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Infection and Immunity, August 2004, p. 4763-4771, Vol. 72, No. 8
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.8.4763-4771.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Experimental Infections of Neonatal Mice with Cysts of Giardia lamblia Clone GS/M-83-H7 Are Associated with an Antigenic Reset of the Parasite

N. von Allmen,1 M. Bienz,2 A. Hemphill,1 and N. Müller1*

Institutes of Parasitology,1 Hematology, University of Berne, CH-3001 Berne, Switzerland2

Received 22 February 2004/ Returned for modification 21 March 2004/ Accepted 15 April 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transmission of the protozoan parasite Giardia lamblia from one to another host individuum occurs through peroral ingestion of cysts which, following excystation in the small intestine, release two trophozoites each. Many studies have focused on the major surface antigen, VSP (for variant surface protein), which is responsible for the antigenic variability of the parasite. By using trophozoites of G. lamblia clone GS/M-83-H7 (expressing VSP H7) and the neonatal mouse model for experimental infections, we quantitatively assessed the process of antigenic variation of the parasite on the transcriptional level. In the present study, variant-specific regions identified on different GS/M-83-H7 vsp sequences served as targets for quantitative reverse transcription-PCR to monitor alterations in vsp mRNA levels during infection. Respective results demonstrated that antigenic switching of both the duodenal trophozoite and the cecal cyst populations was associated with a massive reduction in vsp H7 mRNA levels but not with a simultaneous increase in transcripts of any of the subvariant vsp genes analyzed. Most importantly, we also explored giardial variant-type formation and vsp mRNA levels after infection of mice with cysts. This infection mode led to an antigenic reset of the parasite in that a VSP H7-negative inoculum "converted" into a population of intestinal trophozoites that essentially consisted of the original VSP H7 type. This antigenic reset appears to be associated with excystation rather than with a selective process which favors expansion of a residual population of VSP H7 types within the antigenically diversified cyst inoculum. Based on these findings, the VSP H7 type has to be regarded as a predominant variant of G. lamblia clone GS/M-83-H7 which (re-)emerges during early-stage infection and may contribute to an optimal establishment of the parasite within the intestine of the experimental murine host.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Giardia lamblia is an intestinal protozoan parasite of humans and various animals. Manifestation of the infection varies from asymptomatic carriage to severe diarrhea and malabsorption. G. lamblia has a two-stage life cycle. The nonproliferating, quadrinucleated cysts are ingested through consumption of contaminated freshwater or food. Passage of the cysts through the stomach triggers excystation. This excystation process continues in the small intestine, where cysts release two binucleated trophozoites each. The life cycle is completed when proliferating trophozoites convert into infective cysts. Cysts are excreted in the feces and are able to persist for a long time under humid conditions until they are ingested by a susceptible host.

In the last few years, multiple studies have addressed the ability of G. lamblia to alter its surface antigen properties (10, 12). These studies have revealed that antigenic variation is associated with a unique family of surface antigens, named VSP (for variant surface protein). VSP has a potential function in antigenic variation to circumvent the host immune response, and it may exhibit physiological functions important for the intestinal survival of the parasite within the host (14). Surface antigen alterations were observed within proliferating populations of intestinal trophozoites (2, 4, 6, 7, 19), as well as among individual trophozoites upon the release from nonproliferative cysts (9, 21). The latter phenomenon was investigated by monitoring vsp gene expression during in vitro en- and excystation of G. lamblia isolate WB, clone C6 (21). This investigation demonstrated that major surface antigen VSP C6 predominates in cysts but is downregulated during the process of excystation. Conversely, in vitro-maintained G. lamblia clone GS/M-83-H7 (expressing VSP H7) did not change its surface antigen properties during life cycle stage transition (21).

Antigenic variation has been extensively studied by using the G. lamblia clone GS/M-83-H7 as a model parasite. The VSP repertoire of this clone was estimated to be encoded by about 60 to 80 distinct genes (13). Experimental infections in a combined mother-offspring mouse system revealed that a predominant VSP H7-type population can be replaced by a mixture of different new variant types during infection (11). This initial antigenic switching of the parasite population inside the suckling offspring is under the growth-selective influence of lactogenic anti-VSP H7 antibodies maternally produced in response to the parasite infection (19).

By using G. lamblia clone GS/M-83-H7 and the mother-offspring mouse model for experimental infections, we recently investigated the process of antigenic variation of the parasite on the molecular level (4). For this purpose, trophozoites collected from the intestines of individual animals at different time points postinfection (p.i.) were analyzed directly for their vsp gene transcription patterns, i.e., without cultivating the recovered parasites in vitro. This was done by using a combined 5' rapid amplification of cDNA ends (5'RACE) reverse transcription-PCR (RT-PCR) approach which allowed detection, and subsequent sequence analysis, of vsp gene transcripts after the generation of amplified cDNA analogues. The same PCR approach was applied for analysis of vsp gene transcripts in variants obtained after negative selection of axenic trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. In an overall view on the entire panel of in vivo- and in vitro-derived parasite populations, the transcription of 29 different vsp genes was demonstrated.

