Mammalian bile promotes growth of Giardia lamblia in axenic culture

TKAXSAC~IONS

01’ THE ROYAL SOCIETY OP TROPICAL MEUKINE

Mammalia’n

bile promotes M.

Division

of Geographic

Giardia

J.

G.

Medicine,

AND Hucrrr~,

growth

FARTHING,

S. R.

VOL. 77, No. 4, 467.469 (1983:

of Giardia VARON

AND

lamblia

467

in axenic

culture

G. T. KEUSCH

Tufts University School of Medicine, MA 02111, USA

136 Harrison

Avenue,

Boston,

Summary lamblia wasadaptedto grow in modifiedDiamond’sTYI-S culture mediumwith a mean

generationtime of 12.0 -t 1.1 hours. Addition of bovine or porcinebile to this mediumdramatically promotedgrowth with markedreduction in generationtime. The shortestmeangenerationtime (7.5 hours) was obtained with fresh bovine bile and to our knowledgeis the lowest reported time for axenicallvcultivated G. Zamblia. Trophozoite numberswereincreasedby a factor of 3.4 after 48 hours cultivatidn. Generationtime lengthenedas bile concentrationwasin&eased,the effect being most markedwith commercialbile nrenarations.We considerthat this bile-sunnlemented TYI-S medium will support growth more coisisiently than the usualGiardia culture r;l’ediumTPS-1, and is also cheaper.This trophic effect of bile on Giardia growth may partly explainwhy Giardia trophozoitesare found mainly in the duodenum and proximal jejunum where bile is plentiful. Introduction

Since the establishmentof methods for axenic cultivation of Giardia lamblia knowledgeabout parasitemetabolism(WEINBACHet al., 1980;JARROLLet al., 1981), surface membrane(HILL et al., 1981; FARTHINGet al., 1982),cytoskeleton(HOLBERTON & WARD, 1981), and somegrowth factors (GILLIN & DIAMOND, 1980, 1981),has increasedsubstantially. Further clarification of the growth requirementsin culture may improve our understandingof parasite biology and is critical for developmentof a defined culture medium.The first mediumusedsuccessfully, HSP-1, describedby MEYER(1976)supportedgrowth of Giardia but inconsistently, a failing which was attributed to batch variations in human serum, an essentialcomponent of the medium (VISVESVARA, 1980).VISVESVARA(1980) then modified Diamond’s TPS-1 Entamoeba histolytica medium to which he successfullyadaptedG. lamblia. A major constituent of the TPS-1 mediumis Panmede,a papaindigestof ox liver (Paines& Barnes, Ltd., Greenford, Middlesex, England). We have recently had difficulty in obtaining lots of Diamond’s TPS-1 medium base broth (North American Biologicals,Miami, Florida, USA) which would reliably support growth of G. lamblia and have therefore soughtfor other culture media. Becausedifferent lots of Panmedevaried considerablyin their ability to support growth of E. histolytica, DIAMOND et al. (1978) developed an alternative, the TYI-S medium which dependson Trypticase and yeast extract (BiosatePeptone,BBL, Cockeysville,Maryland, USA) rather than Panmede. Although G. lamblia multiplied in a slightly modified versionof this medium,generationtimeswerelonger than with satisfactorybatchesof Panmede-containing TPS-1. We reasonedthat constituentswhich might be present in the ox liver digest but not in the casein hydrolysate and yeast extract might alter generation time in TYI-S medium. We report here our observations on the stimulatory effectsof mammalianbile on the growth of G. lamblia in axenic culture. Method TYI-S medium nl. (1978) with

was prepared the following

as described by DIAMOND ef minor modifications: (1)

