Introduction
In Bangladesh, almost 80% of the rural population is engaged in livestock husbandry either directly or indirectly, and the livestock sector contributes of 1.85% to the overall gross domestic product. At present, the livestock population consists of around 3.8 million sheep, 26.9 million goats, 1.56 million buffaloes, and 24.8 million cattle [1]. Sheep constitute about 6.7% of the total ruminants in Bangladesh and the popularity of sheep rearing is increasing gradually in Bangladesh to fulfill the growing demand for animal protein [1]. Among many factors of production, various diseases are the major constraints for better economic return in sheep farming. In addition, Bangladesh’s hot and humid climate provides favorable conditions for many diseases, which adversely affect animal productivity and result in high veterinary expenses [2]. Among other diseases, brucellosis is one of the major diseases that hinder the economic return of sheep farming in Bangladesh. Brucellosis is a deadly contagious and zoonotic disease of major concern caused by several species of the Brucella genus and these are tiny, rod-shaped, non-spore-producing, non-motile Gram-negative bacteria [3,4]. These microorganisms contain pathogenic capabilities that can affect various animal species, including humans. Brucella spp. are facultative intracellular organisms that cause persistent infections that can continue throughout the entire life of the infected individual. There are now nine identified species of Brucella, seven affecting terrestrial animals, which are: Brucella abortus, Brucella melitensis, Brucella suis, Brucella ovis, Brucella canis, Brucella neotomae, and Brucella microti [5]. Sheep and goats are primarily infected by B. melitensis, while infections caused by B. abortus or B. ovis are rare [6]. Among the nine recognized Brucella species, five can cause infection in humans. The most harmful and invasive species for humans is B. melitensis, followed by B. suis, B. abortus, and B. canis in decreasing order of pathogenicity [7]. Brucellosis has been widely reported in sheep from different parts of the world. Now, brucellosis is prevalent in both the human and animal populations of Bangladesh [8]. Animal brucellosis is transmitted through the following routes: ingesting contaminated aborted contents (placenta, fetal fluid, fetus, and so on), close interaction with an infected animal, and sexual interaction with mature animals that are infected [9]. Latent infection can be acquired by the consumption of contaminated colostrum and milk, which could explain the high occurrence of positive reactors among younger animals [10]. Sheep having brucellosis exhibit clinical symptoms such as reproductive problems, abortion, retained placenta, orchitis, and epididymitis. In females, the disease appears as placentitis in pregnant animals, with the organisms being excreted in milk and uterine discharges. In males, the primary characteristics of the condition are epididymitis and orchitis [7,9]. Brucellosis might have major consequences on both animal and human health, and also on socioeconomic aspects, which might be a serious impediment to the development of the livestock industry [11]. Humans can become infected with brucellosis by direct contact with sick animals or through consuming any of their byproducts, particularly milk and milk-based products such as cheese derived from unpasteurized goat and sheep milk and rennet from affected young sheep [12]. It primarily affects the reproductive system of animals and causes significant economic detriment in the livestock industry as a result of abortion, being infertile, premature birth, placental retention, and reduced milk yields [4,13]. Diagnosing brucellosis is often difficult and relies primarily on bacteriological and serological assays. Although bacterial culture is considered the most reliable method for diagnosing brucellosis, it is often not successful because of suboptimal conditions and its time-consuming nature. Hence, serological assays are frequently employed in diagnosing and screening for brucellosis [14].
Several studies have been conducted by several researchers in Bangladesh on the prevalence of brucellosis in goats, cattle, buffaloes, and humans. However, there has been limited investigation regarding the seroprevalence of brucellosis in sheep, and the analysis of risk factors has been rare in Bangladesh. Risk factor analysis is very important for Brucella infection [15]. The risk factors related to brucellosis can be classified into three categories: environmental, animal, and management factors. Management risk factors include examination of newly introduced animals, hygienic conditions, flock size, vaccination, mating procedures, and the farming method. Risk factors for animals consist of their age, breed, sex, history of abortion, and method of lactating. Environmental factors include the agro-ecology and locations [9]. To the best of our knowledge, no investigation has been conducted regarding ovine brucellosis on a large scale throughout the various districts of Bangladesh associated with risk factors analysis. Therefore, the current study was conducted to determine the seroprevalence of ovine brucellosis and to identify potential risk factors related to ovine brucellosis in selected sheep-prone areas of Bangladesh. The results of the study will assist government and veterinary officials in monitoring and controlling ovine brucellosis.
