Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish.

Sterilized seawater was used to assess the effects of temperature and salinity on the survival of Vibrio vulnificus. In the temperature range of 13 to 22 degrees C, numbers of V. vulnificus increased during the 6-day incubation. Temperatures outside this range reduced the time of V. vulnificus survival in sterile 10-ppt seawater. At these restrictive temperatures, V. vulnificus numbers were reduced by 90% after 6 days of incubation. Incubation between 0.5 and 10.5 degrees C demonstrated that V. vulnificus survives poorly below 8.5 degrees C. At salinities between 5 and 25 ppt and at 14 degrees C, V. vulnificus numbers actually increased or remained unchanged after 6 days of incubation. At salinities of 30, 35, and 38 ppt, numbers of V. vulnificus decreased 58, 88, and 83%, respectively. V. vulnificus could not be recovered from deionized water, indicating lysis. When a rifampin-resistant strain of V. vulnificus was used to inoculate sterilized and unsterilized seawater (20 ppt, 20 degrees C), numbers increased in sterile seawater but decreased to undetectable levels in 14 days in the unsterilized seawater, indicating that biological factors may play a role in the survival of V. vulnificus in the environment. Since our studies demonstrated sensitivity to low temperatures, the survival of V. vulnificus in naturally contaminated oysters at temperatures of 0, 2, and 4 degrees C was also determined. Numbers of endogenous V. vulnificus in oyster shellstock increased by more than 100-fold in shellstock stored at 30 degrees C but were reduced approximately 10- and 100-fold after 14 days at 2 to 4 degrees C and 0 degrees C, respectively. We conclude that both biological and physicochemical factors are important to the survival of V. vulnificus in the environment and that temperature is critical to controlling its growth in oyster shellstock.     

Evaluation of lactic acid bacterium fermentation products and food-grade chemicals to control Listeria monocytogenes in blue crab (Callinectes sapidus) meat.

Fresh blue crab (Callinectes sapidus) meat was obtained from retail markets in Florida and sampled for viable Listeria monocytogenes. The pathogen was found in crabmeat in three of four different lots tested by enrichment and at levels of 75 CFU/g in one of the same four lots by direct plating. Next, crabmeat was steam sterilized, inoculated with a three-strain mixture of L. monocytogenes (ca. 5.5 log10 CFU/g), washed with various lactic acid bacterium fermentation products (2,000 to 20,000 arbitrary units [AU]/ml of wash) or food-grade chemicals (0.25 to 4 M), and stored at 4 degrees C. Counts of the pathogen remained relatively constant in control samples during storage for 6 days, whereas in crabmeat washed with Perlac 1911 or MicroGard (10,000 to 20,000 AU), numbers initially decreased (0.5 to 1.0 log10 unit/g) but recovered to original levels within 6 days. Numbers of L. monocytogenes cells decreased 1.5 to 2.7 log10 units/g of crabmeat within 0.04 day when washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083, or Nisin per ml. Thereafter, counts increased 0.5 to 1.6 log10 units within 6 days. After washing with food-grade chemicals, modest reductions (0.4 to 0.8 log10 unit/g) were observed with sodium acetate (4 M), sodium diacetate (0.5 or 1 M), sodium lactate (1 M), or sodium nitrite (1.5 M). However, Listeria counts in crabmeat washed with 2 M sodium diacetate decreased 2.6 log10 units/g within 6 days. In addition, trisodium phosphate reduced L. monocytogenes counts from 1.7 (0.25 M) to > 4.6 (1 M) log10 units/g within 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Loss of meiosis in Aspergillus

If strictly mitotic asexual fungi lack recombination, the conventional view predicts that they are recent derivatives from older meiotic lineages. We tested this by inferring phylogenetic relationships among closely related meiotic and strictly mitotic taxa with Aspergillus conidial (mitotic) states. Phylogenies were constructed by using DNA sequences from the mitochondrial small ribosomal subunit, the nuclear ribosomal internal transcribed spacers, and the nuclear 5.8S ribosomal gene. Over 920 bp of sequence was analyzed for each taxon. Phylogenetic analysis of both the mitochondrial and nuclear data sets showed at least four clades that possess both meiotic and strictly mitotic taxa. These results support the hypothesis that strictly mitotic lineages arise frequently from more ancient meiotic lineages with Aspergillus conidial states. Many of the strictly mitotic species examined retained characters that may be vestiges of a meiotic state, including the production of sclerotia, sclerotium-like structures, and hulle cells.      

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Occurrence and characteristics of agglutination of Vibrio cholerae by serum from the eastern oyster, Crassostrea virginica

Cell-free hemolymph (serum) of the eastern oyster, Crassostrea virginica, agglutinated Vibrio cholerae, including all O1 serovars and biovars. Seventy-nine other strains of bacteria, including 14 genera and 26 species, were not agglutinated. The A, B, and C factors of O1 antigen were not involved in agglutination. Bacterial agglutinating (BA) activity was demonstrated for oysters inhabiting different environments of the U.S. Atlantic and Gulf coasts. Oyster serum BA titers showed high individual variation. The serum component(s) involved in BA was inhibited by 80 degrees C heat, pronase, EDTA, mucin, and fetuin treatments. N-Acetylneuraminic acid (10 mg/ml) weakly inhibited BA activity. Ligands of V. cholerae were sensitive to neuraminidase and resistant to 80 degrees C and pronase. High salinities (24 and 30%) enhanced BA. Cross-adsorption tests with V. cholerae and human O+ erythrocytes indicated that BA and hemagglutinating activities may involve different serum components. These results imply that the ecology of V. cholerae in C. virginica is influenced by agglutinating activity of oyster serum.     

Variation in epitopes of the B subunit of El Tor and classical biotype Vibrio cholerae O1 cholera toxin

Variation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae O1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classical-like CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM1 ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined.     

Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.