In the present study, the immunofluorescence method and a quantitative 3'RACE RT-PCR approach were applied to investigate variant-type formation and levels of vsp gene transcripts during the course of a G. lamblia GS/M-83-H7 infection in mice. The contents of vsp transcripts in intestinal trophozoite populations were analyzed after experimental infection of mice with trophozoites. Respective analyses were performed by intragastric inoculation of mice with trophozoites but also for the first time by intragastric inoculation of the animals with cysts, thus approaching conditions that model a natural route of infection.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Gravid 10- to 12-week-old outbred CD-1(ICR)BR mice were obtained from Charles River GmbH, Sulzfeld, Germany. Animals were kept according to the Swiss regulations of animal experiments with free access to germfree food and sterile water.

Parasite. The origin, axenization, and cloning of G. lamblia clone GS/M-83-H7 has been described by Aggarwal et al. (1). This clone expresses a major 72-kDa antigen (VSP H7) on its surface that is recognized by monoclonal antibody (MAb) G10/4. Trophozoites from G. lamblia clone GS/M-83-H7 were cultivated in modified TYI-S-33 medium with antibiotics as previously described (8).

Experimental parasite infection, sample collection, and total RNA extraction. Three-day-old offspring and respective mothers were infected with 5 x 104 in vitro-cultivated G. lamblia clone GS/M-83-H7 trophozoites as previously described (20).

The in vivo-derived trophozoite inoculum was prepared as follows. Sections of about 1 cm from the upper part of the duodenum were slit longitudinally and subsequently incubated for 20 min in 1 ml of phosphate-buffered saline (PBS; containing 0.15 M NaCl [pH 7.2]) on ice to detach trophozoites from the intestinal surface. Then, 0.5 ml of PBS supernatant containing detached trophozoites (ca. 5 x 104 to 105 parasites) was transferred into a well of a microtiter plate (Cellstar TC-Plate, 24 wells; Greiner Labortechnik GmbH, Frickenhausen, Switzerland), and viable parasites were allowed to adhere to the bottom of the well by incubation for 20 min at 37°C. After two washes with 1 ml of prewarmed (37°C) PBS, the trophozoites were detached from the bottom of the well by a 15-min incubation step on ice. These trophozoites were further processed (i.e., without prior expansion of the recovered parasite populations by in vitro cultivation) for infection of mice as previously described (20).

The in vivo-derived cyst inoculi were prepared as follows. Cecal content from six mice per experimental time point was pooled in 10 ml of distilled H2O, and cysts were purified and concentrated by using sucrose flotation according to the method of Belosevic et al. (3). Briefly, this method is based on the principle that, after centrifugation in a highly concentrated sucrose solution (1.12 g/cm3), the intestinal content separates into a precipitate consisting of high-density fecal particles, suspended medium-density particles, and low-density particles such as giardial cysts floating on the surface of the solution. The floating cysts were collected, and residual trophozoites were removed from water-resistant cysts through hypoosmotic lysis by repeated incubation and washing in distilled H2O. Finally, cysts were resuspended at a concentration of 5 x 105 parasites per ml of distilled H2O, and 50 µl (corresponding to ca. 2.5 x 104 cysts) from this suspension was used as an inoculum.

Total RNA from in vitro-cultivated GS-M-83-H7 trophozoites was isolated as previously described (4). For total RNA extraction (see below) from intestinal trophozoites, parasites were isolated as follows. Sections of about 1 cm from the upper part of the duodenum were slit longitudinally and subsequently incubated for 20 min in 1 ml of PBS on ice to detach trophozoites from the intestinal surface. Then, 0.5 ml of PBS supernatant containing detached trophozoites (ca. 104 to 105 parasites) was transferred into a well of a microtiter plate (Cellstar TC-Plate), and viable parasites were allowed to adhere to the bottom of the well by incubation for 20 min at 37°C. After three washes with 1 ml of prewarmed (37°C) PBS, residual, adherent trophozoites were resuspended in 100 µl of lysis buffer-ß-mercaptoethanol mixture from the StrataPrep Total RNA Microprep kit (Stratagene, La Jolla, Calif.). For total RNA extraction (see below) from cysts, ca. 5 x 104 to 105 parasites (for concentrated cyst samples, see above) were resuspended in 1 ml of QIAzol lysis reagent from the RNeasy lipid minikit (Qiagen, Basel, Switzerland). The lysates from both trophozoites and cysts were processed for extraction of total RNA as instructed, including treatment with RNase-free DNase I. Finally, total RNA preparations (solubilized in 50 µl of RNase-free distilled H2O) were stored at –80°C until further use.

Analysis of vsp mRNA by quantitative RT-PCR. cDNA was synthesized by RT from total RNA, prepared from intestinal trophozoite and cyst populations by using 17.5 µM T-ANC primer (Invitrogen, Basel, Switzerland) (see Fig. 1), as well as Moloney murine leukemia virus reverse transcriptase (Promega, Madison, Wis.) and other components as instructed by the manufacturer of the reverse transcriptase.



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  		FIG. 1. Schematic illustration of the 3'RACE RT-PCR-based quantification of vsp mRNA. First, mRNA (within total RNA preparations) from G. lamblia clone GS/M-83-H7 trophozoites or cysts was reverse transcribed into cDNA by using Moloney murine leukemia virus reverse transcriptase and the T-ANC primer. This primer contains a 3'-terminal oligonucleotide (dT) stretch (upper cases) which anneals to the poly(A) tail (lower case) of the mRNA, and a 5'terminal anchor (ANC) sequence as indicated. The T-ANC primer consists of a mixture of molecules containing either base A, or C, or G (V = A/C/G) at the very 3'-terminal position in order for the primer to anneal to the inner end of the poly(A) tail. The cDNA was taken as a template for a 3'RACE PCR which allowed specific amplification of vsp cDNA molecules. Amplification of vsp cDNA was achieved by performing the PCR with forward vsp primers listed in Fig. 2A or forward primer MM16 (targeted to a highly conserved 3'-terminal sequence of the GS/M-83-H7 vsp genes [see reference 15]) and a reverse anchor (ANC) primer as indicated. For amplification of cDNA from "housekeeping" gene gdh, forward gdh primer (see Materials and Methods) was used instead of a forward vsp primer.

