Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wamsley, H. L. Articles by Barbet, A. F. Search for Related Content PubMed PubMed Citation Articles by Wamsley, H. L. Articles by Barbet, A. F.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, July 2008, p. 2314-2319, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.02197-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor

Heather L. Wamsley^* and Anthony F. Barbet

University of Florida, College of Veterinary Medicine, Department of Infectious Diseases and Pathology, Gainesville, Florida 32610

Received 13 November 2007/ Returned for modification 26 December 2007/ Accepted 13 May 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by using rolling-circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The life cycle of Anaplasma spp. involves mammalian peripheral blood cells and arthropod epithelial cells (4). Steps in the tick-feeding-associated establishment and persistence of Anaplasma infection within mammalian hosts are incompletely characterized. Recent in vitro and preliminary in vivo immunofluorescence studies suggest that endothelial cells may be a nidus of Anaplasma infection in mammals and a source of organisms to infect circulating blood cells (3, 8, 12). The purpose of this investigation was to develop a specific and sensitive technique for in situ detection of Anaplasma within tissues of infected hosts, with special attention to mammalian endothelial cells. In the future, this method could be used to determine the in vivo cellular localization of potentially cryptic infection nidi and to provide nucleotide sequence information in situ.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cultivation of Anaplasma spp. The NY18 isolate of Anaplasma phagocytophilum and the Virginia isolate of Anaplasma marginale were cultivated in fetal rhesus monkey (Macaca mulatta) RF/6A endothelial cells (ATCC CRL-1780; American Type Culture Collection, Manassas, VA), and the HZ isolate of A. phagocytophilum was cultivated in human (Homo sapiens) HL-60 myeloblastic leukemia cells (ATCC CCL-240; American Type Culture Collection, Manassas, VA) as described previously (7, 12). Dulbecco modified Eagle medium was used for the RF/6A endothelial cells (HyClone, Logan, UT) and RPMI 1640 medium for the HL-60 myeloblastic leukemia cells (HyClone, Logan, UT), both supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 2 mM Gibco L-glutamine (200 mM [100-fold-concentrated] liquid; final concentration, 4 mM) (Invitrogen, Carlsbad, CA), 0.25% NaHCO₃ (Sigma, St. Louis, MO), and 25 mM HEPES (Sigma, St. Louis, MO) (pH 7.5). Cultures were maintained in vented flasks in a 37°C incubator with 5% CO₂. Uninfected RF/6A endothelial cells and uninfected HL-60 myeloblastic leukemia cells were similarly maintained. When at least 80% of the cells were A. phagocytophilum infected or at least 20% of the cells were A. marginale infected, as determined by light microscopy, cell suspensions were diluted and cytocentrifuged onto Bond-Rite glass microscope slides (Richard-Allan Scientific, Kalamazoo, MI). To form a cell suspension, the RF/6A endothelial cell culture monolayers were detached from the culture flask by using 0.25% trypsin (HyClone, Logan, UT). The HL-60 myeloblastic leukemia cell line is a nonadherent cell line which grows as a cell suspension; therefore, treatment with trypsin was not necessary.

In situ rolling-circle amplification of padlock probes. In situ DNA target-primed rolling-circle amplification of padlock probes was performed with modifications of a previously described technique (11). All reactions were performed on microscope slides without coverslips. The final volume of all reaction mixtures was 40 µl. All heated reactions were performed in a 16/16 dual block slide chamber mounted on a DNA engine (PTC-200) Peltier thermal cycler (Bio-Rad Laboratories, Hercules, CA). Cytospin culture material on the microscope slides was uniformly treated as follows. The slides were washed twice in 1x phosphate-buffered saline (PBS) (pH 7.4, 2 min), fixed in 70% denatured ethanol (20 min), and subsequently washed twice in 1x PBS (2 min). The cells were permeabilized in HCl (pH 3.6, 37°C)—RF/6A endothelial cells for 3 min and HL-60 myeloblastic leukemia cells for 1.5 min. Afterward, the slides were washed three times in 1x PBS (2 min).

