Trop Anim Health Prod (2014) 46:7378

DOI 10.1007/s11250-013-0448-6

REGULAR ARTICLES

Isolation and identification of bovine Brucella isolates

from Pakistan by biochemical tests and PCR
Shahzad Ali & Qurban Ali & Falk Melzer & Iahtasham Khan &
Shamim Akhter & Heinrich Neubauer & Syed M. Jamal

Accepted: 2 July 2013 / Published online: 19 July 2013

# Springer Science+Business Media Dordrecht 2013

Abstract Brucellosis is endemic in bovines in Pakistan. The

Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate
and characterize brucellae from seropositive milk samples,
aborted fetuses, and vaginal swabs of cattle and buffaloes
which had recently aborted. The seropositive milk samples,
aborted fetuses, and vaginal swabs of cattle and buffaloes
were collected from the Potohar Plateau, Pakistan. Isolation
of brucellae was done on modified Farrells serum dextrose
S. Ali : S. Akhter
Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi 46300,
Pakistan
S. Akhter
e-mail: sashraf1993@gmail.com
S. Ali : F. Melzer : I. Khan : H. Neubauer
Friedrich-Loeffler-Institut, Bundesforschungsinstitut fr
Tiergesundheit, Naumburger Strae 96a, 07743 Jena, Germany
F. Melzer
e-mail: Falk.Melzer@fli.bund.de
I. Khan
e-mail: drkhan_uaf@yahoo.com
H. Neubauer
e-mail: Heinrich.Neubauer@fli.bund.de
S. Ali (*) : Q. Ali : S. M. Jamal
National Veterinary Laboratory, Park Road, Islamabad, Pakistan
e-mail: shahzaduaar772@yahoo.com
Q. Ali
e-mail: drqurban@yahoo.com
S. M. Jamal
e-mail: jamal115@yahoo.com
I. Khan
University of Veterinary and Animal Sciences, Lahore, Pakistan
S. M. Jamal
Ministry of National Food Security and Research, Islamabad,
Pakistan

agar. Isolates were characterized by conventional biotyping

methods, while molecular typing was done by genus
(B4/B5) and species-specific (Brucella abortus, Brucella
melitensis, Brucella ovis, and Brucella suis) polymerase
chain reaction (PCR). A total of 30 isolates were recovered
from milk (n=5), aborted fetuses (n=13), and vaginal swabs
(n=12). Most isolates were from cattle (56.7 %). All of them
were identified as B. abortus biovar 1 based on conventional
biotyping methods and genus and species-specific PCR. This
preliminary study provides the first report on the prevalence
of B. abortus biovar 1 in cattle and buffaloes in Pakistan.
Keywords Brucella abortus . Pakistan . Biochemical tests .
PCR

Introduction
Brucellosis is a widespread zoonotic disease affecting both
humans and animals (Shahaza et al. 2009). Brucella abortus
is usually considered as the causative agent of bovine brucellosis, but Brucella melitensis may also cause disease,
especially in areas where bovines have close contact with
sheep and goats (Ahmed et al. 2010). Apart from these two
species, Brucella suis has also been reported in bovines (Tae
et al. 2012).
Pakistan is bordered by Afghanistan and Iran in the west,
India in the east, and China in the far northeast. In most of the
neighboring countries of Pakistan, brucellosis outbreaks
have been reported (Abubakar et al. 2012).
According to the World Health Organization, brucellosis
is considered a neglected disease, particularly in Pakistan
(World Health Organization (WHO) 2012). In Pakistan,
23.9 % of the population lives below the poverty line. Cattle
or buffaloes are mainly kept by the poor rural farmers as a
subsistence farming (The World Bank 2008). Brucellosis has a
negative impact on animal health, pregnancy, and milk

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Trop Anim Health Prod (2014) 46:7378

