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Previous Article | Next Article 
Journal of Clinical Microbiology, June 2000, p. 2317-2323, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Intestinal Lesions Associated with Disseminated
Candidiasis in an Experimental Animal Model
Karl A.
Andrutis,1
Perry J.
Riggle,2
Carol A.
Kumamoto,2 and
Saul
Tzipori1,*
Division of Infectious Diseases, Tufts
University School of Veterinary Medicine, North Grafton, Massachusetts
01536,1 and Department of Molecular
Biology and Microbiology, Tufts University School of Medicine,
Boston, Massachusetts 021112
Received 18 January 2000/Returned for modification 6 March
2000/Accepted 3 April 2000
 |
ABSTRACT |
In human patients, disseminated candidiasis, a life-threatening
disease for immunocompromised patients, is often associated with
intestinal lesions. In this study, we demonstrate that immunosuppressed gnotobiotic (IGB) piglets orally inoculated with wild-type
Candida albicans developed extensive intestinal lesions and
disseminated infection. Severe ulceration of the ileal mucosa was
observed overlying regions of colonization and necrosis of the
gut-associated lymphoid tissue. Despite the high susceptibility of IGB
piglets to many microbial pathogens, an avirulent mutant strain of
C. albicans failed to produce intestinal lesions and
exhibited poor dissemination, demonstrating that these effects required
virulent organisms. It is likely that in IGB piglets, as in human
patients, intestinal lesions provide the mechanism for escape of
C. albicans from the gastrointestinal tract. Multinucleated
giant cells containing fungal organisms were observed within lymph
nodes and lymphatic vessels, and as with other pathogens, such cells
could provide a mechanism for dissemination of C. albicans.
| |
INTRODUCTION |
Candida albicans is an
important opportunistic pathogen of immunocompromised individuals
causing diseases that range from superficial mucocutaneous infections
to life-threatening systemic candidiasis. As a result of modern medical
interventions, the incidence of candidiasis has been increasing over
the past decade (19). Populations at risk for serious
disseminated disease include immunosuppressed patients, such as cancer
patients, particularly leukemia patients, and transplant recipients.
C. albicans also causes mucocutaneous infections, such as
oropharyngeal candidiasis, which are particularly pronounced in AIDS
patients and are typically recurrent (22).
In disseminated candidiasis, C. albicans is thought to
originate from the gut (6, 28), where the organism is
frequently found as a component of normal flora (5). Autopsy
studies of cancer patients with fungal infections indicate that 20% of
such patients exhibit lesions in the small or large intestine
(9). Spontaneous perforation of the ileum is highly
associated with systemic candidiasis in very-low-birth-weight infants,
another susceptible population (1). Despite the association
between intestinal lesions and systemic disease (9),
gastrointestinal candidiasis has not been well studied. The
interactions of C. albicans with the gastrointestinal mucosa
are not well defined, and the mechanisms of invasion and systemic
dissemination have not been identified.
In order to study candidiasis arising from the gastrointestinal tract,
orally inoculated animals have been used. Oral inoculation of infant
mice (10, 30), mice treated with antibiotics or immunosuppressive drugs (7, 8, 13), or immunodeficient mice
(4, 20) has resulted in gastrointestinal colonization and
some level of dissemination of the organism to internal organs. Invasion of the cardiac-atrial fold of the stomach was seen in infant
mice, but intestinal lesions were not observed. Immunosuppressed rabbits were also susceptible to disseminated candidiasis following oral inoculation (38), but pathology of the gastrointestinal (GI) tract was not reported in this study. Thus, the interactions of
C. albicans with the intestinal tract have not been studied in these models.
In this study, oral inoculation of a highly susceptible animal was used
to develop an experimental model for gastrointestinal and disseminated
candidiasis. Gnotobiotic (GB) infant piglets were chosen for this work,
because these animals are susceptible to a variety of human enteric
pathogens and suffer disease that closely resembles human disease. For
example, rotaviruses (33), diarrheogenic Escherichia
coli (12, 34, 35, 36), Shigella sp. (S. Tzipori, unpublished observations), Cryptosporidium parvum (37), and, recently, Enterocytozoon bieneusi
(23) produce disease in the GB piglet with pathology that
closely mimics what is seen in infected humans. In this study, we
demonstrated that immunosuppression of newborn piglets orally
inoculated with C. albicans led to the development of
extensive mucocutaneous candidiasis, intestinal lesions, and
disseminated disease. The intestinal lesions likely represent a
significant portal of entry for the organism into the bloodstream. In
addition, multinucleated giant cells (MNGC) containing C. albicans cells were observed and may contribute to dissemination.
