J. Comp. Path. 2009, Vol. 140, 149e157 Available online at www.sciencedirect.

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Pathological, Immunohistochemical and

Bacteriological Study of Tissues and Milk of Cows
and Fetuses Experimentally Infected
with Brucella abortus
M. N. Xavier, T. A. Paixão, F. P. Poester, A. P. Lage and R. L. Santos
Departamento de Clı´nica e Cirurgia Veterina´ria, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antonio
Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil

Summary
This report describes a pathological, immunohistochemical and bacteriological study of 42 cows and their
progeny (aborted fetuses, weak premature calves, and healthy full-term calves) infected at 6e7 months of ges-
tation by conjunctival inoculation with Brucella abortus. Samples were collected at necropsy within 48 h of abor-
tion or parturition. The most significant lesions were necrotizing and suppurative placentitis and
lymphohistiocytic mastitis in cows, and fibrinous pleuritis, fibrinous pericarditis and bronchopneumonia in
aborted fetuses. B. abortus was isolated more frequently from milk samples than from mammary tissues, and
milk samples from cows with mastitis were often infected. Organisms were often demonstrated immunohisto-
chemically and by culture in tissues showing moderate to severe histological changes.
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: bacterial infection; Brucella abortus; brucellosis; cattle

Introduction where, as facultative intracellular microorganisms,

Bovine brucellosis is an economically important dis-
ease caused by the gram-negative bacterium Brucella
abortus. In addition, human brucellosis is well known
in numerous countries throughout the world. Late
gestation abortion is the predominant clinical sign
of B. abortus infection in cows, resulting in reproduc-
tive failure and consequently a decrease in milk pro-
duction (Enright et al., 1984; Meador et al., 1989a,b;
Silva et al., 2005). Clinical manifestations of bovine
brucellosis depend on age, reproductive and immuno-
logical status, route of infection, and virulence and
dose (natural or experimental) of the Brucella strain
(Adams, 2002).
Infection occurs most commonly via the digestive
tract, the usual source of the organism being an in-
fected placenta or aborted fetus. After ingestion, the
bacteria are internalized by the M cells in the Peyer’s
patches; they then spread to the regional lymph nodes
Correspondence to: R.L. Santos (e-mail: rsantos@vet.ufmg.br).
0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2008.10.004
they proliferate within macrophages (Ackermann
et al., 1988). Subsequently, they spread via the blood-
stream to other tissues, particularly the pregnant
uterus (Silva et al., 2005).
In cattle, B. abortus has a marked tropism for pla-
cental tissues, resulting in gross lesions particularly
in placentomes, which become friable, covered with
a fibrinous exudate, and fetid. Usually, there are no
gross lesions in the mammary glands (Meador et al.,
1988, 1989a,b; Schlafer and Miller, 2007). Aborted
fetuses show variable degrees of autolysis and may
be oedematous, and in some cases abdominal organs
may be covered with fibrin (Enright et al., 1984; Gor-
ham et al., 1986). Pneumonia has been described as
the most common lesion in fetuses aborted due to B.
abortus infection (López et al., 1984; Adams, 2002).
Microscopical changes in experimental brucellosis
include a diffuse lymphocytic and neutrophilic endo-
metritis with ulceration of the luminal epithelium,
whereas the caruncular surface is necrotic, with an in-
tense neutrophilic infiltrate (Payne, 1959; Meador
Ó 2008 Elsevier Ltd. All rights reserved.
150 M.N. Xavier et al.
et al., 1988). A multifocal to diffuse, mild interstitial
mastitis with infiltration of macrophages has been de-
scribed in the mammary glands of experimentally in-
fected goats (Meador et al., 1988). The most common
microscopical lesion in aborted fetuses is pleuropneu-
monia, with diffuse infiltration of mononuclear cells
and multifocal infiltration of neutrophils (López
et al., 1984; Hong et al., 1991). Foci of mononuclear in-
flammatory infiltrate associated with focal necrotiz-
ing granulomas may be observed in the liver, kidney
and spleen. There is also often lymphoid hyperplasia
in the spleen (Enright et al., 1984; Meador et al.,
1988).
The diagnosis of brucellosis is based on direct or in-
direct laboratory methods. Direct methods such as
bacterial isolation have high specificity but are
time-consuming and require facilities with an appro-
priate degree of biosafety (Poester et al., 2005). Sero-
logical methods are used more often, being quicker
and less expensive. Alternative methods for the detec-
