African Journal of Microbiology Research Vol. 6(13), pp. 3242-3247, 9 April, 2012
Available online at http://www.academicjournals.org/AJMR
ISSN 1996-0808 ©2012 Academic Journals
Full Length Research Paper
First notification on the presence of brucellosis in water
buffalo (Bubalus bubalis) in Mexico by serological tests
Rafael Suazo-Cortez, Dora Romero-Salas, José Alfredo Villagómez-Cortés* and David Itzcoatl
Faculty of Veterinary Medicine, Universidad Veracruzana. Ring and Yañez, Col. Veracruzana Unit. 91710 Veracruz,
Accepted 10 January, 2012
A cross-sectional study was conducted to determine the seroprevalence of brucellosis in water
buffaloes (Bubalus bubalis) of three farms located in the south of Veracruz, México. Card test and
rivanol were used in serial for detection of antibodies against Brucella spp. From a total of 565
buffaloes, 99 were tested and the overall seroprevalence of brucellosis was 13% by card test and 7% by
rivanol. By farm, seroprevalence was 2.94% for farm 1. 4% for farm 2, and 12.5% for farm 3. Brucellosis
seroprevalence showed an increasing trend with age, with adult cattle (>6 years) recording the highest
seroprevalence (11.1%), but differences were not statistically significant (p>0.05). In two farms,
buffaloes shared grazing land and water sources with bovine cattle, so 75 head were tested for
brucellosis resulting negative. Because clinical signs suggestive of brucellosis were not observed,
isolation was not attempted. This is the first known report on the presence of brucellosis in water
buffalo in Mexico; thus, public awareness and further epidemiological studies of the disease in wildlife,
livestock, and humans in the study area are of great importance.
Key words: Brucellosis, domestic water buffalo, epidemiology, Mexico.
Brucellosis, a zoonosis of worldwide importance, is
caused by Gram-negative bacteria of the genus Brucella
(Moreno et al., 2002; López and Contreras, 2004; Cutler
et al., 2005). Brucellosis in domestic water buffalo
(Bubalus bubalis) is generally caused by infection with
Brucella abortus (Mohan, 1968; Godfroid, 2002).
Brucellosis is primarily a reproductive disease of cattle,
characterized by late-term abortions, retained placentas,
epididymitis, and orchitis (Nicoletti, 2001). Clinical signs
in buffaloes include abortion, decreased fertility and milk
production, and testicular degeneration in the bull, as a
result of epididymitis - orchitis (Acha and Szyfres, 2003);
because of this, it is considered one of the most
damaging diseases to livestock and disturbing rates of
productivity in buffalo herds (Borriello et al., 2006;
*Corresponding author. E-mail: firstname.lastname@example.org. Tel: 52-
229-9342075, Fax: 52-229-9344053.
Martínez et al., 2006), since prevention and control
involves a high economic impact.
Water buffalo is well adapted to tropical and subtropical
regions, particularly to flooded areas where bovine cattle
thrive with difficulty, while buffalo uses efficiently pasture
resources (Bhat, 1992; Mahadevan, 1992; Borghese and
Mazzi, 2005; Borghese, 2006). The introduction of
buffaloes to tropical states of Mexico like Veracruz and
Tabasco is a relatively recent phenomenon and there is
still a lot unknown about this species. In some farms, the
interaction of water buffalo with other domestic ruminant
species leads to the possibility of cross- species
infections (Bengis et al., 2002).
Brucellosis in the water buffalo generally is caused by
B. abortus, however, since little of its epidemiology has
been studied; it is unclear how the species transmission
may occur (Fosgate et al., 2011). In Mexico, health status
in regard to brucellosis in water buffalo remains unknown.
Therefore, it is necessary to conduct proper studies,
which should start with determining the presence of
Mexico uses the card test as the official screening
procedure for brucellosis,
confirmation (SAGDR, 1996). These tests have been
studied in cattle (Alton et al., 1988; Abdoel et al., 2008)
and subsequently evaluated for use in water buffalo and
other species (Nicoletti, 1992; Fosgate et al., 2002, 2003;
Godfroid et al., 2010). Card test is fast, easy to perform
and allows processing a large numbers of samples per
day (Aricapa, 2006). This test allows to classify animals
as positive or negative, and then perform rivanol test to
positive samples (Acha and Zyfres, 2003), which
functions as complementary and confirmatory, as it
allows to differentiate infected animals from those who
have been vaccinated (OIE, 2004).
