Sero-Prevalence of Brucellosis in Food Animals in the Punjab, Pakistan

Abstract

Aim of the present study was to know the prevalence of brucellosis in food animals in relation to various risk factors through different diagnostic tests. For this purpose, 2375 serum samples were collected from December, 2010 to December, 2012. Rose Bengal Plate Test (RBPT) and Serum Agglutination Test (SAT) were applied for initial screening, positive samples were subjected to enzyme linked immunosorbant assays (i-ELISA and c-ELISA) for confirmation. The data thus collected was interpreted and subjected to Binary Logistic Regression Analysis to know the difference among various groups based on species, sex, age, body weight and parity. Through RBPT and SAT, the higher prevalence of caprine brucellosis was recorded as compared to the buffaloes, camel, cattle and sheep. Prevalence of brucellosis was higher in buffaloes through both types of ELISAs as compared to other food animals. No animal was found positive for brucellosis in camel population. The prevalence of brucellosis was not associated with sex of animals. Sero-prevalence was higher in mature animals as compared to the younger ones in all food animals. The other risk factors like body weight and parity also affected the sero-prevalence of brucellosis in all species.

Full text

454
Pakistan Veterinary Journal
ISSN: 0253-8318 (PRINT), 2074-7764 (ONLINE)
Accessible at: www.pvj.com.pk
Sero-Prevalence of Brucellosis in Food Animals in the Punjab, Pakistan
Shafia Tehseen Gul, Ahrar Khan*, Farzana Rizvi and Iftikhar Hussain1
Department of Pathology; 1Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture,
Faisalabad, Pakistan
*Corresponding author: ahrar1122@uaf.edu.pk
ARTICLE HISTORY (14-248)
ABSTRACT
Received:
Revised:
Accepted:
May 17, 2014
June 13, 2014
June 19, 2014
Aim of the present study was to know the prevalence of brucellosis in food animals
in relation to various risk factors through different diagnostic tests. For this purpose,
2375 serum samples were collected from December, 2010 to December, 2012. Rose
Bengal Plate Test (RBPT) and Serum Agglutination Test (SAT) were applied for
initial screening, positive samples were subjected to enzyme linked immunosorbant
assays (i-ELISA and c-ELISA) for confirmation. The data thus collected was
interpreted and subjected to Binary Logistic Regression Analysis to know the
difference among various groups based on species, sex, age, body weight and parity.
Through RBPT and SAT, the higher prevalence of caprine brucellosis was recorded
as compared to the buffaloes, camel, cattle and sheep. Prevalence of brucellosis was
higher in buffaloes through both types of ELISAs as compared to other food
animals. No animal was found positive for brucellosis in camel population. The
prevalence of brucellosis was not associated with sex of animals. Sero-prevalence
was higher in mature animals as compared to the younger ones in all food animals.
The other risk factors like body weight and parity also affected the sero-prevalence
of brucellosis in all species.
©2014 PVJ. All rights reserved
Key words:
Brucellosis
c-ELISA
Food animals
i-ELISA
RBPT
SAT
Sero-prevalence
To Cite This Article: Gul ST, A Khan, F Rizvi and I Hussain, 2014. Sero-prevalence of brucellosis in food animals
in the Punjab, Pakistan. Pak Vet J, 34(4): 454-458.
INTRODUCTION
Brucellosis, is an important zoonotic disease after
rabies, causes significant reproductive losses in sexually
mature animals and is a major barrier for the trade (Lopes
et al., 2010; Abubakar et al., 2012). Although, brucellosis
has been controlled or eradicated in many developed
countries; yet it still remains an uncontrolled problem in
regions of high endemicity such as the Mediterranean,
Middle East, Africa, Central and Latin America, Eastern
Europe, Caribbean and parts of Asia (Maurin and Maurin,
2005). The disease burden is more profound in the
developing countries due to lack of effective public health
measures, domestic animal health programs and
appropriate diagnostic facilities. As no characteristic
constellation of symptoms and signs exists, the diagnosis
is usually missed (Gul and Khan, 2007).
From public health view point, brucellosis is one of
the world’s major zoonotic problems, categorized as class
B bioterrorist agent and accounting for the annual
occurrence of more than 500,000 cases (Seleem et al.,
2010). This disease is considered to be an occupational
disease that mainly affects slaughter-house workers,
butchers, livestock producers, shepherds, farmers,
veterinarians, and laboratory technicians (Behzadi and
Mogheiseh, 2011). This disease has been imported from
brucellosis-endemic countries into non-endemic areas due
to increase in business and leisure travel (Gwida et al.,
2010).
