Sero‐prevalence and risk factors associated with brucellosis in dairy cattle of Sylhet District, Bangladesh: A cross‐sectional study

Abstract Background Brucellosis is an emerging disease that causes a significant impact on productive and reproductive performance in dairy cattle. Though Brucella is a pivotal microorganism for dairy cattle, the scenario of brucellosis in Sylhet District is unknown. Objectives A cross‐sectional study was carried out to assess the prevalence and determinants associated with brucellosis in dairy cattle of Sylhet District. Methods A total of 386 sera and data on determinants from 63 dairy herds were collected from 12 sub‐districts using simple random sampling. The sera were tested with Rose Bengal Brucella antigen test, Brucella abortus plate agglutination test and serum agglutination test to find out the sero‐positivity. Results Overall, 17.09% (95% CI: 13.67–21.18) prevalence in cows were calculated. Relatively higher prevalence (56.08%; 95% CI: 42.23–70.32) was recorded in cows having parity ≥4 and were at higher risk (OR = 7.28) than the other cows with parity 0–3. Prevalence was significantly higher in cows with history of abortion 90.63% (95% CI: 75.79–96.76), repeat breeding 79.17% (95% CI: 65.74–88.27) and reproductive abnormalities 48.54% (95% CI: 39.12–58.07). Farm‐level prevalence was high in farms with the previous history of abortion 95.45% (95% CI: 78.20–99.19) and repeat breeding 90.00% (95% CI: 74.38–96.54). Conclusions The prevalence was high in Sylhet district, which might be a public health concern. Therefore, this study would represent the baseline information for guiding brucellosis control and prevention.

Brucellosis may cause 30%-70% abortions in the last trimester in fully susceptible herds (Godfroid et al., 2005). Infection may last long in cows during subsequent pregnancies; the recurrence of abortion is rare, but uterine and mammary infections commonly recur (Pappas et al., 2005).
It also prolongs inter-calving intervals and reduces calves' lifetime production. Thus, the economic impact of the disease at the national level is substantial (Radostits et al., 2006). Some countries successfully controlled or eradicated bovine brucellosis through test and slaughter programmes (Godfroid & Käsbohrer, 2002;Ragan, 2002). However, the disease still occurs at varying prevalences, particularly in developing countries, including Bangladesh (Kadohira et al., 1997;Omer et al., 2000;Moreno, 2002;Poester et al., 2002;Hegazy et al., 2011;Rahman, 2015;Logonda et al., 2022). The countries at high risk of brucellosis are countries in the Mediterranean Sea Basin (Portugal, Spain, South France, Italy, Greece, Turkey, Algeria, Tunisia, Egypt and Morocco), likewise nations of South and Central America, Asia, Africa, the Caribbean and the Middle East. The most prevalent endemic countries of bovine brucellosis are Libya, Georgia, Algeria, Nigeria, Zambia, Turkey, India, Pakistan and so on (Minas, 2006;Pappas et al., 2006;Galinska & Zagórski, 2013).
The prevalence of bovine brucellosis varies widely across Bangladesh, with a reported sero-prevalence of 0.3%-20.5% (Rahman et al., 2011;Rahman, 2015). The prevalence of brucellosis of cattle in Mymensingh, Central Cattle Breeding and Dairy Farm, Dhaka and Sirajganj was estimated at 0.3%, 20.5% and 2.66%, respectively (Rahman et al., 2011;Rahman, 2015). Many cases of abortion, retention of placenta, metritis and stillbirth, which may be due to brucellosis, remain undiagnosed and untreated in Bangladesh (Rahman et al., 2010). As a result, the chance of having brucellosis has increased tremendously.
The risk factors responsible for the occurrence of bovine brucellosis include the purchase of infected cattle for replacement, the nature of animal production, demographic factors, regulatory issues, climate and wildlife interaction (Muma et al., 2007;ZareBidaki et al., 2022). Furthermore, one major contributing factor to the spreading of the disease is free movement of animals (Ocholi et al., 2004). In addition, management systems, herding of different species together, use of common pastures and water sources, age, breed, sex, lactation status and season are some other factors that may influence the prevalence of brucellosis (Junaidu et al., 2010;Bertu et al., 2012;Boukary et al., 2013).
Sylhet is a hilly and hoar (wetland ecosystem) based district situated in the northeastern part of Bangladesh, and several sub-districts share borders with the neighbouring country India. Many legal and illegal trades of cattle occur between India and Bangladesh through crossborder cattle movements in this area. This may increase the risk of brucellosis in cattle as it is a transboundary animal disease. Most of the farms in Sylhet region experienced a lot of abortion and stillbirth, which might be due to brucellosis. Unfortunately, information regarding the sero-prevalence and determinants of bovine brucellosis in the Sylhet district has remained unknown. Therefore, the present study aimed to explore the burden and identify the determinants of bovine brucellosis in the Sylhet district.

