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Saturday, 20 January 2007


Brief Communications and Case Reports
In vivo Endothelial Cell Infection by Anaplasma marginale

Department of Pathobiology, Auburn University, Auburn, AL (ADC, CMJ); Department of
Pathobiology, University of Florida, Gainesville, FL (ARA, AFB); and Department of Microbiology
and Pathology, Washington State University, Pullman, WA (GHP, SMN)
Abstract. Anaplasma marginale has recently been shown to infect endothelial cells in vitro, but it
remains unknown as to whether endothelial infection also occurs in vivo. In this report, we demonstrate
through dual fluorescence microscopy that
marginale , d
etected by the monoclonal antibody
ANAF16C1, co-localizes with the endothelial cell marker, von Willebrand factor, in tissue sections from
an experimentally inoculated calf. The results indicate that
A marginale
infection includes endothelial
cells and has implications for both pathogenesis and immune mechanisms.
Key words:
Anaplasmosis is an arthropod-borne hemoparasitic disease of cattle and other ruminants caused by the
gram-negative bacterium,
Anaplasma marginale
, belonging to the order Rickettsiales and the family Anaplasmataceae.
The disease is characterized by severe anemia associated with intraerythrocytic parasitism, resulting in
fever, depression, and weakness.
Cattle that recover clinically remain persistently infected and serve as reservoir for transmission to uninfected cattle.
Anaplasmosis severely reduces the production of meat, milk, and fiber in tropical and subtropical areas of the
8 The family Anaplasmataceae includes the genera Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia,
encompassing a group of obligate intracellular bacteria that reside within a cytoplasmic vacuole in eukaryotic
cells, including erythrocytes, macrophages, endothelial cells, cells of insect, helminth, and arthropod reproductive
tissues, in addition to other tissues.
A marginale
enters red blood cells by endocytosis after which it divides by binary fission.2,11 Presently, A marginale is documented to infect in vivo only mature, circulating erythrocytes of its mammalian hosts, domestic and wild ruminants.
In contrast, the organism invades and replicates in the nucleated cells of the midgut and salivary gland epithelium of the ixodid tick vectors, including Rhipicephalus (Boophilus) microplus, R annulatus, Dermacentor andersoni, and D variabilis.
Recently, Munderloh et al. (2004)
10 have used tick cell cultures of the Virginia isolate of A marginale
, Am291,
to inoculate, in vitro, the cell line BCE C/D1-b (derived from bovine vascular endothelial cells), primate Vero
cells, and RF/6A cells (derived from rhesus monkey microvascular endothelium). The formation of intracellular
A marginale
colonies was associated with the death of endothelial cells within a week of inoculation and the
release of bacteria into the culture supernatant. It is unknown whether or not
A marginale
can infect
endothelial cells in vivo. If endothelial cells can be infected in vivo, these cells might represent a site of
initial replication after tick-borne transmission or be a reservoir for the organism during persistent infection.
In addition, endothelial cells, due to the expression of MHC class-I, could initiate a cell-mediated immune
response, whereas intraerythrocytic infection could not because of the lack of MHC Class-I expression on
A splenectomized calf (animal No. C1058) was inoculated intravenously with 10
organisms of the St.
Maries strain of
A marginale.3 T
he calf was euthanized at 47 days postinoculation when organisms were detected
in 44.5% of circulating red blood cells.
Samples of kidney (taken along the longitudinal axis of the renal cortex, approximately 1
3 1 3 0.1 cm) were harvested immediately after death and frozen in Neg-50 tissuefreezing
compound (Richard Allan Scientific, Kalamazoo, Michigan) using isopentane cooled in liquid
nitrogen. Samples were held at
270u C until they were sectioned at 5 m
m and applied to Superfrost Plus slides
(Fisher Scientific). The slides were fixed for 5 minutes in
acetone (4u
C) before staining.
Tissue sections and RF/6A cells were first blocked with 5% normal rabbit serum (Biomeda, Foster City,
CA) for 30 minutes at room temperature, rinsed in phosphate buffered saline (PBS), and then co-incubated
with the
A marginale monoclonal antibody, anti-ANAF16C1 (100 m
g/ml) and polyclonal rabbit antihuman
von Willebrand factor (vWF, Dako, Carpinteria,CA) at a 1 : 2 dilution of the concentration, supplied by
the manufacturer, for 30 minutes at room temperature, then rinsed for 3 minutes in PBS. Next, the tissue
sections were coincubated with 200m
l of Alexa Fluor 488-conjugated goat antimouse IgG and Alexa Fluor
Veterinary Pathology vetp-44-01-06.3d
8/10/06 14:39:01 1 Cust # 06-VP-0100-J-BCR Vet Pathol 44 :000–000 (2007) 568-conjugated goat antirabbit IgG (Molecular Probes, Carlsbad, CA), both at a concentration of 2m g/ml for 30 minutes at room temperature.
