Vol. 60, No. 6
INFECTION AND IMMUNITY, June 1992, p. 2274-2280 0019-9567/92/062274-07$02.00/0 Copyright © 1992, American Society for Microbiology
Isolation of Two Giardia lamblia (WB Strain) Clones with Distinct Surface Protein and Antigenic Profiles and Differing Infectivity and Virulence IFEANYI A. UDEZULU,lt* GOVINDA S. VISVESVARA,1 DELYNN M. MOSS,' AND GORDON J. LEITCH2 Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 303102 Received 16 December 1991/Accepted 10 March 1992
To determine the relationship between antigenic profiles and pathogenicity among Giardia lamblia clones (WB strain), trophozoites were cloned by the technique of limiting dilution. The phenotype of each clone was determined by an indirect immunofluorescence test using a polyclonal rabbit anti-G. lamblia trophozoite serum made against the parent strain. Two clones were chosen for further studies: a highly fluorescent clone, F+, in which more than 95% of the trophozoites fluoresced, and a low-fluorescence clone, F-, in which fewer than 5% fluoresced. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and enzyme-linked immunotransfer blot studies of the membrane fractions of the two clones and parent strain revealed differences in both the total protein and antigenic profiles. A serum cytotoxicity test with the polyclonal serum showed that the F+ clones were more susceptible to immobilization and killing, while the majority of cells of the F- clones were resistant to such killing. Assessment of the infectivity of the two clones in the Mongolian gerbil animal model indicated that the F- clone more readily initiated infections, produced more cysts, had a higher intestinal trophozoite load, and produced a more severe clinical syndrome, while the F+ clone was less phenotypically stable in vivo and in some cases took longer to be cleared from the intestine. MATERIALS AND METHODS
Giardiasis is a disease caused by the infection of the proximal small intestine with the protozoan parasite Giardia lamblia. This enteropathogen exists in two distinct morphological forms: the motile trophozoite, or feeding stage, that colonizes the intestine and the infective cyst form that is voided with feces (19, 34). The major mode of transmission is via the oral route (19, 28, 34), usually in food or drink. However, person-to-person transmission in day-care centers and among homosexually active men has also been reported (9, 15). Although G. lamblia strains have been characterized by their isoenzyme (1, 8) and surface antigen (26, 29) profiles, little is known about the relationship between such profiles and strain pathogenicity. Nash and coworkers (2, 23, 24) have shown that G. lamblia trophozoites originating from a single cell demonstrate marked variability in their surface antigenic profiles. When clones of G. lamblia trophozoites were used to infect animals or human volunteers, the trophozoites that were recovered from the intestine showed changes in their surface antigenic profiles (3, 25). It is not yet known if such changes were a result of host-induced trophozoite selection or parasite gene rearrangement (22). Similarly, surface antigen variability has been shown to occur in Trypanosoma brucei, and such variability is believed to be involved in this protozoan parasite's evasion of the host's immune surveillance (12). Several animal models have been established to study Giardia infection and pathogenesis (7, 11, 27, 32). Here, we describe the surface protein and antigenic profiles of G. lamblia clones isolated from trophozoites (WB strain) and compare the virulence and infectivity of these clones in gerbils.
Trophozoite cultivation. G. lamblia trophozoites, uncloned (parent strain) and cloned WB strain (ATCC 30957) as well as a WB (A6) clone (obtained from T. E. Nash), were grown and maintained axenically at 37°C in filter-sterilized Diamond's TYI-S-33 medium as modified by Keister (17). G. lamblia clones. Trophozoites were cloned in a 96-well microtiter plate by the method of limiting dilution (5). This technique allowed us to obtain a dilution of 0.5 and 1.0 cell per 0.1 ml to ensure the growth of a trophozoite colony from a single cell. Medium containing either 0.5 or 1.0 cell from each well was pipetted into 2.0-ml vials, and the final volume was adjusted to 1.8 ml with Keister's medium. The vials were incubated at 37°C for 14 days and examined microscopically every other day, starting on day 3. Each positive clone was transferred to a 15-ml screw-cap sterile glass tube and recloned an additional four times. IIF. The phenotype of each clone was determined by an indirect immunofluorescence (IIF) test using polyclonal rabbit anti-G. lamblia trophozoite (uncloned WB parent strain) serum at a dilution of 1:64. Preimmune rabbit serum was used as a negative control (33). Each clone was characterized as either fluorescence negative (clone F-) or fluorescence positive (clone F+) depending on the percentage of fluorescent cells in a colony. Preparation of membrane-rich (MR) protein fractions. Axenic cultures of the WB-cloned, uncloned, and WB (A6) trophozoites were harvested in the log growth phase. Each harvest consisted of trophozoites attached to glass culture tubes, trophozoites not attached to tubes, and unsegregated trophozoites (combined attached and nonattached) except for the WB (A6) clone, from which only attached cells were harvested. The nonattached trophozoites were harvested by decanting the medium from several tubes and centrifuging the pooled medium to sediment the trophozoites. The attached trophozoites from these tubes were detached by
* Corresponding author. t Present address: Department of Biology, Winston-Salem State University, Winston-Salem, NC 27110.
2274
VOL. 60, 1992
chilling tubes filled with 0.85% NaCl (saline) in wet ice for 5 min and then centrifuging the tubes at 250 x g for 10 min at 4°C. Each trophozoite pellet was washed three times in saline. Attached, nonattached, and unsegregated trophozoites were resuspended at 109 trophozoites per 1.6 ml in enzyme-stabilizing solution containing 1 mM EDTA, 1 mM 6-amino-n-caproic acid, and 1 mM dithiothreitol and stored in liquid nitrogen. Washed trophozoite suspensions were thawed at room temperature and further processed by the method described by Moss et al. (20). In brief, the trophozoites were lysed by snap freezing and thawing three times in a methanol-dry-ice slurry and a 37°C water bath. The lysates were fractionated by centrifugation at 4°C at 24,000 x g into cytosol and membrane fractions. The supernatant (cytosol) was removed, and the sediment (membrane) was collected. The sediments were washed three times in saline, solubilized overnight at 4°C in 8.0 M urea-0.05 M Tris HCI-0.3 M KCl-0.2 mM EDTA, pH 8.0, and mixed intermittently every 30 min with a vortex mixer. The sample was further solubilized by ultrasonic disruption with a W-375 Sonifier (Heat System-Ultrasonics, Inc., Plainview, N.Y.) at 40% power, 15% duty cycle, and pulse mode for 3 min at 4°C. After the sonicates were centrifuged at 24,000 x g for 30 min at 4°C, the supernatant solutions were collected, dialyzed, and concentrated against 40 mM Tris HCl-54 mM boric acid-1 mM EDTA, pH 8.3, with a YM-10 membrane (W. R. Grace and Co., Amicon Div., Danvers, Mass.). The dialyzed, MR protein fractions were labeled and stored in liquid nitrogen until needed. The protein contents of the MR protein dialysate fractions were determined by the method of Bradford (10) with dye reagent from Bio-Rad Laboratories (Rockville Centre, N.Y.) and human albumin and globulin from Sigma Chemical Co. (St. Louis, Mo.) as standards. SDS-polyacrylamide gradient gel electrophoresis. Electrophoretic analysis of the MR protein fractions was performed in a discontinuous buffer system as described by Moss et al. (20), except that the samples were diluted with 0.5 mM Tris HCl, pH 8.0, prior to sodium dodecyl sulfate (SDS) treatment. All MR protein fractions were treated with SDS under nonreducing conditions. The SDS-treated MR proteins were separated on 3 to 25% linear gradient polyacrylamide gels with 3% stacking gels at concentrations of 0.15 and 0.2 ,ug of protein per mm of lane for the silver stain and enzyme-linked immunotransfer blot (EITB), respectively. Silver staining was performed as described by Tsang et al. (30). EITB. After MR proteins were electrotransferred to nitrocellulose sheets as described by Tsang et al. (31), the sheets were exposed overnight at 4°C to either polyclonal rabbit anti-G. lamblia trophozoite (uncloned WB parent strain) serum or preimmune negative control serum at a dilution of 1:500. In another experiment, the nitrocellulose was exposed to monoclonal antibody 6E7 (courtesy of T. E. Nash) at a dilution of 1:1,000. Monoclonal antibody 6E7 was previously shown to be specific for the G. lamblia 170-kDa surface protein (23, 24). The nitrocellulose was washed to remove unbound antibodies and exposed to goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (1:1,000 dilution) (Bio-Rad Laboratories, Richmond, Calif.) or goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase for monoclonal antibody 6E7. SDS-treated high-molecular-weight protein (Bio-Rad) and low-molecular-weight protein standards (Pharmacia) were used in all electrophoretic separations. In all EITBs, protein
