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Veterinary World, EISSN: 2231-0916 2080
Veterinary World, EISSN: 2231-0916
Available at www.veterinaryworld.org/Vol.16/October-2023/9.pdf
RESEARCH ARTICLE
Open Access
Comparison of diagnostic tests for detecting bovine brucellosis in
animals vaccinated with S19 and RB51 strain vaccines
Marcelo Ibarra1,2 , Martin Campos1,2 , Benavides Hernán1 , Anthony Loor-Giler3 , Andrea Chamorro4 ,
and Luis Nuñez5,6
1. Facultad de Industrias Agropecuarias y Ciencias Ambientales, Carrera Agropecuaria, Universidad Politécnica Estatal
del Carchi, Antisana S/N y Av Universitaria, Tulcán Ecuador 040102; 2. Facultad de Ciencias Veterinarias, Universidad
Nacional de Rosario, Boulevard Ovidio Lagos y Ruta 33 Casilda-Santa Fe-Argentina; 3. Facultad de Ingeniería y Ciencias
Aplicadas, Carrera de Ingeniería en Biotecnología, Universidad de Las Américas, Antigua Vía a Nayón S/N, Quito EC
170124 Ecuador; 4. Facultad de Industrias Agropecuarias y Ciencias Ambientales, Carrera de Enfermeria, Universidad
Politécnica Estatal del Carchi, Antisana S/N y Av Universitaria, Tulcán Ecuador 040102; 5. Facultad de Ciencias de
la Salud, Carrera de Medicina Veterinaria, Universidad de Las Américas, Antigua Vía a Nayón S/N, Quito EC 170124
Ecuador; 6. One Health Research Group, Universidad de Las Américas, Quito, Ecuador.
Corresponding author: Luis Nuñez, e-mail: fabiann7@yahoo.es
Co-authors: MI: marcelo.ibarra@upec.edu.ec, MC: rolando.campos@upec.edu.ec, BH: hernan.benavides@upec.edu.ec,
AL: a.abel.loor.giler@gmail.com, AC: andreaf.chamorro@upec.edu.ec
Received: 06-06-2023, Accepted: 31-08-2023, Published online: 14-10-2023
doi: www.doi.org/10.14202/vetworld.2023.2080-2085 How to cite this article: Ibarra M, Campos M, Hernán B,
Loor-Giler A, Chamorro A, and Nuñez L. (2023) Comparison of diagnostic tests for detecting bovine brucellosis in animals
vaccinated with S19 and RB51 strain vaccines, Veterinary World, 16(10): 2080–2085.
Abstract
Background and Aim: The diagnosis of bovine brucellosis in animals vaccinated with strain-19 (S19) and Rose Bengal
(RB)-51 strain vaccines can be misinterpreted due to false positives. This study aimed to compare diagnostic tests for
detecting bovine brucellosis in animals vaccinated with S19 and RB51 vaccine strains.
Materials and Methods: Two groups of 12 crossbred Holstein calves between 6 and 8 months of age were used. On day
0, blood samples were collected from the animals, and the competitive enzyme-linked immunosorbent assay was used
for serological diagnosis of bovine Brucellosis. All animals tested negative. After the first blood collection, the animals
were subcutaneously vaccinated: one group received the S19 vaccine and the other received the RB51 vaccine. From the
3rd month after vaccination, all animals were sampled. Sampling was repeated every 2 months until the 7th month. Serological
diagnosis of bovine brucellosis was performed using RB, tube serum agglutination test (SAT), SAT with 2-mercaptoethanol
(SAT-2Me), and fluorescence polarization assay (FPA).
Results: Animals vaccinated with S19 showed positive results with the RB, SAT, and SAT-2Me tests in all months of post-
vaccination diagnosis. In animals vaccinated with S19, FPA showed positive results at months 3 and 5 and negative results
at month 7, indicating that this test discriminates vaccinated animals from infected animals 7 months after vaccination. Rose
Bengal, SAT, SAT-2Me, and FPA tests showed negative results in animals vaccinated with RB51 in all months of diagnosis.
Conclusion: Animals vaccinated with S19 may test positive for brucellosis using RB, SAT, or SAT-2Me tests 7 months later.
Fluorescence polarization assay is an optimal alternative for diagnosing animals in the field, thereby preventing false positives, and
consequently, unnecessary confiscations of animals. Animals vaccinated with RB51 tested negative with RB, SAT, SAT-2Me, and
FPA tests in all months of diagnosis, confirming that the tests are ineffective for diagnosing brucellosis caused by rough strains.