tion of B. abortus in tissues include immunofluores-
cence, which has high specificity but is of low
sensitivity and, moreover, gives an inadequate picture
of tissue morphology (Meyer, 1966). Immunohisto-
chemical examination of paraffin wax-embedded tis-
sues for B. abortus antigens is not only both sensitive
and specific but also clearly shows tissue morphology;
it is, therefore, capable of demonstrating the distribu-
tion of organisms in the tissues, a valuable attribute
for the study of pathogenesis of B. abortus infection
(Meador et al., 1986, 1988; Hong et al., 1991; Pérez
et al., 1998; Santos et al., 1998).
Although lesions caused by B. abortus have been
described previously, most studies were performed
in goats or sheep (Anderson et al., 1986; Gorham
et al., 1986; Meador et al., 1988, 1989a,b). The pres-
ent report describes the pathological, immunohisto-
chemical and bacteriological findings in pregnant
cows and fetuses experimentally infected with B.
abortus.
Material and Methods
Animals
Forty-two crossbred heifers aged 20e30 months were
included in this study. Of these animals, 17 had been
vaccinated at 3e8 months of age with the S19 vaccine
strain and 12 had been vaccinated with the RB51 vac-
cine strain (Professional Biological Company, Den-
ver, CO, USA) at ca 24 months of age (i.e.,
approximately 6 months before pregnancy). The re-
maining 13 heifers had not been vaccinated. All
heifers were serologically negative for brucellosis be-
fore challenge, as assessed by the Rose Bengal plate
agglutination test (RBPAT).
Breeding Procedure and Experimental Challenge
The experimental infection procedure employed in
this study has been described previously (Paixão
et al., 2007). The heifers were subjected to synchroni-
zation of oestrus, followed by artificial insemination.
Pregnancy was confirmed by ultrasonography 35
days after insemination. Between 6 and 7 months of
pregnancy, the animals were challenged by conjunc-
tival inoculation of a virulent B. abortus strain (2308).
Each heifer was inoculated with 3.0  107 colony-
forming units by treating each eye with 50 ml of
bacterial suspension. The animals were then closely
observed until abortion or calving. This experimental
procedure was approved by the University Commit-
tee for Ethical Use of Experimental Animals. On
the basis of the clinical outcome, the animals were di-
vided into two groups, namely, those that either
aborted or gave birth to weak premature calves (G1
cows, n ¼ 18), and those giving birth to full-term
healthy calves (G2 cows, n ¼ 24).
Histopathology and Immunohistochemistry (IHC)
Cows, aborted fetuses and newborn calves were
subjected to necropsy within 48 h of abortion or partu-
rition. Cows and calves were humanely killed by intra-
venous administration of xylazine (15e20 mg/kg)
(Coopazine; Cooper, São Paulo, Brazil) followed by
electrocution. Samples of lymph nodes (mammary,
internal iliac), mammary gland, placentome, endo-
metrium, liver and spleen were collected from
cows, whereas samples of bronchial lymph nodes,
lung, spleen and liver were collected from fetuses
and calves. Mammary gland was sampled from the
right caudal gland. Tissue samples were fixed for
24 h in 10% neutral buffered formalin and further
processed for paraffin wax-embedding, sectioning
(4 mm), and staining with haematoxylin and eosin
(HE). The lesions were scored according to the se-
verity of the inflammation (1 ¼ mild, 2 ¼ moderate,
and 3 ¼ severe).
The immunohistochemical procedure was a modifi-
cation of the method previously described by Santos
et al. (1998). Slides were treated with the primary an-
tibody, a polyclonal anti-B. abortus antibody diluted 1
in 100, for 30 min in a humidified chamber at room
temperature. After washing in phosphate-buffered sa-
line (PBS), the slides were incubated with biotiny-
lated secondary antibody for 20 min at room
temperature, washed in PBS, and incubated with
streptavidineperoxidase complex (LSAB1 Kit;
Dako, Carpinteria, CA, USA) for 20 min at room
temperature. The reaction was developed with
a 0.024% diaminobenzidine solution (Dako). Tissues
from experimentally infected mice (Rolán and Tsolis,
 Brucella abortus Infection in Cattle
2007) were used as positive controls, and bovine tis-
sues from Brucella-free cattle were used as negative
controls. In addition, negative controls included re-
placement of the primary antibody by PBS.
Bacteriology
Samples collected aseptically from cows (mammary
and internal iliac lymph nodes, mammary gland,
spleen, liver, milk, placentome) and from aborted fe-
tuses and calves (bronchial lymph nodes, lung, spleen
and liver) were examined after storage at 20 C.
Thawed tissues were macerated in a stomacher (Sew-
