Seroprevalence of Brucellosis among people in contact with livestock in suburban Khartoum, Sudan

Background and Objective: The objectives of the present study were to survey brucellosis and to determine risk factors among humans in occupational contacts with dairy cattle in suburban and peri-urban regions in Khartoum State, Sudan. Materials and Methods: Serum samples (n = 362) were collected from people in association with 11 farms with history of brucellosis in Khartoum State between August 2009 and February 2010. Results: Out of the 362 serum samples 14 (3.9%) were positive for brucella infection using competitive ELISA (cELISA). A significant association was observed between the seropositivity and community living in association with brucella-infected livestock notably: Illiterate males (P = 0.002), middle age group (20-40 yrs) (P = 0.003), and consumers of raw milk and milk products (P = 0.004) are at risk of infection. Conclusions: A significant association was observed between the seropositivity and community living in association with brucella-infected livestock notably: Illiterate males, middle age group (20-40 years), and consumers of raw milk and milk products are at risk of infection. The study recommends routine serological screening for brucellosis among herders and workers and their livestock particularly in regions with history of brucellosis.

Keywords: Brucella infection, ELISA, Khartoum state, livestock, Sudan

Brucellosis is an important zoonotic disease, which affect a variety of domestic and non-domestic animals and humans, causing high economic loss and public health burden especially in countries with no effective control programs. ^[1] It is caused by bacteria in the genus Brucella, which contains several species that are defined mainly based on animal host specificity. ^[1],[2] Brucellosis is commonly transmitted from infected animals to human by consumption of raw milk infected with Brucella or by direct contact with infected materials. ^[3] Dairy workers, shepherds, veterinarians, abattoir workers and animal husbandry personnel are particularly at risk. Epidemiological evidence indicates that at least 90% of human brucella infections can be attributed to direct contact with infected livestock and consumption of contaminated raw milk and raw milk products. ^[1],[4]

Human brucellosis is now a rare disease in countries where eradication programs directed toward livestock have been successfully implemented. Human brucellosis, however, remains endemic in the Mediterranean basin, Middle East, Western Asia, Africa, and South America. ^[5] Information about human brucellosis in Sudan is insufficient and sporadic. ^[6],[7]

The objectives of the present study were to establish the seroprevalence and to identify the risk factors of brucellosis among humans living in contact with dairy cattle in Khartoum State, Sudan.

Study design and ethical issues

The study protocol was assessed and approved by the National Health Laboratory, Ministry of Health, Khartoum, Sudan. In the present study, investigations using interviews with participants (farmers and livestock herders and associates) as well as blood sampling from them were undertaken following their approval.

Study population and data collection

This study was a cross-sectional study undertaken in 11 farms from around suburban regions in Khartoum State between August 2009 and February 2010 [Figure 1]. A total of 362 blood samples were collected from people in association with dairy cattle. {Figure 1}

All of the investigated farms have history of bovine brucellosis. Data regarding demographic characteristics of the human and animal patients were collected using a questionnaire format. A questionnaire was designed for data collection.

ELISA

Competitive ELISA kit for brucellosis (cELISA) was used in the present study to analyze the samples following procedures described by Stack et al. ^[8] A positive and a negative control were used in each test. The test sera and aliquots of control sera were gently agitated to ensure homogeneity. A total of 20 μl pre-warmed sera were added to each well. Then anti-bovine IgG alkaline phosphatase (Veterinary Laboratories Agency, The AHVLA office, Exeter, U.K.) C34 were added to each well and the resultant preparations were then vigorously shaken for 2 min and cover with a lid, then incubated at room temperature in a humid chamber for 30 min at rotary shaker, at 160 revs/ mins. The plates were then washed five times with phosphate buffer saline (PBS) – Tween solution.

Finally, the substrate solution which was prepared by dissolving one tablet urea Hydrogen peroxides in 12 ml distilled water when dissolved add the orthophenyldiamine (POD) tablet and mixed thoroughly using magnetic stirrer. 100 ul were added in volume immediately after the final cycle of washing in the previous step. Timing of the color development began immediately after the addition of the substrate solution to the wells of the first row of the micro plates. The reaction was stopped by adding 100 μl of stop solution (citric acid) to each well.

Optical density readings were performed using a microplate reader with an interference filter of 450 nm. The plates were read after 60 min. Readings were automatically transferred from the reader to the computer. The cut-off optical density (OD) value was decided at 1.8 decided from readings of known positive sera.

Data analysis

Analyses were done using EPI Info statistical package version 3.5.3 2011. Statistical tests included t-test and analysis of variance (ANOVA) for quantitative variables and chi-square test for qualitative variables. A P-value ≤0.05 in two-sided testing was used as the criterion for statistical significance.

The prevalence of brucellosis among people living in association with dairy cattle in 11 farms in Khartoum suburban areas is shown in [Figure 1].