The diagnosis of brucellosis is usually performed by a
combination of serological and molecular methods.
Definitive diagnosis is usually carried out through
isolation and identification of the causative organism, but
drawback is that it is time-consuming, must be performed
by highly skilled personnel, and is hazardous. For these
reasons, serological tests like RBPT and serum
agglutination test are normally preferred. For confirmation
of findings of RBPT and SAT, ELISA based tests are used
(Gul and Khan, 2007; Poester et al., 2010).
In Pakistan, the prevalence of brucellosis has been
reported to vary from 0 to 32.5% and work has been
carried out on the seroprevalence of brucellosis in almost
all domestic species like cattle and buffaloes (Asif et al.,
2009), sheep and goats (Ghani et al., 1995; Iqbal et al.,
2013), camel (Nasrin et al., 1998), horses (Gul et al.,
2013) and humans (Mukhtar and Kokab, 2008; Asif et al.,
RESEARCH ARTICLE

Pak Vet J, 2014, 34(4): 454-458.
455
2014). Recently, it was observed that the prevalence of
brucellosis in animals is increasing day by day (Abubakar
et al., 2012). Mostly, this prevalence is based on RBPT
and SAT. No further confirmation has been made.
However, serological cross reactions have been
demonstrated between Brucella species and other bacteria.
The present project, therefore was planned to investigate
the sero-prevalence of brucellosis in food animals through
basic serological reactions and also through latest tests
like i-ELISA and c-ELISA.
MATERIALS AND METHODS
Experimental animals: For this study, 2375 food animals
including cattle (n=475), buffaloes (n=212), sheep
(n=1306), goats (n=282) and camels (n=100) of both
sexes were selected randomly from different Government
farms in Punjab, Pakistan from December, 2010 to
December, 2012. Two well-organized private farms of
cattle and buffalo were also included in this study. About
5ml blood without anticoagulant was collected from
above mentioned different food animals, serum was
separated and stored at –20˚C till analysis for sero-
diagnosis.
Sero-diagnosis: The Rose Bengal plate test (RBPT) and
serum agglutination test (SAT) were performed following
the procedure described by Aldomy et al. (2009). The i-
Elisa (Brucella i-ELISA Antibody Test, Kit # 10-2700-
10) and c-ELISA (Brucella Ab C-ELISA Test, Kit # 10-
2701-10) were performed by following the procedures
described by the manufacturers (Brucellosis commercial
ELISA kit manual procured from Svanova, Sweden).
Various risk factors like species, age, sex, body weight
and parity were considered to affect the prevalence of
brucellosis.
Statistical analysis: Animals were divided into different
groups based upon species (cattle, buffaloes, goats and
sheep), sex (female, male), age {cattle (1-7, 8-14 and
above 14 years), buffaloes (1-5, 6-10 and above 10 years),
goats and sheep (0-12, 13-24, 25-36 and above 36
months)}, body weight (cattle, 100-300, 301-600 and
above 600kg; buffaloes, 200-600 and 601-1000kg; goats,
10-30 and 31-60kg; and sheep, 10-40, 41-80kg) and parity
(cattle and buffaloes, 0-5 and 6-10; goats and sheep, 0-3
and 4-7). Binary logistic regression analysis was applied
through a statistical software MINITAB 16.0 version to
know the difference in sero-prevalence among different
groups on the basis of all four diagnostic tests applied.
RESULTS
Overall sero-prevalence of brucellosis: Overall sero-
prevalence of brucellosis in different food animals was
12.29 and 4.58% through RBPT and SAT, respectively.
Initial screening through RBPT showed that highest sero-
prevalence was in goats, followed by buffaloes, cattle and
sheep. Sero-prevalence of brucellosis through SAT was
8.49, 7.57, 9.57 and 2.14% in buffaloes, cattle, goats and
sheep, respectively (Table 1). The statistical analysis
indicated that difference in sero-prevalence among
various species was statistically significant (P<0.001)
through RBPT and SAT. Odds ratio indicated that chances
of brucellosis sero-prevalence were higher in goats and
lower in cattle and sheep as compared to buffaloes
through both tests (Table 1).
The i-ELISA based sero-prevalence of brucellosis
was 8.01, 16.31, 0, 6.73 and 1.76% in buffaloes, cattle,
camels, goats and sheep, respectively and the difference
among these species was statistically significant (Table 2).