Keywords: agglutination, bovine, brucellosis, vaccination.
Introduction
Bovine brucellosis is a contagious disease
caused by Brucella spp. (phylum: α-2 Proteobacteria),
with worldwide distribution, and reproductive condi-
tions that affect both males and females [1]. Brucella
causes productive and reproductive loss in livestock.
Notably, in dairies, Brucella causes abortion, accom-
panied by retained placenta, birth of weak calves, low
milk production in females [2, 3], and epididymitis
and orchitis in males [4].
Bovine brucellosis has been eradicated from
many parts of the world, especially North America and
Western Europe, but remains endemic to certain areas,
particularly in Asia, Africa, and Latin America [5],
where key control strategies include mass vaccination of
animals at risk, with serological diagnosis. The Brucella
abortus strain-19 (S19) vaccine was developed in 1923
from a natural attenuation [6] and has been used for
~50 years. However, it presents drawbacks such as the
inference in conventional diagnostic tests, the non-pos-
sibility of vaccination of adult animals, and the risks to
the veterinarians [1]. A mutant strain of B. abortus strain
2308 Rose Bengal (RB)-51 was isolated in 1982 from
B. abortus biovar 1 [7], to generate a vaccine that can
be used for bovines of all ages and does not infer in the
conventional serological diagnosis [8]. Several studies
have shown that the S19 and RB51 vaccines provide
65%–75% protection against infection [7–9].
Copyright: Ibarra, et al. Open Access. This article is distributed under
the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons
license, and indicate if changes were made. The Creative Commons
Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
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The World Organization for Animal Health
(WOAH) recommends using multiple serological
tests for diagnosing brucellosis to overcome indi-
vidual limitations of each test. Performing multiple
serological tests increases sensitivity and reduces per-
centage of false negatives. Due to these drawbacks,
population screening in control programs requires
the use of combined serological tests [10]. Although
conventional serological tests are key in bovine bru-
cellosis control and eradication programs, they cannot
discriminate between naturally infected and S19-
vaccinated animals, because they detect antibodies
produced against the O chain of lipopolysaccharide
(LPS-O) on the membrane of Brucella spp., which is
present in both field strains and vaccines [11]. This
has led to the development of several diagnostic tests
for bovine brucellosis, including agglutination, enzy-
matic, and cellular immunity tests, but none is consid-
ered a gold standard. The characteristics of tests used
in eradication plans worldwide are being investigated,
because they generate false-positive and false-neg-
ative outcomes [12]. The most common screening
tests for diagnosing brucellosis include: The RB test,
which is a qualitative agglutination test that can be
rapidly observed; the tube serum agglutination test
(SAT); and SAT with 2-mercaptoethanol (SAT-2Me).
Rapid tests specifically detect antibodies against
Brucella spp. LPS [13]. Competitive enzyme-linked
immunosorbent assay (cELISA) is used as a confir-
matory serological test because of its high specific-
ity to distinguish antibodies produced in response to
a vaccine or natural infection [14]. This test uses the
LPS-O-specific monoclonal antibody M-84, and the
antigen–antibody reaction is detected that is quanti-
fied through enzymatic meters [13]. However, due
to its high specificity, the fluorescence polarization
assay (FPA) can be considered a confirmatory test for
bovine brucellosis [15]. Although available B. abor-
tus vaccines are effective against brucellosis, they
have a number of drawbacks, including interference
with diagnostic tests, pathogenicity to humans, and
potential to cause abortions in expectant animals. Due
to the presence of LPS in the S19 vaccine, vaccina-
tion of animals with this strain induces an immune
response against LPS-O that is strikingly similar to
that induced by natural infection. Therefore, distin-
guishing between vaccinated and infected animals is
impossible [16]. In contrast, vaccination with B. abor-
tus strain RB51 did not induce antibodies detected by
the conventional assays used to diagnose brucellosis.
In addition, there is no commercially available test
to detect RB51- or S19-vaccinated animals, which
would be beneficial for evaluating vaccination pro-
grams [17]. Bovine brucellosis is endemic to Ecuador,
which has a bovine brucellosis control and eradica-
tion plan based on epidemiological surveillance, sero-
logical diagnosis, slaughter of seropositive animals,
vaccination, and training. Vaccination is one of the
fundamental pillars of disease eradication; however,
it is performed without the care required by govern-
ment entities. Vaccine strains S19 and RB51 are used
without control. In certain cases, the two strains have
been used in the same animal, without an adequate
cold chain and sanitary records [10]. Therefore, the
diagnostic tests used in Ecuador cannot identify bru-
cellosis-free farms due to interference from false-pos-
itive and negative results [18–20].
