Advances in Infectious Diseases, 2013, 3, 60-62
http://dx.doi.org/10.4236/aid.2013.31008 Published Online March 2013 (http://www.scirp.org/journal/aid)
Brucella melitensis Differs from B. suis in Growth and
Urease Activity In-Vitro, and Infectivity in Fisher-344
Rats In-Vivo
Aloka B. Bandara1*, Stephen M. Boyle1, Araceli Contreras-Rodriguez1, Ana M. Martins2,
Rajiv Prasad1, Christopher M. Reilly1,3
1The Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia
Polytechnic Institute & State University, Blacksburg, USA; 212 Virginia Bioinformatics Institute, Virginia Polytechnic Institute &
State University, Blacksburg, USA; 3Edward Via College of Osteopathic Medicine, Virginia Polytechnic Institute & State University,
Blacksburg, USA.
Received December 26th, 2012; revised January 28th, 2013; accepted February 27th, 2013
ABSTRACT
Importance of urease activity on pathogenic differences among Brucella species was evaluated. In cell-free extracts, the
B. suis urease showed 12 times greater specific activity than the B. melitensis urease. When Fisher-344 rats were inocu-
lated intraperitoneally (IP), at 1 week post-inoculation (PI), B. melitensis wild type 16 M was recovered from spleens
and livers in greater numbers than B. suis wild type 1330. At 8 weeks PI, spleens were clear of B. melitensis, whereas B.
suis remained. The wild type and the urease deficient strains of B. suis did not differ from each other in terms of recov-
ery from spleen or liver. Our observations suggest that B. meliten sis induces greater acute infectivity in Fisher-344 rats,
whereas B. suis causes chronic infectivity; and urease activity has no influence on Brucella infection using an IP route.
Keywords: Brucella; Urease Activity; Splenomegaly; Infectivity; Pathogenicity
1. Introduction
Brucellosis is a disease in humans and animals, resulting
from infection with bacteria belonging to the genus
Brucella [1]. The genus Brucella consists of 10 known
species designated on the basis of host preference, and
antigenic and biochemical characteristics. These include
B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis,
B. suis, B. ceti, B. pinnipedialis, B. microti, and B. ino-
pinata [2-4]. Abortion and sterility are the major mani-
festations of brucellosis among livestock. Fever, sweats,
malaise, weight loss, arthralgia, splenomegaly, and heap-
tomegaly are common clinical presentations in humans
[2,3]. Relatively little is known about the genetic ele-
ments regulating pathogenicity or host-preference among
Brucella species.
Microbial ureases are multi-subunit metalloenzymes
that hydrolyze urea to form carbon dioxide and two
molecules of ammonia that protonate to form ammonium
causing the pH to increase. Thus, the hydrolysis of urea
provides ammonium for incorporation into intracellular
metabolites and facilitates survival in acidic environ-
ments. Among several functional differences among Bru-
cella species, the difference in urease enzyme activity is
prominent [5].
In this study, we sought to determine whether the pa-
thogenicity between two major Brucella species differs
as a function of urease enzyme activity. We compared B.
melitensis and B. suis in terms of their in vitro growth,
urease activity and infectivity in rats.
2. Materials and Methods
B. melitensis wild type strain 16M, B. suis wild type
1330, and the urease-deficient B. suis mutant 1330Δure1K
[6] were obtained from our bacterial culture collection.
The attenuated ctpA mutant of B. suis (133 0ΔctpA) [7]
was used as a control. Brucella was grown in trypticase
soy broth (TSB) or on trypticase soy agar (TSA) (Difco)
at 37˚C in the presence of 5% CO2 as previously de-
scribed [8]. The cultures were grown in 25 mL TSB at
37˚C with shaking at 180 rpm, and Klett units were re-
corded every three hours using a Klett-Summerson col-
orimeter. The specific activity of urease was determined
using the extracts prepared from the strains grown in
TSB and harvested during logarithmic growth, as de-
scribed elsewhere [6,9].
*Corresponding author.

Brucella melitensis Differs from B. suis in Growth and Urease Activity In-Vitro, and Infectivity in Fisher-344 Rats In-Vivo 61
All experiments with animals were approved by the
Virginia Tech Institutional Animal Care and Use Com-
mittee. Five to six week old female Fisher-344 rats
(Charles River Laboratory) were injected intraperito-
neally (IP) with 4.3 × 104 to 4.6 × 104 colony forming
units (cfu)/animal of Brucella strains. Groups of five rats
inoculated with each Brucella strain were humanly sacri-
ficed by exposing to excess CO2 at 1, 4, and 8 weeks
post-inoculation (PI). Parts of liver and spleen were used
to determine the Brucella cfu as described previously [8].
3. Results
After 4 days of growth on TSA, colonies of B. suis
strains 1330 and 1330Δure1K appeared approximately
twice the size of colonies of B. melitensis 16 M (data is
not shown). In TSB media during the logarithmic phase,
the doubling time of the strain 1330 (11.5 h) and
1330Δure1K (11.3 h) were almost twice as long as that
of the strain 16 M (6.5 h). As expected, the urease mutant
1330Δure1K displayed no measurable enzyme activity.
The strain 1330 displayed twelve times greater urease
specific activity than the strain 16M (9.12 and 0.73
μmoles/min/mg of protein, respectively).