Sections were then
rinsed for 3 minutes in 1X PBS. RF/6A cells infected
A marginale
were used as a positive control for
organism staining.
In addition, RF/6A cells and tissue
sections were labeled with the lectin, FITC-conjugated
Ulex europaeus Agglutinin I (UEAI) (Vector Laboratories,
Burlingame, CA), in the same manner as the
primary antibodies. UEAI is reported to label bovine
endothelial cells.
All procedures after the application of fluorochromes
were performed in a dark chamber. Tissue sections were
then incubated with 200
ml of 49
, 6-diamidino-2-phenylindole,
dihydrochloride (DAPI) (Molecular Probes,
Carlsbad, CA) at a concentration of 284 nM, for
30 minutes at room temperature. Negative control
sections were incubated with secondary antibodies alone
and counterstained with DAPI. Tissue sections were
coverslipped with Citifluor (Electron Microscopy
Sciences, Hatfield, PA) and maintained in the dark at
C prior to examination.
Tissue sections and the RF/6A cells were analyzed
with a Nikon Eclipse E800 microscope fitted with 3
Nikon filter cubes for widefield fluorescence capture.
The UV2A filter cube (330–380 nm excitation) was used
for DAPI capture; the G2A filter cube (510–570 nm),
for Alexa 568 capture; and the B3A filter cube (420–
495 nm), for Alexa 488 capture. Images were captured
using the Spot Advanced software (Diagnostic Instru
ments, Sterling Heights, MI).
Widefield fluorescence microscopy of kidney sections
revealed colocalization of
A marginale
and vWF along
vascular endothelium in a granular pattern of fluorescence
(Fig. 1). Similar patterns of fluorescence were
identified in the lung and hemal lymph nodes (data not
shown). The granular pattern was attributable to the
localization of vWF in Weibel-Palade bodies, which are
reported to occur at a lower density in endothelial cells
Veterinary Pathology vetp-44-01-06.3d
8/10/06 14:39:03 2 Cust # 06-VP-0100-J-BCR
Fig. 1.
Kidney; calf No. C1058. Green fluorescence
A marginale
(antibody ANAF16C1) along the
endothelial surface of a vein when detected by indirect
immunofluorescence method with Alexa 488 fluorochrome.
Red fluorescence shows endothelial cells as
identified by vWF expression in the same section using
indirect immunofluorescence method with Alexa 568
fluorochrome. Bar
5 20 mm.
Fig. 2.
Kidney; calf No. C1058. Green fluorescence
with FITC-conjugated Ulex europaeus Agglutinin I
(UEAI) shows endothelial labeling within a glomerulus.
Red fluorescence identifies
A marginale
ANAF16C1) in the same section using an indirect
immunofluorescence method with Alexa 568 fluorochrome.
Fig. 3.
RF/6A cell culture. Green fluorescence with
UEAI identifies the endothelial cell line RF/6A by direct
lectin-FITC labeling and DAPI counterstaining. Red
fluorescence shows
A marginale
labeling of RF/6A cells
incubated with antibody ANAF16C1 and detected by
indirect immunofluorescence method using Alexa
568 fluorochrome.
Brief Communications and Case Reports Vet Pathol 44:1, 2007
of capillaries when compared with endothelial cells of
small arteries.
As a confirmation of endothelial
identification, the kidney sections were also labeled with
ANAF16C1 and the lectin, UEAI (Fig. 2). Negative
controls, consisting of sections from the same tissue
without the application of primary antibody or lectin,
were devoid of specific staining of both
A marginale
vWF. RF/6A cells expressed low levels of vWF, as
and therefore UEAI (expressed highly in
these cells) was used as an endothelial cell marker. These
cells demonstrated co-labeling with the endothelial celllectin
UEAI-FITC and mAb ANAF16C1 conjugated to
Alexa 568 (counterstained with DAPI) (Fig. 3).
These data demonstrate endothelial cell infection in
vivo after experimental infection of a calf with
and extend the finding that endothelial cells
are susceptible to infection after in vitro challenge.
Endothelial cells may serve as an early reservoir for
infection at the site of tick attachment and,
through their expression of MHC Class-I, could be
important in the initiation of a cytotoxic T-lymphocyte
response during the early stages of infection. Further
studies are needed to define the role of endothelial cells
in these key components of
A marginale
infection and
This work was funded by the United States Department
of Agriculture, Grant number 9922.
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Request reprints from Dr. Calvin M. Johnson, Department
of Pathobiology, 166 Greene Hall, College of
Veterinary Medicine, Auburn University, AL 36849-
5519. E-mail: johncal@auburn.edu.
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Vet Pathol 44:1, 2007
Brief Communications and Case Reports 0