Prevalence of Brucellosis in Human Population of District Swat, Pakistan

Hussain Ahmad1, Inamullah1, Ijaz Ali2, Tauseef Ahmad3, Muhammad Tufail4, Kabir Ahmad5 and Bibi Nazia Murtaza3,6*

1Department of Genetics, Hazara University, Khyber Pakhtunkhwa, Pakistan

2Institute of Biotechnology and Genetic Engineering, Khyber Pakhtunkhwa Agricultural University, Peshawar, Pakistan

3Department of Microbiology, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan

4Department of Health, Khyber Pakhtunkhwa, Pakistan

5Department of Zoology, Hazara University, Mansehra, Khyber Pakhtunkhwa

6Department of Zoology, Kinnaird College for Women, Lahore, Pakistan

ABSTRACT

Brucellosis is a zoonotic disease affecting several animal species and human beings. It is caused by Brucella abortus in animals and Brucella melitensis in human. The current study aimed to find out the prevalence of Brucella and active brucellosis in the human population of district Swat. Total 300 individuals (both female and male of all ages, meeting our inclusion/exclusion criteria) with informed consent were included in the study. Blood samples were collected and brucellosis was detected by Standard Plate Agglutination Test (SPAT) and Serum Tube Agglutination Test (STAT) and later on confirmed by PCR. Disease was detected in 3.66% (11/300) individuals by SPAT and 2% (6/300) by STAT and confirmed in 2.66 % (8/300) by PCR. The prevalence of disease in females was found to be higher as compared to males (in current studied group). The age group 31-40 years, was found to be more susceptible for this infection.

Article Information

Received 08 March 2016

Revised 23 June 2016

Accepted 30 June 2016

Available online 11 January 2017

Authors’ Contributions

HA, I and BNM conceived and designed the study. HA, IA, TA, MT and KA collected samples from the hospitals and performed the experiments. MA, IA and BNM analyzed the data. HA and BNM wrote the article. HA and BNM supervised the work.

Key words

Brucellosis, Prevalence, Standard Plate Agglutination test, Serum tube agglutination test.

* Corresponding author: nazia.murtaza@gmail.com

0030-9923/2016/0004-1161 $ 8.00/0

Copyright 2016 Zoological Society of Pakistan

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.1.sc11

Brucellosis is a zoonotic disease affecting several animal species and human beings. It is caused by Brucella abortus in animals and Brucella melitensis in human. The current study aimed to find out the prevalence of Brucella and active brucellosis in the human population of district Swat. Total 300 individuals (both female and male of all ages, meeting our inclusion/exclusion criteria) with informed consent were included in the study. Blood samples were collected and brucellosis was detected by Standard Plate Agglutination Test (SPAT) and Serum Tube Agglutination Test (STAT) and later on confirmed by PCR. Disease was detected in 3.66% (11/300) individuals by SPAT and 2% (6/300) by STAT and confirmed in 2.66 % (8/300) by PCR. The prevalence of disease in females was found to be higher as compared to males (in current studied group). The age group 31-40 years, was found to be more susceptible for this infection.

Brucellosis caused by bacterial genus Brucella, is one of the common zoonotic infections. Pathogenic Brucella species in humans are B. melitensis (animal reservoirs is sheep and goats), B. abortus (animal reservoirs is in cattle), and B. suis (animal reservoirs is in swine). B. abortus and B. melitensis species are genetically very similar to each other. In humans, it causes undulant fever and male sterility and in animals, B. abortus causes abortion. Aerobic gram-negative Brucella has the ability to invade both phagocytic and nonphagocytic cells. It has the ability to survive in the intracellular environment by finding ways to avoid the immune system (Lecaroz et al., 2006). Transmission of infection can be through direct contact with infected animals, inhalation of airborne agents or usage of unpasteurized milk or cheese (Pappas et al., 2006). The disease causes various symptoms including weakness, periodic fever, muscle aches, malaise, weight loss and fatigue (WHO, 2006). Generally brucellosis is diagnosed serologically by detection of agglutinating antibodies (IgM, IgG, and IgA classes) (Godfroid et al., 2005). The most frequently used serological tests are Rose Bengal test (RBT), the tube agglutination test (TAT) and the complement fixation test (CFT) (Manat et al., 2016). Control of the disease depends on the system of animal management in the region and available epidemiological information about the disease. For effective control of this disease in Pakistan, Brucellosis control programs should be initiated (Ali et al., 2013). Current study was designed to estimate the prevalence of disease in human population of District Swat, Pakistan.

