Epidemiological Investigation of Human Brucellosis in Pakistan


avatar Sultan Ali 1 , avatar Zeeshan Nawaz ORCID 2 , * , avatar Azeem Akhtar 1 , avatar Rizwan Aslam 1 , avatar Muhammad Asif Zahoor ORCID 2 , avatar Muhammad Ashraf 1

Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan
Department of Microbiology, Government College University, Faisalabad, Pakistan

how to cite: Ali S, Nawaz Z, Akhtar A, Aslam R, Zahoor M A, et al. Epidemiological Investigation of Human Brucellosis in Pakistan. Jundishapur J Microbiol. 2018;11(7):e61764. doi: 10.5812/jjm.61764.



Human brucellosis is a neglected and zoonotic disease also known as Malta fever or undulant fever.


A cross sectional study was conducted to determine the seroprevalence of brucellosis and to access the role of risk factors associated with this disease in humans of Punjab, Pakistan.


A total of 250 serum samples were collected and subjected to Rose Bengal Plate Test and Enzyme Linked Immunosorbent Assay for screening of Brucella. A predesigned questionnaire was filled prior to sampling to collect data regarding socio - demographic and suspected risk factors of human brucellosis. Descriptive and bivariate statistical analysis was performed using the STATA software version 12.


The study showed 16% seroprevalence of brucellosis. The prevalence was statistically higher in males (24%), age group of 20 to 30 years (26.92%), rural residents, (23%) and individuals with animals at home (22.50%). Among the related risk factors, exposure to animals (OR = 1.87, 95% CI: 0.9459, 3.6973) and consuming raw milk (OR = 2.36, 95% CI: 1.1713, 4.7760) were strongly associated with the disease.


Awareness programs in the rural population should be provided about the disease and its associated risk factors. Consuming unpasteurized milk and products should be avoided to control this disease.

1. Background

Brucellosis is a public health problem and a neglected bacterial disease with zoonotic potential. It has infected human beings and animals for decades. The causative agent behind this disease is a Gram negative coccobacilli bacterium that belongs to the genus Brucella. Infection of human beings is mainly due to Brucella abortus, B. Suis and B. melitensis, through direct interaction with infected animals and using their contaminated products, such as milk and meat, etc. (1, 2). Animals involved in the transmission of brucellosis to humans are buffaloes, cattle, goats, sheep, and pigs (3). Brucellosis remains a big hazard to people, who are in direct contact with animals, such as veterinary staff, laboratory persons, farmers, and workforce of a slaughter house (4). Aerosol and various secretions from animals act as a source of transmission to humans (5). However, human to human transmission is very rare (6).

The alternative names used for animal brucellosis are Epizootic Abortion, Contagious Abortion and Bang’s disease. The various names for human brucellosis are Undulant Fever, Malta fever, and Mediterranean fever. It causes great reproductive losses in mature animals and it has a zoonotic potential (7). The clinical sign of human brucellosis includes headache, irregular fever, chills, profuse sweating, weakness, hepatomegaly and splenomegaly. A few cases of arthritis, epididymitis and orchitis have also been reported in humans (8). Chronic cases are due to the ability of Brucella to survive and multiply in macrophages (9).

Brucella infection occurs more frequently in persons with reduced level of immune response, which may be due to any stress factors or HIV. Although HIV increases vulnerability of infected patients towards many opportunistic pathogens, yet in such patients, brucellosis is much more elevated and is the fatal form of the disease (10).

2. Objectives

In Pakistan, brucellosis is still an important and neglected problem and seroprevalence of brucellosis has been rarely investigated in Pakistan. Therefore, keeping in mind these facts, the present study was conducted with the aim of detecting the seroprevalence and risk factors associated with brucellosis in Pakistan.

3. Methods

3.1. Ethics Statement

This study was approved by the Institutional Biosafety/ Bioethics Committee (IBC) of University of Agriculture, Faisalabad under Code No: 36/ORIC and the samples were collected according to Pakistan biosafety rules (S.R.O 336(1)/2004) and the bioethics were followed during the whole span of the study.

