Chemical analysis and giardicidal effectiveness of the aqueous extract of Cymbopogon citratus Stapf.

Searching for new effective and safe treatment of Giardia lamblia (G. lamblia) parasite is mandatory. The aim was to evaluate the in vitro and in vivo effectiveness of an aqueous extract prepared from the leaves of Cymbagogon citratus (CcAE) against G. lamblia and to reveal the phenolic and antioxidant properties of CcAE. METHODS: CcAE (25, 50, 100, 200, 400, and 500 µg/ml) was in vitro incubated with G. lamblia trophozoites in comparison with metronidazole (MTZ 10 and 25 µg/ml). Growth inhibition was evaluated after 3, 24, and 48 h of drug exposure. Infected groups of mice were orally treated for 7 days with CcAE at 125, 250, and 500 mg/kg/day/mouse, in comparison with a group treated with 15 mg/kg/day/mouse MTZ for the same period. The total phenolic components (TPC), the total flavonoid components (TFC), the 2,2,diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, and the high-performance liquid chromatography (HPLC) for quantitative and qualitative phenolic content were chemically estimated. After 24 and 48 h of in vitro incubation, the estimated minimal inhibitory concentrations (MIC) were 500 and 400 µg/ml, respectively, and the concentrations that induced 50% growth inhibition (IC50) were 93.8 and 60.4 µg/ml, respectively (P < 0.001). Mice given 500 mg/kg CcAE showed 100% stool clearance of G. lamblia stages, similar to MTZ-treated control group (P < 0.001). The TPC was 10.7 ± 0.2 mg GAE/g and the TFC was 23.9 ± 0.3 mg quercetin/g, and the estimated IC50 for DPPH free radical scavenging was 16.4 ± 0.1 mg/ml. HPLC revealed the major phenolic components of CcAE to be carnosic acid, p-coumaric acid, cinnamiac acid, quercetin, rutin, and chlorogenic acid. In conclusion, CcAE is significantly effective against G. lamblia in vitro and in vivo, and has considerable phenolic and antioxidant properties.

Piqueria trinervia as a source of metabolites against Giardia intestinalis.

CONTEXT: Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders. OBJECTIVE: The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia. MATERIALS AND METHODS: P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P & T). Remnants were the red oil, fractions 1 and 2 (RO, F1 & F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78-200 µg/mL) for 48 h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts. RESULTS: Trinervinol and piquerol showed antigiardial activity with an IC50 = 2.03 and 2.42 µg/mL, and IC90 = 13.03 and 8.74 µg/mL, respectively. The concentrations of trinervinol (CC50 = 590 µg/mL) and piquerol (CC50 = 501 µg/mL) were not cytotoxic to human fibroblasts. CONCLUSIONS: Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.

Chemical composition and biological activities of Helicteres vegae and Heliopsis sinaloensis.

CONTEXT: Helicteres vegae Cristóbal (Sterculiaceae) (Hv) and Heliopsis sinaloensis B.L. Turner (Asteraceae) (Hs) are endangered and poorly studied plant species; related plants have been used against chronic-degenerative and infectious diseases. Therefore, Hv and Hs could be sources of bioactive compounds against these illnesses. OBJECTIVE: To determine the chemical composition and biological activities (antioxidant, antimutagenic and antimicrobial) of Hv and Hs leaves (L) and stems (S). MATERIALS AND METHODS: Methanol extracts (ME) of each plant/tissue were evaluated for their phytochemicals; phenolics (HPLC-DAD-ESI-MS); antioxidant activity (AA) (0.125-4 mg/mL) (DPPH, ABTS, ORAC and ß-carotene discoloration); antimutagenicity (0.5 and 1 mg/plate) (Ames assay, tester strain Salmonella enterica serovar Typhimurium YG1024, 1-nitropyrene as mutagen); activity against human pathogens (1 mg/mL); and toxicity (0.01-2 mg/mL) (Artemia salina assay). RESULTS: All ME showed flavonoids and triterpenes/steroids. The ME-SHv had the highest content of total phenolics (TP) (2245.82 ± 21.45 mg GAE/100 g d.w.) and condensed tannins (603.71 ± 1.115 mg CE/100 g d.w.). The compounds identified were flavonoids (kaempferol 7-O-coumaroylhexoside, and two kaempferol 7-O-rhamnosylhexosides) and phenolics [rosmarinic acid, and 3'-O-(8″-Z-caffeoyl) rosmarinic acid]. The ME-LHs showed the highest content of flavonoids (357.88 mg RE/g d.w.) and phenolic acids (238.58 mg CAE/g d.w.) by HPLC. The ME-SHv showed the highest AA. All ME were strong antimutagens (63.3-85.7%). Only the Hs extracts were toxic (ME-LHs, LC50 = 94.9 ± 1.7 µg/mL; ME-SHs, LC50 = 89.03 ± 4.42 µg/mL). DISCUSSION AND CONCLUSIONS: Both Hv and Hs are potential sources of preventive and therapeutic agents against chronic-degenerative diseases.