Materials and methods

Blood samples were collected from Saidu Group of Teaching Hospitals, Swat and Tajwar Sultana Hospital Swat, Khyber Pakhtunkhwa (KP), Pakistan (Table I). The study was conducted during March 2012 to Jan. 2013.

Patients of both genders of all age groups, visiting to seek the medical help with the symptoms suspected for brucellosis (periodic fever, muscle aches etc.), selected by the clinicians, were included in the study. Patients having tuberculosis or with the history of tuberculosis were not included in the study.

To collect the necessary information about the participants, a questionnaire was designed. An informed consent was signed by all the participants and they were ensured about the confidentiality of results and privacy of the hospital record.

From each participant, 5 ml blood was drawn out and transported to Institute of Biotechnology and Genetic Engineering, Peshawar. Each sample was centrifuged for 5 min at 5514 x g and separated serum was stored at -20°C for further analysis.

Two serological tests, SPAT (Co., Inc. Morganville, N.J. 07751) and STAT (Co., Inc. Morganville, N.J. 07751) were used for the diagnosis of brucellosis.

Samples showing positive results with SPAT and STAT were further processed for molecular analysis by PCR. Amplification of small fragment of Brucella genome was done by using Brucella PCR kit (Shangai ZJ Bio-tech Co. Ltd China) according to the recommended procedure established by manufactures with little modifications.

DNA was extracted out from 50µl blood serum. Master mix of volume 34.4µl was prepared by adding 34µl reaction mix and 0.4µl enzyme (provided in the kit). To 35µl of master mix, 5µl DNA sample was added. Initial denaturation was carried out at 94°C for 2 min followed by 35 cycles with denaturation at 93°C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 30 seconds. Final extension was carried out at 72°C for 10 min.

All categorical variables such as age and gender were compared and analyzed by chi-square test. Results were considered significant when p value was less than 0.05.

Table I.- Sample collection from different regions of District Swat.

Name of Tehsil | Male | Female | Total
Babuzai | 10 | 20 | 30
Charbagh | 15 | 25 | 40
Khuwza khelaa | 25 | 35 | 60
Mata | 15 | 39 | 54
Bahrain | 8 | 16 | 24
Kalam | 4 | 12 | 16
Kabal | 15 | 35 | 50
Barikot | 8 | 18 | 26
Total | 100 | 200 | 300

Results

Total 300 serum samples were examined for the prevalence of brucellosis (Table I). Brucellosis was detected in 3.66 % (11/300) samples by SPAT and 2% (6/300) by STAT. The presence of diseases was confirmed by PCR in 2.66 % (8/300) of total studied population. The registered patients were categorized into two age groups. Individuals ˂30 years of age were placed in group 1 and ≥31years of age were in group 2. In group 1, 0.62 % (1/160) samples showed positive results by SPAT while STAT failed to detect brucellosis in this group. In group 2, 7% (10/140) samples showed positive result by SPAT while 4 % (6/140) of samples showed positive result by STAT. It was observed that 3% of male patients were brucellosis positive by SPAT and 2% by STAT (Table II). When area wise distribution of disease was assessed, out of total 8 confirmed brucellosis patients, 6 were from Khuwza khelaa (K. khela), 1 from Babuzai and 1 from Kalam (Table III).

Table II.- Prevalence of brucellosis in association with different demographic characteristics.