3.2. Study Design

A cross - sectional epidemiological study was conducted on 250 individuals from July 2016 to December 2016. The sample size was obtained using 20% expected prevalence and 95% Confidence Interval (CI). Samples were collected using the non - probability convenience sampling technique (11). Expected prevalence was kept as 20%, as reported previously (12). A pre - designed questionnaire was administered to all participants to collect information about socio - demographic characters (gender, age, residence, marital status, occupation, education, religion, and presence of animals at home) and hypothesized factors (exposure to animals, milking, slaughtering, consuming milk products, and consuming raw milk) to detect their influence on spread and persistence of brucellosis.

3.3. Sample Collection

A total of 250 blood samples were collected from humans of Punjab, Pakistan. Equal number of samples were collected from each gender. The blood samples were collected after obtaining verbal and written approval from participants and their legal guardians. After collection, serum was separated from each sample and was stored in a freezer till further processing.

3.4. Brucella Detection

A total of 5 to 7 mL of blood was obtained from each participant following venipuncture in sterile disposable syringes and labelled properly. Serum was separated and screened for anti - Brucella antibodies along with positive and negative control sera using Rose Bengal Plate Test (RBPT) (13) and IgM - ELISA (NovaLisa, GmbH, Germany) as described previously (14). The samples that gave positive reactions for both tests were considered as positive due to the variation in the sensitivity and specificity of each test (15).

3.5. Statistical Analysis

The data obtained was tabulated in the Microsoft Excel spreadsheet and analyzed using STATA version 12 (Stata Corp., USA). Descriptive analysis was used to summarize the data on the basis of percentages and chi square. Bivariate analysis was conducted to establish the association of risk factors with brucellosis in humans. Odds Ratios (OR) were calculated at 95% Confidence Intervals (CI).

4. Results

4.1. Socio - demographic Characteristics

A total of 250 individuals participated in the present study with age range of 10 to 70 years old. The socio - demographic characteristics and number of participants involved in this study are shown in Table 1.

Table 1. Descriptive Socio - demographic Factors and Brucella Sero - prevalence in Human Participants
CharacteristicsNo of ParticipantsParticipants PercentageBrucella PositivePositive PercentageP Value
Overall Prevalence2501004016
Age group (years)0.036
10 - 20187.200316.66
20 - 305220.81426.92
30 - 406526.001421.53
40 - 7011546.000907.80
Marital status0.790
Graduate and above4016.000512.50
Animals at home0.021

4.2. Prevalence of Brucellosis in Humans

The gathered sero - prevalence of brucellosis among the 250 human participants was 16% (N = 40) and it varied among different genders. In males it was 24% and in females 8%. The highest prevalence was detected in participants ranging from 20 to 30 years of age (26.92%, N = 14). The residents of rural areas showed higher prevalence (23%) as compared to urban areas (10%). The individuals, who kept animals in their home displayed greater prevalence of brucellosis (22.50%) in comparison to those, who did not kept animals (10%). The above mentioned socio - demographic factors (gender, age groups, residence and animals at home) showed statistically significant (P < 0.05) results. On the basis of marital status and education level, the highest prevalence of brucellosis was observed as 23.07% in widowed and 18.95% in uneducated participants. Farmers and Christians showed higher prevalence (20.40% and 16.66%) on the basis of occupation and religion, respectively. These factors were none statistically associated (P > 0.05) with the disease as shown in Table 1.

4.3. Risk Factors for Occurrence of Brucellosis in Humans

The potential risk factors for human brucellosis included exposure to animals (22.85%), milking (20%), slaughtering (24%), consuming milk products (17%), and consuming raw milk (25.37%). At bivariate analysis, the exposure to animals (OR = 1.87, 95% CI: 0.945, 3.697) and consumption of raw milk (OR = 2.36, 95% CI: 1.1713, 4.7760) were statistically linked with the occurrence of brucellosis (P < 0.05) (Table 2).