production, and its zoonotic implications cannot be ignored

due to lack of awareness and poor literacy rate of the dairy
farmers.
Pakistan is an agricultural country, and livestock sector is
the backbone of agriculture as it has 55.1 % stake in agriculture (Economic Survey 201112). Livestock is subsistence
sector dominated by small holders to meet their needs of
milk, food and cash income on daily basis. Livestock is
considered as a more secure source of income in rural areas.
The livestock population in Pakistan is assessed at 35.6
million cattle, 31.7 million buffaloes, 28.1 million sheep,
61.5 million goats, 1.0 million camels, 0.4 million horses,
0.2 million mules and 4.7 million donkeys (Economic
Survey 20112012). Apart from other diseases, bovine brucellosis is the major disease of livestock in Pakistan. The
prevalence of brucellosis in the country has been ascertained
using only sero-surveillance (Hamidullah et al. 2009;
Mukhtar 2010; Abubakar et al. 2010, 2012). However, no
report on isolation and characterization of prevalent Brucella
species and biovars based on biochemical tests and polymerase chain reaction (PCR) is available from Pakistan.
Serological data may be misleading due to cross-reactivity
between Brucella and other Gram-negative bacteria.
Furthermore, serological methods cannot differentiate between vaccinated and infected animals. Thus, the isolation
of bacteria is considered as a gold standard for the diagnosis
of brucellosis (Yu and Nielsen 2010). Moreover, molecular
methods like PCR assay enable safe identification of the
bacteria at species level (Huber et al. 2009).
The aim of this study was to isolate brucellae from milk,
aborted fetuses, and vaginal swabs of cattle and buffaloes
and to characterize these isolates using the genus (B4/B5)
and species-specific (AMOS) PCR, respectively, and to

ascertain biovar of Brucella species, employing biochemical

tests, growth in the presence of certain dyes, and agglutination with specific sera.

Materials and methods

Sample collection
A total of 2,330 milk (1,168 cattle and 1,162 buffaloes)
samples were randomly collected from different ecological
regions of three districts located on the Potohar Plateau of
Pakistan. These milk samples were initially screened using
Brucella milk ring test (MRT). Positive milk samples were
cultured. In addition, 49 aborted fetuses and 95 vaginal
swabs from cattle and buffaloes with a history of recent
abortion were also investigated (Table 1).
Isolation of bacteria
Isolation of Brucella was conducted on modified Farrells
serum dextrose agar according to standard procedures
(Farrell and Robinson 1972; Alton et al. 1988). Modified
Farrells serum dextrose agar with 5 % horse serum, 1 %
dextrose, and the following antibiotics (added to 1-l medium): cycloheximide (100 mg), bacitracin (25,000 IU), polymyxin B sulfate (5,000 IU), vancomycin (20 mg), nalidixic
acid (5 mg), and nystatin (100,000 IU) were used for primary
selective isolation of brucellae. Plates were inoculated with
sample material and incubated aerobically and in the presence of 510 % carbon dioxide at 37 C. These plates were
examined 37 days post-inoculation for bacterial growth.

Table 1 List of samples from milk, aborted fetuses, and vaginal swabs collected from different sampling sites of the Potohar Plateau, Pakistan
Sampling sites

ICTb
Chak Shahzad
Rawat
Rawalpindi
Kallar
Chountra
Kahuta
Attock
Kherimurat
Attock
Ahmadal

Milka

Aborted fetuses

Vaginal swabs

Cattle

Buffalo

Cattle

Buffalo

Cattle

Buffalo

30
10

12
8

8
15

7
13

28
17

11
12

9
5
5

7
10
4

4
0
0

1
0
0

5
0
7

8
0
3

8
10
7

13
6
12

1
0
0

0
0
0

3
0
0

1
0
0

Positive milk samples in MRT are given in table in front of each sampling site

Islamabad Capital Territory

Trop Anim Health Prod (2014) 46:7378

75

Table 2 PCR type along with primers type, sequence (5 to 3), target gene, and product length (in base pair) used for Brucella genus-specific (B4/
B5) PCR
PCR type

Primers type

Sequence (5 to 3)

Target gene

Product length (bp)

Brucella
genus

Forward
Reverse

TGG CTC GGT TGC CAA TAT CAA

CGC GCT TGC CTT TCA GGT CTG

bcsp31

223

Biovar typing and Brucella genus-specific (B4/B5)

and species-specific AMOS PCR
Suspected colonies were subcultured for purity on serum
dextrose agar. Identification of these isolates was done
according to standard procedures (Alton et al. 1988). The
isolates were initially examined for Gram and modified
ZiehlNeelsen (MZN) staining. Subsequent biochemical
tests for oxidase, catalase, urease production, hydrogen sulfide production, carbon dioxide requirement, growth on media containing basic fuchsin and thionin (20 g/ml), and
agglutination by monospecific antisera (A, M, R; Anses,
Paris) were carried out. DNA was extracted from colonies
using the High Pure PCR Template Preparation Kit (Roche
Diagnostics, Mannheim, Germany) according to manufacturers instructions. Purity and concentrations of DNA were
assessed using the Nano-Drop ND-1000 UVVis
Spectrophotometer (Nano-Drop Technologies, Wilmington,
DE, USA), and preparations were stored at 20 C for
subsequent analysis.
The extracted DNA preparations were screened with
genus-specific Brucella PCR using B4/B5 primers
(Table 2). Briefly, PCR was done in a 25 l reaction mix
containing 18.3 l HPLC water, 2.5 l PCR puffer (10),
1 l deoxyribonucleotide triphosphate (dNTP) (10 mM),
1 l each primer (10 pmol/l), 0.2 l (5 U/l) Taq polymerase, and 1 l of DNA template. Amplification was done with
initial denaturation for 5 min at 93 C, followed by 35 cycles
for denaturation for 1 min at 90 C, annealing for 1 min at
60 C, elongation for 1 min at 72 C, and final elongation for
5 min at 72 C. Positive samples were subjected to AMOS