Sonoran propolis and some of its chemical constituents inhibit in vitro growth of Giardia lamblia trophozoites.

Propolis is a cereus resin with a complex chemical composition that possesses a wide range of biological activities. The aim of this study was to evaluate the in vitro anti-Giardia lamblia activity of Sonoran propolis collected from three different areas of Sonoran Desert in northwestern Mexico (Caborca, Pueblo de Alamos, and Ures) and some of its chemical constituents. Additionally, we also analyzed the seasonal effect on the anti-G. lamblia activity of propolis. G. lamblia trophozoite cultures were treated with different concentrations of Sonoran propolis or chemical compounds during 48 h cell proliferation and cell viability were determined. Ures propolis showed the highest inhibitory activity against G. lamblia (IC50 63.8 ± 7.1 µg/mL) in a dose-dependent manner (Ures > Pueblo de Alamos > Caborca). Season had a significant effect on the in vitro anti-G. lamblia activity of Ures propolis. Summer propolis showed the highest inhibitory effect on the G. lamblia trophozoite growth (IC50 23.8 ± 2.3 µg/mL), followed by propolis collected during winter (IC50 59.2 ± 34.7 µg/mL), spring (IC50 102.5 ± 15.3 µg/mL), and autumn (IC50 125.0 ± 3.1 µg/mL). Caffeic acid phenethyl ester, an Ures propolis exclusive constituent, had the highest growth-inhibitory activity towards G. lamblia [IC50 63.1 ± 0.9 µg/mL (222.1 ± 3.2 µM)]. To our knowledge, this is the first study showing that caffeic acid phenethyl ester possesses antiparasitic activity against G. lamblia. Naringenin [IC50 125.7 ± 20.7 µg/mL (461.8 ± 76.3 µM)], hesperetin [IC50 149.6 ± 24.8 µg/mL (494.9 ± 82.2 µM)], and pinocembrin [IC50 174.4 ± 26.0 µg/mL (680.6 ± 101.7 µM)] showed weak anti-G. lamblia activity. On the other hand, chrysin and rutin did not show significant antiparasitic activity. In conclusion, our results suggest that Sonoran propolis and some of its chemical constituents had inhibitory effects on the in vitro growth of G. lamblia trophozoites.

Terminalia ferdinandiana extracts as inhibitors of Giardia duodenalis proliferation: a new treatment for giardiasis.

Giardisis is a debilitating disease caused by gastrointestinal parasites of the genus Giardia. High-antioxidant T. ferdinandiana fruit extracts were investigated for the ability to block Giardia duodenalis growth. Methanolic and aqueous extracts had the most potent growth inhibitory activity (IC50 values of approximately 700 and 140 µg/ml, respectively). Ethyl acetate and chloroform extracts also inhibited G. duodenalis growth, albeit with lower potency. The hexane extract was completely devoid of G. duodenalis growth inhibitory activity. All extracts were nontoxic in the Artemia fransiscana bioassay. Nontargeted HPLC-quadrupole time-of-flight (QTOF) mass spectroscopy (with screening against three compound databases) putatively identified 17 compounds in all of the inhibitory extracts but not in the inactive hexane extract. The low toxicity of the Terminalia ferdinandiana fruit extracts and their potent G. duodenalis growth inhibitory bioactivity indicate their potential as medicinal agents in the treatment and prevention of this disease.