As far as the authors are aware, no investigation on the
presence of brucellosis in water buffalo in Mexico has
hitherto been attempted. This study was carried out
therefore to investigate the presence of brucellosis in
water buffalo (B. bubalis) in three farms located in the
south of the state of Veracruz, Mexico.
MATERIALS AND METHODS
Study site and animal population
The study was conducted in the contiguous municipalities of Isla
and Juan Rodríguez Clara, located at the south of the state of
Veracruz, Mexico. Climate is hot humid and annual rainfall range
from 1200 to 2300 mm. Three buffalo’s commercial farms were
identified in the area, showed willingness to participate in the study,
and were surveyed for brucellosis (Table 1).
Sample collection techniques
Population in the three farms was 565 head. At each farm,
systematic random sampling (that is, 1/6 animals interval) was used
to select individual animals. In order to minimize possible false
positive reactors due to maternal antibodies in younger animals,
only those older than six months were sampled. Blood samples for
the detection of antibodies against Brucella spp. were collected
from all sampled animals. The survey covered the period from May
to June 2011 and a total of 99 serum samples were collected in the
three farms from animals older than six months. Information about
each animal such as sex and age was collected and entered into a
data sheet. A second data sheet containing information from each
farm was also constructed.
Testing for brucellosis
Antibodies to Brucella spp. were detected by using the card test as
a screening test, and the rivanol test as a confirmatory test,
according to the official Mexican regulations (SAGDR, 1996). Card
test was performed on plates where 25 μl of the serum was mixed
with equal amounts of a stained, buffered, whole cell suspension of
B. abortus strain 1119-3 antigen (pH 3.6, concentration of 8%). The
samples were mixed on a rocker for 5 min. Depending on the
presence of agglutination; the test is interpreted as either negative
or positive without a "suspicious" category. Rivanol test was
performed by mixing 0.5 ml of serum with 0.5ml of a 1% solution of
rivanol. The tube was then allowed to stand for at least five minutes
with rivanol test for
Suazo-Cortez et al. 3243
for allowing the pack to precipitate. The tube was centrifuged and
the supernatant tested by pipetting 0.08, 0.04, 0.02 and 0.01 ml
onto a glass plate and mixing with 0.03 ml of rivanol plate test
antigen, consisting of inactivated B. abortus strain 1119-3 stained
with brilliant green and crystal violet (pH 5.8 to 6.2, concentration of
4%). The mixtures were incubated for 12 min and sera from non
vaccinated animals showing a complete agglutination reaction at
any dilutions from 1:25 to 1:400 were considered positive.
The overall number of seropositive animals was calculated from the
total number of samples tested over the study period, expressed as
a percentage. Seropositive animals were examined in relation to
age, sex, and farm. Vassarstats software was used to analyze data
to evaluate differences in seroprevalence and to calculate
confidence intervals. Chi-square test was used to measure
differences between categories. Values of p<0.05 were considered
RESULTS AND DISCUSSION
Seropositivity by card test
For the test card, the overall seropositivity was 13%. All
three farms had animals testing positive, but with varying
proportions, as shown in Table 2. According to the farms’
veterinarians, none of the cattle from the studied areas
had been vaccinated against brucellosis, implying that
the antibodies detected were more likely to be due to
natural infection with Brucella spp. rather than by B.
abortus S19. Furthermore, the rivanol test used for
confirmation of seropositive animals has been reported to
differentiate vaccinal antibodies from those of natural
infection (Nielsen and Yu, 2010). Since Yersinia
enterocolitica is assumed to be rare or absent in the
tropics, cross-reactions with B. abortus were unlikely to
have an impact on the results (Nielsen et al., 2004).
Moreover, card test for screening has a high sensitivity
(>90%), thus reducing the possibility of false negative
reactors (OIE, 2004). Hence, it is unlikely that the tested
animals in the present study were wrongly classified as
false negative or false positive due to cross-reactions
with other Brucella spp.