Materials and Methods
Ethical approval
The Animal Experimentation Ethics Committee of Bangladesh Livestock Research Institute (BLRI) approved this research project (Reference no.: AEEC/ BLRI00118/2023). During sample collection, all the guidelines for animal care were carefully followed.
Study area, animals, and period
This investigation was carried out at eight districts (Fig. 1) known for their high sheep population in Bangladesh, namely Faridpur, Noakhali, Rajshahi, Tangail, Dhaka, Thakurgaon, Bandarban and Meherpur, from July 2021 to June 2022. A total of 394 blood samples were collected from sheep using a simple random sampling technique in the research locale, i.e., Faridpur (n=52), Noakhali (n=60), Meherpur (n=48), Thakurgaon (n=50), Rajshahi (n=46), Dhaka (n=46), Tangail (n=48), and Bandarban (n=44). Sheep owners were interviewed directly to identify risk factors utilizing a structured questionnaire. Samples were collected from both male and female sheep from small (1–9 sheep), medium (10–20 sheep), and large (>40 sheep) size farms.
Figure 1.
Map of Bangladesh indicating sampling locations.
Calculation of sample size
The formula for random sampling [16] was implemented to get the sample size for this investigation. Sample size calculation was done with an absolute precision of 5% and a 95% confidence interval. To calculate the maximum sample size, a 50% predicted prevalence was applied. For this, the study needed 384 sheep; however, 394 were sampled to increase precision.
n=1.962 × Pexp (1—Pexp)/d2
Here, n=total number of samples; Pexp=expected prevalence; d=absolute precision.
Collection of sample
In total, 394 blood samples were obtained from sheep of both sexes, categorized by age into three groups: less than 1 year, 1–3years, and older than 3 years. All animals had been properly restrained and then a volume of 5 ml blood was sterilely taken from the jugular vein via standard vacutainer tubes. All samples were serially identified and labeled properly using a permanent marker pen. The blood sample was clotted in an upright position at room temperature and transferred in an ice box to the laboratory for further investigation. After a 24-hour period, the serum was centrifuged at 3,000 rpm for 5 minutes and subsequently separated carefully by pouring it into 1.5 ml eppendorf tubes. The separated serum was then maintained at −20°C in the laboratory until further analysis.
Serological tests
All serum samples were analyzed at the Small Ruminants Research Laboratory of Bangladesh Livestock Research Institute, following the World Organisation for Animal Health instructions for diagnosing brucellosis in sheep. The analysis included applying the Rose Bengal Plate Test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA).
Rose Bengal Plate Test
At first, the existence of antibodies towards Brucella antigens in all serum samples was determined using the RBPT. On a glass slide, RBPT was performed by mixing an equal amount (30 μl) of serum and antigen (which contained a suspension of Brucella spp. stained with Rose Bengal dye, IDvet, France). Subsequently, the antigen and test serum were mixed completely using a pipette tip, shaking for 4 minutes, and the degree of agglutination was visually inspected (Fig. 2). If any agglutination is found then the sample is marked as positive or if no agglutination is found then marked as negative [17].
Figure 2.
Pictures of positive and negative samples found in RBPT. A and B negative samples; C, D, E, and F positive samples showing agglutination.
