved in poultry diagnostics.

A study was undertaken to evaluate the accuracy of diagnostic laboratories at isolation of common avian viral pathogens. Fourteen "unknown" samples were submitted to eight laboratories in seven states. All positive and negative samples were guaranteed pure by SPAFAS. Virus-isolation results were erroneous in many cases. Based on this study, it appears that protocol for virus isolation of avian pathogens should be standardized throughout the United States.

High-performance liquid chromatographic method for the determination of spectinomycin in turkey plasma.

A selective high-performance liquid chromatographic (HPLC) method with ultraviolet-visible (UV-VIS) detection was developed to measure therapeutic concentrations of spectinomycin in turkey plasma. Treatment of plasma samples with 3% trifluoroacetic acid in acetonitrile facilitated spectinomycin extraction and protein precipitation. After centrifugation, the stable derivatization reagent, 2,4-dinitrophenyl-hydrazine, was added to an aliquot of the supernatant, and the mixture was incubated for 30 min at 70 degrees C. Excess reagent was quenched with acetone and additional heating. The resulting derivative, a proposed spectinomycin-hydrazone, was separated from other compounds by reversed-phase HPLC during a short gradient run. The absorbance of the effluent was monitored spectrophotometrically with the UV-VIS detector set at 205 nm. The detector response was linear through the range of interest, 2-100 micrograms/ml.

Mechanism of T cell immunosuppression by infectious bursal disease virus of chickens.

Spleen cells of chickens infected with IBDV responded poorly to in vitro stimulation with Con A. The mitogenic hyporesponsiveness was due to the presence of suppressor cells that could be removed by pretreatment of IS cells with carbonyl iron or cytodex-3 microcarrier beads. Addition of suppressor cells to normal spleen cells prevented the normal cells from responding to mitogen. Cell-to-cell contact between responder and suppressor cells was not necessary for suppression to occur; the effect was mediated by soluble product(s) released by the suppressor cells. IS cells were also deficient in producing mitogen-induced IL-2 and addition of exogenous IL-2 did not restore mitogenic response of IS.

Purification of avian T cell growth factor and immune interferon using gel filtration high resolution chromatography.

A protocol was outlined to separate and purify chicken T cell growth factor or IL-2 and gamma interferon from conditioned medium of Con A activated spleen cells. IL-2 was associated primarily with a protein of 30 kDa in SDS-PAGE and in high resolution gel filtration chromatography. Chicken IL-2 was of low hydrophobicity. A second species of molecular weight 14 kDa was also identified by high resolution gel filtration chromatography. Purified IL-2 gave only 50% of the maximum growth response obtained with IL-2 in crude conditioned medium. Gamma interferon activity was associated primarily with protein of 17 kDa and a second peak of activity was detected with protein of 36 kDa. I-IFN was not cross reactive with human cells and was pH sensitive.

Genetic interaction between non-MHC T- and B-cell alloantigens in response to Rous sarcomas in chickens.

Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6(3) regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line RPRL 100, although these lines are identical for the major histocompatibility B complex. They differ, however, at three independent autosomal loci: Ly-4 and Th-1 determine the surface alloantigens of partly overlapping subsets of T lymphocytes, and Bu-1 determines a surface alloantigen of B lymphocytes. The association of genotypes at these loci with quantitative variation in their ability to regress Rous sarcomas was tested in segregating F4 generation progeny derived from crosses of lines 100 and 6(3). The Ly-4 and Bu-1 genotypes showed association with Rous sarcoma regression, but the Th-1 genotype did not. Chickens of the Ly-4a/Ly-4a, Bu-1b/Bu-1b and Ly-4b/Ly-4b, Bu-1a/Bu-1a genotypes had a significantly higher regressor ability than the other two double homozygous genotypes. These results indicate that higher regression is associated with (1) interaction between the Ly-4 and Bu-1 loci, and (2) complementation between either the line 6 Ly-4a allele and the line 100 Bu-1b allele, or the line 100 Ly-4b allele and the line 6 Bu-1a allele.

Strong suppression by monocytes of T cell mitogenesis in chicken peripheral blood leukocytes.