ard Medical, Worthing, UK), plated on tryptose agar
containing an antibiotic supplement (Brucella Selec-
tive Supplement SR83; Oxoid, Basingstoke, UK)
(Farrel’s medium), and incubated for 12 days at
37 C in an atmosphere containing CO2 5%. Milk
samples were centrifuged at 2500 g for 15 min. The
intermediate phase was then discarded and the super-
nate was mixed with the pellet. One drop of this mix-
ture was plated on tryptose agar supplemented with
antibiotics and incubated as described above. All iso-
lates were identified by routine methods (Mac Fad-
din, 1980; Alton et al., 1988; Schurig et al., 1991).
Statistical Analysis
Comparisons of the frequency of occurrence of posi-
tive results (bacteriological and immunohistochemi-
cal) were assessed by Fisher’s exact text, and
comparisons of scores by the ManneWhitney test,
with Graphpad Instat Software, version 3.05 (Graph-
pad Software Inc., CA, USA).
Results
Abortion was defined as the premature expulsion of
a non-viable fetus more than 15 days before the pre-
dicted date of delivery, whereas premature live calves
which were hypoactive and did not suck colostrum
were regarded as ‘‘weak calves’’. Among the 42 heifers
studied, 12 (28.6%) aborted, six (14.3%) gave birth to
weak calves and 24 (57.1%) gave birth to healthy calves
(Paixão et al., 2007). Although vaccinated animals
tended to have fewer abortions and fewer weak calves
than did non-vaccinated animals (Table 1), the differ-
ences were not statistically significant. In view of this,
the animals were divided into two groups (G1 and G2,
defined above), regardless of vaccination status, to en-
able the intensity of lesions and distribution of organisms
to be assessed in relation to the presence (G1) or absence
(G2) of parturition abnormalities.
The most common macroscopical lesions in in-
fected cows were observed in the uteri, which were
distended with variable quantities of fetid brownish,
151
Table 1
Frequency of occurrence of abortion, birth of weak
calves, or birth of healthy calves in relation to the
vaccination status of the cows
Experimental group Abortion, % Weak calves, % Healthy calves, %
Vaccinated (S19) 17.6 (3/17) 17.6 (3/17) 64.8 (11/17)
Vaccinated (RB51) 33.3 (4/12) 0.0 (0/12) 66.6 (8/12)
Non-vaccinated 38.4 (5/13) 23.0 (3/13) 38.4 (5/13)
Vaccination had no significant effect on the frequency of occurrence of
abortion or birth of weak calves (Fisher’s exact test; P > 0.05).
flocculent exudate containing necrotic material
(Fig. 1A). Caruncular lesions were heterogeneous,
some placentomes being normal and others haemor-
rhagic and necrotic (Fig. 1B). Grossly, 83% (35/42)
of infected cows had placentitis, which was necrotiz-
ing in 29 (69%) animals, necrohaemorrhagic in three
(7%) and haemorrhagic in three (7%). The superfi-
cial and internal lymph nodes, and in some cases
the spleen, showed variable degrees of enlargement.
No macroscopical lesions were observed in the mam-
mary glands, liver, or other organs. G1 cows that
aborted had severe necrotic placentitis, but inflam-
mation, albeit comparatively slight, was also observed
in several G2 cows.
Grossly, the most common lesion in 37.5% (6/16) of
the fetuses and weak newborn calves from G1 cows
was a fibrinous pleuritis (Fig. 1C), without lesions in
the pulmonary parenchyma. Two fetuses (12.5%)
had lesions in the lungs, characterized by focal to dif-
fuse, reddish to greyish, firm areas in the parenchyma,
in addition to fibrinous exudate on the pleural sur-
face. In 37.5% of the fetuses and weak newborn calves
(6/16) the abdominal organs were covered with
a small amount of fibrinous exudate, resulting from
mild peritonitis. In 25% (4/16) there was also abun-
dant fibrinous exudate in the pericardium (Fig. 1D).
The most common microscopical lesion observed in
infected cows was necrotic placentitis, which occurred
in 95% of the cases (40/42) and was characterized by su-
perficial to deep necrosis of the carunculae, associated
with haemorrhage, neutrophilic exudate, intralesional
bacterial colonies, and retained fetal tissues in the carun-
cular crypts (Fig. 2A). Perivascular infiltration of neu-
trophils, lymphocytes, plasma cells and histiocytes,
with a few small foci of necrosis, was observed in the car-
uncular peduncles. In 50% of the 42 cows, mammary
glands showed a multifocal interstitial lymphohistiocytic
infiltrate, with neutrophils in the acinar lumen (Fig. 2B).
A diffuse neutrophilic mastitis was observed in only two
(4.7%) cows. Multifocal to diffuse endometritis, ob-
served in 38 (95%) of 40 cows examined, was character-
ized by multifocal ulceration of the superficial
endometrium and cellular debris on the endometrial
152 M.N. Xavier et al.