Indirect ELISA test
All the sera were retested by a commercial indirect ELISA kit, following the instructions provided by the manufacturer (ID Screen® Brucellosis Serum Indirect Multi-species, IDvet, France; Product code: BRUS-MS-5P, Lot I44). The diagnostic kit was designed specifically to identify antibodies targeting B. melitensis, B. abortus, and B. suis from the sera of sheep, cattle, goats, and pigs. Furthermore, it reduces the risk of cross-reactivity with other Gram-negative bacteria. Initially, the technique involved ensuring that all essential reagents and sera to be tested were allowed to reach the ambient temperature. Subsequently, 190 ml (μl) of dilution buffer 2 were added to all wells, followed by the addition of 10 µl of negative, positive, and test sera. The mixture was then incubated at room temperature for 45 minutes. Following the incubation period, the contents of all wells were removed and rinsed three times with 300 μl of a freshly prepared wash solution. Subsequently, 100 μl of a prepared conjugate solution was added and incubated at room temperature for 30 minutes. Following a 30-minute incubation period, the contents of each well were removed and rinsed three times with 300 μl of wash solution. Subsequently, 100 μl of substrate solution was added and incubated for 15 minutes in a light-restricted environment. Finally, a stop solution was added to each well and the optical density (OD) was quickly measured and recorded using an ELISA reader with a wavelength of 450 nm. The formula used to compute each sample percentage (S/P %) is as follows:
S/P%=(OD sample – OD negative control)/(OD positive control – OD negative control) × 100
If the S/P % is below or equivalent to 110, those samples are classified as negative. If the S/P % is higher than 120, those samples are classified as positive. Otherwise, the samples are considered doubtful.
Risk factors
The variables of sex, parity, flock size, age, rearing method, and biosecurity were assessed to identify the association with the seroprevalence of ovine brucellosis. Statistical significance was attained when p ≤ 0.05 within a 95% confidence interval for the results.
Statistical analysis
Microsoft Excel spread sheet program was used for data storage and the frequency and percentage of different values obtained from results were calculated in Microsoft Excel sheet. Analysis was conducted utilizing MS Excel and STATA 18 (StataCorp LLC, USA) version. Descriptive statistics were applied to assess the correlation between Brucella seropositivity and variables such as location, sex, parity, age, flock size, rearing system, and biosecurity. The relationship between the occurrence of brucellosis infection and the risk factors was examined using univariate logistic regression analysis. After that, a multivariable logistic regression model was utilized to identify possible risk factors by considering the variables having a p-value of less than 0.20 in the univariate analysis. The factors that explain seropositivity were identified using a backward stepwise method. The findings were presented as a p-value and odds ratio (OR) along with a 95% confidence interval (95% CI).
Results
Descriptive statistics
This study involved testing 394 serum samples to estimate the prevalence of ovine brucellosis in Bangladesh. The seroprevalence of ovine brucellosis was found at 5.84% (95% CI: 3.7–8.6) using the RBPT and 3.55% (95% CI: 1.9–5.8) using the iELISA method. Meherpur district had the highest prevalence of brucellosis at 8.33% based on the RBPT method, while Tangail district had the lowest prevalence at 4.17%. Using iELISA method, Rajshahi district had the highest prevalence at 6.52%, while Tangail district had the lowest prevalence at 2.08% but no antibodies were found in sheep from Dhaka district. The prevalence of ovine brucellosis among the eight districts is presented in Table 1. The RBPT test demonstrated superior performance compared to the iELISA test, hence it was selected for subsequent statistical analysis to establish correlations between Brucella occurrence and related risk factors.
Table 1.
Seroprevalence of ovine brucellosis among different districts of Bangladesh by RBPT and iELISA.