An integrated microfludic device for culturing and screening of Giardia lamblia.

In vitro culturing of trophozoites was important for research of Giardia lamblia (G. lamblia), especially in discovery of anti-Giardia agents. The current culture methods mainly suffer from lab-intension or the obstacle in standardizing the gas condition. Thus, it could benefit from a more streamlined and integrated approach. Microfluidics offers a way to accomplish this goal. Here we presented an integrated microfluidic device for culturing and screening of G. lamblia. The device consisted of a polydimethylsiloxane (PDMS) microchip with an aerobic culture system. In the microchip, the functionality of integrated concentration gradient generator (CGG) with micro-scale cell culture enables dose-response experiment to be performed in a simple and reagent-saving way. The diffusion-based culture chambers allowed growing G. lamblia at the in vivo like environment. It notable that the highly air permeable material of parallel chambers maintain uniform anaerobic environment in different chambers easily. Using this device, G. lamblia were successfully cultured and stressed on-chip. In all cases, a dose-related inhibitory response was detected. The application of this device for these purposes represents the first step in developing a completely integrated microfluidic platform for high-throughput screening and might be expanded to other assays based on in vitro culture of G. lamblia with further tests.

Giardia intestinalis: effects of Pulsatilla chinensis extracts on trophozoites.

Pulsatilla chinensis is a medicinal root plant that has been used to treat a wide range of disease conditions. Our study determined the antiprotozoal activity of various P. chinensis extracts and fractions against Giardia intestinalis including their effects on parasite growth, cell viability, adherence, and morphology. Ethyl acetate extracts (IC50 = 257.081 µg/ml) were the most active to inhibit the growth of G. intestinalis followed by aqueous extract (PWE), saponins, and n-butanol extract. The PWE and ethyl acetate extract inhibited G. intestinalis trophozoites adherence after 3 h of incubation and killed almost 50 % of the parasite population in a time-dependent manner. Changes in morphology, presence of precipitates in the cytoplasm, dissolved cytoplasm with large vacuole, break of flagella and ventral disk, membrane blebs, and intracellular and nuclear clearance of the treated trophozoites were observed by scanning and transmission electron microscopy. We demonstrated that P. chinensis induced these changes in G. intestinalis morphology and consequently has potential therapeutic use against giardiasis.

Differential effectiveness of berry polyphenols as anti-giardial agents.

Following previous work on the anti-giardial effect of blueberry polyphenols, a range of polyphenol-rich extracts from berries and other fruits was screened for their ability to kill Giardia duodenalis, an intestinal parasite of humans. Polyphenol-rich extracts were prepared from berries using solid-phase extraction and applied to trophozoites of Giardia duodenalis grown in vitro. All berry extracts caused inhibition at 166 µg gallic acid equivalents (GAE)/ml phenol content but extracts from strawberry, arctic bramble, blackberry and cloudberry were as effective as the currently used drug, metronidazole, causing complete trophozoite mortality in vitro. Cloudberry extracts were found to be the most effective causing effectively complete trophozoite mortality at 66 µg GAE/ml. The polyphenol composition of the more effective berry extracts suggested that the presence of ellagitannins could be an important factor. However, the potency of cloudberry could be related to high ellagitannin content but also to the presence of substantial amounts of unconjugated p-coumaric acid and benzoic acid. These in vitro effects occur at concentrations easily achievable in the gut after berry ingestion and we discuss the likelihood that berry extracts could be effective anti-giardial agents in vivo.

Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: effects on growth, viability, adherence and ultrastructure.

The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC(50)=134 µg/ml) and eugenol (IC(50)=101 µg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis.

An image-based assay for high throughput screening of Giardia lamblia.

Giardia lamblia is a protozoan parasite that causes widespread gastrointestinal illness. Drugs to treat giardiasis are limited, but efforts to discover new anti-giardial compounds are constrained by the lack of a facile system for cell culture and inhibitor testing. We achieved robust and reproducible growth of G. lamblia in 384-well tissue culture plates in a modified TYI-S-33 medium. A high throughput assay for the screening of potential anti-giardial compounds was developed utilizing the WB strain of G. lamblia and automated optical detection of parasites after growth with tested inhibitors. We screened a library of 1600 known bioactive molecules and identified 12 compounds that inhibited growth of G. lamblia at low- or sub-micromolar concentrations. Our high throughput assay should facilitate evaluation of available chemical libraries for novel drugs to treat giardiasis.

