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INFECTION AND IMMUNITY, Feb. 2005, p. 1265–1269 0019-9567/05/$08.00ϩ0 doi:10.1128/IAI.73.2.1265–1269.2005Copyright 2005, American Society for Microbiology. All Rights Reserved.
Lactobacillus johnsonii La1 Antagonizes Giardia intestinalis In Vivo Martı´n A. Humen,1 Graciela L. De Antoni,1,2 Jalil Benyacoub,3 Marı´a E. Costas,4 Marta I. Cardozo,4 Leonora Kozubsky,4 Kim-Yen Saudan,3 ´n y Desarrollo en Criotecnologı´a de Alimentos,1 and Ca ´tedra de Microbiologı´a2 and Ca Parasitologı´a,4 Departamento de Ciencias Biolo ´gicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina, and Nestle´ Research Center, Vers-Chez-les-Blanc, Received 23 August 2004/Returned for modification 16 September 2004/Accepted 18 October 2004 This study describes the in vivo activity of Lactobacillus johnsonii La1 (NCC533) in Giardia intestinalis-
infected gerbils (Meriones unguiculatus). Daily administration of lactobacilli in the drinking water from 7 days
before inoculation with Giardia trophozoites efficiently prevented G. intestinalis strain WB clone C6 from
infecting gerbils. More specifically, shedding of fecal Giardia antigens (GSA65 protein) was diminished in the
La1-treated group, and resolution of infection was observed by 21 days postinoculation. Histology and analysis
of enzymatic markers of microvillus membrane integrity revealed that probiotic administration also protected
against parasite-induced mucosal damage. In addition, a cellular response to Giardia antigens was stimulated
in spleen cells from La1-treated gerbils. Results show for the first time the antigiardial effect of probiotic
lactobacilli in vivo and provide further insight into the antagonistic properties of lactic acid bacteria against
protozoa involved in intestinal infections.
Giardia intestinalis, a flagellated protozoan, is the etiological vivo model of giardiasis (Meriones unguiculatus) to gain further agent of giardiasis, an intestinal infection which causes diar- insight into the antigiardial properties of L. johnsonii La1 rhea and malabsorption in humans and animals (19, 22). This (NCC533; Nestec Culture Collection). The protocol was ap- parasite has a biphasic developmental cycle with two morpho- ´ Research Center and state ethical com- logically different forms: cysts and trophozoites. The latter is the vegetative form of the parasite which is responsible for Frozen suspensions of L. johnsonii (hereafter La1) were infection and pathological manifestations (19). Infection fol- thawed and reactivated in MRS broth before the study. The lows the ingestion of cysts which then give rise to trophozoites bacteria were then subcultured in MRS broth for 16 h at 37°C after passage through the stomach and the upper intestinal and harvested by centrifugation at 10,000 ϫ g for 10 min.
Concentrated suspensions were frozen at Ϫ80°C with 10% Giardia is generally treated with antibiotics (e.g., nitroimid- (vol/vol) glycerol until required. After thawing, bacterial azoles and nitrofurans). However, clinical failures, occurrence counts were determined by plating serial dilutions on MRS of resistant strains, and side effects of anti-Giardia drugs (21, 26, 30, 44) have encouraged research on alternative therapeu- Trophozoites of clone C6 of the strain WB of G. intestinalis, tic strategies which have included the use of plant extracts and products derived from bees (12, 21, 25). However, since pro- doba, Argentina, were grown in Keister’s modified TYI-S-33 biotic microorganisms provide health benefits to the host by medium (28) as previously described (36) and then frozen in antagonizing pathogens and modulating both innate and ac- quired immunity at local and systemic levels (13, 23, 24, 33, 40), Suspensions of La1 were prepared daily by diluting thawed we wondered if such organisms could also be used in the concentrated suspensions in mineral water. Young adult Mon- treatment of parasitic infections. Certainly, colonization of the golian gerbils (Meriones unguiculatus) ingested bacterial sus- intestine by Giardia strongly depends on the intestinal micro- pensions ad libitum in their drinking water each day beginning flora (43). Furthermore, in vitro studies show that some pro- 7 days prior to inoculation with Giardia trophozoites. Based on biotic lactobacilli (e.g., Lactobacillus johnsonii La1) are able to the daily water intake and the bacterial concentration in drink- interfere with the cell cycle of G. intestinalis (36). However, ing water, the daily bacterial intake was estimated to be 108 since this antigiardial effect does not seem to be a general CFU per animal. Control animals received mineral water in- property of lactobacilli, further research is required to select stead of the bacterial suspensions throughout the course of the probiotic strains which can be used in the prevention and study. Seven days after probiotic treatment had begun, frozen treatment of such an infection. For this reason, we used an in trophozoites were thawed in a water bath at 37°C, and a vol-ume of 200 ␮l (5 ϫ 105 trophozoites) was administered bygavage to each animal in the two treatment groups. At various * Corresponding author. Mailing address: CIDCA, Universidad Na- time points thereafter, animals were killed by cervical disloca- cional de La Plata, Calle 47 y 116 (CC 553), 1900 La Plata, Argentina.