CHARACTERISTICS OF G. LAMBLIA CLONES
2275
standard lanes were stained with Aurodye solution (Janssen Life Sciences, Piscataway, N.J.). Serum cytotoxicity test. Trophozoites from each clone were suspended in Keister's medium and added to 96-well round-bottom microtiter plates containing the polyclonal serum to obtain a trophozoite concentration of 106 cells per well and a final serum dilution of 1:2. The plates were incubated in an anaerobic chamber at 37°C for 1 and 24 h. At the end of each test period, the cells were visualized microscopically and chilled in ice water. Samples were taken, and counts were made of live and dead cells (trypan blue exclusion) by using a hemocytometer. Gerbil infectivity studies. Young adult male Mongolian gerbils (Menones unguiculatus) obtained from Tumblebrook Farm (West Brookfield, Mass.) were used throughout. Before being infected, the animals were starved for 24 h. Eight gerbils in each of 12 groups were inoculated orally with 104, 105, or 106 attached or nonattached F- or F+ trophozoites suspended in 0.3 ml of saline. During the postinoculation period, the animals were housed individually in wire-floored cages. Feces were collected daily from each cage from day 3 postinfection through day 35. Each animal was killed, and the intestinal mucosa was scraped and examined for the presence of trophozoites. The fecal pellets were processed, and cysts were isolated by using a 0.75 M sucrose gradient flotation method (32). The daily cyst excretion was quantitated microscopically by using a hemocytometer. In another study, the animals were killed 14 days postinoculation. Both the body and small-intestine weights of each animal were measured to assess enteropooling (18). The proximal one-third of the small intestine was excised, and the contents were collected. The segment was then opened at the antimesenteric border, and the mucosa was removed. After the mucosa and intestinal contents were fixed in 1% formalin-saline and stained with methylene blue, trophozoites were counted with a hemocytometer. Histologic studies on tissue sections obtained from animals inoculated with 104 and 106 trophozoites were also performed. Animals were killed 14 days postinoculation, and 2.5-cm-long segments of proximal duodenum from 0.5 cm distal to the pylorus were removed. Tissue sections were fixed in neutral formalin, embedded in paraffin, and stained with hematoxylin and eosin. Reisolation of trophozoites from infected animals and IIF test. Two groups of five Mongolian gerbils each were infected with 106 trophozoites (attached and nonattached combined) from each clone. At 14 days postinfection, the animals were killed, a laparotomy was immediately performed, and the duodenum was excised. A longitudinal section was made through the duodenum, and the mucosa and luminal contents were harvested. A sample was immediately mounted on a slide and examined microscopically for the presence of trophozoites. Positive samples were then inoculated into 15-ml sterile screw-cap tubes containing warm Keister's medium with 50 ,ug of gentamicin, 1,000 IU of penicillin, 1,000 ,ug of streptomycin, and 5 ,ug of amphotericin B per ml. After 1 h, the recovered trophozoites were washed repeatedly in saline to reduce the chyme and finally suspended in phosphate-buffered saline, pH 7.2, for phenotypic characterization by the IIF test. It was not possible to perform the IIF test on trophozoites taken directly from the intestinal lumen owing to the high background fluorescence of the luminal content. Trophozoite viability assay and growth curve. In one group of studies, cryopreserved trophozoites of clones F- and F+ were recultivated in Keister's medium at a final concentra-
2276
UDEZULU ET AL.
tion of 104 trophozoites per ml in a 15-ml screw-cap sterile glass tube. Samples were observed on alternate days for 11 days starting from day 1, and the cell number and viability were determined by using trypan blue dye exclusion. Viability data were generated for both attached and nonattached trophozoites of clones F- and F+. In another group of studies, cells were suspended in Keister's medium at a concentration of 104 trophozoites per ml in duplicate 15-ml sterile screw-cap tubes. These cells were used for growth curve studies. Trophozoite counts were obtained at zero time and every 12 h thereafter until late stationary phase. In all of the groups, a total of six counts were taken, and the means of the counts were recorded. The cells were maintained at 37°C throughout the period of study. Statistical evaluations. When populations were homogeneous, either Student's t tests or one-way analysis of variance was used for statistical evaluation. However, when populations were nonhomogeneous, either Wilcoxon matched-pair signed-rank tests or Wilcoxon rank sum tests were employed. RESULTS
G. lamblia trophozoites (WB strain) were cloned by the technique of limiting dilution. This method enabled us to obtain a cloning efficiency of more than 37.5% for 0.5 cell per ml and 62.5% for 1.0 cell per ml. Immunofluorescence. All trophozoite cultures, cloned and uncloned, were phenotypically characterized by IIF. Two clones, a highly fluorescent clone, F+, in which more than 95% of the cells fluoresced, and a low-fluorescence clone, F-, in which fewer than 5% of the cells fluoresced, were chosen for further studies. SDS-polyacrylamide gradient gel electrophoresis. The protein profiles of MR fractions obtained from cloned and uncloned trophozoites, including those of the attached cells of the WB (A6) clone, were examined by SDS-polyacrylamide gradient gel electrophoresis. All exhibited in the silver-stained preparations a large number of bands ranging in molecular mass from 14 to 250 kDa. Obvious differences, however, were seen between the three clones. For example, the F- clone exhibited an intensely stained diffuse band at about 150 to 170 kDa that was not seen in the parent strain or in the F+ clone. The WB (A6) clone resembled the F- clone and also exhibited a marked diffuse band at 150 to 170 kDa (Fig. 1, lanes 7, 8, 9, and 10). EITB. Figure 2 shows the EITB profiles of the parent WB strain, F+ clone, F- clone, and WB (A6) clone trophozoites. Nitrocellulose sheets with electrotransferred proteins were exposed to the same polyclonal rabbit anti-Giardia trophozoite serum that was used in the IIF test. Characteristic banding patterns for each of the MR fraction preparations were seen. The anti-G. lamblia (WB) antibody recognized more bands in both the parent strain and the F+ clones than in the F- clones (Fig. 2). Clone F-, however, differed from the F+ clone and the parent strain in possessing a diffuse band with a molecular mass of approximately 150 kDa (Fig. 2, lanes 7, 8, and 9). The bands of the WB (A6) clone again resembled those of the F- clone. However, bands at 21 to 24, 33, 104, and 112 kDa were apparent in the F- clone but not in the WB (A6) clone sample (Fig. 2, lanes 7, 8, 9, and 10). Additionally, there were quantitative differences between the antigenic patterns of attached and nonattached cells of both clones. In the F- clone, bands at 33 and 55 kDa were more evident in the nonattached than in the attached
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-_ 2 3 4 5 6 7 8 9 10 11 FIG. 1. Protein profiles (silver stain) of MR protein fractions. Lanes: 1, unsegregated (attached and nonattached) cells of the uncloned WB parent strain; 2, nonattached cells of the uncloned WB parent strain; 3, attached cells of the uncloned WB parent strain; 4, unsegregated cells of clone F+; 5, nonattached cells of clone F+; 6, attached cells of clone F+; 7, unsegregated cells of clone F-; 8, nonattached cells of clone F-; 9, attached cells of clone F-; 10, attached cells of WB (A6) clone (courtesy of T. E. Nash); 11, molecular weight standards (sizes on the right in kilodaltons). -am