PCR for species identification (Bricker and Halling 1994).

Primers are given in Table 3. AMOS PCR was performed in
25 l reaction volume having 19.3 l of HPLC water, 2.5 l
of PCR puffer (10), 1 l dNTP mix (10 mM), 1 l primer
mix (10 pmol/l), 0.2 l Taq polymerase (5 U/l), and 1 l
of DNA template. Amplification was carried out with initial
denaturation for 5 min at 95 C, followed by 30 cycles
(denaturation for 1 min at 95 C, annealing for 2 min at
58 C, elongation for 2 min at 72 C, and final elongation
for minutes at 72 C). The PCR product was run on a 1.5 %
agarose gel along with DNA ladder for 90 min at 105 V,
stained with ethidium bromide (1 mg/ml) and visualized
under UV light using a gel documentation system (Syngene,
UK).

Results
Out of 2,330 milk samples, 156 (6.7 %) were found positive
in MRT. Among these MRT positive samples, 84 (53.8 %)
were from cattle, and 72 (46.2 %) were from buffaloes.
Thirty Brucella isolates were recovered from these samples,
including 5 from milk, 13 from aborted fetuses, and 12 from
vaginal swabs (Table 4).
All the 30 isolates were Gram-negative and MZN-positive
coccobacilli. They were positive in the Brucella genusspecific (B4/B5) PCR and identified as B. abortus by
AMOS PCR (Fig. 1). Biochemical tests showed that these
isolates were positive for oxidase and catalase production,
but variation was seen in the strength of reaction; some
isolates showed strong reaction, and others, weak reaction.

Table 3 List of primers along with sequence (5 to 3), target gene, and product length (in base pair) used for AMOS PCR
PCR type

Primer type

Sequence (5 to 3)

Target gene

Product length (bp)

AMOS

BA (F)
BA (R)
BM (F)
BM (R)
BO (F)
BO (R)
BS (F)
BS (R)

GAC GAA CGG AAT TTT TCC AAT CCC

TGC CGA TCA CTT AAG GGC CTT CAT
AAA TCG CGT CCT TGC TGG TCT GA
TGC CGA TCA CTT AAG GGC CTT CAT
CGG GTT CTG GCA CCA TCG TCG
TGC CGA TCA CTT AAG GGC CTT CAT
GCG CGG TTT TCT GAA GGT TCA GG
TGC CGA TCA CTT AAG GGC CTT CAT

IS711

498

IS711

731

IS711

976

IS711

285

BA B. abortus, BM B. melitensis, BO Brucella ovis, BS B. suis, F forward, R reverse

76

Trop Anim Health Prod (2014) 46:7378

Table 4 Source and characterization of B. abortus isolates of cattle and buffaloes from the Potohar Plateau, Pakistan
Isolate number Animal species Sample type District

Oxidase Catalase Urease H2S CO2 Growth

on media

Agglutination Species/biovar

THa BFb A

AF

Attock

+c

B. abortus/1

2
34
58
910
11
1218
1920
2122
23
2426
27
28
2930

C
C
C
B
C
B
C
B
C
C
C
B
C

AF
AF
VS
VS
VS
AF
M
VS
VS
AF
M
AF
M

Attock
Rawalpindi
ICT
ICT
ICT
ICT
ICT
ICT
ICT
ICT
ICT
ICT
ICT

+
+
+
+
+
+
++e
+
+
+
++
+
++

+
+
+
+
+
+
++
+
+
+
++
+
++

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
++
+
+
+
++
+
++

+
+
+
+
+
+

+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1
B. abortus/1

B buffalo, C cattle, AF aborted fetus, VS vaginal swab, M milk

a

Thionin (20 g/ml)

Basic fuchsin (20 g/ml)

Weak positive reaction

Negative reaction

Strong positive reaction

The isolates were positive for urease and hydrogen sulfide

production. The isolates grew on media containing basic
fuchsin but failed to grow on thionin media. Most colonies
(83.3 %) required carbon dioxide for growth, while few
isolates (16.7 %) grew aerobically. All the isolates showed
agglutination with Brucella-monospecific antiserum A,
while no agglutination was observed with antisera M and R
(Table 4). On the basis of these tests, the isolates were
confirmed to be B. abortus biovar 1.

