Diversity at the Locus Associated with Transcription of a Variable Surface Antigen of Pneumocystis carinii as an Index of Population Structure and Dynamics in Infected Rats

Abstract

Pneumocystis carinii expresses a surface glycoprotein called MSG. Different isoforms of MSG are encoded by a gene family spread over at least
15 telomeric sites. Only one locus, called UCS, supports the production of MSG mRNA. Previous studies showed that P. carinii populations from individual rats exhibited high degrees of diversity with respect to the MSG genes attached to the UCS locus.
This diversity could have been generated primarily in the rats studied. Alternatively, the rats may have been infected by
P. carinii organisms that were already different at the UCS locus. To investigate this issue, we examined the UCS locus in P. carinii from rats that had been exposed to few of the microbes at a specified time, which produced a bottleneck in the microbial
population. Some of the rats with bottlenecks produced P. carinii populations in which a single MSG sequence resided at the UCS locus in 80 to 90% of the organisms, showing that P. carinii can proliferate within a rat without generating the very high levels of UCS diversity previously seen. From the degree of
diversity observed in the bottlenecked populations, the maximum rate of switching appeared to be 0.01 event per generation.
These data also suggest that the infectious dose is as low as one organism, that rats that share a cage readily infect each
other, and that the doubling time of P. carinii in vivo is ∼3 days. In addition, we found that inoculation with 107 P. carinii organisms from a population highly heterogeneous at the UCS locus reproduced this heterogeneity. By contrast, shifts in population
structure occurred in rats given 104 P. carinii organisms, suggesting that a small fraction of these proliferated.

Full text

INFECTION AND IMMUNITY, Jan. 2003, p. 47–60 Vol. 71, No. 1
0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.1.47–60.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Diversity at the Locus Associated with Transcription of a Variable
Surface Antigen of Pneumocystis carinii as an Index of Population
Structure and Dynamics in Infected Rats
Scott P. Keely,
1
Melanie T. Cushion,
2
and James R. Stringer
1
*
Department of Molecular Genetics, Biochemistry and Microbiology
1
and Department of Internal Medicine,
2
University
of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524
Received 13 June 2002/Returned for modification 6 August 2002/Accepted 10 October 2002
Pneumocystis carinii expresses a surface glycoprotein called MSG. Different isoforms of MSG are encoded by
a gene family spread over at least 15 telomeric sites. Only one locus, called UCS, supports the production of
MSG mRNA. Previous studies showed that P. carinii populations from individual rats exhibited high degrees
of diversity with respect to the MSG genes attached to the UCS locus. This diversity could have been generated
primarily in the rats studied. Alternatively, the rats may have been infected by P. carinii organisms that were
already different at the UCS locus. To investigate this issue, we examined the UCS locus in P. carinii from rats
that had been exposed to few of the microbes at a specified time, which produced a bottleneck in the microbial
population. Some of the rats with bottlenecks produced P. carinii populations in which a single MSG sequence
resided at the UCS locus in 80 to 90% of the organisms, showing that P. carinii can proliferate within a rat
without generating the very high levels of UCS diversity previously seen. From the degree of diversity observed
in the bottlenecked populations, the maximum rate of switching appeared to be 0.01 event per generation.
These data also suggest that the infectious dose is as low as one organism, that rats that share a cage readily
infect each other, and that the doubling time of P. carinii in vivo is ⬃3 days. In addition, we found that
inoculation with 10
7
P. carinii organisms from a population highly heterogeneous at the UCS locus reproduced
this heterogeneity. By contrast, shifts in population structure occurred in rats given 10
4
P. carinii organisms,
suggesting that a small fraction of these proliferated.
Pneumocystis pneumonia is a well-known problem in AIDS
patients. Pneumocystis organisms are also found in a variety of
nonhuman host species, including nonhuman primates, ferrets,
rabbits, horses, shrews, pigs, mice, and rats. Until approxi-
mately 1990, a single genus and species name, Pneumocystis
carinii, was used in reference to all Pneumocystis organisms
regardless of the host species in which they are found. Then,
DNA sequence analysis showed that the Pneumocystis organ-
isms found in different host species are quite different from
one another and appear to be host species specific. To distin-
guish these different organisms, a trinomial nomenclature sys-
tem was adopted (2, 38; S. Keely, H. J. Pai, R. Baughman, C.
Sidman, S. M. Sunkin, J. R. Stringer, and S. L. Stringer, ab-
stract from Third International Workshops on Pneumocystis,
Cryptosporidium, Microsporidia, and Toxoplasma 1994, J. Eu-
karyot. Microbiol. 41:94S, 1994). Under this system, Pneumo-
cystis organisms are distinguished as special forms of P. carinii.
For example, the Pneumocystis organism used in the studies
described in this report was named Pneumocystis carinii f. sp.
carinii. The idea that special forms are actually separate spe-
cies has gained support from the large amount of DNA se-
quence data now available, and the trinomial names are being
phased out. In 1999, two special forms were renamed. P. carinii
f. sp. carinii was renamed P. carinii, and the human pathogen
was renamed Pneumocystis jiroveci (16). Thus, the organism we
call P. carinii here is the same organism previously called P.
carinii f. sp. carinii but not the same organism present in hosts
other than rats, even though all of those microbes were called
P. carinii in the past.
DNA sequence comparisons place the genus Pneumocystis in
the kingdom Fungi (14, 15, 39). While the sequence data firmly
establish that it is a fungus and strongly suggest that it is an
ascomycete, P. carinii is unlike other major fungal pathogens of
mammals in several respects, including a lack of ergosterol,
fragility of the cell wall, and poor proliferation in culture, the
last greatly hampering research progress (37, 38). Culture sys-
tems have been described that can transiently sustain popula-
tions of P. carinii, but only when the culture is inoculated with
millions of cells obtained from an infected animal (9). A cul-
ture system that supports clonal proliferation has not been
described (30).
Because in vitro cultivation of P. carinii is difficult and lim-
ited, researchers have primarily studied organisms obtained
from chemically immunosuppressed laboratory rats (3).
Heavily infected rats can be obtained in three ways. In the
natural transmission method, rats are exposed to airborne P.
carinii from birth by being bred in a colony containing infected
animals. Immunosuppression of animals in such a colony gen-
erally leads to severe infection, making possible the routine
recovery of ⬎100 million P. carinii per animal (8, 10, 17, 20).
Rats infected by the natural-transmission method probably
encounter P. carinii continuously. The second method for pro-
ducing rats with heavy P. carinii infections is to allow latent P.
carinii organisms to proliferate by suppressing the immune
system (5). This can be done while keeping animals isolated
* Corresponding author. Mailing address: Department of Molecular
Genetics, Biochemistry and Microbiology, University of Cincinnati
College of Medicine, Cincinnati, OH 45267-0524. Phone: (513) 558-
0097. Fax: (513) 558-8474. E-mail: stringjr@ucmail.uc.edu.
47

