Brucellosis in livestock: First study on seroepidemiology, risk factors, and preventive strategies to manage the disease in Famenin, Iran

Background and Aim: Brucellosis is an infectious disease in humans and livestock. The disease is endemic in many regions of Iran, for example, Hamedan Province. Knowledge of infection rate and associated risk factors is essential to control and prevent the disease. The study aimed to estimate the prevalence of brucellosis and associated risk factors in cattle, sheep, and goats in Famenin, Hamedan Province, West of Iran. Materials and Methods: Blood samples of 1758 animals (1470 sheep, 190 goats, and 98 cattle) were obtained in different rural regions of Famenin. The samples were evaluated to detect of Brucella-antibodies using rose Bengal plate test (RBPT), Wright standard tube agglutination test (SAT), and 2-Mercapto-Ethanol (2-ME) techniques. The risk factors associated with brucellosis such as age, gender, history of vaccination against brucellosis, and abortion history in animals were evaluated. In the sampling process, the critical gaps related to the distribution of brucellosis in the herds and regions are identified for designing the strategies to prevent and control the disease. Results: About 6.88% and 89.31% of animals had a history of abortion and vaccination against brucellosis, respectively. Most of the animals were female (92.49%) and in the range of 2–3 age old (39.8%). The antibodies to the Brucella-infection in animals were 2.73% with RBPT and 1.30% with SAT and 2-ME. The prevalence of brucellosis was detected 1.3% among individual animals and 11% among herds. This rate was 1.43% for sheep and 1.05% for goats, with no significant statistical difference. No seropositive case was detected in cattle samples using RBPT, STAT, and 2-ME. The highest rate of brucellosis (6.25%) was detected in Emamzadeh-Pirnahan region (22.2% goats and 5.6% sheep). In sheep, most cases of the disease were in 3–4 age-old group (1.92%), animals without a history of abortion (1.58%), and without a history of vaccination against brucellosis (2.80%). Furthermore, 5.94% of males and 1.11% of females were detected positive for brucellosis (p < 0.001). The chance of brucellosis in rams was 5.6 folds higher than in others (odds ratio = 5.64). Brucellosis in goats was detected 2.94% and 1.89% in the age groups <1 and 2–3 year-old. Furthermore, 1.22% of females and 1.34% of animals without a history of abortion were positive. Brucellosis was found in 0.61% of vaccinated and 3.85% of non-vaccinated goats. Except for gender in sheep, no significant statistical correlation (p > 0.05) was observed between prevalence of brucellosis and risk factors. In farmers, low level of information about the transmission and also control and preventive methods of the disease was dominant. Consumption of traditional and unpasteurized dairy products is also very common in the studied regions. Conclusion: This is a comprehensive evaluation of animal brucellosis parallel to humans’ cohort study in the Famenin region for the first time. Although the rate of brucellosis in animals is low in the region, explaining the risk factors to farmers, mass vaccination, regular screening of animals, and culling the positive animals are very important for controlling and reducing the disease in the region.


Introduction
Brucellosis is one of the most important zoonotic diseases in the world, especially in the Middle East and North Africa [1]. The disease is prevalent in many places, especially in developing countries. It has been a great impact on public health and economy [2]. The World Health Organization (WHO) reports that more than 500,000 new cases of brucellosis are recognized in humans on a global scale annually [3]. Brucellosis caused by the various species of Brucella, an intracellular coco-bacilli Gram-negative bacteria, can be transmitted to humans through consumption of unpasteurized dairy products and/or direct contact with secretory materials of infected animals [4]. Brucella melitensis and Brucella abortus are common species presenting a risk to humans and livestock. Brucella pinnipedialis, Brucella ceti, and Brucella microti are newly described species isolated from seals, dolphins, and wolves [5]. Brucellosis has multiple manifestations in the hosts and can affect the different organs in human. In animals such as livestock, Brucella has a high tendency to infect genital organs. The main clinical features in livestock is abortion, reproductive failures (in both gender), and reduction of milk production, which is a top manifestation in domestic animals [6]. Esmaeili [7] reports that 20% loss of milk production, 2-3 fold abortion, and 10% of infertility happen due to brucellosis in animals. Abortion related brucellosis is estimated to be between 30% and 80% in dairy herds with traditional management [8].
There are many reports of human disabilities, economic losses, and obligatory culling due to Brucellainfection in animals on a global scale. The economic losses due to these complications are very significant for farmers and at the national level in the countries where the disease is endemic. Hence, it needs careful attention of authorities [1]. There is a correlation between brucellosis in livestock and humans [6]. No treatment is recommended for brucellosis in animals; thus, culling is the best strategy to stop the transmission of the disease from animals to humans [9].
Brucellosis is an endemic zoonotic disease in many parts of Iran [10]. In Iran, the seven-year incidence rate of brucellosis (2011-2018) was 21.78% (95% confidence interval [CI]: 21.66-21.91%) [11]. Mirnejad et al. [3] reported the annual incidence of brucellosis is 0.001% in Iran. Unlike in many countries, the disease is a public health problem in Iran [7]. This problem is common in Hamedan Province, West of Iran, with an incidence rate of 31-41 per 100,000 people [12]. Consumption of non-pasteurized dairy products, especially from ewes, is common in most rural areas, so the prevalence of brucellosis is high compared to urban areas. Brucellosis usually occurs in grasslands at a moderate elevation and during spring, where sheep and goats are the dominant livestock [13]. In studies about Hamedan Province, 8.1% of veterinarians, 15% of abattoir staff, and 17% of butchers were reported to be Brucella-infection positive [14]. In addition, according to Gharekhani et al. [15], Gharekhani and Sazmand [16], 3.3% of dogs had antibodies to Brucella-infection. Interestingly, no antibody was detected in the studied horses.
For a comprehensive evaluation of brucellosis in the animal population of Famenin, Hamedan Province, we designed a project parallel to the human cohort study in this area [17,18]. This study aimed to estimate the prevalence and risk factors of brucellosis in sheep, goats, and cattle in this region.

