Preliminary Screening of Brucella Antibodies in Sera of Persons in Contact with Animals in Mahlab 2-Hilat Koukou, Khartoum, Sudan in 2022

Brucellosis remains a major zoonosis worldwide. This study was intended to estimate Brucellosis prevalence among people living and working in a high stocking density area (Mahlab 2) and risk factors. A cross-sectional study was done among randomly selected males and females in the area of Mahlab 2. Data was collected using questionnaires and laboratory investigations. Blood samples were collected from participants. The Rose Bengal Plate (RBP) test was used for the detection of Brucella antibodies in serum samples and a modified Rose Bengal Plate (mRBP) test was used for confirmation. A total of 69 participants were enrolled in this study. The majority of those participants were between 18–29 years of age. The seropositivity of patients’ blood samples using these tests, was 28.4%. Socio-demographic characteristics, occupation, clinical signs of disease, types, and size of herds reared by animal owners, sanitary practices, and awareness about zoonosis, Brucellosis, and symptoms of Brucellosis showed no significant correlation with seropositivity among patients’ blood samples for Brucellosis.

It is recommended that surveillance and extension programs should be carried out to evaluate the magnitude of Brucellosis and to increase awareness about it. For proper control and prevention to decrease the incidence in animals, hence decreasing the incidence in humans, animal vaccination is essential.

Introduction

Brucellosis is an endemic globally significant zoonotic disease [1]. It is classified among the top seven world neglected zoonotic diseases and public health problems in developing countries with adverse negative effects on both humans and animals as well as economic implications [2], [3]. The main species of the genus Brucella affecting domestic animals are Brucella abortus, Brucella suis, Brucella melitensis, Brucella canis, and Brucella ovis [4]. However, the moderately to significantly pathogenic to humans are Brucella melitensis (from goats), Brucella suis (from pigs), Brucella abortus (from cattle), and Brucella canis (from dogs) [5]. Infection by Brucella species is worldwide in several animal species and in humans. It tends to be host-adapted, although infections of other animal species, including humans, may occur sporadically [6]. In animals, the disease is characterized by bacteraemia followed by localization of organisms in the reproductive organs, reticuloendothelial tissues, and sometimes joints [4]. In men, it is known as Malta fever and is characterized by fever, headache, night sweats and insomnia, pain in muscles and joints including the lumbar region of the spine, testicular pain in men, nausea, and vomiting [7], [8]. The disease poses a barrier to the trade of animals and animal products and causes losses due to abortion, breeding failure, and diminished animal production. Brucellosis reduces the work capacity through sickness of affected humans [9], [10].

Records of the prevalence of the disease in persons in contact with animals vary greatly in the previous studies. It was reported to be 1% in occupational contacts in one study, while another reported it to be 23% in occupational contacts and 60% in nomads [11], [12]. In a study done in Wau state (once, a part of Sudan) 32.1% of the workers were found to be seropositive for Brucellosis [13]. Together with the level of education of the persons in contact with animals in the area of our study and the absence of governmental programs of extension that raise awareness of the disease, a high prevalence of Brucellosis in humans is expected.

The prevalence of Brucellosis in animals reported in previous studies in Khartoum state ranges from 56–97% in dogs [14] and around 25.7% in cattle [15]. Putting these high prevalence percentages in mind, a high prevalence of the disease in humans is expected. Mahlab 2 is an area of intense animal farming. Humans are in close contact with animals in this area, hence increasing the chance of zoonosis. The disease is a serious one that has a considerable impact on people’s lives because it may pass unnoticed or misdiagnosed. The study is expected to raise awareness about the risk of zoonosis in farms and nearby areas. The aim of this study is to determine the prevalence and risk factors of Brucellosis in persons in contact with animals in Mahlab 2, Sharg Alneel locality, Khartoum, Sudan in 2022.

Materials and Methods

A cross-sectional study was done in Mahlab 2 which is located in Hilat Koukou, Sharg Alneel Locality, Khartoum, Sudan. It consists of 273 farms in an area of 90 Fedans. This area was established in 1970 for the nomads to settle and supply the Alban Koukou milk factory with milk. The factory had been built at that time in Hilat Koukou [16].