Parameters | Total out of 300 individuals | Cases positive by SPAT (n= 11) | Cases positive by STAT (n=6 ) | P value
Age G1 ˂30 G 2 ≥31 | 160 140 | 1 10 | 0 6 | 0.44
Gender (Men) (Women) | 100 200 | 3 8 | 2 4 | 0.81
Education Illiterate Literate | 250 50 | 10 1 | 5 1 | 0.04

Table III.- Analysis of brucellosis in Swat.

Code No | Age | Gender | Area | SPAT | STAT | PCR
B22 | 30 | M | Babuzai | + | - | +
B37 | 40 | M | K. Khelaa | + | + | +
B101 | 35 | F | Kabal | + | - | -
B105 | 37 | F | Matta | + | - | -
B138 | 35 | F | Matta | + | + | +
B144 | 40 | F | K. Khelaa | + | + | +
B153 | 40 | F | K. Khelaa | + | + | +
B175 | 32 | F | K. Khelaa | + | - | -
B190 | 45 | F | K. Khelaa | + | + | +
B211 | 40 | F | Kalam | + | + | +
B213 | 50 | M | K. Khelaa | + | - | +

Discussion

Scientists and clinicians are still facing several challenges in understanding the pathogenic mechanisms of human brucellosis and the identification of markers for disease severity and progression (Franco et al., 2007). Untreated human brucellosis can be fatal or ends with serious health complications. Infection in brucellosis is primarily controlled through cell-mediated immunity system of the body. To control reinfection, IgM levels rise, followed by IgG titers (Lecaroz et al., 2005; Bouza et al., 2005).

In present study, brucellosis was detected in 3.66% (11/300) samples by SPAT and 2% (6/300) by STAT. The presence of diseases was confirmed by PCR in 2.66% (8/300) of total studied population. The highest active human brucellosis was observed in the individuals between 31-40 years of age. The disease is more common in female as compared to male. Generally it is found to be more common in males than in females. Fallatah et al. (2005) found male-to-female ratio of 1.7:1 individuals aged 13-40 years in Saudi Arabia. High seroprevalence of brucellosis was observed in humans aged above 30 years by Nahar and Ahmed (2009). Shafee et al. (2012) studied the prevalence of the disease in cattle and buffaloes by RBPT (3%) and through i-ELISA (3.20%) in Quetta Balochistan. Hussain et al. (2008) observed 4%, 3% and 3.33% seroprevalence of brucellosis in goats by Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT) and Milk Ring Test (MRT), respectively.

Lack of education and proper awareness about health and diet is the major reason for infections in the area. When presence of infection was assessed with education level, 83% of total patients (seeking medical help with related symptoms) found to be illiterate. As the presence of brucellosis in animals has direct effect on human brucellosis so vaccination in animals should be carried out to control the disease. People living in rural areas are consistently exposed to infected animal excreta and secretions during raising, milking and slaughtering of domestic animals (Smith and Kadri, 2005). There are different diagnostic tests being used in Pakistan. We used Standard Plate Agglutination Test (SPAT) and Serum Tube Agglutination Test (STAT) for initial diagnosis and confirmation was done by PCR. The variation in results highlights the need to establish an accurate reference diagnostic method to confirm the actual presence of brucellosis to avoid side effects of prolonged antimicrobial therapy. All the brucellosis patients should be tested every 3 to 6 month interval. Recently established molecular techniques should be used for accurate diagnosis (Azizpour et al., 2013; Cha et al., 2012; Christopher et al., 2010). Also we need to develop a coherent and long-term strategy for to increase the awareness about the transmission of disease.

Conclusions

The study showed that prevalence of the disease is more in female as compared to male. It can be inferred that brucellosis continues to be a public health concern in the rural area of Swat district such as Khuwzakhelaa and need a good applicable strategy to control and eradicate the disease from the area.

Statement of conflict of interest

The authors declare no conflict of interest regarding this paper.

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