Diamond’s TPS-1 40X vitamin solution (North American Bioloeical. Miami, Florida. USA), ,, 25 ml/litre renlaced the “itar&-Tween 80 solution, and (2) the medium was not autoclaved but filter-sterilized by passage through a 0.22 pm filter (Millipore Corporation, Bedford, Mass., USA). The TPS-1 medium was prepared using Diamond’s TPS-1 medium base broth, 10% heat-inactivated bovine serum (Biofluids Inc., Rockville, Maryland, USA), and supplemented with cysteine and TPS-1 medium vitamin solution as described previously (FARTHING et al., 1982). G. lumblia trophozoites, Portland 1 strain, obtained initially from Dr. L. Diamond and Dr. F. Gillin (Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland) were grown in stationary tube culture as described previously (FARTHING et al., 1982). Growth of G. ZumbZiu in axenic culture was assessed by serial determinations of the concentration of trophozoites in each tube in order to calculate the generation time (VISVESVARA, 1980; FARTHING et al.! 1982). Initially growth curves were constructed by determming trophozoite concentration at 0, 24, 48, 72 and 96 hours. Because growth was consistently logarithmic in native and bile-supplemented media up to 48 hours, subsequent experiments were performed over a 48-hour period from which a mean generation time was determined. TYI-S medium was supplemented with the following areparations: (1) TPS-1 medium (O-100% volivol) usine base broth (lot No. iO2023) which we knew suppoited growth poorly, (2) dried commercial bovine and porcine bile (Sigma Chenucal Company, St. Louis, Missouri, USA), from 0 to 4 gilitre, (3) fresh bovine and porcine gall-blader bile and (4) lyophilized fresh bile as in (3) that had been reconstituted. Fresh bile was obtained from local Boston abattoirs. Gall-bladders were excised immediately after death and their contents allowed to drain into a sterile container kept on ice. Bile was centrifuged at 1000 g for 30 min, the supernatant was passed sequentially through Whatman No. 4 and then No. 1 filter papers and finally was filter-sterilized through a 0.2 Km nitrocellulose membrane (Sybron/Nalge, Rochester, New York, USA). Total solids in the filtered bile were determined by desiccation of a known volume. Experiments were performed in triplicate and differences in G. lumbliu growth between native and supplemented media were examined by the Student’s t test.

Results G. lnmblia

consistentlv grew in UnsuDDlemented

TYI-S medium with 1.1 hours (meant

a meanieneration

t&i

of 12.0 &

SEM. n = 32X Growth in TPS-1 medium(l%amond’sbasi broth, iot No. 202023)was

468

GROWTH

OF

G. lamblia

(cl)

c--a

bovine porcine

bile bile

61 -1%

861 L 0

1.0

Bile concentration,

”

1 4.0

2.0

g/L

Fig. 1. Giardra lamblio trophozoite generation times in native and bile-supplemented TYI-S culture medium; (a) commercial dried bovine and porcine bile, (b) fresh bile and (c) lyophilized fresh bile. dramatically slowed, with a mean generation time of 43.8 f 18 hours (n = 7). In contrast, an earlier batch of base broth, lot No. 000839) had produced generation times of about 10 hours (FAR~HING et al., 1982), all other constituents of the medium being the same. Supplementation with TPS-I medium Mixing experiments were then performed to discover if TPS-1 medium contained a growth factor which was absent from, or present only in low concentration in TYI-S medium. Supplementation of TYI-S with TPS-1 (10% vol/vol) decreased generation time from 10.7 +- 0.3 hours (native) to 9.1 & 0.1 hours (PtO.05). Further increments in the proportion of TPS-1 resulted in a progressive slowing of generation time (100% TPS-1, 29.8 + 3.7 hours). Supplementation with mammalian Addition of low concentration bovine and porcine bile to TYI-S reduced generation time (Fig. 1)