Collection of Samples

The sample size was calculated according to [17] using the following formula:

n = Z² X P (1-P)/d²

where n is the required sample size, z is the confidence level (1.96), p is the proportion in the target population (that is obtained from a pilot study conducted by the researchers and found to be 13.3) and d is the level of significance (0.05)

n = 177

Simple random samples were collected from both males and females over 18 years old in contact with animals and animal products, living or working in Mahlab 2. There was a call to provide samples. Of all those living in the study area, only 69 agreed to participate in the study. They were categorized as veterinarians (vets), milkmen, and other occupations including feeder, feed distributor, milk distributor, shopkeeper, and owner.

Data Collection

The data was collected using structured, standardized, self-designed, questionnaires Interviews with the participants were held to fill in the questionnaire which included questions about demographic data, herd data, sanitation, and hygiene measures applied, awareness on zoonosis and brucellosis and contact with the animal, poorly processed dairy products, and meat.

Laboratory Investigation

Blood samples of about one ml were collected in disposable syringes from the population under investigation in Mahlab 2. Samples were transported to the laboratory at Sudan University of Science and Technology, College of Veterinary Medicine, Department of Preventive Medicine, and Public Health alongside ice bags to maintain a suitable temperature. Although farm areas are considered areas of low risk of COVID-19 infection, all participants in this study were provided with face masks and antiseptic solutions to ensure safety from the COVID-19 virus. The samples were preserved at room temperature for 1 hour and serum was collected in Eppendorf tubes and preserved at 4 C in a refrigerator till tested.

Rose Bengal Plate Test (RBPT) and Modified Rose Bengal Plate Test (mRBPT) were performed. Both tests were simple spot agglutination tests using antigen stained with Rose Bengal. The antigen was obtained from Lilydale, London. The tests were performed according to the instructions of the manufacturer and OIE terrestrial manual [18] and (RBPT) and (mRBPT) respectively. The test was done within 12 hours of sample collection. Agglutination was immediately read after the five-minute period was completed. Any visible reaction was considered positive. The participants who tested positive were contacted and results were delivered to each of them individually.

Data Analysis and Presentation

The data collected was analysed using SPSS (Statistical Package for the Social Science) version 24 software program. The data was analysed using Chi Squire. P value < 0.05 was considered significant. Descriptive analysis was also done, and results were presented as frequencies and percentages. A scoring system was used to score awareness of people with the disease using the number of questions they answered out of a total of four questions, where bad awareness is a score between 0–1/4, acceptable awareness is a score of 2/4 and good awareness is a score of 3–4/4.

Results

Sixty-nine samples were collected from people living or working in Mahlab 2. The samples were tested for the presence of Brucella antibodies using the Rose Bengal Plate test and modified Rose Bengal Plate test.

The demographic part of the questionnaire revealed that the majority (94%) of the participants were working in Mahlab 2 only, and 6% of the participants were working in Mahlab 2 and other neighboring animal high-stocking density areas. 18.8% (12) of the respondents were veterinarians (vets), 52.2% (36) were milkmen while other occupations constituted the remaining 29% (21) of the respondents. Out of the 10 tested vets’ valid samples, 16.7% were found to be positive for Brucellosis. The prevalence of Brucellosis was found to be about 28.4% in the study population (Fig. 1).

Fig. 1. Occupation of participants (n = 69) of Brucella antibodies screening program in Mahlab 2, Sharg Alneel Locality, Khartoum State, Sudan.

About fifty-four percent of the study population were in the age group 18–29 years. The age of 30–39 years constitutes about 20% of the population as shown in Fig. 2. 91% were males while the remaining 9% were females. About 17% of the participants were illiterate and 39% were of primary education. Secondary and higher education constitute 15% and 26%, respectively. 3% of the participants refused to answer the question of the level of education (Fig. 3).

Fig. 2. The age range of participants (n = 69) of Brucella antibodies screening program in Mahlab 2, Sharg Alneel Locality, Khartoum State, Sudan.

Fig. 3. Educational levels of participants (n = 69) of Brucella antibodies screening program in Mahlab 2, Sharg Alneel Locality, Khartoum State, Sudan.

