British Journal of Dairy Sciences 2(3): 35-39, 2011
ISSN: 2044-2440
© Maxwell Scientific Organization, 2011
Submitted: November 21, 2011
Accepted: December 20, 2011
Published: December 20, 2011
Sero-prevalence of Brucellosis in Bovines at Farms under
Different Management Conditions
1
Rakhshinda Munir (Late), 1Umer Farooq, 1Zahida Fatima, 1Muhammad Afzal,
2
Zubair Anwar and 1Muhammad Jahangir
1
Animal Health Program, Animal Science Institute, National Agricultural Research Centre,
Park Road, Islamabad, 45500, Pakistan
2
Department of Bioinformatics and Biotechnology, International Islamic University,
Islamabad, Pakistan
Abstract: Brucellosis is endemic in Pakistan. Keeping in view the importance of the disease and non
availability of scientific information, a study was designed to carry out the sero-prevalence of brucellosis using
an indirect ELISA and Rose Bengal Precipitation Test (RBPT). Serum samples were collected from three types
of farm categories having different husbandry practices. (1) Government livestock farms located in Punjab
province (2) Privately owned farms located in out skirts of Lahore and (3) Dairy cattle colony (Gawala colony)
located in Lahore. I-ELISA detected sero-conversion in 15.2% buffaloes and 9% cattle, whereas, by using
RBPT it was 8 and 6.5% in buffaloes and cattle, respectively. In cattle, more abortions were recorded at private
farms (17.86%) followed by gawala colonies (11.61%) and government livestock farms (8.92%). Also, I-ELISA
detected sero-conversion against brucellosis in 13.2% female and 1.3% male animals, while by using RBPT
it was 7.9% in female and 1.6% in male animals, respectively.
Key words: Brucellosis, ELISA, gawala, RBPT, sero-prevalence of brucellosis
While earlier studies indicated prevalence rates between
0.33 to 0.65 % (Sheikh et al., 1967; Mohiyudin, 1979),
more recent studies indicate prevalence 21.05 to 26.1%
(Munir et al., 2008; Sarwar, 2000; Akhtar et al., 1990;
Lodhi et al., 1995; Ramzan, 1996). Among animal
handlers, prevalence of brucellosis ranging from 0.95 to
8.58 % has been reported by various workers (Masoumi
et al., 1992; Afridi et al., 1993; Qazilbash, 1996).
Brucellosis is endemic in Pakistan. However data
regarding the magnitude of the disease in cattle and
buffaloes is scanty and inconsistent. Keeping in view, the
importance of the disease and non availability of scientific
information, a study was designed to carry out the seroprevalence of brucellosis using an indirect ELISA and
Rose Bengal Precipitation Test (RBPT). Sera were
collected from three types of farm categories having
different husbandry practices.
INTRODUCTION
Bovine brucellosis is considered as the most wide
spread zoonotic disease in the world (WHO, 1998). The
importance of this highly contagious disease is due to its
economic impact on livestock industry resulting in the
form of abortions, loss of calves, retention of placenta,
prolonged calving interval, reduced milk yield and
infertility (McDermott and Arimi, 2002). The disease is
also a serious public health hazard as it is communicable
to human beings through milk or as occupational hazard
to farmers, butchers and veterinarians. Two main species
associated with brucellosis in food animals i.e. Brucella
abortus and Brucella melitensis have been reported from
Pakistan (Akhtar et al., 1974; Mohiyudin, 1979) and the
bacterium is capable of surviving and multiplying inside
mononuclear phagocytic system (Munir et al., 2008;
Jarvis et al., 2002).
Prevalence of brucellosis in farm animals depends on
a number of factors, the most important being the
production system and chances of spread during
parturition and breeding. Livestock production system in
Pakistan is changing and more livestock farmers are now
maintaining larger herds, thus increasing the chances of
spread of brucellosis in Pakistan (Afzal, 1997). Studies on
brucellosis have mainly been restricted to prevalence rates
in Pakistan. These studies clearly indicate that the
increasing prevalence rate of brucellosis in the country.
C
C
Government livestock farms located in Punjab
province, where the animals are mainly kept for
breeding improvement and conservation. These farms
are also involved in selling milk and surplus stock to
farmers and hence have a great influence on the
livestock population of the Province.
Privately owned farms located in out skirts of
Lahore. This is peri-urban market oriented dairying
from where milk supply can mostly be made directly
to consumers.