The bacterial genome was made irreversibly linear by the endonuclease digestion of A. phagocytophilum with AfeI (New England BioLabs, Ipswich, MA) and of A. marginale with ZraI (New England BioLabs, Ipswich, MA); each endonuclease was used at 0.5 U/µl in 1x supplied enzyme buffer plus 0.2 µg/µl bovine serum albumin (New England BioLabs, Ipswich, MA) (37°C, 30 min), followed by a brief rinse in buffer A (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl, and 0.05% Tween 20). The genomic DNA target was made single stranded by 5'-to-3' exonucleolysis (Lambda exonuclease; New England BioLabs, Ipswich, MA). The exonuclease was used at 0.2 U/µl in 1x supplied enzyme buffer plus 0.2 µg/µl bovine serum albumin and 10% glycerol (Sigma, St. Louis, MO) (37°C, 15 min), followed by a brief rinse in buffer A.

The genomic DNA target was detected by hybridization to a circularizable, linear, oligonucleotide padlock probe (Fig. 1). Either an A. phagocytophilum-specific probe, an A. marginale-specific probe, or a nonspecific probe was used at 0.10 µM in 2x SSC (pH 7.0) (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-20% formamide (Fisher Scientific, Pittsburg, PA), and 0.5 µg/µl sonicated fish sperm (DNA, MB-grade; Roche Applied Science, Indianapolis, IN) (37°C, 15 min). Subsequently, the slides were washed in prewarmed buffer B (2x SSC [pH 7.0] and 0.05% Tween 20; 37°C, 5 min) and rinsed briefly in buffer A.

View larger version (15K): [in this window] [in a new window]

FIG. 1. Three padlock probes were used: AB1251 (A. phagocytophilum specific), AB1270 (A. marginale specific), and AB1253 (nonspecific). The genomic DNA target-specific sequences of AB1251 and AB1270 are within the underlined 5' and 3' arms of the padlock probes. When the single-stranded genomic complement of each probe's target-specific sequence is detected, the probe hybridizes as a nicked circle, which is subsequently locked in place by ligase as a partially double-helical, closed circle. The circularized padlock probe is the template for in situ DNA target-primed rolling-circle amplification. The 5' and 3' arms of the nonspecific probe, AB1253, had the same nucleotide composition as AB1251; however, the sequence of the nucleotides was randomized within the underlined regions. The 5' and 3' arms of the padlock probes were joined by identical intervening linker regions. The amplification product of the italicized portion of the probe linker region is the complement of the fluorescently labeled oligonucleotide tags, AB1252 and AB1279 (also known as Lin 33 [11]). All oligonucleotides contained a 5'-end modification, designated P (phosphate), FAM (phosphoramidite-coupled fluorescein), or Alexa 555 (Alexa Fluor 555; Invitrogen, Carlsbad, CA).

The A. phagocytophilum-specific probe or the A. marginale-specific padlock probe, which should hybridize as a nicked circle to its complementary genomic DNA target, was then irreversibly locked into place by enzymatic formation of a phosphodiester bond between the juxtaposed 5' phosphate and 3' hydroxyl termini of the padlock probe (T4 ligase; New England BioLabs, Ipswich, MA). The ligase was used at 100 U/µl in 1x supplied enzyme buffer plus 0.2 µg/µl bovine serum albumin (16°C, 15 min). The nonspecific padlock probe should remain linear and be washed away during this step and subsequent steps since this probe should fail to form the appropriate conformation required for ligase recognition and activity. Afterward, the slides were washed in prewarmed buffer B (37°C, 5 min), rinsed once in buffer A, and dehydrated in graded denatured ethanols (75%, 85%, 100%; 3 min each).

After exonucleolysis of any remaining 3' single-stranded genomic DNA, the genomic DNA target was used to prime isothermal in situ rolling-circle amplification of the specifically bound padlock probe (phi29 DNA polymerase; New England BioLabs, Ipswich, MA). The polymerase was used at 1.0 U/µl in 1x supplied enzyme buffer plus 0.25 mM each deoxynucleoside triphosphate (deoxynucleotide solution mix; New England BioLabs, Ipswich, MA), 0.2 µg/µl bovine serum albumin, and 10% glycerol (37°C, 30 min). During the 30-min DNA polymerization, the slides were removed from the slide chamber every 10 min to gently agitate the reaction mixture. The slides were then rinsed briefly in buffer A before the single-stranded, loosely coiled, concatameric amplification product was hybridized to a fluorescently labeled, linear oligonucleotide tag (oligonucleotide AB1252 [green] or oligonucleotide AB1279 [red; also known as Lin 33 {11}]), used at 0.25 µM in 2x SSC, 20% formamide, and 0.5 µg/µl sonicated fish sperm (37°C, 15 min).

