Sero–Prevalence of Brucellosis in Cattle in Southern Area of Khyber Pakhtunkhwa, Pakistan

Abstract

Brucellosis considered as the most important zoonotic disease worldwide. The diagnosis of brucellosis in bovines requires the use of more than one serological test for routine diagnosis. Present study was conducted to estimate the occurrence and prevalence of brucellosis in the lactating dairy cattle located in Bannu and Lakki Marwat districts of Khyber PakhtunKhwa, Pakistan. A total of 302 blood and milk samples were collected from different breed of cattle. The blood and milk samples were screened for brucellosis on the basis of serum plate agglutination test (SPAT) and Milk Ring Test (MRT), respectively. The overall prevalence of brucellosis was found 5.2% and 10.9% in District Bannu and Lakki Marwat, respectively, moreover prevalence of brucellosis on basis of SPAT and MRT showed 7.94% and 3.97% respectively. On the basis of breeding pattern, prevalence of brucellosis was found 10.2%, 19% and 13% in local breed, Friesian cross and Jersey cross respectively and no significant association was found among them (P > 0.05). Higher prevalence of brucellosis was found in cattle with age group of greater than five years with statistically significant association (P<0.05). Moreover, in case of breeding practice the prevalence of brucellosis was found higher within unknown source of breeding followed by community bull and artificial insemination (P > 0.05). Furthermore, higher prevalence of brucellosis was found in animals that were set free for grazing compared to stall feeder animals (P > 0.05). It can be therefore concluded that regular screening of animals for brucellosis in these areas is required to control this disease. All copyrights reserved to Nexus® academic publishers Raqeebullah, Rahim K (2014). Sero– prevalence of brucellosis in cattle in southern area of Khyber Pakhtunkhwa, Pakistan. Res. J. Vet. Pract. 2 (4): 63 – 66.

Full text

Research Journal for Veterinary Practitioners 2 (4): 63 – 66
http://dx.doi.org/10.14737/journal.rjvp/2014/2.4.63.66
Bakhtullah et al (2014). Brucellosis in Cattle
63
ISSN 2308–2798
Short Communication
Bakhtullah1, Farzana Parveen1, Muhammad Shahid2, Abdul Basit3*, Mirza Ali Khan2, Saddiqa Gul4, Inamullah
Wazir5, Raqeebullah1, Kashif Rahim3
1Department of Zoology, Hazara University Mansehra, Khyber PakhtunKhwa, Pakistan; 2Microbiology and Biotechnology Center,
Veterinary Research Institute, Khyber PakhtunKhwa, Peshawar, 25000, Pakistan; 3Department of Microbiology, Kohat University of
Science and Technology, Khyber PakhtunKhwa, 26000, Pakistan ;4Department of Microbiology, Quid –e–Azam University Islamabad,
Pakistan; 5Department of Animal Health, The University of Agriculture Peshawar, Pakistan
*Corresponding author: aabdul.9090@gmail.com
ARTICLE HISTORY
ABSTRACT
Received:
Revised:
Accepted:
2014–03–25
2014–04–11
2014–04–13
Brucellosis considered as the most important zoonotic disease worldwide. The diagnosis of
brucellosis in bovines requires the use of more than one serological test for routine diagnosis.
Present study was conducted to estimate the occurrence and prevalence of brucellosis in the
lactating dairy cattle located in Bannu and Lakki Marwat districts of Khyber PakhtunKhwa,
Pakistan. A total of 302 blood and milk samples were collected from different breed of cattle.
The blood and milk samples were screened for brucellosis on the basis of serum plate
agglutination test (SPAT) and Milk Ring Test (MRT), respectively. The overall prevalence of
brucellosis was found 5.2% and 10.9% in District Bannu and Lakki Marwat, respectively,
moreover prevalence of brucellosis on basis of SPAT and MRT showed 7.94% and 3.97%
respectively. On the basis of breeding pattern, prevalence of brucellosis was found 10.2%, 19%
and 13% in local breed, Friesian cross and Jersey cross respectively and no significant
association was found among them (P > 0.05). Higher prevalence of brucellosis was found in
cattle with age group of greater than five years with statistically significant association
(P<0.05). Moreover, in case of breeding practice the prevalence of brucellosis was found
higher within unknown source of breeding followed by community bull and artificial
insemination (P > 0.05). Furthermore, higher prevalence of brucellosis was found in animals
that were set free for grazing compared to stall feeder animals (P > 0.05). It can be therefore
concluded that regular screening of animals for brucellosis in these areas is required to
control this disease.