Fig. 1 AeD. Gross pathology of cows and fetuses experimentally infected with Brucella abortus. (A) Uterus containing brownish fluid, with
fibrinous exudate on the caruncular surface. (B) Cut surface of a placentome with large amounts of fibrinous necrotic exudate
and multifocal haemorrhage (necrotizing placentitis). (C) Aborted fetus with a large amount of fibrinous exudate on the pleu-
ral surface of the lung and a moderate amount of fluid in the thoracic cavity (fibrinous pleuritis). (D) Aborted fetus with an
opened pericardial sac containing a large amount of fibrinous exudate (fibrinous pericarditis).

surface (Fig. 2C), with oedema and periglandular and
perivascular infiltration of lymphocytes, plasma cells
and neutrophils. Severe neutrophilic vasculitis with fibri-
noid degeneration was sometimes seen in the endome-
trium and caruncular peduncles (Fig. 2D). The
mammary and internal iliac lymph nodes showed vari-
able degrees of lymphoid hyperplasia associated with
neutrophilic (Fig. 2E) and histiocytic infiltrates in the
medulla and paracortex. There was also lymphoid hy-
perplasia and mild perifollicular neutrophilic infiltrate
in the spleen.
Microscopical fetal lesions of pericarditis and pleur-
itis were characterized by severe fibrinous exudate and
infiltration of neutrophils, lymphocytes and plasma
cells, with diffuse congestion in the pericardium and
visceral pleura (Fig. 2F). The lungs of 61.5% (24/
39) of the fetuses and calves showed a multifocal neu-
trophilic infiltrate in the bronchioles and alveoli,
sometimes associated with multifocal interstitial pneu-
monia (Fig. 2G and H). Some cases showed vasculitis
and thrombosis in the pulmonary parenchyma. Peri-
splenitis was observed in 25% (10/40) and perihepati-
tis in 22.5% (9/40) of the fetuses and weak newborn
calves. These changes were characterized by infiltra-
tion of lymphocytes and neutrophils, with small
amounts of fibrin on the surface of the hepatic
and splenic capsule. Multifocal to diffuse, mild to
moderate, neutrophilic or mixed hepatitis was ob-
served in 55% (22/40) of the cases. Splenic and bron-
chial lymph nodes showed mild to moderate lymphoid
hyperplasia and a multifocal neutrophilic infiltrate.
Splenitis was observed in 71% (27/38) and lymphad-
enitis in 56% (22/39) of the fetuses and calves.
The histopathological, immunohistochemical and
bacteriological results in cows are summarized in Ta-
ble 2. G1 and G2 cows differed significantly in respect
of the average score of histopathological changes in
the carunculae and spleen. As expected, placentitis
was more severe in G1 cows than in G2 cows; con-
versely, however, inflammation of the spleen was
more severe in G2 cows. The highest inflammation
scores (>1.5) were those recorded in the endome-
trium and carunculae. The number of isolations of
B. abortus from samples of carunculae, iliac lymph no-
des and milk was significantly greater in G1 cows than
in G2 cows. However, these samples, together with
samples of mammary gland and mammary lymph
node, were the main source of B. abortus isolates in
both groups of cows (Table 2).
Immunolabelling of B. abortus in the carunculae
and endometrium (Fig. 2A) was positive in 94.9%
of cows with placentitis (37/39). In these sites, B. abor-
tus was observed both extracellularly in necrotic areas
and intracellularly within macrophages, neutrophils
 Brucella abortus Infection in Cattle 153