Table I shows the frequencies and percentages of different herd types and small and large herd sizes owned by the participants in the study. Frequencies and percentages of other risk factors of Brucellosis were shown in Table II. 50.7% of the respondents had a bad awareness score between (0–1out of 4) while 47.8% had an acceptable awareness score (2 out of 4) and 41.8% had a good awareness score (3–4 out of 4). Many of the risk factors studied showed significant association with each other (Table III), while all the risk factors were found to be insignificantly related to Brucellosis (Table IV).

Table I. Frequency and Percentages of Herd Type and Size Owned by Participants (n=69) Live or Work in Mahlab 2, Sharg Alneel Locality, Khartoum State, Sudan

Risk factor	Response	Frequency	Percentage
Herd type	Mixed	25	36.2
	Cattle	41	59.4
	Owned dogs	2	2.9
	Stray dogs	69	100.0
	Goat	1	1.4
	Total	69	100.0
Herd size	Small (≤10)	27	39.1
	Large (>10)	42	60.9
	Total	69	100.0

Table II. Frequency and Percentages of Risk Factors of Brucellosis among Participants (n=69) Live or Work in Mahlab 2, Sharg Alneel Locality, Khartoum State, Sudan

Risk factor	Response	Frequency	Percentage
Sanitation and hygiene
Cleaning hands before milking	No	22	31.9
	Yes	25	36.2
	System	22	31.9
	Total	69	100.0
Cleaning hands after milking	No	19	27.5
	Yes	26	37.7
	System	24	34.8
	Total	69	100.0
Disposal of foetal tissues after delivery	No	54	78.3
	Yes	4	5.8
	System	11	15.9
	Total	69	100.0
Disinfection of floor after delivery	No	18	26.1
	Yes	4	5.8
	System	47	68.1
	Total	69	100.0
Water status	Unavailable	2	2.9
	Available and adequate	67	97.1
	Total	69	100.0
	Total	33	100.0
Previously diagnosed disease
Typhoid	No	54	78.3
	Yes	14	20.3
	Missing	1	1.4
	Total	69	100.0
Malaria	No	48	69.6
	Yes	21	30.4
	Total	69	100.0
Tuberculosis	No	68	98.6
	Yes	1	1.4
	Total	69	100.0
Symptoms
Fever	No	56	81.2
	Yes	13	18.8
	Total	69	100.0
Headache	No	53	76.8
	Yes	16	23.2
	Total	69	100.0
Arthralgia	No	44	63.8
	Yes	25	36.2
	Total	69	100.0

Table III. Association of Brucellosis Potential Risk Factors among Participants (n=69) Live or Work in Mahlab 2, Sharg Alneel Locality, Khartoum State, Sudan

Association	Significance
Age * arthralgia	0.003
Level of education * awareness of symptoms	0.000
Awareness of zoonosis * awareness of symptoms	0.001
Awareness of source of infection * awareness of symptoms	0.000

Table IV. Association of Herd Potential Risk Factors and Brucellosis among Participants (n=69) Live or Work in Mahlab 2, Sharg Alneel Locality, Khartoum State, Sudan

Category	Risk factor	Significance
Herd type and	Herd size	0.506
size	Herd type	0.545
Previously	Typhoid	0.749
diagnosed	Malaria	0.681
diseases	Tuberculosis	0.809
	Fever	0.451
Symptoms of	Headache	0.592
Brucellosis	Arthralgia	0.683
Sanitary	Cleaning hands before milking	0.362
measures	Cleaning hands after milking	0.418
	Disposal of fetal tissues after delivery	0.949
	Disinfection of floors after delivery	0.857
	Water status	0.651
Awareness	Aware that diseases can be transmitted from animals to humans	0.629
	If yes, aware that Brucella is transmitted to humans	0.556
	If yes, awareness about sources of infection	0.555
	Awareness about symptoms of the disease	0.083