All copyrights reserved to Nexus® academic publishers
Key Words: Brucellosis,
Cattle, SPAT, MRT, Bannu,
Lakki Marwat
ARTICLE CITATION: Bakhtullah, Basit A, Shahid M, Parveen F, Gul S, Khan MA, Wazir I, Raqeebullah, Rahim K (2014). Sero–
prevalence of brucellosis in cattle in southern area of Khyber Pakhtunkhwa, Pakistan. Res. J. Vet. Pract. 2 (4): 63 – 66.
Brucellosis is the contagious and zoonotic disease of
humans and animals worldwide especially in the Middle
East, Africa, Asia, South and Central America (Yohannes et
al., 2012 and Munir et al., 2010). It is an infectious bacterial
disease caused by member of genus Brucella, Brucella melitensis
and Brucella abortus are the principal cause of brucellosis in
bovines (Karaca et al., 2007 and Radostits et al., 2000).
Brucellosis is primary reproductive disease with potential of
great economic loss in bovines resulting abortion, death of
young stock, birth of weak calves, stillbirth, delayed calving
and infertility in males and reduced milk production in
females (Abubakar et al., 2011 and Maadi et al., 2011). This
disease occurs by means of contaminated water or feed,
inhalation and via conjunctiva, and it can be transmitted
through direct or indirect contact with infected animals or
animal secretions. Moreover ingestion of the excretion in
uterine discharge and milk of infected animals also the
source of transmission (Yohannes et al., 2012).
Signs and symptoms of this disease are nonspecific or
may be atypical. Laboratory confirmation is therefore
essential for the diagnosis of brucellosis. Moreover,
combinations of serological, cultural and molecular
techniques are essential for diagnosis. Serological blood
tests and Milk ring test (MRT) are mostly used for
diagnosis of brucellosis (Al–Mariri and Haj–Mahmoud,
2010). Detection of brucella antibodies in milk considered the
principal technique for screening and monitoring the
infected dairy herds or an individual animal for diagnosing
brucellosis at regular intervals (Godfroid and Kasbohrer,
2002). Serological tests are comparatively easy to carry out
and provide a practical benefit in estimating the prevalence
of Brucella infection in bovines (Abubakar et al., 2011).
Brucellosis is one of the major disease problems in
Pakistan. Many studies documented the prevalence of
brucellosis in livestock of different districts and provinces of
Pakistan (Rabab et al., 2000; Iftikhar et al., 2008; Mukhtar
and Kokab, 2008; Abubakar et al., 2010; Shafee et al., 2011;
Ullah et al., 2013). Moreover, livestock populations of
Pakistan are large and well adapted to the local
environmental conditions. Therefore this disease has a
Sero–Prevalence of Brucellosis in Cattle in Southern Area of Khyber
Pakhtunkhwa, Pakistan

Research Journal for Veterinary Practitioners 2 (4): 63 – 66
http://nexusacademicpublishers.com/journal/13
Bakhtullah et al (2014). Brucellosis in Cattle
64
ISSN 2308–2798
significant impact on human, animal health and
socioeconomic aspects; where the people of rural areas of
Pakistan mainly dependent on livestock, land cultivation
and their domestic animals (Shafee et al., 2011 and Maadi et
al., 2011). Due to the lack of diagnostic facilities and
financial limitations in Pakistan, veterinarians generally
prefer conventional tests for screening of brucellosis. Serum
Agglutination Test, Rose Bengal Test and Milk Ring Test
are usually performed at both private and government
livestock laboratories (Asif et al., 2009; Gul and Khan,
2007).
This study was carried to estimate the prevalence of
brucellosis in milk and serum samples collected from of
different breed of cattle located in Bannu and Laki Marwat
area of Khyber PakhtunKhwa region of Pakistan.
The study was conducted on the lactating dairy cattle in
District Bannu and Lakki Marwat, Khyber PakhtunKhwa
Pakistan, from May to July, 2011(Figure 1).
Figure 1: Map of Khyber PakhtunKhwa province showing the study area
(Highlighted Red)
A total (n = 302) blood sera and milk samples of Cattle were
randomly collected. Approximately 5 mL of milk was
collected from four quarters of each cattle into sterile glass
bottle. All the blood serum and milk samples were then kept
in an ice box and transported directly to the VRI, Peshawar.
The serum samples were subjected to SPAT for screening
brucella antibodies as described by Alton et al., (1975). The
results of agglutination in SPAT were recorded. A titer of
1:80 or above was considered positive for brucellosis
according to the instruction of the antigen manufacturer
(Global invitro.LLP Sudbury Hill, London UK).