This study aimed to compare diagnostic tests for
detecting bovine brucellosis in animals vaccinated
with S19 and RB51 vaccine strains.
Materials and Methods
Ethical approval
All procedures conducted in the present study
were approved by the Committee on the Care and Use
of Laboratory and Domestic Animal resources of the
Agency of Regulation and Control of Phytosanitary
and Animal Health of Ecuador (AGROCALIDAD),
under the approval serial number #INT/DA/019.
Study period and location
The study was conducted from February to
November 2022 at a farm in San Vicente, El Carmelo
parish, Tulcan Canton, Carchi Province, Ecuador.
Experimental design
Two groups of total 12 (six in each group) cross-
bred Holstein calves between 6 and 8 months of age
were used. The animals were kept in paddocks with
forage and water ad libitum. Blood samples were
taken from the coccygeal vein in sterile tubes with-
out anticoagulant (BD, New York, United States), to
obtain serum for the serological diagnosis of Brucella
spp., before and after vaccination. Serological diagno-
sis (day 0) was made using the cELISA test (Svanova
by Indical Bioscience, Uppsala, Sweden), which is
considered a confirmatory test in Ecuador for the diag-
nosis of bovine brucellosis, according to resolution
No. 025 Art. 8 (AGROCALIDAD, Regulation and
Control Agency for Plant and Animal Health. On day
1, six animals each were vaccinated subcutaneously
with a single dose of S19 (5–8 × 1010 colony-forming
unit [CFU]) and RB51 (1.6 × 1010 CFU) [10].
In the 3rd month after vaccination, blood samples
(10 mL) were taken to obtain serum from all animals.
This procedure was repeated every 2 months until the
7th month. The samples were serologically tested for
antibodies against Brucella spp. using RB, SAT, SAT-
2Me, and FPA, at the veterinary diagnostic laboratory
of the State Polytechnic University of Carchi.
Serological tests
Competitive enzyme-linked immunosorbent
assay was performed in duplicate using Svanovir
Brucella-Ab C-ELISA (Svanova Biotech AB). Percent
inhibition was calculated using the following formula:
Subtracting 100 for the division of the average
optical densities (OD) of the samples with the OD of
the conjugate cutoff ≥30% inhibition was considered
positive and <30% inhibition was considered negative.
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Rose Bengal
The RB test was performed according to the pro-
tocol established by the OIE. Rose Bengal Brucellosis
Antigen (Idexx, Hoofddorp, The Netherlands), which
is a bacterial suspension of Brucella stained with RB,
was used as the antigen. The antigen and sera were
placed at room temperature (23°C) for 60 min before
use. Next, 30 µL of serum was placed on a glass plate
and mixed with 30 µL of the antigen. The plate was
homogenized for 4 min at 23°C. The presence or
absence of agglutination was considered positive or
negative, respectively (Figure-1).
Serum agglutination test and SAT-2Me
Serum agglutination test and SAT-2Me used
a 4.5% suspension of B. abortus 1119-3 as antigen,
as well as 0.5% phenolated saline and 0.1 M 2Me as
diluents, respectively. The protocol followed was pro-
posed in 2009 by the WOAH [21]. Considerations for
the interpretation of results include: Complete agglu-
tination is the degree of agglutination in each test,
where the liquid of the serum-antigen mixture appears
transparent and translucent and gentle agitation does
not disperse the aggregates. For incomplete aggluti-
nation, the serum-antigen mixture is partially cloudy
and moderate shaking fails to disperse the clumps.
Negative agglutination occurs when the serum-antigen
mixture appears cloudy and moderate shaking does
not reveal lumps. Reading and interpreting results of
the slow agglutination test in a tube in the presence
of “SAT-2Me” must be performed according to the
same criteria as the slow agglutination test in a tube
(“SAT”) (Figure-2).
Fluorescence polarization assay
Fluorescence polarization assay was performed
according to the specifications of Brucella Antibody
Test Kit FPA (EllieLab, Milwaukee, United States). The
FPA kit uses fluorescein-conjugated O-polysaccharide
from B. abortus. The sera and controls (20 µL) were
placed inside borosilicate tubes with the diluent pro-
vided by the kit (1 mL) and incubated for 3 min at
23°C , to make blank reading of all the samples and
controls. Then, 10 µL of the antigen was incubated
with fluorescein for 3 min at 23°C. Milli-polarization
(mP) values of all samples and controls were obtained.