Ethical approval
The study was approved by the ethics committee of Hamedan University of Medical Sciences (ID: IR.UMSHA.REC.1398.4).

Study period and location
The study was conducted from September 2020 to March 2021 in Famenin. Famenin is a region in the North-East of Hamedan province. Hamedan province, with an area of 19,546 km2 (34.77° N and 48.58° E) is located in the West part of Iran (Figure-1). The main occupation of the people in region is agriculture and animal husbandry. Much of animal husbandry is traditional in rural regions, and usually there are both sheep and goats in the herds. There are 37,000 cattle, 4,000 goats, and 101,000 sheep in Famenin.

Study design and sampling method
This study is part of a cohort project to estimate the prevalence of brucellosis and its risk factors among people and domestic animals in the Famenin region [17,18]. Considering the estimated prevalence of 2.5% of animal brucellosis in this region, taking into account the 95% confidence interval and also the margin of error equal to 25% of p, the estimated sample size was obtained 1167 samples using the Cochran formula [19]. Moreover, due to the fact that we used cluster sampling in the sampling stages, so the design effect (DE = 1.5) was included in the estimation of the sample size and finally, 1750 animals were studied. The method of allocating the estimated sample size to the covered population units was in accordance with Table-1.
The size of clusters was considered as 160 (150 sheep and goats and 10 cattle) and therefore, 11 clusters were selected according to the livestock population in each area. The number of samples from each location is shown in Table-2. The number of clusters and distribution in Famenin was based on the proportional number of livestock in each location to the total livestock of Famenin. A total of 1758 jugular blood samples (1470 sheep, 190 goats, and 98 cattle) were obtained by disposable needles and Venoject tubes using regular simple cluster sampling from 109 herds of eleven locations (A-K) in the studied area ( Figure-1).
In the sampling process, the demographic information of animals and the risk factors associated to brucellosis such as age (<1, 1-2, 2-3, and 3-4 year age-old groups), gender (Male or Female), history of vaccination against brucellosis (Yes or No), and abortion history (Yes or No) in animals was recorded after inserting an era tag (Figure-2 and Table-3). In addition, the critical gaps related to the distribution of brucellosis in the herds and regions are identified for designing the strategies to prevent and control the disease.

Serology
The sera samples were separated after whole blood centrifugation (1400× g for 12 min) and kept under −18°C until the examination [20]. At first, all samples were screened by rose Bengal plate test (RBPT). Then, the positive samples were re-evaluated using Wright standard tube agglutination test (SAT) and 2-Mercapto-Ethanol (2-ME) techniques. The final interpretation of serological results (positive or negative for brucellosis) was performed according to the protocol presented in Table-4 [20].