Overall seropositivity by card test was 13% in this
study, which is close to the 12% reported in the
municipality of Lorica, Department of Córdoba, Colombia
by card test, ELISA (Calderón et al., 2010), but higher
than the 2% observed in four farms in the Departments of
Antioquia and Cordoba, Colombia (Aricapa, 2006), and
closer to the 9.38% obtained in Pakistan (Hussain et al.,
2008), and the 9.59% reported in Gujarat, India
(Ghodasara et al., 2010), all by the test card. In turn,
Nowroozi et al. (2007) in Khoozestan province, Iran
determined in 400 buffaloes a seroprevalence of 20.5 %
by card test, 19.5% by agglutination, and 11 % by 2-
mercaptoethanol. In Egypt, in a study that also included
bovine, sheep, and goats, Samaha et al. (2008) reported
a seroprevalence in buffaloes of 3.52% by card test, 3.44%
3244 Afr. J. Microbiol. Res.
Table 1. Characteristics of buffalo’s commercial farms enrolled in brucellosis study. Veracruz, Mexico. 2011.
Item Farm 1
Juan Rodriguez Clara
Juan Rodriguez Clara
700 300 500
Gentle hills and lowlands with ponds
and a stream
Sloppy hills, but there are
some ponds and a stream.
435 head, Murrah breed is
dominant, but there is also a
small number of animals of
Mediterranean and Carabao
breeds. The initial herd came
from the nearby municipality of
Las Choapas in 2006. Most
animals were born in farm, but
bulls came from other farms.
Animals graze under pasture
rotation but are not
Records and animal inventory
are kept, but not all animals are
identified. Because some
diarrhea cases were observed
in calves, now internal and
external parasites control is
routine. Scheduled vaccination
is only carried out against
brucellosis. Only if an animal is
sick the veterinary is called for
care. Some abortion and
difficulties for pregnancy had
been observed in the herd.
Terrain Mainly low hills with some small ponds.
60 head Murrah breed. The initial
herd came from another farm in the
same municipality, but still some 75
% of the animals were not born in this
70 head, dominated by the Murrah
breed. The Initial herd came from the
neighboring municipality of Acayucan.
Herd is mostly composed by females
three to five year-old.
Extensive management conditions. No
further protein or mineral
supplementation is done.
Continuous grazing. Animals are kept
year-round in a restricted area
Minimum. Neither deworming nor
vaccination is performed. Animals are
unidentified and regular records are
not kept. Except for the presence of
sick animals, the herd remains
unchecked. Every four months
females are inspected by rectal
palpation for pregnancy diagnosis.
Calf delivery is unattended so
personnel are unaware of abortions.
A bull that came with the initial herd is
still used for reproduction.
Most animals remain unidentified and
records are not regularly kept. A bull that
arrived with the initial herd is still used
for natural mating. Herd monitoring is
occasional, veterinary services are only
required if an animal is sick. Females
are not followed up by rectal palpation;
hence the situation on pregnancies or
abortions is unknown. Animals are not
dewormed or vaccinated against
brucellosis or any other disease.
During a short period every year, a
600 bovine herd shares common
grazing and watering sources with
Pasture grazing is shared with a 150
head dual purpose bovine herd, but
unlike the buffalo, bovine herd is rotated
through the paddocks.
by agglutination,and 3.37% by rivanol.
Seropositivity by rivanol test
Herd seroprevalence obtained by rivanol test was 100%,
because in the three herds at least one seropositive
animal was found, however, within each herd seropre-
valence is different, so it was 12.5% in herd 3.4% in herd
2, and 2.94% in herd 1. Overall seropositivity by rivanol
test was 7% in this study. One out of four water buffalo
farms tested in the province of Corrientes, Argentina by
2-mercaptoethanol test was affected by brucellosis with a
seroprevalence of 30%; however, the overall seropre-
valence for the animals was 4.8% (Martínez et al., 2006).
