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

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-Loeffler- Institut, Jena, Germany.

Address of corresponding author: Shahzad Ali, Friedrich-Loeffler-Institut, Naumburger Strasse 96a, 07743 Jena; email: shahzaduaar772@yahoo.com

INTRODUCTION

MATERIALS AND METHODS

RESULTS

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.

DISCUSSION

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.

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 ND-

1000 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 RT-

PCR and/or serology.

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

Variables Samples Positive Samples

Examined RBPT SAT BCSP31 IS711

(RT-PCR)

Sex (n = 262)

Male 220 17 9 14 14

Female 42 1 0 1 1

Age (n = 262)

Upto 20 (*Y) 25 4 3 4 4

21-30 85 5 3 5 5

31-40 49 2 0 0 0

41-50 48 4 3 3 3

51-60 35 1 0 1 1

Above 60 20 2 0 2 2

Occupational groups (n = 262)

Veterinary

Personnels 31 0 0 0 0

Milkers 53 1 0 1 1

Abattoir workers 54 10 5 9 9

Livestock farmers 107 7 4 5 5

Others 17 0 0 0 0

Contact with animals (n = 262)

Yes 245 18 9 15 15

No 17 0 0 0 0

Symptoms (n = 262)

Yes 18 18 9 15 15

No 244 0 0 0 0

Regions (n = 262)

*ICT 82 10 8 9 9

Rawalpindi 110 6 1 4 4

Attock 70 2 0 2 2

*ICT = Islamabad Capital Territory, *Y = years

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 (Abo-

Shehada 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).

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Probert, W. S., K. N. Schrader., N. Y. Khuong., S. L. Bystrom and M. H. Graves. 2004. Real-time multiples PCR assay for detection of Brucella spp., Brucella abortus and Brucella melitensis. J. Clin. Microbiol., 42: 1290-1293.

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Swai, E. S. and L. Schoonman. 2009. Human Brucellosis: Seroprevalence and risk factors related to high risk occupational groups in Tanga Municipality, Tanzania. Zoonosis. Publ. Healt., 56: 183-187.

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Figure 1: Aborted buffalo fetus collected

from human brucellosis sampling site

Figure 2: Screening of farm animals for

brucellosis in Pakistan

Figure 3: Location of sampling sites

in Pakistan

Figure 4: High Pure PCR Template Preparation

Kit (Roche Diagnostics, Mannheim, Germany)

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

Arabian sea

India

Afghanistan

China

Blochistan Province

Punjab Province

Khyber

Pakhtunkhwa

Province

Gilgit-Baltistan

Sindh Province

1

3 2

1. Islamabad Capital Territory (ICT)

2. Rawalpindi

3. Attock

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.