| Location | Total no. of sera tested | No. Positive by RBPT | Prevalence in RBPT (95% CI) | No. Positive by iELISA | Prevalence in iELISA (95% CI) |
|---|---|---|---|---|---|
| Meherpur | 48 | 4 | 8.33% (2.3–19.9) | 2 | 4.17% (0.5–14.2) |
| Dhaka | 46 | 2 | 4.35% (0.5–14.8) | 0 | 0 |
| Bandarban | 44 | 2 | 4.55% (0.5–15.4) | 1 | 2.27% (0.05–12.0) |
| Thakurgaon | 50 | 3 | 6.00% (1.2–16.5) | 2 | 4.00% (0.4–13.7) |
| Tangail | 48 | 2 | 4.17% (0.5–14.2) | 1 | 2.08% (0.05–11.0) |
| Rajshahi | 46 | 3 | 6.52% (1.4–19.0) | 3 | 6.52% (1.3–17.8) |
| Noakhali | 60 | 4 | 6.67% (1.8–16.9) | 2 | 3.33% (0.4–11.5) |
| Faridpur | 52 | 3 | 5.77% (1.2–15.94) | 3 | 5.77% (1.2–15.94) |
| Total | 394 | 23 | 5.84% (3.7–8.6) | 14 | 3.55% (1.9–5.8) |
Univariate analysis
A significant difference in the seropositivity of Brucella infection between different age groups of sheep (p < 0.05) was found. Sheep aged over 3 years (10.71%; 95% CI: 5.0–19.3, OR: 6.17) were more susceptible to Brucella infection compared to those aged 1–3 years (5.85%) and below 1 year (1.90%)). There was a statistically significant difference in the seropositivity recorded between the parity number ewes (p < 0.05). Ewes that had given birth more than 3 times (15.29%, 95% CI: 8.4–24.7, OR: 12.63) were more susceptible to Brucella infection compared to those that had given birth 1 to 3 times or had not given birth at all. The study also found that female sheep (6.09%) were more affected than male sheep (4.88%) and sheep reared in the free-ranging method had higher seropositivity (6.69%) than semi-intensive rearing system (4.52%) but the relation was non-significant. The seropositivity rates for the different flock sizes, namely small, medium, and big, were 5.88%, 4.92%, and 6.25%, respectively. Regarding the biosecurity of sheep farms, it was shown that farms with inadequate biosecurity measures had a higher prevalence rate (6.42%) compared to farms with strong biosecurity practices (4.65%). However, this relationship was not statistically significant (Table 2).
Table 2.
Univariate logistic regression analysis of seropositivity of Brucella with associated risk factors.
| Variable | Category | No. of tested (% positive) | 95% CI | OR (95% CI) | p-value |
|---|---|---|---|---|---|
| Age (year) | Below 1 | 105 (1.90) | 0.2–6.7 | Ref. | |
| 1–3 | 205 (5.85) | 3.0–10.0 | 3.20 (0.70–14.58) | 0.138 | |
| over 3 | 84 (10.71) | 5.0–19.3 | 6.17 (1.29–29.43) | 0.022 | |
| Sex | Male | 82 (4.88) | 1.1–12.0 | Ref. | |
| Female | 312 (6.09) | 3.7–9.3 | 1.26 (0.42–3.82) | 0.678 | |
| Rearing system | Free | 239 (6.69) | 3.8–10.6 | Ref. | |
| Semi Intensive | 155 (4.52) | 1.8–9.0 | 1.52 (0.61–3.78) | 0.371 | |
| Flock size | Small | 221 (5.88) | 3.1–9.8 | 1.21 (0.33–4.38) | 0.773 |
| Medium | 61 (4.92) | 1.0–13.7 | Ref. | ||
| Large | 112 (6.25) | 2.5–12.4 | 1.29 (0.32–5.17) | 0.720 | |
| No. of parity | 0 | 142 (1.41) | 0.17–4.9 | Ref. | |
| 1–3 times | 85 (4.71) | 1.2–11.61 | 3.45 (0.62–19.29) | 0.157 | |
| > 3 times | 85 (15.29) | 8.4–24.7 | 12.63 (2.77–57.53) | 0.001 | |
| Biosecurity | Good | 129 (4.65) | 1.7–9.8 | Ref. | |
| Poor | 265 (6.42) | 3.7–10.0 | 1.40 (0.54–3.65) | 0.485 |
OR, odds ratio; CI, confidence interval.