Cutoff ≥ 89.9 mP indicated a positive result [10].
Statistical analysis
The results were analyzed using descriptive sta-
tistics in R program 4.3.1 version (R Foundation for
Statistical Computing, Vienna, Austria).
Results
Serological diagnosis revealed that on day 0, the
animals did not present antibodies against Brucella
spp. The animals vaccinated with S19 showed the
presence of antibodies (positive diagnosis) for
Brucella spp. using SAT in all collection periods
(months 3, 5, and 7). Rose Bengal and SAT-2Me pre-
sented a positive diagnosis in months 3 and 5 in all
animals; however, in month 7, positive results were
observed in five animals and negative results in one
animal. In months 3 and 5, FPA revealed a positive
diagnosis in all animals, but by month 7, all animals
had a negative diagnosis (Table-1). The animals that
received the RB51 vaccine did not exhibit any pos-
itive results during any of the sampling periods or
diagnostic procedures.
Discussion
Our study demonstrates that unlike the screen-
ing tests (RB, SAT, and SAT-2Me) used for the sero-
logical detection of Brucella spp., FPA has great
potential for detecting animals that are not infected
with Brucella spp., because it can distinguish
between antibodies produced in infected and vacci-
nated animals. This is due to the test’s principle, in
which all the molecules in solution rotate randomly.
The size of the molecules determines the rotation
range, which refers to the formation of immune com-
plexes between antigen and immunoglobulin (Ig)
G-type antibodies. In periods where the concentra-
tion of IgM begins to decrease, negative results are
observed in animals experimentally vaccinated with
S19 at 7 month post-vaccination. In contrast, screen-
ing tests, being general agglutination tests, continue
to identify antibodies generated in response to vac-
cines several months post-vaccination [22–24].
Figure-2: Sero Agglutination Tube with two Mercaptoethanol
test – Positive (left); Negative (right).
Figure-1: Rose Bengal test-Positive (left); Negative
(right).
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Moreover, SAT is a highly sensitive test that
allows detection of IgM-type antibodies, which are the
first to appear after an infection. However, SAT pres-
ents problems of low specificity due to non-specific
antigen–antibody reactions. Serum agglutination test
variants (SAT-Rivanol, SAT-EDTA, and SAT-2Me) use
acidified antigens, allowing for the formation of immu-
nocomplexes between antigens and IgG-type antibod-
ies. Similarly, the RB test (at pH 3.65) allows detection
of IgG-type antibodies, reducing non-specific binding
associated with IgM-type antibodies [15].
Similar results have been obtained with RB,
SAT, and SAT-2Me because they use the same prin-
ciple of agglutination on slides and the antigen uses a
suspension of B. abortus [25]. According to Aparicio
Bahena et al. [26], routine or screening tests, such as
RB, SAT, buffered plate antigen, and milk ring test,
have high diagnostic sensitivity; however, their spec-
ificity is low when differentiating vaccinated from
infected animals [26].
Positive results from RB, SAT, and SAT-2Me
tests can be considered false positives due to the low
specificity of these tests, which is caused by cross-re-
actions with other LPS-O-containing bacteria. Similar
results were described by Nielsen et al. [27], where
the bacteria causing this cross-reaction included
Escherichia hermanni and Escherichia coli O157,
as well as Salmonella O:30 and Stenotrophomonas
maltophilia. According to Ron-Román et al. [25],
vaccination of cattle with S19 produces agglutinating
antibodies that interfere with diagnostic tests based on
the principle of agglutination.
In animals vaccinated with S19, FPA presented
positive results for up to 5 months of sampling.
This diagnosis should be considered a false-positive
because it is attributable to cross-reactions due to
S19 vaccination, where after vaccination with strains
with epitopes similar to the causal agent, very high
levels of antibodies are produced, especially IgM
and IgG [28].
Because FPA has high specificity in animals vac-
cinated with S19 [11, 21, 29], it is possible to observe
negative results for FPA beginning in the 7th month,
where the specificity of FPA, and consequently,
its ability to distinguish vaccinated from naturally
infected animals are 98.60% and 99.80%, respec-
tively, as long as the estimated duration is 6–7-month
post-vaccination at the time of the test.