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samples (Fig. 2, lanes 8 and 9). Similarly, in the F+ clone, bands at 33, 36, and 42 kDa were more apparent in the nonattached samples than in the attached samples (Fig. 2, lanes 5 and 6). In another EITB, monoclonal antibody 6E7 recognized a distinct band at 150 to 170 kDa in the F- and WB (A6) clones but not in the F+ clone or the parent strain (Fig. 3). This monoclonal antibody revealed additional differences between attached and nonattached surface antigens of the Fclone by visualizing diffuse bands in the region of 220 to 250 kDa and a single band at 300 kDa in only the F- attached preparation (Fig. 3, lane 9). Infectivity. Oral inoculation of gerbils with attached and nonattached trophozoites of either F- or F+ clones resulted in reproducible patterns of infection as assessed by the number of cysts excreted, the number of trophozoites in the intestine, and the number of animals infected. Results indicated that with a small inoculum (104 trophozoites), the infection patterns of attached and nonattached cells of the F- clone differed significantly from those of the F+ clone (P < 0.001; Fig. 4a and b). With a large inoculum (106 trophozoites), the F- clone infections were cleared significantly faster than the F+ clone infections (P < 0.002; Fig. 4e and f). A one-way analysis of variance indicated that the number of cysts produced during the course of all the infections was greater in animals infected with the F- clone than in those infected with the F+ clone (Fig. 5). However, this difference was significant only at the lowest trophozoite number (104) (P < 0.01). A one-way analysis of variance also indicated that the total number of cysts excreted increased significantly with inoculum size. This increase was statistically significant for both groups of animals infected with attached (P < 0.01) and nonattached (P < 0.001) F- clone organisms
CHARACTERISTICS OF G. LAMBLI4 CLONES
VOL. 60, 1992
2277
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as well as for those infected with attached (P < 0.01) and nonattached (P < 0.03) F+ clone trophozoites. When the trophozoite loads of the animals were determined on day 14, gerbils infected with the F- clone had a higher trophozoite count than animals infected with the F+ clone. This difference was significant for the small inoculum (104 trophozoites) (attached, P < 0.02, and nonattached, P < 0.03; Fig. 6). In all groups at all inoculum sizes, the smallintestine weight normalized to the body weight was significantly greater in infected animals than in either noninoculated controls (P < 0.05) or inoculated but uninfected animals (104-trophozoite inoculum, P < 0.01 for clone Fand P < 0.02 for clone F+, and 106-trophozoite inoculum, P < 0.01 for clone F- and P < 0.01 for clone F+). In the infected animals, the F- clone infections were associated with significantly higher small-intestine weights normalized to body weight than were the F+ clone infections (104trophozoite inoculum, P < 0.001, and 106-trophozoite inoculum, P < 0.01; Table 1). As there were no significant differences in body weight between groups, increases in intestinal weight were considered indications of enteropooling. The subjective observation of fluid accumulation, particularly in the duodenum, supported this interpretation. Duodenal villus-to-crypt ratios were measured by using appropriately oriented villi in animals inoculated with either 10 or 106 trophozoites. In all groups, the villus-to-crypt ratios were lower in infected animals than in uninfected animals (104 and 106, P < 0.05; Table 2). The villus-to-crypt ratios of the F- clone-infected animals were significantly
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FIG. 3. EITB profiles of MR protein fractions of G. lamblia trophozoites using 170-kDa MAb 6E7 ascites (courtesy of T. E. Nash). Lanes: 1, unsegregated cells of the uncloned WB parent strain; 2, nonattached cells of the uncloned WB parent strain; 3, attached cells of the uncloned WB parent strain; 4, unsegregated cells of clone F+; 5, nonattached cells of clone F+; 6, attached cells of clone F+; 7, unsegregated cells of clone F-; 8, nonattached cells of clone F-; 9, attached cells of clone F-; 10, attached cells of WB (A6) clone; 11, molecular weight standards (sizes on the right in kilodaltons).
lower than those of the F+ clone-infected animals for all the corresponding groups (104 and 106, P < 0.05). Growth and viability studies. In another study, cryopreserved cells from the two clones were recultivated, and their viability, attachment to glass, and growth curves were determined. Results of trypan blue exclusion indicated that F- clones were initially more viable. Larger numbers of Fcells attached to the tubes, and the F- clone adapted to the growth medium faster. However, once the cell colonies had adapted to the medium, the generation time was the same for trophozoites from both clones. Reisolation of trophozoites from infected animals and IEF test. Trophozoites from gerbils infected with trophozoites of the F- and F+ clones were reisolated 14 days postinoculation and immediately processed for IIF studies with the same polyclonal serum that was used in the initial phenotypic characterizations. Results revealed that the phenotypic character of the F- clone was apparently conserved; fewer than 5% of the cells were fluorescence positive. Before being inoculated, the F+ clone was more than 95% immunofluorescence positive. After the F+ clone was reisolated from infected gerbils, the IIF test results showed three distinctly different phenotypic subpopulations of bright (5 to 10%), medium (15 to 20%), and nonfluorescent (65 to 70%) cells. The relative numbers of these three subpopulations varied with the animals from which the trophozoites were reisolated (n = 6).
2278
INFECTr. I MMUN .
UDEZULU ET AL.
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FIG. 4. Percentages of gerbils excreting cysts daily following inoculation with attached or nonattached trophozoites from clone F- or F'. Inocula were of 104 (a and b), 105 (c and d), or 106 (e and f) trophozoites. Values are daily means.
FIG. 5. Total numbers of cysts excreted by gerbils during the period the animals were infected with attached or nonattached trophozoites from clone F- or F+. Means + standard errors of the
DISCUSSION
membrane antigens in both the F- and WB (A6) clones but not in the F+ clone or the parent strain. This observation suggests that the subpopulation from which the F- clone originated could represent a small fraction of the trophozoites in the parent strain, i.e., probably less than the estimated 10% that appeared to be phenotypically fluorescence negative. The next group of studies was conducted to determine whether these clones that were so distinct in their protein and antigenic profiles also differed in their infectivity and pathogenesis or if they demonstrated other biological differences. The Mongolian gerbil was used as the experimental host. Both G. lamblia cysts and trophozoites have been shown to infect this model (7, 14, 18, 32). The F- clone trophozoites were significantly more infectious and produced a more severe clinical syndrome as assessed by enteropooling (18) and reduction of the villus-to-crypt ratio (6, 35). Consistent with the apparent greater pathogenicity, infections with the F- clone produced a significantly larger parasite load on day 14, and infected animals excreted a larger number of cysts over the course of the infection than animals infected with the F+ clone. With a smaller inoculum, the attached cells of the F- clone were more infectious than the nonattached. To the degree that the in vivo and in vitro data can be compared, this greater infection and pathogenesis observed with F- clone trophozoites in vivo is consis-
Genetically controlled strain variation and antigenic variability within strains and clones originating from a single cell in G. lamblia trophozoites have been described previously (2, 23, 26). In this study, an IIF test to screen G. lamblia WB for antigenic heterogeneity revealed that the trophozoite populations were predominantly (90%) made up of fluorescent (F+) cells. The technique of limiting dilution was used to obtain low- and high-fluorescence clones from the parent WB strain. Substantial differences in the membrane proteins and antigens in these groups of trophozoites were noted in both the silver stain and EITB tests. The F+ clone closely resembled the parent strain. This result was expected, as the parent strain contained more than 90% immunofluorescent cells and the F+ clone cells were more than 95% fluorescence positive. The polyclonal antibody recognized fewer bands in the F- and the WB (A6) clones than in the F+ clone and the parent strain. The F- and WB (A6) clones shared bands. Overall, the differences observed between the clones, including the parent strain, were qualitative, while those observed within the subgroups (attached versus nonattached) of a clone were quantitative. The monoclonal antibody directed against the 170-kDa antigen of the WB (A6) clone as described by Nash et al. (23, 24) recognized
means.