Peripheral blood leukocytes (PBL) isolated from chicken blood by flotation on Ficoll-Hypaque (FH) have much lower proliferative responses to Con A and PHA than PBL isolated by slow-speed (SS) centrifuging at 60 X G. FH preparations contain all categories of blood cells except erythrocytes, whereas SS are almost devoid of nonlymphoid cells. FH responses approach SS levels after filtration through Sephadex G-10, which removes almost all monocytes detectable by neutral red and nonspecific esterase methods, while only partially depleting granulocytes and thrombocytes. Thus, the low responses of FH are probably associated with suppressor activity of monocytes in these preparations, numbering 6 to 8% of mononuclear cells. G-10 filtration decreases responses of SS preparations, indicating that the few (less than 0.1%) monocytes in SS function mainly as helper cells. In co-cultures, irradiated FH cells (FHX) but not SSX produce as much as 90% suppression of SS or FH responses. Suppression by FHX is totally inhibited by heat killing (56 degrees C for 45 min) or monocyte inactivation by preincubation with Trypan blue, and is removed by G-10 filtration, although not completely. Adherent cells isolated from unirradiated FH on Cytodex beads (ADC-CY) produce up to 99% suppression in co-culture with SS or nonadherent cells derived from FH. Soluble suppressor factors can be detected in conditioned media from supernatants of Con A-stimulated cocultures containing suppressor monocytes, but their suppressor activity is partially opposed by stimulatory factors, possibly interleukin 2, also present in supernatants. It is concluded that in FH preparations and therefore in blood, but not in SS, monocytes that have not been activated exert strong suppressor activity on mitogen-induced T cell proliferation. Occasional chickens of one inbred line, RPRL 72, had exceptionally high suppressor activity of monocytes, as shown by very low FH responses and very high suppressor effects of FHX well outside the normal range of variation found in this line or in line RPRL 63. This abnormal suppressor activity may have resulted from activation of suppressor monocytes as a response to subclinical infection.

Influence of genotypes at a non-MHC B lymphocyte alloantigen locus (Bu-1) on expression of Ia (B-L) antigen on chicken bursal lymphocytes.

Two inbred lines of chickens known to be identical at the MHC differ in their expression of Ia antigen on cells of the bursa. Line 6 bursas had 23% of intensely staining Ia+ cells while line 7 bursas had a much higher level, 85%. Studies of F4 progeny of line 6(3) X 100 crosses showed that genetic control of the high bursal proportion of Ia+ cells was determined by the Bu-1 alloantigen system. Line 100 is identical to line 7 for the lymphocyte alloantigens identified by the T and B cell reagents used in this study. Tests of F4 heterozygotes at the Bu-1 locus show a dominant effect of the Bu-1b gene in control of a high proportion of strongly staining Ia+ cells in the bursa.

Tests of association of lymphocyte alloantigen genotypes with resistance to viral oncogenesis in chickens. 2. Rous sarcoma and lymphoid leukosis in progeny derived from 6(3) X 15(1) and 100 X 6(3) crosses.

Chickens from inbred line RPRL 6(3) are resistant to virally-induced Marek's disease (MD) and lymphoid leukosis (LL) and are relatively strong regressors of virally induced Rous sarcomas. Line 6(3) chickens are homozygous for three independent loci determining surface alloantigens of lymphocytes, Th-1 and Ly-4 on thymus-derived (T) cells and Bu-1 on bursa-derived (B) cells. In contrast, RPRL Lines 100 and 15(1) are highly susceptible to MD and LL and are weaker regressors of Rous sarcomas than 6(3). RPRL Lines 100 and 15(1) are homozygous for different alleles at the Th-1 and Ly-4 loci than 6(3), whereas only Line 100 differs from 6(3) at Bu-1. To test the possible association of these three antigen loci with variations in resistance to virally-induced tumors, homozygous genotypes among later generations derived from 6(3) X 15(1) and 100 X 6(3) crosses were identified by means of immunofluorescence testing of peripheral blood lymphocytes. Test progeny were then bred from matings of homozygous parents. No association was found between Rous sarcoma regression and the Th-1 and Ly-4 T cell alloantigen loci in F6 (6(3) X 15(1)) chickens, but the Bu-1 locus influenced rate of regression in F3 (100 X 6(3)) chickens. The Ly-4 in F6 (6(3) X 15(1)) and Bu-1 in the F3 (100 X 6(3)) chickens did not influence the incidence of LL. However, Th-1 influenced LL in F3 (100 X 6(3)) chickens (P less than .05) and possibly influenced LL in F6 (6(3) X 15(1)) chickens (P less than .09).