The rats inoculated with all three test strains displayed
substantial splenomegaly compared to those inoculated
with the control 1330ΔctpA (Figure 1). At 1 week PI,
rats injected with the strain 16 M had nearly two-fold
larger spleens than those injected with the strain 1330 or
1330Δure1K. At 4 and 8 weeks PI, rats in all test groups
displayed moderate spleen weights.
At 1 week PI, the strain 16 M was recovered in greater
numbers than strains 1330 and 1330Δure1K from spleens
of rats (Figure 2). At 4 weeks PI, all three test strains
Figure 1. Splenomegaly in Fisher-344 rats. Rats were in-
oculated IP with B. melitensiswild type 16M (_____♦_____),
B. suis wild type 1330 (_____●_____), B. suis urease-defi-
cient mutant 1330Δure1K (_____▲_____ ), or B. suis at-
tenuated mutant 1330ΔctpA (control) (____▄_____). Groups
of rats were euthanized at 1, 4, and 8 weeks PI, and the av-
erage weight of spleens (g) was determined. P values for the
difference among mean values were: <0.005 at 1 week
<0.005 at 4 weeks, and <0.025 at 8 weeks.
Figure 2. Recovery of Brucella from spleens of Fisher-344
rats. Rats were inoculated IP with B. melitensiswild type
16M (_____♦_____), B. suis wild type 1330 (_____●_____),
B. suis urease-deficient mutant 1330Δure1K (_____▲
_____), or B. suis attenuated mutant 1330ΔctpA (control)
(_____▄_____ ). Groups of rats were euthanized at 1, 4, and
8 weeks PI, and the recovery of Brucella from spleens
(log10cfu/spleen) was determined. P values for the differ-
ence among mean values were: <0.01 at 1 week, <0.005 at 4
weeks, and <0.005 at 8 weeks.
Figure 3. Recovery of Brucella from livers of Fisher-344
rats. Rats were inoculated IP with B. melitensiswild type
16M (_____♦_____), B. suis wild type 1330 (_____●_____),
B. suis urease-deficient mutant 1330Δure1K (_____▲_____),
or B. suis attenuated mutant 1330ΔctpA (control) (____▄
______). Groups of rats were euthanized at 1, 4, and 8
weeks PI, and the recovery of Brucella in livers was deter-
mined. P values for the difference among mean values were:
<0.01 at 1 week, and <0.90 at 4 weeks for cfu/gram liver.
were recovered in similar numbers from spleens. How-
ever, at 8 weeks PI, the strain 16M completely cleared
from spleens, but strains 1330 and 1330Δure1K were.
recovered in substantial numbers. The attenuated B. suis
mutant 1330ΔctpA was recovered in significantly smaller
numbers than other strains at 1 and 4 weeks PI, and was
completely cleared by 8 weeks PI.
At 1 week PI, the strain 16M was recovered from the
livers of infected rats in significantly greater number than

Brucella melitensis Differs from B. suis in Growth and Urease Activity In-Vitro, and Infectivity in Fisher-344 Rats In-Vivo 62
the strains 1330 and 1330Δure1K (Figure 3). Neverthe-
less, all three strains were recovered in similar numbers
at 4 weeks PI, and completely cleared from livers by 8
weeks PI.
4. Discussion
The aim of our study was to determine the importance of
urease enzyme activity to the species-specific pathoge-
nicity among Brucella species. We compared two of the
most pathogenic species of Brucella in terms of their in
vitro growth and urease activity, and in vivo infectivity.
We report that in spite of its relatively very low urease
activity, B. melitensis wild type induced greater spleno-
megaly and was recovered from liver and spleen in
greater numbers during the early phase of infection in
Fisher-344 rats. These observations suggest that infectiv-
ity of this species is not related to its low urease activity.
Nevertheless, B. melitensis was cleared from spleens and
livers of rats in less than 8 weeks. Young et al., [10] re-
ported that in C3H female mice, B. melitensis strain EP
cleared from spleens and liver 30 days after IP inocula-
tion, whereas nearly 5.0 log10 cfu per organ (spleen or
liver) of B. abortus strain 2308 was still present. Thus,
our observations support those of Young et al., [10] in
that B. melitensis is less persistent albeit in a different
rodent i.e. Fisher-344 rats and with respect to B. suis.
B. suis wild type exhibited relatively less splenome-
galy and recovered from spleens and livers in smaller
numbers at an early phase of infection, but managed to
persist longer in spleens (past 8 weeks PI). Based on
these observations, one may speculate that the relatively
greater urease enzyme activity of this strain enables its
longer persistence in spleens. Nevertheless, the B. suis
mutant 1330Δure1K that displayed zero urease enzyme
activity also persisted in spleens for longer time periods
exactly as the wild type B. suis did. These observations
suggest that the infectivity of B. suis is not related to its
greater urease activity.
5. Conclusion
B. melitensis differs from B. suis in growth and urease
activity in vitro and persistence in vivo. B. melitensis
induces greater acute infectivity in rats, whereas B. suis
causes chronic infectivity. The urease enzyme activity
does not have an influence on Brucella infectivity in
Fisher-344 rats when inoculated IP. The findings on dif-
ferences between B. melitensis and B. suis in urease en-
zyme activity and pathogenicity will be useful in devel-
opment of measures to prevent and control brucellosis.
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