CHARACTERISTICS OF G. LAMBLIA CLONES
VOL. 60, 1992
TABLE 2. Gerbil duodenum villus-to-crypt ratios
ATTACHED
No. of trophozoites inoculated an inoulaed and condition of animal
Infected
Uninfected 106a Infected
0 **
x
Uninfected
la 0 0
a
NON-ATrACHED
.)
2279
Villus-to-crypt ratio after inoculation with trophozoites Nonattached Attached
F-
F+
F-
F+
3.0 ± 0.4 (n =6) 0.0
4.3 ± 0.1 (n =2) 7.3 ± 0.1 (n =4)
4.4 ± 0.1 (n =4) 6.5 ± 0.1 (n =2)
5.0 + 0.1 (n =2) 8.3 ± 0.2 (n =4)
3.2 ± 0.5 (n =6) 0.0
4.4 ± 0.8 (n =6) 0.0
4.1 ± 0.6 (n =6) 0.0
6.7 ± 1.0 (n =6) 0.0
Mean ± standard error of the mean for controls, 10.3 ± 0.1 (n
=
6).
L. 0
a.
**
104 Trophozoite inoculum FIG. 6. Numbers of G. lamblia trophozoites recovered from the small intestines of gerbils infected with 104 or 106 attached or nonattached trophozoites from clone F- or F+. Means standard errors of the mean. ±
tent with their high resistance to immobilization and killing by our polyclonal serum in vitro. When the inoculum size was increased in the F- clone (attached and nonattached trophozoites), the rate of parasite clearance as assessed by the duration of cyst excretion decreased, while that for the F+ clones increased. At a 106 trophozoite inoculum with the attached and nonattached TABLE 1. Small-intestine weight as a percentage of body weight in animals inoculated with cloned trophozoites as % of body wt after inoculation with trophozoites
Small-intestine wt No. of trophozoites inoculated and condition of animal
F+
F-
104a Infected
Uninfected
106 infectedb
3.6 0.1 (n =15) 2.5 (n =1) 4.5 0.1
(n =16) a b
Mean Mean
-
Nonattached
Attached
F+
F-
2.9
3.8
(n
(n
0.1 =12)
2.4
0.1
0.1 =7) 2.3 0.1 (n =9) 3.7 + 0.1 (n =16)
=4) 3.8 0.1 (n =16) (n
3.0 + 0.1 (n =6) 2.3 + 0.1 (n =10) 3.2 0.2 (n =16)
standard error of the mean for control animals, 2.2 0.1 (n standard error of the mean for control animals, 2.1 + 0.1 (n
= =
6). 6).
cells of the F+ clone, cyst excretion stopped at day 22 and day 24, respectively. However, 2 of the 10 animals in each group began to excrete cysts for an additional 6 to 8 days. It is tempting to suggest that in these four animals, some subset of the trophozoites evaded immunosurveillance and killing by the host. This suggestion is supported by the observation that when reisolated trophozoites from the F+ clone-infected host intestine were phenotypically characterized, three distinct immunofluorescent cell subpopulations were observed. Thus, it is possible that the host immune system, natural selection, and/or possible parasite gene rearrangements allowed the production of additional variants. The persistent infection observed with the F+ clone in some infected animals may therefore not have been a result of continued infection by trophozoites of the original phenotype but may have been due to different variants of this clone. The process of antigenic heterogeneity is not a new phenomenon in parasitic infections. It has been described for Tichomonas (4), Trypanosoma (13, 21), and Borrelia (16) spp. In each case, the organisms have undergone surface antigenic changes, making it possible for them to evade the host immune system during the parasitemia of the host. In this study, host immunoselection and parasite antigenic variation might have played roles in both the degree of infection and the duration of clearance. For example, the Fclone trophozoites, which were more resistant to immobilization and killing by antibody in vivo, were more infectious, sustained infections with higher trophozoite loads and cyst excretion rates, and elicited more host pathophysiology, while the F+ clone was less phenotypically stable and sometimes cleared more slowly. Other biologic differences were observed when cryopreserved cells were recultivated. The F- clones were more viable, attached better to glass, and adapted faster to the growth media. However, when the cell colonies had become established, the generation times were the same for trophozoites from both clones. The role of these findings in the host-parasite interaction is not known. In this study, we have confirmed the presence of antigenic heterogeneity in G. lamblia WB trophozoites and obtained clones with different membrane protein and antigenic profiles, infectivity, and virulence. Studies using these clones have led us to postulate that there is a direct correlation between resistance to antibody killing in vitro and parasite load and pathphysiology and between antigenic variability in vivo and the time required to clear trophozoites from the intestine.
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REFERENCES 1. Abaza, S. M., J. J. Sullivan, and G. S. Visvesvara. 1991. Isoenzyme profiles of four strains of Giardia lamblia and their infectivity to jirds. Am. J. Trop. Med. Hyg. 44:63-68. 2. Adam, R. D., A. Aggarwal, A. A. Lal, V. F. De La Cruz, T. McCutchan, and T. E. Nash. 1988. Antigenic variation of a cysteine-rich protein in Giardia lamblia. J. Exp. Med. 167:109118. 3. Aggarwal, A., and T. E. Nash. 1988. Antigenic variation of Giardia lamblia in vivo. Infect. Immun. 56:1420-1423. 4. Alderete, J. F., L. Suprun-Brown, L. Kasmala, J. Smith, and M. Spence. 1985. Heterogeneity of Trichomonas vaginalis and discrimination among trichomonal isolates and subpopulations with sera of patients and experimentally infected mice. Infect. Immun. 49:463-468. 5. Baum, K. F., R. L. Berens, R. H. Jones, and J. J. Marr. 1988. A new method for cloning Giardia lamblia, with a discussion of the statistical considerations of limiting dilution. J. Parasitol. 74:267-269. 6. Belosevic, M., G. M. Faubert, and J. D. Maclean. 1989. Disaccharidase activity in the small intestine of gerbils (Meriones unguiculatus) during primary and challenge infections with Giardia lamblia. Gut 30:1213-1219. 7. Belosevic, M., G. M. Faubert, J. D. Maclean, C. Law, and N. A. Croll. 1983. Giardia lamblia infections in Mongolian gerbils: an animal model. J. Infect. Dis. 147:222-226. 8. Bertram, M. A., E. A. Meyer, J. D. Lile, and S. A. Morse. 1983. A comparison of isozymes of five axenic Giardia isolates. J. Parasitol. 69:793-801. 9. Black, R. E., A. C. Dykes, S. P. Sinclair, and J. G. Walls. 1977. Giardiasis in day-care centers: evidence of person to person transmission. Pediatrics 360:486-491. 10. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 11. Craft, J. C. 1982. Experimental infection with Giardia lamblia in rats. J. Infect. Dis. 145:495-498. 12. Cross, G. A. M. 1978. Antigenic variation in Trypanosomes. Proc. R. Soc. London B Biol. Sci. 202:55-72. 13. Doyle, J. J., H. Hirumi, K. Hirumi, E. N. Lupton, and G. A. M. Cross. 1980. Antigenic variation in clones of animal-infective Trypanosoma brucei derived and maintained in vitro. Parasitology 80:359-369. 14. Faubert, G. M., M. Belosevic, T. S. Walker, J. D. MacLean, and E. Meerovitch. 1983. Comparative studies on the pattern of infection with Giardia spp. in Mongolian gerbils. J. Parasitol. 69:802-805. 15. Kean, B. H., D. C. William, and S. K. Luminais. 1979. Epidemic of amoebiasis and giardiasis in a biased population. Br. J. Vener. Dis. 55:375-378. 16. Kehl, K. S. C., S. G. Farmer, R. A. Komorowski, and K. K. Knox. 1986. Antigenic variation among Borrelia spp. in relapsing fever. Infect. Immun. 54:899-902. 17. Keister, D. B. 1983. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans. R. Soc. Trop. Med. Hyg. 77:487-488. 18. Leitch, G. J., G. S. Visvesvara, S. P. Wahlquist, and C. T. Harmon. 1989. Dietary fiber and giardiasis: dietary fiber reduces
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33. 34. 35.