 
Quantitative RT-PCR was carried out on a LightCycler (Roche Diagnostics, Rotkreuz, Switzerland) by using SYBR Green I as a double-stranded DNA (dsDNA)-specific fluorescent dye and continuous fluorescence monitoring as previously described (22). Amplifications of cDNA molecules representing analogues of mRNA from either different vsp genes or "housekeeping" gene gdh (coding for glutamate dehydrogenase) were performed by 3'RACE PCR (see Fig. 1). Amplification reaction mixes included forward primers either specific for individual vsp genes and/or gene groups (indicated in Fig. 1 as forward vsp primer and listed in Fig. 2A) or complementary to the MM16 vsp region (primer sequence, 5'-GGCTTCCTCTGCTGGTGGTTC-3'), which has been assumed to be shared by the entire GS/M-83-H7 vsp gene repertoire (15), or specific for gdh (5'-CCTCAAGTTCCTCGGC-3') and reverse anchor (ANC) primer as indicated in Fig. 1. Quantitative PCR was done with 4 µl of 1:100-diluted (cDNA prepared from trophozoites) or 1:10-diluted (cDNA prepared from cysts) cDNA (see above) by using the Quanti-Tect SYBR Green PCR kit (Qiagen) in 10 µl of standard reaction containing a 0.5 µM concentration of forward and reverse primers (Invitrogen). All PCRs containing cDNA were performed either in triplicates (with cDNA from trophozoite and cyst pools, see Fig. 3) or in duplicates (with cDNA of trophozoites sampled from individual animals) (see Fig. 4 and 6). Furthermore, a control PCR included RNA equivalents from samples that had not been reverse transcribed into cDNA (not shown) to confirm that no DNA was amplified from any residual genomic DNA that might have resisted DNase I digestion (see above). PCR was started by initiating a "hot-start" Taq DNA polymerase reaction at 95°C (15 min). Subsequent DNA amplification was done in 50 cycles consisting of 7 cycles of denaturation (94°C for 15 s), annealing with a stepwise temperature shift (2°C per cycle [first cycle at 48°C, second cycle at 50°C, etc., to the sixth cycle at 60°C]; with each cycle lasting 30 s), and extension (72°C for 30 s) and an additional 43 cycles of denaturation (94°C for 15 s), annealing (60°C for 30 s), and extension (72°C for 30 s). The temperature transition rates in all cycle steps were 20°C/s. Fluorescence was measured at 82°C during the temperature shift after each annealing phase in the "single" mode with the channel setting F1. Fluorescence signals from the amplification products were quantitatively assessed by applying the standard software (version 3.5.3) of the LightCycler. Quantification of PCR products was performed during the log phase of the reaction and was achieved by using the secondary derivative maximum mode for plotting of the fluorescence signals versus the cycle numbers. As external standards, serial 10-fold dilutions (4-µl aliquots) of previously generated amplification products from the different target sequences were included in the quantitative PCR analyses. The standard curves from the different assays (vsp- and gdh-PCRs) were run in duplicates and contained 4 log units within a linear range that essentially covered the maximal and minimal concentrations of the vsp- and gdh-cDNA sequences within the different samples. Linearity among the standard reactions was reflected by the correlation coefficient that was calculated by the computer program to be extremely high (between 0.97 and 1.0) for all PCR assays applied. Furthermore, the efficiencies of the quantitative PCRs were revealed to be nearly identical and exhibited high amplification rates that ranged between 1.81 (with forward vsp H7 primer [see below]) and 2.08 (with forward vsp V12 primer [see below]) per cycle.



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  		FIG. 2. Determination of target sequence (A) and specificity (B) of forward vsp primers used for quantitative RT-PCR-based assessment of relative vsp mRNA levels in different G. lamblia GS/M-83-H7 populations (see Fig. 3, 4, and 6). (A) Primers specific for different vsp genes are targeted to a relatively variable vsp stretch as identified by alignment of nucleotide sequences from 3'-terminal regions of vsp H7 (indicated as H7) and subvariant GS/M-83-H7 vsp genes that had previously been described (see reference 4). The figure shows nucleotide (nt) positions of partial sequences from cDNA clones that had been isolated from either trophozoites representing parasite populations at days 0 (original inoculum) (clone H7, clone 0a), 7 (clones 7a to 7c), 14 (clones 14a to 14g), 21 (clones 21a to 21f), and 42 (clones 42a to 42e) p.i. or trophozoites representing an in vitro-switched parasite population (clones IVa to IVh). The asterisk labeling clone 14d in box V8 indicates that this clone shares 100% sequence identity with clones 21b and IVd that are both not included in the figure. Positions demonstrating a high (light gray type), medium (dark gray type) or low (boldface) degree of similarity among vsp H7 and the corresponding nt regions from the subvariant vsp genes identified, as well as the highly diversified target sequences (open boxes) of forward vsp primers (see Fig. 1) are indicated. Dashes within the sequences indicate alignment gaps. Individual vsp genes, or groups consisting of closely related vsp genes with identical forward vsp primer target sequences (H7 and V1 to V18), are indicated within boxes in front of the sequences. Behind the sequences, corresponding GenBank accession numbers are given. The specificity of forward vsp H7-specific primer H7a (amplifying a partial sequence of vsp gene H7) and forward subvariant vsp-specific primers (amplifying a partial sequence of vsp genes V17, V12, and V18) were assessed by applying each of these primers in combination with reverse ANC primer (see Fig. 1) in a quantitative PCR with an equimolar (1 nM) mixture of molecules (4-µl aliquots) of previously generated amplification products from the vsp sequences H7, V17, V12, and V18 as a template (B). These PCRs turned out to be vsp gene- or gene group-specific because each of the reactions exclusively amplified the corresponding vsp gene sequence (vsp H7, V17, V12, or V18) from the template mixture. The template mixture was also included in PCRs with forward primer MM16 (targeted to a highly conserved 3'-terminal sequence of the GS/M-83-H7 vsp genes [see reference 15]) that was subsequently used to monitor total vsp mRNA levels [V(tot)] in G. lamblia GS/M-83-H7 populations (see Fig. 3). The PCR amplification rates are given as arbitrary amplification units and represent logarithmic mean values (plus standard deviations) from triplicate determinations.

