TRANSACTING

Human

OF THE ROYAL SOCIETY OF TROPICAL. MEDICINE

urine stimulates

growth

AND HYGIENE (1991) 85, 477-479

of Leishmania

477

in vitro

Howard’, Mark M. Pharoah’, Frank Ashall’ and Michael A. Miles’ ‘Departmentof Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK; ‘Department of Pure and Applied Biology, Imperial Collegeof Scienceand Technology,Prince ConsortRoad, London, SW7 2AZ, UK M. Keith

Abstract

Enhancementof trypanosomatidmetacyclogenesis by insecturine componentsled us to test the effect of human urine as a culture additive. The addition of l-S% urine to Schneider’sDrosophilamedium containing 10%foetal calf serumenhancedthe growth of 11Leishmaniastrainsrepresenting8 different taxonomic groups. Cell division wasstimulatedin cultures with non-dividing organisms.Peak cell density was increased, as was the efficiency with which L. donovanicould be isolated from infected hamsters. Preliminary work suggested that the modified medium would be useful for field isolation of L. donovaniand L. braziliensis.The active nutrients or growth factors are not known.

serum (FCS) and PGAB (200 units/ml penicillin, 200 yglrnl streptomycin sulphate, 2 mu pyruvate, 2 mM glutamine)plus 5 pg/ml haemin).L. donovani amastigotes of the HU3 strain (MHOM/ET/67/HU3) were isolated from infected hamstersas described previously (HOWARD et al., 1987). Urine from 4 healthy Caucasianvolunteers(3 male, 1 female) was prefiltered through Whatman 3 MM 10;

Introduction

We have previously usedsodiumurate, uric acid, and cysteic acid, known componentsof insecturine, in a culture medium for the rapid production in vitro of a high proportion of Leishmaniadonovanimetacyclic promastigotes(HOWARD et al., 1987).This led to the suggestionthat human urine might provide a readily available substitute medium constituent for the enrichment of trypanosomatid growth and perhapsfor the induction of metacyclogenesis. Here we show that human urine stimulatesthe multiplication of Leishmaniain Schneider’sDrosophilamedium (SDM; HENDRICKS et al., 1987) and reduces the number of organismsrequired to establisha primary culture. Materials

and Methods

The 11 strainsof Leishmaniausedare listed in the Table. Promastigoteswere maintainedat 23°Cby passage in SDM (Gibco) supplementedwith 10% foetal calf Table.

Leishmania

strains

Stock

Zymodeme

MHOMiBRl74lI’P75

LON LON LON LON

49 49 49 46

Disease Type visceral visceral visceral visceral

LON LON LON LON

1 64 4 25

C”faneo”S cutaneous c”taneous cutaneous

LON

123 cutaneous diffuse

MHOM/TNI80/IPTl MCANiTNl78lLEM78 MHOMIET/67/HU3 MHOMlSU173I5ASKH MPSMlSAl83IJISH224 MHOMISAl84IJISH118 MRHOICNI6OIGERBILLI MHOMlBW7SlM2903 MHOMlBRI75iM4147 MHOMNEI57ILLI

cutaneous

c”taneouS

Organism L. chagasi L. infanturn

L. infanmm L. donovani se?Lm lam L. major L. arabica L. major L. gerbilli L. brazilimsis L. guyanemir L. pifanoi

r 0

I

2

4

6 URINE

8

10

CONCENTRATION

12

14

16

(%I

Fig. 1. An example of the stimulation of promastigote division by human urine. Stationary-phase Leishmania dmmvani (HU3) pmmastigotes were sub-cultured into fresh Schneider’s DrosophiZa medium with 10% foetal calf serum at 5x10’ cells/ml in the presence of varying concentrations of human urine. Cell densities were determined on day 6 of sub-culture. The cultures were performed in duplicate: A, with human urine, q , control (phosphate-buffered saline).

t

\ 0

1.0

0.5 1%

LOG

URINE

CONCENTRATION

I

1.5 (%I

2.0-F 100%

Fig. 2. An example of Leirhmania doncruani (HU3) promastigote growth curves in Schneider’s Drosophila medium with 10% foetal calf serum and medium containing increasing concentrations of human urine. Cultures were performed in duplicate: 0, day 2 of culture; n , day 4; A, day 6; 0, day 8; Cl, cell density, at day 8, in a control culture with no urine added (C).

paper, filter-sterilized (0.2 urn), and stored at 4°C with PGAB. Growth curveswere initiated by suspendingpromastigotesfrom stationaryphaseprimary culturesat up to 5x 106/ml(seeResults)in FCS and 10x PGAB plus haemin (5 uglml). Aliquot suspensions (100 pl) of promastigoteswere dispensedinto Leighton tubes and the volumesweremadeup to 1 ml with 900 ul of SDM containing various concentrationsof urine, to give cultures of 5X 10’ organisms/ml.Cultures for growth curve comparisonswere incubated at 28°C. All organismcounts were by haemocytometer. Results Urine from eachdonor enhancedthe growth of L. dunovaniin axenic culture. Growth enhancementwas alsonoted with Ttypanosomacruzi, but this hasnot been studied in detail. The comparativegrowth of suchculturesat day6 in the presenceof between0 and 32%of humanurine or with corresponding proportions of phosphate-buffered saline,pH 7.2, (PBS) is shownin Fig. 1; the inclusion of 1% of human urine in the cultures induced promastigotesto divide (Fig. 1). Both the rate of cell division and the peak cell density achieved before the stationary phasewere dependenton the concentrationof urine addedto the medium.