Milk ring test was conducted on milk as describe by Alton
et al., (1988) with slight modification. The antigen
hematoxylin used in this test was supplied by Veterinary
Research Institute (VRI) Peshawar. The positive samples
were differentiated on the basis of blue ring present on the
top of milk after overnight reaction.
Statistical analysis was executed by using SPSS (version
16.0) software. By using chi square test significance of
difference was determined. Value of p < 0.05 considered as
statistically significant.
In the present study, from 302 samples total 170 and 132
blood and milk samples were collected from District Bannu
and District Lakki Marwat respectively. Among 170 samples
from District Bannu, 13 (7.64 %) and 5 (2.94%) cattle were
found positive for brucellosis on the basis of SPAT and MRT
respectively (Table 1). Similarly from 132 samples from
District Lakki Marwat, (11 8.33%) and 7 (5.30%) samples
were positive on the bases of SPAT and MRT respectively
(Table 1). With respect to the districts, incidence rate of
brucellosis was found higher in Lakki Marwat (10.8%)
compared to Bannu district (5.64%) (Table 1).
Table 1: Prevalence of brucellosis in the cattle located in Bannu and Lakki Marwat Districts, Khyber PakhtunKhwa
Pakistan
Technique used
District Bannu
District Lakki Marwat
Total no of samples
No of Positive Sample
Total no of samples
No of Positive Sample
SPAT
170
13(7.6%)
132
11(8.33%)
MRT
170
5 (2.9%)
132
7 (5.30%)
Total
340
18 (5.29%)
164
18 (10.9%)
Table 2: Prevalence of brucellosis in locally available breed of the cattle, in Bannu and Lakki Marwat distracts, Khyber
PakhtunKhwa Pakistan
Breed
Total sample
Positive on SPAT
Positive on MRT
Total
p– value
Local cross
176
14 (7.95%)
4 (2.27%)
18 (10.2 %)
P > 0.05
Friesian cross
68
7 (10.29%)
6 (8.82%)
13 (19 %)
Jersey cross
37
3 (8.10%)
2 (5.40%)
5 (13%)
Sahiwal
21
0
0
0
Total
302
24 (7.94%)
12 (3.97%)
36 (11.9%)
From total 302 Cattles, 176 were local breed, 68 were
Friesian cross, 37 were Jersey cross and 21 were Sahiwal
breed. From 176 Local Breed cattle, 14 (7.95%) and 4
(2.27%) were found positive for brucellosis on bases of
SPAT and MRT respectively. Similarly in Frisian cross, 7
(10.29%) and 6 (8.82%) and in Jersey cross, 3 (8.10%) and 2
(5.40%) animals were found positive on SPAT and MRT
respectively. No significant association was found among
different breeds of cattle (P > 0.05) (Table 2).
Present study also distributed the cattle in to different age
groups. In age group of 2–4 year, 5–7 year, 8–10 years and 11–
16 years the incidence rate of brucellosis were found 2.6%,
20.3%, 22% and 21.4% respectively (Table 3).

Research Journal for Veterinary Practitioners 2 (4): 63 – 66
http://nexusacademicpublishers.com/journal/13
Bakhtullah et al (2014). Brucellosis in Cattle
65
ISSN 2308–2798
From total 176 artificial inseminated (AI) cattle, 12 (6.81%)
and 6 (3.40%) having brucella infection on the bases of SPAT
and MRT respectively. Similarly in 103 naturally mated
cattle with community bull, 9 (8.73%) and 4 (3.88%) were
positive for brucellosis on the bases of SPAT and MRT
respectively.
The rest of 23 cattle were with unknown source of
breeding pattern, showed 3 (13.04%) and 2 (8.69%) samples
positive on SPAT and MRT, respectively. The incidence of
brucellosis was found higher in cattle mated with unknown
source followed by community bull and then artificially
inseminated (Figure 1). Moreover no significant association
of brucella infection in cattle with respect to breeding
practices were found (P > 0.05).
Feeding Practice
Total Samples
SPAT
MRT
p– value
Stall feeding
246
18 (7.31%)
9 (3.65%)
P > 0.05
Grazing
56
6 (10.71%)
3 (5.35%)
In the present study a total 246 cattle were found stall
feeders and 56 were grazed. In case of stall feeders, 18
(7.31%) and 9 (3.65%) samples were found positive on the
basis of SPAT and MRT respectively.
Similarly from total 56 samples, 6 (10.71%) and 3
(5.35%) were positive for brucellosis on SPAT and MRT,
respectively. No significant association was found in them
(P > 0.05) (Table 4).
Brucellosis is considered most important zoonotic disease.
As this disease make significant economic losses to livestock
industry, therefore common diagnostic techniques are
essential for its efficient diagnosis. Diagnostic laboratories
mostly utilize serological test and MRT for identification of
Brucella species in the host.