Afterward, the slides were rinsed briefly in buffer A, dehydrated in graded denatured ethanols (75%, 85%, 100%; 3 min each), and either immunofluorescently stained for von Willebrand factor or immediately air dried and coverslip mounted using Vectashield HardSet mounting medium with 1.5 µg/ml DAPI (4',6'-diamidino-2-phenylindole) (Vector Laboratories, Burlingame, CA). The slides were examined using a Leica DMI 3000B inverted microscope fitted for epifluorescence and equipped with a digital camera (MicroPublisher 3.3 RTV; QImaging Corporation, Surrey, BC, Canada) and then stored at 4°C. Digital images were collected using QCapture Pro 5.1.1.14 (QImaging Corporation, Surrey, BC, Canada) and uniformly processed using SPOT Advanced Windows version 4.0.9 (Diagnostic Instruments, Sterling Heights, MI) and Adobe Photoshop Elements 2.0 (Adobe Systems Incorporated, San Jose, CA).

Padlock probe design. Three circularizable, linear, oligonucleotide padlock probes were used (Fig. 1) (MWG Biotech, High Point, NC). The A. phagocytophilum-specific probe (AB1251) was designed to include an A. phagocytophilum genomic DNA target-specific sequence whose cognate is a conserved 5' region of msp2 (p44) that is present in the expression sites of three geographic isolates from the United States (HZ isolate) (5), Norway (1), and Sweden (1) and in 81 different pseudogenes of the HZ isolate (5). Of these target sequences within the A. phagocytophilum genome (HZ isolate), there are 29 potential targets (the expression site and 28 pseudogenes) that are associated with a 3' AfeI endonuclease recognition site within 100 bp of the genomic target and could, therefore, be detected by the technique described here.

The A. marginale-specific probe (AB1270) was designed to include an A. marginale genomic DNA target-specific sequence, whose cognate is orfY, which is repeated eight times in the genome (2). Of these target sequences within the A. marginale genome (St. Maries isolate), there are seven potential targets that are associated with a 3' ZraI endonuclease recognition site within 1 bp of the genomic target and could, therefore, be detected by the technique described here. The use of repetitive sequences [msp2 (p44) for A. phagocytophilum and orfY for A. marginale] as genomic DNA targets for padlock probe hybridization was expected to increase the sensitivity beyond that provided by rolling-circle amplification alone.

The target-specific sequence of the padlock probe is within the 5' and 3' arms of the probe, which are joined by an intervening linker region. The rolling-circle amplification product is detected by a fluorescently labeled oligonucleotide tag (AB1252 or AB1279; also known as Lin 33 [11]) that is the complement of the padlock probe linker region amplification product. The linker region of the nonspecific padlock probe (AB1253) was identical to those of the A. phagocytophilum-specific and A. marginale-specific padlock probes. The 5' and 3' arms of the nonspecific probe had the same nucleotide composition as that of the A. phagocytophilum-specific probe; however, the sequence of the nucleotides was randomized.

Indirect immunofluorescent staining of von Willebrand factor in cultured endothelial cells. After the final graded-alcohol dehydration of the in situ rolling-circle amplification procedure, slides that had been reacted with the A. phagocytophilum-specific padlock probe or the nonspecific padlock probe were immunofluorescently stained for von Willebrand factor. All reactions were performed on microscope slides without coverslips; the final volume of all reaction mixtures was 150 µl. The cytospin culture material was blocked with normal rabbit serum (X0902; Dako, Carpinteria, CA), 5% in 1x PBS (pH 7.4, 30 min), and subsequently washed once in 1x PBS (5 min). The cytospin culture material was then incubated with either antibody-free diluent or rabbit polyclonal anti-human von Willebrand factor antibody (N1505; Dako, Carpinteria, CA) diluted 1:1 in 0.05 M Tris-HCl (pH 7.5)-1% bovine serum albumin fraction V (Fisher Scientific, Pittsburg, PA) (30 min). The slides were washed twice in 1x PBS (5 min) prior to secondary antibody labeling. The cytospin culture material was subsequently incubated with a highly cross-adsorbed goat polyclonal anti-rabbit immunoglobulin G antibody-Alexa Fluor 568 conjugate (A11036; Invitrogen Molecular Probes, Carlsbad, CA) used at 2 µg/ml in 0.05 M Tris-HCl (pH 7.5)-1% bovine serum albumin fraction V (30 min). Afterward, the slides were washed twice in 1x PBS (5 min), air dried, coverslip mounted using Vectashield HardSet mounting medium with DAPI, and examined as described above.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In situ detection of Anaplasma spp. by DNA target-primed rolling-circle amplification of padlock probes. During persistent, latent infections that can be caused by Anaplasma spp., it would be valuable to be able to sensitively and specifically detect organisms in infected tissues by using an isothermal DNA amplification technique that has the potential to provide information about organism genotype and host cellular localization. To determine whether such a technique could be used to detect A. phagocytophilum and A. marginale, in situ DNA target-primed rolling-circle amplification of padlock probes was developed based upon previous in situ genotyping of human mitochondrial DNA by rolling-circle amplification of padlock probes (11).