The difference in sero-prevalence on the basis of c-ELISA
was also significant among different species. c-ELISA
based sero-prevalence was 8.01, 6.94, 6.73 and 1.91% in
buffaloes, cattle, goats and sheep, respectively. Odds ratio
indicated that chances of brucellosis sero-prevalence were
lower in cattle, goats and sheep as compared to buffaloes
through both tests (Table 2).
Sex based sero-prevalence of brucellosis: In relation to
sex, sero-prevalence of brucellosis in cattle was higher in
females as compared to males, while in buffaloes, it was
higher in males than females and the differences based
upon sex in case of cattle and buffaloes were statistically
non-significant through all four tests (Table 3). In goats,
prevalence of brucellosis was higher in bucks as
compared to does and the difference was statistically
significant through all tests except RBPT. In sheep, sero-
prevalence of brucellosis was lower in ewes as compared
to rams and the difference was statistically significant
(P<0.001) through all four tests (Table 3).
Age based sero-prevalence of brucellosis: In cattle,
sero-prevalence of brucellosis was higher in sexually
mature animals as compared to younger animals through
all four diagnostic tests. But, the difference among
different age groups was statistically non-significant. In
buffaloes, sero-prevalence of brucellosis was also higher
in sexually mature animals as compared to younger
animals through all four diagnostic tests and the
difference among three different groups was statistically
significant on the basis of all tests (Table 4).
Sero-prevalence of brucellosis in small ruminants was
higher in mature animals as compared to younger animals
as it was observed in large animals. In goats, the
difference in sero-prevalence among these four age groups
was statistically significant (P<0.002) through RBPT,
while it was non-significant through all other tests. In
sheep, the difference among different age groups was
statistically non-significant through all tests except c-
ELISA, where difference was statistically significant
(P<0.04) (Table 4).
Body weight based sero-prevalence of brucellosis: The
difference in sero-prevalence of brucellosis in cattle was
statistically significant among three body weight groups
depending upon all the four diagnostic tests (Table 5) and
highest prevalence was recorded in animals having >600
kg body weight as compared to the other two groups.
While, in buffaloes it was non-significant through all the
tests. In goats and sheep, statistically the difference
among two groups was significant only through RBPT
(Table 5) and prevalence was higher in animals having
higher body weights.

Pak Vet J, 2014, 34(4): 454-458.
456
Table 1: Sero-prevalence of brucellosis in food animals through RBPT and SAT
Species Total Animals Rose Bengal Plate Test Serum Agglutination Test
Positive (%) Coefficient±SE Odds Ratio Positive (%) Coefficient±SE Odds Ratio
Buffalo 212 27 (12.73) -1.924±0.206 - 18 (8.49) -2.377±0.246 -
Cattle 475 49 (10.32) -0.238±0.255 0.79 36 (7.57) -0.123±0.301 0.49
Goat 282 99 (35.10) 1.310±0.240 3.71 27 (9.57) 0.132±0.318 0.61
Sheep 1306 117 (8.95) -0.394±0.227 0.67 28 (2.14) -1.443±0.311 0.13
Chi-square Value 125.095 41.917
P-value 0.001 0.001
Values in parenthesis indicate percentage.
Table 2: ELISA based sero-prevalence of brucellosis in food animals
Species Total Animals i-ELISA c-ELISA
Positive (%) Coefficient±SE Odds Ratio Positive (%) Coefficient±SE Odds Ratio
Buffalo 212 17 (8.01) -2.439±0.252 - 17 (8.01) -2.439±0.252 -
Cattle 475 30 (6.31) -0.257±0.315 0.77 33 (6.94) -0.155±0.310 0.86
Goat 282 19 (6.73) -0.187±0.346 0.83 19 (6.73) -0.187±0.346 0.83
Sheep 1306 23 (1.76) -1.581±0.328 0.21 25 (1.91) -1.496±0.323 0.22
Chi-square Value 33.5135 33.4682
P-value 0.001 0.001
Values in parenthesis indicate percentage.
Parity based sero-prevalence of brucellosis: Sero-
prevalence of brucellosis was higher in animals having 6-
10 parity as compared to those having 0-5 parity in cattle
and buffaloes, the difference was statistically significant
(P<0.004) through RBPT only in cattle, but it was non-
significant through all other tests in animals of the two
species (Table 6).
In goats, sero-prevalence of brucellosis in two groups
based upon parity was higher in animals having 4-7 parity
as compared to those having 0-3 parity the difference
between these two groups was statistically significant on
the basis of all diagnostic tests, except RBPT (Table 6).