bile of dried and fresh medium markedly and concomitantly

IN

AXENIC

CULTURE

increased trophozoite yield (Table I). With the exception of commercially produced dried bovine bile, all preparations had their peak effect at a concentration of 0.1 gllitre (Table I). Further increments in bile concentration resulted in a progressive prolongation of generation time, particularly evident with the commercial bile preparations (Fig. la) when values eventually exceeded control generation times. Hence, both the stimulatory and the inhibitory effects of bile on trophozoite growth are concentrationdependent. The lowest mean generation time (7.5 ri: O-2 hours) was obtained with fresh bovine bile which reoresented a 30% reduction compared to control (Fig. *lb). This improvement in generation time increased mean trophozoite yield by a factor of 3.4. Supplementation of TYS-1 medium with bile did not alter the pH of the culture medium. No differences were found by light and scanning electron microscopy between trophozoites grown in TPS-1 and bile-supplemented TYI-S medium. Discussion These experiments indicate that bovine and porcine bile, obtained either fresh from the gall-bladder or dry from commercial sources, reduce generation time and thus promote growth of G. lamblia in TYI-S culture medium. A previous report indicated that Giardia arew uoorlv in native TYI-S medium (GILLIN & DIAMOND, 1981). Fresh bovine bile suppiementation, however, produced a mean generation time as low as 7.5 hours, which to our knowledge, is the shortest generation time ever reported for G. lamblia in axenic culture. This stimulatory effect of bile was clearly concentration dependent. At higher bile concentrations growth promotion diminished and! with further increases, growth was eventually inhibited to the extent that generation times were greater than unsupplemented controls. This was particularly evident in experiments involving the dried commercial bile preparations. The mechanism by which bile stimulates parasite growth is not clear from these experiments, although it was certainly not sim~lv due to an alteration in the pH of the sup$ementedmedia. Bile is a complex fluid of manv diverse constituents. Biliarv-, lioids. mainlv > cholesterol and lecithin, are potential candidates since there is evidence that Giardia does not synthesize its own lipids de novo, but relies on absorption from the external milieu (TARROLL et al.. 1981). Whether an essential lipid mo:ety is absent from TYI-S or present at critically low concentrations is currently unknown. Alternative candidate growth-promoting constituents of bile are the bile acids. Absorption of lipids bv the parasite is likely to be facilitatedby micelle formation in the nresence of bile acids (CAREY & SMALL. 1972). a step- which may be of snecial imnortance to an organism which is-unable to synthesize its own lipids. The view is supported by the report of HEGNER & ESKRIDGE in 1937, in which feeding rats with the trihydroxy bile salt sodium glycocholate increased the number of intraluminal Giardia in infected animals. Biliary diversion studies in the rat also support the importance of bile in maintaining Giardia infection (BEMRICK, 1963), although interpretation of those experiments is difficult since there was marked structural deterioration in the intestinal mucosa of

M.

Table

I-Giardia

lamblia

trophozoite

concentration

FARTHING

after 48 h incubation

Porcine Fresh Bovine Porcine

with increasing

Concentration

(X

concentrations

of mammalian

bile

105/ml, mean f SE)

0.1

0.2

0.4

0.8

I.0

2.0

4.0

6.7 io.4

11.4 f0.8

12.6 k1.5

13.2 k1.5

14.2 kO.6

15.1 kO.6

12.2 51.1

7.8 50.6

3.7 kO.8

5.2 f0.6

7.7 20.8

11.9 to.1

10.5 20.6

9.6 fl.2

4.2 to.7

1.9 +0.2

0.07 f0.03

0

5.4 to.2

15.7 21.9

18.1 +1.3

15.1 k2.5

14.5 f0.8

11.4 kO.6

5.3 so.9

3.3 kO.1

4.4 f0.3

4.9 f0.6

13.6 k1.4

13.7 to.6

12.4 +0.9

12.9 +0.8

11.1 k1.3

11.3 kO.3

7.8 20.9

7.4 k1.0

4.1 +0.3

10.1 40.7

11.0 kO.5

11.0 +0.3

10.7 +0.6

10.4 f0.7

4.3 kO.5

3.2 f0.4

4.2 kO.6

4.1 kO.3

8.5 +1.1

10.3 11.4

10.1 If-O.8

10.0 TO.7

5.9 f0.7

7.7 fO.2

9.7 20.6

8.7 t1.3

0

Dried (Commercial) Bovine

469

t?t al.

0.05

gilitre

Porcine

G.

Trophozoite

Bile Supplement

Lyophilized Bovine

J.

