Seroprevalence of Brucellosis in High Risk Professionals in Pakistan

Shahzad Ali1, Falk Melzer2, Iahtasham Khan2, Qurban Ali3, Nemat Ullah1, Muhammad Irfan1, Ali Muhammad1, Gamal Wareth 2, Shamim Akhter1, Heinrich Neubauer2
1Pir Mehr Ali Shah Arid Agriculture University (PMAS-AAUR), Rawalpindi, 46300, Pakistan; 2Friedrich-Loeffler-Institut, Bundesforschungsinstitut fr Tiergesundheit Naumburger Strae 96a, 07743,Jena, Germany;
3National Veterinary Laboratories, Park Road, Islamabad, Pakistan

INTRODUCTION

RESULTS

Brucellosis is an important zoonotic disease of animals and humans. It causes

high economic losses worldwide, particularly in developing countries (Nikokar et
al., 2011). Brucellosis is transmitted from infected animals to humans, who are in
close contact with infected material i.e. aborted fetus (Figure 1), secretions, or
consume infected milk or milk products. Shepherds, milkmen, butchers, veterinary
assistants, abattoirs workers e.g. are at high risk (Figure 2) (Agasthya et al.,
2007). Pakistan is an agricultural country, where mostly illiterate people
particularly from rural areas depend on livestock for their livelihood. No report on
the current status of human brucellosis in Pakistan is available. Only
seroprevalence studies were conducted (Hussain et al., 2008). Nowadays, PCR
and realtime PCR (RT-PCR) assays are applied having several advantages when
compared to traditional serological techniques. This study was designed to
evaluate the seroprevalence of brucellosis by using serology and genus and
species specific RT-PCR in the human population of Pakistan.

Out of 262 samples 15 (5.73%) were found to be positive for brucellosis on the basis of both serology and confirmatory
genus specific/species specific RT-PCR (Table 1). The prevalence of brucellosis was higher 14 (6.36%) in male than
women 1 (2.38%). Almost individuals of all age groups were positive for brucellosis. Individual in category milkers 1
(1.89%), livestock farmers 5 (4.67%) and abattoir workers 9 (16.7%) were found to be seropositive. People having
contact with animals were more seropositive 15 (6.12%) compared to people having no contact with animals. Moreover,
sero-positivity was high 15 (83.3%) in peoples having brucellosis associated symptoms. People in ICT were found to be
more positive (11%) compared to other regions. Among 18 individual who were positive in serological tests, 15 (Figure 5)
were found to be positive in genus specific BCSP31 and species specific IS711 for Brucella abortus RT-PCR. Moreover,
randomly selected serologically negative samples (n = 20) were always negative in BCSP31 genus specific RT-PCR.

Figure 1: Aborted buffalo fetus collected

from human brucellosis sampling site

Figure 2: Screening of farm animals for

brucellosis in Pakistan

MATERIALS AND METHODS

A total of 262 human blood samples were collected randomly from high risk
professionals including veterinary personnel, milkers, abattoir workers, livestock
farmers and others (housewives, drivers, cooks, security guards) on the Potohar
Plateau including Islamabad Capital Territory (ICT), Rawalpindi and Attock
districts, Pakistan (Figure 3). This study started in July 2011 and ended in
December 2011 and was approved by the ethical committee of Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi, Pakistan. The RBPT (Rose-Bengal-Test)
and SAT (Slow-Agglutination-Test) were performed according to standard
procedures (Alton et al., 1988). Then DNA was extracted from all seropositive
serum samples (n = 18) and randomly selected negative samples (n = 20).
Extraction was done by Highly Pure PCR Template Preparation kit (Roche
Diagnostics, Mannheim, Germany) according to the manufacturers instructions
(Figure 4). DNA purity and concentration was checked using a Nano-Drop ND1000 UV-Vis spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA).
After that all DNA samples were examined by a Brucella genus-specific and two
species-specific (Brucella abortus and Brucella melitensis) realtime PCR assays
(Probert et al., 2004). A sample was considered positive if it was positive in RTPCR and/or serology.