RBPT
An equal level (30 μL) of RBPT antigen (Vaccine and Serum Research Institute of Razi, Karaj, Iran) and sera samples were mixed by a disposable applicator on a white ceramic tile and shaken for 4 min. A positive result was reported after observing the pink agglutination [20][21][22].
Available at www.veterinaryworld.org/Vol.15/August-2022/26.pdf   Briefly, 0.8 mL of phosphate buffer saline (PBS) was dispensed to the first agglutination tube. Then, 0.5 mL PBS was applied to the 2 nd , 3 rd , 4 th , and 5 th tubes. In the next stage, 0.2 mL of sera sample was added to the 1 st tube and shaken correctly. Serial dilution was carried out by pipetting 0.5 mL of the mixture of the 1 st tube to 2 nd and then to other tubes. In the end, 0.5 mL of materials from the 5 th tube was thrown away. Then, 0.5 mL of Wright antigen with 1/10 concentration (Vaccine and Serum Research Institute of Razi) was added to the contents of all test tubes and incubated at 37°C for 24 h after shaking. The agglutination titers were recorded using guidelines [20][21][22].
Available at www.veterinaryworld.org/Vol.15/August-2022/26.pdf brucellosis in sheep was higher than in goats; but there was no significant difference between positive samples of sheep and goats (p = 0.67). Antibodies to Brucella-infection in animals were 2.73% using RBPT and 1.30% using SAT and 2-ME (Table-5). The prevalence rate regarding animals and different serologic techniques is summarized in Table-5. There were no cattle in some of the sampling locations (Table-2). In addition, there was not any confirmed brucellosis in locations of Asleh, Morgh-Abad, Sadeghloo, Sanaj, and Yengijeh. The highest rate of abortion was seen in Morgh-Abad and Yengijeh regions, with 25 cases in each location. The minimum belonged to Ghale-joogh and Sadeghloo regions, with no reported cases of abortion. Furthermore, most cases of abortion were seen in animals aged 2-3 years old. The highest rate of brucellosis (6.25%) was detected in Emamzadeh-Pirnahan region (22.2% goats and 5.6% sheep) ( Table-2).

Risk factors
Prevalence rate of brucellosis with different evaluated risk factors and also animal type is presented in Table-3.

Goats
In regard to serology, two out of 190 (1.05%) samples were positive for brucellosis. We could not calculate OR or the risk factors due to a low number of positive cases. Brucellosis was detected only in the age groups of less than 1 (2.94%, 1/34) and 2-3 yearold (1.89%, 1/53), female animals (1.22%, 2/164), and animals without a history of abortion (1.34%, 2/149). About 86.32% of goats had a history of vaccination

2-Mercapto-Ethanol
This technique is similar to SAT. A volume of 0.3 mL and 0.5 mL of PBS were dispensed in the first and other tubes, respectively. Then, 0.2 mL of sera sample was added to the first tube. In the experiment, we added 0.5 mL of 2-ME solution (Merck, Germany: 68 μl of 2-ME solution in 5 mL distilled water) to the first tube and shaken it completely and incubated it at 37°C for 1 h. Serial dilution was carried out by pipetting 0.5 mL of the mixture of the 1 st tube to 2 nd and then to the following tubes, respectively. In the end, 0.5 mL of materials from the 5 th tube was transferred out. Then, 0.5 mL of Wright antigen with 1/10 concentration (Vaccine and Serum Research Institute of Razi) was added to the contents of all test tubes and incubated at 37°C for 24 h after shaking. The agglutination titers were recorded using guidelines [20][21][22].

Statistical analysis
The qualitative data were summarized with frequencies and percentages and the quantitative variables with the mean and the standard deviation. The association between demographic variables of the animals with their disease status was assessed by Chi-square test. Moreover, the effect of gender on brucellosis disease was assessed using the logistic regression model. Data were analyzed using Stata software version 14 (www.stata.com/stata14). The significant level was considered <0.05.

Prevention gaps
Most of farmers in the studied regions did not have enough information about the transmission and also control and preventive methods of the disease. Consumption of traditional and unpasteurized dairy products is also very common.