Despite the high susceptibility of the GB piglet to infection,
development of GI tract lesions and dissemination of C. albicans required virulent organisms, because a mutant strain known to be avirulent following intravenous inoculation of mice (27) failed to produce lesions. Therefore, we conclude that in this animal model, intestinal lesions likely represent an important mechanism for hematogenous dissemination.
| |
MATERIALS AND METHODS |
Strains.
The wild-type strain used in this study was the
clinical isolate strain SC5314 (11). Strain can36
(cph1/cph1 efg1/efg1 ura3/ura3), derived from SC5314, was
the kind gift of G. Fink. This strain lacks two putative transcription
factors, Cph1p and Efg1p, and is defective in filamentous growth and
avirulent in the intravenously inoculated mouse model (27).
Strain can36 was transformed with either pDBI52
(URA3+) or pHLB134 (URA3+
EFG1+) by lithium acetate transformation, generating
CKY138 and HLC84, respectively.
Media and growth conditions.
C. albicans strains were
inoculated into YPD (1% yeast extract, 2% Bacto Peptone, 2% glucose)
and grown overnight to saturation at 37°C. The cultures were
centrifuged and washed three times with 1× phosphate-buffered saline
(PBS). The concentration of the inoculum was determined by
hemocytometer counts and confirmed by dilution plating on YPD.
Experimental animals.
The derivation and maintenance of GB
piglets have been described previously (23). Briefly, 20 piglets derived from five litters were divided into three groups, one
for each C. albicans strain, with each strain except strain
HLC84 tested at least twice in animals from separate litters. Piglets
were inoculated 24 h postdelivery, when it was clear that they
were healthy and drinking well. The inoculum consisted of
109 cells in a 5-ml total volume delivered orally with a
16-gauge feeding needle (Popper & Sons, New Hyde Park, N.Y.). Piglets
were housed individually and observed regularly for clinical signs of
candidiasis (i.e., thrush and ocular lesions), diarrhea, and morbidity.
When severe illness developed, or at approximately 3 weeks
postinoculation, piglets were euthanized by intravenous injection of
euthanasia solution (Beuthanasia-D Special; Schering-Plough Animal
Health Corp., Kenilworth, N.J.).
Immunosuppression.
Eighteen of the 20 newborn GB piglets
were immunosuppressed, beginning when they were 1 day old and
continuing for the duration of the experiment as described previously
(23). Briefly, once a day, piglets received 25 mg of
intramuscular methylprednisolone sodium succinate per kg of body weight
(Solu-Medrol; Pharmacia & Upjohn, Kalamazoo, Mich.) and 15 mg of oral
cyclosporine per kg of body weight (Sandimmune Oral Solution; Sandoz
Pharmaceuticals Corp., East Hanover, N.J.) for the duration of the study.
Necropsy procedure.
The piglets were examined for gross
pathological changes, and samples were taken from the oral and ocular
regions, including eyelids and cornea. Animals were dissected
aseptically. The GI tract was removed and further dissected into the
esophagus, stomach, duodenum, jejunum, ileum, cecum, spiral colon, and
distal colon. The following internal organs were also removed: kidney,
liver, spleen, heart, lungs, and mesenteric lymph nodes. After fixation in 10% neutral buffered formalin, tissues were sectioned at 5 µm and
stained with hematoxylin and eosin for histopathological examination by
light microscopy. Tissues were also sectioned at 5 µm for
immunohistochemistry (IHC) by using the polyclonal antibody described
below and a standard immunoperoxidase technique (18). Microscopic photography was performed with an Olympus BX40 microscope with an Olympus PM20 automatic photographic system (Olympus America, Melville, N.Y.).
Tissue samples were also taken for quantitation of C. albicans. Organ pieces were minced with a scalpel, diluted 10-fold
(wt/wt) in 1× PBS, and homogenized with a handheld tissue tearer
(BioSpec Products, Bartlesville, Okla.). Dilutions of organ homogenates were plated on YPD agar plus streptomycin and ampicillin (each at 100 µg/ml), and the numbers of CFU per gram (wet weight) were determined.
Generation of polyclonal antiserum against C. albicans.