Giardia duodenalis: effects of an ozonized sunflower oil product (Oleozon) on in vitro trophozoites.

The ozonized sunflower oil product (Oleozon) was investigated to explore its cytotoxic activity on Giardia duodenalis in vitro cultivated trophozites. Oleozon produced inactivation of Giardia trophozoites in a dose- and cell density-dependent manner. Thirty microliter of Oleozon with peroxide index value of 500 equivalent-mmol of activated oxygen per kilogram were used to achieve a 100% inhibition (<-4.00 log unit) of trophozoites from an initial inoculum of 15x10(4) cells. This potent effect was confirmed by transmission electron microscopy where morphological deterioration of superficial structures mainly in the ventral disc, and formation of a great number of micro vesicles in the cytoplasm were found. We concluded that a direct chemical-oxidation attack by the active substances from Oleozon is one of the causes of the parasitocidal effect of this product. We suggest that the dose and cell density-dependent effect must be taken into account when prescription of this product for giardiasis treatment in humans.

Impact of Giardia intestinalis on vitamin a status in schoolchildren from northwest Mexico.

We conducted a cross-sectional study in northwest Mexico in order to investigate the association between giardiasis and serum vitamin A in 40 Giardia-infected and 70 Giardia-free schoolchildren who were covered by a regional school breakfast program. There were no significant differences in age, Z-scores for nutritional indices of height for age, weight for age, or weight for height, socioeconomic conditions (employment and education of the parents, household conditions, sanitation facilities, type of drinking water, and family income), and mean daily intakes of vitamin A in the Giardia-free (899 +/- 887 microg) and the Giardia-infected (711 +/- 433 microg) groups. A higher concentration of serum retinol was found in the Giardia-free group than in the Giardia-infected group (0.75 micromol/L versus 0.61 micromol/L, respectively; p < 0.0001). Giardia-infected children were more likely to be vitamin A-deficient than the Giardia-free children (OR = 3.2; 95% CI = 1.2-8.5). Although 95% of the children met the daily-recommended intakes of vitamin A, half of them showed subclinical vitamin A deficiency. It is recognized that vitamin A deficiency is multifactorial and giardiasis was a factor significantly associated with this deficiency in this study. Mexican program developers and policymakers should be aware about the distinction between dietary deficiencies and deficiency diseases when current national program strategies for parasitic control and vitamin A supplementation are redesigned.

Effect of propolis versus metronidazole and their combined use in treatment of acute experimental giardiasis.

Propolis, a honey bee product, gained popularity in alternative medicine. Its prophylactic and therapeutic effects were experimentally evaluated. One hundred and fifty immunocompetent mice were orally infected by 5 x 10(5) axenically cultivated Giardia lamblia trophozoites. The trophozoite count in intestine, interferon-gamma serum level, histopathological examination of duodenal and jejunal sections were assessed for evaluation of propolis and metronidazole (MTZ) effect after 6 & 12 days post infection (p.i). Also, T-lymphocyte profile in blood was investigated 12 days p.i using flow cytometry (FCM). Propolis as prophylaxis showed a significant decrease in intensity of infection, together with a significant increase in IF-gamma serum level and increase in CD4+: CD8+T-cell ratio. In treatment it gave a highly significant decrease in trophozoite count than that obtained by MTZ 6 days after infection but the efficacy was almost equal after 12 days. The mice treated with propolis alone showed a reversed CD4+: CD8+ T-lymphocyte ratio, such strong immune enhancing effect resulted in an undesirable increase in inflammatory response at intestinal level. The combined therapy showed a stronger efficacy in reducing the parasite count than that gained by each drug alone. Their combined use caused an immunological balance as shown by the T-lymphocyte profile that saved the intestinal homeostasis and histological architecture.

Giardia lamblia: the effects of extracts and fractions from Mentha x piperita Lin. (Lamiaceae) on trophozoites.

