ISRAEL JOURNAL OF
VETERINARY MEDICINE
PATHOLOGICAL AND IMMUNOPEROXIDASE STUDIES OF THE
PLACENTAL LESIONS OF OVINE BRUCELLOSIS
O. Yazicioglu1, R. Haziroglu2
1. Bornova Veterinary Control and Research Enstitute, 35010 Izmir
2. Department of Pathology, Faculty of Veterinary Medicine, University of Ankara, 06110 Ankara, Turkey
Summary
In this study, the lesions in ten placentas from cases of ovine abortion associated with
brucellosis were morphologically and immunohistochemically evaluated. All placentas showed
necrotizing placentitis characterized by trophoblast necrosis and chorioallantoic ulceration.
Three placentas had necrotizing vasculitis characterized by necrosis and cell infiltration in the
vessel walls. Brucella antigen was detected intracellularly in trophoblasts, macrophages, and
neutrophils and extracellularly in the interstitium and vascular lumen by immunoperoxidase
staining.
Key Words: Brucellosis; abortion; placenta; immunoperoxidase; sheep.
Introduction
Brucella infections cause important economical losses such as abortion, premature
delivery and reduction of milk production in domestic animals (1,2). It is known that
brucellae are among the most important infectious agents causing in abortion in
sheep, and B.melitensis has been isolated from such cases in Turkey. Until now, there
are few reports on placental lesions in ovine brucellosis, and these are limited to
experimental studies (3-6).
Recently, immunoenzymatic techniques have been introduced to detect the location of
brucella organisms in formalin-fixed tissues of goats (7-9), cows (9, 10), and mice (9,
11). The objective of the present study was to evaluate placental lesions from field
cases of ovine abortion and to detect the presence and the location of brucella in tissue
sections by immunoperoxidase staining.
Materials and methods
Histology and lmmunohistochemistry
The placentas were collected from 10 aborting ewes from different herds in Ankara
Province during the lambing season in 1993 and 1994. Placental tissues were fixed in
10 % neutral buffered formalin, processed routinely for histologic examination, cut at
5 to 6 µm thick, and stained with hematoxylin and eosin. Other selected sections were
also stained with Brown-Hopps modified Gram stain. Best's Carmin for fibrin,
Tumbull blue for hemosiderin, and Hall's method for bilirubin.
Unstained sections were used for immunohistochemical staining of brucella antigen
using a streptavidin-biotin-peroxidase complex staining technique and a commercially
available kit (Shandon: lmmunon, MaxiTags Universal Anti-rabbit Kit, Life Sciences
International Ltd, Hampshire, UK) according to manufacturer's instructions. Sera
obtained from New Zealand white rabbits hyperimmunised with multiple injections of
killed whole B.melitensis strain 16 M ( Dr. Uysal Y., Pendik Veterinary Control and
Researh Institute, Brucella Laboratory, Istanbul) served as the primary polyclonal
antibody. The serum had an agglutinating titer of 1: 160, B. melitensis, and
agglutinated B. abortus, and B. suis. Primary antibody was used at a dilution of 1:
4000. Deparaffinized and endogenous peroxidase-blocked placental sections were
incubated sequentially with normal goat serum, diluted polyclonal primary antibody,
biotinylated goat anti-rabbit lgG as the secondary antibody, and the streptavidin-biotin
peroxidase complex. The peroxidase was localized with AEC chromogen. Sections
were counterstained with Gill's hematoxylin. Control sections were incubated with
normal rabbit serum instead of primary antibody.
Bacteriology
Four placentas were obtained with their fetuses. Fetal livers, spleens, stomach
contents, and placental cotyledons were cultured on 7 % sheep blood agar and
Brucella agar (Difco), and incubated for 3 to 5 days at 370C in 10% CO2 incubator.
Brucella colonies were identified by colony morphology and microscopic characteristics.
Results
Gross Lesions
Chorioallantoic membranes were focally or diffusely thickened with edematous fluid
in all placentas. One placenta (No. 9) had grossly visible yellow areas of necrosis on
the intercotyledonary zone.
Microscopic Lesions
Histologically, a necrotizing placentitis was seen. Placentitis in placentas numbers 1
and 10 were classified respectively as necro-purulent, and necro-haemorrhagic,
depending on the exudate. Since placentas were obtained from field cases, they also
were autolytic to some degree but not extensive enough to destroy the histologic
lesions.
In affected cotyledons, the trophoblastic epithelium lining the chorionic villi was
necrotic and partially or completely sloughed while the undesquamated necrotic
epithelium had a focal infiltration of neutrophils. In some villi, necrosis of epithelium
and neutrophil infiltration extended into the stroma and involved the whole villus.
