Tropical Biomedicine 30(2): 277–280 (2013)
Serologic detection of antibodies to Brucella spp. using a
commercial ELISA in cattle in Grenada, West Indies
Chikweto, A.1, Tiwari, K.1, Kumthekar, S.1, Stone, D.1, Louison, B.2, Thomas, D.2, Sharma, R.1 and
1Pathobiology Academic Program, School of Veterinary Medicine, P.O Box 7, St. George’s University,
St. George’s, Grenada, West Indies
2Ministry of Agriculture, Forestry and Fisheries, St. George’s, Grenada, West Indies
*Corresponding author e-mail: email@example.com
Received 28 November 2012; received in revised form 19 March 2013; accepted 20 March 2013
Abstract. Bovine brucellosis, caused mainly by Brucella abortus, a zoonotic bacterium, has
been reported from many areas of the world, including Central and South America, and the
Caribbean island state of Trinidad and Tobago. Although brucellosis has been eradicated from
domestic cattle in Canada it still exists in one or two herds in the United States. Serological
tests are important in estimating prevalence of Brucella exposure in order to target eradication
programmes. In this study, serum samples from 150 cattle were tested using a commercial
competitive enzyme linked immunosorbent assay (SVANOVIR®Brucella-Ab C-ELISA) which
detects antibodies to both B. abortus and Brucella melitensis. All cattle tested were greater
than 6 months old and were unvaccinated. Sampled cattle were from 35 herds representing
animals from all 6 parishes of Grenada. Nine of the 150 animals (6%) were positive for
antibodies to B. abortus and/or melitensis by the C-ELISA. Of the 35 herds, 7 (20%) had C-
ELISA-positive animals. Three of the 6 parishes contained positive herds. Based on the high
sensitivity (98%) and specificity (99.7%) of the C-ELISA, these results strongly indicate the
presence of cattle exposed to B. abortus and/or melitensis in Grenada.
Brucellosis in cattle is mainly caused by
Brucella abortus, and less commonly by
Brucella melitensis (Verger, 1985; Jimenez
de Bagues et al., 1991). Occasionally,
Brucella suis may cause infection in cattle,
but it has not been known to cause abortion
or to spread among cattle (Ewalt et al., 1997).
In the last decade bovine brucellosis was
reported to be endemic in several countries
in Central and South America with individual
cow prevalence ranging from 3% to 11% and
herd prevalence as high as 15% (Radostits
et al., 2007). Information on brucellosis from
Caribbean islands is limited. Serological
evidence for Brucella exposure in goats and
sheep was reported from Saint Croix (Ahl
et al., 1993). A 1997 review of the literature
indicated that brucellosis was not detected
in Barbados, Surinam, or St. Kitts/Nevis
(Corbel, 1997). However, more recently
serologic detection of Brucella exposure
and culture-confirmed infection in cattle
has been reported in Grenada’s neighboring
island of Trinidad (Fosgate et al., 2002), and
seropositive sheep have been detected in
Grenada itself (Stone et al., 2012). In Grenada
the estimated cattle population is around
4000 and typical herd size is from 2 to 10
animals. Cattle are frequently pastured along
with sheep and goats. Thus, the recent
detection of sheep positive for antibodies
against Brucella spp. is an important rationale
for testing cattle on this island. The objective
of this study was to serologically test cattle
from all 6 parishes in Grenada using a
competitive ELISA (C-ELISA), a test with a
high performance index compared to other
serologic tests (Gall & Nielsen, 2004). The
C-ELISA used in this study detects serum
antibodies to both B. abortus and B.
melitensis. Our results will facilitate
implementation of control programmes for
this important zoonotic disease.
MATERIALS AND METHODS
A total of 150 cattle were randomly selected
from 35 herds representing all 6 parishes of
the island of Grenada. Herd size ranged from
2 to 10 animals and only animals greater than
6 months of age were selected. Both males
and females were tested. The cattle were of
Holstein/Friesian-local mixed breed and
clinically normal. None of the animals tested
had received any vaccinations, nor was there
any herd history of abortion. Cattle in
Grenada are reared in a free-range pasture
environment often together with sheep and
goats. Ten mls of venous blood was collected
from each animal in a sterile, red-top tube
and allowed to clot. The samples were
transported on ice to the diagnostic
laboratory of the St. George’s University
School of Veterinary Medicine. Serum
samples were collected after centrifugation
(at 3000 rpm for 10 minutes) and stored at -
80ºC until tested for antibodies to Brucella
spp. The SVANOVIR®Brucella C-ELISA
antibody test kit (SVANOVA Biotech AB,
Uppsala, Sweden) was used to test the serum
samples for antibodies to B. abortus and B.