tion, and the duodenal contents were analyzed for the presence Phone: 54 221 424 92 87. Fax: 54 221 425 48 53. E-mail: [email protected].
of trophozoites. To this end, 5-cm-long segments of the small intestine were excised at a distance of 11 cm from the pylorus.
TABLE 1. Infection of gerbils at different time points after These samples were then placed in 2 ml of ice-cold culture inoculation with G. intestinalis WB clone C6a medium, opened longitudinally, and vortexed. Enumeration of trophozoites was done by using a hemocytometer with a de- tection limit of 800 trophozoites/cm.
In order to determine fecal giardial antigens, five fecal pel- lets (0.27 [Ϯ0.05] g [wet weight]) per gerbil were homogenized in 0.5 ml of extraction buffer (50 mM EDTA, 100 ␮g of soy- bean trypsin inhibitor [Sigma, St. Louis, Mo.]/ml in phos- phate-buffered saline), vortexed for 2 min, and centrifuged at Gerbils received either La1 or placebo daily from 7 days before inoculation with the trophozoites of G. intestinalis WB clone C6 (5 ϫ 105 trophozoites per 13,000 ϫ g for 15 min. Detection of GSA65 antigen in the animal). Results are from four independent experiments.
supernatants (1) was performed by using a ProSpecT kit ac- b Number of gerbils with detectable parasites in the small intestine/total num- cording to the instructions of the manufacturer (Alexon- ber of gerbils studied. The statistical significance of the infection rate of La1-treated animals compared to that of controls was calculated using Fisher’s exact Trend). Cyst determination was performed microscopically on test. P values are shown in parentheses.
c Averages Ϯ standard deviations of the log of parasites per infected gerbil.
ND, no parasites were detected (Ͻ800 trophozoites/cm).
For histological analysis, 1 cm of the small intestine collected 10 cm from the pylorus was fixed in Bouin fixative and dehy-drated in a graded series of ethanol. Paraffin-embedded sec-tions of 5 ␮m were rehydrated and stained with hematoxylin pare infection rates, and the two-tailed t test with equal vari- ances was used to compare mucosal sucrase activity and cell Intestinal sucrase activity was measured in 5-cm-long sec- proliferation indices of the two groups (Software InfoStat, tions of the small intestine which were collected 5 cm below the 2004; Grupo InfoStat, FCA, Universidad Nacional de Co pylorus. The intestinal samples were placed in ice-cold phos- phate-buffered saline and flushed with 50 ml of ice-cold dis- As shown in Table 1, there was a peak in the Giardia infec- tilled water using a syringe. The segments were then opened tion rate 7 days postinoculation. At this time point, 10 of the 12 longitudinally, and the mucosa was collected by scraping with gerbils (83%) in the placebo group and 3 out of 11 gerbils in a glass microscope slide. Scrapings were stored at Ϫ20°C until the La1-treated group had trophozoites in their small intestine analysis. Intestinal disaccharidase activity was determined as (P ϭ 0.01). By day 14, the infection was resolved in the La1- previously reported (16). Protein concentrations were deter- treated group, but 6 out of 14 gerbils in the placebo group were mined using the Bradford assay, and the results are expressed still infected (Table 1) (P ϭ 0.02). Of these gerbils, three were as arbitrary units per gram of protein (3).
still infected 21 days after inoculation. Although probiotic To determine cell proliferation indices, spleens were placed treatment reduced the infection rate, it had no significant effect in 2 ml of complete RPMI 1640 medium (10% fetal calf serum, on the intensity of infection (Table 1).