 


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  		FIG. 3. Quantitative RT-PCR-based assessment of relative vsp mRNA levels (vsp mRNA level/gdh mRNA level) in duodenal trophozoite (A) and cecal cyst (B) pools composed of samples from six G. lamblia clone GS/M-83-H7-infected mice (infection with in vitro-cultivated trophozoites). Samples were taken at days 0 (inoculum), 7, 14, and 21 p.i. as indicated. The relative levels of mRNA from vsp genes or gene groups H7, V17, V12, and V18 (see Fig. 2A), as well as relative levels of total vsp mRNA [V(tot)] represent logarithmic mean values (plus standard deviations) from triplicate determinations.

 


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  		FIG. 4. Quantitative RT-PCR-based assessment of relative vsp mRNA levels (vsp mRNA level/gdh mRNA level) in duodenal trophozoite samples taken from six G. lamblia clone GS/M-83-H7-infected mice (infection with in vitro cultivated trophozoites) at days 7 (A), 14 (B), and 21 (C) p.i. The relative levels of mRNA from vsp genes or gene groups H7, V17, V12, and V18 (see Fig. 2A) represent logarithmic mean values from duplicate determinations.

 


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  		FIG. 6. Quantitative RT-PCR-based assessment of relative vsp mRNA levels (vsp mRNA level/gdh mRNA level) in mice upon infection of mice with cysts and trophozoites. Three mice were infected with in vivo-derived VSP H7-type (isolated in the previous infection experiment at day 7 p.i.,) (A, open circle) and non-VSP H7-type (isolated in the previous infection experiment at day 21 p.i.) (B, open circle) cysts, or with in vivo-derived non-VSP H7-type trophozoites (isolated in the previous infection experiment at day 21 p.i.) (C, open circle). From these mice, duodenal trophozoites (closed circles) were sampled at day 7 p.i. and analyzed by quantitative RT-PCR for relative levels of mRNA from vsp genes or gene groups H7, V17, V12, and V18 (see Fig. 2A). Relative vsp mRNA levels are given as logarithmic mean values from triplicate (cyst inocula) or duplicate (duodenal trophozoites isolated from infected animals) determinations.

 
Lack of PCR-inhibitory effects and overall comparability of the different standard and sample reactions were evidenced by the quasi-identity of the slopes from the amplification plots (monitoring amplification rates).

Control experiments for identification of PCR products included a DNA melting-point analysis (16; data not shown). The DNA melting profile assay was run after the final PCR cycle by gradually increasing the temperature to 95°C at a transition rate of 0.1°C/s with continuous acquisition (determination of the melting profile by measuring loss of fluorescence). The data from the DNA melting profile assay were processed by using the standard software (version 3.5.3). In all PCR tests performed, identical melting temperatures of amplicons from samples and respective standards indicated identical and specific amplification reactions without unwanted primer-dimer formation (not shown). This overall identity and specificity of reactions was confirmed by subsequent agarose gel electrophoresis (2% gels) (17) that monitored the PCR products as single DNA bands of expected sizes (not shown). In the cases of the analyses of the trophozoite and cyst pool samples shown in Fig. 3, positions H7 and V17, nucleotide sequence authenticity of the amplification products of the vsp H7- and vsp V17-specific PCRs was confirmed by automated sequencing through a commercial sequencing service (Mycrosynth, Balgach, Switzerland).

In order to compensate for variations in input RNA amounts and efficiencies of RT, mRNA of the housekeeping gene gdh was quantitated. Respective mean values from triplicate (pool samples [see above]) or duplicate (samples from individual animals [see above]) determinations were taken for the calculation of the relative vsp mRNA levels (vsp-mRNA level/gdh mRNA level).