Difference in sero-prevalence of ovine brucellosis in two
groups based upon parity was statistically non-significant
(Table 6). However, prevalence of brucellosis in ewes
having more than 4 lactations was more as compared to
those having less than 4 number of lactations. Parity based
results indicated that animals having more parity were
more prone to the brucellosis.
DISCUSSION
Brucellosis is a highly contagious bacterial disease
which is not only of zoonotic importance but is also a
disease of economic importance. It adversely affects the
productive and reproductive potential of animals in terms
of reduction or complete cessation of milk production
after abortion, loss of young ones and temporary or
permanent infertility (Gul and Khan, 2007; Shabbir et al.,
2013).
Overall sero-prevalence of brucellosis in different
food animals was 12.29 and 4.58% through RBPT and
SAT, respectively. The highest sero-prevalence of
brucellosis was recorded in goats, followed by buffaloes,
cattle and sheep on the basis of RBPT and SAT. The odds
ratio indicated that the brucellosis occurrence chances
were higher in goats as compared to the other species. The
published literature also indicated that the prevalence of
caprine brucellosis was higher as compared to the other
types of brucellosis (Akbarmehr and Ghiyamirad, 2011).
It might be due to fact that in husbandry practices, these
animals are usually kept overcrowded and reared in open
system with different ages and without differentiation of
aborted and pregnant ones and even males and females are
housed together with high stocking density, all these
factors play important role in the spread of the infection.
Another reason could be that in traditional farming,
farmer does not have knowledge about brucellosis and
usually keep Brucella infected animals for breeding
purpose which serves as source of infection.
Through i-ELISA, the highest sero-prevalence was
recorded in buffaloes, followed by goats, cattle and sheep.
The i-ELISA based sero-prevalence of brucellosis in cattle
was lower than the recently reported prevalence in
Pakistan which was 20%, but in buffaloes it was higher
which was 0.0-7.74% (Abubakar et al., 2010). Overall
prevalence of brucellosis in camel population was
recorded 0% which is in accordance to the previous
results in which prevalence of brucellosis in camel had
been reported to be 0.0-17.20% (Gul and Khan, 2007). A
wide variation in brucellosis prevalence among different
food animal species could be due to difference in
prevalence of the disease in that geographic region,
diagnostic tests applied, close contact with infected
domestic and wild animals, population intensity or
husbandry system being practiced (Sikder et al., 2012;
Gul et al., 2013).
In this study, a non-significant difference was
observed in sero-prevalence of brucellosis on the basis of
sex in bovines and it was in accordance to the results
previously reported that the prevalence of brucellosis
appeared not to be associated with sex and disease
prevalence was as frequent in males as in females
(Akbarmehr and Ghiyamirad, 2011; Asmare et al., 2013).
In case of caprine and ovine brucellosis, prevalence was
higher in males as compared to females as has also been
expressed by Rahman et al. (2011). However, these
results contradict with previous reports, where it was
stated that prevalence in females was significantly higher
than males (Khan et al., 2009; Omer et al., 2010; Junaidu
et al., 2011).
In the present study, sero-prevalence of brucellosis
was higher in sexually mature animals as compared to
younger animals in buffaloes. The same was true in case
of caprine brucellosis on the basis of RBPT and in ovine
brucellosis on the basis of c-ELISA. These results are in
accordance to the previous reports which stated that the
prevalence of brucellosis appears to be associated with

Pak Vet J, 2014, 34(4): 454-458.
457
Table 3: Sex based sero-prevalence of brucellosis in food animals (%)
Test Sex Species
Cattle Buffaloes Goats Sheep
RBPT Female 10.65 12.37 34.27 8.25
Male 0 20 41.17 24.1
P-value 0.068 0.508 0.434 0.001
SAT Female 7.82 8.41 7.25 1.52
Male 0 10 26.47 15.51
P-value 0.121 0.864 0.002 0.001
i-ELISA Female 6.52 7.92 4.83 1.36
Male 0 10 20.58 10.34
P-value 0.158 0.819 0.002 0.001
c-ELISA Female 7.17 7.92 5.24 1.44
Male 0 10 17.64 12.06
P-value 0.138 0.819 0.019 0.001
Table 4: Age based sero-prevalence of brucellosis in food animals (%)
Species Age Groups
Test
RBPT SAT i-ELISA c-ELISA
Cattle 1-7 7.48 7.08 5.51 6.29
8-14 13.17 7.80 7.31 7.80
Above 14 18.75 12.5 6.25 6.25
P-value 0.078 0.751 0.733 0.816
Buffaloes
1-5 6.75 2.70 1.35 1.35
6-10 12.12 10.10 10.10 10.10
Above 10 25.64 15.38 15.38 15.38
P-value 0.023 0.038 0.009 0.009
Goats
0-12 7.69 0 0 0
13-24 29.03 8.06 4.83 4.83
25-36 41.97 8.64 6.17 8.64
Above 36 39.82 13.27 9.73 7.96
P-value 0.002 0.069 0.139 0.195
Sheep
0-12 6.31 2.10 0 0
13-24 12.85 5.0 3.57 4.28
25-36 7.37 2.45 2.18 2.45
Above 36 9.36 1.41 1.41 1.41
P-value 0.208 0.105 0.098 0.040
Age of cattle/buffaloes in years while age of goats/sheep in months.