from environmental P. carinii by housing them under a physical
barrier. The provocation of latent P. carinii is sometimes prob-
lematical because the proportion of rats that develop heavy
infections can be more variable than in the natural-transmis-
sion model. A third method of producing Pneumocystis pneu-
monia in rats is inoculation. Previous reports have established
that inoculation of an immunosuppressed rat with a million or
more P. carinii organisms reliably produces a heavy infection
(4, 7; M. T. Cushion, M. J. Linke, M. Collins, S. P. Keely, and
J. R. Stringer, abstract from Sixth International Workshops on
Opportunistic Protists 1999, J. Eukaryot. Microbiol. 46:111S,
1999).
While all three methods (natural transmission, provocation
of latency, and inoculation) can produce Pneumocystis pneu-
monia in laboratory rats and are therefore useful for studies of
organism burden and the possible benefits of treatment, these
methods may not be equivalent when it comes to studies of the
nature of the P. carinii organisms in a given rat. This issue
became particularly important as we sought to pursue studies
of the genetic system that controls expression of the P. carinii
major surface glycoprotein (MSG) gene family. Different iso-
forms of MSG are encoded by the members of a gene family
containing as many as 100 genes (18, 19, 24–29, 31–33, 35, 42,
43, 46). Only one MSG appears to be expressed in an individ-
ual organism at any given time (40, 41; J. K. Schaffzin and J. R.
Stringer, abstract from Sixth International Workshops on Op-
portunistic Protists 1999, J. Eukaryot. Microbiol. 46:127S,
1999). Previous studies of rats in an open-air colony showed
that P. carinii populations taken from individual rats exhibited
a high degree of variation at the locus involved in MSG gene
transcription (the UCS locus) (Fig. 1).
The presence of many different MSG genes at the UCS locus
implies that MSG genes can move from a silent site to the UCS
locus by recombination (41, 44). However, the frequency of
such MSG switches has not been determined because such a
determination would require knowing the numbers and kinds
of organisms contributing to an infection and the time when
each contributing organism entered the rat. Hence, while the
variation observed at the UCS locus can be attributed to
switching the MSG gene at that locus, it is not possible to
determine when the switch occurred. A switch might have
occurred in the rat from which the P. carinii organisms were
harvested, in a rat that contributed to the cloud of airborne
organisms that pervaded the animal room, or in a previous rat,
ad infinitum.
In order to examine the stability of the UCS locus as a
function of time, it is necessary to study populations of P.
carinii formed within a single rat. Theoretically, it should be
possible to obtain such populations in either of two ways:
provocation of latent organism or inoculation. Here, we report
results obtained using both of these methods.
We found that P. carinii organisms from latently infected
rats varied with respect to UCS locus diversity. Some were
similar to P. carinii organisms from rats kept in open-air col-
onies and had many different MSG genes at the UCS locus.
Others were much simpler at the UCS locus, showing that
pneumonia can develop without great diversity at the UCS
locus. Experiments with inoculation used a population of P.
carinii that had at least 27 different MSG genes at the UCS
locus. Three different doses of P. carinii were given. Rats that
received the highest dose, 10
7
organisms, produced P. carinii
populations that were as heterogeneous at the UCS locus as
the population that contributed the inocula. By contrast, rats
inoculated with the lowest dose, 10 organisms, tended to have
a single MSG gene sequence predominant at the expression
locus, suggesting that the infectious dose may be as small as 1
organism. An intermediate dose, 10
4
organisms, produced P.
carinii populations heterogeneous at the UCS locus but with
signs of possible genetic drift. From the degree of diversity
observed in the populations that went through the bottleneck
imposed by low-dose inoculation, the maximum rate of switch-
ing appeared to be 0.01 event per generation. The data from
inoculated rats also suggest that rats that share a cage readily
infect each other and that the doubling time of P. carinii in vivo
is ⬃3 days.
We conclude that heterogeneity at the UCS locus can be
slow to develop within a population founded by a low number
of P. carinii. Hence, it is possible that the high levels of heter-
ogeneity at the UCS locus previously seen in P. carinii from
rats that have been exposed to a high number of airborne P.
carinii organisms for a long time were due to infection with P.
carinii that were already different at this locus. The results
obtained with low-dose rats suggest that it may be possible to
clone P. carinii via this method.
MATERIALS AND METHODS
Acquisition and housing of rats. For latency studies, rats presumed to have
been infected with P. carinii as neonates were purchased from either Charles
River Laboratories, Inc. (Hollister, Calif.), Hilltop Inc. (Scottsdale, Pa.), or
Harlan Inc. (Indianapolis, Ind.). The animals in the study included males and
females from five strains, Lewis CD, Sprague-Dawley, Long Evans, Wistar, and
Fisher. Inoculation studies used Lewis CD rats (125 to 150 g) from a colony
believed to be free of P. carinii (Charles River Laboratories, Inc.). The animals
were shipped in filtered transport cages. Upon receipt, the animals were imme-
diately placed in shoebox cages fitted with microisolator tops (3-␮m exclusion)
on horizontal-flow filter racks. Two animals were placed in each cage, and they
received irradiated food (Tekmar Irradiated Chow; Harlan Industries) and au-
toclaved water. To prevent the rats from contracting secondary bacterial infec-
tions, cephadrine (Velosef; ER Squibb and Sons, Inc., Princeton, N.J.) was added
to the water to achieve a final concentration of 0.200 mg/ml. All animals to be
FIG. 1. Map of the UCS-MSG locus and locations of PCR primers.
The UCS region of the locus is invariant and marks a unique telomeric
site in the genome. Adjacent to the UCS, there is always a sequence
encoding a protein called MSG. The MSG gene sequence at the UCS
locus can be different in different organisms, presumably because re-
combination installs different MSG-encoding sequences at the UCS
locus. The genome carries many copies of MSG-encoding sequences,
which reside at the ends of all chromosomes and differ from one
another. Only the MSG-encoding sequence that is linked to the UCS
locus is represented by mRNA. The CRJE is a 25-bp conserved se-
quence located between the UCS and its adjacent MSG gene. A copy
of the CRJE is also at the beginning of all known MSG-encoding
sequences not attached to the UCS locus.
48 KEELY ET AL. INFECT.IMMUN.

