Alastair C. Wardlaw

Echinochrome-A as a bactericidal substance in the coelomic fluid of Echinus esculentus (L.)

Abstract 1. 1. The concentrations of echinochrome-A in coelomic fluid from healthy specimens of the sea urchin Echinus esculentus (L.) ranged from 3 to 60 μg/ml, with a geometric mean of 14 μg/ml. 2. 2. Echinochrome-A (50 μg/ml) dissolved in sea water, with the aid of mammalian proteins as dispersants, was bactericidal or bacteriostatic towards six out of seven strains of marine gram-negative and gram-positive bacteria. 3. 3. Echinochrome-A is suggested as a major factor in the bactericidal activity of coelomic fluid from E. esculentus .

Bactericidal activity of coelomic fluid from the sea urchin Echinus Esculentus

Abstract Coelomic fluid from the common British sea urchin Echinus esculentus was shown to kill in vitro a black-colonied, agar-digesting marine Pseudomonas sp. (strain No. 111). The bactericidal reaction, which was monitored by viability counting on marine agar plates, took 48 hr to reach full expression. An incubation temperature of 4°C gave the most complete and consistent killing. Clotting of the coelomocytes did not influence bactericidal activity. The vast majority of nearly 200 specimens of E. esculentus from the Clyde Sea area of Scotland yielded coelomic fluid with bactericidal activity, and there was no apparent seasonal variation. Activity was localized in the coelomocytes and could be obtained in cell-free extracts by ultrasonic disruption of these cells. The bactericidal activity in these extracts was lost during dialysis but was only partially destroyed by heating for 30 min at 56° or 100°C.

Bactericidal action of high-power Nd:YAG laser light on Escherichia coli in saline suspension

Infra‐red light (1064 nm) from a high‐power Nd:YAG laser caused more than 90% loss of viability of Escherichia coli during exposures that raised the temperature of PBS suspensions of the bacteria to 50 °C in a thermocouple‐equipped cuvette. In contrast, there was minimal loss of viability after heating the same suspensions to 50 °C in a water‐bath, or in a PCR thermal cycler. The mechanism of laser killing at 50 °C was explored by differential scanning calorimetry, by laser treatment of transparent and turbid bacterial suspensions, and by optical absorbancy studies of E. coli suspensions at 1064 nm. Taken together, the data suggested that the bactericidal action of Nd:YAG laser light at 50 °C was due partly to thermal heating and partly to an additional, as yet undefined, mechanism. Scanning electron microscopy revealed localized areas of surface damage on laser‐exposed E. coli cells.

Inactivation of bacteria and yeasts on agar surfaces with high power Nd : YAG laser light

G.D. WARD, I.A. WATSON, D.E.S. STEWART‐TULL, A.C. WARDLAW AND C.R. CHATWIN. 1996. Near infrared light from a high‐powered, 1064 nm, Neodymium : Yttrium Aluminium Garnet (Nd : YAG) laser killed a variety of Gram‐positive and Gramnegative bacteria and two yeasts, lawned on nutrient agar plates. A beam (crosssectional area, 1.65 cm2) of laser light was delivered in 10 J, 8 ms pulses at 10 Hz, in a series of exposure times. For each microbial species, a dose/response curve was obtained of area of inactivation vs energy density (J cm−2). The energy density that gave an inactivation area (IA) equal to 50% of the beam area was designated the IA50‐value and was plotted together with its 95% confidence limits. Average IA50‐values were all within a threefold range and varied from 1768 J cm−2 for Serratia marcescens to 4489 J cm−2 for vegetative cells of Bacillus stearothermophilus. There were no systematic differences in sensitivity attributable to cell shape, size, pigmentation or Gram reaction. At the lowest energy densities where inactivation was achieved for the majority of organisms (around 2000 J cm−2), no effect was observed on the nutrient agar surface, but as the energy density was increased, a depression in the agar surface was formed, followed by localized melting of the agar.

Responses to Bordetella pertussis mutant strains and to vaccination in the coughing rat model of pertussis.

Phase I strains 18-323, Tohama and L-84 of Bordetella pertussis produced paroxysmal coughing when encased in agarose beads and administered intrabronchially to adult Sprague-Dawley rats. In contrast, the Phase IV variant of strain L-84 was inactive in cough induction, as was strain BP 357, a transposon-insertion mutant which is deficient only in pertussis toxin (PT). Strain BPM 1809, which lacks only the heat-labile toxin, was similar to the unmodified Phase I strains for cough induction, indicating that this toxin is not needed to induce coughing. B. parapertussis also was inactive as a cough inducer. These results indicate that PT, present in Phase I strains of B. pertussis, and absent from Phase IV strains, strain BP 357 and B. parapertussis, is essential for the induction of paroxysmal coughing in this rat model of whooping cough. Prior injection of DTP (whole-cell) vaccine greatly reduced the incidence of coughing in rats challenged subsequently with Phase I B. pertussis. Serological responses were monitored after intrabronchial infection with the various bacterial strains and after vaccination and challenge. The PT-positive or -negative status of the strains in vivo was confirmed by the appropriate presence or absence of anti-PT IgG in the convalescent sera.