Fig. 1 AMOS PCR for B. abortus. Lane M 100-bp marker; lanes 115
B. abortus DNA in samples from Potohar Plateau, Pakistan; lane 16
positive control B. melitensis 16 M (10 ng/l); lane 17 B. abortus
BA544 (10 ng/l); lane 18 B. suis (10 ng/l); lane 19 non-template
control

Discussion
Seroprevalence studies in animals show that brucellosis is
endemic in Pakistan (Hamidullah et al. 2009; Munir et al.
2011; Shafee et al. 2011). However, the Brucella species and
their biovars endemic in Pakistan are unknown. The present
study provides the first evidence for the presence of B.
abortus biovar 1 in cattle and buffaloes on the basis of
species-specific PCR and biochemical tests.
Isolation of Brucella is cumbersome, labor-intensive, and
time-consuming but can be achieved even with conventional
techniques using the candle jar method to obtain
microaerophilic conditions (Shareef 2006). Hence, up to
our knowledge, no Pakistani investigation was made in the
past to culture brucellae from animals. Applying the best
possible culture conditions including 510 % CO2 atmosphere, these authors cultured 43.3 % of brucellae from
aborted fetuses and 40 % from vaginal excretions/swabs.
These findings are in accordance to the previous reports from
Sri Lanka and Kenya (Priyantha 2011; Muendo et al. 2012).
Ocholi et al. (2004) isolated brucellae from aborted fetuses,
hygroma fluids, milk, and vaginal swabs obtained from
aborting cattle, sheep, goats, pigs, and horses in Nigeria.
They reported a rate of isolation from milk to be 7.2 %.

Trop Anim Health Prod (2014) 46:7378

The recovery of B. abortus from milk samples in the present

study was low, i.e., 3.2 %.
Shedding of Brucella in the milk of infected animals is an
important source of transmission of disease to humans if the
raw milk is consumed. Hence, pasteurized or boiled milk is
recommended to be used. Further, dairy product prepared
from Brucella-contaminated milk have also been implicated
for human brucellosis (Tantillo et al. 2003). The isolation of
Brucella from milk samples may be improved if more than
one culture medium is used.
All isolates showed a phenotype typically for B. abortus
biovar 1 except requirements of carbon dioxide. However,
variation in carbon dioxide requirement has been reported
previously (Ocholi et al. 2004). B. abortus isolates positive for catalase, oxidase, and urease were recovered
from the semen of breeding bulls in the neighboring
country India, but among these isolates, some were negative for hydrogen sulfide production in contrast to our
isolates (Nagalingam et al. 2012). All the isolates of B.
abortus showed agglutination reaction with monospecific
antiserum A. None of them agglutinated against monospecific antisera M and R. These are characteristics of
smooth strains of Brucella.
Although, B. abortus is identified as the major cause of
brucellosis in bovines in neighboring countries, i.e., India
and Iran (Kumar and Basanti 2004; Doosti and Dehkordi
2011) and worldwide (Adesiyun et al. 2011; Priyantha 2011;
Martinez-Herrera et al. 2012), B. melitensis and B. suis have
also been reported from cattle (Tae et al. 2012; World Health
Organization (WHO) 2012). This may be due to rearing of
different species of animals together.
B. abortus biovar 1 is the most prevalent biovar in countries where bovine brucellosis is endemic. Characterization
of B. abortus from India revealed the presence of B. abortus
biovars 1, 2, and 4 in India (Kanani 2007). Results of the
biochemical tests and growth of Brucella in the presence of
dyes revealed that all the isolates belonged to B. abortus
biovar 1 in the present study. To the best of our knowledge,
there is no report of isolation of B. abortus so far from
Pakistan. In this study, B. abortus was isolated from
bovines and characterized to be biovar 1. This appears
to be the first report of isolation of B. abortus from
Pakistan. Because the present study is restricted to limited geographical area, study in other areas is needed to
explore the presence of B. abortus in bovine population
in other parts of the country.
Acknowledgments SA is a recipient of Indigenous PhD Fellowship
and International Research Support Initiative Program from the Higher
Education Commission, Pakistan, at the Friedrich-Loeffler-Institut,
Jena, Germany.
Conflict of interest The authors declare that they have no conflict of
interest.

77

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