Corresponding Author: Zubair Anwar, Department of Bioinformatics and Biotechnology, International Islamic University,
Islamabad, Pakistan
35
Br. J. Dairy Sci., 2(3): 35-39, 2011
2362
1533
1500
1000
500
MATERIALS AND METHODS
146
244
390
2000
1735
1294
2500
829
Number of animals
3000
3029
Cattle
Buffalo
Total
3500
56
221
277
Dairy cattle colony (Gawala colony) located in
Lahore. This is unique aggregation of cattle and buffaloes
maintained on commercial basis for selling milk. Dry
animals are continuously replaced with the animals
coming from the entire country. The study will help us to
chalk out control strategies for the rational control of
disease.
Private
Gawala
0
Government
A total of 3029 serum samples from adult cattle and
buffaloes were collected between the year 2007 to 2009,
from three types of farm categories; government farms
(Livestock Research Station, National Agricultural
Research Centre, Park Road, Islamabad; Government
Livestock farms located at Jahingirabad, District
Khanewal; Khizerabad, District Sargodha; Rakh
Ghulaman, District Mianwali; Chak Katora, District
Vehari) and semen production units (Barani Livestock
Production and Research Institute, Kherimurat, District
Attock and Semen Production Unit Qadirabad, District
Sahiwal) located in Punjab province; peri-urban private
farms around Lahore; and Gawala colony Lahore. Of
these samples 2362 sera were collected from eleven
government farms, 390 from ten private owned farms and
277 from Gawala colony. Among these sera 376 were
from cattle and buffalo from two semen production units.
All necessary information regarding number, type of
animals kept at farm, vaccination schedule and history of
abortion were recorded on a specially designed Performa.
Sera were tested by an indirect ELISA and Rose Bengal
Precipitation Test (RBPT). Rose Bengal precipitation test
was performed following the method Alton et al. (1988).
Briefly, 30 :L of serum was mixed with 30 :L RBPT
antigen (N-vitro Diagnostikum, Germany) on glass slide.
It was rocked at 22ºC for 4 min. The reaction was
interpreted as positive when agglutination was observed
and declared negative when the mixture was homogenous
after 4 min.
For further labortary analysis Indirect Enzyme
Linked Immunosorbent Assay (ELISA) was performed
using a commercial indirect ELISA kit produced by
Chekit, Bommeli Laboratories, Switzerland. Polystyrene
flat bottomed 96 wells micro titration were pre-coated
with an activated antigen. The test samples and
appropriate sera controls (strong positive, weak positive,
strong negative and weak negative) were diluted 1:200 in
phosphate buffered saline Tween-20 (PBST) and added in
the wells. These plates were incubated at 25ºC for 90 min
in a humidified chamber and were washed three times.
Then anti-ruminant IgG conjugate 1:200 dilution was
added to each well. The plates were again incubated at
25ºC in a moist chamber for 30 min. After three washings
chromogen ABTS (2, 2 azinobis 3-ethyle benzthiazolin)
was added. All the washings were carried out using
PBST.
Total
0
Buffalo Cattle
Total
ELISA positive
216
153
38
25
91
14
7
104
62
24
18
25
59
18
11
50
115
100
48
150
157
200
112
197
250
243
Government
Private
Gawala
Total
300
34
Number of animals
400
354
Fig. 1: Blood collected from cattle and buffalo farms under
different managemental conditions
Buffalo Cattle
Total
RBPT positive
Fig. 2: Comparison of ELISA and RBPT for brucellosis in
buffalo and cattle at farms under different
managemental conditions
Number of animals
400
350
ELISA positive
RBPT positive
354
300
250
200
243
216
153
150
100
59
25
50
0
Government
Private
52 38
Gawala
Total
Fig. 3: Comparison of ELISA and RBPR for brucellosis at
farms under different managemental conditions
The plates were kept for 15 minutes in dark for color
development. The reaction was stopped by adding 4%
SDS solution and optical density of plates were recorded
using 405 and 492 nm differential filters (Biancifiori
et al., 1997; OIE, 2000; Wright et al., 1993).