Specificity of forward vsp H7-specific primer H7a (amplifying partial sequences of vsp gene H7, see Fig. 2A) and forward subvariant vsp-specific primers (amplifying partial sequences of vsp genes V17, V12, and V18, see Fig. 2A) were assessed by applying each of these primers in combination with reverse ANC primer (see Fig. 1) in a quantitative PCR with an equimolar (1 nM) mixture of molecules (4-µl aliquots) of previously generated amplification products from the vsp sequences H7, V17, V12, and V18 as a template (see Fig. 2B). The specific character of the PCRs with the forward vsp primers H7a, V17, V12, or V18 was determined by demonstrating that each of the reactions exclusively amplified the corresponding vsp gene sequence from the template mixture. The specificity of the different amplification products was assessed by comparative DNA melting-point analysis (see above). The above-mentioned template mixture was also used for a quantitative PCR with forward primer MM16 annealing to a highly conserved region close to the 3' terminus of the GS/M-83-H7 vsp genes (15) (see Fig. 2B). This amplification reaction differed from the PCRs listed above in that it was performed with a slightly modified temperature profile that included measurement of the fluorescence signal at 72°C (instead of 82°C [see above]) after each annealing phase.

Sequence alignments. Alignment of the vsp nucleotide sequences derived from a previous study (4) and accessible in GenBank (for accession numbers, see Fig. 2A) was done by using MultAlin and ESPript1.9 computer software, which are available at the ExPASy molecular biology server.

Immunofluorescence assays. The kinetics of expression of the major surface antigen on trophozoites isolated from the duodenum of experimentally infected mice (see below) were assessed for immunofluorescence by using MAb G10/4 as described previously (6). For staining of nuclei, trophozoites were incubated for 3 min in presence of the 1:300-diluted dsDNA-specific fluorescent dye Hoechst 33258 (Sigma, Steinheim, Germany) stock solution (1 mg/ml in distilled H2O) and subsequently washed twice in PBS and once in distilled H2O.

In order to perform the immunofluorescence assays with cysts, ca. 5 x 104 to 105 parasites (for discussion of concentrated cyst samples, see above) were fixed in 100 µl of 3% paraformaldehyde solution (in PBS) for 1 h at room temperature, followed by one wash in PBS, and a 3-h incubation with 100 mM glycine in PBS. Fixed cysts were permeabilized with 0.1% Triton X-100 in PBS for 1 h and blocked for 2 h in 5% bovine serum albumin (BSA) in PBS. Fixed and permeabilized cells were incubated with MAb G10/4 diluted 1:100 in 5% BSA-0.1% Triton X-100 in PBS for 1 h. After three washes with ice-cold PBS, the cysts were incubated for 1 h with goat anti-mouse immunoglobulin G-fluorescein isothiocyanate (Fc specific; Sigma). Cysts were then washed three times in ice-cold PBS and incubated in presence of 1:20 Texas red-conjugated mouse MAb A300-TR (Waterborne, New Orleans, La.), an anti-cyst wall protein antibody.

Specimens containing trophozoites and cysts were inspected on a Nikon Eclipse E800 digital confocal fluorescence microscope at a 600-fold magnification. Processing of the images was performed by using the Openlab 3.11 software (Improvision, Heidelberg, Germany). The percentages of VSP H7-positive versus negative parasites were determined by inspection of 300 parasites. Since the immunofluorescence analyses of cysts included several washing steps, which reduced the final recovery of parasitic material (<100 cysts per specimen), often only a few parasites (<10 cysts per microscopic field) were detected within an individual specimen. In these cases, more than one specimen was inspected.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunofluorescence-based assessment of in vivo antigenic switching of G. lamblia clone GS/M-83-H7 after infection of mice with in vitro-cultivated trophozoites. In order to study in vivo antigenic switching of G. lamblia clone GS/M-83-H7, we used a previously described protocol (19) for infection of 3-day-old murine offspring and respective mothers with trophozoites. From these animals, we collected duodenal trophozoites as well as cecal cysts from 6 animals at days 0 (representing the original inoculum), 7 and 14 (representing parasite populations at time points during the lactation phase of mice), and 21 (representing parasite populations at a time point after the lactation phase of the animals) p.i. Immunofluorescence analysis with VSP H7-specific MAb G10/4 (19) demonstrated that both trophozoites from the inoculum (representing day 0 p.i.) and the trophozoite populations isolated from the duodenum at day 7 p.i. were mostly (ca. 92 to 95%) VSP H7 positive. In contrast, the duodenal parasite populations recovered after day 7 p.i., i.e., on days 14 and 21 p.i., only possessed few (ca. 1% at day 14 p.i.) or no (0% at day 21 p.i.) detectable VSP H7-type trophozoites. These results are in agreement with our previous findings (4), indicating that antigenic switching of duodenal GS/M-83-H7 trophozoite populations in offspring occurred between day 7 and 14 p.i. Immunofluorescence testing of cecal cyst populations in the present study revealed ca. 81% VSP H7-type parasites at day 7 p.i. and no detectable parasites of this variant-type at days 14 and 21 p.i. This observation demonstrated that in vivo antigenic switching of the trophozoite and corresponding cyst populations occurred between day 7 and 14 p.i.

