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Research Article Agrobiological Records
Open Access Journal
Khan UD, Khan A, Gul ST, Saleemi MK and Du XX, 2020. Seroprevalence of brucellosis in cattle (Bos taurus) kept in peri urban
areas of Pakistan. Agrobiological Records 1: 6-10.
6
SEROPREVALENCE OF BRUCELLOSIS IN CATTLE (BOS TAURUS) KEPT IN PERI
URBAN AREAS OF PAKISTAN
Umar Daraz Khan1, Ahrar Khan1,2, Shafia Tehseen Gul1, M. Kashif Saleemi1 and Xiaoxia Du2,3*
1Department of Pathology, University of Agriculture, Faisalabad 38040, Pakistan
2Shandong Vocational Animal Science and Veterinary College, Weifang, China
3Department of Biology, Central Washington University, Ellensburg, WA 98926, USA
*Correspondence: duxiaoxia0931@hotmail.com; duxiaoxia0931@126.com
ABSTRACT
Brucellosis is a highly contagious bacterial disease of zoonotic importance that causes significant
economic losses. Many factors affect the prevalence of brucellosis and variable with the diagnostic tests
applied. These all necessitates to carry out seroprevalence frequently to suggest control measures, this study
was thus carried out to the seroprevalence and various factors affecting its prevalence. Total of 335 cows of
various age groups, breeds (Cross-bred (Sahiwal X Friesian), Friesian, Sahiwal, Cholistani and Jersey) and
stages (pregnant/non-pregnant, lactating/non-lactating) were selected randomly for sampling. Blood was
collected and serum was separated and subjected to Rose Bengal Precipitation Test (RBPT). RBPT positive
samples were confirmed by c-ELISA for further analysis. The seroprevalence was 12.53% and 2.40%
through RBPT and c-ELISA, respectively. The prevalence of the disease based on RBPT was higher in cross-
bred, females, mature, good condition, pregnant and lactating animals as compared to their counterparts. It
was concluded that the cross-bred cattle, pregnancy and lactation are playing important roles for the disease
prevalence. Specific preventive measures be opted for control of the disease.
Keywords: Brucellosis, Cattle, Prevalence, Faisalabad, Pakistan
Article History (2020-0303): Received: 06 Mar 2020 || Revised: 14 Mar 2020 || Accepted: 17 Mar 2020 || Published Online: 22 Mar 2020
INTRODUCTION
Pakistan's economy is mainly based on Agriculture. This sector directly supports the country's population and
accounts for 26% of gross domestic product (GDP). Being subsector agriculture, livestock contributes approximately
56% of value addition in agriculture and nearly 11% to the GDP (Rehman et al. 2017). Livestock production makes
a major contribution to agriculture value added services, however, many diseases hamper its production, e.g.,
brucellosis, Peste Des Petits Ruminants (PPR), foot and mouth disease (FMD), trypanosomiasis, etc. (Hussain et al.
2018, Khan et al., 2018, Imtiaz et al. 2018, Hussain et al. 2020).
Brucellosis is a highly contagious bacterial disease having significant zoonotic importance that causes abortion,
low fertility, decreased milk production and cost of replacement (Shafee et al. 2011, Khan and Zahoor 2018). Serious
socioeconomic issues can be posed by the disease to livestock owners (Megersa et al. 2011). Due to lack of hygienic
measures, public health measures, national animal health and management strategies, the disease is more common in
developing countries (Thakur et al. 2002, Farouk et al. 2017, Hassan et al. 2020). Due to rapidly increasing
intercontinental tourism and animal trade, there are more chances that disease could spread in developed countries
(Greenfield et al. 2002).
The way of transmission is either through infected animal discharges like milk, urine, blood, semen or contact
with the placenta, fetal fluids, fetus, and vaginal discharge. The organism can also spread by equipment and clothing.
The more chances of infection are in abattoir workers, laboratory technicians, farmers, veterinarians and the persons
working with animal products (Mustafa et al. 2011). The entry of Brucella in the body is mainly via intact skin,
mucosal layers, lungs, and digestive tract. Then it goes via the blood and lymphatic system to tissues causing a local
infection (Lapaque et al. 2005).