(Fresh)

operated control animals. The bile acid hypothesis might also explain the inhibitory effects of bile since at higher concentrations, conjugated and, particularly, deconjugated bile acids are disruptive to cells. The deleterious effects of some batches of Panmedecontaining TPS-1 medium could, perhaps, be due to a preponderance of deconjugated bile acids resulting from bacterial contamination of the bile. Our observation that mammalian bile promotes growth of G. lamblia in vitro has several important implications. First, the possibility of cultivating the organism in TYI-S medium is economically advantageous and may prove to be more reliable than growth in TPS-I medium. For bile supplementation we recommend using either commercially prepared dried bovine bile (0.8 g/l) or fresh bovine bile which can be centrifuged, filtered and lyophilized or stored frozen in appropriate aliquots at -20°C. Second, this study suggests the possibility that bile may be an important intraluminal determinant of the site of intestinal colonization by Giardia. Except in rare circumstances when infection is overwhelming, Giardia trophozoites are usually restricted to the duodenum and proximal jejunum (OLVEDA et al., 1982), the region where bile concentrations are greatest. It remains to be seen whether this observation can be used in the future to gain a therapeutic advantage over the parasite. Acknowledgements

MJGF is a Wellcome Tropical Lecturer. This work was supported by a grant in Geographic Medicine from the Rockefeller Foundation. References

Bemrick, W. J. (1963). The effect of bile flow on Giardia duodenalis race Simoni in the intestine of a laboratory strain of Rattus norvegicus. Journal of Parasitology, 49, 956-959. Carey, M. C. & Small, D. M. (1972). Micelle formation by bile salts. Physical-chemical and thermodynamic considerations. Archives of Internal Medicine, 130, 506-527.

Diamond, L. S., Harlow, D. R. & Cunnick, C. C. (1978). A new medium for the axenic cultivation of Entamoeba histolytica and other entamoeba. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72, 431-432. Farthing, M. J. G., Pereira, M. E. A. & Keusch, G. T. (1982). The cell surface lectin of Giardia lamblia. Clinical Research, 30, 496 A. Farthing, M. J. G., Pereira, M. E. A. & Keusch, G. T. (1982). Giardia lamblia: Evaluation of roller bottle cultivation. Experimental Parasitology, (in press). Gillin, F. D. & Diamond, L. S. (1980). Clonal growth of Giardia lamblia trophozoites in a semi-solid agarose medium. Journal of Parasitology, 66, 350-352. Gillin, F. D. & Diamond, L. S. (1981). Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents. Experimental Parasitology, 51, 382-391. Hegner, R. W. & Eskridge, L. (1937). The influence of bile salts on Giardia infection in rats. American Journal of Hygiene, 26, 186-192. Hill, D. R., Hewlett, E. L. & Pearson, R. D. (1981). Lectin binding by Giardia lamblia. Infection and Immunity, 34, 733-738. Holberton, D. V. & Ward, A. P. (1981). Isolation of the cytoskeleton from Giardia. Tubulin and a low-molecular weight protein associated with microribbon structures. Journal of Cell Science, 47, 139-166. Jarrol, E. L., Muller, P. J., Meyer, E. A. & Morse, S. A. (1981). Lipid and carbohydrate metabolism of Giardia lamblia. Molecular and Biochemical Parasitology, 2, 187196. Mayer, E. A. (1976). Giardia lamblia: isolation and axenic cultivation. Experimental Parasitology, 39, 101-105. Olveda, R. K., Andrews, J. S. & Hewlett, E. L. (1982). Murine giardiasis: localization of trophozoites and small bowel histopathology during the course of infection. American

-Youma

of Tropical

Medicine

and Hygiene,

31,

60-66. Visvesvara, G. (1980). Axenic growth of Giardia lamblia in Diamond’s TPS-1 medium. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 213-215. Weinbach, E. C., Claggett, C. E., Keister, D. B., Diamond, L. S. & Ken,, H. (1980). Respiratory metabolism of Cilardia lambba. Journal of Parasitology, 66, 347-350. Accepted

for

publication

3rd

December,

1982.