Multivariate analysis
The multivariable logistic regression analysis method identified two possible risk factors for ovine brucellosis which included age and parity of ewes. Sheep aged over 3 years (OR: 6.56, 95% CI: 1.73–31.48) and parity of ewes (OR: 13.11, 95% CI: 2.82–60.92) have been identified as more likely to become seropositive for brucellosis as shown in Table 3.
Table 3.
Multivariate analysis of seropositivity of Brucella with associated risk factors.
| Variable | Category | Adjusted OR (95% CI) | p-value |
|---|---|---|---|
| Age (year) | Below 1 | Ref. | |
| 1–3 | 3.36 (1.14–15.40) | 0.118 | |
| Over 3 | 6.56 (1.73–31.48) | 0.019 | |
| No. of parity | 0 | Ref. | |
| 1–3 times | 3.65 (1.19–20.94) | 0.147 | |
| > 3 times | 13.11 (2.82–60.92) | 0.001 |
Discussion
Brucellosis is a serious infection that leads to abortion in infected pregnant animals and is a major public health issue in several nations [18,19], including Bangladesh [8,11]. Bangladesh was recognized as a highly endemic country for brucellosis due to the significant annual exposure of both animal and human populations to the infection [11]. In Bangladesh, there is a high level of interaction and close proximity among humans and animals, particularly in rural locales. This is mostly because the livelihood of people there depends heavily on livestock. The problem becomes worse because of the inadequate understanding and knowledge of the transmission of infection and abortion control by impoverished farmers. This study presents new information on the seroprevalence of ovine brucellosis and enhances our knowledge of the related risk factors in Bangladesh.
An accurate serological approach is necessary for the detection of brucellosis in endemic areas to effectively control and eradicate the disease [18]. The RBPT and i-ELISA employed in this investigation are widely accepted and reliable serological assays that are commonly recommended for conducting epidemiological research on brucellosis [4]. It is important to use sequential tests to increase the precision of the results. RBPT is very sensitive, but ELISA is highly specific and applied to confirm brucellosis in animals [9]. The seroprevalence of brucellosis exhibits considerable variation across various animal species, countries, flocks, and geographic regions. The current investigation found the overall seroprevalence of ovine brucellosis was 5.84% using RBPT and 3.55% using iELISA. This result is consistent with several prior investigations carried out in various geographical areas, including Southern Ethiopia (3.2%) [20], the Gaibandha district of Bangladesh (3.39%) [11], Oman (3.6%) [21], Niger (3.6%) [22], Bena Tsemay district of Ethiopia (3.69%) [9], and Algeria (3.8%) [23], using iELISA. These might be ascribed to the similar animal management practices and ecological circumstances present in these regions. According to other studies, this study’s result is 5 times lower than in Kashmir, India (17.38%) [24] and Tunisia (16. 10%) [25], 4 times lower than Rajasthan, India (15%) [26], 3 times lower than India (11.55%) [13] and Mymensingh region of Bangladesh (9.92%) [27], and 2 times lower than Southern Cameroon (8.04%) [4], Dakahlia governorate, Egypt (7.21%) [19], and Khyber Pakhtunkhwa, Pakistan (7%) [18]. On the other hand, it is higher than previous reports from the Arabian Gulf region (0.71%) [28], eastern Ethiopia (0.9%) [7], Mymensingh, Bangladesh (1.2%) [29], Northern Ethiopia (1.38%) [17], Tanzania (1.6%) [30], Central Ethiopia (2%) [31], and Sudan (2.5%) [32]. The seroprevalence of ovine brucellosis may vary across different locations because of some factors such as climatic circumstances, animal raising methods, and their housing system. These factors have previously been associated with the development of different infectious diseases [33]. However, earlier researches is not comparable to the present study due to differences in sampling locations and time, sample size, sheep breeds, husbandry practices, testing methods, and agro-ecology of the study area.