Various tests have been developed for screening and confirmation of the disease. The samples of blood, serum,
milk, nasal secretions, vaginal excretions are screened and confirmed by RBPT, complement fixation test, and
polymerase chain reaction (PCR) (Gupta et al. 2010). A novel, rapid and simple method i.e., rapid vertical flow
technology has been introduced for detecting brucellosis but still needs field trials (Shi et al. 2020). In farm animals,
serological, bacteriological, and molecular methods are used. Some molecular and bacteriological methods, such as
PCR, are economical but time-consuming. In dairy cows, RBPT and Milk Ring Test (MRT) are used for monitoring
and screening of the disease (Ali et al. 2013). For diagnostic confirmation, serological tests such as Serum
Agglutination Test (SAT), RBPT, and Enzyme-Linked Immunosorbent Assay (ELISA) are used (Imtiaz et al. 2018).
Research Article Agrobiological Records
Open Access Journal
Khan UD, Khan A, Gul ST, Saleemi MK and Du XX, 2020. Seroprevalence of brucellosis in cattle (Bos taurus) kept in peri urban
areas of Pakistan. Agrobiological Records 1: 6-10.
7
Brucellosis is still uncontrolled and endemic in Asia, Middle East, Africa and Latin America (Refai 2003,
Shahzad et al. 2017). The highest incidence reported in bovines around the world ranges from 0.85% to 76% (Pandeya
2013, Khan et al. 2018). Overall, the seroprevalence of brucellosis in Pakistan ranged from zero to 76% in all livestock
species (Gul et al. 2014, Ahmad et al. 2017, Arif et al. 2018). The prevalence of brucellosis was affected by many
factors, such as different climatic conditions, sex, age, species, geography, and diagnostic test applied (Gul et al. 2014,
Gul et al. 2015), thus it necessitates to carry out seroprevalence frequently. This study was carried out on various
breeds of cattle on different forms to know the present status of brucellosis so that preventive measures could be
suggested.
MATERIALS AND METHODS
Experimental animals: For this study, 335 cows from various age groups (1-5, 6-10, 11-15, and above 15 years), body
condition (poor, fair, and good), and breed (cross-bred: Sahiwal X Friesian, Cholistani, Sahiwal, and Jersey) were
selected randomly to investigate seroprevalence of brucellosis from two cattle colonies located in peri-urban areas of
Faisalabad, Punjab, Pakistan. Approximately 5 ml blood without anticoagulant was collected from randomly selected
animals. Serum after separation was stored at -20ºC till further analysis (Bertu et al. 2010).
Sero-diagnosis: For screening of brucellosis, serum was screened RBPT (Veterinary Research Institute, Lahore)
following the standard procedure described by Aldomy et al. (2009). These samples were also subjected to c-ELISA
(Brucella Ab C-ELISA test, kit # 10-2701-10) following the procedures described by the manufacturers (Brucellosis
commercial ELISA kit, Svanova, Sweden).
Statistical analysis: The data thus collected regarding various breeds, body weight, age, sex and history of abortion
and parity were analyzed to find out the seroprevalence of brucellosis using software MINITAB 16.0 version.
RESULTS
The overall prevalence recorded through RBPT was 12.53% whereas by c-ELISA prevalence was 2.39%. On
breed basis, the highest prevalence of the disease was recorded in cross-bred animals 15.76% followed in Jersey
(11.11%), Cholistani (10.52%), Sahiwal (8.43%) and Friesian (4.54%), by RBPT. However, statistical analysis
indicated non-significant difference among various breeds in terms of disease prevalence as shown in Table 1.
Through c-ELISA highest prevalence of brucellosis was recorded in the Friesian breed (4.54%) followed by
cross-bred and Sahiwal breed. No animal was found positive in Cholistani and Jersey breed. This difference in
prevalence was non-significant statistically (Table 1). However, the probability of the brucellosis was higher in
Friesian followed by cross-bred and Sahiwal cows.