Bacterial colonies were found in the necrotic areas of villi and were surrounded by
necrotic cell debris and neutrophils. In partly intact villi, bacterial colonies were seen
within the epithelium and connective tissue (Fig. 1).
Spaces between the chorionic villi contained sloughed epithelial cells, necrotic cell
debris, neutrophils, and bacteria. Gram-stained sections showed gram-negative
bacterial colonies within the trophoblastic epithelium, stroma, vascular lumen, and
spaces between the villi. In the sections stained by Tumbull blue and Hall's bilirubin
stain, hemosiderin-hematoidin pigments were found within the cytoplasm of
trophoblasts lining the base of the chorionic villi.
Hilar mesenchyme of the cotyledon was edematous, and had infiltration of
macrophages especially between and around vessels in 4 placentas (Nos. 2,5,8, and
10). Extensive areas of haemorrhage were also present around placental vessels.
Fig. 1.
Placenta No.4. Chorionic villus
section showing bacterial
colonies (arrows) within the
epithelial layer and
inflammatory leucocyte
infiltration into the underlying
stroma. HE x 400.
Fig.2. Placenta No. 1.
Chorioallantoic membrane.
Focal necrotizing panarteritis in
the wall of an arterial vessel
and the severe inflammation in
surrounding connective tissue.
Hyalinous thrombus is present
within the lumen. HE x 200.
All placentas had degenerative and necrotic changes in the chorioallantoic
trophoblasts at the edges of the cotyledons. In some areas, the integrity of the
trophoblastic epithelium was compromised, and the superficial necrosis extended
deeply into the underlying connective tissue (chorioallantoic ulceration). The
intercotyledonary zone had similar necrotic lesions in 3 placentas (Nos. 1,4, and 9).
There were large areas of necrosis, and numerous large bacterial colonies
accompanied by severe infiltration of macrophages and neutrophils extending from
the hilar zone of the cotyledons along the intercotyledonary mesenchyme in 2 of 3
placentas (Nos. 4, and 9). In one placenta (No. 1) these was dense and difflise
neutrophil infiltration extending from the chorioallantoic surface into the underlying
mesenchyme, areas of necrosis and bacterial colonies along the allantoic surface and
around the vessels, and multifocal microabcesses in the intercotyledonary placenta.
Moreover, changes in the of vessel walls in chorioallantoic membranes were the most
remarkable feature in these 3 placentas. Some of the large arteries within and near
these lesions had segmental necrosis largely in the tunica media and occasionally
between the tunica intima and media, and bacterial colonies in the tunica media and
adventitia, accompanied by focal infiltrations of macrophages and neutrophils. In
addition, focal necrotizing panarteritis (Fig.2) was seen in the medium and small sized
arteries in placenta nos. I, and 4. In placenta no. 1, most of the medium and small
sized arteries had degeneration of the endothelium, and hyaline thrombi (Fig.2). Best's
carmin-stained sections showed fibrinoid degeneration in the tunica media of some of
the medium and large arteries. Otherwise, calcification was seen in the tunica media
of a few large sized arteries in this placenta, and also in the cotyledonary mesenchyme
in 4 placentas (Nos. 2,5,9, and 10) (Table 1).
Table 1. Placentitis and Immunoperoxidase Results.
Placenta No.
1
2
3
4
5
6
7
8
9
10
Placentitis
+++
+
++
+++
+++
+
+
+
++
++
Immunoperoxidase
Staining
+
++
+++
+++
+++
+++
+
+++
+
++
Lesions and degree of staining: +: mild, ++: moderate, +++: severe
Immunoperoxidase staining
Brucella-specific staining was seen within the cytoplasm of trophoblastic cells (Figs.
3 - 6), macrophages, and neutrophils (Figs. 6, 7). Cytoplasmic staining was the most
intense in trophoblasts, especially lining the base of the chorionic villi (Figs. 3, 4).
Extracellular brucellae were obvious in the interstitium (Figs. 6, 7) and spaces
between the chorionic villi in all placentas, in addition, in vascular lumens in placenta
no. 3, and 6, and the inflammed walls of the vessels (Fig. 7) in placenta no. 4, and 9
with vacular lesions.
In 2 placentas (no. l and 7), some of the bacterial colonies were gram-negative with
Gram's stain and especially, respectively those observed within the inflamed vessel
walls and surrounding connective tissue, while the vascular lumens were not stained
specifically by immunoperoxidase (Table 1).