Rabbit antiserum was produced to perform IHC on paraffin
sections. C. albicans strain SC5314 cells grown at 37°C in
YPD were washed three times with 1× PBS, resuspended in 2% neutral
formalin (vol/vol), and incubated overnight at room temperature. The
cells were then washed extensively in 1× PBS and resuspended at a
concentration of 109/ml. Two young adult female New Zealand
White rabbits (Millbrook Farms, Amherst, Mass.) were used for
polyclonal antibody production as previously described (15).
Briefly, rabbits were inoculated four or five times at 4-week intervals
with 109 CFU of killed C. albicans mixed with
incomplete Freund's adjuvant. Blood was obtained at euthanasia, and
serum was prepared by standard methods (15).
| |
RESULTS |
Intestinal candidiasis in immunosuppressed GB infant piglets.
Oral inoculation of two GB piglets with 109 CFU of a
wild-type C. albicans strain, SC5314 (11), led to
C. albicans colonization throughout the GI tract, as shown
by quantitative culture of organ homogenates (data not shown). In
addition, both piglets developed mild oral thrush by day 10 after
inoculation. However, histologic examination by IHC of the intestinal
tract and internal organs failed to detect lesions or organisms in
tissue. Except for a few organisms detected in kidney homogenates, none
were recovered from homogenates of other internal organs. Therefore,
although C. albicans colonized the GI tract of GB piglets,
only a few organisms disseminated to the kidney, with no apparent lesions.
Since immunosuppression is a well known predisposing factor for
candidiasis, orally inoculated piglets were administered
immunosuppressive agents as described in Materials and Methods. Piglets
inoculated with wild-type strain SC5314 developed clinical signs of
thrush and conjunctivitis beginning at 4 days postinoculation. The
dorsal surface of the tongue showed small, white foci that progressed to larger white or yellow coalescing foci to form large plaques on the
dorsal and ventral surfaces of the tongue and oropharyngeal mucosa
(Fig. 1A). Most of these piglets also
developed bilateral mild-to-moderate conjunctivitis and had corneal
involvement initially consisting of focal opacity followed by
ulceration of the corneal epithelium and keratitis.
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FIG. 1.
Gross lesions of mucocutaneous candidiasis. IGB piglets
were orally inoculated with C. albicans and euthanized after
morbidity was observed. Tongue tissue was dissected and photographed.
(A) Inoculation with SC5314. Severe thrush with complete coverage by
fungal plaques is shown (magnification, ×1). (B) Inoculation with
CKY138. A normal surface with no evidence of thrush is shown
(magnification, ×1).
|
|
Upon sacrifice, quantitative culture of organ homogenates showed that
the GI tract was extensively colonized by C. albicans (Table
1). The tongue, esophagus, stomach, and
colon exhibited the highest colonization levels. Thus, immunosuppressed
GB (IGB) piglets were readily colonized by C. albicans in
the GI tract and were susceptible to mucocutaneous candidiasis.
Histological and IHC analysis of tissues from IGB piglets orally
inoculated with the wild-type strain demonstrated the presence of ileal
lesions, which ranged from moderate submucosal mixed inflammation to
segmental ulceration or complete obliteration of the mucosa overlying
the gut-associated lymphoid tissue (GALT) (Fig. 2A). Lesions consisted
of extensive necrosis and suppurative inflammation of the GALT and
contained yeast cells and filamentous forms of C. albicans
detected by IHC. Similar lesions were found in the mesenteric lymph
nodes associated with the ileum. Foci of similar but less extensive
invasive lesions were also seen in the cecum, spiral colon, and distal
colon, with numerous organisms and cellular debris in the lumen (Fig.
2B). In contrast, the glandular and pyloric regions of the stomach,
duodenum, and jujunum usually exhibited only casual association of
organisms with surface enterocytes, with no evidence of invasion,
ulceration, or inflammation. These results demonstrated the presence of
extensive intestinal lesions in the orally inoculated IGB piglet.
In addition to GI tract lesions, multifocal lesions containing
organisms were observed in the kidney, lung, liver, spleen, and heart
(Fig. 2C and D), demonstrating the
presence of disseminated disease. Organisms were also cultured from
homogenates of these organs (Table 1). Thus, in this animal model, as
in many human patients, disseminated candidiasis accompanied the
extensive lesions in the intestinal tract.
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FIG. 2.