Table 1: Breed based seroprevalence of brucellosis in cattle in Faisalabad, Punjab, Pakistan
Breed
Total Animals
Positive
Percentage
P Value
RBPT
Cholistani
19
2
10.52
0.004
Cross-Bred
184
29
15.76
0.549
Friesian
22
1
4.54
0.475
Jersey
27
3
11.11
0.772
Sahiwal
83
7
8.43
0.950
Overall
335
42
12.53
c-ELISA
Cholistani
19
0
0
0.999
Cross-Bred
184
6
3.26
0.999
Friesian
22
1
4.54
0.999
Jersey
27
0
0
1.000
Sahiwal
83
1
1.20
0.999
Overall
335
8
2.39
*As the number of animals in most groups is less than five (5) so that’s why the statistical analysis is not showing this data.
On the RBPT basis, seroprevalence was higher in females (12.73%) as compared to male animals (9.52%).
Similarly, seroprevalence was higher in non-lactating (13.38%) than lactating animals (12.20%). Pregnant cows
showed higher (9.68%) seroprevalence than non-pregnant (4.52%) cows (Table 2). P value indicated that differences
among various groups based on sex, pregnancy and lactation status were non-significant in this study. While based
on the confirmatory test (c-ELISA), more prevalence in lactating animals 4.09% as compared to non-lactating, dry
and pregnant animals was found (Data not shown).
On the RBPT basis, more (P>0.05) seroprevalence was in 6-10 years group (13.63%) followed by 12.96%,
11.45% and 0% in age groups 11-15, 1-5 and >15 years, respectively (Table 3). On confirmation through c-ELISA,
seroprevalence was more (P>0.05) in the age group of 11-15 years than other age groups (Table 3). The prevalence
Research Article Agrobiological Records
Open Access Journal
Khan UD, Khan A, Gul ST, Saleemi MK and Du XX, 2020. Seroprevalence of brucellosis in cattle (Bos taurus) kept in peri urban
areas of Pakistan. Agrobiological Records 1: 6-10.
8
in poor condition animals was 13.33%, as compared to good body condition animals (Table 3). No animal with fair
body condition was found positive for Brucella antibodies. The seroprevalence was more in poor condition animals
(3.33%) as compared to good condition animals, however, the difference was non-significant.
Table 2: Seroprevalence of brucellosis in relation to sex, lactating and pregnancy status in cattle by applying RBPT
Parameters
Sex
Lactating Status
Pregnancy Status
Male
Female
Lactating
Non-Lactating
Pregnant
Non-Pregnant
Total
21
314
172
142
93
221
Positive
2
40
21
19
9
10
Prevalence %
9.52
12.73
12.20
13.38
9.68
4.52
Chi sq. = 0.148;
P-Value = 0.70; df = 1
Chi sq. = 0.065;
P-Value = 0.798; df = 1
Chi sq. = 2.657;
P-Value = 0.103; df = 1
Table 3: Age and body condition-based prevalence of brucellosis in cattle
Test
Age (Years)
Body Condition
1-5
6-10
11-15
<15
Good
Poor
Fair
RBPT
Total
96
176
54
9
295
30
10
Positive
11
24
7
0
38
4
0
Prevalence %
11.4
13.63
12.96
0
12.88
13.33
0
Chi square = 1.364; P-Value = 0.713; df = 3
Chi square = 1.292; P-Value = 0.52; df = 2
c-ELISA
Total
96
176
54
9
295
30
10
Positive
2
4
2
0
7
1
0
Prevalence %
2.08
2.27
3.7
0
2.37
3.33
0
Chi square = 0.639; P-Value = 0.887; df = 3
Chi square = 0.348; P-Value = 0.84; df = 2
DISCUSSION
Brucellosis is regarded as one of the most important zoonotic diseases in the world (Abubakar et al. 2011, Zeng
et al. 2019, Alkahtani et al. 2020). The economic losses arising from brucellosis in animals are mainly due to the
clinical manifestation of abortions during the last trimester of gestation, the decrease in milk yield, temporary
infertility and perinatal mortalities (Munir et al. 2011, Gul et al. 2015). To prevent the losses, routine monitoring of
animals and early diagnosis is necessary. Controlling the disease in livestock is an important task in many countries.