Bacteriological results
Since placentas were obtained from field cases, no brucella organisms were isolated
from placental cotyledons because of contamination. However, fetal organs in four
cases (Nos.l,2,3, and 4) yielded Brucella spp. on the basis of colony morphology and
microscopic characteristics. The remaining placentas were without fetuses. Sera
obtained from these aborting ewes three weeks after abortion were examined
serologically, and those with an agglutinating titre of 1:40 or more were considered
positive. Sera were negative for campylobacter antibodies.
Fig.3.
Placenta No.4. Placental
cotyledon. Dense brucellaspecific staining of trophoblasts
lining the base of the chorionic
villi. Immunoperoxidase x 100.
Fig.4.
Placenta No.6. Higher
magnification of trophoblasts at
the base of a chorionic villus.
Note densely cytoplasmic
staining. Immunoperoxidase x
400.
Fig.5.
Placenta No.8. Stained brucella
antigen within cytoplasm of
chorioallantoic trophoblasts at
the edge of a placental
cotyledon. Immunoperoxidase x
400.
Fig.6.
Placenta No.4. Chorioallantoic
membran. Stained brucella
antigen in trophoblasts (arrow),
phagocytes and interstitium.
Immunoperoxidase x 200.
Fig. 7.
Placenta no.4. Chorioallantoic
membrane. Stained brocella
antigen in the inflamed wall of
an artery, and in the
surrounding interstitium and
phagocytes.
lmmunoperoxidase x 200.
Discussion
In this study, placenta lesions found in field cases of ovine brucellosis were
morphologically evaluated. Streptavidin-biotin peroxidase complex technique was
used to detect the brucella antigen in placental sections. In addition, the relationship
between the organism and morphologic lesions was determined (Table 1).
Morphological changes in the placenta were similar to those described previously in
experimentally infected sheep (3-6). The thickening of chorioallantoic membranes
with edema was the most consistently gross alteration present, except for one (No. 9)
which had visible areas of necrosis in the intercotyledonary placenta. This finding has
been reported by Molello et al. (3-5) who first described placental lesions related to
three species of brucella in experimentally infected sheep. Histological lesions in
placentas with necrotizing placentitis recorded in our study, except for vasculitis were
also correlated with placental lesions in these experimental studies. Molello et al. (4)
suggested that B. melitensis and B. abortus exerted their primary necrotizing effects
on the placentome, but B. melitensis also caused necrosis throughout the placentome.
Accordingly (3), the fact that the degenerative changes were manifested so
extensively and severely throughout the placentome emphasize the virulence of B
.melitensis and explained the severe fetal mortality associated with infection. In our
study, the necrotic changes in 3 placentas (Nos. 1,4, and 9) were so extensive that
they also involved the intercotyledonary chorioallantoic membranes,while necrosis of
membranes was visibly apparent in one of these (No.9). A remarkable feature in these
placentas was necrotizing vasculitis of chorioallantoic vessels. Chorioallantoic
vasculitis indicates haematogenous spread of infection to the fetus. Placental
vasculitis has been reported in experimentally infected caprine (8, 12), bovine (10)
and ovine (6) placentas. Vasculitis was either segmental or total, involving the entire
vessel wall, in the present study. However, in placenta no. 1 with more severe
vascular lesions, some of the bacterial colonies stained negative with Gram's stain
were not stained brucella-specific by immunoperoxidase. This shows both the
presence of secondary gram-negative bacterias involved in of inflammation and also
explains the necro-purulent placentitis observed. In addition, it shows that
immunoperoxidase techniques have an advantage over routine histologic techniques.
A similar staining was seen in placenta no. 7 without vasular lesions.
Another change observed histologically was calcification in the mesenchyme of
cotyledons. Calcification was also recorded in 147 placentas in a study of the causes
of bovine abortion (13).
The intracellular localization of brucella in the placental trophoblast has been reported
in the placenta of bovine (9, 10, 14), caprine (7, 8, 9, 12), ovine (3-5), and murine (9,
11) species in many experimental studies. Also, brucella antigen was detected in the
cytoplasm of phagocytic cells, and extracellularly in the interstitium, exudate, and
lumen of the inflamed vessels by immunoperoxidase technique (7-11).
Brucella-specific staining was the most intense in trophoblasts, especially lining the
base of the chorionic villi. In some sections, staining was so intense that it interfered
with visualization of cellular structures, while the nuclei of cells were obscured by
antigen (9).
Acknowledgements
This paper is part of the thesis submitted by the senior author for the PhD degree in
the Department of Veterinary Pathology, Ankara University, Veterinary Faculty,
Ankara, Turkey.
We thank Dr. Yavuz Uysal for help in preparing brucella suspension.
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