119

Biological Selection of Variant-Specific Surface Proteins in Giardia lamblia

Steven M. Singer,a Heidi G. Elmendorf,a Laboratory of Parasitic Diseases, National Institutes of Health,
John T. Conrad, and Theodore E. Nash Bethesda, Maryland

Immune evasion is frequently cited as the main reason for antigenic variation in pathogenic
microorganisms. To better understand the role of switching of variant-specific surface proteins
(VSPs) in Giardia lamblia–host interactions, antigenic variation during infections of mice and
gerbils was examined, using clones that predominantly expressed unique VSPs. As expected,
VSPs were selected against during infections of immunocompetent hosts. In contrast, in im-
munodeficient hosts, some VSPs were selected for and others were selected against. These diverse
patterns of selection demonstrate that there are host-VSP interactions that exert both positive
and negative selective pressures on parasites, independent of the adaptive immune response.
Furthermore, selection was dependent on both the particular VSP and the host. Thus, the large
number of VSP genes in G. lamblia may allow the parasite to infect multiple different hosts,
and antigenic variation could be a mechanism to expand the parasite’s host range.

Surface antigenic variation occurs in a wide variety of organ-
isms, including protists, bacteria, and viruses (reviewed in [1]).
The biological role of antigenic variation among organisms dif-
fers and, in some cases, may be complex. The most commonly
cited biological role is immunological escape, in which antibodies
produced by the host against the dominant antigen destroy those
organisms bearing this antigen, resulting in replacement by the
organisms that possessed a variant form of the antigen. In some
instances the varying surface antigens have other specific bio-
logical activities [1, 2]. Antigenic variation in response to envi-
ronmental stimuli also occurs in free-living organisms [3], a phe-
nomenon that is difficult to reconcile with the often-mentioned
role of antigenic variation to evade the immune responses of the
host.
Giardia lamblia is a protozoan pathogen that infects the gas-
trointestinal tract of many species of animals. G. lamblia un-
dergoes antigenic variation by changing the expression of its
variant-specific surface proteins (VSPs; reviewed in [4]). Each
parasite contains ∼150 different VSP genes, totaling ∼2% of
the parasite genome, although each parasite expresses only 1
VSP at a time [5, 6]. Surface labeling and electron microscopy
demonstrate that VSPs are the major surface protein of the
parasite and form a dense coat on its surface [7–9]. VSPs have
several conserved cysteine-rich motifs containing the sequence
CXXC, similar to LIM and RING domains, which suggests
Received 17 July 2000; revised 14 September 2000; electronically published
16 November 2000.
Presented in part: Woods Hole Immunoparasitology Meeting, Woods
Hole, Massachusetts, 27–30 September 1997 (abstract 73).
a a host antibody response, then VSP switching in antibody-
Present affiliation: Department of Biology, Georgetown University,
Washington, DC.
Reprints or correspondence: Dr. Theodore E. Nash, Laboratory of Para-
sitic Diseases, National Institutes of Health, 9000 Rockville Pike, Bldg. 4,
Rm. B1-31, Bethesda, MD 20892-0425 (tnash@niaid.nih.gov).
The Journal of Infectious Diseases 2001; 183:119–24
q 2001 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2001/18301-0015$02.00
that VSPs may participate in protein-protein interactions [10,
11]. The substantial sequence diversity among VSPs suggests
that they have the potential to fulfill multiple biological roles
on the surface of the parasite, including evading innate and
adaptive host immune responses and interacting with host cells.
Immune responses to G. lamblia are important for control
of infections (reviewed in [12]). Antigenic variation in G. lamblia
is thought to allow increased parasite persistence by evasion of
the host immune response, similar to antigenic variation in