In Pakistan, the prevalence of brucellosis is increasing especially in large-sized dairy herds. There is no official
strategy for brucellosis control and eradication. Consequently, there has not been taken any necessary action to restrict
the spread of the disease in various private and government farms (Akhtar et al. 1990).
One of the rapid and sensitive techniques for screening animals against Brucella infection is RBPT (Ruiz-Mesa
et al. 2005). The RBPT has low specificity, to overcome this problem an indirect ELISA (i-ELISA) is used which has
high specificity (Nielson 2002). In the present study, we opted for the same pattern to screen cows against brucellosis
in the study area.
In the present study, a higher prevalence of brucellosis was detected in cross-bred (Sahiwal X Friesian) cattle of
more than 5 years of age. A cross-sectional type epidemiological survey carried out by Gumi et al. (2013) indicated
higher seroprevalence of brucellosis in older animals as compared to the younger ones. Swiss and Schoonman (2010)
also suggested that the animals of more than 6 years of age were more prone to seropositivity than animals having
less than 6 years of age. That could be due to impaired immune system rendered by the disease, aging and even in
pregnant animals, production of erythritol within the placenta also allows rapid multiplication of bacteria that leads
to endometritis, infections of cotyledons and placentitis (Gul et al. 2007).
The presence of Brucella infection in animals having abortion history has also been supported by Dhandet et al.
(2005). While Ibrahim et al. (2010) reported that the occurrence of abortion is positively correlated with the
seropositivity of brucellosis. Similarly, a positive correlation was observed among seroprevalence and the occurrence
of abortion as reported by Ahmad et al. (2009). In the present study, seroprevalence was higher in females as compared
to males that are in accordance to the previous report by Deselegn et al. (2011) who stated that there was a significant
variation in the prevalence of brucellosis based on sex with a higher prevalence in females as compared to males.
In the present study, there was higher prevalence in lactating animals as compared to non-lactating animals.
Similar results were obtained by Ibrahim et al. (2010) through serological survey of brucellosis, they indicated higher
seroprevalence in milking animals than non-lactating animals that could be due to the reason that lactation period
reduces the resistance of animals, thus making them valuable to attach of Brucella abortus infection (Hashim et al.
2007).
Conclusion: The results of the current study indicated the risk factors considered for this study are playing an
important role in the prevalence of brucellosis. Indigenous breeds like Sahiwal and Cholistani cattle less susceptible
Research Article Agrobiological Records
Open Access Journal
Khan UD, Khan A, Gul ST, Saleemi MK and Du XX, 2020. Seroprevalence of brucellosis in cattle (Bos taurus) kept in peri urban
areas of Pakistan. Agrobiological Records 1: 6-10.
9
as compared to the exotic breeds. Similarly, sexually matured animals are more prone to brucellosis as compared to
the younger and aged animals. A lot of other risk factors like poor management and hygienic practices observed in
these farms need to be explored to control the disease.
Contribution of Authors: AK and STG conceived and designed experiments. UDK performed the experiments.
AK and MKS carried out the supervision of studies. UDK performed statistical analyses of experimental data and
prepared the draft of the manuscript. XXD edited the manuscript. All authors critically revised the manuscript and
approved the final version.
ORCID
Ahrar Khan https://orcid.org/0000-0001-5492-4266
Shafia Tehseen Gul https://orcid.org/0000-0003-4667-0117
M. Kashif Saleemi https://orcid.org/0000-0002-2329-7228
Xiaoxia Du https://orcid.org/0000-0002-6245-9138
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Research Article Agrobiological Records
Open Access Journal
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areas of Pakistan. Agrobiological Records 1: 6-10.
10
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