3'RACE RT-PCR-based quantification of vsp mRNA levels associated with in vivo antigenic switching of G. lamblia clone GS/M-83-H7 after infection of mice with in vitro-cultivated trophozoites. For 3'RACE RT-PCR-based quantitative assessment of vsp mRNA levels in different G. lamblia clone GS/M-83-H7 populations during in vivo antigenic switching, duodenal trophozoite and cecal cyst pools were generated by combining respective parasite populations from six animals representing the experimental time points at days 7, 14, and 21 p.i. From the different sample groups, total RNA was prepared and then used as a template for cDNA synthesis (Fig. 1). The cDNA molecules were generated by applying an oligo(dT) primer containing an anchor sequence at its 5' end. In order to establish a PCR system for quantitative and differential vsp gene amplification, 3' regions of gene vsp H7 and subvariant vsp genes from clone GS/M-83-H7 were subjected to a nucleotide sequence alignment (Fig. 2A). This comparative sequence analysis led to the identification of a relatively variable vsp region, which allowed us to design forward primers that were specific for either individual vsp genes or at least small groups consisting of two to four closely related vsp genes. Each of these forward vsp primers in combination with universal reverse ANC primer (see Fig. 1) were used for PCR-based specific quantification of corresponding vsp cDNA molecules generated from the different trophozoite and cyst samples outlined above. In order to validate our approach, specificities of experimentally most relevant (see below) forward vsp primers amplifying a partial sequence of either vsp H7 (sequence H7a; see Fig. 2A) or subvariant vsp genes V17, V12, and V18 (see Fig. 2A) were assessed. This was achieved by testing each of the four primers plus the reverse ANC primer in a quantitative PCR with an equimolar mixture of previously generated amplification products from the vsp sequences H7a, V17, V12, and V18 as a template. The specificities of the primers were confirmed in that the individual PCRs amplified the homologous, but not the three heterologous, vsp sequences within the template mixture (Fig. 2B). Respective amplification rates were ca. 5.0 (vsp V17-specific PCR) to 2.3 (vsp H18-specific PCR) times lower than the amplification rate achieved with forward primer MM16 (see above and reference 15) annealing to all four vsp sequences included in the template mixture.

By applying different forward vsp primers and reverse ANC primer for the quantification of the individual vsp cDNAs, we assessed the relative amounts of vsp mRNA within intestinal trophozoite and cyst populations that had consecutively emerged in infected offspring (Fig. 3 and 4). Analyses of trophozoites were performed with both pool samples and samples separately collected from individual animals. Conversely, cysts were exclusively analyzed as pool samples because the extremely low mRNA content of the cysts did not allow differential RT-PCR-based testing of individual parasite populations. As can be seen for both pool samples (Fig. 3) and samples from individual animals (Fig. 4), duodenal trophozoites at day 7 p.i. contained >500 times more vsp H7 mRNA than trophozoites at days 14 and 21 p.i. In an analogous assay of the corresponding cyst pools, samples taken at day 7 p.i. exhibited >250 more vsp H7 mRNA than those taken at days 14 and 21 p.i. These findings confirmed that antigenic switching had occcured between day 7 and 14 p.i. on the transcriptional level.

An overview on the non-vsp H7 gene transcription in both VSP H7- and non-VSP H7-type parasites (trophozoites and cysts) revealed that mRNA derived from vsp genes and/or gene groups V17, V12, and V18 was synthesized on an extremely low level (Fig. 3). In this case, mRNA production was determined to be >200 (trophozoites) or >150 (cysts) times lower than vsp H7 mRNA production in VSP H7-type populations. Within the pooled trophozoite and cyst samples, the mRNA levels derived from the other subvariant vsp genes and/or gene groups (V1 to V11 and V13 to V16 [see Fig. 2A]) included in the study (not shown) turned out to be consistently below the mRNA levels derived from vsp genes or gene groups H7, V17, V12, and V18. As assessed by RT-PCR with a primer complementary to the highly conserved vsp region MM16 (15), total vsp mRNA levels (Vtot) were consistently high in all trophozoite (Fig. 3A) and cyst (Fig. 3B) populations investigated. Respective values were similar to those determined for the vsp H7 transcripts in VSP H7-type trophozoite or cyst populations.

Taken together, the above-mentioned results indicated that downshift of the vsp H7 mRNA level during in vivo antigenic switching of both trophozoites and cysts of G. lamblia clone GS/M-83-H7 was not associated with a significant upshift of the mRNA levels derived from the 18 subvariant vsp genes, or gene groups, tested.

Immunofluorescence-based assessment of in vivo antigenic switching of G. lamblia clone GS/M-83-H7 after infection of mice with in vivo-derived cysts and trophozoites. In a further experiment, we infected 3-day-old murine offspring and respective mothers with cecal cysts which had been sampled at days 7 (identified as VSP H7-type cysts [see above]) and 21 (identified as non-VSP H7-type cysts [see above]) p.i. from mice that belonged to the previous G. lamblia GS/M-83-H7 infection experiment (see above). In parallel, a control infection with non-VSP H7-type duodenal trophozoites sampled at day 21 p.i. of the previous infection experiment was performed. In order to exactly monitor the initial parasite populations emerging during these infections, three animals per experimental group were sacrificed daily between days 1 and 7 p.i. and were assessed for the presence of duodenal trophozoites. For all three types of infection, this monitoring exhibited detectable numbers of parasites starting from day 6 p.i. and substantial amounts of parasites (>104 trophozoites per 1 cm of duodenal section) at day 7 p.i. As can be seen in the immunofluorescence assay shown in Fig. 5, infection of mice with VSP H7-type cysts (Fig. 5Aa) resulted in an initial duodenal trophozoite population (at day 7 p.i.) that was essentially VSP H7 positive (91 to 94% positivity) (Fig. 5Ba). Surprisingly, a similar VSP H7-type positivity (90 to 92%) (Fig. 5Bb) was also observed within the initial duodenal trophozoite population that resulted from the infection with non-VSP H7-type cysts (sampled at day 21 during the previous infection [see above]) (Fig. 5Ab). Immunofluorescence analyses with samples taken at later time points (days 14 and 21 p.i) after infection of mice with cysts consistently demonstrated predominance (>99%) of non-VSP H7-type trophozoites. This indicated that antigenic switching of the trophozoite populations had occurred between days 7 and 14 p.i (not shown).