rate of intestinal infection by Giardia lamblia in the gerbil. Am. J. Trop. Med. Hyg. 41:512-520. Meyer, E. A., and E. L. Jarrall. 198t). Giardiasis. Am. J. Epidemiol. 111:1-12. Moss, D. M., H. M. Mathews, G. S. Visvesvara, J. W. Dickerson, and E. M. Walker. 1990. Antigenic variation of Giardia lamblia in the feces of Mongolian gerbils. J. Clin. Microbiol. 28:254-257. Myler, P. J., A. L. Allen, N. Agabian, and K. Stuart. 1985. Antigenic variation in clones of Trypaniosoma brucei grown in immune-deficient mice. Infect. Immun. 47:684-690. Nash, T. E. 1989. Antigenic variation in Giardia lamblia. Exp. Parasitol. 68:238-241. Nash, T. E., A. Aggarwal, R. D. Adam, J. T. Conrad, and J. W. Marritt, Jr. 1988. Antigenic variation in Giardia lamblia. J. Immunol. 141:636-641. Nash, T. E., F. D. Gillin, and P. D. Smith. 1983. Excretorysecretory products of Giardia lamnblia. J. Immunol. 131:20042010. Nash, T. E., D. A. Herrington, M. M. Levine, J. T. Conrad, and J. W. Marritt. 1990. Antigenic variation of Giardia lamblia in experimental human infections. J. Immunol. 144:4362-4369. Nash, T. E., and D. B. Keister. 1985. Differences in excretorysecretory products and surface antigens among 19 isolates of Giardia. J. Infect. Dis. 152:1166-1171. Roberts-Thomson, 1. C., D. P. Stevens, A. A. F. Mahmoud, and K. S. Warren. 1976. Giardiasis in the mouse: an animal model. Gastroenterology 71:57-61. Shaw, P. T., R. E. Brodsky, D. 0. Lyman, B. T. Wood, C. P. Hibler, G. R. Healy, K. I. E. Macleod, W. Stahl, and M. G. Shultz. 1977. A community outbreak of giardiasis with evidence of transmission by a municipal water supply. Ann. Intern. Med. 87:426-432. Smith, P. D., F. D. Gillin, N. A. Kaushal, and T. E. Nash. 1982. Antigenic analysis of Giardia lamblia from Afghanistan, Puerto Rico, Ecuador, and Oregon. Infect. Immun. 36:714-719. Tsang, V. C. W., K. Hancock, S. E. Madison, A. L. Beatty, and D. M. Moss. 1984. Demonstration of the adult microsomal antigens from Schistosoma mnansoni and S. japoniicin (MAMA and JAMA). J. Immunol. 132:2607-2613. Tsang, V. C. W., K. Hancock, M. Wilson, D. F. Parmer, S. D. Whaley, J. S. McDougal, and S. Kennedy. 1986. Enzyme-linked immunoelectrotransfer blot technique (Western blot) for human T-lymphotropic virus type ll/lymphadenopathy-associated virus (HTLV-111/LAV) antibodies. Immunology series no. 15. Procedural guide. U.S. Department of Health and Human Services, Atlanta. Visvesvara, G. S., J. W. Dickerson, and G. R. Healy. 1988. Variable infectivity of human-derived Giardia lamblia cysts for Mongolian gerbils (Meriones unguiiclulatus). J. Clin. Microbiol. 26:837-841. Visvesvara, G. S., P. D. Smith, G. R. Healy, and W. R. Brown. 1980. An immunofluorescence test to detect serum antibodies to Giardia lamblia. Ann. Intern. Med. 93:802-805. Wolfe, M. S. 1978. Current concepts in parasitology. Giardiasis. N. Engl. J. Med. 298:319-321. Yardley, J. H., J. Takano, and T. R. Hendrix. 1964. Epithelial and other mucosal lesions of the jejunum in giardiasis: jejunal biopsy studies. Bull. Johns Hopkins Hosp. 115:389-406.
INFECTION AND IMMUNITY, June 1992, p. 2274-2280 0019-9567/92/062274-07$02.00/0 Copyright © 1992, American Society for Microbiology
Isolation of Two Giardia lamblia (WB Strain) Clones with Distinct Surface Protein and Antigenic Profiles and Differing Infectivity and Virulence IFEANYI A. UDEZULU,lt* GOVINDA S. VISVESVARA,1 DELYNN M. MOSS,' AND GORDON J. LEITCH2 Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 303102 Received 16 December 1991/Accepted 10 March 1992
To determine the relationship between antigenic profiles and pathogenicity among Giardia lamblia clones (WB strain), trophozoites were cloned by the technique of limiting dilution. The phenotype of each clone was determined by an indirect immunofluorescence test using a polyclonal rabbit anti-G. lamblia trophozoite serum made against the parent strain. Two clones were chosen for further studies: a highly fluorescent clone, F+, in which more than 95% of the trophozoites fluoresced, and a low-fluorescence clone, F-, in which fewer than 5% fluoresced. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and enzyme-linked immunotransfer blot studies of the membrane fractions of the two clones and parent strain revealed differences in both the total protein and antigenic profiles. A serum cytotoxicity test with the polyclonal serum showed that the F+ clones were more susceptible to immobilization and killing, while the majority of cells of the F- clones were resistant to such killing. Assessment of the infectivity of the two clones in the Mongolian gerbil animal model indicated that the F- clone more readily initiated infections, produced more cysts, had a higher intestinal trophozoite load, and produced a more severe clinical syndrome, while the F+ clone was less phenotypically stable in vivo and in some cases took longer to be cleared from the intestine. MATERIALS AND METHODS
Giardiasis is a disease caused by the infection of the proximal small intestine with the protozoan parasite Giardia lamblia. This enteropathogen exists in two distinct morphological forms: the motile trophozoite, or feeding stage, that colonizes the intestine and the infective cyst form that is voided with feces (19, 34). The major mode of transmission is via the oral route (19, 28, 34), usually in food or drink. However, person-to-person transmission in day-care centers and among homosexually active men has also been reported (9, 15). Although G. lamblia strains have been characterized by their isoenzyme (1, 8) and surface antigen (26, 29) profiles, little is known about the relationship between such profiles and strain pathogenicity. Nash and coworkers (2, 23, 24) have shown that G. lamblia trophozoites originating from a single cell demonstrate marked variability in their surface antigenic profiles. When clones of G. lamblia trophozoites were used to infect animals or human volunteers, the trophozoites that were recovered from the intestine showed changes in their surface antigenic profiles (3, 25). It is not yet known if such changes were a result of host-induced trophozoite selection or parasite gene rearrangement (22). Similarly, surface antigen variability has been shown to occur in Trypanosoma brucei, and such variability is believed to be involved in this protozoan parasite's evasion of the host's immune surveillance (12). Several animal models have been established to study Giardia infection and pathogenesis (7, 11, 27, 32). Here, we describe the surface protein and antigenic profiles of G. lamblia clones isolated from trophozoites (WB strain) and compare the virulence and infectivity of these clones in gerbils.
Trophozoite cultivation. G. lamblia trophozoites, uncloned (parent strain) and cloned WB strain (ATCC 30957) as well as a WB (A6) clone (obtained from T. E. Nash), were grown and maintained axenically at 37°C in filter-sterilized Diamond's TYI-S-33 medium as modified by Keister (17). G. lamblia clones. Trophozoites were cloned in a 96-well microtiter plate by the method of limiting dilution (5). This technique allowed us to obtain a dilution of 0.5 and 1.0 cell per 0.1 ml to ensure the growth of a trophozoite colony from a single cell. Medium containing either 0.5 or 1.0 cell from each well was pipetted into 2.0-ml vials, and the final volume was adjusted to 1.8 ml with Keister's medium. The vials were incubated at 37°C for 14 days and examined microscopically every other day, starting on day 3. Each positive clone was transferred to a 15-ml screw-cap sterile glass tube and recloned an additional four times. IIF. The phenotype of each clone was determined by an indirect immunofluorescence (IIF) test using polyclonal rabbit anti-G. lamblia trophozoite (uncloned WB parent strain) serum at a dilution of 1:64. Preimmune rabbit serum was used as a negative control (33). Each clone was characterized as either fluorescence negative (clone F-) or fluorescence positive (clone F+) depending on the percentage of fluorescent cells in a colony. Preparation of membrane-rich (MR) protein fractions. Axenic cultures of the WB-cloned, uncloned, and WB (A6) trophozoites were harvested in the log growth phase. Each harvest consisted of trophozoites attached to glass culture tubes, trophozoites not attached to tubes, and unsegregated trophozoites (combined attached and nonattached) except for the WB (A6) clone, from which only attached cells were harvested. The nonattached trophozoites were harvested by decanting the medium from several tubes and centrifuging the pooled medium to sediment the trophozoites. The attached trophozoites from these tubes were detached by
* Corresponding author. t Present address: Department of Biology, Winston-Salem State University, Winston-Salem, NC 27110.