Cough production, leucocytosis and serology of rats infected intrabronchially with Bordetella pertussis.

Adult Sprague-Dawley rats infected intrabronchially with Bordetella pertussis strain 18-323 encased in agarose beads (BP-beads), developed a paroxysmal cough and leucocytosis, both of which peaked at around day 10. When animals were exposed to ether for 2 min after delivery of the beads, there was an enhancement of the number of subsequent coughing episodes. Inclusion of carrageenan in the beads also enhanced coughing. Control rats, given sterile beads or left untreated, showed only a low level of coughing or no coughing, depending upon their source. Rats challenged by the same route with heat-killed B. pertussis in beads, or with live organisms in suspension (without beads) showed no cough induction or leucocytosis. However, intranasal delivery of B. pertussis suspension gave rise to a moderate amount of coughing and leucocytosis. Serum IgG responses to B. pertussis antigens were greatest in rats infected with BP-beads and antibodies against both pertussis toxin and filamentous haemagglutinin were detected. Since the rat is the only conveniently accessible laboratory animal species in which B. pertussis induces an intermittent paroxysmal cough, as in man, it merits further study for determining the mechanisms of pathogenesis and immunity in pertussis.

Iodination of a mixture of soluble proteins by the [125I]-lactoperoxidase technique

A mixture of 4 purified proteins at equal concentrations was radiolabelled with [125I] and lactoperoxidase and analysed by SDS-polyacrylamide gel electrophoresis. Bovine serum albumin took up 9 times as much label as ovalbumin or lysozyme and 3.3 times as much as α− chymotrypsinogen A. These results suggest that when applying the [125I]- lactoperoxidase technique to labelling unknown mixtures of proteins, such as may exist on the surfaces of cells, caution should be exercised in interpreting the degree of labelling of particular proteins in terms only of surface abundance or accessibility.

Effect of hyperreactivity to endotoxin on the toxicity of pertussis vaccine and pertussis toxin in mice

In mice, greatly enhanced susceptibility to the lethal toxicity of whole-cell pertussis vaccine (PV) was produced by agents known to induce hypersusceptibility to endotoxin (LPS). The decreases in LD50 were 100-fold, 125-fold and 16-fold with galactosamine (GalN), actinomycin D (AcD) and lead acetate (PbAc) respectively and the animals died within 1-2 days. However, these decreases were less than those observed with extracted E. coli LPS, the LD50 of which was reduced approximately 500-fold, 800-fold and 50-fold respectively by these agents. In control mice, without drugs, the main lethal factor in the PV used here seemed to be pertussis toxin (PT), since deaths occurred at 3-5 days after injection, and heating the vaccine at 80 degrees C for 30 min raised the LD50 from 4 to greater than 6 single human doses (SHD) per mouse. In GalN and PbAc-treated mice, the toxicity of PV can be explained by its LPS content in view of the failure of heating at 80 degrees C to reduce toxicity. However, in AcD-treated mice, the 80 degrees C heated vaccine was threefold less toxic than the unheated material, suggesting a contribution of PT to vaccine toxicity in these animals. Indeed the toxicity of PT was increased by AcD. The possible bearing of these observations on children who appear to show serious adverse reactions to PV is discussed. Two acellular vaccines were devoid of lethal toxicity in either normal mice or in mice treated with any of the three drugs.

Time-course of infection and responses in a coughing rat model of pertussis

Adult female Sprague-Dawley rats were challenged intrabronchially with Bordetella pertussis strain 18-323 embedded in fine agarose beads and the time-course of infection and other events was determined. There was a steady decline in the numbers of B. pertussis recovered from the rat lungs, with clearance of the infection in most animals by day 12. Leucocytosis, lung inflammation and an increase in total serum IgE in the rats as a result of the challenge were highest around day 10, which was coincident with the highest incidence of coughing in such animals. IgG and IgA antibodies to the B. pertussis antigens pertussis toxin and filamentous haemagglutinin were not detected until after this period. The coughing rat model of pertussis resembles the human disease in the relationship between the time course of infection and cough production.

Responses to acellular pertussis vaccines and component antigens in a coughing-rat model of pertussis

Two acellular pertussis vaccines (SmithKline Beecham 3-component and Connaught 5-component), and a whole-cell pertussis vaccine (Evans), were similarly protective against paroxysmal coughing and leukocytosis in a coughing-rat model of pertussis. A two-dose immunization schedule was followed by sublethal intrabronchial challenge with Bordetella pertussis strain 18-323, encased in fine agarose beads, and the coughing monitored by sound-activated tape recorders. Pertussis toxoid by itself gave some protection against coughing, but lower than that afforded by the vaccines, despite inducing a higher serum anti-PT titre. The other component antigens, given individually, failed to protect against coughing although inducing antibodies. Immunization with the whole-cell and acellular vaccines and with their component antigens, as well as challenge with B. pertussis, caused significant elevation of total serum IgE antibodies. Antigen-specific IgG and IgA were detected in tracheobronchial washings from rats recovering from B. pertussis challenge, but vaccination prior to challenge had little influence on these antibody levels. The coughing-rat model of pertussis may be useful for the comparative testing of different formulations of pertussis vaccines before trials in human infants.