Cytospin preparations of uninfected or A. phagocytophilum HZ-infected human myeloblastic leukemia cell cultures and a padlock probe, either a nonspecific probe or an A. phagocytophilum-specific probe, were used for in situ DNA target-primed rolling-circle amplification. When A. phagocytophilum HZ-infected cytospin preparations were microscopically examined after in situ DNA target-primed rolling-circle amplification of an A. phagocytophilum-specific padlock probe, numerous stippled, green or red fluorescent aggregates, which represented the fluorescently labeled, localized amplification product, were frequently identified within intact cultured myeloblastic leukemia cells. The intracellular location of fluorescence correlated well with microscopic observations of A. phagocytophilum morulae within intact myeloblastic leukemia cells in a Wright-Giemsa-stained infected cytospin culture (Fig. 2A, B, and D). Similar fluorescence was not observed when either an uninfected cytospin culture (not shown) or a nonspecific padlock probe (Fig. 2C and E) was used. The results were confirmed in five independent experiments.

View larger version (44K): [in this window] [in a new window]

FIG. 2. Numerous intracellular morulae were observed within heavily A. phagocytophilum HZ-infected, intact human myeloblasts in cytospin preparations stained with Wright-Giemsa (bright field) (A) or when detected as a stippled, green fluorescent product (B) or a stippled, red fluorescent product (D) by in situ DNA target-primed rolling-circle amplification of an A. phagocytophilum-specific padlock probe (63x objective; differential interference contrast with blue and green emissions [B] or with blue and red emissions [D]). Similar fluorescence was not detected when a nonspecific padlock probe was used in an otherwise identical procedure (C and E) (63x objective; differential interference contrast with blue and green emissions [C] or with blue and red emissions [E]).

Cytospin preparations of uninfected or A. marginale Virginia-infected fetal rhesus monkey endothelial cell cultures and a padlock probe, either a nonspecific probe or an A. marginale-specific probe, were used for in situ DNA target-primed rolling-circle amplification. When A. marginale Virginia-infected cytospin preparations were microscopically examined after in situ DNA target-primed rolling-circle amplification of an A. marginale-specific padlock probe, stippled, green or red fluorescent round aggregates, which represented the fluorescently labeled, localized amplification product, were frequently identified within intact cultured endothelial cells. The intracellular location of fluorescence correlated well with microscopic observations of A. marginale morulae within intact endothelial cells in a Wright-Giemsa-stained infected cytospin culture (Fig. 3A, B, and D). Similar fluorescence was not observed when either an uninfected cytospin culture (not shown) or a nonspecific padlock probe (Fig. 3C and E) was used. The results were confirmed in three independent experiments.

View larger version (45K): [in this window] [in a new window]

FIG. 3. Intracellular morulae were observed within A. marginale-infected, intact fetal rhesus monkey endothelial cells in cytospin preparations stained with Wright-Giemsa (bright field) (A) or when detected as a stippled, green fluorescent product (B) or a stippled, red fluorescent product (D) by in situ DNA target-primed rolling-circle amplification of an A. marginale-specific padlock probe (63x objective; differential interference contrast with blue and green emissions [B] or with blue and red emissions [D]). Similar fluorescence was not detected when a nonspecific padlock probe was used in an otherwise identical procedure (C and E) (63x objective; differential interference contrast with blue and green emissions [C] or with blue and red emissions [E]).