Fig. 2 AeI. Histopathology and immunohistochemistry of cows, aborted fetuses and weak premature calves experimentally infected with B.
abortus. (A) Cow; placentome showing caruncular crypts filled with necrotic debris, multifocal haemorrhage, intense inflamma-
tory infiltrate, and several bacterial colonies (acute necrotizing placentitis). HE. Inset: immunolabelled colonies of B. abortus.
IHC. Bar, 167 mm. (B) Cow; mammary gland with focal interstitial infiltration of lymphocytes, macrophages and neutrophils,
and neutrophils in an acinar lumen. HE. Inset: immunolabelled B. abortus mainly associated with macrophages. IHC. Bar,
50 mm. (C) Cow; endometrium with an extensive area of erosion of the luminal epithelium, and accumulations of fibrin, cell
debris, and bacterial colonies. HE. Bar, 250 mm. (D) Cow; endometrial arteriole with a mural and perivascular inflammatory
infiltrate, and fibrinoid degeneration (vasculitis). HE. Bar, 100 mm. (E) Cow; mammary lymph node with medullary sinuses
filled with macrophages and neutrophils (suppurative lymphadenitis). HE. Bar, 33.5 mm. (F) Aborted fetus; severely thickened
visceral pleura with fibrin accumulation and diffuse inflammatory infiltrate (fibrinous pleuritis). HE. Bar, 100 mm. (G) Weak
premature calf: lung showing diffusely thickened alveolar walls with interstitial inflammatory infiltrate (interstitial pneumonia).
HE. Bar, 33.5 mm. (H) Weak premature calf; bronchiole with large amount of fibrinous exudate in the lumen and mild inflam-
matory infiltrate in the adjacent parenchyma (bronchopneumonia). HE. Bar, 80 mm. (I) Weak premature calf; lung showing
immunolabelled B. abortus in the interstitium and alveolar lumen, mainly associated with macrophages. IHC. Bar, 25 mm.
154 M.N. Xavier et al.