Giardia lamblia is a parasite that causes giardiasis in humans and other mammals. The common treatment includes different classes of drugs, which were described to produce unpleasant side effects. Mentha x piperita, popularly known as peppermint, is a plant that is frequently used in the popular medicine to treat gastrointestinal symptoms. We examined the effects of crude extracts and fractions from peppermint against G. lamblia (ATCC 30888) on the basis of trophozoite growth, morphology and adherence studies. The methanolic, dichloromethane and hexanic extracts presented IC(50) values of 0.8, 2.5 and 9.0microg/ml after 48h of incubation, respectively. The aqueous extract showed no effect against the trophozoites with an IC(50)>100microg/ml. The aqueous fraction presented a moderate activity with an IC(50) of 45.5microg/ml. The dichloromethane fraction showed the best antigiardial activity, with an IC(50) of 0.75microg/ml after 48h of incubation. The morphological and adhesion assays showed that this fraction caused several alterations on plasma membrane surface of the parasite and inhibited the adhesion of G. lamblia trophozoites. Cytotoxic assays showed that Mentha x piperita presented no toxic effects on the intestinal cell line IEC-6. Our results demonstrated antigiardial activity of Mentha x piperita, indicating its potential value as therapeutic agent against G. lamblia infections.

Susceptibility of Giardia lamblia to Hovenia dulcis extracts.

Giardia lamblia is the causative agent of giardiasis, a common parasitic infection of the human and animal digestive tract. Although several drugs have been available to treat this infection, they present unpleasant side effects or cytotoxicity. In order to find a more natural treatment for the disease, we analyzed the effects of the methanolic extract and three fractions obtained from Hovenia dulcis Thunb. (Rhamnaceae) leaves on G. lamblia. Comparing all fractions, dichloromethane was more efficient in reducing Giardia growth. The exposition of G. lamblia to this fraction lead to degenerations in the surface, modifications in the cell shape and alterations in the localization of nuclei. Besides that, the adhesion of G. lamblia was also altered. Experiments revealed that the obtained fraction did not present cytotoxic effects in mammalian cells. In summary, dichloromethane fraction has strong antigiardial effects and could become an important new substance for the treatment of giardiasis.

Validación del uso tradicional de plantas medicinales cultivadas en Cuba / Validation of the traditional use of medicinal plants cultivated in Cuba

Se evaluó la actividad antimicrobiana y antigiardiósica in vitro de extractos fluidos de Artemisia absinthium L., Stachitarpheta jamaicensis L. y Teloxis ambrosioides L., empleando los ensayos de diluciones en medio líquido y producción de formazán, respectivamente. Fue calculada la concentración mínima inhibitoria y mínima microbicida de microorganismos de interés clínico humano y el por ciento de inhibición del crecimiento de G. Lamblia. Los extractos mostraron actividad antimicrobiana y antigiardiásica, lo que guarda relación con su uso tradicional. No obstante, otros aspectos deben ser analizados con detenimiento para proponer el uso de estos extractos en el desarrollo de fitofármaco(AU)

The microaerophilic flagellate Giardia intestinalis: Allium sativum (garlic) is an effective antigiardial.

Whole garlic (Allium sativum L.) extract and some of its components were assayed for antigiardial activity. Whole garlic extract gave an IC(50) at 24 h of 0.3 mg ml(-1). Most of the components assayed were inhibitory to the organism, especially allyl alcohol and allyl mercaptan, with IC(50) values of 7 microg ml(-1) and 37 microg ml(-1) respectively. Studies with calcofluor white indicated that whole garlic and allyl alcohol collapse the transmembrane electrochemical membrane potential (Deltapsi) of the organism, as indicated by uptake of the fluorochrome. Electron microscopy allowed the morphological changes that occur with garlic inhibition to be recorded. Both the surface topography and internal architecture of the organism changed during incubation with the biocides. Both whole garlic and allyl alcohol resulted in fragmentation of the disc and an overexpression of disc microribbons, internalization of flagella, vacuole formation and an increase in distended vesicles. Allyl mercaptan, however, only gave an increase in distended vesicles, suggesting that this biocide has a different mode of action.

Giardia lamblia: incorporation of free and conjugated fatty acids into glycerol-based phospholipids.