Table 2. Potential Risk Factor Assessment Using Bivariate Analysis for Brucellosis Sero - prevalence in Human Participants
VariablesNo of ParticipantsBrucella PositivePositive (%)OR (95% CI)P Value
Exposure to animals1.87 (0.9459, 3.6973)0.03
Milking1.50 (0.7399, 3.0409)0.26
Slaughtering1.77 (0.6607, 4.7633)0.25
Consuming milk products1.50 (0.5930, 3.8044)0.39
Consuming raw milk2.36 (1.1713, 4.7760)0.01

5. Discussion

Brucellosis is an animal disease, which has a zoonotic potential through interaction with infected animals and consuming their products, such as meat, milk and milk products (16, 17). The best way for Brucella diagnosis is via the isolation and identification of microorganisms, yet this method has several drawbacks. Sero - diagnosis remains an important tool for rapid detection of this disease (18). The sensitivity of RBPT is high (99%) yet specificity is low (19).

In the present study the cumulative prevalence of Brucella was recorded as 16% from the study area, which was near the findings of 17% in Uganda (12) and 18% in Turkey (20). The observed prevalence was bit higher compared to previous studies conducted in Pakistan showing 9.33% and 10%, respectively (21, 22). The difference in prevalence of brucellosis might be due to the difference in livestock population and environmental conditions, while the increasing pattern of disease is due to lack of knowledge regarding disease and pasteurization procedures of dairy products.

The prevalence of brucellosis was higher in males (24%; 30/125) as compared to females (8%; 10/125). Similar results were also recorded in Uganda (12), Pakistan (22), and Libya (23), which showed that brucellosis is more prominent in male participants. On the other hand, the present study represents that age group ranging between 20 and 30 years was highly involved in Brucella sero positivity, which coincides with a study conducted in Turkey that reported comparatively high prevalence among the younger population (24). Same results were also depicted from a study conducted in Pakistan (22). The traditional role of male and young members in livestock management and common habit of these males to take milk directly from animals is the prominent reasons behind these facts. Another factor associated with higher prevalence of Brucella, was rural area residency; these individuals were 2.3 times more likely to be Brucella seropositive as compared to urban areas. This record is in concordance with previous studies, which reported high incidences in rural areas (25-27).

The result of the study elaborates (20.40%) prevalence in farmers as compared to other employers, which is in agreement with the findings of Shahid, who reported higher Brucella positivity in farmers (33%) and Tumwine (20.5%) and also coincides the fact that Brucella is more prominent in individuals, who kept animals at their homes and who had direct contact with animals (12, 28). The possible grounds of such results are that rural residents and farmers are in close contact with animals, which act as a reservoir of brucellosis. Other factors studied were religion, marital status, and education level of owners, which were not studied previously.

The individuals involved in milking and slaughtering of animals showed non statistically higher Brucella prevalence in this study (20% and 24%, respectively), yet some other studies also favoured this fact (12, 29). On the other hand, these results are in contrast with the findings that slaughtering is a significant factor for Brucella prevalence (30). The milkers and slaughterers are in direct and frequent contact with animals and the chances of carrying infection are much more in individuals belonging to these groups. The prevalence of Brucella is 17% in participants consuming milk products as compared to 12%, in those who do not. Similarly, raw milk consumers showed significantly higher (25.37%) sero prevalence than their counter group. There is high similarity between these findings and the results of a study conducted in Palestine (26). The results of a study conducted in Bangladesh were also on the same page as that of the current study (31). This consolidates the fact that humans become infected by consuming contaminated animal products, such as milk, butter, meat, etc. (32). This might be due to very poor understanding of the disease, lack of hygiene and safety of food products, and avoidance of pasteurized milk and milk products in Pakistan.

6. Conclusions

Brucella infection, particularly in rural areas of Pakistan, is an important public health concern. This study revealed that consumption of raw milk, keeping animals at home, direct contact with animals, and living in rural areas are risk factors associated with Brucella sero positivity among humans. Awareness programs in the rural population should be provided about the disease and its associated risk factors. Consumption of unpasteurized milk and products should be highly discouraged and personal care should be adopted before dealing with the animals.


  • 1.