Study area
The study area includes all 12 sub-districts in Sylhet district, which are

Sample size determination
The target population includes local indigenous and crossbred cows from all 12 sub-districts of Sylhet district. The sample size was calculated by using the formula for estimating prevalence according to Thursfield (2005).
As no reliable prior information on the prevalence of bovine brucellosis in Sylhet was available, an expected prevalence (Pexp) of 50% was used to maximize the sample size. Using this Pexp with a desired absolute precision d = 0.05 and Z = 1.96 for a 95% confidence interval, a required sample size of at least 384 was determined.

Study design
A cross-sectional study was conducted for 6 months in the study area.
Lists of the farms were collected from the local sub-district livestock offices. A minimum of five farms from each sub-district and six cows from each farm were selected using a simple random sampling technique. However, in some sub-districts, it was difficult to select 5 farms to fulfil the inclusion criteria, and finally, 63 farms from 12 sub-districts were included in the study. Then, blood samples from the lactating cows of selected farms were collected to achieve the desired number of samples size. Finally, 386 serum samples from 63 selected farms were processed from the blood samples for this study.

Serum tested by buffered Brucella antigen tests (BBATs)
The protocols used for two BBATs; Rose Bengal Test (RBT) and Plate Agglutination Test for Brucella abortus (BPAT) were essentially the same described by Alton et al. (1988). First, the preserved serum samples were brought to room temperature from −20 • C and allowed to thaw. After thawing, an equal volume (30 μL) of serum and antigen (containing Bacterial suspension of Brucella organisms stained with Rose Bengal dye and buffered at pH 3.6 and 0.95 g/L of sodium aside) was taken in a glass slide. Then, by a small glass rod, the mixture of serum and antigen was allowed to rotate for 4 min. The result was considered positive when any agglutination was observed within the required period.

Serum agglutination test (SAT)
Serum agglutination test (SAT) was another serological test used to detect the antibody from the serum of Brucella infected animals, which was described by Rahman (2015) and Rahman et al. (2013). Briefly, 168 μL of Serum Agglutination de Wright (SAW) buffer in the 1st well, and 100 μL were added to the 2nd and 3rd well of 96 well plates. Then 32 μL of preserved serum samples were added to the 1st well (dilution 1/6.25). After mixing, transferred 100 μL from the 1st to 2nd well (dilution 1/12.5) and followed by the 3rd (dilution 1/25) and finally discarded 100 μL from 3rd well. Each serum sample was duplicated, and a total of 15 serum samples were loaded in the 96 wells microtitre plate.
Next, 100 μL of standardized SAW antigen was added to each well giving the serial dilution of 1/12.5, 1/25 and 1/50. Then the plates were incubated at 37 • C for 20-24 h. Reading was done based on agglutination and expressed as International Unit (IU). A sample was considered to be Brucella-positive if it contained ≥30 IU/mL.

Case definition
If a cow's serum sample showed a positive reaction in any of the three tests (RBT, BPAT, SAT), the cow was identified as positive for bovine brucellosis. On the other hand, if the serum sample of a cow has shown negative results in all three tests, then that cow was considered negative. However, in the case of farm level, if at least one cow from a particular farm was classified positive for bovine brucellosis, that farm was selected as bovine brucellosis-positive.

Data collection and management
The owners of the selected farms were interviewed face to face using a well-designed questionnaire to record the farm and cow-level information. The questionnaire included different types of farm and individual animal-level information related to risk factors. Type of house barn, sanitary condition, replacement stock, history of abortion, abnormal uterine discharge, retention of placenta, problem in heat and problemrelated to repeat breeding were associated with brucellosis were captured for both farm-and animal-level questionnaires to find out the relationship with the sero-positivity of brucellosis. Then collected individual level, farm level and laboratory information was initially entered into a Microsoft Excel spreadsheet and coded for analysis. Prelimi-nary descriptive analysis was done with 10 variables, including one pseudo-continuous variable (herd size) and nine categorical variables for estimating prevalence and identifying the determinants of bovine brucellosis. Herd size was categorized into two levels (≤20 and >20) categorical variable by taking the mean of the herd size as cut-off to make a homogenous distribution in each category. Furthermore, different stages of pregnant animals were found among the lactating cows and categorized as parity 1-4 (when the animals were in parity ≥4 were considered parity number four).

Statistical analysis
The results obtained from the cross-sectional study were used for prevalence and risk factors analysis. Prevalence was calcu-

Prevalence and distribution of bovine brucellosis in dairy animals
Out of 386 individual serum samples, 36 (9.33%), 46 (11.92%) and 58 (15.03%) were identified positive for bovine brucellosis TA B L E 1 Prevalence of bovine brucellosis in cows estimated from 386 randomly collected serum samples from 63 cattle farms of 12 sub-districts.

F I G U R E 1 Prevalence map of bovine brucellosis at cow level in Sylhet district estimated in a cross-sectional study.
lowest in Golapganj (3.70%; 95% CI: 1.45%-6.22%) sub-district ( Figure 1).

