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Infection and Immunity, March 2007, p. 1502-1506, Vol. 75, No. 3
0019-9567/07/$08.00+0 doi:10.1128/IAI.01801-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Selection for Simple Major Surface Protein 2 Variants during Anaplasma marginale Transmission to Immunologically Naïve Animals
Guy H. Palmer,1*
James E. Futse,1
Christina K. Leverich,1
Donald P. Knowles Jr.,2
Fred R. Rurangirwa,1 and
Kelly A. Brayton1
Program in Vector-borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University,1
Animal Diseases Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington2
Received 10 November 2006/
Returned for modification 1 December 2006/
Accepted 7 December 2006

ABSTRACT
Anaplasma marginale, a rickettsial pathogen, evades clearance
in the animal host by antigenic variation. Under immune selection,
A. marginale expresses complex major surface protein 2 mosaics,
derived from multiple donor sequences. However, these mosaics
have a selective advantage only in the presence of adaptive
immunity and are rapidly replaced by simple variants following
transmission.

TEXT
Persistent infection is fundamental to the epidemiology of vector-borne
pathogens, providing a reservoir for transmission during periods
of peak arthropod activity. African trypanosomes, which use
a large repertoire of variable surface glycoprotein (
vsg) sequences
to generate diversity in their bloodstream form surface coats,
classically illustrate how antigenic variation allows evasion
of the host immune response and persistence within an immunocompetent
mammalian reservoir host (
4,
18). The importance of this process
in vector-borne pathogen persistence, and thus transmission,
is evident by the use of a very similar mechanism in evolutionarily
distinct pathogens, most notably the tick-borne bacteria in
the genera
Anaplasma and
Borrelia (
3).
Anaplasma marginale,
the most globally prevalent tick-borne pathogen of livestock,
closely mimics the trypanosome in that it uses duplication of
chromosomal donor sequences into the expression site (
7,
8).
A. marginale, with a 1.2-Mb genome, less than 1/20 the size
of the
Trypanosoma brucei genome (
5,
6), represents a simplified
model of this convergent mechanism of antigenic variation, since
all major surface protein-2 (MSP2) surface coat variants are
generated by recombination from 5 to 7 chromosomal donor sequences,
termed functional pseudogenes, into a single expression site
(Fig.
1A). The evolutionary selection for gene duplication,
resulting in the repertoire of the donor
msp2 and
vsg sequences
in
A. marginale and
T. brucei, respectively, is evident: this
repository must be sufficiently diverse to generate numerous
antigenic variants and allow persistent infection in the face
of a rapidly responding immune system.
In addition to this selective pressure for surface coat diversity,
there is a balancing selection for growth fitness. At a structural
level, this is evident by conservation of the transmembrane
domains among MSP2 variants in
A. marginale and among VSG variants
in
T. brucei (
7,
10,
23); however, the extracellular domains
are also very likely under growth fitness selection. While the
large genome size, the complexity of the
vsg families, and the
multiple expression sites have, to date, precluded comprehensive
testing in
T. brucei,
A. marginale variants expressing the complete
extracellular domain derived from each of the unique pseudogenes
of the St. Maries strain can be identified early in infection
(
14). This expression indicates that each of the chromosomally
encoded extracellular MSP2 domains confers in vivo fitness.
A. marginale also generates expression site mosaics following
recombination of oligonucleotide segments derived from two or
more donor sequence loci (Fig.
1B) (
1,
8,
14). These mosaics,
quantified in terms of complexity based on the number of expression
site segments derived from different donor pseudogene sequences,
emerge over time during infection and dominate the variant population
following months of persistence (
14). Similar mosaics are generated
by
T. brucei and the related
Trypanosoma equiperdum, in which
mosaics emerge over time during infection (
16,
20,
23). The
ability to generate mosaics tremendously expands the repertoire