AMASTIGOTE (PER

‘CELL ML)

(DAY

DENSITY 01

Fig. 3. The establishment of primary Leishmanin donmuni (HU3) ctdNres in medium containing urine. L. donovani amastigotes, freshly isolated from hamster spleen, were seeded at a range of cell densities into Schneider’s Drosophila medium with 10% foetal calf serum (duplicate cultures). 0, Cul~res with 2% human urine; 0, cultures without human urine.

Growth curvesfor a seriesof cultureswith increasing concentrationsof addedurine areshownin Fig. 2. Promastigotecounts were performed on days 2, 4, 6 and 8. Maximal stimulation of cell division was obtained by incorporating between1 and 10%urine. Peak cell densitiesreachedapproximately lo8 organisms/ml with l-2% urine, whereasthe density in urine-free medium did not usually exceed 3~ 10’ organisms/mland was frequently lower (Fig. 1 and HOWARD,

1989).

There were slight variations in the growth curves producedby urine samplesfrom different donors(not shown).Sequentialsamplesof diluted urine produced by a donor after increasedfluid intake (3 litres of water) were lesseffective in stimulatingthe growth of Leishmuniu(data not shown).The marked effect of urine on the easeof establishingL. donovaniHU3 cultures with amastigotesfreshly isolatedfrom infected hamster spleenis shown in Fig. 3. In the absenceof urine cultures were initiated with amastigotesat cell densitiesof lo4 cells/ml,whereaswith 2% urine in the mediumcultures could be initiated with 10 organisms/ml.

479

Discussion

Supplementationwith human urine has greatly aidedthe growth of severalLeishmaniustrainsstudied in this laboratory (BISHOP& MILES, 1987;HOWARDet al., 1991; BISHOP81AKINSEHINRJA,1989)and 2% urine has been used to induce proliferation in non-dividing cultures of both Old and New World Leishmaniaspecies,including agentsof both visceral and cutaneousdisease.Primary isolationin culture of Leishmuniapromastigotesfrom patients, reservoir hosts,or sandflyvectors can be difficult (GITHURE et al., 1984; LAINSONet al., 1985; PETERSON, 1988). The urine additive may also prove to be of routine value for the primary culture of new isolates.Successful growth is dependentupon the initial organism deniity, regardiess of which medium is used (LEMESREet al.. 1988): substantialreduction of the density required to i&ate growth (Fig. 3) should enhanceisolationof parasitesfrom tissues,and may benefit the diagnosisof leishmaniasis,especiallyof cutaneousor a&pica1infections (MEI~A~TU et-al., 1989; ZELED~N et al.. 1989;SANTRICHet ul., 19901. The isolationof pan&es into culture hasthe advantageof both speedand cost, comparedwith isolation into hamsters(CUBA CUBA et al., 1986; WEIGLE et al., 1987).Urine-supplementedSDM plus 10% FCS has beenusedto enhancethe isolationof L. infanturn from human liver biopsiesand bone marrow aspirates,of L. chugusi from canine splenic and shin lesion aspirates,andof L. bruziliensis from humancutaneous and mucocutaneouslesions(Howard et.ul., unpublished observations). The factor or factors in the urine that stimulate(s) growth of Lekhmuniu hasor havenot beenidentified. Although human urine contains epidermal growth factor (EGF) and trypanosomesare known to possess a receptorfor this factor (HIDE et al:, 1989),we have been unable to detect any mitogemcactivity due to purified EGF alone (data not shown). Whatever the active componentmay be.,it is clearthat humanurine is a cheapandreadily avarlablegrowth supplementfor the culture of all speciesof Leishmuniu. Acknowledgements These experimentswere part of an initial training programme for M.M.P. and were financed by the Medical Research Council and the Wellcome Trust. References

Bishop, R. P. & Akinsehinwa, F. (1989). Characterization of Leishmania donovani stocks by genomic DNA heterogeneity and molecular karyotypc. Trunsuctions of the ~,~&alociety of Tropical Medicine and Hygiene, 83, “_,

-“*

.

.

Bishop, R. P. & Miles, M. A. (1987). Chromosome size polymorphisms of Leishmania donovani. Molecular and Biochemical Parasitology, Cuba Cuba, C. A., Netto,

24, 263-272.

E. M., Marsden, P. D., Rosa, A. de C, Llanos Cuentas, E. A. & Costa, J. L. M. (1986).

Cultivation of Leishmunia braeihensis braziliensis from skin ulcers in man under field conditions. Trunsuczions of the Royal AFM57 .