Kemet D. Spence

The peritrophic membrane: Ultrastructural analysis and function as a mechanical barrier to microbial infection in Orgyia pseudotsugata

Abstract The feasibility of freeze-etching as a method for structural analysis of the peritrophic membrane (PM) has been demonstrated, and this method and other electron microscopical techniques have been employed to investigate the possible function of the PM as a mechanical barrier to infection of the larval midgut. Thin sections show that the PM consists of granular and microfibrous layers typical of PMs from other insects. Freeze-etch studies show that the membrane consists of at least three structurally distinct layers. The major component is a layer of chitin microfibrils that appear to be arranged in oriented layers. The fibrils exhibit a periodicity of 4 nm and are embedded in a granular matrix. The fibrils coalesce into 100-nm fibers which run throughout the microfibrillar layer. Adjacent to this layer is a very rough layer, with randomly dispersed granules 100 nm in diameter and covered with particles 32 nm in diameter. Adjacent to the midgut lumen is a finely granular layer which gives the PM a smooth appearance. No pores or other discontinuities through which a bacterial or viral pathogen could penetrate were detected in the PM, indicating the PM of the Douglas fir tussock moth could function as a mechanical barrier to microbial infection. The importance of chitin and protein to the apparent strength of the Douglas fir tussock moth PM was partially elucidated by studies with chitinases and proteases. Both types of enzymes degraded the PM, causing release of the products of hyrolysis or structural changes in the membrane.

Rapid Detection and Identification of Lactobacillus spp. in Dairy Products by Using the Polymerase Chain Reaction

Species-specific primers for use in the polymerase chain reaction (PCR) were designed to differentially amplify DNA from the common dairy lactobacillus species Lactobacillus casei , Lactobacillus delbrueckii , Lactobacillus helveticus , and Lactobacillus acidophilus . A method for rapid extraction of bacterial DNA from dairy products was developed. The sensitivity of bacterial DNA extraction from food and subsequent amplification by PCR was 100 cells total. Lactobacillus DNA was extracted and identified from commercial yoghurts, acidophilus milk, and cheeses. The methodology allows the presumptive identification of dairy lactobacilli in less than 6 hours.

Effect of sublethal Bacillus thuringiensis crystal endotoxin treatment on the larval midgut of a moth, Manduca: SEM study.

The effect of a single, sublethal dose of B. thuringiensis crystal endotoxin on the midgut of the moth Manduca sexta larvae was monitored during acute and recovery stages. Initially both goblet and columnar cells swelled. Many columnar cells produced membrane extrusions. In some cases the affected cells ruptured, extruding cellular debris into the midgut lumen. Following the acute stage, the midgut tissue recovered, the damaged cells being extruded into the midgut lumen apparently as newly regenerated cells rose to take their place. The insects appeared to recover completely and continue normal development.

A BACTERIAL-INDUCED LECTIN WHICH TRIGGERS HEMOCYTE COAGULATION IN MANDUCA SEXTA

An inducible hemagglutinin termed M13, was purified from M. sexta hemolymph. M13 is a glucose-specific lectin which in addition to erythrocyte agglutination, can activate dedifferentiation of various hemocytes into a filamentous coagulation network. When lectin activity was inhibited with glucose or antiserum, neither erythrocyte agglutination or hemocyte coagulation occurred. When M13 was boiled or trypsin treated, hemocyte activation was lost, but erythrocyte agglutination remained. Hence M13 activity appears to be bimodal, possessing both a lectin activity and a hemocyte-coagulating activity.

Chemical and sensory effects of a Lactobacillus adjunct in Cheddar cheese

Cheddar cheeses were made using lactic starter and traditional methods with 0 (control), 102, or 103 CFU of Lactobacillus helveticus WSU19 added as an adjunct. Cheeses containing the higher concentration of the Lactobacillus adjunct received significantly higher flavor scores from the dairy judges than the other cheeses after 3 and 6 months aging (P < 0.05). The attribute panelists perceived both cheeses with the Lactobacillus adjunct as more crumbly and higher in oaky/ nutty flavors than control Cheddar cheeses (P < 0.05). Difference panelists could detect significant differences among the cheeses after 6 months aging (P < 0.05). Consumer acceptance scores were significantly higher (P < 0.05) for cheeses containing the adjunct after 6 months aging. Cheeses containing the Lactobacillus adjunct exhibited significantly greater rates of proteolysis than control Cheddar cheeses. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of cheeses with the Lactobacillus adjunct contained an 11 kD band after 3 and 6 months aging that was not present in control Cheddar cheeses. L. helveticus WSU19 enhances protein breakdown and increases oaky/nutty flavor and consumer acceptance in Cheddar cheeses.