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chilling tubes filled with 0.85% NaCl (saline) in wet ice for 5 min and then centrifuging the tubes at 250 x g for 10 min at 4°C. Each trophozoite pellet was washed three times in saline. Attached, nonattached, and unsegregated trophozoites were resuspended at 109 trophozoites per 1.6 ml in enzyme-stabilizing solution containing 1 mM EDTA, 1 mM 6-amino-n-caproic acid, and 1 mM dithiothreitol and stored in liquid nitrogen. Washed trophozoite suspensions were thawed at room temperature and further processed by the method described by Moss et al. (20). In brief, the trophozoites were lysed by snap freezing and thawing three times in a methanol-dry-ice slurry and a 37°C water bath. The lysates were fractionated by centrifugation at 4°C at 24,000 x g into cytosol and membrane fractions. The supernatant (cytosol) was removed, and the sediment (membrane) was collected. The sediments were washed three times in saline, solubilized overnight at 4°C in 8.0 M urea-0.05 M Tris HCI-0.3 M KCl-0.2 mM EDTA, pH 8.0, and mixed intermittently every 30 min with a vortex mixer. The sample was further solubilized by ultrasonic disruption with a W-375 Sonifier (Heat System-Ultrasonics, Inc., Plainview, N.Y.) at 40% power, 15% duty cycle, and pulse mode for 3 min at 4°C. After the sonicates were centrifuged at 24,000 x g for 30 min at 4°C, the supernatant solutions were collected, dialyzed, and concentrated against 40 mM Tris HCl-54 mM boric acid-1 mM EDTA, pH 8.3, with a YM-10 membrane (W. R. Grace and Co., Amicon Div., Danvers, Mass.). The dialyzed, MR protein fractions were labeled and stored in liquid nitrogen until needed. The protein contents of the MR protein dialysate fractions were determined by the method of Bradford (10) with dye reagent from Bio-Rad Laboratories (Rockville Centre, N.Y.) and human albumin and globulin from Sigma Chemical Co. (St. Louis, Mo.) as standards. SDS-polyacrylamide gradient gel electrophoresis. Electrophoretic analysis of the MR protein fractions was performed in a discontinuous buffer system as described by Moss et al. (20), except that the samples were diluted with 0.5 mM Tris HCl, pH 8.0, prior to sodium dodecyl sulfate (SDS) treatment. All MR protein fractions were treated with SDS under nonreducing conditions. The SDS-treated MR proteins were separated on 3 to 25% linear gradient polyacrylamide gels with 3% stacking gels at concentrations of 0.15 and 0.2 ,ug of protein per mm of lane for the silver stain and enzyme-linked immunotransfer blot (EITB), respectively. Silver staining was performed as described by Tsang et al. (30). EITB. After MR proteins were electrotransferred to nitrocellulose sheets as described by Tsang et al. (31), the sheets were exposed overnight at 4°C to either polyclonal rabbit anti-G. lamblia trophozoite (uncloned WB parent strain) serum or preimmune negative control serum at a dilution of 1:500. In another experiment, the nitrocellulose was exposed to monoclonal antibody 6E7 (courtesy of T. E. Nash) at a dilution of 1:1,000. Monoclonal antibody 6E7 was previously shown to be specific for the G. lamblia 170-kDa surface protein (23, 24). The nitrocellulose was washed to remove unbound antibodies and exposed to goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (1:1,000 dilution) (Bio-Rad Laboratories, Richmond, Calif.) or goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase for monoclonal antibody 6E7. SDS-treated high-molecular-weight protein (Bio-Rad) and low-molecular-weight protein standards (Pharmacia) were used in all electrophoretic separations. In all EITBs, protein
CHARACTERISTICS OF G. LAMBLIA CLONES
2275
standard lanes were stained with Aurodye solution (Janssen Life Sciences, Piscataway, N.J.). Serum cytotoxicity test. Trophozoites from each clone were suspended in Keister's medium and added to 96-well round-bottom microtiter plates containing the polyclonal serum to obtain a trophozoite concentration of 106 cells per well and a final serum dilution of 1:2. The plates were incubated in an anaerobic chamber at 37°C for 1 and 24 h. At the end of each test period, the cells were visualized microscopically and chilled in ice water. Samples were taken, and counts were made of live and dead cells (trypan blue exclusion) by using a hemocytometer. Gerbil infectivity studies. Young adult male Mongolian gerbils (Menones unguiculatus) obtained from Tumblebrook Farm (West Brookfield, Mass.) were used throughout. Before being infected, the animals were starved for 24 h. Eight gerbils in each of 12 groups were inoculated orally with 104, 105, or 106 attached or nonattached F- or F+ trophozoites suspended in 0.3 ml of saline. During the postinoculation period, the animals were housed individually in wire-floored cages. Feces were collected daily from each cage from day 3 postinfection through day 35. Each animal was killed, and the intestinal mucosa was scraped and examined for the presence of trophozoites. The fecal pellets were processed, and cysts were isolated by using a 0.75 M sucrose gradient flotation method (32). The daily cyst excretion was quantitated microscopically by using a hemocytometer. In another study, the animals were killed 14 days postinoculation. Both the body and small-intestine weights of each animal were measured to assess enteropooling (18). The proximal one-third of the small intestine was excised, and the contents were collected. The segment was then opened at the antimesenteric border, and the mucosa was removed. After the mucosa and intestinal contents were fixed in 1% formalin-saline and stained with methylene blue, trophozoites were counted with a hemocytometer. Histologic studies on tissue sections obtained from animals inoculated with 104 and 106 trophozoites were also performed. Animals were killed 14 days postinoculation, and 2.5-cm-long segments of proximal duodenum from 0.5 cm distal to the pylorus were removed. Tissue sections were fixed in neutral formalin, embedded in paraffin, and stained with hematoxylin and eosin. Reisolation of trophozoites from infected animals and IIF test. Two groups of five Mongolian gerbils each were infected with 106 trophozoites (attached and nonattached combined) from each clone. At 14 days postinfection, the animals were killed, a laparotomy was immediately performed, and the duodenum was excised. A longitudinal section was made through the duodenum, and the mucosa and luminal contents were harvested. A sample was immediately mounted on a slide and examined microscopically for the presence of trophozoites. Positive samples were then inoculated into 15-ml sterile screw-cap tubes containing warm Keister's medium with 50 ,ug of gentamicin, 1,000 IU of penicillin, 1,000 ,ug of streptomycin, and 5 ,ug of amphotericin B per ml. After 1 h, the recovered trophozoites were washed repeatedly in saline to reduce the chyme and finally suspended in phosphate-buffered saline, pH 7.2, for phenotypic characterization by the IIF test. It was not possible to perform the IIF test on trophozoites taken directly from the intestinal lumen owing to the high background fluorescence of the luminal content. Trophozoite viability assay and growth curve. In one group of studies, cryopreserved trophozoites of clones F- and F+ were recultivated in Keister's medium at a final concentra-
2276
UDEZULU ET AL.