of possible antigenic variants, allowing continual immune evasion
and persistence. However, unlike the stable chromosomal donor
sequence loci,
A. marginale expression site mosaics themselves
are not subject to long-term selection, since they are not permanently
represented in the genome and are lost from the genome when
they are replaced in the expression site by a subsequent recombination
event (
1,
8,
12,
13). Thus, we hypothesize that while complex
mosaics emerge under strong immune selective pressure, they
do not represent in vivo growth fitness selection, and in the
absence of immunity, simple type variants will predominate.
To test this hypothesis, we established persistent A. marginale infection in a natural animal reservoir host, confirmed that the msp2 expression site encoded complex mosaic variants, and transmitted these complex MSP2 variants to immunologically naïve animals. Briefly, infection of calf 983 was initiated by transmission feeding of adult male Dermacentor andersoni ticks infected with the St. Maries strain of A. marginale (22). This strain was selected because it has been completely sequenced (6), and thus each expression site sequence can be mapped to one or more specific msp2 donor pseudogenes. In this analysis, msp2 expression site variants are amplified, sequenced, and either mapped to a whole-donor msp2 pseudogene or, in the case of mosaics, each oligonucleotide segment in the expression site is mapped to its specific-donor pseudogene (see Fig. 1B) (14). Acute infection of calf 983 with high-level bacteremia (
109 A. marginale parasites per ml) was confirmed by microscopic detection of infected erythrocytes in Giemsa-stained blood smears. Expression of the unique extracellular domains derived from recombination of each intact whole pseudogene (Fig. 1A) was detected within the first 60 days of infection (Fig. 2), consistent with their preferential use early in infection. Persistent infection was tracked using quantitative real-time msp5 PCR, as previously described in detail (15), and was maintained at levels of
107 organisms per ml of blood from 3 months post-tick transmission through the end of the study. During persistent infection, the population structure of the expressed MSP2 was confirmed to be mosaic (Fig. 2) (14). Analysis of at least 30 sequences provides a >90% probability that all sequences representing at least 5% of the variant population will be detected (14). A complexity score of zero reflects the presence of a single whole pseudogene sequence in the expression site, a score of 1 indicates the recombination of a segment derived from a distinct donor pseudogene, and scores of 2 and 3 apply when, respectively, two and three segments from distinct pseudogenes are recombined into the expression site (Fig. 1B). The complexity score of calf 983 progressively increased during infection to a mean score of >2.0 (Fig. 2), indicating that the expressed MSP2 was derived from a mosaic of multiple distinct pseudogenes, a result consistent with the prior analysis of multiple calves during long-term persistent infection (14).
Dermacentor andersoni adult males were then allowed to feed
on calf 983 for 7 days to acquire infection. The mean complexity
of MSP2 variants in the blood during acquisition tick feeding
was 2.2 ± 0.69. Following 48 h of incubation at 26°C
and 98% relative humidity, ticks were transmission fed on each
of four age-matched calves (calves 1104, 1113, 1118, and 1121),
which had been found seronegative by competitive indirect enzyme-linked
immunosorbent assay (VMRD, Pullman, WA), for an additional 7
days. From the earliest detectable time point following transmission
through the first 3 weeks of acute infection, the complexity
score for all calves was <1.0, reflecting a significantly
less complex variant structure (Fig.
3). Both the overall complexity
scores for the emergent organisms in the four calves combined
(0.45 ± 0.29) and those for the emergent organisms in
each calf (Fig.
3) were significantly lower (
P < 0.05) than
those present in the blood of persistently infected calf 983
during tick acquisition feeding. For each of the transmission
calves and at each time point, 100% of the 121 total variants
examined were simple variants (complexity score,