    Kutlu M, Ergonul O, Sayin-Kutlu S, Guven T, Ustun C, Alp-Cavus S, et al. Risk factors for occupational brucellosis among veterinary personnel in Turkey. Prev Vet Med. 2014;117(1):52-8. doi: 10.1016/j.prevetmed.2014.07.010. [PubMed: 25132061].

  • 2.

    Makita K, Fevre EM, Waiswa C, Kaboyo W, De Clare Bronsvoort BM, Eisler MC, et al. Human brucellosis in urban and peri-urban areas of Kampala, Uganda. Ann N Y Acad Sci. 2008;1149:309-11. doi: 10.1196/annals.1428.015. [PubMed: 19120236].

  • 3.

    Corbel MJ. Brucellosis in humans and animals. World Health Organization; 2006.

  • 4.

    Pappas G, Akritidis N, Bosilkovski M, Tsianos E. Brucellosis. N Engl J Med. 2005;352(22):2325-36. doi: 10.1056/NEJMra050570. [PubMed: 15930423].

  • 5.

    Lapaque N, Forquet F, de Chastellier C, Mishal Z, Jolly G, Moreno E, et al. Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts. Cell Microbiol. 2006;8(2):197-206. doi: 10.1111/j.1462-5822.2005.00609.x. [PubMed: 16441431].

  • 6.

    Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, Walravens K, et al. From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Vet Res. 2005;36(3):313-26. doi: 10.1051/vetres:2005003. [PubMed: 15845228].

  • 7.

    Wadood F, Ahmad M, Khan A, Gul ST, Rehman N. Seroprevalence of brucellosis in horses in and around Faisalabad. Pakistan Vet J. 2009;29(4):196-8.

  • 8.

    Franco MP, Mulder M, Gilman RH, Smits HL. Human brucellosis. Lancet Infect Dis. 2007;7(12):775-86. doi: 10.1016/S1473-3099(07)70286-4. [PubMed: 18045560].

  • 9.

    Roop RM 2nd, Gaines JM, Anderson ES, Caswell CC, Martin DW. Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host. Med Microbiol Immunol. 2009;198(4):221-38. doi: 10.1007/s00430-009-0123-8. [PubMed: 19830453]. [PubMed Central: PMC3814008].

  • 10.

    Al-Anazi KA, Al-Jasser AM. Brucella bacteremia in patients with acute leukemia: a case series. J Med Case Rep. 2007;1:144. doi: 10.1186/1752-1947-1-144. [PubMed: 18036218]. [PubMed Central: PMC2174500].

  • 11.

    Thrusfield MV. Veterinary Epidemiology. 3rd ed. Iowa, USA: Blackwell Publishers; 2007. p. 228-42.

  • 12.

    Tumwine G, Matovu E, Kabasa JD, Owiny DO, Majalija S. Human brucellosis: sero-prevalence and associated risk factors in agro-pastoral communities of Kiboga District, Central Uganda. BMC Public Health. 2015;15:900. doi: 10.1186/s12889-015-2242-z. [PubMed: 26374402]. [PubMed Central: PMC4572625].

  • 13.

    Edelsten RM. Techniques for the brucellosis laboratory. Vet Res Comm. 1989;13(6):420. doi: 10.1007/bf00402562.

  • 14.

    Alrodhan M. Serological Investigation Of Caprine Brucellosis At Saniyah District. Kufa J Vet Med Sci. 2017;8(1):94-9.

  • 15.

    Oie A. Manual of diagnostic tests and vaccines for terrestrial animals. Paris, France: Office international des epizooties; 2008. 14 p.

  • 16.

    Corbel MJ. Brucellosis: an overview. Emerg Infect Dis. 1997;3(2):213-21. doi: 10.3201/eid0302.970219. [PubMed: 9204307]. [PubMed Central: PMC2627605].

  • 17.

    Abdussalam M, Fein DA. Brucellosis as a world problem. Dev Biol Stand. 1976;31:9-23. [PubMed: 1261753].

  • 18.