In situ intracellular colocalization of the A. phagocytophilum rolling-circle amplification product and von Willebrand factor immunofluorescence. Since A. phagocytophilum can be continuously cultivated in endothelial cells (12) and preliminary immunofluorescent staining of SCID mouse tissues suggests that endothelial cells may also be infected in vivo (8), there has been heightened interested in determining more conclusively whether endothelial cells are an in vivo nidus of A. phagocytophilum in naturally or experimentally infected, immunocompetent mammals. To that end, in situ A. phagocytophilum DNA target-primed rolling-circle amplification of a padlock probe was combined with indirect immunofluorescent staining of von Willebrand factor, which is present within Weibel-Palade bodies of endothelial cells (16). Cytospin preparations of uninfected or A. phagocytophilum NY18-infected fetal rhesus monkey endothelial cell cultures, a padlock probe (a nonspecific probe or an A. phagocytophilum-specific probe), and one of two antibody staining variations (secondary antibody only or primary and secondary antibodies together) were examined using these combined techniques.

When A. phagocytophilum NY18-infected cytospin cultures were microscopically examined after in situ DNA target-primed rolling-circle amplification of an A. phagocytophilum-specific padlock probe, stippled, green fluorescent focal aggregates, which represented the fluorescently labeled, localized amplification product, were often identified perinuclearly within intact cultured endothelial cells. The intracellular location of green fluorescence correlated well with microscopic observations of A. phagocytophilum morulae within intact endothelial cells in a Wright-Giemsa-stained infected cytospin culture (Fig. 4A and B). Similar green fluorescence was not observed when either an uninfected cytospin culture (not shown) or a nonspecific padlock probe (Fig. 4C) was used. The results were confirmed in five independent experiments.

View larger version (36K): [in this window] [in a new window]

FIG. 4. A few perinuclear morulae were observed within intact A. phagocytophilum NY18-infected fetal rhesus monkey endothelial cells in cytospin preparations when stained with Wright-Giemsa (bright field) (A) or when detected as a stippled, green fluorescent product by in situ DNA target-primed rolling-circle amplification of an A. phagocytophilum-specific padlock probe (63x objective; phase contrast with blue and green emissions) (B). A similar green fluorescent product was not detected when a nonspecific padlock probe was used in the otherwise identical procedure (40x objective; phase contrast with blue and green emissions) (C). A. phagocytophilum-infected fetal rhesus monkey endothelial cells in cytospin preparations were subjected to combined in situ DNA target-primed rolling-circle amplification of an A. phagocytophilum-specific padlock probe and indirect immunofluorescent staining of von Willebrand factor. Stippled, green fluorescent focal aggregates were identified perinuclearly and juxtaposed with focal areas of red fluorescence, which represented the fluorescently labeled von Willebrand factor (D); similar green and red fluorescence was not observed when the combined procedure was performed using a nonspecific padlock probe and only the fluorescently labeled secondary antibody (40x objective; blue, red, and green emissions) (E).

When A. phagocytophilum-infected cytospin cultures were examined microscopically after combined in situ DNA target-primed rolling-circle amplification of an A. phagocytophilum-specific padlock probe and indirect immunofluorescent staining of von Willebrand factor, stippled, green fluorescent focal aggregates were identified perinuclearly, juxtaposed with focal areas of red fluorescence, which represented the fluorescently labeled von Willebrand factor within Weibel-Palade bodies (Fig. 4D). Similar green and red fluorescence was not observed when the combined procedures were performed using a nonspecific padlock probe and only the fluorescently labeled secondary antibody (Fig. 4E).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The in situ DNA target-primed rolling-circle amplification of a padlock probe technique adapted here for Anaplasma detection was first described as a method to distinguish single nucleotide polymorphisms in human mitochondrial DNA (11). We considered it likely that this technique could be used for in situ detection of intracellular microorganisms. Here, it is demonstrated that it is possible to detect and localize Anaplasma spp. within intact cultured mammalian cells by using in situ DNA target-primed rolling-circle amplification of padlock probes and that this technique can be combined with immunofluorescent staining to identify A. phagocytophilum and an endothelial cell antigen within a single cultured endothelial cell. These observations suggest that this technique could be applied to natural Anaplasma sp. isolates in tissues obtained from naturally or experimentally infected mammalian or arthropod hosts to determine the cellular localization of potentially cryptic infections in vivo and to provide nucleotide sequence information in situ.