1.0) derived
from recombination of a whole pseudogene hypervariable region
or single segment into the expression site, in contrast to the
overwhelming predominance (>90%) of complex variants (complexity
score,

2.0) in the blood of persistently infected calf 983 at
the time of acquisition tick feeding.
The emergence of
A. marginale simple variants during early infection
in immunologically naïve animals is consistent with the
progressive development of complex mosaic variants during persistent
infection only under the selective pressure of immunity: in
the absence of immune memory, these complex variants have no
intrinsic advantage. To the contrary, the data indicates that
these variants are at a disadvantage, since neutral selection
would predict that the population early in infection of the
naïve animals would reflect that of calf 983 at the time
of acquisition feeding. To address where this selection for
simple variants occurs, we examined two nonmutually exclusive
possibilities: (i) during invasion, replication, and transmission
by the tick vector and (ii) during invasion and replication
in the naïve calf. For the first,
A. marginale msp2 expression
site variants were amplified and sequenced from the tick salivary
glands (
21) obtained on the last day of transmission feeding
on calves 1104, 1113, 1118, and 1121, since these organisms
should best represent the cumulative selective events during
replication and development prior to and at the time of transmission.
The
msp2 expression site variants from individual ticks fed
on each animal were amplified and sequenced. The mean complexity
score from all expressed variant sequences (
n = 200) was 0.49,
and that of ticks fed on each individual calf ranged from a
mean of 0.26 to 0.74. These salivary gland variants were of
the simple type, reflected in the significantly lower complexity
score (
P < 0.05), compared to the MSP2 variants present during
the tick acquisition feeding (Fig.
4). For the second possibility,
10 ml of blood, collected from calf 983 during tick acquisition
feeding and containing MSP2 variants with a mean complexity
score of 2.2, was inoculated intravenously directly into calf
1125, and the
msp2 expression site variants early in infection
were determined as described above for the calves following
tick transmission. Examination of 192 variants collected in
the first month of acute infection revealed a mean complexity
score of 0.42 ± 0.47, with a score of 0.29 ± 0.47
at the earliest time point examined (7 days postinoculation).
All time points were again significantly different (
P < 0.05)
from the inoculated
A. marginale both in complexity score (Fig.
5) and in the usage of recombined whole pseudogenes.
We accept the hypothesis that in the absence of the selective
pressure of the mammalian adaptive immune response, MSP2 simple
variants have a competitive fitness advantage and predominate.
The rapid emergence of very simple variants within the first
week of infection following direct inoculation demonstrates
the rigor of selection for optimal in vivo growth. Unlike the
mosaics, which are not maintained in the genome and thus are
not under specific evolutionary selection, the individual whole-pseudogene
sequences, originally generated by gene duplication, can be
under selection both for ability to generate antigenic variants
and for growth fitness following recombination and expression.
Consistent with this dual selection, one or more identical
msp2 pseudogenes are conserved among otherwise genetically different
A. marginale strains (
6-
8,
19).
Interestingly, the selection for growth of simple variants occurred both within the tick and in the mammalian host following transmission. Previously, we proposed that there is selection for specific MSP2 variants within the tick (21). While our new data cannot exclude a role for specific variants in development within the tick, the most parsimonious explanation for the results is that A. marginale simple variants have a fitness advantage that is manifest in the absence of adaptive immunity, be it in the tick or in the immunologically naïve animal host. This explanation is consistent with prior studies, although previously interpreted differently, using two additional strains of A. marginale. Variants that can now be identified as simple type MSP2 variants in the South Idaho strain (following sequencing of the pseudogene repertoire in this strain) were detected in the tick midgut within 3 days following acquisition feeding (17). In a separate study, these South Idaho strain simple variants were shown to predominate in the tick salivary gland and, following transmission, in naïve calves (21). Independent studies using the Oklahoma strain of A. marginale revealed that for splenectomized calves, in which immune pressure is abrogated, the same MSP2 types were identified in the splenectomized calf used for tick acquisition feeding, the salivary glands of ticks fed on this calf, and, again following transmission, in the naïve calf (2). Although not examined, our results would predict that these represent simple variants with a fitness advantage in the absence of immune selection.
Whether the fitness disadvantage of organisms expressing complex mosaic variant coats contributes to the lower bacteremia levels observed during persistent A. marginale infection is unknown. Unlike the peak bacteremia levels of
109 per ml during acute infection, A. marginale variants emergent during persistent infection of immunocompetent animals are controlled at levels below 107 per ml (11-13). This has been proposed to reflect more rapid development of variant-specific immune responses due to linked conserved epitopes (9); however, a significantly lower replication rate would also provide more time for the immune system to respond to and control emergent variants. We emphasize that it remains unknown if A. marginale parasites expressing simple MSP2 variants have a significantly higher replication rate that those expressing complex mosaics, since in vivo fitness reflects both growth and survival. African trypanosomiasis follows a similar course, with initial peak parasitemia levels followed by a gradual reduction in the peak of subsequent cycles in which novel VSG variants sequentially arise and are controlled. Whether duration of persistence and amplitude of parasitemic peaks are associated with the emergence of complex VSG mosaics in trypanosomiasis awaits investigation. However, the development of mosaics as a mechanism to generate sufficient antigenic variants to evade immune clearance, even when this is associated with a significant fitness disadvantage, illustrates the overriding importance of persistent infection for efficient onward transmission of vector-borne pathogens.

ACKNOWLEDGMENTS
This research was supported by NIH R01 AI44005, USDA ARS-CRIS
5348-32000-016-00D, and USDA-ARS Cooperative Agreement 58-5348-3-212.
The technical assistance of Ralph Horn, Beverly Hunter, and James Allison is gratefully acknowledged.

FOOTNOTES
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-6033. Fax: (509) 335-8529. E-mail:
gpalmer{at}vetmed.wsu.edu.

Published ahead of print on 18 December 2006. 
Editor: R. P. Morrison

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Infection and Immunity, March 2007, p. 1502-1506, Vol. 75, No. 3
0019-9567/07/$08.00+0 doi:10.1128/IAI.01801-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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