Table 1: Seroprevalence and PCR Results for Brucellosis in High Risk Groups
Variables

Samples
Examined

Positive Samples
SAT
BCSP31
(RT-PCR)

RBPT

IS711

Sex (n = 262)
Male
Female

220
42

17
1

9
0

14
1

14
1

Age (n = 262)
Upto 20 (*Y)
21-30
31-40
41-50
51-60
Above 60

25
85
49
48
35
20

4
5
2
4
1
2

3
3
0
3
0
0

4
5
0
3
1
2

4
5
0
3
1
2

Occupational groups (n = 262)

Veterinary
Personnels
31
Milkers
53
Abattoir workers
54
Livestock farmers
107
Others
17

0
1
10
7
0

0
0
5
4
0

0
1
9
5
0

0
1
9
5
0

Contact with animals (n = 262)

Yes
245
No
17

18
0

9
0

15
0

15
0

Symptoms (n = 262)
Yes
No

18
244

18
0

9
0

15
0

15
0

Regions (n = 262)
*ICT
Rawalpindi
Attock

82
110
70

10
6
2

8
1
0

9
4
2

9
4
2

*ICT = Islamabad Capital Territory, *Y = years

DISCUSSION

China

1. Islamabad Capital Territory (ICT)

2. Rawalpindi

Gilgit-Baltistan
Khyber
Pakhtunkhwa
Province

3. Attock

1
3

Afghanistan

Punjab Province

Blochistan Province
India
Sindh Province

Arabian sea

Figure 3: Location of sampling sites

in Pakistan

Figure 4: High Pure PCR Template Preparation

Kit (Roche Diagnostics, Mannheim, Germany)

In present study male were found to be more seropositive (6.36%) as compared to female (2.38%). Similar male gender
related high seroprevalence were reported in previous studies from neighbour countries i.e. India and Bangladesh
(Thakur and Thapliyal, 2002; Rahman et al., 2011). Seropositive case was found almost in all age groups in present
study. Our finding that brucellosis is present in all age groups regarding present study is inline with study done in Algeria
(Habib et al., 2003). Sero-positivity varies in five occupational groups and abattoir workers recorded highly infected. The
reason of high seroprevalence of brucellosis in abattoir workers was due to their direct contact with animals and their
body secretion/fluid. Our finding is in line with investigations done in high risk groups from Tanzania (Swai and
Schoonman, 2009). After abattoir workers, livestock farm workers were identified as more sero-positive (4.67%) for
brucellosis. Sero-positivity among these peoples is due to milk of infected animals is a source of infection. Moreover,
consumption of raw milk, washing of animal teat before milking, hands washing before and especially after milking are
not generally practiced. This group having contact with other body secretion and excretions of animals apart from milk,
handling of calves during parturition and aborted fetuses are also their duty. It is well known that milk and abortion
related animal material have significant number of brucella in it which can infect human having contact with these animal
commodities (Nikokar et al., 2011). Sero-positivity was found in milkers during our study like previous study (AboShehada et al., 1996). No seropositive case was identified in case of veterinary personnel. More or less symptoms of
brucellosis were noted in more of these seropositive cases in present study and likewise brucellosis related symptoms
were noted in high risk professionals from previous finding (Rezaee et al., 2012). Variation in sero-positive cases were
seen in three regions. Higher prevalence (11%) was seen from Islamabad capital territory (ICT) because many abbattoir
workers are from this region. Similarly, variation in sero-positivity in different localities was found to in a study in Georgia
related to human brucellosis (Havas et al., 2012).

Figure 5: Amplification plots of serum DNA samples positive in realtime PCR

CONCLUSION
Although most of the studies related to human brucellosis especially in case of high risk personnels relay on serological tests, an first attempt was made to support serological evidence of brucellosis in
human with brucella genus and species specific RT-PCR in this study. Using IS711 species specific realtime PCR it was confirmed that Brucella abortus is present in human population of Pakistan and
especially in high risk professionals.
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Acknowledgement: This research work was supported by Indigenous PhD Fellowship Program, International Research Support Initiative Program (IRSIP) of Higher Education Commission Pakistan and the Friedrich-LoefflerInstitut, Jena, Germany.
Address of corresponding author: Shahzad Ali, Friedrich-Loeffler-Institut, Naumburger Strasse 96a, 07743 Jena; email: shahzaduaar772@yahoo.com