The specificity of this technique arises from the use of a padlock probe, which depends upon hybridization specific to the genomic DNA target sequence. The use of ligase allows interrogation of the hybridization quality between the target-specific sequence of the padlock probe and the genomic target. If the quality of the hybridization is not optimal, ligation of the padlock probe to form a closed, partially double-stranded circle involving the genomic target will not occur. The use of ligase in this procedure is the basis for the distinction of genomic single nucleotide polymorphisms based on the quality of hybridization with the 3' terminus of the padlock probe (9, 10, 11, 13, 14, 15). The exponential amplification of the bound padlock probe is one basis for the sensitivity of this technique (11, 15). The amplification product remains bound to the target sequence and is subsequently detected by the addition of a second fluorescently labeled oligonucleotide tag that specifically binds repeated linker sequences within the concatameric amplification product. The fact that the amplification product remains tightly bound as a 3' extension of the genomic target limits background fluorescence, which also contributes to the sensitivity and specificity of this technique and is another improvement over other in situ nucleotide detection techniques (11, 14).

An additional benefit of the in situ DNA amplification technique applied here over other methods of in situ DNA detection is that the reactions are isothermal, which preserves tissue architecture and allows subsequent immunostaining of host cell antigens, such as the endothelial cell antigen von Willebrand factor. This is of particular import, given the recently heightened interest in endothelial cells as a potential natural mammalian infection nidus in light of the ability to continuously cultivate A. phagocytophilum and A. marginale in fetal primate endothelial cells (12) and the preliminary immunofluorescent colocalization of A. phagocytophilum or A. marginale with endothelial cell antigens in vivo (3, 8).

Transformation of A. phagocytophilum that renders it able to express green fluorescent protein and subsequent experimental infection of mice with A. phagocytophilum transformants have been recently described (6). Such transformants may facilitate temporal tracking of organism tissue distribution during experimental infection, including host cellular binding, entry, and intracellular development; however, their use is dependent upon an experimental model of mammalian infection and the ability to maintain stable transformants in long-term culture and during the full course of acute and chronic infections. The fluorescence microscopy-based Anaplasma sp. detection method described here does not depend upon a genetic transformant that may be unstable or attenuated and could be applied to naturally or experimentally infected mammalian or arthropod host tissues to detect potentially cryptic sites of infection. Additionally, with the knowledge of variable Anaplasma genomic DNA sequences that may be present in host tissues during an infection, multiple padlock probes that bind to differentially labeled fluorescent oligonucleotide tags could be designed and used in a single isothermal reaction to provide information about Anaplasma nucleotide sequences in situ.

In summary, the observations presented here show that in situ DNA target-primed rolling-circle amplification of padlock probes can be used to detect Anaplasma spp. within intact cultured mammalian cells. This demonstrates that the amplification technique can be used for in situ detection of an intracellular microorganism. Also, this technique can be combined with immunofluorescent staining in order to identify a host cell antigen and the fluorescently labeled rolling-circle amplification product within a single cell. This work forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected hosts and in cell cultures. There is also the potential to apply the technique described here to investigate prospective cryptic host cellular localization of other microorganisms and to further elucidate the etiopathogenesis of disease associated with infection by other obligate or facultative intracellular microorganisms.

 
This work was supported by grant R01-AI45580 from the National Institute of Allergy and Infectious Disease and grant 2003-35204-13825 from the United States Department of Agriculture.

We thank Basima Al-Khedery and A. Rick Alleman for the provision of uninfected, A. phagocytophilum-infected, and A. marginale-infected cell cultures.

 
* Corresponding author. Mailing address: University of Florida, College of Veterinary Medicine, 2015 SW 16th AV, Box 100103C, Gainesville, FL 32610. Phone: (352) 392-2235, ext. 5848. Fax: (352) 392-2938. E-mail: wamsleyh{at}vetmed.ufl.edu