Table 2
Score of histological lesions and frequency of detection of Brucella abortus by bacteriology and immunohistochemistry in
groups G1 and G2 of experimentally infected cows

Sample Histopathology score Bacteriology (%) Immunohistochemistry (%)

G1 G2 P G1 G2 P G1 G2 P

Caruncula 2.27  0.82 1.66  0.86 0.039 100 (18/18) 39.1 (9/23) <0.0001 100 (18/18) 79.3 (18/23) NS
Endometrium 1.94  0.74 1.60  0.89 NS ND ND ND 88.8 (16/18) 50 (10/20) 0.0149
Mammary lymph node 0.50  0.71 0.71  0.75 NS 38.9 (7/18) 17.4 (4/23) NS 16.7 (3/18) 4.4 (1/23) NS
Iliac lymph node 0.44  0.51 0.50  0.59 NS 38.9 (7/18) 8.7 (2/23) 0.0283 11.1 (2/18) 13.0 (3/23) NS
Liver 0.56  0.78 0.50  0.51 NS 5.9 (1/17) 0.0 (0/22) NS 0.0 (0/17) 9.1 (2/22) NS
Spleen 0.50  0.61 1.20  0.77 0.006 5.9 (1/17) 0.0 (0/19) NS 35.3 (6/17) 31.6 (6/19) NS
Mammary gland 0.89  0.96 0.50  0.72 NS 33.3 (6/18) 19.0 (4/21) NS 33.3 (6/18) 4.8 (1/21) 0.0328
Milk NA NA NA 61.1 (11/18) 30.4 (7/23) 0.0063 NA NA NA

G1 ¼ cows that aborted or gave birth to weak premature calves; G2 ¼ cows that gave birth to normal calves; P ¼ P value (histopathology scores
were compared by ManneWhitney’s test, and frequencies of positivity by immunohistochemistry and bacteriology were compared by Fisher’s
exact test); NS ¼ non-significant (P > 0.05); NA ¼ not applicable; ND ¼ not determined.

and trophoblastic cells. B abortus was also frequently
isolated from the exudate on the surface of ulcerated
areas of endometrium, 77.1% (27/35) of cows with
endometritis giving positive results. Immunolabelling
of B. abortus in the endometrium and mammary gland
occurred significantly more frequently in G1 cows
than in G2 cows.
B. abortus was isolated more often from milk samples
(43.9% of cows; 18/41) than from samples of mammary
tissue (24.4%; 10/41); 22% (9/41) of cows yielded the or-
ganism from both samples. Immunolabelling of B. abor-
tus in macrophages in the interstitium of the mammary
gland or in the acinar lumen was observed in 22.2%
(4/18) of cows with lymphohistiocytic interstitial mastitis
(Fig. 2B); however, bacterial culture gave a greater
number of positive results (mammary tissue, 38% of
cows [8/21]; milk, 57.1% of cows [12/21]).
Table 3 shows the histopathological, immunohisto-
chemical and bacteriological results in (1) progeny of
G1 cows (aborted fetuses and weak premature
calves), and (2) progeny of G2 cows (full-term healthy
calves). In G1 progeny the histopathological changes
were mild, as reflected by low inflammation scores;
the only difference between such changes in G1 and
G2 progeny was a more intense inflammation in the
spleen in G2 progeny. B. abortus was detected bacteri-
ologically and immunohistochemically more fre-
quently in G1 progeny than in G2 progeny, and
more frequently in lung and spleen than in other sites
examined. Immunolabelled bacteria were observed
inside inflammatory cells, free in the parenchyma,
and inside leucocytes within blood vessels (Fig. 2I).
Discussion
This report describes a comprehensive pathological,
immunohistochemical and bacteriological evaluation
of cows and fetuses experimentally infected with B.
abortus. The most common gross lesions observed
were a fibrinous necrotizing placentitis in cows,
and a fibrinous pleuritis and peritonitis in fetuses,
in accordance with previous reports (Payne, 1959;
Palmer et al., 1996; Pérez et al., 1998). However,
most of the previously published studies describe le-
sions caused by B. abortus in goats and sheep, as
models of bovine infection (Molello et al., 1963;

Table 3
Score of histological lesions and frequency of detection of Brucella abortus by bacteriology and immunohistochemistry in
progeny of experimentally infected cows of groups G1 and G2