Microscopic lesions in IGB piglets orally inoculated
with C. albicans. IGB piglets were orally inoculated with
C. albicans and euthanized after morbidity was observed. IHC
analysis of fixed tissue with antiserum directed against surface
antigens of C. albicans was performed as described in
Materials and Methods. (A) Ileum of IGB piglet inoculated with SC5314.
Segmental colonization of the ileum with mucosal ulceration and
transmural necrosis and inflammation is shown (magnification, ×40).
(B) Colon of IGB piglet inoculated with SC5314. Segmental colonization
of the colon with ulceration, necrosis, and inflammation is shown
(×40). (C) Kidney of IGB piglet inoculated with SC5314. Focus of
colonization with mild inflammation is shown (×200). (D) Liver of IGB
piglet inoculated with SC5314. Focus of colonization with marked
inflammation is shown (×200). (E) Large intestine of IGB piglet
inoculated with CKY138. Pneumatosis intestinalis of the colon with
large cysts in the gut wall is shown (×40).
|
|
In tissues from orally inoculated IGB piglets, a few MNGC containing
fungal organisms were seen in the parenchyma and in the lymphatic
vessels of the mesenteric lymphoid tissue (Fig.
3). MNGC are a common feature of
granulomas that develop in response to certain inflammatory reactions.
The presence of MNGC containing C. albicans cells in the
orally inoculated IGB piglet suggests that MNGC may provide a vehicle
for dissemination of C. albicans into the bloodstream and
localization in internal organs.
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FIG. 3.
MNGC containing C. albicans. IGB piglets were
inoculated orally with HLC84 [cph1/cph1 efg1/efg1
(EFG1+)], a strain that exhibits a wild-type
phenotype. Tissue was processed as described in the legend to Fig. 2.
(A) Mesenteric lymph node. Several MNGC containing organisms are
present in lymphatic vessels (magnification, ×200). (B) Mesenteric
lymph node. An MNGC containing several organisms in a lymphatic vessel
is shown (×400).
|
|
A mutant strain of C. albicans defective in regulation
of filamentous growth fails to produce GI tract lesions and exhibits
poor dissemination.
Because IGB piglets are highly susceptible to
infection by a variety of pathogens, we sought to demonstrate that the
lesions and dissemination observed above required virulent C. albicans. For this purpose, a mutant strain that had been
previously shown to be avirulent following intravenous inoculation of
mice (27) was used. This strain, CKY138 (cph1/cph1
efg1/efg1), lacks the putative transcription factors Efg1p and
Cph1p and is extremely defective in filamentous growth under standard
laboratory conditions (27).
In IGB piglets, mutant strain CKY138 (cph1/cph1 efg1/efg1)
was observed to colonize the GI tract (Table 1) and cause mild mucosal
disease. Five of eight piglets developed grossly visible thrush,
although the extent and distribution of lesions were reduced (Fig. 1B).
The development of clinical signs of thrush was also delayed compared
to that in the wild-type strain (9 days versus 4 days). Upon sacrifice,
quantitative culture of organ homogenates demonstrated that
colonization levels for the esophagus and GI tract ranged from
105 to 106 CFU/g (Table 1), very similar to the
levels observed with wild-type C. albicans. However, in most
of the piglets, no organisms were detected in the liver, spleen, heart,
or lungs (Table 1). Approximately half of the piglets were colonized in
the kidney, but the level of colonization was usually low. Thus,
despite the fact that CKY138 colonized the lower GI tract at close to
wild-type levels, dissemination was significantly attenuated.
In IGB piglets orally inoculated with CKY138 (cph1/cph1
efg1/efg1), infection of the GI tract was limited to superficial
colonization of the mucosa, except for two piglets in which the ileal
GALT was involved. In these piglets, the GALT lesions consisted of limited necrosis and mixed inflammation with predominantly yeast-form organisms. These lesions were much less severe than in infection with
the wild-type strain (data not shown). Organisms were also present in
the mesenteric lymph nodes of all piglets infected with CKY138. These
results demonstrate that a mutant strain with low virulence in the
intravenous mouse model similarly exhibited reduced virulence in the
IGB piglet model. Neither the extensive lesions in the intestinal tract
nor extensive systemic disease was observed with this mutant strain.
Histological analysis of mesenteric lymphoid tissue demonstrated that
MNGC containing organisms were commonly seen within the parenchyma and
in surrounding lymphatics. These results suggest that MNGC may commonly
form during infection with C. albicans.
