Pericarp anatomy and hormone profiles of cypselas in dormant and non-dormant inbred sunflower lines.

The pericarp anatomy and the effects of storage after harvest, storage temperature and early cypsela imbibition on phytohormone profiles were studied in inbred sunflower lines B123 and B91. On day 0, germination of B123 cypselas was near 0%, indicating dormancy, whereas that of B91 cypselas was near 100%, indicating non-dormancy. The germination of B123 and B91 on day 33 at room temperature (25 °C) storage was similar. Cell wall thickness and sclerification of the pericarp were higher in B123 than B91, suggesting that structural characteristics may contribute to physical dormancy in B123. Jasmonates (JAs), salicylic acid (SA) and abscisic acid (ABA) were measured in dry and imbibed pericarps. SA content of dry pericarp was higher on day 33 than day 0. SA content during imbibition on day 33 was similar for room and low (-20 °C) storage temperatures. ABA content after 12 h imbibition was similar on days 0 and 33 at low temperature, but it increased on day 33 at room temperature for B123. 12-Oxo-phytodienoic acid (OPDA) was maximal on day 0 for B123, but peaked at day 33 at low temperature for B91. JA was higher on days 0 and 33 at room temperature as compared with low temperature. Our findings indicate that pericarp hormone profiles are affected in the two lines with different dormancy degree depending on storage conditions and imbibition processes.

Immunochemical studies on a Brucella ovis specific protein antigen.

A Brucella ovis surface protein antigen (P-II), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-II labeled with 125I, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains (B. abortus and B. melitensis).

Characterization of Brucella ovis surface antigens.

A rough antigen (SRA) extracted from Brucella ovis in hot saline by Myers procedure, showed three precipitation lines when tested in immunodiffusion against sera from experimentally infected rams. The components responsible for the lines could be isolated by ultracentrifugation or gel filtration which gave 3 fractions, named PI, PII and PIII. The lipopolysaccharide (LPS) appeared in the pellet (SRA-pp) after ultracentrifugation as judged by the presence of lipids, sugar composition, 2 keto-2deoxyoctulosonic acid (KDO), and its characteristic immunoelectrophoretic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns. SRA-pp contained the antigen responsible for one of the immunoprecipitation lines of SRA and the supernatant (SRA-sn) contained only antigens responsible for the other two. Gel filtration of SRA-pp showed the presence of PI, while SRA-sn gave PII with high protein content and PIII with high carbohydrate content. Immunological activity in gel diffusion (GD) of the Fraction PII and PIII was specific for sera of B. ovis infected rams. Sera from rams experimentally infected with smooth strains (Brucella abortus and melitensis), were not able to react with these antigens.

Immunochemical studies of oligosaccharides obtained from the lipopolysaccharide of Brucella ovis.

Brucella ovis rough lipopolysaccharide (R-LPS) was studied with respect to its heterogeneity, chain length, sugar composition and immunological activity. R-LPS was mildly hydrolysed and oligosaccharides were recovered in the upper phase after partition with chloroform-methanol. Gel-filtration of the upper phase in a column of Bio-Gel P-2 yielded oligosaccharides of 2, 4, 6 and 7 monosaccharide units, 2-keto-deoxy-octulosonic acid (KDO), and monosaccharides. Strong acid hydrolysis followed by paper chromatography showed that the hexa- and heptasaccharides are both composed of glucose, KDO and an unidentified sugar while tetrasaccharide is composed of glucose, mannose and glucosamine. These three oligosaccharides were able to inhibit the LPS-antibody reaction in a solid phase radioimmunoassay, suggesting the oligosaccharides bear antigenic determinants of LPS.

Development and validation of an indirect enzyme immunoassay for detection of ovine antibody to Brucella ovis.

An indirect enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Brucella ovis infection was developed. The assay uses a mouse monoclonal antibody to bovine IgG1 horseradish peroxidase (HRPO) conjugate that cross-reacts with immunoglobulin from sheep and a purified antigen from Brucella ovis. The ELISA data were read and analyzed according to a targeting procedure. The ELISA results were compared with a cold complement fixation test (CFT). Sera from 675 rams from three uninfected flocks were used to determine the ELISA cut-off value (O.D. 405 nm: 0.095) and the diagnostic specificity of the ELISA (100%) and the CFT (99.69% +/- 0.42). The ELISA cut-off value was corroborated by receiver operating characteristic (ROC) analysis. Six hundred and forty semen and serum samples from 419 rams from two naturally infected flocks were collected before and after mating-time during two consecutive years. All semen samples were cultured and Brucella ovis was isolated from 28 samples. Sera from the 28 rams with positive semen were used to determine the diagnostic sensitivity of the ELISA (96.43% +/- 6.8) and of the CFT (including suspected positive samples with titers of 1:5; 88.89% +/- 11.85). Considering the CFT suspicious and the anti-complementary reactions as positive resulted in a diagnostic sensitivity value of 89.28% +/- 11.46. Six hundred and ten serum samples from the 640 sera were used to determine relative sensitivity (excluding sera with 1:5) at: ELISA/CFT 97.26% +/- 3.74 and CFT/ELISA was 71.72% +/- 8.87. The percent agreement, beyond chance measured by the Kappa index was 79.7. Relative sensitivity ELISA/CFT (including 1:5 titers in the CFT as positive) was 94.9% +/- 4.83 and CFT/ELISA was 72.84% +/- 8.59. The Kappa index was 79.4.

Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis.

Sera from Canadian pigs (brucellosis free, n = 14037) and sera from pigs infected with Brucella suis (n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.

Enzyme immunoassay for the diagnosis of brucellosis: chimeric Protein A-Protein G as a common enzyme labeled detection reagent for sera for different animal species.

A recombinant protein combining the immunoglobulin binding sites of Proteins A and G, conjugated with horseradish peroxidase was used as a universal detection reagent for the assessment of antibodies against Brucella spp. The reagent was applied in an indirect enzyme immunoassay for detection of antibodies to smooth lipopolysaccharide antigen in sera from Brucella spp. exposed and non-exposed cattle, sheep, goats and pigs and to antibodies to rough lipopolysaccharide in sheep, dogs and cattle. The results were similar to those obtained when murine monoclonal antibody-enzyme conjugates were used. An added advantage was that a universal cut-off for all tests using the proteins A and G detection reagent could be established, simplifying diagnostic interpretation of the data.

Stimulation of jasmonic acid production in Zea mays L. infected by the maize rough dwarf virus-Río Cuarto. Reversion of symptoms by salicylic acid.

In the present paper we study the possible biological relevance of endogenous jasmonic acid (JA) and exogenous salicylic acid (SA) in a plant-microbial system maize-virus. The virus disease "Mal de Río Cuarto" is caused by the maize rough dwarf virus-Río Cuarto. The characteristic symptoms are the appearance of galls or "enations" in leaves, shortening of the stem internodes, poor radical system and general stunting. Changes in JA and protein pattern in maize control and infected plants of a virus-tolerant cultivar were investigated. Healthy and infected-leaf discs were collected for JA measurement at different post-infection times (20, 40, 60 and 68 days). JA was also measured in roots on day 60 after infection. For SDS-PAGE protein analysis, leaf discs were also harvested on day 60 after infection. Infected leaves showed higher levels of JA than healthy leaves, and the rise in endogenous JA coincided with the enation formation. The soluble protein amount did not show differences between infected and healthy leaves; moreover, no difference in the expression of soluble protein was revealed by SDS-PAGE. Our results show that the octadecanoid pathway was stimulated in leaves and roots of the tolerant maize cultivar when infected by this virus. This finding, together with fewer plants with the disease symptoms, suggest that higher foliar and roots JA content may be related to disease tolerance. SA exogenous treatment caused the reversion of the dwarfism symptom.

Vector development for the expression of foreign proteins in the vaccine strain Brucella abortus S19.

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.

Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells.

Null cyclic beta-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic beta-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.

Evaluation of primary binding assays for presumptive serodiagnosis of swine brucellosis in Argentina.

An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which Brucella suis was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98. 3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which B. suis was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%.

Antibody dynamics in Holstein Friesian heifers vaccinated with Brucella abortus strain 19, using seven serological tests.

The serological response induced by Brucella abortus strain 19 was evaluated in 52 Holstein females from a brucellosis-free herd using seven serological tests. Each calf was vaccinated at an age of 4 and 8 months old with 3 x 10(10) CFU B. abortus S19 and the antibody response was determined as the proportion of positive results to each test. The antibody dynamics, measured with the buffered plate antigen (BPA) test and the rapid automated presumptive (RAP) test, were similar. The proportion of positive reactions in these tests reached 100% one week after vaccination and remained at this level for seven weeks, after which the proportion of positive samples slowly declined to 8% (BPA) and 2% (RAP) at week 50. The response in the indirect enzyme immunoassay (i-ELISA) was similar, but shorter than that observed with the BPA/RAP. The antibody dynamic, measured using the seroagglutination test (SAT) in parallel with the 2-mercaptoethanol (2-Me) test and the complement fixation test (CFT) were similar to the RAP/BPA, but of slightly shorter duration. The competitive ELISA (c-ELISA) was positive in all animals for 3 weeks, followed by a rapid decline. The fluorescence polarization assay (FPA) reached a maximum of 68.5% positive animals at week 4 and then declined. Based on these data, the c-ELISA and FPA discriminated residual antibody activity due to vaccination more efficiently than the other tests.