1% nonessential amino acids), homogenized through a cell Fecal shedding of GSA65 antigen followed different kinetics strainer (70 ␮m; BD Falcon, Bedford, Mass.), and centrifuged from that of live trophozoites in the small intestine. In the at 250 ϫ g for 5 min. The pellets were then rapidly lysed with placebo group, the number of GSA65-positive gerbils progres- 1 ml of sterile distilled water and centrifuged at 250 ϫ g for 5 sively increased from day 7 to day 21 post-Giardia inoculation min. Cells were resuspended in complete RPMI 1640 medium (Fig. 1). In contrast, the single gerbil positive for GSA65 an- at a concentration of 106 cells/ml before testing cell prolifera- tigen in the La1-treated group at day 14 was no longer positive tion in the presence of the T-cell mitogen concanavalin A (ConA) at a concentration of 3 ␮g/ml or the B-cell mitogen G. intestinalis infection in gerbils has a profound effect on pokeweed mitogen (PWM) at a concentration of 1 ␮g/ml (both the structure and function of the intestinal mucosa (Fig. 2).
from Sigma). Stimulation in the presence of Giardia antigens Villus shortening and hypercellularity of the lamina propria was also evaluated. These membrane antigens were extracted due to increased numbers of mononuclear, polymorphonu- as reported previously (17) and used at a concentration of 10 clear, and eosinophilic cells were observed in control animals.
In contrast, no morphological damage of the gut mucosa was Spleen cells (105 cells/well) were incubated with complete observed in La1-treated animals. These findings correlated RPMI 1640 medium (control) supplemented with either well with the difference in mucosal sucrase activity observed in ConA, PWM, or Giardia antigen in a final volume of 200 ␮l of the two groups. While the intestines of La1-treated animals medium. After incubation at 37°C for 72 h, the cells were pulse maintained an enzymatic activity of approximately 200 arbi- labeled with 1.5 ␮Ci of [3H]thymidine (Amersham Pharmacia trary units/g of protein throughout the study, the intestines of ¨bendorf, Switzerland) for a further 16 h. The cells control animals had decreasing enzymatic activity over time were then harvested on nitrocellulose filters (Packard Bio- (Fig. 3). Differences between the two groups were statistically sciences B.V., Groningen, The Netherlands), and the incorpo- significant at both 14 and 21 days postinoculation (P Ͻ 0.05).
ration of [3H]thymidine was measured by scintillation counting No differences in spleen cell proliferation were observed (TopCount; Packard Biosciences B.V.). Stimulation indices between the two treatment groups 7 days after inoculation with (SI) were determined as the severalfold increase in titrated Giardia trophozoites (data not shown). However, at day 14, thymidine incorporation in stimulated cells compared to that splenocytes of La1-treated animals showed a greater response to ConA (placebo group SI, 7.8 Ϯ 2.2; La1-treated group SI, Fisher’s two-tailed exact statistical analysis was used to com- 22.8 Ϯ 11.6 [P ϭ 0.09]), to PWM, and to Giardia antigen FIG. 3. Intestinal sucrase activity. Gerbils received either La1 or FIG. 1. Shedding of fecal GSA65 antigen. Gerbils received either placebo daily from 7 days before inoculation with the trophozoites of La1 (■) or placebo (E) daily from 7 days before inoculation with the G. intestinalis WB clone C6 (5 ϫ 105 trophozoites per animal). Values trophozoites of G. intestinalis WB clone C6 (5 ϫ 105 trophozoites per (arbitrary units [AU] per gram of protein) represent averages from six animal). Values represent the ratio between GSA65-positive (GSA65 gerbils. Error bars indicate standard deviations. Differences between ϩ) gerbils and the total number of gerbils at various times after La1 and placebo groups are statistically significant (P ϭ 0.05 and P ϭ Giardia infection. The number of animals per group was 18, 12, and 5 0.009 at 14 and 21 days, respectively).
at 7, 14, and 21 days post-Giardia infection, respectively.
Ϯ 0.9 [P ϭ 0.007]) and to the B-cell mitogen PWM (placebo (placebo group SI, 1.2 Ϯ 0.2; La1-treated group SI, 3.7 Ϯ 0.8 group SI, 2.1 Ϯ 0.6; La1-treated group SI, 3.6 Ϯ 0.6 [P ϭ 0.03]) [P ϭ 0.006]) than those of control animals. At 21 days post- was observed, but there was no longer a difference in the Giardia infection, a significantly greater response to Giardia cellular response to ConA between the two groups (data not antigen (placebo group SI, 0.9 Ϯ 0.2; La1-treated group SI, 3.6 shown). Research on the efficacy of probiotic bacteria against FIG. 2. Histological sections of small intestine. Gerbils received either La1 (A) or placebo (B) daily from 7 days before inoculation with the trophozoites of G. intestinalis WB clone C6 (5 ϫ 105 trophozoites per animal). Intestinal segments were collected 7 days post-Giardia infection.