other pathogens, although infections usually resolve sponta-
neously. Immune responses to G. lamblia are characterized by
a strong antibody response, particularly against the VSPs [13,
14]. Inclusion of anti-VSP antibodies in culture is reported to
cause a rapid accumulation of parasites expressing other VSPs
by selecting against those parasites expressing the VSP recog-
nized by these antibodies [15–17]. In immunocompetent mice
infected with the GS/M H7 clone of G. lamblia, parasites ex-
pressing the H7 VSP were very rare after 1–2 weeks of infection,
coincident with the appearance of H7-specific antibodies and
parasites expressing other VSPs and consistent with antibody-
mediated selection for antigenic variation [18, 19]. This suggests
that antibodies are the major mechanism for controlling Giardia
infections. However, mice that are unable to make antibodies
are still able to control acute G. lamblia infections [19]. Thus,
control of acute infections occurs independently of selection for
antigenic variants, and, therefore, evasion of the host antibody
response cannot explain antigenic variation during this stage
of infection [19].
If the only role of VSPs in G. lamblia infections were to evade
deficient mice should be similar to switching observed without
antibody selection during in vitro culture. In vitro studies of
antigenic variation of several G. lamblia isolates and clones,
including the GS/M H7 clone, have shown that switching be-
tween VSPs occurs rapidly and without any predefined order,
resulting in a gradual decline in the number of parasites ex-
120 Singer et al.
pressing the VSP found on the original clone and the appear-
ance of a population of parasites with heterogeneous VSP ex-
pression [15–17]. In contrast, in antibody-deficient mice 195%
of the parasites continue to express the H7 VSP for several
weeks [18–20]. This suggests that an antibody-independent
mechanism selects for parasites that express the H7 VSP in
immunodeficient mice.
The persistence of VSP H7–expressing cells in immunodeficient
mice, but their disappearance in vitro, could be explained by
different rates of VSP switching in animals versus axenic culture,
positive selection for parasites expressing this VSP in the mice,
or some unusual property of the parasite occurring only in the
H7 clone. To better understand these differences between VSP
switching observed in vitro and in vivo, immunocompetent and
immunodeficient mice and gerbils were infected with recently
isolated clones of GS/M expressing unique VSPs, and the pro-
portion of VSPs expressed during infections was determined,
using monoclonal antibodies (MAbs) specific for each of these
VSPs.
Materials and Methods
Parasites and MAbs. The G. lamblia isolate GS/M, the H7
clone, and the MAb G10/4 that is specific for the VSP expressed
by H7 (and referred to as the H7 VSP) have all been described
elsewhere [17]. Parasites were cultured anaerobically in Diamond’s
medium supplemented with antibiotics [21] and were cloned by
limiting dilution, as described elsewhere [15]. To select against VSP
H7–expressing cells, parasites were grown in medium supplemented
with 1% G10/4 ascites.
MAb production. A membrane protein fraction was prepared
from G10/4-negative parasites, as described elsewhere [22], and
protein concentration was determined (Bio-Rad Protein Assay;
Bio-Rad). Protein was emulsified in complete Freund’s adjuvant
(Sigma-Aldrich) and was injected intraperitoneally into BALB/c
mice. Two weeks later, mice were given booster injections of protein
emulsified in incomplete Freund’s adjuvant, and 3 days later spleen
cells were fused to myeloma cells, as described elsewhere [23]. Hy-
bridomas secreting antibodies reactive with G10/4-negative para-
sites were recloned by limiting dilution. By screening G10/4-neg-
ative parasite clones, we identified MAbs 6D8, 7D12, 17C11, and
12G2, which were specific for the VSPs expressed by the clones
A7, F6, D11, and A5, respectively. Thus, each clone expressed a
new VSP, and each MAb was specific for that VSP. Alternately,