The study found a substantially higher brucellosis seroprevalence (p < 0.05) in the older aged sheep compared to the medium and young-aged sheep. This is consistent with Kamga et al. [4] and Hussen et al. [7], who found a greater seroprevalence of brucellosis in sheep over 2–3 years old. In a similar way, some other researchers also found higher seroprevalence in old aged sheep than in younger sheep, but the variation had no statistical significance [18,25,27]. In contrast, Tadeg et al. [34] found greater prevalence in young sheep compared to older sheep in the Afar region, Ethiopia. This could be attributed to the collection of a higher number of samples from younger sheep.
Brucellosis seroprevalence was also higher in female sheep than males, but not statistically significant (p > 0.05). This finding agrees with Hussen et al. [7], Guesmi et al. [25], and Shafy et al. [27], who found higher seroprevalence in female sheep. Male animals might be less susceptible to getting Brucella due to the lack of erythritol [18,35]. However, females remain in the herd for longer periods for reproduction; they may be more susceptible to diseases compared to males [4].
Seroprevalence and parity stage of ewes were also significantly associated (p > 0.05). This is in agreement with many researchers, who observed higher parity stage females had increased seropositivity of brucellosis [7,9,18]. Sexually mature and pregnant sheep are more likely to contract Brucella than immature sheep. This may be because of erythritol and sex hormones, which stimulate Brucella species growth and reproduction and increase with age and sexual maturity [7].
Ovine brucellosis seroprevalence was greater in large flocks compared to medium and small size flocks, and this finding was not statistically significant. The flock size and animal density directly affect disease incidence as well as complicate the situation for preventing infection [7,9,34]. In addition, overpopulation and confinement promote disease spread in the sheep herd.
The investigation indicated that free-ranging sheep had a higher brucellosis seroprevalence compared to semi-intensive animals. This phenomenon could be attributed to the fact that a majority of the sheep owners in the research area practiced sheep rearing using a free-ranging method. However, the unrestricted migration of sheep and the increased number of samples obtained from these sheep farmers may also lead to a higher prevalence of brucellosis. The study found that farms with inadequate biosecurity management had a higher prevalence of brucellosis infection compared to farms with adequate biosecurity measures. Effective implementation of biosecurity measures can help prevent the spread of various infectious infections including brucellosis [36]. Farms that have proper biosecurity measures experienced minimal outside exposure, resulting in lower disease prevalence.
The present investigation focused solely on the seroprevalence of ovine brucellosis and excluded other susceptible animals such as goats, buffaloes, and cattle. Furthermore, no attempt was made to determine the prevalence of the disease in humans to correlate the results obtained from the animals. It is suggested that there may be a need to conduct a nationwide epidemiological study utilizing both PCR and serological tests or other reliable diagnostic methods to determine the prevalence of brucellosis in the livestock population of Bangladesh.
Conclusion
The current research has shown that the seroprevalence of ovine brucellosis is quite low in Bangladesh. According to the multivariable logistic regression model, sheep age and parity of ewes were highly associated with Brucella seropositivity. Furthermore, it is important to immediately initiate an awareness campaign within the community to inform sheep farmers about the extent of the causes, transmission routes, signs and symptoms, risk hazards, and preventive measures associated with the disease. Frequent diagnostic testing is crucial to understand the progression of brucellosis and minimize its economic and public health impacts on the livestock sector.
Acknowledgments
The authors would like to express their sincere gratitude to the lab personnel’s of the Small Ruminant Research Laboratory, Goat Production Research Division at BLRI, Savar, Dhaka 1341, Bangladesh, for their invaluable support throughout the course of this study.
Conflict of interest
The authors declare they have no competing interests concerning this investigation.
Author contributions
Md Habibur Rahman: conceptualization, methodology, data analysis, and interpretation, writing original draft and revised the manuscript. Sonia Akther: conceptualization and methodology. Md Nurul Haque: methodology and writing original draft. Md Zulfiker Ali: Data analysis and interpretation, critical review. Md Asief Hossain Zihadi: revised the manuscript and critical review. Md Zillur Rahman: conceptualization and supervision. All authors have read, reviewed, and approved the final manuscript.