Animals vaccinated with the RB51 strain pre-
sented negative results in all the diagnostic tests under
study (RB, SAT, SAT-2Me, and FPA) and in all the
sampling months (Table-1).
The negative results of the RB, SAT, SAT-2Me,
and FPA diagnostic tests are attributed to the use of sus-
pensions of B. abortus as antigen, which is a smooth
bacterium, due to the presence of LPS-O, Vargas [30],
whereas the vaccine applied in this group of animals,
which was RB51, is a rough mutant strain lacking the
side chain “O” of the LPS, which induces the production
of other types of antibodies, in which the antigen–anti-
body ratio of the diagnostic tests under study is null, as
mentioned by Schurig et al. [12] and Cheville et al. [31].
Two types of commercial vaccines are available
worldwide for bovine brucellosis: S19 and RB51,
which are live vaccines, with similar degrees of
immunity; however, they sometimes trigger the pro-
duction of agglutinating antibodies that interfere with
all serological diagnostic tests [32, 33]. In the case of
the RB, SAT, and SAT-2Me diagnostic tests performed
on animals vaccinated with S19, positive results were
obtained up to the 7th month of sampling, whereas for
FPA, negative results were seen at 7th month of diag-
nosis. Thus, FPA can differentiate vaccinated animals,
after the highest peak of vaccine immunity passes in
6- and 7-month post-vaccination. A comparison of the
diagnostic capacity of FPA with cELISA (test recog-
nized by AGROCALIDAD) revealed a high diagnos-
tic correlation [9].
In animals vaccinated with RB51, the RB, SAT,
SAT-2Me, and FPA tests showed negative results in
Table-1: Determination of antibodies to Brucella spp. by screening agglutination tests.
Vaccine Animal Detection of antibodies for Brucella spp. at different post-vaccination times
Month 3 Month 5 Month 7
RB SAT SAT-2Me FPA RB SAT SAT-2Me FPA RB SAT SAT-2Me FPA
S19 1 + + + + + + + + + + + -
2 + + + + + + + + + + + -
3 + + + + + + + + - + - -
4 + + + + + + + + + + + -
5 + + + + + + + + + + + -
6 + + + + + + + + + + + -
RB51 7 - - - - - - - - - - - -
8 - - - - - - - - - - - -
9 - - - - - - - - - - - -
10 - - - - - - - - - - - -
11 - - - - - - - - - - - -
12 - - - - - - - - - - - -
RB=Rose Bengal test, SAT=Sero agglutination tube, SAT-2Me=Sero agglutination tube with 2 Mercaptoethanol,
FPA=Fluorescence polarization assay
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all months of diagnosis, proving that the antibodies
induced by RB51 cannot be detected (there is no anti-
gen–antibody interaction) by the diagnostic screening
tests for bovine brucellosis [34].
Thus, in cattle herds where the RB51 vaccine
strain is used correctly, the use of diagnostic screening
tests, such as RB, could be considered unequivocally.
In addition, because the RB51 strain is a rough strain,
its immunological efficiency cannot be determined
with traditional diagnostic tests, because these tests
use antigens obtained from smooth strains. Therefore,
antigens from rough strains must be used to evaluate
the immunological efficiency of RB51.
Conclusion
In this study, FPA was shown to be a useful tool
for the rapid and effective detection of antibodies
against Brucella spp. in cattle, contributing signifi-
cantly to the differentiation between animals that are
infected and those that appear healthy or are vacci-
nated (7 months after vaccination). However, FPA is
not useful for detecting antibodies produced by rough
strains. Therefore, FPA is the most recommended test
in Ecuador and other countries for detecting animals
infected with Brucella spp.
Authors’ Contributions
MI and MC: Sample and data collection. MI,
MC, BH, AL, AC, and LN: Study design, drafted the
manuscript, laboratory work, and data analysis. All
authors have read, reviewed, and approved the final
manuscript.
Acknowledgments
The authors are thankful to Universidad
Politecnica Estatal del Carchi, for the technical and
funding support – Grant: Bases para el mejoramiento
de la competitividad de la cadena de valor lácteo de
la provincia del Carchi and also to Universidad de
Las Americas, Quito, Ecuador, for the payment of the
copyediting.
Competing Interests
The authors declare that they have no competing
interests.
Publisher’s Note
Veterinary World remains neutral with regard
to jurisdictional claims in published institutional
affiliation.
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