tion of 104 trophozoites per ml in a 15-ml screw-cap sterile glass tube. Samples were observed on alternate days for 11 days starting from day 1, and the cell number and viability were determined by using trypan blue dye exclusion. Viability data were generated for both attached and nonattached trophozoites of clones F- and F+. In another group of studies, cells were suspended in Keister's medium at a concentration of 104 trophozoites per ml in duplicate 15-ml sterile screw-cap tubes. These cells were used for growth curve studies. Trophozoite counts were obtained at zero time and every 12 h thereafter until late stationary phase. In all of the groups, a total of six counts were taken, and the means of the counts were recorded. The cells were maintained at 37°C throughout the period of study. Statistical evaluations. When populations were homogeneous, either Student's t tests or one-way analysis of variance was used for statistical evaluation. However, when populations were nonhomogeneous, either Wilcoxon matched-pair signed-rank tests or Wilcoxon rank sum tests were employed. RESULTS
G. lamblia trophozoites (WB strain) were cloned by the technique of limiting dilution. This method enabled us to obtain a cloning efficiency of more than 37.5% for 0.5 cell per ml and 62.5% for 1.0 cell per ml. Immunofluorescence. All trophozoite cultures, cloned and uncloned, were phenotypically characterized by IIF. Two clones, a highly fluorescent clone, F+, in which more than 95% of the cells fluoresced, and a low-fluorescence clone, F-, in which fewer than 5% of the cells fluoresced, were chosen for further studies. SDS-polyacrylamide gradient gel electrophoresis. The protein profiles of MR fractions obtained from cloned and uncloned trophozoites, including those of the attached cells of the WB (A6) clone, were examined by SDS-polyacrylamide gradient gel electrophoresis. All exhibited in the silver-stained preparations a large number of bands ranging in molecular mass from 14 to 250 kDa. Obvious differences, however, were seen between the three clones. For example, the F- clone exhibited an intensely stained diffuse band at about 150 to 170 kDa that was not seen in the parent strain or in the F+ clone. The WB (A6) clone resembled the F- clone and also exhibited a marked diffuse band at 150 to 170 kDa (Fig. 1, lanes 7, 8, 9, and 10). EITB. Figure 2 shows the EITB profiles of the parent WB strain, F+ clone, F- clone, and WB (A6) clone trophozoites. Nitrocellulose sheets with electrotransferred proteins were exposed to the same polyclonal rabbit anti-Giardia trophozoite serum that was used in the IIF test. Characteristic banding patterns for each of the MR fraction preparations were seen. The anti-G. lamblia (WB) antibody recognized more bands in both the parent strain and the F+ clones than in the F- clones (Fig. 2). Clone F-, however, differed from the F+ clone and the parent strain in possessing a diffuse band with a molecular mass of approximately 150 kDa (Fig. 2, lanes 7, 8, and 9). The bands of the WB (A6) clone again resembled those of the F- clone. However, bands at 21 to 24, 33, 104, and 112 kDa were apparent in the F- clone but not in the WB (A6) clone sample (Fig. 2, lanes 7, 8, 9, and 10). Additionally, there were quantitative differences between the antigenic patterns of attached and nonattached cells of both clones. In the F- clone, bands at 33 and 55 kDa were more evident in the nonattached than in the attached
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samples (Fig. 2, lanes 8 and 9). Similarly, in the F+ clone, bands at 33, 36, and 42 kDa were more apparent in the nonattached samples than in the attached samples (Fig. 2, lanes 5 and 6). In another EITB, monoclonal antibody 6E7 recognized a distinct band at 150 to 170 kDa in the F- and WB (A6) clones but not in the F+ clone or the parent strain (Fig. 3). This monoclonal antibody revealed additional differences between attached and nonattached surface antigens of the Fclone by visualizing diffuse bands in the region of 220 to 250 kDa and a single band at 300 kDa in only the F- attached preparation (Fig. 3, lane 9). Infectivity. Oral inoculation of gerbils with attached and nonattached trophozoites of either F- or F+ clones resulted in reproducible patterns of infection as assessed by the number of cysts excreted, the number of trophozoites in the intestine, and the number of animals infected. Results indicated that with a small inoculum (104 trophozoites), the infection patterns of attached and nonattached cells of the F- clone differed significantly from those of the F+ clone (P < 0.001; Fig. 4a and b). With a large inoculum (106 trophozoites), the F- clone infections were cleared significantly faster than the F+ clone infections (P < 0.002; Fig. 4e and f). A one-way analysis of variance indicated that the number of cysts produced during the course of all the infections was greater in animals infected with the F- clone than in those infected with the F+ clone (Fig. 5). However, this difference was significant only at the lowest trophozoite number (104) (P < 0.01). A one-way analysis of variance also indicated that the total number of cysts excreted increased significantly with inoculum size. This increase was statistically significant for both groups of animals infected with attached (P < 0.01) and nonattached (P < 0.001) F- clone organisms
CHARACTERISTICS OF G. LAMBLI4 CLONES
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as well as for those infected with attached (P < 0.01) and nonattached (P < 0.03) F+ clone trophozoites. When the trophozoite loads of the animals were determined on day 14, gerbils infected with the F- clone had a higher trophozoite count than animals infected with the F+ clone. This difference was significant for the small inoculum (104 trophozoites) (attached, P < 0.02, and nonattached, P < 0.03; Fig. 6). In all groups at all inoculum sizes, the smallintestine weight normalized to the body weight was significantly greater in infected animals than in either noninoculated controls (P < 0.05) or inoculated but uninfected animals (104-trophozoite inoculum, P < 0.01 for clone Fand P < 0.02 for clone F+, and 106-trophozoite inoculum, P < 0.01 for clone F- and P < 0.01 for clone F+). In the infected animals, the F- clone infections were associated with significantly higher small-intestine weights normalized to body weight than were the F+ clone infections (104trophozoite inoculum, P < 0.001, and 106-trophozoite inoculum, P < 0.01; Table 1). As there were no significant differences in body weight between groups, increases in intestinal weight were considered indications of enteropooling. The subjective observation of fluid accumulation, particularly in the duodenum, supported this interpretation. Duodenal villus-to-crypt ratios were measured by using appropriately oriented villi in animals inoculated with either 10 or 106 trophozoites. In all groups, the villus-to-crypt ratios were lower in infected animals than in uninfected animals (104 and 106, P < 0.05; Table 2). The villus-to-crypt ratios of the F- clone-infected animals were significantly
2 3 4
5 6
7
891011
FIG. 3. EITB profiles of MR protein fractions of G. lamblia trophozoites using 170-kDa MAb 6E7 ascites (courtesy of T. E. Nash). Lanes: 1, unsegregated cells of the uncloned WB parent strain; 2, nonattached cells of the uncloned WB parent strain; 3, attached cells of the uncloned WB parent strain; 4, unsegregated cells of clone F+; 5, nonattached cells of clone F+; 6, attached cells of clone F+; 7, unsegregated cells of clone F-; 8, nonattached cells of clone F-; 9, attached cells of clone F-; 10, attached cells of WB (A6) clone; 11, molecular weight standards (sizes on the right in kilodaltons).
lower than those of the F+ clone-infected animals for all the corresponding groups (104 and 106, P < 0.05). Growth and viability studies. In another study, cryopreserved cells from the two clones were recultivated, and their viability, attachment to glass, and growth curves were determined. Results of trypan blue exclusion indicated that F- clones were initially more viable. Larger numbers of Fcells attached to the tubes, and the F- clone adapted to the growth medium faster. However, once the cell colonies had adapted to the medium, the generation time was the same for trophozoites from both clones. Reisolation of trophozoites from infected animals and IEF test. Trophozoites from gerbils infected with trophozoites of the F- and F+ clones were reisolated 14 days postinoculation and immediately processed for IIF studies with the same polyclonal serum that was used in the initial phenotypic characterizations. Results revealed that the phenotypic character of the F- clone was apparently conserved; fewer than 5% of the cells were fluorescence positive. Before being inoculated, the F+ clone was more than 95% immunofluorescence positive. After the F+ clone was reisolated from infected gerbils, the IIF test results showed three distinctly different phenotypic subpopulations of bright (5 to 10%), medium (15 to 20%), and nonfluorescent (65 to 70%) cells. The relative numbers of these three subpopulations varied with the animals from which the trophozoites were reisolated (n = 6).