Published ahead of print on 21 May 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Barbet, A. F., A. M. Lundgren, A. R. Alleman, S. Stuen, A. Bjoersdorff, R. N. Brown, N. L. Drazenovich, and J. E. Foley. 2006. Structure of the expression site reveals global diversity in MSP2 (P44) variants in Anaplasma phagocytophilum. Infect. Immun. 74:6429-6437.[Abstract/Free Full Text]
2
2. Brayton, K. A., L. S. Kappmeyer, D. R. Herndon, M. J. Dark, D. L. Tibbals, G. H. Palmer, T. C. McGuire, and D. P. Knowles, Jr. 2005. Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. Proc. Natl. Acad. Sci. USA 102:844-849.[Abstract/Free Full Text]
3
3. Carreño, A. D., A. R. Alleman, A. F. Barbet, G. H. Palmer, S. M. Noh, and C. M. Johnson. 2007. In vivo endothelial cell infection by Anaplasma marginale. Vet. Pathol. 44:116-118. (Erratum, 44:427.)[Abstract/Free Full Text]
4
4. Dumler, J. S., A. F. Barbet, C. P. J. Bekker, G. A. Dasch, G. H. Palmer, S. C. Ray, Y. Rikihisa, and F. R. Rurangirwa. 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int. J. Sys. Evol. Microbiol. 51:2145-2165. (Erratum, 52:5-6, 2002.)
5
5. Dunning Hotopp, J. C., M. Lin, R. Madupu, J. Crabtree, S. V. Angiuoli, J. Eisen, R. Seshadri, Q. Ren, M. Wu, T. R. Utterback, S. Smith, M. Lewis, H. Khouri, C. Zhang, H. Niu, Q. Lin, N. Ohashi, N. Zhi, W. Nelson, L. M. Brinkac, R. J. Dodson, M. J. Rosovitz, J. Sundaram, S. C. Daugherty, T. Davidsen, A. S. Durkin, M. Gwinn, D. H. Haft, J. D. Selengut, S. A. Sullivan, N. Zafar, L. Zhou, F. Benahmed, H. Forberger, R. Halpin, S. Mulligan, J. Robinson, O. White, Y. Rikihisa, and H. Tettelin. 2006. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genet. 2:208-223.[CrossRef]
6
6. Felsheim, R. F., M. J. Herron, C. M. Nelson, N. Y. Burkhardt, A. F. Barbet, T. J. Kurtti, and U. G. Munderloh. 2006. Transformation of Anaplasma phagocytophilum. BMC Biotechnol. 6:42.[CrossRef][Medline]
7
7. Goodman, J. L., C. Nelson, B. Vitale, J. E. Madigan, J. S. Dumler, T. J. Kurtti, and U. G. Munderloh. 1996. Direct cultivation of the causative agent of human granulocytic ehrlichiosis. N. Engl. J. Med. 334:209-215. (Erratum, 335:361.)[Abstract/Free Full Text]
8
8. Herron, M. J., M. E. Ericson, T. J. Kurtti, and U. G. Munderloh. 2005. The interactions of Anaplasma phagocytophilum, endothelial cells, and human neutrophils. Ann. N. Y. Acad. Sci. 1063:374-382.[CrossRef][Medline]
9
9. Landegren, U., R. Kaiser, J. Sanders, and L. Hood. 1988. A ligase-mediated gene detection technique. Science 241:1077-1080.[Abstract/Free Full Text]
10
10. Landegren, U., M. Samiotaki, M. Nilsson, H. Malmgren, and M. Kwiatkowski. 1996. Detecting genes with ligases. Methods 9:84-90.[CrossRef][Medline]
11
11. Larsson, C., J. Koch, A. Nygren, G. Janssen, A. K. Raap, U. Landegren, and M. Nilsson. 2004. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat. Methods 1:227-232.[CrossRef][Medline]
12
12. Munderloh, U. G., M. J. Lynch, M. J. Herron, A. T. Palmer, T. J. Kurtti, R. D. Nelson, and J. L. Goodman. 2004. Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum. Vet. Microbiol. 101:53-64.[CrossRef][Medline]
13
13. Nilsson, M., H. Malmgren, M. Samiotaki, M. Kwiatkowski, B. P. Chowdhary, and U. Landegren. 1994. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265:2085-2088.[Abstract/Free Full Text]
14
14. Nilsson, M., F. Dahl, C. Larsson, M. Gullberg, and J. Stenberg. 2006. Analyzing genes using closing and replicating circles. Trends Biotechnol. 24:83-88.[CrossRef][Medline]
15
15. Nilsson, M. 2006. Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification. Histochem. Cell Biol. 126:159-164.[CrossRef][Medline]
16
16. Weibel, E. R., and G. E. Palade. 1964. New cytoplasmic components in arterial endothelia. J. Cell Biol. 23:101-112.[Abstract/Free Full Text]

---

Journal of Clinical Microbiology, July 2008, p. 2314-2319, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.02197-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wamsley, H. L. Articles by Barbet, A. F. Search for Related Content PubMed PubMed Citation Articles by Wamsley, H. L. Articles by Barbet, A. F.