Sample Histopathology score Bacteriology (%) Immunohistochemistry (%)

G1 progeny G2 progeny P G1 progeny G2 progeny P G1 progeny G2 progeny P

Lung 0.92  0.91 0.75  0.79 NS 71.4 (10/14) 30.4 (7/23) 0.0210 64.3 (9/14) 17.4 (4/23) 0.0058
Bronchial lymph node 0.64  0.92 1.09  1.01 NS 57.1 (8/14) 18.2 (4/22) 0.0286 42.9 (6/14) 4.6 (1/22) 0.0083
Liver 0.93  0.77 0.45  0.50 NS 50 (8/16) 4.4 (1/23) 0.0015 37.5 (6/16) 0.0 (0/23) 0.0025
Spleen 0.66  1.11 1.65  0.83 0.003 81.3 (13/16) 31.8 (7/22) 0.0036 56.3 (9/16) 22.7 (5/22) 0.0468

G1 ¼ cows that aborted or gave birth to weak premature calves; G2 ¼ cows that gave birth to normal calves; P ¼ P-value (histopathology scores
were compared by ManneWhitney’s test, and frequencies of positivity by immunohistochemistry and bacteriology were compared by Fisher’s
exact test); NS ¼ non-significant (P > 0.05).
 Brucella abortus Infection in Cattle
Anderson et al., 1986; Meador et al., 1988, 1989a,b).
Similar lesions have been described in B. abortus-in-
fected bison (Bison bison) (Rhyan et al., 2001). The
mouse has been used extensively as a model for Bru-
cella infection (Enright et al., 1990), in which studies
on pathogenesis and immunity have proved possible
(Sun et al., 2002; Roux et al., 2007). However, infec-
tion in mice does not mimic the reproductive disease
observed in cattle, on which transmission of infection
depends.
Necrotic neutrophilic placentitis with perivascular
infiltrate, which was the most frequently observed mi-
croscopical change in experimentally infected cows,
was associated with large numbers of B. abortus intra-
cellularly in macrophages and trophoblasts and also
extracellularly in necrotic tissues. Trophoblasts are
thought to be the primary target cell for invasion
and multiplication of B. abortus in the placenta (An-
derson et al., 1986). This tropism may be due to the
presence of erythritol, or to hormone synthesis by tro-
phoblastic cells (Samartino and Enright, 1993). How-
ever, immunolabelling demonstrated that the
ulcerative lymphoplasmacytic endometritis observed
in most of the cows was not associated with intracellu-
lar localization of B. abortus. These lesions in the preg-
nant uterus were consistent with those previously
reported, although most previous studies were per-
formed in small ruminants (Payne, 1959; Molello
et al., 1963; Anderson et al., 1986; Meador et al.,
1988, 1989a,b).
The microscopical lesions observed in the fetuses
and calves, e.g., fibrinous pleuritis and peritonitis,
bronchopneumonia and splenitis, have been reported
previously (López et al., 1984; Hong et al., 1991).
However, fibrinous pericarditis, frequently observed
in the present study, has not previously been de-
scribed as a significant fetal lesion in brucellosis.
Inflammation in the caruncular tissue was signifi-
cantly greater in G1 cows than in G2 cows, an obser-
vation supported by immunolabelling and bacterial
culture. These results support the notion that abor-
tion due to B. abortus is dependent on the severity
and distribution of caruncular lesions, which suppos-
edly cause fetal oxygen and nutrient deprivation. In
the spleen, however, the inflammation score was sig-
nificantly higher in G2 cows than in G1 cows, yet
there was no corresponding difference in the immuno-
labelling and cultural results; this suggests that the
splenic inflammation was not directly triggered by
the organism. Moreover, the inflammation score
was higher in the progeny of G2 cows (healthy calves)
than in the progeny of G1 cows (aborted fetuses and
premature, weak calves). It would seem possible
that the increased splenic inflammation was in fact
a manifestation of an effective immune response. It
155
was recently demonstrated in mice that virulent Bru-
cella induced a strong pro-inflammatory response in
the spleen, as assessed by evaluating the gene expres-
sion profile (Roux et al., 2007).
B. abortus-induced mastitis has been described in
experimental infection in goats (Meador et al., 1986,