used in inoculation studies were tested for evidence of latent P. carinii infection
by screening sera for anti-Pneumocystis antibodies via immunoblotting. The rat
sera were diluted 1:10 and 1:40 and incubated with a blot containing separated
proteins from a standard preparation of P. carinii organisms, as described pre-
viously (46, 48). Three of the 31 animals received were sacrificed, and lung tissues
were harvested and processed for DNA analysis, which was performed by PCR
using primers targeted to the large-subunit rRNA of the mitochondrial genome
(34). The PCR assay could detect as few as 10
4
P. carinii organisms per rat (22).
Provocation of latent P. carinii. Rats were immunosuppressed for 12 weeks by
weekly subcutaneous injections of 4 mg of methylprednisolone (Pharmacia and
Upjohn Co., Kalamazoo, Mich.). Each injection produces a dose of approxi-
mately 20 mg/kg of animal weight. The animals were kept under barrier housing
conditions at all times prior to and during immunosuppression.
Inoculation. The P. carinii organisms to be used to inoculate rats were from a
cryopreserved stock of P. carinii karyotype form 1 (stock M46-5) (10). To deter-
mine the number of P. carinii organisms in the stock, nuclei were counted by light
microscopy after being stained with HEMA-3 (Curtis Matheson, Inc.), a rapid
variant of the Giemsa stain. Cyst forms were visualized by staining them with
cresyl echt violet (6). Once counted, the P. carinii preparation was serially diluted
with RPMI 1640 (GIBCO) to produce suspensions with different numbers of
organisms per milliliter. Prior to inoculation, the rats were immunosuppressed
for 2 weeks by administration of methylprednisolone as described above. Intra-
tracheal inoculation was performed on anesthetized animals by instilling 0.2 ml
of RPMI 1640 containing P. carinii per animal into the trachea using a feeding
canula introduced through the oral cavity according to the method of Boylan and
Current (7). The inoculated rats were maintained in an immunosuppressed state
by weekly subcutaneous injections of 4 mg of methylprednisolone.
Assessment of infection by microscopy. After 12 weeks of immunosuppression,
the rats were euthanized and lung tissues were homogenized, as previously
described (10). Once homogenized, the tissue from a pair of rat lungs typically
occupies a volume of 10 ml. A small aliquot was removed for staining of P. carinii
with cresyl echt violet and HEMA-3. The organism burdens per animal were
measured by counting the average number of P. carinii cysts in at least 30 oil
immersion fields, which is approximately 1.4 ⫻10
⫺5
ml (47). Enumeration of
cysts rather than nuclei is preferred because of their unambiguous morphology in
lung homogenates (11). The limit of detection was approximately 10,000 cysts per
rat (12, 22). Another aliquot of the lung homogenate was processed for pulsed-
field gel electrophoresis, and karyotype profiles were produced by contour-
clamped homogeneous electric field as described previously (21). A third aliquot
of the lung homogenate was processed for PCR as described below.
Assessment of infection and DNA recovery using PCR. The primers used in
PCR are listed in Table 1. A region of the gene encoding the large-subunit
mitochondrial rRNA was amplified with primers PAZ102-H and PAZ102-E
under the following conditions: 95°C hot start for 5 min and 30 cycles of incu-
bation at 95°C for 60 s, 50°C for 120 s, and 72°C for 60 s. The internal transcribed
spacer region 2 of the nuclear rRNA locus was amplified using primers ITS2U
and ITS2L under the same conditions described above, except that the annealing
temperature was 50°C and the number of cycles was 40 (23). To measure the
number of UCS templates in P. carinii preparations, DNA was analyzed with a
Cepheid (Sunnyvale, Calif.) Smart Cycler using Smart Cycler software version
1.01. Real-time PCR was performed under the following conditions: 95°C hot
start for 120 s and 40 cycles of incubation at 95°C for 15 s, 45°C for 15 s, 72°C for
15 s, and 80°C for 10 s with optics set to detect SYBR Green fluorescence. The
reaction mixture volumes were 25 ␮l and contained 100 ␮M (each) dATP, dCTP,
dGTP, dTTP;3UofTfl polymerase (Epicenter, Madison, Wis.); 5 mM MgCl
2
;
1␮l of a 1:20,000 dilution of SYBR Green I (BioWhitaker Molecular Applica-
tions); and 20 ng each of primers ⫺145 and anti-AUG (Table 1 and Fig. 1). The
amplicons produced from this primer pair were 165 bp in size. The specificity of
amplification was monitored in two ways. First, SYBR Green fluorescence was
measured in the Smart Cycler as a function of increasing temperature (60 to 95°C
at 0.2°C/s). Such melting curves reveal when SYBR Green fluorescence is not
attributable to the production of double-stranded DNA of the expected base
composition and size. None of the real-time PCR results reported here failed the
melting curve test. Second, the amplified DNA was subjected to electrophoresis
through an agarose gel and detected by staining it with SYBR Green. The
concentration of UCS DNA in samples was calculated by comparing the number
of cycles required to reach a threshold level of SYBR Green fluorescence to the
same parameter for various amounts of a standard plasmid containing the UCS
locus. To prepare the standard curve, the amount of DNA in the standard
plasmid was measured by optical absorbance at 260 nm. The plasmid was then
diluted by serial 10-fold dilutions. A standard curve relating amplification to the
DNA amount was produced by observing the number of cycles required to reach
a threshold level of SYBR Green fluorescence in reactions containing diminish-
ing amounts of standard plasmid.
Amplification of UCS-MSG junctions. An aliquot of lung homogenate (1 ml)
was treated with proteinase K, and genomic DNA was isolated by isopropanol
extraction (13). The DNA was dissolved in 0.01 M Tris–0.001 M EDTA, pH 8.
One microliter of the DNA was subjected to PCR using primer ⫺145 paired with
C2 (Table 1 and Fig. 1) under the following conditions: 95°C hot start for 5 min
and 40 cycles of incubation at 95°C for 60 s, 45°C for 120 s, and 72°C for 60 s. The
reaction mixture volumes were 25 ␮l and contained dATP, dCTP, dGTP, and
dTTP, each at 100 ␮M;1␮lof[␣-
32
P]dATP (10 mCi/ml; 3,000 Ci/mmol); 1.25 U
of Tfl polymerase (Epicenter); 1.5 mM MgCl
2
; and 20 ng of each primer (40).
In some experiments, a heminested PCR assay was used, in which the first
amplification was with primers ⫺145 and C2 (Table 1 and Fig. 1), followed by
reamplification with primers AUG and C2 as previously described (40). How-
ever, most of the data in this study were acquired without the heminested-PCR
step because this step was rendered unnecessary by using a 4% polyacrylamide
gel to separate amplicons. The radioactive amplicons were heated at 95°C for 3
min and then cooled on ice prior to being loaded on a fresh 4% sequencing gel
(36). The gels were run at 80 W for 6 h, dried, and exposed to X-ray film and to
PhosphorImager plates (Molecular Dynamics).
For PCR amplification targeted to the 271.2 sequence, a putative allele-
specific primer (primer ASP [Table 1]) was made by synthesizing an oligonucle-
otide that matched a site in sequence 271.2 but did not match any of the
sequences already known to be in the input population. PCRs were performed
using the ASP primer paired with the ⫺145 primer (Table 1), and the results
were monitored in real time using the Cepheid Smart Cycler. Reactions were
performed under the following conditions: 95°C hot start for 120 s and 40 cycles
of incubation at 95°C for 15 s, 59°C for 15 s, 72°C for 15 s, and 80°C for 10 s with
optics set to detect SYBR Green fluorescence. Measured amounts of DNA from
P. carinii were subjected to amplification, and SYBR Green fluorescence was
monitored as a function of reaction cycles. The abundance of DNA templates
amplified by the ⫺145-ASP primer pair was calculated by comparison to reac-
tions performed with known amounts of a plasmid carrying a copy of the 271.2
sequence.
Sequence analysis. When total PCR products were to be cloned, the PCR was
performed in the absence of radioactive nucleotide triphosphate, and the am-
plicons were purified by agarose gel electrophoresis (1.5%; 3 parts Nusieve, 1
part SeaPlaque [FMC, Rockland, Maine]) and Geneclean (Bio 101, Vista, Cal-
TABLE 1. Primers used in PCR amplification experiments
Primer Sequence Target Reference
⫺145 5⬘-TAGACGATATGAAGGGAGAAT-3⬘UCS 41
Anti-AUG 5⬘-CTCTTAACCGGCCGTGCCAT-3⬘CRJE 41
AUG 5⬘-ATGGCACGGCCGGTTAAGAG-3⬘CRJE 41
C2 5⬘-ATACATTTTTCTTCATGTTTT-3⬘MSG 41
C5 5⬘-CATGAAAGACTTGAGAAATGT-3⬘MSG
C7 5⬘-GTCTTGTCCCTTTTTATAGCA-3⬘MSG
ASP 5⬘-GTTCTTTAACTTTTTCATCC-3⬘MSG 271.2
ITS2U 5⬘-GTGGCAAAATGCGATAAGTA-3⬘5.8S rRNA gene
ITS2L 5⬘-TATGCTTAAGTTCAGCGGGT-3⬘26S rRNA gene
PAZ102H 5⬘-GTGTACGTTGCAAGTACTC-3⬘Mitochondrial rRNA gene 34
PAZ102E 5⬘-GATGGCTGTTTCCAAGCCCA-3⬘Mitochondrial rRNA gene 34
VOL. 71, 2003 P. CARINII POPULATION STRUCTURE 49

if.). The amplicons were cloned into the plasmid pCRII-TOPO (Invitrogen,
Carlsbad, Calif.), which was introduced into the strain of Escherichia coli pro-
vided with the vector.
When the goal was to determine the sequence of the MSG genes in the
radioactive DNA in bands separated on the 4% sequence gels, the bands were
excised and then eluted in distilled sterile water and reamplified with primers
AUG and C2 (Table 1) under the following conditions: 95°C hot start for 5 min
and 40 cycles of incubation at 95°C for 60 s, 48°C for 120 s, and 72°C for 60 s.
These PCR products were then cloned in pCRII-TOPO.
We also produced MSG gene sequences by amplifying genomic DNA with an
AUG primer paired with primers C5 and C7 (Table 1). These reactions were
performed under the conditions described above for amplification of the excised
gel bands.
DNA sequences were determined by the Sequencing Facility at the University
of Cincinnati College of Medicine. Most sequences were determined from both
strands. Sequences were aligned with DNAMAN software (Lynnon BioSoft,
Vaudreuil, Quebec, Canada) using the default settings. The alignments were
optimized by introducing a limited number of gaps, which were not counted in
relatedness calculations. The relatedness of pairs of aligned sequences was cal-
culated using the observed divergence method, which counts the number of
directly unmatched residues and divides this number by the total number of
residues compared. The calculated values were used to construct a distance
matrix. A homology tree was made from the distance matrix using the un-
weighted pair group method with arithmetic mean.
Statistical analysis. The statistical significance of differences in diversity of
UCS-MSG junctions among P. carinii from latently infected rats was tested by
Fisher’s exact test. The data from each rat were categorized as follows. Plasmids
with a sequence seen once in a given rat were placed in one category, and
plasmids with a sequence seen more than once in that rat were placed in another
category. For each test, the number of plasmids in each category in the test rat
was compared to the number of plasmids in each category in rat LAT 24, the rat
with the most homogeneous UCS locus, in which only one sequence was seen
among the eight plasmids analyzed. To illustrate, the numbers when LAT 3 was
compared to LAT 24 were nine and zero (LAT 3) and zero and eight (LAT 24).
RESULTS
PCR detects a broad spectrum of UCS-MSG junctions. PCR
was the method of choice to obtain UCS-MSG junctions be-
cause it is allows rapid analysis of many P. carinii populations,
even in cases where the number of microbes in the sample is
too low to support other techniques, such as cloning from
genomic libraries. Furthermore, PCR can amplify numerous
UCS-MSG junctions because the members of the MSG gene
family share certain nucleotide sequences, and these conserved
sequences can be used as primer sites. In previous studies, one
such primer, C2, had been used (41).
The C2 primer had previously supported amplification of
numerous MSG sequences, implying that a large fraction of
MSG genes have the C2 sequence. To test this idea, we in-
spected the GenBank database. To identify MSG genes in the
database, we searched for entries that had the 25-bp conserved
sequence called the CRJE (conserved recombination junction
element), which is located at the 5⬘end of all MSG genes and
defines the border between MSG and UCS sequences (Fig. 1)
(44). There were 41 different CRJE-linked MSG sequences in
the database. All but 3 of these 41 sequences had a sequence
that matched at least 24 of the 25 bp in the canonical C2
sequence. The other three MSG gene sequences matched 23 of
the 25 bp in the C2 sequence.
We also produced an additional 22 MSG gene sequences by
amplifying genomic DNA with an AUG primer paired with
primers that target conserved sequences downstream of the C2
region (C5 and C7 [Fig. 1]). Ten of these MSG gene sequences
had a sequence identical to the C2 sequence. Ten of the re-
maining 12 sequences matched 24 of the 25 bp in C2. The other
two sequences matched 23 of 25 bp, but one mismatched po-
sition was at the 5⬘end of the C2 primer binding site, reducing
the chance that it adversely affected the amplification of such
genes.
We tested the capacity of the C2 primer to amplify cloned
genes that matched only 24 of the 25 bp in the primer. Two
MSG genes of this type and two that matched C2 perfectly
were tested, and all four genes were amplified (data not
shown). These data predicted that the C2 primer will support
amplification of at least 90% of the MSG genes in the genome.
Previous amplification experiments had produced results con-
sistent with this expectation. We had observed that amplifica-
tion using the C2 primer paired with primer ⫺145 (Table 1 and
Fig. 1) produced an amplicon containing ⬎26 different MSG
sequences (41). Further work along the same lines, some of
which is described below, has shown that these two primers can
amplify ⬎80 different MSG sequences.
P. carinii populations that emerge from latency can be rel-
atively uniform at the UCS locus. To obtain populations of P.
carinii formed in a single rat, 10 rats that were presumed to be
latently infected were kept under a physical barrier and treated
with immunosuppressive drugs. Our studies included animals
obtained from three commercial animal vendors (see Materials
and Methods). Nine of the 10 rats developed lung burdens on
the order of 10
9
cysts per animal. The 10th rat had ⬎10
8
cysts.
To characterize the UCS locus, the region was amplified and
the amplicons were cloned into a plasmid vector to produce
libraries. Multiple colonies were picked from each library, and
the plasmid inserts were sequenced.
The collection of latently infected rats produced P. carinii
organisms that exhibited different degrees of diversity at the
UCS locus (Table 2). In three cases, LAT 3, LAT 9, and LAT
16, all of the sequenced inserts were different from each other.
In three others, LAT 7, LAT 1, and LAT 5, the same insert was
seen in more than one plasmid, but no single sequence was
TABLE 2. Diversity of UCS-MSG junctions in P. carinii
from latently infected rats
Rat
No. of
plasmids
sequenced
No. of
sequences
observed
%
Plasmids with a
sequence that
was observed
once
%
Plasmids with a
sequence that
was observed
more than once
P
g
Lat 3 9 9 100 0 ⬍0.001
Lat 9 5 5 100 0 ⬍0.001
Lat 16 5 5 100 0 ⬍0.001
Lat 7 8 7 75 25
a
0.003
Lat 1 8 6 50 50
b
0.029
Lat 5 10 7 50 50
c
0.038
Lat 19 7 4 43 57
d
0.077
Lat 21 8 4 38 62
e
0.099
Lat 14 9 3 23 77
f
0.260
Lat 24 8 1 0 100
Total 77 51
a
One sequence was in two plasmids.
b
Two sequences were each in two plasmids.
c
One sequence was in three plasmids; another was in two plasmids.
d
One sequence was in four plasmids.
e
One sequence was in five plasmids.
f
One sequence was in seven plasmids.
g
Pvalues calculated via Fisher’s exact test by comparing data from each rat to
those from rat Lat 24.
50 KEELY ET AL. INFECT.IMMUN.