In present study, sero–prevalence of brucellosis was
found higher on SPAT (7.94%) as compared to MRT
(3.97%). These findings are in agreement of Saleha et al.,
(2014), they also revealed in their study that SPAT test
showed more positive results compared to MRT. This might
be due to fact that milk proteins hinder the brucella
antibodies isolation (Akhtar et al., 2010).
In present study overall prevalence of brucellosis in District
of Bannu and Lakki Marwat was found 5.29% and 10.9%
respectively on the bases of SPAT and MRT. Shafee et al.,
(2011), in their study reported the prevalence of 8.5% in
Quetta Pakistan. Similar study was also conducted by Song
et al., (2009) who estimated the sero–prevalence brucellosis
5.0% and 0.5% in cattle located in Bangladesh Agriculture
University, by using antigens of Brucella abortus and Brucella
melitensis. The same patterns of results were observed by
Wali et al., (2005), who assessed the sero–prevalence of
brucellosis in livestock at different districts of Khyber
PakhtunKhwa. The variation in results might be due to
sample size and different habitat of animals.
In the present study the prevalence of brucellosis in age
group of > 5 showed higher prevalence compared to the
other age group of Cattle (Table 3). These findings are in
agreement with Ahmad et al., (2009), who reported the high
prevalence of brucellosis in age group of more than five
compared to the younger animals. The reason might be due
to fact that with the passage of time animals are likely to be
exposed to the Brucella infection. Radostits et al., (2000) also
revealed that this disease is chronic and increases with age.
Age
Total Sample
SPAT
MRT
Total
2–4 years
151
3 (1.98%)
1 (0.66%)
4 (2.6%)
5–7 years
64
8 (12.5%)
5 (7.81%)*
13 (20.3%)
8–10 years
59
9 (15.25%)
4 (6.77%) *
13 (22%)
11–16 years
28
4 (14.28%)
2 (7.14%)
6 (21.4%)
Table 3: Age wise distribution of brucella infection in
the cattle in Bannu and Lakki Marwat distracts,
Khyber PakhtunKhwa Pakistan
Table 4: Prevalence of brucella infection in the cattle
on the basis of feeding practices in Bannu and Lakki
Marwat distracts, Khyber PakhtunKhwa Pakistan
Figure 1: Prevalence of brucella infection in the c attle with respect to breeding practice in Bannu and L akki Marwat distracts, Khyber PakhtunKhwa Pakistan

Research Journal for Veterinary Practitioners 2 (4): 63 – 66
http://nexusacademicpublishers.com/journal/13
Bakhtullah et al (2014). Brucellosis in Cattle
66
ISSN 2308–2798
The present study also revealed that Sahiwal (Native) and
local breed of Cattle showed relative low prevalence of
brucellosis 0.0% and 7.95% respectively, compared to
Friesian cross and Jersey cross breeds (Table 2). It might be
due to fact that the native and local cross breeds have
developed immunity and more resistance to brucella
infection as compared to Friesian and Jersey cross breeds.
These findings are in agreement with the result of
Akbarmehr and Ghiyamirad (2011), revealed that the
brucellosis was dominant in non–local breeds of Cattle
compared to native and local cross breeds.
Moreover the present study also studied the breeding
pattern. It was determined that naturally matted samples
have high risk of brucellosis with respect to artificial
insemination. It may be due to the regular use of community
bull not for single cattle or herd. In the present study high
prevalence of brucellosis was also reported in grazing cattle
followed by stall feeder. It might be due to congregation of
the animals feeding and drinking from common source.
It is concluded that brucellosis is prevalent in these
districts and this disease can be diagnosed through regular
testing of the animals and awareness should be created to
lower the risk of this disease.
REFERENCES
Abubakar M, Arshed MJ, Hussain M, Ehtisham–ul–Haq, Ali Q (2010).
Serological evidence of brucella abortus prevalence in Punjab province,
Pakistan–a cross–sectional study. Transbound Emerg Dis. 57: 443 –447.
Abubakar M, Mansoor M, Arshed MJ (2011). Bovine Brucellosis. Old and new
concepts with Pakistan perspective. Pak Vet .J. 32: 1 –9.
Ahmad M, Al–Majali, Talfha AQ, Ababneh MM, Ababneh M (2009).
Seroprevalence and risk factors for bovine brucellosis in Jordan.Vet. J.
of Sci. 10: 61– 65.
Akbarmehr J, Ghiyamird M ( 2011). Serological survey in livestock animals in
Sarab city, Iran. African J. of Micro. 5: 1220 –1223.