Finally, three of the eight IGB piglets orally inoculated with CKY138
developed pneumatosis intestinalis with severe mesenteric edema and
gas-filled bullae surrounding the cecum and spiral colon. No organisms
were found associated with these lesions (Fig. 2E). Although similar
lesions were not observed histologically in piglets inoculated with
wild-type organisms, gas in the bowel wall was observed in one such
piglet, whose tissues were not examined due to death of the animal
prior to euthanasia.
To demonstrate that the reduced virulence observed with CKY138
(cph1/cph1 efg1/efg1) reflected the absence of Efg1p and
Cph1p, studies were conducted with strain HLC84 [cph1/cph1
efg1/efg1 (EFG1+)], which carries a
wild-type copy of the EFG1 gene. Introduction of wild-type
EFG1 restores the ability of the double-null mutant to form
hyphae at 37°C in serum (27) and restores the virulence of
the double-null mutant in mice (P. J. Riggle and C. A. Kumamoto, unpublished observations). Only two piglets were inoculated
with HLC84, but both developed mucocutaneous and disseminated
infections. Both piglets developed significant thrush lesions by 4 days
postinoculation, and one piglet had severe corneal involvement.
Histologic examination also showed invasion of the ileal GALT and
mesenteric lymph nodes and dissemination to internal organs (data not
shown). Colonization levels for the tongue and esophagus approached
107 CFU/g, while those in the GI tract ranged from
106 to 107 CFU/g and those in the internal
organs ranged from 102 to 104 CFU/g (Table 1).
Again, the kidney yielded the highest numbers of colonies. These levels
of colonization were similar to those of the wild-type strain and were
10- to 100-fold greater than those of strain CKY138 (cph1/cph1
efg1/efg1). Thus, this strain produced clinical signs and
pathologic lesions similar in time course and severity to those of the
wild-type strain of C. albicans, demonstrating that the
reduced virulence of CKY138 was due to the absence of Efg1p.
| |
DISCUSSION |
In this study, we demonstrated extensive intestinal lesions
associated with systemic candidiasis in a highly susceptible animal model. In the GI tract, mucosal invasion by C. albicans and
proliferation within the GALT were extensive in segments of the ileum
and occasionally in the large intestine. The mucosal surfaces overlying
these areas of necrosis and inflammation in the GALT were either
severely ulcerated or completely absent and were often replaced by a
pseudomembrane of cellular debris and fungal organisms.
Although intestinal candidiasis is not commonly diagnosed, human cases
of infection have been reported and studied (9, 14, 21, 24,
31). In addition, an association between invasive enteritis and
candidemia has been noted (14). In leukemia patients, the
lungs and bowel were the organs most commonly involved in patients
suffering from severe candidiasis (3). Autopsy studies of
infants and children infected with Candida demonstrate that the small intestine is one of the most common sites of infection (24), and spontaneous perforation of the intestine in
very-low-birth-weight infants is associated with systemic candidiasis
(1). Thus, the events observed in IGB piglets resemble
events that occur in human patients.
In human patients, systemic dissemination of C. albicans is
believed to occur by dissemination from the GI tract (6,
28), and gastrointestinal disease frequently precedes systemic
disease (9). In IGB piglets too, invasion of the gut mucosa
occurs long before dissemination is apparent (data not shown). However, the mechanism of translocation from the GI tract is unclear. The results obtained in IGB piglets suggest that organisms invading the gut
and proliferating in the GALT could spread to adjacent mesenteric lymph
nodes, where they would continue to multiply and then spread through
the lymphatics into the bloodstream and internal organs. In IGB
piglets, C. albicans organisms were observed in large
numbers in the adjacent mesenteric lymph nodes.
Studies with dogs have demonstrated that a major mechanism for
clearance of C. albicans from the portal vein is
phagocytosis by Kupffer cells in the liver (32). This
mechanism may clear low levels of organisms that cross the GI tract by
persorption. Persorption of C. albicans in a healthy human
has been demonstrated (25). In this study, the avirulent
strain CKY138 did not produce lesions in the GI tract. In the absence
of lesions, the mutant organisms may utilize only low-efficiency
mechanisms such as persorption to cross the GI tract and hence may be
readily eliminated by phagocytic cells. The mutant organisms may also
be more effectively killed by phagocytic cells (27). These
effects may account for the poor dissemination observed with the mutant
strain. The results support the hypothesis that virulent organisms
create lesions and significantly damage the GI tract, allowing them to
escape the GI tract, overwhelm the host defense mechanisms, and become hematogenously disseminated.