present in ⬎50% of the plasmids. In LAT 19, LAT 21, and
LAT 14, one sequence was present in ⬎50% of the plasmids.
In one of the 10 latently infected rats, LAT 24, a single UCS-
MSG junction was present in eight of eight plasmids se-
quenced.
The data from LAT 24 indicate that the UCS locus in the P.
carinii organisms dwelling in this animal was not highly heter-
ogeneous and might have been homogeneous. Probability
analysis suggests that at least 70% of the organisms in this rat
had the same UCS-MSG sequence, because the probability of
seeing a minority sequence in eight trials when this sequence
comprises 30% of the population is 95% (1 ⫺0.7
8
⫽0.95).
However, the sample of eight plasmids was not large enough to
rule out the possibility of a smaller minority fraction. To illus-
trate, there is a 43% chance of seeing the observed result (eight
plasmids with the same sequence) when the population of
plasmids sampled is 90% homogeneous.
When compared to LAT 24 by Fisher’s exact test, P. carinii
populations from six rats (LAT 1, 3, 5, 7, 9, and 16) were found
to be statistically significantly less homogeneous at the UCS
locus (the Pvalues are shown in Table 2). The other three P.
carinii populations (LAT 14, 19, and 21) were clearly not ho-
mogeneous, but the differences between the data from these
populations and those from LAT 24 were not statistically sig-
nificant.
Comparison of the UCS-MSG sequences from different rats
showed that each population of P. carinii was different from the
others (Fig. 2). This observation suggests that emergence from
FIG. 2. Relatedness of MSG genes adjacent to the UCS locus in populations of P. carinii from latently infected rats. MSG gene sequences were
aligned by pairwise comparisons. The horizontal branch lengths represent divergence, and the bar above the dendrogram shows percent identity.
VOL. 71, 2003 P. CARINII POPULATION STRUCTURE 51

latency is not associated with expression of any particular MSG.
In summary, the data on rats with latent infections showed
that emergence from latency can be accompanied by high
diversity at the UCS locus. However, emergence from latency
does not require such diversity, nor is it associated with the
presence of a particular MSG at the UCS locus.
While not required, heterogeneity at the UCS locus was
common among the P. carinii populations emergent from the
rats with latent infections studied. The source of this hetero-
geneity is not clear. One possibility is that rats can become
latently infected by multiple P. carinii organisms that differ at
the UCS locus. An alternative is that infection is established by
a single P. carinii organism and that changes occur at the UCS
locus as the population of fungi expands. Distinguishing be-
tween these alternatives requires control over the nature of the
progenitor P. carinii. The latently infected rat model does not
lend itself to such control. Therefore, we explored the utility of
inoculation as a means to control infection.
Rats inoculated with as few as 10 organisms developed ful-
minate pneumonia. Rats were obtained from a pathogen-free
commercial colony, which should be free of latent P. carinii.
The rats were tested for evidence of latent P. carinii infection
immediately after their arrival. All animals were tested by
screening their sera for anti-Pneumocystis antibodies. In addi-
tion, 3 of the 31 animals in the shipment were sacrificed, and
lung tissues were taken and analyzed by PCR. Both of these
tests indicated that the animals were not carrying P. carinii.
Prior to inoculation, animals were rendered susceptible to P.
carinii infection by injections of methylprednisolone acetate.
The animals were then inoculated by the method of Boylan
and Current (7), which entails injection of P. carinii organisms
into the trachea using a feeding canula introduced through the
oral cavity. The animals were inoculated with either 10
7
,10
4
,
10, or 0 organisms. The number of organisms introduced was
estimated from the total number of nuclei in an aliquot from
the most concentrated suspension of organisms. As is usually
the case for P. carinii, about half of the nuclei were in cysts,
which carry up to eight nuclei each, and the other half were in
the uninucleate trophic form of the organism. The inoculated
animals were housed (two per cage) under barrier conditions
to prevent exposure to other P. carinii organisms. The animals
were sacrificed either when they appeared moribund or after
12 weeks, whichever came first. The lungs were homogenized
and screened for P. carinii by light microscopy.
Inspection of the lung homogenates by microscopy showed
that all of the animals given P. carinii were heavily infected.
TABLE 3. P. carinii organisms and DNA in inoculated rats
Rat Dose of
P. carinii
a
Results
No. of cysts
per lung
Standard PCR
b
Quantitative PCR
(no. of P. carinii
genomes per
lung)
ITS2
c
mtrRNA
d
UCS-MSG
255 0 7 ⫻10
5
Negative Negative Negative ⬍1⫻10
4
256 0 ⬍1⫻10
4
Negative Negative Negative ⬍1⫻10
4
257 0 2 ⫻10
6
Negative Negative Negative ⬍1⫻10
4
260 0 ⬍1⫻10
4
Negative Negative Negative ⬍1⫻10
4
261 0 ⬍1⫻10
4
Negative Negative Negative ⬍1⫻10
4
262 0 ⬍1⫻10
4
Negative Negative Negative ⬍1⫻10
4
267 10 3 ⫻10
7
Positive Positive Positive 3 ⫻10
7
268 10 8 ⫻10
8
Positive Positive Positive 4 ⫻10
9
269 10 3 ⫻10
5
ND ND Positive ND
270 10 5 ⫻10
6
ND ND Negative ND
271 10 2 ⫻10
7
Positive ND Positive 8 ⫻10
6
272 10 2 ⫻10
8
Positive ND Positive 3 ⫻10
8
273 10 2 ⫻10
8
ND ND Positive ND
274 10 1 ⫻10
7
ND ND Positive 7 ⫻10
7
282 10
4
8⫻10
8
Positive Positive Positive ND
283 10
4
2⫻10
8
Positive Positive Positive ND
284 10
4
6⫻10
7
Positive ND Positive ND
285 10
4
1⫻10
8
Positive ND Positive ND
286 10
4
3⫻10
8
Positive ND Positive ND
287 10
7
5⫻10
9
Positive ND Positive ND
288 10
7
2⫻10
9
Positive ND Positive ND
290 10
7
3⫻10
9
Positive ND Positive ND
291 10
7
6⫻10
8
Positive ND Positive ND
292 10
7
6⫻10
7
Positive ND Positive ND
294 10
7
6⫻10
7
Positive ND Positive ND
295 10
7
3⫻10
8
Positive ND Positive ND
296 10
7
9⫻10
8
Positive ND Positive ND
298 10
7
2⫻10
9
Positive ND Positive ND
a
Counts are total number of P. carinii nuclei in 0.200 ml, which was the volume injected into each rat. Nuclei in both cysts and trophic forms of P. carinii were counted
with a light microscope.
b
Standard PCR was performed for 40 rounds under conditions described in Materials and Methods. The primers used are listed in Table 1. ND, not done. The results
of a standard PCR were scored as positive if a band of the expected size was visible after an agarose gel was stained with ethidium bromide.
c
ITS2 is a single-copy locus located in the rRNA gene of P. carinii. The primers used are listed in Table 1.
d
mtrRNA is the gene in the P. carinii mitochondrial genome encoding the RNA in the large ribosome subunit of mitochondria. The primers used are listed in
Table 1.
52 KEELY ET AL. INFECT.IMMUN.