Peyer’s patch cells were collected from 20 BALB/c mice infected
for 1 week with GS/M H7 parasites [24]. These cells were fused to
myeloma cells, and the anti-VSP antibody producing hybridoma
1G1 was recloned by limiting dilution. For fluorescence-activated
cell sorting (FACS; FACStar Plus; Becton Dickinson), we modified
a protocol described elsewhere [25]. To generate a clone expressing
the VSP reactive with this new MAb, a heterogeneous parasite
population was incubated with 1:500 dilutions of 1G1 ascites in
PBS on ice for 15 min, washed 3 times with cold PBS, and stained
with fluorescein isothiocyanate (FITC)–conjugated goat anti–
mouse IgG, IgA, and IgM (Cappel-ICN) diluted 1:1000 in PBS
on ice for 15 min. After being washed 3 times with cold PBS, cells
JID 2001;183 (1 January)
were sorted on a FACStar Plus (Becton Dickinson) and were im-
mediately cloned.
VSP detection. VSP expression on fixed parasites was analyzed
as described elsewhere [19]. In brief, parasites recovered from the
small intestines of the animals were purified by adherence to glass,
adhered to slides, fixed with methanol:acetone, and stained with
MAb and FITC-conjugated goat anti–mouse IgG, IgA, and IgM
(Cappel-ICN). The slides were coded, and the percentage of pos-
itive parasites in each sample was noted by one of us (H.G.E.),
who was unaware of the code.
Animals and infections. Four-week-old female BALB/cJ or
C57BL/6J-SCID mice were obtained from Jackson Laboratories
(Bar Harbor, ME), 4-week-old C57BL/6Tac mice were obtained
from Taconic Farms (Germantown, NY), and 4–6-week-old female
gerbils were obtained from Charles River Laboratories (Wilming-
ton, NJ). Mice were each given 500,000 trophozoites by gavage,
as described elsewhere [26]. To immunosuppress the gerbils [27],
they were irradiated (600 R) before infection with 500,000 tropho-
zoites by gavage. Neomycin was added to the drinking water of
the irradiated animals, to prevent secondary infections, as described
elsewhere [28]. Parasites were counted as described elsewhere [19].
Results
We generated 5 new MAbs specific for VSPs expressed by the
GS/M isolate of G. lamblia and the clones of the parasite ex-
pressing each of these VSPs. None of the MAbs reacted with
VSPs expressed by clones other than the corresponding clone.
Initial characterization of these new clones showed that they all
grew at similar rates in vitro and that none of the VSPs that they
expressed were present on the WB isolate of G. lamblia (data not
shown). Immediately after isolation, each clone predominantly
expressed a single VSP, but extended culturing (2–4 weeks, with
30–60 cell doublings) led to a decrease in the proportion of para-
sites expressing this VSP (figure 1 and data not shown). Although
little VSP switching was seen for the A5 clone in the experiment
presented in figure 1, this clone switched as rapidly as other clones
in different experiments. Thus, spontaneous switching of VSPs
occurred at a high frequency in most cultures, as has been noted
elsewhere for the GS/M H7 clone [29].
To determine whether differences in VSP expression led to
differences in the outcome of infection or in the nature of anti-
genic variation, immunocompetent animals were infected with
clones expressing different VSPs. Parasites were used shortly
after cloning, so that a single VSP was present on 50%–90%
of the cells in each culture. Infection of wild-type C57BL/6J
mice with each of these clones resulted in recovery of similar
numbers of parasites (data not shown). Interestingly, whereas
most of the parasites continued to express the VSP present on
the original clone 1 week after infection, the D11 clone switched
to other VSPs (figure 2) before there was any significant re-
duction in the total number of parasites recovered. Because the
switch occurred before the usual decrease in parasite numbers
and before the development of an adapted immune response,
this suggested that mechanisms other than immune mechanisms
JID 2001;183 (1 January) Antigenic Variation in Giardia lamblia 121