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  		FIG. 5. Immunofluorescence-based assessment of VSP H7 (MAb G10/4) positivity of parasite populations related to an infection of mice with in vivo-derived cysts or trophozoites. Analyses of both inocula used for infection of mice (A) and duodenal trophozoites sampled from infected mice at day 7 p.i. (B) are shown. For infection, VSP H7-type cysts (isolated in the previous infection experiment at day 7 p.i.) (a), non-VSP H7-type cysts (isolated in the previous infection experiment at day 21 p.i.) (b), and non-VSP H7-type trophozoites (isolated in the previous infection experiment at day 21 p.i.) (c) were used as inocula. VSP H7-type parasites were immunostained by a two-step incubation with VSP H7-specific mouse MAb G10/4 and anti-mouse immunoglobulin G-fluorescein isothiocyanate visible as yellow-green (cysts) or green (trophozoites) stain. In order to localize the MAb G10/4 (VSP H7)-negative parasites, cysts were stained with a Texas red-conjugated anti-cyst wall protein MAb (red stain) and trophozoites were visualized by staining of nuclei with dsDNA-specific fluorescent dye Hoechst 33258 (blue stain). Approximate percentages of VSP H7-positive parasites within the inocula (indicated in box on the top of the figure) and approximate mean values (plus ranges) representing percentages of VSP H7-positive parasites within duodenal trophozoites populations from three infected mice (indicated in box at the bottom of the figure) are indicated.

 
Substantially different results were obtained by performing immunofluorescence analyses with the duodenal trophozoite populations sampled at days 7 (Fig. 5Bc) and 14 and 21 p.i. (data not shown) from individual animals that had been infected with non-VSP H7-type trophozoites (sampled at day 21 during the previous infection [see above]) (Fig. 5Ac). Here, all trophozoite populations including the one taken at day 7 p.i. exhibited a VSP H7-negative phenotype.

3'RACE RT-PCR-based quantification of vsp mRNA levels associated with in vivo antigenic switching of G. lamblia clone GS/M-83-H7 after infection of mice with in vivo-derived cysts and trophozoites. The immunofluorescence results shown in Fig. 5 were essentially confirmed on the transcriptional level by using the 3'RACE RT-PCR approach as outlined above (Fig. 6). A highly abundant level of vsp H7 mRNA was detectable in initial duodenal trophozoite populations taken at day 7 p.i. from mice that had been inoculated with either VSP H7-type (Fig. 6A) or non-VSP H7-type (Fig. 6B) cysts. Conversely, only low vsp H7 mRNA levels were found in the initial trophozoite population samples from mice that had received a non-VSP H7-type trophozoite inoculum.

Compared to intestinal trophozoite populations emerging at day 7 upon infection with VSP H7-type trophozoites (Fig. 4A), populations emerging upon infection with VSP H7-type and non-VSP H7-type cysts (Fig. 6A and B) exhibited a reduced (~4-fold reduction) content of transcripts concerning both vsp H7 and subvariant vsp (V17, V12, and V18) mRNA. This finding suggested differences in steady-state levels of vsp mRNA within the first and the two latter parasite populations mentioned above. However, the absence of significant differences in the ratios between vsp H7 and subvariant vsp (V17, V12, and V18) mRNA levels did not indicate a differential vsp mRNA composition within these populations. The ratios were consistently high (>100) and indicated predominance of vsp H7 mRNA within the parasites.

In summary, infection of mice with cysts of G. lamblia clone GS/M-83-H7 resulted in VSP H7-type-dominated initial trophozoite populations irrespective of the variant type(s) used as the inoculum. In contrast, inoculation of animals with non-VSP-type trophozoites did not result in detectable VSP H7 positivity of the intestinal parasite populations at any experimental time point of the infection.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
G. lamblia clone GS/M-83-H7 expresses the well-characterized surface antigen VSP H7 and represents one of the few members of G. lamblia isolates that develops an active infection in the experimental murine host (e.g., reviewed in references 10 and 12). Especially the mother-offspring infection model had previously proven to be well suited for investigating those parameters that are related to antigenic variation of the parasite (e.g., reviewed in references 10 and 12). The goal of our study was to assess, in a quantitative manner, vsp mRNA levels in intestinal trophozoite populations emerging during an experimental G. lamblia clone GS/M-83-H7 infection in mice. By using a quantitative RT-PCR approach, we were able to quantitatively assess levels of mRNA representing both vsp H7 and a set of 18 subvariant vsp genes that had been identified in one of our recent studies (4). In particular, we demonstrated that vsp H7 mRNA was predominant in VSP H7-type parasites. Our observations from this analysis indicated that initial antigenic switching of clone GS/M-83-H7 was associated with a downshift of the vsp H7 mRNA level but was not accompanied by a significant upshift of the mRNA levels representing any of the subvariant vsp genes tested. In this context, it is important to note that our study only assessed the mRNA levels from <30% of the entire GS/M-83-H7 vsp gene repertoire (18 of ca. 60 to 80 genes estimated to constitute the entire vsp repertoire [see reference 13]). The primer design of our present quantitative RT-PCR approach relied on the limited vsp gene sequence information that had been generated in a previous study which in a nonquantitative manner allowed detection of vsp mRNA molecules in VSP H7- and non-VSP H7-type trophozoites (4). In that investigation, vsp gene transcripts had been identified as cDNA analogues by performing a multistep 5'RACE RT-PCR that included degenerate vsp primers for amplification of vsp cDNA molecules. By applying this experimental strategy, a selective process during the different amplification steps (e.g., caused by inefficient priming or lack of priming of degenerate vsp primers to a subset of vsp gene sequences) might have excluded detection of, and subsequent generation of respective sequence information on, cDNA analogues from predominant vsp mRNA molecules within the non-VSP H7-type parasite populations. Due to this possible lack of important sequence information it is possible that our study was not able to demonstrate the entire composition of the non-vsp H7 gene transcripts emerging during antigenic switching of the parasite. This possibility has to be taken into account because, in contrast to the vsp H7 mRNA levels, total vsp mRNA levels turned out to be similar in VSP H7-type and non-VSP H7-type intestinal parasite populations (see Fig. 3).