Hussain et al. (2008) in Pakistan found a seroprevalence
in buffaloes of 9.38% by card test, and 6.9% by ELISA;
for bovine cattle, the seroprevalence was 0.1 and 8%,
and for humans, 14 and 11%, respectively. Another study
in Baluchistan, Pakistan, found that the seroprevalence of
brucellosis in buffaloes was 1.7% milk ring test and 0%
by indirect ELISA, whereas in bovine cattle, the
seroprevalence was 4.6 and 20%, respectively (Shafee et
al., 2011). Ghodasara et al. (2010) in Gujarat, India
sampled 73 buffaloes and found a brucellosis
seroprevalence of 9.59% by card test, 12.33% by
agglutination, and 14.45% by indirect ELISA.
Seroprevalence by age
Age-specific seroprevalence through rivanol confirmatory
Suazo-Cortez et al. 3245
Table 2. Seropositivity (%) to Brucella abortus by card test and rivanol in water buffalo on farms in Veracruz, Mexico.
Farm Total sampled Card test positives, No (%) C. I. 95% Rivanol test positives, No (%) C. I. 95%
2 – 24
7 – 41
4 – 27
7 – 21
0 - 17
0 - 22
4 - 27
3 - 14 Total
Table 3. Seroprevalence (%) of antibodies to Brucella abortus by age in water buffalo in Veracuz, Mexico.
Age (year) Positive No. (%)
0.5 1 (11)
2 1 (2.63)
3 4 (11.43)
5 1 (50)
Total 7 (7)
0 - 49
0 - 15
3 - 27
2 - 97
3 - 14
test was found ranging from 0%, in one and four year-old
animals, to 50% in those of five year-old (Table 3). There
are some reports claiming that older animals had
increased chances of testing Brucella positive (Muma et
al., 2007; Matope et al., 2010), what seems logic
because as an animal ages its chances of contact with an
infectious agent may increase. Also, the onset of sexual
maturity is associated with a significant increase in the
risk of infection with Brucella spp. and such animals are
likely to seroconvert. Even though age is not clearly
precised, Nowroozi et al. (2007) in Khoozestan province,
Iran found a variation in seroprevalence according to age
group and sex; in females, the seroprevalence was
12.9% for adults, 10.7% in subadult, and 3% for the
youngest animals; however, the seoprevalence in males
was 15% in adults, 10.6% in subadults and 5.3% in
In this study, because of the small number of males,
animals were not analyzed by sex, but Kubuafor et al.
(2000) state that sex and brucellosis risk association can
vary with different cattle populations.
Prevalence in bovine herds
Because two out of three herds have bovine cattle
sharing the habitat with water buffaloes, it was decided to
determine their health status in relation to brucellosis.
Forty head were sampled in herd 1 and 35 in herd 2 to
research herd status, but all animals were negative to
Brucella abortus. Two recent studies in the region agree
with this finding. Martínez (2008) found a seroprevalence
of 0% for bovine cattle from the municipalities of Juan
Rodríguez Clara, Tierra Blanca, and Tres Valles. Torres
(2010), in the neighboring municipalities of Minatitlán,
Mecayapan, and Agua Dulce reports a seroprevalence of
0.3% in bovine cattle. Finding buffalo herds positive
whereas bovine herds remain negative seems to
contradict Adesiyun et al. (2011) suggestion of water
buffalo being more resistant to infection than bovine
The epidemiology of Brucella infection has not been
studied extensively in domestic water buffalo, but
differences between the epidemiology of brucellosis in
water buffalo and bovine may complicate control
(Borriello et al., 2006; Fosgate et al., 2011). Transmission
of B. abortus is mainly by direct and mucosal contact with
fluids or tissues associated with the birth or abortion of
infected fetuses (World Health Organization, 2006).
Probably the most important spread of brucellosis takes
place from animal to animal, when those infected
contaminate the pasture and uninfected animals become
infected by ingestion when grazing. Bovine cattle sharing
grazing pastures and watering points with buffaloes are
likely to facilitate transmission of the disease in both
directions (Nicoletti, 1980; Bengis et al., 2002; Muma et
al., 2007). Fosgate et al. (2011) demonstrated that the
ingestion of B. abortus causes infection in buffaloes and
their natural behavior of commingling in a small area
facilitates disease transmission, and congregation of
water buffalo in wallows may be an important factor for
spread of brucellosis. In our study, two farms have
buffaloes sharing ponds and pasture land with bovines,
but not evidence of infection in the latter was found,
probably due to the fact that the final prevalence rate is
determined by the intensity of contacts within and
3246 Afr. J. Microbiol. Res.
between herds and with infected pasture and water, or
because bovine herds are subjected to a vaccination
program for brucellosis.