In the end qualitative data of ELISA and RBPT was
analyzed by Chi-square test (Steel and Torie, 1984) for
statistical analysis
RESULTS
Blood samples collected from cattle and buffalo at
farms under different managemental conditions is
summarized in Fig. 1. Further comparison of ELISA and
RBPT for Brucellosis in Buffalo and cattle at farms under
36
Br. J. Dairy Sci., 2(3): 35-39, 2011
Table 1: Comparison and P2 distribution of ELISA and RBPT for brucellosis in buffalo and cattle at farms under different managemental conditions
Brucellosis (ELISA)
RBPT
-------------------------------------------------------------------------------Animal
Farms
Negative
Positive
Not sure
Total
None
Positive
Total
Buffalo
Government 698
115
16
829
767
62
829
(0.14,84.2) (1.00,13.9)
(0.11,1.9)
(1.24,100)
(0.03,92.5)
(0.32,7.5)
(0.34,100)
Gawala
171
48
2
221
203
18
221
(0.84,77.4) (6.12,21.7)
(3.93,0.9)
(7.91,100)
(0.00,91.9)
(0.00,18.1)
(0.00,100)
Private
205
34
5
244
220
24
224
(0.03,84)
(0.27,13.9)
(0.10,2)
(0.39,100)
(0.09,90.2)
(0.98,9.8)
(1.06,100)
Total
1074
197
23
1294
1190
104
1294
(1.01,83)
(7.36,15.2)
(1.15,1.8)
(-,100)
(0.11,92)
(1.30,8)
(-,100)
2
Total P
9.55*
1.42
Probability
(0.04)
(0.49)
Cattle
Government 1379
128
26
1533
1440
91
1531
(0.10,90)
(0.81,8.3)
(0.01,1.7)
(0.91,100)
(0.04,94.1)
(0.64, 5.9)
(0.68,100)
Gawala
43
11
2
56
49
7
56
(0.97,76.8) (6.96,19.6)
(1.21,3.6)
(9.14,100)
(0.22,87.5)
(3.16,12.5)
(3.37,100)
Private
127
18
1
146
132
14
146
(0.08,87)
(1.75,12.3)
(0.85,0.7)
(2.67,100)
(0.15,90.4)
(2.21,9.6)
(2.36,100)
Total
1549
157
29
1735
1621
112
1733
(1.14,89.3) (9.51,9)
(2.06,1.7)
(-,100)
(0.41,93.5)
(6.00,6.5)
(-,100)
Total P2
12.72**
6.41*
Probability %
0.01
0.04
2
The vales in parenthesis are (P , percentages); *: Significance level
Table 2: Comparison and P2 distribution of ELISA and RBPT for brucellosis at farms under different managemental conditions
Brucellosis (ELISA)
RBPT
----------------------------------------------------------------------------------------Farms
Negative
Positive
Not sure
Total
None
Positive
Government
2077
243
42
2362
2207
153
(0.49,87.9)
(3.96,10.3)
(0.05,1.8)
(4.49,100)
(0.11,93.5)
(1.41,6.5)
Gawala
214
59
4
277
252
25
(2.79,77.3)
(21.90,21.3) (0.12,1.4)
(24.81,100)
(0.11,91)
(1.39,9)
Private
332
52
6
390
352
38
(0.10,85.1)
(0.90,13.3)
(0.07,1.5)
(1.07,100)
(0.29,90.3)
(3.72,9.7)
Total
2623
354
52
3029
2811
216
(3.37,86.6)
(26.76,11.7) (0.24,1.7)
(-,100)
(0.50,92.9)
(6.51,7.1)
Total P2
30.38**
Probability %
1
2
The vales in parenthesis are (P , percentages); *: Significance level
Table 3: Comparison and P2 distribution of ELISA and RBPT for brucellosis in buffalo and cattle
Brucellosis (ELISA)
RBPT
-------------------------------------------------------------------------------------------Animal
Negative
Positive
Not sure
Total
None
Positive
Buffalo
1074
197
23
1294
1190
104
(1.93,83)
(13.85,15.2) (0.03,1.8)
(15.81,100)
(0.10,92)
(1.35,8)
Cow
1549
157
29
1735
1621
112
(1.44,89.3)
(10.33,9)
(0.02,1.7)
(11.79,100)
(0.08,93.5)
(1.01,6.5)
Total
2623
354
52
3029
2811
216
(3.37,86.6)
(24.18,11.69) (0.04,1.72)
(-,100)
(0.18,92.9)
(2.35,7.1)
2
Total P
27.60**
Probability %
1
The vales in parenthesis are (P2, percentages); *: Significance level
Table 4: Comparison and P2 distribution of ELISA and RBPT for brucellosis in females and males
Brucellosis (ELISA)
RBPT
-------------------------------------------------------------------------------------------Sex
Negative
Positive
Not sure
Total
None
Positive
Female
2254
349
50
2653
2441
210
(0.82,85)
(4.89,13.2)
(0.44,1.9)
(6.14,100)
(0.17,92.1)
(2.18,7.9)
Male
369
5
2
376
370
6
(5.78,98.1)
(34.51,1.3)
(3.07,0.5)
(43.37,100)
(1.18,98.4)
(15.41,1.6)
Total
2623
354
52
3029
2811
216
(6.60,86.6)
(39.40,11.7)
(3.51,1.7)
(-,100)
(1.35,92.9)
(17.59,7.1)
Total P2
49.51**
Probability %
1
2
The vales in parenthesis are (P , percentages); *: Significance level
37
Total
2360
(1.51,100 )
277
(1.49,100)
390
(4.00,100)
3027
(-,100)
7.01*
0.03
Total
1294
(1.45,100)
1733
(1.08,100)
3027
(-,100)
2.53
0.11
Total
2651
(2.35,100)
376
(16.58,100)
3027
(-,100)
18.94**
1
Br. J. Dairy Sci., 2(3): 35-39, 2011
than 50 % animals from each farms category were bled
for detecting sero-conversion against brucellosis.