Discussion

Brucellosis is one of the most frequent zoonoses in the world, wreaking havoc on the animal industry and posing a severe health risk to humans. It is regarded as one of the most serious zoonoses in the world, with over 500,000 human cases reported each year [9], [19], [20] and classified among the top seven world neglected zoonosis and public health problems in developing countries, with a negative effect on both human and animals as well as economic implications [21], [22]. The disease is of importance in sub-Saharan Africa [23]. Most studies in Sub-Saharan Africa focused on Brucella antibody seroprevalence in humans and livestock, with data that provides insight into the inferred high burden of Brucellosis particularly among rural populations who heavily rely on livestock for their livelihood [24]. There is a paucity of information concerning the prevalence of Brucellosis in humans in Mahlab 2 in specific, and Sudan in general. This study was conducted in Mahlab 2, Khartoum State with a sample size of 69 participants aiming to measure the seroprevalence and risk factors of Brucellosis in persons in contact with animals.

The prevalence of Brucellosis in this study was found to be 28.4%. The seroprevalence of Brucellosis was found to be 9% among veterinarians and 28% among the whole screened population, about the same prevalence of Brucellosis in veterinarians in Northern State, Sudan (9.5%) reported by [24]. The prevalence in animal handlers in the same state was reported to be 26.5% [24], which is lower than the prevalence in Mahlab 2. This may be due to the high stocking density of ruminants in this area.

Compared to other Sub-Saharan African countries, the seroprevalence of Brucellosis in Mahlab 2 was found to be higher than that recorded in previous studies in Ethiopia [25], Central Uganda [26], and Kenya [27], ranging from 3.78, 4.8 and 5.2 in three different areas in Ethiopia to about 17.0 in Central Uganda and Kenya. However, it was approximately the same as that reported in Tanzania [26].

Due to erroneous diagnosis, limited surveillance, and imprecise reporting, brucellosis in humans may go unnoticed or underestimated. The World Health Organization (WHO) estimates that the true incidence is at least one order of magnitude greater [19]. In this study, the probability of erroneous diagnosis is decreased as there is an insignificant correlation between brucellosis and some of the other diseases with similar symptoms such as typhoid, malaria, and tuberculosis while the sample size which was controlled with the agreement of the population to participate affects negatively the degree of confidence on the results.

Although there is no significant association between Brucellosis and the symptoms suggestive of it, the prevalence of these symptoms was high enough to draw attention to the underlined causes in the area of our study. Arthralgia is one of these symptoms. It was found to have no significant correlation with Brucellosis among the participants surveyed and to be more common in old age which dictates that investigation of its cause is a priority in the elderly.

It was noted that the correlation between the level of education and awareness of symptoms is highly significant. A lower awareness of the symptoms in the participants in respondents with lower levels of education was detected. This suggests that schooling and education should be considered as one of the promising methods of control and prevention of Brucellosis. Henceforth, introducing night schooling may be of considerable help to those people living in poverty and in need of work at a young age. Awareness of the symptoms was associated also with the awareness of the source of infection. This significant association and the insignificance of both variables with Brucellosis should not be neglected as it may change the approach to the prevention and control of the disease. It is important to raise awareness of both symptoms and sources of infection of brucellosis, together with the implication of getting infected for better control of the disease in humans.

In this study, the awareness was scored into good, acceptable, and bad awareness levels, where good and acceptable awareness levels represented 47.8% and bad awareness represented 50.7% from a total of 69 participants. These results are in line with the analysed pooled data from 79 other original articles in a total of 22 countries that revealed 55.5% of surveyed people were aware of Brucellosis [28].

According to the socio-demographic data, about 54% of the study population are at age of 18–29 years and 20% are at the age of 30–39 years. The age range of 18–39 is considered to be the active working age. The prevalence of Brucellosis among these two categories was found to be about 29%. This is an obvious handicap to the workforce in the area.

There is a high prevalence of Brucellosis in Mahlab 2. However, there is no association between brucellosis and the risk factor investigated. Brucellosis leads to a decrease in the workforce and complicates the social and financial status of those people living in poverty and in great need of their daily income. Surveillance should be carried out in areas where there is considerable contact between humans and animals. For proper control and prevention to decrease the incidence in animals, hence decreasing the incidence in humans, animal vaccination is essential.