Akhtar R, Chaudhry ZI, Shako ori AR, Ahmad M, Aslam A (2010).
Comparative efficacy of conventi onal diagnostic methods and
evaluation of polymerase chain re action for the diagnosis of bovine
brucellosis. Vet World. 3: 53–56.
Al–Mariri A, Haj–Mahmoud N (2010). Detection of Brucella abortus in Bovine
Milk by Polymerase Chain Reaction. ACTA VET. BRN O. 79: 277–280.
Alton GG, Jones LM, Angus RD , Verger JM ( 1988). Techniques for the
Brucellosis Laboratory. Institute National de la Recherche
Agronomique, Paris, France.13–61.
Alton G G, Jones L M, Pietz DE (1975). Laboratory techniques in brucellosis.
In: world health organization monograph series Nº 55, Geneva,
Switzerland. Volume 2: 1 – 163.
Asif M, A wan AR, Babar ME, Al i A, Firy al S, Khan QM (2009). Development
of genetic m arker for molecular detection of Brucella abortus. Pak J Zool
Suppl Ser. 9: 267–271.
Godfroid J, Käsbohrer A (2002). Brucellosis in the European Union and
Norway at the turn of the t wenty–first century. Vet Microb iol. 90: 135–
45.
Gul ST, Khan A (2007). Epidemiology and epizootology of b rucellosis; a
review. Pak. Vet. J. 27: 145–151.
Iftikhar H, Arshad MI, Mahmood MS, Akhtar M (2008). Seroprevalence of
Brucellosis in Human, Cattle, and Buffalo Populations in Pakistan .
Turk. J. Vet. Anim. Sci. 32(4): 315–318.
Karaca M, abur CB, Ueleb i B, Akkan HA, Tutuncu M, Keleu II, Atalay UB,
Kiliu S (2007). Investigation on the seroprev alence of toxoplasm osis,
listeriosis and brucellosis in goats living in the region of Van, Turkey.
Yuzuncu Yil universitesi Veteriner Fakultesi Derg isi. 18: 45–49.
Maadi H, Moharamnejad M, Haghi M (2011). Prevalence of Brucellosis in
Cattle in Urmia, Iran. Pak. Vet. 31(1): 81–82.
Mukhtar F, Kokab F. Brucella serology in abattoir workers (2008). J. Ayub
Med. Coll. Abbottabad. 20(3): 57–61.
Munir R, Af zal M, Hussain M, Naqvi SMS, Khanum A ( 2010). Outer
membrane proteins of B. abortus vaccinal and field str ains and their
immune response in buffaloes. Pak. Vet. J. 30: 110–114.
Rabab A, Att as A, Khalifa MA, Al Qurashi AR, Badawy M, Al Gul ay N
(2000). Evaluation of PCR, culture and serology for the diagnosis of
acute human Brucellosis. Ann. Saudi. Med. 20(4): 224 –228.
Radostits OM, Gay CC, Blood DC, and Hinchcliff KW (2000). Veterinary
Medicine ELBS. Bailliere Tindall, London, UK. 870–871.
Saleha S, Basit A, Rahim K, Shahid M, Khan MA (2014). Comparison of milk
ring test; serum plate agglutination test and polymerase chain reaction
for the detection of bovine brucellosis. Res. j. vet. pract. 2(1): 5 – 8.
Shafee M, Rabbani M, Sheikh AA, Ahmad MD, Razzaq A (2011). Prevalence of
bovine b rucellosis in org anized d airy f arms, using m ilk ELI SA, in
Quetta City, Balochistan,Pakistan. Vet. Med. Int. 1–3.
Song HJ, Ahasan MS, Hugue AF, Rahm an AA, Alam N, Rahman MS
(2009).Seroprevalence of sp ecific bruce llosis infection of cattle in
Bangladesh Agriculture University, Korean. Vet. J. 32: 219 – 225.
Ullah RW, Shirazi JH, Abub akar M, Zahur AB, Latif A, Alam T (2013).
Genetic diversity, zoonotic risk and “One Health” initiative of b ovine
brucellosis. Res. J. Vet. Pract. 1 (1): 5–9.
Wali A (2005). Seroprevalence of brucellosis in various districts of Khyber
Pakhtunkhwa, (KP) M.Phil Thesis, Department of Biological Sciences
Quaid–e–Azam University Islamabad. 22–54.
Yohannes M, Mersha T, Degefu H, Tolosa T, Woyes a M (2012). Bovine
Brucellosis: Serological Survey in Guto–Gida District, East Wollega
Zone, Ethiopia. Global Veterinaria. 8 (2): 139–143.