The spread of Brucella spp. from one herd and one
area to another is often due to the movement of an
infected animal into a non-infected susceptible herd
(Crawford et al., 1990). The purchase of unknown
Brucella-status animals for the purpose of restocking
herds can be suspected as the source of spread of
brucellosis into the herds. In our study that may happen
even though herds are closed if some of the animals in
the starter herd were infected. A number of other factors
may be associated with the outcome of infection in cattle
such as age, reproductive and immunological status,
natural resistance, route of infection, infectious challenge,
and virulence of the strain (Borriello et al., 2006; Carvalho
Neta et al., 2010).
Bovine brucellosis control programs have effectively
reduced and eliminated the prevalence of diseases in
livestock, but spillover of the disease from domestic
livestock to wildlife has complicated regulatory efforts. It
is difficult to eradicate brucellosis in cattle without
resolution of the disease in wildlife (Olsen and Tatum,
The milk produced by infected females is the most
important source of spread of Brucella spp. for animals to
man (Corbel, 2006). Infected animals shed viable
brucellae in milk, but dam-to-calf transmission has not
been evaluated directly. However, buffalo calves born to
seropositive dams on an infected farm are more likely to
become seropositive themselves compared with calves
born to seronegative dams (Akhtar and Mirza, 1995).
Even though in our study buffaloes are not milked and
contact with personnel is minimal, the results of the
present study established the presence of brucellosis in
buffaloes and hence as a potential threat to public health
as brucellosis could be a serious zoonosis (Lucero et al.,
2008; Selem et al., 2010). Although no human brucellosis
information was available when this study was
conducted, we strongly suggest further studies to
investigate the impact of this zoonosis on human
populations. Follow-up studies will be necessary to
confirm the possible presence of brucellosis in these
The source or origin of brucellosis in the present study
area could not be accurately ascertained as there have
been no previous studies on the disease in the area.
Also, because no sick animals were observed during the
study, isolation of Brucella sp. was not attempted. It is
suggested to monitor the reproductive performance of
buffaloes, particularly older females, in order to identify
reproduction abnormalities suggestive of the presence of
brucellosis. Infected water buffalo expel the bacterium
during abortion, and this may serve as a source of
infection for herd mates. Experimental studies have
demonstrated that ingestion of virulent B. abortus causes
infection in female water buffalo (Mohan, 1968). Also,
although buffalo are not included within the Mexican law on
brucellosis (SAGDR, 1996), it is suggested to start
monitoring buffalo herds in the country by the
implementation of approved serological tests to identify
and remove reactors preventing the spread of disease
inside and outside their herds. Finally, although there is
no history of brucellosis vaccine application in the water
buffalo in Mexico, other nations have chosen to follow the
same vaccination protocol performed in cattle with a
favorable outcome, based on this argument it would be
possible to apply the vaccination schedule used in cattle
based on the official regulation (SAGDR, 1996), with the
use of strain 19 classical vaccine doses to prevent
disease in females three to six month-old (Afzal et al.,
2000), and reduce dose strain 19 vaccine for those over
six month-old, even in pregnant animals. Apparently, the
RB51 vaccine does not adequately protect against
brucellosis infection in water buffalo (Fosgate et al.,
Brucellosis was present in three water buffalo herds in
the state of Veracruz, Mexico, but apparently not in the
bovine herds, as determined by card test and rivanol test.
Brucellosis in the sampled animals had its higher
prevalence in individuals between three and five year-old.
The source of brucellosis in buffaloes in the study area
could not be accurately ascertained. Future studies
should be directed to determining factors affecting
susceptibility to brucellosis among different domestic
animal species including water buffalos.
This study is part of the Project “Emerging and re-
emerging animal diseases in water buffalo (B. bubalis) in
Veracruz, México” sponsored by the Animal Population
Health Institute, College of Veterinary Medicine &
Biomedical Sciences. Colorado State University and the
Caesar Kleberg Wildlife Research Institute. Texas A and
M University-Kingsville. Thanks to Dr. Maximino
Diagnostic Laboratory for proving official confirmation for
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