1988, 1989a,b; Meador and Deyoe, 1991), and in
one early bovine study, which lacked immunohisto-
chemical analysis (Emminger and Schalm, 1943).
In the present study, immunolabelling demonstrated
B. abortus intracellularly in macrophages as well as in
the acinar lumina in a relatively small number of
cows, and this was supported by bacterial isolation
from mammary tissues. The results suggest that in
cattle B. abortus sometimes causes a mild interstitial in-
flammatory reaction in the mammary gland, associ-
ated with small numbers of organisms. It is
noteworthy that positive cultural results were ob-
tained more frequently from a pooled milk sample
from all four glands than from the mammary tissue it-
self. It is also noteworthy that 57.1% of cows with
mammary lesions shed B. abortus in the milk; more-
over, 90% (9/10) cows that yielded positive cultures
from mammary tissue also shed bacteria in the milk.
This suggests that brucella mastitis is highly likely to
be associated with the presence of organisms in the
milk, a point of public health importance. In a report
on Brucella suis infection of cattle, Ewalt et al. (1997)
described mastitis similar to that observed in the pres-
ent study.
There were no significant differences between the
moderate histopathology scores of the healthy prog-
eny of G2 cows and the abnormal progeny of G1
cows, despite the greater number of positive immuno-
labelling and cultural results in the latter. These re-
sults support the notion that the primary cause of
abortion in B. abortus infection is not the severity of fe-
tal lesions, but the lack of oxygen and nutrients to the
fetus due to placental lesions at late stages of gestation
(Enright et al., 1984; López et al., 1984). It has been
reported previously that B. abortus may often be iso-
lated from fetal tissues showing no histological
changes (Meador et al., 1988). Supposedly, fetal infec-
tion results from aspiration of amniotic fluid contain-
ing B. abortus, a process exacerbated by anoxia
secondary to placentitis. Following invasion through
the respiratory tract, bacteraemia may develop,
spreading the organism to other organs (López
et al., 1984). In the present study, immunolabelled
bacteria were observed within blood vessels in some
infected fetuses.
Immunolabelling of B. abortus was stronger and oc-
curred more frequently in tissues with moderate to se-
vere lesions than in those with mild lesions, indicating
that these lesions, which consisted mainly of
156 M.N. Xavier et al.
inflammation and necrosis, were in fact due to B. abor-
tus infection. Immunohistochemistry would appear to
be an excellent alternative to bacterial culture as
a method of diagnosis.
The results showed no statistically significant differ-
ences between the abortion rates in vaccinated and
non-vaccinated cattle, probably because of the small
number of animals used in this study, which was not
designed to assess vaccine protection. Indeed, in a re-
cent study with larger numbers of animals under sim-
ilar experimental conditions (Poester et al., 2006),
RB51 vaccine proved to be effective. Lack of positive
serological results (data not reported) confirmed that
RB51 vaccination does not interfere with antibody
tests. As the antibodies detected by such tests are di-
rected to the O side-chain of smooth brucellae, the
lack of antibody response after RB51 vaccination is
most likely due to the absence of O chain in RB51
(Schurig et al., 1991; Stevens et al., 1994). As expected,
heifers vaccinated with S19 at 3e8 months of age
were serologically negative at 24 months.
Acknowledgments
This study was supported by FAPEMIG (‘‘Fundação
de Amparo à Pesquisa do Estado de Minas Gerais’’,
Belo Horizonte, Brazil; grants EDT-2007/03 and
CAG-1092/05). Four of the authors (RLS, MNX,
TAP and APL) are also supported by CNPq (‘‘Con-
selho Nacional de Desenvolvimento Cientı́fico e Tec-
nológico, Brası́lia, Brazil).
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½ Received,
Accepted, October 5th, 2008 
October 26th, 2007