melitensis according to the manufacturer’s
instructions. The optical density (OD) values
for each of the controls provided in the kit
and serum samples in the wells were read at
450 nm using a microplate photometer
(Universal Microplate Reader, Bio-Tek
Instruments, Inc.). The percent inhibition (PI)
values were calculated according to the
manufacturer’s instructions. The results were
expressed as negative for Brucella antibodies
(PI <30%) or positive for Brucella antibodies
Nine of the 150 cattle sampled were positive
by the C-ELSA test, thus giving a sero-
prevalence of 6% for Brucella exposure in
cattle in this study. The seroprevalence was
highest in St. David parish at 16.3% (5 of
31 animals positive) followed by a 5.26%
seroprevalence for cattle from St. Andrew
parish (3 of 57 animals positive). Of 33 cattle
sampled from St. John parish, only one was
positive (3.03% seropositive). None of the
cattle sampled from the remaining 3 parishes
tested positive (Table 1).
The SVANOVIR®Brucella-Ab C-ELISA
detects serum antibodies to both B. abortus
and B. melitensis. In this assay, serum
samples are exposed to B. abortus smooth
lipopolysaccharide (S-LPS) coated wells on
microtiter plates together with a mouse
monoclonal antibody (mAb) specific for an
epitope on the o-polysaccharide side chain
of the S-LPS antigen and labeled with
horseradish peroxidase. If the test serum
contains B. abortus- and/or B. melitensis-
specific antibodies (positive) they compete
Table 1. Seroprevalence of brucellosis in cattle in Grenada, based on C-ELISA
ParishNo. of herdsNo. of samplesNo. positive% positive
with the mouse mAb binding to the o-
polysaccharide portion of S-LPS antigen
resulting in a decrease or lack of colour
change after addition of substrate. C-ELISAs
using the Brucella LPS antigen significantly
increased the specificity of antibody tests
and allowed for the differentiation between
Strain-19-vaccinated animals (low affinity
antibody) and naturally exposed animals.
(Nielsen et al., 1996). Prior vaccination was
not an issue in the present study as cattle in
Grenada are not vaccinated against Brucella.
The sensitivity and specificity of the C-ELISA
as reported by the manufacture are 98% and
99.7% respectively (SVNOVIR®Brucella
C-ELISA (Svanova, 2012), which is higher
than most other serological tests used in
screening cattle for exposure to Brucella spp.
The sensitivity was based on 331 samples
confirmed positive by culture. The specificity
was based on 1144 samples from negative
herds in Canada. The competitive Brucella
ELISA is a marked improvement over
conventional ELISAs where false-positive
reactions were common (Hariharan et al.,
1986). Importantly, the C-ELISA has been
demonstrated to be an effective tool for
detecting exposure to Brucella spp. in both
cattle and buffaloes (Fosgate et al., 2011).
In this study, 6% of cattle tested were
serologically positive for Brucella antibodies.
This reflects natural exposure and not current
infection or disease. The actual prevalence
of Brucella exposure, however, may be higher
because animals can lose their antibody titers
over a period of time and there may be a few
false negative due to the C-ELISA having a
sensitivity of less than 100% (Godfroid et al.,
2010). However, C-ELISA is an excellent
confirmatory assay for detecting exposure
to Brucella in most mammalian species
(Nielsen & Yu, 2010). Although the sero-
prevelance of bovine brucellosis in regions
of the world varies considerably, our results
are in agreement with other seroprevalence
reports from the Americas (3-11%) reflecting
data from Argentina, Brazil, Guatemala,
Paraguay, and Venezuela (Radostits et al.,
2007). Importantly, brucellosis has been
confirmed in cattle and water buffaloes in
Trinidad (Fosgate et al., 2002).
The C-ELISA kit used in this study is
designed to detect serum antibodies to B.
melitensis as well as B. abortus. In Latin
America, of the 1377 strains isolated in
1968-1991, nearly 32% were from cattle, most
of them being B. abortus, but a few B.
melitensis as well (Lucero et al., 2008). A
recent study conducted in Grenada showed
a seroprevalence of 3.6% for brucellosis in
sheep, which used an indirect ELISA with the
smooth LPS antigens of B. abortus strain S-
99 (Stone et al., 2012). In Trinidad, B. abortus
has been cultured from the lymph nodes of
both cattle and buffaloes (Fosgate et al.,
2011). The ‘gold standard’ diagnostic test for
brucellosis continues to be based on isolation
of the organism from the organs and lymph
nodes of the fetus, the placenta, milk, vaginal
mucus or uterine exudate (Radostits et al.,
2007). Further studies are required to confirm
brucellosis in cattle in Grenada by culture or
PCR, and to determine the species of Brucella
associated with positive antibody titers.
Acknowledgements. The authors would like
to thank Ms Abigail Noel and Mr. Aaron Morris
for technical assistance.
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