Giardia lamblia trophozoites are flagellated protozoa that inhabit the human small intestine, where they are exposed to various dietary lipids and fatty acids. It is believed that G. lamblia, which colonizes a lipid-rich environment of the human small intestine, is unable to synthesize phospholipids, long-chain fatty acids, and sterols de novo. Therefore, it is possible that this protozoan has developed a special process for acquiring lipids from its host. We have previously shown that G. lamblia can take up saturated fatty acids and incorporate them into phosphatidylglycerol (PG) and other glycerol-based phospholipids (Stevens et al., Experimental Parasitology, 86, 133-143, 1997). In the present study, an attempt has been made to investigate the underlying mechanisms of transesterification and interesterification reactions of giardial phospholipids by free and conjugated fatty acids. Results show that exogenously supplied, unsaturated, fatty acids were taken up by Giardia and incorporated into various phosphoglycerides, including PG. To test whether this intestinal pathogen can utilize conjugated fatty acids, live trophozoites were exposed to either [3]H;cbphosphatidylcholine (PC), where the fatty acid was 3H-labeled at its sn2 position, or to [14C]lyso-PC (fatty acid was 14C-labeled at the sn1 position) for 90 min, followed by phospholipid analysis using thin-layer chromatography. The results suggest that conjugated fatty acids, like free fatty acids, were incorporated into PG. It was also observed that aristolochic acid, an inhibitor of Ca2+-ionophore-stimulated phospholipase A2, decreased the transfer of fatty acids from [3H]PC to PG, indicating that giardial phospholipases were involved in these esterification reactions. Additional experiments, which include culturing trophozoites in serum-supplemented and serum-deprived medium, along with numerous biochemical analyses suggest that (i) PG is a major transesterified and interesterified product, (ii) it is likely that giardial phospholipases are involved in esterification reactions, (iii) in G. lamblia, PG is localized in perinuclear membranes, as well as intracellularly, but not in the plasma membrane, and (iv) various synthetic analogs of PG inhibit the growth of the parasite in vitro. These studies suggest that PG is an important phospholipid of Giardia and a potential target for lipid-based chemotherapy against giardiasis.

Cloning of Thai strain Giardia intestinalis.

Axenic cultures of Giardia intestinalis trophozoites were successfully established from human fecal specimens and rectal swabs from dogs using sucrose gradient centrifugation to separate the cysts from fecal material, the excystation method of Robert-Thompson et al. and culture of the preparation in TYI-S-33 medium supplemented with human serum, vitamin mixture and piper acillin and amikacin antibiotics, respectively. Fungal contamination could be controlled by amphotericin B at 10 micrograms per ml of medium. Clones of the parasites were obtained using a combination of dilution method and micromanipulation technique.

Arginine metabolism during culture of Giardia intestinalis.

The effect of arginine on the growth and metabolism of Giardia intestinalis trophozoites was determined. Supplementation of the normal growth medium (Diamond's TYI-S-33) with 5 or 10 mM arginine accelerated trophozoite growth over the first 2 days. There was a corresponding rapid utilisation of arginine, with none being detectable after this time. The decrease was associated with the appearance in the growth medium of 1 mol of ornithine and 2 mol of ammonia per mol of arginine utilised, the stoichiometry being consistent with the operation of the arginine dihydrolase pathway. Subsequently, there was a decrease in the ammonia concentration in the medium. Removal of arginine from the medium by pretreatment with arginase substantially decreased cell growth. In TYI-S-33 medium containing no added glucose, instead of the normal 50 mM glucose concentration, arginine supplementation also increased cell growth over the first 2 days, with concurrent stoichiometric production of ornithine and ammonia. However, in these conditions, the ammonia concentration remained elevated. This suggests that under normal conditions there is re-uptake of ammonia, which is glucose dependent. The observations confirm the operation of a functional arginine dihydrolase pathway in G. intestinalis. The concordance of cessation of rapid growth with the depletion of arginine, and the beneficial effect on growth of arginine supplementation suggests that arginine availability is a limiting factor during the initial stages of rapid growth. It would appear that arginine is a major potential energy source during the initial stages of giardial growth, and that supplementation of Diamond's TYI-S-33 medium with additional arginine may provide an improved in vitro culture medium.