Table 3 lists the number of P. carinii cysts per animal as de-
termined by counting an aliquot of lung homogenate. The
average numbers of P. carinii cysts per lung were 1 ⫻10
9
,3⫻
10
8
, and 2 ⫻10
8
in animals inoculated with 10
7
,10
4
, and 10
organisms, respectively. Hence, the average number of cysts
was only weakly related to the dose, suggesting that as few as
10 organisms can initiate infections that give rise to lung bur-
dens as great as those in rats given 10
7
organisms.
While infection was easily discernible in rats given only 10 P.
carinii organisms, no cysts were seen in four of the six rats that
had been inoculated with medium lacking P. carinii (sham
inoculated). One putative cyst was seen in the lung homoge-
nate from one of the other two sham-inoculated rats, and three
were seen in the other. The identity of these cyst-like objects is
uncertain because the PCR showed no evidence of P. carinii
DNA in the lung homogenates from any of the sham-inocu-
lated rats (see below). Nevertheless, it is possible that the
observed objects were P. carinii cysts, presumably derived from
latent organisms. If it is assumed that latent infections were the
source of the putative cysts, then these latent organisms pro-
duced very few progeny compared to the number seen in the
rats given 10 or more P. carinii organisms.
To estimate the amount of P. carinii DNA recovered from
lung homogenates, at least one sample from each rat was
tested for P. carinii DNA by standard PCR, which entailed 40
rounds of cycling followed by analysis of the products on an
agarose gel. Every animal that had received at least 10
4
P.
carinii organisms produced a PCR product detectable by
ethidium bromide staining (Table 3). All but one of the rats
given 10 P. carinii organisms produced a PCR product visible
by ethidium bromide staining. By contrast, lung homogenates
from the six sham-inoculated rats did not produce a PCR
product from any of the three loci tested, including the gene
encoding the large-subunit mitochondrial rRNA, which is the
most sensitive target for PCR, presumably because there are
multiple copies of mitochondrial DNA per organism (45).
Similar results were obtained when the PCR was performed
in real time, which allowed estimation of the amount of am-
plifiable DNA. In this assay, a 165-bp region of the UCS locus
was amplified using primers ⫺145 and anti-AUG (Table 1 and
Fig. 1). As shown in Table 3, none of the sham-inoculated rats
produced a detectable signal in the real-time PCR assay, which
in our hands detected as few as 10 templates per reaction. The
real-time PCR results showed that the numbers of UCS DNA
molecules in the lungs of rats that were inoculated with 10 P.
carinii were high, ranging from approximately 8 ⫻10
6
to 4 ⫻
10
9
genomes per lung. In general, the organism burdens esti-
mated by counting cysts were consistent with the results of
real-time PCR. Deviations from perfect congruence may have
been caused by factors such as inefficiency in DNA recovery
and variation in the ratio of the cysts to trophic forms.
High-dose inoculation produced P. carinii populations that
closely resembled the population from which the inocula were
drawn. To study UCS-MSG junctions, the C2 and ⫺145 prim-
ers were used to amplify this region (Table 1). The amplifica-
tion products were first analyzed by gel electrophoresis. As
shown in Fig. 3, lanes 1 and 20, the UCS-MSG junction in the
P. carinii population used to inoculate rats (input population)
was quite heterogeneous. There were 10 obvious bands, as well
as several fainter bands. These bands ranged in size from ⬃510
to 450 bp. Analysis of DNA sequences present in these ampli-
cons showed that there were at least 27 different MSG gene
sequences at the UCS locus in the input P. carinii population
(see below). All of the animals inoculated with 10
7
organisms
produced the same band pattern as the input population (Fig.
3, lanes 11 to 19).
The animals inoculated with 10
4
organisms also produced a
UCS-MSG band pattern that had all of the bands seen in the
input population (Fig. 3, lanes 6 to 10). However, the band
intensities within patterns varied more than when animals were
inoculated with 10
7
P. carinii. One band that exhibited this trait
is marked in Fig. 3. Such fluctuations are expected in small
populations, which are more subject to the effects of genetic
drift.
Low-dose inoculation produced P. carinii with little hetero-
FIG. 3. Sizes of UCS-MSG amplicons from rats inoculated with different doses of P. carinii. UCS-MSG junctions were amplified from genomic
DNA extracted from different P. carinii populations. The radiolabeled amplicons were separated by electrophoresis through 4% acrylamide. Lanes
1 and 20, template DNA from the population from which inocula were drawn. The dots in lane 1 indicate the 10 strongest bands in the input
pattern. Lanes 2 to 5, 6 to 10, and 11 to 19, template DNAs from rats inoculated with 10
1
,10
4
, and 10
7
organisms, respectively. The DNA bands
in lanes 2, 3, 4, and 5 were produced from animals 267, 268, 271, and 272. The arrowhead next to lane 6 marks band 8, which was one of several
bands that varied in intensity among the rats inoculated with 10
4
organisms. The band in lane 21 was generated by amplification from a plasmid
containing a single UCS-MSG junction. The numbers on the left are DNA sizes in base pairs.
VOL. 71, 2003 P. CARINII POPULATION STRUCTURE 53