Figure 1. Selection of variant-specific surface proteins (VSPs) in immunodeficient mice and gerbils. Groups of 4 C57BL/6J SCID mice and
600 R–irradiated gerbils were infected for 15 days with GS/M clones H7, A7, A5, F6, D11, and 1G1. VSP expression on the parasites used for
infection was determined on day 0 (l). Parasites were also maintained in vitro by passage every 3 days for the duration of the experiment. VSP
expression 15 days after infection was determined for parasites maintained in vitro (m), as well as for parasites in the small intestines of the mice
(m) and gerbils (v). Each data point represents an individual animal. Some gerbil groups (A7 and A5) have only 3 data points because of gerbils’
dying before the end of the experiment, presumably from radiation sickness. Data are representative of >2 individual experiments.

select against parasites expressing this VSP in mice. Two weeks
after infection, the numbers of parasites found in the small
intestines were greatly reduced for each of the clones, as is
normal in immunocompetent mice ([19] and data not shown),
and the parasites that remained no longer expressed the original
VSP (figure 2). A heterogeneous population with regard to VSP
expression was observed instead (see below).
To observe the roles of VSPs during infections in the absence
of an antibody response, we used each of these clones to infect
SCID mice, which lack both T and B cells, cannot make adap-
tive immune responses, and cannot control G. lamblia infections
[19, 26]. For all of the clones, the number of parasites in these
mice was very high, as has been observed before in SCID mice
([19, 26], and data not shown). The H7 clone switched from
∼80% G10/4 reactive cells at the time of infection to 199% G10/
4 reactive cells after only 2 weeks in SCID mice, similar to
findings of earlier reports (figure 1 and [20, 26]). These parasites
remained homogeneous with regard to VSP expression for >1
month, in contrast to parallel cultures maintained in vitro,
which steadily accumulated parasites expressing other VSPs
(figure 1 and data not shown). This is strong evidence for pos-
itive selection of parasites expressing the H7 VSP in mice. Sim-
ilar positive selection was observed for parasites expressing the
A7, A5, and F6 VSPs while the parasite population remained
homogeneous during 2 weeks of infection (figure 1). In contrast,
infections of SCID mice with parasites expressing the 1G1 VSP
resulted in heterogeneous populations with regard to VSP ex-
pression. Because this clone became quite heterogeneous during
parallel culture in vitro, it is impossible to distinguish between
negative and neutral selection of parasites expressing this VSP.
Infection of 4 SCID mice with parasites expressing the D11
VSP resulted in negative selection of D11-expressing parasites
in 3 mice and neutral selection in 1 mouse, in comparison to
VSP switching in vitro (figure 1). Thus, in the absence of an
adaptive immune response, G. lamblia parasites are still subject
to either positive or negative selection, based on the VSPs that
they express. These parasites must, therefore, have been selected
on the basis of other biological roles of VSPs.
Because parasite numbers did not decrease during negative
selection of the D11 VSP, it was obvious that new VSPs must
be present on the parasites. Indeed, we observed positive se-
lection for parasites expressing the H7, A7, and A5 VSPs during
infections with the D11 clone in SCID mice (figure 3). Although
!5% of the cells in the D11 clone expressed the H7, A7, or A5
VSP at the time of inoculation, after 2 weeks these VSPs were
now enriched in SCID mice. This suggests that the positive
selection observed during infections with clones was due to the
particular VSPs being expressed and not to some other mu-
tation that might have occurred coincidentally during the orig-
inal isolation of the parasite clones.
To further demonstrate that the apparent negative selection
of parasites expressing the D11 VSP was also due solely to
122 Singer et al. JID 2001;183 (1 January)

A7 VSP. Also similar to the results in SCID mice, 2 gerbils

infected with parasites expressing the D11 VSP contained very
heterogeneous populations of parasites after 2 weeks, consistent
with negative selection, whereas 2 others had much less het-
erogeneous populations (figure 1). In contrast to what was seen
in SCID mice, parasites expressing the A5 VSP were clearly
negatively selected in 2 of 3 gerbils. Importantly, parasites ex-
pressing the 1G1 VSP were clearly positively selected in gerbils
but not in mice, when compared with growth in vitro. Thus,
the direction of selection (positive or negative) of parasites is
dependent on both VSP and host.