The prevalence of brucellosis in Khartoum suburban communities according to their ages is shown in [Figure 2]. Of the 14 brucella positive patients, 3 (21.4%) aged 1-20 years and this comprised 0.8% of the total cases (362) examined. Eight (57%) aged 21-40 years, and this comprised 2.2% of the total cases examined. While two (14.3%) aged 41-60 years and this comprised 0.6% of the total cases; whereas one (7.1%) was from the group aged 60 and over, which comprised 0.3% of the total individuals surveyed. With reference to age, the main affected group aged between 20 and 40 years (P = 0.003), which are the core of the effective class in these communities with regard to diary production and herding. The majority of positive cases (92.9%) were males, this comprised 3.6% of the total cases (362) examined. The risk of infections for those using milk and milk products is three times higher (78.6%) than for those who did not (21.4%) (P = 0.004). {Figure 2}

The prevalence of brucellosis among study population according to the level of education is shown in [Figure 3]. The illiterates represented a high risk of brucella infection (1.9%; P = 0.002) followed by primary school students.{Figure 3}

Human brucellosis is principally an occupational disease related to jobs in direct contact with farm animals. Currently, there are no practical schemes of preventing human brucellosis from occurring among pastorals and livestock workers, attention should be directed toward efficiently controlling the disease in animals. Brucellosis in traditional livestock husbandry practices in many sub-Sahara African countries causes a zoonotic risk to the public, because of raw milk consumption, close contact with animals and provision of help during parturition where the risk comes from direct inoculation by skin abrasion, mucous membranes and inhalations. In these communities, usual eating habits including the consumption of unpasteurized milk and fresh cheese and butter is particularly common in Sudan rural areas. These products are the primary causes of the spread of brucellosis. ^[9] Slaughterhouse workers and others involved in animal handling are at a higher risk of direct inoculation by skin abrasion, mucous membranes, and inhalations.

The role of risks on people living or on those working in direct or indirect contact with dairy animals has not been given much importance. In the present study, the prevalence of brucellosis using cELISA among the studied population was found 3.9%. This figure is likely to be lower among other community sectors, since the screened individuals were selected for the history of bovine brucellosis in their herds. Human brucellosis is principally an occupational disease related to jobs in direct contact with farm animals. Currently, there are no practical schemes of preventing human brucellosis from occurring among pastorals and livestock workers, attention should be directed toward efficiently controlling the disease in animals. In these communities, usual eating habits including the consumption of unpasteurized milk and fresh cheese and butter are particularly common in Sudan rural areas. These products are the primary causes of the spread of brucellosis. ^[9]

Due to lack of diagnostic facilities and information on its occurrence, human brucellosis is most likely misdiagnosed for other febrile diseases prevailing in the areas, which are, treated empirically. ^[10] The results of the present study identified males, middle age group (21-40 years) and consumers of raw milk and milk products as the liable and more groups at risk of brucella infection. The rate reported in this study (2.2%) was comparable to that of Mudaliar et al. ^[11] who reported 5.33% prevalence of brucellosis among animal handlers in India. These rates were, however, significantly lower than that of Ahmed et al. ^[12] who reported 40% seropositive brucellosis in two municipalities in Libya.

At rural pastoral communities in Sudan and among households that own livestock for their living, male practice shepherding, and herding. Livestock owners in suburban Khartoum are practicing intensive and semi-intensive production due to a high demand of milk by the city. These factors have been identified in other countries. ^[13] Different strategies in different socioeconomic and epidemiologic situations can be applied to either control or eradicate brucellosis animals. ^[4]

Our results indicated a significance risk and a high prevalence of brucellosis among people working with or living in close contact with dairy cattle. Brucellosis prevalence represents a serious health hazard notably among the productive age group (21-40 years). 30.9% of those were found positive for brucellosis. Such results reflect either negligence from these farmers and animal herders of the disease or an overlook from authorities to provide protection means and people, or both factors might be existing! Illiterates have constituted 50% of all the positive cases detected in this study (P = 0.002). This is not surprising since education at Khalwa or primary school [Figure 3] could be sufficient to provide minimum health education that would reduce exposure of people to risk of infections. Of the 11 farms examined in Khartoum suburban areas, Dairy Farm Number 3 (Khartoum North) represented a contagious spot since more cases were found brucella seropositive than in the other locations. Most of these farms are located in Khartoum North. Also these locations are the parts of the Butana cattle zone. Some of Butana cattle herders settled around Khartoum North and intensified their production with a good level of cross breeding between the local Butana and the foreign Friesian breed.

The study concluded that the threat of pathogen spillover and interactions between livestock and man are the potential hazards. The study identified males; middle age group (21-40 years) and consumers of unpasteurized dairy products especially in endemic areas are at risk of food-borne brucellosis. The study recommends routine testing for brucellosis among herders. Control will be best accomplished at the animal level since people have a social and cultural tendency to consume and manufacture raw milk.

The authors acknowledge the cooperation of the Ministry of Health (Khartoum State) and the Federal Ministry of the Animal Resources and Fisheries. We thank Kawthar Salih for help with data analysis.

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1755-6783.140241