geneity at the UCS locus. By contrast with the results obtained
in rats inoculated with 10
4
or more organisms, seven of the
eight rats inoculated with 10 P. carinii organisms produced a
band pattern that was much simpler than that of the input
population. (The eighth rat in the group, rat 270, did not
produce a PCR product.) Amplification of lung homogenates
from five of the seven rats, 271 and 272 (Fig. 3, lanes 4 and 5),
and 269, 273, and 274 (data not shown) yielded a single strong
band. Rats 267 and 268 produced three and two strong bands,
respectively (Fig. 3, lanes 2 and 3). No bands were present after
amplification of DNA prepared from lung homogenates of
sham-inoculated rats (data not shown). These results are con-
sistent with the results of quantitative PCR described above.
To confirm that the amplified UCS-MSG junctions accu-
rately represent the UCS locus, we explored the possible in-
fluence of PCR artifacts in these experiments. One possibility
that must be excluded in any PCR experiment is DNA con-
tamination, which is usually caused by DNA from previous
PCR experiments. We can rule out DNA contamination for
the following reasons. (i) Real-time PCR showed that the
number of P. carinii genomes in the lung homogenates corre-
sponded to the number of cysts seen in the microscope. There-
fore, the amplicons were not produced from a rare template, as
would be expected if contamination were the source of that
template. (ii) Contamination would not be expected to pro-
duce the many different UCS-MSG junction bands produced
by amplification of lung homogenates. (iii) Control reactions
that lacked lung homogenates did not produce a product. (iv)
No P. carinii DNA was detected in sham-inoculated rats. (v)
The sequences produced from the low-dose rats did not match
anything we had amplified and sequenced before.
A second possible source of artifacts would be failure of the
PCR to amplify all of the UCS-MSG junctions present in the
sample. This was a particular concern in the experiments with
rats that had been inoculated with ⬍10
7
organisms, because
these animals tended to be somewhat less infected (Table 3).
Taken to the extreme, a low template concentration might
cause only the most common UCS-MSG junction to amplify.
We tested this possibility by amplifying diminishing amounts of
DNA from the lung homogenate of rat 292, which had rela-
tively few cysts (Table 3) but had produced a pattern with 10
major bands when undiluted DNA was amplified (Fig. 3, lane
15). These experiments showed that a reaction mixture that
contained a 32-fold dilution of sample 292 produced the pat-
tern of 10 bands. Rats 267, 268, 271, 272, 273, and 274 all
contained at least five times as many organisms as the 32-fold
dilution of sample 292 (Table 3). Therefore, the simple UCS-
MSG band patterns produced from these rats were not arti-
facts imposed by low DNA template concentrations
Another concern associated with PCR is the possibility of
production of artifactual bands. This possibility was examined
by experiments on plasmids carrying UCS-MSG junctions of
various sizes. As expected, amplifications of cloned UCS-MSG
junctions each produced a single band, which migrated in the
expected manner (Fig. 4). In addition, amplification of a mix-
ture of these plasmids produced the pattern expected, and no
new bands were generated (data not shown). These control
experiments showed that amplification of genomic DNA from
P. carinii would be expected to faithfully reproduce the UCS-
MSG junctions in the genome and not to generate junctions
that do not exist. Further evidence in support of the fidelity of
the PCR assay was obtained by sequencing the amplicons,
which showed that all of them had an open reading frame
encoding an MSG (see below). Preservation of the open read-
ing frame is inconsistent with the hypothesis that fragments of
different sizes were produced by PCR mistakes that either
deleted, added, or recombined DNA, because such mistakes
would be expected to often disrupt the reading frame.
Each UCS-MSG junction band from low-dose-inoculated
rats contained predominantly one sequence. To quickly deter-
mine if bands from low-dose rats might contain a single se-
quence, the largest and smallest bands from 267, the smallest
band from 268, and the band in 271 (shown in Fig. 3, lanes 2,
3, and 4) were excised from the gel, reamplified, and se-
quenced directly. In each case, an unambiguous sequence
could be read, indicating that each band contained predomi-
nantly one UCS-MSG sequence. By contrast, the same analysis
of bands from the input population indicated that these bands
were comprised of multiple sequences. To confirm the se-
quence obtained by direct sequencing, DNA molecules from
the four bands from rats 267, 268, and 271 were reamplified
with primer AUG and primer C2, and the products were
cloned by insertion into a plasmid vector. At least three plas-
mid clones from each band were sequenced. All plasmids made
from a given band contained the same sequence, and the
cloned DNA sequence matched that read directly from the
DNA purified from the gel.
UCS-MSG sequences predominant in inoculated rats
matched sequences in the input organism population. The
inoculation model is based on the premise that the procedure
causes P. carinii infections. If this is the case, then the se-
quences of UCS-MSG junctions found in inoculated rats would
be expected to match the UCS-MSG sequences in the input
population. To test this hypothesis, the UCS-MSG junctions in
the input organism population were subjected to sequence
analysis. In the first stage of this analysis, we cloned the DNA
in four of the bands (bands 2, 3, 4, and 6) in the input band
pattern (Fig. 3, lanes 1 and 20). We sequenced 11 plasmid
inserts, 3 each from bands 2, 3, and 4 and 2 from band 6. These
11 plasmid inserts were all different in sequence, but all 11
FIG. 4. Sizes of UCS-MSG amplicons produced from control plas-
mids. UCS-MSG junctions were amplified from six plasmids, each of
which carried a different sequence. The radiolabeled amplicons were
separated by electrophoresis through 4% acrylamide. Lanes 1 to 6,
amplicons from six different plasmids, each carrying a different UCS-
MSG junction. On the right are DNA sizes in base pairs.
54 KEELY ET AL. INFECT.IMMUN.

sequences encoded an MSG protein. The percent identities of
the MSG genes from different bands were 85.9 to 86.9 (band
2), 83 to 96 (band 3), 85.2 to 98.4 (band 4), and 99% (band 6).
To further evaluate the UCS-MSG junctions in the input
population, an aliquot of the organisms in this population was
amplified and the amplicons were cloned directly into plasmids
without first purifying bands of different sizes via electro-
phoretic separation. Plasmids from 25 bacterial clones were
sequenced. When these sequences were pooled with the data
obtained by sequencing plasmids made from gel-purified
bands, 36 sequences were available for comparison. Figure 5
shows the relatedness of these 36 sequences. One sequence
was found four times, two sequences were found three times,
and two sequences were found twice. Twenty-two sequences
were present a single time. Therefore, there were 27 different
MSG genes among the 36 UCS-MSG junctions sequenced.
The sequences in Fig. 5 include those previously seen in five of
the low-dose rats (267, 271, 272, 273, and 274). The absence of
sequences seen in the other two low-dose rats may have been
due to the incompleteness of the data from the input popula-
tion. Very few sequences were seen in more than one plasmid,
indicating that the plasmid library made from the input pop-
ulation contains more than the 27 sequences observed in the 36
plasmids sequenced.
Low-dose inoculation radically reduced but did not elimi-
nate variation at the UCS locus. Analysis of gel-purified bands
produced by amplification of the UCS locus in homogenates
from rats inoculated with 10 organisms had detected just one
sequence in each band. However, these data did not rule out
the possibility of minor heterogeneity at the UCS loci in these
samples. For example, minor junctions might be a different size
than the gel-purified bands. To address this issue, the PCR
products produced from rats 271, 272, and 273 were inserted
directly into plasmids without first being subjected to gel elec-
trophoresis. Multiple plasmids were isolated and sequenced
(eight from 271 and seven each from 272 and 273).
This procedure showed that the UCS locus from each of the
three rats was not homogeneous (Fig. 6). Six of the eight
cloned PCR products from rat 271 had the sequence expected
from previous sequencing analyses, but the other two plasmids
each carried a different MSG sequence. Similarly, one of the
seven plasmids carrying the PCR product from rat 272 was
different from the other six. In rat 273, one of the seven plas-
mids was different from the other six.
In each of the three rats, the minority sequence(s) was quite
different from the predominant sequence (13 to 22% diver-
gent). To illustrate this divergence, Fig. 7 shows a nucleotide
alignment of majority and minority sequences from rat 271.
The two sequences differ at 44 positions. This degree of diver-
gence cannot be due to PCR or sequencing error, the fre-
quency of which, in our hands, was one mistake per 4,000 bp
amplified (data not shown).
Minority sequences were not found among the 36 plasmids
bearing UCS-MSG junctions amplified from the input popu-
lation. A sample size of 36 is large enough to suggest that none
of these sequences comprised more than 10% of that popula-
tion. To confirm this suggestion, a more sensitive test was
applied. Allele-specific PCR (ASP) was used to test the input
population for the presence of sequence 271.2, one of the two
minority sequences found in rat 271. Sequence 271.2 was se-
lected as the target of this experiment because it contained an
appropriately located region in which the sequence was differ-
ent from the 27 sequences observed in the input population.
Hence, an oligonucleotide that matched this region of 271.2
FIG. 5. Relatedness of MSG genes adjacent to the UCS locus in
the population of P. carinii used to prepare inocula. Inserts in 36
plasmids carrying UCS-MSG junctions amplified from the input pop-
ulation were sequenced. Twenty-seven different sequences were
present. Five sequences were present in more than one plasmid. These
are numbered, e.g., 1.1 and 1.2. The sequences were aligned by pair-
wise comparisons, and percent identities were calculated. These are
displayed in the dendrogram. The horizontal branch lengths represent
divergence, and the bar above the dendrogram shows percent identity.
To illustrate, sequences and 17 and 18 differed by ⬃17%. Sequence 13
matched that predominant in inoculated rats 271 and 272. Sequence 23
matched 13, which matched that predominant in inoculated rats 273
and 274. The sequence in plasmids 15.1, 15.2, and 15.3 was the same as
that seen in inoculated rat 267.
VOL. 71, 2003 P. CARINII POPULATION STRUCTURE 55