    Abubakar M, Mansoor M, Arshed MJ. Bovine Brucellosis: Old and New Concepts with Pakistan Perspective. Pakistan Vet J. 2012;32(2):147-55.

  • 19.

    Barroso PG, Rodriguez-Contreras RP, Gil BE, Maldonado AM, Guijarro GH, Martin AS, et al. Study of 1,595 brucellosis cases in the Almeria province (1972-1998) based on epidemiological data from disease reporting. Revista clinica espanola. 2002;202(11):577-82. [PubMed: 12392643].

  • 20.

    Arvas G, Akkoyunlu Y, Berktas M, Kaya B, Aslan T. The Prevalence of Brucellosis in Adults in Northeastern Region of Turkey. Jundishapur J Microbiol. 2013;6(3):262-4. doi: 10.5812/jjm.5147.

  • 21.

    Din AMU, Khan SA, Ahmad I, Rind R, Hussain T, Shahid M, et al. A study on the seroprevalence of brucellosis in human and goat populations of district Bhimber, Azad Jammu and Kashmir. J Anim Plant Sci. 2013;23:113-8.

  • 22.

    Perveen F, Raqeebullah MS. Sero-prevalence of brucellosis in humans population of Charsasdda, Khyber Pakhtunkhwa, Pakistan. Int J Med Invest. 2015;4(2):232-40.

  • 23.

    Ahmed MO, Elmeshri SE, Abuzweda AR, Blauo M, Abouzeed YM, Ibrahim A, et al. Seroprevalence of brucellosis in animals and human populations in the western mountains region in Libya, December 2006-January 2008. Euro Surveill. 2010;15(30). [PubMed: 20684813].

  • 24.

    Gur A, Geyik MF, Dikici B, Nas K, Cevik R, Sarac J, et al. Complications of brucellosis in different age groups: a study of 283 cases in southeastern Anatolia of Turkey. Yonsei Med J. 2003;44(1):33-44. doi: 10.3349/ymj.2003.44.1.33. [PubMed: 12619173].

  • 25.

    Elbeltagy KE. An epidemiological profile of brucellosis in Tabuk Province, Saudi Arabia. East Mediterr Health J. 2001;7(4-5):791-8. [PubMed: 15332781].

  • 26.

    Husseini AS, Ramlawi AM. Brucellosis in the West Bank, Palestine. Saudi Med J. 2004;25(11):1640-3. [PubMed: 15573193].

  • 27.

    Sofian M, Aghakhani A, Velayati AA, Banifazl M, Eslamifar A, Ramezani A. Risk factors for human brucellosis in Iran: a case-control study. Int J Infect Dis. 2008;12(2):157-61. doi: 10.1016/j.ijid.2007.04.019. [PubMed: 17698385].

  • 28.

    Shahid M. Prevalence of Brucellosis among the Hospital Patients of Peshawar, Khyber Pakhtunkhwa. J Infect Mol Biol. 2014;2(2):19-21. doi: 10.14737/jimb.2307-5465/

  • 29.

    Abo-Shehada MN, Odeh JS, Abu-Essud M, Abuharfeil N. Seroprevalence of brucellosis among high risk people in northern Jordan. Int J Epidemiol. 1996;25(2):450-4. [PubMed: 9119573].

  • 30.

    Mukhtar F. Brucellosis in a high risk occupational group: seroprevalence and analysis of risk factors. J Pak Med Assoc. 2010;60(12):1031-4. [PubMed: 21381558].

  • 31.

    Rahman AK, Dirk B, Fretin D, Saegerman C, Ahmed MU, Muhammad N, et al. Seroprevalence and risk factors for brucellosis in a high-risk group of individuals in Bangladesh. Foodborne Pathog Dis. 2012;9(3):190-7. doi: 10.1089/fpd.2011.1029. [PubMed: 22300225].

  • 32.

    Mishal J, Ben-Israel N, Levin Y, Sherf S, Jafari J, Embon E, et al. Brucellosis outbreak: analysis of risk factors and serologic screening. Int J Mol Med. 1999;4(6):655-8. [PubMed: 10567679].

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