of blood will be decreased because of an increase of thickness of

plasma layer near the vessel wall. Our experiments do not contradict these ideas, but neither do they confirm them-another space

experiment would be needed to do so. The potential importance of this possibility is in the mystery of abnormal calcium metabolism in astronauts’ that was noted after the astronauts’ return from orbital flights, and which might be related to an increased viscosity of blood; this increase can affect perfusion of the bone, bone resorption and necrosis, and leaching of calcium out of the bone. It is known that abnormal calcium metabolism and rise in parathyroid hormone are related to increased blood viscosity and increased rigidity of the red cells.8 Both were suggested to have a connection with the space sickness.

Jones Lang Wootton, COSSA, QANTAS, Rebecca Cooper Medical Research Foundation, and Hawker de Havilland gave financial support. Team at Cape Canaveral included Mr B. Maguire, Mr S. Cherry, Mr H. Jedrzejczyk, Miss Amal Halou, and Mrs Barbara Jedrzejczyk. Department of Medicine, University of Sydney, Sydney, NSW 2006, Australia


1. Dintenfass L. Red cells under zero gravity. Lancet 1985; i: 747-48. 2. Dintenfass L, Osman P, Maguire B, Jedrzejczyk H. Experiment on aggregation of red cells under microgravity on STS 51-C. Adv Space Res 1986; 6: 81-84. 3. Drost-Hansen W, Singleton JL. Liquid asset. The Sciences 1989; Sept/Oct: 38-42. 4. Oka S. Effect of gravity on microcirculation. Biorheology 1986; 23: 534. 5. Oka S. Microcirculatory dysfunction in an environment of weightlessness. In: Manabe H, Zweifach BW, Messmer K, eds. Microcirculation in circulatory disorders. Berlin: Springer-Verlag, 1988: 63-67. 6. Takano Y. The flow rate of blood in an environment of weightlessness. Biorheology

1989; 26: 703-10 7. Hoffler SL. Cardiovascular studies of US space crews: an overview and perspectives. In Hwang NHC, Norman NA, eds. Cardiovascular flow dynamics and measurements. Baltimore. University Park Press, 1975: 335-63. 8. Dintenfass L, Ibels LS. Blood viscosity factors and occlusive arterial disease in renal transplant recipients. Nephron 1975; 15: 456-65.

Estimation of Legionella pneumophila virulence by nitroblue-tetrazolium reduction SIR,-Most of the methods for the estimation of the virulence of Legionella pneumophila, the causative agent of legionnaires’ disease, are expensive, time-consuming, or difficult to do because they require animal mortality estimates (LDso),’ tissue culture,2 or an assessment of their interaction with protozoa.3 We present a method based on the capacity of L pneumophila cells to reduce nitrobluetetrazolium (NET). The resulting formation of formazan as a blue-violet precipitate produces an increase in the optical density of the reaction mixture. The speed and sensitivity of the reaction is increased in the presence of either free-living ameobae or human polymorphonuclear leucocytes (PMNs) phagocytosing the legionellae. Six strains of L pneumophila serogroup I with estimates of their virulence, as measured in respect of LDso for guineapigs, were kindly provided by PHLS Centre for Applied Microbiology and Research, Porton Down. Of these strains, four were clinical isolates isolated from environments associated with the of legionnaires’ disease. In our laboratory they were passaged twice on buffered charcoal yeast extract agar,’ harvested, and stored at - 70°C on glass beads. Acanthamoeba polyphaga, a free-living aquatic amoeba, was obtained from the Public Health Laboratory Service, Bath, and maintained in axenic culture.s PMNs were isolated from venous blood. Microtitre plates were used to study the reduction of NBT by legionella cells alone and in the presence of either amoebae or PMNs. In each of the three experiments, heat-killed legionellae were placed in the first six wells as a control. The six wells of the next six columns contained the viable bacterial strains. In the first experiment the effect of bacteria alone (50 ul at 5 x 107/ml) on NET (50 III at 1 mg/ml) was tested in the presence of 50 (il Page’s amoebal saline (PAS).’ In the second experiment, amoebae at a concentration of 510’’/ml were included in PAS. For the control wells, the amoebal suspension was pre-incubated for 2 h at 37°C

and two



Reduction of NBT as a function of logarithm of LDso after 3 h incubation with L pneumophila alone CÁ.) and in presence of A polyphaga (.) and PMNs (8).

Correlation coefficients —094, —090, and - 0-95, respectively Each mean of six readings. SEMs ranged from 6.8 x 10-5

data point represents to 1.0x10-3

with 50 µl of 10 mmol/1 iodoacetamide to inhibit phagocytosis. After this time the amoebae settled to the bottom of the wells and the supernatant solution was removed and replaced with NBT, PAS, and heat-inactivated bacteria in the quantities and concentrations indicated. The final experiment was identical to the second except that PMNs at a concentration of 5x 106 /rnl in Hank’s balanced salt solution were used instead of amoebae. In all experiments the microtitre plates were incubated at 37°C and the optical density of the reaction mixtures were measured at timed intervals with an ELISA reader fitted with a 570 nm filter. As seen in the figure, after 3 h incubation NBT reduction seemed to be proportional to the logarithm of the LD so of the legionella strains. Reduction was enhanced in the presence of both amoebae and PMNs, although even in their absence, a good correlation was obtained between virulence and optical density. The results indicate that the methods we have outlined afford a potentially useful test for legionella virulence. The simplest system, consisting only of legionella cells and NET, could be readily used in most routine diagnostic laboratories. Results can be obtained in as little as 3 h, and if amoebae are used to increase the reaction rate the method does not require PMN isolation from blood. We thank the Hariri Foundation for financial support, Dr J. V. Lee and Dr A. A. West, CAMR Porton Down, for assistance, and Mr S. Kelvington (PHLS Bath) and Dr D. Warhurst (LSHTM) for providing cultures of amoebae. Microbial

Physiology Research Group, King’s College, London W8 7AH, UK

1. Baskerville A,



Fitzgeorge PB, Brosten M, et al. Experimental transmission of disease by exposure to aerosols of Legionella pneumophila. Lancet

1981; ii: 1389-90. RE, Lee SHS, Haldane D, et al. Plaque assay for virulent Legionella pneumophila. J Clin Microbiol 1989; 27: 1961-64. 3. Fields BS, Barbaree JM, Emmett M, et al. Comparison of guinea pig and protozoan models for determining virulence of Legionella species. Infect Immun 1986; 53: 2 Fernandez


Improved semiselective medium for isolation of L pneumophila from contaminated clinical and environmental specimens. J Clin Microbiol 1981; 14: 298-305. 5. Page FC An illustrated key to freshwater and soil amoebae with notes on cultivation and ecology. Ambleside: Freshwater Biological Association, 1976.

4. Edelstein PH.

Estimation of Legionella pneumophila virulence by nitroblue-tetrazolium reduction.

240 rate of blood will be decreased because of an increase of thickness of plasma layer near the vessel wall. Our experiments do not contradict the...
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