Fortunately, the above-mentioned experimental constraints did not substantially affect the outcome of those investigations, which were focused on the primary goal of the present study. Our major efforts were dedicated to a series of experimental infections which were aimed at the examination of eventual surface antigen alterations of G. lamblia clone GS/M-83-H7 in association with transmission of the parasite from one to another host individuum. In one of our experiments, we simulated the natural infection mode in that we applied a novel protocol that allowed intragastric inoculation of in vivo-isolated cysts into suckling mice. In this infection experiment, VSP H7-type trophozoites were revealed to dominate the initial intestinal parasite population irrespective of the variant-type composition of the cyst inoculum applied for infection of the animals. As a consequence of these findings, we first addressed the question of whether such an antigenic reset after transmission of the parasite via cysts was due to a growth-selective process that had occurred during establishment of the trophozoite population inside the intestinal habitat of the murine host. Such a selection could have been promoted through overgrowth of subvariant types by a few VSP H7 types that had eventually resided within the inoculum. However, this scenario was rather unlikely because immunofluorescence analysis of the inoculum in question did not detect VSP H7-type cysts that could have initiated such a growth-selective process after in vivo excystation. The existence of such a process could be largely excluded by demonstrating that infection of suckling mice with in vivo-derived non-VSP H7-type trophozoites did not exhibit the antigenic reset phenomenon.

The findings described above raise the question of whether the antigenic reset had occurred in direct association with in vivo excystation of the parasite. Surface antigen alterations related to en- and/or excystation of G. lamblia is a known phenomenon that was particularly demonstrated for clone WB C6 (9, 21). In the case of clone WB C6, in vitro stage conversion was shown to trigger switching of vsp gene transcription. Conversely, testing of G. lamblia clone GS/M-83-H7 (expressing VSP H7) under analogous experimental conditions did not exhibit such a switching effect (21). In the present study, we repeatedly tried to follow the same protocol to achieve in vitro en- and/or excystation with non-VSP H7-type cultures from clone GS/M-83-H7 (not shown). We intended to do these in vitro experiments to definitively confirm that the antigenic reset phenomenon is both temporally and causally linked to the process of excystation. Unfortunately, all of these trials failed because, at least in our studies, the excystation efficiency of non-VSP H7-type cultures was extremely low.

Based on our data, the antigenic reset mechanism has to be regarded as an integral part of transmission (via cysts) of G. lamblia clone GS/M-83-H7 from one to another murine host individuum. Clone GS/M-83-H7 may have evolved the VSP H7-type as a predominant variant that achieves an optimal initiation of the parasite infection and/or maintenance of the parasite within an affected host population. Further infection experiments in different animal systems will reveal whether the VSP H7 type is able to establish a G. lamblia GS/M-83-H7 infection not only in mice but also in other species representing potential experimental (e.g., gerbils) or natural hosts (e.g., dogs) of the parasite. Investigations in these host systems will also address the question of whether the strategy of such an antigenic reset is compatible with both (i) a participation of surface antigen alterations in an adaptive process facilitating transmission of G. lamblia from one to another host species, as considered by Singer et al. (18), and (ii) the occurrence of repeated G. lamblia infections in the same individuum, as suggested by Gilman et al. (5).


   ACKNOWLEDGMENTS
 
We thank A. Hehl and M. Marti (Institute of Parasitology, Zürich, Switzerland) and N. Keller (Institute of Parasitology, Berne, Switzerland) for technical support and T. E. Nash (National Institutes of Health, Bethesda, Md.) for the gift of MAb G10/4 and G. lamblia clone GS/M-83-H7.

This study was financed through a grant obtained from the Swiss National Science Foundation (31-066795.01).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Parasitology, P.O. Box 8466, CH-3001 Berne, Switzerland. Phone: (4131) 6312474. Fax: (4131) 6312622. E-mail: nmueller{at}ipa.unibe.ch. Back

Editor: W. A. Petri, Jr.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1. Aggarwal, A., J. W. Merritt, and T. E. Nash. 1989. Cysteine-rich variant surface proteins of Giardia lamblia. Mol. Biochem. Parasitol. 32:39-48.[CrossRef][Medline]
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6. Gottstein, B., G. R. Harriman, J. T. Conrad, and T. E. Nash. 1990. Antigenic variation in Giardia lamblia: cellular and humoral immune response in a mouse model. Parasite Immunol. 12:659-673.[Medline]
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Infection and Immunity, August 2004, p. 4763-4771, Vol. 72, No. 8
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.8.4763-4771.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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