Discussion

In the present paper, we show that, in the absence of an

adaptive immune response, clones of G. lamblia expressing
Figure 2. Negative selection of variant-specific surface proteins unique VSPs behave differently in the host and that the patterns
(VSPs) by antibodies in immunocompetent mice. Four C57BL/6J mice of selection in different host species are not the same. Growth
each were infected with the GS/M clones H7, A7, A5, F6, and D11. in vitro, however, was similar among all the clones, and VSPs
Each clone was 80%–90% positive for the predominant VSP, except
D11, which was ∼60% positive. Groups of 2 mice were killed on days
eventually became heterogeneous for all clones after prolonged
7 (stippled bars) and 14 (solid bars) after infection, and VSP expression in vitro growth (figure 1 and data not shown). Thus, VSPs have
on parasites in the small intestine was analyzed. Data are presented as a role in mediating host-parasite interactions that is distinct
the means for each clone. Similar data were obtained in a second from evasion of the host adaptive immune response. Further-
independent experiment. more, the fact that a single VSP can be positively selected in
mice and negatively selected in gerbils, or vice versa (figure 1,
expression of this particular VSP, we generated a new subclone clones A5 and 1G1), suggests that some VSPs cannot success-
from the D11 clone that predominantly expressed the H7 VSP. fully interact with both hosts. Thus, our data suggest that an-
To accomplish this, we used the D11 clone to infect a SCID tigenic variation diversifies the host range of the parasite, per-
mouse, and 4 weeks later parasites recovered from the small haps by increasing transmission between different species.
intestine were cloned by limiting dilution. These subclones were Selection for and against VSPs in immunodeficient hosts
screened for expression of the H7 VSP, and a new clone that
predominantly expressed the H7 VSP was selected and called
D12. Infection of SCID mice with D12 parasites resulted in a
pattern of infection indistinguishable from that of the original
H7 parasites (data not shown). Thus, the negative selection
observed for the D11 clone was due to the VSP being expressed,
and not to other differences that might have arisen during the
initial cloning. Together, these data clearly show distinct pat-
terns of selection for VSPs in immunodeficient mice.
To determine why the parasite would express VSPs that were
negatively selected in vivo, we hypothesized that VSPs might
show different patterns of selection depending on the host spe-
cies. To test this hypothesis, we infected gerbils with these
clones, since gerbils are highly susceptible to infections with G.
lamblia [30]. Gerbils were sublethally irradiated to compromise
their immune systems [27] and minimize immune selection
against VSPs. As evidence of this, the irradiated gerbils failed
to produce any serum antibodies capable of reacting with par- Figure 3. Positive selection of H7, A7, and A5 variant–specific
asite antigens during the 2 weeks of infection, as determined surface proteins (VSPs) in SCID mice infected with the D11 clone of
by indirect immunofluorescence assays on fixed parasites (data GS/M. Clone D11 was used to infect a group of 4 C57BL/6J SCID
mice, and VSP expression was analyzed on parasites recovered from
not shown). Parasites expressing the H7, A7, and F6 VSPs were the small intestines on day 15 after infection (m). Data are from the
positively selected in gerbils, as they were in SCID mice (figure same mice used in figure 1. Each data point represents an individual
1), although 1 gerbil negatively selected parasites expressing the mouse.
JID 2001;183 (1 January) Antigenic Variation in Giardia lamblia
demonstrates that VSPs play a key role in the biology of the
host-parasite interaction, because such selection was not found
in vitro. One possible role for VSPs is in interactions with the
host epithelium and/or mucous layer in the small intestine. No
direct evidence exists to suggest that the VSPs are important
in parasite attachment [31]. However, when parasites detach
from epithelial cells in vitro, VSPs can clearly be seen remaining
attached to the host cells, potentially allowing VSPs to modify
the normal functions of these cells (T.E.N., unpublished data).
Indeed, the presence of LIM- and RING-like domains in VSPs
further suggests that they could participate in protein-protein
interactions with the host [10, 11]. These domains could also
help form a barrier on the parasite surface by providing inter-
actions between VSP molecules. Thus, a second possible role
for VSPs is to maintain the infection via resistance to host
cytotoxic mechanisms. Sensitivity of G. lamblia to host intes-
tinal proteases has been correlated with expression of certain
VSPs [32]. G. lamblia has also been shown to be sensitive to
antimicrobial peptides such as cryptdins or defensins, which act