In infected lymph nodes, MNGC containing C. albicans were
seen scattered throughout the parenchyma and in the surrounding lymphatic vessels. Interestingly, we found that MNGC were more common
in IGB piglets infected with the mutant strain CKY138 than in those
infected with the wild-type strain. This observation is consistent with
the finding that the cph1/cph1 efg1/efg1 double mutant
organisms were defective in escaping from macrophage-like cells in
tissue culture (27).
MNGC originate from fusion of monocytes or macrophages, but the exact
mechanism of their formation is unclear. They are a characteristic
feature of several infectious diseases such as tuberculosis, Crohn's
disease, some viral infections, and some fungal infections, including
candidiasis in humans (29). MNGC have also been observed in
hyperplastic GALT of a patient with AIDS (26). MNGC
containing C. albicans may contribute to dissemination by
delivering organisms directly into the bloodstream. The organisms within these giant cells appeared to be morphologically intact and
presumably were viable.
Pneumatosis intestinalis was observed in the ileum and colon of several
piglets. Despite the presence of gas in the gut wall, C. albicans antigen was not detected in association with the lesions. Similar conditions have been observed in human patients. For example, pneumatosis intestinalis was observed in association with necrotizing enterocolitis attributed to candidiasis in a patient with AIDS (2). Gas in the bowel wall was detected in a patient
suffering from unusually severe infection with Candida
glabrata (16). Emphysematous pyelonephritis and
cystitis, diseases which may have a similar mechanism, have also been
reported in humans (17).
The newborn piglet is able to mount an immune response, but the level
of the response is suboptimal and increases gradually over the first 4 weeks of life. Therefore, it was not surprising that immunocompetent GB
piglets developed only transient mild signs of superficial candidiasis
with limited dissemination. The combination of daily oral cyclosporine
and parenteral methylprednisolone induces a state of moderate
immunosuppression in newborn piglets, as determined by marked reduction
in the proliferative response of mesenteric lymph node and peripheral
blood lymphocytes to stimulation with concanavalin A and
lipopolysaccharide mitogens (23). This immunosuppressive
regime led to the establishment of severe candidiasis in IGB piglets
inoculated with wild-type strain SC5314. The immunosuppressive regimen
appeared to mimic T-cell immunodeficiency and, as in AIDS patients,
predisposed the piglets to extensive mucocutaneous disease.
In summary, the IGB piglet exhibited severe forms of mucocutaneous
invasion, intestinal candidiasis, and systemic dissemination. The
locally invasive form of candidiasis that most commonly afflicts patients with immunodeficiencies includes ulcerations of the
respiratory, intestinal, and genitourinary tracts. The most serious
form, disseminated candidiasis, includes intraparenchymal lesions in
all major internal organs. The IGB piglet model exhibited many of the
features of candidiasis seen in humans. Features such as MNGC formation
in mesenteric lymph nodes, corneal lesions, and pneumatosis
intestinalis were also observed in this model. We believe the IGB
piglet model will facilitate future studies of the expression and role
of virulence factors in vivo and facilitate preclinical evaluation of
therapeutic agents against C. albicans infection.
| |
ACKNOWLEDGMENTS |
We thank Honorine Ward, Andrew Camilli, and Matt Waldor for
helpful discussion and Ralph Isberg for discussion and comments on the
manuscript. We are grateful to Gerry Fink for strains and plasmids. We
also thank Jessica Brisben, Melissa Paris, and Susan Chapman for
assistance with animal care and Barry Stein and Cheryl Sibley for
assistance with histology and immunohistochemistry. The use of
resources and facilities of the Division of Infectious Disease at Tufts
University School of Veterinary Medicine is greatly appreciated.
This work was supported in part by National Institute of Allergy and
Infectious Diseases grants K08 AI01407 (to K.A.A.) and R01 AI38591 (to
C.A.K.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, Tufts University School of Veterinary Medicine,
North Grafton, MA 01536. Phone: (508) 839-7955. Fax: (508) 839-7977. E-mail: Stzipori{at}infonet.tufts.edu.
| |
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Journal of Clinical Microbiology, June 2000, p. 2317-2323, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
