Quantification of conidial density of Aspergillus flavus and A. parasiticus in soil from almond orchards using real-time PCR.

AIMS: To design the Aspergillus flavus and Aspergillus parasiticus-specific primers and a real-time PCR assay for quantification of the conidial density in soil. METHODS AND RESULTS: Aspergillus flavus and A. parasiticus-specific DNA primers were designed based on internal transcribed spacer sequences to distinguish these two species and from other Aspergillus and other fungal species. A method of pathogen DNA extraction directly from soil samples was developed. Using the designed primers, a real-time PCR assay was developed to quantitatively determine the conidial density of each A. flavus and A. parasiticus in soil, after generating corresponding standard curves. Known conidial densities of each A. flavus or A. parasiticus in soil significantly correlated with those tested with the real-time PCR. CONCLUSIONS: This study demonstrated the applicability of the real-time PCR assay in studies of quantifying A. flavus and A. parasiticus in soil as inoculum sources. SIGNIFICANCE AND IMPACT OF THE STUDY: The A. flacus and A. parasitic-specific primers can be widely used in aflatoxin research. The real-time PCR assay developed in this study provides a potential approach to quantify the plant pathogen density from not only soil but also other sources in relation to aflatoxin contamination from environment, food and feed commodities.

School Health Education Evaluation. Implication of results: factors for success.

The four-year evaluation study summarized in this issue brings new evidence and directions for more successful health education programs. Two major factors for success stand out. The implementation of the program requires administrative support to assure adequate teacher preparation, and, fidelity to the curriculum must be maintained. The authors will describe these factors.

CD4+ T-cell loss and delayed expression of modulators of immune responses at mucosal sites of vaccinated macaques following SIV(mac251) infection.

Systemic immunization of macaques with a combination of DNA-poxvirus-based vaccines confers protection from high level of both systemic and mucosal viral replication following rectal exposure to the pathogenic SIV(mac251). Here we investigated early post-infection events in rectal and vaginal tissues, and found that the loss of CCR5+CD4+ T cells was equivalent in vaccinated and control macaques, despite a three logs reduction at mucosal sites of simian immunodeficiency virus (SIV) RNA in the vaccinated group. Even though a normal CD4+ T cell number is not reconstituted at mucosal sites in either group, vaccination appeared to confer a better preservation of the CD4+ CCR5+ T cells that replenish these sites. Analysis of rectal tissues RNA following challenge exposure demonstrated a decreased expression in vaccinated macaques of transforming growth factor-beta, cytotoxic T lymphocyte antigen-4, FoxP3, and indoleamine 2,3-dioxygenase, an immune suppressive enzyme expressed by dendritic cells that converts tryptophan to kynurenine and limits T-cell responses. Accordingly, the ratio of kynurenine and tryptophan in the plasma was significantly reduced in the vaccinated animals respect to the controls. Thus, preexisting adaptive immune responses induced by these vaccine modalities, although they do not protect from CD4+ T-cell depletion, nevertheless, they contain SIV(mac251) replication and delay expression of markers of T-cell activation and/or suppression at mucosal sites.

An evaluation of three techniques improving immunization levels in elementary schools.

Three techniques for improving immunization levels among school-age children were tested and then compared for most effective use of school nurses' time. Method A involved reviewing school immunization records, specifically inviting immunization-deficient children to a school-based clinic, with some follow-up to achieve good response. Method B involved sending out permission slips for a school-based clinic to all students without additional investment of nursing time. Method C involved a health education program encouraging parents to have their children immunized on their own. Using an average of 38 hours of school nurse time, Method A succeeded significantly better then Method B in immunizing more immunization-deficient children and raising immunization levels , while giving fewer unnecessary immunizations. Method C did not produce significant improvement of immunization levels.