(primer ASP in Table 1) would be expected to amplify the
271.2 sequence and not to amplify most, if not all, of the other
sequences in the input population DNA.
PCRs were performed using primer ASP paired with the
⫺145 primer (Table 1), and the results were monitored in real
time. The ASP produced a product, but only after ⬎30 cycles.
By contrast, control experiments showed that the UCS locus
amplified much faster. The delay in ASP product formation
indicated that the template for this product constituted no
more than 0.6% of the UCS-MSG junctions in the input pop-
ulation. Subsequent sequence analysis, however, showed that
the DNA amplified in the ASP did not match the 271.2 se-
quence but instead matched a sequence that had been seen
once in the collection of plasmids carrying UCS-MSG junc-
tions amplified from the input population. This sequence
matched the ASP primer at all but one position, which was
near the 5⬘end of the primer, so it is not surprising, in retro-
spect, that the ASP supported amplification of this gene. Be-
cause the 271.2 sequence was not detected under conditions
that allowed amplification of a related gene that constituted no
more than 0.6% of the UCS-MSG junctions, we conclude that
if the 271.2 sequence is in the input population, it is less
abundant than 0.6%.
Cagemates tended to have the same UCS-MSG junction.
Before and after inoculation, rats had been kept two to a cage.
The eight rats in the low-dose group were housed as four pairs
of cagemates as follows: 267 with 268, 269 with 270, 271 with
272, and 273 with 274. Rat 270 did not produce a PCR product,
leaving three pairs of cagemates for analysis. In two of the
three pairs of cagemates, 271-272 (Fig. 3, lanes 4 and 5) and
273-274 (data not shown), the UCS-MSG junction band was
the same size in both members of the pair. In the remaining
pair (267-268), the band patterns were more complex, with
three bands in 267 and two in its cagemate, 268, but the two
smaller bands of 267 comigrated with the bands in 268 (Fig. 3,
lanes 2 and 3). Sequence analysis showed that each cage had a
different MSG predominant at the UCS locus but that the two
rats in a cage had the same predominant UCS-MSG junction.
These results are unlikely to occur by chance. To illustrate,
in rat pair 271-272 one UCS-MSG junction predominated. The
probability that this particular genotype will be the one to
expand to form the predominant USC-MSG genotype in a rat
is no greater than 1/27, or 0.037, because there were at least 27
different UCS-MSG junctions in the input population. The
probability that both rats in a cage will have this particular
genotype predominate is 0.037
2
. However, because there are
FIG. 6. Relatedness of UCS-linked MSG genes from rats inoculated with 10 P. carinii organisms and housed either together or separately.
Twenty-five plasmids carrying UCS-MSG junctions amplified from three rats (271 and 272, which were cagemates, and 273, which was housed in
a different cage) were aligned by pairwise comparisons. Percent identities were calculated and are displayed as a dendrogram. The horizontal
branch lengths represent divergence, and the bar above the dendrogram shows percent identity. To illustrate, sequences 271.1 and 271.2 differed
by ⬃17%.
56 KEELY ET AL. INFECT.IMMUN.

27 different genotypes to choose from, the probability that any
two rats will have the same genotype is the sum of all proba-
bilities of this happening for a particular genotype,
冘
1
27 0.037
2
⫽
0.037. Hence, the probability of seeing two rats with the same
genotype is low, and the probability of seeing two sets of
cagemates that share a genotype is extremely unlikely to occur
by chance.
Sharing of genotypes between cagemates was also seen by
comparing the minority sequences. One of the minority DNA
sequences in rat 271 was 99.7% identical to the minority se-
quence of rat 272.
DISCUSSION
A previous study of rats from open-air colonies had shown
that variation at the UCS locus was pronounced and common
(41). In the previous study, all 37 rats analyzed contained P.
carinii organisms that were heterogeneous at the UCS locus. In
exhibiting this high degree of variation, the UCS locus is ex-
traordinary compared to the remainder of the P. carinii ge-
nome, where sequence variation is very rarely observed, even
at loci that would be expected to be prone to mutation due to
their structure, such as the presence of microsatellites. The
studies presented here showed that, in contrast to those in rats
from the open-air colony, the UCS locus was much less com-
plex in some of the rats that developed pneumonia following
the provocation of latent P. carinii organisms and in all of the
rats that received a low-dose of P. carinii via inoculation. The
association between high variation at the UCS locus and the
natural-transmission method suggests that high variation may
be caused by infection of a given rat by numerous P. carinii
organisms, each with a different UCS-MSG junction. The pres-
ence of P. carinii with different UCS-MSG junctions may be
due to the accumulation of different UCS-MSG junctions in
the population of P. carinii being produced by the open-air
colony as a whole. A long period of accumulation is possible,
because this colony has been maintained for years, during
which time animals from different commercial colonies have
been introduced. Given the high frequency of latency in com-
mercial colonies, most, if not all, of the rats introduced into the
open-air colony can be assumed to have been harboring latent
P. carinii organisms.
The UCS diversity in P. carinii obtained by immunosuppres-
sion of latently infected rats may have come from infection
with multiple P. carinii organisms prior to being placed under
a barrier. Alternatively, diversity may be generated after infec-
tion. Latent infections are probably initiated in neonates,
which are immunonaive but immunocompetent (1). With time,
the animals might detect the presence of P. carinii and mount
a response, which could select for switching at the UCS locus.
This scenario differs from what would be expected in the rats
given a low dose of P. carinii, because inoculated animals were
immunosuppressed before and after inoculation.
To determine whether the cause of UCS diversity in latent
infections is due to events that happen before or after infec-
tion, it is necessary to control the source of the P. carinii
FIG. 7. Alignment of two UCS-linked MSG genes from rat 271. The sequence labeled 271.12 was in six of eight sequenced plasmids produced
by cloning the amplicons from rat 271. Sequence 271.1 was in one of these plasmids. In both sequences, the first 25 bases are the CRJE (44), which
is a conserved sequence found at the beginning of all known MSG genes and at all known USC-MSG junctions. The dots represent identity, and
the dashes represent gaps introduced to optimize the number of matches. The number at the end of each line of sequence indicates the number
of nucleotides from the beginning of that sequence. The two sequences differ at 54 positions (17%).
VOL. 71, 2003 P. CARINII POPULATION STRUCTURE 57