Discussion
The most important concern about brucellosis is health. It is also responsible for significant economic losses in the livestock industry, especially in endemic areas. Brucellosis is one of the most important infectious diseases in Iran [1]. All domestic and wild animals as well as pets, act as the reservoir of transmitting the Brucella-infection in humans [6]. Brucellosis is transmitted through both vertical and horizontal ways in animals. There is a high concentration of Brucella in the vaginal discharge of infected animals [23]. Risk factors and epidemiological data on brucellosis are needed for designing a comprehensive program to prevent and control it in any region [20]. In Iran, most sheep and goats are bred traditionally with few hygienic guidelines. Farmers use mobile and semi-mobile herd management practices and move periodically among different pasture areas and seasonally. In some countries with a high incidence of brucellosis, the frequent and unrestricted transfer of animals, especially within the national and international borders, poses a problem for the control of the disease. Therefore, international collaborations are needed to improve border security. Furthermore, in this region, the trade of live animals is much more common than the trade of animal products due to religious slaughtering practices and Muslim festivals such as Hajj, Ramadan, Eid ul-Fitr, and Eid ul-Adha. Animal transferring and slaughtering during this time result in increased direct contact with animals, posing a risk of transmitting zoonotic diseases to humans.
There are different reports on animal brucellosis in Iran. With regard to the animal population in Iran, much work has been concentrated on cattle followed by sheep, goats, camel, and buffalo. Furthermore, the species of B. melitensis, B. abortus, and co-infection of B. melitensis and B. abortus are more dominant, respectively [1]. Initially, the rate of brucellosis among Iranian cattle was reported to be 17.6% [24]. Recent study [22] shows that seroprevalence of cattle brucellosis in different regions of Iran is 5.6%, 3.9%, and 4.9% using RBPT, SAT, and enzyme-linked immunosorbent assay, respectively. In addition, the prevalence of brucellosis in animals in rural areas is higher than peri-urban areas [22]. In a meta-epidemiological investigation by Dadar et al. [1], brucellosis was calculated to affect 10.18% of Iranian livestock; this rate was 2.2-5.7% in Hamedan Province. Regarding animal species, brucellosis rate is reported 14.66% in cattle, 12.83% in sheep, 11.97% in buffalo, 4.34% in goats, and 3.39% in camels. The infection rate in animals with clinical manifestations (38.65%) is significantly higher than in healthy animals (8.38%).
The RBPT, a rapid, simple, low-cost, and userfriendly method, is used for screening and detecting the antibodies to Brucella-infection such as immunoglobulin (Ig) M, IgG, and IgA. SAT and 2-ME techniques are applied routinely to detect the titer of antibodies in non-vaccinated and vaccinated animals, respectively. In serological tests, SAT remained popular and is used on a global scale [8]. We also used these serological methods based on previous studies. Using serological methods, we detected 1.3% and 11% of brucellosis in individual animals and herds, respectively. This rate was reported 1.43% and 1.05% for sheep and goats, respectively. In the cohort study conducted in Famenin, antibodies to Brucellainfection were 6.6% and 3.5% using SAT and 2-ME techniques, respectively [18]. About 43.4% of the positive cases were confirmed by molecular tools and 28.5% were positive for the species of B. melitensis and B. abortus [17]. In this work, contact with domestic animals and consumption of non-pasteurized dairy products, especially curd, were strong risk factors for brucellosis [2].
In our study, the infection rate in sheep was more than in goats (p > 0.05). In a previous report Available at www.veterinaryworld.org/Vol.15/August-2022/26.pdf from Hamedan Province [20], the brucellosis rate was 3% and 4.6% in sheep and goats, respectively. In addition, the infection rate in goats was significantly higher than in sheep (OR = 1.8). Reviriego et al. [36] and Suryawanshi et al. [37] results are different from the present study. In contrast, some of the researchers reported equal levels of the infection rate in sheep and goat flocks [38,39].
The principal manifestation of brucellosis in livestock is abortion and also genitally disorders. Usually, shedding of the infected materials occurs after delivery. Therefore, the secretory materials are so harmful to public health [6] because of releasing highly infectious uterine secretions during the abortion and shortly after that could be an important source of contamination of the environment, grounds, and pastures that can spread the infection to other animals and humans [17]. About 0.93% of ewes with abortion history were positive for brucellosis; while all of the goats' sample (n = 15) with a history of abortion was negative. In Iran, 14.2-25.7% of animals with a history of abortion were positive for Brucella-infection (p < 0.05) [1]. In Gharekhani et al. [20] investigation, the rate of brucellosis in livestock with an abortion history was significantly high (p < 0.05). Furthermore, the researchers confirmed a strong correlation between brucellosis and the incidence of abortion in animals such as sheep and goats [1]. An outbreak of abortion can lead to more incidence of Brucella-infection in animals and also farmers [10]. A relationship between abortion and prevalence rate of brucellosis was reported in different investigations [1]. In our work, the prevalence of brucellosis in animals with a history of abortion was not statistically significant. The finding highlighted the role of other risk factors (infectious and/or non-infectious) for abortion. Furthermore, farmers' information may also be incomplete while obtaining the livestock history due to the traditional way of animal husbandry in the region.