The highest ELISA positive buffaloes (21.7%) were
observed in Gawala colonies followed by government
(13.9%) and private farms (13.9%), while in cattle it was
also highest (19.6%) in Gawala colonies followed by
private (12.3%) and government farms (8.3%).
In case of RBPT the highest positive buffaloes
(18.1%) were detected at Gawala colony followed by
private farms (9.8%) and government farms (7.5%), while
highest RBPT positive cattle (12.5%) was observed in
Gawala colony followed private farms (9.6%) and
government farms (5.9%).
Interestingly, the highest ELISA positive (21.3%)
animals were at gawala colonies followed by private
(13.3%) government farms (10.3%), while highest RBPT
positive animals (9.7%) were at private farms followed by
gawala colony (9%) and government farms (6.5%).
When I-ELISA and RBPT were compared for the
detection of brucella infection, it was observed that
ELISA detected a higher number of positive cases as
compared to RBPT which detected higher number of
negative cases. In a campaign for the control of a disease
like brucellosis, it is desirable that the screening test
should be reliable and detects almost all positive cases in
a herd. In our studies I-ELISA detected more positive
samples as compared with RBPT and therefore, it would
be a better choice to launching a campaign for the control
and eradication of brucellosis. Also acceptable method for
screening should minimize false negatives results
especially when used for the detection of infection with
serious consequences.
In the present study, it was observed that I-ELISA
detected more positive sera as compared to RBPT this
observation was recorded by Nielsen et al. (1996) and
Dajer et al. (1998), who compared I-ELISA with different
serological tests and reported its sensitivity up to 100%.
Molnar et al. (1998) and Poester et al. (1998), have also
observed the higher sensitivity of I-ELISA.
It was observed that I-ELISA detected seroconversion in 15.2% buffaloes and 9% cattle, whereas, by
using RBPT it was 8% and 6.5% in buffaloes and cattle,
respectively. In cattle, more abortions were recorded at
private farms (17.86%) followed by gawala colonies
(11.61%) and government livestock farms (8.92%); this
might be due to their husbandry practices and adopted
hygienic measures.
I-ELISA detected sero-conversion against brucellosis
in 13.2% female and 1.3% male animals, whereas it was
7.9 and 1.6% positive by RBPT for female and male
animals, respectively. Most of the male animals were
kept by the government farms while only few male
animals were kept at private and gawala colonies.
Number of animals
250
200
197
157
150
112
104
100
50
0
RBPT
positive
ELISA
positive
ELISA
positive
RBPT
positive
1049
629
Cattle
abortion
Cattle tolal
31
35
45
111
200
0
56
146
800
600
400
221
244
1400
1200
1000
Government
Private
Gawala
Total
121
10
17
148
Number of animals
1600
1357
1800
1559
Fig. 4: Comparison of ELISA and RBPR for brucellosis in
Buffalo and Cattle
Buffalo
abortion
Buffalo total
Fig. 5: Comparison of abortions in buffalo and cattle
Number of animals
400
349
350
300
250
210
200
150
100
50
6
5
0
ELISA
positive
Male
RBPT
positive
ELISA
positive
RBPT
positive
Female
Fig. 6: Comparison of ELISA and RBPR for brucellosis in
female and male animals
different managemental conditions with their statistical
analysis is presented in Table 1 and Fig. 2. Next
comparison of ELISA and RBPT for Brucellosis at farms
under different managemental conditions and their
statistical analysis is presented in Table 2 and Fig. 3.
Comparative analysis of ELISA and RBPT for Brucellosis
in Buffaloes and cattle and their statistical analysis is
shown in Table 3 and Fig. 4. And the comparison of
abortion in cattle and buffaloes and their statistical
analysis is shown in Table 4 and Fig. 5. In the comparison
of ELISA and RBPT for Brucellosis in females and males
is presented in Fig. 6.
DISCUSSION AND CONCLUSION
The multi fold herd size was present at government
farms as compared to private and gawala colony. More
38
Br. J. Dairy Sci., 2(3): 35-39, 2011
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