organisms that cause infections. The results of the low-dose
inoculation experiments suggest that such control can be ex-
erted via inoculation.
The data obtained from the low-dose inoculation experi-
ments suggest a maximum frequency for switching the MSG
gene at the UCS locus. This rate, r, can be estimated from the
relationship f⫽(1 ⫺r)
d
, where fis the fraction of organisms
that have the predominant MSG at the UCS locus and dis the
number of population doublings. fis known, and dcan be
estimated from the difference between the number of organ-
isms introduced and the final population size. Inoculation with
10 P. carinii organisms typically produced a population num-
bering approximately 10
8
organisms. To increase the popula-
tion size from 10 to 10
8
would require at least 23 doublings (2
23
⫽0.84 ⫻10
7
). However, it is probable that ⬎23 doublings
transpired, because most of the 10 P. carinii organisms intro-
duced did not appear to measurably contribute to the popula-
tion present at the end of the experiment. (That few organisms
proliferated can be inferred from the relative uniformity of the
UCS locus at the end of the experiment compared to the
heterogeneity at the UCS locus in the input population from
which inocula were drawn.) If only one organism proliferated,
then dwould be 27 doublings. When 23 ⬍d⬍27 and f(the
fraction of UCS-MSG junctions with the predominant MSG) is
0.8, the calculated rate of switching, r, is approximately 0.01
events per P. carinii organism per doubling, or one switch for
every 100 organisms. It could be argued that it is inappropriate
to base such calculations on population doublings because the
manner by which P. carinii reproduces is not known and may
not involve simple mitotic cell division. If mitotic reproduction
were not the proliferation mechanism, the presumptive alter-
native would be via mating followed by sporulation, in which
case proliferation would proceed by using two mating partners
to produce eight spores, thereby increasing the population in
fourfold increments rather than twofold. If such a mating pro-
cess were the sole means of reproduction, the population size
(p) would be related to the number of mating generations (m)
as follows: p⫽2
2
m
⫺1
. To increase from 10 to 10
8
organisms
via mating would require 11 mating generations. Therefore,
since f⫽(1 ⫺r)
m
, the calculated rate of change at the UCS (r),
as a function of mating generations (m), is 0.02 event per
mating generation, or one switch for every 50 mating events.
Admittedly, estimating the switching rate is speculative at
this point, especially since the minority sequences seen at the
UCS locus need not have been formed by switching in the
inoculated rat. Alternative sources of the minority sequences
seen at the UCS locus include proliferation of more than one
of the organisms introduced by inoculation and a contribution
by latent P. carinii organisms. While we cannot conclude that
switching was observed, it is interesting to consider the theo-
retical long-term ramifications of a switching rate of 0.01 event
per organism per doubling. Starting from a single P. carinii
organism, switching at this rate over the course of 100 dou-
blings would generate a population in which most (two-thirds)
of the members would have an MSG at the UCS locus that is
different from that in the P. carinii organism that founded the
population. Hence, switching at this rate for a period of
months or years would seem to be sufficient to generate the
high levels of heterogeneity at the UCS locus observed in the
open-air colony.
While the UCS locus can be highly heterogeneous in certain
populations of P. carinii, the rate at which it changes is low
enough to allow UCS diversity to be used as a tool to under-
stand population dynamics in inoculated rats. It has been
known for years that inoculation with high doses of P. carinii
(on the order of 10
6
organisms) causes heavy infection in most
rats, but the details of the infection process had not been
examined. The possibilities span two extremes. At one extreme
is the possibility that only one organism reproduces to even-
tually cause disease. At the other extreme is the possibility that
all of the input organisms reproduce themselves. Tracking the
UCS-MSG junctions present in input and output populations
in rats that received at least 10
4
P. carinii organisms suggests
that neither of these two extremes is the case. Rather, it seems
that multiple organisms contribute to the infection when the
dose is in the tens of thousands. However, the fraction of
introduced organisms that contributes to proliferation may be
as low as 1 in 1,000.
Multiple organisms must have contributed to population
growth in rats inoculated with 10
7
P. carinii, because these rats
produced populations with the same complex collection of
UCS-MSG junctions as the input population; at least this ap-
peared to be true based on the sizes of the UCS-MSG junc-
tions amplified. It is not possible to say at this point if the
populations produced in the nine rats given 10
7
P. carinii or-
ganisms were identical to the input, but it seems probable. This
issue can be addressed by subjecting the output P. carinii pop-
ulations to further analysis by sequencing UCS-MSG junctions.
However, such an analysis would require the sequencing of
hundreds of cloned UCS-MSG junctions, which was beyond
the scope of this study.
While all of the rats inoculated with 10
7
P. carinii organisms
produced the same band pattern upon amplification of the
UCS-MSG junction, the patterns produced from rats inocu-
lated with 10
4
organisms varied with respect to the intensities
of the bands. Fig. 3, lane 8 shows an example of intensity
variation. Band 8 is very faint in this lane. The near loss of this
band suggests that only a small fraction of the input organisms
proliferated. This fraction can be estimated from the following.
In the input band pattern, band 8 is one of five bands that stand
out as being most intense. Therefore, the fraction of P. carinii
organisms in the input population that have a UCS-MSG junc-
tion that produces a PCR product the size of band 8 is between
10 and 20%. If we assume that this fraction is 15%, then the
probability that this band would not be represented among the
organisms that proliferated is given by P⫽0.85
x
, where xis the
number of organisms that proliferate. If we assume that 1,000
of the 10
4
input organisms contributed to the population, then
P⫽0.85
1,000
⫽2⫻10
⫺71
. Hence, it is very improbable that the
band in question would be missing if 1,000 organisms were to
proliferate. It follows that the chance of seeing variation in the
intensity of this band would also be negligibly small. By con-
trast, if only 10 of the 10
4
input organisms founded the popu-
lation, then P⫽0.85
10
⫽0.2, and it is probable that the band
would be missing in ⬃20% of the rats. Therefore, it is reason-
able to speculate that few of the input P. carinii organisms,
perhaps only 10, proliferated in rats inoculated with 10
4
organ-
isms. It is interesting that if the efficiency of proliferation in
rats given 10
7
P. carinii organisms were similarly low, i.e., 10 in
10
4
, approximately 1,000 organisms would contribute to the
58 KEELY ET AL. INFECT.IMMUN.

infection, in which case one would expect to see no variation in
band intensities, as was observed.
While multiple organisms appear to have proliferated in the
rats that received 10
4
or more P. carinii organisms, results with
rats that received only 10 P. carinii organisms suggest that only
1 may be required to cause disease. In four of these animals
(rats 271, 272, 273, and 274), the P. carinii organisms exhibited
characteristics that would be expected of a clonally derived
population, i.e., a single predominant UCS-MSG junction,
which was also present in the input population. On the other
hand, the UCS-MSG junction was not completely homoge-
neous, which would seem to argue against clonality. However,
the small fraction of organisms with a different UCS-MSG
junction may have been generated by switching at the UCS
locus. The possibility of switching is supported by the obser-
vation that the minority UCS-MSG junctions were not found
in the input population. This observation is as would be ex-
pected if new MSG genes were to be installed at the UCS in
some organisms as the population expanded. While it would be
premature to conclude that switching generated the heteroge-
neity at the UCS locus, the consistent production of popula-
tions of P. carinii in which most (80 to 90%) of the members
have the same UCS-MSG junction fits well with what would be
expected from switching. By contrast, proliferation of two or-
ganisms might be expected to produce populations in which
two genotypes are present in roughly equal numbers. Regard-
less of whether switching occurred, the heterogeneity at the
UCS locus was low enough to suggest that just a few of the 10
P. carinii organisms introduced proliferated.
The rate at which the P. carinii population increases in vivo
has not been well studied. Inoculation helps address this ques-
tion by delivering a measured number of organisms at a fixed
time. The rats that received 10 P. carinii organisms carried an
average of 2 ⫻10
8
P. carinii organisms per animal at the end
of the experiments (12 weeks [84 days] postinoculation). The
minimum number of doublings required to increase from 1 to
10
8
organisms is approximately 27. Therefore, if only one input
organism contributed to the infection, then the apparent dou-
bling time was approximately 3.1 days (84 days/27 doublings).
If all 10 of the input organisms proliferated, then the calcu-
lated doubling time increases to 3.5 days. These doubling time
estimates are subject to caveats. The doubling time could have
been ⬍3.1 days if the proliferation period were shorter than 12
weeks. This parameter can be analyzed in the future. The only
way that the true doubling time could have been much more
than 3.5 days would be if many more than 10 P. carinii con-
tributed to population growth. For example, if doubling the
population actually required 5 days, then the production of 10
8
P. carinii organisms in 12 weeks would require a founding
population of 1,000 P. carinii organisms.Since only 10 P. carinii
organisms were introduced, the only way the founding popu-
lation could have been larger would be via the contribution of
latent organisms. The presence of such a large number of
latent P. carinii organisms seems very unlikely. The sham-
inoculated rats produced no detectable P. carinii DNA, indi-
cating that if latent organisms were present, they were at a very
low level. Therefore, all indications are that the doubling time
of P. carinii in low-dose rats was ⬍3.5 days.
Another interesting observation that emerged from the in-
oculation studies was the tendency of low-dose rats kept in the
same cage to have the same MSG gene at the UCS locus. This
observation suggests that one animal may have infected the
other in the cage. Even though both animals in each pair of
cagemates were inoculated, it is possible that only one was
infected by the inoculation because the dose was low. The
transmission scenario fits with the large difference in organism
burden between rats 271 and 272, for instance. The alternative
to the transmission scenario is that the sharing of genotypes in
cagemates was due to chance. Indeed, it is nearly certain that
the two aliquots of P. carinii used to infect the pair of rats in a
given cage shared at least one genotype. Nevertheless, the
probability that both rats in a cage would have the same pre-
dominant genotype by chance is only 0.037. Hence, the pres-
ence of the same predominant UCS-MSG junction in multiple
pairs of cagemates seems unlikely to have been due to chance.
A more surprising aspect of the cagemate data was the
presence of the same minority UCS-MSG junctions in one pair
of cagemates. This situation may have been caused by trans-
mission of the majority UCS-MSG junction, followed by a
programmed switch to the minority MSG gene. Alternatively,
it is possible that one cagemate was infected by one UCS-MSG
genotype and the other by the other genotype, followed by
transmission between the cagemates in both directions. An-
other possibility is that both UCS-MSG junctions were trans-
mitted from one rat to the other, perhaps independently, per-
haps by a transmissible form of the organism that carries both
genotypes.
In summary, the P. carinii populations in rats that received
10 organisms were much less complex at the UCS locus than
the input population. The populations of P. carinii observed in
the low-dose rats were at least 80% homogeneous with respect
to the UCS locus. One explanation of these results is that only
one organism founded the population and that switching oc-
curred as the population grew. Whether or not this explanation
proves to be correct, the presence of populations that are at
least quasiclonal strongly suggests that inoculation with one
organism can be used to clone P. carinii, provided that one can
obtain rats that are free of P. carinii prior to being inoculated.
In any event, our data suggest that when the objective is to
obtain P. carinii that are relatively homogeneous with respect
to MSG, it would be advantageous to use low-dose inoculation
rather than rats infected via constant exposure to airborne P.
carinii. Alternatively, latently infected rats can be screened for
populations of P. carinii that are relatively simple at the UCS
locus.
ACKNOWLEDGMENTS
We thank Mario Medvedovic and Gary Weiss for advice on statistics
and probability.
This work was supported by grants R01AI36701 (J.R.S.) and
R01AI29839 (M.T.C.), both from the National Institute of Allergy and
Infectious Diseases.
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Editor: J. M. Mansfield
60 KEELY ET AL. INFECT.IMMUN.