In the case of brucellosis, the importance of producing preventive immunity in animals has always been considered. However, as the development of active immunity in humans is still in the experimental stages, and control of the disease in humans is possible by limiting the spread of infection in animals [6]. Vaccination is one of the most effective and practical methods for controlling brucellosis, which is applied successfully in most endemic areas in the world [4]. In our work, the brucellosis rate was less common in vaccinated livestock, parallel to the previous report in this area [20]. Fortunately, the rate of vaccination in Famenin was approximately 90% that might be better in the future. As seen in this study, vaccination could be one of the most important factors in decreasing the rate of infection among livestock. In Iran, vaccination with attenuated B. melitensis Rev.1 strain is used for immunization of sheep and goats and RB51 (attenuated B. abortus) for cows. The full and reduced doses were injected into the heifers or lambs and adults. They have been suggested as safe and effective approaches to eliminate brucellosis in ruminants [1]. In Emamzadeh-Pirnahan, one of the studied area, brucellosis was high compared to other locations. Therefore, preventive measures, especially vaccination of livestock against brucellosis in this area, should be prioritized compared to other regions.
In our findings, the prevalence of brucellosis in sheep aged 3-4 years was high compared to other groups. There was a different level of infection in age groups in sheep and goats with no significant statistical differences, similar to Teklue et al. [40] reports from Ethiopia. The results of age impact as a risk factor for brucellosis are high in relation to sample size and method of sampling. Negash et al. [39] believe that the infection rate in younger animals is high due to the low level of antibodies in the immune system. The chance of infection increases parallel to the age of animals; therefore, some researchers reported high infection in adult animals [41]. The age of animals was also reported as a significant risk option in the other works [10].
Brucellosis rate in rams was 5.6 times higher than in ewes in the Famenin region in accordance with a previous report in Hamedan Province [20]. Kiros et al [8] reportedthat brucellosis in female animals was 2.1 folds higher than in males. Furthermore, in a meta-analysis project from Iran, the prevalence rate in female animals (8.7-13.3%) was significantly more than in males (5.6-11.2%) [1]. In pregnant animals, Erythritol (an alcoholic material) is used in the fetus that provides suitable conditions for the growth of Brucella [20]. In Iran, male animals are less immune to brucellosis due to a lack of vaccination in some areas; therefore, males are more susceptible than females. Males are often used to mate, so they have a significant role in transmitting the infection.
In Gharekhani et al. [15] study from Hamedan, all of the milk samples from dairy farm tested were negative for Brucella-infection using molecular methods. Unlike this, wild and vaccinated of Brucella strains were isolated in milk samples of dairy farms in different regions of Iran [10]. However, vaccination against brucellosis must be the main goal in endemic regions because contact with livestock is a strong risk factor for human brucellosis [1,22].
Detection of mixed infections of B. abortus and B. melitensis could be related to keeping different species of livestock together [10]. Farmers should avoid keeping different livestock together which is common in Iran. In ZareBidaki et al.'s [10] report, the presence of non-vaccinated and different types of animals on the farm, increase of animals' age, incidence of abortion, and contact of livestock with wild animals were strong risk options for brucellosis. This may be due to differences in study design and protocols, type of samples and sample size, laboratory techniques, geographical regions, ecological factors, livestock density, and herd management [20]. To reduce brucellosis in the human community, the infection should be eliminated in reservoir hosts. In addition, using Available at www.veterinaryworld.org/Vol.15/August-2022/26.pdf the vaccination and test-and-slaughter projects are so effective for this purpose.
To prevent the disease, the following 11 practical points were taught to the local farmers, who were mostly illiterate: 1. Vaccinate all of your livestock regularly 2. Keep livestock species (e.g., sheep and cattle) and different genders (i.e., rams and ewes) separately 3. Allocate specific pastures for each flock 4. Do not cross-move livestock between flocks 5. Test the rams for brucellosis regularly 6. Avoid slaughtering out of the registered abattoir 7. Remove the aborted/dead fetuses as soon as observed to avoid their consumption by carnivores, and destroy them preferably by incineration 8. Slaughter livestock at younger ages to decrease the infection risk 9. Gradually switch from traditional to industrial husbandry systems with higher hygienic measures 10. Avoid consumption of unpasteurized milk and dairy products 11. Cull the infected animals from the herds.

Conclusion
This was a comprehensive evaluation of animals' brucellosis parallel to humans' cohort study in the region for the first time. We developed a program to cull positive animals in all of the sampled locations to cut the chain of infection transmission. The rate of brucellosis in animals was low in comparison to previous reports. Furthermore, cattle have no significant role in transmitting the infection to humans in the area. Educating farmers to learn about a better definition of risk factors, mass vaccination, regular screening of animals, and culling the positive animals are highly recommended. These are very effective in controlling and decrease the rate of the disease and risk factors. Future studies with emphasis on molecular methods are proposed to determine circulating strains in the region.