Stimulation of jasmonic acid production in Zea mays L. infected by the maize rough dwarf virus-Río Cuarto: reversion of symptoms by salicylic acid

In the present paper we study the possible biological relevance of endogenous jasmonic acid (JA) and exogenous salicylic acid (SA) in a plant-microbial system maize-virus. The virus disease "Mal de Río Cuarto" is caused by the maize rough dwarf virus-Río Cuarto. The characteristic symptoms are the appearance of galls or enations in leaves, shortening of the stem internodes, poor radical system and general stunting. Changes in JA and protein pattern in maize control and infected plants of a virus-tolerant cultivar were investigated. Healthy and infected-leaf discs were collected for JA measurement at different post-infection times (20, 40, 60 and 68 days). JA was also measured in roots on day 60 after infection. For SDS-PAGE protein analysis, leaf discs were also harvested on day 60 after infection. Infected leaves showed higher levels of JA than healthy leaves, and the rise in endogenous JA coincided with the enation formation. The soluble protein amount did not show differences between infected and healthy leaves; moreover, no difference in the expression of soluble protein was revealed by SDS-PAGE. Our results show that the octadecanoid pathway was stimulated in leaves and roots of the tolerant maize cultivar when infected by this virus. This finding, together with fewer plants with the disease symptoms, suggest that higher foliar and roots JA content may be related to disease tolerance. SA exogenous treatment caused the reversion of the dwarfism symptom. (AU)

Stimulation of jasmonic acid production in Zea mays L. infected by the maize rough dwarf virus-Río Cuarto: reversion of symptoms by salicylic acid

In the present paper we study the possible biological relevance of endogenous jasmonic acid (JA) and exogenous salicylic acid (SA) in a plant-microbial system maize-virus. The virus disease "Mal de Río Cuarto" is caused by the maize rough dwarf virus-Río Cuarto. The characteristic symptoms are the appearance of galls or enations in leaves, shortening of the stem internodes, poor radical system and general stunting. Changes in JA and protein pattern in maize control and infected plants of a virus-tolerant cultivar were investigated. Healthy and infected-leaf discs were collected for JA measurement at different post-infection times (20, 40, 60 and 68 days). JA was also measured in roots on day 60 after infection. For SDS-PAGE protein analysis, leaf discs were also harvested on day 60 after infection. Infected leaves showed higher levels of JA than healthy leaves, and the rise in endogenous JA coincided with the enation formation. The soluble protein amount did not show differences between infected and healthy leaves; moreover, no difference in the expression of soluble protein was revealed by SDS-PAGE. Our results show that the octadecanoid pathway was stimulated in leaves and roots of the tolerant maize cultivar when infected by this virus. This finding, together with fewer plants with the disease symptoms, suggest that higher foliar and roots JA content may be related to disease tolerance. SA exogenous treatment caused the reversion of the dwarfism symptom.

Stimulation of jasmonic acid production in Zea mays L. infected by the maize rough dwarf virus-Río Cuarto. Reversion of symptoms by salicylic acid.

In the present paper we study the possible biological relevance of endogenous jasmonic acid (JA) and exogenous salicylic acid (SA) in a plant-microbial system maize-virus. The virus disease [quot ]Mal de Río Cuarto[quot ] is caused by the maize rough dwarf virus-Río Cuarto. The characteristic symptoms are the appearance of galls or [quot ]enations[quot ] in leaves, shortening of the stem internodes, poor radical system and general stunting. Changes in JA and protein pattern in maize control and infected plants of a virus-tolerant cultivar were investigated. Healthy and infected-leaf discs were collected for JA measurement at different post-infection times (20, 40, 60 and 68 days). JA was also measured in roots on day 60 after infection. For SDS-PAGE protein analysis, leaf discs were also harvested on day 60 after infection. Infected leaves showed higher levels of JA than healthy leaves, and the rise in endogenous JA coincided with the enation formation. The soluble protein amount did not show differences between infected and healthy leaves; moreover, no difference in the expression of soluble protein was revealed by SDS-PAGE. Our results show that the octadecanoid pathway was stimulated in leaves and roots of the tolerant maize cultivar when infected by this virus. This finding, together with fewer plants with the disease symptoms, suggest that higher foliar and roots JA content may be related to disease tolerance. SA exogenous treatment caused the reversion of the dwarfism symptom.