2278
INFECTr. I MMUN .
UDEZULU ET AL.
a
Attached
ATTACHED
Non-attached
b
E FM F+
FF+
0 x
-aaL) a) Lx
a)
Cl) Cl)
4Ji
I
C) o~~~~~~~~~~~~~~
NON-ATTACHED
.0
0 o-
.0
E
i' ' 20
Po
0
1
'
10o
Days, Postinoculation
Trophozoite inoculum
FIG. 4. Percentages of gerbils excreting cysts daily following inoculation with attached or nonattached trophozoites from clone F- or F'. Inocula were of 104 (a and b), 105 (c and d), or 106 (e and f) trophozoites. Values are daily means.
FIG. 5. Total numbers of cysts excreted by gerbils during the period the animals were infected with attached or nonattached trophozoites from clone F- or F+. Means + standard errors of the
DISCUSSION
membrane antigens in both the F- and WB (A6) clones but not in the F+ clone or the parent strain. This observation suggests that the subpopulation from which the F- clone originated could represent a small fraction of the trophozoites in the parent strain, i.e., probably less than the estimated 10% that appeared to be phenotypically fluorescence negative. The next group of studies was conducted to determine whether these clones that were so distinct in their protein and antigenic profiles also differed in their infectivity and pathogenesis or if they demonstrated other biological differences. The Mongolian gerbil was used as the experimental host. Both G. lamblia cysts and trophozoites have been shown to infect this model (7, 14, 18, 32). The F- clone trophozoites were significantly more infectious and produced a more severe clinical syndrome as assessed by enteropooling (18) and reduction of the villus-to-crypt ratio (6, 35). Consistent with the apparent greater pathogenicity, infections with the F- clone produced a significantly larger parasite load on day 14, and infected animals excreted a larger number of cysts over the course of the infection than animals infected with the F+ clone. With a smaller inoculum, the attached cells of the F- clone were more infectious than the nonattached. To the degree that the in vivo and in vitro data can be compared, this greater infection and pathogenesis observed with F- clone trophozoites in vivo is consis-
Genetically controlled strain variation and antigenic variability within strains and clones originating from a single cell in G. lamblia trophozoites have been described previously (2, 23, 26). In this study, an IIF test to screen G. lamblia WB for antigenic heterogeneity revealed that the trophozoite populations were predominantly (90%) made up of fluorescent (F+) cells. The technique of limiting dilution was used to obtain low- and high-fluorescence clones from the parent WB strain. Substantial differences in the membrane proteins and antigens in these groups of trophozoites were noted in both the silver stain and EITB tests. The F+ clone closely resembled the parent strain. This result was expected, as the parent strain contained more than 90% immunofluorescent cells and the F+ clone cells were more than 95% fluorescence positive. The polyclonal antibody recognized fewer bands in the F- and the WB (A6) clones than in the F+ clone and the parent strain. The F- and WB (A6) clones shared bands. Overall, the differences observed between the clones, including the parent strain, were qualitative, while those observed within the subgroups (attached versus nonattached) of a clone were quantitative. The monoclonal antibody directed against the 170-kDa antigen of the WB (A6) clone as described by Nash et al. (23, 24) recognized
means.
CHARACTERISTICS OF G. LAMBLIA CLONES
VOL. 60, 1992
TABLE 2. Gerbil duodenum villus-to-crypt ratios
ATTACHED
No. of trophozoites inoculated an inoulaed and condition of animal
Infected
Uninfected 106a Infected
0 **
x
Uninfected
la 0 0
a
NON-ATrACHED
.)
2279
Villus-to-crypt ratio after inoculation with trophozoites Nonattached Attached
F-
F+
F-
F+
3.0 ± 0.4 (n =6) 0.0
4.3 ± 0.1 (n =2) 7.3 ± 0.1 (n =4)
4.4 ± 0.1 (n =4) 6.5 ± 0.1 (n =2)
5.0 + 0.1 (n =2) 8.3 ± 0.2 (n =4)
3.2 ± 0.5 (n =6) 0.0
4.4 ± 0.8 (n =6) 0.0
4.1 ± 0.6 (n =6) 0.0
6.7 ± 1.0 (n =6) 0.0
Mean ± standard error of the mean for controls, 10.3 ± 0.1 (n
=
6).
L. 0
a.
**
104 Trophozoite inoculum FIG. 6. Numbers of G. lamblia trophozoites recovered from the small intestines of gerbils infected with 104 or 106 attached or nonattached trophozoites from clone F- or F+. Means standard errors of the mean. ±
tent with their high resistance to immobilization and killing by our polyclonal serum in vitro. When the inoculum size was increased in the F- clone (attached and nonattached trophozoites), the rate of parasite clearance as assessed by the duration of cyst excretion decreased, while that for the F+ clones increased. At a 106 trophozoite inoculum with the attached and nonattached TABLE 1. Small-intestine weight as a percentage of body weight in animals inoculated with cloned trophozoites as % of body wt after inoculation with trophozoites
Small-intestine wt No. of trophozoites inoculated and condition of animal
F+
F-
104a Infected
Uninfected
106 infectedb
3.6 0.1 (n =15) 2.5 (n =1) 4.5 0.1
(n =16) a b
Mean Mean
-
Nonattached
Attached
F+
F-
2.9
3.8
(n
(n
0.1 =12)
2.4
0.1
0.1 =7) 2.3 0.1 (n =9) 3.7 + 0.1 (n =16)
=4) 3.8 0.1 (n =16) (n
3.0 + 0.1 (n =6) 2.3 + 0.1 (n =10) 3.2 0.2 (n =16)
standard error of the mean for control animals, 2.2 0.1 (n standard error of the mean for control animals, 2.1 + 0.1 (n
= =
6). 6).
cells of the F+ clone, cyst excretion stopped at day 22 and day 24, respectively. However, 2 of the 10 animals in each group began to excrete cysts for an additional 6 to 8 days. It is tempting to suggest that in these four animals, some subset of the trophozoites evaded immunosurveillance and killing by the host. This suggestion is supported by the observation that when reisolated trophozoites from the F+ clone-infected host intestine were phenotypically characterized, three distinct immunofluorescent cell subpopulations were observed. Thus, it is possible that the host immune system, natural selection, and/or possible parasite gene rearrangements allowed the production of additional variants. The persistent infection observed with the F+ clone in some infected animals may therefore not have been a result of continued infection by trophozoites of the original phenotype but may have been due to different variants of this clone. The process of antigenic heterogeneity is not a new phenomenon in parasitic infections. It has been described for Tichomonas (4), Trypanosoma (13, 21), and Borrelia (16) spp. In each case, the organisms have undergone surface antigenic changes, making it possible for them to evade the host immune system during the parasitemia of the host. In this study, host immunoselection and parasite antigenic variation might have played roles in both the degree of infection and the duration of clearance. For example, the Fclone trophozoites, which were more resistant to immobilization and killing by antibody in vivo, were more infectious, sustained infections with higher trophozoite loads and cyst excretion rates, and elicited more host pathophysiology, while the F+ clone was less phenotypically stable and sometimes cleared more slowly. Other biologic differences were observed when cryopreserved cells were recultivated. The F- clones were more viable, attached better to glass, and adapted faster to the growth media. However, when the cell colonies had become established, the generation times were the same for trophozoites from both clones. The role of these findings in the host-parasite interaction is not known. In this study, we have confirmed the presence of antigenic heterogeneity in G. lamblia WB trophozoites and obtained clones with different membrane protein and antigenic profiles, infectivity, and virulence. Studies using these clones have led us to postulate that there is a direct correlation between resistance to antibody killing in vitro and parasite load and pathphysiology and between antigenic variability in vivo and the time required to clear trophozoites from the intestine.
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