Inhibition of active K+ transport in the tobacco hornworm (Manduca sexta) midgut after ingestion of Bacillus thuringiensis endotoxin

The effect of Bacillus thuringiensis endotoxin after ingestion on the K+ transport of Manduca sexta midgut is described. Direct measurements of short circuit current and transepithelial potential demonstrate that the transport is inhibited at the end of 1 hr with many of the insects exhibiting both negative short circuit current and transepithelial potential by the end of a 4-hr ingestion period.

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.

Improvement of sensory quality of reduced fat Cheddar cheese by a Lactobacillus adjunct

Abstract A Lactobacillus helveticus adjunct was added to 33% reduced fat Cheddar cheeses at concentrations of 10 2 or 10 3 CFU. Full fat and reduced fat control Cheddar cheeses were also made according to traditional methods. Reduced fat cheeses containing the Lactobacillus helveticus adjunct were significantly less bitter and contained greater oaky/nutty flavor intensity after three and six months aging than control reduced fat cheeses ( P ≤ 0.05). Trained dairy judge flavor scores for the reduced fat cheeses with the adjunct were not significantly different from flavor scores for the full fat Cheddar cheese ( P > 0.05). Acceptance scores after six months aging for reduced fat cheeses containing the adjunct culture were significantly higher than acceptance scores for the full fat Cheddar cheese ( P ≤ 0.05). Reduced fat cheeses containing Lactobacillus helveticus as an adjunct culture exhibited significantly greater rates of proteolysis than control cheeses ( P ≤ 0.05).

Ultrastructural changes in Staphylococcus aureus treated with pulsed electric fields / Cambios ultraestructurales en Staphylococcus aureus sometida a campos eléctricos pulsantes

Early stationary phase cells of Staphylococcus aureus were inoculated into a model food, simulated milk ultrafiltrate (SMUF) and subjected to 16, 32, and 64 pulses at electric field intensities of 20, 40 and 60 kV/cm at 13 °C. In addition temperatures of 20, 25 and 30 °C were also tested with 32 pulses and an electric field of 60 kV/cm. The temperature of the SMUF increased by 1-2 ° C at the end of the 64 pulses. Cells subjected to 64 pulses at 20, 40 and 60 kV/cm were observed for ultrastructural changes using scanning and transmission electron microscopy techniques. The cell surface was rough after treatment with electric field when observed by scanning electron microscopy (SEM). The cell wall was broken, and the cytoplasmic contents were leaking out of the cell after exposure to 64 pulses at 60 kV/cm when observed by transmission electron microscopy (TEM). The breaking of the cell wall is an indication of electro-mechanical breakdown of the cell. The increase in inactivation with an increase in the electric field strength can be related to the increase in the damage to the cells. Cells subjected to 32 pulses at 60 kV/cm and 13, 20 or 25 °C were compared microscopically with the untreated control cells. Cells subjected to heat treat ment (10 min, at 66 °C) were compared with electric field-treated and untreated control cells. Although important changes were observed in the protoplast, no cell wall breakdown was observed in heat-treated cells when compared to the electric field-treated cells. This result indi cates a different mechanism of inactivation of cells with heat treatment.

Differentiation of Lactobacillus helveticus strains using molecular typing methods

Abstract Three molecular typing methods: SDS-PAGE, amplification of the 16S-23S internal transcribed spacer (ITS) loci by the polymerase chain reaction (PCR), and random amplification of polymorphic DNA (RAPD), were examined for their ability to type sixteen strains of Lactobadllus helveticus . Strains were grouped based on SDS-PAGE and ITS amplification patterns. All sixteen strains were distinguished by RAPD. Molecular typing methods allow rapid and relatively inexpensive ways to strain type L. helveticus and potentially other dairy lactobacilli.