Ontogeny of con A and PHA responses of chicken blood cells in MHC-compatible lines 6(3) and 7(2).

The development of T cell responsiveness to Con A and PHA was examined in two MHC-compatible inbred chicken lines, RPRL 6(3) and 7(2), at ages 2 to 118 days posthatching. These lines are respectively resistant or susceptible to Marek's disease, a naturally occurring, virally induced T cell lymphoma. Between-line comparisons were made of optimal in vitro responses of diluted serum-free blood cells to each mitogen in two groups of chicks tested over ages 2 to 63 and 41 to 118 days. Over 2 to 63 days, Con A responses increased with age at the same rate in each line, but 7(2) responses averaged 2.3 times higher than 6(3). The increase with age was dependent on blood lymphocyte counts, which also increased with age in parallel in both lines. In contrast, the between-line difference in responsiveness was dependent on intrinsic reactivity of cells as well as lymphocyte counts. Covariance analysis was used to estimate that line 7(2) was 1.4 times higher than 6(3) in intrinsic cell reactivity, after accounting for the effect of the twofold higher blood lymphocyte counts in 7(2), and that this intrinsic difference contributed almost one-half the total difference. Over 41 to 118 days Con A responses no longer increased with age, although lymphocyte counts were still increasing, and the line difference (2.6 times) was now almost entirely contributed by a 2.3-fold superiority of 7(2) blood cells in intrinsic reactivity. The line difference in PHA responses was the reverse of the above in young chicks, with 6(3) responses greater than 7(2) in spite of lower lymphocyte counts. In additional chicks tested over 5 to 26 days, intrinsic reactivity of 6(3) cells to PHA averaged 4.5 times higher than 7(2). There was an abrupt decline in intrinsic reactivity of line 6(3) blood cells between 26 and 41 days to a level equal with 7(2). After this age, line 7(2) responses were 1.8 times greater than those of 6(3), and this difference was dependent solely on lymphocyte count differences. The results suggest that different gene systems mediate blood cell responses to PHA as compared with Con A. The pattern of developmental differences between inbred lines indicates the existence of distinct or partly overlapping T cell subsets with different reactivities to PHA or Con A, and of higher suppressor activity of adherent cells in line 6(3) blood. Both these differences may be related to line 6(3) inherited resistance to Marek's disease.

Influence of non-MHC T lymphocyte alloantigens on regression of Rous sarcomas in the chicken.

Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6(3) regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line 7(2), although these lines are identical for the major histocompatibility complex (MHC, B complex). They differ, however, at two independent autosomal loci, Ly-4 and Th-1, which determine surface alloantigens of partly overlapping subsets of T lymphocytes. Association of genotypes at these loci with quantitative variation in ability to regress Rous sarcomas was tested in segregating progeny derived from crosses of lines 6(3) and 7(2). In the F4 generation chickens of the Ly-4a/Ly-4a, Th-1a/Th-1a genotype (symbolized aa/aa) had significantly higher regressor ability than any of the other three double homozygous genotypes. In F5, all nine genotypes formed by combinations of homozygotes and heterozygotes were tested, and higher regressor ability was shown by the aa/aa, ab/aa, and aa/ab genotypes. These results indicate that higher regression is associated with: (1) interaction between the line 6(3) Ly-4a and Th-1a alleles in homozygous form; and (2) dominance x dominance interaction, in that the a allele at each locus is dominant for higher regression only within the homozygous aa genotype at the other locus.

Tests of association of lymphocyte alloantigen genotypes with resistance to viral oncogenesis in chickens. 1. Marek's disease in F7 progeny derived from 6(3) X 15(1) crosses.

Marek's disease (MD)-susceptible progeny of the B5B5 genotype from the F7 generation derived from crosses of Regional Poultry Research Laboratory inbred lines 6(3) and 15(1) were tested for variation in MD resistance associated with the T lymphocyte antigen loci Ly-4 and Th-1. The four double homozygous genotypic classes of Ly-4 and Th-1 were tested in a 2 X 2 design. The Ly-4bb/Th-1aa chickens were significantly more resistant to MD oncogenesis than the other three genotypes. Thus, resistance within B5B5 genotypes appears to be associated with genetic interaction between two T cell antigen loci, which are not linked with the B complex.