Aspergillus flavus Infection and Aflatoxin Production in Corn: Influence of Trace Elements

Distribution of trace element levels in corn germ fractions from kernels naturally infected with Aspergillus flavus and from kernels free of the fungus demonstrated an association between the presence of A. flavus and higher levels of metals. A. flavus production of aflatoxin on various autoclaved corn media showed that ground, whole corn was an excellent substrate; similar high levels of toxin were observed on full-fat corn germ but endosperm and defatted corn germ supported reduced yields. The influence of trace elements and their availability in defatted corn germ to A. flavus-mediated aflatoxin biosynthesis were measured. Enrichment of the substrate with 5 to 10 mug of manganese, copper, cadmium, or chromium per g of germ increased toxin yields. Addition of lead or zinc (50 to 250 mug/g) also enhanced toxin accumulation. Aflatoxin elaboration was reduced by the addition of 25 mug of cadmium per g or 500 mug of copper per g of germ.     

Viriditoxin Production by Aspergillus viridi-nutans and Related Species

Bioproduction of viriditoxin on various substrates by strains of the Aspergillus fumigatus group was determined under several incubation conditions. Aspergillus viridi-nutans strains NRRL 4365 and 576 produced the largest quantities of toxin, A. brevipes gave reduced yields, and there was no detectable synthesis by isolates of four related species. After 30 days in static culture at 20 C on various autoclaved agricultural commodities, optimal yields of 440 and 380 mg of toxin were observed per kilogram of sorghum and rice. Toxin levels were reduced on corn, rye, and wheat (40-200 mg/kg); yields were low on cottonseed, barley, and oats. Incubation at 10 C restricted biosynthesis of viriditoxin, and no toxin accumulated on substrates maintained at 5 C for 120 days. In a liquid, yeast extract-sucrose medium, maximal mycotoxin production developed in shake flasks; after 156 h, 10 mg of toxin accumulated per gram of mycelium. Viriditoxin produced in submerged culture was associated with the mycelium; less than 1% was detected in the filtered broth after 156 h of incubation.     

Development and characterization of a recombinant chicken single-chain Fv antibody detecting <Emphasis Type="Italic">Eimeria acervulina</Emphasis> sporozoite antigen

Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the V_H and V_L genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l^–1. The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken V_L genes suggested that the gene conversion with V pseudogenes might contribute to the diversification of V_L genes in chickens.     

Effect of thyroxine and chicken growth hormone on immune function in autoimmune thyroiditis (obese) strain chicks.

The effect of thyroxine (T4) and/or recombinant chicken growth hormone (rcGH) supplementation on immune function and on immune cell maturation was examined in Obese strain chickens. Day-old Obese strain chicks received the control treatments or were treated with either T4 (supplemented in the diet), T4-rcGH, or rcGH (by daily injection) in a full factorial design. At 4 weeks of age, the proliferative activity of peripheral blood T cells to either mitogenic or allogenic cell (mixed lymphocyte response) challenge was assessed. At the same time, peripheral blood lymphocytes and thymocytes were collected and prepared for flow cytometry analysis. Proliferative responses to both T cell mitogens and allogeneic splenocytes were significantly increased (P less than 0.05) by rcGH treatment, while the combined T4-rcGH treatment resulted in a significant increase in allogeneic and in concanavalin A responsiveness, but not in the response to phytohemagglutinin. All supplemented groups showed a significant decrease in the mean fluorescent intensity for CT-1a+ thymocytes, while thymocytes from birds receiving either T4 or rcGH alone had higher proportions of CD4+ and CD8+ cells. The monoclonal antibody staining of thymocytes from T4-rcGH-supplemented animals more closely resembled that of the unsupplemented controls. Among the peripheral blood lymphocytes, there were no changes in the numbers of CD4+, CD8+, or sIg+ cells as a result of treatment. The mean fluorescent intensity of sIg+ cells was significantly decreased, however, as a result of T4 supplementation when given either alone or in combination with rcGH. Finally, the mean fluorescent intensity ratios of CD4+ to CD8+ cells was significantly increased as a result of rcGH supplementation. These results strongly support a role for both the thyroid hormones and growth hormone in regulating and/or enhancing immune function, with changes in functional responses paralleled by concomitant changes in the T cell populations as expressed by shifts in T cell surface marker expression.     

Relationship of byssinosis to the generation of oxygen radicals by bract tissues of cotton plants

Summary Byssinosis is a hazardous respiratory disorder of workers in natural fiber processing industries and, in the case of cotton, is caused by exposure to respirable dust generated from leafy trash associated with raw fibers. To understand the chemical characteristics of involucral trash components that might contribute to byssinosis, we examined the human airway constricting activity and oxygen radical generating activity of dry, frost-killed cotton bracts. In response to inhalation of aerosolized bract extracts, the expiratory flow rates of human volunteers at 40% of vital capacity during partial forced expiration decreased by 3 to 32%. These values enabled us to identify two potentially byssinogenically active bract specimens, a specimen virtually inactive, and a fourth intermediately so. Using spin trapping techniques of electron spin resonance spectrometry, we found that all specimens catalyzed the generation of hydroxyl (preponderantly) and superoxide radicals from hydrogen peroxide. However, the weakest constrictor was the most potent catalyst, and vice versa. This was consistent with transition metal content of the specimens; the most potent catalyst also contained the largest amounts of those metals, suggesting a Fenton-type reaction mechanism. Other possibilities for the inverse relationship of airway constricting (byssinogenic) activity with oxygen radical generation are discussed.We also found that neither aflatoxin nor endotoxin, contingent contaminants of bracts, catalyzed oxygen radical production from hydrogen peroxide.     

Enzymes in atlatoxin B1 biosynthesis: Strategies for identifying pertinent genes

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B₁ (AFB₁) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain ofAspergillus parasiticus (SRRC 163): 1)A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB₁. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated fromAspergillus parasiticus mycelia during the onset of AFB₁ biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.     

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.