Farm-level prevalence
A total of 46 farms out of 63 had at least 1 cow showing a positive reaction against Brucella antigens and were considered positive farms; therefore, the prevalence of bovine brucellosis at the farm level was 73.02% with 95% CI: 60.97%-82.42%. Farm-level prevalence was significantly high in farms with the previous history of abortion and repeat breeding. It is worth noting that prevalence was comparatively higher in large farms (88.89%) than in small farms (64.44%). High prevalences at farm level were observed in almost all sub-districts of Sylhet district, and it ranged from 20% (95% CI: 11.56%-28.32%) to 100% (95% CI: 56.55%-100%). The highest farm prevalence was found in Gowainghat and Jaintapur sub-districts and the lowest in Golapganj sub-district ( Figure 2).

Determinants at cow level
The univariable association of each determinant with bovine brucellosis among dairy animals is shown in

Determinants at the farm level
In the univariable analysis, herd size and farms' history of repeat breeding had a significant association (

DISCUSSION
The study investigated the prevalence and risk factors of bovine brucellosis in Sylhet district of Bangladesh. The burden of bovine brucellosis may vary in the Sylhet region, but little is known about the prevalence, determinants and geographic disparities. Therefore, the findings of the current study are pivotal to identifying the risk factors and sub-districts with high prevalence so as to provide proper prevention and control measures to combat the bovine brucellosis burden.

Prevalence of bovine brucellosis
This study confirmed a high prevalence of bovine brucellosis in the dairy cows of Sylhet district. Similar to our findings, few other earlier studies in Bangladesh also reported such a high prevalence in individual cattle (Rahman et al., 2011;Rahman, 2015). In addition, the prevalence (20.5%) of brucellosis in Government Central Cattle Breeding and Dairy Farm (CCBDF) in Savar, Dhaka, Bangladesh reported by Rahman (2015) was comparable with the findings of the present study.
It is worth noting that Sylhet district is topographically a hoar based area with high rainfall, and most of the grazing lands remain damp for a considerable period of a year which might be a possible reason behind this observed high prevalence. Wetlands near the farm area were identified as a contributing factor to increasing prevalence in a study in Brazil (Borba et al., 2013). Moreover, Sylhet district shares a border with the neighbouring country India. Many legal and illegal cattle trade happen between India and Bangladesh through cross-border cattle movements. A previous study by Kubuafor et al. (2000) reported that cross-border cattle movement significantly impacts brucellosis transmission. However, on the other hand, the findings of the present study is in disagreement with Rahman et al. (2006), who reported low animal-level sero-prevalence of brucellosis in cattle with 2.4%-8.4%.
Another later investigation on bovine brucellosis in the Mymensingh region by Rahman (2015) recorded only 0.3% prevalence in cattle.
Geo-ecological differences along with the difference in study design could be a probable reason behind the differences in prevalence estimates.

Risk factors of bovine brucellosis
This study revealed that the risk of bovine brucellosis in dairy cows increased with increasing age (age was indicated by the number of parity), and the highest odds was observed in cows with ≥4 parity. The  Radostits et al., 2006;Mai et al., 2012;Aulakh et al., 2008). Age is one of the intrinsic factors which influence susceptibility to Brucella infection.
Brucellosis may increase with age due to prolonged exposure to the pathogen and prolonged antibody response duration in infected animals, particularly in traditional husbandry practice where females are maintained in herds over a long period of time.
In this study, it is found that cows with a history of abortion were significantly associated with Brucella sero-positivity, which is in agreement with other previous studies (Makita et al., 2011;Megersa et al., 2011;Patel et al., 2014). In addition, a previous study conducted by Magona et al. (2009) in Uganda also reported the history of abortion at the herd level as a significant factor for brucellosis. This might be due to the fact that the Brucella organism may present in the aborted foetus and play a pivotal role in the transmission of brucellosis through aborted materials and contaminated milk and urine. It is worth noting that the prevalence of brucellosis was comparatively similar in crossbred and local bred cows, and no significant association exists between breeds, as a similar result was documented by Mai et al. (2012). It might be due to the fact that genetic variations between breeds do not significantly influence brucellosis. Moreover, better management practices in the farms might be another possible factor for the low prevalence of brucellosis in both the crossbred and local bred.
It was observed that the previous history of repeat breeding of cow and farms was also significantly associated with bovine brucellosis at animal and farms level, respectively. A study in Gujarat reported that farms with a history of repeat breeding had a higher risk of brucellosis than farms without that history (Patel et al., 2014). This study also identified that cows' previous history of reproductive abnormalities other than abortion and repeat breeding, like stillbirth, retained placenta and so on increases the risk of brucellosis in individual cows. Moreover, Patel et al. (2014) also described a similar association between brucellosis and the history of repeat breeding and retained placenta, however, at herd level. Therefore, it is worth considering that there is a causal relationship between bovine brucellosis and reproductive abnormalities. However, it needs to be clarified, out of the scope of this study, whether the previous history of reproductive abnormalities increases the risk of cows being Brucella-positive or whether these previous episodes of reproductive abnormalities resulted from exposure to Brucella organisms.

CONCLUSION
The study showed the importance of direct contact with the aborted