by forming pores in cell membranes [33]. Prior studies have
noted survival differences among various G. lamblia isolates
and clones during in vitro culture and in infections of gerbils
and suckling mice [34–36]. While they did not correlate these
differences with VSP expression or immune selection, our data
suggest a possible explanation for these observations. Reso-
lution of potential roles for VSPs during infections will require
a better understanding of the actual pathways used by the host
to eliminate the parasite.
Equally interesting is the different patterns of VSP-dependent
selection observed between mice and gerbils. Although multiple
VSPs were positively selected in any single host, clear preferences
exist for specific VSPs in different hosts. A possible explanation
for the large repertoire of VSPs present in the G. lamblia genome,
then, is that antigenic variation allows the parasite to express
VSPs that enhance survival in many different hosts and envi-
ronmental conditions. The differences between hosts that affect
VSP selection remain to be determined. The finding that switch-
ing of VSP expression is augmented during passage through the
cyst stage and completion of the parasite life cycle is consistent
with the idea that VSP switching has evolved to promote trans-
mission of the parasite to new hosts of the same or different
species [37, 38]. This may be particularly important for G. lamblia,
given the ability of the parasite to generate a large number of
cysts in each infection and the small number of cysts necessary
to initiate subsequent infections. If the trophozoites that emerge
from these cysts express VSPs distinct from the ones expressed
in the previous host, they may be better able to initiate an in-
fection in multiple new species of hosts. If each cyst is preadapted
to infect one or a few species of hosts, on the basis of the par-
ticular VSP the trophozoites within it express, then antigenic
variation could thereby promote transmission to many different
species, rather than only to the same species that was previously
infected. However, completion of the life cycle in vitro did not
123
augment VSP switching of all isolates tested [37], notably the
GS/M isolate used in this paper. This may help explain why cross-
species transmission was not as efficient as intraspecies trans-
mission in some studies [39].
Although patterns of antigenic variation were generally con-
sistent among animals infected with any single clone, some
variability was seen, particularly when examining the irradiated
gerbils. This is likely due to variable responses to irradiation
in these animals that were not reflected fully in immunofluo-
rescence assays using sera from these animals. Nevertheless,
parasites were clearly selected in immunodeficient hosts on the
basis of the VSPs that they expressed, and the patterns of se-
lection were different among the clones analyzed and, for some
clones, between hosts as well.
There are other examples of antigenic variation adding di-
versity to the biology of a pathogen. The malaria parasite,
Plasmodium falciparum, undergoes immune-selected antigenic
variation of the PfEMP1 protein found on the surface of in-
fected erythrocytes ([2] and reviewed in [1]). This variation is
thought to allow the parasites to sequester in different locations
throughout the body, based on binding to different host ligands.
T. brucei may also link antigenic variation to infections of dif-
ferent hosts. One mechanism of antigenic variation in this par-
asite involves switching expression from one locus containing
a variant surface glycoprotein (VSG) gene to a different locus
with a different VSG gene (reviewed in [40]). This parasite also
encodes a different isoform of transferrin receptor (TfR) at each
of these loci. Selection of which locus is used for VSG and TfR
expression involves matching the TfR isoform to the particular
host to ensure adequate uptake of host transferrin [41].
Selection for antigenic variation in G. lamblia infections can
occur via an antibody-independent mechanism. This result de-
pends on the particular species of host and can produce both
positive and negative selection of parasites. During acute in-
fection, it is likely that antigenic variation is not meant to evade
the antibody response, because antibody-independent mecha-
nisms can control this phase of the infection. Instead, antigenic
variation may serve to increase the diversity of the excreted
cyst population and the likelihood of transmission downstream
to the next host, thus diversifying the host range of the parasite.
Acknowledgments
We thank Kevin Holmes and Susan Barbieri (Resource Technology
Branch, National Institute of Allergy and Infectious Diseases [NIAID],
National Institutes of Health [NIH]) for assistance with fluorescence-
activated cell sorting of Giardia lamblia and Howard Adams and Sandy
Cooper (Animal Care Branch, NIAID, NIH) for animal care.
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