Table 5: Body weight based sero-prevalence of brucellosis in food
animals (%)
Species Body Weight (kg) Test
RBPT SAT i-ELISA c-ELISA
Cattle 100-300 6.97 4.65 3.87 3.87
301-600 10.54 7.53 6.32 7.53
Above 600 35.71 35.71 28.57 21.42
P-value 0.000 0.001 0.003 0.002
Buffaloes 200-600 10 7.9 6.92 6.92
601-1000 17.07 9.75 9.75 9.75
P-value 0.137 0.602 0.464 0.464
Goat 10-30 19.23 6.15 3.84 4.61
31-60 48.68 12.5 9.21 8.55
P-value 0.000 0.066 0.067 0.182
Sheep 10-40 6.87 1.87 1.56 1.40
41-80 10.96 2.40 1.95 2.40
P-value 0.009 0.510 0.592 0.186
Table 6: Parity based sero-prevalence of brucellosis in food animals (%)
Species Parity Test
RBPT SAT i-ELISA c-ELISA
Cattle 0-5 8.33 6.66 5.55 6.38
6-10 19 12 10 10
P-value 0.004 0.094 0.129 0.233
Buffaloes 0-5 12 8 6.4 7.2
6-10 12.98 9.09 10.38 9.09
P-value 0.837 0.787 0.314 0.631
Goats 0-3 33.48 5.04 3.21 2.75
4-7 40 23.33 16.66 23.33
P-value 0.485 0.002 0.008 0.000
Sheep 0-3 8.34 1.07 0.94 0.94
4-7 8.11 2.17 1.98 2.17
P-value 0.887 0.123 0.125 0.076
age (Bekele et al., 2011) and prevalence was low in young
stock than the adults (Sanogo et al., 2012). Age is known
as one of the intrinsic factors influencing the sero-
positivity of brucellosis (Megersa et al., 2011). This
influence can be explained by the fact that brucellosis is
essentially a disease of the sexually mature animals, the
predilection site being the reproductive tract, especially
the gravid uterus (Abubakar et al., 2012). So,
susceptibility of an animal increases after sexual maturity
in both sexes, because sex hormones and erythritol
stimulate the growth of Brucella organism. Younger
animals tend to be more resistant to Brucella infections;
however, latent infections can occur in these animals (Gul
et al., 2013).
In relation to body weight, sero-prevalence of
brucellosis in cattle with >600 kg body weight was higher
than other groups. In goats and sheep, such difference was
observed only through RBPT. This might be due to the
fact that the body weight of mature animals was higher as
compared to immature or younger calves, so the
prevalence was higher in these animals.
A significant difference (P<0.05) was observed in
sero-prevalence of brucellosis based upon parity through
RBPT in cattle and in goats through SAT, i-ELISA and c-
ELISA. Sero-prevalence based upon parity was higher in
animals having more parity. It is due to the reason that
prevalence of brucellosis increases with repeated exposure
to parturition and other physiological stresses during
gestation (Matope et al., 2011; Hadush et al., 2013).
Conclusion: Brucellosis is a highly contagious and
zoonotic disease, resulting in heavy economic losses. The
prevalence of the disease is associated with species, age,
body weight and parity. Brucellosis is more prevalent in
buffaloes as compared to the other food animals and also
mature animals are at higher risk as compared to younger
animals. However, it is not associated with the sex of the
animal.
Acknowledgment: Financial support of the Higher
Education Commission, Islamabad through Project # 20-
1795/R&D, titled "Molecular Pathobiology of Brucellosis
in Food Animals and Humans" is highly appreciated.
Author’s contribution: STG and AK conceived the idea,
designed the project, executed the experiment and
analyzed the sera and data. All authors were involved in
the interpretation of the data, write up and revision of the
manuscript.
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