Sections were stained with hematoxylin and eosin and observed at ϫ100 original magnification. (C) Further magnification of the intestine froma control animal. Arrows indicate the presence of trophozoites.
intestinal infections has focused primarily on protection different variant surface proteins in control and La1-treated against bacterial pathogens. The present in vivo study consol- groups could not be ruled out (35), our results suggest a prim- idates recent reports describing the capacity of some lactoba- ing of the immune system by La1 that leads to clearance of cilli of human and canine origin to antagonize Giardia infec- both trophozoites and Giardia antigens from the intestines tion which interferes with the growth and cell cycle of G.
(Table 1 and Fig. 1). It is possible that this reflects the devel- intestinalis in vitro (36). Mongolian gerbils (Meriones unguicu- opment of a memory response following probiotic administra- latus) are a good animal model for studying Giardia infection with strains of human origin (3, 4, 11). Their susceptibility to Although the mechanisms underlying the virulence of G.
giardiasis has been attributed to a deficient antibody response intestinalis remain poorly understood, both direct interaction to specific Giardia antigens (34). We have observed that gerbils with enterocytes and secretion of soluble mediators are are readily infected with trophozoites administered by gavage thought to contribute to the pathogenesis (14). The immune and begin to eliminate cysts in their feces 1 week after inocu- response of the host and inflammatory status of the intestinal lation (data not shown). As a consequence of this infection, the mucosa are also key determinants (41). It is therefore inter- animals have reduced disaccharidase activity in their intestinal esting that a lower cellular infiltration was observed in the intestinal villi of La1-treated animals than that of controls (Fig.
The presence of viable trophozoites in the small intestine is 2). This result indicates the absence of inflammatory processes a recognized marker of Giardia infection (37, 42). We found in the intestine of La1-treated gerbils.
that administration of L. johnsonii La1 dramatically reduces In the present study, it is unlikely that metabolic products in the proportion of gerbils with active trophozoites in the gut the bacterial suspensions contribute to the protective effects and leads to resolution of infection within 14 days. In contrast, observed. Although lactic acid is the main product of La1 viable trophozoites were consistently observed in the intestines metabolism, only traces of this organic acid were detected in of the placebo group at all the time points studied (Table 1).
the bacterial suspensions administered (data not shown). Thus, Furthermore, we have found a time-dependent increase in the the antigiardial effect of L. johnsonii La1 in vivo is probably proportion of GSA65-positive gerbils in this group (Fig. 1).
due to the production of inhibitory substances in situ and/or to Since GSA65 antigen is present on both trophozoites and cysts the modulation of the immune response of the host. Certainly, (38), it is important that the increased proportion of GSA65- previously published results have shown that L. johnsonii La1 positive gerbils with time may reflect changes in the numbers mediates its anti-Giardia effect through the production of sol- of both trophozoites and cysts reaching the distal small intes- uble mediators (36) and that it is able to modulate the immune tine. Certainly, differences in the kinetics of both GSA65 shed- ding and trophozoite load in the small intestine are related to To summarize, we report for the first time an in vivo effect the distribution of trophozoites and cysts along the orad- of a probiotic lactobacillus against G. intestinalis. Our results provide scientific evidence that the applications of probiotic Progression of Giardia infection in gerbils correlates with a organisms can be extended to include alternative strategies in progressive impairment in intestinal sucrase activity (3), which the prevention of a widespread parasitic infection.
may account for the diminished nutrient absorption and failureto thrive associated with giardiasis (11, 19). However, admin- We are indebted to the Agencia de Promocio istration of L. johnsonii La1 protects against the impaired ´gica for financial support. M.A.H. is a fellow at the Consejo Nacional sucrase activity and the histological changes found in infected member of the Carrera del Investigador Cientı´fico y Tecnolo animals. (Fig. 2). It is noteworthy that cysts were eliminated in ´n de Investigaciones Cientı´ficas de la Provincia de Buenos only a few of the gerbils in the placebo group (data not shown).
Aires (CIC-PBA), Buenos Aires, Argentina; and P.F.P. is a member of This is an important observation because cyst production is the the Carrera del Investigador Cientı´fico y Tecnolo only means by which the parasite is known to spread. This observation is also in agreement with previous studies in vitro C6. We also thank Martine Rouvet for her help in the preparation of which have demonstrated that extracellular factors from La1 photographic images, Samuel Steffen and Lucı´a Brandy for technical arrest G. intestinalis in the G phase of the cell cycle (36).
assistance, and Alicia N. Califano for statistical assessment. Revision According to Bernander et al. (6), only trophozoites in G of the manuscript by Anne Donnet is greatly acknowledged.
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